Transcriptionally mediated inhibition of telomerase of fungal immunomodulatory protein from Ganoderma tsugae in A549 human lung adenocarcinoma cell line

Abstract

Telomerase expression is the hallmark of tumor cells, and activation of this ribonucleoprotein complex may be a rate-limiting or critical step in cellular immortalization and oncogenesis. Fungal immunomodulatory protein, FIP-gts, has been isolated from Ganoderma tsugae. In the present study, we expressed and purified the recombinant fungal immunomodulatory protein reFIP-gts in E. coli. We found that reFIP-gts significantly and selectively inhibits the growth of A549 cancer cells while not affecting the growth of normal MRC-5 fibroblasts. The reFIP-gts suppression of telomerase activity is concentration-dependent, due to the downregulation of the telomerase catalytic subunit (hTERT). It also happens at the mRNA level. These results were confirmed by transient transfections of A549 cells with pGL3-Basic plasmid constructs containing the functional hTERT promoter and its E-box-deleted sequences cloned upstream of a luciferase reporter gene. With electrophoretic mobility shift assays and Western blotting, we demonstrated that in response to reFIP-gts, binding of c-myc transcriptional factor to the E-box sequence on the hTERT promoter is inhibited. These results show that reFIP-gts suppresses telomerase activity and inhibits transcriptional regulation of hTERT via a c-myc-responsive element-dependent mechanism. Our findings provide new insight into both the anticancer function of reFIP-gts and the regulation of hTERT/telomerase expression, which may be valuable in the development of a promising chemopreventive agent. (c) 2006 Wiley-Liss, Inc.