A Low Cost Antibody Signal Enhancer Improves Immunolabeling in Cell Culture, Primate Brain and Human Cancer Biopsy.

The use of antibodies to identify neuronal receptors, neurotransmitters, cytoskeletal elements or pathologic protein aggregates, ion channels, adhesion molecules or other cell-type specific markers, is common practice in neuroscience. Antibody detection systems are often based on confocal, epifluorescence or brightfield microscopy. Three types of technical issues can interfere with immunolabeling: low abundance of the target protein, low specific affinity of the antibody and/or signal background sometimes related to tissue fixation. Here, giving tribute to Professor Miledi's mentorship, we propose the application of an antibody signal enhancer (ASE) solution based on glycine, hydrogen peroxide and a detergent mix as a simple, low cost, protocol variation that significantly and specifically improves the signal to noise ratio during immunostaining experiments. We describe three new settings in which ASE improves the detection of a variety of antibodies applied on long-time stored non-human primate brain sections, cell culture monolayers and on squamous carcinomas retrieved from cervical cancer patients. The significant improvement of ASE over optimized immunohistochemical protocols used in clinical practice (i.e. cancer detection) combined with its simplicity and low cost makes it an attractive method for biomedical applications.

Inhibition of RAD51 by siRNA and Resveratrol Sensitizes Cancer Stem Cells Derived from HeLa Cell Cultures to Apoptosis.

Cervical cancer is the second most frequent tumor type in women worldwide with cases developing clinical recurrence, metastasis, and chemoresistance. The cancer stem cells (CSC) may be implicated in tumor resistance to therapy. RESveratrol (RES), a natural compound, is an antioxidant with multiple beneficial activities. We previously determined that the expression of RAD51 is decreased by RES. The aim of our study was to examine molecular mechanism by which CSC from HeLa cultures exhibit chemoresistance. We hypothesized CSC repair more efficiently DNA breaks and that RAD51 plays an important role in this mechanism. We found that CSC, derived from cervical cancer cell lines, overexpress RAD51 and are less sensitive to Etoposide (VP16). We inhibited RAD51 in CSC-enriched cultures using RES or siRNA against RAD51 messenger RNA and observed a decrease in cell viability and induction of apoptosis when treated simultaneously with VP16. In addition, we found that inhibition of RAD51 expression using RES also sensitizes CSC to VP16 treatment. Our results suggest that resveratrol is effective to sensitize cervical CSC because of RAD51 inhibition, targeting high RAD51 expressing CD49f-positive cells, which supports the possible therapeutic application of RES as a novel agent to treat cancer.

Resveratrol induces downregulation of DNA repair genes in MCF-7 human breast cancer cells.

To gain insights into the antitumor mechanisms of resveratrol (RES), we carried out a DNA microarray analysis in the breast cancer cell line MCF-7 to study the global gene expression profile induced by RES treatment. The mRNA expression level of 19 734 well-characterized human genes from MCF-7 cells was determined using Affymetrix microarrays under two different RES treatments: 150 µmol/l (IC(50)) and 250 µmol/l during 48 h. A total of 1211 genes were found to have altered mRNA expression levels of two-fold or more in the 150 µmol/l RES-treated group (518 upregulated and 693 downregulated genes). However, 2412 genes were found to have altered expression levels of two-fold or more in the 250 µmol/l RES-treated group (651 genes upregulated and 1761 downregulated). Under both conditions of RES treatment, several genes of mismatch repair, DNA replication, homologous recombination (HR), and cell cycle were strongly inhibited. Consistently, we found decreased protein levels of the MRN complex (MRE11-NBS1-RAD50), an important complex of the HR DNA repair pathway. The ability to inhibit the expression of DNA repair genes by RES could help to overcome drug resistance commonly shown by transformed cells and to provide a solid basis for carrying out clinical trials with RES, alone or in combination with other agents, to enhance treatment efficacy, reduce toxicity, and overcome chemoresistance. Remarkably, after RES treatment, we found a decrease in NBS1 and MRE11 protein levels, two major proteins involved in HR, which suggests that RES could be used to sensitize cancer cells to cell death in combination with anticancer drugs.