Natural Compound Alternol Induces Apoptosis Of Prostate Cancer Cells By Activating ROS-Bax Dependent Pathway

PurposeDespite advances in medical science and technology, castration-resistant prostate cancer (CRPC) continues to be a major clinical problem. Treatment options for CRPC are still limited. Chemotherapy is always the first-line treatment of CRPC, but there are many inadequacies. It is the desperate need to develop new drugs for CRPC. In this study, a natural compound Alternol was evalutated its anti-prostate cancer effect in vitro and vivo, and the pharmacological mechanism of Alternol was investigated in prostate cancer cell lines with the properties of CRPC.Materials and MethodsProstate cancer and normal prostate cell lines, RWPE-1, LNCaP, C4-2, DU145, PC-3, BPH1, and22RV1were used. Cell death was detected by trypan blue exclusion assay. The hallmarks of cell signaling pathways were evaluated by Immunohistochemistry (IH), Western blot assay, flow cytometry, immunoprecipitation (IP), DNA fragmentation assay, JC-1staining, and fluorescence detection. In addition, the in vivo suppressive effects of Alternol against PC-3and DU145xenograft models were also tested in athymic nude mice.ResultsAlternol induced cell death in a time-dependent manner, and there is relative selectivity for tumor cells. Further analysis revealed that Alternol treatment caused cell death was a typical apoptotic response, as evidenced by change of mitochondrial membrane potential, release of mitochondrial cytochrome C, caspase-3and caspase-9activation, PARP cleavage, and DNA fragmentation. Bax appeared obvious cleavage after Alternol treatment. Knocking down Bax with gene-specific siRNA or blocking Bax-based channel formation effectively suppressed Alternol-induced apoptosis, which verified the involvement of Bax activation in Alternol-induced cell death. Later we verified that Alternol can induce intracellular oxidative stress, and this process could be almost completely inhibited by N-Acetyl-L-cysteine (NAC). NAC also blocked change of mitochondrial membrane potential, release of mitochondrial cytochrome C, caspase-3and caspase-9activation, PARP cleavage, and DNA fragmentation caused by Alternol treatment. Animal experiments showed that Alternol significantly suppressed tumor growth of xenografts derived from PC-3cells and induced apoptosis, but not DU145cells.ConclusionThese data suggest that Alternol can increase intracellular oxidative stress level. And it activated the Bax dependent intrinsic pathway to induce apoptotic cell death in prostate cancer cells although the clinical use of Alternol awaits further determination. PurposeFunctional failure of smooth muscle cells and endothelial cells in corpus cavernosum contributes to erectile dysfunction (ED) in aging men. Given that VEGF may improve the function of smooth muscle cells and endothelial cells through different mechanisms, it is thus expected that increasing the expression of VEGF may have beneficial effects on erectile function. The aim if this article is to explore the possibility that VEGF can be induced by RNA activation (RNAa) technology, and VEGF induction by RNAa has the potential of treating ED.Materials and MethodsPrimary human corpus cavernosum smooth muscle cells (CCSMCs) were isolated and cultured in vitro. The expression of a-smooth muscle actin was detected by immunohistochemistry to identify CCSMCs. A previously identified VEGF promoter-targeted small activator RNA (saRNA, dsVEGF-706) and a negative control dsRNA were chemically synthesized. Cultured human CCSMCs were transfected with the saRNAs. The expression of VEGF mRNA and protein in transfected CCSMCs was evaluated by RT-PCR and Western blotting assay, respectively. Immunofluorescent staining was also used to confirm VEGF protein expression in cultured CCSMCs.ResultsAfter transfection, real-time quantitative PCR analysis showed that the expression of VEGF mRNA was significantly induced in dsVEGF-706transfected cells compared to cells receiving control treatments (P<0.05). Consistent with mRNA induction, Western blotting and immunofluorescence analysis showed VEGF protein expression was also induced by dsVEGF-706.ConclusionVEGF expression can be activated by RNAa in primary human CCSMCs, suggesting a potential application of RNAa-mediated VEGF activation for the treatment of ED.