In vitro study of the effects of arsenic trioxide and vitis vinifera (grape) skin extracts on proliferation, differentiation, and apoptosis of myeloblast cells

This study was conducted with the aim to prepare suitable native Acute Myelocytic Leukemia (AML) blast cells for in vitro study and to detect the effect of the anti-leukemic drug arsenic trioxide and the local variant of Vitis vinifera (grape) skin extracts individually and in combination on proliferation, differentiation, and apoptosis of the A ML blast cells. A separation system of three steps (centrifugation. depletion of monocytes and lymphocytes) was used for the preparation of highly pure native AML blast cells from patients' blood samples with moderate blast percentages. This system was inefficient and a significant decrease in total cell number and contamination with other cells after each separation step was observed. The proliferation of native AML blast cells in short-term cultures required several factors, a suitable starting cell number (about 10/ml), the presence of colony-stimulating factor and the absence of neutrophil. The colony-stimulating factor provided by phytoheamoagglutinin-leucocytes conditioned medium (PHA-LCM) and Hep-2 conditioned medium. The plasmacytoma cell line conditioned medium did not stimulate the proliferation of native AML blast cells. The kinetics of growth under the stimulation of 10% PHA-LCM was increasing in the cell number up to the third day of culture, then remained on the same level for two days, during the days 5 to 7, the cell number was decreased. Two types of grape skin extracts were prepared, alcoholic and aqueous. The active compounds detected were polyphenols, flavonoids, tannins, and glycosides. The cytotoxic assays for arsenic trioxide and grape skin extracts were performed against native AML blast cells and normal human lymphocytes by using MTT method. Whereas Against plasmacytoma cell line and normal mouse embryo fibroblast cells, the neutral red method was used. The results indicated that arsenic trioxide had cytotoxic effects on AML blast cells and on plasmacytoma cell line. The alcoholic extract of grape skin showed cytotoxic effects on AMI, blast cells and on plasmacytoma cell line, while the aqueous extract of grape skin showed cytotoxic effects on AML blast cells and on plasmacytoma cell line, but with little activity as compared with the grape skin alcoholic extract. The combination of grape skin extracts enhanced the cytotoxic effects of arsenic trioxide The results of the cytotoxic effects showed that there was a difference in the toxicity of grape skin extracts against native AML blast cells and plasmacytoma cell line. The results of DNA fragmentation test showed that arsenic trioxide caused DNA fragmentation of AML blast cells at concentration of 20 (.ig/ml. Although the alcoholic extract of grape skin induced DNA fragmentation at different concentrations (50, 75,100, 125, and 150 ug/ml), while aqueous extract did not induce DNA fragmentation. The results of apoptotic activity assessment illustrate that combination of grape skin alcoholic extract significantly potentate the effect of arsenic trioxide induction of apoptosis, while the combination of grape skin aqueous extract did not enhance the apoptotic activity of arsenic trioxide. Moreover, the cell differentiation test revealed no blast cell differentiation during the treatment of M3, and M2 subtypes with arsenic trioxide and grape skin extracts individually and in combination.