| Summary: | 博士 === 中山醫學大學 === 醫學分子毒理學研究所 === 96 ===
ABSTRACT
Telomerase expression is the hallmark of tumor cells in which this ribonucleoprotein complex activation may therefore be a critical step in cellular immortalization and oncogenesis. Fungal immunomodulatory proteins, FIP-gts, were found in Ganoderma tsugae. This protein has been implicated in activating human peripheral blood mononuclear cells. However, the effect of FIP-gts on cancer cells has not clearly been described. In the present study, we expressed and purified the recombinant fungal immunomodulatory protein (reFIP-gts) in E. coli, and demonstrated its role in tumor suppressor activity. Treatment of A549 cells with reFIP-gts significantly inhibited their growth but did not affect MRC-5, which is human normal lung fibroblast. We further demonstrated that reFIP-gts suppressed telomerase activity in a concentration-dependent manner. Repression of telomerase activity can be accounted by a decrease in expression of the telomerase catalytic subunit (hTERT) as revealed by decreasing its mRNA level, suggesting that the reFIP-gts may directly or indirectly regulate trnascirption of telomerase subunit. These results were also confirmed by luciferase reporter assay, in which transient transfection of A549 cells with a construct containing the luciferase reporter gene driven by a functional hTERT promoter. Further deletion analyses of hTERT promoter revealed a 19-bp region (-196 to -177) that clearly showed transrepression activiy of FIP-gts . Since this region contains an E-box, we performed electrophoretic mobility shift assays and Chromatin immunoprecipitation assay to demonstrate that the binding activity of c-Myc transcriptional factor to the E-box sequence of the hTERT promoter was inhibited in response to reFIP-gts treatment. `
Here, we proved that reFIP-gts entry into the cell and localization in endoplasmic reticulum can result in ER stress, thereby increasing ER stress markers (CHOP/GADD153) and intracellular calcium release in A549 cells. The use of calcium chelator restores reFIP-gts–mediated reduction in telomerase activity. These results strongly suggest that ER stress induces intracellular calcium release and results in inhibition of telomerase activity. Although reFIP-gts decreased hTERT mRNA level in both A549 and H1299 cells, only the telomerase activity in A549 cells was inhibited. Surprisingly, we found that reFIP-gts induces translocation of hTERT from the nucleus into the cytosol in A549 cells but not in H1299 cells. Using leptomycin B, nuclear export inhibitor, we showed that hTERT is not transported. Using MG132, a proteasome inhibitor, reFIP-gts also prevents hTERT translocation from proteasome degradation. Based on these findings, the posttranslational modifications of hTERT protein might play more important role than down-regulation of hTERT transcriptional activity in mediating reFIP-gts-induced suppression of telomerase activity in lung cancer cells. We also demonstrated that reFIP-gts-treated lung cancer cells were arrested at G1 phase by flow cytometry and its morphological phenotype was consistent with cellular senescence. The premature senescent nature of these cells was supported by positive staining for senescence-associated β-galactosidase activity , increased lysosomal content, and up regulation of cyclin-dependent kinase 2 inhibitor p27, but lacking of telomere shortening in A549 lung cancer cells. Finally, reFIP-gts grew more slowly and formed significantly fewer A549 cell colonies in soft agar. Importantly, in mouse model, A549 cells treated with reFIP-gts grew significantly slower than the cells treated with PBS alone. This study evidences that reFIP-gts strongly inhibits tumorigenesis of lung cancer, and thus it may turn into an attractive agent for chemoprevention and antineoplastic therapy.
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