Effect and mechanism of ubiquitin-specific processing peptidase 10 on gemcitabine resistance of pancreatic cancer

Abstract

Objective To investigate the effect and mechanism of ubiquitin-specific processing peptidase 10 (USP10) on gemcitabine resistance of pancreatic cancer. Methods (1) The 50% inhibiting concentration (IC50) of USP10 in the PANC-1, BxPC-3 and SW1990 cell lines of pancreatic cancer was detected by CCK8 assay. (2) The expression of USP10 in the BxPC-3, PANC-1, SW1990 and SW1990-GEM cell lines of pancreatic cancer was detected by Western blot. (3) Small interfering RNA (siRNA) of SW1900 cell lines was conducted, siRNA and USP10siRNA were transected and then were respectively allocated into the control group and USP10 group. (4) IC50 of gemcitabine in the cancer cell lines between the 2 groups was detected by CCK8 assay. (5) Colony-forming unit assay: number of cloned cells was counted and colony-forming efficiency (CFE) was calculated. (6) Cells in the 2 groups were interfered with 0.3 μg/mL gemcitabine for 48 hours, and cell apoptosis was detected by flow cytometry and percentage of apoptosis was calculated. (7) Cells in the 2 groups were interfered with 0.3 μg/mL gemcitabine for 48 hours, and cell cycle was detected by flow cytometry. (8) The relative expressions of proteins in the 2 groups were detected by Western blot: ① the relative expressions of USP10 and P21 of cell lines interfered with 0, 0.30, 0.60, 1.20 μg/mL gemcitabine for 72 hours in the 2 groups were detected. ② The relative expressions of USP10 and P21 of cell lines interfered with 0.30 μg/mL gemcitabine at 0, 24, 48, 72 hours in the 2 groups were detected. ③ The relative expression of P53 in SW1990 and SW1990-GEM cell lines was detected. ④ The relative expression of P53 in SW1990 cell line interfered with 0, 0.30, 0.60, 1.20 μg/mL gemcitabine for 72 hours was detected. ⑤ The relative expression of P53 in SW1990 cell line interfered with 0.30 μg/mL gemcitabine at 0, 24, 48, 72 hours was detected. (9) The interaction between the proteins was detected by co-immunoprecipitation. Measurement data was presented as ±s. The comparisons among groups were evaluated with the one-way ANOVA, and parirwise comparison was analyzed by the LSD test. Comparison between groups was analyzed by t test. Results (1) The IC50 of gemcitabine and relative expression of USP10 in the cancer cell lines: ① the IC50 of BxPC-3, PANC-1 and SW1990 cell lines interfered with gemcitabine for 72 hours which was detected using CCK8 assay was (0.070±0.040)μg, (0.120±0.010)μg and (0.350±0.050)μg, with a statistically significant difference among them (F=10.765, P 0.05), and there was a statistically significant difference in the expression of P21 between the 2 groups (t=3.765, P 0.05). ③ The relative expressions of P53 in SW1990 and SW1990-GEM cell lines were 2.650±0.050 and 1.450±0.060, respectively, showing a statistically significant difference (t=19.075, P<0.05). The relative expressions of P53 in the control and USP10 groups were 3.250±0.050 and 1.550±0.050, respectively, showing a statistically significant difference (t=9.240, P<0.05). ④ The relative expressions of P53 in SW1990 cell line interfered with 0, 0.30, 0.60, 1.20 μg/mL gemcitabine for 72 hours were 0.590±0.050, 1.015±0.050, 2.050±0.050 and 2.850±0.050, with a significant concentration-dependence (F=34.088, P<0.05). ⑤ The relative expressions of P53 in SW1990 cell line interfered with 0.30 μg/mL gemcitabine at 0, 24, 48, 72 hours were 0.890±0.050, 1.225±0.030, 2.180±0.150 and 3.030±0.150, with a significant time dependence (F=29.650, P<0.05). (7) The results of co-immunoprecipitation: there was expression of USP10 protein in P53 protein complexes and expression of P53 protein in USP10 protein complexes. Conclusion USP10 promotes primary and acquired resistance of gemcitabine in pancreatic cancer via activation of P53/P21 pathway. Key words: Pancreatic neoplasms; Gemcitabine; Resistance