Key roles for GRB2-associated-binding protein 1, phosphatidylinositol-3-kinase, cyclooxygenase 2, prostaglandin E2 and transforming growth factor alpha in linoleic acid-induced upregulation of lung and breast cancer cell growth.

The distribution of omega-6 and omega-3 polyunsaturated fatty acid (PUFA) intake in Western diets is disproportionate, containing an overabundance of the omega-6 PUFA, linoleic acid (LA; C18:2). Increased enrichment with LA has been shown to contribute to the enhancement of tumorigenesis in several cancer models. Previous work has indicated that phosphatidylinositol 3-kinase (PI3K) may play a key role in LA-induced tumorigenesis. However, the modes by which LA affects carcinogenesis have not been fully elucidated. In this study, a mechanism for LA-induced upregulation of cancer cell growth is defined. LA treatment enhanced cellular proliferation in BT-474 human breast ductal carcinoma and A549 human lung adenocarcinoma cell lines. Enrichment of LA increased cyclooxygenase (COX) activity and led to increases in prostaglandin E2 (PGE2), followed by increases in matrix metalloproteinase (MMP) and transforming growth factor alpha (TGF-α) levels, which are all key elements involved in the enhancement of cancer cell growth. Further investigation revealed that LA supplementation in both BT-474 breast and A549 lung cancer cell lines greatly increased the association between the scaffolding protein GRB2-associated-binding protein 1 (Gab1) and epidermal growth factor receptor (EGFR), although Gab1 protein levels were significantly decreased. These LA-induced changes were associated with increases in activated Akt (pAkt), a downstream signaling component in the PI3K pathway. Treatment with inhibitors of EGFR, PI3K and Gab1-specific siRNAs reversed the upregulation of pAkt, as well as the observed increases in cell proliferation by LA in both cell lines. A549 xenograft assessment in athymic nude mice fed high levels of LA exhibited similar increases in EGFR-Gab1 association and increased levels of pAkt, while mice fed with high levels of the omega-3 PUFA, docosahexaenoic acid (DHA; C22:6), demonstrated an opposite response. The involvement of Gab1 in LA-induced tumorigenesis was further defined utilizing murine cell lines that express high levels of Gab1. Significant increases in cell proliferation were observed with the addition of increasing concentrations of LA. However, no changes in cell proliferation were detected in the murine paired cell lines expressing little or no Gab1 protein, establishing Gab1 as major target in LA-induced enhancement of tumorigenesis.

Induced oxidative stress and cell death in the A549 lung adenocarcinoma cell line by ionizing radiation is enhanced by supplementation with docosahexaenoic acid.

Both ionizing radiation and docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid (PUFA), have been shown to inhibit tumor cell growth at least in part by increasing oxidative stress. In this study, the effects of ionizing radiation, DHA, or a combination of the two on cell proliferation, anchorage-independent growth, apoptosis, and lipid peroxidation in A549 lung adenocarcinoma cells were examined. In this study, significant decreases in cell proliferation and colony formation were noted for ionizing radiation or DHA treatments, whereas a combination of the two showed significant reductions over either treatment alone. Conversely, lipid peroxidation and apoptotic cell death showed significant increases with ionizing radiation and DHA treatments, whereas cells receiving both treatments demonstrated further significant increases. Moreover, addition of vitamin E, an antioxidant, was able to completely reverse lipid peroxidation and cell death due to ionizing radiation and partially reverse these changes in DHA treatments. Finally, the preferential incorporation of DHA into lung and xenograft compared to liver tissue is demonstrated in an in vivo model. These findings confirm the potential of DHA supplementation to enhance the treatment of lung cancer using ionizing radiation by increasing oxidative stress and enhancing tumor cell death.

Tumor development and cytokine production by human colon tissues and carcinoma cell lines.

