Combination Of Hydralazine And Valproic Acid Inhibits Tamoxifen-resistant Breast Cancer:Effectiveness And Mechanism

Objective:To investigate the effect of combination of hydralazine and valproic acid in tamoxifen-resistant?TAM-resistant?breast cancer.Methods:WST-1 proliferation assay,scratch assay,cell transwell assay,ELISA apoptosis assay,and flow cytometry assay were used in TAM-resistant breast cancer cell line—LCC2 cells to investigate the effects of combination of hydralazine and valproic acid on the proliferation,migration,invasion,and apoptosis.Infinium MethylationEPIC BeadChip and Illumina Hiseq Xten technologies were used to collect the data of global-genomic methylation and gene expression.Variation analysis was performed using EXCEL and IBM SPSS 24.0 statistical software based on the raw data achieved.The chromosome distribution figure for differentially methylated sites,cluster analysis,GO analysis,and KEGG analysis were performed using R3.5.2.Correlation between DNA methylation and gene expression was performed using chi-square test.Survival analysis was performed using Kaplan-Meier plotter.The statistical significance was defined by P<0.05.Results:1.Effect of combination treatment with hydralazine and valproic acid on the function of TAM-resistant breast cancer cellsAverage value of survival LCC2 cells?OD?after treated with 5?M hydralazine and 0.5mM valproic acid for 5 days was 0.483±0.005,with an inhibition rate of 40%.The average OD value of LCC2 cells treated with 10?M hydralazine and 1mM valproic acid was 0.537±0.014,with an inhibition rate of 33%.The ability of inhibiting proliferation with 5?M hydralazine and 0.5mM valproic acid was significantly greater than that with 10?M hydralazine and 1mM valproic acid?P=0.001?.In the cell scratch assay,the free area(627290.667±83950.209 pixel^{^2})in control group was significantly smaller than that in hydralazine and valproic acid group(874265.667±51858.378 pixel^{^2})at 24 hour?P=0.012?.In cell transwell assay,the number of successfully invaded cells in control group was 2.6 times higher than that in hydralazine and valproic acid group?P<0.001?.In the ELISA apoptosis assay,the number of apoptotic cells in hydralazine and valproic acid group was higher?2.4 times?than that in control group?P=0.002?.In flow cytometry assay,although no statistical significance was found?P>0.05?,the number of apoptotic cells in hydralazine and valproic acid group was as 1.5 times as that in control group.In addition,we found that average OD value of the LCC2 cells treated with combination of hydralazine,valproic acid and TAM was 0.345±0.014,with a higher inhibition rate?about 57%?than the control group?0.807±0.046,P<0.001?as well as the TAM treatment group?0.776±0.042,P<0.001?or combination treatment of hydralazine and valproic acid group?0.565±0.016,P<0.001?.2.Effect of combination treatment with hydralazine and valproic acid on global DNA methylation in TAM-resistant breast cancer cellsWe identified 28573 differentially methylated sites.Among them,methylation level in 1759 sites were increased;however,methylation level in 26814 sites were decreased.A total of 9969 differentially methylated genes?DMGs?were identified?9148 of them were hypomethylation,and 821 genes were hypermethylation?.A total of 5329DMGs were found on proximal promotor regions?TSS200,TSS1500,and 5'UTR?.GO analysis of 9969 DMGs showed that these genes were related to negative regulation of phosphorylation,regulation of cell-cell adhesion,and regulation of hormone secretion.KEGG analysis showed that DMGs were associated with MAPK signaling pathway,estrogen regulation signaling pathway,and osteoclast differentiation.3.Effect of combination treatment with hydralazine and valproic acid on gene expression in TAM-resistant breast cancer cellsWe identified that the expression of 4386 genes were differentially regulated by hydralazine and valproic acid combination treatment?the expression level of 2091genes were upregulated and 2295 genes were downregulated?.GO analysis of the 4386 genes showed that they were involved in adherens junction,negative regulation of intracellular signal transduction,and hormonal regulation.Moreover,KEGG analysis showed that these genes were associated with FoxO signaling pathway,p53 signaling pathway,and apoptosis.4.Correlation analysis between DNA methylation and gene expressionCombining the DNA methylation data with the gene expression data,we identified1616 differentially methylated and expressed genes?DMEGs?.Among them,69 from117 hypermethylated genes were downregulated expression level,and 776 from 1499hypomethylated genes were upregulated expression level??²=5.01,P=0.03?.In our study,853 DMEGs located in the proximal promoter region.Among them,the expression level 42 from 67 hypermethylated genes was downregulated,and the expression level 398 from 786 hypomethylated genes was upregulated??²=4.38,P=0.04?.A total of 776 hypomethylated genes with upregulated expression level were assigned to GO analysis,we found that these genes were associated with adherens junction,negative regulation of phosphorylation,and negative regulation of intracellular signaling.In addition,KEGG analysis showed that these genes related to MAPK signaling pathway,osteoclast differentiation,and TNF signaling pathway.5.Survival analysis of five years on hypomethylated genes with upregulated expression level in ER-positive breast cancer patients treated with TAMWe selected the five genes?TRIM29,TP53INP1,ABAT,SOCS3,and PTPRR?to perform the survival analysis.Patients with high expression level of TRIM29 showed better distance metastasis free survival?DMFS??HR=0.49,95%CI:0.29–0.85,P=0.009?;patients with high expression level of ABAT showed better recurrence-free survival?RFS??HR=0.67,95%CI:0.47–0.97,P=0.033?;patients with high expression level of SOCS3?HR=0.32,95%CI:0.13–0.8,P=0.011?showed better overall survival?OS?.Expression level of TP53INP1 and PTPRR was not associated with prognosis of ER-positive breast cancer patients treated with TAM.Conclusion:1.Combination treatment with hydralazine and valproic acid inhibits the cell proliferation,migration,invasion,induced the cell apoptosis,and sensitive TAM-resistant cells to TAM.2.Combination treatment with hydralazine and valproic acid contributes to the global DNA hypomethylation in TAM-resistant breast cancer cells.DMGs in the proximal promoter region account for more than half of the total DMGs.There is a correlation between the level of methylation and gene expression after the combination treatment with hydralazine and valproate acid.3.After combination treatment with hydralazine and valproic acid,hypomethylated genes with upregulated expression level were associated with adherens junction,negative regulation of phosphorylation,negative regulation of intracellular signaling,MAPK signaling pathway,osteoclast differentiation,and TNF signaling pathway.4.The higher expression level of TRIM29,ABAT,and SOCS3 is associated with better prognosis in ER-positive breast cancer patients treated with TAM.