The Pathological Characteristics Of Metastasis And Anti-Metastasis Effects Of BRACs On HER-2 Positive Cancer

Objective Breast cancer is the most frequently diagnosed cancer among females.HER-2 positive breast cancer is accounted for 30% of the total breast cancer.Regulating of cell pathway, including survive and death signaling pathway, is play an important role in mammary cancer on growth and invasion. RAS/RAF/MAPK pathway is one of the most major signaling pathway about cancer growth and metastases. Higher intakes of BRACs consumption was associated with lower rate of growth and invasion on breast cancer, but the mechanism is unclear. Additional prospective studies are required to confirm these findings. Our study would investigate the pathology mechanism for metastases of HER-2 positive breast cancer, and the anti-metastasis effects of BRACs on HER-2 positive breast cancer cells in vitro.Methods Black rice anthocyanins(BRACs), RAF/MAPK inhibitor(ZM33627, U0126,SP600125) or RAF/MAPK gene silencing(RAF-si RNA, MEK-si RNA,JNK-si RNA) were used individually or together with HER-2 positive breast cancer cell MDA-MB-453. Cell morphology was observed by phase contrast electron microscope. Migration and invasion were detected by healing assay, cell migration assay and transwell method. Phosphorylation level of RAF/MAPK was analyzed by western blot. Interaction between HER-2 receptor and the protein of RAF/MAPK was reflected by immunoprecipitation. The expression level of RAF/MAPK gene m RNA(raf, mek and jnk m RNA) was detected by PCR.Results1. MDA-MB-453 cells were flattened epithelial and Polygonal-like cells, which adhered to the dish in L-I5 medium. It had more migration/invasion than MCF-10A(P<0.05).2. Compared with control group, BRACs group, RAF/MAPK inhibitors group and RAF/MAPK Si RNA group suppressed migration/invasion of MDA-MB-453 cells. Interestingly, compared with BRACs group, statistically significant decrease was observed in BRACs+RAF/MAPK inhibitor group and BRACs+RAF/ MAPK-Si RNA group on items of migration and invasion in MDA-MB-453 cell(p <0.05).3. BRACs significantly inhibited the phosphorylation levels of RAF, MEK,ERK, JNK, decreased the protein of K-RAS and MMP-2/MMP-9. BRACs also could weaken the interaction between HER-2 and the protein of RAF/MAPK pathway, and down regulated the interaction between MMP-2/MMP-9 and the protein of ERK/JNK. While BRACs combined with RAF/MAPK inhibitors or RAF/MAPK Si RNA performed a more remarkable inhibiting effect than BRACs on the phosphorylation levels of RAF, MEK,ERK, JNK.4. Although BRACs and RAF/MAPK Si RNA suppressed the m RNA expression of raf, mek and jnk(P<0.05), BRACs used together to RAF/MAPK Si RNA were more effective at inhibiting m RNA expression of raf, mek and jnk(P<0.05).Conclusion1. HER-2 positive cancer cells are flattened epithelial, which have more growth and metastasis.2. In vitro, RAS/RAF/MAPK activities can greatly promote the migration/invasion of HER-2 positive breast cancer, which lays an experimental foundation for a new treatment method for anti-metastasis of BRACs in HER-2 positive breast cancer.3. RAF/MAPK inhibitor(ZM33627, U0126, SP600125) or RAF/MAPK gene silencing(RAF-si RNA, MEK-si RNA, JNK-si RNA) would enhance the suppression of BRACs in HER-2 positive cancer.4. These findings suggest that the targeted inhibition of RAS/RAF/MAPK activities by BRACs contribute to the chemopreventive potential of fruits and vegetables.