Alpha-Tocopherol Induces Apoptosis in Acute Promyelocytic Leukemia Cells through a P53-Independent Pathway.

Succinate of α tocopherol (VES) is a derivative of vitamin E with well-known antioxidant and pro-apoptotic activities. It has been demonstrated that VES induces apoptosis in different solid tumor cells and preliminary clinical data suggest that the use of vitamin E in association to first-line chemotherapy may improve the efficacy of therapy, without significant adverse effects. About one third of the patients with acute promyelocytic leukemia (APL) relapse after treatment with all trans retinoic acid (ATRA) and many of them present resistance to further treatment with ATRA. Therefore, the development of new strategies effective to the treatment of APL would be relevant to improve survival of ATRA-resistant patients. In order to determine whether VES would induce apoptosis and/or differentiation of APL cells, we treated in vitro NB4 and leukemic cells from 11 patients with APL harboring PML/RARα rearragements with 20, 40, 80 or 120μg/mL of VES or vehicle (DMSO, control) for 3, 6, 12, 24 and 72h. At each time point, cells were harvested, stained with Propidium Iodite and an anti-annexin V antibody, and the percentage of apoptotic cells was determined by flow cytometry (FC). Differentiation after 72h of treatment was evaluated morphologically on Leishman stained cytospin preparations and by quantification of CD11+ cells by FC. Compared to control, a significant increase of the number of apoptotic cells was detected in all samples treated with VES for at least 6h. VES inhibitory concentration 50% (IC50%) was calculated to be 40μg/mL. The incubation for 12h with 40μg/mL VES resulted in a median percentage of apoptotic cells of 45% (25 - 75 percentiles: 40.6–55.3%) and of 24% (12.7 – 31.2%) in NB4 and primary cell samples, respectively, whereas in control samples these values were inferior to 15%. On the hand, no differentiation was detected. In order to test whether VES synergized with As₂O₃ in the induction of apoptosis, NB4 cells were treated with As₂O₃ (1μM), VES (40μg/mL) or their association at the same doses. After 24h of incubation, the median percentage of apoptotic cells in samples treated with the association of As₂O₃+VES was 78.3% (p25–p75: 77.2–83.5%) and those treated with VES or As₂O₃ alone it was of 72.7% (59.7 – 85.5%) and 68.3 (61.2–96.5%), respectively (P>0.05). Therefore, no synergism was detected between As₂O₃ and VES. To elucidate if the proapoptotic activity of VES was associated with lipid peroxidation and/or free radicals generation, the concentration of malondialdehyde (MDA) and glutathione (GSH) was determined. No significant differences in MDA (0.14 versus 0.13μM/mL in treated and control samples, respectively) or GSH (11.6 versus 11.3nM/mL) concentrations were detected after 24h of VES treatment. Finally, we evaluated the expression of the HDM2 and p21 genes by semi-quantitative RT-PCR, since these genes are known to play a key role in the control of apoptosis. Wild-type p53 regulates HDM2 and p21 transcription, nevertheless bi-allelic mutations in p53 gene abrogate this function in NB4 cells. Treatment with VES increased HDM2 and decreased p21 expression. In conclusion, our results demonstrate that VES is a potent proapoptotic agent in APL which act in a p53-independent fashion. Considering that serum concentrations of VES similar to those used here have been safely achieved in cancer patients, the therapeutic use of VES for APL may represent a promising alternative.