Experimental results using 3-bromopyruvate in mesothelioma: in vitro and in vivo studies

Over many years we have taken advantage of the special metabolism of cancer cells involving an increased consumption of glucose associated with lactic acid production even in the presence of oxygen, a phenomenon referred to as the “Warburg effect”, to counteract cancer cell growth. We have tested 3-bromopyruvate (3-BrPA), an inhibitor of pyruvate-associated reactions. Firstly, we tested this agent, in vitro, in two mesothelioma cell lines. Cellular response would appear to depend on the mode of administration (immediately or 24 h after seeding). Depending on the line, 3-BrPA induced a cytostatic or cytotoxic effect. This effect was accompanied by cell death induction even in cells highly refractory to cisplatin. Mitochondrial apoptotic death appeared to involve both lines; however, a different death pathway such as necrosis cannot be excluded. Interestingly, 3-BrPA leads to a diminution of the expression of the anti-apotptoic protein Mcl-1. We then tested 3-BrPA in vivo. Survival of nude mice bearing human mesothelioma was significantly prolonged (p < 0.0001). Toxicity and clinical studies should be performed to test 3- BrPA as local therapy for patients suffering from pleural or peritoneal mesothelioma. Association with cisplatin should be particularly considered.     

Combined arginine and ascorbic acid treatment induces apoptosis in the hepatoma cell line HA22T/VGH and changes in redox status involving the pentose phosphate pathway and reactive oxygen and nitrogen species

Arginine is a physiological substrate for nitric oxide synthase to generate nitric oxide (NO), which can influence tumor cell survival, while ascorbic acid is selectively toxic for cancer cells. This study explored the effect of an arginine/ascorbic acid combination on human cancer cell lines. The hepatoma cell line HA22T/VGH was the most sensitive of the tested cells to combination treatment. A combination of 5.74 mM of arginine and 0.57 mM of ascorbic acid induced HA22T/VGH cell death through apoptosis and an increase in levels of reactive oxygen species and NO, as well as its stable products NO₂⁻ and NO₃⁻. The combination also reduced the activity of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and transaldolase in the pentose phosphate pathway, a major mechanism for producing NADPH, resulting in a marked decrease in intracellular NADPH levels. A dramatic decrease in intracellular glutathione (GSH) levels, a decrease in the mitochondrial membrane potential, ATP depletion and release of cytochrome c were also seen. Caspase-9 and caspase-3 were activated, apoptotic protein Bax expression increased and the expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL decreased. These results suggest that this combination induced HA22T/VGH cell death by interfering with redox state regulation by a reduction in pentose phosphate pathway activity and increasing oxidative and nitrosative stress.     

Dependence of leukemic cell proliferation and survival on H2O2 and l-arginine

The proliferation and/or survival of a variety of cells is dependent on cellular hydrogen peroxide (H₂O₂) production. We tested whether this was true of leukemic cells, using cell lines from leukemic patients (CEM, 697, Mn-60, and Tanoue). We found that addition of catalase inhibited proliferation of all cell lines and induced death in two. However, this turned out to be due to arginase contamination of the catalase. Pure arginase inhibited cell proliferation and survival, which was reversible by adding l-arginine, demonstrating the l-arginine dependency of these cells. The glutathione peroxidase mimetic ebselen killed the cells by a novel, rapid form of death, preceded by cell blebbing and prevented by N-acetylcysteine, suggesting toxicity is not due to ebselen's antioxidant activity. Addition of N-acetylcysteine to remove endogenous H₂O₂ stimulated survival and proliferation, suggesting that basal levels of H₂O₂ promoted cell death. Consistent with this, leukemic cell death was induced by adding as little as 5 μM H₂O₂. Ascorbic acid, even at 100 μM, induced death through H₂O₂ production. Thus H₂O₂ does not promote proliferation and survival, rather the opposite, and previous literature may have misinterpreted the effects of antioxidants. Arginase, H₂O₂, ascorbic acid, and ebselen might be useful in the treatment of leukemia.     

