Abstract:

Objective：To clarify the possible mechanism of suboptimal efficacy of enhancer of zeste homolog 2 （EZH2） inhibitors in pancreatic cancer， and to provide basis for exploring a new treatment plan for pancreatic cancer. Methods The pancreatic cancer PANC-1 cells， CFPAC-1 cells and pancreatic metastasis cancer SW1990 cells were treated with different concentrations（0－50 μmol·L^－1）of EZH2 inhibitor GSK126. CCK8 method was used to detect the survival rates of the cells in various groups. Then the cells were divided into control group and GSK126 group. After 48 h of GSK126 incubation， the apoptotic rates of the cells in various groups were detected by flow cytometry，the lipid droplet formation in the cells in various groups were observed with oil red O staining，and the levels of triglyceride（TG） in the cells in various groups were detected.The expression levels of apoptosis genes cysteine-aspartic acid protease（1，3，7 and 9） （Caspase-1， Caspase-3， Caspase-7， Caspase-9）， vascular endothelial growth factor A （VEGFA）， E-cadherin and stemness markers CD133， CD24 and CD44 mRNA and the expression levels of fat acid synthase （FASN）， acetyl CoA carboxylase alpha （ACACA）， citrate synthase （CS）， ATP citrate lyase （ACLY）， stearoyl-CoA desaturase （SCD） and acyl-CoA synthetase short chain family member 2（ACSS2） mRNA were detected by real-time fluorescence quantitative PCR（RT-qPCR） method. The three kinds of cells were divided into control group， GSK126 group， FASN inhibitor Orlistat （Orlistat） group and GSK126+Orlistat group，respectively. After incubated for 48 h， the survival rates of cells in various groups were detected by CCK-8 method， and the combination index （CI） value of the two drugs was calculated.The lipid droplet formation in the cells in various groups were observed with oil red O staining，and the levels of TG in the cells in various groups were detected； the apoptotic rates of cells in various groups were detected by flow cytometry，and the expression levels of Caspase-1， Caspase-3， Caspase-7 and Caspase-9 and CD133， CD24 and CD44 mRNA in the cells in various groups were detected by RT-qPCR method. Results GSK126 inhibited the proliferation of pancreatic cancer cells in a concentration dependent manner. There were no significant differences in the apoptotic rates and apoptosis gene mRNA expression levels in the three kinds of cells between control group and GSK126 group（P>0.05）. Compared with control group， the expression levels of VEGFA mRNA in the SW1900 cells and E-cadherin mRNA in the PANC-1 cells in GSK126 group were significantly decreased （P<0.05）；the expression levels of CD133 in three kinds of cells and the expression levels of CD24 mRNA in the CFPAC-1 cells and SW1990 cells were signicantly increased （P<0.05）.Compared with control group， the number of lipid droplets in three kinds of cells in GSK126 group were significantly increased，the TG levels and the expression levels of FASN mRNA were significantly increased （P<0.05 or P<0.01）， and the expression levels of other lipid metabolism-related genes were also increased to varying degrees.Compared with control group， GSK126 group and Orlistat group， the survival rates of the cells in GSK126+Orlistat group were significantly decreased（P<0.05 or P<0.01）； the CI of two drugs was less than 1， which meaned synergistic effect of GSK126 and Orlistat.Compared with control group，GSK126 group and the number of lipid droplets in the cells in GSK126+Orlistat group was signifieantly decreased；compared with control group and GSK126 group，the TG level in the cells in GSK126+Orlistat group was significantly decreased（P<0.01），and the expression levels of apoptosis-related gene and stemness gene mRNA were significantly increased（P<0.05 or P<0.01）；compared with control group，GSK126 group and Orlistat group，the apoptotic rates of the cells in GSK126+Orlistat group was significantly increased（P<0.01）. Conclusion GSK126 inhibits the proliferation of pancreatic cancer cells and induces reprogramming of lipid metabolism， which weakens its anticancer effect. The combined use of GSK126 and FASN inhibitor Orlistat can partially reverse this effect， synergistically inhibit the cell proliferation， enhance apoptosis and reduce the expression of stemness genes.

Key words: Pancreas neoplasms, Chemotherapy, EZH2 inhibitor, GSK126, Lipid metabolism reprogramming, Fatty acid synthase