| Aminopeptidase N (GD13) inhibitor Bestatin is a low molecular weight peptide obtained from the culture medium of Streptomyces Olivoreticuli which shows unequivocal antitumor effects through augmentation of the host immune system. It has already been reported that Bestatin or in combination with chemicals can prolongate the disease-free interval and survival period in adult acute or chronic leukemia. Recently, there were a few reports abroad about Bestatin which shows directly antitumor effects with the mechanism unknown. In this study we tried to elucidate the direct antitumor mechanisms of Bestatin in order to expand the train of thought of the clinical application of Bestatin and to some extent, to provide a theoretical and experimental basis for designing new antitumor strategies.We examined the effect of Bestatin on the growth of human acute granulocytic leukemia cell line HL60 cells and human acute erythroleukemiacell line K562 cells by MTT assay. The results showed that the growth of HL60 cells and K562 cells was remarkably inhibited by Bestatin in a dose dependent manner. The growth of HL60 cells was significantly inhibited after being treated with Bestatin at 0.01ug/ml and the ICso value of Bestatin in HL60 cells was 13.03ug/ml. But K562 cells were less sensitive than HL60. The growth of K562 cells was significantly inhibited after being treated with Bestatin at 5u.g/ml and the rate of growth inhibit was less than 50% in K562 cells being treated with Bestatin at 400(ag/ml for 72 hours. These results suggested that the sensitivity of different original leukemic cells to Bestatin was different, and HL60 cells were more sensitive than K562 cells. The possible cause was that the expression of CD 13 on HL60 cells and K562 cells was different, and we found that the expression of CD 13 on HL60 was 70.10% but only 1.58% on K562. This result indicated that there might be a correlation between the CD 13 expression and the growth-suppressive effect. It was found that the growth of the bone marrow mononucleus cells of healthy donor was also inhibited by Bestatin and the rate of growth inhibit was 7.8% after being treated with Bestatin at 100p.g/ml for 24 hours, but the rate of inhibit was remarkably lower than the HL60 cells (59.2%).In order to explore the mechanism of the growth-suppression caused by Bestatin, apoptosis of leukemic cells induced by Bestatin was analyzed by morphlolgical observation, DNA agarose gel electrophoresis, terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method and flow cytometry assay. Characteristic changes for apoptosis emerged in HL60 cells and K562 cells after exposure to Bestatin at a certain concentration. DNA ladder was also observed in the DNA agarose gel electrophoresis and there were positive cells when evaluated by TUNELmethod. Apoptosis of lekemic cells HL60 and K562 could be induced by Bestatin, showing the apoptotic peak before Gl phase cells and the positive of Annexin VFITC on the cells membrane by flow cytometry. Besides that, Bestatin induced apoptosis in HL60 cells and K562 cells in a dose- and time-dependent manner. These results indicated that inducing cells apoptosis is an important mechanism of the antitumor effect of Bestatin.The cell cycle of HL60 cells analysis measured by flow cytometry showed that the percentage of S phase cells raised a little and then descended, the G2 phase cells remarkably descended and the Gl phase cells remarkably raised after treated with Bestatin at 100p.g/ml for 6 hours, and the cell cycle was blocked at Gl phase after being treated for 12 hours.In order to explore the possibile pathway of the signal conduction of the cell apoptosis induced by Bestatin, the mitochondrial transmembrane potentials ( A Wm) of HL60 cells treated or untreated with Bestatin were detected through staining of Rhodaminel23 , of which fluorescent intensities were measured on flow cytometry, the caspase-3 activity was evaluated with a spectrophotometric enzyme assay to measure the cleavage of pept...
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