Study On The Effect And Mechanism Of Cyperotundone On Anthracycline Chemotherapy Drugs For Breast Cancer Based On NRF2/ARE Pathway

Objective:Our study by observing the effect of the intervention of cyperotundone on anthracycline chemotherapeutic drugs in breast cancer cells in vivo and in vitro,the mechanism of cyperotundone increasing the sensitivity of anthracycline chemotherapeutic drugs was discussed,in order to provide some theoretical basis for finding new treatment methods for breast cancer patients who are not sensitive to or resistant to anthracycline chemotherapeutic drugs clinically.Methods:1.Cell experiment:CCK-8 experiment was used to detect the effect of cyperotundone and adriamycin on the viability of MCF-7 and MCF-7/ADR cells.MCF-7 and MCF-7/ADR cells were treated with 0.1,0.2,0.3,0.4,0.5 or 0.6 m M cyperotundone and 0.078,0.156,0.313,0.625,1,25,2.5,5,10,20 or 40μg/m L adriamycin respectively for 24 h,48 h or 72h,and the IC50 of cells at different time points were measured,and the optimal concentration of action was selected for subsequent experiments.CCK-8 experiment was used to detect the effect of cyperotundone combined with adriamycin on the viability of MCF-7 and MCF-7/ADR cells.Clone form assay was used to detect the effect of cyperotundone combined with adriamycin on the proliferation of MCF-7 and MCF-7/ADR cells.Flow cytometry was used to detect the effect of cyperotundone combined with adriamycin on the apoptosis and cycle of MCF-7 and MCF-7/ADR cells,and Western blots were used to detect the expression of apoptosis related proteins BAX,Bcl2,Cleaved-caspase 3,and cycle related proteins Cyclin-D1 and P16.Flow cytometry was used to detect the effect of cyperotundone combined with adriamycin on the expression of ROS in MCF-7 and MCF-7/ADR cells and after adding ROS inhibitor NAC,the effect of cyperotundone combined with adriamycin on the expression of ROS in MCF-7 and MCF-7/ADR cells.After adding ROS inhibitor NAC,the effect of cyperotundone combined with adriamycin on the apoptosis of MCF-7 and MCF-7/ADR cells was detected by flow cytometry.Weston blots were used to detect the expression of P62,NRF2,HO-1 in MCF-7 and MCF-7/ADR cells after the combination of cyperotundone and adriamycin.Flow cytometry was used to detect the effect of cyperotundone combined with adriamycin on the apoptosis of MCF-7 and MCF-7/ADR cells after adding Brusatol,the NRF2inhibitor.2.Animal experiment:The xenograft tumor models in nude mice of MCF-7 and MCF-7/ADR cells were established.When the tumor volume reached about 100m~3,the mice were randomly divided into four groups,three in each group,and injected intraperitoneally with normal saline,adriamycin,cyperotundone or cyperotundone combined with adriamycin,respectively.The tumor size and body weight of nude mice after administration were measured at different time periods.Biochemical experiment was used to detect the effect of cyperotundone on hepatotoxicity and nephrotoxicity in nude mice.The expression of Ki67 in the xenograft tumor models in nude mice of MCF-7 and MCF-7/ADR cells was detected by immunohistochemistry.The expression of ROS in the xenograft tumor in nude mice of MCF-7 and MCF-7/ADR cells was detected by immunofluorescence assay.Weston blots were used to detect the protein expression of P62,NRF2,HO-1 in the xenograft tumor models of MCF-7 and MCF-7/ADR cells treated with cyperotundone combined with adriamycin.Results:1.Cell experiment:(1)Effects of cyperotundone and adriamycin on the viability of MCF-7 and MCF-7/ADR cellsThe results of CCK-8 assay to detect the activity of cyperotundone and adriamycin on MCF-7 and MCF-7/ADR cells showed that cyperotundone and adriamycin inhibited the activity of MCF-7 and MCF-7/ADR cells in a concentration and time dependent manner,respectively.The concentration was selected according to the cell activity curve of cyperotundone and adriamycin,where the cell activity was about 70%of the single drug concentration for subsequent experiments.Therefore,the drug concentration used by MCF-7 cells is ADR 0.5μg/ml,CYT 0.3 m M,the drug concentration used by MCF-7/ADR cells is ADR 4μg/ml,CYT 0.3 m M.Compared with the control group,the effect of cells in the single drug group on the viability of MCF-7 and MCF-7/ADR cells was significantly higher than in the control group.Compared with the single drug group,the effect of cyperotundone combined with adriamycin on the viability of MCF-7 and MCF-7/ADR cells was significantly higher than that in the single drug group,and the difference was statistically significant.