Objective: To evaluate the influence of Everolimus (RAD001) on chemically induced urothelial lesions in mice and its influence on in vitro human bladder cancer cell lines.Methods: ICR male mice were given N-butyl-N-(4-hydroxybutyl) nitrosamine in drinking water for a period of 12 weeks. Subsequently, RAD001 was administered via oral gavage, for 6 weeks. At the end of the experiment, all the animals were sacrificed and tumor development was determined by means of histopathologic evaluation; mammalian target of rapamycin (mTOR) expressivity was evaluated by immunohistochemistry. Three human bladder cancer cell lines (T24, HT1376, and 5637) were treated using a range of RAD001 concentrations. MTT assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and flow cytometry were used to assess cell proliferation, apoptosis index, and cell cycle analysis, respectively. Immunoblotting analysis of 3 cell line extracts using mTOR and Akt antibodies was performed in order to study the expression of Akt and mTOR proteins and their phosphorylated forms.Results: The incidence of urothelial lesions in animals treated with RAD001 was similar to those animals not treated. RAD001 did not block T24 and HT1376 cell proliferation or induce apoptosis. A reduction in cell proliferation rate and therefore G 0 /G 1 phase arrest, as well as a statistically significant induction of apoptosis (P ϭ 0.001), was only observed in the 5637 cell line.Conclusion: RAD001 seems not to have a significant effect on chemically induced murine bladder tumors. The effect of RAD001 on tumor proliferation and apoptosis was achieved only in superficial derived bladder cancer cell line, no effect was observed in invasive cell lines.
The free use of rice in the phenylketonurics diet needs the reduction of phenylalanine (Phe) content from its proteins. This procedure involves protein extraction and hydrolysis followed by Phe removal. Enzymatic processes were then used for extracting and hydrolyzing rice proteins. In the former, a protease from Bacillus licheniformis was used while for the second case, six different enzymes were tested (pancreatin, papain, proteases from Aspergillus sojae, Bacillus stearothermophilus and two from Bacillus subtilis). Also, the activated carbon (AC) was employed as the adsorptive medium for removing Phe. The efficiency of Phe removal was evaluated by second derivative spectrophotometry. The hydrolysate prepared with papain showed the highest Phe removal (94.1%), using an E:S ratio of 4:100, a protein:AC ratio of 1:88 and three different meshes of AC simultaneously.
COX2 expression in HPV16-induced lesions may be modulated by nutraceuticals, reducing tumor-associated inflammation. However, this was not sufficient to block carcinogenesis, calling for additional studies focused on combination therapies.
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