Effects Of Chidamide And Imatinib On Proliferatio And Apoptosis Of SUP-B15 Cells And Its Related Mechanism

Background:Acute lymphoblastic leukemia?ALL?is a type of malignant clonal tumor that originates from lymphocyte precursor cells^[1].Among them,Philadelphia?Ph?chromosome can be detected in 30%-40%of adult ALL and more than 50%of elderly ALL patients,whose translocation forms the BCR/ABL fusion gene,which encodes the BCR/ABL fusion protein.The oncoprotein BCR/ABL has sustained tyrosine kinase activity,abnormally activates multiple signaling pathways,promotes cell proliferation,and inhibits apoptosis.It is a high-risk subtype with a poor prognosis.The long-term survival rate of chemotherapy alone does not exceed 10%.Although allogeneic hematopoietic stem cell transplantation?allo-HSCT?based on tyrosine kinase inhibitor?TKI?such as imatinib and targeted inhibition of BCR/ABL combined with chemotherapy and first complete response?CR?was Ph⁺ALL Patients bring significant results.However,due to the high transplant-related mortality and the lack of HLA-matched donors in most patients,allo-HSCT is difficult to implement clinically on a large scale.Relapse and drug resistance are important challenges in current treatment.Therefore,we urgently need to explore new treatment strategies to improve the prognosis of patients.Most scholars believe that the occurrence of malignant hematological disease is the result of the combined effect of cytogenetics and epigenetics.Epigenetics mainly changes transcription and post-transcriptional gene expression through DNA methylation,histone modification,and microRNA.Histone decatlase?HDAC?expression was increased in most tumor cells,and histones showed a low acetylation state.The appearance of histone deacetylase inhibitor?HDACI?can inhibit the high expression of HDAC activity in tumor cells,and then up-regulate the level of histone acetylation,regulate the transcription of related genes,and thus hinder tumor progression.As a class of chemotherapeutic drugs,HDACi can inhibit the proliferation of a variety of tumor cells,promote cell differentiation and induce apoptosis,and overcome some of the drug resistance of tumor drugs.Therefore,it is possible to combine HDACi with other antitumor drugs such as TKIs.It provides new ideas for targeted treatment of hematological malignancies.Objective:In this study,we investigate the effects of Imatinib?IM?,Chidamide?CS055?single drug and combination on human acute lymphoblastic leukemia cell line SUP-B15?Ph⁺?,and to observe its proliferation inhibition and induction of apoptosis The preliminary research on STAT3-related pathways provides experimental basis for finding effective methods for treating Ph⁺ALL.Methods:1.CCK-8 method was used to determine the cell growth inhibition of Ph⁺ALL cell line SUP-B15 at 24h,48h and 72h after IM,CS055 and two drugs combined intervention.2.FCM was used to detect the apoptosis of single-agent IM,CS055 single-agent and the combination of two drugs for SUP-B15 cells after 48h.3.Observe the expressions of BCR-ABL,STAT3,P-STAT3,SOCS3 mRNA by RT-PCR method after the single drug IM,CS055 and the two drugs are used for SUP-B15 cells48h.4.Western blot analysis was used to determine the expression of Ac-H3,STAT3,P-STAT3,and SOCS3 protein in single-agent IM,CS055 and the combination of two drugs in SUP-B15 cells 48h.Results:1.CCK-8 test results showed that IM and CS055 monotherapy had inhibitory effect on the proliferation of SUP-B15 cells and had a concentration-time dependence?P<0.001?.At the 48h time point,the IM concentration was 5.0?mol/L?less than IC50?,and the CS055concentration was 5.0?mol/L?less than IC50?.The single drug and the combination of the two drugs?IM 5.0?mol/L+CS055 5.0?mol/L?It has obvious proliferation inhibition effect on SUP-B15 cell line,and the inhibition rate of the two drug combination group is higher than that of each single drug group,respectively.According to the Calcusyn 2.0 software,the combined effect index of 0.45 was highly synergistic.2.FCM results showed that after different drugs were applied to SUP-B15 cells for 48hours,the average apoptosis rate of the blank control group was?0.45±1.16?%,and the average apoptosis rate of the CS055 group?5.0?mol/L?was?8.98±1.68?.%,IM group?5.0?mol/L?had an average apoptotic rate of?23.59±1.01?%,and the two drugs combined in the group?IM 5.0?mol/L+CS055 5.0?mol/L?had an average apoptotic rate?34.12±2.98?)%,The apoptotic rate of the combined drug group was significantly higher than that of the control group and each single drug group?P<0.05?.3.Real-time fluorescence quantitative polymerase chain reaction?RT-PCR?results showed:CS055 group?5.0?mol/L?,IM group?5.0?mol/L?,and combined group?IM 5.0?mol/L+CS055 5.0?mol/L?after 48 hours of action on SUP-B15 cells,the expressions of STAT3 mRNA and BCR-ABLmRNA in the combined drug group were significantly lower than those of the single drug group,and the SOCS3 mRNA was significantly higher than that of the control and single drug group?P<0.05?4.Western Blot test results showed that CS055 group?5.0?mol/L?,IM group?5.0?mol/L?and combination group?IM 5.0?mol+CS055 5.0?mol/L?acted on SUP-B15 cells for48h.After that,the expression of STAT3 and its phosphorylated active form P-STAT3 protein was significantly down-regulated compared with the blank control group,and the levels of Ac-H3 and SOCS3 protein were significantly up-regulated,and the difference was statistically significant?P<0.01?.In the two-drug combination group,the levels of STAT3and its phosphorylated active form P-STAT3 protein were down-regulated,and the levels of Ac-H3 and SOCS3 proteins were up-regulated more significantly than those of the single-agent group,and the difference was statistically significant?P<0.01?.Conclusions:1.CS055 and IM alone can inhibit the proliferation and promote apoptosis of Ph⁺ALL cell line SUP-B15.The two have a synergistic effect.2.After CS055 combined with IM intervention in SUP-B15 cells,the expression of STAT3,BCR-ABL decreased,and the expression of Ac-H3 and SOCS3 increased.