RNF138 confers cisplatin resistance in gastric cancer cells via activating Chk1 signaling pathway

Abstract

Chemotherapy resistance represents a major issue associated with gastric cancer (GC) treatment, and arises through multiple mechanisms, including modulation of the cell-cycle check point. Several ubiquitin kinases, including RING finger protein 138 (RNF138), have been reported to mediate the G2/M phase arrest. In this study, we investigated the role of RNF138 in the development of cisplatin resistance of two GC cell lines. We show that RNF138 levels are higher in cisplatin-resistant cell lines, compared with cisplatin-sensitive cells, and RNF138 expression was elevated during drug withdrawal following the cisplatin treatment. Using gene overexpression and silencing, we analyzed the impact of altering RNF138 level on GC cell viability, apoptosis, and cell cycle phenotypes in two isogenic cisplatin-sensitive and resistant cell lines. We show that RNF138 overexpression increased GC cell viability, decreased apoptosis and delayed cell cycle progression in the cisplatin-sensitive GC cells. Conversely, RNF138 silencing produced opposite phenotypes in the cisplatin-resistant cells. Moreover, RNF138-dependent phosphorylation of Chk1 was seen in GC cells, indicating a novel connection between cisplatin-induced DNA damage and apoptosis. Collectively, these data suggest that RNF138 modulates the cisplatin resistance in the GC cells, thus serving as a potential drug target to challenge chemotherapy failure. In addition, RNF138 can also be used as a marker to monitor the development of cisplatin resistance in GC treatment.

Keywords: Chk1; RNF138; apoptosis; cell cycle; cisplatin resistance; gastric cancer; viability.

Figure 1.

RNF138 is upregulated during acquiring cisplatin resistance in GC cells. (A and B) Cluster heatmap of RNF138 mRNA expression profiles were detected with real-time qPCR using 0.5 μg/ml in AGS and 0.25 μg/ml in SGC7901 cells for the indicated cisplatin treatment time (A) and detected with the indicated cisplatin doses for continuous 24 h treatment or 24h after replacement in AGS and SGC7901 GC cells (B). Cisplatin treatment represented as cisplatin continuous stress for indicated time. Withdrawal represented as cisplatin continuous stress for 24 hours, and then replaced to normal medium for indicated time. β-actin served as loading control. (C) The expression of RNF138 was determined in AGS and SGC7901 cells with indicated cisplatin treatment time by immunoblotting analysis. α-Tubulin was used as the loading control. (D and E) The expression of RNF138 was determined in AGS and AGS/DDP cell lines by immunoblotting analysis (D) and real-time qPCR analysis (E). α-Tubulin and β-actin were used as loading controls, respectively. (F and G) The expression of RNF138 was determined in SGC7901 and SGC7901/DDP cell lines by immunoblotting analysis (F) and real-time qPCR analysis (G). α-Tubulin and β-actin were used as loading controls, respectively. Graphs show the mean of three experiments, and error bars represent SD. Statistically significant differences are shown by *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 2.

RNF138 level determines the sensitivity of GC cells to cisplatin. (A, B) IC50 values were calculated in AGS (A) and SGC7901 (B) cells transfected with the empty vector or RNF138 using cytotoxicity assay. (C) Colony formation was detected in AGS and SGC7901 cells transfected with RNF138 compared with empty control vector. The normalized ratio of clonogenic assay was shown in the histogram. (D, E) IC50 values were calculated in AGS/DDP (D) and SGC7901/DDP (E) cells transfected with the control or siRNF138 using cytotoxicity assay. (F) Colony formation was detected in AGS/DDP and SGC7901/DDP cells transfected with the control or siRNF138. The normalized ratio of clonogenic assay was shown in the histogram and error bars represent SD. Statistically significant differences are shown by *, P < 0.05.

Figure 3.

RNF138 level determines cisplatin-induced cell apoptosis. (A, B) Percentage of apoptosis was detected in AGS (A) and SGC7901 (B) cells transfected with control vector or RNF138 with or without cisplatin treatment. Early apoptotic cells can be seen in the bottom right quadrant, late apoptotic cells can be seen in the top right quadrant, and the sum is the total apoptosis. The normalized ratio of apoptosis assay is shown in the histogram. (C, D) Cell apoptosis percentages of AGS/DDP and SGC7901/DDP cells transfected with control or siRNF138 combined with cisplatin treatment were detected. The normalized ratio of apoptosis assay was shown in the histogram and error bars represent SD. Statistically significant differences are shown by *, P < 0.05; **, P < 0.01.

Figure 4.

RNF138 participates in the cisplatin-induced G2/M cell cycle arrest. (A, B) The cell cycle distribution in AGS (A) and SGC7901 (B) cells transfected with control vector or RNF138 with cisplatin treatment was analyzed by flow cytometry. Normalized ratio of the cell cycle assay is shown in the histogram. (C, D) The cell cycle distribution of AGS/DDP (C) and SGC7901/DDP (D) cells transfected with control or siRNF138 was detected. Normalized ratio of the cell cycle assay was shown in the histogram and error bars represent SD. Statistically significant differences are shown by *, P < 0.05.

Figure 5.

Cisplatin induced the phosphorylation of Chk1 and Chk1 phosphorylation is RNF138 dependent. (A, B) Immunoblotting analysis of the RNF138 and phosphorylation of Chk1 signaling pathway level in AGS and SGC7901 cells treated with cisplatin. α-Tubulin was used as the loading control. (C, D) Immunoblotting analysis of the RNF138 and Chk1 signaling pathway level in isogenic AGS and AGS/DDP (C) and SGC7901 and SGC7901/DDP (D) cell lines treated with cisplatin. GAPDH was used as the loading control. (E) Chk1 signaling pathway was detected in AGS and SGC7901 stable cell lines treated with cisplatin by immunoblotting detection. α-Tubulin was used as the loading control. (F) Chk1 signaling pathway was detected in AGS/DDP and SGC7901/DDP cells transfected with siRNA treated with cisplatin by immunoblotting detection. α-Tubulin was used as the loading control. Data were normalized to control-treated cells. The numbers below the panels represent the normalized protein expression levels.

Figure 6.

The stability of RNF138 is increased in cisplatin-resistant GC cells. (A, B) Half-life of RNF138 was detected in isogenic AGS, AGS/DDP and SGC7901, SGC7901/DDP GC cell lines by CHX assay. RNF138 and α-Tubulin signals in the immunoblotting detection were quantified by densitometry. The ratios of RNF138/α-Tubulin were normalized to time zero and plotted against time. (C) Schematic model for the mechanism by which RNF138 contributes to cisplatin resistance by facilitating activation of the Chk1 pathway in GC cells.