Knockdown of COPA,Identified by Loss-of-Function Screen,Induces Apoptosis and Suppresses Tumor Growth in Mesothelioma Mouse Model

Malignant mesothelioma is a highly aggressive tumor arising from serosal surfaces of the pleura and is triggered by past exposure to asbestos. Currently, there is no widely accepted treatment for mesothelioma. Development of effective drug treatments for human cancers requires identification of therapeutic molecular targets. We therefore conducted a large-scale functional screening of mesothelioma cells using a genome-wide small interfering RNA library. We determined that knockdown of 39 genes suppressed mesothelioma cell proliferation. At least seven of the 39 genes—COPA, COPB2, EIF3D, POLR2A, PSMA6, RBM8A, and RPL18A—would be involved in anti-apoptotic function. In particular, the COPA protein was highly expressed in some mesothelioma cell lines but not in a pleural mesothelial cell line. COPA knockdown induced apoptosis and suppressed tumor growth in a mesothelioma mouse model. Therefore, COPA may have the potential of a therapeutic target and a new diagnostic marker of mesothelioma.     

Ascorbic acid maintenance in HaCaT cells prevents radical formation and apoptosis by UV-B.

We have investigated the enzymatic reduction and accumulation of vitamin C in HaCaT epithelial cells. The subcellular localization and the activities of ascorbyl free radical reductase and dehydroascorbate reductase showed that mitochondrial, microsomal and plasma membranes fractions express high levels of ascorbyl free radical reductase activity, whereas dehydroascorbate reductase activity was found at low levels only in the post microsomal supernatant. We have also investigated cell proliferation and vitamin C accumulation induced by ascorbic acid 2-phosphate. This derivative caused no inhibition of cell growth, was uptaken from the extracellular medium and accumulated as ascorbic acid in mM concentrations. These results show that HaCaT cells possess very efficient systems to maintain high levels of both intracellular and extracellular ascorbic acid. The regeneration and uptake of ascorbic acid from extracellular medium contributes to the intracellular antioxidant capacity, as evaluated by 2',7'-dihydrodichlorofluorescein staining. Consequently, cells became more resistant to free radical generation and cell death induced by UV-B irradiation.     

DNA degradation and repair in human laryngeal carcinoma HEp-2 cells after combined exposure to vitamin B12b and ascorbic acid

The formation and accumulation of DNA fragments containing no more than 23,000 pairs of bases were observed under exposure of human larynx epidermoid carcinoma cells (Hep-2) to "chemical nuclease", oxycobalamin (vitamin B12b) and ascorbic acid (vitamin C). The obtained DNA damages were repaired more slowly than those induced by gamma-irradiation in the dose adequate to the level of DNA damages. DNA reparation was not revealed after washing the cells from vitamin B12b and ascorbic acid, and in the course of cell incubation with ascorbic acid. Vitamin B12b and ascorbic acid separately did not induce degradation of DNA. DNA damages induced by "chemical nuclease" action precede the cell death observed later.     

Preclinical Demonstration of Synergistic Active Nutrients/Drug (AND) Combination as a Potential Treatment for Malignant Pleural 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.     

Wnt inhibitory factor-1, a Wnt antagonist, is silenced by promoter hypermethylation in malignant pleural mesothelioma

Wnt inhibitory factor-1 (WIF-1) is a secreted protein that antagonizes Wnt signaling. We recently demonstrated the importance of aberrant activation of the Wnt signaling pathway in various cancers including malignant pleural mesothelioma. In this study, we revealed downregulated WIF-1 expression in cell lines and primary tissue when compared to normal mesothelial cell lines and adjacent pleura, respectively. We observed hypermethylation in four of four mesothelioma cell lines, but not in two normal mesothelial cell lines. In primary tissue samples, we observed methylation in three paired tumor specimens compared to their adjacent normal pleura and methylation in eight of nine unpaired tumor tissue samples. Taken together, our studies suggest that WIF-1 silencing due to its promoter hypermethylation is an important mechanism underlying the constitutively activated Wnt signaling in mesothelioma. New therapies toward inhibition of the Wnt pathway through WIF-1 might be promising for the future treatment of malignant mesothelioma.     

Juglone–ascorbic acid synergy inhibits metastasis and induces apoptotic cell death in poorly differentiated thyroid carcinoma by perturbing SOD and catalase activities

Anaplastic thyroid carcinoma (ATC) requires more innovative approaches as the current regimes for therapy are inadequate, also most anticancer drugs cause general suppression of physiological functions. However, therapy with limited nontarget tissue damage is desirable. In the present study, we show prooxidant ability of ascorbic acid, which enhances cytotoxicity induced by juglone. We decipher that juglone–ascorbate combination induces reactive oxygen species‐mediated apoptosis leading to cell death in ARO cell line originated from ATC. This combination also affects enzyme activity of catalase, glutathione reductase, and superoxide dismutase destabilizing redox balance in cell and thereby making juglone effective at a lower dose. We also show that juglone–ascorbate combination suppresses cell migration, invasion, and expression of tumor‐promoting, and angiogenic genes in ARO cell line, thereby disrupting epithelial–mesenchymal transition ability of the cells. Overall, we show that ascorbic acid increases cytotoxic potency of juglone through redox cycling when used in synergy.     

