Objective The aim of the current study was to establish an oxaliplatin-resistant hepatoma cell line(HepG2/OXA) and investigate the potential mechanisms of its drug resistance.Methods The hepatoma cell subline, HepG2/O...Objective The aim of the current study was to establish an oxaliplatin-resistant hepatoma cell line(HepG2/OXA) and investigate the potential mechanisms of its drug resistance.Methods The hepatoma cell subline, HepG2/OXA, resistant to oxaliplatin(OXA), was established from a parent cell line HepG2, by stepwise exposure to gradually increasing concentrations of OXA over a half-year period. Chemosenstivity of the cytotoxic drugs, OXA, cisplatin(CDDP), adriamycin(ADM), and 5-fuorouracil(5-FU), was determined in HepG2 and HepG2/OXA cells, by the Cell counting kit-8(CCK8) assay. Cell cycle distribution of HepG2 and HepG2/OXA cells was analyzed by Flow cytometry(FCM). The expression levels of several drug resistance-related proteins, such as P-glycoprotein(P-gp), multidrug resistant protein 1(MRP1), and excision repair-cross complementing 1(ERCC1) protein in the two cell lines were tested by the western blot assay.Results The IC50 of OXA in HepG2/OXA and HepG2 were 136.84 μmol/L and 23.86 μmol/L, respectively. The resistance index(RI) was 5.34. HepG2 was also demonstrated to be cross-resistant to other antitumor agents, such as 5-FU, ADM, and CDDP. The percentage of HepG2/OXA cells in the S phase was significantly decreased compared to HepG2 cells(25.58% ± 2.36% vs 14.37% ± 2.54%, P < 0.05), while the percentage of cells in the G0/G1 and G2/M phases showed no statistical difference(respectively 55.29% ± 4.98% vs 56.73% ± 4.56%, P > 0.05, and 24.63% ± 4.81% vs 28.26% ± 3.82%, P > 0.05). The ERCC1 was found to be over expressed in HepG2/OXA cells, while there was no difference in the expressions of P-gp and MRP1 between the multiple drug resistance(MDR) phenotype cell line and its parental cell line.Conclusion HepG2/OXA showed an MDR ability; the over expression of ERCC1 might be associated with the platinum resistance of the cells, but P-gp and MRP1 are not.展开更多
AIM: To investigate the molecular mechanisms underlying the reversal effect of emodin on platinum resistance in hepatocellular carcinoma. METHODS: After the addition of 10 μmol/L emodin to HepG2/oxaliplatin (OXA) cel...AIM: To investigate the molecular mechanisms underlying the reversal effect of emodin on platinum resistance in hepatocellular carcinoma. METHODS: After the addition of 10 μmol/L emodin to HepG2/oxaliplatin (OXA) cells, the inhibition rate (IR), 50% inhibitory concentration (IC 50 ) and reversal index (IC 50 in experimental group/IC 50 in control group) were calculated. For HepG2, HepG2/OXA, HepG2/OXA/T, each cell line was divided into a control group, OXA group, OXA + fibroblast growth factor 7 (FGF7) group and OXA + emodin group, and the final concentrations of FGF7, emodin and OXA in each group were 5 ng/mL, 10 μg/mL and 10 μmol/L, respectively. Single-cell gel electrophoresis was conducted to detect DNA damage, and the fibroblast growth factor receptor 2 (FGFR2), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and excision repair cross-complementing gene 1 (ERCC1) protein expression levels in each group were examined by Western blotting. RESULTS: Compared with the IC50 of 120.78 μmol/L in HepG2/OXA cells, the IC 50 decreased to 39.65 μmol/L after treatment with 10 μmol/L emodin; thus, the reversal index was 3.05. Compared with the control group, the tail length and Olive tail length in the OXA group, OXA + FGF7 group and OXA + emodin group were significantly increased, and the differences were statistically significant (P < 0.01). The tail length and Olive tail length were lower in the OXA + FGF7 group than in the OXA group, and this difference was also statistically significant. Compared with the OXA + FGF7 group, the tail extent, the Olive tail moment and the percentage of tail DNA were significantly increased in the OXA + emodin group, and these differences were statistically significant (P < 0.01). In comparison with its parental cell line HepG2, the HepG2/OXA cells demonstrated significantly increased FGFR2, p-ERK1/2 and ERCC1 expression levels, whereas the expression of all three molecules was significantly inhibited in HepG2/ OXA/T cells, in which FGFR2 was silenced by FGFR2 shRNA. In the examined HepG2 cells, the FGFR2, p-ERK1/2 and ERCC1 expression levels demonstrated increasing trends in the OXA group and OXA + FGF7 group. Compared with the OXA group and OXA + FGF7 group, the FGFR2, p-ERK1/2, and ERCC1 expression levels were significantly lower in the OXA + emodin group, and these differences were statistically significant. In the HepG2/OXA/T cell line that was transfected with FGFR2 shRNA, the FGFR2, p-ERK1/2 and ERCC1 expression levels were significantly inhibited, but there were no significant differences in these expression levels among the OXA, OXA + FGF7 and OXA + emodin groups. CONCLUSION: Emodin markedly reversed OXA resistance by enhancing OXA DNA damage in HepG2/OXA cells, and the molecular mechanism was related to the inhibitory effect on ERCC1 expression being mediated by the FGFR2/ERK1/2 signaling pathway.展开更多
