| Breast cancer is a common malignant tumor which has the highest attack rate among women in many western countries. Breast cancer has also become common to Chinese women in recent days with its attack rate elevated year by year, threatening the health of Chinese women severely.HER-2/neu, a member of the epidermal growth factor (EGF) family, is a membrane receptor encoded by the oncogene erbB-2/neu with PTK activity. HER-2/neu is over-expressed in approximately 20~30% percent of the breast cancers, responsible for the high malignant potent, fast vascular angiogenesis and tumor growth, easy metastasis, as well as the high recurrence rate of the breast cancer. Vascular angiogenesis has been reported as the key point in tumor growth and metastasis. However, currently applied drugs have only limited influence on the vascular angiogenesis of breast cancer over-expressing HER-2/neu, often with severe side effects. HER-2/neu therefore has been accepted as the label of drug resistance as well as prognosis mala of the breast tumor. Also, HER-2/neu has become the target of numerous anti-tumor studies.Black rice anthocyanins are a group of anthocyanins naturally located in the skin of black rice, mainly comprised of cyanidin-3-glucose and peonidin-3-glucose. Anthocyanins are phytochemicals classified as flavonoids. Anthocyanins have shown powerful ability to inhibit tumor growth through various mechanisms, drawing lots of studies focused on them. It has been reported that anthocyanins could inhibit the PTK activity, through which we assumed that anthocyanins may have the ability to limit the initiation and development of breast cancers over-expressing HER-2/neu by inhibiting its PTK activity.This study was established according to newly reported research data as well as the assumptions above. The inhibitory effects of black rice anthocyanins on angiogenesis were firstly evaluated in transplanted tumor models, which were established with the Balb/c nude mice and the human breast cancer cell line MDA-MB-453 over-expressing HER-2/neu. Primary analysis on molecular mechanisms of these effects was also carried out by immunohistochemistry and Western blot. More laboratory approach, including MTT, Western blot, cellular immunoflueroscence and RT-PCR, were then applied to analyze the influence of black rice anthocyanins on the receptor HER-2/neu, the key points in its downstream signal paths including ERK-1/-2 and NF-κB, as well as the expression of angiogenesis-promoting factors including VEGF, MMP-2/-9 and uPA in the MDA-MB-453 cells. Assumptions of the molecular mechanisms through which black rice anthocyanins inhibited the angiogenesis in the transplanted tumor of breast cancer over-expressing HER-2/neu were form, bringing a new thought to the clinical treatment on breast cancers over-expressing HER-2/neu.The main results and final conclusions were summarized as followings:1. The transplanted tumor models, which were established with the Balb/c nude mice and human breast cancer cell line MDA-MB-453, were intervened by black rice anthocyanins-rich extract (100mg-1·kg-1·d-1, ig) and/or VEGF (5μg·kg-1·d-1, ip). Compared to the control group, statistically significant decrease was observed in the black rice anthocyanins group on items of tumor volume, MVD value and the expression levels of tumor angiogenesis-promoting factors, with each P value lower than 0.05. Also, statistically significant increase was observed in the VEGF group on the items described above, with each P value lower than 0.05. There are no significant differences between the co-treatment group and the control group on these items. On the other hand, significant decrease of these items described above in black rice anthocyanins group as well as the co-treatment group were observed in comparison against the VEGF group, with each P values lower than 0.05. Besides, the amount of cells with strong positive Ki-67 staining dramatically increased in the VEGF group and decreased in the black rice anthocyanins group compared to the control group. These data suggest that black rice anthocyanins could significantly inhibit the growth of human breast cancer cell line MDA-MB-453 transplanted tumor, presumably by inhibiting angiogenesis in tumor tissue as a result of down-regulating the expression of tumor tissue angiogenesis-promoting factors including MMP-2/-9 and uPA.2. MDA-MB-453 cells were intervened by EGF at the concentration of 40 ng/ml and/or black rice anthocyanins-rich extract at the concentration of 0, 25, 50 and 100μg/ml separately. The phoslporylation level of HER-2/neu, also indicating its PTK activity, was inhibited by black rice anthocyanins in a dose-dependent manner. Phosphorylation of ERK-1/-2, the key protein of HER-2/neu downstream signal pathway, was similarly inhibited. Positioning of NF-κB p65 in nuclease dramatically decreased in cells treated by black rice anthocyanins-rich extract versus those in untreated group, suggesting a down-regulating of transcription level. RT-PCR analysis showed significant decrease of expression of angiogenesis-promoting factors including VEGF, MMP-2/-9 and uPA at the mRNA level, also in a dose-dependent manner. The results suggest that black rice anthocyanins could inhibit the HER-2/neu PTK activity, as well as the expression of tumor angiogenesis-promoting factors. This inhibitory effect was closely related to the down-regulation of HER-2/neu downstream EGFR/Ras/MAPK (ERK-1/-2) pathway.The final conclusion of the whole paper could be drawn based on the results described above as followings: The black rice anthocyanins could inhibit the angiogenesis of the transplanted tumor of human breast cancer over-expressing HER-2/neu as well as the proliferation activity of the tumor tissue, and then slow down the growth of the transplanted tumor. This inhibitory effect was probably achieved by black rice anthocyanins inhibiting HER-2/neu PTK activity of the tumor cells, down-regulating its downstream EGFR/Ras/MAPK (ERK-1/-2) pathway, and finally leading to the decrease of expression of tumor angiogenesis-promoting factors. |
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