BACKGROUND AND OBJECTIVES: Solid tumors evade the host immunologic responses they initiate by unknown mechanisms. The authors investigated patterns of cytokine content in human colon carcinomas, colon cancer cell lines in vitro, and nude mouse xenografts from those lines in order to clarify those mechanisms. METHODS: Epithelial tumor cell lines were developed from specimens of human colon adenocarcinoma. Aliquots of these cells were then xenografted into female heterozygous BALB/c nu/+ immunologically deficient mice and serially passaged. Original tumors, cell lines, and resultant xenografts were then analyzed for histology/cytology and for levels of TGF-beta and TNF-alpha by enzyme linked immunoassay. RESULTS: Cytokine levels were elevated beyond baseline mucosal levels in original tumors and xenograft mouse tumors but not detectable in extracts from epithelial cultures. CONCLUSIONS: While the precise source of cytokine production remains unclear, these data suggest tumor/host interactions not found in pure epithelial cancer cells in culture.

An atraumatic method to establish human colon carcinoma in long-term culture.

Techniques for creation of colon carcinoma epithelial cells lines in long-term culture have been available for years, but these techniques have involved mechanical or enzymatic methods to separate epithelial cells from surrounding tissues. While this practice has been intermittently successful, the effect of these traumatic methods on long-term cellular behavior is unknown. Samples of colon carcinoma from patient volunteers were subjected to serial nonenzymatic disruptions of carcinoma cells from surrounding fibrous tissues. Cells were collected, allowed to proliferate, and then tested for their epithelial characteristics (mucin, vimentin, cytokeratin, colon-specific antigen, carcinoembryonic antigen) by immunohistochemistry and flow cytometry. Growth characteristics were determined by phase-contrast microscopy, multiple passage, and freeze/thaw effects. Tumorigenicity was proven in nude mice. Of 11 initial attempts, three resulted in stable long-term culture lines of cells which are demonstrated to behave similarly to the original tumors from which they were derived. This technique adds another reliable in vitro tool for the study of colon carcinoma.

Time-course of hypericin phototoxicity and effect on mitochondrial energies in EMT6 mouse mammary carcinoma cells.

Photoactivated hypericin produces singlet oxygen and superoxide anion radical; however, the intracellular events contributing to toxicity are unknown. Clonogenic assays of oxygen-dependent hypericin phototoxicity to EMT6 cells have previously shown that 0.5 microM hypericin + 1.5 J cm(-2) fluorescent light is non-toxic and that 1.0 microM hypericin + 1.5 J cm(-2) fluorescent light produces LD40 toxicity. Intracellular events leading to toxicity were revealed at these doses. Lactate dehydrogenase leakage was elevated for both 0.5 microM and 1.0 microM hypericin + light immediately following irradiation. While values eventually returned to control levels for 0.5 microM hypericin + light, leakage increased over time for 1.0 microM hypericin indicating reversible and irreversible toxicity, respectively. Increases in lipid and protein oxidation were measured immediately following irradiation; however, these parameters return to control levels within 0.5 h for both doses. Both total cellular ATP levels and cellular respiration were depressed by approximately 50% of control values for 1.0 microM hypericin + light. These values were unchanged for 0.5 microM hypericin + light. Along with previously reported data demonstrating that light-activated hypericin can inhibit mitochondial succinoxidase in beef heart mitochondria in vitro, these data support oxidative stress-initiated mitochondrial damage as a key target in hypericin phototoxicity.

Antioxidant enzyme response to hypericin in EMT6 mouse mammary carcinoma cells.

Antioxidant enzyme activities were measured following exposure to hypericin +/- irradiation in EMT6 cells. CuZnSOD and catalase activities peaked within 0.5 h following irradiation for nontoxic 0.5 microM hypericin and toxic 1.0 microM hypericin. Catalase remained elevated up to 3 h for 1.0 microM hypericin + light. MnSOD activity was elevated immediately following irradiation for both doses. These levels returned to control by 1 h for 0.5 microM hypericin, but were depressed after 1 h for 1.0 microM hypericin. This suggests that mitochondria impairment may be a critical factor in hypericin phototoxicity. Glutathione reductase was inhibited immediately following irradiation with 1.0 microM hypericin, suggesting that an altered status of the glutathione pool contributed to cytotoxicity. Glutathione peroxidase activities were elevated following irradiation but returned to control levels within 0.5 h for both doses, implicating hydroperoxide formation as an early event in hypericin phototoxicity. Inhibition by hypericin in the dark was demonstrated for purified CuZnSOD, Se-dependent glutathione peroxidase, glutathione S-transferase, and glutathione reductase activities in vitro. Irradiation did not potentiate hypericin-mediated glutathione reductase inhibition and decrease inhibition for the other enzymes. Collectively, these data demonstrate an antioxidant enzyme response to hypericin photoactivation and confirm a role for oxygen in hypericin phototoxicity.

