Study On The Anti-Proliferation And Apoptosis-Inducing Effect Of Flavonoids From Seed Residues Of Hippophae Rhamnoides L.on Human Breast Cancer Cell Line Bcap-37

The breast cancer is one of women's most common malignant tumor in American and European countries. Recent years, its incidence increases year by year in the developed areas of our country such as Shanghai and Beijing etc. The statistical data shows that women's breast cancer case in urban area of Shanghai is increased by 2.7 percent per year, it has been the first women's malignant tumor in the urban area of Shanghai. So it is becoming the urgent task to research and develop the new medicine to prevent and cure the disease.Flavonoids can suppress the proliferation of breast cancer cell and induce it to undergo apoptosis, so there is a potential value worthy of prevention and cure for breast cancer. In this dissertation we used the flavonoids from seed residues of Hippophae rhamnoides L.(FHR) and human breast cancer cell line Bcap-37 to investigate the effects and mechanisms of FHR-induced growth inhibition and apoptisis in human breast cancer cell line Bcap-37. The study will offer the theoretical base for the anti-tumor function of FHR and application in the breast cancer's prevention and remedy, meanwhile, it'll set up the basis for reusage of the seed residues of Hippophae rhamnoides L..To investigate the effects of FHR-induced growth inhibition in human breast cancer cell line Bcap-37, the anti-proliferative effect of FHR against Bcap-37 cell was tested by CCK-8 method, the cell phase distribution of Bcap-37 was measured by flow cytometric assay. A microarray contained 13824 human 14K cDNA was used to investigate the anti-proliferation related genes expressed differentially in human breast cancer cell line Bcap-37 that were induced by FHR, and to reveal the possible mechanism of anti-proliferation. The reliability of the microarray data was tested by semi-quantitative RT-PCR and Western Blot. Then the result of the microarray was analyzed to search the anti-proliferation related genes. The results showed a dose-dependent effect of proliferation inhibition in Bcap-37 cells, IC₅₀ was 600μg/ml. After treatment with different concentration of FHR for 24 h, the percentage of G2/M phase increased from 10.49 % to 25.74 %, and S phase of Bcap-37 cells increased, but at the same time, the percentage of G0/G1 phase decreased obviously. It showed a G2/M cell cycle arrest. The result of cDNA microarray assay showed that after treatment with FHR(1000μg/ml) for 24 h , the transcription of Cyclin B1 and cyclin-dependent kinase 1(CDK1) were down-regulated, ratio value were 0.465 and 0.380 respectively. Also, the transcription of one of the cell cycle protein-Nek2 wasdown-regulated, ratio value was 0.484. GADD45A was up-regulated to 2.241, which can inhibits the activity of Cyclin B1-cdk1.Not only the expression of DNA replication-related genes such as DNA polymerase α, topoisomerase II and proliferating cell nuclear antigen (PCNA) are undermined, ratio value were 0.5, 0.45 and 0.35 respectively, but also the transcription of several oncogenes including c-fos(0.42) and c-fos(0.45) are attenuated. While the transcription of the anti-oncogenes TSSC3 and GADD34 were up-regulated, The expression of p53 was up-regulated to 1.454-fold compared with control group. In summary, the inhibitory mechanisms of FHR in Bcap-37 cells were regulated through many way and multi-gene.To investigate the effects of FHR-induced apoptosis in human breast cancer cell line Bcap-37, the apoptotic rate of Bcap-37 cells was measured by flow cytometric assay, apoptotic phenotype of Bcap-37 was observed by transmission electron microscopy, the DNA fragmentation was demonstrated by incubating Bcap-37 cells with FHR (1000 ug/ml) for 24 h. After treatment with FHR for 24 h, the percentage of Sub-Gl phase increased from 1.63 % to 8.34 %, and G2/M phase in cultured Bcap-37 cells increased. In testing group (FHR, 1000 ug/ml), the percentage of G0/G1 phase decreased to 32.63 %, but the percentage of S phase increased to 35.62 %. The characteristic morphological change of apoptosis in Bcap-37 was observed after treatment with different concentration FHR for 24h.