Proteomic Profiling of Chemotherapy Responses in FOLFOX-Resistant Colorectal Cancer Cells

Abstract

Chemoresistance mechanisms of colorectal cancer remain largely elusive. We aim to compare the difference of chemotherapy responses between FOLFOX-resistant and wild-type colorectal cancer cells by proteomic profiling to suggest novel treatment targets. FOLFOX-resistant colorectal cancer cells DLD1-R and HCT116-R were developed by chronic exposure to progressive FOLFOX doses. Proteomic profiling of FOLFOX-resistant and wild-type cells under FOLFOX exposure were conducted by mass-spectrometry-based protein-analysis technology. Verification of selected KEGG pathways was conducted by Western blot. DLD1-R had significantly higher FOLFOX-chemoresistance (10.81 times) than its wild-type counterpart. A total of 309 and 90 differentially expressed proteins were identified in DLD1-R and HCT116-R, respectively. In terms of gene ontology molecular function, RNA binding and cadherin binding ranked first for DLD1 and HCT116 groups, respectively. For gene set enrichment analysis, ribosome pathway and DNA replication were significantly up-regulated and down-regulated in DLD1-R, respectively. The most significantly up-regulated pathway in HCT116-R was regulation of the actin cytoskeleton. Up-regulations in the ribosome pathway (DLD1-R) and actin cytoskeleton (HCT116-R) were verified by Western blot. There were several significantly altered signaling pathways in FOLFOX-resistant colorectal cancer cells under FOLFOX with notable up-regulations in the ribosomal process and actin cytoskeleton.

Keywords: FOLFOX; chemoresistance; colorectal cancer; cytoskeleton; proteomic profiling; ribosome.

Figure A1

Heat maps of selected KEGG pathways in GSEA analysis. For DLD1-R, significant up-regulation of ribosome (a) and down-regulation of DNA replication (b) were found. While for HCT116-R, regulation of actin cytoskeleton (c) was significantly up-regulated in HCT116-R. N = 3–4.

Figure A1

Heat maps of selected KEGG pathways in GSEA analysis. For DLD1-R, significant up-regulation of ribosome (a) and down-regulation of DNA replication (b) were found. While for HCT116-R, regulation of actin cytoskeleton (c) was significantly up-regulated in HCT116-R. N = 3–4.

Figure 1

CCK8 test for the verification of chemoresistance. (a) DLD1-R had significantly higher chemoresistance than DLD1; (b) HCT116-R did not have significantly higher chemoresistance than HCT116. FOLFOX has a 5-FU:LEU:OXA ratio of 10:1:1. Data in mean ± SD. N = 3–4, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

Overall view of proteomics data. The volcano plots of the proteomics data of (a) DLD1-R vs. DLD1 and (b) HCT116-R vs. HCT116 with red and blue points showing the up-regulated and down-regulated DEPs, respectively. Selected DEPs were marked. The cluster heat maps of DEPs of (c) DLD1-R vs. DLD1 and (d) HCT116-R vs. HCT116. N = 3–4.

Figure 2

Overall view of proteomics data. The volcano plots of the proteomics data of (a) DLD1-R vs. DLD1 and (b) HCT116-R vs. HCT116 with red and blue points showing the up-regulated and down-regulated DEPs, respectively. Selected DEPs were marked. The cluster heat maps of DEPs of (c) DLD1-R vs. DLD1 and (d) HCT116-R vs. HCT116. N = 3–4.

Figure 3

GO analysis of DEPs. GO analysis of biological process, molecular function and cellular component were conducted for the DEPs in DLD1 (a,c,e) and HCT116 (b,d,f) groups. The graphs show the top 10 GO terms according to p value with the more significant terms in lighter color. N = 3–4.

Figure 4

Overview of GSEA analysis. GSEA analysis of GO molecular function for DLD1 (a) and HCT116 (b) groups showed 9 and 7 processes were significantly dysregulated. For the KEGG pathway, 3 and 13 pathways were significantly dysregulated in DLD1-R (c) and HCT116-R (d), respectively. N = 3–4.

Figure 5

Enrichment plots of selected KEGG pathways in GSEA analysis. For DLD1-R, significant up-regulation of ribosome (a) and down-regulation of DNA replication (b) were found. For HCT116-R, regulation of actin cytoskeleton was significantly up-regulated (c). N = 3–4.

Figure 6

Western blot verification of the proteomics results. Key ribosome markers RPL26 (a) and RPS3 (b) were significantly up-regulated in DLD1-R. No significant alteration was found for the DNA replication marker MCM4 for DLD1-R (c). Key actin-skeleton markers RAC (d) and p-RAC (e) had up-regulation trends in HCT116-R. Data in mean ± SEM, N = 3–4, * p < 0.05, ** p < 0.01.