Preliminary Analysis Of Bisbibenzyls-induced Autophagy In Apoptosis And Differentiation

Background:Bisbibenzyls display various biological activities, such as antitumor, antifungal and anti-inflammation effects. However, the roles of bisbibenzyls in autophagy and bisbibenzyls-induced autophagy in apoptosis and differentiation remained unclear. Autophagy may lead to cell death or serve as a cell survival pathway, and in other cases, it occurs in parallel with apoptosis. The role of autophagy in bone has been concerned in recent years. Autophagy may promote or inhibit osteoclastogenesis under different conditions. Therefore, we screened the autophagy induction activity of several bisbibenzyls, and studied the effects of RD-induced autophagy in apoptosis of human androgen-independent prostate cancer PC-3cells, and analyzed the effects of MC-induced autophagy in osteoclastogenesis. Methods: Human astrocytoma U87cells stably expressing GFP-LC3were treated with the bisbibenzyls, and GFP-LC3punctate dots were observed with a fluorescence microscope, compounds which were able to induce autophagy were screened out. Western blotting was used to analyze the expression levels of autophagy related proteins LC3, Atg5, Beclinl, p62and apoptosis related protein poly(ADP-ribose) polymerase in PC-3cells exposed to chemicals. Autophagy inhibitors or Atg5-targeting siRNA was employed to examine the effect of chemicals on autophagy induction. Cellular apoptosis was analyzed by Flow cytometry, and cell viability was detected by MTT assay. CD14+monocytes were isolated from PBMC with a bead-labeled anti-CD14mAb using the MACS magnetic cell sorting system. Generation of human CD14+monocytes-derived OCs or mouse macrophagy RAW264.7cells-derived OCs were conducted in vitro, and TRAP staining was used to detect the OCs. Rhodamine phalloidin staining was used to detect the actin rings in mature OCs. The mRNA level of RANK, M-CSFR and NFATcl was detected with RT-PCR, and the LC3and NFATc1protein levels were detected with western blotting.Results:The screening data showed that all the6bisbibenzyls displayed autophagy induction activity, and RD and MC induced LC3-II in PC-3cells. RD significantly induced the processing of LC3-I to LC3-II in PC-3cells, and moderately up-regulated Atg5and Beclinl expressions, which in turn leading to reduction of p62. Blockage of autophagy resulted in enhancement of RD-induced apoptosis as evidenced by increased cleavage of PARP and reduced cell viability. The LC3-II protein expression induced by RD was not altered in the presence of an apoptosis inhibitor Z-VAD-FMK. High dose MC (10μmol/L) significantly inhibited OCs formation, in contrast, RD of the same dose promoted OCs formation. MC inhibited the formation of actin rings, which was associated with OCs function. MC also suppressed the RASF-induced osteoclastogenesis. After MC treatment, NFATc1and M-CSFR mRNA were reduced, and NFATc1protein expression was inhibited, meanwhile LC3-II was induced.Conclusion:All bisbibenzyls induced autophagy, and RD induced protetive autophagy in PC-3cells. RD enhanced OCs formation, while MC inhibited osteoclastogenesis. Actin rings formation of OCs and RASF-induced osteoclastogenesis were inhibited after MC treatment. Osteoclastogenesis-related factors were inhibited, while autophagy was induced.