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Title: Characterisation of the effects and mechanism of action of rapamycin and genistein on acute myeloid leukemia using high-throughput techniques
Keywords: Acute Myeloid Leukemia, Genistein, Rapamycin, Microarray, iTRAQ, Mechanism, High-throughput
Issue Date: 1-Jul-2010
Citation: KARTHIK NARASIMHAN (2010-07-01). Characterisation of the effects and mechanism of action of rapamycin and genistein on acute myeloid leukemia using high-throughput techniques. ScholarBank@NUS Repository.
Abstract: Acute Myeloid Leukemia (AML), caused by the uncontrolled proliferation of the leukocytes of the myeloid lineage, is a cancer with a very high mortality rate. Present therapies to treat AML include chemotherapy and bone marrow transplant. These methods suffer from certain inherent limitations such as heavy cytotoxicity and innocent bystander effects in the case of the former and acute allograft rejection in the latter. Hence there is an urgent need for more effective therapeutic strategies. In this study, we have evaluated the efficacy of two such potential therapeutic drugs, namely Rapamycin (rapa) and Genistein (GEN) and have characterised their mechanism of action using high-throughput strategies. A combination of microarray and 4-plex iTRAQ based approach was adopted to study the effects of rapa on MV4-11 and THP-1 cells and an 8-plex iTRAQ based methodology was employed to profile the proteome of the MV4-11 and HL-60 cells treated with GEN. We found that rapa had potent anti-proliferative effect on all the AML cell lines tested. We chose the cell lines with the lowest and highest IC50, MV4-11 and THP-1 respectively, for functional characterisation. High-throughput studies indicated that rapa regulates Cell cycle, IGF-1 and FGF signalling, death receptor signalling, protein ubiquitination and hypoxia signalling pathways. Functional studies showed that rapa did not induce apoptosis but effected a time-dependent G1 arrest, with the peak inhibitory effect at 16 h. Interestingly, rapa down-regulated IGFBP2, usually elevated in AML patients. Our study showed that rapa represses Skp2, an important constituent of the protein ubiquitination pathway. Working on this clue, we identified that the time dependent G1 arrest is in fact the result of the inhibition of Skp2, leading to the accumulation of p27, which in turn causes repression of Cdk2 and Cdk4. In the second study, we found that GEN had inhibitory effects on both the MV4-11 (IC50 20µM) and HL-60 (IC50 30µM) cells. We discovered that GEN inhibited the constitutive phosphorylation of FLT3 in the MV4-11 cells, which carry the FLT3-ITD (Internal Tandem Duplication) mutations. However, GEN had potent anti-leukemic effects on the HL-60 cells too, in spite of them possessing the wild-type version of the gene. A purely proteomic-based approach, using the 8-plex iTRAQ strategy, was employed to understand the dynamics of GEN¿s effects on the two subsets of AML. We found that GEN down-regulated the mTOR pathway, thus arresting protein synthesis in the AML cells. GEN up-regulated Akt, leading to elevation in the reactive oxygen species (ROS) levels which in turn caused apoptosis. While HL-60 underwent a caspase- mediated cell death, the apoptosis in MV4-11 was caspase independent. GEN induced arrest at the G2/M phase of the cell cycle in HL-60 while it caused a moderate G1 arrest in MV4-11. We can attribute these differences in the mechanism of action of GEN to the FLT3 mutational status of the two cell lines. Hence, we conclude that GEN has an all encompassing anti-proliferative effect on AML irrespective of its FLT3 mutational status and is an ideal candidate for clinical trials.
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