The Expressions Of TWEAK And Its Receptor Fn14 And Its Relationship With MVD In Breast Cancer

INTRODUCTIONTWEAK ( TNF - related weak inducer of apoptosis ) is a member of the tumor necrosis factor (TNF) superfamily, and a type II transmember protein , but can be cleaved to generate a 18kDa soluble factor with biological activity. TWEAK has pleiotropic biological effects to induce apoptosis and cytotoxicity, proliferation, migration, expression of cytokine and angiogenesis. Fnl4( fibro-blast growth factor — inducible 14 ) is a member of the tumor necrosis factor receptor superfamily and a type I transmember protein, and switch series of signal transduction pathway, initiation homologous gene activation by linking different receptor proteins with TRAF ( TNF receptor - associated factor) structure domain or intracytoplasm protein. TWEAK/Fnl4 signal transduction pathway implies many progress of pathological disease, but the true mechanism is presently unknown. TWEAK expresses at low levels in many normal tissue and non-lymphocyte tumor cells, but significantly higher levels in a variety of tumor than original tissue, at the same time Fnl4 expresses higher in tumor than normal tissue. However, the alterative expression universalism of TWEAK and the significance to prognostic evaluation will need further to study.At present, investigation is very few about the expression of TWEAK and its receptor Fnl4 in tumor tissues and its relationship with MVD. So we assessed the expressions of TWEAK and its receptor Fnl4 in breast cancer and its relationship with MVD by immunohistochemical staining, western blot, ELISA.MATERIALS AND METHODS1. Tissue samplesThirty - five cases of breast ductal infiltrating carcinoma and another thirty - five cases of ductal carcinoma in situ with neighboring thirty cases of noncan-cerous tissue samples were obtained from patients underwent surgical resection in the First Affiliated Clinical hospital of Chinese Medical University between 2003 and 2004, fixed in 4% paraformaldehyde, embedded in paraffin. All the patients did not received radiotherapy and chemotherapy.Human breast adenocarcinoma cell lines MCF - 7 (low metastasis ) and MDA -MB -231 (high metastasis )were purchased from Chinese Science Institutes Cell Company. MCF -7 was cultured in Dulbecco's modified Eagles medium and MDA -MB -231 was cultured in RPMI 1640 in humidified 5%C02air2. ReagentsRabbit anti - TWEAK polyclonal antibody and goat anti - Fnl4 polyclonal antibody were purchased from Santa Cruz Company, and mouse anti - CD34 monoclonal antibody was purchased from Daco Company. The Ultro - sensitive S - P kit and DAB agent kit were bought from Fujian Maxin Biological Company. ELISA Instant Assay kit was purchased from Bender MedSystems.3. Methods3. 1 Immunohistochemical staining for TWEAK was performed on 70 surgically resected breast cancer tissues. Briefly, deparaffinized sections were heated to reactivate the antigen and treated with 0. 3% H2O2 in methanol for 20 min to abolish endogenous peroxidase activity. Sections were blocked with normal goat serum or normal rabbit serum in phosphate - buffered saline, and incubated with a 1:50 dilution ( in phosphate - buffered saline) of rabbit anti - TWEAK polyclonal antibody, goat anti - Fnl4 polyclonal antibody and mouse anti -CD34 monoclonal antibody overnight at 4 C. The sections were incubated with a second biotinylated antibody, followed by avidin - biotin - peroxidase complex for 30 min. After washing, they were developed in a substrate solution of 0.01% 3, 3'— diaminobenzidene -hydrogen peroxide and counterstained with he-matoxylin. The staining was performed according to the manufacture instructions of The Ultro - sensitive S - P kit.3. 2 The amount of secreted TWEAK in the culture medium of the two human breast cancer cells was measured by ELISA. The culture supernatant was collected, centrifuged for 10 min (3,000 rpm at 4 C) , and the obtained supernatant was used for experiments. The protein content was measured using an ELISA Instant Assay kit. Color development was measured at 450nm with a mi-crotiter reader.3. 3 Expression of TWEAK in human breast adenocarcinoma cell lines MCF-7 and MDA -MB -231 was analyzed by Western blot. Briefly, collect the cell . After 13000/minute centrifugation for half an hour at 4^ , supernatants were collected, the protein content was measured using a Bio - Rad Protein Assay kit. Equal amounts of protein from each extract were separated by 15% sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( SDS - PAGE ) and transferred onto nitrocellulose membranes. Blots were blocked by incubating in 5% nonfat milk, overnight at 4 C with rabbit anti — TWEAK polyclonal antibody or goat anti - Fnl4 polyclonal antibody. Antibodies were diluted 1:200 with 5% nonfat milk in Tris — HC1 ( pH 7.5) and 0. 1% Tween 20. After the final wash, signal was detected with a DAB kit for three to ten minutes, and nitrocellulose membranes were dried and preserved without light.4. Statistical analysis:Statistical Product and Services Solutions ( SPSS) statistical software ( version 13. 0) was applied for data analysis. T - test was used to compare the expression of TWEAK and Fnl4 and the relationship with MVD. Pearson - test was used to test the correlation of TWEAK and Fnl4. A P -value less than 0. 05 were considered significant.Results1. Expression of TWEAK protein in breast cancer.( 1) Immunohistochemically, the expression of TWEAK in breast cancer ishigher than in normal tissue (P < 0. 05) , the expression of TWEAK in breast ductal infiltrating carcinoma is higher than in breast ductal carcinoma in situ (P <0.05).(2)The expression of TWEAK in cell line MDA - MB -231 is higher than cell line MCF - 7 by Western Blot ( P < 0.05).(3)The secretion of soluble TWEAK in cell line MDA - MB -231 is higher than ceU line MCF -7 by ELISA (P < 0. 05 ).2. Expression of Fnl4 protein in breast cancer. Immunohistochemically , the expressions of Fnl4 in breast cancer is higherthan in normal breast tissue(P <0. 05) , the expressions of Fnl4 in breast ductal infiltrating carcinoma is higher than in breast ductal carcinoma in situ ( P < 0. 05).3. The relationship of the expressions of TWEAK and its receptor Fnl4 with MVD in breast cancer.(1) In breast ductal infiltrating carcinoma, the expression of TWEAK is significantly correlation with MVD ( P < 0. 05 ) . In breast ductal carcinoma in situ is not so ( P >0. 05). But the MVD in breast ductal infiltrating carcinoma is higher than in breast ductal carcinoma in situ ( P <0. 05) .(2) In breast ductal infiltrating carcinoma, the expression of Fnl4 is not correlated with MVD (P >0. 05) . In breast ductal carcinoma in situ the expression of Fnl4 is not correlated with MVD (P >0. 05).4. The correlation of TWEAK and Fnl4.The expression of Tweak is significantly positive correlation with the expressions of Fnl4 in breast ductal infiltrating carcinoma (P <0. 05) , but the correlation is not in breast ductal carcinoma in situ (P >0.05).Conclusion1. The expression of TWEAK and its receptor Fnl4 is significantly higher in breast cancer than in normal breast tissues. The expression of TWEAK and its receptor Fnl4 is higher in breast ductal infiltrating carcinoma than in ductal carcinoma in situ.2. The expression of membrane TWEAK and the secretion of soluble TWEAK are both higher in high metastasis cell line than low metastasis cell line.3. The expression of TWEAK is significantly correlation with MVD in breast ductal infiltrating carcinoma. The MVD is higher in breast ductal infiltrating carcinoma than in ductal carcinoma in situ.4. The expression of TWEAK is significantly positive correlated with the expressions of Fnl4 in breast ductal infiltrating carcinoma.