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.

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.

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.

A redox cycling mechanism of action for 2,3-dichloro-1,4-naphthoquinone with mitochondrial membranes and the role of sulfhydryl groups.

The addition of 2,3-dichloro-1,4-naphthoquinone (CNQ) to substrate-depleted, GSH-supplemented rat liver mitochondria resulted in a dose-dependent depletion of reactable suflhydryl groups and a concomitant increase in mitochondrial disulfide content at a ratio of 2 thiols depleted/disulfide generated. The molar ratio of thiol depleted/CNQ added approached 20 at low CNQ concentrations and was unity at higher doses. The addition of CNQ to substrate-depleted mitochondrial suspensions resulted in O2 consumption which increased with increasing concentrations of mitochondria and was sensitive to N-ethylmaleimide (NEM) which establishes the ability of CNQ to interact with mitochondrial thiol redox centers. The CNQ-mediated large amplitude swelling of rat liver mitochondria was exacerbated by thiol oxidizing agents and depressed by disulfide reducing agents. A redox cycling mechanism between mitochondrial thiol groups, CNQ and oxygen was proposed to lower the matrix glutathione pool and make the mitochondria more susceptable to toxic oxygen radicals which induce swelling in isolated mitochondrial suspensions. In support of this mechanism, alpha-tocopherol was shown to prevent the CNQ-mediated swelling process. Beef heart mitochondrial NADH was oxidized by CNQ in a 1/1 molar ratio anaerobically and in a 3/1 molar ratio under aerobic conditions, whereas the fully reduced quinone, CNQH2, oxidized NADH aerobically but not anaerobically. Thus, CNQ is capable of interacting with NADH of the mitochondrial electron transport chain in a redox cycling fashion.

Anti-neoplastic activity of C205: a new topical anti-cancer formulation.

C205, a topical ointment currently in clinical trial, was tested by a method developed to evaluate the topical activity of new drugs. Subcutaneous B-16 melanoma was used as the primary tumor model. Tumor size of treated animals as percentage of tumor size of control animals (T/C ratio) was found to be 14, with 50% of the animals tested free of viable and palpable tumor at 60 days post treatment. The anti-neoplastic activity of C205 was superior to 5-fluorouracil using the same method of evaluation. Only minimal loss of animal body weight occurred after C205. C205 also showed anti-tumor activity against subcutaneously implanted Lewis lung carcinoma.

Influence of medium-chain triglycerides on lipid metabolism in the chick.

The effect of corn oil, coconut oil, and medium-chain triglyceride (MCT, a glyceride mixture consisting almost exclusively of fatty acids of 8 and 10 carbons in length) ingestion on lipid metabolism was studied in chicks. In chicks fed cholesterol-free diets, MCT ingestion elevated plasma total lipids and cholesterol and depressed liver total lipids and cholesterol when compared to chicks receiving the corn oil diet. As a consequence of the opposite effects of MCT ingestion on plasma and liver cholesterol and total lipids, the plasma-liver cholesterol pool was not altered. When cholesterol was included in the diets, dietary MCT depressed liver and plasma total lipids and cholesterol as compared with corn oil, consequently also lowered the plasmaliver cholesterol pool.The in vitro cholesterol and fatty acid synthesis from acetate-1-(14)C was higher in liver slices from chicks fed MCT than in those from chicks fed corn oil. The percentage of radioactivity from acetate-1-(14)C incorporated into the carboxyl carbon of fatty acids by liver slices was not altered by MCT feeding, indicating that the increased acetate incorporation represented de novo fatty acid synthesis. The conversion of palmitate-1-(14)C to C(18) acids was increased in liver of chicks fed MCT, implying that fatty acid chain elongating activity was also increased. Studies on the conversion of stearate-2-(14)C to mono- and di-unsaturated C(18) acids showed that hepatic fatty acid desaturation activity was enhanced by MCT feeding. Data are presented on the plasma and liver fatty acid composition of chicks fed MCT-, corn oil-, or coconut oil-supplemented diets.

Influence of medium-chain triglycerides on lipid metabolism in the rat.

Lipid metabolism was studied in rats fed diets containing corn oil, coconut oil, or medium-chain triglyceride (MCT), a glyceride mixture containing fatty acids of 8 and 10 carbons in length. The ingestion of MCT-supplemented, cholesterolfree diets depressed plasma and liver total lipids and cholesterol as compared with corn oil-supplemented diets. In rats fed cholesterol-containing diets, plasma cholesterol levels were not influenced by dietary MCT, but liver cholesterol levels were significantly lower than in animals fed corn oil. In vitro cholesterol synthesis from acetate-1-(14)C was lower in liver slices of rats that consumed MCT than in similar preparations from corn oil-fed rats. Studies of fatty acid carboxyl labeling from acetate-1-(14)C and the conversion of palmitate-1-(14)C to C(18) acids by liver slices showed that chain-lengthening activity is greater in the liver tissue of rats fed MCT than in the liver of animals fed corn oil. The hepatic fatty acid desaturation mechanisms, evaluated by measuring the conversion of stearate-2-(14)C to oleate, was also enhanced by feeding MCT.Adipose tissue of rats fed MCT converts acetate-1-(14)C to fatty acids at a much faster rate than does tissue from animals fed corn oil. Evidence is presented to show that the enhanced incorporation of acetate into fatty acids by the adipose tissue of rats fed MCT represents de novo synthesis of fatty acids and not chain-lengthening activity. Data are also presented on the fatty acid composition of plasma, liver, and adipose tissue lipids of rats fed the different fats under study.