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The X-linked inhibitor of apoptosis (XIAP), the most potent member of the inhibitor of apoptosis protein (IAP) family of endogenous caspase inhibitors, blocks the initiation and execution phases of the apoptotic cascade. As such, XIAP represents an attractive target for treating apoptosis-resistant forms of cancer. Here, we demonstrate that treatment with the membrane-permeable zinc chelator, N,N,N′,N′,-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces a rapid depletion of XIAP at the post-translational level in human PC-3 prostate cancer cells and several non-prostate cell lines. The depletion of XIAP is selective, as TPEN has no effect on the expression of other zinc-binding members of the IAP family, including cIAP1, cIAP2 and survivin. The downregulation of XIAP in TPEN-treated cells occurs via proteasome- and caspase-independent mechanisms and is completely prevented by the serine protease inhibitor, Pefabloc. Finally, our studies demonstrate that TPEN promotes activation of caspases-3 and -9 and sensitizes PC-3 prostate cancer cells to TRAIL-mediated apoptosis. Taken together, our findings indicate that zinc-chelating agents may be used to sensitize malignant cells to established cytotoxic agents via downregulation of XIAP.Cell Death and Differentiation (2008) 15, 1745–1751; doi:10.1038/cdd.2008.106; published online 11 July 2008 [ABSTRACT FROM AUTHOR]

Abstract Background Cadmium has been classified as a human carcinogen, affecting health through occupational and environmental exposure. Cadmium has a long biological half-life (>25 years), due to the flat kinetics of its excretion. The prostate is one of the organs with highest levels of cadmium accumulation. Importantly, patients with prostate cancer appear to have higher levels of cadmium both in the circulation and in prostatic tissues. Results In the current report, we demonstrate for the first time that cadmium down-regulates expression of the X-linked inhibitor of apoptosis protein (XIAP) in prostate cancer cells. Cadmium-mediated XIAP depletion occurs at the post-transcriptional level via an NF-κB-independent, proteasome-mediated mechanism and coincides with an increased sensitivity of prostate cancer cells to TNF-α-mediated apoptosis. Prolonged treatment with cadmium results in selection of prostate cancer cells with apoptosis-resistant phenotype. Development of apoptosis-resistance coincides with restoration of XIAP expression in cadmium-selected PC-3 cells. Conclusions Selection of cadmium-resistant cells could represent an adaptive survival mechanism that may contribute to progression of prostatic malignancies.

Leukemia cells accumulate DNA damage, but altered DNA repair mechanisms protect them from apoptosis. We showed here that formaldehyde generated by serine/1-carbon cycle metabolism contributed to the accumulation of toxic DNA-protein crosslinks (DPCs) in leukemia cells, especially in driver clones harboring oncogenic tyrosine kinases (OTKs: FLT3(internal tandem duplication [ITD]), JAK2(V617F), BCR-ABL1). To counteract this effect, OTKs enhanced the expression of DNA polymerase theta (POLθ) via ERK1/2 serine/threonine kinase-dependent inhibition of c-CBL E3 ligase-mediated ubiquitination of POLθ and its proteasomal degradation. Overexpression of POLθ in OTK-positive cells resulted in the efficient repair of DPC-containing DNA double-strand breaks by POLθ-mediated end-joining. The transforming activities of OTKs and other leukemia-inducing oncogenes, especially of those causing the inhibition of BRCA1/2-mediated homologous recombination with and without concomitant inhibition of DNA-PK-dependent nonhomologous end-joining, was abrogated in Polq-/- murine bone marrow cells. Genetic and pharmacological targeting of POLθ polymerase and helicase activities revealed that both activities are promising targets in leukemia cells. Moreover, OTK inhibitors or DPC-inducing drug etoposide enhanced the antileukemia effect of POLθ inhibitor in vitro and in vivo. In conclusion, we demonstrated that POLθ plays an essential role in protecting leukemia cells from metabolically induced toxic DNA lesions triggered by formaldehyde, and it can be targeted to achieve a therapeutic effect., (© 2023 by The American Society of Hematology.)

