Effects Of 2-deoxyglucose And Metformin On Proliferation, Apoptosis And Metabolism Of Cystic Epithelial Cells From Human Polycystic Kidney Disease

Objective: Autosomal dominant polycystic kidney disease (ADPKD) is a renal disease characterized by abnormal proliferation of renal tubular epithelial cells.Polycystic kidney cystic epithelial cells are similar to tumor cells in the presence of abnormal glycolysis. Metformin and 2- deoxyglucose are metabolic drugs that inhibit tumor proliferation. In this study, we investigated the effects of metformin and 2-deoxyglucose on the proliferation and apoptosis of human polycystic kidney cystic cells (WT9-7),and explored the possible molecular mechanisms.Methods:1. WT9-7 cells were divided into control group, 2-DG group, MET group and 2-DG+MET group. CCK-8 method and EdU nucleic acid labeling technique were used to detect the proliferation of polycystic kidney cystic epithelial cells in each group.2. The activation levels of AMPK, mTOR, 4E-BP1, p70 S6K, Akt, PI3K, B-Raf,MEK1/2, MAPK (Erk1/2) were analyzed in each groups of polycystic kidney cystic epithelial cells.3. To detect the changes of cell cycle and the expression levels of CyclinD3,CyclinE1, CDK4, CDK2, P27 in each group.4. The activity of Caspase-3 and apoptosis rate in each group were analyzed.5. To analyze the metabolic phenotypes of the polycystic kidney cystic epithelial cells and to detect the expression levels of hexokinase (HK2) and complex I in four groups.Results:1. The results of CCK-8 and EdU nucleic acid labeling showed that both 2-DG and MET could inhibit the proliferation of polycystic kidney cystic epithelial cells, but the combination of two drugs with low dose could significantly inhibit the proliferation of polycystic kidney cystic epithelial cells (P < 0.001).2. Compared with the control group, 2-DG and MET had an inhibitory effect on the activation of mTOR and ERK signal pathway, but this effects were significantly induced in the combined group (P < 0.01). In addition, the combination of the two drugs significantly inhibited the expression of some cell cycle related molecules,inhibited the replication of cells and make them stay in the G0/G1 phase.3. 2-DG or MET alone all have little effects on apoptosis in polycystic kidney cystic epithelial cells, but the combined use of double medicine can increase the activity levels of Caspase-3 and early apoptosis rate in polycystic kidney cystic epithelial cells (P< 0.001).4. 2-DG can downregulated the protein expression level of HK2 and inhibit glycolysis and the Synthetic ATP of polycystic kidney cystic epithelial cells, MET can decrese the protein expression level of ComplexI and inhibit oxidative phosphorylation and the synthetic ATP in cells. However, the protein expression level of HK2 and Complex I can downregulated in the combined group, at the same time, two kinds of metabolism phenotypes were inhibitied and reduced the synthetic ATP in cells significantly (P < 0.001).Conclusions: Combined use of 2-DG and MET can inhibit proliferation related signaling pathways of cAMP-PKA, mTOR and ERK in polycystic kidney cystic epithelial cells, inhibiting cell replication and evetually inhibit cell proliferation.Combinative use of two drug can also promote overexpression of activited Caspase-3 and induce cystic epithelial cells apoptosis. On one hand, 2-DG and MET can restrain glycolysis and mitochondrial oxidative phosphorylation metabolisms respectively in polycystic kidney cystic epithelial cells, 2-DG and MET can also inhibited the production of ATP significantly, so as to upregulate the activation of energy sensor AMPK,downregulate the activation of mTOR proliferation signal pathway. In conclude, 2-DG and MET decrease over-accumulation of polycystic kidney cystic epithelial cells through inhibit over activation of multiple intracellular proliferation signaling pathway and promote cell apoptosis.