ascorbic-acid and Mesothelioma

Non-thermal plasma (NTP) is a recently developed technology that elicits a variety of biological effects. This includes cancer cell-specific cytotoxicity, which is mainly attributed to the regional generation of reactive oxygen species (ROS). We studied the effects of NTP on malignant mesothelioma (MM) and its modulation by l-ascorbate. l-ascorbate is a major water-soluble anti-oxidant in vivo, but its pro-oxidant activity in vitro has been well recognized. Thus, the effects of ascorbate on the efficacy of NTP is important to examine. NTP exposure dose-dependently killed MM cells, whereas MM cells tolerated 1 mM l-ascorbate. However, brief pre-treatment with a pharmacological dose (250-750 μM) of l-ascorbate immediately prior to NTP exposure significantly increased its cytotoxicity in a dose-dependent manner, which was inhibited by the iron chelator, deferoxamine. However, paradoxically, this potentiating effect of l-ascorbate was completely abolished by a prolonged 4 h pre-incubation with l-ascorbate (500 μM). MM cytotoxicity induced by NTP was associated with immediate oxidative stress evaluated by 2',7'-dichlorodihydrofluorecein diacetate, which was followed by an increase in the expression of the autophagosome marker, LC3B-II. In conclusion, MM can be a target for NTP treatment and l-ascorbate can increase or decrease its efficacy depending on the length of the pre-incubation period.

Topics: Antineoplastic Agents; Antioxidants; Apoptosis; Ascorbic Acid; Autophagy; Cell Line, Tumor; Cell Survival; Deferoxamine; Dose-Response Relationship, Drug; Humans; Lung Neoplasms; Mesothelioma; Mesothelioma, Malignant; Microscopy, Fluorescence; Oxidative Stress; Plasma Gases; Reactive Oxygen Species; Temperature

Malignant mesothelioma (MMe) is a poor-prognosis tumor in need of innovative therapies. In a previous in vivo study, we showed synergistic anti-MMe properties of the ascorbate/epigallocatechin-3-gallate/gemcitabine combination. We have now focused on the mechanism of action, showing the induction of apoptosis and cell cycle arrest through measurements of caspase 3, intracellular Ca(2+), annexin V, and DNA content. StellArray™ PCR technology and Western immunoblotting revealed DAPK2-dependent apoptosis, upregulation of cell cycle promoters, downregulation of cell cycle checkpoints and repression of NFκB expression. The complex of data indicates that the mixture is synergistic in inducing cell cycle deregulation and non-inflammatory apoptosis, suggesting its possible use in MMe treatment.

Topics: Apoptosis; Ascorbic Acid; Catechin; Cell Cycle; Cell Line, Tumor; Deoxycytidine; Drug Combinations; Drug Synergism; Gemcitabine; Humans; Mesothelioma

Malignant pleural mesothelioma (MPM) is a poor prognosis disease lacking adequate therapy. We have previously shown that ascorbic acid administration is toxic to MPM cells. Here we evaluated a new combined therapy consisting of ascorbate/epigallocatechin-3-gallate/gemcitabine mixture (called AND, for Active Nutrients/Drug). In vitro effects of AND therapy on various MPM cell lines revealed a synergistic cytotoxic mechanism. In vivo experiments on a xenograft mouse model for MPM, obtained by REN cells injection in immunocompromised mice, showed that AND strongly reduced the size of primary tumor as well as the number and size of metastases, and prevented abdominal hemorrhage. Kaplan Meier curves and the log-rank test indicated a marked increase in the survival of AND-treated animals. Histochemical analysis of dissected tumors showed that AND induced a shift from cell proliferation to apoptosis in cancer cells. Lysates of tumors from AND-treated mice, analyzed with an antibody array, revealed decreased TIMP-1 and -2 expressions and no effects on angiogenesis regulating factors. Multiplex analysis for signaling protein phosphorylation exhibited inactivation of cell proliferation pathways. The complex of data showed that the AND treatment is synergistic in vitro on MPM cells, and blocks in vivo tumor progression and metastasization in REN-based xenografts. Hence, the AND combination is proposed as a new treatment for MPM.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Ascorbic Acid; Catechin; Cell Line, Tumor; Cell Proliferation; Deoxycytidine; Drug Synergism; Gemcitabine; Humans; Lung Neoplasms; Male; Mesothelioma; Mesothelioma, Malignant; Mice; Mice, Inbred NOD; Mice, SCID; Pleural Neoplasms; Signal Transduction; Xenograft Model Antitumor Assays

