Expression Of Caveolin-1 In Cancer Associated Fibroblasts And Bcl-2 Or TIGAR In Breast Cancer Cells And Its Effect On Cancer Cell Apoptosis

ObjectiveThe correlation between the expression of Caveolin-1 in fibroblasts and B-cell lymphoma-2（Bcl-2）or tumor protein 53?induced glycolysis and apoptosis regulator（TIGAR）in breast cancer cells was researched through establishing a co-culture model of human breast cancer cells with fibroblasts in vitro,and the effect of this expression on breast cancer cell apoptosis was investigated.Also this correlation was investigated in breast tumor-burdened nude mice model.This study could provide theoretical and experimental basis for molecular targeted therapy of breast cancer.Methods1.Cell culture and co-culture in vitroHuman breast cancer cell line MDA-MB-231 and human fibroblast line CCC-ESF-1（ESF）were cultured,respectively,in DMEM-H medium with 10%fetal bovine serum,at 37?C in a humidified atmosphere with 5%CO₂.When cells grew to 80%～90%confluence,cells were subcultured.MDA-MB-231 and ESF cells were co-cultured using the culture plates and trans wells to detecte the expression of Caveolin-1 in fibroblasts and Bcl-2 and TIGAR in breast cancer cells.2.RT-q PCR We used Trizol method to extract total cellular RNA,judged the quality of RNA extraction.c DNA synthesized by reverse transcription was stored at-20℃.PCR amplification reaction was processed.40 cycles were used and melting curve was from 65℃to 95℃.The experimental data were automatically calculated and analyzed by fluorescence quantitative PCR analysis software named Bio-Rad CFX Manager.3.Western-Blot We washed and collected the cultured cells,and added cell lysis buffer.According to the instruction of BCA protein assay kit,protein standart was prepared.20μg protein of each lane were put for protein polyacrylamide gel electrophoresis（10%separated gel,10%concentrated gel）.The electrophoresprotein was transferred to the PVDF membrane.After incubation in a blocking buffer（5%non-fat dry milk）,the membranes were immunoblotted with the first antibody.After washing,the membranes were incubated with the secondary antibody.Finally,the membranes were developed using ECL.The optical density of each electrophoresis band of western blotting was analyzed using Image J software.4.Establishing a breast cancer model in nude mice 100μL cell suspension（cell concentration:2×10⁶MDA-MB-231 cells,1×10⁶ESF cells）were injected into the breast of right side of 6-7 week old female nude mice.Every other day we used vernier caliper to measure the length-diameter（a）and the short-diameter（b）of the tumors,calculated the tumor size according to tumor volume formula:V（mm3）=1/6πab2.Length-diameter of tumor was 5 mm which were regarded as tumor occurrence in tumor-burdened nude mice.The nude mice were killed by cervical dislocation at the 30th day of implanting MDA-MB-231 and ESF cells.The tumor tissues were put into 10%formalin to fix tissues.5.Experiments of cell apoptosis?Annexin V experiment Cells（1×10⁶in each sample）were incubated with Annexin V-Biotin for15 min,then incubated with streptavidin-FITC for15 min.7-amino-actinomycin 7（AAD）was used to distinguish apoptotic cells from other cell death.Finally,the samples were detected by flow cytometry.?Fluorescence detection of caspase3 activity Total protein was extracted from the cultured cells.We set up blank goups and experimental groups.Caspase 3 reaction buffer,which contained fluorescent substrate AC-DEVD-AMC,was added into blank goups and experimental groups.Normal cell lysis solution was added into blank goups.The extracted proteins from cells were added into experiments groups.The amount of released AMC was detected by fluorescence spectrophotometer,then the fluorescent intensity was analyzed.6.Immunofluorescence detectionTumor tissue was made into paraffin embedding sections.The paraffin sections were labeled by immunofluorescence dye after dewaxing process.Main steps were as follows:repairing antigen in 0.01 M citrate buffer in microwave oven;blocking with 1%BSA at 37℃for 30 min;incubating sections with primary antibody at 4℃overnight;placing them at room temperature for 30 min;incubating sections with fluorescent secondary antibody in dark at 37℃for 60 min.The results were examined by laser confocal microscope,photographed,and analyzed by Image-Pro Plus software.7 Immunohistochemical experiment Dewaxing section;incubating sections with 3%H₂O₂at 37℃for 10 min;repairing antigen in microwave oven;incubating sections with the first antibodies overnight;incubating sections with the second antibodies in dark at 37℃for 30 min;showing color with DAB;redyeing nuclei with hematoxylin.The results were examined by microscope,photographed,and analyzed by Image-Pro Plus software.8.Statistical analysisData are presented as mean±SD.SPSS Statistics software（17.0）was used to analyze the data.Statistical analysis between two groups was performed by Student’s t-test.P values of<0.05 were considered to be statistical significance,P values of>0.05 were considered to be statistical no-significance.Results1.The results of Caveolin-1 expressed in ESF and MDA-MB-231 cells RT-q