56 results on '"Deborah J. Kuhn"'
Search Results
2. Supplementary Table 3 from Drug Resistance to Inhibitors of the Human Double Minute-2 E3 Ligase Is Mediated by Point Mutations of p53, but Can Be Overcome with the p53 Targeting Agent RITA
- Author
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Robert Z. Orlowski, Deborah J. Kuhn, Veerabhadran Baladandayuthapani, Chad C. Bjorklund, and Richard J. Jones
- Abstract
PDF file, 53K, p53 mutations in MI63R and NutlinR cells.
- Published
- 2023
3. Supplementary Table 1 from Drug Resistance to Inhibitors of the Human Double Minute-2 E3 Ligase Is Mediated by Point Mutations of p53, but Can Be Overcome with the p53 Targeting Agent RITA
- Author
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Robert Z. Orlowski, Deborah J. Kuhn, Veerabhadran Baladandayuthapani, Chad C. Bjorklund, and Richard J. Jones
- Abstract
PDF file, 65K, IC50 values of MI63R and NutlinR cell lines to HDM-2 Inhibitors.
- Published
- 2023
4. Data from Inhibition of Interleukin-6 Signaling with CNTO 328 Enhances the Activity of Bortezomib in Preclinical Models of Multiple Myeloma
- Author
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Robert Z. Orlowski, Jeffrey A. Nemeth, Mohamed H. Zaki, Robert E. Corringham, John S. Strader, Sally A. Hunsucker, George W. Small, Deborah J. Kuhn, Qing Chen, and Peter M. Voorhees
- Abstract
Purpose: Inhibition of the proteasome leads to the activation of survival pathways in addition to those that promote cell death. We hypothesized that down-regulation of interleukin-6 (IL-6) signaling using the monoclonal antibody CNTO 328 would enhance the antitumor activity of the proteasome inhibitor bortezomib in multiple myeloma by attenuating inducible chemoresistance.Experimental Design: The cytotoxicity of bortezomib, CNTO 328, and the combination, along with the associated molecular changes, was assessed in IL-6–dependent and IL-6–independent multiple myeloma cell lines, both in suspension and in the presence of bone marrow stromal cells and in patient-derived myeloma samples.Results: Treatment of IL-6–dependent and IL-6–independent multiple myeloma cell lines with CNTO 328 enhanced the cytotoxicity of bortezomib in a sequence-dependent fashion. This effect was additive to synergistic and was preserved in the presence of bone marrow stromal cells and in CD138+ myeloma samples derived from patients with relative clinical resistance to bortezomib. CNTO 328 potentiated bortezomib-mediated activation of caspase-8 and caspase-9 and the common downstream effector caspase-3; attenuated bortezomib-mediated induction of antiapoptotic heat shock protein-70, which correlated with down-regulation of phosphorylated signal transducer and activator of transcription-1; and inhibited bortezomib-mediated accumulation of myeloid cell leukemia-1, an effect that was associated with down-regulation of phosphorylated signal transducer and activator of transcription-3.Conclusions: Taken together, our results provide a strong preclinical rationale for the clinical development of the bortezomib/CNTO 328 combination for patients with myeloma.
- Published
- 2023
5. Supplementary Figures S1-S2 from Inhibition of Interleukin-6 Signaling with CNTO 328 Enhances the Activity of Bortezomib in Preclinical Models of Multiple Myeloma
- Author
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Robert Z. Orlowski, Jeffrey A. Nemeth, Mohamed H. Zaki, Robert E. Corringham, John S. Strader, Sally A. Hunsucker, George W. Small, Deborah J. Kuhn, Qing Chen, and Peter M. Voorhees
- Abstract
Supplementary Figures S1-S2 from Inhibition of Interleukin-6 Signaling with CNTO 328 Enhances the Activity of Bortezomib in Preclinical Models of Multiple Myeloma
- Published
- 2023
6. Targeting the insulin-like growth factor-1 receptor to overcome bortezomib resistance in preclinical models of multiple myeloma
- Author
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Donna M. Weber, Caimiao Wei, R. Eric Davis, Deborah J. Kuhn, Zuzana Berkova, Timothy Madden, Sheeba K. Thomas, Veerabhadran Baladandayuthapani, Robert Z. Orlowski, Richard Woessner, Michael Wang, Hua Wang, Chad C. Bjorklund, Richard J. Jones, Jatin J. Shah, Wencai Ma, and Pei Lin
- Subjects
medicine.medical_treatment ,Blotting, Western ,Immunology ,Antineoplastic Agents ,Apoptosis ,Enzyme-Linked Immunosorbent Assay ,Mice, SCID ,Pharmacology ,Biochemistry ,Receptor, IGF Type 1 ,Bortezomib ,Mice ,Paracrine signalling ,Insulin-like growth factor ,immune system diseases ,hemic and lymphatic diseases ,Antineoplastic Combined Chemotherapy Protocols ,Biomarkers, Tumor ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,Insulin-Like Growth Factor I ,Autocrine signalling ,Receptor ,Multiple myeloma ,Cell Proliferation ,Oligonucleotide Array Sequence Analysis ,Lymphoid Neoplasia ,biology ,Cell growth ,Gene Expression Profiling ,Imidazoles ,Drug Synergism ,Cell Biology ,Hematology ,Flow Cytometry ,medicine.disease ,Boronic Acids ,Xenograft Model Antitumor Assays ,Survival Rate ,Insulin receptor ,Drug Resistance, Neoplasm ,Pyrazines ,biology.protein ,Multiple Myeloma ,medicine.drug - Abstract
Proteasome inhibition with bortezomib is a validated approach to the treatment of multiple myeloma, but drug resistance often emerges and limits its utility in the retreatment setting. To begin to identify some of the mechanisms involved, we developed bortezomib-resistant myeloma cell lines that, unlike previously reported models, showed no β5 subunit mutations. Instead, up-regulation of the insulin-like growth factor (IGF)–1 axis was identified, with increased autocrine and paracrine secretion of IGF-1, leading to increased activation of the IGF-1 receptor (IGF-1R). Exogenous IGF-1 reduced cellular sensitivity to bortezomib, whereas pharmacologic or small hairpin RNA–mediated IGF-1R suppression enhanced bortezomib sensitivity in cell lines and patient samples. In vitro studies with OSI-906, a clinically relevant dual IGF-1R and insulin receptor inhibitor, showed it acted synergistically with bortezomib, and potently resensitized bortezomib-resistant cell lines and patient samples to bor-tezomib. Importantly, OSI-906 in combination with bortezomib also overcame bor-tezomib resistance in an in vivo model of myeloma. Taken together, these data support the hypothesis that signaling through the IGF-1/IGF-1R axis contributes to acquired bortezomib resistance, and provide a rationale for combining bortezomib with IGF-1R inhibitors like OSI-906 to overcome or possibly prevent the emergence of bortezomib-refractory disease in the clinic.
- Published
- 2012
7. In Vitro and In Vivo Selective Antitumor Activity of a Novel Orally Bioavailable Proteasome Inhibitor MLN9708 against Multiple Myeloma Cells
- Author
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Paul G. Richardson, Ze Tian, Kenneth C. Anderson, Bin Zhou, Robert Z. Orlowski, Noopur Raje, Deborah J. Kuhn, and Dharminder Chauhan
- Subjects
Boron Compounds ,Proteasome Endopeptidase Complex ,Cancer Research ,Cell Survival ,medicine.drug_class ,Blotting, Western ,Glycine ,Administration, Oral ,Biological Availability ,Antineoplastic Agents ,Apoptosis ,Mice, SCID ,Pharmacology ,Biology ,Article ,Dexamethasone ,Ixazomib ,Bortezomib ,Mice ,chemistry.chemical_compound ,In vivo ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Lenalidomide ,Multiple myeloma ,Cell Proliferation ,Caspase 8 ,Caspase 3 ,Histone deacetylase inhibitor ,NF-kappa B ,Drug Synergism ,medicine.disease ,Boronic Acids ,Xenograft Model Antitumor Assays ,Caspase 9 ,Thalidomide ,Enzyme Activation ,Oncology ,chemistry ,Proteasome ,Pyrazines ,Proteasome inhibitor ,Growth inhibition ,Multiple Myeloma ,Proteasome Inhibitors ,medicine.drug - Abstract
Purpose: The success of bortezomib therapy for treatment of multiple myeloma (MM) led to the development of structurally and pharmacologically distinct novel proteasome inhibitors. In the present study, we evaluated the efficacy of one such novel orally bioactive proteasome inhibitor MLN9708/MLN2238 in MM using well-established in vitro and in vivo models. Experimental Design: MM cell lines, primary patient cells, and the human MM xenograft animal model were used to study the antitumor activity of MN2238. Results: Treatment of MM cells with MLN2238 predominantly inhibits chymotrypsin-like activity of the proteasome and induces accumulation of ubiquitinated proteins. MLN2238 inhibits growth and induces apoptosis in MM cells resistant to conventional and bortezomib therapies without affecting the viability of normal cells. In animal tumor model studies, MLN2238 is well tolerated and inhibits tumor growth with significantly reduced tumor recurrence. A head-to-head analysis of MLN2238 versus bortezomib showed a significantly longer survival time in mice treated with MLN2238 than mice receiving bortezomib. Immununostaining of MM tumors from MLN2238-treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. Mechanistic studies showed that MLN2238-triggered apoptosis is associated with activation of caspase-3, caspase-8, and caspase-9; increase in p53, p21, NOXA, PUMA, and E2F; induction of endoplasmic reticulum (ER) stress response proteins Bip, phospho-eIF2-α, and CHOP; and inhibition of nuclear factor kappa B. Finally, combining MLN2238 with lenalidomide, histone deacetylase inhibitor suberoylanilide hydroxamic acid, or dexamethasone triggers synergistic anti-MM activity. Conclusion: Our preclinical study supports clinical evaluation of MLN9708, alone or in combination, as a potential MM therapy. Clin Cancer Res; 17(16); 5311–21. ©2011 AACR.
- Published
- 2011
8. Second Generation Proteasome Inhibitors: Carfilzomib and Immunoproteasome-Specific Inhibitors (IPSIs)
- Author
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Deborah J. Kuhn, Robert Z. Orlowski, and Chad C. Bjorklund
- Subjects
Proteasome Endopeptidase Complex ,Cancer Research ,Antineoplastic Agents ,Pharmacology ,Biology ,chemistry.chemical_compound ,In vivo ,Neoplasms ,Drug Discovery ,medicine ,Animals ,Humans ,Protease Inhibitors ,Multiple myeloma ,Bortezomib ,Cell cycle ,medicine.disease ,Carfilzomib ,Oncology ,Proteasome ,chemistry ,Apoptosis ,Proteasome inhibitor ,Oligopeptides ,Proteasome Inhibitors ,medicine.drug - Abstract
The ubiquitin-proteasome pathway (UPP) is an attractive chemotherapeutic target due to its intrinsically stringent regulation of cell cycle, pro-survival, and anti-apoptotic regulators that disproportionately favor survival and proliferation in malignant cells. A reversible first-in-class proteasome inhibitor, bortezomib, is Food and Drug Administration approved for multiple myeloma and relapsed/refractory mantle cell lymphoma and has proven to be extremely effective, both as a single agent and in combination. An irreversible second generation proteasome inhibitor, carfilzomib, has shown preclinical effectiveness against hematological and solid malignancies both in vitro and in vivo. Carfilzomib, a peptidyl-epoxyketone functions similarly to bortezomib through primary inhibition of chymotrypsin-like (ChT-L) activity at the b5 subunits of the core 20S proteasome. Carfilzomib is also currently achieving successful response rates within the clinical setting. In addition to conventional proteasome inhibitors, a novel approach may be to specifically target the hematological-specific immunoproteasome, thereby increasing overall effectiveness and reducing negative off-target effects. The immunoproteasome-specific inhibitor, IPSI-001, was shown to have inhibitory preference over the constitutive proteasome, and display enhanced efficiency of apoptotic induction of tumor cells from a hematologic origin. Herein, we discuss the preclinical and clinical development of carfilzomib and explore the potential of immunoproteasome-specific inhibitors, like IPSI-001, as a rational approach to exclusively target hematological malignancies.
- Published
- 2011
9. Blockade of interleukin-6 signalling with siltuximab enhances melphalan cytotoxicity in preclinical models of multiple myeloma
- Author
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Mark Cornfeld, Jeffrey A. Nemeth, Steven M. Kornblau, Deborah J. Kuhn, Jatin J. Shah, Donna M. Weber, Robert Z. Orlowski, Peter M. Voorhees, Michael Wang, Sheeba K. Thomas, Valeria Magarotto, Hong Xie, and Sally A. Hunsucker
- Subjects
Melphalan ,medicine.medical_treatment ,Hematology ,Biology ,medicine.disease ,Siltuximab ,chemistry.chemical_compound ,Cytokine ,chemistry ,hemic and lymphatic diseases ,medicine ,Cancer research ,Protein kinase B ,PI3K/AKT/mTOR pathway ,Monoclonal gammopathy of undetermined significance ,Bcl-2 Homologous Antagonist-Killer Protein ,Multiple myeloma ,medicine.drug - Abstract
Signalling through the interleukin (IL)-6 pathway induces proliferation and drug resistance of multiple myeloma cells. We therefore sought to determine whether the IL-6-neutralizing monoclonal antibody siltuximab, formerly CNTO 328, could enhance the activity of melphalan, and to examine some of the mechanisms underlying this interaction. Siltuximab increased the cytotoxicity of melphalan in KAS-6/1, INA-6, ANBL-6, and RPMI 8226 human myeloma cell lines (HMCLs) in an additive-to-synergistic manner, and sensitized resistant RPMI 8226.LR5 cells to melphalan. These anti-proliferative effects were accompanied by enhanced activation of drug-specific apoptosis in HMCLs grown in suspension, and in HMCLs co-cultured with a human-derived stromal cell line. Siltuximab with melphalan enhanced activation of caspase-8, caspase-9, and the downstream effector caspase-3 compared with either of the single agents. This increased induction of cell death occurred in association with enhanced Bak activation. Neutralization of IL-6 also suppressed signalling through the phosphoinositide 3-kinase/Akt pathway, as evidenced by decreased phosphorylation of Akt, p70 S6 kinase and 4E-BP1. Importantly, the siltuximab/melphalan regimen demonstrated enhanced anti-proliferative effects against primary plasma cells derived from patients with myeloma, monoclonal gammopathy of undetermined significance, and amyloidosis. These studies provide a rationale for translation of siltuximab into the clinic in combination with melphalan-based therapies.
