Fig. 1. Effects of d-glucosamine on cell proliferation and cell cycle progression in DU145 cells. (A) Effects of d-glucosamine on cell proliferation in DU145 cells were determined after 3 days of treatment using MTS assay. Percent growth inhibition was calculated as described in Materials and methods. The data shown are the mean of triplicates and representative of the three independent experiments. (B) Effects of d-glucosamine on cell cycle distribution in DU145 cells. Exponentially growing cells were treated for 72 h with d-glucosamine at the indicated concentrations, and their cell cycle distribution was determined by flow cytometric analysis of the DNA content after staining with propidium iodide as described in Materials and methods.

Fig. 2. Effects of d-glucosamine on the phosphorylation of p70S6K and its substrates in DU145 cells. (A) Time dependent effect of d-glucosamine. DU145 cells were treated with 5 mM d-glucosamine and whole cell extracts were prepared after incubation of the indicated times. The whole cell lysates were analyzed by immunoblotting. (B) Dose dependent effect of d-glucosamine. DU145 cells were treated with the indicated concentrations of d-glucosamine for 24 h. The whole cell lysates were subjected to immunoblotting. (C) Time dependent effect of PUGNAc. DU145 cells were treated with 100 μM PUGNAc and whole cell lysates were subjected to immunoblotting. (D) Effects of alloxan and streptozocin. DU145 cells were treated with 5 mM d-glucosamine, 5 mM alloxan, or 5 mM streptozocin for 24 h. The whole cell lysates were subjected to immunoblotting. (E) Effects of mannitol. DU145 cells were treated with indicated concentrations of mannitol for 24 h. The whole cell lysates were subjected to immunoblotting.

Fig. 3. Effects of d-glucosamine on MDA-MB-231, PC-3 and human skin fibroblast (HSF) cells. (A) Effects of d-glucosamine on cellular proliferation were determined after 3 days of treatment using MTS assay. Percent growth inhibition was calculated as described in Materials and methods. The data shown are the mean of triplicates and representative of the three independent experiments. (B) Effects of d-glucosamine on the phosphorylation of p70S6K and RPS6. The cells were treated with the indicated concentrations of d-glucosamine for 24 h. The whole cell lysates were subjected to immunoblotting.

Fig. 4. Combinatorial effect of d-glucosamine with rapamycin on the proliferation of DU145 cells. The cells were treated with a mixture of d-glucosamine and rapamycin for 3 days. Cell growth was examined using MTS assay and % GI was calculated as described in Materials and methods. The data shown are the mean of triplicates and representative of the two independent experiments.