Effects Of Astragalus Polysaccharides On The Proliferation And Apoptosis Of Human Erythroleukemia K562Cells And Its Mechanisms

BACKGROUND AND OBJECTIVESNowadays, more and more attention has been drawn to active exploration into the new treatment strategies of leukemia.Leukemia is one of the most common malignant tumors endangering human health. Leukemia cells originate from hematopoietic stem cells, and its growth characteristics are uncontrolled proliferation, blocked apoptosis and impaired differentiation. There have been no effective therapies for leukemia so far. In addition, leukemia cells may invade the human circulatory system, blood forming organs and encroach on tissues, which are different from solid tumor, so its treatment options are limited. Therefore, to explore traditional Chinese medicine and its effective components, which could inhibit proliferation of leukemia cells, induce apoptosis and promote their differentiation and maturation, and to investigate its mechanisms has become one of the highly dynamic projects by using modern molecular biology technology.Anti-tumor effects of traditional Chinese MedicineChemotherapy drugs are limited in clinical application due to the toxic side effects and drug resistance. In recent years, researchers have developed some traditional Chinese medicines with anti-tumor activity, which have the advantages of less toxic side effects and are easily accepted by patients. However, the mechanisms of anti-tumor effect of traditional Chinese medicine are still a major problem plaguing researchers.The active components, biological activity and anti-tumor effects of Astragalus polysaccharidesAstragalus is a dried root belonging to legume genus--Mongolia Astragalus amd Astragalus membranaceus. Its main ingredients include Astragalus saponin, Astragalus polydsaccharide (APS), amino acids, trace elements and calcium. APS is the main active ingredient of Astragalus, which has high abundance and strongest pharmacological effects. APS have many pharmacological effects such as enhancing immune function, strengthening cardiac function and lowering blood pressure, regulating blood sugar level, anti-stress, anti-virus, anti-radiation, anti-oxidation and so on. It is worth noting that more and more studies have confirmed its anti-tumor effects. Studies have found that APS has obvious inhibitory effects on liver cancer, stomach cancer, colon cancer, lung cancer, nasopharyngeal cancer, breast cancer, S180sarcoma and other malignancies in vivo and vitro.Anti-tumor mechanisms and defects of Astragalus PolysaccharidesThe studies on the mechanisms of anti-tumor effects of APS have been mainly focused on enhancing immune functions like promoting the expression of interleukin-2(IL-2), interleukin-6(IL-6), factor expression of interleukin-12(IL-12) and tumor necrosis factor-α (TNF-α) and other cytokines, and enhancing the phagocytosis of dendritic cells (DC), macrophages and natural killer cells. In addition, studies have also found that APS can inhibit tumor growth by affecting telomerase activity and angiogenesis. However, further study is still needed to explore the anti-tumor mechanisms of APS at molecular level by using modern molecular biology techniques. Xu Dujuan’s study, in which solid tumor models in mice and liver cancer cell lines were subjects, found that Astragalus extract significantly inhibited tumor growth in mice solid tumor and led to apoptosis of hepatoma cell lines. Liu Rongbing’s study found that through inhibition of the expression of nuclear factor NF-κB, APS could inhibit apoptosis after cerebral hemorrhage in rats. These studies indicate that the relationship between APS and apoptosis and its related mechanisms deserve further study.Research content, research hypothesis and its significanceBased on successful extraction of APS, our research team intended to carry out further studies whether APS could inhibit proliferation and induce apoptosis of human erythroleukemia K562cells from different perspectives and in different ways, and explore further the molecular mechanisms of APS in inhibiting proliferation of K562cells and inducing apoptosis by using molecular biology techniques so as to provide scientific basis of APS for treatment of leukemia.METHODS1Cell linesHuman erythroleukemia K562cells were purchased from Shanghai Cell Bank of Chinese Academy of Science.2Effects of APS on the proliferation of K562cellsThe cell growth curve was drawn by methyl thiazolyl tetra-zolium assay (MTT) and cell growth rate and changes in proliferation were observed, and cell cycle distribution was detected by flow cytometry in order to investigate whether APS could inhibit proliferation of human erythroleukemia K562cells.3Molecular mechanisms of APS in inhibiting the proliferation