Effect Of Tetrandrine Derivative LYY-47 On Proliferation And Apoptosis Of Triple Negative Breast Cancer Polyploid Giant Cancer Cells By Inhibiting Its BLM DNA Helicases

Objective:The expression of BLM DNA helicase in triple-negative breast cancer MDA-MB-231 cells,polyploid giant cancer cells(PGCCs)and PGCCs with budding will be observed.The mechanism and effects of tetrandrine derivative LYY-47 on the proliferation and apoptosis of MDA-MB-231 cells and PGCCs with budding will be investigated.Methods:The paclitaxel(PTX)was used to induce MDA-MB-231 cells to form PGCCs and PGCCs with budding.H&E staining,flow cytometry,and Western blot were used to identify the morphology,polyploidy characteristics,and stem cell characteristics of PGCCs.Immunofluorescence was used to detect the expression of γ-H2 AX in MDAMB-231 cells and PGCCs with budding.Western blot was used to detect the expression of BLM,BRCA1 and Rad51 proteins in MDA-MB-231 cells and PGCCs with budding.Crystal violet staining was used to observe the effect of ML216,a specific inhibitor of BLM,on PGCCs formation.The tumor formation ability of MDA-MB-231 cells and PGCCs with budding was compared in nude mice.The H&E was used to stain the tumor tissues to observe their cell morphology,and the immunohistochemical was used to detect expression of BLM and Ki-67 in the tumor tissues.Tetrandrine derivatives were screened by fluorescence polarization assay to inhibit the enzymatic activity of BLM DNA helicase.MTT assay was used to detect the effects of tetrandrine derivatives LYY-47 and ML216 on the proliferation of MDA-MB-231 cells and PGCCs with budding.Colony formation assay was used to detect the effects of LYY-47 and ML216 on the colony formation of the above two groups of cells.The effects of LYY-47 and ML216 on the cell cycle and apoptosis of MDA-MB-231 cells and PGCCs with budding were detected by flow cytometry.Western blot was used to detect the effect of LYY-47 on the expression of cycle-related proteins and apoptosis-related proteins in MDA-MB-231 cells and PGCCs with budding.Results:1.PGCCs formed with paclitaxel induction showed unique morphological features,including enlarged nuclear and cytoplasmic regions,tumor stem cell properties,and the ability to generate progeny cells through asymmetric division.Compared with MDAMB-231 cells,the number of S(p<0.01)and G2/M phase cells increased in PGCCs(p<0.05),while the number of S phase cells in the PGCCs with budding increased(p<0.01),G2/M phase cells decreased(p<0.01).The expression of CDK1 and Cyclin B1 was significantly down-regulated in PGCCs(p<0.05),while the expression of CDK1 and Cyclin B1 was up-regulated in the PGCCs with budding(p<0.05).The expressions of stemness-related proteins ALDH1A1,CD44,CD133 in PGCCs and PGCCs with budding were higher than those in MDA-MB-231 cells(p<0.05).The proliferation and clonal formation ability of the PGCCs with budding were significantly higher than those of MDA-MB-231 cells(p<0.05).The expression of γ-H2 AX was increased.The expression of BLM,BRCA1 and Rad51 proteins was up-regulated(p<0.05).ML216 inhibited PGCCs formation(p<0.01).Tumor formation in nude mice showed that the tumor growth rate,tumor volume and weight of PGCCs with budding were significantly higher than those of the MDA-MB-231 cells(p<0.01).Immunohistochemical results showed that BLM and Ki-67 were highly expressed in PGCCs with budding(p<0.01).2.The results of fluorescence polarization assay showed that tetrandrine derivative LYY-47 significantly inhibited the enzymatic activity of BLM DNA helicase and inhibited the expression of BLM,Rad51 and BRCA1 proteins in MDA-MB-231 cells and PGCCs with budding(p<0.05).LYY-47 and ML216 inhibited the proliferation and colony formation of MDA-MB-231 cells and PGCCs with budding in a concentrationdependent manner(p<0.05).LYY-47 could increase G2/M phase cells(p<0.05),decrease S phase cells(p<0.05),and the cells were arrested in G2/M phase in MDAMB-231 cells and PGCCs with budding.The expressions of CDK1 and Cyclin B1 were down-regulated.ML216 increased G1 phase cells and decreased S phase cells in MDAMB-231 cells and PGCCs with budding(p<0.05).LYY-47 promoted the apoptosis of MDA-MB-231 cells and PGCCs with budding.Compared with the control group,the expression of apoptosis-related proteins Bax,cleaved Caspase-3 and cleaved Caspase-8 was up-regulated in LYY-47 treatment group(p<0.05),and the expression of Bcl-2was down-regulated(p<0.05).Conclusions:PGCCs derived from MDA-MB-231 cells have the characteristics of tumor stem cells.Compared with MDA-MB-231 cells,the proliferation,migration and invasion abilities of PGCCs with budding were enhanced,and the expression of γ-H2 AX,BLM,BRCA1 and Rad51 proteins was up-regulated.The formation of PGCCs is associated with BLM DNA helicase,LYY-47 and ML216 affect the proliferation of PGCCs with budding by inducing apoptosis and arresting the cell cycle by inhibiting BLM DNA helicase.