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Sub-millimolar concentration of the novel phenol-based compound, 2-hydroxy benzoate zinc, induces apoptosis in human HT-1080 fibrosarcoma cells.

ABSTRACT: OBJECTIVES:To examine the effect of a novel phenolic-based compound, 2-hydroxy benzoate zinc (2HBZ), and acetylsalicylic acid (ASA) on human HT-1080 fibrosarcoma cells. MATERIALS AND METHODS:MTT assay was used to assess cell proliferation while different methods were used to detect apoptosis morphologically and immunohistochemically in Human HT-1080 fibrosarcoma cells. Apoptosis was determined by Annexine-V labelling, and caspase-3 activation. In addition, western blot was used to analyse p21, p53 and Bax and flow cytometry was to analyse the cell cycle. RESULTS:2HBZ exhibited a more than 5-fold increase in cytotoxic potency when compared with ASA with mean LD50 values of 210 and 1100 lM respectively (P < 0.0001). The cytotoxic effects of 2HBZ were both time- and dosedependent with marked apoptosis being evident only after 24 h at concentrations as low as 200 mM. In contrast, ASA-induced apoptosis was observed only at concentrations in excess of 1000 mM at the same time point. Both 2HBZ and ASA induced caspase-3 activation in the cells, which confirmed that their cytotoxic effects were the result of apoptotic cell death. These findings were further confirmed by immunomorphological studies for the detection of apoptosis including haematoxylineosin, methyl green/pyronin Y staining and scanning electron microscopy. In addition, 2HBZ caused a marked increase in p21, p53 and Bax protein expressions and these effects were associated with an increase in G1 and G2 arrest of the cell cycle and a reduction in S-phase. CONCLUSIONS:These results demonstrate that the novel phenolic compound 2HBZ is a potent apoptosis-inducing agent in HT-1080 cells and warrants further investigation as a potential chemotherapeutic agent in primary cancer cell models.

SUBMITTER: Mahdi JG

PROVIDER: S-EPMC6496447 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Sub-millimolar concentration of the novel phenol-based compound, 2-hydroxy benzoate zinc, induces apoptosis in human HT-1080 fibrosarcoma cells.

Mahdi J G JG Pepper C J CJ Alkarrawi M A MA Mahdi A J AJ Bowen I D ID

Cell proliferation 20091117 1

<h4>Objectives</h4>To examine the effect of a novel phenolic-based compound, 2-hydroxy benzoate zinc (2HBZ), and acetylsalicylic acid (ASA) on human HT-1080 fibrosarcoma cells.<h4>Materials and methods</h4>MTT assay was used to assess cell proliferation while different methods were used to detect apoptosis morphologically and immunohistochemically in Human HT-1080 fibrosarcoma cells. Apoptosis was determined by Annexine-V labelling, and caspase-3 activation. In addition, western blot was used to ...[more]

PMID: 19922491

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Project description:BackgroundCancer cachexia is a complex metabolic disease with unmet medical need. Although many rodent models are available, none are identical to the human disease. Therefore, the development of new preclinical models that simulate some of the physiological, biochemical, and clinical characteristics of the human disease is valuable. The HT-1080 human fibrosarcoma tumour cell line was reported to induce cachexia in mice. Therefore, the purpose of this work was to determine how well the HT-1080 tumour model could recapitulate human cachexia and to examine its technical performance. Furthermore, the efficacy of ghrelin receptor activation via anamorelin treatment was evaluated, because it is one of few clinically validated mechanisms.MethodsFemale severe combined immunodeficient mice were implanted subcutaneously or heterotopically (renal capsule) with HT-1080 tumour cells. The cachectic phenotype was evaluated during tumour development, including body weight, body composition, food intake, muscle function (force and fatigue), grip strength, and physical activity measurements. Heterotopic and subcutaneous tumour histology was also compared. Energy balance was evaluated at standard and thermoneutral housing temperatures in the subcutaneous model. The effect of anamorelin (ghrelin analogue) treatment was also examined.ResultsThe HT-1080 tumour model had excellent technical performance and was reproducible across multiple experimental conditions. Heterotopic and subcutaneous tumour cell implantation resulted in similar cachexia phenotypes independent of housing temperature. Tumour weight and histology was comparable between both routes of administration with minimal inflammation. Subcutaneous HT-1080 tumour-bearing mice presented with weight loss (decreased fat mass and skeletal muscle mass/fibre cross-sectional area), reduced food intake, impaired muscle function (reduced force and grip strength), and decreased spontaneous activity and voluntary wheel running. Key circulating inflammatory biomarkers were produced by the tumour, including growth differentiation factor 15, Activin A, interleukin 6, and TNF alpha. Anamorelin prevented but did not reverse anorexia and weight loss in the subcutaneous model.ConclusionsThe subcutaneous HT-1080 tumour model displays many of the perturbations of energy balance and physical performance described in human cachexia, consistent with the production of key inflammatory factors. Anamorelin was most effective when administered early in disease progression. The HT-1080 tumour model is valuable for studying potential therapeutic targets for the treatment of cachexia.