Deletion of ABL1 was detected in a cohort of hematologic malignancies carrying AML1-ETO and NUP98 fusion proteins. Abl1-/- murine hematopoietic cells transduced with AML1-ETO and NUP98-PMX1 gained proliferation advantage when compared to Abl1 + /+ counterparts. Conversely, overexpression and pharmacological stimulation of ABL1 kinase resulted in reduced proliferation. To pinpoint mechanisms facilitating the transformation of ABL1-deficient cells, Abl1 was knocked down in 32Dcl3-Abl1ko cells by CRISPR/Cas9 followed by the challenge of growth factor withdrawal. 32Dcl3-Abl1ko cells but not 32Dcl3-Abl1wt cells generated growth factor-independent clones. RNA-seq implicated PI3K signaling as one of the dominant mechanisms contributing to growth factor independence in 32Dcl3-Abl1ko cells. PI3K inhibitor buparlisib exerted selective activity against Lin-cKit+ NUP98-PMX1;Abl1-/- cells when compared to the Abl1 + /+ counterparts. Since the role of ABL1 in DNA damage response (DDR) is well established, we also tested the inhibitors of ATM (ATMi), ATR (ATRi) and DNA-PKcs (DNA-PKi). AML1-ETO;Abl1-/- and NUP98-PMX1;Abl1-/- cells were hypersensitive to DNA-PKi and ATRi, respectively, when compared to Abl1 + /+ counterparts. Moreover, ABL1 kinase inhibitor enhanced the sensitivity to PI3K, DNA-PKcs and ATR inhibitors. In conclusion, we showed that ABL1 kinase plays a tumor suppressor role in hematological malignancies induced by AML1-ETO and NUP98-PMX1 and modulates the response to PI3K and/or DDR inhibitors., (© 2023. The Author(s).)

Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that affect the epigenetic regulation of DNA methylation. Mutations in both genes often co-occur with activating mutations in genes encoding oncogenic tyrosine kinases such as FLT3 ITD , BCR-ABL1, JAK2 V617F , and MPL W515L , or with mutations affecting related signaling pathways such as NRAS G12D and CALR del52 . Here, we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing these oncogenes. Malignant TET2-deficient cells displayed downregulation of BRCA1 and LIG4, resulting in reduced activity of BRCA1/2-mediated homologous recombination (HR) and DNA-PK-mediated non-homologous end-joining (D-NHEJ), respectively. TET2-deficient cells relied on PARP1-mediated alternative NHEJ (Alt-NHEJ) for protection from the toxic effects of spontaneous and drug-induced DNA double-strand breaks. Conversely, DNMT3A-deficient cells favored HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells were sensitive to PARP inhibitor (PARPi) treatment in vitro and in vivo , whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity or TET2-Wilms' tumor 1 (WT1)-binding ability was responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of WT1 mimicked the effect of TET2 mutation on DSB repair activity and sensitivity to PARPi. Collectively, these findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically. SIGNIFICANCE: TET2 and DNMT3A mutations affect distinct DNA repair mechanisms and govern the differential sensitivities of oncogenic tyrosine kinase-positive malignant hematopoietic cells to PARP inhibitors., (©2021 American Association for Cancer Research.)

Synthetic lethality triggered by PARP inhibitor (PARPi) yields promising therapeutic results. Unfortunately, tumor cells acquire PARPi resistance, which is usually associated with the restoration of homologous recombination, loss of PARP1 expression, and/or loss of DNA double-strand break (DSB) end resection regulation. Here, we identify a constitutive mechanism of resistance to PARPi. We report that the bone marrow microenvironment (BMM) facilitates DSB repair activity in leukemia cells to protect them against PARPi-mediated synthetic lethality. This effect depends on the hypoxia-induced overexpression of transforming growth factor beta receptor (TGFβR) kinase on malignant cells, which is activated by bone marrow stromal cells-derived transforming growth factor beta 1 (TGF-β1). Genetic and/or pharmacological targeting of the TGF-β1-TGFβR kinase axis results in the restoration of the sensitivity of malignant cells to PARPi in BMM and prolongs the survival of leukemia-bearing mice. Our finding may lead to the therapeutic application of the TGFβR inhibitor in patients receiving PARPis., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Treatment with tyrosine kinase inhibitors (TKIs) significantly improves survival of patients with renal cell carcinoma (RCC). However, about one-quarter of the RCC patients are primarily refractory to treatment with TKIs., Methods: We examined viability of RCC and endothelial cells treated with low-density lipoprotein (LDL) and/or TKIs. Next, we validated the potential role of PI3K/AKT signalling in LDL-mediated TKI resistance. Finally, we examined the effect of a high-fat/high-cholesterol diet on the response of RCC xenograft tumours to sunitinib., Results: The addition of LDL cholesterol increases activation of PI3K/AKT signalling and compromises the antitumour efficacy of TKIs against RCC and endothelial cells. Furthermore, RCC xenograft tumours resist TKIs in mice fed a high-fat/high-cholesterol diet., Conclusions: The ability of renal tumours to maintain their cholesterol homoeostasis may be a critical component of TKI resistance in RCC patients.

