Molecular Mechanism Of PBITC-induced Autophagy,G2/M Arrest And Apoptosis In Breast Cancer Cells

BackgroundBreast cancer ranks first in female's cancer incidence and mortality and it is a major health risk to women's lives.The targeted chemotherapy of breast cancer depends on the expression of estrogen receptor(ERs),progesterone receptor(PRs)and human epidermal growth factor receptor-2(HER-2).Due to the lack of these three targeted proteins,the chemotherapy for three negative breast cancer(TNBC)faces an unbeatable challenge.Anticarcinogenic effect of cruciferous vegetables is attributed to organic isothiocyanates(ITCs).It has been reported that several ITCs derived from cruciferous vegetables,such as sulforaphane(SFN),phenethyl isothiocyanate(PEITC)and benzyl isothiocyanate(BITC),have attracted a great deal of interest because of their anti-cancer activities.4-phenybutyl isothiocyanate(PBITC),a potential chemopreventive agent synthesized based on naturally occurring isothiocyanates,has been reported to inhibit growth of pancreatic cancer(adenocarcinomas)and lung cancer(adenomas and adenocarcinomas).However,the molecular mechanism of PBITC-exhibited anti-cancer activity in breast cancer remains unclear.Microtubules,the central constituents of the cytoskeleton,are composed of ?-and ?-tubulins.With highly dynamic structures,microtubules are involved in a variety of fundamental cell functions.Because of their importance in the formation of the mitotic spindle and in regulating the mitotic apparatus,disruptions of microtubule dynamics can induce cell cycle arrest at G2/M phase,resulting in the formation of abnormal mitotic spindles and induction of apoptosis.Agents that affect microtubule functions can be divided into two groups based on their effects on microtubule assembly.The first group is the microtubule-stabilizing agents.These compounds stabilize microtubules,enhance microtubule polymerization,and induce the formation of microtubule bundles in cells.The second group is microtubule-destabilizing agents,which inhibit microtubule polymerization and decrease the length of microtubules in cells.Both types of microtubule agents alter microtubule dynamics via direct binding to tubulin.The strong ability of these agents,such as paclitaxel and vinblastine,to disrupt microtubules,inhibit proliferation,and induce programmed cell death have made them very effective in clinical therapy for treatment of cancers.Bcl2-associated athanogene 3(BAG3),a member of the BAG family of co-chaperones which contains WW domain,plays a critical role in the regulation of apoptosis,development,cell motility,autophagy,and tumor metastasis,which is involved in mediating cell adaptive responses to stress.BAG3 exerts an anti-apoptotic effect.Overexpression of BAG3 may lead to resistance to chemotherapy.In this study,we explored the effect and molecular mechanism of PBITC-mediated autophagy,cell cycle arrest and apoptosis in tri-negative breast cancer MDA-MB-231 cells,and to provide reference for the evaluation of PBITC.Methods and Results1.PBITC induced autophagy,cell cycle arrest and apoptosis in MDA-MB-231 cellsIn this chapter,we examined the effect of PBITC on autophagy,cell cycle and apoptosis in MDA-MB-231 cells by using Western blot,flow cytometry and laser confocal microscopy.We found that PBITC at low doses induced autophagy and G2/M cell cycle arrest and induced apoptosis at high dose in MDA-MB-231 cells.PBITC-induced autophagy may be a chaperone assisted selective autophagy(CASA).2.Comparative proteomic detection of breast cancer cells after treatment with PBITCIn this part,we investigated the effect of PBITC on the protein expression of MDA-MB-231 cells through comparative proteomic analysis.We found that the expressions of tubulin,heat shock protein,ubiquitination protein,zinc finger protein and transcription factor were significantly changed after treatment with PBITC.The levels of most proteins were reduced by more than 50% in cells treated with PBITC compared to that in control cells.In contrast,the levels of BAG3 and HspB8 were significantly elevated.3.PBITC inhibits tubulin depolymerizationLaser confocal microscopy was used to observe the formation of microtubules.The effect of PBITC on microtubule aggregation was analyzed by immunoblotting after subcellular segregation.The binding of PBITC to tubulin was predicted by computer molecular docking.We found that PBITC may inhibit tubulin depolymerization.4.PBITC activates the BAG3-HspB8 complex,which binds to the microtubulesImmunoprecipitation was used to detect the interaction between BAG3,HspB8,TUBA and Ub protein.We found that HspB8 interacts with either BAG3 or TUBA.The TUBA interacting with HspB8 may be a dimer and accompanied by a ubiquitination.ConlusionsIn this study,we found that PBITC induced autophagy,G2/M cell cycle arrest and apoptosis in the three-negative breast cancer MDA-MB-231 cells in dose-and time-dependent manners.We found for the first time that PBITC inhibits tubulin depolymerization which may be the cause of PBITC-induced cycle arrest and apoptosis.We found for the first time that PBITC activates the BAG3-HspB8 complex,which may bind to the tubulin in the polymerized state.This study could provide references for the evaluation of druggability of PBITC.