Effect of sinomenine on apoptosis of colorectal cancer cells by binding to key glycolytic enzyme
Objective To investigate the correlation between sinomenine-induced apoptosis and glycolysis in colorectal cancer cells and explore the underlying mechanism.Methods Different treatment methods were used to result in the following groups:control group,sinomenine treatment group,overexpression control group,HXK2 overexpression group,knockdown control group,HXK2 knockdown group,control cell group,HXK2 knockout cell group.Apoptosis rate and glycolysis levels(2-deoxy-D-glucose uptake and lactate production levels)of colorectal cancer cells were assessed using flow cytometry and microplate reader.Real-time quantitative PCR and immunoblotting were used to detect the mRNA and protein expression levels of HXK2,respectively.Pearson correlation coefficient analysis was conducted to examine the relationship between cell apoptosis and glycolysis after sinomenine treatment.Results Compared to the control group,the sinomenine treatment group exhibited increased apoptosis levels in colorectal cancer cells(14.06±1.09 vs 62.29±4.07,t=19.81,P<0.05),decreased 2-deoxy-D-glucose uptake levels(544.46±32.06 vs 46.34±2.47,t=7.71,P<0.05),and reduced lactate production levels(14.29±1.34 vs 1.20±0.04,t=8.60,P<0.05).Pearson correlation coefficient analysis revealed a correlation between sinomenine-induced cell apoptosis levels and both 2-DG uptake levels and lactate production levels.Overexpression of HXK2 was able to counteract the inhibitory effect of 25 μmol·L-1 sinomenine on lactate production levels(1.66±0.47 vs 15.44±1.14,t=14.27,P<0.05).However,when sinomenine was excessively administered,the lactate production levels in the HXK2 overexpression group decreased as compared with the overexpression control group(13.66±0.39 vs 1.41±0.11,t=8.22,P<0.05).Moreover,compared to the overexpression control group,the apoptosis levels in colorectal cancer cells were decreased in the HXK2 overexpression group(15.19±1.03 vs 8.44±0.09,t=7.68,P<0.05).Similarly,compared to the knockdown control group,the apoptosis levels in colorectal cancer cells were increased in the HXK2 knockdown group(14.88±1.10 vs 65.83±2.20,t=6.36,P<0.05).Following the construction of HXK2 knockout HT29 cell lines,sinomenine did not affect the apoptosis levels of HXK2 knockout HT29 cell lines.However,when wild-type HXK2 was overexpressed in the HXK2 knockout cell lines,sinomenine could significantly enhance the apoptosis levels(10.71±1.21 vs 67.89±4.31,t=11.29,P<0.05).Conclusion Sinomenine can inhibit the function of HXK2 to reduce glycolysis and promote the apoptosis of colorectal cancer cells.
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