In Vivo Analysis of the Anti-Leukemic Activity of Alpha-Tocopherol in Acute Promyelocytic Leukemia.

As2O3 has been successfully been employed in the treatment of patients with acute promyelocytic leukemia (APL), however it is unavailable to most APL patients in Latin America, and the lack of alternative treatments lead to a very poor prognosis of relapsed/ATRA resistant patients. Here we investigate the anti-leukemic effect of alpha tocopherol succinate (VES) (a vitamin E analog with antiproliferative activity against several cancer cell types) which was shown to activate the intrinsic cell death mediators of caspase-9 and -3 by both its strong redox activity and inhibition of protein kinase C (PKC) and target Akt and JNK pathways. We used a syngenic transplantation model of APL: blasts from hCG-PML/RARalpha transgenic mice (TM) were IV injected in irradiated non-transgenic littermates. Massive infiltration of bone marrow (BM), spleen and liver was detected by 21st day post transplant. Recipient mice were treated with: VES (50UI/g/d) (n=15), As2O3 (2.5μg/g/d) (n=15), VES + As2O3 (n= 15) at the same doses, or vehicle (Control; n=15) starting from Day 4 for 21 days. Molecular remission was determined by RT-PCR for PML/RARalpha. The mean survival time in the Control group was of only 25,6 days (95% C.I. = 20,9 – 30,3), whereas in the VES it was 160,40 (95%CI = 134,2 – 185,6); in the As2O3 of 162,1 (95% CI = 137,97 −186,3) and in the VES+ As2O3 of 163 (95% CI = 139,93 – 186,1). Compared to controls, all treatments significantly prolonged survival. Molecular remission was attained in 86,5% of mice in the VES arm, 80% in the As2O3, and 86,5% in VES + As2O3. No significant organ toxicity was found by histopathological analyses. Another group of twelve recipients was treated with VES or DMSO for 6 days, and the percentage of apoptotic CD117+ leukemic cells in spleen and liver was determined by flow cytometry. Differentiation was evaluated morphologically on Leishman stained BM cytospin preparations after 72h of treatment with VES. The mean percentage of CD117+ apoptotic cells in VES treated mice was significantly higher (41,17 ± 5 versus 62,11 ± 4%, in spleen p<0,03; and 94,65 ± 1% versus 12,94 ± 11,95%, in liver, p<0,01). No significant difference in the number of mature granulocytic cells was detected. Furthermore, we compared the gene expression profile of BM cells obtained from leukemic mice treated with VES or DMSO (n=6, per group) using nylon microarrays representing 598 genes. Data mining from our microarray results revealed the higher expression of Mitogen Activated Protein Kinase 3, Protein Kinase C delta and BCL2 antagonist Killer-1 genes in VES treated samples. These were confirmed by Real Time-PCR. In conclusion, our results demonstrate that VES induces prolonged remissions and that it activates of MAPK and PKC signaling pathways leading to apoptosis.