254 results on '"Susan Band Horwitz"'
Search Results
2. Taxol®: The First Microtubule Stabilizing Agent
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Chia-Ping Huang Yang and Susan Band Horwitz
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Taxol® ,drug binding site ,photoaffinity labeling ,drug resistance ,tubulin isotypes ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Taxol®, an antitumor drug with significant activity, is the first microtubule stabilizing agent described in the literature. This short review of the mechanism of action of Taxol® emphasizes the research done in the Horwitz’ laboratory. It discusses the contribution of photoaffinity labeled analogues of Taxol® toward our understanding of the binding site of the drug on the microtubule. The importance of hydrogen/deuterium exchange experiments to further our insights into the stabilization of microtubules by Taxol® is addressed. The development of drug resistance, a major problem that arises in the clinic, is discussed. Studies describing differential drug binding to distinct β-tubulin isotypes are presented. Looking forward, it is suggested that the β-tubulin isotype content of a tumor may influence its responses to Taxol®.
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- 2017
- Full Text
- View/download PDF
3. Exploiting MEK inhibitor-mediated activation of ERα for therapeutic intervention in ER-positive ovarian carcinoma.
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June Y Hou, Alicia Rodriguez-Gabin, Leleesha Samaraweera, Rachel Hazan, Gary L Goldberg, Susan Band Horwitz, and Hayley M McDaid
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Medicine ,Science - Abstract
While the clinical benefit of MEK inhibitor (MEKi)-based therapy is well established in Raf mutant malignancies, its utility as a suppressor of hyperactive MAPK signaling in the absence of mutated Raf or Ras, is an area of ongoing research. MAPK activation is associated with loss of ERα expression and hormonal resistance in numerous malignancies. Herein, we demonstrate that MEKi induces a feedback response that results in ERα overexpression, phosphorylation and transcriptional activation of ER-regulated genes. Mechanistically, MEKi-mediated ERα overexpression is largely independent of erbB2 and AKT feedback activation, but is ERK-dependent. We subsequently exploit this phenomenon therapeutically by combining the ER-antagonist, fulvestrant with MEKi. This results in synergistic suppression of tumor growth, in vitro and potentiation of single agent activity in vivo in nude mice bearing xenografts. Thus, we demonstrate that exploiting adaptive feedback after MEKi can be used to sensitize ERα-positive tumors to hormonal therapy, and propose that this strategy may have broader clinical utility in ERα-positive ovarian carcinoma.
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- 2013
- Full Text
- View/download PDF
4. Supplementary Tables 1 and 2 and Supplementary Figures 1 through 4 from Proteomics of Cancer Cell Lines Resistant to Microtubule-Stabilizing Agents
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Chia-Ping Huang Yang, Susan Band Horwitz, Ruth H. Angeletti, and Jakob Albrethsen
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PDF - 489K, Table S1. Antibodies for Western Blot Analyses. Table S2. Identification of proteins differentially expressed in drug-sensitive and -resistant cells. Fig. S1. Expression levels of stathmin, Ser16-P-stathmin (A) and galectin-1 (B) in drug-sensitive MDA231 and Taxol-resistant K20T cells. Fig. S2. Expression levels of CLIC1, Ezrin and NM23-H1 in drug-resistant A549 and Hey cells. Fig. S3. Suppression of galectin-1 expression in drug-resistant cells. Fig. S4. Suppression of galectin-1 expression increased drug sensitivity in both drug-sensitive and drug-resistant Hey cells.
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- 2023
5. Supplementary Data from Insulin-like Growth Factor 2 Expression Modulates Taxol Resistance and Is a Candidate Biomarker for Reduced Disease-Free Survival in Ovarian Cancer
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Susan Band Horwitz, Harriet O. Smith, Gary L. Goldberg, Eric R. Prossnitz, Clifford R. Qualls, Hugo Arias-Pulido, Juan Lin, Tiffany M. Hebert, Christine H. Kim, Marissa J. Ramirez, Jurriaan Brouwer-Visser, and Gloria S. Huang
- Abstract
Supplementary Data from Insulin-like Growth Factor 2 Expression Modulates Taxol Resistance and Is a Candidate Biomarker for Reduced Disease-Free Survival in Ovarian Cancer
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- 2023
6. Data from Potentiation of Taxol Efficacy by Discodermolide in Ovarian Carcinoma Xenograft-Bearing Mice
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Hayley M. McDaid, Susan Band Horwitz, Gary L. Goldberg, Amos B. Smith, B. Scott Freeze, Lluis Lopez-Barcons, and Gloria S. Huang
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Purpose: To evaluate the drug combination of discodermolide and Taxol in human ovarian cancer cells and in an in vivo model of ovarian carcinoma.Experimental Design: The combination index method was used to evaluate the interaction of Taxol and discodermolide in human ovarian SKOV-3 carcinoma cells. Data were correlated with alterations in cell cycle distribution and caspase activation. In addition, SKOV-3 xenograft-bearing mice were treated with either Taxol, discodermolide, or a combination of both drugs given concurrently to evaluate the antitumor efficacy and toxicity of this combination. The Matrigel plug assay and CD31 immunohistochemistry were done to assess antiangiogenic effects.Results: Taxol and discodermolide interact synergistically over a range of concentrations and molar ratios that cause drug-induced aneuploidy in ovarian carcinoma cells. In SKOV-3 xenograft-bearing mice, the combination is significantly superior to either single agent, and induces tumor regressions without notable toxicities. Immunohistochemical analysis of CD31 and Matrigel plug analysis show decreased vessel formation in mice treated with the combination relative to either drug alone.Conclusions: The synergistic activity of Taxol and discodermolide in cells is most potent at drug concentrations that result in drug-induced aneuploidy rather than mitotic arrest. Moreover, in an animal model of ovarian carcinoma, this is a well-tolerated combination that induces tumor regressions and suppresses angiogenesis. These data confirm the potency of this combination and support the use of concurrent low doses of Taxol and discodermolide for potential use in cancer therapeutics.
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- 2023
7. Supplementary Figure S1 from Potentiation of Taxol Efficacy by Discodermolide in Ovarian Carcinoma Xenograft-Bearing Mice
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Hayley M. McDaid, Susan Band Horwitz, Gary L. Goldberg, Amos B. Smith, B. Scott Freeze, Lluis Lopez-Barcons, and Gloria S. Huang
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Supplementary Figure S1 from Potentiation of Taxol Efficacy by Discodermolide in Ovarian Carcinoma Xenograft-Bearing Mice
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- 2023
8. Data from Insulin-like Growth Factor 2 Expression Modulates Taxol Resistance and Is a Candidate Biomarker for Reduced Disease-Free Survival in Ovarian Cancer
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Susan Band Horwitz, Harriet O. Smith, Gary L. Goldberg, Eric R. Prossnitz, Clifford R. Qualls, Hugo Arias-Pulido, Juan Lin, Tiffany M. Hebert, Christine H. Kim, Marissa J. Ramirez, Jurriaan Brouwer-Visser, and Gloria S. Huang
- Abstract
Purpose: This study was undertaken to examine the role of the insulin-like growth factor (IGF) signaling pathway in the response of ovarian cancer cells to Taxol and to evaluate the significance of this pathway in human epithelial ovarian tumors.Experimental Design: The effect of Taxol treatment on AKT activation in A2780 ovarian carcinoma cells was evaluated using antibodies specific for phospho-AKT. To study the drug-resistant phenotype, we developed a Taxol-resistant cell line, HEY-T30, derived from HEY ovarian carcinoma cells. IGF2 expression was measured by real-time PCR. A type 1 IGF receptor (IGF1R) inhibitor, NVP-AEW541, and IGF2 small interfering RNA were used to evaluate the effect of IGF pathway inhibition on proliferation and Taxol sensitivity. IGF2 protein expression was evaluated by immunohistochemistry in 115 epithelial ovarian tumors and analyzed in relation to clinical/pathologic factors using the χ2 or Fisher's exact tests. The influence of IGF2 expression on survival was studied with Cox regression.Results: Taxol-induced AKT phosphorylation required IGF1R tyrosine kinase activity and was associated with upregulation of IGF2. Resistant cells had higher IGF2 expression compared with sensitive cells, and IGF pathway inhibition restored sensitivity to Taxol. High IGF2 tumor expression correlated with advanced stage (P < 0.001) and tumor grade (P < 0.01) and reduced disease-free survival (P < 0.05).Conclusions: IGF2 modulates Taxol resistance, and tumor IGF2 expression is a candidate prognostic biomarker in epithelial ovarian tumors. IGF pathway inhibition sensitizes drug-resistant ovarian carcinoma cells to Taxol. Such novel findings suggest that IGF2 represents a therapeutic target in ovarian cancer, particularly in the setting of Taxol resistance. Clin Cancer Res; 16(11); 2999–3010. ©2010 AACR.
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- 2023
9. Supplementary Figures S1 & S2 from Enhancement of the Therapeutic Efficacy of Taxol by the Mitogen-Activated Protein Kinase Kinase Inhibitor CI-1040 in Nude Mice Bearing Human Heterotransplants
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Susan Band Horwitz, Román Pérez-Soler, Steven Keller, Marie Lia, Aaron Grossman, Lluis Lopez-Barcons, and Hayley M. McDaid
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Supplementary Figures S1 & S2 from Enhancement of the Therapeutic Efficacy of Taxol by the Mitogen-Activated Protein Kinase Kinase Inhibitor CI-1040 in Nude Mice Bearing Human Heterotransplants
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- 2023
10. Supplementary Figure 2 from Targeting Protein Translation in Human Non–Small Cell Lung Cancer via Combined MEK and Mammalian Target of Rapamycin Suppression
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Hayley M. McDaid, Susan Band Horwitz, Roman Pérez-Soler, Steven M. Keller, Lluis Lopez-Barcons, Han-Guang Yan, Chia-Ping Huang Yang, and Marie-Emmanuelle Legrier
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Supplementary Figure 2 from Targeting Protein Translation in Human Non–Small Cell Lung Cancer via Combined MEK and Mammalian Target of Rapamycin Suppression
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- 2023
11. Supplementary Figure 1 from Targeting Protein Translation in Human Non–Small Cell Lung Cancer via Combined MEK and Mammalian Target of Rapamycin Suppression
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Hayley M. McDaid, Susan Band Horwitz, Roman Pérez-Soler, Steven M. Keller, Lluis Lopez-Barcons, Han-Guang Yan, Chia-Ping Huang Yang, and Marie-Emmanuelle Legrier
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Supplementary Figure 1 from Targeting Protein Translation in Human Non–Small Cell Lung Cancer via Combined MEK and Mammalian Target of Rapamycin Suppression
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- 2023
12. Data from Enhancement of the Therapeutic Efficacy of Taxol by the Mitogen-Activated Protein Kinase Kinase Inhibitor CI-1040 in Nude Mice Bearing Human Heterotransplants
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Susan Band Horwitz, Román Pérez-Soler, Steven Keller, Marie Lia, Aaron Grossman, Lluis Lopez-Barcons, and Hayley M. McDaid
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Taxol may contribute to intrinsic chemoresistance by activating the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) cytoprotective pathway in human cancer cell lines and tumors. We have previously shown additivity between Taxol and the MEK inhibitor, U0126 in human cancer cell lines. Here, the combination of Taxol with an orally bioavailable MEK inhibitor, CI-1040, was evaluated in human lung tumors heterotransplanted into nude mice. Unlike xenograft models that are derived from cells with multiple genetic alterations due to prolonged passage, heterotransplanted tumor models are more clinically relevant. Combined treatment with both drugs resulted in inhibition of tumor growth in all models and tumor regressions in three of four models tested, supporting our previous observation that Taxol's efficacy is potentiated by MEK inhibition. Concurrent administration was superior to intermittent dosing. Pharmacodynamic assessments of tumors indicated that suppression of MEK was associated with induction of S473 phosphorylated Akt and reduced proliferation in the combination groups relative to single agents, in addition to suppression of fibroblast growth factor–mediated angiogenesis and reduced expression of vascular endothelial growth factor. These findings are significant and indicate that this combination may have broad therapeutic applications in a diverse range of lung tumors with different intrinsic chemosensitivities.