The clinical potential of PARP-1 inhibitors has been recognized >10years ago, prompting intensive research on their pharmacological application in several branches of medicine, particularly in oncology. However, natural or acquired resistance of tumors to known PARP-1 inhibitors poses a serious problem for their clinical implementation. Present study aims to reignite clinical interest to PARP-1 inhibitors by introducing a new method of identifying highly potent inhibitors and presenting the largest known collection of structurally diverse inhibitors. The majority of PARP-1 inhibitors known to date have been developed as NAD competitors. NAD is utilized by many enzymes other than PARP-1, resulting in a trade-off trap between their specificity and efficacy. To circumvent this problem, we have developed a new strategy to blindly screen a small molecule library for PARP-1 inhibitors by targeting a highly specific rout of its activation. Based on this screen, we present a collection of PARP-1 inhibitors and provide their structural classification. In addition to compounds that show structural similarity to NAD or known PARP-1 inhibitors, the screen identified structurally new non-NAD-like inhibitors that block PARP-1 activity in cancer cells with greater efficacy and potency than classical PARP-1 inhibitors currently used in clinic. These non-NAD-like PARP-1 inhibitors are effective against several types of human cancer xenografts, including kidney, prostate, and breast tumors in vivo. Our pre-clinical testing of these inhibitors using laboratory animals has established a strong foundation for advancing the new inhibitors to clinical trials., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

The c-Met protein, a transmembrane receptor tyrosine kinase, is the product of a proto-oncogene. Its only known ligand, hepatocyte growth factor (HGF), regulates cell growth, motility, migration, invasion, proliferation, and angiogenesis. The aberrant expression of c-Met is often associated with poor prognosis in multiple cancers, including renal cell carcinoma (RCC). Silencing or inactivation of c-Met leads to decreased viability of cancer cells, thereby making ablation of c-Met signaling an attractive concept for developing novel strategies for the treatment of renal tumors. Naturally-occurring products or substances are the most consistent source of drug development. As such, we investigated the functional impact of piperlongumine (PL), a naturally occurring alkaloid present in the Long pepper (Piper longum) on c-Met expression in RCC cells and demonstrated that PL and its analogs rapidly reduce c-Met protein and RNA levels in RCC cells via ROS-dependent mechanism. PL-mediated c-Met depletion coincided with the inhibition of downstream c-Met signaling; namely Erk/MAPK, STAT3, NF-κB and Akt/mTOR. As such, PL and PL analogs hold promise as potential therapeutic agents for the treatment of metastatic RCC and the prevention of postoperative RCC recurrence.

PARP-1 is a nuclear enzyme regulating transcription, chromatin restructuring, and DNA repair. PARP-1 is activated by interaction with NAD+, DNA, and core histones. Each route of PARP-1 activation leads to somewhat different outcomes. PARP-1 interactions with core histones control PARP-1 functions during transcriptional activation in euchromatin. DNA-dependent regulation of PARP-1 determines its localization in heterochromatin and PARP-1-dependent silencing. Here we address the biological significance of DNA-dependent PARP-1 regulation in vitro and in vivo. We report that minor grove binding ligands (MGBLs) specifically target PARP-1 interaction with DNA, and, hence, the DNA-dependent pathway of PARP-1 activation. By obstructing its interaction with DNA molecules, MGBLs block PARP-1 activity in vitro and in vivo, as we demonstrate using Drosophila, as well as human cancer-derived cells. We also demonstrate synergistic inhibition of PARP-1, combining MGBLs with conventional NAD+-dependent inhibitors in human cancer cells. These results suggest that combining different classes of PARP-1 inhibitors can precisely modulate PARP-1 activity in living cells, thus holding promise for new avenues of cancer treatment.

