Mechanism Underlying The Reversal Of Drug Resistance In Human Breast Cancer MCF-7/ADR Cells By Chelidonine

The traditional Chinese medicine celandine（Chelidonium majus L.）is a dried whole grass of the celandine perennial herbaceous plant.Celandine It has a bitter taste,mild temperature,and poisonous;it has analgesic,diuretic,cough,clearing heat and detoxifying effects.The main components of Chelidonium majus are alkaloids,chelidonine is one of them.Studies have shown that chelidonine has significant anti-tumor effects on breast cancer,liver cancer,bladder cancer,cervical cancer,pancreatic cancer,rectal cancer,etc.In recent years,it has attracted the attention of a large number of scholars at home and abroad the study.In the previous study,our research group found that chelidonine not only has a good antitumor effect on Human gastric carcinoma SGC-7901 cells and human breast cancer MCF-7 cells,but also has a significant inhibitory effect on the proliferation of adriamycin resistant human breast cancer MCF-7/ADR cells,reducing the expression of P-gp protein,and reversing the drug resistance of cells.In this paper,network pharmacology and experimental verification were used to further explore how chelidonine reverses the drug resistant human breast cancer cell MCF-7/ADR to adriamycin（ADR）and the related mechanism based on the previous studies,so as to provide a scientific basis for the development and clinical application of resistance reversal agent.This experiment first used network pharmacology to predict the core targets and action mechanisms of chelidonine against breast cancer.The results showed that 65 chelidonine targets were obtained from the TCMSP database,Swiss Target Prediction database and literature mining.A total of 653 breast cancer related targets were obtained from OMIM data,GAD database and Pharm GKB database.Draw chelidonine-disease intersection PPI network,screen out core targets,and use DAVID database to perform GO and KEGG analysis of chelidonine acted breast cancer core targets.A total of 30 pathways are involved,including 14cancer pathways.16 signal paths.Of the 16 signaling pathways,10 are related to breast cancer resistance.Based on previous studies,chelidonine can reverse the resistance of breast cancer cells by reducing P-gp.Therefore,this article will further study its mechanism of action.Some literatures indicate that PI3K/AKT,MAPK,Foxo,Cell cycle,HIF-1 predicted by network pharmacology can regulate P-gp.The number of core targets involved in the pathway is PI3K/AKT>MAPK>Foxo>Cell cycle>HIF-1.Combining the experimental purpose with the results of network pharmacology,we choose the first two pathways for further experimental verification.On this basis,after verifying the effect of chelidonine on reversing the resistance of breast cancer to ADR,whether chelidonine reverses the resistance of MCF-7/ADR cells through acting on PI3K/AKT pathway was further studied,the prediction results of network pharmacology was verified,and the possible mechanism of chelidonine reversing the resistance of breast cancer was clarified.MTT method was used to determine the inhibitory effect of chelidonine combined with ADR on proliferation of MCF-7/ADR cells.Among them,IC₅₀ of MCF-7/ADR cell lines treated with ADR alone was 27.96±1.10μmol-L^-1in the control group,and IC₅₀ of MCF-7/ADR treated with chelidonine(1.5μmol?L^-1,3μmol?L^-1 and 6μmol?L^-1)combined with ADR dropped to14.69±0.28,8.37±0.28 and 6.21±0.43μmol?L^-1 respectively,by calculation,the reversal times are 1.90,3.34 and 4.50 times,respectively.The fluorescence intensity of ADR in each group detected by flow cytometry was as follows:15.7%?0.49 in the ADR alone group,48.2%?2.7,54.6%?0.05 and 56.6%?1.3 in the ADR combined with chelidonine(1.5μmol?L^-1,3μmol?L^-1 and 6μmol?L^-1)group（P<0.05）.The results showed that chelidonine could reduce the resistance of cells to ADR and increase the absorption of ADR in a dose-dependent manner within a certain concentration range.Western blotting was used to detect the expression change of P-gp,AKT,p-AKT,PI3K,p-PI3K,PTEN,p-IKK,p-IKB and NF-?B protein in MCF-7/ADR cells treated with ADR alone or chelidonine with different doses combined with ADR.The results showed that compared with the blank control group,there was no significant change in the expression level of each protein in the group treated with ADR alone（P>0.05）.Compared with the blank control group,the expression of P-gp、p-AKT and p-PI3K protein in chelidonine with different concentrations group was significantly lower（P<0.01）;while the expression of PTEN decreased significantly（P<0.01）,and the expression of AKT and PI3K protein in the cells did not change significantly（P>0.05）;compared with the blank control group,p-IKK,p-IKB and NF-?B in the cells from chelidonine with different concentrations group decreased significantly（P<0.05）,showing a certain dose-dependent relationship.The results showed that chelidonine could reverse the resistance of MCF-7/ADR cells to ADR through PI3K/AKT pathway,and the decreased expression of P-gp might be related to the activation of PI3K/AKT pathway.Due to the diverse and complex signaling pathways and mechanisms of drug regulation on tumor cells,the expression of P-gp protein was determined again after AKT phosphorylation was blocked by AKT inhibitor in this study.Whether there are other mechanisms involved in the regulation of P-gp protein expression by chelidine and the reversal of drug resistance.By using fluorescence microscopy after 24h culture of AKT inhibitor MK-2206,the fluorescence intensity of Rh123 and ADR in the cells from the blank control group and the ADR alone group was lower,while the fluorescence intensity of ADR combined with chelidonine was enhanced.Western blot was used to determine the effect of MK2206 addition on P-gp protein expression in cells,and it was found that the expression of P-gp was still significantly decreased in the chelidonine combined with ADR group（P<0.01）,which indicated that in addition to PI3K/AKT pathway,there are other pathways involved in the regulation of P-gp expression by chelidonine to reverse drug resistance.According to the speculative results of network pharmacology,the second ranked MAPK signal pathway was verified.Western blotting was used to detect the expression change of p38,p-p38,ERK,p-ERK,JNK,p-JNKprotein in MCF-7/ADR cells treated with ADR alone or chelidonine with different doses combined with ADR.The results showed that compared with the blank control group,there was no significant change in the expression level of each protein in the group treated with ADR alone（P>0.05）.The expression of p-p38 and p-ERK protein in MCF-7/ADR cells decreased significantly with the increase of chelidonine concentration（P<0.01）;meanwhile,the expression of p-JNK protein in MCF-7/ADR cells treated with chelidonine(3μmol?L^-1,6μmol?L^-1)and ADR(6μmol?L^-1)also decreased significantly（P<0.01）.The results indicate that MAPK pathway may also be one of the mechanisms by which chelidonine reverses human breast cancer resistant cells MCF-7/ADR and reduces P-gp expression.In summary,chelidonine can effectively reverse the resistance of MCF-7/ADR cells to ADR.The mechanism might be related to the downregulation effect of chelidonine on P-gp expression by inhibiting PI3K/AKT and MAPK pathway.However,whether its effect of reversing drug resistance involves other cell signaling pathways and mechanisms remains to be further investigated.