Study On The Anti-myeloma Effect And Mechanism Of Metformin Based On PI3K/AKT/mTOR And AMPK/mTOR Signaling Pathway

BackgroundMultiple myeloma is an abnormal proliferated malignancy of monoclonal plasma cell which is characterized by producing monoclonal immunoglobulin. The treatment of MM mainly include chemotherapy, drug targeting therapy and immune therapy,etal at present. The median survival time is3-5years although with the targeted therapy significantly improved the efficacy of MM, MM is still considered an incurable disease. So, looking for the new mechanism of effecting MM cells survival and the corresponding treatment methods is becoming urgent need to resolved clinical problems.Recently, the relationship between diabetes and cancer has been attracted more and more interseting. Researches have shown that there is also an association between diabetes and MM. Insulin resistance of diabetic patients leads accumulation of insulin and insulin growth factor(I/IGF) in vivo. I/IGF mediated activation of PI3K/AKT/mTOR signaling pathway has important role in tumor cells proliferation and drug resistance including MM cells. Metformin is a safe and efficient biguanides. Acts as insulin sensitizers, metformin effectively reduce the blood insulin levels mainly through reducing hepatic gluconeogenesis, increasing the sensitivity of peripheral tissues to insulin and inhibiting intestinal absorption of glucose. So, metformin may affect the I/IGF pathway to inhibit the proliferation of MM cells. Meanwhile, metformin has important role in cell energy metabolism and could activate AMP-activated protein kinase(AMPK). Activated AMPK could negative regulate mTOR which is another mechanism of metformin exerting antitu,or effect. There is still controversial about whether AMPK is an oncogene or a tumor suppressor. AMPK is controlled by upstream kinase such as live kinase B1/serine/threonine protein kinase11(LKB1/STK11). Loss expression or mutation resulted the lack of function of LKB1/STK11could effect activation of AMPK by metformin. The role of LKB1/AMPK in MM is also still less known. In this study, we will focus on the effect of metformin for MM cells and its mechanism. The role of AMPK in MM will also be discussed.ObjectiveThe study is aimed to explore the effect of metformin to MM cell lines and primary MM cells proliferation, apoptosis, cell cycle arrest and find the combined effect of metformin with other anti-myeloma drugs. Then, the mechanisms of metformin inhibiting MM cells proliferation will be discussed mainly focus on PI3K/AKT/mTOR and AMPK/mTOR signaling pathway. The in vitro anti-myeloma effect of metformin alone or combined with other drugs will be confirmed in vivo by SCID mice. Act as an AMPK activator, the effect of metformin to AMPK and the role of AMPK in MM cells behavior will also be discussed through knockdown AMPK with small interfering RNA packing in lentivirus.Methods1. The effect of metformin to the proliferation of MM cells was detected by MTT. Flow cytometry(FCM) was used to detect the cell cycle and apoptosis after MMcells were exposed to metformin.2. The combined effect of metformin with dexamethasone or bortezomib was tested by MTT. The quantification of the combination were analyzed using the Chou-Talalay method with CalcuSyn software. Combination index (CI) offers quantitative definition of interaction between different drugs with synergism (CI<1), additive effect (CI=1), and antagonism (CI>1)3. Western blot and FCM were used to detect the changes of apoptotic-related and cell cycle-related proteins to verify the anti-myeloma effect of metformin alone or combined with dexamethasone or bortezomib. 