Objective: To examine whether pharmacologically relevant zinc-binding agents are capable of depleting X-linked inhibitor of apoptosis protein in tumor cells. Our prior work reveals that treatment with zinc-chelating agents induces selective downregulation of the X-linked inhibitor of apoptosis protein in cancer cells of various origins. A precursor of the heme synthetic pathway, 5-aminolevulinic acid, is metabolized to protoporphyrin IX, which is highly reactive with zinc. We assessed whether modified versions of 5-aminolevulinic acid with lipophilic side chains can enhance efficacy and selectivity with respect to protoporphyrin IX accumulation, X-linked inhibitor of apoptosis protein depletion, and tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in human castration-resistant prostate cancer cells., Methods: Seven modified versions of 5-aminolevulinic acid (5 esters and 2 amides) were synthesized. Levels of endogenous protoporphyrin IX were examined by flow cytometry. X-linked inhibitor of apoptosis protein expression was examined by Western blotting. terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay was used to assess cell apoptosis. Results were compared qualitatively., Results: Accumulation of endogenous protoporphyrin IX by castration-resistant prostate cancer cells was shown to be directly related to the carbon chain length of the esterified 5-aminolevulinic acid derivatives. In fact, treatment with 5-aminolevulinic acid-HE was superior to that achieved by 5-aminolevulinic acid with respect to X-linked inhibitor of apoptosis protein downregulation. 5-aminolevulinic acid and 5-aminolevulinic acid-HE in combination with tumor necrosis factor-related apoptosis-inducing ligand significantly enhanced apoptotic cell death in castration-resistant prostate cancer cell lines., Conclusion: Esterified derivatives of 5-aminolevulinic acid alone or in combination with other agents may provide therapeutic opportunities in the treatment of castration-resistant prostate cancer by harnessing apoptotic pathways that are triggered by cellular zinc imbalance., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Tyrosine kinase inhibitors exhibit impressive activity against advanced renal cell carcinoma. However, recent clinical studies have shown an equivocal response to sunitinib in patients with castration-resistant prostate cancer. The tumor suppressor PTEN acts as a gatekeeper of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR cell-survival pathway. Our experiments showed that PTEN expression inversely correlates with sunitinib resistance in renal and prostate cancer cells. Restoration of PTEN expression markedly increases sensitivity of tumor cells to sunitinib both in vitro and in vivo. In addition, pharmacologic manipulation of PI3K/Akt/mTOR signaling with PI3K/mTOR inhibitor, GDC-0980, mTOR inhibitor, temsirolimus, or pan-Akt inhibitor, GSK690693, was able to overcome sunitinib resistance in cancer cells. Our findings underscore the importance of PTEN expression in relation to sunitinib resistance and imply a direct cytotoxic effect by sunitinib on tumor cells in addition to its antiangiogenic actions., (©2012 AACR.)

Background: Docetaxel is the mainline treatment approved by the FDA for castration-resistant prostate cancer (CRPC) yet its administration only increases median survival by 2-4 months. Docetaxel is metabolized in the liver by hepatic CYP3A4 activity. Piperine, a major plant alkaloid/amide, has been shown to inhibit the CYP3A4 enzymatic activity in a cell-free system. Thus, we investigated whether the co-administration of piperine and docetaxel could increase docetaxel's pharmacokinetic activity in vitro and in vivo., Methods: Liver CYP3A4 enzymatic activity was measured by fluorescence. In vivo docetaxel pharmacokinetic activity was analyzed by liquid chromatography. An in vivo xenograft model of human CRPC was utilized to assess the anti-tumor effect of docetaxel when co-administered with piperine., Results: Inhibition of hepatic CYP3A4 activity resulted in an increased area under the curve, half-life and maximum plasma concentration of docetaxel when compared to docetaxel alone administration. The synergistic administration of piperine and docetaxel significantly improved the anti-tumor efficacy of docetaxel in a xenograft model of human CRPC., Conclusions: Docetaxel is one of the most widely used cytotoxic chemotherapeutic agents and is currently the mainstay treatment for metastatic CRPC. Dietary constituents are important agents modifying drug metabolism and transport. In our studies, dietary consumption of piperine increases the therapeutic efficacy of docetaxel in a xenograft model without inducing more adverse effects on the treated mice., (Copyright © 2011 Wiley Periodicals, Inc.)