Intervention of transplantable human mammary carcinoma MX-1 chemotherapy with dietary menhaden oil in athymic mice: increased therapeutic effects and decreased toxicity of cyclophosphamide.

We investigated the effects of dietary menhaden oil on cyclophosphamide (CP) antineoplastic activity and its protective effect against CP toxicity. We found that dietary menhaden oil (HMO, 20% menhaden oil + 5% corn oil) enhanced the CP antitumor effect at the lowest dose tested (50 mg/kg) compared with the control group (LCO, 5% corn oil). Dietary HMO and CP treatment had a significant effect on the activities of tumor and liver microsomal cytochrome P-450 (CYP) over the controls. Activity of one of the key CP activating enzymes, CYP2B1 (which is similar to human CYP2B6), was significantly enhanced in the liver and tumor by the HMO diet, which could result in the formation of more pharmacologically active CP metabolites and, therefore, increased CP antitumor response. Moreover, the HMO diet exhibited a very significant protective effect against CP acute toxicity. The activity of the CP detoxifying enzyme aldehyde dehydrogenase (ADH) was significantly increased in the liver after HMO feeding; thus the observed protective effect of HMO feeding against CP toxicity may be partially the result of induction of ADH activity in the liver. In summary, our findings suggested that dietary menhaden oil can modulate ADH and CYP activities in a manner that may alter the metabolism of CP and, therefore, improve its therapeutic index by increasing its therapeutic effect and decreasing its toxicity.

Dichlone-induced oxidative stress in a model insect species, Spodoptera eridania.

Southern armyworm, Spodoptera eridania, larvae were provided ad libitum 0.002-0.25% w/w dichlone, 2,3-dichloro-1,4-naphthoquinone (CNQ). Larval mortality occurred in a time-and-dose dependent manner, with an LC17 of 0.01% and an LC50 of 0.26% CNQ at day-5. Extracts of larvae fed control, 0.01, and 0.25% CNQ diets for 5 days were assayed for antioxidant enzymes. While 0.01% CNQ had a mild effect, 0.25% CNQ profoundly increased levels of all antioxidant enzymes that were examined. The increases as compared to control were: 5.3-, 1.9-, 3.2-, 2.6-, 2.8-, and 3.5-fold higher for superoxide dismutase, catalase, glutathione transferase and its peroxidase activity, glutathione reductase and DT-diaphorase, respectively. At 0.01% CNQ, the thiobarbituric acid reactive substances (TBARS) were similar to the control group. However, despite the induction from 0.25% CNQ of all enzymes examined, the lipid peroxidation was not attenuated; the TBARS were 29.7% over the control value. High mortalities and CNQ-induced pathologies reflected in retarded growth, wasting syndrome, and diuresis clearly indicated that the insect sustained severe oxidant-induced injuries before appropriate defenses were fully mobilized. Thus, this quinone causes an oxidative stress in a model insect species analogous to that observed in mammalian species.

Dietary menhaden oil enhances mitomycin C antitumor activity toward human mammary carcinoma MX-1.

