The Role Of Raf-1 In HUVECs And In The Angiogenesis Ability Induced By Breast Cancer Cells

Breast cancer is the most common form of cancer and the second most common cause of cancer death in women.According to estimates in 2002,there were 1 151 298 new cases of breast cancer diagnosed,410 712 deaths caused by breast cancer,and more than 4.4 million women living with breast cancer worldwide.By far,the treatment of breast cancer is still a great challenge in medical field.Like most solid tumors,breast cancers require new blood vessel growth if they are to grow beyond a few millimeters in diameter.The new vessels not only supply additional nutrients to meet the growing demands of the tumor, but also provide potential routes for tumor dissemination and metastasis.Poor prognosis of breast cancer has been shown to correlate with increasing micro-vascular density and with factors that stimulate new vessel growth.With the hope of limiting neovascularization and ultimately tumor growth and metastasis,anti-angiogenic therapy is particularly attractive.A number of agents in current clinical trials have been identified using in anti-tumor therapy. However,it is hard to get approving effect just by one of them because of the complexity of the angiogenic process as it happens in cancer.Thus,it is necessary to find a new upstream molecule that controls many processes or factors which play important roles in tumor angiogenesis.Raf-1 is a 74 kDa cytosolic serine-threonine kinase,ubiquitously expressed. There is a higher expression of Raf-1 in cancerous human tissues than in the surrounding noncancerous glands in breast cancer.Raf-1 is a key intermediate in MAPK signal transduction pathway and also involves in the other signaling pathways.These pathways interact with each other through Raf-1 and form a complicated signaling net.Raf-1 plays an important role in tumor cell growth, proliferation,cell cycle and apoptosis.To inhibit Raf-1 can inhibit tumor growth. Raf-1 can protect endothelial cell from apoptosis by involving in different signaling pathway.Thus,Raf-1 maybe involves in the function processes of several proangiogenesis-related factors in tumor angiogenesis.Upregulated Raf-1 activity has also been implicated in tumor angiogenesis and metastasis.【Aims】1.To observe the effect of Raf-1 in cell growth,cell cycle,apoptosis and the ability of tube formation of HUVECs;2.To detect the expression of Raf-1 in breast cancer cell lines and to observe the relationship between Raf-1 and the ability of angiogenesis induced by breast cancer cells;3.To observe the effect of over-expression of Raf-lin the ability of tumor angiogenesis induced by breast cancer cells and to observe the change of the ability after the expression of Raf-1 inhibited by RNAi especially.【Methods】1.To culture HUVECs primarily which were obtained from the fresh umbilical cord;To synthesize siRNAs according to raf-1 gene sequence and to construct RNAi vector pSilencer3.1-H1-Raf-1;2.To transfect HUVECs transiently by Raf-1 siRNA and pCDNA3.0-Raf-1-flag respectively and to transfect MCF-7 stablely by pSilencer3.1-H1-Raf-1 and pCDNA3.0-Raf-1-flag respectively;3.To detect the expression of Raf-1 mRNA,phosphorylation and protein by RT-PCR and Western blot in all cells needed in the experiment.4.To measure the cell growth of following cells by MTT assay:HUVECs transiently transfected by Raf-1 siRNA and pCDNA3.0-Raf-1-flag respectively;HUVECs treated by the supernatant of conditional culture medium from four kinds of breast cancer cells or from MCF-7s with different Raf-1 expression;5.To determine cell cycle and apoptosis by Flow cytometry in HUVECs transfected by Raf-1 siRNA and pCDNA3.0-Raf-1-flag respectively;6.To determine the concentration of VEGF in the supernatant of conditional culture medium from breast cancer cells;7.To use in vitro tube formation assay kit determining the ability of tube formation of all HUVECs in step 4;8.To establish xenograft tumors in nude mice and to observe tumor growth;To determine the expression of Raf-1 and CD34 in xenograft tumors by immunohistochemistry and to observe the relativity between them.【Results】1.The expression of Raf-1 mRNA,phosphorylation and protein in HUVECs transfected by pCDNA3.0-Raf-1-flag was higher than in HUVECs transfected by the lipofectin control.Sequently,HUVECs showed rapid cell proliferation,increased ratio of cells in S stage,decreased apoptosis ratio and enhanced ability of tube formation;2.To inhibit Raf-1 by siRNA specially,the expression of Raf-1 mRNA, phosphorylation and protein in HUVECs decreased obviously compared with the control.And then,the cell proliferation of HUVECs slowed down,cell cycle arrested in G1 stage,the number of apoptosis cell increased,and the ability of tube formation of HUVECs was poor.3.There weren't statistical difference among the expression of Raf-1 mRNA, phosphorylation and protein in breast cancer cells MCF-7,MDA-MB-231, Bcap-37,except SK-Br-3.The expression of Raf-1 mRNA,phosphorylation and protein in SK-Br-3 was lower than in other three kinds of breast cancer cells.4.The concentration of VEGF in the supernatant of conditional culture medium from SK-Br-3 was lower than in MCF-7,MDA-MB-231,Bcap-37.5.The tube formation induced ability in HUVECs treated by the supernatant of conditional culture medium from SK-Br-3 was poorer than in MCF-7 in vitro: and nude mice tumor formation assay in vivo showed that the MVD in SK-Br-3 xenograft tumors was lower than in MCF-7 xenograft tumors.6.The expression of Raf-1 mRNA,phosphorylation and protein in MCF-7 stablely transfected by pCDNA3.0-Raf-1-flag was higher than the control, and the former showed the high concentration of VEGF in the supernatant of conditional culture medium and good tube formation induced ability.The overexpression of Raf-1 resulted in high MVD of xenograft tumors in nude mice tumor formation assay in vivo.7.To inhibit the expression of Raf-1 mRNA,phosphorylation and protein in MCF-7s using pSilencer3.1-H1-Raf-1,the less concentration of VEGF in the supernatant of conditional culture medium and poor tube formation induced ability were observed.The inhibition of Raf-1 expression resulted in less MVD of xenograft tumors in nude mice tumor formation assay in vivo,and then,inhibited tumor growth..【Conclusion】The expression of Raf-1 is positively associated with cell proliferation,cell cycle progression and the tube formation ability of HUVECs,and negatively associated with apoptosis;The level of intrinsic expression of Raf-1 in cancer cells can influence the production of VEGF and,sequently,influence cell proliferation and tube formation of HUVECs;The specific inhibiton of Raf-1 by RNAi in HUVECs and cancer cells results in the inhibition of angiogenesis.All of above give us a clue to treat breast cancer by anti-angiogenic therapy.