Objective: To determine if cellular interleukin-6 production predicts response to tyrosine kinase inhibitors (TKIs). As clinical experience using TKIs in patients with castration-resistant prostate cancer (CRPC) matures, Phase II trials show a heterogeneous response to sunitinib in CRPC patients. Change in serum prostate-specific antigen (PSA) level has proven unreliable for prediction of CRPC response to TKIs. Interleukin-6 (IL-6), a critical mediator of prostate cancer pathogenesis, has been shown to rise in patients with disease progression. As such, we investigated whether cellular IL-6 production can predict TKI response in both in vitro and in vivo models., Methods: IL-6 mRNA levels and protein expression were examined by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Apoptosis was examined using the terminal dUTP nick-end labeling assay. For in vivo studies, a CRPC xenograft model in C.B17/Icr-scid mice was used., Results: PC-3 and DU-145 CRPC cell lines exhibited a heterogeneous response to sunitinib and pazopanib. Dose-dependent reduction of IL-6 was observed in TKI-sensitive DU-145 cells. In contrast, the TKI-resistant PC-3 cells failed to suppress IL-6 secretion. Instead, in the presence of tumor necrosis factor-alpha, IL-6 rose significantly upon administration of TKIs. Findings of in vitro experiments were confirmed in an in vivo mouse model of CRPC., Conclusion: Sensitivity of CRPC cells to TKIs is heterogeneous. These findings are consistent with results of recently published Phase II clinical trials using sunitinib in patients with CRPC. A substantial rise in IL-6 occurs both in vitro and in vivo in the presence of TKIs in resistant PC-3 cells but not in TKI-sensitive DU-145 cells. These findings suggest that IL-6 may represent a biomarker for TKI resistance in patients with CRPC., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Docetaxel-based combination chemotherapy is approved by the FDA for the treatment of metastatic castration-resistant prostate cancer. Unfortunately, docetaxel's efficacy is significantly limited by its considerable toxicity on hematopoietic progenitor cells, thus necessitating dose reduction or even discontinuation of the chemotherapy. Induction of pre-mitotic arrest protects cells against docetaxel-mediated toxicity and affords therapeutic opportunities., Methods: Cell cycle progression was examined by propidium iodide staining. Zinc uptake was determined by FluoZin-3 AM staining. Apoptotic DNA fragmentation was detected using APO-BRDU kit., Results: In the course of our current work, we treated the myeloid progenitor TF-1 cells and the castration-resistant PC-3 and DU-145 prostate cancer cells with physiologically relevant concentrations of zinc. In doing so, we were able to prevent docetaxel-mediated mitotic arrest in zinc accumulating myeloid progenitor TF-1 cells but not in castration-resistant PC-3 and DU-145 prostate cancer cells. Moreover, pre-treatment with zinc abolished docetaxel-induced apoptosis in TF-1 cells, whereas such treatment had no effect on apoptosis in PC-3 and DU-145 prostate cancer cells., Conclusions: Our results suggest that zinc can protect myeloid progenitor cells against docetaxel-induced toxicity without compromising the drug's anti-tumor activity., (Copyright © 2011 Wiley-Liss, Inc.)

