The role and mechanism of HMGB1 improving the chemosensitivity of gemcitabine-resistant pancreatic cancer PANC1 cells

Abstract

Objective To investigate the role and potential mechanism of high mobility group box-1 protein (HMGB1) on improving the chemosensitivity of gemcitabine-resistant pancreatic cancer PANC1 cells. Methods Gemcitabine-resistant pancreatic cancer PANC1 (PANC1-GR) cell line was established by using increased gradient concentration of gemcitabine. The si-HMGB1-PANC1 and si-HMGB1-PANC1-GR cells were established by the transfection with HMGB1 siRNA using liposome. The 50% inhibitory concentration (IC50) and Resistance index (RI) of gemcitabine in 4 PANC1 cell lines with or without HMGB1 siRNA transfection were determined and calculated by CCK-8 assay. Western blot assay was used to detect the protein expression of HMGB1 in PANC1 and PANC1-GR cells and the expression of autophagy marker protein Beclin1 in the 4 PANC1 cell lines. Flow cytometry assay was used to evaluate the apoptosis rate of 4 pancreatic caner cell lines. Results The gemcitabine-resistant pancreatic cancer cell line PANC1-GR was successfully established, which could grow stably and passage in media with 100 μmol/L gemcitabine. The IC50 of gemcitabine in PANC1, PANC1-GR, si-HMGB1-PANC1, and si-HMGB1-PANC1-GR cells lines were (4.7±0.4)μmol/L, (166.5±13.6)μmol/L, (3.2±0.3)μmol/L, and (52.4±8.4)μmol/L, respectively. The IC50 in PANC1-GR was significantly higher than that in PANC1, while the IC50 in the transfected cells was significantly lower than that in untransfected cells, and the differences were both statistically significant (both P<0.01). The RI value of gemcitabine in transfected and untransfected PANC1-GR cells was 35.4 and 16.4. The relative protein levels of HMGB1 in PANC1 and PANC1-GR were 0.17±0.08 and 0.38±0.11. The expression of HMGB1 in PANC1-GR was obviously higher than that in PANC1, and the difference was statistically significant (P<0.01). The relative protein levels of Beclin1 in PANC1, PANC1-GR, si-HMGB1-PANC1 and si-HMGB1-PANC1-GR cells were 2.68±0.23, 3.28±0.15, 0.68±0.23 and 0.78±0.11, which in two transfected cells was greatly lower than those in untransfected cells. The apoptosis level was (34.58±3.14)%, (79.56±3.58)%, (19.41±1.53)%, and (34.57±2.94)%. The apoptosis level in the 2 transfected cell lines were significantly higher than those in the 2 untransfected cell lines, and the differences were both statistically significant (P<0.01). Conclusions The inhibition of HMGB1 could improve the chemosensitivity of gemcitabine in pancreatic cancer PANC1 cells, which might be mediated by the activation of autophagy. Key words: Pancreatic neoplasms; Cell line, tumor; High mobility group proteins; Autophagy; Apoptosis; Gemcitabine