Metformin Inhibits Glycolysis Of Multiple Myeloma Cells Through Hexokinase 2

Objective:To study the pathway of metformin to inhibit the glycolysis of multiple myeloma cells and provide a possible theoretical basis for targeted metabolic therapy of multiple myeloma.Methods:Human multiple myeloma cell lines ARP-1 and CAG cell lines were cultured in vitro,treated with different concentrations of metformin,and their effects on cell viability were detected by CCK8 method.Western blotting technique was used to detect HK2,C-MYC and Bcl-2 Expression level,colorimetric method to detect glucose uptake rate and intracellular and intracellular lactic acid levels,CellTiter-Lumi ? luminescence to detect intracellular adenosine triphosphate(ATP)level,and ELISA method to detect intracellular reduced nicotinamide adenine dinucleotide Phosphoric acid(NADPH)content.Furthermore,after knocking out ARP-1 cells HK2 using siRNA,the changes of glycolysis-related products and possible signal pathway changes in ARP-1 cells were observed.Results:Using the CCK8 method,it was found that different concentrations of metformin can inhibit the proliferation of multiple myeloma cell lines ARP-1 and CAG cells,and with the increase of drug concentration,the inhibitory effect increased,with statistical significance(P<0.05).Western blotting technology detected that the expression levels of HK2 and C-MYC in ARP-1 and CAG after metformin treatment decreased and decreased with the increase of drug concentration,while the expression level of Bcl-2 increased.The relative glucose uptake rate of the above cell lines detected by colorimetry after treatment with metformin increased with the increase of metformin concentration,which was statistically significant(P<0.05).The lactic acid production of ARP-1 and CAG cell lines treated with metformin detected by colorimetry was decreased,which was drug concentration-dependent and had statistical significance(P<0.05).Using CellTiter-Lumi? luminescence to detect intracellular ATP levels,it was found that the above-mentioned cell lines were treated with metformin and the intracellular ATP level decreased,and decreased with the increase of drug concentration,which was statistically significant(P<0.05).Using ELISA method to detect the content of NADPH in cells,it was found that the content of the above cell lines decreased after treatment with metformin,which was statistically significant(P<0.05).The ARP-1 cell line that simply knocked down HK2 had increased C-MYC expression levels,but was inhibited by metformin.The expression level of Bcl-2 increased in ARP-1 cell line that simply knocked down HK2,and after metformin treatment,the expression increased gradually,which was statistically significant(P<0.05).After knocking down HK2,the glucose uptake rate of the cells increased and the content of ATP and NADPH in the cells decreased,which was statistically significant(P<0.05).After knocking down HK2,RNA sequencing revealed that it clustered in the PI3K/AKT signaling pathway.Conclusions:1.Metformin has a proliferation inhibitory effect on multiple myeloma cell lines;2.Metformin inhibits glycolysis of multiple myeloma cell lines;3.HK2 regulates the glycolysis of multiple myeloma cell lines,the mechanism may be related to PI3 K / AKT signaling pathway;4.HK2 may become a target for metabolic therapy of multiple myeloma.