Objectives: We examined the in vitro cellular effects of the multi-targeted receptor tyrosine kinase inhibitors (TKIs) sunitinib and pazopanib on a series of human renal cell carcinoma (RCC) cell lines., Methods: The human RCC cell lines 769-P, 786-O, HRC-24, HRC-31, HRC-45, HRC-78, SK-26B, and SK-45 were treated with varying concentrations of sunitinib and pazopanib. Cellular proliferation and cellular death were assessed using the CellTiter-Blue Cell Viability Assay and the TUNEL assay, respectively. Effective doses (ED) for inhibition of cellular proliferation or induction of apoptosis were calculated for sunitinib and pazopanib in each RCC cell line., Results: Both sunitinib and pazopanib exhibited anti-proliferative activity to varying degree against all human RCC cell lines; however, sunitinib's effects were achieved at significantly lower concentrations. Moreover, sunitinib had a direct pro-apoptotic effect on all tested cell lines, while pazopanib failed to induce apoptosis in any of the examined human RCC cell lines even at maximal concentrations., Conclusions: Although sunitinib and pazopanib are often used interchangeably in the clinical setting, our results suggest that in-vitro biological activity of the two agents differs. Sunitinib exhibits a cytotoxic effect on RCC cell lines, while pazopanib's activity is solely cytostatic. These data may be clinically relevant given the current lack of comparative in-vivo studies between the two agents.

Background: Cadmium has been classified as a human carcinogen, affecting health through occupational and environmental exposure. Cadmium has a long biological half-life (>25 years), due to the flat kinetics of its excretion. The prostate is one of the organs with highest levels of cadmium accumulation. Importantly, patients with prostate cancer appear to have higher levels of cadmium both in the circulation and in prostatic tissues., Results: In the current report, we demonstrate for the first time that cadmium down-regulates expression of the X-linked inhibitor of apoptosis protein (XIAP) in prostate cancer cells. Cadmium-mediated XIAP depletion occurs at the post-transcriptional level via an NF-kappaB-independent, proteasome-mediated mechanism and coincides with an increased sensitivity of prostate cancer cells to TNF-alpha-mediated apoptosis. Prolonged treatment with cadmium results in selection of prostate cancer cells with apoptosis-resistant phenotype. Development of apoptosis-resistance coincides with restoration of XIAP expression in cadmium-selected PC-3 cells., Conclusions: Selection of cadmium-resistant cells could represent an adaptive survival mechanism that may contribute to progression of prostatic malignancies.

hZip1 has been characterized as the major zinc uptake transporter regulating the accumulation of zinc in prostate cells. The mechanisms regulating expression of hZip1 have not been described. To explore the mechanisms of transcriptional regulation of the hZip1 gene, we determined the putative promoter sequence for hZip1 and identified the potential transcription start site within the predicted hZip1 promoter region. To further characterize the promoter region for basal hZip1 transcription, 3' and 5' deletion constructs and constructs with mutated binding sites for putative transcription factors were generated by PCR amplification and assessed for transcriptional activity with a luciferase reporter assay in PC-3 prostate cancer cells. The ability of the specific transcription factors to bind the hZip1 core promoter was confirmed by EMSA, GelSupershift and ChIP assays. Our experiments identified the core promoter region responsible for constitutive expression of hZip1 and demonstrated critical roles for SP1 and CREB1 in transcriptional regulation of the hZip1 gene in prostate cancer cells.

Background: Zinc accumulation diminishes early in the course of prostate malignancy and continues to decline during progression toward hormone-independent growth. In contrast, constitutive levels of NF-kappaB activity increase during progression of prostate cells toward greater tumorigenic potential. We have reported previously that physiological levels of zinc suppress NF-kappaB activity in prostate cancer cells and reduce expression of pro-angiogenic and pro-metastatic cytokines VEGF, IL-6, IL-8, and MMP-9 associated with negative prognostic features in prostate cancer., Methods: Intracellular zinc levels were examined by atomic absorption spectroscopy. NF-kappaB activity was examined by TransAm and Luciferase reporter assays, and Western blot analysis of p50 nuclear translocation. VEGF, IL-6 and IL-8 levels were assessed by ELISA., Results: Selective zinc deficiency induced by the membrane-permeable zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) increases activation of NF-kappaB and up-regulates expression of the NF-kappaB controlled pro-angiogenic and pro-metastatic cytokines VEGF, IL-6 and IL-8 in androgen-independent PC-3 and DU-145 prostate cancer cells. Pre-incubation with I kappaB alpha dominant mutant adenovirus efficiently blocks expression of these cytokines in zinc deficient cells indicating that the observed effects are NF-kappaB dependent., Conclusions: Our findings suggest that zinc deficiency may contribute to the tumor progression via augmented expression of the NF-kappaB-dependent pro-tumorigenic cytokines., ((c) 2008 Wiley-Liss, Inc.)

