Killing Effect And Its Mechanisms Of Micro-tubule Protcin Inhibitor CYT997on Acute Myeloid Leukemia Cell

Aim: To study cytotoxic effects and apoptosis induced by microtubule targeting agentsCYT997on acute myeloid leukemia cells lines,so as to provide the theory for the clinicalapplication of acute myeloid leukemia.Methods: The effect of CYT997on AML cells was measured by MTT assay. AnnexinV-FITC/propidium iodide flow cytometry technique was used for detecting apoptosis. Hoechststaining was used to observe the morphological changes of HL-60cells. Cell cycle retardationwas detected by flow cytometry. The expression of caspase-3,-8,-9, PARP and other signalpathway associated proteins in AMLcell lines were examined by Western blotting.Results:（1）The MTT colorimetic assay showed that CYT997significantly inhibited thegrowth of AML cell lines HL-60、KG-1、THP-1、Kasumi-1and HEL from12.5nM to200nM.24h IC50was98.69、125.84、160.61、141.42、213.98nM. And48h IC50was60.75、111.38、96.06、71.43、134.33nM.（2）Treated with different concentrations of CYT997for48h, apoptoticbody appeared obviously in HL-60cells nucleus and in a dose-dependent manner, whichconfirmed that CYT997could induce apoptosis in AML cell lines.（3）After treatment withdifferent concentrations of CYT997for24h, the flow cytometry showed that the apoptosis ratioin HL-60cells was2.81%、3.3%、3.59%、16.15%, respectively, which indicated that CYT997could effectively induce apoptosis in HL-60cell.（4）Treated with CYT997for24h, PI staining,flow cytometry displayed the cells ratio in G₂/M phase was significantly increased withincreasing concentrations of CYT997. The ratio was9.87%,11.61%,51.88%and68.11%,respectively which indicated that CYT997could block cells at the G₂/M phase.（5）As measuredby western-blot, we found that caspase-3、-8、-9and PARP were cleaved and activated in HL-60cells. Also, the cdc25c and PP2A B subunit were down regulated and the mTOR and PI3Kâ…¢were inhibited. In addition, it could control NF-κB and XIAP.（6）After treated with thecombination of CYT997with Caspase-8inhibitor Z-IETD-FMK, flow cytometry showed theapoptosis rates of the combined treatment group was lower than the drug alone treatment groupin HL-60cells. From the opposite side, it showed CYT997could induce AML cells to undergoapoptosis by activating the exogenous pathway of apoptosis.Conclusions: CYT997activated both the exogenous pathway and mitochondrial pathway to induce apoptosis of HL-60cell. It blocked HL-60cells at the G₂/M phase via inhibiting cellcycle-related proteins and inhibited cell growth. Also, it could inhibit the expression of mTORand NF-κB.