Ginsenoside Rk3 Inhibits Glycolysis By Targeting Glyceraldehyde-3-phosphate Dehydrogenase In HepG2 Cells

Ginsenoside is the main active component of ginseng,which has broad-spectrum pharmacological activity and can effectively inhibit the growth of various tumor cells such as breast cancer,lung cancer,liver cancer,and colon cancer.Ginsenoside Rg1(G-Rg1)inhibits the invasion and migration of HepG2 cells;(20S)-ginsenoside Rg3((20S)G-Rg3)exerts anti-tumor effects by inducing apoptosis,inhibiting tumor cell proliferation,metastasis and angiogenesis.Our previous study showed that(20S)-ginsenoside Rh2((20S)G-Rh2)kills tumor cells rapidly by initiating mitochondria-mediated endogenous apoptotic pathway and membrane death receptor-mediated extrinsic apoptotic pathway together.(20S)G-Rh2 induces cell cycle arrest and apoptosis in hepatoma HepG2 cells by targeting HSP90A and Annexin A2.Cancer cells rely on the glycolytic pathway for glucose metabolism,unlike normal differentiated cells who rely on tricarboxylic acid cycle.Cancer cells reduce the glycolysis product pyruvate to lactate,which is secreted into the extracellular matrix,resulting in increased lactate in the tumor microenvironment.The increase of lactate promotes the immune escape and migration of tumor cells.The abnormal enhancement of the glycolytic pathway in tumor cells provides material and energy for the rapid growth of tumor cells.Therefore,inhibition of the glycolytic pathway is one of the important means of cancer treatment.Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)is an important enzyme in the process of glycolysis.It uses NAD~+as a coenzyme to catalyze the phosphorylation and oxidation of glyceraldehyde-3-phosphate(G3P)to generate 1,3-Diphosphoglycerate(1,3-BPG)and NADH.Large data on clinical tumor showed that GAPDH is highly expressed in hepatocellular carcinoma,and its expression level is negatively correlated with the prognosis of patients.It is expected to inhibit tumor cell growth and tumor progression by reducing glycolysis level in tumor cells by inhibiting GAPDH.Liver cancer causes the sixth most common cancer and the third leading cause of cancer-related death worldwide.The incidence of liver cancer is higher than the world average in our country,while the incidence and mortality of liver cancer are on the rise.Although the vaccination of hepatitis B vaccine has reduced the incidence of liver cancer caused by viral infection to a certain extent,the incidence of liver cancer has been continuously increased due to poor living habits such as drinking and obesity.Because there is no obvious clinical symptoms on early-stage liver cancer,patients are diagnosed on the middle-advanced stage usually,where liver tumor cannot be surgically removed or transplanted.There is no effective drug for patients with advanced liver cancer at present.In our previous study,we used(20S)G-Rh2-PEGA to screen the T7 human liver cancer c DNA library by phage display technology,found that GAPDH may be the target of(20S)G-Rh2 luckily.In this paper we found that 1)6 kinds of ginsenosides(G-Rg1,(20S)G-Rg3,(20S)G-Rh2,G-Rh4,G-Rk1,G-Rk3)was found bind to GAPDH through molecular docking based on Auto Dock software,the binding free energies are-1.26,-2.07,-4.89,-6.52,-2.79,-6.19 kcal/mol,respectively;2)The interaction between these saponins and GAPDH was confirmed by thermal Shift Assay then;3)GAPDH catalyzes the reaction of glyceraldehyde-3-phosphate(G3P),inorganic phosphorus,and NAD+to generate 1,3-bisphosphoglycerate(1,3-BPG)and NADH,we determinate the effect of ginsenosides on GAPDH in this reaction,and found that G-Rk3 significantly inhibited GAPDH enzyme activity,while other saponins did not;5)It was further proved that G-Rk3 inhibited the glycolysis of HepG2 cells;6)We used MTT colorimetric assay found that Rk3 significantly inhibited the proliferation of HepG2cells.In conclusion,this study revealed the molecular mechanism that G-Rk3 inhibiting glycolysis in HepG2 cells by targeting GAPDH.Metabolic reprogramming of cells is a markable character of cancer and therefore an important target for the development of antitumor drugs.This study shows that ginsenoside Rk3 inhibits the enzymatic activity of GAPDH by targeting GAPDH,inhibiting the glycolysis and cell proliferation of HepG2 cells thereby.