The Study Of Trichostatin A On Imatinib Resistant Chronic Myeloid Leukemia Cells

Background Imatinb mesylate(IM) selectively inhibits the tyrosine kinase activity of BCR/ABL and is successfully used in the treatment of BCR/ABL(+) chronic myeloid leukemia(CML). But with the long-term observation, the resistance to IM is an increasing clinical problem, the drug-resisitant rate was higher than25%.and IM has limited benefit in the accelerated phase and blast crisis. To develop novel therapeutic strategies to override the obstacle is becoming more and more important.Epigenetics is to study the modulation of gene expression via DNA modification, which involved in DNA methylation, histone acetylation, chromatin remodeling and RNA interference.Considerable research efforts have focused on potentially reversible alterations in chromatin structure to modulate tumor-associated genes. The histone modifications coincide with aberrant gene expression and malignant transformation, which provides the impetus for the development of agents that target histone modifiers for cancer therapy.Previous studies have shown that treatment with HDACIs alone can inhibit IM-sensitive and IM-resistant cell lines including the T3151mutation of BCR/ABL, HDACIs can deplete the BCR/ABL expression in multiple levels.How do HDACIs modulate the tumor-associated genes expression? And if the modulations involve the DNA methylation? More information is still unclear.On the other hand, because of the modulation is uncontrolled, to date, many reports have shown that HDACIs induce multiple ATP-binding cassette transporters expression including ABCG2, which results in a very broad anticancer drug resisitant phenotype and is a big obstacle for HDACIs' utilization.The mechanism of drug transporter gene expression induced by HDACIs and how to deal with it is deserved to further exploration.TSA, which belongs to the group of hydroxamic acids, has been demonstrated to exert antileukemia effects in vitro and in vivo. Our research aimed to assess the effect of TSA on the growth of K562R (imatinib-resistance) cells, and focused on the expression and epigenetic modification of BCR/ABL, ABCG2gene promoter in TSA-inhibited K562and K562R cells.Part1:Effects of trichostatin alone and combination withimatinib on proliferation and apoptosis of the K562R cellsObjective:To explore the effects of trichostatin alone and combination with imatinib on proliferation and apoptosis of K562R cells.Method:3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) assay was used to observe the proliferation of K562R cells and apoptosis was analyzed by annexin-Ⅴ/propidium iodide(PI) staining.Result:The50%inhibition concentration(IC50) of K562cells to imatinib was0.58±0.21μmol/L, While the IC50of K562R cells to imatinib was23.88±3.45μmol/L, The drug resistant fold was41.5. After exposure to TSA, The proliferation of K562R cells was inhibited, Combined with imatinib, the effect of proliferation inhibition was better. And the effect was in time-and dose-dependent manner. There is a synergistic effect of TSA and imatinib on the proliferation inhibition of K562R cells. The apoptosis rate of imatinib group,TSA group and TSA combined with imatinib group was (4.7±1.2)%,(12.0±1.8)%and (22.1±3.5)%, The apoptosis rate of TSA group was significant higher than that of imatinib group and the apoptosis rate of TSA combined with imatinib group was significant higher than that of TSA group.Conclusion:TSA alone can inhibit the proliferation of K562R cells and induce its apopotosis. Combined with imatinib, the effectsof proliferation inhibition and apoptosis induction were better. Thereis a synergistic effect of TSA and imatinib on the proliferation inhibition of K562R cells.TSA maybe provide a new theoretical drug for imatinib-resistant patients. Part2:The expression and epigenetic modification of BCR/ABL gene in trichostatin A inhibited K562R cells.Objective:To explore the effect of TSA on the expression of BCR/ABL gene and epigenetic modification of BCR/ABL gene promoter.Methods:Real-time PCR was applied to test mRNA expression. Western blot was applied to detect the protein expression. Histone modification was investigated with chromatin immunoprecipitation (ChIP) assay. Methylation specific PCR (MSP) assay was applied to detect CpG island methylation staus.Results:Compared with K562cells, the BCR/ABL mRNA and protein were up-regulated in K562R cells. There were no significant differences of global AcH3,AcH4levels and AcH3,AcH4levels of BCR/ABL promoter between K562and K562R cells. The CpG island status of BCR/ABL promoter didn't change as compared with K562cells. After treatment with TSA on K562R cells, the BCR/ABL mRNA and protein were down-regulated. Combined with imatinib, the effect was more obviously. After treatment with TSA, the global AcH3,AcH4levels of K562R cells was up-regulated, but the AcH3,AcH4levels of BCR/ABL promoter had no significant difference. The CpG island status of BCR/ABL promoter didn't change. Conclusion:TSA combined with imatinib down-regulated the BCR/ABL mRNA and protein expression obviously, TSA maybe provide a new theoretical drug for imatinib-resistant patients. TSA had no effects on AcH3,AcH4levels of BCR/ABL promoter and the CpG island status of BCR/ABL promoter, the mechanism of TSA on BCR/ABL expression deserved further exploration. Part3:The effct of TSA on the expression and epigenetic modification of ABCG2geneObjective:To explore the effect of TSA on the expression of ABCG2gene and epigenetic modification of ABCG2gene promoter. To explore the effect of ABCG2inhibitor on the proliferation inhibition of TSA.Methods:Real-time PCR was applied to test mRNA expression. Western blot was applied to detect the protein expression. Histone modification was investigated with chromatin immunoprecipitation (ChIP) assay. Methylation specific PCR (MSP) assay was applied to detect CpG island methylation staus. MTS assay was used to observe the cell proliferation. Result:Compared with K562cells,the ABCG2mRNA and protein were up-regulated in K562R cells. There was no significant difference of global AcH3,AcH4levels between K562and K562R cells. The CpG island status of ABCG2promoter of K562R didn't change as compared with K562cells. But the AcH3level of ABCG2promoter of K562R cells was higher than that of K562cells. After treatment with TSA on K562and K562R cells, the ABCG2mRNA and protein were up-regulated. the global AcH3,AcH4levels of K562R cells was up-regulated, the AcH3, AcH4levels of ABCG2promoter were higher than those of untreated groups. Further, CpG island methylation level of ABCG2gene promoter of K562R is lower than that of K562.Conclusion:The AcH3level of ABCG2promoter of K562R cells was higher than that of K562cells; Hyperacetylation of ABCG2gene promoter may be contributed to the high expression of ABCG2gene of K562R.Higher levels of ACH3, ACH4and lower level of CpG island methylation of ABCG2promoter were observed in both cell lines after treatment with TSA. Hyper-acetylation and hypo-methylation of ABCG2promoter may co-affect to lead to up-regulation of ABCG2expression. Verapamil can enhance the proliferation inhibition role of TSA and imatinib, which implied that inhibition of ABCG2can enhance positive role and reduce the adverse effect of TSA.