Tacrolimus causes reduced GLI1 expression and phenotypic changes in the TE 354.T basal cell carcinoma cell line.

Objective The product of GLI-1 gene is a key protein in the intracellular transduction pathway of the intercellular signaling molecule Sonic hedgehog (SHH). SHH controls the proliferation and differentiation of several cells populations during development and in mature organism. Overexpression of GLI-1 is typical of basal cell carcinoma and is presumed to be a key event in the cell biology of this tumor. Increased proliferation and expression of BCL-2 and reduced expression of laminin are believed to be relevant events downstreams of GLI-1. Tacrolimus (FK-506) has extensive homology with rapamycin, an immunomodulator molecule which can antagonise the cellular transformation caused by GLI-1 overexpression. We have addressed here whether Tacrolimus affects the expression of GLI-1 and which consequences this bears on the behavior of a basal carcinoma cell line, TE354.T. Methods Upon extraction of mRNA with Trizol, the expression of GLI-1 was evacuate by quantitative RT-PCR with TaqMan polymerase. GLI-1 and BCL-2 protein expression was analyzed in cell lysates by gel eleectrophoresis and Western blotting. Suspended and adherent cells were in part fixed with periodate-lysine-paraformaldeyde and analyzed by immunohistochemistry, in part they were fixed with Karnovsky and analyzed by electron microscopy. Results TE354.T cells are flat and rich in organelles of the secretory pathway and in intermediate filaments; the cell surface adhering to the substrate is coated in part by a basal lamina. These cells express GLI-1 mRNA; BCL-2 and laminin are expressed at the protein level with variable intensity among cells. Three and seven day treatment with Tacrolimus (0,5–5-50ng/ml) led to a significant, dose-related drop in the expression of GLI-1mRNA. The cell growth rate remained unaltered. The decrease in GLI1-mRNA was accompanied by a decrease in the antiapoptotic protein BCL-2 and in the fraction of cells immunolabeled for this protein, and by an increase in the fraction of cells immunolabeled for laminin, as well as by an increased amount of basal lamina material close to cells, as shown by electron microscopy. Conclusions The data indicate that Tacrolimus causes partial inhibition of GLI-1 transcription in basal carcinoma cells spontaneously expressing this molecule and that this inhibition is accompanied by reduced expression of BCL-2 and increased expression of laminin and presumably other basal lamin molecole. These results support the hyphotesis of a role of GLI-1 in the regulation of the expression of BCL-2 and basal lamina proteins and also lead to propose tacrolimus as a suitable tool to address issues related to SHH signaling pathway in neoplastic cells and presumably in normal cells as well.