- Published
- 2011
10. Inhibition of Interleukin-6 Signaling with CNTO 328 Enhances the Activity of Bortezomib in Preclinical Models of Multiple Myeloma
- Author
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Deborah J. Kuhn, Jeffrey A. Nemeth, Peter M. Voorhees, Mohamed H. Zaki, Robert Corringham, Sally A. Hunsucker, George W. Small, John S. Strader, Qing Chen, and Robert Z. Orlowski
- Subjects
STAT3 Transcription Factor ,Cancer Research ,Stromal cell ,Myeloid ,Cell ,Antineoplastic Agents ,Apoptosis ,Bone Marrow Cells ,DNA Fragmentation ,Biology ,Bortezomib ,immune system diseases ,hemic and lymphatic diseases ,medicine ,Humans ,HSP70 Heat-Shock Proteins ,Multiple myeloma ,Interleukin-6 ,Antibodies, Monoclonal ,medicine.disease ,Boronic Acids ,Gene Expression Regulation, Neoplastic ,STAT1 Transcription Factor ,medicine.anatomical_structure ,Oncology ,Proteasome ,Drug Resistance, Neoplasm ,Pyrazines ,Proteasome inhibitor ,Cancer research ,Immunotherapy ,Syndecan-1 ,Bone marrow ,Multiple Myeloma ,Signal Transduction ,medicine.drug - Abstract
Purpose: Inhibition of the proteasome leads to the activation of survival pathways in addition to those that promote cell death. We hypothesized that down-regulation of interleukin-6 (IL-6) signaling using the monoclonal antibody CNTO 328 would enhance the antitumor activity of the proteasome inhibitor bortezomib in multiple myeloma by attenuating inducible chemoresistance.Experimental Design: The cytotoxicity of bortezomib, CNTO 328, and the combination, along with the associated molecular changes, was assessed in IL-6–dependent and IL-6–independent multiple myeloma cell lines, both in suspension and in the presence of bone marrow stromal cells and in patient-derived myeloma samples.Results: Treatment of IL-6–dependent and IL-6–independent multiple myeloma cell lines with CNTO 328 enhanced the cytotoxicity of bortezomib in a sequence-dependent fashion. This effect was additive to synergistic and was preserved in the presence of bone marrow stromal cells and in CD138+ myeloma samples derived from patients with relative clinical resistance to bortezomib. CNTO 328 potentiated bortezomib-mediated activation of caspase-8 and caspase-9 and the common downstream effector caspase-3; attenuated bortezomib-mediated induction of antiapoptotic heat shock protein-70, which correlated with down-regulation of phosphorylated signal transducer and activator of transcription-1; and inhibited bortezomib-mediated accumulation of myeloid cell leukemia-1, an effect that was associated with down-regulation of phosphorylated signal transducer and activator of transcription-3.Conclusions: Taken together, our results provide a strong preclinical rationale for the clinical development of the bortezomib/CNTO 328 combination for patients with myeloma.
- Published
- 2007
11. Proteasome inhibitors and modulators of heat shock protein function
- Author
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Deborah J. Kuhn, Robert Z. Orlowski, and Erik L. Zeger
- Subjects
MAPK/ERK pathway ,Cancer Research ,medicine.diagnostic_test ,Bortezomib ,Proteolysis ,NF-κB ,Biology ,medicine.disease ,Bioinformatics ,chemistry.chemical_compound ,Oncology ,Proteasome ,chemistry ,Heat shock protein ,medicine ,Proteasome inhibitor ,Cancer research ,Multiple myeloma ,medicine.drug - Abstract
The proteasome is a multi-catalytic proteinase complex that is integral to intracellular proteolysis, and plays a key role in many critical functions. Inhibition of the proteasome induces cell cycle arrest and apoptosis in a variety of tumor cell types in vitro and in vivo, thus making it an attractive therapeutic agent for the treatment of cancer. Bortezomib (VELCADE®; formerly PS-341) is the first proteasome inhibitor to enter into clinical practice. Phases I–III clinical trials have shown bortezomib and proteasome inhibition to be well tolerated. Impressive anti-tumor activity against relapsed and refractory multiple myeloma has been seen, leading to the approval of bortezomib for this indication, where it seems poised to change the natural history of the disease. Additional studies are ongoing to define its role in initial therapy of myeloma, as well as in other areas, such as non-Hodgkin's lymphoma. While bortezomib has displayed less activity as a single agent against solid tumors, new research into combination regimens with other chemotherapeutics holds promise to increase efficacy against both solid tumors and hematologic malignancies. Heat shock proteins (HSPs) are molecular chaperones that interact with the ubiquitin-proteasome pathway, and function to aid in de novo protein folding and to stabilize key intracellular signaling proteins. Many tumors overexpress high-affinity HSPs that form unusually stable interactions with oncogenic client proteins, while normal, adjacent tissues only exhibit a latent, low-affinity form. Phase I/II clinical trials are ongoing with two potent HSP-90 inhibitors, which have so far been well tolerated in patients with some activity in hematologic malignancies. Drugs that modulate protein turnover through the proteasome, and protein folding through HSPs, seem destined to form an important part of our therapeutic armamentarium against a variety of malignancies.
- Published
- 2006
12. Anti-Angiogenic and Anti-Tumor Properties of Proteasome Inhibitors
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Qing Ping Dou, Aslamuzzaman Kazi, Deborah J. Kuhn, and Kenyon G. Daniel
- Subjects
Cancer Research ,Angiogenesis ,Lactacystin ,Angiogenesis Inhibitors ,Antineoplastic Agents ,Pharmacology ,Biology ,Metastasis ,Bortezomib ,Neovascularization ,chemistry.chemical_compound ,Neoplasms ,Drug Discovery ,medicine ,Humans ,Protease Inhibitors ,Neovascularization, Pathologic ,medicine.disease ,Boronic Acids ,Acetylcysteine ,medicine.anatomical_structure ,Oncology ,chemistry ,Proteasome ,Apoptosis ,Pyrazines ,Cancer research ,medicine.symptom ,Proteasome Inhibitors ,medicine.drug ,Blood vessel - Abstract
Tumor growth and metastasis depend on the formation of blood vessels, angiogenesis, to supply the developing mass with nutrients, oxygen, and waste removal. The proteasome, a massive multisubunit catabolic body, exerts a regulatory influence on angiogenesis. Inhibition of the proteasome activity has been found to inhibit angiogenesis and induce apoptosis in human cancer cells with limited toxicity to normal cells. Therefore, the dual action of angiogenesis inhibition and cell death induction makes proteasome inhibition an attractive modality for chemotherapy. A variety of proteasome inhibitors have been studied including: antibiotics such as lactacystin, the green tea polyphenols, and the boronic acid Velcade (MLN-341). Most recently, certain classes of copper compounds have been found to act as potent proteasome inhibitors. The potential of particular organic compounds, such as 8-hydroxyquinoline, to spontaneously bind with tumor cellular copper and form proteasome inhibitors provides a new modality of anti-proteasome and anti-angiogenesis chemotherapy. This review examines angiogenesis, the proteasome, representative proteasome inhibitors, and the emerging role of copper. The formation of new blood vessels, or angiogenesis, is an important and necessary function in both embryonic development and wound repair. Therefore, the ability to regenerate or form new vessels for blood flow is essential. The control of angiogenic pathways is tightly regulated in normal differentiated adult cells, which generally do not stimulate blood vessel growth unless injury occurs. However, cancerous tissues stimulate angiogenesis that in turn leads to increased tumor formation and possible metastases. Many of the factors involved in angiogenesis are regulated by the proteasome, which recently has become a focus in anti-cancer therapies due to its involvement in cell cycle and apoptosis control. Here we discuss angiogenesis and its relation to the proteasome. Additionally, current modalities of anti-angiogenic treatment, mainly proteasome inhibitory strategies, are reviewed. Furthermore, proteasome inhibitors, both natural and synthetic, and their anti-angiogenic effects as well as future approaches to anti-angiogenic chemotherapies are also discussed.
- Published
- 2005
13. A potential prodrug for a green tea polyphenol proteasome inhibitor: evaluation of the peracetate ester of (−)-epigallocatechin gallate [(−)-EGCG]
- Author
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Deborah J. Kuhn, Albert S. C. Chan, Larry M.C. Chow, Aslamuzzaman Kazi, Wai Har Lam, Q. Ping Dou, and Tak Hang Chan
- Subjects
Proteasome Endopeptidase Complex ,Clinical Biochemistry ,Flavonoid ,Drug Evaluation, Preclinical ,Pharmaceutical Science ,Epigallocatechin gallate ,complex mixtures ,Biochemistry ,Catechin ,Jurkat Cells ,chemistry.chemical_compound ,Phenols ,Drug Discovery ,medicine ,Humans ,Prodrugs ,Protease Inhibitors ,heterocyclic compounds ,Peracetic Acid ,Molecular Biology ,Flavonoids ,chemistry.chemical_classification ,Tea ,biology ,Organic Chemistry ,Polyphenols ,food and beverages ,Esters ,Biological activity ,Prodrug ,chemistry ,Enzyme inhibitor ,Polyphenol ,biology.protein ,Proteasome inhibitor ,Molecular Medicine ,sense organs ,Proteasome Inhibitors ,medicine.drug - Abstract
Green tea has been shown to have many biological effects, including effects on metabolism, angiogenesis, oxidation, and cell proliferation. Unfortunately, the most abundant green tea polyphenol (-)-epigallocatechin gallate or (-)-EGCG is very unstable in neutral or alkaline medium. This instability leads to a low bioavailability. In an attempt to enhance the stability of (-)-EGCG, we introduced peracetate protection groups on the reactive hydroxyls of (-)-EGCG (noted in text as 1). HPLC analysis shows that the protected (-)-EGCG analog is six times more stable than natural (-)-EGCG under slightly alkaline conditions. A series of bioassays show that 1 has no inhibitory activity against a purified 20S proteasome in vitro, but exhibits increased proteasome-inhibitory activity in intact leukemic cells over natural (-)-EGCG, indicating an intercellular conversion. Inhibition of cellular proteasome activity by 1 is associated with induction of cell death. Therefore, our results indicate that the protected analog 1 may function as a prodrug of the green tea polyphenol proteasome inhibitor (-)-EGCG.
- Published
- 2004
14. Novel N-thiolated β-lactam antibiotics selectively induce apoptosis in human tumor and transformed, but not normal or nontransformed, cells
- Author
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Timothy E. Long, Aslamuzzaman Kazi, Q. Ping Dou, Randy J Hill, Deborah J. Kuhn, and Edward Turos
- Subjects
Male ,Programmed cell death ,DNA damage ,Cell ,Apoptosis ,Biology ,beta-Lactams ,Biochemistry ,Jurkat cells ,Jurkat Cells ,chemistry.chemical_compound ,Tumor Cells, Cultured ,polycyclic compounds ,medicine ,Humans ,Fibroblast ,Cell Line, Transformed ,Pharmacology ,Prostatic Neoplasms ,Fibroblasts ,Cell Transformation, Viral ,Killer Cells, Natural ,medicine.anatomical_structure ,chemistry ,Cancer cell ,Cancer research ,Lactam - Abstract
Historically, it has been shown that the β-lactam antibiotics play an essential role in treating bacterial infections while demonstrating selectivity for prokaryotic cells. We recently reported that certain N -methylthio-substituted β-lactam antibiotics had DNA-damaging and apoptosis-inducing activities in various tumor cells. However, whether these compounds affect human normal or nontransformed cells was unknown. In the current study, we first show that a lead compound (lactam 1 ) selectively induces apoptosis in human leukemic Jurkat T, but not in the nontransformed, immortalized human natural killer (NK) cells. Additionally, we screened a library of other N -methylthiolated β-lactams to determine their structure–activity relationships (SARs), and found lactam 12 to have the highest apoptosis-inducing activity against human leukemic Jurkat T cells, associated with increased DNA-damaging potency. Furthermore, we demonstrate that lactam 12 , as well as lactam 1 , potently inhibits colony formation of human prostate cancer cells. We also show that lactam 12 induces apoptosis in human breast, prostate, and head-and-neck cancer cells. Finally, lactam 12 induces apoptosis selectively in Jurkat T and simian virus 40-transformed, but not in nontransformed NK and parental normal fibroblast, cells. Our results suggest that there is potential for developing this class of β-lactams into novel anticancer agents.
- Published
- 2004
15. The Immunoproteasome as a Target in Hematologic Malignancies
- Author
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Robert Z. Orlowski and Deborah J. Kuhn
- Subjects
Proteasome Endopeptidase Complex ,Bortezomib ,Antineoplastic Agents ,Hematology ,Pharmacology ,Biology ,medicine.disease ,Carfilzomib ,Article ,Ixazomib ,Clinical trial ,chemistry.chemical_compound ,Therapeutic index ,chemistry ,Proteasome ,Hematologic Neoplasms ,medicine ,Cancer research ,Animals ,Humans ,Mantle cell lymphoma ,Proteasome Inhibitors ,Multiple myeloma ,medicine.drug - Abstract
Suppression of proteasome function with the first-in-class small molecule inhibitor bortezomib is a rational therapeutic strategy against several hematologic malignancies, including multiple myeloma and mantle cell lymphoma. Second-generation inhibitors such as carfilzomib, ixazomib, and marizomib that, like bortezomib, target both the constitutive proteasome and the immunoproteasome, are also in clinical trials and showing encouraging activity. While the efficacy of these agents is well documented, toxicities associated with their use, such as peripheral neuropathy and gastrointestinal effects, can necessitate dose reductions or even discontinuations, possibly hampering their anti-neoplastic effects. These findings suggested that it could be possible to improve the therapeutic index of this class of drugs by specifically targeting only the immunoproteasome. Since the immunoproteasome is a unique target found in lymphoid-derived cells, immunoproteasome-specific inhibitors (IPSIs) could preserve efficacy while reducing treatment-emergent toxicities since they would spare other tissues with little to no immunoproteasome expression. This review discusses the current state of development of IPSIs, and the potential of using such agents for the treatment of hematologic malignancies.
- Published
- 2012
16. Blockade of interleukin-6 signalling with siltuximab enhances melphalan cytotoxicity in preclinical models of multiple myeloma
- Author
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Sally A, Hunsucker, Valeria, Magarotto, Deborah J, Kuhn, Steven M, Kornblau, Michael, Wang, Donna M, Weber, Sheeba K, Thomas, Jatin J, Shah, Peter M, Voorhees, Hong, Xie, Mark, Cornfeld, Jeffrey A, Nemeth, and Robert Z, Orlowski
- Subjects
Dose-Response Relationship, Drug ,Cell Survival ,Interleukin-6 ,Plasma Cells ,Antibodies, Monoclonal ,Apoptosis ,Article ,Neoplasm Proteins ,bcl-2 Homologous Antagonist-Killer Protein ,hemic and lymphatic diseases ,Antineoplastic Combined Chemotherapy Protocols ,Tumor Cells, Cultured ,Humans ,Drug Screening Assays, Antitumor ,Multiple Myeloma ,Melphalan ,Proto-Oncogene Proteins c-akt ,BH3 Interacting Domain Death Agonist Protein ,Signal Transduction ,bcl-2-Associated X Protein - Abstract
Signalling through the interleukin (IL)-6 pathway induces proliferation and drug resistance of multiple myeloma cells. We therefore sought to determine whether the IL-6-neutralizing monoclonal antibody siltuximab, formerly CNTO 328, could enhance the activity of melphalan, and to examine some of the mechanisms underlying this interaction. Siltuximab increased the cytotoxicity of melphalan in KAS-6/1, INA-6, ANBL-6, and RPMI 8226 human myeloma cell lines (HMCLs) in an additive-to-synergistic manner, and sensitized resistant RPMI 8226.LR5 cells to melphalan. These anti-proliferative effects were accompanied by enhanced activation of drug-specific apoptosis in HMCLs grown in suspension, and in HMCLs co-cultured with a human-derived stromal cell line. Siltuximab with melphalan enhanced activation of caspase-8, caspase-9, and the downstream effector caspase-3 compared with either of the single agents. This increased induction of cell death occurred in association with enhanced Bak activation. Neutralization of IL-6 also suppressed signalling through the phosphoinositide 3-kinase/Akt pathway, as evidenced by decreased phosphorylation of Akt, p70 S6 kinase and 4E-BP1. Importantly, the siltuximab/melphalan regimen demonstrated enhanced anti-proliferative effects against primary plasma cells derived from patients with myeloma, monoclonal gammopathy of undetermined significance, and amyloidosis. These studies provide a rationale for translation of siltuximab into the clinic in combination with melphalan-based therapies.