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- 2023
13. Data from Targeting Protein Translation in Human Non–Small Cell Lung Cancer via Combined MEK and Mammalian Target of Rapamycin Suppression
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Hayley M. McDaid, Susan Band Horwitz, Roman Pérez-Soler, Steven M. Keller, Lluis Lopez-Barcons, Han-Guang Yan, Chia-Ping Huang Yang, and Marie-Emmanuelle Legrier
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Lung cancer is a genetically heterogeneous disease characterized by the acquisition of somatic mutations in numerous protein kinases, including components of the rat sarcoma viral oncogene homolog (RAS) and AKT signaling cascades. These pathways intersect at various points, rendering this network highly redundant and suggesting that combined mitogen-activated protein/extracellular signal-regulated kinase (MEK) and mammalian target of rapamycin (mTOR) inhibition may be a promising drug combination that can overcome its intrinsic plasticity. The MEK inhibitors, CI-1040 or PD0325901, in combination with the mTOR inhibitor, rapamycin, or its analogue AP23573, exhibited dose-dependent synergism in human lung cancer cell lines that was associated with suppression of proliferation rather than enhancement of cell death. Concurrent suppression of MEK and mTOR inhibited ribosomal biogenesis by 40% within 24 h and was associated with a decreased polysome/monosome ratio that is indicative of reduced protein translation efficiency. Furthermore, the combination of PD0325901 and rapamycin was significantly superior to either drug alone or PD0325901 at the maximum tolerated dose in nude mice bearing human lung tumor xenografts or heterotransplants. Except for a PTEN mutant, all tumor models had sustained tumor regressions and minimal toxicity. These data (a) provide evidence that both pathways converge on factors that regulate translation initiation and (b) support therapeutic strategies in lung cancer that simultaneously suppress the RAS and AKT signaling network. [Cancer Res 2007;67(23):11300–8]
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- 2023
14. Supplementary Figure S3 from Enhancement of the Therapeutic Efficacy of Taxol by the Mitogen-Activated Protein Kinase Kinase Inhibitor CI-1040 in Nude Mice Bearing Human Heterotransplants
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Susan Band Horwitz, Román Pérez-Soler, Steven Keller, Marie Lia, Aaron Grossman, Lluis Lopez-Barcons, and Hayley M. McDaid
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Supplementary Figure S3 from Enhancement of the Therapeutic Efficacy of Taxol by the Mitogen-Activated Protein Kinase Kinase Inhibitor CI-1040 in Nude Mice Bearing Human Heterotransplants
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- 2023
15. Data from Synergistic Suppression of Microtubule Dynamics by Discodermolide and Paclitaxel in Non-Small Cell Lung Carcinoma Cells
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Mary Ann Jordan, Claudette Briand, Leslie Wilson, Susan Band Horwitz, Diane Braguer, Kathy Kamath, and Stéphane Honore
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Discodermolide is a new microtubule-targeted antimitotic drug in Phase I clinical trials that, like paclitaxel, stabilizes microtubule dynamics and enhances microtubule polymer mass in vitro and in cells. Despite their apparently similar binding sites on microtubules, discodermolide acts synergistically with paclitaxel to inhibit proliferation of A549 human lung cancer cells (L. Martello et al., Clin. Cancer Res., 6: 1978–1987, 2000). To understand their synergy, we examined the effects of the two drugs singly and in combination in A549 cells and found that, surprisingly, their antiproliferative synergy is related to their ability to synergistically inhibit microtubule dynamic instability and mitosis. The combination of discodermolide and paclitaxel at their antiproliferative IC50s (7 nm for discodermolide and 2 nm for paclitaxel) altered all of the parameters of dynamic instability synergistically except the time-based rescue frequency. For example, together the drugs inhibited overall microtubule dynamicity by 71%, but each drug individually inhibited dynamicity by only 24%, giving a combination index (CI) of 0.23. Discodermolide and paclitaxel also synergistically blocked cell cycle progression at G2-M (41, 9.6, and 16% for both drugs together, for discodermolide alone, and for paclitaxel alone, respectively; CI = 0.59), and they synergistically enhanced apoptosis (CI = 0.85). Microtubules are unique receptors for drugs. The results suggest that ligands that bind to large numbers of binding sites on an individual microtubule can interact in a poorly understood manner to synergistically suppress microtubule dynamic instability and inhibit both mitosis and cell proliferation, with important consequences for combination clinical therapy with microtubule-targeted drugs.
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- 2023
16. Supplementary Figure 3 from Targeting Protein Translation in Human Non–Small Cell Lung Cancer via Combined MEK and Mammalian Target of Rapamycin Suppression
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Hayley M. McDaid, Susan Band Horwitz, Roman Pérez-Soler, Steven M. Keller, Lluis Lopez-Barcons, Han-Guang Yan, Chia-Ping Huang Yang, and Marie-Emmanuelle Legrier
- Abstract
Supplementary Figure 3 from Targeting Protein Translation in Human Non–Small Cell Lung Cancer via Combined MEK and Mammalian Target of Rapamycin Suppression
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- 2023
17. Supplementary Tables 1-2 from Synergistic Suppression of Microtubule Dynamics by Discodermolide and Paclitaxel in Non-Small Cell Lung Carcinoma Cells
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Mary Ann Jordan, Claudette Briand, Leslie Wilson, Susan Band Horwitz, Diane Braguer, Kathy Kamath, and Stéphane Honore
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Supplementary Tables 1-2 from Synergistic Suppression of Microtubule Dynamics by Discodermolide and Paclitaxel in Non-Small Cell Lung Carcinoma Cells
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- 2023
18. Utilization of Photoaffinity Labeling to Investigate Binding of Microtubule Stabilizing Agents to P-Glycoprotein and β-Tubulin
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Chia-Ping Huang Yang, Susan Band Horwitz, and Hayley M. McDaid
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Excipients ,Pharmacology ,Paclitaxel ,Complementary and alternative medicine ,Tubulin ,Organic Chemistry ,Drug Discovery ,Pharmaceutical Science ,Molecular Medicine ,ATP Binding Cassette Transporter, Subfamily B, Member 1 ,Microtubules ,Article ,Analytical Chemistry - Abstract
Photoaffinity labeling approaches have historically been used in pharmacology to identify molecular targets. This methodology has played a pivotal role in identifying drug-binding domains and searching for novel compounds that may interact at these domains. In this review we focus on studies of Microtubule Stabilizing Agents (MSAs) of natural product origin, specifically taxol (paclitaxel). Taxol and other microtubule interacting agents bind to both P-glycoprotein (ABCB1), a drug efflux pump that reduces intracellular drug accumulation, and the tubulin/microtubule system. Both binding relationships modulate drug efficacy and are of immense interest to basic and translational scientists, primarily because of their association with drug resistance for this class of molecules. We present this body of work and acknowledge its value as fundamental to understanding the mechanisms of taxol and elucidation of the taxol pharmacophore. Furthermore, we highlight the ability to multiplex photoaffinity approaches with other technologies to further enhance our understanding of pharmacologic interactions at an atomic level. Thus, photoaffinity approaches offer a relatively inexpensive and robust technique that will continue to play an important role in drug discovery for the foreseeable future.
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- 2022
19. Structural Refinement of the Tubulin Ligand (+)-Discodermolide to Attenuate Chemotherapy-Mediated Senescence
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Hayley M. McDaid, Nancy R. Zhang, Andrea E. Prota, Alicia Rodriguez-Gabin, Tobias Mühlethaler, Michel O. Steinmetz, Kenny Ye, Amos B. Smith, Boying Guo, and Susan Band Horwitz
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0301 basic medicine ,Senescence ,Cell Survival ,Triple Negative Breast Neoplasms ,Crystallography, X-Ray ,Lactones ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Cell Line, Tumor ,Alkanes ,Humans ,Ovarian Neoplasms ,Pharmacology ,Taxane ,Natural product ,Dose-Response Relationship, Drug ,Molecular Structure ,biology ,Chemistry ,Articles ,Discodermolide ,Ligand (biochemistry) ,Tubulin Modulators ,030104 developmental biology ,Tubulin ,A549 Cells ,Drug Resistance, Neoplasm ,Pyrones ,Cell culture ,Area Under Curve ,Cancer cell ,biology.protein ,Cancer research ,Molecular Medicine ,Female ,Taxoids ,Carbamates ,030217 neurology & neurosurgery - Abstract
The natural product (+)-discodermolide (DDM) is a microtubule stabilizing agent and potent inducer of senescence. We refined the structure of DDM and evaluated the activity of novel congeners in triple negative breast and ovarian cancers, malignancies that typically succumb to taxane resistance. Previous structure-activity analyses identified the lactone and diene as moieties conferring anticancer activity, thus identifying priorities for the structural refinement studies described herein. Congeners possessing the monodiene with a simplified lactone had superior anticancer efficacy relative to taxol, particularly in resistant models. Specifically, one of these congeners, B2, demonstrated 1) improved pharmacologic properties, specifically increased maximum response achievable and area under the curve, and decreased EC(50); 2) a uniform dose-response profile across genetically heterogeneous cancer cell lines relative to taxol or DDM; 3) reduced propensity for senescence induction relative to DDM; 4) superior long-term activity in cancer cells versus taxol or DDM; and 5) attenuation of metastatic characteristics in treated cancer cells. To contrast the binding of B2 versus DDM in tubulin, X-ray crystallography studies revealed a shift in the position of the lactone ring associated with removal of the C2-methyl and C3-hydroxyl. Thus, B2 may be more adaptable to changes in the taxane site relative to DDM that could account for its favorable properties. In conclusion, we have identified a DDM congener with broad range anticancer efficacy that also has decreased risk of inducing chemotherapy-mediated senescence. SIGNIFICANCE STATEMENT: Here, we describe the anticancer activity of novel congeners of the tubulin-polymerizing molecule (+)-discodermolide. A lead molecule is identified that exhibits an improved dose-response profile in taxane-sensitive and taxane-resistant cancer cell models, diminished risk of chemotherapy-mediated senescence, and suppression of tumor cell invasion endpoints. X-ray crystallography studies identify subtle changes in the pose of binding to β-tubulin that could account for the improved anticancer activity. These findings support continued preclinical development of discodermolide, particularly in the chemorefractory setting.
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- 2020
20. Reflections on My Life with Taxol
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Susan Band Horwitz
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Binding Sites ,Paclitaxel ,MEDLINE ,Breast Neoplasms ,Biology ,Pharmacology ,Antineoplastic Agents, Phytogenic ,Microtubules ,General Biochemistry, Genetics and Molecular Biology ,Autobiographies as Topic ,Paclitaxel metabolism ,Humans ,M Phase Cell Cycle Checkpoints ,Female ,HeLa Cells - Published
- 2019
21. Taxol Analogues Exhibit Differential Effects on Photoaffinity Labeling of β-Tubulin and the Multidrug Resistance Associated P-Glycoprotein
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Iwao Ojima, Changwei Wang, Chia Ping Huang Yang, and Susan Band Horwitz
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0301 basic medicine ,Paclitaxel ,Pharmaceutical Science ,macromolecular substances ,Drug resistance ,Vinblastine ,Microtubules ,Article ,Analytical Chemistry ,03 medical and health sciences ,0302 clinical medicine ,Tubulin ,Drug Discovery ,Tumor Cells, Cultured ,medicine ,Humans ,ATP Binding Cassette Transporter, Subfamily B, Member 1 ,P-glycoprotein ,Ovarian Neoplasms ,Pharmacology ,Photoaffinity labeling ,biology ,Chemistry ,Organic Chemistry ,Isotype ,Molecular biology ,Drug Resistance, Multiple ,Multiple drug resistance ,030104 developmental biology ,Verapamil ,Complementary and alternative medicine ,Drug Resistance, Neoplasm ,Cell culture ,030220 oncology & carcinogenesis ,biology.protein ,Molecular Medicine ,Female ,Taxoids ,medicine.drug - Abstract
Several next-generation taxanes have been reported to possess high potency against Taxol-resistant cancer cell lines overexpressing βIII-tubulin and/or P-glycoprotein (P-gp), both of which are involved in drug resistance. Using a photoaffinity Taxol analogue, 2-(m-azidobenzoyl)-taxol, two potent next-generation taxanes, SB-T-1214 and SB-CST-10202, exhibited distinct inhibitory effects on photolabeling of β-tubulin from different eukaryotic sources that differ in β-tubulin isotype composition. They also specifically inhibited photolabeling of P-gp, and the inhibitory effect correlated well with the steady-state accumulation of [(3)H]vinblastine in a multidrug resistant (MDR) cell line, SKVLB1. Several microtubule-stabilizing agents (MSAs)-resistant cell lines from the human ovarian cancer cell line Hey were isolated, and their MDR1 and βIII-tubulin levels determined. Distinct potencies of the two taxanes against different MSA-resistant cells expressing unique levels of MDR1 and βIII-tubulin were found. Cytotoxicity assays, done in the presence of verapamil, indicated that SB-T-1214 is a substrate, although not as good as Taxol, for P-gp. The mechanisms involved in drug resistance are multifactorial, and the effectiveness of new Taxol analogues depends on the interaction between the drugs and all possible targets; in this case the two major cellular targets are β-tubulin and P-gp.
