Three dimensional reconstruction of skin with melanoma: A model for study of invasion in vitro.

Melanoma is a type of tumor skin with high metastatic potential. Reconstructed human skin, development for pre-clinic assay, are make using primary human cells, but with same limitations. The aim this study was to characterize a cell culture model, with structure similar to human skin containing melanoma cells entirely from cell lines. Reconstructed skin with melanoma were development using human fibroblasts (MRC5), human epidermal keratinocytes (HaCat), and human melanoma (SK-MEL-28) embedded in collagen type I. The structure was characterized by hematoxylin-eosin stained, as well as points of melanoma cell invasion, which was associated with activity of MMPs (MMP-2 and MMP-9) by zymographic method. Then, the gene expression of the target molecular mechanisms involved in melanoma progression were evaluated. Here, the model development showed a region epidermis organized and separated from the dermis, with fibroblast cells confined and melanoma cells form delimited area invasion. MMP-2 and MMP-9 were identified during of cell culture and gene expression of BRAF, NRAS, and Vimentin was confirmed. The proposed model provides one more opportunity to study in vitro tumor biology of melanoma and also to allows the study of new drugs with more reliable results then whats we would find in vivo.

Differences in glucose concentration shows new perspectives in gastric cancer metabolism.

Gastric cancer (GC) is among the deadliest cancers worldwide despite available therapies, highlighting the need for novel therapies and pharmacological agents. Metabolic deregulation is a potential study area for new anticancer targets, but the in vitro metabolic studies are controversial, as different ranges of glucose used in the culture media can influence results. In this study, we evaluated cellular viability, glucose uptake, and LDH activity in gastric cancer cell lines when exposed to different glucose concentrations: high (HG, 25mM), low (LG, 5.5mM), and free (FG, 0mM) glucose media. Moreover, we evaluated how glucose variations may influence cellular phenotype and the expression of genes related to epithelial-mesenchymal transition (EMT), metabolism, and cancer development in metastatic GC cells (AGP-01). Results showed that metastatic cells exposed to FG medium evidenced higher alterations when compared to other cell lines. Most phenotypic assays did not show difference when exposed to either HG or LG media. However, gene expression profile of cells exposed to LG revealed differences in mRNA levels of metabolism-related genes when compared to HG medium. According to our results, we recommend using LG medium for metabolic studies since the glucose concentration is closer to physiological levels. These findings point out new relevant targets in metabolic reprogramming that can be alternatives to current chemotherapies in patients with metastatic GC.

22ß-hydroxytingenone induces apoptosis and suppresses invasiveness of melanoma cells by inhibiting MMP-9 activity and MAPK signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: 22ß-hydroxytingenone (22-HTG) is a quinonemethide triterpene isolated from Salacia impressifolia (Miers) A. C. Smith (family Celastraceae), which has been used in traditional medicine to treat a variety of diseases, including dengue, renal infections, rheumatism and cancer. However, the anticancer effects of 22-HTG and the underlying molecular mechanisms in melanoma cells have not yet been elucidated. AIM OF THE STUDY: The present study investigated apoptosis induction and antimetastatic potencial of 22-HTG in SK-MEL-28 human melanoma cells. MATERIALS AND METHODS: First, the in vitro cytotoxic activity of 22-HTG in cultured cancer cells was evaluated. Then, cell viability was determined using the trypan blue assay in melanoma cells (SK-MEL-28), which was followed by cell cycle, annexin V-FITC/propidium iodide assays (Annexin/PI), as well as assays to evaluate mitochondrial membrane potential, production of reactive oxygen species (ROS) using flow cytometry. Fluorescence microscopy using acridine orange/ethidium bromide (AO/BE) staining was also performed. RT-qPCR was carried out to evaluate the expression of BRAF, NRAS, and KRAS genes. The anti-invasiveness potential of 22-HTG was evaluated in a three-dimensional (3D) model of reconstructed human skin. RESULTS: 22-HTG reduced viability of SK-MEL-28 cells and caused morphological changes, as cell shrinkage, chromatin condensation, and nuclear fragmentation. Furthermore, 22-HTG caused apoptosis, which was demonstrated by increased staining with AO/BE and Annexin/PI. The apoptosis may have been caused by mitochondrial instability without the involvement of ROS production. The expression of BRAF, NRAS, and KRAS, which are important biomarkers in melanoma development, was reduced by the 22-HTG treatment. In the reconstructed skin model, 22-HTG was able to decrease the invasion capacity of melanoma cells in the dermis. CONCLUSIONS: Our data indicate that 22-HTG has anti-tumorigenic properties against melanoma cells through the induction of cell cycle arrest, apoptosis and inhibition of invasiveness potential, as observed in the 3D model. As such, the results provide new insights for future work on the utilization of 22-HTG in malignant melanoma treatment.

22ß-hydroxytingenone reduces proliferation and invasion of human melanoma cells.

Melanoma is a skin cancer with high invasive potential and high lethality. Considering that quinonemethide triterpenes are described as promising anticancer agents, the aim of this study was to evaluate the effect of 22ß-hydroxytingenone (22-HTG) against human melanoma cells. Alamar blue assay was performed in order to evaluate its cytotoxic effect. Thus, subtoxic concentrations (1.0, 2.0, and 2.5 µM) were used to evaluate the effect of this compound on proliferation, migration, metabolism, and invasion. IC50 value against SK-MEL-28 cell line was 4.35, 3.72, and 3.29 µM after 24, 48, and 72 h of incubation, respectively. 22-HTG reduced proliferation, migration and invasion by melanoma cells, with decreased activity of metalloproteinases (MMP-2 and MMP-9). Futhermore, 22-HTG decreased expression of lactate dehydrogenase (LDHA), an enzyme associated with cell metabolism. Howerver, the small reduction in LDHA enzyme activity must have occurred by the cytotoxic effect of 22-HTG. According to the results, 22-HTG interferes with important characteristics of cancer, with anti-proliferative, and anti-invasive effect against melanoma cells. The data suggest that 22-HTG is an effective substance against melanoma cells and it should be considered as a potential anticancer agent.