Re-Expression of Bone Marrow Proteoglycan-2 by 5-Azacytidine is associated with STAT3 Inactivation and Sensitivity Response to Imatinib in Resistant CML Cells

Abstract

Background: Epigenetic silencing of tumor suppressor genes (TSG) is involved in development and progression of cancers. Re-expression of TSG is inversely proportionate with STAT3 signaling pathways. Demethylation of DNA by 5-Azacytidine (5-Aza) results in re-expression of silenced TSG. Forced expression of PRG2 by 5-Aza induced apoptosis in cancer cells. Imatinib is a tyrosine kinase inhibitor that potently inhibits BCR/ ABL tyrosine kinase resulting in hematological remission in CML patients. However, majority of CML patients treated with imatinib would develop resistance under prolonged therapy. Methods: CML cells resistant to imatinib were treated with 5-Aza and cytotoxicity of imatinib and apoptosis were determined by MTS and annexin-V, respectively. Gene expression analysis was detected by real time-PCR, STATs activity examined using Western blot and methylation status of PRG2 was determined by pyrosequencing analysis. Result: Expression of PRG2 was significantly higher in K562-R+5-Aza cells compared to K562 and K562-R (p=0.001). Methylation of PRG2 gene was significantly decreased in K562-R+5-Aza cells compared to other cells (p=0.021). STAT3 was inactivated in K562-R+5-Aza cells which showed higher sensitivity to imatinib. Conclusion: PRG2 gene is a TSG and its overexpression might induce sensitivity to imatinib. However, further studies are required to evaluate the negative regulations of PRG2 on STAT3 signaling.

Keywords: CML; PRG2; imatinib; 5-Azacytidine; STAT3.

Figure 1

Cell Growth Inhibition by Imatinib in K562, K562-R and K562-R+5-Aza Cells. MTS result shows a significant lower of IC50 of imatinib on K562-R+5-Aza cells (290 nM) compared to K562-R cells (3953 nM). The cells were exposed to serial concentration of imatinib for 72 hours and the IC50 was quantified by cell proliferation assay. Each result is presented as the median percentage of proliferation to unexposed control cultures.

Figure 2

The Profile Plot of Apoptotic Cells for Serial Concentrations of Imatinib. Repeated measure ANOVA between groups based on concentrations was applied. The profile plot shows the adjusted mean (estimated marginal means) of apoptotic cells for serial concentrations imatinib. Although the mean percentage of apoptotic cells before incubations with imatinib were almost equal for K562, K562-R-pkc and K562-R+5-Aza cells, there was a sharp increase in K562 and K562-R+5-Aza apoptotic cells with an increase in imatinib concentrations to reach 49 and 79%, respectively at 400 nM. In contrast, there was no significant increase in the apoptotic cells in K562-R with an increase in concentration to reach only 19% apoptosis at 400 nM.

Figure 3

Expression of PRG2 Gene was Restored in K562-R+5-Aza Cells. Real-time quantitative PCR (RQ-PCR) results revealed re-expression of PRG2 with the highest fold changes in K562-R+5-Aza cells compared to in K562-R cells. The results shows more than 5000 times higher of PRG2 expression in K562-R+5-Aza cells compared to K562-R cells but no restoration of expression of SOCS-1 or SOCS-3 in these cells.

Figure 4

Low Methylation of PRG2 Gene in K562-R+5-Aza Cells. Pyrosequencing results show a significant lower in methylation level of PRG2 genes in K562-R+5-Aza cells compared to K562 and K562-R cells (p=0.021). However, there was no significant difference in methylation of PRG2 genes between K562 and K562-R cells (p=0.284).

Figure 5

Activation of STAT3 in Resistant Cells. Western blot analysis depicts expression of STAT1, STAT3 and STAT5 proteins in K562, K562-R and K562-R+5-Aza cells. Although, STAT3 was phosphorylated in K562-R cells, it showed de-phosphorylation in K562-R+5-Aza cell. However, STAT1 and STAT5 display no differences in phosphorylation status in all cell lines.