- Published
- 2018
22. Expression of βV-Tubulin in Secretory Cells of the Fallopian Tube Epithelium Marks Cellular Atypia
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Deepti Mathew, Yanhua Wang, Anne Van Arsdale, Hayley M. McDaid, and Susan Band Horwitz
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0301 basic medicine ,Pathology ,endocrine system diseases ,medicine.medical_treatment ,BRCA ,Pilot Projects ,Epithelium ,Cohort Studies ,0302 clinical medicine ,Basic Science ,Tubulin ,Salpingectomy ,Atypia ,Ovarian Neoplasms ,Secretory Pathway ,Cystadenoma, Serous ,Obstetrics and Gynecology ,Middle Aged ,Immunohistochemistry ,female genital diseases and pregnancy complications ,3. Good health ,Serous fluid ,medicine.anatomical_structure ,Oncology ,030220 oncology & carcinogenesis ,ComputingMethodologies_DOCUMENTANDTEXTPROCESSING ,Female ,Adult ,medicine.medical_specialty ,Fallopian tube ,Young Adult ,03 medical and health sciences ,Ovarian cancer ,medicine ,Humans ,Fallopian Tubes ,Secretion ,Aged ,β-Tubulin ,business.industry ,medicine.disease ,Serous Cystadenoma ,Cystadenocarcinoma, Serous ,Preneoplastic condition ,030104 developmental biology ,business ,Precancerous Conditions - Abstract
Supplemental digital content is available in the text., Objectives Class V Beta tubulin isotype (βV-tubulin) was recently found to have tissue-specific expression patterns in epithelial tissues with secretory function and aberrant expression in tumors. The aims of this pilot study were (a) to examine expression of βV-tubulin in the fallopian tube epithelium (FTE) of patients who underwent salpingectomy, (b) to characterize FTE atypia in high-risk patients with BRCA mutations, and (c) to determine expression of βV-tubulin in serous ovarian neoplasms. Methods Immunohistochemistry, with a highly specific antibody developed in our laboratory against human βV-tubulin, was used to evaluate expression in paraffin-embedded sections of the fallopian tube (n = 82) and tumors (n = 13), from prospectively selected cases, categorized by reason for salpingectomy. Results βV-tubulin, when present, was expressed in secretory cells and essentially never in ciliated cells of the FTE. Histologically “normal” FTE had very rare, scattered βV-tubulin–positive cells; percentage positivity increased in cases of serous ovarian neoplasms. The highest expression was observed in FTE from patients with BRCA mutant breast cancer. Four distinct types of FTE atypia were delineated in patients with known BRCA mutations. In a few additional test cases of ovarian neoplasms, βV-tubulin was highly expressed, with the extent and intensity of staining elevated in high-grade serous carcinomas compared with serous borderline tumors. Conclusions In summary, βV-tubulin was localized to secretory cells of the distal FTE and its expression varied according to the clinical diagnosis. The frequency of these cells and thus expression of βV-tubulin were dramatically enriched in tissue obtained from BRCA mutant cases, which also exhibited pronounced histologic atypia indicative of early predysplastic aberrations. Furthermore, elevated expression of βV-tubulin correlated with poor differentiation status in serous ovarian neoplasms.
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- 2018
23. Structural basis of microtubule stabilization by discodermolide
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Michel O. Steinmetz, Amos B. Smith, Ian Paterson, Katja Bargsten, Chia Ping H. Yang, Andrea E. Prota, Mariano Redondo-Horcajo, Hayley M. McDaid, José Fernando Díaz, Susan Band Horwitz, Ministerio de Economía y Competitividad (España), National Cancer Institute (US), Breast Cancer Research Foundation, Swiss National Science Foundation, Prota, Andrea E. [0000-0003-0875-5339], Redondo-Horcajo, Mariano [0000-0002-9329-4786], Horwitz, Susan [0000-0002-2392-1211], Díaz, José Fernando [0000-0003-2743-3319], Steinmetz, Michel O. [0000-0001-6157-3687], Prota, Andrea E., Redondo-Horcajo, Mariano, Horwitz, Susan, Díaz, José Fernando, and Steinmetz, Michel O.
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0301 basic medicine ,Bridged-Ring Compounds ,Stereochemistry ,Context (language use) ,Crystallography, X-Ray ,01 natural sciences ,Biochemistry ,Microtubules ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Lactones ,Microtubule ,Tubulin ,Alkanes ,Humans ,Binding site ,Molecular mechanism of action ,Molecular Biology ,X-ray crystallography ,Binding Sites ,biology ,010405 organic chemistry ,Tubulin Modulators ,Drug discovery ,Structure elucidation ,Organic Chemistry ,Discodermolide ,0104 chemical sciences ,3. Good health ,030104 developmental biology ,chemistry ,Paclitaxel ,Pyrones ,biology.protein ,Molecular Medicine ,Taxoids ,Carbamates - Abstract
5 p.-4 fig.-1 schem., Microtubule-stabilizing agents (MSAs) are widely used in chemotherapy. Here, using X-ray crystallography we describe the detailed binding modes of two potent MSAs, (+)-discodermolide (DDM) and the DDM-paclitaxel-hybrid KS-1-199-32, in the taxane pocket of B-tubulin. Both compounds bind in a very similar hairpin conformation as previously observed in solution. However, they differentially stabilize the M-loop of B-tubulin: KS-1-199-32 induces an M-loop helical conformation that is not observed for DDM. In the context of the microtubule structure, both MSAs connect the B-tubulin helices H6 and H7 and loop S9-S10 with the M-loop, which is similar to the structural effects elicited by epothilone A, but distinct from paclitaxel. Together, our data rationalize a differential binding mechanism of DDM and KS-1-199-32 on tubulin., This work was supported by grants from the Ministerio de Economia y Competitividad (BFU2016‐75319‐R (AEI/FEDER, UE), to J.F.D), the National Cancer Institute Grant CA077263 (A.B.S, H.M.D. and S.B.H), the Breast Cancer Research Foundation (H.M.D. and S.B.H.) and the Swiss National Science Foundation (31003A_166608; to M.O.S.). The authors acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery” and the COST action CM1470.
- Published
- 2017
24. Eribulin disrupts EB1-microtubule plus-tip complex formation
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Brian P. O’Rourke, Susan Band Horwitz, Chia Ping Huang Yang, and David J. Sharp
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Eribulin Mesylate ,Halichondrin B ,biology ,Microtubule-associated protein ,Cell Biology ,Ketones ,Pharmacology ,Microtubules ,chemistry.chemical_compound ,Tubulin ,chemistry ,Microtubule ,Cell Line, Tumor ,biology.protein ,Biophysics ,Humans ,Binding site ,Furans ,Microtubule-Associated Proteins ,Molecular Biology ,Mitosis ,Reports ,Developmental Biology ,Eribulin - Abstract
Eribulin mesylate is a synthetic analog of halichondrin B known to bind tubulin and microtubules, specifically at their protein rich plus-ends, thereby dampening microtubule (MT) dynamics, arresting cells in mitosis, and inducing apoptosis. The proteins which bind to the MT plus-end are known as microtubule plus-end tracking proteins (+TIPs) and have been shown to promote MT growth and stabilization. Eribulin's plus-end binding suggests it may compete for binding sites with known +TIP proteins such as End-binding 1 (EB1). To better understand the impact of eribulin plus-end binding in regard to the proteins which normally bind there, cells expressing GFP-EB1 were treated with various concentrations of eribulin. In a concentration dependent manner, GFP-EB1 became dissociated from the MT plus-ends following drug addition. Similar results were found with immuno-stained fixed cells. Cells treated with low concentrations of eribulin also showed decreased ability to migrate, suggesting the decrease in MT dynamics may have a downstream effect. Extended exposure of eribulin to cells leads to total depolymerization of the MT array. Taken together, these data show eribulin effectively disrupts EB1 +TIP complex formation, providing mechanistic insights into the impact of eribulin on MT dynamics.
- Published
- 2014
25. 2-(m-Azidobenzoyl)taxol binds differentially to distinct β-tubulin isotypes
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Hui Xiao, Eng Hui Yap, Andras Fiser, Susan Band Horwitz, and Chia Ping Huang Yang
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0301 basic medicine ,macromolecular substances ,Polymerization ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Protein Domains ,Microtubule ,Tubulin ,Animals ,Humans ,Protein Isoforms ,Amino Acid Sequence ,Threonine ,Binding site ,Peptide sequence ,Alanine ,Multidisciplinary ,Binding Sites ,biology ,organic chemicals ,Biological Sciences ,Isotype ,Molecular biology ,Antineoplastic Agents, Phytogenic ,030104 developmental biology ,chemistry ,Biochemistry ,030220 oncology & carcinogenesis ,Mutation ,biology.protein ,Cyanogen bromide ,Taxoids ,Sequence Alignment ,HeLa Cells - Abstract
There are seven β-tubulin isotypes present in distinct quantities in mammalian cells of different origin. Altered expression of β-tubulin isotypes has been reported in cancer cell lines resistant to microtubule stabilizing agents (MSAs) and in human tumors resistant to Taxol. To study the relative binding affinities of MSAs, tubulin from different sources, with distinct β-tubulin isotype content, were specifically photolabeled with a tritium-labeled Taxol analog, 2-(m-azidobenzoyl)taxol, alone or in the presence of MSAs. The inhibitory effects elicited by these MSAs on photolabeling were distinct for β-tubulin from different sources. To determine the exact amount of drug that binds to different β-tubulin isotypes, bovine brain tubulin was photolabeled and the isotypes resolved by high-resolution isoelectrofocusing. All bands were analyzed by mass spectrometry following cyanogen bromide digestion, and the identity and relative quantity of each β-tubulin isotype determined. It was found that compared with other β-tubulin isotypes, βIII-tubulin bound the least amount of 2-(m-azidobenzoyl)taxol. Analysis of the sequences of β-tubulin near the Taxol binding site indicated that, in addition to the M-loop that is known to be involved in drug binding, the leucine cluster region of βIII-tubulin contains a unique residue, alanine, at 218, compared with other isotypes that contain threonine. Molecular dynamic simulations indicated that the frequency of Taxol-accommodating conformations decreased dramatically in the T218A variant, compared with other β-tubulins. Our results indicate that the difference in residue 218 in βIII-tubulin may be responsible for inhibition of drug binding to this isotype, which could influence downstream cellular events.
- Published
- 2016
26. A label-free mass spectrometry method for relative quantitation of β-tubulin isotype expression in human tumor tissue
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Hui Xiao, Charles Dumontet, Sylvie Isaac, Ruth Hogue Angeletti, Pascal Sève, Chia Ping Huang Yang, Susan Band Horwitz, and Leah M. Miller
- Subjects
Bridged-Ring Compounds ,Taxane ,biology ,Molecular Sequence Data ,Clinical Biochemistry ,macromolecular substances ,Molecular biology ,Isotype ,Article ,Label-free quantification ,Tubulin ,Drug Resistance, Neoplasm ,Cell culture ,Cell Line, Tumor ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,biology.protein ,Humans ,Protein Isoforms ,Immunohistochemistry ,Taxoids ,Trypsin ,Amino Acid Sequence ,Antibody ,Peptide sequence - Abstract
Purpose Quantitation of β-tubulin isotype expression in taxane resistant human tumor tissue has been difficult to achieve because of the limited availability of validated antibodies. Here we present a label-free MS method to quantitate relative expression levels of β-tubulin isotypes. Experimental design Using isotype-specific reporter peptides, we determined relative β-tubulin isotype expression levels in human lung tumor tissue. Results Four reporter peptides were chosen to quantitate the βI/βII, βIV, βIII, and βV tubulin isotypes. These peptides were validated using human cancer cell lines. The label-free method was then used to determine β-tubulin isotype expression in nine human lung tumor samples, which had been described as high or low βIII-tubulin expressing using immunohistochemistry. It was found that βI/βII (accounting for 18.7–65.7% of total β-tubulin) and βIVa/βIVb (26.3–79.1%) were the most abundant isotypes and that the βIII (0–8.9%) and βV (1.0–10.4%) were less abundant in the tissue. We also categorized the samples as high or low βIII-tubulin expressing. Conclusion and clinical relevance With this method we can determine the relative expression levels of β-tubulin isotypes in human tumor tissue. This method will facilitate studies assessing the use of tubulin isotypes as biomarkers of taxane resistance.
