Study On Antitumor Activity And Mechanism Of A New Generation Of Tetrahydronaphthalene Amides Pocyclotinib(CY03529B) To Drug-resistant Cells K562/G01 And GIST-1210

At present,the development and marketing of tyrosine kinase inhibitors targeting Bcr-Abl fusion genes for chronic myeloid leukemia(CML)has revolutionized the treatment of CML.As the first generation of tyrosine kinase inhibitors,imatinib can significantly improve the efficacy and prognosis of CML patients,but with the treatment process,drug resistance gradually appeared.In this regard,the second generation of tyrosine kinase inhibitors,nilotinib and dasatinib,have been developed,but they still have no effect on T315I mutation,and imatinib resistance has also appeared in the treatment of gastrointestinal stromal tumor(GIST).Therefore,the development of new small molecule targeted drugs to solve the problem of Imatinib resistance has become the focus of research.The novel small molecule compound CY03529B was a new type of mother nucleus structure by combining the theory of computer-aided drug design and analyzing the structure and actin of protein.The aim of this study was to investigate the antitumor activity and its mechanism of K562/G01 cell line and GIST-1210 cell line resistant to imatinib.Firstly,the MTT method was used to draw the growth curve of tumor cells,and to understand the logarithmic growth period of cells.And the drug resistance multiple of the two tumor cells was detected,the resistance multiple of K562/G01cells was 9.35 times,and the resistance multiple of GIST-1210 cells was 6.18 times.The quantitative effect curve of the two drug-resistant cells was plotted according to the tumor cell proliferation inhibition rate in vitro,and the selectivity of the compounds to other common tumor cells was further detected.The inhibition of Bcr-Abl and c-Kit and their associated mutant kinases was detected by MSA method.the results showed that CY03529B had a strong inhibitory effect on both kinases and their associated mutant kinases at 1μM concentration.The effect of CY03529B on colony formation rate of drug-resistant tumor cells K562/G01 and GIST-1210 was examined by soft agar clone formation experiment.The results showed that the two drug-resistant tumor cells were highly sensitive to CY03529B and significantly superior to Imatinib at the same concentration.The ability of tumor cells to migrate was determined by examining the effect of CY03529B on the migration ability of GIST-1210 cells in the degree of"scratch"healing in the scratch test.The results showed that CY03529B significantly inhibited the migration ability of GIST-1210 cells with time and concentration dependence.Based on the above in vitro antitumor activity experiments,we further examined the antitumor effect in CY03529B in vivo.A model of resistant human-derived chronic myeloid leukemia NOD/SCID mices were established.the volume of solid tumor reached more than160 mm~3.After 24 days of administration of CY03529B,the volume of solid tumor in the mice in the CY03529B administration group became significantly smaller,and the effect of the low-dose group of 50 mg/kg was better than that in the high-dose group ofImatinib 100 mg/kg,and there was no significant loss of death or weight in the mice.The results of in vivo experiments are basically consistent with those of in vitro activity,indicating that CY03529B has good in vivo and in vitro activity and good in vivo safety for drug-resistant tumor cells.To further investigate the mechanism of CY03529B on drug-resistant tumor cells K562/G01 and GIST-1210,we first observed the effect of CY03529B on cell morphology by AO-EB double staining,indicating that CY03529B can induce apoptosis of two drug-resistant tumor cells.Then the effect of CY03529B on apoptosis of the two drug-resistant tumor cells was quantitatively detected by flow cytometry(FCM).The apoptosis rate of CY03529B on K562/G01 cells and GIST-1210 cells was significantly higher than that of the positive drugImatinib group,and was consistent with the results of AO-EB staining.Flow cytometry was used to detect cell cycle changes of drug-resistant tumor after administration.The results of the study found that the prognosis of CY03529B stem increased the G0\G1 phase of the two drug-resistant tumor cells significantly,and could not enter the stage of synthesis,inhibit the proliferation of tumor cells,and the effect was better than that of Imatinib,and showed a concentration-dependent effect.At the same time,western blot found that CY03529B could inhibit the expression of Bcr-Abl protein and c-Kit protein on the one hand and regulate the intracellular signaling pathway on the other.And RT-PCR experiments proved that CY03529B significantly up-regulated the expression of Caspase-3 and Caspase-8 genes induced apoptosis,and simultaneously down-regulated Bcr-Abl and c-Kit genes to inhibit cell proliferation.In conclusion,the results showed that CY03529B had good antitumor activity against imatinib resistant K562/G01 cell line and GIST-1210 cell line in vitro and in vivo.The mechanism of action was to down regulate pathogenic genes and proteins,up regulate apoptosis genes caspase-3 and caspase-8,and inhibit phosphorylation of P-STAT5 and P-Crkl by inducing apoptosis,blocking G0/G1 phase.These results provide a theoretical basis for further clinical research and development of a more safe and effective imatinib resistant tyrosine kinase inhibitor.