Protection by α-tocopherol but not ascorbic acid from hydrogen peroxide induced cell death in normal human breast epithelial cells in culture

α-Tocopherol and ascorbic acid have been suggested to play a role in breast cancer prevention due to their antioxidative capacity. Increased exposure to endogenous and exogenous sex steroids is a known risk factor for breast cancer. We have studied the effects of α-tocopherol and ascorbic acid on hydrogen peroxide induced cell death in sex hormone treated normal breast epithelial cells in culture. We found that α-tocopherol but not ascorbic acid alone protected the cells. The effect of α-tocopherol increased when ascorbic acid was added to the cultures. The hydrogen peroxide degradation rate decreased in cultures treated with α-tocopherol alone and in combination with ascorbic acid compared to cells grown in medium or with ascorbic acid only. Oestradiol and progesterone treatment did not influence the results. Possible beneficial effects of combining various antioxidants, endogenous as well as exogenous, on human breast tissue need to be investigated further both in vivo and in vitro.     

Superoxide dictates the mode of U937 cell ascorbic acid uptake and prevents the enhancing effects of the vitamin to otherwise nontoxic levels of reactive oxygen/nitrogen species

Exposure of U937 cells to low micromolar levels of ascorbic acid or dehydroascorbic acid, while resulting in identical ascorbic acid accumulation, is unexpectedly associated with remarkably different responses to exogenous oxidants. We observed that otherwise nontoxic levels of hydrogen peroxide, tert-butylhydroperoxide or peroxynitrite promote toxicity in cells preloaded with ascorbic acid, whereas hardly any effect was detected in cells pretreated with dehydroascorbic acid. Further experiments performed with peroxynitrite in cells preloaded with ascorbic acid provided evidence for a very rapid nonapoptotic death, preceded by early Bax mitochondrial translocation and by mitochondrial permeability transition. The notion that conversion of extracellular ascorbic acid to dehydroascorbic acid prevents the enhancing effects on oxidant toxicity and nevertheless preserves the net amount of vitamin C accumulated was also established using ascorbate oxidase as well as various sources of superoxide, namely, xanthine/xanthine oxidase or ATP-driven NADPH oxidase activation. These findings suggest that superoxide-dependent conversion of extracellular ascorbic acid to dehydroascorbic acid represents an important component of the overall survival strategy of some cell types to reactive oxygen/nitrogen species.     

Vitamin C homeostasis in skeletal muscle cells

In skeletal muscle, vitamin C not only enhances carnitine biosynthesis but also protects cells against ROS generation induced by physical exercise. The ability to take up both ascorbic and dehydroascorbic acid from the extracellular environment, together with the ability to recycle the intracellular vitamin, maintains high cellular stores of ascorbate. In this study, we examined vitamin C transport and recycling, by using the mouse C2C12 and rat L6C5 muscle cell lines, which exhibit different sensitivity to oxidative stress and GSH metabolism. We found that: (1) both cell lines express SVCT2, whereas SVCT1 is expressed at very low levels only in proliferating L6C5 cells; furthermore L6C5 myoblasts are more efficient in ascorbic acid transport than C2C12 myoblasts; (2) C2C12 cells are more efficient in dehydroascorbic acid transport and ascorbyl free radical/dehydroascorbic acid reduction; (3) differentiation is paralleled by decreased ascorbic acid and dehydroascorbic acid transport and reduction and increased ascorbyl free radical reduction; (4) differentiated cells are more responsive to oxidative stress induced by glutathione depletion; indeed, myotubes showed increased SVCT2 expression and thioredoxin reductase-mediated dehydroascorbic acid reduction. From our data, SVCT2 and NADPH-thioredoxin-dependent DHA reduction appears to belong to an inducible system activated in response to oxidative stress.     

