PO-076 HMNQ induces apoptosis and autophagy dependent on reactive oxygen species through activation of JNK signalling pathway

Abstract

Material and methods Effect of metformin on A498 human renal cell carcinoma cells was studied. Morphological changes were visualised using a LM. Cellular DNA was stained by applying PI and the relative DNA contents of the stained cells were analysed using FACS. Proteins such as anti-cFLIPL, anti-PARP, anti-Bcl-2, anti-Bcl-xL, anti-Mcl-1, anti-cIAP-2, anti-XIAP and anti-actin antibodies were detected using by Imaging System. c-FLIPL mRNA expression was determined by RT-PCR. ROS generation was assessed by the dichlorofluoresence in fluorescence intensity of the cells using flow cytometer. Data were analysed using one-way ANOVA followed by posthoc comparisons using the SPSS 8.0. Results and discussions We found that degradation of cellular FADD-like interleukin-1-converting enzyme (FLICE) inhibitory protein (c-FLIP) and activation of procaspase-8 were associated with metformin-mediated apoptosis. In contrast, treatment with metformin did not affect the mRNA level of c-FLIPL in A498 cells. Treatment with benzyloxycarbonyl-Val-Ala-Aspfluoromethyl ketone (z-VAD-fmk, a pan-caspase inhibitor) almost completely blocked metformin-induced apoptosis and degradation of c-FLIPL protein. However, N-acetyl-l-cysteine (NAC), a reactive oxygen species (ROS) scavenger, did not inhibit metformin-mediated apoptosis in A498 cells. Conclusion These results demonstrated that metformin-induced apoptosis was mediated by the degradation of c-FLIPL protein via activation of caspase-8 in A498 human renal cell carcinoma cells. This suggests that metformin can play the role of a chemotherapeutic agent for diabetes, as well as an anti-cancer agent. Introduction Apoptosis is down regulated in most forms of cancer. Mitochondria are central to the apoptotic process and are targeted in cancer therapy by novel drug candidates. Calliandra portoricensis (CP) is used in the management of prostate enlargement in folk medicine. This study was designed to investigate the effects of CP on mitochondrial-mediated apoptosis and cell proliferation using prostate cancer cells. Material and methods Prostate cancer cells were treated with methanol fraction of CP (MFCP), cell cycle analysis was evaluated by flow cytometry and levels of pro-apoptotic Bax, antiapoptotic Bcl-2, Cytochrome C Release and activation of caspases 3and 9 were determined using ELISA kits. Results and discussions The MFCP inhibited (p<0.05) proliferation of prostatic cancer cells. The growth inhibition by MFCP (10 mg/mL) correlated with a 3-fold decreased expression of Bcl-2 and a 4-fold increase in Bax levels in LNCaP cells. The MFCP (10 mg/mL) activated C3 and C9 at by 4.2 and 5.1 folds over control, respectively which prompted cancer cells to arrest at S phase. The LC-MS analysis revealed the presence of polyphenols in MFCP. Conclusion Taken together, MFCP-induced cell death is mediated by alteration of mitochondrial integrity and cell cycle arrest. Hence, MFCP may be effective for cancer pharmacotherapy. Introduction 8-hydroxy-2-methoxy-1,4-naphthoquinone (HMNQ), a natural compound isol...