- Published
- 2012
27. Structural Evidence for Cooperative Microtubule Stabilization by Taxol and the Endogenous Dynamics Regulator MAP4
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Hui Xiao, Marina Khrapunovich-Baine, Susan Band Horwitz, Ruth Hogue Angeletti, Chloe Bulinski, Zongcai Tu, Hui Wang, and Xue-Chun Zhang
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Paclitaxel ,Protein Conformation ,Protein Stability ,Microtubule-associated protein ,Stathmin ,macromolecular substances ,General Medicine ,Plasma protein binding ,Biology ,Microtubules ,Biochemistry ,Article ,Cell biology ,Spindle apparatus ,Tubulin ,Protein structure ,Microtubule ,biology.protein ,Humans ,Molecular Medicine ,Microtubule-Associated Proteins ,Mitosis ,Protein Binding - Abstract
MTs are highly dynamic protein polymers that are crucial in many aspects of cellular function, including mitosis. Mitosis can be disrupted by arresting microtubule dynamics, which may be accomplished with drugs that interact with MTs (1). One such class of drugs is the microtubule stabilizing agents, which bind to the MT polymer and prevent its disassembly, thereby inducing loss of dynamicity of the mitotic spindle required for progression of mitosis (2). The prototype of this class of drugs is Taxol, whose activity and properties have been studied extensively (3, 4). The dynamic behavior of MTs is also regulated in vivo by microtubule associated proteins (MAPs) (5). Some of these regulatory proteins, including stathmin, sequester tubulin dimers and promote microtubule disassembly, whereas others stimulate assembly and favor stable MTs (5, 6). Tau, MAP2 and MAP4 belong to a family of MAPs that promotes microtubule assembly and stabilization. Tau and MAP2 are found predominantly in neuronal and some glial cells, whereas MAP4 is ubiquitously expressed (7, 8). All members of this family have a similar primary structure, with each including an N-terminal projection (PJ) domain and a C-terminal microtubule-binding (MTB) domain (9–11). This latter MTB domain is responsible for binding to and regulating MT assembly. It can be subdivided into three distinct regions: a proline-rich region, an assembly promoting region, and a highly hydrophobic tail region (12). The assembly promoting region, which contains three, four, or five 18-residue imperfect repeats, is homologous in all family members and can bind to MTs and promote microtubule elongation (13, 14). The proline-rich regions of tau, MAP2 and MAP4 are similar in structure, but display no significant sequence homology. While sequences of tau and MAP2 tail regions bear a close resemblance to each other, MAP4 differs significantly from the other family members (12). Clearly, despite the sequence similarities found in this family of proteins, there may be differences in their mechanisms of microtubule stabilization, not least because MAP4 has not been studied as intensely as MAP2 and tau. As the predominant MAP in normal and cancer cells, MAP4 was selected in our study to investigate the structural changes in the αβ-tubulin heterodimer upon binding of endogenous microtubule stabilizers. Since MAP4 is ubiquitously expressed and shares characteristic structures with neural MAP2 and tau, analysis of structural changes in MTs induced by MAP4 binding will further our understanding of the role MAPs play in the regulation of MT dynamics. While we have obtained valuable information concerning the interaction of different types of microtubule-stabilizing drugs with tubulin and MTs (15, 16), our experimental system thus far has been limited by the absence of endogenous factors that further regulate MT dynamics, altering their conformation in vivo. Thus, here we expand our current HDX-MS studies to include MAP4 that is an endogenous dynamic regulator. The HDX-MS method is ideal for probing the interactions between protein-protein and protein-ligand complexes. A typical HDX-MS experiment involves the comparison of HDX rates of peptic peptides before and after interacting with its binding partners. A reduced HDX rate indicates that the peptide is either involved in the direct interactions with its partners or its dynamicity has been reduced by the binding interactions. Understanding how MAPs stabilize MTs in the presence and absence of Taxol may provide important insights into their role in modulating drug effects and MT dynamics. In the present study, we demonstrate that MTB-MAP4 significantly reduces deuterium incorporation into MTs, suggesting that it stabilizes the polymer via its effects on the multiple interfaces that comprise a stable MT polymer. This effect of MAP4 is further enhanced in the presence of Taxol, indicating cooperative action of these two stabilizers on MT dynamics.
- Published
- 2012
28. Hallmarks of Molecular Action of Microtubule Stabilizing Agents
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Vilas Menon, John H. Miller, Susan Band Horwitz, Peter T. Northcote, Chia Ping Huang Yang, Hui Xiao, Ruth Hogue Angeletti, Marina Khrapunovich-Baine, and Andras Fiser
- Subjects
biology ,Tubulin Modulators ,Ixabepilone ,Cell Biology ,Discodermolide ,Biochemistry ,Motor protein ,chemistry.chemical_compound ,Protein structure ,Tubulin ,chemistry ,Microtubule ,biology.protein ,Biophysics ,Binding site ,Molecular Biology - Abstract
Microtubule stabilizing agents (MSAs) comprise a class of drugs that bind to microtubule (MT) polymers and stabilize them against disassembly. Several of these agents are currently in clinical use as anticancer drugs, whereas others are in various stages of development. Nonetheless, there is insufficient knowledge about the molecular modes of their action. Recent studies from our laboratory utilizing hydrogen-deuterium exchange in combination with mass spectrometry (MS) provide new information on the conformational effects of Taxol and discodermolide on microtubules isolated from chicken erythrocytes (CET). We report here a comprehensive analysis of the effects of epothilone B, ixabepilone (IXEMPRATM), laulimalide, and peloruside A on CET conformation. The results of our comparative hydrogen-deuterium exchange MS studies indicate that all MSAs have significant conformational effects on the C-terminal H12 helix of α-tubulin, which is a likely molecular mechanism for the previously observed modulations of MT interactions with microtubule-associated and motor proteins. More importantly, the major mode of MT stabilization by MSAs is the tightening of the longitudinal interactions between two adjacent αβ-tubulin heterodimers at the interdimer interface. In contrast to previous observations reported with bovine brain tubulin, the lateral interactions between the adjacent protofilaments in CET are particularly strongly stabilized by peloruside A and laulimalide, drugs that bind outside the taxane site. This not only highlights the significance of tubulin isotype composition in modulating drug effects on MT conformation and stability but also provides a potential explanation for the synergy observed when combinations of taxane and alternative site binding drugs are used.
- Published
- 2011
29. Insights into 4E-BP1 and p53 mediated regulation of accelerated cell senescence
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Suzan K. Chao, Susan Band Horwitz, and Hayley M. McDaid
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p53 ,Senescence ,4E-BP1 ,Cell ,Cell Cycle Proteins ,mTORC1 ,Biology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Cell Line, Tumor ,Neoplasms ,medicine ,Humans ,Doxorubicin ,Molecular Targeted Therapy ,Transcription factor ,Cellular Senescence ,Adaptor Proteins, Signal Transducing ,030304 developmental biology ,0303 health sciences ,Molecular Pharmacology ,Discodermolide ,Genes, p53 ,Phosphoproteins ,3. Good health ,Cell biology ,discodermolide ,medicine.anatomical_structure ,Oncology ,chemistry ,030220 oncology & carcinogenesis ,Research Perspectives ,Tumor Suppressor Protein p53 ,Cell aging ,medicine.drug - Abstract
Suzan K. Chao 1 , Susan Band Horwitz 1 , and Hayley M. McDaid 1,2 1 Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461 2 Department of Medicine (Oncology) Albert Einstein College of Medicine, Bronx, NY 10461 Keywords: Senescence, 4E-BP1, mTORC1, p53, discodermolide Received: February 22, 2011; Accepted: February 22, 2011; Published: February 22, 2011; Correspondence: Susan Band Horwitz, e-mail: // Hayley McDaid, e-mail: // // Abstract Senescence is a valid tumor suppressive mechanism in cancer. Accelerated cell senescence describes the growth arrested state of cells that have been treated with anti-tumor drugs, such as doxorubicin that induce a DNA damage response. Discodermolide, a microtubule-stabilizing agent, is a potent inducer of accelerated cell senescence. Resistance to discodermolide is mediated via resistance to accelerated cell senescence, and is associated with reduced expression of the mTORC1 substrate, 4E-BP1 and increased expression of p53 [1]. Although the association of p53 with senescence induction is well-characterized, senescence reversion in the presence of high expression of p53 has not been well-documented. Furthermore, studies addressing the role of mTOR signaling in regulating senescence have been limited and recent data implicate a novel, senescence-associated role for 4E-BP1 in crosstalk with the transcription factor p53. This research perspective will address these somewhat contradictory findings and summarize recent research regarding senescence and mTORC1 signaling.
- Published
- 2011
30. The interaction between mitotic checkpoint proteins, CENP-E and BubR1, is diminished in epothilone B-resistant A549 cells
- Author
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Lingling Liu, Susan Band Horwitz, Amy E. Ikui, and Chia Ping Huang Yang
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Chromosomal Proteins, Non-Histone ,Mitosis ,macromolecular substances ,Protein Serine-Threonine Kinases ,Biology ,Cell Line, Tumor ,Centromere ,Humans ,Kinetochores ,Molecular Biology ,Metaphase ,Centrosome ,A549 cell ,Kinetochore ,Cell Biology ,Transfection ,Molecular biology ,Cell biology ,Protein Transport ,Drug Resistance, Neoplasm ,Epothilones ,Cell culture ,Protein Binding ,Developmental Biology - Abstract
Centromere associated protein-E (CENP-E), a mitotic checkpoint protein, is required for efficient, stable microtubule capture at kinetochores during mitosis. Absence of CENP-E results in misaligned chromosomes leading to metaphase arrest. Microtubule-interacting agents such as Taxol and epothilone B (EpoB), at concentrations that induce mitotic arrest, transiently increase expression of CENP-E in a variety of cancer cell lines. The CENP-E level in an EpoB-resistant A549 cell line, EpoB40, is ~ 2-fold higher than in A549 cells. CENP-E overexpression, after transfection with CENP-E cDNA into drug sensitive cells, does not alter Taxol or EpoB sensitivity. However, suppression of CENP-E expression by CENP-E siRNA results in a moderate increase in drug sensitivity, suggesting that a minimal quantity of CENP-E is required for maintaining its function. It is known that CENP-E binds to BubR1 and enhances its recruitment to each unattached kinetochore. Suppression of CENP-E results in a decrease in BubR1 levels in EpoB40 cells. During metaphase, both targeting of CENP-E and BubR1 to the kinetochores and the interaction between CENP-E and BubR1 are significantly reduced in EpoB40 cells, compared to A549 cells. In addition, the distance between the two centrosomes during metaphase is shorter in EpoB40 than in A549 cells, suggesting that defects in the spindle-assembly checkpoint have occurred in EpoB40 cells during the development of drug resistance. These results indicate that defects in the mitotic checkpoint may have a role in, or be the result of, the development of EpoB resistance.
