A Study Of Total Flavonoids Of Astragalus And It's Constituents On Inhibiting Effects And Mechanisms For Carcinoma Cells

IntroductionHuman hepatocellular carcinoma(HCC) is the fifth most common malignancy in the world and is estimated to cause several millions deaths annually. The annual number of new cases worldwide is approximately 550,000, representing more than 5%of human cancers and is the third leading cause of cancer-related deaths. About 11 millions people die from HCC each year in China and accounting for 45%of deaths worldwide. Curative treatment options are represented mainly by surgery (ie, resection or transplantation), but most patients are not candidates for a curative option, and only palliative treatment could be given to these patients. Among palliative treatments, only chemoembolization has been proven to be effective, but other options are currently being investigated.Leukemia is a malignant tumor of hematopoietic system, which can seriouly destroy the health of human being. K562 cell is a high degree of deterioration in the human chronic mylogenous leukemia cell line and a pluripotential cell line in vitro. So far the treatment of leukemia, which uses a great dose of combination chemotherapy, is not satisfactory because of the side effects such as the damage of immune system and harmfulness to hematopoietic system. The investigation of the mechanism of maglignant tumor, especially for retroconversion of leukemia cells is becoming one of the most intersting topic of the biomedicine. It is becoming an important hope for cure of leukemia that there are some useful medicine in traditional Chinese medicine which may restrain the maligant generation of leukemia cells.Astragalus is a traditional Chinese medicine which has been used in clinical practice for thousand of years. It has proved that Astragalus can be fuctioning effectively in cardiovascular system, immune system and has effect of anti-virus, antioxidant and anti-mutation. It is also found that Astragalus have an important effect in preventing and curing cancer. Total flavonoids of Astragalus is main active ingredient separated from Astragalus which has effect of antioxidant and scavenging the free radical. Total flavonoids of Astragalus have six kinds of constituents indentificated by the institute of radiation medcine of Military medical science. The six constituents are (3-sitosterol, formononetin, calycosin,β-sitosterol-3-O-β-D-glucoside, formononetin-7-O-β-D-glucopyranoside and calycosin-7-O-β-D-glucopyranoside.Therefore, the purpose of this study is to identify the inhibitory effect of human hepatocellular carcinoma cell and erythroleukemia cell growth by total flavonoids of Astragalus and its constituents and related mechanisms in vitro, BEL-7402 cell line and K562 cell line are chosen as the research models.Objectives1. To study the effects of total flavonoids of Astragalus and its constituents on the proliferation of human hepatocellular carcinoma BEL-7402 cells and erythroleukemia K562 cells in vitro,and make the relative between dosage and effects, then choose proper concentrations for the following research.2. To explore the influence of total flavonoids of Astragalus and its constituents on the cell cycle distribution of BEL-7402 cells, and further to detect the changes of genes and proteins, therefore reveal the possible mechanisms.3. To evaluate the effects of total flavonoids of Astragalus and its constituents inducing apoptosis of K562 cells, and make sure the changes of genes related to cell cycle such as cyclin D1. 