- Published
- 2011
17. Evidence of a role for activation of Wnt/beta-catenin signaling in the resistance of plasma cells to lenalidomide
- Author
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R. Eric Davis, Jatin J. Shah, Zhiqiang Wang, Michael Wang, Steven M. Kornblau, Deborah J. Kuhn, Wencai Ma, Chad C. Bjorklund, and Robert Z. Orlowski
- Subjects
Beta-catenin ,Transcription, Genetic ,Plasma Cells ,Antineoplastic Agents ,Biology ,Biochemistry ,Gene Expression Regulation, Enzymologic ,Proto-Oncogene Proteins c-myc ,Wnt3 Protein ,Glycogen Synthase Kinase 3 ,GSK-3 ,Cell Line, Tumor ,Wnt3A Protein ,medicine ,Humans ,Cyclin D1 ,Enhancer ,Molecular Biology ,Lenalidomide ,Multiple myeloma ,beta Catenin ,Glycogen Synthase Kinase 3 beta ,Wnt signaling pathway ,Casein Kinase Ialpha ,Cell Biology ,medicine.disease ,Thalidomide ,Gene Expression Regulation, Neoplastic ,Wnt Proteins ,Drug Resistance, Neoplasm ,Cancer research ,biology.protein ,Casein kinase 1 ,Signal transduction ,Multiple Myeloma ,medicine.drug ,Signal Transduction - Abstract
Lenalidomide plays an important role in our chemotherapeutic armamentarium against multiple myeloma, in part by exerting direct anti-proliferative and pro-apoptotic effects. Unfortunately, long-term exposure leads to the development of drug resistance through unknown mechanisms, and we therefore sought to identify pathways that could be responsible for this phenotype. Chronic drug exposure produced myeloma cell lines that were tolerant of the direct effects of lenalidomide, with a degree of resistance of up to 2,500-fold. Gene expression profiling and pathway analysis identified dysregulation of the Wnt/β-catenin pathway as a consistent change across four independent cell isolates, and a pair of primary plasma cell samples. Acute drug treatment also increased β-catenin transcription by 3-fold or more, and both acute and chronic exposure resulted in enhanced accumulation of β-catenin protein by up to 20-fold or more. This produced Wnt/β-catenin pathway activation, as judged by increased activity of a lymphoid enhancer factor/T-cell factor promoter reporter, and enhanced accumulation of the downstream targets cyclin D1 and c-Myc. Components of the β-catenin destruction complex were also impacted by lenalidomide, which suppressed casein kinase 1α expression while augmenting glycogen synthase kinase 3α/β phosphorylation. Stimulation of Wnt/β-catenin signaling with recombinant Wnt-3a, or by overexpression of β-catenin, reduced the anti-proliferative activity of lenalidomide. Conversely, suppression of β-catenin with small hairpin RNAs restored plasma cell sensitivity to lenalidomide. Together, these findings support the hypothesis that lenalidomide mediates activation of Wnt/β-catenin signaling in plasma cells as a mechanism of inducible chemoresistance through effects at the transcriptional and post-translational levels.
- Published
- 2010
18. Bortezomib and EGCG: no green tea for you?
- Author
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Jatin J. Shah, Deborah J. Kuhn, and Robert Z. Orlowski
- Subjects
Oncology ,Gerontology ,medicine.medical_specialty ,Chemotherapy ,business.industry ,Bortezomib ,Extramural ,medicine.medical_treatment ,Immunology ,MEDLINE ,Cell Biology ,Hematology ,Green tea ,Biochemistry ,Internal medicine ,hemic and lymphatic diseases ,Inside Blood ,medicine ,business ,medicine.drug - Abstract
In this issue of Blood, Golden and colleagues report findings that indicate patients undergoing bortezomib-containing chemotherapy should avoid consuming green tea products.
- Published
- 2009
19. Targeted inhibition of interleukin-6 with CNTO 328 sensitizes pre-clinical models of multiple myeloma to dexamethasone-mediated cell death
- Author
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Peter M. Voorhees, Robert Z. Orlowski, Qing Chen, Jeffrey A. Nemeth, Deborah J. Kuhn, Sally A. Hunsucker, and George W. Small
- Subjects
Programmed cell death ,medicine.medical_treatment ,Dexamethasone ,Article ,Bortezomib ,Recurrence ,Cell Line, Tumor ,medicine ,polycyclic compounds ,Humans ,Protease Inhibitors ,Interleukin 6 ,Protein kinase B ,Glucocorticoids ,Multiple myeloma ,biology ,Cell Death ,Interleukin-6 ,Interleukin ,Antibodies, Monoclonal ,Drug Synergism ,Hematology ,medicine.disease ,Boronic Acids ,Cytokine ,Apoptosis ,Drug Resistance, Neoplasm ,Pyrazines ,biology.protein ,Cancer research ,Multiple Myeloma ,hormones, hormone substitutes, and hormone antagonists ,medicine.drug - Abstract
Interleukin (IL)-6-mediated signaling attenuates the anti-myeloma activity of glucocorticoids (GCs). We therefore sought to evaluate whether CNTO 328, an anti-IL-6 monoclonal antibody in clinical development, could enhance the apoptotic activity of dexamethasone (dex) in pre-clinical models of myeloma. CNTO 328 potently increased the cytotoxicity of dex in IL-6-dependent and –independent human myeloma cell lines (HMCLs), including a bortezomib-resistant HMCL. Isobologram analysis revealed that the CNTO 328/dex combination was highly synergistic. Addition of bortezomib to CNTO 328/dex further enhanced the cytotoxicity of the combination. Experiments with pharmacologic inhibitors revealed a role for the p44/42 mitogen-activated protein kinase pathway in IL-6-mediated GC resistance. Although CNTO 328 alone induced minimal cell death, it potentiated dex-mediated apoptosis, as evidenced by increased activation of caspases-8, -9, and -3, Annexin-V staining, and DNA fragmentation. The ability of CNTO 328 to sensitize HMCLs to dex-mediated apoptosis was preserved in the presence of human bone marrow stromal cells. Importantly, the increased activity of the combination was also seen in plasma cells from patients with GC-resistant myeloma. Taken together, our data provide a strong rationale for the clinical development of the CNTO 328/dex regimen for patients with myeloma.
- Published
- 2009
20. Targeted inhibition of the immunoproteasome is a potent strategy against models of multiple myeloma that overcomes resistance to conventional drugs and nonspecific proteasome inhibitors
- Author
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Sally A. Hunsucker, Deborah J. Kuhn, Marian Orlowski, Peter M. Voorhees, Qing Chen, and Robert Z. Orlowski
- Subjects
medicine.medical_specialty ,Umbilical Veins ,Immunology ,Immunoblotting ,Anti-Inflammatory Agents ,Apoptosis ,Drug resistance ,Biochemistry ,Binding, Competitive ,Dexamethasone ,Bortezomib ,Ubiquitin ,Internal medicine ,medicine ,Humans ,Protease Inhibitors ,RNA, Messenger ,Multiple myeloma ,Cells, Cultured ,Hematology ,biology ,Chromosomes, Human, Pair 13 ,Reverse Transcriptase Polymerase Chain Reaction ,Drug Synergism ,Cell Biology ,Dipeptides ,medicine.disease ,Boronic Acids ,In vitro ,Pancreatic Neoplasms ,Proteasome ,Drug Resistance, Neoplasm ,Pyrazines ,Proteasome inhibitor ,biology.protein ,Cancer research ,Endothelium, Vascular ,Chromosome Deletion ,Multiple Myeloma ,Proteasome Inhibitors ,medicine.drug - Abstract
Proteasome inhibition is a validated strategy for therapy of multiple myeloma, but this disease remains challenging as relapses are common, and often associated with increasing chemoresistance. Moreover, nonspecific proteasome inhibitors such as bortezomib can induce peripheral neuropathy and other toxicities that may compromise the ability to deliver therapy at full doses, thereby decreasing efficacy. One novel approach may be to target the immunoproteasome, a proteasomal variant found predominantly in cells of hematopoietic origin that differs from the constitutive proteasome found in most other cell types. Using purified preparations of constitutive and immunoproteasomes, we screened a rationally designed series of peptidyl-aldehydes and identified several with relative specificity for the immunoproteasome. The most potent immunoproteasome-specific inhibitor, IPSI-001, preferentially targeted the β1i subunit of the immunoproteasome in vitro and in cellulo in a dose-dependent manner. This agent induced accumulation of ubiquitin-protein conjugates, proapoptotic proteins, and activated caspase-mediated apoptosis. IPSI-001 potently inhibited proliferation in myeloma patient samples and other hematologic malignancies. Importantly, IPSI-001 was able to overcome conventional and novel drug resistance, including resistance to bortezomib. These findings provide a rationale for the translation of IPSIs to the clinic, where they may provide antimyeloma activity with greater specificity and less toxicity than current inhibitors.
- Published
- 2008
21. Targeting the p27 E3 ligase SCFSkp2 results in p27- and Skp2-mediated cell-cycle arrest and activation of autophagy
- Author
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Deborah J. Kuhn, Keiko Nakayama, David R. Webb, Peter M. Voorhees, Qing Chen, Laure Moutouh de Parseval, Veronique Plantevin Krenitsky, Antonia Lopez-Girona, Robert Z. Orlowski, Weilin Xie, Derek Mendy, Laura G. Corral, Weiming Xu, Frank Mercurio, and Keiichi I. Nakayama
- Subjects
Programmed cell death ,Ubiquitin-Protein Ligases ,Immunology ,Antineoplastic Agents ,Biochemistry ,Drug Delivery Systems ,Cell Line, Tumor ,Autophagy ,medicine ,Humans ,S-Phase Kinase-Associated Proteins ,chemistry.chemical_classification ,DNA ligase ,biology ,Neoplasia ,Bortezomib ,Cell Cycle ,Cell Biology ,Hematology ,Cell cycle ,Ubiquitin ligase ,Cell biology ,Cell killing ,Proteasome ,chemistry ,Drug Resistance, Neoplasm ,biology.protein ,Cancer research ,Proteasome inhibitor ,Multiple Myeloma ,Cyclin-Dependent Kinase Inhibitor p27 ,medicine.drug - Abstract
Decreased p27Kip1 levels are a poor prognostic factor in many malignancies, and can occur through up-regulation of SCFSkp2 E3 ligase function, resulting in enhanced p27 ubiquitination and proteasome-mediated degradation. While proteasome inhibitors stabilize p27Kip1, agents inhibiting SCFSkp2 may represent more directly targeted drugs with the promise of enhanced efficacy and reduced toxicity. Using high-throughput screening, we identified Compound A (CpdA), which interfered with SCFSkp2 ligase function in vitro, and induced specific accumulation of p21 and other SCFSkp2 substrates in cells without activating a heat-shock protein response. CpdA prevented incorporation of Skp2 into the SCFSkp2 ligase, and induced G1/S cell-cycle arrest as well as SCFSkp2- and p27-dependent cell killing. This programmed cell death was caspase-independent, and instead occurred through activation of autophagy. In models of multiple myeloma, CpdA overcame resistance to dexamethasone, doxorubicin, and melphalan, as well as to bortezomib, and also acted synergistically with this proteasome inhibitor. Importantly, CpdA was active against patient-derived plasma cells and both myeloid and lymphoblastoid leukemia blasts, and showed preferential activity against neoplastic cells while relatively sparing other marrow components. These findings provide a rational framework for further development of SCFSkp2 inhibitors as a novel class of antitumor agents.
- Published
- 2008
22. Potent activity of carfilzomib, a novel, irreversible inhibitor of the ubiquitin-proteasome pathway, against preclinical models of multiple myeloma
- Author
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Mark K. Bennett, Asher A. Chanan-Khan, Kevin D. Shenk, Qing Chen, Fijs W. B. van Leeuwen, Robert Z. Orlowski, Susan Demo, John S. Strader, Congcong M. Sun, Peter M. Voorhees, and Deborah J. Kuhn
- Subjects
Immunology ,Drug Evaluation, Preclinical ,Antineoplastic Agents ,Apoptosis ,Pharmacology ,Caspase 8 ,Biochemistry ,Models, Biological ,Bortezomib ,chemistry.chemical_compound ,medicine ,Tumor Cells, Cultured ,Humans ,Protease Inhibitors ,Caspase ,Multiple myeloma ,Cell Proliferation ,biology ,Neoplasia ,Ubiquitin ,Cell Biology ,Hematology ,medicine.disease ,Carfilzomib ,Boronic Acids ,chemistry ,Proteasome ,Drug Resistance, Neoplasm ,Pyrazines ,Proteasome inhibitor ,biology.protein ,Cancer research ,Multiple Myeloma ,Oligopeptides ,Proteasome Inhibitors ,medicine.drug ,Signal Transduction - Abstract
The proteasome has emerged as an important target for cancer therapy with the approval of bortezomib, a first-in-class, reversible proteasome inhibitor, for relapsed/refractory multiple myeloma (MM). However, many patients have disease that does not respond to bortezomib, whereas others develop resistance, suggesting the need for other inhibitors with enhanced activity. We therefore evaluated a novel, irreversible, epoxomicin-related proteasome inhibitor, carfilzomib. In models of MM, this agent potently bound and specifically inhibited the chymotrypsin-like proteasome and immunoproteasome activities, resulting in accumulation of ubiquitinated substrates. Carfilzomib induced a dose- and time-dependent inhibition of proliferation, ultimately leading to apoptosis. Programmed cell death was associated with activation of c-Jun-N-terminal kinase, mitochondrial membrane depolarization, release of cytochrome c, and activation of both intrinsic and extrinsic caspase pathways. This agent also inhibited proliferation and activated apoptosis in patient-derived MM cells and neoplastic cells from patients with other hematologic malignancies. Importantly, carfilzomib showed increased efficacy compared with bortezomib and was active against bortezomib-resistant MM cell lines and samples from patients with clinical bortezomib resistance. Carfilzomib also overcame resistance to other conventional agents and acted synergistically with dexamethasone to enhance cell death. Taken together, these data provide a rationale for the clinical evaluation of carfilzomib in MM.
- Published
- 2007
23. Methylation suppresses the proteasome-inhibitory function of green tea polyphenols
- Author
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Deborah J. Kuhn, Richard Wiegand, Tak Hang Chan, Kristin R. Landis-Piwowar, Sheng Biao Wan, and Q. Ping Dou
- Subjects
Programmed cell death ,Physiology ,Clinical Biochemistry ,Apoptosis ,Biology ,complex mixtures ,Jurkat cells ,Methylation ,Catechin ,chemistry.chemical_compound ,Jurkat Cells ,Phenols ,In vivo ,Anticarcinogenic Agents ,Humans ,Protease Inhibitors ,Flavonoids ,Cell Death ,Tea ,food and beverages ,Polyphenols ,Cell Biology ,Molecular biology ,Biochemistry ,Proteasome ,chemistry ,Proteasome Inhibitors ,Intracellular - Abstract
Under physiological conditions, biotransformation reactions, such as methylation, can modify green tea polyphenols (GTPs) and therefore limit their in vivo cancer-preventive activity. Although a recent study suggested that methylated polyphenols are less cancer-protective, the molecular basis is unknown. We previously reported that ester bond-containing GTPs, for example (-)-epigallocatechin-3-gallate [(-)-EGCG] or (-)-epicatechin-3-gallate [(-)-ECG], potently and specifically inhibit the proteasomal chymotrypsin-like activity. In this study, we hypothesize that methylated GTPs have decreased proteasome-inhibitory abilities. To test this hypothesis, methylated (-)-EGCG and (-)-ECG analogs that can be found in vivo were synthesized and studied for their structure-activity relationships (SARs) using a purified 20S proteasome. The addition of a single methyl group on (-)-EGCG or (-)-ECG led to decreased proteasome inhibition and, as the number of methyl groups increased, the inhibitory potencies further decreased. These SARs were supported by our findings from in silico docking analysis published recently. Previously, we synthesized a peracetate-protected (-)-EGCG molecule, Pro-EGCG (1), to enhance its cellular permeability and stability, and current HPLC analysis confirms conversion of Pro-EGCG (1) to (-)-EGCG in cultured human leukemic Jurkat T cells. Furthermore, in this study, peracetate-protected forms of methylated GTPs were added in intact Jurkat T cells to observe the intracellular effects of methylation. Peracetate-protected, monomethylated (-)-EGCG induced greater cellular proteasome inhibition and apoptosis than did peracetate-protected, trimethylated (-)-EGCG, consistent with the potencies of the parent methylated analogs against a purified 20S proteasome. Therefore, methylation on GTPs, under physiological conditions, could decrease their proteasome-inhibitory activity, contributing to decreased cancer-preventive effects of tea consumption.