- Published
- 2010
31. Distinct Pose of Discodermolide in Taxol Binding Pocket Drives a Complementary Mode of Microtubule Stabilization
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Hui Xiao, Marina Khrapunovich-Baine, Andras Fiser, Vilas Menon, Amos B. Smith, Susan Band Horwitz, Pascal Verdier-Pinard, and Ruth Hogue Angeletti
- Subjects
Paclitaxel ,Binding pocket ,macromolecular substances ,Biology ,Microtubules ,Biochemistry ,Microtubule stabilization ,Article ,Lactones ,chemistry.chemical_compound ,Tubulin ,Microtubule ,Alkanes ,Animals ,Protein Isoforms ,Binding site ,Binding Sites ,Photoaffinity labeling ,Protein Stability ,Electron crystallography ,Deuterium Exchange Measurement ,Drug Synergism ,Discodermolide ,Peptide Fragments ,Tubulin Modulators ,chemistry ,Pyrones ,Biophysics ,biology.protein ,Cattle ,Carbamates ,Chickens ,Dimerization ,Protein Binding - Abstract
The microtubule cytoskeleton has proven to be an effective target for cancer therapeutics. One class of drugs, known as microtubule stabilizing agents (MSAs), binds to microtubule polymers and stabilizes them against depolymerization. The prototype of this group of drugs, Taxol, is an effective chemotherapeutic agent used extensively in the treatment of human ovarian, breast, and lung carcinomas. Although electron crystallography and photoaffinity labeling experiments determined that the binding site for Taxol is in a hydrophobic pocket in beta-tubulin, little was known about the effects of this drug on the conformation of the entire microtubule. A recent study from our laboratory utilizing hydrogen-deuterium exchange (HDX) in concert with various mass spectrometry (MS) techniques has provided new information on the structure of microtubules upon Taxol binding. In the current study we apply this technique to determine the binding mode and the conformational effects on chicken erythrocyte tubulin (CET) of another MSA, discodermolide, whose synthetic analogues may have potential use in the clinic. We confirmed that, like Taxol, discodermolide binds to the taxane binding pocket in beta-tubulin. However, as opposed to Taxol, which has major interactions with the M-loop, discodermolide orients itself away from this loop and toward the N-terminal H1-S2 loop. Additionally, discodermolide stabilizes microtubules mainly via its effects on interdimer contacts, specifically on the alpha-tubulin side, and to a lesser extent on interprotofilament contacts between adjacent beta-tubulin subunits. Also, our results indicate complementary stabilizing effects of Taxol and discodermolide on the microtubules, which may explain the synergy observed between the two drugs in vivo.
- Published
- 2009
32. Tubulin proteomics: Towards breaking the code
- Author
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Eddy Pasquier, Daniel Lafitte, Hui Xiao, Claude Villard, Diane Braguer, Berta Burd, Leah M. Miller, Pascal Verdier-Pinard, Ruth Hogue Angeletti, and Susan Band Horwitz
- Subjects
Proteomics ,biology ,Phylum ,Microtubule-associated protein ,Electrospray ionization ,Biophysics ,Cell Biology ,Computational biology ,Biochemistry ,Molecular biology ,Mass Spectrometry ,Article ,Tubulin ,Microtubule ,Organelle ,biology.protein ,Animals ,Humans ,Functional significance ,Molecular Biology - Abstract
Since the discovery of tubulin as the major component of microtubules over 40 years ago, its diversity of forms has raised a continuum of fundamental questions about its regulation and functions in a variety of organisms across phyla. Its high abundance in the brain or in specialized organelles such as cilia has allowed early characterization of this important target for anticancer drugs. However, it was only when matrix-assisted laser desorption ionization and electrospray ionization mass spectrometry technologies became available in the late 1980's that the full complexity of tubulin expression patterns became more obvious. This contributed in a major way to the idea that due to increasing and conserved tubulin heterogeneity during evolution, a tubulin code read by microtubule associated proteins might exist and be of functional significance. We review here the merging of recent genetic and cell biology studies with proteomics to decipher this code and illustrate some of the tubulin proteomic approaches with new data generated in our laboratories.
- Published
- 2009
33. Increased Levels of a Unique Post-Translationally Modified βIVb-Tubulin Isotype in Liver Cancer
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Susan Band Horwitz, Anuradha Menthena, Pascal Verdier-Pinard, Leah M. Miller, Phyllis M. Novikoff, Champak Chatterjee, and Ruth Hogue Angeletti
- Subjects
Male ,Paclitaxel ,Microtubule-associated protein ,Molecular Sequence Data ,macromolecular substances ,Biology ,Biochemistry ,Article ,Tubulin ,Microtubule ,Detyrosination ,Animals ,Amino Acid Sequence ,Cytoskeleton ,Liver Neoplasms ,Isotype ,Peptide Fragments ,Rats, Inbred F344 ,Rats ,Polyglycylation ,Acetylation ,Hepatocytes ,biology.protein ,Isoelectric Focusing ,Protein Processing, Post-Translational - Abstract
Microtubules are dynamic protein polymers that play an essential role in cell division, maintenance of cell shape, transport of vesicles and cell motility (1). These hollow cylinders are constructed of heterodimers of α- and β-tubulin, which bind head-to-tail to form protofilaments that associate laterally to form microtubules (2). There are 6 α- and 7 β-tubulin genes described in mammals, with each gene being highly conserved across species (3). The majority of the diversity in tubulin gene products, termed isotypes, occurs in the last 15–20 residues of the C-terminus, which is known as the isotype defining region and is located on the outer surface of microtubules (4). Further increasing the diversity of the tubulin isotypes is their ability to undergo a range of post-translational modifications. Common post-translational modifications to tubulin are polyglutamylation, tyrosination/detyrosination, polyglycylation, acetylation and phosphorylation, most of which occur in the C-terminal region (5). Although there is no consensus regarding the specific role or function of individual tubulin isotypes, tissue-specific changes in the expression of tubulin isotypes have been observed, and isotype composition has been demonstrated to affect the dynamics of microtubule assembly (6–8). It has been suggested that divergent C-termini may provide a mechanism for isotype-specific microtubule associated protein (MAP) binding (9). Recently, a tubulin code, similar to the histone code, was proposed whereby tubulin isotypes and their modifications could serve as discrete signals to modulate cellular events, suggesting a mechanistic role for the observed diversity in microtubules (10). The integrity of the cytoskeleton is essential for the proper functioning of all cells. Therefore, changes in the composition of microtubules, a major component of the cytoskeleton, could contribute to tumorigenesis. Previous studies have examined the α- and β-tubulin isotypes in both tumors and cancer cell lines. An increase in βIII tubulin is the most commonly described alteration in tubulin expression in cancer, with an increase of this neuronal isotype observed in a variety of tumors (11–15). An increase in the expression of the βII tubulin in cancer has also been described (16). While a majority of studies have focused on β-tubulins, a recent report demonstrated a shift in post-translational modifications of α-tubulin isotypes in prostate cancer cell lines (17). In these studies, isotypes were assigned either at the protein level using isotype-specific antibodies or at the mRNA level by RT-PCR. Both of these methods continue to provide important qualitative and quantitative data about tubulin isotypes. However, neither method is able to completely describe the tubulin isotypes at the protein level. Mass spectrometry (MS) complements traditional methods of characterizing tubulin isotypes (18). It is an ideal tool for the identification of post-translational modifications to tubulins, with the ability to characterize multiple modifications in one experiment (19, 20). Unlike experiments employing antibodies, mass spectrometry can be used to detect mutations in isotypes as well as to identify new isotypes (21, 22). Also, when combined with “stable isotope labeling with amino acids in culture” (SILAC), mass spectrometry provides information regarding the relative expression levels of tubulin isotypes in drug resistant cell lines as compared to the tubulin isotypes present in drug sensitive cell lines (23). Therefore, the application of mass spectrometry to tubulin isotype characterization can provide unique information about tubulin isotypes at the protein level that cannot readily be achieved with other methods. Using a highly reproducible rat liver cancer model, the resistant hepatocyte model, we profiled the tubulin isotypes present at early, middle and late stages of cancer development (24). Hepatocellular carcinoma (HCC) is one of the most common and fatal forms of cancer worldwide (25). Detection of HCC at early stages is difficult and treatment options after identification are limited and often ineffective (26). In order to gain a better understanding of the changes occurring at the molecular level in carcinogenesis, we employed high resolution isoelectric focusing along with cyanogen bromide (CNBr) cleavage and MS to identify the tubulin isotypes present in the liver. Our methodology allowed for the identification of a novel C-terminal modification to βIVb-tubulin. We quantitated the relative amount of the peptide harboring this novel post-translational modification compared to all β-tubulin isotypes, and when comparing the levels in rat liver which underwent chemical carcinogen treatment relative to control liver, we demonstrated a 3-fold increase in modification during later stages of cancer. Such changes in a post-translational modification of a tubulin isotype during progression of liver cancer suggest that alterations to microtubules may be a useful indicator for the detection and treatment of liver cancer.
- Published
- 2008
34. Exploring the Mechanisms of Action of the Novel Microtubule Inhibitor Vinflunine
- Author
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Sharon Lobert, Mary Ann Jordan, Susan Band Horwitz, and John J. Correia
- Subjects
Vinca ,medicine.drug_class ,Mitosis ,Pharmacology ,Vinblastine ,Microtubules ,Vinca alkaloid ,chemistry.chemical_compound ,Drug Delivery Systems ,medicine ,Humans ,Vinflunine ,Cell Death ,biology ,Cell Cycle ,Hematology ,Cell cycle ,biology.organism_classification ,Antineoplastic Agents, Phytogenic ,Tubulin Modulators ,Spindle apparatus ,Oncology ,chemistry ,Cancer cell ,medicine.drug - Abstract
Microtubules have been identified as a suitable target for anticancer therapy, primarily based on their biological importance in coordinating chromosomal segregation at mitosis. Two main classes of microtubule-targeted agents, the taxanes and vinca alkaloids, suppress the dynamic behavior of spindle microtubules, inducing mitotic arrest and subsequent apoptotic cell death. Clinical activity of taxanes and first-generation vinca alkaloids in the treatment of solid tumors and hematologic malignancies, respectively, has prompted further research for novel analogs with improved clinical efficacy and safety. Such efforts have led to the development of vinflunine, a bifluorinated vinca alkaloid endowed with unique antitumor properties. Highlighted in this review are the key features of vinflunine that lead to effective suppression of microtubule dynamics and induction of cell death in cancer cells.
- Published
- 2008
35. Design, Synthesis, and Biological Evaluation of New-Generation Taxoids
- Author
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Ilaria Zanardi, Jon Mallen-St. Clair, Iwao Ojima, Susan Band Horwitz, Shujun Xia, Xianrui Zhao, Dafna Bar-Sagi, David Gallager, Jin Chen, Tao Wang, Xudong Geng, Michael L. Miller, Ralph J. Bernacki, Larisa V. Kuznetsova, Songnian Lin, Jean M. Veith, Jennifer L. Guerriero, Chuanxing Qu, Liang Sun, Christopher P. Borella, and Paula Pera
- Subjects
Male ,Paclitaxel ,Transplantation, Heterologous ,Mice, Nude ,Antineoplastic Agents ,Article ,Taxoid ,Mice ,Structure-Activity Relationship ,chemistry.chemical_compound ,Biopolymers ,Tubulin ,In vivo ,Cell Line, Tumor ,Drug Discovery ,Animals ,Humans ,Point Mutation ,Structure–activity relationship ,Cytotoxicity ,biology ,Chemistry ,Discodermolide ,Biochemistry ,Drug Resistance, Neoplasm ,Cell culture ,Drug Design ,biology.protein ,Molecular Medicine ,Female ,Taxoids ,Drug Screening Assays, Antitumor ,Neoplasm Transplantation - Abstract
Novel second-generation taxoids with systematic modifications at the C2, C10, and C3'N positions were synthesized and their structure-activity relationships studied. A number of these taxoids exhibited exceptionally high potency against multidrug-resistant cell lines, and several taxoids exhibited virtually no difference in potency against the drug-sensitive and drug-resistant cell lines. These exceptionally potent taxoids were termed "third-generation taxoids". 19 (SB-T-1214), 14g (SB-T-121303), and 14i (SB-T-1213031) exhibited excellent activity against paclitaxel-resistant ovarian cancer cell lines with mutations in beta-tubulin as well, wherein the drug resistance is mediated by the beta-tubulin mutation. These taxoids were found to possess exceptional activity in promoting tubulin assembly, forming numerous very short microtubules similar to those formed by discodermolide. Taxoids 19 and 14g also showed excellent cytotoxicity against four pancreatic cancer cell lines, expressing three to four multidrug-resistant genes. Moreover, taxoid 19 exhibited excellent in vivo efficacy against highly drug-resistant CFPAC-1 pancreatic as well as DLD-1 human colon tumor xenografts in mice.
