Vladimir M. Kolenko

Renal cell carcinoma–derived gangliosides suppress nuclear factor-κB activation in T cells

Activation of the transcription factor nuclear factor-kappaB (NFkappaB) is impaired in T cells from patients with renal cell carcinomas (RCCs). In circulating T cells from a subset of patients with RCCs, the suppression of NFkappaB binding activity is downstream from the stimulus-induced degradation of the cytoplasmic factor IkappaBalpha. Tumor-derived soluble products from cultured RCC explants inhibit NFkappaB activity in T cells from healthy volunteers, despite a normal level of stimulus-induced IkappaBalpha degradation in these cells. The inhibitory agent has several features characteristic of a ganglioside, including sensitivity to neuraminidase but not protease treatment; hydrophobicity; and molecular weight less than 3 kDa. Indeed, we detected gangliosides in supernatants from RCC explants and not from adjacent normal kidney tissue. Gangliosides prepared from RCC supernatants, as well as the purified bovine gangliosides G(m1) and G(d1a), suppressed NFkappaB binding activity in T cells and reduced expression of the cytokines IL-2 and IFN-gamma. Taken together, our findings suggest that tumor-derived gangliosides may blunt antitumor immune responses in patients with RCCs.

Methylseleninic acid sensitizes prostate cancer cells to TRAIL-mediated apoptosis

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.

Caspase-dependent and -independent death pathways in cancer therapy

The majority of current anticancer therapies induce tumor cell death through the induction of apoptosis. Alterations in the apoptotic pathways may determine tumor resistance to these therapies. Activation of the proteolytic cascade involving caspase family members is a critical component of the execution of cell death in apoptotic cells. However, recent studies suggest that cell death can proceed in the absence of caspases. In this review we describe the role of caspase-dependent and -independent pathways as targets for anticancer treatment; better understanding of diverse modes of tumor cell death will help to avoid ineffective treatment and provide a molecular basis for the new strategies targeting caspase-independent death pathways in apoptosis-resistant forms of cancer.

Zinc and zinc transporters in prostate carcinogenesis

The healthy human prostate accumulates the highest level of zinc of any soft tissue in the body. This unique property is retained in BPH, but is lost in prostatic malignancy, which implicates changes in zinc and its transporters in carcinogenesis. Indeed, zinc concentrations diminish early in the course of prostate carcinogenesis, preceding histopathological changes, and continue to decline during progression toward castration-resistant disease. Numerous studies suggest that increased zinc intake might protect against progression of prostatic malignancy. In spite of increased dietary intake, zinc accumulation might be limited by the diminished expression of zinc uptake transporters, resulting in decreased intratumoural zinc levels. This finding can explain the conflicting results of various epidemiological studies evaluating the role of zinc supplementation on primary and secondary prostate cancer prevention. Overall, more research into the mechanisms of zinc homeostasis are needed to fully understand its impact on prostate carcinogenesis. Only then can the potential of zinc and zinc transport proteins be harnessed in the diagnosis and treatment of men with prostate cancer.

Zinc chelation induces rapid depletion of the X-linked inhibitor of apoptosis and sensitizes prostate cancer cells to TRAIL-mediated apoptosis

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.

Piperlongumine promotes autophagy via inhibition of Akt/mTOR signalling and mediates cancer cell death