基金Supported by grants from the National Natural Sciences Foundation of China(No.81001067)Ministry of Science and Technology International Cooperation Project(No.S2010GR0991)Astrazeneca Special Research Foundation for Targeted Therapy of Wu Jieping Medical Foundation(No.320.6700.09068)
文摘Objective The aim of the current study was to establish an oxaliplatin-resistant hepatoma cell line(HepG2/OXA) and investigate the potential mechanisms of its drug resistance.Methods The hepatoma cell subline, HepG2/OXA, resistant to oxaliplatin(OXA), was established from a parent cell line HepG2, by stepwise exposure to gradually increasing concentrations of OXA over a half-year period. Chemosenstivity of the cytotoxic drugs, OXA, cisplatin(CDDP), adriamycin(ADM), and 5-fuorouracil(5-FU), was determined in HepG2 and HepG2/OXA cells, by the Cell counting kit-8(CCK8) assay. Cell cycle distribution of HepG2 and HepG2/OXA cells was analyzed by Flow cytometry(FCM). The expression levels of several drug resistance-related proteins, such as P-glycoprotein(P-gp), multidrug resistant protein 1(MRP1), and excision repair-cross complementing 1(ERCC1) protein in the two cell lines were tested by the western blot assay.Results The IC50 of OXA in HepG2/OXA and HepG2 were 136.84 μmol/L and 23.86 μmol/L, respectively. The resistance index(RI) was 5.34. HepG2 was also demonstrated to be cross-resistant to other antitumor agents, such as 5-FU, ADM, and CDDP. The percentage of HepG2/OXA cells in the S phase was significantly decreased compared to HepG2 cells(25.58% ± 2.36% vs 14.37% ± 2.54%, P < 0.05), while the percentage of cells in the G0/G1 and G2/M phases showed no statistical difference(respectively 55.29% ± 4.98% vs 56.73% ± 4.56%, P > 0.05, and 24.63% ± 4.81% vs 28.26% ± 3.82%, P > 0.05). The ERCC1 was found to be over expressed in HepG2/OXA cells, while there was no difference in the expressions of P-gp and MRP1 between the multiple drug resistance(MDR) phenotype cell line and its parental cell line.Conclusion HepG2/OXA showed an MDR ability; the over expression of ERCC1 might be associated with the platinum resistance of the cells, but P-gp and MRP1 are not.
基金Supported by National Natural Sciences Foundation of China,No. 81001067the Ministry of Science and Technology International Cooperation Project, No. 2010DFA31870the AstraZeneca Special Research Foundation for Targeted Therapy of the Wu Jieping Medical Foundation, No. 320.6700.09068
文摘AIM: To investigate the molecular mechanisms underlying the reversal effect of emodin on platinum resistance in hepatocellular carcinoma. METHODS: After the addition of 10 μmol/L emodin to HepG2/oxaliplatin (OXA) cells, the inhibition rate (IR), 50% inhibitory concentration (IC 50 ) and reversal index (IC 50 in experimental group/IC 50 in control group) were calculated. For HepG2, HepG2/OXA, HepG2/OXA/T, each cell line was divided into a control group, OXA group, OXA + fibroblast growth factor 7 (FGF7) group and OXA + emodin group, and the final concentrations of FGF7, emodin and OXA in each group were 5 ng/mL, 10 μg/mL and 10 μmol/L, respectively. Single-cell gel electrophoresis was conducted to detect DNA damage, and the fibroblast growth factor receptor 2 (FGFR2), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and excision repair cross-complementing gene 1 (ERCC1) protein expression levels in each group were examined by Western blotting. RESULTS: Compared with the IC50 of 120.78 μmol/L in HepG2/OXA cells, the IC 50 decreased to 39.65 μmol/L after treatment with 10 μmol/L emodin; thus, the reversal index was 3.05. Compared with the control group, the tail length and Olive tail length in the OXA group, OXA + FGF7 group and OXA + emodin group were significantly increased, and the differences were statistically significant (P < 0.01). The tail length and Olive tail length were lower in the OXA + FGF7 group than in the OXA group, and this difference was also statistically significant. Compared with the OXA + FGF7 group, the tail extent, the Olive tail moment and the percentage of tail DNA were significantly increased in the OXA + emodin group, and these differences were statistically significant (P < 0.01). In comparison with its parental cell line HepG2, the HepG2/OXA cells demonstrated significantly increased FGFR2, p-ERK1/2 and ERCC1 expression levels, whereas the expression of all three molecules was significantly inhibited in HepG2/ OXA/T cells, in which FGFR2 was silenced by FGFR2 shRNA. In the examined HepG2 cells, the FGFR2, p-ERK1/2 and ERCC1 expression levels demonstrated increasing trends in the OXA group and OXA + FGF7 group. Compared with the OXA group and OXA + FGF7 group, the FGFR2, p-ERK1/2, and ERCC1 expression levels were significantly lower in the OXA + emodin group, and these differences were statistically significant. In the HepG2/OXA/T cell line that was transfected with FGFR2 shRNA, the FGFR2, p-ERK1/2 and ERCC1 expression levels were significantly inhibited, but there were no significant differences in these expression levels among the OXA, OXA + FGF7 and OXA + emodin groups. CONCLUSION: Emodin markedly reversed OXA resistance by enhancing OXA DNA damage in HepG2/OXA cells, and the molecular mechanism was related to the inhibitory effect on ERCC1 expression being mediated by the FGFR2/ERK1/2 signaling pathway.