- Published
- 2008
36. The Effect of Ketoconazole on the Pharmacokinetics and Pharmacodynamics of Ixabepilone: A First in Class Epothilone B Analogue in Late-Phase Clinical Development
- Author
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Lisa Iacono, Sanjay Goel, Carrie Xu, Gary L. Goldberg, S. Nilgun Comezoglu, François André, Marvin B. Cohen, David Jayabalan, Donglu Zhang, Susan Band Horwitz, Hayley M. McDaid, W. Griffith Humphreys, Sridhar Mani, Adriana Comprelli, Van T. Ly, Lionel Perrin, Lentz, Celine, Système membranaires, photobiologie, stress et détoxication (SMPSD), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
Adult ,Male ,Cancer Research ,Microtubule bundle formation ,Genital Neoplasms, Female ,[CHIM.THER] Chemical Sciences/Medicinal Chemistry ,Antineoplastic Agents ,[CHIM.THER]Chemical Sciences/Medicinal Chemistry ,Biology ,Pharmacology ,chemistry.chemical_compound ,Pharmacokinetics ,Neoplasms ,medicine ,Cytochrome P-450 CYP3A ,Humans ,Aged ,Aged, 80 and over ,CYP3A4 ,Liver Neoplasms ,Ixabepilone ,Computational Biology ,Prostatic Neoplasms ,Middle Aged ,Drug interaction ,Tubulin Modulators ,Ketoconazole ,Liver ,Oncology ,chemistry ,Epothilones ,Pharmacodynamics ,Leukocytes, Mononuclear ,Microsomes, Liver ,Epothilone B Analogue ,Cytochrome P-450 CYP3A Inhibitors ,Female ,medicine.drug - Abstract
Purpose: To determine if ixabepilone is a substrate for cytochrome P450 3A4 (CYP3A4) and if its metabolism by this cytochrome is clinically important, we did a clinical drug interaction study in humans using ketoconazole as an inhibitor of CYP3A4. Experimental Design: Human microsomes were used to determine the cytochrome P450 enzyme(s) involved in the metabolism of ixabepilone. Computational docking (CYP3A4) studies were done for epothilone B and ixabepilone. A follow-up clinical study was done in patients with cancer to determine if 400 mg/d ketoconazole (inhibitor of CYP3A4) altered the pharmacokinetics, drug-target interactions, and pharmacodynamics of ixabepilone. Results: Molecular modeling and human microsomal studies predicted ixabepilone to be a good substrate for CYP3A4. In patients, ketoconazole coadministration resulted in a maximum ixabepilone dose administration to 25 mg/m2 when compared with single-agent therapy of 40 mg/m2. Coadministration of ketoconazole with ixabepilone resulted in a 79% increase in AUC0-∞. The relationship of microtubule bundle formation in peripheral blood mononuclear cells to plasma ixabepilone concentration was well described by the Hill equation. Microtubule bundle formation in peripheral blood mononuclear cells correlated with neutropenia. Conclusions: Ixabepilone is a good CYP3A4 substrate in vitro; however, in humans, it is likely to be cleared by multiple mechanisms. Furthermore, our results provide evidence that there is a direct relationship between ixabepilone pharmacokinetics, neutrophil counts, and microtubule bundle formation in PBMCs. Strong inhibitors of CYP3A4 should be used cautiously in the context of ixabepilone dosing.
- Published
- 2008
37. Targeting Protein Translation in Human Non–Small Cell Lung Cancer via Combined MEK and Mammalian Target of Rapamycin Suppression
- Author
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Marie Emmanuelle Legrier, Steven M. Keller, Chia Ping Huang Yang, Lluis Lopez-Barcons, Roman Perez-Soler, Hayley M. McDaid, Susan Band Horwitz, and Han Guang Yan
- Subjects
Cancer Research ,Lung Neoplasms ,Immunoblotting ,Mice, Nude ,Biology ,Mice ,Carcinoma, Non-Small-Cell Lung ,medicine ,Animals ,Humans ,PTEN ,Peptide Chain Initiation, Translational ,Lung cancer ,Protein kinase B ,PI3K/AKT/mTOR pathway ,Cell Proliferation ,Feedback, Physiological ,Mitogen-Activated Protein Kinase Kinases ,Sirolimus ,Cell Death ,Kinase ,TOR Serine-Threonine Kinases ,RPTOR ,Diphenylamine ,PTEN Phosphohydrolase ,Cancer ,Drug Synergism ,medicine.disease ,Genetic translation ,Oncology ,Benzamides ,Immunology ,biology.protein ,Cancer research ,Drug Therapy, Combination ,Protein Kinases ,Proto-Oncogene Proteins c-akt ,Immunosuppressive Agents ,Signal Transduction - Abstract
Lung cancer is a genetically heterogeneous disease characterized by the acquisition of somatic mutations in numerous protein kinases, including components of the rat sarcoma viral oncogene homolog (RAS) and AKT signaling cascades. These pathways intersect at various points, rendering this network highly redundant and suggesting that combined mitogen-activated protein/extracellular signal-regulated kinase (MEK) and mammalian target of rapamycin (mTOR) inhibition may be a promising drug combination that can overcome its intrinsic plasticity. The MEK inhibitors, CI-1040 or PD0325901, in combination with the mTOR inhibitor, rapamycin, or its analogue AP23573, exhibited dose-dependent synergism in human lung cancer cell lines that was associated with suppression of proliferation rather than enhancement of cell death. Concurrent suppression of MEK and mTOR inhibited ribosomal biogenesis by 40% within 24 h and was associated with a decreased polysome/monosome ratio that is indicative of reduced protein translation efficiency. Furthermore, the combination of PD0325901 and rapamycin was significantly superior to either drug alone or PD0325901 at the maximum tolerated dose in nude mice bearing human lung tumor xenografts or heterotransplants. Except for a PTEN mutant, all tumor models had sustained tumor regressions and minimal toxicity. These data (a) provide evidence that both pathways converge on factors that regulate translation initiation and (b) support therapeutic strategies in lung cancer that simultaneously suppress the RAS and AKT signaling network. [Cancer Res 2007;67(23):11300–8]
- Published
- 2007
38. Wild-type class I β-tubulin sensitizes Taxol-resistant breast adenocarcinoma cells harboring a β-tubulin mutation
- Author
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Kenneth M. Wiesen, Shujun Xia, Chia Ping Huang Yang, and Susan Band Horwitz
- Subjects
Models, Molecular ,Cancer Research ,Paclitaxel ,Cell Survival ,Glycine ,Mutation, Missense ,Glutamic Acid ,Breast Neoplasms ,Docetaxel ,macromolecular substances ,Adenocarcinoma ,Transfection ,Lactones ,chemistry.chemical_compound ,Tubulin ,Microtubule ,Cell Line, Tumor ,Alkanes ,Humans ,Fluorescent Antibody Technique, Indirect ,Protein Structure, Quaternary ,P-glycoprotein ,Expression vector ,biology ,Wild type ,Discodermolide ,Antineoplastic Agents, Phytogenic ,Molecular biology ,Tubulin Modulators ,Oncology ,chemistry ,Drug Resistance, Neoplasm ,Epothilones ,Pyrones ,Cell culture ,biology.protein ,Taxoids ,Carbamates ,Dimerization - Abstract
A Taxol-resistant cell line, K20T, which does not express P-glycoprotein, was selected with Taxol from human MDA-MB-231 breast adenocarcinoma cells and maintained in the presence of 20nM Taxol. K20T cells were approximately 18-fold resistant to Taxol, displayed cross-resistance to Taxotere and the epothilones, but little cross-resistance to discodermolide. Sequence analysis of the class I beta-tubulin indicated that it harbored an A593G mutation resulting in a change from glutamate to glycine at amino acid 198, which is near the intradimer interface within the alpha/beta-tubulin heterodimer. An HA-tagged wild-type class I beta-tubulin expression vector was transfected into the K20T cells. Immunofluorescence studies demonstrated that this exogenous tubulin was incorporated into cellular microtubules and Western blot analysis indicated that the K20T transfectants predominantly expressed the exogenous wild-type class I beta-tubulin. The transfected cells were only approximately 5-fold resistant to Taxol. Our results, plus the knowledge that Glu198 is the target for other anti-tubulin agents, suggest that glutamate198 in beta-tubulin is a critical determinant for microtubule stability and Taxol resistance.
- Published
- 2007
39. A Symposium in Honor of Richard G. Powell, a Long-Time Associate Editor of the Journal of Natural Products
- Author
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A. Douglas Kinghorn and Susan Band Horwitz
- Subjects
Pharmacology ,Biological Products ,Molecular Structure ,Philosophy ,Organic Chemistry ,Pharmaceutical Science ,Environmental ethics ,Analytical Chemistry ,Associate editor ,Complementary and alternative medicine ,Honor ,Drug Discovery ,Molecular Medicine ,Natural (music) ,Classics - Published
- 2015
40. Germline mutations of the DNA repair pathways in uterine serous carcinoma
- Author
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Marina Frimer, Gary L. Goldberg, June Y. Hou, Alicia Rodriguez-Gabin, Yanhua Wang, Kelly S. Levano, and Susan Band Horwitz
- Subjects
0301 basic medicine ,DNA Repair ,DNA repair ,Genes, BRCA2 ,Genes, BRCA1 ,medicine.disease_cause ,Bioinformatics ,Germline ,Uterine serous carcinoma ,03 medical and health sciences ,0302 clinical medicine ,Germline mutation ,medicine ,Humans ,Homologous Recombination ,Exome sequencing ,Germ-Line Mutation ,Aged ,Mutation ,business.industry ,Obstetrics and Gynecology ,medicine.disease ,Cystadenocarcinoma, Serous ,030104 developmental biology ,Oncology ,030220 oncology & carcinogenesis ,Uterine Neoplasms ,Cancer research ,DNA mismatch repair ,Female ,Carcinogenesis ,business - Abstract
Objective Treatment options are limited for patients with uterine serous carcinoma (USC). Knowledge of USC's somatic mutation landscape is rapidly increasing, but its role in hereditary cancers remains unclear. We aim to evaluate the frequency and characteristics of germline mutations in genes commonly implicated in carcinogenesis, including those within homologous recombination (HR) and mismatch repair (MMR) pathways in patients with pure USC. Methods By using targeted capture exome sequencing, 43 genes were analyzed in a cohort of 7 consecutive patients with paired tumor and non-tumor USC samples in our institutional tumor repository. Mutations predicted to have damaging effects on protein function are validated by Sanger Sequencing. Results We found 21 germline mutations in 11 genes in our USC cohort. Five patients harbored 7 germline mutations (33.3%) within genes involved in the HR pathway, RAD51D being the most common. Four patients had 9 (42.8%) germline mutations in hereditary colon cancer genes, most commonly MLH. All patients (42.7%) who are platinum-sensitive had HR germline mutations (RAD50, NBN, ATM). Patients with HER2 overexpression (2/7, 28.6%) had germline HR mutations and were platinum-sensitive. Three patients in our cohort reported a personal history of breast cancer, one with HR germline mutation, and 2 in patients with germline mutations in HCC genes. In addition, 5 out of 7 patients had germline mutations in genes associated with growth factor signaling pathway. Conclusions A significant proportion of our cohort harbor germline mutations in DNA repair genes. This may be associated with the high rate of breast cancer in our patients and their family, and suggests a targeted cohort for genetic counseling. If validated in a larger cohort, our findings may allow clinicians to expand therapeutic options to include targeted therapies and inclusion of USC patient in preventative and genetic counseling.
