The Effect Of Caveolin-1 On The Growth And Phenotype Change Of Laryngeal Squamous Cell Carcinoma And Its Relative Signal Transduction Pathway

Caveolae are morphologically identifiable plasma membrane invaginations that are distinct from the large electron-dense clathrin-coated pits. It plays an important role in cellular processes, including molecule transport, cell adhesion and signal transduction. Caveolin-1(CAV1) is an essential structural component of caveolae and functionally regulates the activity of many signaling molecules, such as G-proteins, Src family kinases, H-Ras, protein kinase C, epidermal growth factor receptor (EGFR), endothelial nitric oxide synthase and integrins, which are potentially involved in the development of human cancer. Thus, caveolin-1 could be a key molecule for growth-related signaling and cancer development.Caveolin-1 has been considered as a candidate tumor suppressor. Down-regulation of caveolin-1 expression has been observed in breast, lung, sarcomas and ovarian cancer. Exogenous expression of caveolin-1 in oncogenically transformed cells and cancer cell lines inhibits cell growth and tumorigenesis. These evidences indicate that caveolin-1 can act as a tumor suppressor. But elevated expression of caveolin-1 is associated with development of prostate and pancreatic cancers; therefore, the role of caveolin-1 may vary considerably, depending on the tissue involved. Whether caveolin-1 plays as a tumor suppressor in human laryngeal squamous cell carcinoma (LSCC) is unknown so far.Thus, in the present study we have observed the biological effects of stable overexpression of caveolin-1 in human laryngeal squamous cell carcinoma HEp2 cell lines in vitro and in vivo; and also examined caveolin-1 expression in human laryngeal squamous cell carcinoma and its relation with clinic characters. Part I The Construction of HEp2 Transfectants Overexpressed CAV-1 GeneObjectives To construct HEp2 transfectants overexpressed caveolin-1 gene.Methods 632 bp cDNA fragment containing the complete coding region of human caveolin-1 was amplified by reverse transcriptase-polymerase chain reaction from pc-3 cells; the PCR product was subcloned into the pcDNA3.0 expression vector. Human laryngeal squamous cell carcinoma cell line HEp2 were transfected stably with recombinant plasmids of pcDNA3.0-CAV1 by Lipofectamine^TM 2000. The positive clones were determined by Real-time Quantitative RT-PCR, Western blot analysis and immunofluorescence respectively.Results The plasmids pcDNA3.0-CAV1 were transfected into HEp2 cells, and stable overexpressing caveolin-1 clones were obtained. The transfected cells, namely HEp2-CAV1-1, HEp2-CAV1-2 and HEp2-CAV1-3, were detected respectively about 6.89, 108.73 and 75.03 fold higher caveolin-1 gene expression than that in parental HEp2 cells. Western blots results showed that there were respectively 3.49, 11.56 and 7.56 fold increases in the level of caveolin-1 protein expression.Conclusion Successfully transfecting recombinant plasmid containing caveolin-1 to HEp2 cell by lipofectin method, three positive cell clones overexpressing markedly caveolin-1 mRNA and protein were established.Part II The Effects of CAV-1 Overexpression on Growth and Phenotype Change of Laryngeal Squamous Cell Carcinoma in vivo and in vitroObjective To study the influence of CAV-1 overexpression on the growth and phenotype change of laryngeal squamous cell carcinoma in vivo and in vitro.Methods Cells proliferation viability was tested by MTT assay; anchorage independent growth was determined by assaying colony formation in soft agar; flow cytometry was used to assay the cell cycle and apoptosis; Bcl-2, p53 and p21 proteins were examined by Westernblot; the in vivo antitumor activity of caveolin-1 was tested in HEp2 xenograft tumor models in athymic nude mice. Results MTT assay results showed that the proliferation of Caveolin-1 overexpression HEp2 cell clones decreased significantly comparing with the control. The tranfected cells exhibited a slower rate of growth and formed fewer colonies in soft agar. FACS analysis results revealed that overexpression of caveolin-1 resulted in the cell cycle arrest in the G₀/G₁ phase and increased the apoptotic cell fraction. After transfected, Bcl-2 protein expression was downregulated in HEp2 cells, without affecting p53 and p21 protein levels. In the xenograft tumor models in nude mice, Caveolin-1 overexpression HEp2 cell clones show the less tumor incidence, slower growth and smaller tumor weight.Conclusions Overexpression of caveolin-1 suppresses the growth of HEp2 cells in vivo and in vitro, and resulted in the cell cycle arrest in G₀/G₁ phase and increased the apoptotic cell fraction, downregulation of Bcl-2 may be included in its mechanism of pro-apoptosis.Part III caveolin-1 interacted with EGFR and downregulated activity of MAPKs signal transduction pathwayObjective To explore the interaction between caveolin-1 and EGFR, and inhibitory effect of caveolin-1 on MAPKs pathway.Methods Activities of Erk1/2 and EGFR were determined by Western blots, and the interaction between EGFR and caveolin-1 was determined by Co-Immunoprecipitation assay and localization of caveolin-1 and EGFR was studied by laser confocal microscopy. Immunohistochemistry was used to examine the protein expression of p-EGFR, p-Erk1/2 and caveolin-1 in HEp2 xenograft tumor models.Results Compared to the wild HEp2 cells, the phosphorylation of EGFR and Erk1/2 was reduced by 66% and 83% in HEp2-CAV1-2 cells and by 58% and 78% in HEp2-CAV1-3 cells. The interaction between EGFR and caveolin-1 was found by co-immunoprecipitation, EGFR and caveolin-1 were colocalized on cell membrane and cytoplasm. Four tumors of the transfected cells group (HEp2-CAV1-3) showed stronger stains of caveolin-1 than HEp2 or HEp2-pcDNA3.0 group, whereas reduced phosphorylation of EGFR and Erk1/2 were found.Conclusions Exogenous expression of caveolin-1 interacts with EGFR in HEp2 cells via its scaffolding domain and negatively regulates the activity of EGFR-MAPK signaling pathway.Part IV Expression of caveolin-1 and hTERT in LSCC and its relation to cliniccharactersObjective To explore the expression of caveolin-1 and hTERT in LSCC and its relation to clinic charactersMethod Immunohistochemistry was used to detect the expression of caveolin-1 in human laryngeal squamous cell carcinoma (LSCC). In situ hybridization used to examine the expression level of hTERT mRNA.Result Positive rates of hTERT mRNA in normal laryngeal tissue, laryngeal hyperplasia and LSCC were 0%(0/10), 10%(1/10), 80%(32/40), respectively. The prevalence and intensity of hTERT mRNA expression were much greater in LSCC than those in normal laryngeal tissue or laryngeal hyperplasia (P < 0.05). The expression level of hTERT was not correlated with age, gender, lymph node metastasis, TNM category and clinicopathological stage (P < 0.05). Positive rates of caveolin-1 in LSCC (82%, 33/40) was less than normal laryngeal tissue (100%, 10/10) and laryngeal hyperplasia (100%, 10/10), but there was no significance in statistics. The expression of caveolin-1 was not correlated with hTERT expression, age, gender, lymph node metastasis, TNM category and clinicopathological stage (P > 0.05).Conclusion Level of hTERT mRNA was obvious higher than laryngeal normal tissues and hyperplasia, Semi-quantitative detection of hTERT mRNA expression in situ is helpful in differentiated diagnosis of malignant tissues and non-malignant tissues of larynx. Positive rate of caveolin-1 expression in LSCC was less than normal laryngeal tissues and laryngeal hyperplasia, but there was no significance in stastistics.