Molecular Mechanisms And Functions Of Cell Death Induced By Corilagin In Human Breast Cancer Cells

Breast cancer is the most common life-threatening malignant tumor among women.In recent decades,although the treatment effect has made considerable progress,the morbidity of breast cancer still rise sharply in developing countries.Therefore,the development of new drugs for treatment breast cancer is very important.Corilagin is a kind of natural polyphenols.Researches showed that corilagin with anti-tumor,anti-hyperalgesia,anti-virus,treatment cardiovascular disease and low adverse reaction,has attracted the attention of scholars in recent years,but its biological role in breast cancer and the potential mechanism are rarely reported.Programmed cell death mainly includes apoptosis,autophagy and necroptosis.Apoptosis is a common pathway.Autophagy regulate cell survival,whereas autophagic cell death may be evoked after excessive autophagy.Necroptosis is one special type of necrosis.The aim of the present study is to explore anti-tumor activity of corilagin and its molecular mechanism in human breast cancer cells.Firstly,proliferation was detected by MTT and EdU assay,results showed that corilagin could inhibit proliferation in breast cancer MCF-7 and SK-BR3 cells but had little effect in several normal cell lines,including human mammary epithelial MCF-10A cells,human normal liver L02 cells,human gastric mucosal GES-1 cells.Apoptosis was detected by western blotting and enzyme activity,results showed that corilagin activited caspase-dependent and-independent apoptosis in breast cancer MCF-7 cells.Secondly,the expression of autophagy-related protein were detected by western blotting,meanwhile,we used transmission electron microscopy and immunofluorescence technique to observe autophagy and the mottled LC3 expression respectively,results suggested that corilagin activited Akt/mTOR pathway regulating autophagy in breast cancer MCF-7 cells.Besides,we employed CQ,a late-stage inhibitor,results suggested that inhibition of autophagy can enhance apoptosis by performing MTT and western blotting,inferring autophagy may play a protective role.In addition,the production of ROS and its effect were tested by DCFH-DA probe and western blotting respectively,results revealed that corilagin increased intracellular ROS generation and NAC,scavenger of ROS,partial diminished apoptosis and autophagy in breast cancer MCF-7 cells,inferring corilagin induced ROS dependent apoptosis and autophagy.Next,to explore the influence of Nec-1,scavenger of necroptosis,in breast cancer SK-BR3 cells by MTT assay,results revealed that Nec-1 could not inhibit cell death in SK-BR3 cells but could inhibit cell death in HT-29 cells(positive control).At the same time,the expression of RIP 1,RIP3 were detected by western blotting,results displayed that the level of RIP1,RIP3 were not increased in SK-BR3 cells.The results demonstrated corilagin could not activated necroptosis in breast cancer SK-BR3cells,which express high level of RIP3.Finally,we validate anti-tumor effect of corilagin in vivo.We found corilagin could suppress tumor growth by counting volume.Western blotting and immunohistochemical were performing to check expression of caspase-3,PARP and PCNA,results showed that corilagin could increase apoptosis and expression of PCNA.Taken together,for the first time,we find corilagin can activate apoptosis and autophagy,which is dependent on ROS in breast cancer MCF-7 cells and corilagin can not induce necroptosis in breast cancer SK-BR3 cells.Furthermore,corilagin can suppress the progress of tumor in vivo.The above results can provide theoretical basis for treatment of corilagin used in breast cancer.