Establish cisplatin-resistant gastric cancer cell line MGC-803/cisplatin and reveal its mechanism

Abstract

Objective To establish a cisplatin-resistant gastric cancer cell line MGC-803/cisplatin (DDP) and to explore its drug resistance mechanism. Methods The cisplatin was used to induce the drug resistance of MGC-803 cells. The half maximal inhibitory concentration (IC50) of the drug-resistant strains was detected by the cell counting kit-8 (CCK-8) method. The expression of chloride intracellular channel 1 (CLIC1) was detected by Western blotting analyses. The wild-type CLIC1 plasmid and the short hairpin RNA (shRNA) plasmid targeting CLIC1 were constructed. After Lipo3000 was transfected into gastric cancer cells, the IC50 of gastric cancer cells to cisplatin was detected, and then the intracellular Cl-concentration was detected by MQAE fluorescent probe. Results The cisplatin-resistant cell line MGC-803/DDP was successfully induced with an IC50 of (7.02±0.13) mg/L, while the IC50 of MGC803 was (1.29±0.09) mg/L, (t=36.090, P<0.05). The CLIC1 protein of MGC803/DDP was up-regulated by (2.27±0.15) times relative to MGC803 (t=7.841, P<0.05). After silencing the CLIC1 gene of MGC803/DDP, the IC50 of the CON, CN, and KD groups were (6.96±0.09), (6.93±0.15) and (3.02±0.20) mg/L, respectively, which was significantly decreased in the KD group (F=0.209, P<0.05). After overexpression of the CLIC1 gene in MGC803 cells, the IC50 of the CON, CN, and OE groups were (1.35±0.07), (1.25±0.07) and (4.77±0.12) mg/L, respectively, and the OE group was significantly elevated (F=0.508, P<0.05). The concentration of chloride ion was detected by MQAE. The relative fluorescence intensity of MGC-803, MGC-803/OE, MGC-803/DDP and MGC-803/DDP-KD were (1.02±0.03), (0.61±0.02), (0.67±0.01) and (1.39±0.02), respectively. The concentration of Cl- in MGC-803/OE cells was higher than that in MGC-803 (t=10.800, P<0.05), while the concentration of Cl- in MGC-803/DDP was higher than that in MGC-803 and MGC-803/DDP-KD, and the concentration of Cl- in KD group was the lowest (F=229, P<0.05). Conclusion The CLIC1 gene can induce the resistance of gastric cancer cell line MGC-803 to cisplatin in vitro. The mechanism may be related to the up-regulation of CLIC1 expression, which leads to an increase of intracellular chloride ion concentration. Key words: Gastric cancer; Chloride intracellular channel 1; Cisplatin; Drug resistance