Research On The Synergistic Inhibitory Effects And Mechanism Of PFT?/daidzein Combined With Topotecan On Breast Cancer

Topotecan(TPT)is a semi-synthetic analog of camptotecin and it has been widely used as the second-line drugs for treatment of solid tumors because of its low price and obvious anticancer effect.TPT has poor selectivity to cancer cells and it has strong toxic and side effects.At the same time,TPT has a single target that is topomerase I(Topo I)and drug resistance is easy to occur.These two shortcomings seriously limit the clinical application of TPT.Therefore,the aim of this study is to reduce the dosage of TPT,improve its efficacy and reverse its drug resistance through the combination of drugs.This paper mainly includes three parts:the first part is that TPT is reguided as the main drug and the anti-tumor effect of combination with p53 inhibitor Pifithrin-alpha(PFT?)is studied;the second part is that TPT is reguided as the main drug and the anti-tumor effect of combination with DAI is studied;the third part is to reverse the resistance of drug-resistant cells to TPT by DAI.The main experimental results and conclusions are as follows:(1)MTT assay was used to detect the effect of TPT combined with PFTa on the proliferation of tumor cells.The results showed that PFT? enhanced the inhibitory effect of TPT on the proliferation of MCF7,BGC823 and HepG2 cells at 48 h significantly.DNA relaxation test and comet assay were used to detect the effects of combination of the two drugs on Topo I activity and DNA damage.The results showed that PFTa enhanced the inhibitory effect of TPT on Topo I activity,resulting in more DNA damage.The effects of combination on cell cycle and apoptosis were analysed by PI single staining,AV/PI double staining,qPCR and flow cytometry.The results showed that cell cycle were arrested in S phase and more cell apoptosis appeared in the combined drug group.10 ?M PFT? itself did not inhibit the expression of p53 and p21,but inhibited the expression of mdm2 and phosphorylated p53.The expression of p53 and p21 in the combination group increased significantly,the expression of mdm2 decreased significantly,while the expression of phosphorylated p53 in combination grroup was similar to that in TPT group.Then the accumulation of p53 and TPT in the nucleus was detected by laser confocal method.The results showed that the accumulation of p53 and TPT in the nucleus increased significantly in the combination group.These results indicate that on the one hand,PFT? increases the accumulation of TPT in cells to increase the inhibition of TPT on Topo I and induce more DNA damage.On the other hand,PFT? reduces the degradation of p53 by inhibiting the expression of mdm2,and increases the accumulation of p53 in the nucleus,which leads to more cell undergo cell cycle arrest or cell apoptosis.(2)MTT method was used to screen drugs who has a synergistic inhibitory effect on proliferation of cancer cells with TPT.The results showed that DAI and TPT had a strong synergistic effect when administered simultaneously in MCF7 at 48 h.DNA relaxation test and comet assay showed that DAI enhanced the inhibitory effect of TPT on Topo I activity,resulting in more DNA damage when the TPT was combined with DAI.PI single staining,AV/PI double staining and western blot methods were used to detect the effect of the combination on cell cycle and cell apoptosis.The results showed that the cell cycle of MCF7 was blocked in G2/M phase and more cells were induced to apoptosis by activating the mitochondrial apoptotic pathway.MCF7 xenotransplantation model in nude mice was constructed to evaluate the pharmacodynamics of the combination of two drugs in vivo.TPT and DAI were administered intraperitoneally and intragastrically respectively.The results showed that the inhibitory rate of the drug group(3 mg/kg TPT+5 mg/kg DAI)drug group on tumors was 95.98±1.50%after 15 days of administration.Compared with TPT alone,the drug concentration of TPT in the combined drug group could be reduced three times to reach the same pharmacodynamics.Immunohistochemical results further proved that the combination of the two drugs could induce cell cycle arrest by up-regulating p21 and p53,and cell apoptosis by adjusting the proportion of Bax/Bcl2.(3)The cell model of MCF7/ADR resistant to TPT was successfully constructed by increasing concentration gradient and the drug resistance index was 7.17.MTT assay was used to analyze the inhibitory effect on MCF7/ADR cells when DAI was combined with TPT.The results showed that DAI could reverse the resistance of MCF7/ADR to TPT.The expression of related genes was detected by qPCR.The results showed that the expression of ERa and BCRP in combination group was significantly decreased,but the expression of P-gp was not significantly different from that in TPT group.The distribution of BCRP on MCF7/ADR cell membrane and the accumulation of TPT in cell nucleus were detected by confocal laser scanning.The results showed that DAI significantly reduced the distribution of BCRP on cell membrane and increased the accumulation of TPT in cell nucleus.These results suggest that DAI reverses TPT resistance in drug-resistant cells by inhibiting the expression of ERa,decreasing the expression and distribution of BCRP on cell membrane and increasing the accumulation of TPT in cells.MCF7/ADR xenotransplantation model in nude mice was constructed to evaluate the pharmacodynamics of the combination of two drugs in vivo.TPT and DAI were administered intraperitoneally and intragastrically respectively.The results showed that the inhibitory rate of the drug group(1 mg/kg TPT+5 mg/kg DAI)drug group on tumors was 81.29±3.72%after 15 days of administration,reaching the same pharmacodynamics,and the drug concentration of TPT in the combined drug group could be reduced nine times.Immunohistochemical results showed that the combined use of the two drugs in vivo also reduced the expression of ERa and BCRP,which was consistent with the conclusion reached at the cellular level.In conclusion,the synergistic effect between PFTa and TPT was found,and the mechanism of the combination of PFTa and TPT was revealed.At the same time,we found that DAI and TPT have a synergistic anti-cancer effect,and DAI can reverse the resistance of drug-resistant cells to TPT.We also revealed the mechanism of the synergistic anti-cancer effect of DAI and TPT,and the mechanism of DAI reversed the drug resistance to TPT.The evaluation of tumor animal models proved that the combination of DAI and TPT had a significant synergistic anti-tumor effect.