Background:The Akt/mammalian target of rapamycin (mTOR) signalling pathway serves as a critical regulator of cellular growth, proliferation and survival. Akt aberrant activation has been implicated in carcinogenesis and anticancer therapy resistance. Piperlongumine (PL), a natural alkaloid present in the fruit of the Long pepper, is known to exhibit notable anticancer effects. Here we investigate the impact of PL on Akt/mTOR signalling.Methods:We examined Akt/mTOR signalling in cancer cells of various origins including prostate, kidney and breast after PL treatment. Furthermore, cell viability after concomitant treatment with PL and the autophagy inhibitor, Chloroquine (CQ) was assessed. We then examined the efficacy of in vivo combination treatment using a mouse xenograft tumour model.Results:We demonstrate for the first time that PL effectively inhibits phosphorylation of Akt target proteins in all tested cells. Furthermore, the downregulation of Akt downstream signalling resulted in decrease of mTORC1 activity and autophagy stimulation. Using the autophagy inhibitor, CQ, the level of PL-induced cellular death was significantly increased. Moreover, concomitant treatment with PL and CQ demonstrated notable antitumour effect in a xenograft mouse model.Conclusions:Our data provide novel therapeutic opportunities to mediate cancer cellular death using PL. As such, PL may afford a novel paradigm for both prevention and treatment of malignancy.

Functional significance of protein kinase A activation by endothelin-1 and ATP: negative regulation of SRF-dependent gene expression by PKA.

Endothelin-1 (ET1) and ATP stimulate contraction and hypertrophy of vascular smooth muscle cells (VSMC) by activating diverse signalling pathways. In this study, we show that in VSMC, ET1 and ATP stimulate transient and sustained activation of protein kinase A (PKA), respectively. Using a dominant negative PKA mutant (PKA-DN), we examined the functional significance of PKA activation in the signalling of ET1 and ATP. Overexpression of PKA-DN did not alter the ET1- or ATP-induced phosphorylation of the extracellular signal-regulated protein kinase, Erk2. ATP stimulated a profound, PKA-dependent activation of cAMP-response element (CRE), whereas the effect of ET1 was negligible. Both ET1 and ATP stimulated serum response factor (SRF)-dependent gene expression. Overexpression of PKA-DN potentiated the effects of ET1 and ATP on SRF activity, whereas stimulation of PKA by isoproterenol, forskolin or by overexpression of the PKA catalytic subunit decreased SRF activity. These data demonstrate that (i) PKA negatively regulates SRF activity and (ii) ET1 and ATP stimulate opposing pathways, whose balance determines the net activity of SRF.

Overexpression of the zinc uptake transporter hZIP1 inhibits nuclear factor-kappaB and reduces the malignant potential of prostate cancer cells in vitro and in vivo.

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-κB (NF-κB) 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-κB activity and tumorigenic potential in human prostate cancer cells in vitro and in vivo. Experimental Design: NF-κB 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-κB activity in the presence of physiologic levels of zinc. NF-κB inhibition coincides with a reduction in expression of several NF-κB 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-κB-dependent pathways and support the concept that hZIP1 may function as a tumor suppressor gene in prostate cancer.

Mechanism of apoptosis induced by zinc deficiency in peripheral blood T lymphocytes.

Alterations in intracellular Zn2+ concentrations are believed to play a crucial role in modulating apoptosis. The observation that Zn2+ deficiency can induce cell death both in vivo and in vitro has been attributed to the fact that exchange of Zn2+ for Ca2+ and Mg2+ within the nuclei may directly activate endogenous endonucleases therefore inducing DNA fragmentation independent of cytoplasmic factors. Here we show that the membrane-permeable zinc chelator, N,N′,N′-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces translocation of cytochrome c from the mitochondrial intramembranous space into the cytosol in human peripheral blood T lymphocytes (PBL) with subsequent activation of caspases-3, -8, and -9. Pretreatment of T lymphocytes with caspase inhibitors Z-VAD.fmk or DEVD.fmk prevented DNA fragmentation in response to TPEN indicating that apoptosis triggered by zinc deficiency is entirely dependent on activation of caspase family members. The release of cytochrome c and activation of downstream caspases precedes changes in the mitochondrial transmembrane potential (Δ Ψm). Therefore, cytoplasmic and mitochondrial events are critical to this process.

Modulation of Akt/mTOR Signaling Overcomes Sunitinib Resistance in Renal and Prostate Cancer Cells

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. Mol Cancer Ther; 11(7); 1510–7. ©2012 AACR.