Gamma and Delta Tocopherol downregulate HOXA9, PBX1, AND E2A-PBX1 in human leukemia cells, KG-1 Free

A109

Vitamin E is a fat-soluble vitamin that exists in eight different isoforms: four tocopherols (alpha-, beta-, gamma- and delta-), and four tocotrienols (also alpha-, beta-, gamma- and delta-). Studies are underway to determine whether vitamin E, through its ability to limit production of free radicals, might help prevent or delay the development of “cancer”. In our study, we examined the effect of vitamin E isotypes on some genes involved in acute leukemia such as HOXA9 or Homeobox A9, a transcription factor with a central role in both hemopoiesis and leukemia, a high level of HOXA9 is a characteristic feature of acute myeloid leukemia (AML) and may be sufficient to cause this disease; PBX1, also called Pre-B cell leukemia transcription factor 1, PBX1 is involved in a form of pre-B-cell acute lymphoblastic leukemia (B-ALL); and E2A-PBX1, a Chromosomal translocation t(1;19)(q23;p13.3) which involves PBX1 and E2A genes, E2A-PBX1 transforms cells by constitutively activating transcription of genes regulated by PBX1 or by other members of the PBX protein family. Five round dishes, containing 7 million KG-1 cells each, were used to treat cells with different vitamin E isotypes: Gamma Tocotrienol (γT3) at 20 micromolar (µM) concentration, Delta Tocotrienol (δT3) at 20 µM, Gamma Tocopherol (γT) at 50µM and Delta Tocopherol (δT) at 50 µM; The fifth dish was used as a vehicle (V), where alcohol 70% was added instead of a vitamin E. Specific KG-1 media was added to promote cell growth in 5% CO2 at 37°C for 72 hours. After treatment, the cells were separated from their media, washed with 1 ml of Phosphate Buffered Saline (PBS) each, then transferred to a 1.5ml centrifuge tubes used to separate and remove the PBS from the cells. One hundred µl of protein lysis buffer and the 1 µl of protease inhibitor cocktail were added to each tube for protein isolation. Bicinchoninic Acid (BCA) Protein Assay for protein quantification was done after incubation on ice for one hour of the specimens. Western Blot was performed using a NuPAGE Bis-Tris Gel, and the Sea-blue as a protein molecular weight marker; Then the proteins were transferred to a membrane using the XCell IITM Blot Module. The membrane then was removed from the electroblotter, incubated in TBST for 1 hour and blocked with 5% Milk solution. The membrane was incubated with the primary antibody (Ab) overnight, and then with the secondary antibody. Before and after each step, the membrane was washed in TBST 3 times, for 10 minutes each. The primary antibodies used are anti BETA ACTIN Ab, anti HOXA9 Ab and anti PBX1 Ab. The secondary Ab is the anti mouse Ab. Our results showed that both δT and γT have a downregulatory effect on HOXA9, PBX1 and E2A-PBX1 genes expression in the leukemic KG-1 cells, when these cells were treated with 50 µM concentration for 72 hours.