Effect and mechanism of tetrandrine derivative LYY-47 on proliferation and apoptosis of triple negative breast cancer cells and their polyploid giant cancer cells
Effect and mechanism of tetrandrine derivative LYY-47 on proliferation and apoptosis of triple negative breast cancer cells and their polyploid giant cancer cells
Objective To explore the effects and mechanism of tetrandrine derivative LYY-47 on the proliferation and apoptosis of triple negative breast cancer(TNBC)cells and polyploid giant cancer cells(PGCCs)with budding.Methods TNBC MDA-MB-231 cells and MDA-MB-436 cells were cultured to logarithmic growth phase to induce PGCCs.After 35 days of culture,H&E staining was used to observe the morphological characteristics of PGCCs of the two types at different culture time,and the PGCCs were counted.MDA-MB-231 cells,PGCCs and their PGCCs with budding were collected and analyzed for cell cycle by Flow cytometry.Western blot was used to detect the expression of cyclin-related proteins[cyclin dependent kinase 1(CDK1)and Cell cycle proteinB1(CyclinB1)],stemness related protein[acetaldehyde dehydrogenase1A1(ALDH1A1),cluster of differentiation 44(CD44)and cluster of differentiation 133(CD133)],DNA damage repair related protein[Bloom(BLM),(Rad51)and breast cancer susceptibility gene 1(BRCA1)]and apoptosis-related proteins[Bcl-2-associated X protein(Bax),B-cell lymphoma protein2(Bcl-2),cleaved Caspase-3,and cleaved Caspase-8].Methyl thiazol tetrazolium(MTT)assay and colony formation assay were used to detect cell viability and colony number.Immunofluorescence assay was used to detect the expression of phosphorylated histone 2AX(γ-H2AX).The activity of BLM DNA helicase was detected by fluorescence polarization assay.MDA-MB-231 cells in logarithmic growth phase were collected.They were divided into the control group(the same volume of complete medium),PTX group(500 nmol/L PTX),3-CF3,4-F-phenyl analog(ML216)group(3 µmol/L ML216)and ML216+PTX group(500 nmol/L PTX and 3 µmol/L ML216).Image J software was used to count the number of cells in each group.Logarithmic growth MDA-MB-231 cells and PGCCs with budding were collected and divided into the control group(0.00 μmol/L)and LYY-47 treatment group(2.50 µmol/L,5.00 µmol/L,and 6.50 µmol/L).The apoptosis of the above cells was detected by flow cytometry.Twelve 6-week-old female nude mice of specific pathogen free(SPF)athymic BALB/c were subcutaneously injected with 5 × 106 MDA-MB-231 cells and their PGCCs with budding,respectively.The tumor volume was measured every 4 days for 29 consecutive days.The tumor tissues were taken for H&E staining and immunohistochemical staining to observe cell morphology and detect the expression of BLM and Ki-67.Results PGCCs induced by PTX showed enlarged nuclear and cytoplasmic areas compared with TNBC MDA-MB-231 cells and MDA-MB-436 cells,which produced progeny cells through asymmetric division.Compared with MDA-MB-231 cells,the number of S and G2/M phase cells increased in PGCCs,and the expression of CDK1 and CyclinB1 was significantly down-regulated(P＜0.05 or P＜0.01),and the number of S phase cells in PGCCs with budding increased,but G2/M phase cells decreased,while the expression of CDK1 and CyclinB1 was up-regulated(P＜0.05 or P＜0.01).The expressions of stemness-related proteins ALDH1A1,CD44 and CD133 were significantly up-regulated in PGCCs with budding(P＜0.05)and the proliferation and colony formation ability of PGCCs with budding were significantly enhanced(P＜0.05);The protein expression of γ-H2AX,BLM,BRCA1 and Rad51 was up-regulated(P＜0.05).Compared with the PTX group,the number of PGCCs formed in the ML216+PTX group was significantly reduced(P＜0.01).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),and BLM,Ki-67 were highly expressed in the tumor(P＜0.01).Compared with the control group,The ATPase activity of DNA helicase,dsDNA unfolding activity and DNA binding activity of BLM642-1290 were down-regulated in LYY-47 treatment group,and the expression of BLM,Rad51 and BRCA1 proteins in MDA-MB-231 cells and PGCCs with budding was also down-regulated(P＜0.05).Compared with the control group,treatment with LYY-47 and ML216 significantly decreased the proliferation and colony formation ability of MDA-MB-231 cells and PGCCs with budding(P＜0.05).Treatment with LYY-47 significantly increased the number of cells in G2/M phase(P＜0.05)and decreased the number of cells in S phase(P＜0.05).The expression of cycle-related proteins CDK1 and CyclinB1 in the cells was down-regulated(P＜0.05)in MDA-MB-231 and PGCCs with budding.ML216 treatment significantly increased the number of cells in G1 phase(P＜0.05)and decreased the number of cells in S phase(P＜0.05)in MDA-MB-231 and PGCCs with budding.Compared with the control group,LYY-47 treatment significantly increased the proportion of total apoptosis and up-regulated the expression of apoptosis-related proteins Bax,cleaved Caspase-3 and cleaved Caspase-8 in MDA-MB-231 cells and PGCCs with budding(P＜0.05).The expression of Bcl-2 was down-regulated(P＜0.05).Conclusions LYY-47 and ML216 can affect the proliferation of triple-negative breast cancer cells and PGCCs with budding,which may be related to the inhibition of BLM DNA helicase induced apoptosis and cell cycle arrest.
维普
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