There has been a growing interest in the beneficial effects of simple phenolic acids and their ability to exhibit various biological activities. The aim of this study was to assess in vitro biological activities of 2-, 3-, and 4-hydroxybenzoate lithium (HBLi) complexes on HT-1080 human fibrosarcoma cells by methods of using a metabolic activity assay, immunochemical and morphological techniques. Results showed that HBLi complexes exert their cytotoxic activities in a concentration-and chemical structuredependent manner in the following order: 4-HBLi > 3-HBLi > 2-HBLi. Flow cytometry displayed evidence of apoptosis induced by 3-HBLi (21.8%) and 4-HBLi (33.2%). These results were verified by SEM, which revealed the formation of apoptotic bodies. In addition, these 3-HBLi and 4-HBLi caused an increase in HT-1080 cell cycle arrest in G0/G1 phase when compared to the controls (25% and 30.6%, resp.) when cells were treated with 6 mM for 24 hours. Immunochemical studies related to the molecular mechanism of apoptosis indicated that HBLi complexes downregulated the expression of Bcl-2 and upregulated Bax, p53, and caspases-3 in a concentration-dependent manner. HBLi complexes lowered Bcl-2/Bax ratios and induced the expression of p53 and caspase-3. These results suggest that HBLi complexes may exert their apoptotic effects through mitochondrial-mediated, caspasedependent, apoptotic mechanisms.
A novel immunocytochemical method is presented for the qualitative detection of DNA fragmentation in apoptosis. Anti-histone antibody is employed to localize exposed nucleosomal histones (H1, H2a, H2b, H3 and H4) rather than tagging the cut ends of fragmenting DNA as in conventional technique. The method was tested on squamous cell carcinoma of the larynx routinely fixed in formaldehyde and embedded in paraffin wax and compared with results obtained employing Apop-Tag kit (Oncor).
A series of techniques based on LR White resin are described, which permit the use of an anti-histone antibody for the in situ localization of DNA fragmentation characteristic of apoptosis at both the light and the electron microscope level. The methods, applied to an untreated squamous carcinoma of the pharynx, allow direct comparison of light microscopic localization of exposed nucleosomal histones using 3,3'-diaminobenzidine (DAB) and silver-enhanced techniques with a colloidal gold-based anti-histone technique at the electron microscope level. Parallel histochemical localization of acid phosphatase activity is also presented.
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