In Vitro Studies On The Mechanisms Of Estrogen Receptor In Silibinin-Induced Growth Inhibition Of Human Breast Cancer MCF-7 Cells

The expressions of estrogen receptors alpha(ERa)and beta(ER?)have important impacts on the progression and therapeutic treatment of breast cancer and gynecological tumors.From a functionality perspect,ERa ptromotes the growth and proliferation but ER?serves as a growth suppressor in tumor cells.Silibinin,a natural flavonoid,is a major bioactive component of silymarin isolated from the plant milk thistle Silybum marianum(L.)Gaertn.Silibinin has a steroidal structure.The structural similarity appears to be a cause of silibinin to bind and activate ERs on mammalian target cells.This dissertation reports the mechanisms of ERs in silibinin-induced death in human breast cancer MCF-7 cells.The results showed that silibinin induced apoptosis in breast cancer cells MCF-7 via the down-regulation of ERa expression.Upon ERa activation,series of downstream signalling pathways were activated.We found that silibinin reduced the expressions of protein kinase B(AKT)/mammalian target of ramycin(mTOR)and extracellular-signal-related kinase(ERK),which respectively accounted for the induction of autophagy and apoptosis.Moreover,up-regulation of autophagy induced by silibinin accounted for apoptotic exacerbation,being further augmented by co-treatment with ERa inhibitor MPP dihydrochloride hydrate(MPP).Silibinin induced a loss of cell viability in MCF-7 cells not only by ERa down-regulation but also by ERP up-regulation.The incubation with silibinin,elevated ER? expression,resulting in the tumor growth inhibition.The cytotoxic effect of silibinin was diminished by ER?-selective antagonist PHTPP and enhanced by ER?-selective agonist Diarylprepionitrile(DPN).Silencing of ER? by siRNA confirmed these results.Apoptotic cascades,including the sequential activation of caspase-9 and-6,and finally the cleavage of caspase substrates,PARP and ICAD,caused by treatment with silibinin,were all repressed by PHTPP pre-treatment but exacerbated by DPN.Unlike ERa,ER? was not involved in the regulation of autophagic process,since neither autophagic inhibitor 3-methyladenine(3-MA)nor the inducer rapamycin affected the cell survival rates modulated by ER? activity.Exposure of MCF-7 cells to different concentrations of silibinin up-regulated the generation of inducible protective reactive oxygen species(ROS)and reactive nitrogen species(RNS).Silibinin-induced generation of ROS/RNS was significantly promoted by ERa activation,while ER? elicited no obvious effect on this aspect.ROS/RNS induced by silibinin or corresponding donors tBHP/SNP repressed autophagy pathway.Meanwhile,inhibition of autophagy with 3MA obviously increased the production of ROS/RNS.ROS/RNS and autophagy formed a negative feedback loop in silibinin-treated MCF-7 cells.The generation of pro-survival ROS/RNS partly attenuated the cytotoxicity of slibinin by repressing the destructive autophagy.Furthermore,scavenging ROS/RNS and stimulating autophagy enhanced silibinin's cytotoxicity under the background of ERa activation.