Expression And Significance Of Caveolin-1 In Lung Cancer

IntroductionCaveolae are 50 - 100nm invaginated plasma membrane domains present in some kinds of terminally differentiated cells, such as fibroblasts, alveolar epithelial cells and endothelial cells. It has been proposed that caveolae functions as a key position to organize and concentrate specific lipids and lipidmodified signaling molecules within caveolae membrane. Caveolin - 1 is the principal component of caveolae membrane in vivo, and is found to suppress the kinase activity of Src family tyrosine kinase ( c - Src) , epidermal growth factor receptor , Neu, and protein kinase C through the caveolin - 1 scaffolding domain, a modular protein domain that recognizes a specific caveolin binding motif in various signaling molecules. The expression of caveolin — 1 is frequently lost in human lung cancer and breast cancer cell lines and other cancer cell lines, while the re - expression of caveolin - 1 in oncogenically transformed cell lines inhibits tumor cell growth and colony formation, so caveolin - 1 may function as a tumor suppressor. However, the expression and significance of caveolin - 1 is controversial in different tumors. This study was to detect the expression of caveolin -1 in lung cancer and human lung giant cell lines, and investigate whether there is the relationship between its expression and some clinicopathological factors and MVD.Materials and Methods1. Materials154 primary lung cancer tissue samples and the neighboring noncanceroustissue samples were obtained from patients who had surgery in the First Affiliated Hospital, China Medical University between 2001 and 2005. The histological diagnosis and grade of differentiation were evaluated by examination of hematoxylin and eosin - stained sections, according to the classification system of the World Health Organization (2004). All tumors were staged according to the p -TNM classification of the International Union Against Cancer (2002). 55 lung SCCs, differentiation: well in 12, moderately in 18, poorly in 25;clinical stage: I in 24 patients, II in 13, HI in 18;30 cases showed lymphatic metastasis. 60 lung adenocarcinomas, differentiation;well in 21, moderately in 20, poorly in 19;clinical stage;I in 24 patients, II in 10, HI in 23 and IV in 3;30 cases showed lymphatic metastasis. 13 LCLC, clinical stage;I in 6 patients, II in 1, HI in 5 and IV in 1;7 cases showed lymphatic metastasis. 3 adenosquamous carcinomas, 3 carcinoids, 5 adenoid cystic carcinomas , 1 epidermoid carcinoma and 14 SCLCs. 63 fresh tissue samples among 154 samples were adopted for western blot assay, which contained 25 lung SCCs, 25 lung adenocarcinomas and 13 neighboring noncancerous tissues. 36 lymph node metastasis including 15 lung SCCs, 16 lung adenocarcinomas and 5 LCLCs.Cell culture;Human high invasive pulmonary giant cell carcinoma, sub-series of PG cell lines BE1 and LH7 were obtained from Medical College of Beijing University as a gift. LH7 and BE1 cells lines were propagated in RPMI medium 1640 supplemented with 15% fetal bovine serum, 2g/L NaHCO2, PH 7. 2, 100 U/ml penicillin, and 100|xg/ml streptomycin at 37T! , 5% CO2, in a humidified incubator.2. ReagentsAntibodies and their sources were as follows;anti - caveolin - 1 IgG ( rabbit polyclonal antibody;Santa Cruz Biotech, Santa Cruz Biotech, CA);anti -CD34 IgG ( mouse monoclonal antibody;Maixin Biological Company, Fujian China);anti - (3 - actin IgG (rabbit polyclonal antibody;Zhongshan Biological Company, Zhongshan, China);goat- anti- rabbit second antibody ( Huamei Biological Company);ultrasensitive S - P kit and DAB agent kit( Maxin Biological Company, Fujian, China);RPMI 1640 and fetal bovine serum( GiBCO). FITC conjugated goat - anti - rabbit second antibody (ZF - 0311 ) was pur-chased from Zhongshan Biological Company, (Zhongshan, China);4, 6 - Dia-midino -2 - phenylindole (DAPI) (236276) was purchased from Roche Molecular Biochemicals. V3. Methods3. 1 ImmunohistochemistryTissue sections (4jxm thick) of formalin - fixed, paraffin - embedded specimens were deparaffinized in xylene, rehydrated in graded alcohol, and transferred to PBS. The slides were rinsed 3 times with PBS, and antigen retrieval was performed with pressure - cooker for 75 second. Endogenous peroxi-dase was blocked by the use of 3% hydrogen peroxide in PBS for 20 min. The samples were washed three times with PBS and incubated for 30 min at room temperature with a protein blocking solution. Excess blocking solution was drained, and the samples were incubated overnight at 4 C with anti - caveolin -1 antibody and anti - CD34 antibody. Immunodetection was performed with dia-minobenzidine as the chromogen and counterstained with hematoxylin and dehydrated in alcohol before mounting.Immunohistochemical evaluationCaveolin — 1 was a membrane/plasm staining protein. The type I pneumo-cytes or endothelial cell of blood vessels, both known