In vitro and in vivo effect of carmustine and selenite combination on EGFR signaling in androgen-independent prostate cancer

Introduction: Despite the use of androgen deprivation therapy, the majority of prostate cancer patients will progress to castration resistant disease within 2-3 years, driven by aberrant androgen receptor activation. We have previously shown that combination of carmustine and selenite effectively induces apoptosis by completely reducing AR and AR-variants in AR-dependent castration resistant prostate cancer cells both in vitro and in vivo. Although studies have demonstrated that AR signaling is a central mechanism of castration resistant prostate cancer progression, the cell growth mediated by AR-independent signaling is also operative. EGFR signaling has been implicated in the survival, invasion, and metastasis of prostate cancer cells in an AR-independent manner. Therefore, in this study, we tested whether the combination of carmustine and selenite could inhibit EGFR signaling, induce apoptosis, and inhibit growth of AR-independent prostate cancer cells in an in vitro cell culture and in vivo Methods: AR negative PC-3 prostate cancer cell line was used to study the effect of carmustine and selenite alone or in combination on EGFR signaling. Cell viability, proliferation, and apoptosis in the presence or absence of EGF (50ng/ml) were determined. Dose and time dependent effect of EGF on the activation of EGFR was studied. The carmustine and/or selenite effect on EGF stimulated phospho-EGFR, and its downstream signaling pathways, such as AKT and MAPK, were studied in comparison with inhibitors of EGFR (AG1478) or PI3 kinase (LY294002) or MAPK (PD98059). The in vivo effect of carmustine and selenite was examined on PC-3 tumor growth in athymic nude mice. Results: Combination of carmustine and selenite treatment in the presence of EGF markedly suppressed cell viability and proliferation of PC-3 cells. EGF exposure increased phospho-EGFR (Tyr845, Tyr992, Tyr1068, and Tyr1045), pAkt (Ser473), and pERK1/2 (Thr204/Tyr202) protein expression levels in PC-3 cells in a dose and time dependent manner. The combination treatment exhibited a strong inhibitory effect on EGF stimulated EGFR, Akt, and ERK1/2 phosphorylation. Combination treatment was able to induce apoptosis even in the presence of EGF. Consistent with in vitro results, combination treatment for 3 weeks reduced PC-3 xenograft tumors in nude mice by 88% without any toxicity. Whereas individual agent treatment showed only partial effect both in vitro and in vivo. Conclusions: Our pre-clinical data demonstrate that the combination treatment of carmustine and selenite substantially inhibits EGFR signaling, induces apoptosis, and reduces tumor growth of AR-independent prostate cancer cells in vitro and in vivo. Our novel findings suggest that the combination of carmustine and selenite is an effective therapeutic agent for successful treatment, survival, and improved quality of life for patients with prostate cancer

Oxalate-induced activation of PKC-α and -δ regulates NADPH oxidase-mediated oxidative injury in renal tubular epithelial cells

Oxalate-induced oxidative stress contributes to cell injury and promotes renal deposition of calcium oxalate crystals. However, we do not know how oxalate stimulates reactive oxygen species (ROS) in renal tubular epithelial cells. We investigated the signaling mechanism of oxalate-induced ROS formation in these cells and found that oxalate significantly increased membrane-associated protein kinase C (PKC) activity while at the same time lowering cytosolic PKC activity. Oxalate markedly translocated PKC-α and -δ from the cytosol to the cell membrane. Pretreatment of LLC-PK1 cells with specific inhibitors of PKC-α or -δ significantly blocked oxalate-induced generation of superoxide and hydrogen peroxide along with NADPH oxidase activity, LDH release, lipid hydroperoxide formation, and apoptosis. The PKC activator PMA mimicked oxalate's effect on oxidative stress in LLC-PK1 cells as well as cytosol-to-membrane translocation of PKC-α and -δ. Silencing of PKC-α expression by PKC-α-specific small interfering RNA significantly attenuated oxalate-induced cell injury by decreasing hydrogen peroxide generation and LDH release. We believe this is the first demonstration that PKC-α- and -δ-dependent activation of NADPH oxidase is one of the mechanisms responsible for oxalate-induced oxidative injury in renal tubular epithelial cells. The study suggests that the therapeutic approach might be considered toward attenuating oxalate-induced PKC signaling-mediated oxidative injury in recurrent stone formers