Effect Of Artesunate Water Extract On Inhibiting Proliferation And Inducing Differentiation Of Human Leukemia Line

Objective:Leukemia is a malignant hematopoietic tumor which threatens the health of human being. Up to now, traditional chemotherapy and radiotherapy are still the main methods to treat leukemia. But they have many side effects and ratio of relapse is very high, especially, it greatly damages the immunity and hematopoiesis. So looking for nature effective ingredients extracted from Chinese Traditional Medicine, which has both higher effective and lower poisonous, has become the popular topic in research of anti-tumor drug now.Artemisinin is the anti-malaria active principle which is separated from the leaf of artemisiae annuae, it has obvious anti-malaria function, and its toxicity is small. The research confirms that artemisinin has the better functions of anti-malaria and anti-tumor. Up to date, there is no research that artemisinine derivative can inhibit the proliferation of leukemia cells and induce their differentiation.The human erythroleukemia K562cells is a low-differentiated blast cell with differentiating potentialities towards erythrocyte, monocyte, granulocyte or megakarryocyte lines, so it is the ideal model to research the proliferation and differentiation of leukemia cells. In this study, we choose artesunate water extract as a research drug, which is an anti-tumor ingredient separated and purified by ourselves. By using techniques of modern biology, we explore the effect of artesunate water extract on the proliferation and differentiation of K562cells, our purpose is to clarify whether artesunate water extract has specific selectivity on inhibiting proliferation of tumor cells, and it can provide the theoretical basis and the experimental evidence for studying natural anti-tumor drug.Methods:Artemisinin residual is extracted in hydrosolvent, artesunate water extract was made after being purified and crystallized. The effects of artesunate water extract on proliferation and cell cycle distribution of K562are detected by using techniques of trypan blue exclusion, the colorimetric5-diphenyl tetrazolium bromide (MTT) and flow cytometry (FCM). Under light, electron microscopy and with image analysis, we observe the morphological changes on K562cells induced by artesunate water extract (including nucleus mature condition, nucleus/cytoplasm ratio, cell diameter, cell volume et al). Immunocytochemistry is used to study the effects of artesunate water extract on the expressions of myeloperoxidase (POX), a-naphtholacetate eacerase (a-NAE). The effects of artesunate water extract on the proliferation and differentiation of CFU-Mix from mice are observed by using the techniques of hematopoietic progenitor cell culture ex vivo. Results:1. Artesunate water extract was extracted successfully.2. The effects of artesunate water extract on inhibiting proliferation and inducing differentiation of human leukemia cells line (K562).2.1Artesunate water extract has two different effects on normal CFU-Mix and K562cells:Artesunate water extract has little effect ex vivo of CFU-Mix at the concentration of3.125-6.25mg/L; Artesunate water extract can also significantly inhibit the proliferation of K562at the concentration of3.125-25.00mg/L (MTT method), the inhibiting extent is paralled to the concentration of artesunate water extract and the inductive time.2.2Artesunate water extract may prevent K562cells of the silent phase (GO) from entering the active proliferation phase (S/G2+M):By using FCM techniques, the number of the active proliferation phase K562cells are significantly decrease after they are treated with artesunate water extract for2days. The result suggested that artesunate water extract can prevent K562cells of the silent phase from entering the active proliferation phase to prohibit the proliferation of the leukemia cells.2.3Artesunate water extract can induce the apoptosis of K562cells. By using FCM techniques, it has been found that the apoptotic ratio of K562cells reaches10%-20%after it are treated with artesunate water extract for2days. Using electron microscopy we observed apoptosis body in K562cells induced by artesunate water extract.2.4Artesunate water extract can induced K562cells to differentiate into mature cells:①The morphological observation indicated:after induced by artesunate water extract, the volume, diameter and ratio of nucleus/cytoplasm of K562cells are decreased, while the heterochromatin is condensed in nucleus. It differentiate toward mature direction.②The immunocytochemistry indicated:Artesunate water extract can markedly increase the expression of hemoglobin and myeloperoxidase in K562cells.Summary:Artesunate water extract can markedly inhibit the proliferation of K562cells. We speculate that artesunate water extract can selectively inhibit the proliferation of tumor cells, it has lower poisonous function, which is a natural anti-tumor drug that need the full research and development.