(2)Cyperotundone combined with adriamycin can inhibit the proliferation of MCF-7and MCF-7/ADR cellsIn MCF-7 and MCF-7/ADR cells,compared with the control group,the single drug group effectively inhibited the proliferation of MCF-7 and MCF-7/ADR cells.Compared with the single drug treatment group,the combined drug group more effectively inhibited the proliferation of MCF-7 and MCF-7/ADR cells,with a statistically significant difference.(3)Apoptosis of MCF-7 and MCF-7/ADR cells can be increased by combination of cyperotundone and adriamycinIn MCF-7 and MCF-7/ADR cells,compared with the control group,the single drug group effectively promoted the apoptosis of MCF-7 and MCF-7/ADR cells.Compared with the single drug treatment group,the combined drug group more effectively promoted the apoptosis of MCF-7 and MCF-7/ADR cells.Western blots showed that in MCF-7 and MCF-7/ADR cells,compared with the control group,the single drug group effectively promoted the expression of proapoptotic protein BAX and Cleaved caspased 3 in MCF-7and MCF-7/ADR cells.Compared with the single drug treatment group,the combined drug group more effectively promoted the expression of proapoptotic protein BAX and Cleaved caspased 3 in MCF-7 and MCF-7/ADR cells.In MCF-7 and MCF-7/ADR cells,compared with the control group,the single drug group effectively reduced the expression of antiapoptotic protein Bcl-2.Compared with the single drug treatment group,the combined drug group more effectively reduced the expression of antiapoptotic protein Bcl-2 in MCF-7 and MCF-7/ADR cells,and the difference was statistically significant.(4)Cyperotundone combined with adriamycin can enhance the inhibition of G0/G1phase cell cycle of MCF-7 and MCF-7/ADR cellsIn MCF-7 and MCF-7/ADR cells,compared with the control group,the single-drug group effectively inhibited the G0/G1 phase of the cycle of MCF-7 and MCF-7/ADR cells.Compared with the single drug treatment group,the combined drug group more effectively inhibited the G0/G1 phase of the cycle of MCF-7 and MCF-7/ADR cells.Western blots were used to detect the expression of cyclin related proteins.In MCF-7 and MCF-7/ADR cells,compared with the control group,the single drug group effectively inhibited the expression of Cyclin D1 and increased the expression of P16.Compared with the single drug treatment group,the combined drug group more effectively inhibited the expression of Cyclin D1,and increased the expression of P16,with statistically significant differences.(5)Cyperotundone combined with adriamycin increases ROS production to induce apoptosis in MCF-7 and MCF-7/ADR cellsIn MCF-7 and MCF-7/ADR cells,compared with the control group,the single drug group effectively increased the expression of ROS in MCF-7 and MCF-7/ADR cells after0.5 h incubation with DCFH-DA.Compared with the single drug group,the combination of cyperotundone and adriamycin significantly increased the production of ROS in MCF-7and MCF-7/ADR cells after 0.5 h incubation with DCFH-DA.NAC(N-acetyl-1-cysteine)is a ROS inhibitor.In MCF-7 and MCF-7/ADR cells,compared with the control group,the combined drug group effectively reduced the expression of ROS in MCF-7 and MCF-7/ADR cells after adding NAC.These results showed that the combination of cyperotundone and adriamycin could induce oxidative damage in MCF-7 and MCF-7/ADR cells,while NAC could significantly reverse the ROS production induced by the combination of CYT and ADR.We used flow cytometry to detect the apoptosis rate of each group after adding NAC to further verify the relationship between ROS and apoptosis.The results showed that compared with the control group,the addition of NAC effectively reduced the apoptosis of MCF-7 and MCF-7/ADR cells.Compared with the combination group,the combination group significantly reduced the apoptosis of MCF-7 and MCF-7/ADR cells after the addition of NAC,indicating that the production of ROS may be one of the key mechanisms of cyperotundone combined with adriamycin leading to apoptosis and enhancing chemosensitivity.