to be abundant in caveolin - 1, were used as positive controls. For a negative control, phosphate buffered saline was used for the primary antibody. When over 50% of all cancer cells were stained, the tumor was considered caveolin - 1 positive;and less than 50% of all caner cells were stained , the tumor was considered caveolin - 1 negative.Quantification of MVDMVD was determined according to the procedure described by Weidnerby light microscopy after sections were immunostained with anti - CD34 antibodies. Clusters of stained endothelial cells distinct from adjacent microvessels, tumor cells, or other stromal cells were counted as one microvessel. The MVD was expressed as the average count of the 3 areas with the highest MVD identified within a single 200 field.3.2 ImmunofluorescenceLH7 and BE1 cells were cultured in 24 pores plate with a density of 2 x10 /ml for 48 hours, then fixed in 95% alcohol for 10 minutes, penetrated with 0. 1% Triton X - 100 for 10 minutes and incubated with 2% BSA for 1 hour. Then cells were incubated with polyclonal anti - caveolin - 1 antibody ( 1:125) overnight at humidified 4^ environment. The secondary antibody was labeled with FITC fluorescein. Incubated with second antibody for 1 hour at 37X1 , with 0. 1% DAPI for 10 minutes, then cells were rinsed in PBS and Triton(10:1) and then in PBS. Immunofluorescence was observed with immunofluorescence microscope (BX60, OLYMPUS). When the shape of cells can be identified by FITC fluorescence, it was judged positive expression.3. 3 Western blot assayProtein extracts were prepared by incubation of 1 x 10 cells in 500 ul of RIPA buffer for 30 minutes on ice. Tissue samples (1 -2g) were homogenized in the same buffer. After electrophoresis with 15% sodium dodecyl sulfate -polyacrylamide gel electrophoresis, the protein samples were electrotransferred to PVDF membrane, then incubated with polyclonal anti - caveolin - 1 antibody (1;400) overnight at 4CC. After incubated with Horseradish peroxidase - conjugated second antibodies, the bands were detected with automatic electrophoresis gel analytic system ( Chemilmager 5500, Alpha InnCh). To ensure equal loading amounts, f$ - actin was used as internal control, and all the density value of bands were compared with ($ - actin before analysis.4. Statistic analysisAll the data were analyzed with SPSS for Windows 13.0 software . The Chi - square test and precise probability method were adopted to compare the clini-copathological characteristics with the expressions of caveolin - 1. McNemar' s test was used for the trend of caveolin -1 expression in primary sites and nodal metastases. Student' s test was used to analyse the data from western blot and the correlation between caveolin - 1 expression and MVD. Statistical significance was defined as P <0.05.Results1. Immunohistochemistry1. 1 Caveolin - 1 was localized in membrane/plasm. It showed a strong expression in bronchial epithelial cells, alveolar epithelial cells and vascular en-dothelial cells. The positive percentage of caveolin - 1 in lung cancer tissues was 59. 1% , much lower than that in normal lung tissues (P <0. 01) , caveolin - 1 expression in small cell lung cancers (SCLC) (7.1%) was much lower than that in non - small cell lung cancers ( NSCLC) (64. 3% , P < 0. 01). In NSCLC, the expression of caveolin — 1 was much higher in patients with lymph node metastasis than that without lymph node metastasis (P =0. 005);and the expression of caveolin - 1 was much higher in HI ^ IV stage than that in I N II stage (P =0.042). .1. 2 The association between caveolin - 1 expression and MVD. In NSCLC, MVD was positively related to lymph node metastasis(t =2. 356,P =0. 020)and p - TNM stage (t = 2. 280, P = 0. 024). There was no difference for MVD between positive caveolin -1 staining cases and negative caveolin - 1 staining cases. In SCLC, there was no difference between MVD and clinicopathological profiles.2. ImmunofluorescenceThe immunofluorescence stain of caveolin - 1 protein was abundantly expressed in high - invasive BE1 and LH7 cells.3. Western blotCaveolin - 1 protein expression was observed in all the normal lung tissues. Caveolin - 1 protein in tumor tissues was noticeably lower than that in normal tissues (P =0. 0002, P =0. 0009) . And it was abundantly expressed in high -invasive BE1 and LH7, and the result was correspondently with the immunofluorescence result.DiscussionA number of signaling molecules inhibited by caveolin - 1 are involved in cell proliferation, apoptosis, adhesion and movement. It is well established that caveolin - 1 is a tumor suppressor gene. Caveolin - 1 mRNA and protein expression are frequently lost in human cancer cell lines, including lung cancer celllines and breast cancer cell lines. Re - expression of caveolin - 1 in oncogeni-cally transformed cell lines inhibits tumor cell growth and reduces tumorigenici-ty. Our study showed immunostaining for caveolin -1 was positive in 59. 