- Published
- 2007
24. Comparison of Free Light Chain Assay with Protein Electrophoresis for Screening and Monitoring PTLD
- Author
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Jie Xuan, Barbara Savoldo, Zheng Cao, Ping Wang, Irene Shu, and Deborah J. Kuhn
- Subjects
Immunofixation ,medicine.medical_specialty ,Pathology ,medicine.medical_treatment ,Immunology ,Population ,Liver transplantation ,Biochemistry ,Gastroenterology ,hemic and lymphatic diseases ,Gammopathy ,Internal medicine ,medicine ,education ,education.field_of_study ,biology ,medicine.diagnostic_test ,business.industry ,Cell Biology ,Hematology ,Gel electrophoresis of proteins ,Immunoglobulin M ,Serum protein electrophoresis ,Monoclonal ,biology.protein ,business - Abstract
Background: Post-transplant lymphoproliferative disorder (PTLD) is primarily diagnosed histologically using tissue biopsy. Free light chain (FLC) assay and serum protein electrophoresis (SPE) have both been studied as tools to screen and monitor PTLD. However, limited data are available to compare these two assays in a well characterized patient population. It is also not clear what reference ranges should be adopted for the FLC assay in a post-transplant population. Method: Blood samples from 169 patients receiving a variety of solid organ transplants were analyzed for FLCs and screened for gammopathies by SPE/IFE. Results: Compared with non-PTLD patients, PTLD patients had higher mean, median and upper 95 percentile range of both κ and λ FLCs (p ranging from 0.0002 to 0.024). The mean, median and 95 percentile range of κ:λ ratio were similar between the two groups. PTLD patients were more likely to have polyclonal or monoclonal FLC elevations (p = 0.04). They also showed a higher frequency of gammopathy abnormalities (p = 0.0052). Nonetheless, neither FLC assay nor SPE demonstrated a clear association with the timing of PTLD diagnosis. FLC concentrations in non-PTLD recipients were higher than those in the general healthy population (95 percentile range: κ, 0.60-8.33 mg/dL vs. 0.33-1.94 mg/dL; λ, 0.77-7.08 mg/dL vs. 0.571-2.63 mg/dL) but the κ:λ ratio was similar to that of the healthy group (0.26-1.65). Conclusions: Our results suggested that elevated FLC concentrations and gammopathy abnormalities were both associated with PTLD. Therefore, FLC assay and SPE should be used conjunctively for screening PTLD among solid organ transplant recipients. For this application, the data showed that a higher upper limit of κ and λ FLC levels and normal κ:λ ratio should be used as diagnostic reference ranges. Additionally, neither method was clearly associated with the timing of PTLD diagnosis, indicating that they may be unsuitable for monitoring PTLD in the post-transplant population. Table 1. Longitudinal measurements of serum/plasma free light chains and SPE/IFE in eight PTLD cases. Type of transplant and type of PTLD Samples Days from PTLD diagnosisa κ FLC, mg/dL λ FLC, mg/dL κ /λ SPE/IFE Liver transplant, B-cell PTLD 1.1 1.2 – 616 2.33b 1.82 4.96 6.01 0.47 0.303 no band –c Liver transplant, B-cell PTLD 2.1 2.2 2.3 2.4 2.5 -145* -126 84 141 428 0.338 0.79 0.335 0.476 2.53 0.62 1.02 1.06 1.15 1.98 0.545 0.775 0.316 0.414 1.28 no band no band 1 IgG ©µ, 1 IgG λ 1 IgG ©µ, 1 IgG λ no band Liver transplant, polymorphic hyperplasia 3.1 3.2 -77 208 4.19 7.18 5.9 3.38 0.71 2.12 2 IgG ©µ, 2 λ FLC– Liver transplant, B-cell PTLD 4.1 4.2 4.3 61 272 537 4.64 3.25 7.1 11.1 6.65 10.96 0.418 0.489 0.648 no band no band no band Liver transplant, B-cell PTLD 5.1 5.2 5.3 5.4 5.5 9 12 393 429 476 305.5 957 0.721 1.01 1.24 74.25 192 1.67 1.72 2.56 4.11 4.98 0.432 0.587 0.484 1 IgG ©µ, 1 λ FLC 2 IgM ©µ no band no band no band Liver transplant, B-cell PTLD 6.1 6.2 6.3 6.4 62 153 174 188 3.16 5.58 3.97 2.14 2.92 3.91 3.81 3.37 1.08 1.43 1.04 0.635 – 1 IgG ©µ 1 IgG ©µ 1 IgG ©µ Kidney transplant, PTLD 7.1 7.2 15 30 1.4 1.68 1.58 2.06 0.89 0.82 no band no band Lung transplant, Non-Hodgkin lymphoma 8.1 -60 4.28 4.58 0.94 no band a Positive values indicate time points before PTLD diagnosis, while negative values indicate time points after PTLD diagnosis. b Numbers in bold format indicates values above ULN. c SPE/IFE results not available due to insufficient sample volume. Disclosures Kuhn: The Binding Site, Inc: Employment.
- Published
- 2014
25. Evaluation of proteasome-inhibitory and apoptosis-inducing potencies of novel (-)-EGCG analogs and their prodrugs
- Author
-
Deborah J. Kuhn, Sheng Biao Wan, Q. Ping Dou, Tak Hang Chan, Di Chen, and Kristin R. Landis-Piwowar
- Subjects
Cell ,food and beverages ,General Medicine ,Cell cycle ,Biology ,Prodrug ,In vitro ,Cytosol ,medicine.anatomical_structure ,Proteasome ,Biochemistry ,Cell culture ,Apoptosis ,Genetics ,medicine - Abstract
The anti-cancer and cancer-preventive effects of green tea and its main constituent (-)-epigallocatechin gallate [(-)-EGCG] are well documented by a variety of studies, including epidemiological, cell culture, animal, and clinical studies. While (-)-EGCG remains the most potent polyphenol in green tea, it is very unstable in neutral or alkaline conditions (i.e. physiologic pH). In an effort to discover more stable polyphenol proteasome inhibitors, we synthesized several novel (-)-EGCG analogs with -OH groups eliminated from the B- and/or D-rings. In addition, we also synthesized their putative prodrugs with -OH groups protected by peracetate that can be removed by cellular cytosolic esterases. We first examined the structure-activity relationship of these unprotected and protected compounds to their proteasome-inhibitory potentials. We found that decreasing -OH groups from either the B- or D-ring leads to diminished proteasome-inhibitory activity in vitro. However, in cultured tumor cells only the protected analogs were capable of potently inhibiting the proteasome activity. Furthermore, these protected analogs induced apoptotic cell death in a tumor cell-specific manner. The superior efficacy of the protected (-)-EGCG analogs indicates the formation of an entirely new compound(s) in intact tumor cells. These data suggest that the B-ring/D-ring peracetate-protected EGCG analogs have great potential to be developed into novel anti-cancer and cancer-preventive agents.
- Published
- 2005
26. Direct inhibition of interleukin-2 receptor alpha-mediated signaling pathway induces G1 arrest and apoptosis in human head-and-neck cancer cells
- Author
-
Q. Ping Dou and Deborah J. Kuhn
- Subjects
Interleukin 2 ,Cyclin A ,Cell ,Down-Regulation ,Apoptosis ,Biochemistry ,Daclizumab ,Cell Line, Tumor ,medicine ,Humans ,Enzyme Inhibitors ,Molecular Biology ,biology ,G1 Phase ,Interleukin-2 Receptor alpha Subunit ,Janus Kinase 3 ,Cell Biology ,Receptors, Interleukin ,Cell cycle ,Protein-Tyrosine Kinases ,Cell biology ,medicine.anatomical_structure ,Proto-Oncogene Proteins c-bcl-2 ,Head and Neck Neoplasms ,Cancer cell ,biology.protein ,Carcinoma, Squamous Cell ,Signal transduction ,medicine.drug ,Signal Transduction - Abstract
Overexpression of the interleukin-2 receptor (IL-2R) alpha chain in tumor cells is associated with tumor progression and a poor patient prognosis. IL-2Ralpha is responsible for the high affinity binding of the receptor to IL-2, leading to activation of several proliferative and anti-apoptotic intracellular signaling pathways. We have previously shown that human squamous cell carcinoma of a head-and-neck line (PCI-13) genetically engineered to overexpress IL-2Ralpha exhibit increased transforming activity, proliferation, and drug resistance, compared to the vector control cells (J Cell Biochem 2003;89:824-836). In this study, we report that IL-2Ralpha(+) cells express high levels of total and phosphorylated Jak3 protein and are more resistant to apoptosis induced by a Jak3 inhibitor than the control LacZ cells. Furthermore, we used daclizumab, a monoclonal antibody specific to IL-2Ralpha, and determined the effects of IL-2Ralpha inhibition on cell cycle and apoptosis as well as the involvement of potential cell cycle and apoptosis regulatory proteins. We found that daclizumab induces G(1) arrest, associated with down-regulation of cyclin A protein, preferentially in IL-2Ralpha(+) cells, but not in LacZ cells. In addition, daclizumab activates apoptotic death program via Bcl-2 down-regulation preferentially in IL-2Ralpha(+) cells. Finally, daclizumab also sensitizes IL-2Ralpha(+) cells to other apoptotic stimuli, although the effect is moderate. These results indicate that daclizumab inhibits the proliferative potential of IL-2Ralpha(+) cells via inhibition of cell cycle progression and induction of apoptosis.
- Published
- 2005
27. Structure-activity relationships OF N-methylthiolated beta-lactam antibiotics with C3 substitutions and their selective induction of apoptosis in human cancer cells
- Author
-
Edward Turos, Jeung Yeop Shim, Di Chen, Q. Ping Dou, Kenyon G. Daniel, Yang Wang, Cristina Coates, Fred R. Miller, Vesna Minic, Kristin R. Landis-Piwowar, G. Suresh Kumar Reddy, and Deborah J. Kuhn
- Subjects
medicine.drug_class ,Drug discovery ,Antibiotics ,Apoptosis ,beta-Lactams ,Anti-Bacterial Agents ,Cell membrane ,chemistry.chemical_compound ,Structure-Activity Relationship ,medicine.anatomical_structure ,Biochemistry ,chemistry ,Cell culture ,medicine ,Lactam ,Tumor Cells, Cultured ,Potency ,Humans ,Fibroblast - Abstract
The development of novel anti-cancer drugs that induce apoptosis has long been a focus of drug discovery. Beta-lactam antibiotics have been used for over 60 years to fight bacterial infectious diseases with little or no side effects observed. Recently a new class of N-methylthiolated beta-lactams has been discovered that have potent activity against methicillin resistant Staphylococcus aureas. Most recently, we determined the potential effects of these N-thiolated beta-lactams on tumorigenic cell growth and found that they are apoptosis-inducers in human cancer cell lines. In the current study, we further determined the effects of the substitution of the O-methyl moiety on C3 and stereochemistry of the beta-lactams on the anti-proliferative and apoptosis-inducing abilities. We have found that lactam 18, in which C3 is substituted with an acrylate ester group, is a very effective proliferation inhibitor against human premalignant and malignant breast, leukemic, and simian virus 40-transformed fibroblast cells. Generally speaking, increasing the size of the moiety on C3 decreases its anti-proliferation potency, possibly indicating steric hindrance with the cellular target or decreased permeability through the cell membrane. We also found that the stereochemistry of the beta-lactams plays an important role in their potency. The 3S,4R isomers are more effective than their enantiomers (3R,4S), suggesting that 3S,4R configuration is more favorable for target interaction.
- Published
- 2005
28. Evaluation of proteasome-inhibitory and apoptosis-inducing potencies of novel (-)-EGCG analogs and their prodrugs
- Author
-
Kristin R, Landis-Piwowar, Deborah J, Kuhn, Sheng Biao, Wan, Di, Chen, Tak Hang, Chan, and Q Ping, Dou
- Subjects
Flavonoids ,Jurkat Cells ,Phenols ,Tea ,Anticarcinogenic Agents ,Humans ,Polyphenols ,Apoptosis ,Prodrugs ,Proteasome Inhibitors ,Catechin - Abstract
The anti-cancer and cancer-preventive effects of green tea and its main constituent (-)-epigallocatechin gallate [(-)-EGCG] are well documented by a variety of studies, including epidemiological, cell culture, animal, and clinical studies. While (-)-EGCG remains the most potent polyphenol in green tea, it is very unstable in neutral or alkaline conditions (i.e. physiologic pH). In an effort to discover more stable polyphenol proteasome inhibitors, we synthesized several novel (-)-EGCG analogs with -OH groups eliminated from the B- and/or D-rings. In addition, we also synthesized their putative prodrugs with -OH groups protected by peracetate that can be removed by cellular cytosolic esterases. We first examined the structure-activity relationship of these unprotected and protected compounds to their proteasome-inhibitory potentials. We found that decreasing -OH groups from either the B- or D-ring leads to diminished proteasome-inhibitory activity in vitro. However, in cultured tumor cells only the protected analogs were capable of potently inhibiting the proteasome activity. Furthermore, these protected analogs induced apoptotic cell death in a tumor cell-specific manner. The superior efficacy of the protected (-)-EGCG analogs indicates the formation of an entirely new compound(s) in intact tumor cells. These data suggest that the B-ring/D-ring peracetate-protected EGCG analogs have great potential to be developed into novel anti-cancer and cancer-preventive agents.
- Published
- 2005
29. Dietary flavonoids as proteasome inhibitors and apoptosis inducers in human leukemia cells
- Author
-
Kristin R. Landis-Piwowar, Deborah J. Kuhn, Marina S. Chen, Di Chen, Kenyon G. Daniel, and Q. Ping Dou
- Subjects
Programmed cell death ,Apoptosis ,Biology ,Cysteine Proteinase Inhibitors ,Biochemistry ,Jurkat cells ,chemistry.chemical_compound ,Jurkat Cells ,Structure-Activity Relationship ,Ubiquitin ,Anticarcinogenic Agents ,Humans ,Computer Simulation ,Apigenin ,Kaempferols ,Pharmacology ,Flavonoids ,Diet ,Killer Cells, Natural ,Proteasome ,chemistry ,Cancer cell ,biology.protein ,Myricetin ,Quercetin ,Proteasome Inhibitors - Abstract
It has been shown that proteasome activity is required for cancer cell survival and consumption of fruits and vegetables is associated with decreased cancer risk. Previously, we reported that grape extract could inhibit proteasome activity and induce apoptosis in tumor cells. In this study, we examined the flavonoids apigenin, quercetin, kaempferol and myricetin for their proteasome-inhibitory and apoptosis-inducing abilities in human tumor cells. We report that apigenin and quercetin are much more potent than kaempferol and myricetin at: (i) inhibiting chymotrypsin-like activity of purified 20S proteasome and of 26S proteasome in intact leukemia Jurkat T cells; (ii) accumulating putative ubiquitinated forms of two proteasome target proteins, Bax and Inhibitor of nuclear factor kappabeta-alpha in Jurkat T cells and (iii) inducing activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase in Jurkat T cells. The proteasome-inhibitory abilities of these compounds correlated with their apoptosis-inducing potencies. Results from computational modeling of the potential interactions of these flavonoids to the chymotrypsin site (beta5 subunit) of the proteasome were consistent with the obtained proteasome-inhibitory activities. We found that the C(4) carbon may be a site of nucleophilic attack by the OH group of N-terminal threonine of proteasomal beta5 subunit and that the C(3) hydroxyl may alter the ability of these flavonoids to inhibit the proteasome. Finally, apigenin neither effectively inhibited the proteasome activity nor induced apoptosis in non-transformed human natural killer cells. Our results suggested that the proteasome may be a target of these dietary flavonoids in human tumor cells and that inhibition of the proteasome by flavonoids may be one of the mechanisms responsible for their cancer-preventive effects.
