Mechanistic Evaluation and Translational Signature of Gemcitabine-induced Chemoresistance by Quantitative Phosphoproteomics Analysis with iTRAQ Labeling Mass Spectrometry

Abstract

One of the main causations of the poor prognosis of pancreatic cancer is the lack of effective chemotherapies. Gemcitabine is a widely used chemotherapeutic drug, but limited therapeutic efficacy is achieved due to chemoresistance. Recent studies demonstrated that the presence of cancer stem cells may lead to the failure of chemotherapy. Moreover, gemcitabine can promote the stemness of pancreatic cancer cells. We detected the alterations in protein phosphorylation and signaling pathways in pancreatic cancer cells after gemcitabine treatment using iTRAQ labeling LC-MS/MS, because it was featured with the advantages of strong separation ability and analysis range. A total of 232 differentially expressed phosphorylated proteins were identified in this study. Gene Ontology analysis revealed that nuclear lumen, nuclear part and organelle lumen were enriched for cell components and protein binding, poly (A) RNA binding and RNA binding were enriched for molecular function. A variety of signaling pathways were enriched based on KEGG analysis. AMPK, mTOR and PI3K/Akt pathways were verified after gemcitabine exposure. Moreover, we found there were complex interactions of phosphorylated proteins in modulating cancer stemness induced by gemcitabine exposure based on PPIs map. Our experiments may identify potential targets and strategies for sensitizing pancreatic cancer cells to gemcitabine.

Figure 1

Gemcitabine treatment promotes the stemness markers of pancreatic cancer cells. (a) The changes of CD133+ and CD24+ CSCs treated as indicated above were measured with the use of flow cytometry. The numerical values represent the percentage of positive cells. (b) After gemcitabine treated for 48 h, the Patu8988 cells were subjected to the stem cell medium according to the tumor sphere-forming assay. The results were representative of three independent experiments. (c) The expression levels of Bmi1, Nanog and Sox2 were compared with western blot analysis after 2.5 μg/mL gemcitabine treatment for 48 h. Scale bar, 200 μm. *P < 0.05; **P < 0.01.

Figure 2

Workflow of iTRAQ labeling LC-MS/MS. The workflow of LC-MS/MS to identify differentially expressed phosphorylated proteins after gemcitabine treatment in pancreatic cancer cells.

Figure 3

Differentially expressed phosphorylated proteins identified by iTRAQ after gemcitabine treatment. (a) Compared to the control group, the number of up-regulated and down-regulated phosphorylated proteins were shown in the bar graph after gemcitabine treatment. (b) Some differentially expressed phosphorylated proteins. Red stands for up-regulated proteins and green stands for down-regulated proteins.

Figure 4

GO analysis of differentially expressed phosphorylated proteins. This graph showed the first 20 items in the enrichment results of three kinds of basic categories. The horizontal coordinate was the process name, and the vertical coordinate was the percentage of the total number of proteins that were enriched.

Figure 5

Effects of targeted anticancer agents on Patu8988 cells. (a) Patu8988 cells were treated with gemcitabine for 48 h at the concentrations of 2.5 μg/mL, and the expression of phosphorylated proteins were detected by Western Blot. (b,c) The pancreatic CSCs markers CD133-PE, CD24-FITC after gemcitabine and the corresponding inhibitors/activator treatment were measured by flow cytometry. The cells were treatment with gemcitabine (2.5 μg/mL, 48 h), rapamycin (1 μM, 48 h), LY294002 (20 μM, 24 h) and A-769662(20 μM, 2 h). The numerical values represent the percentage of positive cells. (d) After inhibitors or activator treatment, the Patu8988 cells were performed with the tumor sphere-forming experiments in stem cell medium. (e,f) Dot-plots represented the apoptotic status of Patu8988 cells using Annexin V-FITC/PI method. The dot-plots in the upper right and lower right quadrant were considered as the percentage of apoptotic cells. (g) The expression levels of Bmi1, Nanog and Sox2 were compared with western blot analysis after gemcitabine and the corresponding inhibitors/activator combination treatment. The results were representative of three independent experiments. Scale bar, 200 μm. *P < 0.05; **P < 0.01.

Figure 6

Interaction of different phosphorylated proteins. Biological network among phosphorylated proteins were shown by lines, which represented the interactions.