1% of lung cancers, much lower than that in neighboring noncancerous tissues, and Western blot showed that caveolin - 1 expression was much lower than that in neighboring noncancerous tissues. Our results are consistent with the data that caveolin - 1 was down - regulated in lung cancer, gastric carcinoma, ovarian carcinoma, and sarcomas, so caveolin - 1 may be a tumor suppressor gene. To our knowledge, caveolin -1 scaffolding domain negatively regulates the activity of p42/44 MAP kinase with the result that caveolin - 1 dramatically inhibits signalling from EGF - R, Raf, MED - 1 and ERK -2 to the nucleus. These results indicate that down - regulation caveolin -1 expression in lung cancer may lead to an increase in basal activity of signaling pathways, and possibly affects the genesis of tumors.While, many researchers reported that caveolin - 1 may also function as a tumor metastasispromoting molecule, which is unrelated to its obvious function of cell growth inhibition. In our research, caveolin — 1 protein in lung cancer was much lower than that in neighboring noncancerous tissues. However in 140 NSCLC cases, caveolin -1 expression was positively related to lymph node metastasis and advanced stage. In the nodal metastases, the positive staining of caveolin - 1 was 86. 1 % , so in our research, caveolin - 1 also functions as a tumor metastasispromoting molecule. Ho and Yoo have reported that upregulated caveolin - 1 expression was associated with lymph node metastasis, advanced pathologic stage and poor prognosis in lung adenocarcinoma and squamous cell carcinoma respectively. Ho have also reported that elevated caveolin - 1 expression in lung adenocarcinoma cell lines correlated with enhanced cell invasive a-bility, and their study revealed that up - regulated caveolin - 1 in lung adenocarcinoma cells was necessary for mediating filopodia formation, which may enhance cell migration and, in part, the invasive ability of lung adenocarcinoma cells. In our study, immunofluorescence and Western blot showed that caveolin -1 protein were both abundantly expressed in high - invasive cell lines;BE1 and LH7. So we assumed that caveolin -1 may be important in the lymph nodemetastasis in NSCLC and the movement of cells may be affected by the caveolin - 1 during the progression of NSCLC.Angiogenesis is very important during the progression of many cancers. > Furthermore, caveolin - 1 may also be involved in angiogenesis: endothelial cells abundantly express caveolin - 1 and caveolae;caveolin — 1 interacts with a number of signaling proteins including prostacyclin synthase, VEGFR — 2 ^flk -1, which are involved in angiogenesis;caveolin - 1 plays an important positive role in the regulation of endothelial cell differentiation, a prerequisite step in the process of angiogenesis. Joo et al reported that caveolin - 1 may be important in the progression of clear cell RCC and angiogenesis may be affected by caveolin -1 during the progression of RCC. Our study showed that MVD was positively correlated with the lymph node metastasis and p - TNM stage, but there was no difference between positive caveolin -1 staining cases and negative caveolin -1 staining cases ( P > 0. 05 ) . We suppose that there is other mechanism that caveolin - 1 accentuated cancer cell invasiveness.Although there is no significance between caveolin 1 - 1 expression in all types lung cancer of NSCLC, however the expression of caveolin - 1 in SCLC was much lower than that in NSCLC. It is reported that the down - regulation of caveolin — 1 may be caused by hypermethylation of caveolin — 1 promoter. Suna-ga et al found caveolin - 1 methylation in 93% SCLC ( n = 15) and 9% NSCLC cell lines (n = 11) , whereas 5 - aza - 2 deoxyeytidine treatment restored caveolin -1 expression in SCLC cell lines. Wikman et aldidntfound methylation in the lung cancer samples which caveolin - 1 expression was down - regulated. These data indicate that down - regulate caveolin - 1 expression in SCLC may be caused by the promoter methylation of caveolin - 1 , whereas the molecular mechanism in NSCLC must be studied in the future.In conclusion, the expression of caveolin - 1 in lung cancer tissues was much lower than that in normal tissues. The expression of caveolin -1 was significantly correlated with advanced pathologic stage and lymph node metastasis in NSCLC. Caveolin -1 may be important in the progression of NSCLC.Conclusions1. The expression of caveolin -1 in lung cancer was much lower than that in normal lung tissues.2. The expression of caveolin - 1 in SCLC was much lower than that in NSCLC.3. In NSCLC, the expression of caveolin - 1 was positively related to lymph node metastasis and p - TNM stage.4. There was no difference for MVD between positive caveolin -1 staining cases and negative caveolin - 1 staining cases.5. Caveolin - 1 protein was abundantly expressed in high - invasive BE1 and LH7 cell lines.