Down-Regulation of HO-1 Promoted Apoptosis Induced By Decitabine Via Increasing p15^INK4B Promoter Demethylation in myelodysplastic syndrome

Background: Decitabine, reverts hypermethylation of p15^INK4B gene in vitro, was used to improves cytopenias and blast excess in over 50% of patients with high-risk myelodys plastic syndrome (MDS). In this study, over-expression of Heme Oxygenase-1(HO-1) was found in MDS cells line SKM-1 cells, and it was closely related to resistance to apoptosis induced by decitabine.

Objective: we aimed to further investigated what role of HO-1 exactly played in apoptosis induced by low-does of decitabine in MDS.

Method: CCK-8 kits was used to determine the proliferation inhibition of SKM-1 cells. Flow cytometry was used for analyzing cell proliferation rate and apoptosis. The methylation status and expression of P15^INK4B in mRNA and protein levels were measured by methylation-specifc polymerase chain reaction (PCR [MSP]). Apoptosis relative factors expression were detected by real-time transcription and Western blot.

Result: Up-regulation of HO-1 by transfected it into SKM-1 cells via lenti-virus vector promoted proliferation and protected cells against apoptosis. In contrast, down-regulation of HO-1 enhanced decitabine-induced apoptosis but reduced accumulation of S phase in cell cycle. To explore the mechanism, we detected cell cycle relative protein expression after SKM-1 cells were treated by decitabine in each group. As a result, over-expression of p15 ^INK4B and CDK4 were observed in SKM-1 cells which HO-1 was inhibited. And p15 ^INK4B and CDK4 expression-dependent apoptosis was related to caspase3 pathway. Even though HO-1 was silenced, but apoptotic rate never increased as caspase3 pathway was blocked.

Conclusion: As we known that p15 ^INK4B is a keypoint to regulate S phase of cell cycle, in our study, more obvious demethylation of p15 ^INK4B was seen in group of SKM-1 cells in which HO-1 was down-regulated. It’s equally in patients’ mononuclear cells who suffered from MDS. The worse the prognosis of MDS was judged, the more the mRNA level of HO-1 expressed. In conclusion, over-expression of HO-1 indicated resistance to demethylation of p15 ^INK4B induced by decitabine.