Effects Of Artesunate On Proliferation, Apoptosis Of K562 Cells And Its Mechanism

Objective: Chronic myeloid leukemia (CML) originates from the malignant proliferation of hematopoietic stem/progenitor cell. In China, the incidence rate of CML is about 0.36/100,000, account for 20% of all kinds of leukemia. At present, interferon, hematopoietic stem cell transplantation (HSCT) and tyrosine kinase inhibitor (TKI) are the main therapies for chronic myeloid leukemia. However, the main problems of these treatment stratagy are exorbitant prices for TKI and HSCT, restricted source of hematopoietic stem cell, lack of curative effect and drug-resistance, and so on. Finding new natural drugs from Chinese Herbal Medicine, which not only has anti-leukemia activities but also has little side effect, has become an important approach for leukemia therapy.In recent years, many researches are trying to treat different kind of cancers with traditional chinese medicine (TCM), dozens of traditional chinese medicinal materials have been found to have good effect on cancer treatment. Artemisinin is an extract from China Compositae Artemisia annua, and is a novel and effective antimalarial drug developed by Chinese medical workers. Recently, several artemisinin derivatives, such as dihydroartemisinin, artemether and artesunate have been developed with more effectiveness and low toxicity. Laboratory data indicated that artemisinin and its derivatives have strong anti-tumor effects.The most attractive characteristic of artemisinin and its derivatives is the lack of cross-resistance with conventional chemotherapy drugs. It is not only active against multidrug resistant in plasmodium strains, but also active against drug-resistant tumor cells. Another propitious characteristic of artemisinin and its derivatives is lack of significant adverse side effects, so that patients can tolerate with it very well. Artemisinin may be a promising candidate for the treatment of cancers. However, the exact mechanism of the anti-cancer effect for Artemisinin is still unclear. Therefore, in the present study, we try to explore the effect of different concentrations of artesunate on proliferation, apoptosis of K562 cells and its mechanism related with this anti-leukemia effect.Method:1 Cell Culture: K562 cells were cultivated in RPMI 1640 medium conventionally. Culture medium contained 10% viviparous bovine serum (FBS), 100U/ml penicillin and 100μg/ml streptomycin. Cells were cultured and passaged at 37℃and in 5% CO2 saturated environment. The culture medium were changed when the cells were 70% to 80% confluent. The experiments were performed when cells are in logarithmic phase.2 Inhibition of cell proliferation assay. The growth inhibition rate was measured with MTT assay method. After the K562 cells were treated with artesunate, at final concentrations of 4μg/ml, 10μg/ml, 20μg/ml and 40μg/ml, for 48 hours. Inhibition rate was calculated according to the following formula: Inhibition rate(%)=[(the optical density(OD)value of control group-the OD value of experimental group]/the OD value of control group×100%.3 AnnexinV/PI double staining was performed to detect cell apoptosis rate: K562 cells were collected after the cells had been treated with artesunate at final concentrations of 0μg/ml, 4μg/ml, 10μg/ml, 20μg/ml and 40μg/ml for 48 hours. The apoptosis rate was detected by flow cytometry.4 Measurement of cell cycle distribution by flow cytometry. Logarithmic phase K562 cells were collected after the cells had been treated with artesunate at final concentrations of 0μg/ml, 10μg/ml, 20μg/ml and 40μg/ml for 48 hours. Cell cycle was detected immediately by flow cytometry.5 Measurement of protein expression levels by western blotting: K562 cells were collected after the cells treated with artesunate at final concentrations of 0μg/ml, 4μg/ml, 10μg/ml, 20μg/ml and 40μg/ml for 48 hours. The proteins expression levels of Bcl-2, Bax, Bcl-rambo, Caspase-3, and Survivin was measured by western blotting.Result:1 Artesunate inhibited the proliferation of K562 cells in a dose-dependent manner. The inhibition rate of K562 cells increased from 54.29% to 77.98% when the concentration of artesunate increased from 4μg/ml to 40μg/ml.2 Apoptosis inducing effect of Artesunate on K562 cells。Cell apoptosis rate was detected by Annexin V/PI double staining assay. The results showed that apoptotsis rate of K562 cells was increased in a dose-dependent manner after the cells had been treated with artesunate at concentrations of 4μg/ml, 10μg/ml, 20μg/ml and 40μg/ml for 48 hours. When K562 cells were treated with 40μg/ml artesunate, the apoptotic rate was 49.47±5.08%, which was significantly higher than that of the control group and the other experiment groups(P<0.05).3 Effect of Artesunate on cell cycle distribution changing in K562 cells: K562 cells were treated with 0μg/ml, 10μg/ml, 20μg/ml and 40μg/ml of artesunate for 48 hours. Then, cell cycle distribution was detected by flow cytometry. The results showed that with the increasing of artesunate concentration, the proportion of G2/M phase cells increased gradually(P<0.05), and the proportion of G0/G1+S phase cells decreased gradually(P<0.05).4 The result of Western blotting assay showed that, with the increasing in the dosage of artesunate treatment, the expression level of Bcl-2 protein in K562 cells increased significantly when compared with that in control group(P<0.05). The expression level of Bax protein also increased significantly when compared with that in control group(P<0.05). The same results were obtained with expression of Bcl-rambo protein and Caspase-3 protein(P<0.05). The expression level of Survivin protein decreased significantly when compared with that in control group(P<0.05).Conclusion:Artesunate could significantly inhibit growth of K562 cells and induce apoptosis in K562 cells. It probably plays a role in suppressing leukemia progression via activations of the Bcl-2 protein family and caspase family. In vitro studies have shown that artesunate is a potential anti-leukemia drug.