- Published
- 2015
41. TP53 hot spot mutations in ovarian cancer: selective resistance to microtubule stabilizers in vitro and differential survival outcomes from The Cancer Genome Atlas
- Author
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Kevin H. Eng, Chia Ping Huang Yang, Brandon Luke L. Seagle, Shohreh Shahabi, Monica Dandapani, Kunle Odunsi, Gary L. Goldberg, Susan Band Horwitz, and Oluwatosin Odunsi-Akanji
- Subjects
endocrine system diseases ,Paclitaxel ,Biology ,Carcinoma, Ovarian Epithelial ,Article ,Microtubule ,Ovarian carcinoma ,Cancer genome ,Cell Line, Tumor ,medicine ,Differential survival ,Humans ,Neoplasms, Glandular and Epithelial ,Ovarian Neoplasms ,Cell Death ,Obstetrics and Gynecology ,Middle Aged ,medicine.disease ,Genes, p53 ,Molecular biology ,female genital diseases and pregnancy complications ,In vitro ,Tubulin Modulators ,Serous fluid ,Oncology ,Epithelial ovarian carcinoma ,Drug Resistance, Neoplasm ,Epothilones ,Mutation ,Cancer research ,Female ,Tumor Suppressor Protein p53 ,Ovarian cancer - Abstract
To test if TP53 hot spot mutations (HSMs) confer differential chemotherapy resistance or survival outcomes, the effects of microtubule stabilizers on human ovarian carcinoma cells (OCCs) expressing TP53 HSMs were studied in vitro. Survival outcomes of patients with high grade serous epithelial ovarian carcinoma (HGS EOC) expressing matched HSMs were compared using The Cancer Genome Atlas (TCGA) data.Growth inhibition of OCCs transfected with a HSM (m175, m248 or m273) was measured during treatment with paclitaxel, epothilone B (epoB), or ixabepilone. Effects of epoB on p53 expression, phosphorylation, and acetylation, as well as p53-regulated expression of p21 and mdm2 proteins, were determined by Western blot analysis. Expression of p53 target genes P21, GADD45, BAX, PIDD, NF-kB2, PAI-1, and MDR1 was measured by RT-PCR. cBioPortal.org identified patients with codon R175, R248 or R273 HSMs from TCGA data. Survival outcomes were characterized.p53-m248 confers chemoresistance and is not acetylated during epoB treatment. m273 demonstrated high MDR1 expression and resistance to paclitaxel. P21, GADD45 and PAI-1 expression were down-regulated in mutant OCCs. Optimally cytoreduced patients with codon R273 (n=17), R248 (n=13), R175 (n=7) HSMs, or any other TP53 mutation demonstrated median 14.9, 17.6, 17.8 and 16.9months (p=0.806) progression free survival and 84.1, 33.6, 62.1 and 44.5months (p=0.040) overall survival, respectively.Human OCCs harboring different TP53 HSMs were selectively resistant to microtubule stabilizers. Patients with different HSMs had significantly different overall survival. Both in vitro data and clinical experience support further studying the outcomes of particular TP53 HSMs.
- Published
- 2015
42. A conversation with Susan Band Horwitz
- Author
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Susan Band Horwitz and I. David Goldman
- Subjects
Gerontology ,Psychoanalysis ,Biomedical Research ,Paclitaxel ,media_common.quotation_subject ,education ,Toxicology ,History, 21st Century ,Lactones ,Alkanes ,Drug Discovery ,Medicine ,Humans ,Conversation ,Molecular Targeted Therapy ,media_common ,Pharmacology ,Career Choice ,business.industry ,History, 20th Century ,Antineoplastic Agents, Phytogenic ,humanities ,Tubulin Modulators ,Pyrones ,Carbamates ,business - Abstract
Susan Band Horwitz is a Distinguished Professor and holds the Falkenstein Chair in Cancer Research at Albert Einstein College of Medicine in New York. She is co-chair of the Department of Molecular Pharmacology and associate director for therapeutics at the Albert Einstein Cancer Center. After graduating from Bryn Mawr College, Dr. Horwitz received her PhD in biochemistry from Brandeis University. She has had a continuing interest in natural products as a source of new drugs for the treatment of cancer. Her most seminal research contribution has been in the development of Taxol®. Dr. Horwitz and her colleagues made the discovery that Taxol had a unique mechanism of action and suggested that it was a prototype for a new class of antitumor drugs. Although Taxol was an antimitotic agent blocking cells in the metaphase stage of the cell cycle, Dr. Horwitz recognized that Taxol was blocking mitosis in a way different from that of other known agents. Her group demonstrated that the binding site for Taxol was on the β-tubulin subunit. The interaction of Taxol with the β-tubulin subunit resulted in stabilized microtubules, essentially paralyzing the cytoskeleton, thereby preventing cell division. Dr. Horwitz served as president (2002–2003) of the American Association for Cancer Research (AACR). She is a member of the National Academy of Sciences, the Institute of Medicine, the American Academy of Arts and Sciences, and the American Philosophical Society. She has received numerous honors and awards, including the C. Chester Stock Award from Memorial Sloan Kettering Cancer Center, the Warren Alpert Foundation Prize from Harvard Medical School, the Bristol-Myers Squibb Award for Distinguished Achievement in Cancer Research, the American Cancer Society's Medal of Honor, and the AACR Award for Lifetime Achievement in Cancer Research. The following interview was conducted on January 23, 2014.
- Published
- 2015
43. Insights into the mechanism of microtubule stabilization by Taxol
- Author
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Hui Xiao, Narcis Fernandez-Fuentes, Pascal Verdier-Pinard, Berta Burd, Ruth Hogue Angeletti, Susan Band Horwitz, Andras Fiser, and George A. Orr
- Subjects
Models, Molecular ,Paclitaxel ,GTP' ,macromolecular substances ,Plasma protein binding ,Microtubules ,chemistry.chemical_compound ,Protein structure ,Tubulin ,Microtubule ,Animals ,Protein Structure, Quaternary ,Multidisciplinary ,biology ,Deuterium Exchange Measurement ,Biological Sciences ,Peptide Fragments ,Protein Structure, Tertiary ,chemistry ,Biochemistry ,Biophysics ,biology.protein ,Hydrogen–deuterium exchange ,Chickens ,Dimerization - Abstract
The antitumor drug Taxol stabilizes microtubules and reduces their dynamicity, promoting mitotic arrest and cell death. Upon assembly of the alpha/beta-tubulin heterodimer, GTP bound to beta-tubulin is hydrolyzed to GDP reaching a steady-state equilibrium between free tubulin dimers and microtubules. The binding of Taxol to beta-tubulin in the polymer results in cold-stable microtubules at the expense of tubulin dimers, even in the absence of exogenous GTP. However, there is little biochemical insight into the mechanism(s) by which Taxol stabilizes microtubules. Here, we analyze the structural changes occurring in both beta- and alpha-tubulin upon microtubule stabilization by Taxol. Hydrogen/deuterium exchange (HDX) coupled to liquid chromatography-electrospray ionization MS demonstrated a marked reduction in deuterium incorporation in both beta-and alpha-tubulin when Taxol was present. Decreased local HDX in peptic peptides was mapped on the tubulin structure and revealed both expected and new dimer-dimer interactions. The increased rigidity in Taxol microtubules was distinct from and complementary to that due to GTP-induced polymerization. The Taxol-induced changes in tubulin conformation act against microtubule depolymerization in a precise directional way. These results demonstrate that HDX coupled to liquid chromatography-electrospray ionization MS can be effectively used to study conformational effects induced by small ligands on microtubules. The present study also opens avenues for locating drug and protein binding sites and for deciphering the mechanisms by which their interactions alter the conformation of microtubules and tubulin dimers.
- Published
- 2006
44. Potentiation of Taxol Efficacy by Discodermolide in Ovarian Carcinoma Xenograft-Bearing Mice
- Author
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Gary L. Goldberg, Lluis Lopez-Barcons, Amos B. Smith, Hayley M. McDaid, Susan Band Horwitz, B. Scott Freeze, and Gloria S. Huang
- Subjects
Cancer Research ,Pathology ,medicine.medical_specialty ,Paclitaxel ,endocrine system diseases ,Angiogenesis ,Biology ,Article ,Lactones ,Mice ,chemistry.chemical_compound ,In vivo ,Cell Line, Tumor ,Ovarian carcinoma ,Alkanes ,Antineoplastic Combined Chemotherapy Protocols ,Carcinoma ,medicine ,Animals ,Humans ,Ovarian Neoplasms ,Neovascularization, Pathologic ,Cell Cycle ,Drug Synergism ,Discodermolide ,Cell cycle ,medicine.disease ,Immunohistochemistry ,Xenograft Model Antitumor Assays ,Enzyme Activation ,Platelet Endothelial Cell Adhesion Molecule-1 ,Transplantation ,Oncology ,chemistry ,Pyrones ,Caspases ,Cancer research ,Female ,Carbamates - Abstract
Purpose: To evaluate the drug combination of discodermolide and Taxol in human ovarian cancer cells and in an in vivo model of ovarian carcinoma. Experimental Design: The combination index method was used to evaluate the interaction of Taxol and discodermolide in human ovarian SKOV-3 carcinoma cells. Data were correlated with alterations in cell cycle distribution and caspase activation. In addition, SKOV-3 xenograft-bearing mice were treated with either Taxol, discodermolide, or a combination of both drugs given concurrently to evaluate the antitumor efficacy and toxicity of this combination. The Matrigel plug assay and CD31 immunohistochemistry were done to assess antiangiogenic effects. Results: Taxol and discodermolide interact synergistically over a range of concentrations and molar ratios that cause drug-induced aneuploidy in ovarian carcinoma cells. In SKOV-3 xenograft-bearing mice, the combination is significantly superior to either single agent, and induces tumor regressions without notable toxicities. Immunohistochemical analysis of CD31 and Matrigel plug analysis show decreased vessel formation in mice treated with the combination relative to either drug alone. Conclusions: The synergistic activity of Taxol and discodermolide in cells is most potent at drug concentrations that result in drug-induced aneuploidy rather than mitotic arrest. Moreover, in an animal model of ovarian carcinoma, this is a well-tolerated combination that induces tumor regressions and suppresses angiogenesis. These data confirm the potency of this combination and support the use of concurrent low doses of Taxol and discodermolide for potential use in cancer therapeutics.
- Published
- 2006
45. Design, Synthesis, and Biological Evaluation of Potent Discodermolide Fluorescent and Photoaffinity Molecular Probes
- Author
-
Shujun Xia, Ignacio Brouard, Susan Band Horwitz, Paul V. Rucker, Amos B. Smith, and B. Scott Freeze
- Subjects
Molecular Structure ,Stereochemistry ,Chemistry ,viruses ,Organic Chemistry ,Stereoisomerism ,Photoaffinity Labels ,Discodermolide ,Biochemistry ,Combinatorial chemistry ,Fluorescence ,Benzophenones ,Lactones ,chemistry.chemical_compound ,Design synthesis ,Pyrones ,Molecular Probes ,Alkanes ,Carbamates ,Physical and Theoretical Chemistry ,Molecular probe ,Fluorescent Dyes ,Biological evaluation - Abstract
[structure: see text] The design, synthesis, and biological evaluation of a series of (+)-discodermolide molecular probes possessing photoaffinity and fluorescent appendages has been achieved. Stereoselective olefin cross-metathesis comprised a key tactic for construction of two of the molecular probes. Three photoaffinity probes were radiolabeled with tritium.