4. The proteins change of IGF-IR/PI3K/AKT/mTOR and AMPK/mTOR signaling pathway were tested by western blot after MM cells were exposed to metformin with or without IGF-I(100ng/ml) and LY294002(10μM or5μM).5. A xenograft model of human myeloma was established with injecting1×107MM.1S cells subcutaneously to test the anti-myeloma effect of metformin alone or combined with dexamethasone in vivo.6. AMPK of MM cells was knockdown with siRNA packing in lentivirus. Then, MM cells proliferation, apoptosis and cell cycle were detected by MTT and FCM with or without metformin exposure. Gene sequencing of the lkbl/stkll gene of MM cells were detected to find whether there is mutation in MM cells.Results1. Metformin could significantly inhibit MM cells proliferation in a dose-and time-dependent. Concentrations at50%of growth inhibition (IC50) of metformin for MM cell lines at48hours were ranging from5.95mM to32.18mM. Metformin could induce MM cells apoptosis and cell cycle arrest in G1phase. The percentage of G1phase in group of control vs group of metformin were28.09±2.40%vs38.65±2.51%for RPM8226(p=0.0063);52.44±0.89%vs65.75±3.41%for MM.1S(p=0.0028);50.64±3.17%vs81.50±2.71%for MM.1R(p=0.0002) respectively.2. Metformin had a synergistic effect with dexamethasone (CI<1) but not bortezomib (CI>1) after exposure for48hours. The synergistic effect of metformin with dexamethasone or bortezomib was further detected by western blot and FCM. Results showed that the synergistic effect of metformin with dexamethasone mainly through cell cycle arrest in RPMI8226cells and apoptosis in MM.1S cells respectively.3. Metformin can activate caspase3, caspase9, PARP-1and pro-apoptotic proteins, such as Smac and Bak. Anti-apoptotic proteins, such as Mcl-1, survivin and HIAP-1, were reduced after treatment with metformin. Metformin treatment resulted in cell cycle related protein cyclin D1, CDK4, CDK6repressed and p21activated. Further study revealed that metformin could supress the expression of IGF-IR and its downstream proteins PI3K, leading to repression of phosphorylation of AKT and mTOR, which further repressed phosphorylation of4E-BP1and p70S6K. IGF-I(100ng/ml) could block metformin-induced anti-myeloma effect and attenuate the repression effect of metfomin to PI3K, pAKT, and pp70S6K for RPMI8226and MM.1S. While LY294002could exert opposite effect and enhance the repression effect of metfomin to PI3K, pAKT, and pp70S6K.4. MM cells express AMPK and pAMPK detected by western blot. Metformin could repress the expression of pAMPK.5. The human MM-SCID mice model using cell lines MM.1S was established to examine antimyeloma effects of metformin in vivo. Results show that group of metformin combined with dexamethasone could obviously lower tumor burdens as compared with groups of control, metformin alone or dexamethasone alone. the mean±SD of tumor volumes for the control, metformin alone, dexamethasone alone, and combination of metformin and dexamethasone at day21were4381±1617mm3,1816±359mm3,1371±472mm3and699±268mm3respectively (p=0.0098). However, the overall survival of SCID mice were significantly extended in metformin alone group than other groups. The median survivals were57.5,82,71and63days for the control, metformin, dexamethasone and combination groups respectively (p<0.0048).6. Knockdown AMPK could repress the proliferation of MM cells and induce MM cells cell cycle arrest in G1phase, but did not affect the apoptosis of MM cells. Knockdown AMPK could enhance the anti-myeloma effect of metformin.7. There are mutations of Ikbl/stkll gene in MM cells which result in353L>L/H. Whether this mutation has important role in activating AMPK by metformin is still less known.Conclusions1. Metformin could inhibit MM cells proliferation, induce MM cells apoptosis and cell cycle arrest in G1phase.2. Metformin displays anti-myeloma activity and synergistic activity with dexamethasone but not bortezomib.3. Metformin inhibits MM cells proliferation may through IGF-IR/PI3K/AKT/mTOR signaling pathway. Adding IGF-I could reverse metformin-induced MM cells apoptosis.4. Metformin treatment could lower tumor burdens of SCID mice especially combined with dexamethasone. Meftormin treatment had the best overall survival of SCID mice.5. MM cells express AMPK and pAMPK. Metformin treatment could reduce the expression of pAMPK.6. Knockdown AMPK could repress the proliferation of MM cells and induce MM cells cell cycle arrest in G1phase, but did not affect the apoptosis of MM cells. Knockdown AMPK could enhance the inhibition effect of metformin to MM cells.7. lkbl/stkll gene mutation is found in MM cells which results c.353L>L/H. However, whether this mutation affect activation of AMPK induced by metformin still needs further research.