Azacytidine Enhances Sensitivity Response to Imatinib in BCR/ABL positive CML Cell Line

Abstract

Azacytidine (5-Aza) is a chemotherapeutic drug that has been known to restore the expression of Tumour suppressor genes by de-methylation and shown clinical efficacy in Myelodysplastic syndrome (MDS) [1-3]. Currently, 5-Aza is being used in UK for the treatment of some adults with MDS, chronic myelocytic leukemia (CML) and acute myelocytic leukemia (AML) [4]. Majority of CML patients treated with imatinib, a BCR/ABL inhibitor would develop resistance under prolonged therapy. Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor that is constitutively activated in various human cancers including hematological malignancies. Activation of STAT3 represents an important mechanism of imatinib resistant [5]. Methylation of SHP-1 is involved in the constitutive activation of STAT3 [6], and a low level of SHP-1 is not sufficient to inhibit activated STAT3 [7]. Epigenetic silencing of SHP-1 also plays a role in the development of resistance to imatinib in BCR/ABL positive CML cells [8].

Here we evaluated the expression of SHP-1 gene and its methylation status with sensitivity response of resistant CML cell lines to imatinib before and after treatment with 5-Aza. For this purpose, BCR/ABL positive CML cell lines, K562 and K562-R, an imatinib resistant cell lines were treated with 5-Aza. Cytotoxicity of imatinib and apoptosis were determined by MTS and Annexin-V, respectively. Gene expression analysis was detected by real time-PCR, STATs activity using Western blot and methylation status of SHP-1 gene by pyrosequencing analysis. There was a significant hypomethylation of SHP-1 gene in K562-R+5-Aza cells compared to other cells (p=0.003), Table 1.

Gene expression analysis indicates a significant re-expression of SHP-1 gene (p=0.001) after treatment of K562-R cells with 5-Aza (K562-R+5-Aza cells), Fig 1. Interestingly, the re-expression of SHP-1 in K562-R+5-Aza cell lines, was associated with STAT3 inactivation and higher sensitivity to imatinib. In conclusion, 5-Aza could enhance efficacy of imatinib on BCR/ABL CML cells through re-expression of SHP-1 gene and inhibition of STAT3 signaling that could be a new target in cancers treatment.