- Published
- 2004
30. Green tea and tea polyphenols in cancer prevention
- Author
-
Zhigang Wang, Q. Ping Dou, Kenyon G. Daniel, Tak Hang Chan, Di Chen, Wai Har Lam, Mohammad Bhuiyan, Sheng Biao Wan, Deborah J. Kuhn, Aslamuzzaman Kazi, and Lian-Hai Li
- Subjects
MAPK/ERK pathway ,Proteasome Endopeptidase Complex ,Cell cycle checkpoint ,MAP Kinase Signaling System ,Apoptosis ,Pharmacology ,Biology ,complex mixtures ,Phenols ,Neoplasms ,medicine ,Anticarcinogenic Agents ,Humans ,Flavonoids ,Clinical Trials as Topic ,Cancer prevention ,Tea ,Ubiquitin ,food and beverages ,Cancer ,Polyphenols ,medicine.disease ,Biochemistry ,Cell culture ,Animal studies ,Signal transduction ,Signal Transduction - Abstract
The cancer-preventive effects of green tea and its main constituent (-)-epigallocatechin gallate [(-)-EGCG] are widely supported by results from epidemiological, cell culture, animal and clinical studies in the recent decade. In vitro cell culture studies show that tea polyphenols potently induce apoptotic cell death and cell cycle arrest in tumor cells but not in their normal cell counterparts. Green tea polyphenols affect several signal transduction pathways, including growth factor-mediated, the mitogen-activated protein kinase (MAPK)-dependent, and ubiquitin/proteasome degradation pathways. Epidemiological studies have suggested that the consumption of green tea lowers the risk of cancer. Various animal studies have revealed that treatment by green tea inhibits tumor incidence and multiplicity in different organ sites such as skin, lung, liver, stomach, mammary gland and colon. Phase I and II clinical trials were carried out recently to explore the anticancer effects of green tea in patients with cancer. At this time, more mechanistic research, animal studies, and clinical trials are necessary to further evaluate the role of green tea in cancer prevention.
- Published
- 2004
31. Beta-lactams and their potential use as novel anticancer chemotherapeutics drugs
- Author
-
Aslamuzzaman Kazi, Deborah J. Kuhn, Mohammad Bhuiyan, Edward Turos, Di Chen, Qing Ping Dou, Kenyon G. Daniel, and Cristina Coates
- Subjects
medicine.drug_class ,medicine.medical_treatment ,Cephalosporin ,Antibiotics ,Antineoplastic Agents ,Apoptosis ,beta-Lactams ,Neoplasms ,medicine ,Animals ,Anticarcinogenic Agents ,Humans ,Prodrugs ,Monobactams ,Chemistry ,Drug discovery ,DNA ,Prodrug ,Radioimmunotherapy ,Intercalating Agents ,Cell culture ,Drug Design ,Cancer research ,DNA Damage - Abstract
The discovery of natural and synthetic antibiotics is one of the most important medical breakthroughs in human history. Many diseases, such as bacterial meningitis, pneumonia, and septicemia, are now curable with the use of antibiotics. Antibiotics are efficacious, generally well tolerated in patients, and have a low toxicity level. It is for these reasons antibiotics remain an attractive target for drug discovery. Traditional beta-lactam antibiotics (e.g. penicillins, penems, cephalosporins) have a bicyclic ring structure that is conformationally rigid and functions to inhibit bacterial cell wall synthesis. In addition to the bactericidal action of antibiotics, it has been discovered that many antibiotics are capable of inhibiting tumor cell growth. There are currently many antitumor antibiotics approved for cancer therapy, which work to inhibit tumor cell growth by DNA intercalation. The use of beta-lactams as prodrugs has also met with success by aiding delivery of the chemotherapeutic directly to tumor sites. Recently, a novel class of N-thiolated monobactams, so termed because they possess a monocyclic ring instead of the bicyclic ring, has been found to induce apoptosis potently and specifically in many tumor cell lines but not in normal, non-transformed cell lines. Other beta-lactams, such as the polyaromatics, have been found to slow or inhibit tumor cell growth, and the 4-alkylidene beta-lactams are capable of inhibiting matrix metalloproteinases and leukocyte elactase activity. These data indicate that synthesis and evaluation of beta-lactams are a promising area for further development in anticancer research.
- Published
- 2004
32. A natural musaceas plant extract inhibits proteasome activity and induces apoptosis selectively in human tumor and transformed, but not normal and non-transformed, cells
- Author
-
Nagi B. Kumar, Edward R Jackson, Gail P Greenfelder, Q. Ping Dou, Aslamuzzaman Kazi, Daniel A Urbizu, Deborah J. Kuhn, and Abelardo L Acebo
- Subjects
T cell ,Cell ,General Medicine ,Biology ,Cell cycle ,Molecular biology ,Jurkat cells ,medicine.anatomical_structure ,Proteasome ,Cell culture ,Apoptosis ,LNCaP ,Genetics ,medicine - Abstract
Animal studies have demonstrated that a dietary polyphenol known as tannic acid (TA) exhibits anticarcinogenic activity in chemically induced cancers. Most recently, we have reported that TA and ester-bond containing green tea polyphenols are potent proteasome inhibitors in vitro and in vivo. We hypothesize that CellQuest, a patented formula which contains high level of TA obtained from a musaceas (plantain) plant extract, will inhibit the tumor cell proteasome activity. Here, we report that a partially purified CellQuest fraction, S3, potently inhibits the proteasomal chymotrypsin-like activity of Jurkat T cell extracts in a concentration-dependent manner. Inhibition of the proteasome by S3 in leukemia Jurkat T, simian virus 40-transformed and prostate cancer LNCaP cells results in accumulation of ubiquitinated proteins and the natural proteasome substrate p27Kip1, followed by induction of apoptosis. In contrast, non-transformed, immortalized human natural killer cells and normal human fibroblasts are resistant to S3-mediated proteasome inhibition and apoptosis induction. Our present study suggests that CellQuest targets and inhibits the proteasome selectively in tumor cells, which may contribute to the claimed anticancer activity.
- Published
- 2003
33. A natural musaceas plant extract inhibits proteasome activity and induces apoptosis selectively in human tumor and transformed, but not normal and non-transformed, cells
- Author
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Aslamuzzaman, Kazi, Daniel A, Urbizu, Deborah J, Kuhn, Abelardo L, Acebo, Edward R, Jackson, Gail P, Greenfelder, Nagi B, Kumar, and Q Ping, Dou
- Subjects
Male ,Cysteine Endopeptidases ,Jurkat Cells ,Proteasome Endopeptidase Complex ,Musaceae ,Multienzyme Complexes ,Plant Extracts ,Humans ,Prostatic Neoplasms ,Apoptosis ,Cell Line, Transformed - Abstract
Animal studies have demonstrated that a dietary polyphenol known as tannic acid (TA) exhibits anticarcinogenic activity in chemically induced cancers. Most recently, we have reported that TA and ester-bond containing green tea polyphenols are potent proteasome inhibitors in vitro and in vivo. We hypothesize that CellQuest, a patented formula which contains high level of TA obtained from a musaceas (plantain) plant extract, will inhibit the tumor cell proteasome activity. Here, we report that a partially purified CellQuest fraction, S3, potently inhibits the proteasomal chymotrypsin-like activity of Jurkat T cell extracts in a concentration-dependent manner. Inhibition of the proteasome by S3 in leukemia Jurkat T, simian virus 40-transformed and prostate cancer LNCaP cells results in accumulation of ubiquitinated proteins and the natural proteasome substrate p27Kip1, followed by induction of apoptosis. In contrast, non-transformed, immortalized human natural killer cells and normal human fibroblasts are resistant to S3-mediated proteasome inhibition and apoptosis induction. Our present study suggests that CellQuest targets and inhibits the proteasome selectively in tumor cells, which may contribute to the claimed anticancer activity.
- Published
- 2003
34. Direct inhibition of the ubiquitin-proteasome pathway by ester bond-containing green tea polyphenols is associated with increased expression of sterol regulatory element-binding protein 2 and LDL receptor
- Author
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Deborah J. Kuhn, Aslamuzzaman Kazi, Q. Ping Dou, and Audrey C Burns
- Subjects
Proteasome Endopeptidase Complex ,Camellia sinensis ,Catechin ,HeLa ,Ubiquitin ,Multienzyme Complexes ,Humans ,Receptor ,Molecular Biology ,biology ,food and beverages ,Esters ,Cell Biology ,biology.organism_classification ,Sterol ,Sterol regulatory element-binding protein ,DNA-Binding Proteins ,Cysteine Endopeptidases ,Proteasome ,Biochemistry ,Receptors, LDL ,LDL receptor ,biology.protein ,Sterol regulatory element-binding protein 2 ,HeLa Cells ,Sterol Regulatory Element Binding Protein 2 ,Transcription Factors - Abstract
Green tea has been shown to lower plasma cholesterol, associated with up-regulation of the low-density lipoprotein receptor (LDLR) although the responsible molecular mechanism is unknown. Previously, we reported that ester bond-containing green tea polyphenols (GTPs), such as (-)-epigallocatechin-3-gallate [(-)-EGCG], potently inhibit the tumor cellular proteasome activity, which may contribute to the cancer-preventative effect of green tea. In the current study, we hypothesize that the proteasome is a heart disease-associated molecular target of GTPs. We have shown that ester bond-containing GTPs, including (-)-EGCG, potently inhibit the proteasomal activity in intact hepatocellular carcinoma HepG2 and cervical carcinoma HeLa cells, as evident by accumulation of ubiquitinated proteins and three natural proteasome targets (p27, IkappaB-alpha and Bax). (-)-EGCG selectively inhibits the chymotrypsin-like, but not trypsin-like, activity of the proteasome. Associated with proteasome inhibition by ester bond-containing GTPs, there was a significant, time- and concentration-dependent increase in levels of the cleaved, activated, but not the precursor, form of sterol regulatory element-binding protein 2 (SREBP-2), an essential factor for LDLR transcription. Subsequently, LDL receptor expression was increased dramatically in HepG2 and HeLa cells treated with (-)-EGCG. Our results suggest that ester bond-containing GTPs inhibit ubiquitin/proteasome-mediated degradation of the active SREBP-2, resulting in up-regulation of LDLR. This identified molecular mechanism may be related to the previously reported cholesterol-lowering and heart disease-preventative effects of green tea.
- Published
- 2003
35. Regulation of 17-AAG-induced apoptosis: role of Bcl-2, Bcl-XL, and Bax downstream of 17-AAG-mediated down-regulation of Akt, Raf-1, and Src kinases
- Author
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Hong Gang Wang, Ramadevi Nimmanapalli, Kapil N. Bhalla, Hirohito Yamaguchi, Deborah J. Kuhn, and Erica O'Bryan
- Subjects
Lactams, Macrocyclic ,Immunology ,Blotting, Western ,bcl-X Protein ,Down-Regulation ,Apoptosis ,Protein Serine-Threonine Kinases ,Biochemistry ,chemistry.chemical_compound ,Bcl-2-associated X protein ,Heat shock protein ,Proto-Oncogene Proteins ,polycyclic compounds ,Benzoquinones ,Tumor Cells, Cultured ,Humans ,Protein kinase A ,Protein kinase B ,bcl-2-Associated X Protein ,biology ,Kinase ,Cytochrome c ,Cell Biology ,Hematology ,Geldanamycin ,Proto-Oncogene Proteins c-raf ,src-Family Kinases ,chemistry ,Proto-Oncogene Proteins c-bcl-2 ,Rifabutin ,biology.protein ,Cancer research ,Proto-Oncogene Proteins c-akt - Abstract
17-allylamino-demethoxy geldanamycin (17-AAG) inhibits the chaperone function of heat shock protein—90 (Hsp-90) and promotes the proteasomal degradation of its misfolded client proteins. Here, we demonstrate that treatment of the human acute myeloid leukemia HL-60 cells with 17-AAG attenuates the intracellular levels of a number of Hsp-90 client proteins, including Akt, c-Raf-1, and c-Src. Also, 17-AAG induced the mitochondrial release and cytosolic accumulation of cytochrome c (cyt c) and second mitochondria-derived activator of caspases (Smac)/DIABLO, resulting in the activation of caspase-9 and caspase-3 and apoptosis. Treatment with 17-AAG triggered the B-cell lymphoma—2 (Bcl-2)—associated X protein (Bax) conformational change associated with apoptosis, while Bax-deficient cells were resistant to 17-AAG—induced apoptosis. In addition, in HL-60/Bcl-2 and HL-60/Bcl-xL cells, which ectopically express Bcl-2 and Bcl-xL respectively, 17-AAG—induced Bax conformational change, cytosolic accumulation of cyt c and Smac/DIABLO, and apoptosis were markedly inhibited. Although the rate of 17-AAG—mediated decline in Akt, c-Raf-1, and c-Src levels was blunted, the total decline was not compromised in HL-60/Bcl-2 and HL-60/Bcl-xL cells. Cotreatment with HA14-1, a nonpeptidic ligand that can bind and inhibit the antiapoptotic activity of Bcl-2, significantly overcame the resistance to 17-AAG—induced apoptosis in HL-60/Bcl-2 cells. Together, these findings indicate that although 17-AAG treatment causes the levels of a number of survival-signaling protein kinases to decline, the downstream engagement of the mitochondrial pathway of apoptosis is regulated by the activity of the Bcl-2 family of proteins. Also, neutralizing the antiapoptotic effect of Bcl-2 would further enhance the antileukemia activity of 17-AAG. (Blood. 2003;102:269-275)
- Published
- 2003
36. Serum Heavy Light Chain Immunoassays Compared To Traditional Serum Based Laboratory Testing For The Diagnosis and Screening Of Monoclonal Gammopathies
- Author
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Deborah J. Kuhn and Qingli Wu
- Subjects
Immunofixation ,Paraproteinemia ,medicine.diagnostic_test ,biology ,business.industry ,Myeloma protein ,Immunology ,Waldenstrom macroglobulinemia ,Macroglobulinemia ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Immunoglobulin M ,Serum protein electrophoresis ,medicine ,biology.protein ,business ,Monoclonal gammopathy of undetermined significance - Abstract
Background Traditional laboratory tests for the screening and diagnosis of monoclonal gammopathies, such as monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma, and related disorders like paraproteinemia, include serum protein electrophoresis (sPEP), serum immunofixation electrophoresis (sIFE), and serum free light chain (sFLC) ratio. Pilot studies have shown that immunoassays for the combined heavy-light chain (sHLC) molecule (Hevylite®) are useful for the paired isotype matching of intact immunoglobulins (eg. IgGk/IgGl, IgAk/IgAl, IgMk/IgMl) in an automated, sensitive manner as an alternative to sIFE. Methods The primary objective of this study is to compare the results of the quantitative intact immunoglobulin Hevylite™ assay with sPEP and sIFE. Routine requests for the evaluation of monoclonal gammopathy in the Ochsner Medical Center Clinical Chemistry Laboratory were collected prospectively and tested using sPEP and sFLC with reflex sIFE. IgA, IgM and IgG k/λ HLC reagent kits, provided by The Binding Site, Inc, were used on a subset of patients where the serum samples were still available. HLC values were assessed on a SPAplus turbidometer. Results Seventy-six (76) samples were available for patients with multiple myeloma (n=18), MGUS (n=28), Waldenstrom's Macroglobulinemia (n=6), IgM paraproteinemia (n= 5) and normal patient sera (n=20). sHLC k/λ ratios were abnormal in 64% of IgG multiple myeloma samples, 100% IgA multiple myeloma samples and 100% IgM paraproteinemia, indicating a high rate of correlation with traditional laboratory testing. Of the four normal IgG k/λ HLC ratios reported, all patients were on active chemotherapy with sPEP M-spikes Conclusion These data provide support for the use of sHLC k/λ ratios for the screening and diagnosis of monoclonal gammopathies and related disorders. In addition, abnormal ratios were found in a significant subset of MGUS patients which has been shown to be a risk factor for progression. Lastly, the use of sHLC immunoassay in Waldenstrom's macroglobulinemia has not been well studied, but these data indicate that sHLC may be a beneficial in both diagnosis and monitoring of this disease. Disclosures: Wu: Ochsner Medical Center: Employment; The Binding Site Inc: Research Funding, travel fund to ASH meeting, travel fund to ASH meeting Other. Off Label Use: Hevylite® is an immunoassay that is pending FDA approval for the measurement of heavy light chain immunoglobulins. Kuhn:The binding site INC: Employment.
