Study On The Mechanism Of Verapamil Enhancing Chemosensitivity Of MCF-7/ADR Cells To Doxorubicin Based On Metabolomics

Background:Breast cancer is one of the most common malignant tumors in women.According to global cancer statistics for 2018,about 2.1 million new cases of breast cancer have been found,ranking second among all cancers.Chemotherapy is a common clinical method for the treatment of breast cancer,but the occurrence of drug resistance often leads to unsatisfactory chemotherapy effect.P-glycoprotein(P-gp)is one of the main causes of drug resistance.As a classical inhibitor of P-gp,verapamil can enhance the efficacy of chemotherapy drugs by inhibiting the activity of P-gp.However,the molecular mechanism of verapamil reversing chemotherapy resistance in breast cancer remains unclear.Objective:In this study,metabolomics and bioinformatics techniques were used to systematically analyze the effect and its molecular mechanism of verapamil on enhancing the chemosensitivity of breast cancer resistant cell line MCF-7/ADR to doxorubicin.Method:(1)The MTT assay was used to investigate the effect of verapamil on proliferation and drug resistance of breast cancer cells.(2)LC-MS/MS metabolomics technology was used to analyze metabolic reprogramming of endogenous small molecule metabolites in MCF-7/ADR cells and MCF-7 cells,MCF-7/ADR cells and MCF-7 cells treated with doxorubicin,MCF-7/ADR cells and MCF-7 cells treated with co-administration of doxorubicin and verapamil;the original data was preprocessed and analyzed by SIEVE2.2 software;the pattern discriminant analysis was performed by SIMCA14.1 software;the differential compounds were screened according to VIP>1 and P<0.05,and were confirmed by using Metlin and HMDB databases.MetaboAnalyst website was used to analyze the metabolic pathways of differential metabolites.(3)The GSE76540 dataset was download from the GEO database,and the dataset contained gene chip data of breast cancer resistant cell line MCF-7/ADR and corresponding parent cell line MCF-7.Empirical Bayes method was used to screen the differentially expressed genes between MCF-7/ADR and MCF-7 cells.GO and KEGG enrichment analysis of differentially expressed genes were performed.Results:(1)MTT experimental results showed that the IC50 value of doxorubicin on MCF-7/ADR cells was 50.87±1.79 ?M and the IC50 value of doxorubicin on MCF-7 cells was 0.51±0.03 ?M after doxorubicin was applied to MCF-7/ADR and MCF-7 cells for 48 h.The multiple of drug resistance of MCF-7/ADR cells was 99.75.Survival rates of the two groups of cell lines were greater than 90%after 8 ?M verapamil was applied to MCF-7/ADR and MCF-7 cells for 48 h,and 8.00 ?M verapamil had no cytotoxic effect on MCF-7/ADR and MCF-7 cells.The IC50 value of doxorubicin on MCF-7/ADR cells was 9.61 ±0.21 ?M in the united group(doxorubicin plus verapamil)and the IC50 value of doxorubicin on MCF-7/ADR cells was 50.87±1.79 ?M in the control group after administration for 48 h.The reversal fold of verapamil against MCF-7/ADR cells was 5.29.(2)Results of metabonomics showed that a total of 31 differential metabolites were screened between MCF-7 and MCF-7/ADR cells,including sphinganine,sphingosine,sphingomyelin,phosphatidylcholine,choline,L-arginine and L-proline et al.,which were mainly involved in pathways including sphingolipid metabolism,arginine and proline metabolism,and glycerophospholipid metabolism.A total of 17 differential metabolites were screened after doxorubicin was applied to MCF-7/ADR cells,including lecithin,lyso-lecithin,sphingomyelin and sphinganine et al.,which were mainly involved in pathways including sphingolipid metabolism,glycerophospholipid metabolism and phenylalanine metabolism.A total of 18 differential metabolites were screened after doxorubicin was applied to MCF-7 cells,including sphingosine,sphinganine,sphingomyelin and L-phenylalanine et al.,which were mainly involved in pathways including sphingolipid metabolism and phenylalanine metabolism.A total of 28 differential metabolites were screened after doxorubicin combined with verapamil was applied to MCF-7/ADR cells,including sphingomyelin,sphingosine,sphinganine,phosphatidylcholine,choline,lysolecithin et al.,which were mainly involved in pathways including sphingolipid metabolism and glycerophospholipid metabolism.A total of 26 differential metabolites were screened after doxorubicin combined with verapamil was applied to MCF-7 cells,including sphingosine,sphinganine,lecithin,spermidine and L-glutamine et al.,which were mainly involved in pathways including aminoacyl-tRNA biosynthesis,arginine and proline metabolism,alanine,aspartate and glutamate metabolism.(3)Results of Bioinformatics analysis showed 2898 differentially expressed genes between MCF-7/ADR and MCF-7 cells were screened.GO enrichment analysis results indicated that the products of differential genes were mainly located in cell membranes and golgi apparatus,and involved in biological processes including regulation of cell proliferation.The results of KEGG enrichment analysis revealed that the differential genes between MCF-7/ADR and MCF-7 cells were mainly involved in pathways including sphingolipid metabolism.Conclusion:(1)The mechanism of drug resistance in MCF-7/ADR cells may be related to pathways including sphingolipid metabolism,glycerophospholipid metabolism,arginine and proline metabolism.(2)Verapamil may affect sphingolipid metabolism and glycerophospholipid metabolism through increasing the content of different compounds including sphinganine,sphingomyelin,lecithin,lysophosphatidylcholine,and choline,which could affect the lipid environment of P-gp,inhibit transport activity of P-gp,reduce the efflux of drugs,and enhance the sensitivity of MCF-7/ADR cells to doxorubicin.