of K562cellsAfter total cellular RNA and total protein were extracted and quantified, we detected the expression of cyclin A (Cyclin A), cyclin B (Cyclin B), cyclin E (Cyclin E) and p21gene at the mRNA and protein levels by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting.4Effects of APS on the apoptosis of K562cellsResearch was conducted whether APS can induce apoptosis of K562cells by Annexin V-FITC/PI combined with flow cytometry and Caspase-3activity assay.5Molecular mechanisms of APS in inducing apoptosis of K562cellsAfter total cellular RNA and protein were extracted and quantified, we detected the expression of B-cell lymphoma-2(Bcl-2), B-cell lymphoma-extra large (Bcl-XL), Bcl-2associated X protein (Bax), inhibitor of apoptosis protein-1(IAP-1) and second mitochondria-derived activator of caspase (Smac) gene at the mRNA and protein levels by RT-PCR and Western blotting.6Statistical analysisStatistical analysis was performed using SPSS (Statistical Package for the Social Sciences)17.0programs. All data were reported as means±SD. The t test was used for the comparison of two sample means and One-Way ANOVA was used for the comparison of multiple sample means followed by LSD-t when variance of groups is equal or followed by Welch’s F test when variance of groups is not equal. And multiple comparison procedures were conducted by Duncan’s T3method. P<0.05was considered statistically significant.RESULTS1Extraction and quantification of APSAfter extraction of APS, the concentration of APS was calculated as49.340mg/mL according to the standard curve.2Effects of APS on the proliferation of K562cellsCell growth curve was drawn by MTT assay and the proliferation time was calculated. Results showed that K562cell growth rate of K562cells in groups of100μg/mL,200μg/mL and400μg/mL with APS slowed significantly (P<0.01) compared with control group (0μg/mL APS), and the proliferating time was significantly longer (P<0.01), and showed a concentration-dependent manner.As could be seen from the above results,100μg/mL,200μg/mL and400μg/mL with APS could significantly inhibit the growth rate of K562cells and prolong their proliferating time. In view of the cellular state observed in experiments, in subsequent experiments the experimental group was K562cells in200μg/mL with APS induced for48h and the control group was K562cells with no dose given.Cycle distribution, determined by flow cytometry, showed that the G1phase cells in experimental group increased significantly (P<0.01) compared with control group while the S and G2/M phase cells decreased significantly (P<0.01).3Molecular mechanisms of APS in inhibiting the proliferation of K562cellsRT-PCR and Western blotting showed that cyclin B and cyclin E expression of K562cells in the experimental group were significantly lower at the mRNA and protein levels (P<0.01) than that of the control group, whereas p21expression was significantly enhanced at mRNA and protein levels (P<0.01), and cyclin A expression was not significantly different at mRNA and protein levels (P>0.05).4Effects of APS on the apoptosis of K562cellsApoptotic cells detected by Annexin V-FITC/PI in combination with flow cytometry showed that the mean apoptotic K562cells in the experimental group was significantly higher than that in control group (P<0.01), the percentage of apoptotic cells in control group and experimental group was1.010%and32.966%, respectively. Caspase-3activity assay showed that, Caspase-3activity of K562cells increased significantly in the experimental group compared with that in control group (P<0.01). 5Molecular mechanisms of APS in the inhibition of apoptosis of K562cellsRT-PCR and Western blotting results showed that Bcl-2and IAP-1expression of K562cells were significantly lower in the experimental group at mRNA and protein levels (P＜0.01) than that of control group, and Bax mRNA and Smac expression increased significantly at mRNA and protein levels (P<0.01), and Bcl-XL expression differences was not significantly different at mRNA and protein levels (P>0.05).CONCLUSIONS1. APS was confirmed in its ability to inhibit the proliferation of human erythroleukemia K562cells significantly.2. APS was confirmed in its ability to inhibit the proliferation of K562cells by down-regulating the expression of Cyclin B and Cyclin E and upregulating the expression of p21for the first time, and these effects may be not obviously related to Cyclin A.3. APS was confirmed in its ability to induce apoptosis of human erythroleukemia K562cells significantly.4. APS was confirmed in its the ability to induce apoptosis of K562cells by down-regulating the expression of Bcl-2and IAP-1and upregulating the expression of Bax and Smac for the first time, and these effects may be not obviously related to Bcl-XL.