- Published
- 2013
37. Abstract 844: Targeting the pro-survival protein c-MET with ARQ 197 inhibits growth of multiple myeloma cells
- Author
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Chad C. Bjorklund, Varsha Gandhi, Shadia Zaman, Deborah J. Kuhn, Robert Z. Orlowski, Christine M. Stellrecht, and Shujun Shentu
- Subjects
Cancer Research ,medicine.medical_specialty ,Stromal cell ,biology ,Bortezomib ,Cell growth ,medicine.disease ,Receptor tyrosine kinase ,chemistry.chemical_compound ,Endocrinology ,medicine.anatomical_structure ,Oncology ,chemistry ,Internal medicine ,medicine ,Cancer research ,biology.protein ,Hepatocyte growth factor ,Bone marrow ,Growth inhibition ,Multiple myeloma ,medicine.drug - Abstract
Multiple myeloma (MM) is a B-cell disorder characterized by the accumulation of mature plasma cells in the bone marrow. Earlier studies have established that patients with MM have high plasma concentrations of hepatocyte growth factor (HGF) which is correlated with poor prognosis and advanced disease stages. HGF is the ligand for the c-MET receptor tyrosine kinase. Our group has previously shown that inactivation of the c-MET receptor by siRNA and ribozyme approaches inhibited proliferation and induced apoptosis in MM cell lines. Hence, we hypothesized that the HGF/c-MET axis plays a critical role in myeloma cell survival and targeting this pathway would be an effective strategy to treat MM. To further test our hypothesis, we used MM1.S, U266 and OPM-2 myeloma cell lines and ARQ 197 (tivantinib), a small molecule non-ATP-competitive and selective c-MET inhibitor (Ki=355 nM). This drug is orally bioavailable, achieving steady-state levels of median 6-7 µM in plasma of patients with solid tumors. Cell growth was inhibited by at least 50% and annexin V/propidium iodide positive cells increased by at least 60% within 48 hours of treatment with 1 µM (U266 and OPM-2) and 3 µM (MM.1S) ARQ 197. Consistent with this biological response, downstream effectors of c-MET signaling including phospho-GAB1 and phospho-ERK1/2 were decreased. MM patients develop resistance to the current drugs such as lenalidomide and bortezomib. We tested ARQ 197 in paired MM cell lines - RPMI 8226, ANBL-6 and KAS-6/1 - that are sensitive and resistant to lenalidomide or bortezomib. In general, all the tested cell lines expressed readily detectable levels of proHGF as well as detectable albeit low levels of c-MET. Interestingly, the resistant cell lines expressed significantly higher c-MET protein levels compared to drug-naïve controls, but were equally sensitive to ARQ 197 indicating that this c-MET kinase inhibitor overcomes inherent drug resistance. The bone marrow microenvironment is also a source for acquired drug resistance, yet ARQ 197 was effective in inhibiting growth of MM cell lines grown on human stromal cells (NKtert) which mimic the bone marrow microenvironment. Consistent with these data, exogenous addition of the cytokines interleukin-6 (0.5 and 1.0 ng/ml) or HGF (50 ng/ml) did not abrogate ARQ 197-mediated cell death or growth inhibition. Importantly, plasma cells from healthy donors (n = 15) showed no difference in c-MET mRNA expression compared to plasma cells from MM patients (n = 147) but showed relatively lower levels of HGF expression compared to MM patients. These results suggest that the HGF/c-MET pathway is upregulated in MM plasma cells. In conclusion, inhibition of the c-MET receptor tyrosine kinase activity with ARQ 197 is a novel-target based strategy to impact on the pathobiology of MM. Based on these data, we are initiating a clinical trial of ARQ 197 in patients with relapsed/refractory MM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 844. doi:1538-7445.AM2012-844
- Published
- 2012
38. Targeting the Wnt/β-Catenin Signaling Pathway and CD44-Mediated Adhesion As a Rational Approach to Overcome Lenalidomide Resistance in Multiple Myeloma
- Author
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Mohamad A. Hussein, Rajesh Chopra, Caimiao Wei, Sharon L. Aukerman, Robert Z. Orlowski, Deborah J. Kuhn, Isere Kuiatse, Chad C. Bjorklund, and Antonia Lopez-Girona
- Subjects
biology ,business.industry ,Immunology ,CD44 ,Cell ,Wnt signaling pathway ,Cancer ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,medicine.anatomical_structure ,medicine ,biology.protein ,Cancer research ,Extracellular matrix binding ,Signal transduction ,business ,Multiple myeloma ,Lenalidomide ,medicine.drug - Abstract
Abstract 928 Background: Lenalidomide (Len) has been shown to be clinically effective against multiple myeloma (MM) as a single agent and in combination for both newly diagnosed and relapsed/refractory settings, and is increasingly utilized in maintenance therapy. Despite the successful use of Len, some patients display a poor response or become refractory after an initial response. As previously published by our group, hyperactivation of Wnt/β-catenin signaling was identified as a mediating factor for Len-specific resistance in MM cell lines. Additionally, a downstream transcriptional product of Wnt/β-catenin signaling is the extracellular matrix binding protein CD44, which has been implicated in cellular-adhesion-mediated drug-resistance (CAM-DR) in several cancer models, including dexamethasone-resistant MM. These early studies established a rationale to target aberrant Wnt/β-catenin signaling and CD44-mediated CAM-DR to overcome Len resistance. Methods: Wild-type (Wt) and lenalidomide-resistant (LR) MM cell lines were used as models to evaluate the effectiveness of targeting the Wnt/β-catenin signaling pathway and CD44-dependent CAM-DR to overcome Len resistance with Wnt/β-catenin antagonists, shRNA knockdown of CD44, and anti-CD44 neutralization. Results: All-trans-retinoic acid (ATRA) inactivates β-catenin nuclear signaling by altering β-catenin cellular distribution. Combination treatment with Len and ATRA produced a significant (p Conclusions: Collectively, our results suggest that MM cell Len resistance is, at least in part, dependent on β-catenin and CD44 and that selective targeting of these cellular proteins in conjunction with Len treatment represents a rational approach for clinical treatment of Len-resistant MM. Disclosures: Aukerman: Celgene Corporation: Employment. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Hussein:Celgene Corporation: Employment. Chopra:Celgene Corporation: Employment. Orlowski:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
- Published
- 2011
39. Blockade of IGF-1R with OSI-906 Overcomes Bortezomib-Resistance in Multiple Myeloma
- Author
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Robert Z. Orlowski, Deborah J. Kuhn, Chad C. Bjorklund, Richard J. Jones, and Hua Wang
- Subjects
education.field_of_study ,business.industry ,Bortezomib ,Immunology ,Cell ,Population ,Cell Biology ,Hematology ,Pharmacology ,medicine.disease ,Biochemistry ,Lymphoma ,medicine.anatomical_structure ,Apoptosis ,Cell culture ,hemic and lymphatic diseases ,medicine ,Proteasome inhibitor ,Cancer research ,business ,education ,Multiple myeloma ,medicine.drug - Abstract
Abstract 925 Blockade of IGF-1R with OSI-906 Overcomes Bortezomib-resistance in Multiple Myeloma Deborah J. Kuhn, Hua Wang, Richard J. Jones, Chad C. Bjorklund, Robert Z. Orlowski The Department of Lymphoma & Myeloma, The University of Texas M. D. Anderson Cancer Center, Houston, TX Intro: Bortezomib (Velcade®) therapy is now one of the standards of care in the treatment of newly diagnosed and relapsed/refractory multiple myeloma. Unfortunately, like many other novel agents, the emergence of drug resistance often results in a reduced response to any subsequent therapies that contain bortezomib. Identifying the molecular signaling pathways predominant in bortezomib-resistance can lead to the discovery of therapies that can overcome or prevent the emergence of such resistance all together. Design: In order to improve our understanding of the mechanisms responsible for bortezomib-resistance, our group has developed cell line models of interleukin (IL)-6-dependent and –independent bortezomib-resistant multiple myeloma. Results: Gene expression profiling identified insulin-like growth factor (IGF-1) signaling as one pathway that was induced in bortezomib-resistant myeloma cell lines. Its role was validated in molecular studies that showed exogenous IGF-1 protected drug-naïve cells from bortezomib, while shRNA-mediated knockdown of the IGF-1 receptor (IGF-1R) in bortezomib-resistant models restored sensitivity to this proteasome inhibitor. We then evaluated whether targeting IGF-1R with the clinically relevant inhibitor OSI-906 (OSI Pharmaceuticals, Inc.) could be a valid strategy to overcome bortezomib-resistance. OSI-906 alone preferentially induced cell death in bortezomib-resistant cell lines, while drug-naïve cell populations were relatively spared. Simultaneous addition of bortezomib and increasing concentrations of OSI-906 enhanced the amount of cell death. Also, OSI-906 preferentially induced apoptosis as measured by Annexin V staining. Knockout of IGF-1R gene expression using lentiviral shRNAs in bortezomib-resistant cell lines decreased sensitivity to OSI-906 compared to their scrambled control counterparts, underscoring the importance of IGF-1R signaling in these cells. Conversely, lentiviral overexpression of IGF-1R in drug-naïve wild-type cell lines reduced apoptosis when these models were exposed to OSI-906. Next, we determined whether OSI-906 alone would have an effect on MDA-MM-002, a cell line developed from the pleural effusion of a patient with advanced myeloma. MDA-MM-002 cells, which are resistant to a number of chemotherapeutics, including bortezomib, showed no decrease in viable cell populations when treated with OSI-906 alone. However, when combined with bortezomib, there was a dose-dependent decrease in the viable cell population. Importantly, flank xenograft models of bortezomib-resistant myeloma cells in immunodeficient mice remained insensitive to bortezomib treatment, but showed a dose-dependent response to OSI-906 as evidenced by decrease tumor cell growth, and caspase-3 and PARP cleavage. Examination of synergy profiles using isobologram analysis demonstrated a high degree of synergy with OSI-906 and bortezomib over the use of either drug alone. Conclusions: In addition to the important role of IGF-1 in myeloma biology, the findings herein provide an excellent rationale for using OSI-906 to target IGF-1 signaling in combination with bortezomib as an approach to overcome, or possibly even prevent outgrowth of resistance to bortezomib in myeloma patients. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2011
40. Abstract 2550: Combination of the KSP inhibitor ARRY-520 with bortezomib causes sustained tumor regressions and significantly increased time to regrowth in bortezomib sensitive and resistant models of multiple myeloma
- Author
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Richard Woessner, Duncan Walker, Robert Z. Orlowski, Sumeet Rana, Tunquist Brian J, and Deborah J. Kuhn
- Subjects
Cancer Research ,Bortezomib ,business.industry ,Cancer ,Phases of clinical research ,Pharmacology ,medicine.disease ,KSP Inhibitor ARRY-520 ,Oncology ,Apoptosis ,hemic and lymphatic diseases ,Cancer research ,medicine ,MCL1 ,business ,Dexamethasone ,Multiple myeloma ,medicine.drug - Abstract
The allosteric kinesin spindle protein (KSP) inhibitor ARRY-520 has potent antitumor activity as a single agent in xenograft models of multiple myeloma, with complete response rates as high as 100%. It has shown promising signs of activity in a phase 1 trial in patients with advanced / refractory multiple myeloma, and a phase 2 study in this patient population is in progress. We report here on the striking activity of ARRY-520 in combination with bortezomib in multiple myeloma xenograft models. We investigated the activity of the combination of ARRY-520 with bortezomib in several xenograft models of multiple myeloma that are sensitive (Kas-6, ANBL-6, and H929) and resistant (RPMI8226, JJN3, a subline of ANBL-6 made resistant to bortezomib by in vitro treatment, and a subline of RPMI8226 that acquired a PI3K activating mutation) to bortezomib. The combination of ARRY-520 with bortezomib showed additive or superadditive activity, compared to either drug alone, in 6 of the 7 models tested. Complete responses and cures were observed in several models. The Kas-6 model was particularly sensitive, with a 100% cure rate (complete response through day 100). Since previous work from our lab (Mol. Cancer Ther. 9(7), 2046, 2010) implicates MCL1 status as a predictor of response to ARRY-520 in multiple myeloma cell lines, we evaluated the effect of the ARRY-520 + bortezomib combination on MCL1 and other markers of apoptosis. Treatment with the combination resulted in elevation of the cleaved / full length PARP ratio (up to 15X), compared to tumors treated with the single agents, along with a decline in MCL1 levels that coincided with elevation of the level of a 28kd fragment of MCL1 (up to 17X). This shortened form of MCL1 has been reported to be pro-apoptotic. Thus, the cleavage of MCL1 to a proapoptotic form, leading to a decrease in survival signaling and an increase in apoptotic signaling, may play a role in the activity of the ARRY-520 + bortezomib combination. The observation that the combination remained highly active even in models that were poorly responsive to single agent bortezomib suggests that the mechanisms underlying resistance to bortezomib may be distinct from those that contribute to activity of the combination. These results support ARRY-520 + bortezomib as a rational combination for clinical evaluation, including in patients with bortezomib-refractory disease. A phase 1 trial to evaluate the combination of ARRY-520 + bortezomib + dexamethasone is in progress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2550. doi:10.1158/1538-7445.AM2011-2550
- Published
- 2011
41. Abstract B66: Resistance to bortezomib in myeloma cell lines is mediated by increased IGF-1/Akt signaling to activate Cot kinase and NF-κB
- Author
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Deborah J. Kuhn, Chad C. Bjorklund, and Robert Z. Orlowski
- Subjects
Cancer Research ,Bortezomib ,Kinase ,RELB ,Biology ,Oncology ,Proteasome ,Cell culture ,Immunology ,medicine ,Cancer research ,Picropodophyllin ,Phosphorylation ,Protein kinase B ,medicine.drug - Abstract
Background: Targeting the proteasome as an anti-cancer therapy was first realized with bortezomib, now one of the standards used in relapsed/refractory multiple myeloma and mantle cell lymphoma. Despite this advance, response rates have been as low as 23%, indicating the importance of innate or acquired resistance to bortezomib. Methods: To improve our understanding of the mechanisms responsible for bortezomib resistance, we developed cell line models of interleukin (IL)-6-dependent (ANBL-6 and KAS-6/1) and -independent (RPMI 8226 and OPM-2) bortezomib-resistant (BR) myeloma by continuously exposing drug-naïve cells to increasing bortezomib concentrations. All of the lines where characterized by gene expression profiling to identify potential mechanisms of resistance. Results: Cell lines resistant to bortezomib retained their viability in the presence of 50 nM or more of bortezomib with degrees of resistance up to 4.3. Gene expression profiling revealed up-regulation of insulin-like growth factor (IGF)-1 transcripts (5.2-fold), Akt (3.2-fold), and cancer Osaka thyroid (Cot) kinase (2.3-fold). Consistent with our profiling data, BR clones had increased soluble IGF-1 levels, increased IGF-1 protein, and phospho-IGF-1R activation. Next we examined the effect of overexpression of Cot on BR clones. Cot functions to regulate nuclear factor kappa B (NF-κB)-dependent transcription in response to Akt by promoting post-translational processing of inactive NF-κB p105 to active p50. Basel levels of nuclear activated p50 were increased up to 4.7-fold in BR clones. Activated NF-κB dimers were also found to be significantly increased in ANBL-6.BR and 8226.BR cells as evidenced by phosphorylation of p65 (RelB). Our examination into Cot expression in drug-naive and BR cells showed a large increase of Cot protein and phospho-Cot (Thr290) activation in 8226.BR cells that was enhanced with 24-hour bortezomib treatment. A transcriptional target of NF-κB is Bcl-2 was also found to be increased in profiling studies (5.5-fold) and correlates with increased expression in BR cell lines tested. Treatment with 10 nM bortezomib for 6 hours led to a decreased in Bcl-2 protein both drug-naïve and drug-resistant cells. A decrease in Bcl-2 protein was also observed in ANBL-6.BR, RPMI 8226.BR, and OPM-2.BR cells in response to 24-hour treatment with picropodophyllin, a pharmacologic inhibitor of IGF-1R activation, consistent with the hypothesis that the IGF-1/Akt signaling axis in important in Bcl-2 protein expression in bortezomib-resistant clones. Conclusions: Our data support the hypothesis that increased signaling through the IGF-1/Akt axis and enhanced expression of Cot kinase is another mechanism that mediates stable bortezomib resistance in vitro through the activation of NF-κB and its downstream transcriptional targets, including Bcl-2. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B66.
