Research In Vitro And In Vivo About The Inhibition Of Serine On The DEN Intiated Hepatic Carcinogenesis

ObjectiveAs one of the essential 20 amino acids,L-serine is widely involved in the synthesis of cellular and tissue structures and functional proteins in humans.As a major donor of one carbon unit,it is used to synthesize various single nucleotides to meet the needs for neonatal tissue division and proliferation of fetuses and neonates,and the formation and development of organ tissues.As a synthetic precursor of various important functional substances(phosphatidylserine and D-serine,etc.),it participates in the maintenance of central nervous system and brain function.The cardiovascular system is protected by the participation of selenium protease and glutathione in the maintenance of an antioxidant system and a collective transsulfur pathway for the removal of excess homocysteine.The sources of serine in the body include four ways:dietary intake;degradation of macromolecules(including proteins and phospholipids);synthesis of sugar metabolism bypasses-synthesis of the de novo synthesis pathway of serine;and conversion of glycine.Early studies have revealed that,in normal circumstances,when the dietary intake of protein or serine is insufficient,the body mainly relies on endogenous synthetic serine to compensate,and the main pathway of endogenous serine synthesis stems from the conversion of glycine.Since 2011,some scholars have used genomics to discover another pathway for the endogenous synthesis of serine in breast cancer patients,that is,the glucose metabolism pathway.Since the high expressions of the key enzyme in the de novo serine synthesis pathway,3-phosphoglycerate dehydrogenase(PHGDH)has emerged,a new hot spot is propoesed in the study of tumor mechanism and anti-cancer drugs.Deep exploration has found that the metabolism of various tumor cells is not the same.Some tumor cells can only take exogenous serine from the extracellular.But some other tumor cells highly express metabolic enzyme genes by the activation of endogenous serine synthesis pathway,so that they can synthesize a large amount of serine,which can synthesize one carbon units and glycine,to meet the need for unlimited cell division and proliferation of tumor cells.A very early study in vitro enzymology has showed that a large amount of serine can inhibit the activation of 3-phosphoglycerate dehydrogenase(PHGDH),a key enzyme in the de novo serine synthesis pathway in feedback way.Therefore,we propose the following hypothesis:Intracellular signaling pathway changes in DEN-intiated normal cells,which leads to changes of gene expression of the serine de novo pathway metabolic enzyme 3-phosphoglycerate dehydrogenase(PHGDH).Then the normal glucose metabolic pathway changes,and the cells undergo an abnormal division and proliferation,which gradually evolves into tumor cells.However,this is a long-time process.During this evolution,the increase of exogenous serine can in feedback way inhibit the expression of PHGDH and other genes,which makes it possible to block the carcinogenesis.However,in the transition from normal cells to cancer cells,the expression changes of serine de novo pathways and the regulation of serine-related enzymes have not yet been reported in the letters.This study will provide a new idea for cancer chemoprevention and nutritional intervention.Methods(1)In vitro experiments,Human hepatoma cells were selected by artificially adding serine to observe its effect on tumor cell function and explore its molecular mechanism.HepG2 cells were cultured in vitro and treated with different concentrations of serine from 0.01μM to 100μM.The proliferation,migration,and adhesion of HepG2 cells were detected by CCK8 method,scratch test,and in vitro matrix adhesion assay;and the expressions of pERK and pAKT were detected by western blot with 1μM serine treament.(2)In vitro experiments,we selected human normal liver cells,L-02,intiated by chemical carcinogen N-nitrosodiethylamine(DEN),and artificially added serine,then we observed its effect on cellular function during cell transformation and the effect on the de novo synthesis pathway enzymes.L-02 cells were cultured in vitro and divided into four groups,including normal control group(CK group),serine group(Serine group),N-nitrosodiethylamine group(DEN group),and serine +N-nitrosodiethylamine group(S+D group).Serine group and S+D group were pretreated with 0.4 mM serine for a week and DEN group and S+D group were treated with 1 mM DEN for four days.CCK8 assay,scratch test and in vitro matrix adhesion assay were used to detect the proliferation,migration and adhesion of L-02 cells.The cell surface tumor marker CD44 was detected by western blot to determine the degree of cell carcinoma and the expression of the serine de novo pathway enzymes,such as PHGDH.