| ObjectiveChronic myeloid leukemia (CML) originates from the malignant proliferation of hematopoietic stem/progenitor cell. In China, the incidence rate of CML is 0.36/100,000, account for 20% in all kinds of leukemia. At present, interferon, bone marrow transplantation and tyrosine kinase inhibitor are the main therapies of leukemia. However, many pressing problems still pending, such as exorbitant prices, restricted marrow's source, little curative effect and drug-resistance etc. Finding new natural drug from Chinese Herbal Medicine, which not only inhibited leukemia cells proliferation but also has little side effect, has become an important approach for leukemic therapy.Artemisinin that extracte from traditional herbal medicine is a polycyclic sesquiterpene lactone containing special peroxide group. It has been appointed as unique effective drug of treatment for falciparum malaria by WHO. Furthermore, it is a new drug what has been approved widely by the international community in China. Resent studies indicate there are more than 150 types in derivatives of artemisinin. Study on antitumor effect of several derivatives has been taking, while several derivatives majority belong to fat-soluble or alcohol-soluble materials and brings much inconvenience for practical application, greatly influence the effect of drugs. On this basis, we distilled a water-soluble component from Artemisia and investigated the pharmacological effect of artemisia aqueous extract on K562 cells and umbilical cord blood mononuclear cells, the effect on the leukemic model mouse and the molecular mechanism of anti-tumor. Our purpose is to provide the theoretical basis and the experimental evidence for finding out water-soluble derivatives of artemisinin having antitumor activity, little toxic and side effect.Methods1. Inhibition of proliferation was measured with a colorimetric MTT assay. The morphological changes of K562 were observed under a light microscope and an electron microscope. Flow cytometry assay detected the ratio of apoptosis and oncosis of K562 cells and HCMNCs induced three different times by three different concentrations.2. Fluorescence spectrophotometer detected the membrane fluidity of K562 cells by Artemisia aqueous extract treated. Laser scanning confocal microscopy detected in mitochondrial membrane potential and intracellular calcium concentration ([Ca2+] i) by Artemisia aqueous extract treated.3. The leukemic animal model (SCID mouse) established by injecting K562 cells through tail vein. Based on the successful human xenograft leukemic model, artemisia aqueous extract injected intraperitoneally. We studied the effect of tumor-bearing mouse after therapy by means of comparing the incidence, the survival time, analysis of peripheral hemogram and myelogram, the expression level of bcr-ab1 oncogene of tumor cells.Result1. Artemisia aqueous extract had promoted K562 differentiation and had an obvious inhibiting effect on the proliferation of K562. The 50% effective dose, evaluated on 24h,48h,72h of ordinal exposure to the extract, was 65.17μg/ml,31.63μg/ml,10.51μg/ml. It was discovered that artemisia aqueous extract treated cells exhibit morphological changes typical of apoptosis and oncosis. Furthermore, the ratio of apoptosis was equal to the ratio of oncosis. Artemisia aqueous extract less than the dose range of 50μg/ml had little inhibitory effect on HCMNCs.2. Artemisia aqueous extract increased the membrane fluidity of K562 cells and present typical dose-dependency and time-dependency. Artemisia aqueous extract increased K562 [Ca2+]i and present dose-dependency; K562 [Ca2+]i increased when add to Ca2+ concentration (20mM) in the extracellular fluid; K562 [Ca2+]i decreased when add calcium channel blocker in the extracellular fluid. A significant decrease in mitochondrial membrane potential of K562 cells was observed.3. The leukemic animal model (SCID mouse) was established by injecting K562 cells through tail vein. The leukemia cells were found on peripheral blood smear on the fourth week after inoculation. Human leucocyte infiltration was observed in the mouse's liver, spleen, kidney, peripheral blood, bone marrow etc. and solid tumor partly generated.4. Artemisia aqueous extract treatment resulted in improver common condition, increaser weight and a significant prolonged survival in leukemia model mouse. Peripheral leukocyte count was decrease; the ratio of mature granulocyte was increase in the middle of granulocyte of bone marrow; the expression of bcr-ab1 oncogene was detected in the mouse's peripheral blood and wasn't detected in the mouse's liver, spleen, bone marrow etc.Conclusion1. Artemisia aqueous extract could control proliferation of K562 in vitro by two modes: apoptosis and oncosis. The effects were indistinctive between the two modes.2. Artemisia aqueous extract less than the dose range of 50μg/ml had little inhibitory effect on HCMNCs.3. The inhibition of artemisia aqueous extract in proliferation had mainly acted on K562 cell membrane. It could promote the change of membrane permeability. These changes led to an increase in the [Ca2+] i and a decrease in mitochondrial membrane potential and subsequently inducing apoptosis and oncosis.4. The method that injecting K562 cells through tail vein in SCID mouse could triumphantly establish the leukemic animal model.5. Artemisia aqueous extract had therapeutic effect on the human xenograft leukemia model mouse.
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