- Published
- 2009
42. Activation of the Wnt/β-Catenin Pathway Mediates Lenalidomide Resistance in Multiple Myeloma
- Author
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Deborah J. Kuhn, Jairo Matthews, Veerabhadran Baladandayuthapani, Chad C. Bjorklund, Robert Z. Orlowski, and Michael Wang
- Subjects
Immunology ,CD44 ,Wnt signaling pathway ,Cell Biology ,Hematology ,Pharmacology ,Biology ,medicine.disease ,Biochemistry ,LRP1 ,GSK-3 ,Cell culture ,Catenin ,medicine ,biology.protein ,Multiple myeloma ,Lenalidomide ,medicine.drug - Abstract
Abstract 113 Background: Novel drugs such as the immunomodulatory agent lenalidomide have revolutionized the treatment of multiple myeloma, as evidenced by an increasing overall survival for patients with both newly-diagnosed, and relapsed and/or refractory disease. Despite these improvements, myeloma remains incurable, and is still characterized by a trend for increasing chemoresistance at relapse, with a decreasing duration of benefit from each successive line of therapy. By understanding the mechanisms responsible for the emergence of drug resistance, which have so far not been well characterized in the case of lenalidomide, it may be possible to rationally design novel regimens that could either overcome this resistance, or possibly prevent its emergence altogether. Methods: To improve our understanding of the mechanisms responsible for lenalidomide resistance, we developed cell line models of interleukin (IL)-6-dependent (ANBL-6 and KAS-6/1) and –independent (U266 and MM1.S) lenalidomide-resistant multiple myeloma cells. Starting at a concentration that was 1/10 of the IC50 for lenalidomide's anti-proliferative effects in drug-naïve cells, increasing drug concentrations were used until all the cell lines could proliferate and maintain cell membrane integrity in the presence of 10 μM lenalidomide. These cell lines were then used as an in vitro model of lenalidomide-specific drug resistance, and subjected to further characterization, including with gene expression profiling. Results: Resistance to lenalidomide was evidenced by a dramatic, 100-1000-fold increase in the IC50 values of these myeloma cells. In the case of ANBL-6 cells, for example, drug-naïve cells showed an IC50 of 0.14 μM using tetrazolium dye-based viability assays, but this increased to >100 μM in the drug-resistant cells, as was the case in U266 and MM1.S cells. This resistance was a stable phenotype, since removal of lenalidomide for seven to ninety days from cell culture conditions did not re-sensitize them when 10 μM lenalidomide was reintroduced. Gene expression profiling followed by pathway analysis to examine changes at the transcript level between wild-type parental and lenalidomide-resistant cell lines identified the Wnt/β-catenin pathway as the most altered across all cell lines. Increased expression was seen in several members of the low-density-lipoprotein receptor related protein family, including LRP1 and 5; members of the wingless-type MMTV integrations site family, including WNT3 and 4; β-catenin; and downstream Wnt/β-catenin targets such as CD44. Similar changes were detected in primary samples from a patient who developed clinically lenalidomide-refractory disease. Reporter assays revealed an up to 5-fold increase in LEF/TCF-dependent transcription both in drug-naïve cells acutely exposed to lenalidomide, and in their chronically exposed, lenalidomide-resistant clones. Western blotting and flow cytometry confirmed that these lenalidomide-resistant cells had increased expression by 2-20 fold of β-catenin and CD44, as well as other LEF/TCF targets, including Cyclin D1 and c-Myc. Comparable changes occurred after lenalidomide exposure in myeloma cells grown in the context of bone marrow stroma. Notably, lenalidomide-resistant cells showed decreased expression of casein kinase 1 and increased phosphorylation of glycogen synthase kinase 3 at Ser21/9, both of which would reduce the phosphorylation of β-catenin needed for its later proteasome-mediated degradation. Stimulation of the Wnt/β-catenin pathway with recombinant human Wnt3a resulted in resistance to lenalidomide in wild-type, drug-naïve cells, as evidenced by a 10-fold increase in the IC50. Conversely, exposure of lenalidomide-resistant cell lines to quercetin, a known antagonist of the β-catenin/TCF interaction, induced a partial re-sensitization to lenalidomide. Conclusions: These data support the hypothesis that activation of the Wnt/β-catenin pathway represents a mechanism of both acute and chronic resistance to the anti-proliferative effects of lenalidomide in multiple myeloma. Moreover, they support the development of strategies aimed at suppressing Wnt/β-catenin activity to resensitize multiple myeloma to the effects of this immunomodulatory agent in vivo. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2009
43. Bortezomib Resistance Is Mediated by Increased Signaling through the Insulin-Like Growth Factor-1/Akt Axis
- Author
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Michael Wang, Valeria Magarotto, Veerabhadran Baladandayuthapani, Chad C. Bjorklund, Robert Z. Orlowski, Deborah J. Kuhn, and Jairo Matthews
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Bortezomib ,Immunology ,Cell Biology ,Hematology ,Pharmacology ,Biology ,medicine.disease ,Biochemistry ,chemistry.chemical_compound ,Proteasome ,chemistry ,medicine ,Proteasome inhibitor ,Picropodophyllin ,LY294002 ,Protein kinase B ,Multiple myeloma ,PI3K/AKT/mTOR pathway ,medicine.drug - Abstract
Abstract 2739 Poster Board II-715 Background: Inhibition of the proteasome as an anti-cancer strategy was first validated with the approval of bortezomib (VELCADE®), which is now a standard of care in relapsed and/or refractory multiple myeloma, and is also being used in the front-line setting, where it has shown improved efficacy compared to previously available induction therapies. However, myeloma remains incurable, and the use of bortezomib up-front may limit its later utility in the relapsed setting. Indeed, response rates on retreatment of patients with previously bortezomib-sensitive disease have been as low as 23%, indicating the rapid development of resistance. The mechanisms by which plasma cells survive and become resistant to bortezomib therapy have not been fully characterized, in part due to a lack of appropriate model systems. Discovery of these resistance mechanisms will facilitate the development of strategies to circumvent them, and help to identify potential novel therapies for the treatment of patients with disease showing resistance to bortezomib. Methods: To improve our understanding of the mechanisms responsible for bortezomib resistance, we developed cell line models of interleukin (IL)-6-dependent (ANBL-6 and KAS-6/1) and –independent (RPMI 8226 and OPM-2) bortezomib-resistant (BR) myeloma by continuously exposing drug-naïve cells to increasing bortezomib concentrations. One of these pooled lines derived from RPMI 8226 cells was then subjected to single-cell cloning, and all of the lines where characterized by gene expression profiling to identify potential mechanisms of resistance. Results: Cell lines resistant to bortezomib, such as RPMI 8226.BR, retained their viability, and did not demonstrate increased Annexin V staining compared to drug-naïve cells in the presence of 50 nM or more of this proteasome inhibitor. Calculations of the degree of resistance revealed values of up to 4.3 or more. Bortezomib resistance was a stable phenotype, since these cells remained unaffected by bortezomib treatment even after a one month period of drug withdrawal. Mutation of the binding site of the β5 proteasome subunit has been reported as one mechanism of bortezomib resistance, but the chymotrypsin-like proteasome activity in RPMI 8226.BR cells could still be suppressed by bortezomib even in the face of increased β5 expression in some subclones. Increased drug clearance has also been proposed as a resistance pathway, but studies by mass spectrometry indicated increased intracellular concentrations of bortezomib and its metabolites in RPMI 8226.BR cells, suggesting that neither of these mechanisms contributed to resistance in our models. Gene expression profiling revealed evidence of up-regulation of insulin-like growth factor (IGF)-1 transcripts by up to 5.2-fold, along with increased expression of phosphoinositide 3-kinase (by up to 4.4-fold) and protein kinase B/Akt (up to 3.2-fold), but suppression of phosphatase and tensin homolog (by up to 5.3-fold). RPMI 8226.BR clones secreted increased levels of IGF-1 into their media leading, in combination with the above gene expression changes, to increased levels of cellular activated, phosphorylated IGF-1 receptor (IGF-1R) and Akt. Addition of exogenous IGF-1 did reduce bortezomib sensitivity in drug-naïve myeloma cells, but induced a much greater increase in resistance in the BR clones even greater than that present in un-supplemented media. In contrast, inhibition of the IGF-1/IGF-1R/Akt axis using picropodophyllin, a pharmacologic inhibitor of IGF-1R; LY294002, a PI3K inhibitor; or NVP-BEZ-235, a novel Akt inhibitor, enhanced sensitivity to bortezomib, and this enhancement was greatest in the BR clones. Similarly, suppression of IGF-1R expression using Lentiviral-mediated delivery of specific shRNAs re-sensitized bortezomib-resistant cells to bortezomib-induced apoptosis, and indeed returned their sensitivity to that of their drug-naïve counterparts. Conclusions: Our data support the hypothesis that increased signaling through the IGF-1/Akt axis mediated by enhanced plasma cell secretion of IGF-1, and enhanced expression of PI3K and Akt, is another mechanism that mediates stable bortezomib resistance in vitro. Moreover, if validated by ongoing studies of primary patient samples, they provide a rationale for the introduction of combination therapies with bortezomib and either Akt or IGF-1 inhibitors to overcome or prevent the emergence of bortezomib resistance in the clinic. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2009
44. 490 POSTER Examination of the mechanisms associated with bortezomib- resistance in human multiple myeloma cell lines
- Author
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Deborah J. Kuhn and Robert Z. Orlowski
- Subjects
Cancer Research ,Oncology ,Myeloma protein ,Cell culture ,business.industry ,Bortezomib ,Cancer research ,Medicine ,business ,medicine.disease ,Multiple myeloma ,medicine.drug - Published
- 2008
45. Enhancing Activity of Bortezomib Against Multiple Myeloma (MM) through Inhibition of Interleukin (IL)-6 Signaling: A Role for Repression of Myeloid Cell Leukemia (Mcl)-1
- Author
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Jeffrey A. Nemeth, Qing Chen, Sally A. Hunsucker, Deborah J. Kuhn, Robert Corringham, George W. Small, Peter M. Voorhees, Robert Z. Orlowski, Mohamed H. Zaki, and Carlos A. Garay
- Subjects
Myeloid ,Stromal cell ,Bortezomib ,Chemistry ,Immunology ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Leukemia ,medicine.anatomical_structure ,immune system diseases ,hemic and lymphatic diseases ,medicine ,Cancer research ,Bone marrow ,Viability assay ,neoplasms ,Dexamethasone ,Multiple myeloma ,medicine.drug - Abstract
Levels of Mcl-1, an anti-apoptotic Bcl-2 family member that plays an important role in MM cell survival, are tightly regulated by the proteasome, and recent data has demonstrated that proteasome inhibition with bortezomib leads to Mcl-1 accumulation, thereby attenuating its activity. Previously, we reported that inhibition of IL-6 signaling with the chimeric antibody, CNTO328, potentiated the anti-myeloma activity of bortezomib in IL-6-dependent cell line models of MM, and that the enhanced activity was associated with inhibition of downstream IL-6 signaling pathways and repression of bortezomib-mediated induction of the anti-apoptotic heat shock response. Based on our promising results with this combination and the role that IL-6 plays in Mcl-1 regulation, we have extended our preclinical studies and investigated whether CNTO328 could inhibit bortezomib-mediated Mcl-1 induction and how the bone marrow microenvironment affects the activity of the combination. Pre-treatment of the IL-6-dependent MM cell lines KAS-6 and ANBL-6 with CNTO328, but not an isotype control antibody, blunted bortezomib-mediated induction of anti-apoptotic Mcl-1L, and enhanced the cytotoxicity and pro-apoptotic activity of bortezomib. In contrast, CNTO328 did not attenuate bortezomib-mediated Mcl-1L accumulation in the IL-6-independent MM cell line RPMI 8226, nor did it enhance the cytotoxicity of bortezomib in these cells. In the presence of patient-derived bone marrow stromal cells, CNTO328 and bortezomib resulted in a greater reduction of cell viability than with either agent alone in both ANBL-6 and KAS-6 cells. Furthermore, although CNTO328 alone did not lead to an increase in apoptosis of ANBL-6 cells in the presence of bone marrow stroma, it significantly potentiated the pro-apoptotic activity of bortezomib in a synergistic manner at clinically achievable concentrations. As opposed to our cell viability data using the co-culture system, CNTO328 was not able to increase levels of apoptosis in KAS-6 cells either as a single agent or in combination with bortezomib, suggesting that CNTO328 may be inducing cell cycle arrest of KAS-6 cells in the presence of bone marrow stroma but is not able to potentiate the apoptotic activity of bortezomib. Finally, given the important role of IL-6 in glucocorticoid resistance and the additive preclinical and clinical activity of bortezomib and dexamethasone in MM, we evaluated the activity of dexamethasone in combination with CNTO328 and bortezomib in ANBL-6 cells. Whereas the combination of bortezomib (2.5 nM) and CNTO328 reduced viability to 58%, the addition of dexamethasone (10 mM) reduced viability further to 26%. Taken together, the above data demonstrate that CNTO328 enhances the activity of bortezomib in part by attenuating bortezomib-mediated Mcl-1 accumulation, and provide the rationale for clinical evaluation of CNTO328 and bortezomib, with or without dexamethasone, in patients with MM.