In the present study, we investigated the effects of high levels of dietary fish oil on the growth of MX-1 human mammary carcinoma and its response to mitomycin C (MC) treatment in athymic mice. We found that high levels of dietary fish oil (20% menhaden oil + 5% corn oil, w/w) compared to a control diet (5% corn oil, w/w) not only lowered the tumor growth rate, but also increased the tumor response to MC treatment. We also found that high levels of dietary fish oil significantly increased the activities of tumor xanthine oxidase and DT-diaphorase, which are proposed to be involved in the bioreductive activation of MC. Since menhaden oil is highly unsaturated, its intake caused a significant increase in the degree of fatty acid unsaturation in tumor membrane phospholipids. This alteration in tumor membrane phospholipids made the tumor more susceptible to oxidative stress, as indicated by the increased levels of both endogenous lipid peroxidation and protein oxidation after feeding the host animals the menhaden oil diet. In addition, the tumor antioxidant enzyme activities, catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPOx), and glutathione S-transferase peroxidase (GSTPx), were all significantly enhanced by feeding a diet high in fish oil. MC treatment caused further increases in tumor lipid peroxidation and protein oxidation, as well as in the activities of CAT, SOD, GPOx, and GSTPx, suggesting that MC causes oxidative stress in this tumor model which is exacerbated by feeding a diet high in menhaden oil. Thus, feeding a diet rich in menhaden oil decreased the growth of human mammary carcinoma MX-1, increased its responsiveness to MC, and increased its susceptibility to endogenous and MC-induced oxidative stress, and increased the tumor activities of two enzymes proposed to be involved in the bioactivation of MC, that is, DT-diaphorase and xanthine oxidase. These findings support a role of these two enzymes in the bioactivating of MC and indicate that the type of dietary fat may be important in tumor response to therapy.

Effect of NDGA on beef heart mitochondria and EMT6 mouse mammary carcinoma cells.

The inhibition of mitochondrial succinate cytochrome c reductase by nordihydroguaiaretic acid (NDGA) was measured in vitro under aerobic and hypoxic conditions. The photodynamic effect of NDGA on EMT6 mouse mammary carcinoma cells was also investigated in vitro under aerobic and hypoxic conditions. NDGA strongly inhibited succinate cytochrome c reductase, which contains the mitochondrial electron transport Complexes II and III. By comparing the I50 values of aerobic and hypoxic assays, NDGA inhibition was found to be oxygen-independent. This finding suggests that oxygen is not involved in the inhibitory effect of mitochondrial respiration by NDGA. Similarly, our data demonstrate that NDGA-induced cytotoxicity towards EMT6 tumor cells is neither light-dependent nor oxygen-dependent. A dose-dependent loss of viability of EMT6 cell was observed although the toxicity of NDGA towards EMT6 cells was not strong, with an LC50 in the 200 microM range. NDGA caused a depletion of mitochondrial sulfhydryl groups. Sulfhydryl compounds, GSH and cysteine, prevented the inhibition of succinoxidase activity by NDGA. This suggests that NDGA exerts its biological effects by the depletion of sulfhydryl groups in key biochemical systems related to mitochondrial function.

Toxicity of oxygen from naturally occurring redox-active pro-oxidants.

The survival of all aerobic life forms requires the ground-state of molecular oxygen, O2. However, the activation of O2 to reactive oxygen species (ROS) is responsible for universal toxicity. ROS are responsible in deleterious intracellular reactions associated with oxidative stress including membrane lipid peroxidation, and the oxidation of proteins and DNA. Redox-active allelochemicals such as quinones and phenolic compounds are involved in activating O2 to its deleterious forms including superoxide anion free radical, O2.-, hydrogen peroxide, H2O2, and hydroxyl radical, OH. Molecular oxygen is also activated in biologically relevant photosensitizing reactions to the singlet form, 1O2. The insect lifestyle exposes them to a broad diversity of pro-oxidant allelochemicals and, like mammalian species, they have developed an elaborate antioxidant system comprised of chemical antioxidants and a bank of anti-oxidant enzymes. We have found that an insect's antioxidant adaptation to a particular food correlates well with its risk of exposure to potential pro-oxidants.

An insect model for assessing oxidative stress related to arsenic toxicity.

