Study On The Effects Of Gamma-aminobutyric Acid On Colorectal Cancer And Its Molecular Mechanisms

Background and Purpose:Gamma-aminobutyric acid?GABA?is a natural active component,which is widely distributed in plants and animals,such as rice germ,rice bran,tea,lactic acid bacteria,yeast,etc.GABA has many important physiological functions,such as sedation,anti-anxiety,regulating cardiovascular and hormone secretion,lowering blood pressure,anti-aging.Recent studies have showed the anti-tumor activity of GABA on liver cancer,lung cancer,pancreatic cancer and cholangicarcinoma,but the effects of GABA on colorectal cancer and its molecular mechanisms are unclear.Therefore,the present study explored the effects of GABA on the proliferation,cell cycle,migration and invasion of colorectal cancer cells and its potential molecular mechanisms in in vitro experiment;the effects of GABA on the chemosensitivity of colorectal cancer were investigated using both in vitro and in vivo experiment.Methods:Three strains of colorectal cancer cells?SW620,SW480,HCT116?were selected,and the effects of GABA on the proliferation,cell cycle,migration and invasion of these three cells were observed by CCK-8,flow cytometry,transwell migration and invasion assays,respectively;RNA-Seq was used to screen the differentially expressed genes of these three cells treated by GABA,and the results were verified by qRT-PCR,the mRNA expression of GABA A and B receptors in these three colorectal cancer cells treated by GABA was detected by qRT-PCR;the effects of GABA combined with 5-fluorouracil and oxaliplatin on the proliferation of cells were determined with CCK-8 assay and nude-mouse transplanted tumor models.The tumor size and weight were measured,the expression of Ki67 protein and the apoptosis of tumor cells in tumor tissues were observed with immunohistochemistry staining and Tunel staining,respectively.Results:GABA?100??44??significantly inhibited the proliferation of SW620,SW480 and HCT116 cells?P<0.05?,the inhibition rate was32.53?7.34%,33.88?2.60%,41.35?10.48%,respectively,increasing of the concentration of GABA to 400??did not further increase the inhibition rate;the amount of SW620 and SW480 cell in S phase was decreased by 15%,13%,respectively,the cell amount of HCT116 cell in G1/G0 phase decreased by 10%,and that in G2/M phase increased by 30%;GABA effectively inhibited the migration and invasion of SW620 and SW480 cells.RNA-Seq and qRT-PCR verification results showed that GABA downregulated the expression of FOS,FOSB,NR4A1,EGR1,EGR2 in three cells?P?27?0.05?,downregulated the expression of FRMD4A in HCT116cells?P?27?0.0001?,upregulated the expression of FRMD4A in SW620 and SW480 cells?P?27?0.0001?;qRT-PCR results showed that GABA significantly affected the mRNA expression levels of GABA A and B receptors in three cells(SW620:GABA_A?₂ and GABA_A?were upregulated,GABA_A?₂ was downregulated;SW480:GABA_A?were upregulated,GABA_A?and GABA_B11 were downregulated;HCT116:GABA_A?₂,GABA_A?₃,GABA_A?₃,GABA_B2,GABA_A?₂,GABA_A?₂ were downregulated).Finally,the combination of 5-fluorouracil with GABA increased the inhibition rate of SW620,SW480,HCT116 cells proliferation by 6.28?4.13%,2.03?1.71%,2.68?2.19%,respectively,the combination of oxaliplatin with GABA increased the inhibition rate of SW620,SW480,HCT116 cells proliferation by 2.75?2.69%,4.10?2.48%,7.28?1.54%,respectively.Animal experiments showed that combination of GABA with oxaliplatin significantly inhibited the growth of tumors,reduced the expression of Ki67protein in tumor tissues and promote apoptosis of transplanted tumor cells compared with oxaliplatin group.Conclusion:GABA could affect the proliferation,cell cycle distribution,migration and invasion of colorectal cancer cells partially due to the downregulation of FOS,FOSB,NR4A1,EGR1,EGR2 genes,upregulation of FRMD4A gene,and influence of expression of GABA receptors(GABA_A?₂,GABA_A?₃,GABA_A?₂,GABA_A?₃,GABA_A?,GABA_A?,GABA_A?₂,GABA_B1,GABA_B2).GABA could enhance the chemosensitivity of colorectal cancer cells to 5-fluorouracil and oxaliplatin,suggesting that GABA may play an important role in prevention and adjuvant treatment of colorectal cancer.