Effects of lactic acid via cholesterol on Hedgehog signaling activity in non-small cell lung cancer cells
Objectives To explore the effects of lactic acid via cholesterol on Hedgehog(Hh)signaling pathway activity in non-small cell lung cancer cells.Methods Lactic acid(0.0 mM,7.5 mM,15.0 mM,20.0 mM)and cholesterol(0,25 μg/mL,50 μg/mL)were adopted to treat A549 and NCI-H1703 cells for 6 and 24 hours respectively to detect the expression levels of Hh signaling pathway target genes,genes or proteins relat-ed to cholesterol biosynthesis enzymes,and to measure the total cholesterol level in the cells.In order to ex-clude the effects of pH value on the study results,A549 and NCI-H1703 cells were treated with the same con-centration of sodium lactate(7.5 mM)for 24 h.The expression levels of genes related to cholesterol biosynthesis enzymes were detected by qPCR method.A549 cells were treated with a cholesterol synthetase inhibitor simvas-tatin(0.2 μM)and lactic acid(0.0 mM,7.5 mM,15.0 mM,20.0 mM)for 24 h to detect the expression level of Hh signaling pathway target genes.Besides,RTCA DPlus method was used to detect proliferation and migration.Results The mRNA levels of GLI1 and PTCH1 in A549 and NCl-H1703 cells treated with lactic acid and cho-lesterol were up-regulated compared with those in control group(P<0.05).After lactic acid treatment,the total cholesterol level and mRNA levels of HMGCR,FDFT1 and SQLE in A549 cells were increased(P<0.001).The protein levels of HMGCR and FDFT1 were increased as well.The mRNA levels of HMGCR and FDFT1 were in-creased in A549 and NC1-H1703 cells treated with lactic acid and sodium lactate respectively(P<0.01).The mRNA levels of GLI1 and PTCH1 were down-regulated by 0.2 μM simvastatin(P<0.05)after cholesterol level was down-regulated(P<0.01).Lactic acid could reverse the inhibitory effects of simvastatin on GLI1 and PTCH1 mRNA levels,while inhibit the proliferation(F=3.941,P=0.020)and migration(F=5.851,P=0.003)of A549 cells.Conclusions Lactic acid may up-regulate the cholesterol level of NSCLC cells to promote the activity of Hh signaling pathway,and enhance cell proliferation and migration ability,suggesting that inhibiting lactic ac-id may improve the efficacy of simvastatin in NSCLC.