- Published
- 2005
46. Activation of the Steroid and Xenobiotic Receptor (Human Pregnane X Receptor) by Nontaxane Microtubule-Stabilizing Agents
- Author
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Susan Band Horwitz, Haiyan Huang, Amos B. Smith, Sumathy Sundarababu, Ganjam V. Kalpana, Wenjing Liu, and Sridhar Mani
- Subjects
Receptors, Steroid ,Cancer Research ,Transcription, Genetic ,Receptors, Cytoplasmic and Nuclear ,Pharmacology ,Lactones ,Mice ,chemistry.chemical_compound ,Nuclear Receptor Coactivator 1 ,Cytochrome P-450 Enzyme System ,Cytochrome P-450 CYP3A ,Receptor ,Glutathione Transferase ,Histone Acetyltransferases ,Pregnane X receptor ,Pregnane X Receptor ,Nuclear Proteins ,Discodermolide ,DNA-Binding Proteins ,Oncology ,Paclitaxel ,Aryl Hydrocarbon Hydroxylases ,Plasmids ,Transcriptional Activation ,Immunoblotting ,Antineoplastic Agents ,Biology ,Xenobiotics ,In vivo ,Two-Hybrid System Techniques ,Alkanes ,Coactivator ,Animals ,Humans ,Nuclear Receptor Co-Repressor 1 ,Nuclear Receptor Co-Repressor 2 ,Reflex, Abnormal ,CYP3A4 ,Oxidoreductases, N-Demethylating ,Blotting, Northern ,Antineoplastic Agents, Phytogenic ,Mice, Inbred C57BL ,Repressor Proteins ,chemistry ,Epothilones ,Pyrones ,Carbamates ,Corepressor ,Transcription Factors - Abstract
Purpose: Because induction of drug efflux transporters is one of the major underlying mechanisms of drug resistance in cancer chemotherapy, and human pregnane X receptor (hPXR) is one of the principal “xenobiotic” receptors whose activation induces transporter and drug-metabolizing enzyme gene transcription, it would be ideal to develop chemotherapy drugs that do not activate hPXR. This report describes studies undertaken to explore the characteristics of hPXR stimulation and mechanisms of drug-receptor interactions in vitro with new anti-tubulin drugs. Experimental Design: In vitro transient transcription, glutathione S-transferase pull-down assays, and mammalian one-hybrid and two-hybrid systems were used to explore drug-receptor interactions. Loss of righting reflex was used to assess effects of drugs on PXR activity in vivo. Results: The current study showed that paclitaxel, discodermolide, and an analogue of epothilone B, BMS-247550, induced CYP3A4 protein expression in HepG2 hepatoma cells. Transient transcription assays of a luciferase reporter in the presence and absence of a GAL4-steroid and xenobiotic receptor (SXR) plasmid in HepG2 cells showed that these drugs activate hPXR. This was not true for the inactive analogue of paclitaxel, baccatin III, or for an analogue of epothilone A, analogue 5, none of which stabilizes microtubules. To determine the mechanisms by which paclitaxel, discodermolide, and BMS-247550 activate hPXR, a mammalian two-hybrid assay was done using VP16SRC-1 (coactivator) and GAL4-SXR. SRC-1 preferentially augmented the effects of these drugs on hPXR. Expression of SMRT (corepressor) but not NCoR suppressed the drug-induced activation of SXR by ∼50%, indicating a selectivity in corepressor interaction with hPXR. These drugs resulted in shortened duration of loss of righting reflex in vivo, indicating drug-induced activation of PXR in mice. Conclusion: These findings suggest that activation of hPXR with selective displacement of corepressors is an important mechanism by which microtubule-stabilizing drugs induce drug-metabolizing enzymes both in vitro and in vivo.
- Published
- 2005
47. The Microtubule Stabilizing Agent Discodermolide is a Potent Inducer of Accelerated Cell Senescence
- Author
-
B. Scott Freeze, Amos B. Smith, Susan Band Horwitz, and Laura E. Klein
- Subjects
Senescence ,Time Factors ,Paclitaxel ,Cell ,Clone (cell biology) ,Mitosis ,Antineoplastic Agents ,Biology ,Microtubules ,Inhibitory Concentration 50 ,Lactones ,chemistry.chemical_compound ,Microtubule ,Cell Line, Tumor ,Alkanes ,medicine ,Humans ,Molecular Biology ,Mitotic catastrophe ,Cellular Senescence ,Mitogen-Activated Protein Kinase 1 ,A549 cell ,Mitogen-Activated Protein Kinase 3 ,Cell Biology ,Discodermolide ,Hydrogen-Ion Concentration ,Flow Cytometry ,Antineoplastic Agents, Phytogenic ,Cell biology ,Phenotype ,medicine.anatomical_structure ,Bromodeoxyuridine ,Gene Expression Regulation ,chemistry ,Pyrones ,Cancer research ,Carbamates ,Signal Transduction ,Developmental Biology - Abstract
Discodermolide is a microtubule stabilizing agent that suppresses dynamic instability and blocks cells in mitosis. Selection of A549 nonsmall cell lung carcinoma cells with increasing concentrations of discodermolide yielded a clone that proliferated in 8 nM. When these cells were exposed to any concentration greater than 8 nM, replication ceased and the cells developed a flattened, enlarged, granular morphology. Accelerated senescence was demonstrated by a functional beta-galactosidase activity at pH 6. When parental A549 cells were treated with IC50-concentrations of doxorubicin, Taxol or discodermolide, the latter two drugs quickly produced aberrant mitosis. However, discodermolide, but not Taxol, also produced a large increase in senescence-associated beta-galactosidase activity and altered levels of known senescence markers. Although some of these differences between Taxol and discodermolide were dose dependent, only discodermolide produced a doxorubicin-like induction of a senescence phenotype, including a senescence-associated beta-galactosidase activity, up-regulation of PAI-1 and p66Shc, and a strong, sustained, Erk1/2 activation. This research provides insights into the mechanism of action of discodermolide and provides the first demonstration of a microtubule stabilizing agent that inhibits tumor cell growth with a powerful induction of accelerated senescence.
- Published
- 2005
48. Mechanisms of Taxol resistance related to microtubules
- Author
-
George A. Orr, Pascal Verdier-Pinard, Hayley M. McDaid, and Susan Band Horwitz
- Subjects
Cancer Research ,Paclitaxel ,endocrine system diseases ,Molecular Sequence Data ,Context (language use) ,macromolecular substances ,Drug resistance ,medicine.disease_cause ,Microtubules ,Article ,chemistry.chemical_compound ,Tubulin ,Microtubule ,Neoplasms ,Genetics ,medicine ,Humans ,Amino Acid Sequence ,Molecular Biology ,biology ,organic chemicals ,Cancer ,Class III β-tubulin ,medicine.disease ,Cell biology ,Gene Expression Regulation, Neoplastic ,chemistry ,Drug Resistance, Neoplasm ,biology.protein ,Cancer research ,Carcinogenesis - Abstract
Since its approval by the FDA in 1992 for the treatment of ovarian cancer, the use of Taxol has dramatically increased. Although treatment with Taxol has led to improvement in the duration and quality of life for some cancer patients, the majority eventually develop progressive disease after initially responding to Taxol treatment. Drug resistance represents a major obstacle to improving the overall response and survival of cancer patients. This review focuses on mechanisms of Taxol resistance that occur directly at the microtubule, such as mutations, tubulin isotype selection and post-translational modifications, and also at the level of regulatory proteins. A review of tubulin structure, microtubule dynamics, the mechanism of action of Taxol and its binding site on the microtubule are included, so that the reader can evaluate Taxol resistance in context.
- Published
- 2003
49. Direct Analysis of Tubulin Expression in Cancer Cell Lines by Electrospray Ionization Mass Spectrometry
- Author
-
Susan Band Horwitz, George A. Orr, Pascal Verdier-Pinard, Ruth Hogue Angeletti, Berta Burd, and Fang Wang
- Subjects
Spectrometry, Mass, Electrospray Ionization ,Paclitaxel ,Electrospray ionization ,Molecular Sequence Data ,macromolecular substances ,medicine.disease_cause ,Mass spectrometry ,Peptide Mapping ,Biochemistry ,Tubulin ,Microtubule ,Cell Line, Tumor ,medicine ,Humans ,Protein Isoforms ,Trypsin ,Amino Acid Sequence ,Peptide sequence ,Chromatography, High Pressure Liquid ,Mutation ,biology ,Chemistry ,Molecular biology ,Isotype ,Peptide Fragments ,Neoplasm Proteins ,Molecular Weight ,Matrix-assisted laser desorption/ionization ,Drug Resistance, Neoplasm ,Epothilones ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,biology.protein ,HeLa Cells - Abstract
Differential expression of tubulin isotypes, mutations, and/or post-translational modifications in sensitive and Taxol-resistant cell lines suggests the existence of tubulin-based mechanisms of resistance. Since tubulin isotypes are defined by their C-terminal sequence, we previously described a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based analysis of tubulin diversity in human cell lines by analysis of their CNBr-released C-terminal peptides [Rao, S., Aberg, F., Nieves, E., Horwitz, S. B., and Orr, G. A. (2001) Biochemistry 40, 2096-103]. We now describe the liquid chromatography/electrospray ionization mass spectrometry analysis of native tubulins in Taxol-stabilized microtubules from parental and Taxol/epothilone-resistant human cancer cell lines. This method allows the direct determination of tubulin isotype composition, including post-translational modifications and mutations occurring throughout the entire protein. Four major isotypes, betaI-, betaIVb-, Kalpha1-, and alpha6-tubulin, were detected in two human carcinoma cell lines, A549 and HeLa. betaIII-Tubulin represented a minor species, as did alpha4-tubulin which was detected for the first time in both cell lines. The three alpha-tubulins were almost totally tyrosinated, and post-translational modifications were limited to low levels of monoglutamylation of Kalpha1-, betaI-, and betaIII-tubulin. betaII- and betaIVa-tubulins were not detected in either parental or drug-resistant cell lines, in contrast to previous RNA-based studies. Since mutations can occur in a single tubulin allele, the question as to whether the wild-type and mutant transcripts are both translated, and to what levels, is important. Heterozygous expression of Kalpha1- or betaI-tubulin mutants that introduced mass changes as small as 26 Da was readily detected in native tubulins isolated from Taxol- and epothilone-resistant cell lines.
- Published
- 2003
50. Distinct mechanisms of Taxol-induced serine phosphorylation of the 66-kDa Shc isoform in A549 and RAW 264.7 cells
- Author
-
Susan Band Horwitz and Chia Ping Huang Yang
- Subjects
Threonine ,Src Homology 2 Domain-Containing, Transforming Protein 1 ,Paclitaxel ,Pyridines ,Taxol ,Macrophage ,p38 mitogen-activated protein kinases ,Gene Expression ,macromolecular substances ,Biology ,p38 Mitogen-Activated Protein Kinases ,Cell Line ,Serine ,Mice ,Nitriles ,Butadienes ,Animals ,Humans ,Microtubule-interacting agent ,Enzyme Inhibitors ,Phosphorylation ,Molecular Biology ,RAW 264.7 Cells ,Adaptor Proteins, Signal Transducing ,Mitogen-Activated Protein Kinase Kinases ,A549 cell ,Tumor Necrosis Factor-alpha ,Kinase ,Imidazoles ,JNK Mitogen-Activated Protein Kinases ,Proteins ,Cell Biology ,Cell cycle ,Antineoplastic Agents, Phytogenic ,Cell biology ,Adaptor Proteins, Vesicular Transport ,Shc Signaling Adaptor Proteins ,Mitogen-Activated Protein Kinases ,Signal transduction ,p66shc phosphorylation - Abstract
Nanomolar concentrations of Taxol, and other antimitotic agents that interact with microtubules, mediate serine phosphorylation of the 66-kDa Shc isoform (p66shc) in A549 human lung carcinoma cells, 9-18 h after drug treatment. This event coincides with the release of PARP cleavage fragments that are early indicators of apoptosis. Taxol-induced serine phosphorylation of p66shc results from a MEK-independent signaling pathway that is activated in A549 cells that have a prolonged or abnormal mitotic phase of the cell cycle [Cancer Res. 60 (2000) 5171]. In contrast, in murine macrophage RAW 264.7 cells, micromolar concentrations of Taxol but not other microtubule-interacting agents induced serine phosphorylation of p66shc that correlated with the phosphorylation of Raf-1 and extracellular signal-regulated kinase (ERK1/2), within 15-30 min after Taxol treatment. This event also was induced by lipopolysaccharide (LPS). The MEK-inhibitor, U0126, that specifically inhibits the activation of ERK also blocked the phosphorylation of p66shc and Raf-1, suggesting that these processes were MEK-dependent, quite different from that which was observed in A549 cells. Taxol also induced phosphorylation of p38 and JNK MAP kinases within 8-15 min after drug treatment. It is known that Taxol, but not other microtubule-interacting agents, induces the production of cytokines, such as tumor necrosis factor alpha (TNF-alpha) in mouse macrophages. The time course of Taxol-induced TNF-alpha expression coincides with that of Taxol-induced p66shc phosphorylation, and U0126 inhibits significantly Taxol-induced TNF-alpha expression in RAW 264.7 cells. Our data indicate that the Taxol-induced serine phosphorylation of p66shc in RAW 264.7 cells is microtubule-independent and may be related to increased TNF-alpha expression after Taxol and LPS treatment. It is concluded that the mechanisms involved in Taxol-induced p66shc phosphorylation are distinct in A549 and RAW 264.7 cells.
- Published
- 2002
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