Purpose: Intracellular zinc levels and expression of the zinc uptake transporter, hZIP1, are markedly down-regulated in prostate adenocarcinomatous tissue compared with normal prostate tissue. Our previous studies have shown that zinc inhibits nuclear factor-kappaB (NF-kappaB) activity and reduces the malignant potential of prostate cancer cells in vitro. In this study, we investigate the functional effect of hZIP1 overexpression on NF-kappaB activity and tumorigenic potential in human prostate cancer cells in vitro and in vivo., Experimental Design: NF-kappaB activity in PC-3 prostate cancer cells was examined by Western blotting and luciferase assay. ELISA was used to examine the expression of tumorigenic cytokines. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, adhesion, and invasiveness assays were used to assess the malignant potential of tumor cells. The effect of hZIP1 overexpression on prostate tumor progression in vivo was assessed using a xenograft model., Results: Overexpression of the hZIP1 transporter in PC-3 cells results in significant inhibition of NF-kappaB activity in the presence of physiologic levels of zinc. NF-kappaB inhibition coincides with a reduction in expression of several NF-kappaB controlled prometastatic and antiapoptotic factors as well as sensitization of the cells to etoposide and tumor necrosis factor-mediated apoptosis-inducing ligand-mediated cell death. Moreover, overexpression of the hZIP1 transporter induces regression of prostate tumor growth in a xenograft model., Conclusions: Our results show that hZIP1 overexpression has a functional effect on the malignant potential of prostate cancer cells via inhibition of NF-kappaB-dependent pathways and support the concept that hZIP1 may function as a tumor suppressor gene in prostate cancer.

Background: NF-kappaB and AP-1 transcriptional factors contribute to the development and progression of prostate malignancy by regulating the expression of genes involved in proliferation, apoptosis, angiogenesis, and metastasis., Methods: NF-kappaB and AP-1 activities were examined by TransAm assay. Cytokines levels were assessed by ELISA. ICAM-1 and gp130 expression was examined by flow cytometry. Cell adhesion was examined by the ability of cells to adhere to fibronectin-coated plates. Cell viability was determined by propidium iodide staining., Results: Treatment with alpha-tocopherol succinate (VES) inhibits NF-kappaB but augments AP-1 activity, reduces expression of IL-6, IL-8, and VEGF, suppresses cell adhesion, ICAM-1 and gp130 expression in androgen-independent PC-3, DU-145, and CA-HPV-10 cells. VES supplementation also decreases the expression of anti-apoptotic XIAP and Bcl-X(L) proteins and sensitizes androgen-dependent LNCaP cells to androgen deprivation., Conclusions: Our findings propose a potential mechanism of VES-mediated anti-tumor activity and support the role of vitamin E analogs as potential chemopreventative agents against prostate cancer., ((c) 2007 Wiley-Liss, Inc.)

