Abstract
PML, a suppresser of growth and proliferation in many cell types including hematopoetic cells, is localised to nuclear dot like structures known as PML oncogenic domains (POD). PML plays a crucial role in POD dependent and caspase 3 mediated apoptosis. PML-RAR, the causative agent of Acute Promyelocytic Leukaemia (APL), induces the disintegration of PODs, while Retinoid Acid (RA) mediated remission of APL is associated with reorganisation of PODs. These findings led to the concluation that disorganisation of POD is linked with the transforation of APL blasts. We have reported that nuclear hormon receptor co-repressor (N-CoR) is a component of PODs and is essential for PML mediated tumour suppressive function of Mad and Rb. (Khan MM et al. J Biol Chem.279:11814-24). Our most recent findings demonstrate that fusion protein PML-RAR triggers the ER stress and Unfolded Protein Response (UPR) by causing mis-folding and insoluble aggregation of N-CoR protein. (Khan MM et al. J Biol Chem.279:11814-24). These findings suggest that targeting the insoluble aggregation of N-CoR and the ER stress could represent an attractive therapeutic strategy for APL. Based on the hypothesis, we screened compounds known for their inhibitory effect on protein aggregation and ER stress. Among the compounds tested, genistein, a flavonoid, exhibited significant growth inhibitory effect on both RA sensitive and RA resistant APL cell NB4 in dose and time dependant manner (see fig.1 & 2). Genistein inhibited the growth on NB4 cells through collective effects on the cell cycle progression, apoptosis and differentiation. Genistein induced apoptosis in NB4 cells was mediated by the activation of caspase-3 and 9. Genistein also induced differentiation of NB4 cells as marked by appearance of cell surface differentiation marker CD11b. In cell cycle analysis, NB4 cells were accumulated in the G2-M phase after genistein treatment. In Western blotting assay, genistein unregulated and stabilized the level of PML protein in NB4 cells while promoting the degradation of PML-RAR. In protein solubility assay, genistein enhanced the solubility of N-CoR protein and reorganized the micro speckled like distribution of POD to normal dot like pattern in NB4 cells by immunocytochemical assay. In this study, we also identified that genistein abrogates PML-RAR mediated tyrosine phosphorylation of N-CoR protein. These findings, for the first time, have identified the therapeutic potential of genistein in both ATRA sensitive and resistant APL cells.
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