Protection by α-tocopherol but not ascorbic acid from hydrogen peroxide induced cell death in normal human breast epithelial cells in culture

-Tocopherol and ascorbic acid have been suggested to play a role in breast cancer prevention due to their antioxidative capacity. Increased exposure to endogenous and exogenous sex steroids is a known risk factor for breast cancer. We have studied the effects of -tocopherol and ascorbic acid on hydrogen peroxide induced cell death in sex hormone treated normal breast epithelial cells in culture. We found that -tocopherol but not ascorbic acid alone protected the cells. The effect of -tocopherol increased when ascorbic acid was added to the cultures. The hydrogen peroxide degradation rate decreased in cultures treated with -tocopherol alone and in combination with ascorbic acid compared to cells grown in medium or with ascorbic acid only. Oestradiol and progesterone treatment did not influence the results. Possible beneficial effects of combining various antioxidants, endogenous as well as exogenous, on human breast tissue need to be investigated further both in vivo and in vitro.     

Mechanism of vitamin C inhibition of cell death induced by oxidative stress in glutathione-depleted HL-60 cells

Vitamin C is a well known antioxidant whose precise role in protecting cells from oxidative challenge is uncertain. In vitro results have been confounded by pro-oxidant effects of ascorbic acid and an overlapping role of glutathione. We used HL-60 cells as a model to determine the precise and independent role of vitamin C in cellular protection against cell death induced by oxidative stress. HL-60 cells do not depend on glutathione to transport or reduce dehydroascorbic acid. Depletion of glutathione rendered the HL-60 cells highly sensitive to cell death induced by H2O2, an effect that was not mediated by changes in the activities of glutathione reductase, glutathione peroxidase, catalase, or superoxide dismutase. The increased sensitivity to oxidative stress was largely reversed when glutathione-depleted cells were preloaded with ascorbic acid by exposure to dehydroascorbic acid. Resistance to H2O2 treatment in cells loaded with vitamin C was accompanied by intracellular consumption of ascorbic acid, generation of dehydroascorbic acid, and a decrease in the cellular content of reactive oxygen species. Some of the dehydroascorbic acid generated was exported out of the cells via the glucose transporters. Our data indicate that vitamin C is an important independent antioxidant in protecting cells against death from oxidative stress.     

Zebularine exerts its antiproliferative activity through S phase delay and cell death in human malignant mesothelioma cells

Malignant mesothelioma is an asbestos-related aggressive tumor and current therapy remains ineffective. Zebularine as a DNA methyltransferase (DNMT) inhibitor has an anti-tumor effect in several human cancer cells. The aim of the present study was to investigate whether zebularine could induce antiproliferative effect in human malignant mesothelioma cells. Zebularine induced cell growth inhibition in a dose-dependent manner. In addition, zebularine dose-dependently decreased expression of DNMT1 in all malignant mesothelioma cells tested. Cell cycle analysis indicated that zebularine induced S phase delay. Zebularine also induced cell death in malignant mesothelioma cells. In contrast, zebularine did not induce cell growth inhibition and cell death in human normal fibroblast cells. These results suggest that zebularine has a potential for the treatment of malignant mesothelioma by inhibiting cell growth and inducing cell death.     

New highly toxic bile acids derived from deoxycholic acid,chenodeoxycholic acid and lithocholic acid

We have prepared a new panel of 23 BA derivatives of DCA, chenodeoxycholic acid (CDCA) and lithocholic acid (LCA) in order to study the effect of dual substitution with 3-azido and 24-amidation, features individually associated with cytotoxicity in our previous work. The effect of the compounds on cell viability of HT-1080 and Caco-2 was studied using the 3-[4,5-dimethylthizol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Compounds with high potency towards reduction of cell viability were further studied using flow cytometry in order to understand the mechanism of cell death. Several compounds were identified with low micromolar IC₅₀ values for reducing cell viability in the Caco-2 and HT1080 cell lines, making them among the most potent BA apoptotic agents reported to date. There was no evidence of relationship between overall hydrophobicity and cytotoxicity supporting the idea that cell death induction by BAs may be structure–specific. Compounds derived from DCA caused cell death through apoptosis. There was some evidence of selectivity between the two cell lines studied which may be due to differing expression of CD95/FAS. The more toxic compounds increased ROS production in Caco-2 cells, and co-incubation with the antioxidant N-acetyl cysteine blunted pro-apoptotic effects. The properties these compounds suggest that there may be specific mechanism(s) mediating BA induced cell death. Compound 8 could be useful for investigating this phenomenon.     

