Vladimir Kolenko, MD, PhD
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The role of zinc in prostate carcinogenesisKonstantin Golovine & Petr Makhov
Prostate carcinogenesis involves dramatic changes in zinc accumulation. While the normal human prostate accumulates the highest levels of zinc of any soft tissue in the body (1018 μg/g of dry tissue), and this unique capability is retained in benign prostatic hyperplasia (BPH; 1142 μg/g), zinc levels in prostate adenocarcinoma are markedly decreased (146 μg/g). Importantly, zinc concentrations diminish early in the course of prostate cancer, prior to detectable histopathological changes, and continue to decline during progression toward hormone-independent growth. Failure to accumulate zinc is one of the most characteristic features of prostatic malignancy.
The functional role of zinc accumulation is to inhibit citrate oxidation in the highly specialized secretory epithelial cells, which permits the production and secretion of extremely high levels of citrate as a major component of prostatic fluid. In addition to its functional role, effects of high zinc levels include metabolic consequences such as the inhibition of terminal oxidation, truncation of the Krebs cycle, and decreased energy production; inhibition of invasiveness and motility; inhibition of cell growth and proliferation; and induction of mitochondrial apoptogenesis. This complex pattern of growth-limiting properties initially suggested that increasing zinc intake might be protective against prostate carcinogenesis, causing us to study the cell mechanisms regulating intraprostatic zinc levels.
Decreased serum zinc concentrations have been reported in patients with metastatic prostate carcinoma. However, this is not likely to be the major cause for decreased zinc accumulation in malignant prostate cells. Specialized mechanisms are required for both zinc uptake and release. Proteins from two metal transporter families regulate zinc levels. These are the ZIP family, which imports zinc, and the ZnT family, which functions in releasing zinc from the cell, or sequestering zinc internally. Expression of the hZIP1, hZIP2 and hZIP3 genes and proteins is markedly downregulated in adenocarcinomatous glands compared with normal peripheral zone glandular epithelium and benign hyperplastic glands. In addition, high-risk patient populations, such as African-American men, appear to have altered prostatic zinc homeostasis, resulting in a decreased intracellular zinc accumulation that coincides with down-regulation of hZIP1 and hZIP2. Therefore, the reduced expression of zinc uptake transporters may result in decreased intra-prostatic zinc levels despite supplemental zinc intake.
Clinically, tumors that maintain higher levels of intracellular zinc have been shown to have significantly slower growth rates. Data from our laboratory indicate that physiological levels of zinc characteristic of the normal prostate suppress NF-κB activity, reduce expression of proteins involved in angiogenesis and metastasis (e.g. VEGF, IL-6, IL-8, and MMP-9), and decrease the invasive and adhesive properties of prostate cancer cells. Our recent studies also demonstrate that overexpression of hZIP1 transporter reduces the tumorigenic potential and growth of PC-3 prostate cancer cells both in vitro and in vivo. In addition, we were the first to provide experimental evidence that selective zinc deficiency induced by the membrane-permeable zinc chelator N,N,N’,N’-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) increases activation of NF-κB and up-regulates expression of the NF-κB -ontrolled pro-angiogenic and pro-metastatic cytokines VEGF, IL-6, and IL-8 in androgen-independent PC-3 and DU-145 prostate cancer cells.
The fact that expression of zinc uptake transporters in malignant prostate tissue remains weakly detectable, and the retention of such expression in cell lines established from metastatic lesions, together suggest that the absence of zinc uptake transporters in malignant glands in situ is not due to the deletion or fatal mutation of the transporter gene but rather due to an epigenetic silencing event. Indeed, our experiments reveal that treatment of DU-145 and PC-3 prostate cancer cells with the demethylating agent 5-aza-2'-deoxycytidine, or the histone deacetylase inhibitor Trichostatin A, significantly increases cellular zinc uptake. In turn, an increase in intracellular zinc correlates with up-regulation of hZIP1, hZIP2, and hZIP3 expression. The reversibility of epigenetic alterations makes them attractive targets for cancer prevention and treatment. Given that zinc levels diminish early in the course of prostate cancer, prior to histopathological changes, we plan to validate the expression and methylation profiles of zinc transport genes as potential tumor biomarkers for early diagnosis and risk assessment for prostate cancer. Such biomarkers have the potential to usefully complement existing prognostic variables such as the Gleason score and PSA.Top
Therapeutics of XIAP degradation by zinc chelatorsKonstantin Golovine & Petr Makhov, in collaboration with Alexander Kutikov & Robert Uzzo
Androgen ablation is an effective treatment for advanced prostate cancer. However, most treated patients ultimately develop hormonally resistant disease and neither systemic (chemotherapy) nor local (surgery/radiation) regimens have significantly improved overall survival. Therefore, the development of new therapeutic strategies for advanced prostate cancer represents a goal with enormous clinical and scientific merit. Anticancer therapeutic regimens trigger tumor cell death largely through the induction of apoptosis. X-linked inhibitor of apoptosis protein (XIAP) is a member of the inhibitor of apoptosis protein (IAP) family of caspase inhibitors, that selectively binds and inhibits caspases-3, -7 and -9. Importantly, XIAP is the only member of IAP family able to directly inhibit both the initiation and execution phases of the caspase cascade. By inhibiting effector caspases, XIAP blocks the downstream activation of the apoptosis pathway effectors, thus blocking apoptosis induced by multiple stimuli including the mitochondrial and death receptor-mediated pathways of caspase activation. As such, XIAP is an attractive target for novel therapeutic agents for the treatment of advanced prostate malignancy.
