Effects Of Pangolin Protein Extract On Proliferation And Apoptosis Of K562 Cells

[Background]Chronic myeloid leukemia (CML) is one of the common malignant tumors in the blood system. Chemotherapy remains the primary treatment of CML, but its side effects and clinical drug resistance have become major obstacles to chemotherapy. The emergence of Tyrosine kinas inhibitors (TKI) is a new milestone in the treating of CML, but there is an increasing number of drug resistance because of genetic mutations, so looking for more effective and lower toxic drugs against CML in clinical treatment is an urgent need to address this problem.The treatment of cancer using traditional Chinese medicine has a long history. Modern Chinese medicine believes that the pathogenesis of most tumors are" heat-toxicity ", " stasis", "stasis-toxicity ", etc..The results has suggested in recent decades that arsenic (realgar), matrine (Sophora) and indirubin (indigo naturalis) are thus as a " using poison to counteract poison "," clearing away heat and toxic material", " Promoting blood circulation and removing blood stasis " drugs to be applied to clinical medicine, and have had remarkable clinical efficacy. Currently, with modern science and technology, develop strongly on traditional Chinese herbal medicine, understand and elaborate the pharmacology, research and develop new anti-cancer drugs, and carry forward the Chinese traditional medicine vigorously, have become the medical research trends.Modern studies confirmed the role of the treatment of traditional Chinese medicine of leukemia which can be shown in the following areas:direct induction of apoptosis, to some extent to reverse the multi-drug resistance of leukemia, increase the immunity of patients and reduce side effects of chemotherapy. Leukemia is closely related to apoptosis of leukemia cells and using apoptosis mechanism to clear leukemia cells rise new concepts, new methods. Chinese medicine can induce apoptosis of leukemia cells in a variety of ways, including kill leukemia cells directly,arrest cell cycle, influence apoptosis gene expression, induce differentiation and increase levels of cytokines and other hormones.Modern pharmacological studies show that the pangolin has impact on tumor growth including a certain degree of inhibition of tumor, increasing activity of T cell subsets and NK cell, improving the quality of patients'life and immune function, reducing nausea and vomiting, leukopenia, thrombocytopenia and other side effects of chemotherapy. Clinical researches about the treatment of lung cancer, breast cancer, leukemia and other malignancies have been reported. Pangolin has usually applied with other medicine, used alone or its extract is seldom found, and its effect of chronic myeloid leukemia and the mechanism are still can not be seen. This study wants to validate pangolin protein extract has effect of anti-leukemia in vitro, and lays the foundation of clinical use of pangolin.[Objective]To further verify the anti-leukemia effect of pangolin, using chronic myeloid leukemia K562 cell lines as study object, to observe the effect of pangolin protein extract on the proliferation and apoptosis of K562 cells, to explore the mechanism of pangolin which induces apoptosis of K562 cells.[Methods]1. MTT assay was used to observe the effects on growth inhibition induced by pangolin protein extract in K562 cells.2. The morphological changes of K562 cells induced by pangolin protein extract were observed with Hoechst 33258 staining and transmission electron microscope. 3. The percentage of apoptotic cells was detected by flow cytometry, in order to verify the effect of pangolin protein extract on apoptosis.[Results]1. MTT experimental observation of pangolin protein extract on K562 cell growth and proliferationAll kinds of concentrations of pangolin protein extract could inhibit K562 cell proliferation strongly, compared with the control group there were significant differences (P<0.01), and in a certain dose range, with increasing concentration of pangolin protein extract, The inhibition rates were higher,appeared dose-dependent; 24-hour IC50 is 12.07ug/ml; The extract itself had some apoptosis ability, but compared the pangolin protein extract, the difference was statistically significant (P <0.05).2. Hoechst 33258 staining was used to observe the morphological changes of K562 cells20μg/ml dose of pangolin protein extract effected on K562 cells for 48 h,in the microscope,cell shrinkage,deformation,chromatin condensation and marginalization, some cells became smaller and showed DNA fragmentation or appeared fluorescent stain dense block and strong granular fluorescence.3. Apoptosis was analyzed by flow cytometryAnnexin V-PI double staining showed that the 48-hours apoptosis rates of the negative control group,the extract control group and pangolin protein extract group were (3.42±0.69)%, (35.32±1.45)%, (49.04±1.84)%, the apoptosis rate of pangolin protein extract group higher than the negative control group and the extract control group (P<0.05). Pangolin protein extract group and the pure extract group showed no difference in necrosis.[Conclusions]1. Pangolin protein extract could inhibit the K562 cells on some degrees, and inhibition rates appeared dose-dependent. 2. Hoechst 33258 staining assay showed that pangolin protein extract could induce apoptosis, showed characteristic morphology of apoptosis.3. Annexin V-PI double staining further confirmed pangolin protein extract can induce apoptosis of K562 cells.