α-Tocopherol-ascorbic acid hybrid antioxidant based cationic amphiphile for gene delivery: Design,synthesis and transfection

Natural antioxidants and vitamins have potential to protect biological systems from peroxidative damage induced by peroxyl radicals, α-tocopherol (Vitamin E, lipid soluble) and ascorbic acid (vitamin C, water soluble), well known natural antioxidant molecules. In the present study we described the synthesis and biological evaluation of hybrid of these two natural antioxidants with each other via ammonium di-ethylether linker, Toc-As in gene delivery. Two control cationic lipids N14-As and Toc-NOH are designed in such a way that one is with ascorbic acid moiety and no tocopherol moiety; another is with tocopherol moiety and no ascorbic acid moiety respectively. All the three cationic lipids can form self-assembled aggregates. The antioxidant efficiencies of the three lipids were compared with free ascorbic acid. The cationic lipids (Toc-As, N14-As and Toc-NOH) were formulated individually with a well-known fusogenic co-lipid DOPE and characterization studies such as DNA binding, heparin displacement, size, charge, circular dichroism were performed. The biological characterization studies such as cell viability assay and in vitro transfection studies were carried out with the above formulations in HepG2, Neuro-2a, CHO andHEK-293T cell lines. The three formulations showed their transfection efficiencies with highest in Toc-As, moderate inN14-As and least in Toc-NOH. Interestingly, the transfection efficiency observed with the antioxidant based conjugated lipid Toc-As is found to be approximately two and half fold higher than the commercially available lipofectamine 2000 at 4:1 charge ratio in Hep G2 cell lines. In the other cell lines studied the efficiency of Toc-As is found to be either higher or similarly active compared to lipofectamine 2000. The physicochemical characterization results show that Toc-As lipid is showing maximum antioxidant potency, strong binding with pDNA, least size and optimal zeta potential. It is also found to be least toxic in all the cell lines studied especially in Neuro-2a cell lines when compared to other two lipids. In summary, the designed antioxidant lipid can be exploited as a delivering system for treating ROS related diseases such as malignancy, brain stroke, etc.     

Oxidative stress and innate immunity status in chickens exposed to high dose of ascorbic acid

The effects of high dose ascorbic acid (10 000 mg· kg^–1 in the diet) and the transition metal on the presence of oxidative stress in the internal organs of growing chicks, as well as on the innate immune system status, were investigated. Supplementation with a high dose of ascorbic acid had pro‐inflammatory effects on the intestinal mucosa, and lysozyme levels were decreased significantly in all organs studied. High‐dose ascorbic acid caused an imbalance between prooxidative and antioxidative activities and was associated with the generation of semiquinone radicals. We observed that ascorbic acid increased iron and cadmium absorption. When a high dose of ascorbic acid was applied, elevated kidney and intestinal mucosa iron concentrations were observed. The amount of free malondialdehyde in the above organs has increased as well. These data have important implications for the mechanism of the oxidative stress development under the influence of high dose of ascorbic acid, indicating the importance of the side reactions of the mitochondrial electron transport chain with the formation of semiquinone radicals and the role of transition metals in this process. Copyright © 2013 John Wiley & Sons, Ltd.     

Anticarcinogenic effects of halofuginone on lung‐derived cancer cells

Malignant mesothelioma is a rare but aggressive form of malignancy, which is difficult to diagnose and is resistant to current chemotherapeutic treatment options. Molecular techniques have been used to investigate the mechanisms of action and the beneficial therapeutic effects of halofuginone (HF) in several cancers but not malignant mesotheliomas. In this study, the antiproliferative and apoptotic effects of HF were investigated through its ability to deregulate EGFR downstream signalling cascade proteins in the pathologically aggressive malignant mesothelioma and non‐small‐cell lung cancer cells. We showed that administration of HF at nanomolar concentrations induced a dose‐dependent reduction in the viability of cancer cells, made cell cycle arrest, inhibited proliferation of cancer cells via STAT3 and ERK1/2 pathways and triggered the apoptotic cascade via p38MAPK. We demonstrated that the apoptotic cell death mechanism was mediated by enhanced activation of caspase‐3 and concomitant PARP cleavage, downregulation of Bcl‐2 and upregulation of Bax in both malignant mesothelioma and lung cancer cells. In particular, we demonstrated that cancer cells were more sensitive to HF treatment than normal mesothelial cells. Taken together, this study suggests that HF exerts its anticancer effects in lung‐derived cancers by targeting signal transduction pathways mainly through deregulation of ERK1/2, STAT3 and p38MAPK to reduce cancer cell viability, induce cell cycle arrest and apoptotic cell death. Thus, HF might be considered as a potential agent against malignant mesothelioma and/or lung cancer cells.