The Molecular Mechanism Study Of D-limonene On Breast Cancer Metastasis

Breast cancer is the most common malignant neoplasm in female. Lymphatic metastasis is the main metastatic pathway and fatal cause. The anti-lymphatic metastasis treatment on breast cancer will effectively prolong the survival time of patients and increase the cure rate. Invasion is intensely relevant to the metastasis in the process of tumor growth; invasion is the inner factor resulting in tumor metastasis. Invasion and metastasis are the main reasons for most patients suffering from tumor in clinic. The tumor cells can directly invade the lymphatic vessels, both former and neogenetic lymphatic vessels are the pathway of lymphatic metastasis of tumor cells. Tumor cells drop from the primary site, they can reach lymph node valve via vascular circulation and be captured by the lymph node, leading to lymph node involved. So, the expressing level of metastasis relevant molecular, such as MMPs, the density of micro vessels and micro lymphatic vessels of malignant neoplasm, will directly influence the occurring of the lymphatic metastasis. d-limonene is a monomer extracted from traditional Chinese medicine citrus peel, and is a kind of natural monoterpene component. d-limonene has prevention and curative effect on the chemo-induced breast cancer, skin cancer, hepatic cancer, lung cancer, gastric cancer and pancreatic cancer in rodent animal. It receives great concern of researchers from domestic and international field, because of its low toxicity, broad application and edible feature. In the initial stage, d-limonene can induce the expression the metabolic enzyme to detoxify the carcinogen: after the initial stage, it can induce apoptosis and redifferentiation of cancer cell, and can inhibit the Isopropylizasion of protein transferase for cancer cell growth. Our previous data indicates that d-limonene can promote expression of bax gene, prohibit the expression of p53, bcl-2, CD 44v6, PCNA and VEGF gene, inactivate the NF-κB, promote the release of cytochrome-C and activate the caspase-3 related apoptosis pathway. All above are involved in inducing the apoptosis of gastric cancer cell. The experiments on nude mice show that d-limonene can inhibit the tumor growth and metastasis by inhibiting the angiogenesis and lymphangiogenesis of gastric cancer. VEGF-C is the particular growth factor for lymphatic endothelium. It promotes lymphangiogenesis mainly through activating receptor VEGFR-3 peculiarly expressed in endothelium cell of lymphatic vessels. In our study, the VEGF-C expression level in breast cancer will be detected, the relevance between VEGF-C expression and the biological behavior of tumor will be analyzed to explore the metastatic mechanism; The effect of VEGF-C expression on the metastatic potency of breast cancer cells will be detected and the inhibiting effect of d-limonene will be evaluated in vivo (nude mice) and in vitro (cells strain). The anti-metastatic mechanism will be discussed to provide a theoretical basid for the clinical application of d-limonene. Objective: To observe the influence of VEGF-C ASODN mediated by liposomes on the adhesive and invasive ability of breast cancer cell and discuss the relevant mechanisms. Methods: Human breast cancer cell lines MDA-MB-435 routinely cultured and transfected by ASODN designed for human VEGF-C gene initial district mediated by liposomes. RT-PCR was used to defect the expression level of VEGF-C. The adhesive and invasive experiments in vitro were used to detect the metastatic potency change in breast cancer cell; western blot was used to detect the VEGF-C and MMP-2 protein expression blocked by ASODN. Results: RT-PCR results show that treated groups and control group have peculiar VEGF-C gene zone in 300 bp, but the gene expression of ASODN group is significantly lower than that of SODN group. O.D (optical density) value is 0.35±0.12, the difference is significant compared with group SODN and control group (0.79±0.10,0.82±0.07). In ASODN group, the adhesive rates are 30.3±2.8% and 23.3±1.5% respectively, notablyreduced comparing with the cont...