The aim of this study was to investigate the possible relationship between antioxidant vitamin levels and malignant pleural mesothelioma (MPM). For this purpose, we measured the serum levels of 4 antioxidant vitamins, β-carotene, α-tocopherol, retinol, and ascorbic acid, in patients with environmentally induced MPM and in healthy controls from one tremolite village (Kureysler), the biggest erionite village (Tuzkoy) and Ankara. A total of 160 subjects were enrolled in the study, 42 (26.3%) diagnosed with MPM and 118 (73.7%) healthy subjects. A comparison was made between the MPM group and three control groups of which two were exposed and one was unexposed to mineral fibers. The study population consisted of 82 males (51%) and 78 females (49%) with a mean of age of 44.8±14 years (range; 20-65 years). Lowest levels of β-carotene, ascorbic acid, and α-tocopherol were found in MPM patients (MPM vs control groups combined, p<0.0001 for each antioxidant vitamin), without any relation to age or sex. There was no significant difference between the antioxidant levels of healthy controls of Tuzkoy and Ankara. In conclusion; our findings suggested an increased risk of MPM being associated with low levels of α-tocopherol and ascorbic acid in patients with MPM.

Topics: Adult; alpha-Tocopherol; Antioxidants; Ascorbic Acid; beta Carotene; Case-Control Studies; Female; Humans; Male; Mesothelioma; Middle Aged; Pleural Neoplasms; Risk; Vitamin A

We studied the mechanism of ascorbate toxicity in malignant mesothelioma (MMe) cells. Neutral red uptake showed that ascorbate, but not dehydroascorbate, was highly toxic in the MMe cell lines REN and MM98, and less toxic in immortalized (human mesothelial cells-htert) and primary mesothelial cells. Ascorbate transport inhibitors phloretin, sodium azide, and ouabain did not reduce ascorbate toxicity. Ascorbate promoted the formation of H(2)O(2) in the cell medium, and its toxicity was suppressed by extracellular catalase, but the concentration of endogenous catalase was higher in MMe cells than in normal cells. The confocal imaging of cells loaded with the dihydrhodamine 123 reactive oxygen species probe showed that ascorbate caused a strong increase of rhodamine fluorescence in MMe cells, but not in mesothelial cells. MMe cells showed a higher production of superoxide and NADPH oxidase (NOX)4 expression than did mesothelial cells. Two inhibitors of cellular superoxide sources (apocynin and rotenone) reduced ascorbate toxicity and the ascorbate-induced rise in rhodamine fluorescence. NOX4 small interfering RNA also reduced ascorbate toxicity in REN cells. Taken together, the data indicate that ascorbate-induced extracellular H(2)O(2) production induces a strong oxidative stress in MMe cells because of their high rate of superoxide production. This explains the selective toxicity of ascorbate in MMe cells, and suggests its possible use in the clinical treatment of malignant mesothelioma.

Topics: Antineoplastic Agents; Apoptosis; Ascorbic Acid; Catalase; Cell Line, Tumor; Dose-Response Relationship, Drug; Glucose Transporter Type 1; Humans; Hydrogen Peroxide; Inhibitory Concentration 50; Mesothelioma; Microscopy, Confocal; Mutation; NADPH Oxidase 4; NADPH Oxidases; Necrosis; Organic Anion Transporters, Sodium-Dependent; Ouabain; Oxidation-Reduction; Oxidative Stress; Phloretin; RNA Interference; Sodium Azide; Superoxides; Symporters; Tumor Suppressor Protein p53

Malignant mesothelioma (MMe) is a lethal tumor arising from the mesothelium of serous cavities as a result of exposure to asbestos. Current clinical standards consist of combined treatments, but an effective therapy has not been established yet and there is an urgent need for new curative approaches. Ascorbate is a nutrient that is also known as a remedy in the treatment of cancer. In the present study, we have tested the cytotoxicity of ascorbate to MMe cells in combination with drugs used in MMe therapy, such as cisplatin, etoposide, gemcitabine, imatinib, paclitaxel, and raltitrexed, as well as with promising antitumor compounds like taurolidine, α-tocopherol succinate, and epigallocatechin-3-gallate (EGCG). Dose-response curves obtained for each compound by applying the neutral red uptake (NRU) assay to MMe cells growing in vitro, allowed to obtain IC50 values for each compound used singularly. Thereafter, NRU data obtained from each ascorbate/drug combination were analyzed through Tallarida's isobolograms at the IC50 level (Tallarida, 2000), revealing synergistic interactions for ascorbate/gemcitabine and ascorbate/EGCG. These results were further confirmed through comparisons between theoretical additivity IC50 and observed IC50 from fixed-ratio dose-response curves, and over a broad range of IC levels, by using Chou and Talalay's combination index (Chou and Talalay, 1984). Synergistic interactions were also shown by examining apoptosis and necrosis rates, using the caspase 3 and lactic dehydrogenase assays, respectively. Hence, data indicate that ascorbate/gemcitabine and ascorbate/EGCG affect synergistically the viability of MMe cells and suggest their possible use in the clinical treatment of this problematic cancer.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Ascorbic Acid; Catechin; Cell Line, Tumor; Cell Proliferation; Cell Survival; Coloring Agents; Deoxycytidine; Drug Synergism; Gemcitabine; Humans; L-Lactate Dehydrogenase; Lung Neoplasms; Mesothelioma; Neutral Red

