Our research also demonstrated upregulation of several cytochrome P450 genes (CYPs) viz. genes were validated by qRT-PCR. Analysis by the i-pathway revealed membrane transporters including solute carrier proteins, ATP-binding cassette transporters, and drug metabolizing enzymes as the most prominent genes dysregulated in resistant cell lines. RNA-Seq data demonstrated predominance of solute carrier genes during metabolic reprogramming and A-385358 development of drug resistance. Upregulation of these genes were associated with higher uptake of lactic/citric acid and lower glucose intake in resistant cells. Our data suggest the predominance of solute carrier genes during metabolic reprogramming of prostate cancer cells in an androgen-deprived environment, thus signifying them as potentially attractive therapeutic targets. values less than 0.05 were considered as significant. The qRT-PCR data were analyzed using the two tailed unpaired value < 0.0005 and FDR< 0.05. To further visualize the DEGs, a volcano plot was generated displaying the relationship between the magnitude of gene expression change (log2 fold-change; X-axis) and statistical significance of this change [?log10 were in agreement with the expression of the RNA-Seq data. Higher gene expression of (13.9 fold), followed by (13.8), (12.8), (9.8), (6.17) (6.17), (5.16), (4.12), and (3.94) in their expression were noted in the enzalutamide resistant cells compared to the LNCaP parental cells (Figure 2A). The fold change gene expression of these genes in C4-2B cells including (13.0 fold), followed by (10.12), (5.65), (5.6), (5.4), (5.15), (4.77), (4.43), and (2.12) in their expression were noted in C4-2B enzalutamide resistant cells compared to the parental cells (Figure 2B). Open in a separate window Figure 2 Real time PCR validation of genes A-385358 in (A) LNCaP cells. Bars represent mRNA expression analysis of genes differentially expressed between LNCaP enzalutamide-resistant cells compared to the parental cell line. (B) C4-2B cells. Bars represent mRNA expression analysis of genes differentially expressed between C4-2B enzalutamide-resistant cells compared to the parental cell line. The qRT-PCR data were analyzed using REST? (Relative Expression Software Tool), Qiagen, USA. Bar represents the standard error mean (SEM) for three biological and three technical replicates. ** < 0.001, *** < 0.0001 Control versus enzalutamide resistant cells. 3.3. Pathway Enrichment Analysis and Mining of Disease Association We next performed signaling pathway analysis using iPathway on differentially expressed genes to investigate their biological relevance and pathway association. To achieve this, the data were separately analyzed with upregulated (fold change > 2) and downregulated (fold change < ?2) DEGs. Analysis of iPathway showed overrepresented pathways associated with DEGs that included focal adhesion, bile secretion, Hippo signaling, PI3K-Akt signaling, cytokine-cytokine receptor interaction, axon guidance, pathways in cancer, amino acid A-385358 biosynthesis pathway, metabolic pathway, and alanine glutamate pathway in LNCaP cells (Figure 3A). A-385358 In C4-2B cells, DEGs include neuroactive ligand receptor interaction, insulin and bile secretion, cAMP signaling, and cell adhesion pathways (Figure 3B). Signaling pathway associated with cellular metabolism including alterations in amino acid, bile acid biosynthesis, salts, and glucose were noted to be commonly overrepresented in both LNCaP and C4-2B enzalutamide resistant cell lines compared to their parental counterparts. Open in a separate window Figure 3 Pathway enrichment analysis of disease association in (A) LNCaP enzalutamide resistant cells and (B) C4-2B enzalutamide resistant cells compared to their parental counterparts. Overrepresented signaling pathways were analyzed by iPathway. Red color dots represent the pathway after FDR correction and yellow dots represent top hit pathways such as cytokine-cytokine receptor interaction in LNCaP cells and metabolic pathway in C4-2B cells. The colored dots denote the overrepresented pathways with corrected value (FDR < 0.05) (Left panel). The circular plot displays significantly enriched pathways associated with the disease. Both LNCaP and C4-2B enzalutamide resistant cells exhibited DEGs linked with IL7 metabolic disorder. The circular plot of metabolic disease represent the DEGs genes overlaid with International Classification of Diseases, Tenth Revision (ICD-10). The metabolic disease display color magenta is the most significant and cyan is less significant (right panel). Next, we analyzed the DEGs and their disease association through the circular plot. The plot displays significantly enriched pathways associated with the disease. Both LNCaP and C4-2B enzalutamide resistant cells exhibited DEGs that were linked mainly with metabolic disorder (Figure 3A,B, right panel). Further clustering showed disease association with disorder of lipids, carbohydrates, fatty acid, and metabolism of branched chain amino-acids, fatty acids, and glycoproteins (Figure 3A,B, right panel). The data showed that the ratio of the number of genes associated with the metabolic disorder was significantly high compared to other diseases when corrected using FDR. 3.4. Gene Set Enrichment Analysis (GSEA) We analyzed the DEGs with gene set enrichment analysis (GSEA) v3.0 (http://software.broadinstitute.org/gsea/downloads.jsp) to identify genes, their expression,.