The potential usefulness of an insect model to evaluate oxidative stress induced by environmental pollutants was examined with trivalent arsenic (As3+, NaAsO2) and pentavalent arsenic (As5+, Na2HAsO4) in adult female house flies, Musca domestica, and fourth-instar cabbage loopers, Trichoplusia ni. M. domestica was highly susceptible to both forms of arsenic following 48 h exposure in the drinking water with LC50s of 0.008 and 0.011% w/v for As3+ and As5+, respectively. T. ni larvae were susceptible to dietary As3+ with an LC50 of 0.032% w/w but seem to tolerate As5+ well with an LC50 of 0.794% concentration after 48 h exposure. The minimally acute LC5 dose of both As3+ and As5+ varied considerably but averaged 0.005% for both insects. The potential of both valencies of arsenic for inducing oxidative stress in the insects exposed ad libitum to approximately LC5 levels was assessed. The parameters examined were the alterations of the antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT), glutathione transferase (GST), the peroxidase activity of glutathione transferase (GSTPX), and glutathione reductase (GR), and increases in lipid peroxidation and protein oxidation. SOD (1.3-fold), GST (1.6-fold), and GR (1.5-fold) were induced by As3+ in M. domestica but CAT and GSTPX were not affected. As5+ had no effect on M. domestica. In T. ni, the antioxidant enzyme activities were not affected by As3+ except for SOD which was suppressed by 29.4% and GST which was induced by 1.4-fold. As5+ had no effect except the suppression of SOD by 41.2%. Lipid peroxidation and protein oxidation, which represent stronger indices of oxidative stress, were elevated in both insects by up to 2.9-fold. However, based on the antioxidant enzyme response to the arsenic anions, the mode of action of arsenic induced oxidative stress may differ between the two insects. Until this aspect is further clarified, evidence at this time favors the prospect of As3+ as a pro-oxidant, especially for M. domestica.

Enhancement of the antineoplastic effect of mitomycin C by dietary fat.

In the present study, we investigated the effects of high dietary fat on the growth of MX-1 heterotransplanted in athymic mice and its response to mitomycin C (MC) treatment. We found that high fat intake (25% corn oil, w/w) significantly increased tumor growth, but at the same time it also increased the tumor response to MC treatment compared to the control low fat diet (5% corn oil, w/w). In the tumors from mice fed either low (5% w/w) or high (25% w/w) fat, MC treatment induced oxidative challenge, indicated by significantly increased tumor total superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase peroxidase activities, as well as increased tumor lipid peroxidation. On the other hand, glutathione reductase activity was inhibited by MC treatment. Some of the enzymes which are known to activate MC, such as cytochrome b5 reductase and DT-diaphorase, were also induced in the tumor by high dietary fat intake. The enzyme activities in hepatic tissues were also altered by dietary fat and MC treatment but to a lesser extent. We conclude that high dietary fat intake could enhance the chemotherapeutic effect of MC by increasing MC-activating enzyme activities. The observed increase in lipid peroxidation after MC treatment in MX-1 human mammary carcinoma implanted in the nude mice could result from the observed inhibited glutathione reductase activity. It is tempting to speculate that this might be another antineoplastic mechanism for MC in addition to its known role as a bioreductive alkylating agent. Alternatively, glutathione reductase may be a target for bioreductive alkylation.

Mitomycin C induced alterations in antioxidant enzyme levels in a model insect species, Spodoptera eridania.

1. An insect species, the southern armyworm Spodoptera eridania, was used as an in vivo model to examine mitomycin C's (MMC) pro-oxidant effect reflected in alterations of antioxidant enzymes. 2. Following a 2-day exposure to 0.01 and 0.05% w/w dietary concentrations, MMC only induced superoxide dismutase activity. All other enzyme activities were not affected, indicating oxidative stress was mild. 3. Following a 5-day exposure to 0.05% w/w dietary MMC, the activities of superoxide dismutase, glutathione-S-transferase and its peroxidase activity and DT-diaphorase were induced. GR activity was not altered. The high constitutive catalase activity was also not affected. These responses of S. eridania's antioxidant enzymes are analogous to those of mammalian systems in alleviating MMC-induced oxidative stress. 4. S. eridania emerges as an appropriate non-mammalian model for initial and cost-effective screening of drug-induced oxidative stress.

Inhibition of mitochondrial respiration and cyanide-stimulated generation of reactive oxygen species by selected flavonoids.

