AIM: Our objective was to investigate the potential of AOC3 and LRRC17 as biomarkers for fibroblast activation thus predicting their roles in CRC progression.
METHODS: Immunofluorescence (IF) staining of AOC3 and LRRC17 was performed on myofibroblast line (CCD-112CoN), primary fibroblasts from colorectal tumor (CAFs), and adjacent normal tissue (normal fibroblasts-NFs). SW620 (epithelial CRC cell line) was used as a control. Conventional CAF biomarker (alpha-smooth muscle actin - α-SMA) was included in the IF analysis. Fluorescence intensity was compared between groups using ImageJ software. Proliferation and contractility of treated cells were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and collagen gel contraction assays, respectively. Fibroblast contraction under TGF-β1 treatment was compared to those treated with complete medium (addition of 10% serum) and serum free (SF) medium.
RESULTS: Positive AOC3, LRRC17, and α-SMA expression were observed in colonic fibroblasts, more prominent in CAFs, whereas negative staining was found in SW620. Significant downregulation of AOC3, and upregulations in LRRC17 and α-SMA expression was found in TGF-β1-treated fibroblasts compared to SF medium treatment (p-value<0.05). All fibroblasts exhibited higher proliferation in complete medium and under treatment with conditioned medium from SW620 than SF medium. Significant contraction of NFs was recorded in complete medium and TGF-β1 (p-value<0.01).
CONCLUSION: Our results demonstrate AOC3 and LRRC17 as the potential markers of CAF activation which promote CRC progression.
RESULTS: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis fractionated raw snail extract to approximately 24 protein bands, between 9 and 245 kDa. The prominent band at 33 kDa was detected in all raw and processed snail extracts. Immunoblotting tests of the raw extract demonstrated 19 immunoglobulin E (IgE)-binding proteins, and four of them, at 30, 35, 42 and 49 kDa, were revealed as the major IgE-binding proteins of P. polita. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified the 49 and 42 kDa major allergens as actin, whereas the 30 and 35 kDa major allergens were identified as tropomyosin. Immunoblotting revealed that the raw snail had more allergenic proteins than the processed snail. The degree of allergenicity in decreasing order was raw > brine pickled> boiled > roasted > fried > vinegar pickled. The presence of cross-reactivity between P. polita and the shellfish tested was exhibited with either no, complete, or partial inhibitions.
CONCLUSION: Actin and tropomyosin were identified as the major and cross-reactive allergens of P. polita among local patients with snail allergy. Those major allergens are highly stable to high temperatures, acidic pH, and high salt, which might played a crucial role in snail allergy in Malaysia. © 2023 Society of Chemical Industry.
MATERIALS AND METHOD: Eighty-seven paraffin-embedded ameloblastoma cases (20 unicystic, 47 solid/multicystic, 3 desmoplastic and 17 recurrent) were subjected to immunohistochemistry for expression of cortactin, N-WASP, WIP, Src kinase and F-actin, and findings correlated with clinicopathological parameters.
RESULTS: Invadopodia proteins (except Src kinase) and F-actin were widely detected in ameloblastoma (cortactin: n = 73/87, 83.9%; N-WASP: n = 59/87; 67.8%; WIP: n = 77/87; 88.5%; and F-actin: n = 87/87, 100%). Protein localization was mainly cytoplasmic and/or membranous, and occasionally nuclear for F-actin. Cortactin, which functions as an actin-scaffolding protein, demonstrated significantly higher expression levels within ameloblastoma tumoral epithelium than in stroma (P < 0.05). N-WASP, which coordinates actin polymerization and invadopodia-mediated extracellular matrix degradation, was overexpressed in the solid/multicystic subtype (P < 0.05). WIP, an upstream regulator of N-WASP, and F-actin were significantly upregulated along the tumour invasive front compared to tumour centres (P < 0.05). Except for males with cortactin overexpression, other clinical parameters (age, ethnicity and anatomical site) showed no significant correlations.
CONCLUSIONS: Present results suggest that local invasiveness of ameloblastoma is dependent upon the migratory potential of its tumour cells as defined by their distribution of cortactin, N-WASP and WIP in correlation with F-actin cytoskeletal dynamics.
Methods: The mechanism involved in the cytotoxic activities of F5 against MCF7 cells was elucidated by flow cytometry-based apoptosis detection, caspases activity measurement, and expression profiling of apoptosis markers by western blotting. Molecular attributes of F5 were further mined from L. rhinocerus's published genome and transcriptome for future exploration.
Results and Discussion: Apoptosis induction in MCF7 cells by F5 may involve a cross-talk between the extrinsic and intrinsic apoptotic pathways with upregulation of caspase-8 and -9 activities and a marked decrease of Bcl-2. On the other hand, the levels of pro-apoptotic Bax, BID, and cleaved BID were increased accompanied by observable actin cleavage. At gene level, F5 composed of three predicted non-synonymous single nucleotide polymorphisms (T > C) and an alternative 5' splice site.
Conclusions: Findings from this study provide an advanced framework for further investigations on cancer therapeutics development from L. rhinocerus.