Endocrine Abstracts

Introduction: Glucocroticoids (GCs) which are routinely used in the treatment of Acute Lymphocytic Leukemia (ALL), found recently to be beneficial in a subset of AML patients resistant to chemotherapy, by promoting leukemia cells apoptosis. Current studies indicate that vitamin D (VitD) deficiency is highly prevalent among AML patients, posing vitamin D as a therapeutic supplementation. Our previous study demonstrated that VitD exerts inhibitory effect on Dexamethasone-induced apoptosis in AML cells via altering Bcl2/Bax ratio, and inhibits GC-mediated cell cycle arrest through reduction of p21.

Aim: In the present study, we aimed to clarify if 1) these actions are mediated through formation of GR-VDR complex and 2) this complex is driven by VitD (1,25 (OH)2D3) in the promoter of Bax and p21 or not.

Methods: Kasumi-1 cells were incubated for 72 hours with dexamethasone (Dex-10–6M and 10–7M) alone or pre-incubated with VitD (10–8M) for 24 h following by co-incubation with Dex. MTS assay was performed to quantify viable cells whereas apoptosis was evaluated by Annexin V/PI based flow-cytometry assay. The mRNA levels of apoptotic and anti-apoptotic markers such as Mcl-1, Noxa, Bcl-2, Bax, p21 was measured by quantitative real time PCR. Immunoprecipitation was performed in order to examine the formation of GR-VDR complex, while ChIP assay was employed in order to evaluate GR-VDR occupancy at the Bax and p21 promoters.

Results: MTS and FACS analysis indicated that Dex induces apoptosis in Kasumi-1 cells dose-dependetly, while this effect was reduced significantly when cells were pre-incubated with VitD. Bax and p21 mRNA expression was significantly decreased in cells pre-incubated with VitD (24h) following by co-incubation with Dex for further 72 h as compared to cells incubated with Dex alone. Immunoprecipitation showed that GR forms a stable complex with VDR in cells preicubated with VitD . Moreover, ChIP analysis showed that preincubation with VitD increased the formation ofGR-VDR complex. Additionally, pre-incubation ofcells with vitamin D decreased the binding of this complex in p21 promoter.

Conclusions: VitD inhibits Dex induced-apoptosis and induces cell survival in AML cells, at least in part, via formation of VDR-GR complex and attenuation of GR transcriptional activity.