- Published
- 2006
46. Development of Immunoproteasome-Specific Inhibitors as Novel Anti-Cancer Drugs
- Author
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Deborah J. Kuhn, Zhigen Hu, Peter M. Voorhees, Frank J. Bruzzese, Robert Z. Orlowski, John S. Strader, Jefrey P Ciavarri, and Qing Chen
- Subjects
Chemistry ,Bortezomib ,Kinase ,Chronic lymphocytic leukemia ,Immunology ,Myeloid leukemia ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Proteasome ,Apoptosis ,medicine ,Cancer research ,Proteasome inhibitor ,Multiple myeloma ,medicine.drug - Abstract
Introduction: The proteasome is a large (~2.5 MDa), ATP-dependent, intracellular protease responsible for degrading ubiquitinated proteins as part of the ubiquitin-proteasome pathway. The immunoproteasome is a unique proteasomal variant with distinct catalytic subunits termed low molecular mass proteins that functions predominately in cells derived from hematopoietic precursors, and differs from the constitutive proteasome found in most other cells. Bortezomib (VELCADE®; Millennium Pharmaceuticals, Inc.) is a first-in-class proteasome inhibitor, which is approved for treatment of multiple myeloma patients who have received at least one prior therapy. While the overall safety profile of bortezomib is manageable and predictable, some toxicities, such as peripheral neuropathy, associated with bortezomib treatment can be dose-limiting. These toxicities may be due to the inhibition of all isoforms of the proteasome. Development of immunoproteasome-specific inhibitors (IPSIs) would allow for targeted therapy against cancers arising from hematologic origins, thereby sparing normal tissues, such as gastrointestinal and neurological tissues. Methods: We have identified several novel IPSIs, most notably IPSI-001, with selective activity against the immunoproteasome, which we therefore sought to characterize. Results: Expression of proteins associated with the immunoproteasome (low molecular mass protein-2; 11S Reg-α) was found primarily in cell lines of hematopoietic origin, while solid tumor cell lines exhibited expression of constitutive proteasome proteins (β5; 19S S6′). IPSI-001 exposure induced preferential inhibition of the chymotrypsin-like activity, the rate-limiting step of proteolysis, in hematologic cell lines over solid tumors. This inhibition was associated with an increase in ubiquitinated substrates, activation of c-Jun N-terminal kinase, and accumulation of Bax. IPSI-001 treatment led to preferential induction of apoptosis as evidenced by DNA fragmentation assays and cleavage of β-actin by caspase-3 into an apoptotic marker, fractin. Furthermore, IPSI-001 had potent chymotrypsin-like inhibitory activity in patient samples of chronic lymphocytic leukemia and acute myeloid leukemia. A dose-dependent decrease in proliferation was observed in additional patient samples of acute myeloid leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma treated with IPSI-001. Also, IPSI-001 exposure induced apoptosis in multiple myeloma and chronic lymphocytic leukemia patient samples. In an effort to increase the efficacy of IPSIs, a series of boronic acid analogs were made of several IPSIs. Conversion into the boronate analogs increased the potency by up to 1000-fold against the chymotrypsin-like activity of the immunoproteasome in vitro and in cellulo. Dose-dependent inhibition of proliferation was observed in ANBL-6, KAS-6/1, and Ramos cell lines, which was associated with induction of apoptosis. Conclusions: Studies are ongoing to characterize the specificity and molecular effects associated with IPSI-boronic acid derivatives exposure in immunoproteasome- and constitutive proteasome-containing cell types.
- Published
- 2006
47. Inhibition of Kinesin Spindle Protein Induces Apoptosis and Overcomes Drug Resistance in Models of Multiple Myeloma
- Author
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Peter M. Voorhees, Robert Z. Orlowski, John S. Strader, Qing Chen, Vishwan Pamarthi, and Deborah J. Kuhn
- Subjects
education.field_of_study ,Bortezomib ,Immunology ,Population ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Spindle pole body ,Cell biology ,Spindle apparatus ,Cell culture ,hemic and lymphatic diseases ,medicine ,Kinesin ,Viability assay ,education ,Mitosis ,medicine.drug - Abstract
Introduction: Kinesin spindle protein is a mitotic kinesin that is expressed only in proliferating cells and plays a key role in spindle pole separation, formation of a bipolar mitotic spindle, and thus in satisfaction of the mitotic checkpoint. Ispinesib (SB-715992) is a potent and selective inhibitor of kinesin spindle protein with a Ki of 0.6 nM, has cytotoxic activity at less than 10 nM in a spectrum of tumor cell lines, and disrupts the assembly of functional bipolar mitotic spindles. Methods: This study sought to examine whether spindle disruption by inhibition of kinesin spindle protein with ispinesib may have therapeutic potential in the treatment of multiple myeloma. Results: Ispinesib reduced cell viability in both interleukin-6-independent (RPMI 8226 and U266) and interleukin-6-dependent (ANBL-6 and KAS-6/1) models of multiple myeloma in a time- and concentration-dependent fashion. The average IC50 for ispinesib against these cell lines was 3.0 nM, 1.7 nM, 1.8 nM, and 1.8 nM, respectively. Cell cycle analysis showed that ispinesib induced growth arrest of myeloma cells with 4N DNA content (in M phase) within 24-hours. Two days after treatment at a 1 nM concentration, cells were able to recover from M phase arrest and resume normal cycling but, after exposure to 10 nM, treated cells could not escape M phase arrest, and instead entered apoptosis as determined by an increased sub-G1 population. Ispinesib was able to overcome resistance to melphalan in that the IC50 in melphalan-resistant RPMI 8226/LR5 cells (1.6 ± 0.2 nM) was comparable to that in parental RPMI 8226 controls (3.0 ± 0.9 nM). Similarly, ispinesib was also able to overcome dexamethasone resistance and bortezomib resistance. In regard to the latter, KAS-6/VR5 bortezomib-resistant cells (IC50 of 12.5 nM for bortezomib) retained sensitivity to ispinesib (IC50 1.6 ± 0.2 nM). Combination therapy with ispinesib and bortezomib in these cells resulted in enhanced levels of specific apoptosis (24%) that were greater than the sum of either agents alone (7% for ispinesib and 1% for bortezomib), suggesting synergy. Importantly, ispinesib was also active against freshly isolated CD138+ patient-derived multiple myeloma cells, while relatively sparing CD138− cells. Conclusions: Taken together, these studies demonstrate that kinesin spindle protein inhibition with ispinesib was able to induce growth arrest and apoptosis in myeloma cells, and overcome resistance to both conventional drugs and novel agents such as bortezomib. Moreover, the preferential activity against transformed plasma cells with sparing of normal bone marrow cells provides a strong rationale for translation of this agent into the clinic to combat relapsed/refractory multiple myeloma.
- Published
- 2006
48. CNTO328 Abrogates Glucocorticoid (GC) Resistance Conferred by Interleukin (IL)-6 and the Bone Marrow Microenvironment in Multiple Myeloma (MM)
- Author
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Deborah J. Kuhn, George W. Small, Robert Corringham, Peter M. Voorhees, Qing Chen, Mohamed H. Zaki, Carlos A. Garay, Robert Z. Orlowski, Jeffrey A. Nemeth, and Sally A. Hunsucker
- Subjects
medicine.medical_specialty ,Stromal cell ,Cell growth ,Growth factor ,medicine.medical_treatment ,Immunology ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Endocrinology ,medicine.anatomical_structure ,Cell culture ,Internal medicine ,Cancer research ,medicine ,Cytotoxic T cell ,Viability assay ,Bone marrow ,PI3K/AKT/mTOR pathway - Abstract
Given the critical role that IL-6 plays in MM cell proliferation, survival, and resistance to GCs, we evaluated the ability of CNTO328, a chimeric monoclonal IL-6 neutralizing antibody, to overcome GC resistance in cell line models of human MM. In the presence of IL-6, the MM cell lines ANBL-6 and KAS-6 were resistant to the cytotoxic activity of dexamethasone (Dex) as assessed by cell viability assays both in suspension culture and in the context of patient-derived stromal cells. Resistance to dexamethasone was readily reversed by CNTO328, but not an isotype control antibody, in suspension culture. For example, in the case of the ANBL-6 model, viability was reduced by 12% with CNTO328 alone, 8% with Dex, but 74% with the combination, consistent with a synergistic interaction Given the ability of other growth factors in the bone marrow microenvironment to confer GC resistance in preclinical models of MM, we evaluated the activity of the CNTO328 and Dex combination in ANBL-6 and KAS-6 cells using a physiologically-relevant MM cell/patient-derived bone marrow stromal cell co-culture system. Importantly, bone marrow stromal cells rendered ANBL-6 and KAS-6 cells resistant to Dex in cell viability assays, and CNTO328 was able to reestablish Dex sensitivity, thus confirming a central role of IL-6 in bone marrow stroma-mediated GC resistance. Furthermore, treatment of ANBL-6 and KAS-6 cells with Dex alone did not induce apoptosis in this co-culture system, whereas the combination of CNTO328 and Dex led to a synergistic induction of apoptosis. In KAS-6 cells, IL-6-mediated Dex resistance was not overcome using pharmacologic inhibitors to p38, PI-3 kinase, mTor or MEK, suggesting that other IL-6 signaling pathways are likely involved. In contrast, the mTor inhibitor rapamycin was capable of sensitizing ANBL-6 cells to Dex in the presence of IL-6, suggesting that this pathway may be relevant to IL-6-mediated GC resistance in these cells. Induction of the pro-apoptotic Bcl-2 family member, Bim, has been shown to play an important role in GC-mediated cell death in lymphocytes as well as preclinical lymphoma and acute lymphoblastic leukemia models. Interestingly, although treatment of ANBL-6 cells in the presence of IL-6 with either CNTO328 or dexamethasone did not lead to induction of Bim, the combination led to a 3.3-fold increase in its expression. Taken together, the above data demonstrate that inhibition of IL-6 signaling with CNTO328 can effectively overcome IL-6-mediated GC resistance even in the presence of bone marrow stroma, and provide a compelling rationale for translation of this combination into clinical trials for patients suffering from MM. Furthermore, we show that the ability of CNTO328 to overcome GC resistance may be mediated in part by its ability to reverse IL-6-mediated repression of GC-induced Bim expression. Studies evaluating the relevance of Bim modulation in IL-6-mediated GC resistance and the molecular pathways that mediate this effect are on-going.
- Published
- 2006
49. Targeting the p27Kip1 E3 Ubiquitin Ligase for the Treatment of Multiple Myeloma and Other Hematological Malignancies
- Author
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Deborah J. Kuhn, Qing Chen, Emilia Glezer, Kyle W. H. Chan, Frank Mercurio, Derek Mendy, Peter M. Voorhees, Robert Z. Orlowski, Laura G. Corral, Veronique Plantevin, Weiming Xu, Weilin Xie, Antonia Lopez-Girona, and Laura De Parseval
- Subjects
biology ,Bortezomib ,Immunology ,Autophagy ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Molecular biology ,Ubiquitin ligase ,Ubiquitin ,hemic and lymphatic diseases ,Heat shock protein ,medicine ,biology.protein ,Proteasome inhibitor ,Cancer research ,Heat shock ,Multiple myeloma ,medicine.drug - Abstract
Introduction: The proteasome inhibitor bortezomib (VELCADE®; formerly PS-341) has been approved for therapy of relapsed/refractory multiple myeloma but may be limited in its effectiveness by activation of an anti-apoptotic heat shock protein response. In contrast, targeting a unique E3 ubiquitin ligase could selectively stabilize specific cellular proteins whose ubiquitination is regulated by this E3, reducing the likelihood of any unwanted pro-survival effects on other pathways. Methods: To this end, we sought to identify a small molecule that preferentially stabilized the tumor suppressor proteins p21Cip1 and p27Kip1. Results: Here we report the identification of CC-125007, a small molecule that stabilized p21Cip1, p27Kip1, and p57 in models of multiple myeloma. Unlike bortezomib, CC-125007 did not induce accumulation of other cell cycle regulatory proteins such as p53. CC-125007 reduced viability in a time- and concentration-dependent fashion and, again unlike bortezomib, which induced arrest at G2/M, CC-125007 induced cell cycle arrest at the G1/S phase. Decreased viability due to CC-125007 was not accompanied by caspase-mediated apoptosis, however, since it could not be blocked by pre-incubation with a pan-caspase inhibitor. Instead, a puncate monodansylcadaverine staining pattern was observed, demonstrating the formation of autophagic vacuoles consistent with activation of autophagy, or type II programmed cell death. Further supporting this possibility, combination of the autophagy inhibitor bafilomycin A1 and CC-125007 diminished autophagic vacuole formation. Furthermore, Western blotting demonstrated that CC-125007 induced conversion of microtubule-associated protein light chain 3 form I to II, a biochemical marker for autophagy. Meanwhile, Western blotting also showed that CC-125007 did not activate the anti-apoptotic heat shock response proteins 27, 70, and 90, which would be typical of bortezomib. CC-125007 overcame melphalan and bortezomib resistance in multiple myeloma cell lines, and a combination of low dose bortezomib and CC-125007 triggered synergistic anti-myeloma activity. Importantly, in freshly isolated bone marrow mononuclear cells purified from multiple myeloma patients, CC-125007 decreased the viability of the CD138+ population to a much greater extant than that of the CD138− cells. CC-125007 also stabilized p27Kip1 in samples from patients with acute myeloid leukemia, and enhanced bortezomib-induced apoptosis. Studies to elucidate the mechanism by which CC-125007 stabilized p27Kip1 indicate direct targeting of the E3 ligase. Conclusions: Together, these data suggest that p27Kip1 E3 ligase inhibition with agents such as CC-125007 is a rational strategy for the treatment of multiple myeloma and other hematological malignancies, providing a framework for further development of this novel class of agents.
- Published
- 2006
50. The Novel, Irreversible Proteasome Inhibitor PR-171 Demonstrates Potent Anti-Tumor Activity in Pre-Clinical Models of Multiple Myeloma, and Overcomes Bortezomib Resistance
- Author
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Mark K. Bennett, Kevin D. Shenk, Peter M. Voorhees, Qing Chen, John S. Strader, Susan Demo, Congcong M. Sun, Robert Z. Orlowski, Fred W. van Leeuwen, and Deborah J. Kuhn
- Subjects
Kinase ,Bortezomib ,Immunology ,Caspase 3 ,Cell Biology ,Hematology ,Pharmacology ,Biology ,medicine.disease ,Biochemistry ,Mechanism of action ,Proteasome ,In vivo ,Proteasome inhibitor ,medicine ,medicine.symptom ,Multiple myeloma ,medicine.drug - Abstract
Introduction: The ubiquitin-proteasome pathway has been validated as a therapeutic target with the approval of the small molecule proteasome inhibitor, bortezomib (VELCADE®), in multiple myeloma and non-Hodgkin lymphoma. However, the overall response rate of patients with multiple myeloma in phase III clinical trials was 43%, underscoring the need for a next generation of inhibitors with the potential for greater efficacy. Methods: PR-171 is a novel, tetrapeptide epoxomicin-related inhibitor that binds the proteasome irreversibly, and our objectives were to evaluate its activity and mechanism of action in pre-clinical models of multiple myeloma. Results: PR-171 potently bound and inhibited the chymotrypsin-like subunit of the proteasome in vitro, in cellulo, and in vivo at low concentrations. At higher concentrations, however, unlike bortezomib, which targeted the chymotrypsin-like and peptidyl-glutamyl peptide hydrolyzing activities in vivo, PR-171 also displayed significant inhibition of the trypsin-like and the peptidyl-glutamyl peptide hydrolyzing activities. PR-171-induced proteasome inhibition was associated with accumulation of polyubiquitinated substrates and pro-apoptotic Bax. Brief pulse PR-171 exposure, which simulates the in vivo pharmacokinetics of bortezomib, led to PR-171-mediated inhibition of cellular proliferation linked to induction of caspase-3-dependent apoptosis through both intrinsic (caspase-9) and extrinsic (caspase-8-dependent) pathways. Pretreatment with caspase-3, -8, and -9 inhibitors rescued the anti-proliferative effect of PR-171. Furthermore, pulse PR-171 treatment activated c-Jun-N-terminal kinase, a key-signaling molecule in proteasome inhibitor-induced apoptosis, and cleavage of poly-ADP-ribose polymerase, while abrogation of c-Jun-N-terminal kinase signaling with a dominant-negative c-Jun inhibited PR-171-induced effects. PR-171 displayed enhanced anti-proliferative activity compared to bortezomib in multiple myeloma cell lines and freshly isolated patient-derived CD138+ plasma cells, associated with enhanced phosphorylation of c-Jun-N-terminal kinase and capase-3, -8, and -9 activation. Lastly, PR-171 was a potent inhibitor of proliferation in a multiple myeloma cell line model resistant to bortezomib and in isolates from two patients, one with primary and the other with acquired bortezomib-resistance. Conclusions: These data indicate that PR-171 has enhanced activity against preclinical models of multiple myeloma, perhaps owing to its irreversible binding and subunit specificity, and provide a rationale for its translation into the clinic.
- Published
- 2006
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