Effects And Mechanism Of Molecular Targeting Drugs 17-AAG And RO3280 In Acute Myeloid Leukemia

Part one.HSP90 Selective Inhibitor 17-AAG Induces Apoptosisin Acute Myeloid LeukemiaObjective:Investigating theapoptosis effectand molecular mechanism of Heat Shock Protein 90 inhibitor 17-AAG on the human acute myeloid leukemia cell lines, to lay the foundation for further clinical study.Methods:The inhibitory effects of 17-AAG on AML cell lines HL60 and NB4 after 24 h were measured by CCK-8 test. Annexin V-FITC/PIstaining followed by flow cytometric analysis was used to detect apoptosis inexposure to 17-AAG. Propidium iodide staining followed by flow cytometric analysis was used to detect cell cycle disorder. Moreover, to clearly demonstrate that 17-AAG causes apoptosis in AML cells, we assessed the following well-recognized markers of apoptosis:PARP and Caspase-3. After 24 h of treatment with 17-AAG(5μM), we used the SABioscience Human Apoptosis PCR Array PAHS-3012 to analyze the expression of 370 genes associated with apoptosis in DMSO or 17-AAG treated NB4 cells.Results:17-AAG decreased HL60and NB4 cell viability in a dose-dependent manner. Cell cycle assay showed that 17-AAG significantly inducesdcell cycle arrest in phase G2/M,and with the raised concentrations and time some showed significantly cell cycle disorder.Annexin V staining showed that with the increasing concentrations and/or time, the apoptosis rate increased significantly. In NB4 cells the apoptosis rate was:DMSO group 6.0%±0.5%,17-AAG 5μM group 36.5%±2.2%,17-AAG lOμM group 46.9% ±3.2%; In HL60 cells the apoptosis rate was:DMSO group 2.7%±0.7%,17-AAG 5μM group 16.7% ±2.6%,17-AAG lOμM group 30.8%±1.6%.After 24 h of treatment with 17-AAG (5 or 10μM), we found that the expression of c-PARPwas increased following the increased concentration. But we did not found the c-Caspase-3. The real time PCR array data revealed that 56 genes were significantly upregulated and 23 genes were significantly downregulated in 17-AAG treatment group compared with DMSO control group.Conclusion:17-AAG can inhibit the proliferation of leukemia cells, induce apoptosis and arrestcell cycle in G2/Mphase.These effects are mediated by a variety of apoptosis related genes.Part two. Molecular Targeting of the Oncoprotein PLK1 in Pediatric Acute Myeloid Leukemia:RO3280, a Novel PLK1 Inhibitor, Induces Apoptosis in Acute Myeloid Leukemia CellsObjective:To investigate theapoptosis effectand the mechanismofPolo-like kinase 1 inhibitor RO3280on the human acute myeloid leukemia cells, and lay the foundation for further clinical study.Methods:The expression of PLK1 in leukemia cell lines, pediatric AML and control patients was detected via Western Blot.Real-time PCR was used to examine the mRNA transcript levels of PLK1 in 105 pediatric AML samples and 30 Non leukemia patients.The inhibitory effects of RO3280, Rigosertib(ON O191O.Na) and BI2536 on acute leukemia cells were measured by CCK-8 test. We also determined the effectiveness of RO3280 in primary leukemia cells.After 24 h of treatment with RO3280 (50 or 100 nM), we assessed Annexin V-FITC/PI staining, cell cycle and hoechst 33342 stainingin leukemia cells.Moreover, to clearly demonstrate that RO3280 causes apoptosis in leukemia cells, we assessed the following well-recognized markers of apoptosis:PARP, Caspase-3, and Caspase-9. In order to identify apoptosis molecules, we used the SABioscience Human Apoptosis PCR Array PAHS-3012 to analyze and clusterthe expression of 370 genes associated with apoptosis in DMSO or RO3280 treated cells.Finally we validated some genes in the protein level.Results:Our study showed that the expression of PLK1 is very high in leukemia cell lines.And the high protein expression was observed in 73.3%(11/15) of the pediatric AML samples compared to 0%(0/12) of the normal bone marrow (NBM) control samples.Real-time PCR showed that PLK1 expression was significantly higher in the AML samples compared to.the control samples (106 × Log 2 (-ΔCt):82.95 ± 110.28 vs.6.36 ± 6.35; p< 0.001). The novel PLK1 inhibitor RO3280 decreased the viability of leukemia and primary leukemia cells in a dose-dependent manner.The 50% inhibitory concentration (IC50) of RO3280 for acute leukemia cells was between 74 and 797 nM. In primary AMLcells the IC50 of RO3280 was between52.80 and 147.50 nM. In NB4 cells the IC50 of the PLK1 inhibitors were:RO328013.45 nM, ON 01910. Na 13.02 nM, and BI253687.65 nM and in K562 cells:RO3280 301 nM, ON 01910. Na 1606 nM, and BI2536 448 nM, which demonstrated that RO3280 was the most sensitive in NB4 and HL60 cells.Cells treated with RO3280 for 24 h showed higher Annexin V staining compared with untreated cells. This indicates that RO3280 induces apoptosis in leukemia cells. Cell cycle assay showed that RO3280 significantly induces cell cycle arrest in phase G2/M and some showed significantly cell cycle disorder.Hoechst 33342 staining showed that 24 h RO3280 treatment induced DNA fragmentation and the formation of abnormal nuclear cells.After 24 h of treatment with RO3280 (50 or 100 nM), we found that the expression of c-PARP, c-Caspase3^ c-Caspase9 was increased following the increased concentration.The real time PCR array data revealed that 32 genes were significantly upregulated and 16 genes were significantly downregulated in RO3280 treatment group compared with DMSO control group.The RO3280-dependent upregulation of DCC and CDKNIA and downregulation of BTK and SOCS2 were verified by western blot analysisConclusion:RO3280 can inhibit the proliferation of leukemia cells effectivelyand induce apoptosis and cell cycle disorder.These effects are mediated by a variety of apoptosis related genes.