Nuclear factor-kappaB (NF-kappaB) and AP-1 nuclear transcriptional factors regulate expression of multiple genes involved in tumor growth, metastasis and angiogenesis; however, the relative contribution of each factor to cancer initiation and progression has not been established. Prostate carcinogenesis involves transformation of normal zinc-accumulating epithelial cells to malignant cells that do not accumulate zinc. Whereas activation of both NF-kappaB and AP-1 has been implicated in prostate cancer development and growth, we tested the relative effects of zinc supplementation on these important transcriptional factors. Herein, we demonstrate that physiological levels of zinc inhibit NF-kappaB but augment activities of AP-1 in DU-145 and PC-3 human prostate cancer cells. Additionally, we show that chelation of zinc with membrane-permeable zinc chelator, N,N,N',N',-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) abolishes this effect. We further propose a potential mechanism for this observation by demonstrating that zinc supplementation induces phosphorylation of the members of three major MAPK subfamilies regulating AP-1 and NF-kappaB activation (ERK 1/2, JNK and p38) while blocking TNF-alpha-mediated degradation of the inhibitory subunit I kappa B alpha and nuclear translocation of RelA in prostate cancer cells. VEGF, IL-6, IL-8 and MMP-9 are major pro-angiogenic and pro-metastatic molecules whose promoter regions contain binding sites for both NF-kappaB and AP-1. These cytokines have been associated with negative prognostic features in prostate cancer. We demonstrate that treatment of human prostate cancer cell lines with zinc reduces expression of VEGF, IL-6, IL-8 and MMP-9. We further show that zinc reduces expression of intercellular adhesion molecule-1 and functionally suppresses tumor cell invasiveness and adhesion. Therefore, the ability of zinc supplementation to inhibit NF-kappaB supercedes zinc-mediated activation of AP-1 family members. Upregulation of intracellular zinc levels may have important implications for inhibiting the angiogenic and metastatic potentials of malignant cells, predominantly through suppression of NF-kappaB signaling.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytotoxic agent that preferentially induces apoptosis in a variety of human cancer cells. Unfortunately, some tumor cells remain resistant to TRAIL. Therefore, agents that sensitize malignant cells to TRAIL-mediated cell death might be of particular importance for the development of novel antitumor therapeutic regimens. Recent studies establish a critical role of selenium in prostate cancer prevention in vitro and in vivo. Here, we demonstrate that concomitant administration of TRAIL and methylseleninic acid (MSA) produces synergistic effects on the induction of apoptosis in androgen-dependent LNCaP and androgen-independent DU-145 prostate cancer cells. MSA rapidly and specifically downregulates expression of the cellular FLICE inhibitory protein, a negative regulator of death receptor signaling. In addition, we demonstrate that the synergistic effects of MSA and TRAIL result from the activation of the mitochondrial pathway-mediated amplification loop. Addition of MSA effectively blocked TRAIL-mediated BAD phosphorylation at Ser112 and Ser136 in DU-145 cells and was accompanied by induction of the mitochondrial permeability transition and release of apoptogenic cytochrome c and Smac/DIABLO proteins from the mitochondria and into the cytosol. These results suggest that selenium-based dietary compounds may help to overcome resistance to TRAIL-mediated apoptosis in prostate cancer cells.

Aromatase cytochrome P450, the key enzyme of estrogen biosynthesis, is encoded by Cyp19. To elucidate the complex regulation of this gene in mouse gonads (ovary and testis) and brain (thalamic/hypothalamic areas), Cyp19 transcripts were isolated using rapid amplification of cDNA 5' ends and transcript concentrations were estimated in juveniles at different postnatal days (P0, P7, and P14) and in adult animals by real time polymerase chain reaction (PCR). In addition, the murine Cyp19 locus including all known exons and promoters was reconstructed from a recently published sequence of a mouse bacterial artificial chromosome. From each of the tissues investigated, Cyp19 transcripts with a specific 5' untranslated region (5' UTR) were isolated: T(ov) from ovary, T(br) from brain, and T(tes) from testis. T(tes) included a novel 5' UTR that did not show sequence similarities to other Cyp19 transcripts. Real time PCR experiments revealed similar levels of Cyp19 transcript concentrations in neonatal gonads of both sexes. The majority of transcripts were T(ov) in ovaries and T(tes) in testes. During further postnatal development, testicular Cyp19 transcript concentrations transiently decreased, but the contributions of different transcript variants basically remained unchanged. However, ovarian Cyp19 transcript concentrations increased by about 100 times, and almost 100% of all Cyp19 transcripts were identified as T(ov) in adult ovaries. Brains of both sexes showed highest transcript concentrations at P0. However, concentrations in female brains were reduced to adult levels earlier than in male brains. In brains of both sexes, T(br) was found to predominate throughout postnatal life. The results suggest that the mouse Cyp19 gene includes three different promoters that specifically direct expression in ovary, testis, and brain.