Antitumor Activity Of Trifluoromethyl Benzamides XX03529 In Human Chronic Myelogenous Leukemia Cell Line K562 And Its Mechanism Of Action

Imatinib,a first-line drug for the treatment of chronic myeloid leukemia,has some problems such as patient intolerance and frequent occurrence of drug resistance,which has led to the development of new targeted therapeutics for such small molecules.Although some new generation inhibitors applied in patients with mutation,its strong side effects make the patient’s life hardly extended,even inducing more danger.Moreover,there are no small molecule inhibitors on the market available for the CML patients with T315I mutation and other domains mutations are unpredictable.Therefore,we turned our attention to the development of more potent and effective Bcr-Abl tyrosine kinase inhibitors based on a trifluoromethylbenzamide core,which differ from benzamide-core of Imatinib.The trifluoromethylbenzamide compound XX03529 is a novel compound entity,which was obtained by optimizing and reforming the structure of Imatinib through the rational drug design theory and skeleton transition method.The purpose of this study was to investigate the anti-tumor activity of XX03529 and its mechanism of action.The inhibitory effect of XX03529 on the proliferation of chronic myeloid leukemia K562 cell line,human lung cancer A549 cell line,human gastric cancer MKN45 cell line and human gastrointestinal stromal tumor GIST882 cell line were tested by MTT viability assay.The IC₅₀0 values of these four tumor cells were 0.019±0.011μmol/L,8.319±5.361μmol/L,7.577±2.943μmol/L and 5.353±1.742μmol/L,respectively.This result demonstrates that XX03529 is highly selective for chronic myelogenous leukemiaK562cells,itscellproliferationinhibitoryeffectwas concentration-dependent and time-dependent,and this inhibitory effect was 11.9-folds than Imatinib.The results of soft-agar colony formation assay showed that XX03529inhibited the colony formation of K562 cells more strongly than the positive control drug,indicating that K562 cells were highly sensitive to this trifluoromethyl benzamide compound.The results of Wright-Giemsa staining showed that treated K562 cells were differentiated into mature cells after 24 h and cells showed apoptotic morphological changes after 48 h.Therefore,it initially determined that XX03529 can induce apoptosis of cells by inducing differentiation of cells into mature cells.In addition,the change of cell cycle distribution after 24 h administration was detected by FCM,and the results showed that the cells in the administration group were arrested in the G0/G1 phase and the number of cells in the S phase was significantly reduced.It was verified that XX03529 could induce differentiation of K562 cells,and XX03529 had a stronger inhibitory effect on cell cycle than Imatinib.The results of AO-EB staining showed that the induction effect of XX03529 on apoptosis was concentration-dependent,and the apoptosis rate of cells in the XX03529-administered group was significantly higher than that of Imatinib,especially in the treatment of 0.1μM.The results of agarose gel electrophoresis showed that the unique DNA gradient bands of apoptotic cells,which were observed in the treated group.The statistical results of flow cytometry Annexin-V-FITC and PI double labeling assays revealed that the apoptosis rate of XX03529 treated cells was time-dependent,concentration-dependent increased and stronger than the positive drug.The grey level analysis of RT-PCR results showed that XX03529 has a concentration-dependent up-regulation effect of the expression of apoptosis genes Caspase-3 and Caspase-8 in K562 cells,and the effect of the above modulation was significantly higher Imatinib.At the same time,the effect of XX03529 on the expression of BCR-ABL gene and the relative expression level of the encoded pathogenic proteins were investigated in the present study from the gene and protein levels.The results showed that XX03529 showed a concentration-dependent down-regulation of the expression of pathogenic genes and pathogenic proteins in K562 cells,and its effect was significantly higher than that of Imatinib.In addition,by using computer-aided drug design software,molecular docking mimics the conformation of receptor proteins and small molecule compounds.The simulation results predict that XX03529 is selectively bound to the catalytically active domain of Abl kinase in the non-activated state,respectively,with Thr315,Glu286and Asp381 form three hydrogen bonds,and trifluoromethylphenyl groups have obvious extension to hydrophobic regions（Ile293,Leu298,Leu354 and Val379）.Meanwhile,the affinity of XX03529 to the Abl kinase in the"DFG-out"conformation is higher than that of Imatinib.All in all,these experimental results provide a theoretical basis for the future development of a novel tyrosine kinase inhibitors.