Effect Of Lipoxin A₄ On The Metabolism Of S-Methyl-5'-thioadenosine And Related Compounds In HepG2 Cells

Objective: S-methyl-5'-thioadenosine(MTA)is an intermediate product of methionine metabolism.Previous studies indicated that,it plays important roles in cancer,inflammation,cell proliferation and several other pathophysiologic processes.In recent years,our group proved that Lipoxin A4(LXA4),a crucial anti-inflammatory and pre-resolution lipid mediator in inflammation,exerted suppressive effect on hepatocellular carcinoma(HCC)through inhibiting the inflammatory and hypoxic micro-environment in tumor.In order to understand the influence of LXA4 on metabolism of HCC,we investigated the metabonomics,especially the change of MTA and its related compounds in Hep G2 cells,under normoxic or hypoxic condition.Methods:(1)Human HCC cell lines,Hep G2,was cultured in vitro and divided into 4 groups: Cells in C1 and C2 group were cultured under normal oxygen,and cells in T1 and T2 group were cultured under 1% oxygen.Cells in C2 and T2 groups were treated with 200 n M LXA4 for 36 hours,while cells in C1 and T1 groups were treated with 0.7ul anhydrous ethanol as solvent control.(2)Metabolomic analysis: a.Hep G2 cells were collected by using extract solution(methanol: acetonitrile: water = 2:2:1)and then frozen into liquid nitrogen.b.Cells were treated with repeated freeze-thaw method,each cycle accompanied with a ultrasonication.Insoluble impurities were removed with centrifugation.The supernatant was dried with low-temperature vacuum,and re-dissolved in acetonitrile water solution(acetonitrile: water = 1:1).c.Metabolomic analysis was performed using a High-Performance Liquid Chromatography-Mass Spectrometry(HPLC-MS).(3)Levels of MTA and related compounds were measured with HPLC-MS: a.Repeated freeze-thaw treatment was used to extract MTA and related compounds in Hep G2 cells.The insoluble impurities were removed with centrifugation.The supernatant was dried with low temperature vacuum,and re-dissolved in mobile phase.b.Pre-test on HPLC-MS with commercial standard products to obtain the determination conditions and peak time of MTA and related compounds.c.Draw the standard curve with linear regression of target materials.d.The content of MTA and related compounds in each sample was determined.(4)Western blot analysis of the protein levels of MTA metabolism related enzymes.Research results:(1)Metabolomic analysis indicated that,under hypoxic condition,there were obviously higher concentration of SAH,MTA,L-tryptophan,indole,hypotaurine,Nacetylneuraminic acid,D-mannose,D-threitol,2-oxoadipic acid,thiamine,nicotinate,Lproline,L-tryptophan.and obviously lower concentrations of glycine,creatine,CMP,acetylcarnitine in LXA4-treated cells in T2 group compared with cells in T1 group.(2)Under normoxic condition,there were no significant difference in the levels of MTA and related compounds,as well as protein levels of target proteins between cells in C1 group and LXA4-treated cells in C2 group.(3)Compared with the cells in C1 group,HPLC-MS results indicated that the level of S-(5?-Adenosyl)-L-homocysteine(SAH)was obviously lower(p<0.01),while the contents of S-adenosyl-l-methionine(SAM)and MTA were higher in T1 group(p<0.01).At the same time,Western blotting result indicated that the protein level of S-adenosylmethionine decarboxylase(SAMDC)was significantly lower in T1 group(p<0.01).(4)Under hypoxic condition,compared with T1 group,LXA4-treated cells in T2 group had obviously higher MTA,SAH level but lower SAM level(p<0.01).At the same time,the protein level of 5'-methylthioadenosine phosphorylase(MTAP),spermine synthase(SRM)and SAMDC proenzyme were obviously lower but SAMDC level was higher(p<0.01).Conclusions:(1)Under normoxia,LXA4 had no significant effect on the metabolism of MTA and related compounds in Hep G2 cells.(2)Hypoxia could reduce the level of SAH and SAMDC in Hep G2 cells,but increase the level of SRM,SAM and MTA.(3)Under hypoxia,LXA4 might improve the content of MTA in Hep G2 cells by reducing the content of MTAP,promoting the synthesis of SAMDC and activating the enzymatic transformation of SAMDC proenzyme into SAMDC.The protein content of SRM decreased significantly after LXA4 treatment.It could be the result of negative feedback regulation by enhanced level of MTA.This might be one of the mechanisms under which LXA4 inhibit liver cancer.