Shikonin Inhibits The Proliferation Of Breast Cancer Cell Lines In Vitro And In Vivo

Background:Breast cancer is a global health burden and the second leading cause of cancer-related deaths among women.Traditional Chinese Medicine plays an important role in disease treatment.Shikonin is an active herbal monomer isolated from the traditional Chinese herbal medicine Zi Cao.Shikonin and its derivatives are potent pharmaceutical substances widely used for wound healing,and they also possess anti-bacteria,anti-inflammatory,anti-viral,analgesic,immune-stimulatory,free radical scavenging and anti-thrombotic properties.In addition,shikonin is potentially a therapeutic agent for cancer treatment.Research showed that it could induce apoptosis in a variety of tumor cell lines by different molecular mechanism,including colorectal cancer,melanoma,leukemia,and hepatocellular cancer cell lines.Shikonin can also inhibit the growth of a variety of cancer cell lines.Clinical trials demonstrated a shikonin-containing mixture could effectively and safely inhibit the tumor progression of late-stage lung cancer.According to literature,shikonin can inhibit the proliferation and induce breast cancer cell death in a dose-and time-dependent manner.However,only MCF-7,an ER(Estrogen Receptor)positive breast cancer cell line,was tested.As breast cancer is a high heterogeneouds tumor,more evidence for different kinds of cell lines(e.g.ER negative cell lines)was needed.Furthermore,there is no report about the effects of shikonin in breast cancer in vivo.In this study,two ER positive breast cancer cell lines(MCF7,ZR-75-1)and two ER negative breast cancer cell lines(SUM1315,MDA-MB-231)were used to test the anti-cancer effects of shikonin in vitro and in vivo,and explore the potential molecular mechanism of shikonin,so as to provide preliminary evidence on the potential useage of shikonin as a clinical agent for breast cancer therapy.Objective:Detect the inhibitory effect of Shikonin on MCF7,ZR-75-1,SUM1315 and MDA-MB-231.Determine the apoptotic cells and cell cycle fractions by flow cytometry.Study the change of protein expression levels of Cleaved caspase 3,Cyclin D1,MAPK/ERK pathway and PI3K/AKT/MTOR pathway to reveal the underlying mechanism.Detect the inhibitory effect of shikonin on breast cancer tumors in vivo.Methods:CCK8 assay was used to test the effect of shikonin on the viability of 4 breast cancer cell lines(MCF7,ZR-75-1,SUM1315 and MDA-MB-231),then calculate the IC50.MCF7,ZR-75-1,SUM1315 and MDA-MB-231 were exposed to different treatments for 48h.After treatments,the cells were collected.Cell cycle and apoptosis analyses were detected by flow cytometer.After treatments,the protein were collected.Cleaved caspase 3,Cyclin D1,MAPK/ERK pathway andPI3K/AKT/MTOR pathway were detected by Western Blot analysis.Establish xenograft model of breast cancer with 30 female nude mice.Seperated the mice into five groups and intraperitoneally injected different concentritions of shikonin.Tumor volumes were calculated.Results:CCK8 assay revealed shikonin inhibited MCF7,ZR-75-1,SUM1315 and MDA-MB-231 breast cancer cell proliferation in a dose-dependent manner.The IC50 of MCF7,ZR-75-1,SUM1315 and MDA-MB-231 were 1.165 ± 0.025,1.043 ± 0.010,1.595 ± 0.006 and 1.516 ± 0.015 μmol/L,respectively.Flow cytometry showed treatment with shikonin for 48 h significantly increased the combined proportions of early and late apoptotic cells.The apoptosis rate of MCF7,ZR-75-1,SUM1315 and MDA-MB-231 were 15.8±0.7(p<0.05),32.312.0(p<0.05),45.0±2.5(p<0.01),15.8±0.4(p<0.01)，respectively.The difference was regarded as statistically significant.Shikonin significantly increased the percentage of G1 phase cells in MCF7,ZR-75-1,SUM1315 and MDA-MB-231 cells.When compared with control group,the percentages of G1 phase were 15.8±0.7(p<0.05),32.3±2.0(p<0.05),45.01-2.5(p<0.01),15.8±0.4(p<0.01),respectively.Western blot showed shikonin up-regulated the expression of Cleaved caspase 3 in ZR-75-1,SUM1315 and MDA-MB-231 cells.Shikonin down-regulated the expression of Cyclin Dland significantly reduced the levels of phosphorylated p-Akt,p-mTOR and p-ERK,but did not alter the total expression levels of these proteins in all 4 breast cancer cell lines.Tumor volume was significantly smaller in shikonin-treated mice as compared with control mice,which was positive correlation with concentration.A statistical difference was found between Group C and A on the 7th day(p<0.05).On the 9th day,there was statistical difference between Group C and A(p<0.0001),so as Group C and B(p<0.01).On the 11th day,the comparison between any two groups have statistical significance(A vs B:p<0.01;B vs C:p<0.01;A vs C:p<0.0001).Conclusion:Shikonin inhibited the proliferation of breast cancer cell lines in a dose-dependent manner.Shikonin induced apoptosis of breast cancer cells and cell cycle arrest by up-regulating the expression of Cleaved caspase 3 in ZR-75-1,SUM1315 and MDA-MB-231 cells.Shikonin induced cell cycle arrest by down-regulating expression of Cyclin D1 in all 4 breast cancer cell lines.Shikonin reduced the levels of phosphorylated p-Akt,p-mTOR and p-ERK to modulate the growth and apotosis of breast cancers.Shikonin inhibited the proliferation of breast cancer cells in vivo.