Potential Influences Of Berberine Combined With Adriamycin On The Proliferation And Migration Of Triple-negative Breast Cancer Cell

Objective:This study aims to explore the role of Berberine（BBR）combined withAdriamycin（ADR）in regulating the proliferation and migration of the triple-negative breast cancer cell line MDA-MB-231 through altering the acetylation level of H3K56ac.Methods:The triple-negative breast cancer cell line MDA-MB-231 was induced with BBR or ADR at different concentrations,or a combination of BBR and ADR,followed by examination of the proliferation inhibition by CCK-8 assay.In addition,CCK-8 assay was performed to measure the IC₅₀value of MDA-MB-231 cells against BBR or ADR.The combination index（CI）in MDA-MB-231 cells induced with BBR combined with ADR was analyzed using the Compu Syn software,so as to identify the optimal combination regimen.Protein levels of P300,SIRT2 and H3K56ac in MDA-MB-231 cells were detected by Western blot.Finally,potential influences of BBR and ADR on the migration and invasion of MDA-MB-231 cells were assessed by wound healing assay and Transwell assay.Results:1.CCK-8 assay results revealed that after 24-h induction of MDA-MB-231 cells by 20,40,60,80 and 100μmol/L BBR,or 0.5,1,1.5,2 and 2.5μg/m L ADR,inhibitory effects of BBR and ADR on the proliferation of MDA-MB-231 cells were dose-dependently enhanced.The IC₅₀value of MDA-MB-231 cells against BBR and ADR was 78.87±1.52μmol/L and 1.12±0.71μg/m L,respectively.CI in MDA-MB-231 cells induced with BBR at different concentrations combined with low-dose ADR was less than 1,suggesting that the combination of BBR and ADR had a synergistic effect on triple-negative breast cancer cells.2.Western blot was performed to detect protein levels of P300,SIRT2 and H3K56ac in MDA-MB-231cells.Compared with those of control group,SIRT2 was downregulated and P300 was upregulated in MDA-MB-231 cells induced with either BBR or ADR.Protein level of H3K56ac was upregulated in BBR-induced MDA-MB-231 cells.Compared with those induced with BBR only,protein level changes of P300,SIRT2 and H3K56ac were more significant in MDA-MB-231 cells induced with BBR combined with ADR（P<0.05）.3.Traswell assay results showed that compared with the control group,after induction of 1μg/m L ADR and 80μmol/L BBR for 24h,the number of MDA-MB-231cells invading from the upper chamber of the Transwell chamber through the polyesteror polyethylene terephthalate（PET）to the bottom was reduced to 128±1.25and 223±4.14,respectively.After a combination induction of ADR and BBR with the calculated CI,the number of invasive MDA-MB-231 cells was reduced to 68±1.07,which was statistically significant from that of controls（P<0.05）.4.Wound healing assay results showed that the migration rate of MDA-MB-231 cells induced with ADR and BBR for 24 h was significantly lower than that of controls（24±5.21,38±3.25,respectively versus 58±3.55）,which was only 8±2.13 after a combination induction of ADR and BBR with the calculated CI,and the difference was statistically significant（P<0.05）.Conclusions:The combination of BBR and ADR significantly inhibits the proliferation,migration and invasion of triple-negative breast cancer MDA-MB-231 cells,showing a synergistic anti-tumor effect mainly through regulating the acetylation level of H3K56ac by mediating expression levels of the histone acetyltransferase P300 and the histone deacetylase SIRT2.