The Effects And Mechanism Of D-limonene On The Microfilament Skeleton And Invasion Capabiity Of Breast Cancer Cell

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.Adopted to explore the function of VEGF-C gene expression in breast cancer metastasis in vivo and in vitro,to evaluate the inhibitive effect of d-limonene on the metastasis of breast cancer,we came to the following conclusions: 1.VEGF-C gene ASODN Transfection mediated by liposomes can reduce VEGF-C protein expression of MDA-MB-435 cell,and can signi-ficantly inhibit the adhesive and invasive ability of breast cancer cell in vitro;regulating the expression of MMP-2 protein may be the pathway.2.Through constructing VEGF-C eukaryotic expressive plasmid PCDNA3.1-VEGF-C,liposomes mediated transfection,with the use of western blot and RT-PCR, we identified VEGF-C gene positive clone MDA-MB-435-VEGF-C.There was no notable effect of VEGF-C gene on the in vitro growth of breast cancer cell; over-expression of VEGF-C can increase the adhesive and invasive ability of breast cancer cell in vitro;increasing the expression of MMP-2 may be its mechanism.3.D-limonene can reduce the expressions of VEGF-C and MMp-2 protein in VEGF-C over-expressive breast cancer cell,inhibit adhesive and invasive ability induced by VEGF-C gene transfection,and has notable dose-dependent relation.4.Western-blot and immunohistochemical methods was used to detect the expressions of eIF-4E and VEGF-C and MVD,LMVD in 57 specimen of breast cancer.eIF-4E gene may induce the angiogenesis and lymphangio-genesis by up regulating the expression of VEGF-C,and then promote lymphatic metastasis of breast cancer. Blocking the transmission of VEGF-C/VEGF-2/VEGF-3 signals may become the effective way for lymphatic metastasis of breast cancer.eIF-4E can be used as the predictive marker for lymphatic metastasis of breast cancer.5.VEGF-C over-expressive cell MDA-MB-435-VEGF-C was used to construct the metastatic model of human breast cancer in nude mice to observe the effects of d-limonene on lymphangiogenesis of breast cancer and lymphatic metastasis.The result shows the mammary gland of nude mice in orthotopic position of every group all developed tumor.The lymphatic metastatic rate in model control group is 87.5%,the metastatic rate to lung,brain and other internal organs is 75.0%,which proved the success of metastatic model.The experimental treatment of d-limonene can inhibit the in vivo growth and metastasis of human breast cancer in nude mice.The mechanisms by which d-limonene inhibits the lymphatic metastasis of breast cancer are reducing the expression of VEGF-C protein,inhibiting the formation of lymphatic vessel induced by VEGF-C and decreasing the density of lymphatic vessel of breast cancer.Objective:To explore the effects and mechanism of d-limonene on the microfilament skeleton and invasion capabiity of breast cancer cell line-MDA-MB-435.Methods The laser confocal microscopy was used to get the mor-phology of F-actin cytoskeleton of breast cancer cell. Using Matrigel and Fibronectin to construct the artificial basal membrane, the invasion inhib-ition effect of d-limonene was studied in Transwell cabin. The expression level of fascin-1 protein was detected by Western-blot method.Results:The breast cancer cells was well-arranged after the treatment of d-limonene and the microspikes strcture in cell basilar part was hard to found。The invasion capability of breast cancer cell were prohibited by d-limonene significantly; The expression of fascin-1 was down regulated (2.9±0.3,12.4±0.7 and 19.3±0.7 respectively) by d-limonene compared with control groups (28.6±0.5).Conclusion:D-limonene can inhibit the invasion capability of breast cancer cell MDA-MB-435.Down-regulating the expression of Fascin-1 to alter the microfilament cyto-skeleton may be part of the anti-cancer mechanism of d-limonene.