Studies On The Anti Proliferative Effects And Molecular Mechanism Of Bigeloviia

Breast cancer is the most common neoplasm in women.2015 Annual Report by the National Central Cancer Registry of China had displayed that about 249 thousand breast cancer cases occurred in China and its incidence rate had been always increasing in recent 10 years.Therefore,it is important to search for effective lead compounds treating breast cancer.Twelve triterpene saponins and nine nortriterpene saponins were isolated from Salicornia bigelovii and Salicornia europaea by our group.These nortriterpene saponins had the rare aglycone of 30-nor oleanane type.So far,less than thirty 30-nortriterpenoids glycosides were reported in the literature and their studies on the pharmacodynamics were nearly blank.This is the first time to study their antitumor effects and mechanisms with the comprehensive application of animal level,cellular level and molecular level,using gel mobility shift assay(EMSA),Western blot,enzyme-linked immunosorbent assay,flow cytometry.Among twenty-one saponins,eight saponins showed potent cytotoxicity(IC50:10-70?M,24h)on human breast cancer cells MCF7,human cervical cancer cells Hela and human liver cancer cells HepG2.The saponins 8,10,14,15,18-20,with esterification of 3-O-?-D-glucuropyranosyl,exhibited moderate to considerable cytotoxicity.Among them,saponins 18-20,with 3-O-(6-butyl ester)-?-D-glucuropyranosyl,showed the most potent cytotoxicities.A free carboxylic group at C28 was an essential factor for the cytotoxic activity.However,it was less important than esterification of 3-O-?-D-glucuropyranosyl.The saponin 18,with methyl at C23,exhibited stronger cytotoxicity than saponin 19,with formaldehyde at C23.In addition,the cytotoxicities of 30-nortriterpenoid glycosides were similar to their triterpenoids glycosides.Two new nortriterpenoid glycosides,Bigelovii A and Bigelovii C,were chosen to investigate their antitumor activities.Both saponins showed antiproliferative activities against eight cancer cell lines(human breast cancer cells MCF7,human cervical cancer cells Hela,human liver cancer cells HepG2,human lung cancer cells A549,human leukaemia cells HL-60,mouse melanoma cells B16,human colon cancer cells Lovo and human glioblastoma cells LN229),especially MCF7 cell lines.For three different breast cancer cells,the antiproliferative effect of Bigelovii A on MCF7 cells was significantly better than on MDA-MB-231 and MDA-MB-468 while the antiproliferative effect of Bigelovii C on MDA-MB-231 cells was significantly better than on MCF7 and MDA-MB-468.The cytotoxic activities of Bigelovii A and Bigelovii C were time-dependent.MCF7 cells became round and floated after treatment with Bigelovii A and Bigelovii C for 24 h,while the untreated healthy cells displayed a dissimilar cytoskeleton.Bigelovii A and Bigelovii C inhibited clone formation of MCF7 cells.Cytotoxicities of Bigelovii A were more obvious than thato of Bigelovii C.Therefore,the in vivo antitumor activity of Bigelovii A was further exmined.Tumors in xenograft mice treated with Bigelovii A resulted in 54.57%inhibition compared with the control.We then defined the mechanism in Bigelovii A induced cell death,which displayed typical characteristics of apoptotic cells including externalization of phosphatidylserine on the plasma membrane,the increase of cells with hypodiploid DNA,cell condensation,chromatin condensation,as well as activation of caspase-9,caspase-7 and PARP.Bigelovii A inhibited I?Ba kinase complex(IKK)activation and I?Ba phosphorylation,which made NF-?B fixed in cytoplasm.Bigelovii A also suppressed p65 phosphorylation and nuclear translocation,and abrogated NF-KB-dependent reporter activity.NF-?B activates the transcription of several apoptotic genes,such as Bcl-2,Bcl-xl and COX-2.With increasing concentrations of Bigelovii A,the levels of Bcl-2,Bcl-xl and COX-2 reduced,mitochondrial membrane potential decreased and mitochondrial membrane permeability increased,leading to intrinsic apoptotic pathway.Furthermore,we investigated the effect of Bigelovii A on autophagy.Bigelovii A-treated MCF7 cells displayed a dramatic increase in the number of MDC-labeled vesicles,increased the expressions of LC3-?.Atg3 and Atg16L1,then induced cell autophagy.We have proven that in MCF7 cells.Bigelovii A inhibited mTOR signaling by decrease of Akt and p-ERK.Consistently,Bigelovii A decreased phosphorylation levels of mTOR,Akt,p70S6K(Ser371,Thr389)and 4EBP1.Inhibiting Bigelovii A-induced autophagy with the autophagy inhibitor 3-methyladenine significantly decreased cell viability,which suggested that Bigelovii A-induced autophagy played a pro-survival role.In conclusion,Bigelovii A may be a promising chemotherapeutic agent that induced mitochondrial apoptotic pathway involving inhibition of NF-?B,decrease of antiapoptotic proteins Bcl-2,Bcl-xl and COX-2.However,Bigelovii A inhibited the expression of Akt and p-ERK,then inhibited mTOR pathway,induced cell autophagy to pretect human breast cancer cell MCF7.