4. To provide scientific experimental basis of Astragalus for the treatment of HCC and leukemia.Methods1. MTT assay was used to determine the growth inhibition effect on K562 cells of total flavonoids of Astragalus and its constituents at different drug concentrations (20μg/ml,50μg/ml, 100μg/ml and 200μg/ml) at different times(24h,48h,72h). Neutral red method was used to determine the growth inhibition effect on BEL-7402 cells of total flavonoids of Astragalus and its constituents at different drug concentrations (1μg/ml,3.16μg/ml, l0μg/ml, 31.6μg/ml, 100μg/ml and 316μg/ml).2. Cell cycle distribution of BEL-7402 cells and K562 cells were detected by FCM using PI dye at different concentrations.3. Apoptosis of K562 cells was detected by FCM using apoptosis kit including dying with PI and AnnexinⅤ-FITC by the drug concentration of IC50 at different times(2h,4h,6h,12h and 24h).4. Gene chips is used to detect the expression of genes related to cell cycle of BEL-7402 cells after treated with total flavonoids of Astragalus and its constituents.5. Two-dimensional gel electrophoresis and MALDI-TOF-MS were used to detect the expression of proteins of BEL-7402 cells after treated with total flavonoids of Astragalus and its constituents.6. RT-PCR was used to determine the level of cyclin D1 mRNA in K562 cells after treated with total flavonoids of Astragalus and calycosin.Results1. Treatment of K562 and BEL-7402 cells with variable concentrations of total flavonoids of Astragalus and its constituents resulted in an obvious decrease of cell growh in a time and concentration dependent manner in vitro. IC50 of total flavonoids of Astragalus and calycosin in different times are respectively 8.63μg/ml,87.90μg/ml,63.10μg/ml (24h,48h,72h) and 130.32μg/ml, 123.03μg/ml,122.18μg/ml (24h,48h,72h). IC50 of total flavonoids of Astragalus, calycosin, calycosin-7-O-β-D-glucopyranoside, formononetin and formononetin-7-O-β-D-glucopyranoside are respectively 40μg/ml,69.93μg/ml, 100ug/m, 100μg/ml and 100μg/ml.2. Total flavonoids of Astragalus and its constituents can induce G0/G1 phase of K562 cells arrest, the proportion of S phase decrease. While apotosis of K562 cells cannot be induced by total flavonoids of Astragalus and calycosin. 3. There were respctively 45,17,6,4 and 25 genes that differentially expressed in BEL-7402 cells after treated with total flavonoids of Astragalus, calycosin, calycosin-7-O-β-D-glucopyranoside, formononetin and formononetin-7-O-β-D-glucopyranoside.4. Proteomic analysis suggested the 10,14,10,9 and 12 proteins that differentially expressed in BEL-7402 cells after treated with total flavonoids of Astragalus, calycosin, calycosin-7-O-β-D-glucopyranoside, formononetin and formononetin-7-O-β-D-glucopyranoside.5. RT-PCR analysis suggested the level of cyclin D1 mRNA in K562 cells decrease after treated with total flavonoids of Astragalus and calycosin.Conclusions1. Total flavonoids of Astragalus and its constituents could inhibit the growth of K562 and BEL-7402 cells in a concentration and time dependent manner in vitro.2. Total flavonoids of Astragalus could cause G0/G1 cell cycle arrest in BEL-7402 cells through upregulating the level of CDKN3, CDKN1B, CDKN2B, CDKN2C, RBI and downregulating the level of CDK5, Skp2, E2F1, CCNF, CCND3. Calycosin could cause G0/G1 cell cycle arrest in BEL-7402 cells throug upregulating the level of RBX1, CDC2, and downregulating the level of CKS1, CCNA2, Skp2, E2F2, CCND3, CCNC. Calycosin-7-O-β-D-glucopyranoside could cause G0/G1 cell cycle arrest in BEL-7402 cells through downregulating the level of CCNA2, CDK2 and CCNB1. Formononetin could cause G0/G1 cell cycle arrest in BEL-7402 cells through upregulating the level of GADD45A, CKS2 and Skp1A. Formononetin-7-O-β-D-glucopyranoside could cause G0/G1 cell cycle arrest in BEL-7402 cells throug upregulating the level of CDC2, GADD45A, CKS2, CDKN1B, and downregulating the level of Skp1A, RBX1, Skp2, CCNF, CCND3.3. Total flavonoids of Astragalus and its constituents could change the proteins express in BEL-7402 cells, which are transgelin 2, pyridoxine 5'-phosphate, stress-induced-phosphoprotein 1, peroxiredoxin 1, endoplasmic reticulum protein 29, phosphoglycerate mutase 1, thioredoxin peroxidase. It might be related to the mechanism of anti-tumor of the total flavonoids of Astragalus and its constituents.4. Total flavonoids of Astragalus and calycosin could decrease the level of cyclin D1 mRNA of K562 cells, to prevent cell cycle complexes phosphorylation and inhibit the changes of cell cycle control points G1/S, thus inhibit the proliferation of K562 cells.