The role and mechanism of HMGB1 improving the chemosensitivity of gemcitabine-resistant pancreatic cancer PANC1 cells / 中华胰腺病杂志

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 ceils.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 wassignificantly 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 (bothP ＜ 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 siHMGB1-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.