(6)Cyperotundone combined with adriamycin induced Apoptosis of MCF-7 and MCF-7/ADR cells by inhibiting NRF2/ARE signaling pathwayWestern blots showed that compared with the control group,the expression of P62,NRF,HO-1 in MCF-7 and MCF-7/ADR cells was decreased in the single drug group,and compared with the single drug group,the expression of P62,NRF,HO-1 in MCF-7 and MCF-7/ADR cells in the combined drug group was significantly decreased.Flow cytometry results showed that compared with the control group,the addition of Brusatol effectively increased the apoptosis of MCF-7 and MCF-7/ADR cells.Compared with the combination of cyperotundone and adriamycin,the combination of cyperotundone and adriamycin significantly increased the apoptosis of MCF-7 and MCF-7/ADR cells after the addition of Brusatol,indicating that the NRF2/ARE pathway may be one of the key mechanisms of CYT combined with ADR leading to apoptosis and enhancing chemosensitivity.2.Animal experiment(1)Cyperotundone has no obvious toxicity on liver and kidney function on xenograft tumor in nude miceThe hepatorenal toxicity index of the treatment group and the control group were both within the normal range,indicating that there was no obvious hepatorenal toxicity of cyperotundone on xenograft tumor in nude mice.(2)Cyperotundone combined with adriamycin has obvious inhibitory effect on xenograft tumor in nude miceOn xenograft tumor in nude mice with MCF-7 and MCF-7/ADR cells,compared with the control group,the growth of tumor volume in the single drug group was slightly slower than that in the control group;Compared with the single drug group,the combined treatment group showed that the growth of tumor volume was significantly slower than the single drug group,and the tumor volume was significantly reduced.There was no significant change in body weight in all groups.Immunohistochemistry showed that the expression of Ki67 in the single drug group was lower than that in the control group;Compared with the single drug group,the expression of Ki67 in the combined drug group was significantly lower,and the difference was statistically significant.(3)Cyperotundone combined with adriamycin may have a significant inhibitory effect on xenograft tumor in nude mice bearing tumor through ROS productionWe used immunofluorescence to detect ROS production in tumor tissue of nude mice,in order to further study the mechanism of cyperotundone combined with adriamycin inhibiting tumor in nude mice.The results showed that the expression of ROS in the single drug group was higher than that in the control group in the nude mice;Compared with the single drug group,the expression of ROS in the combined drug group was significantly higher,and the difference was statistically significant.(4)Cyperotundone combined with adriamycin may show significant inhibitory effect on xenograft tumor in nude mice bearing tumor through NRF2/ARE pathwayWe used weston blots to detect the production of P62,NRF2 and HO-1 in tumor tissue of nude mice,so as to further study the mechanism of cyperotundone combined with adriamycin inhibiting tumor in nude mice.The results showed that compared with the control group,the expression of P62,NRF2 and HO-1 in the single drug group was lower;Compared with the single drug group,the expression of P62,NRF2 and HO-1 in the combined drug group was significantly lower,with a statistically significant difference.Conclusion:1 Our research results show for the first time that the combination of cyperotundone and adriamycin anti proliferation effect on MCF-7 and MCF-7/ADR breast cancer cells in vivo and in vitro.2 Cyperotundone can increase the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to adriamycin in vivo and in vitro.3 Cyperotundone may increase the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to adriamycin by inducing ROS production and inhibiting P62/NRF2/HO-1 signaling pathway.