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.
breast neoplasmsDNA helicasescell proliferationapoptosistetrandrine derivativespolyploid giant cancer cellsBLM DNA helicase
维普
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