Study On The Effects And Mechanisms Of Eupalinolide J On Human Breast Cancer Cells Through Targeting STAT3 Signalling Pathway

Objective: Eupalinolide J is the major sesquiterpene lactone compound in the Eupatorium lindleyanum DC.It has great effects in reducing blood fat,anti-inflammatory and antibacterial.However,few researches have reported its effect on tumor threapy.The purpose of this study is to study the anti-breast cancer effects and the mechanisms of Eupalinolide J(EJ)via STAT3 signaling pathway.Methods: Several human breast cancer cells(MDA-MB-231,MDA-MB-468)and normal breast epithelial cells MCF-10 A were used to detect the inhibitory effect of Eupalinolide J on tumor growth by MTT assay.A STAT3 low-expression breast cancer cell line was constructed by shRNA lentivirus infection and confirmed by western blotting.And sensitivity of the cells to Eupalinolide J was examined.The effect of Eupalinolide J on cell cycle was measured by flow cytometry.Western Blotting was used to detected the expression of Cyclin D1,c-Myc,and p21 which were in the downstream of STAT3 after Eupalinolide J treatment.Flow cytometry was used to detect the apoptosis rate of EJ-induced breast cancer cells.We used fluorescence microscopy to observe the nucleus of EJ-induced apoptosis of breast cancer cells by DAPI staining.The changes of apoptotic related proteins PARP,Caspase-3,Caspase-8,Caspase-9,and Bcl-2,Bax,and Bad were detected by western blotting.We investigated the effect of Eupalinolide J on the growth of tumors in nude mice.We detected apoptosis in tumor tissue by TUNEL staining.The effect of EJ on STAT3 and related proteins in tumor tissue sections was examined by immunohistochemistry.Results: The expression of STAT3 total protein was also inhibited after administration.MTT assay showed that Eupalinolide J significantly inhibited the proliferation of breast cancer cells(MDA-MB-231 and MDA-MB-468)in a time and dose-dependent manner and had little effect on normal human breast cells(MCF-10A).After the inhibition of STAT3 expression by shRNA,the sensitivity of breast cancer cells to Eupalinolide J was decreased.The results of PI staining alone indicated that Eupalinolide J could block cell cycle at G2/M phase.Western Blotting showed that Eupalinolide J could down-regulate Cyclin B1,c-Myc and up-regulate p21 in the downstream of STAT3 in a dose-dependent manner.Eupalinolide J also significantly increased the rate of apoptosis of breast cancer cells by activating PARP,Caspase-3,Caspase-8,and Caspase-9 proteins.The expression of anti-apoptotic proteins(Bcl-2 and Bcl-xL)in the downstream of STAT3 was down-regulated,and the expression of Bax and Bad was increased,indicating that Eupalinolide J can induce breast cancer cell apoptosis in a dose-dependent manner.After the mude mouse transplanted tumor was treated with Eupalinolide J,the average tumor volume and weight of administration group were smaller than control group.The expression of STAT3 in administration group was also significantly down-regulated compared with control group.Bcl-xL were also inhibited.TUNEL staining showed that Eupalinolide J could induce apoptosis in the tumor.Conclusion: Eupalinolide J can significantly inhibit breast cancer proliferation in vivo and in vitro.The mechanism may be to induce cell cycle arrest and apoptosis by inhibiting the activity of STAT3.As a novel STAT3 inhibitor,it is expected to become a potential drug for the treatment of breast cancer.