| Chronic myelocytic leukemia(CML) is a myeloid hyperplasia as the main manifestation of malignant clonal disease.The CML treatment’s goal is to eliminate symptoms,control blood and genetic abnormalities,mean while prolonging the over all survival. CY03529 is Dihydrogen coumarone amide-type new entity compounds of anti-CML, which is designed on the basis of Imatinib (Glivec, IMA-3),which is a me. In the current study, we investigated CY03529 had inhibition on K562 and induced apoptosis, which provided theoretical foundation for the development of low toxicity and high efficiency anti-CML drugs.In accordance with the mechanism and the targets of imatinib, the bcr/abl kinase, c-Kit kinase, and bcr/ab1T3151 mutant kinase were selected, docking with the tetrahydronaphthalene amides,through the scoring process, CY03529 was proved to be the hit compound. CY03529 docked with the shared targets bcr/abl kinase and c-Kit kinase of imatinib. The results showed that bcr/abl average score (14.597) of CY03529 was higher than bcr/abl kinase interaction average score (8.546) of imatinib; CY03529 interacting with c-Kit (15.077) was also significantly higher than the average scoring (6.361) of imatinib; CY03529 interacting with bcr/ab1T3151 mutant kinase (13.805) was also significant higher than the average scoring (7.581) of imatinib.The theory principle of molecular docking were utilized to finding the better site. In order to examine the growth characteristics of cells, we drew cell growth curves by hematimeter; MTT viability assay, double Soft Agar Cloning and Giemsa’s staining were applied to observe the changes of cell proliferation; Induction of apoptosis and cell cycle were estimated using DNA ladder analysis and flow cytometry with propidium iodide (PI) staining, following 48 h cell treatment with various doses of CY03529; Moreover, the molecular mechanism was detected by the genetic expression of bcr/abl and the protein expression of P210ber-abl using RT-PCR and Western Blotting;The obtained results are as follow:1. The molecular docking results showed that CY03529 combined with Sitel and Site4 of bcr/abl kinase was much better than others.2. The preliminary study of double time about K562 was 1.60 days respectively. Cell inoculation and incubation time was 2～4X 104cell/mL,1～2 days. The IC50 of CY03529 for K562 was 24.83nmol/L. Dose-response curves showed that K562 was inhibited by CY03529 in dose-dependent and time-dependent manners.3. The inhibition rates of colony formations about CY03529 was greater than 70% in double Soft Agar Cloning experiment. The results of Giemsa’s staining indicated that CY03529 could induce K562 to normal cells in concentration-dependent manner. The results of AO-EB double staining showed that apoptotic body increased, when the cells were treated with constantly-increased concentrations of drugs. Apoptosis phenomenon obviously increased.4. The cell cycle distribution by FACS showed: the cells were in the state of G1 phase. Agarose gel electrophoresis showed that significant characteristic DNA ladders appeared (180-200bp)after treated with CY03529 which accorded with the feature of apoptosis. The Western Blotting experiment showed that expression of P210ber-abl decreased after incubation with drugs in different concentrations for 48h.In summary, CY03529 has a good anti-tumor activities and it can be used as a further object of study. |
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