Findings from our laboratory indicate that treatment of prostate cancer cells with the zinc chelating agent N,N,N’,N’,-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces rapid down-regulation of XIAP at posttranslational level. 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. Importantly, the observed effect was not specific to a single cell type, as TPEN reduced expression of XIAP in malignant cell lines derived from leukemia and from colon, ovarian, breast and cervical cancers. The down-regulation 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. Our findings indicate that zinc-chelating agents may be used to sensitize malignant cells to established cytotoxic agents via down-regulation of XIAP.Top
Therapeutic synergy between piperine and docetaxel in prostate cancerDaniel Canter, Konstantin Golovine & Petr Makhov, in collaboration with Robert Uzzo
Docetaxel-based combination chemotherapy is one of the regimens approved by the FDA for treatment of metastatic castration-resistant prostate cancer. Oral chemotherapeutic agents offer advantages over intravenous agents, including patient convenience, reduced complications associated with the need for long-term venous access, and decreased use of health care resources associated with medication administration. Moreover, oral docetaxel offers a practical approach for achieving long-term steady state therapeutic drug levels. Nevertheless, the development of a suitable oral formulation for docetaxel has been impeded by low oral bioavailability (<10%) due to the extensive metabolic inactivation of this drug (both hepatic and intestinal) by CYP3A4, and by outward-directed transport of docetaxel by the P-gp transporter in the gastrointestinal tract. The effectiveness of docetaxel in clinical practice is also compromised by its substantial toxicity.
Recent experimental findings suggest that dietary constituents may play an important role not only in cancer prevention but also in cancer management. Experimental evidence from our group and others reveals that piperine, a major plant alkaloid/amide present in black (Piper nigrum Linn) and long (Piper longum Linn) peppers, inhibits both the P-gp transporter and the drug-metabolizing enzyme CYP3A4. Our recent studies reveal that dietary piperine increases the serum-half-life of docetaxel, and its anti-tumor efficiency in a xenograft model of castration-resistant prostate cancer. Moreover, treatment of PC-3 prostate cancer cells with piperine inhibits NF-κB activity, reduces the expression of several NF-κB controlled anti-apoptotic proteins and synergistically enhances the cytotoxic effect of docetaxel on prostate cancer cells. The overall objective of these studies is to validate the therapeutic potential of piperine in improving bioavailability and therapeutic index of docetaxel.Top
Overcoming resistance to multitargeted receptor tyrosine kinase inhibitors (TKIs) in genitourinary tumorsKonstantin Golovine, Petr Makhov & Daniel Canter, in collaboration with Alexander Kutikov & Robert Uzzo
Angiogenic inhibition using multi-targeted receptor tyrosine kinase inhibitors (TKIs) represents a clinically relevant strategy for patients with advanced tumors such as renal cell carcinoma (RCC) and gastrointestinal stromal tumors (GISTs). Given their efficacy in RCC and GIST, TKIs are being tested clinically for many other solid tumors. Recent studies have confirmed activity of the TKI sunitinib against castration-resistant prostate cancer (CRPC) cells. In Phase II trials, a heterogeneous response to sunitinib was noted in both docetaxel-resistant and chemotherapy-naïve patients, with a significant proportion of treated tumors demonstrating at least partial radiographic response. Notably, clinical response did not correlate well with decreases in prostate specific antigen (PSA), indicating that PSA is a suboptimal biomarker for CRPC response to TKIs. Our studies reveal that TKI-resistance coincides with the loss of phosphatase and tensin homolog (PTEN) protein expression in prostate and renal malignant cells. Furthermore, over-expression of PTEN sensitizes prostate and renal tumors to sunitinib both in vitro and in vivo. In addition, our studies indicate that an increase in expression of IL-6 and IL-8 correlates with resistance to TKIs in prostate and renal tumor cells, suggesting that these cytokines could be harnessed as clinically useful biomarkers for patients who are receiving TKIs.Top