Project description:The effect of dexamethasone on the synthesis and degradation of type IV collagen was studied in human fibrosarcoma cells, HT-1080. A dexamethasone concentration as low as 0.1 microM markedly increased collagen synthesis in HT-1080 cells labelled with [14C]proline. The increase in type IV collagen synthesis was not specific, since total protein synthesis was also increased. Further studies indicated that part of the increase was due to an increase in the specific radioactivity of the intracellular proline pool, after dexamethasone treatment. In fact, with dexamethasone concentrations of 0.1-10 microM the relative collagen synthesis was decreased, indicating that synthesis of other protein was increased more than that of type IV collagen. This was also confirmed by measuring the relative amount of type IV collagen RNA by using recombinant plasmid cDNA specific for the human procollagen pro alpha l (IV) RNA. The results indicated that relative collagen synthesis and the relative amount of type IV collagen messenger RNA was decreased similarly, indicating that dexamethasone affected type IV collagen synthesis at the pre-translational level. The dexamethasone-induced effect on total protein and collagen synthesis was maximal after 12-24 h. Dexamethasone induced a marked accumulation of collagen into the cell layer, leading to diminished deposition of soluble collagen into the medium. Since bacterial-collagenase treatment of the cell layer drastically decreased the collagen content of the dexamethasone-treated cells, this indicates that dexamethasone caused an accumulation of collagen into the extracellular matrix of the cell layer. In contrast, the amount of fibronectin was markedly increased in the medium. Dexamethasone decreased the type IV collagen-degrading activity in HT-1080 cells. The HT-1080 cells contained glucocorticoid receptors, as demonstrated by two different methods: by a whole-cell binding assay and by using a cytosol-gel-filtration method. The number of specific binding sites was similar to that in human skin fibroblasts. In conclusion, glucocorticoids affect the metabolism of type IV collagen and fibronectin in HT-1080 cells, and, since these cells contain specific glucocorticoid receptors, the effects are apparently receptor-mediated.

Project description:Here, we describe an engineering approach to quantitatively compare migration, morphologies, and adhesion for tumorigenic human fibrosarcoma cells (HT-1080s) and primary human dermal fibroblasts (hDFs) with the aim of identifying distinguishing properties of the transformed phenotype. Relative adhesiveness was quantified using self-assembled monolayer (SAM) arrays and proteolytic 3-dimensional (3D) migration was investigated using matrix metalloproteinase (MMP)-degradable poly(ethylene glycol) (PEG) hydrogels ("synthetic extracellular matrix" or "synthetic ECM"). In synthetic ECM, hDFs were characterized by vinculin-containing features on the tips of protrusions, multipolar morphologies, and organized actomyosin filaments. In contrast, HT-1080s were characterized by diffuse vinculin expression, pronounced β1-integrin on the tips of protrusions, a cortically-organized F-actin cytoskeleton, and quantitatively more rounded morphologies, decreased adhesiveness, and increased directional motility compared to hDFs. Further, HT-1080s were characterized by contractility-dependent motility, pronounced blebbing, and cortical contraction waves or constriction rings, while quantified 3D motility was similar in matrices with a wide range of biochemical and biophysical properties (including collagen) despite substantial morphological changes. While HT-1080s were distinct from hDFs for each of the 2D and 3D properties investigated, several features were similar to WM239a melanoma cells, including rounded, proteolytic migration modes, cortical F-actin organization, and prominent uropod-like structures enriched with β1-integrin, F-actin, and melanoma cell adhesion molecule (MCAM/CD146/MUC18). Importantly, many of the features observed for HT-1080s were analogous to cellular changes induced by transformation, including cell rounding, a disorganized F-actin cytoskeleton, altered organization of focal adhesion proteins, and a weakly adherent phenotype. Based on our results, we propose that HT-1080s migrate in synthetic ECM with functional properties that are a direct consequence of their transformed phenotype.

Dataset Information

Sub-millimolar concentration of the novel phenol-based compound, 2-hydroxy benzoate zinc, induces apoptosis in human HT-1080 fibrosarcoma cells.

Publications

Sub-millimolar concentration of the novel phenol-based compound, 2-hydroxy benzoate zinc, induces apoptosis in human HT-1080 fibrosarcoma cells.

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