Malignant mesothelioma is an asbestos-related fatal disease with no effective cure. Recently, high dose of ascorbate in cancer treatment has been reexamined. We studied whether high dose of ascorbic acid induced cell death of four human mesothelioma cell lines. High dose of ascorbic acid induced cell death of all mesothelioma cell lines in a dose-dependent manner. We further clarified the cell killing mechanism that ascorbic acid induced reactive oxygen species and impaired mitochondrial membrane potential. In vivo experiment, intravenous administration of ascorbic acid significantly decreased the growth rate of mesothelioma tumor inoculated in mice. These data suggest that ascorbic acid may have benefits for patients with mesothelioma.

Topics: Animals; Apoptosis; Ascorbic Acid; Cell Line, Tumor; Cell Proliferation; Humans; Membrane Potential, Mitochondrial; Mesothelioma; Mice; Mice, SCID; Reactive Oxygen Species; Xenograft Model Antitumor Assays

In the first part of this study we have shown how the serum levels of four selected tumour markers, namely tissue polypeptide antigen (TPA), carcino-embryonic antigen (CEA), hyaluronic acid (HA) and ferritin, display patterns characteristic of mesothelioma (M) or bronchogenic carcinoma (BC) in asbestos-exposed workers, and we hypothesize that the differences in marker patterns correspond to differences in carcinogenesis mechanisms. In a preliminary study, we found these specific marker patterns in 5/19 exposed workers of whom only one demonstrated any radiological signs of disease. Thus these specific marker patterns may be early events, occurring long (possibly years) before the classical radiological signs of exposure to asbestos. Accordingly they afford an optimal opportunity for prevention which should be adapted to the carcinogenesis mechanism as it is revealed by the marker pattern; it is aimed at antagonizing free radical carcinogenesis in all persons with TPA levels in excess of 100 U/l or Ferritin in excess of 400 ng/ml, and at inhibiting chemical carcinogenesis in those having elevated CEA levels (over 3 ng/ml). The mechanisms involved in these inhibitory processes are described and discussed, as well as the practical implementations that proceed from them. A prevention trial is now being started among 300 active and retired workers of an asbestos-cement works in northern France; the design of the study is presented. This prevention programme should be maintained over many years and holds a strong potential for reducing the untoward effects of exposure to asbestos.

Topics: Acetylcysteine; Antigens, Neoplasm; Asbestos; Ascorbic Acid; beta Carotene; Biomarkers, Tumor; Carcinoembryonic Antigen; Carcinoma, Bronchogenic; Carotenoids; Cohort Studies; Ferritins; Humans; Hyaluronic Acid; Longitudinal Studies; Lung Neoplasms; Male; Mesothelioma; Middle Aged; Occupational Diseases; Occupational Exposure; Peptides; Riboflavin; Selenium; Tissue Polypeptide Antigen; Vitamin A; Vitamin E

Immunological studies are reported showing that ascorbic acid, like selected sulfhydryl inhibitors, can induce immunity against some cancers in mice. Accompanying this immunizing action are changes in the surface structure of the cancer cells, as revealed by scanning electron microscopy. Electron spin resonance measurements show that the ascorbate anion free radical is readily produced in oxygenated cancer tissue and that this radical can react with sulfhydryl groups which are free radical scavengers. It is proposed that ascorbate acts as an effective thiolprive in oxygenated cancer tissues. This action is thought to lead to the observed changes in the cancer cell surface structure and to the concomitant immunological response.

Topics: Animals; Ascorbic Acid; Carcinoma, Ehrlich Tumor; Cell Line; Cell Membrane; Electron Spin Resonance Spectroscopy; Free Radicals; Mesothelioma; Mice; Microscopy, Electron, Scanning; Microvilli; Sarcoma 180; Sulfhydryl Reagents

Topics: Androgens; Antibiotics, Antineoplastic; Antineoplastic Agents; Ascorbic Acid; Azirines; Bronchial Neoplasms; Carcinoma; Carcinoma, Squamous Cell; Cyclophosphamide; Drug Synergism; Fluorouracil; Glucocorticoids; Humans; Hydrazines; Injections, Intramuscular; Injections, Intravenous; Lectins; Lung Neoplasms; Mechlorethamine; Mercaptopurine; Mesothelioma; Methotrexate; Phenylbutazone; Pleural Neoplasms; Thiotepa; Vinblastine