A continuation of our structure-activity study on flavonoids possessing varied hydroxyl ring configurations was conducted. We tested six additional flavonoids for their ability to inhibit beef heart mitochondrial succinoxidase and NADH-oxidase activities. In every case, the IC50 observed for the NADH-oxidase enzyme system was lower than for succinoxidase activity, demonstrating a primary site of inhibition in the complex I (NADH-coenzyme Q reductase) portion of the respiratory chain. The order of potency for inhibition of NADH-oxidase activity was robinetin, rhamnetin, eupatorin, baicalein, 7,8-dihydroxyflavone, and norwogonin with IC50 values of 19, 42, 43, 77, 277 and 340 nmol/mg protein, respectively. Flavonoids with adjacent tri-hydroxyl or para-dihydroxyl groups exhibited a substantial rate of auto-oxidation which was accelerated by the addition of cyanide (CN-). Flavonoids possessing a catechol configuration exhibited a slow rate of auto-oxidation in buffer that was stimulated by the addition of CN-. The addition of superoxide dismutase (SOD) and catalase in the auto-oxidation experiments each decreased the rate of oxygen consumption, indicating that O2- and H2O2 are generated during auto-oxidation. In the CN(-)-stimulated oxidation experiments, the addition of SOD also slowed the rate of oxygen consumption. These findings demonstrate that the CN-/flavonoid interaction generated O2- non-enzymatically, which could have biological implications.

An insect model for assessing mercury toxicity: effect of mercury on antioxidant enzyme activities of the housefly (Musca domestica) and the cabbage looper moth (Trichoplusia ni).

The effect of mercury as Hg2Cl2 and HgCl2 on the antioxidant enzyme levels and its toxicity was investigated in an insect model comprised of adult females of the common housefly, Musca domestica, and fourth-instar larvae of the cabbage looper moth, Trichoplusia ni. HgCl2 was found to be more toxic than Hg2Cl2 to both M. domestica and T. ni. The LC50s for M. domestica were 1.17% and 0.38% w/v concentration for Hg2Cl2 and HgCl2, respectively. For the more tolerant T. ni, the LC50S were 5.15% for Hg2Cl2 and 0.96% w/w concentration for HgCl2. The minimally acute LC5 dose of both oxidation states of Hg was approximately 0.005% for both insects (w/v for M. domestica and w/w for T. ni). At the LC5, both forms of Hg significantly induced the activity of superoxide dismutase in both insect species. Catalase was induced by both Hg2Cl2 and HgCl2 in M. domestica but was only induced by HgCl2 in T. ni. Glutathione-S-transferase, its peroxidase activity, and glutathione reductase activities were also significantly altered in most cases by Hg in both insects although the pattern of alternation was different between the two insects. It is evident that mercury induces oxidative stress in insects as it does in vertebrates. Our findings suggest that insects may serve as a valuable, non-mammalian model species to assess Hg-induced oxidative stress as a component of environmental toxicity.

Inhibition of glutathione reductase by flavonoids. A structure-activity study.

A structure-activity study of fourteen chemically related flavonoids was conducted to evaluate their abilities to inhibit glutathione reductase (GR). By comparing the I50 values of flavonoids from different classes possessing an identical hydroxyl configuration, we determined the following order of potency for inhibition of GR: anthocyanidin > dihydroflavonol = chalcone > flavonol > catechin. Enzyme inhibition by delphinidin chloride and myricetin was partially prevented in a N2 atmosphere which implicates a role for oxygen in the mechanism of inhibition. To determine the role of oxygen species in enzyme inhibition, GR was preincubated with either mannitol, diethylenetriaminepenta-acetic acid (DETAPAC), superoxide dismutase (SOD), catalase (CAT), or SOD and CAT prior to assays for enzyme inhibition by flavonoids. Enzyme inhibition by delphinidin chloride and myricetin was suppressed by the addition of SOD, suggesting that superoxide (O2-.) is involved. However, inhibition by quercetin and morin was not sensitive to antioxidants. To further investigate the role of O2-. in GR inhibition, a superoxide generating system was utilized in the presence and absence of flavonoid. The O2-. generating system failed to inhibit GR in the absence of flavonoid but enhanced the inhibition by myricetin, indicating that the O2-. did not directly inhibit GR but reacted directly with certain flavonoids to form a reactive intermediate which, in turn, inhibited GR. These findings suggest that the mechanism of inhibition of GR by flavonoids is complex and may have oxygen-dependent and oxygen-independent components.