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The Smoothened (SMO) receptor is the most druggable target in the Hedgehog (HH) pathway for anticancer compounds. However, SMO antagonists such as vismodegib rapidly develop drug resistance. In this study, new SMO antagonists having the versatile purine ring as a scaffold were designed, synthesised, and biologically tested to provide an insight to their mechanism of action. Compound 4s was the most active and the best inhibitor of cell growth and selectively cytotoxic to cancer cells. 4s induced cell cycle arrest, apoptosis, a reduction in colony formation and downregulation of PTCH and GLI1 expression. BODIPY-cyclopamine displacement assays confirmed 4s is a SMO antagonist. In vivo, 4s strongly inhibited tumour relapse and metastasis of melanoma cells in mice. In vitro, 4s was more efficient than vismodegib to induce apoptosis in human cancer cells and that might be attributed to its dual ability to function as a SMO antagonist and apoptosis inducer.

Amongst the available methodologies for protein determination, the bicinchoninic acid (BCA) assay highlights for its simplicity, sensitivity, repeatability and reproducibility. Nevertheless, in spite that the general principle behind this methodology is known, there are still unanswered questions regarding the chemistry behind the assay and the experimental conditions commonly employed. The present work explored the kinetics, and the analytical response of the assay to free amino acids, peptides (containing tryptophan and tyrosine), and proteins. Results revealed kinetic profiles characterized by the absence of plateaus, with behaviors depending on the type of the sample. The latter, along with contribution to the BCA index elicited by oxidation products generated at the side chain of tryptophan and tyrosine, as well as pre-oxidized β-casein, evidenced the presence of complex reaction mechanisms. In spite of such complexity, our results showed that the BCA index is not modulated by the incubation time. This applies for responses producing absorbance intensities (at 562 nm) higher than 0.1. Therefore, we propose that the assay can be applied at shorter incubation times (15 min) than those indicated in manufactures specifications, and usually used by researches and industry (30 min at 37 °C)., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Dityrosine and ditryptophan bonds have been implied in protein crosslinking. This is associated with oxidative stress conditions including those involved in neurodegenerative pathologies and age-related processes. Formation of dityrosine and ditryptophan derives from radical-radical reactions involving Tyr˙ and Trp˙ radicals. However, cross reactions of Tyr˙ and Trp˙ leading to Tyr-Trp crosslinks and their biological consequences have been less explored. In the present work we hypothesized that exposure of free Tyr and Trp to a high concentration of carbonate anion radicals (CO 3 ˙ - ), under anaerobic conditions, would result in the formation of Tyr-Trp species, as well as dityrosine and ditryptophan crosslinks. Here we report a simple experimental procedure, employing CO 3 ˙ - generated photochemically by illumination of a Co(iii) complex at 254 nm, that produces micromolar concentrations of Tyr-Trp crosslinks. Analysis by mass spectrometry of solutions containing only the individual amino acids, and the Co(iii) complex, provided evidence for the formation of o , o '-dityrosine and isodityrosine from Tyr, and three ditryptophan dimers from Trp. When mixtures of Tyr and Trp were illuminated in an identical manner, Tyr-Trp crosslinks were detected together with dityrosine and ditryptophan dimers. These results indicate that there is a balance between the formation of these three classes of crosslinks, which is dependent on the Tyr and Trp concentrations. The methods reported here allow the generation of significant yields of isolated Tyr-Trp adducts and their characterization. This technology should facilitate the detection, and examination of the biological consequences of Tyr-Trp crosslink formation in complex systems in future investigations., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

A series of N-acyl-2,5-dimethoxyphenyl-1H-benzimidazoles were designed based on a CoMFA model for cannabinoid receptor type 1 (CB1) ligands. Compounds were synthesized and radioligand binding affinity assays were performed. Eight novel benzimidazoles exhibited affinity for the CB1 receptor in the nanomolar range, and the most promising derivative compound 5 displayed a K(i) value of 1.2 nM when compared to CP55,940. These results confirm our previously reported QSAR model on benzimidazole derivatives, providing new information for the development of small molecules with high CB1 affinity., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The present study evaluates the phenotypic and genotypic changes that take place during early oncogenesis. The submandibular glands of male rats were injected with a 0.5% solution of 9,10-dimethyl-1,2-benzanthracene (DMBA) in acetone. Gland samples were taken at 0, 7, 30 and 150 days post-injection and submitted to histological, biochemical, immunocytochemical and PCR evaluation. Histopathological analysis was performed on hematoxylin-eosin stained slides. Total protein content was assessed by Lowry's method and the protein profile was analyzed by 12% SDS-PAGE. Bcl-2 was demonstrated by silver-enhanced gold immunolabeling. p53 immunolabeling was performed using the streptavidin-biotin system. All the treated animals developed carcinoma-like lesions at 30 and 150 days. Total protein concentration rose significantly (p < 0.05) above control values at 7, 30 and 150 days. The treated glands exhibited positive immunolabeling for p53 in the nuclei of neoplastic cells at 30 and 150 days. Treated glands also showed positive cytoplasmic immunolabeling for Bcl-2, exhibiting statistically significant differences between 7, 30 and 150 days (p = 0.0015), and with controls (p < 0.0001). No p53 mutations were observed whereas a point mutation, C-to-A, of the Bcl-2 gene was detected at 7, 30 and 150 days by PCR amplification. This mutation led to a single aminoacid change (thre --> asn) in the protein molecule. Our results suggest that the early histopathological changes correspond to quantitative and qualitative protein changes. The histopathological, biochemical, immunocytochemical and genetic alterations observed during the course of experimental carcinogenesis in the submandibular gland of the rat could constitute reproducible indices of malignant transformation applicable to human oncogenesis, given the high degree of homology between the oncogenes of mice, rats and human beings.

The aim of this study was immunolabeling oncoproteins Ck14, p53, p21 and Bcl-2 in order to evaluate their expression in premalignant and malignant stomatological lesions in oral epithelial, and to compare this expression with exfoliative cytology alterations in the same patients. It was studied biopsies and cytologies of 13 subjects with oral lichen planus, with or without Human Papilloma Virus (HPV), leukoplakia and squamous cell carcinoma clinically diagnosed and confirmed by anatomopathological studies. The oral lichen planus lesion presented binuclei orange cells; and in leukoplakia lesions only orange stained was observed; meanwhile koilocytes, inflammatory cells, enlarge nuclear volume and pathogenic microorganisms were observed in the HPV infections and squamous cells carcinoma (SCC). The Ck14, p53, p21 and Bcl-2 proteins were found modified in the leukoplakia, oral lichen planus and cancer. Cytological alterations and positive immunolabeling or over-expression of Ck14 cytokeratine in the upper epithelial stratus should be indicator of malignant transformations as doing subsequence exams.