(3)In vivo experiments,ICR mice were used as subjects.Forty experimental animals were divided into four groups,including normal control group(CK group),serine group(Serine group),N-nitrosodiethylamine group(DEN group),and serine +N-nitrosodiethylamine group(S+D group).Serine group and S+D group were given 375 mg/kg serine(2 times/week)from the week before the experiment to the end of the experiment by gavage and 100 mg/kg DEN was injected to DEN group and S+D group in the first week.From the second week DEN group and S+D group were given CC14:olive oil(1:4)(2 times/week)by gavage and supplied with 9%ethanol in drinking water,and then were given 50 mg/kg DEN by gavage every other week.Forty mice were dissected respectively in two months and three months by cervical dislocation.Some sections were made into paraffin sections.HE staining and immunohistochemistry were used to detect the progress of hepatic carcinogenesis in mice and the expression of serine de novo synthesis pathway enzymes,PHGDH,PSAT1,and PSPH.Some other parts were used to extract tissue proteins and do western blot to detect the cell surface tumor marker CD44 and serine de novo pathway enzymes,PHGDH and PSPH.Results(1)In vitro intervention experiment of human hepatocellular carcinoma(HepG2 cells),the proliferation of HepG2 cells was rapidly promoted after treated with 1μM serine,and the difference was statistically significant at 48 h and 72 h(P<0.05).The migration of HepG2 cells was promoted after treated with 1μM serine and the difference was statistically significant at 24 h(P<0.05).After treated with 0.01 and 1μM serine,HepG2 cell adhesion rate increased respectively by 15%(P<0.05)and 20%(P<0.01).After treated with 1μM serine,the ERK and AKT activation(pERK and pAKT)were significantly increased in HepG 2 cells.It is suggested that exogenous addition of serine may activate ERK and AKT signaling pathways to promote malignant proliferation and migration.(2)In vitro intervention experiment of human normal hepatocytes(L-02 cells),we found that 0.4 mM serine can effectively block the canceration of L-02 cells intiated by DEN.Compared with DEN group,proliferation of L-02 cells in S+D group was inhibited at 24 h,72 h,and 96 h,with statistical differences respectively(P<0.01,P<0.001,and P<0.001).The inhibition rate of the migration function was 18%(P<0.05).Unfortunately,it had not shown the expected result that artificially added serine could inhibit of the key enzyme PHGDH gene expression of the serine de novo synthesis pathway in feedback way in L-02 cells.(3)In vivo intervention experiment of ICR mice,we observed that,after two months of DEN induction,DEN group showed poor growth status,no obvious weight gain,and the liver surface was rough and fine-grained while S+D group had better growth status,more weight gain,and less heavier liver granules.Under the microscope,DEN group showed changes in hepatic lobule structure and fibrous tissue around the leaflets and fibrosis lesions were aggravated,whereas in S+D group,the appearance of fibrotic lesions such as hepatic lobular structural changes was rarely seen.Western blot revealed that,compared with CK group and Serine group,DEN group and S+D group had a high expression of CD44,and the expression of CD44 and PSPH in S+D group was a little lower than that in DEN group,suggesting that serine can inhibit the expression of PSPH enzyme,and it may block DEN-intiated liver cancer.Immunohistochemical analysis found that although semi-quantitative analysis of the means of PHGDH and PSPH expression in DEN group and S+D group were similar,within S+D group,the values were scattered,suggesting that because of the existence of individual differences,exogenous serine can partly inhibit the expression of PHGDH and PSPH in feed back way.After three months of DEN induction,the appearance of liver tissue in mice showed that the lesions were aggravated in both DEN group and S+D group,and some individuals in DEN group began to appear white nodules which had not appeared in S+D group,suggesting early stage of liver cancer.Under the microscope,the hepatic lesions of DEN group and S+D group were aggravated.The nucleus of liver cells increased,and a few inflammatory cells infiltrated in the portal area.The DEN group showed phagocytized Kuffer cells.The S+D group showed a slight improvement.Morever,western blot analysis of mouse liver tissue protein found that,compared with CK group and Serine group,CD44 expression in DEN group and S+D group were significantly higher,and PSPH expression in S+D group was lower than DEN group.However,unlike the two-month results,the expression of CD44 in S+D group was not lower than that in DEN group,suggesting that serine can inhibit the expression of PSPH,but the expression of PSPH in the serine de novo pathway and CD44 expression did not keep the same pace.Immunohistochemical analysis revealed that the expression of PHGDH and PSPH was decreased in the S+D group compared with the DEN group,suggesting that exogenously addition of serine can in feedback way inhibit the expression of PHGDH and PSPH.ConclusionIn HepG2 cells,serine can promote the proliferation,migration and adhesion of HepG2 cells by activating ERK and AKT signaling pathways.InDEN-intiatedL-02cells,serine can inhibit the tumor-associated proliferation and migration during the process of carcinogenesis.In vivo,by inhibiting the expression of serine de novo synthesis pathway metabolic enzymes(such as PSPH,etc.),Serine can block the progress of liver cancer in mice to some extent(such as decreasing theexpression of tumor surface marker CD44,reducingthe degree of liver cancer,etc.).