PCR and Western blot experimental results showed that Caveolin-1 m RNA and protein expression levels of ESF cells were higher in ESF mono-culture groups than in MDA-MB-231+ESF co-culture groups（P<0.05）.Caveolin-1 m RNA and protein expression levels of MDA-MB-231 cells were not significant difference between MDA-MB-231 mono-culture groups and MDA-MB-231+ESF co-culture groups（P>0.05）.These results indicated that Caveolin-1 expression of fibroblats could be down-regulated in the co-culture of breast cancer cells with fibroblasts,but the co-culture had not significant effect on Caveolin-1 expression of breast cancer cells.2.The results of anti-apoptotic factors Bcl-2 and TIGAR expressed in MDA-MB-231 cells The results of RT-q PCR and Western blot showed that Bc l-2m RNA and protein expression levels of MDA-MB-231 cells were lower in MDA-MB-231 mono-culture groups than in MDA-MB-231+ESF co-culture groups（P<0.05）,and TIGAR m RNA and protein expression levels of MDA-MB-231 cells were also lower in MDA-MB-231 mono-culture groups than in MDA-MB-231+ESF co-culture groups（P<0.05）.These results indicated that the co-culture of breast cancer cells with fibroblasts could up-regulated anti-apoptotic factors Bcl-2 and TIGAR in breast cancer cells.3.The effect of the co-culture on MDA-MB-231 cell apoptosis The results of caspase3 fluorescence detection showed that caspase3 activity level of MDA-MB-231cells was higher in MDA-MB-231 mono-culture groups than in MDA-MB-231+ESF co-culture groups（P<0.05）.The results of Annexin V-Biotin showed that MDA-MB-231 cells of early apoptosis in MDA-MB-231 mono-culture groups were more than in MDA-MB-231+ESF co-culture groups.These results indicated that the co-culture of breast cancer cells with fibroblasts could inhibite MDA-MB-231 cell apoptosis.4.The experimental results of breast cancer tissues from tumor-burdened nude mice（1）HE staining confirmed the breast cancer tissues from tumor-burdened nude mice.（2）Green fluorescence represented positive expression of Caveolin-1.Immune fluorescent confocal microscopy observation showed that cancer tissues appeared strong green fluorescence in MDA-MB-231 groups and weak green fluorescence in MDA-MB-231+ESF groups.The results of fluorescent intensity analyzed by Image-pro plus software showed that the fluorescent intensity in MDA-MB-231 groups significantly increased compared with MDA-MB-231+ESF groups.This indicated that co-growth of breast cancer cells with fibroblasts could down-regulate Caveolin-1 expression in human tumor-burdened nude mice.（3）Colour tan represented positive expression of Bcl-2 and TIGAR.The results of immunohistochemistry showed that Bcl-2 and TIGAR were expressed in breast cancer tissues of human breast tumor-burdened nude mice.The results of the expressed Bcl-2 and TIGAR optical density analyzed by Image-pro plus software showed that the average optical density of Bcl-2 and TIGAR in the sections of breast cancer tissues was stronger in MDA-MB-231+ESF groups than in MDA-MB-231groups（P<0.05）.These results indicated that the co-growth of breast cancer cells with fibroblasts could promote Bcl-2 and TIGAR expression in human breast tumor-burdened nude mice.5.The experimental results of clinical breast cancer tissues（1）HE staining confirmed the breast cancer tissue from clinical breast cancer tissues.（2）Immunohistochemical optical microscope observation showed that the particles of colour tan were positive expression of Caveolin-1.The results of the expressed Caveolin-1 optical density analyzed by Image-pro plus software showed that the average optical density of Caveolin-1 in the sections of breast cancer tissues was low compared with breast no-cancer tissues（P<0.05）,which indicated Caveolin-1expression was lower in breast cancer tissues than in breast no-cancer tissues.（3）Immunohistochemical optical microscope observation showed that the particles of colour tan were positive expression of Bcl-2 and TIGAR.The results of the expressed Bcl-2 and TIGAR optical density analyzed by Image-pro plus software showed that the average optical density of Bcl-2 and TIGAR in the sections of breast cancer tissues was high compared with breast no-cancer tissues（P<0.05）.This indicated that Bcl-2和TIGAR expression was higher in breast cancer tissues than in breast no-cancer tissues.Conclusions1.The co-culture of breast cancer cells with fibroblasts in vitro could down-regulate Caveolin-1 expression of fibroblasts,but has no obvious effect on Caveolin-1 expression of breast cancer cells.2.The co-culture of breast cancer cells with fibroblasts in vitro could up-regulate the expression of anti-apoptotic factors Bcl-1 and TIGAR of breast cancer cells,wich could inhibite MDA-MB-231 cell apoptosis.3.The co-growth of breast cancer cells with fibroblasts in vivo could donw-regulate Caveolin-1 expression in human breast tumor-burdened nude mice,but up-regulate Bcl-and TIGAR.4.TIGAR positive rate was 80%,Bcl-2 72%,Caveolin-1 52%in 25 eases of clinical breast cancer.tissues.5.The expression of Caveolin-1 in breast cancer associated fibroblasts is negatively correlated with Bcl-2 and TIGAR expression in breast cancer cells.