Suppression Of Phosphoglucose Isomerase/Autocrine Motility Factor Enhances Ginsenoside RH2Pharmacological Effect On KG1A Cells In Vitro

BackgroundLeukemia is malignant tumor of hematopoietic system. The majorinterventions of conventional medicine failed to have its effects because ofits side effects. Thus, it is urgent for us to find another effective therapy.Phosphoglucose isomerase (PGI), a housekeeping gene, plays actritical role in both glycolysis and gluconeogenesis, catalyzing theinterconversion of glucose-6-phosphate and fructose-6-phosphate inintracellular environment, which behaves extracellularly as a cytokinecalled autocrine motility factors (AMF). The over-expressing of PGI/AMFcould accelerate cancer progression and metastasis. On the contrary,down-regulation of PGI/AMF could inhibit the growth of tumor. Ourprevious studies have showed that PGI gene can be significantly inhibitedin leukemia cells by ginseng polysaccharide. Down-regulation of PGI genecould inhibit the proliferation of KG1α cells. Ginseng saponin monomers has been proved have significant anti-cancer effect on leukemia cell lines.To investigate the relationship between PGI and ginsenoside Rh2, we usedsmall interfering RNA to down-regulate the expression of PGI/AMF and toresearch the PGI/AMF affect on the efficacy of ginsenoside Rh2and therelated mechanism.MethodsPart ⅠCCK-8assay was used to investigate the most effective drug on theproliferation of KG1α cells among ginsenoside Rb1, Rg1and Rh2. Theeffects of the ginsenoside on cell cycle and apoptosis were detected by flowcytometry (FCM) combined with PI staining and Annexin V-FITC/PI,respectively. The expressions of P53, P21, Cyclin D1and CleavedCaspase-3were examined by Western blotting.Part ⅡThe constructed lentivirus plasmid were used to transfect the leukemiacell line which is highest expression of PGI, RT-PCR and Western Blot wereapplied to detect the expression of PGI after interference. The growthinhibitory effect on KG1α cells after silencing PGI was determined bytrypan blue dye exclusion. The effect on cell cycle by suppressing theexpression of PGI was detected by FCM. Hochest dyeing was used to testthe effect of ginsenoside Rh2on KG1α group, negative control group andtransfected group. The effects of ginsenoside Rh2and silencing PGI on mTOR pathway were tested by Antibody Array. The expression levels ofAKT、PGI、mTOR、Raptor、Rag were detected by western blotting. Thenwe analyzed the relationship between PGI gene and ginsenoside Rh2.Results1. Ginsenoside Rh2had the most efficient inhibitory effect on leukemiaKG1α cells.2. Cell cycle: Ginsenosede Rh2arrested cell cycle in G0/G1phase(P<0.05). The percentage of cells in G0/G1phase increased significantlyfrom（26.78±3.14）％to （29.26±2.31）%at24h and to （44.77±2.26）％at48h.(P﹤0.05)3. Cell apoptosis: Ginsenoside Rh2showed up-regulated apoptosis ratessignificantly from （2.37±0.02）%to （8.37±0.15）%at24h and to(33.22±1.67）%at48h.(P﹤0.05)4. The results of Western blotting showed the expression of P53, P21and Cleaved Caspase-3went up significantly, meanwhile Cyclin D1proteindropped significantly.5. Hochest dyeing and CCK-8assay demonstrated that down-regulateof PGI gene could suppress the proliferation of KG1α cells, meanwhile itenhanced the sensibility of KG1α cells to ginsenoside Rh2.6. Antibody Array showed ginsenoside Rh2regulated the proliferationof KG1α through decreasing the expression of mTOR, AKT, AMPKα,PARP and Bad proteins. 7. The antibody array indicated that down-regulate the expression ofPGI reduced expression of PARP, State1, SAPK/JNK and Erk1/2, whileP38were up-regulated.8. Down-regulation of PGI/AMF made KG1α more sensitive to Rh2byreducing mTOR、Raptor and Rag expression (P<0.05), and PGI hadsynergistic effect on ginsenside Rh2.Conclusions1. Ginsenoside Rh2had the most efficient inhibitory effect on leukemiaKG1α cells, by arresting cell cycle, inhibiting proliferation and acceleratingapoptosis.2. Ginsenoside Rh2inhibited cell proliferation through up-regulatingthe expression of P53, P21and Cleaved Caspase-3, meanwhiledown-regulating the expression of CyclinD1.3. Down-regulation of PGI/AMF coordinated with ginsenoside Rh2tomodulate the proliferation of KG1α.4. Down-regulation of PGI/AMF enhanced the pharmacological effectsof ginsenoside Rh2on KG1α by reducing AKT/mTOR signaling.