| PART 1: THE EXPRESSION AND SIGNIFICANCE OF cFLIP IN HEAD AND NECK SQUAMOUS CELL CARCINOMAPurpose The Fas (also called APO-1 or CD95) system has been demonstrated to play a major role in cell apoptosis and immune surveillance. Although head and neck squamous cell carcinoma (HNSCC) cells constantly express Fas, they are still resistant to Fas-mediated apoptosis in vitro and in vivo, suggesting that head and neck squamous cell carcinoma cells might develop some intracellular inhibitors to overcome Fas-mediated apoptosis. Recently, cellular FLICE-like inhibitory protein (cFLIP) has been identified as an endogenous inhibitor of Fas or other receptor-mediated apoptosis and its overexpression has a suspected association with tumour development or progression. The aim of this study was to determine the expression of cellular FLICE-like inhibitory protein (cFLIP) in head and neck squamous cell carcinoma and revealed its possible correlation to tumour clinical parameters and Fas protein.Methods The expression of cFLIP was analysed in 58 head and neck squamous cell carcinoma samples and 30 morphologically normal tissues adjacent to the carcinomas using immunohistochemistry; reverse transcription-polymerase chain reaction (RT-PCR) was used to investigate the mRNA expressions of two isoforms of cFLIP in 30 head and neck squamous cell carcinoma and matched normal tissues; western blot analysis was also applied to investigate the protein expressions of two isoforms of cFLIP in 10 head and neck squamous cell carcinoma and matched normal tissues. Furthermore, its possible correlation to the expression of tumour clinical parameters and Fas protein were discussed.Results 1. cFLIP protein was demontrated to be up-regulated in most head and neck squamous cell carcinoma samples than in normal tissues by immunohistochemistry. Because 55 of 58 Head and neck squamous cell carcinoma samples expressed cFLIP protein at various levels, but only 3 of 20 normal tissues expressed cFLIP protein. And the difference in weighted scores between these two group was significant(P<0.01)2. Although the mRNA levels of both isoforms of cFLIP, long form (cFLIPL) and short form (cFLIPs), in head and neck squamous cell carcinoma samples were higher than those in normal tissues (P<0.01), only cFLIPL protein could be detected by western blot.3. Since there was significant correlation between expressions of cFLIPL mRNA and cFLIP protein and only cFLIPL protein could be detected by western blot analysis, cFLIPL could be regarded as the primay isoform of cFLIP in head and neck squamous cell carcinoma.4. Although there were no correlation between expression of cFLIPL protein and some tumour clinical parameters (e.g. age, gender, tumour site and histological grade), the expression of cFLIPL protein was significantly associated with tumour clinical stage (P<0.01) and lymph node metastasis (P=0.01).5. About 89.7% (52/58) head and neck squamous cell carcinoma samples were Fas protein positive , there was no significant difference between normal tissues and head and neck squamous cell carcinoma samples (P=0.376). And the Fas expression level in head and neck squamous cell carcinoma samples has no relation to any tumour clinicopathologic parameters Since all of the tumors with Fas immunostaining also express cFLIP protein, there was no significant correlation between them (P>0.05).Conclusions Overexpression of cFLIPL is a frequent event in head and neck squamous cell carcinoma. And the expression of cFLIPL protein was significantly correlated with tumour clinical stage and lymph node metastasis. So head and neck squamous cell carcinoma cells in vivo may need overexpressed cFLIPL to evade apoptosis mediated by Fas or other receptors, which might contribute to tumour development and progression. PART 2: THE MECHANISMS INVOLVED IN LUTEOLIN INDUCED APOPTOSIS IN HEP-2 CELLS AND ROLE OF cFLIP IN THIS PROCESSPurpose Luteolin, a naturally occurring flavonoid, possesses much biological function. In addition, its anti-cancer activity has attracted more and more attention in recent years. As being reported, Luteolin possesses activities against several human cancers, but its activity against head and neck squamous cell carcinoma (HNSCC) is never mentioned. And the mechanism of its anti-cancer activity needs to be elucidated. Although activation of caspase-8 and up-regulation of Fas and DR5 have been demonstrated to contribute to luteolin induced apoptosis in cancer cells, the possible role of their inhibitor, ie cFLIP, has not been discussed. Overexpression of cFLIPL is a frequent event in head and neck squamous cell carcinoma and head and neck squamous cell carcinoma cells in vivo may need it to evade apoptosis mediated by Fas or other receptors, which might contribute to tumour development and progression. And loss of caspase-8 activation pathway is a possible mechanism for CDDP resistance in human laryngeal squamous cell carcinoma, Hep-2 cells. So the purpose of this study was to evaluate the anti-cancer activity against HNSCC of luteolin and to provide some information about its mechanism using Hep-2 cells.Methods Hep-2 cell cultured in RPMI-1640 medium was treated with luteolin at different time and different concentration. The anti-proliferative effects of Luteolin on Hep-2 cells was evaluated by MTT assay; the cell cycle change of Hep-2 cells was analyzed by flow cytometry; The apoptosis of Hep-2 cells were confirmed by morphological alterations, Annexin-V/PI double staining and DAPI staining; western blot were performed on detecting the expression of cFILP and Fas protein; and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was used to investigate reasons about change of the expression of cFILP and Fas protein.Results 1. Luteolin can inhibite Hep-2 cells proliferation at time-dependent and dose-dependent manner. The concentration required for 50% inhibition of growth (IC50) at 24h, calculated by Bliss's method ,was 52.69μmol/L.2. Luteolin also induced cell cycle arrest in Hep-2 cells, most cells were arrested at G1 phase. And the effect was dose-dependent (P<0.01).3. The results of Annexin V/PI double staining demonstrated that Luteolin could induce apoptosis in Hep-2 cells and the effect was time dependent (P<0.01). And there was a gradual dose-dependent increase in the number of nuclear condensation after treatment with luteolin for 24 h in Hep-2 cells by DAPI staining.4. Caspase-3, caspase-8 and caspase-9 were all activated after luteolin treatment and their activaties increased along luteolin treatment. In addition, the increase of caspase-8 activity was more significant than those of caspase-3 and caspase-9 after 12h incubation.5. cFLIPS bands were so weak that could hardly be detected in normal and luteolin treated Hep-2 cells. cFLIPL was down-regulated at a time-dependent manner after luteolin treatment. On the contrary, Fas protein was up-regulated. Luteolin treatment was also demonstrated to down-regulate cFLIPL and up-regulate Fas mRNA at the same time.Conclusions Luteolin can inhibite proliferation of Hep-2 cells and induce cell cycle arrest at G1 phase in vitro. Both caspase pathways led by caspase-8 and caspase-9 were activated in luteolin induced apoptosis which also involves down-regulateion of cFLIPL protein. So Luteolin could be used as a novel anti-cancer drugs for many kinds of carcinoma including head and neck squamous cell carcinoma.
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