Figure 1.

Rapamycin inhibits the mTOR signaling pathway in HNSCC cells but does not affect cell proliferation in vitro. A, Inhibition of constitutively activate mTOR downstream targets, pS6 and p4E-BP1 by rapamycin (RP) is dose dependent in HNSCC cells without affecting the expression levels of S6, 4E-BP1, and Akt, or the detection of the phosphorylated species of Akt, as judged by Western blotting (WB) with the indicated antibodies. B, Cell cycle distribution represented as the fraction of HNSCC cells in G1, S, and G2/M phase upon rapamycin treatment for 24 h at the indicated concentrations was evaluated by FACS analysis after nuclear labeling of cells with propidium iodide. C and D Cytotoxic effects of rapamycin in HNSCC cell lines were determined by a colorimetric assay, MTT. C, After serum starvation for 24 h, cells were exposed to rapamycin at the indicated dose for 48 h before performing this assay. LY294002 (25 μM, LY) was used throughout as a positive control for PI3K/Akt/mTOR pathway inhibition. D, cells cultured in serum containing media were treated with interleukin 1β (IL-1β; 30 ng/ml) and rapamycin (100 nM) or vehicle for the indicated days. These assays were performed in triplicate. Similar results were obtained in multiple repetitions of these experiments (at least three times). *** p< 0.001