The Effects And Mechanisms Of A Novel PDK1 Inhibitor On Multiple Myeloma Cells

Part I:The effects and the mechanisms of PDK1 inhibitor GSK2334470 alone or combined with mTOR inhibitor PP242 on multiple myeloma cellsObjective:To study the effects and mechanisms of GSK2334470 on multiple myeloma cells;To explore the mechanisms of synergistic apoptosis induced by GSK2334470 and mTOR inhibitor PP242.Methods:Cell growth was determined by MTT assay.Apoptosis was determined by Annexin V-FITC flow cytometry technique.The expression of PARP,caspase-3/8/9 and PI3K/PDK1/Akt/mTOR signaling pathway associated proteins were examined by western blotting.Furthermore,we investigated the effect of GSK2334470 and PP242 in vivo.Results:1.GSK2334470 could inhibit the proliferation of MM cells and primary cells in a dose-dependent manner.Human normal cells were much less sensitive to GSK2334470.IL-6 or IGF-1 could not protect against GSK2334470-induced growth inhibition.3.GSK2334470 could induce apoptosis in MM cells and primary cells by activating Caspase-3/8/9 and PARP.4.GSK2334470 could regulate PI3K/Akt/mTOR pathway by inhibiting the phosphorylation of PDK1.5.PP242 sensitized multiple myeloma cells to GSK2334470-mediated cell death.6.Combined GSK2334470 and PP242 resulted in a complete inhibition of mTORC1/C2 and full activity of Akt.7.Combination treatment with GSK-470 and PP242 was proved very efficacious as shown by significant inhibition of tumor growth compared with GSK2334470 or PP242 alone.Part Ⅱ:Study the importance of PTEN gene in the anti-myeloma effect of PDKl inhibitor GSK2334470Objective:To study and compare the growth inhibition and apoptosis induced by GSK2334470 on cell lines of MM,and explore the relationship with PTEN expression.To explore the mechanisms of synergistic apoptosis induced by GSK2334470 and proteasome inhibitor MG 132.Methods:Cell growth was determined by MTT assay.Apoptosis was determined by Annexin V-FITC flow cytometry technique.The mRNA expression level of PTEN gene were examined by PCR.The expression level of PTEN in MM cells were changed by infection with adenovirus.The expression of PTEN in cytoplasm,in nucleus and PI3K/PDK1/Akt/mTOR signaling pathway associated proteins were examined by western blotting.The subcellular colocalization of PTEN in RPM18226 was examined by laser scanning confocal.Results:1.The cytotoxicity efficacy of GSK2334470 was closely related with the expression of PTEN.ARP-1 and MM.1R cells display more sensitive to GSK2334470 than RPMI8226 and OPM-2 cells that contained lower expression of PTEN.2.PTEN knockdown resulted in drug resistant while restoration of PTEN expression led to increased cell death in response to GSK-470 treatment.3.MG-132 sensitized multiple myeloma cells and primary cells to GSK2334470-mediated cell death.MG 132 enhanced the ability of GSK2334470 to inhibit the PI3K/Akt/mTOR pathway by increasing the expression of PTEN.4.Enhanced PTEN accumulation in the nucleus induced by GSK2334470 play a crucial role in synergistic effect between GSK2334470 and MG-132.Conclusion:GSK2334470 could regulate PI3K/Akt/mTOR pathway by inhibiting the phosphorylation of PDK1,then induced apoptosis in MM cells.Combining PP242,a dual mTORC1/C2 inhibitor,with GSK2334470,had greater antimyeloma activity than either one alone in vitro and in MM xenograft model established in immunodeficient mice.Combined GSK2334470 and PP242 resulted in a complete inhibition of mTORC1/C2 and full activity of Akt.More importantly,we found that the cytotoxicity efficacy of GSK2334470 was closely related with the expression of PTEN.GSK2334470 increased the nuclear accumulation of PTEN via decreasing the phosphorylation of its C-terminal tail.Combination of GSK2334470 with proteasome inhibitor MG 132 increased the expression and nuclear accumulation of PTEN,completely inhibited the activity of Akt and mTOR,thus,promoted cell death.