Influence Of ABL SH3-T79Y Mutant Combined With Imatinib On Proliferation And Apoptosis Of Chronic Myeloid Leukeemia Cells And Its Mechanism

Researches showed the SH3 domain of BCR-ABL fusion protein of chronic myeloid leukemia (CML) cells can interact with RIN1 protein in a high specificity yet low affinitymanner which is responsible for the abnormal activation of tyrosine kinase activity and leads to CML relapse and progression. In our previous works, by using computer drug design technology we obtained ABL SH3 mutant SH3-T79Y and we found its combination with imatinib (IM) could inhibit proliferation and induce apoptosis of K562 and KCL22 cells via interacting with RIN1.In this study, in vitro we first investigated the influence of mutant SH3-T79Y combined with IM on the proliferation and apoptosis of IM-resistant CML cell line K562/G01, and then analyzed the mechanism. At last, subcutaneous tumor model and CML transplantation tumor model were established to explore the influence of SH3-T79Y in combination with IM on proliferation of KCLL22 cells in vivo.The main methods are as follows:1 Western Blot was conducted to detect the expression of p-BCR-ABL induced after the treatment of different concentration of imtinib in K562/G01 cells for determine optimal IM concentration. After combined treatment of SH3-T79Y and IM, cell-colony ability was detected by clone formation assay, changes of cell morphology were observed by Wright’s staining, cell cycles and apoptosis were assessed by flow cytometry.2 After SH3-T79Y and IM combined treatment, the expression of p-BCR-ABL, BCR-ABL, p-CrkL, CrkL, p-Stat5, Stat5, p-Akt, Akt, MEK, p-Erk, Erk, Bcl-2, Bax, c-Myc, Cyclin-D1, Caspase-8, BID and Caspase-3 protein in K562/G01 cells were determined by Western Blot to analyze the underlying mechanism.3 Combined treated KCL22 cells were injected subcutaneously into Balb/c mice for a subcutaneous model. The injection via tail vein into NOD-SCID mice constructed CML transplantation model. PBS treatment and IM alone treatment cells were injected in mice of control groups. General condition was observed. Subcutaneous tumor formation rate was calculated. Wright’s staining was used to observe the changes of blood and marrow, HE staining was carried out to evaluate tumor cell invasion in liver and spleen, PCR was conducted to detect the transcription level of bcr-abl gene and survival curve of mice was made to determine the survival rate changes.The main results and conclusion are as follows:1 K562/G01 cells were resistant to IM, the chosen concentration was 3 μM. After combined treatment, colony forming efficiency significantly decreased, cell cycles arrested at S phase, obvious apoptosis phenomenon and cell apoptosis rate significantly increased.Conclusion:SH3-T79Y in combination with IM could inhibit proliferation and induce apoptosis of K562/G01 cells in vitro.2 In K562/G01 cells SH3-T79Y combined with IM could inhibit BCR-ABL and CrkL phosphorylation, and the expression of p-Stat5/Stat5, p-Akt, MEK and p-Erk/Erk on downstream of BCR-ABL, and decrease Bcl-2, c-Myc, Cyclin-D1, but increase Bax. Besides, SH3-T79Y could induce the activation of caspase-8 and caspase-3, but it had no effects on BID.Conclusion:SH3-T79Y-induced effects were correlated with the altered expression of BCR-ABL, downstream pathway proteins and apoptosis-related proteins.3 Balb/c mice subcutaneous tumor formation rate of PBS group, IM group and combined treatment group was 100%,33.3% and 16.7% respectively. In NOD-SCID mice CML model,4 weeks after injection of KCL22 cells, the mice in PBS group gradually had physical signs of decreased reaction, depression, swollen hindlimb muscles and punctate hemorrhage on hindlimb femur. Peripheral white blood cells (WBC) quantity began to significantly increase after 5 weeks, immature granulocyte could be seen in blood smear, and tumor cells were found in liver, spleen and marrow. High expression of bcr-abl fusion gene was observed in marrow cells, survival time without therapy was about 70 days. Combined treatment group mice had good general condition, normal blood smear, no changes in liver and spleen, decreased bcr-abl gene and more than 90 days survival time. IM treatmen group mice had the results between the two groups.Conclusion:SH3-T79Y in combination with IM could inhibit proliferation of KCL22 cells in vivo, which was obviously better than IM alone.