RESUMO
AIM: To investigate the expression of DNA repair genes and the impact of the breast cancer 1, early onset (BRCA1) protein on chemoresistance of hepatocellular carcinoma (HCC). METHODS: Microarray gene expression datasets were analyzed using the gene set enrichment analysis method. BRCA1 protein was tested by Western blotting. Response of HCC cells to interstrand cross-links was investigated by cell viability assay following exposure to mitomycin C, cisplatin, and melphalan. Effects of BRCA1 ectopic expression were studied in HepG2 cells with BRCA1-expression plasmids. Effects of BRCA1 downregulation were studied in SNU449 cells with BRCA1-specific siRNAs. Response of transfected SNU449 cells to mitomycin C was analyzed by cell viability tests and cell cycle analysis using flow cytometry. RESULTS: Expression of Fanconi anemia and double-stranded DNA break repair genes was significantly upregulated in HCC tumors. This upregulation displayed a gradual amplification during tumor progression. BRCA1 and BRCA2 genes were among consistently upregulated genes. Epithelial-like HCC cells had low BRCA1 expression and low chemoresistance, whereas mesenchymal-like HCC cells had high BRCA1 expression and increased chemoresistance. Ectopic expression of BRCA1 increased the chemoresistance of epithelial-like HepG2 cells. Conversely, BRCA1 knockdown chemosensitized mesenchymal-like SNU449 cells. Chemosensitization of SNU449 cells was due to cell cycle arrest at 4N stage. CONCLUSION: Increased expression of Fanconi anemia and double-stranded DNA repair genes such as BRCA1 is a novel mechanism of HCC chemoresistance. However, functional inactivation of BRCA1 expression is sufficient to reverse such chemoresistance.
RESUMO
Cell surface glycoconjugates present alterations of their structures in chronic diseases and distinct oligosaccharide epitopes have been associated with cancer. Among them, truncated glycans present terminal non-reducing ß-N-acetylglucosamine (GlcNAc) residues that are rare on healthy tissues. Lectins from unconventional sources such as fungi or algi provide novel markers that bind specifically to such epitopes, but their availability may be challenging. A GlcNAc-binding lectin from the fruiting body of the fungus Psathyrella velutina (PVL) has been produced in good yield in bacterial culture. A strong specificity for terminal GlcNAc residues was evidenced by glycan array. Affinity values obtained by microcalorimetry and surface plasmon resonance demonstrated a micromolar affinity for GlcNAcß1-3Gal epitopes and for biantennary N-glycans with GlcNAcß1-2Man capped branches. Crystal structure of PVL complexed with GlcNAcß1-3Gal established the structural basis of the specificity. Labeling of several types of cancer cells and use of inhibitors of glycan metabolism indicated that rPVL binds to terminal GlcNAc but also to sialic acid (Neu5Ac). Analysis of glycosyltransferase expression confirmed the higher amount of GlcNAc present on cancer cells. rPVL binding is specific to cancer tissue and weak or no labeling is observed for healthy ones, except for stomach glands that present unique αGlcNAc-presenting mucins. In lung, breast and colon carcinomas, a clear delineation could be observed between cancer regions and surrounding healthy tissues. PVL is therefore a useful tool for labeling agalacto-glycans in cancer or other diseases.
