Self-Deliverable Peptide-Mediated and Reactive-Oxygen-Species-Amplified Therapeutic Nanoplatform for Highly Effective Bacterial Inhibition.

An "antibiotic-free strategy" provides a viable option to address bacterial infections, especially for the "superbug" challenge. However, the undesirable antibacterial activity of antibiotic-free agents hinders their practical applications. In this study, we developed a combination antibacterial strategy of coupling peptide-drug therapy with chemodynamic therapy (CDT) to achieve the effective bacterial inhibition. An amphiphilic oligopeptide (LAOOH-OPA) containing a therapeutic unit of D(KLAK)2 peptide and a hydrophobic linoleic acid hydroperoxide (LAHP) was designed. The positively charged D(KLAK)2 peptide with an α-helical conformation enabled rapid binding with microbial cells via electrostatic interaction and subsequent membrane insertion to deactivate the bacterial membrane. When triggered by Fe2+, moreover, LAHP could generate singlet oxygen (1O2) to elicit lipid bilayer leakage for enhanced bacteria inhibition. In vitro assays demonstrated that the combination strategy possessed excellent antimicrobial activity not only merely toward susceptible strains (Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli) but also toward methicillin-resistant Staphylococcus aureus (MRSA). On the mouse skin abscess model induced by S. aureus, self-assembled LAOOH-OPA exhibited a more significant bacteria reduction (1.4 log10 reduction) in the bioburden compared to that of the standard vancomycin (0.9 log10 reduction) without apparent systemic side effects. This combination antibacterial strategy shows great potential for effective bacterial inhibition.

Bioactivation of leukotoxins to their toxic diols by epoxide hydrolase.

Leukotoxin is a linoleic acic oxide produced by leukocytes and has been associated with the multiple organ failure and adult respiratory distress syndrome seen in some severe burn patients. Leukotoxin has been reported to be toxic when injected into animals intravenously. Herein, we report that this lipid is not directly cytotoxic in at least two in vitro systems. Using a baculovirus expression system we demonstrate that leukotoxin is only cytotoxic in the presence of epoxide hydrolases. In addition, it is the diol metabolite that proves toxic to pulmonary alveolar epithelial cells, suggesting a critical role for the diol in leukotoxin-associated respiratory disease. In vivo data also support the toxicity of leukotoxin diol. For the first time we demonstrate that soluble epoxide hydrolase can bioactivate epoxides to diols that are apparently cytotoxic. Thus leukotoxin should be regarded as a protoxin corresponding to the more toxic diol. This clearly has implications for designing new clinical interventions.

Dietary Fats High in Linoleic Acids Impair Antitumor T-cell Responses by Inducing E-FABP-Mediated Mitochondrial Dysfunction.

The most recent American Dietary Guidelines (2020-2025) recommend shifting dietary fats from solid saturated fats to unsaturated oils. Dietary oils contain different compositions of unsaturated fatty acids (UFA). Oleic acid (OA) and linoleic acid (LA) are the most common UFA in dietary oils. How individual UFA in oils regulate immune cell function and cancer risk remains unclear. Here we demonstrated that high-fat diets (HFD) rich either in OA or LA induced a similar degree of murine obesity, but the LA-rich HFD specifically promoted mammary tumor growth. LA impaired antitumor T-cell responses by promoting naïve T-cell apoptosis and inhibiting TNFα production. While exogenous OA and LA were taken up by T cells with similar efficacy, only LA induced significant mitochondrial reactive oxygen species production and lipid peroxidation. Importantly, naïve T cells predominantly expressed epidermal fatty acid binding protein (E-FABP), which is central in facilitating LA mitochondrial transport and cardiolipin incorporation. Genetic depletion of E-FABP rescued LA-impaired T-cell responses and suppressed LA-rich HFD-associated mammary tumor growth. Collectively, these data suggest that dietary oils high in LA promote mammary tumors by inducing E-FABP-mediated T-cell dysfunction. SIGNIFICANCE: These findings suggest that modulation of dietary oil composition and inhibition of E-FABP activity may represent novel strategies to enhance T-cell function in the prevention and treatment of obesity-associated cancers.

Amended safety assessment of tall oil acid, sodium tallate, potassium tallate, and ammonium tallate.

Tall oil acid is a mixture of oleic and linoleic acids (fatty acids) and rosin acids derived from tall oil, a by-product of pulp from resinous woods, used in cosmetic products as a surfactant at concentrations up to 8%. Ammonium, potassium, and sodium salts also are listed as cosmetic ingredients. In addition to the studies summarized in this report, extensive toxicity, genotoxicity, and carcinogenicity studies in animals are available for oleic, lauric, palmitic, myristic, and stearic fatty acids as published earlier by the Cosmetic Ingredient Review (CIR). These data may be extrapolated to tall oil acid and its salts. There are no reports of current uses or use concentration data for ammonium tallate, nor are use concentration data available for the other salts. The CIR Expert Panel found tall oil acid, ammonium tallate, potassium tallate, and sodium tallate to be safe cosmetic ingredients in the given practices of use and concentration.

Effect of a conjugated quercetin metabolite, quercetin 3-glucuronide, on lipid hydroperoxide-dependent formation of reactive oxygen species in differentiated PC-12 cells.

To assess the efficacy of conjugated quercetin metabolites as attenuators for oxidative stress in the central nervous system, we measured the 13-hydroperoxyoctadecadienoic acid (13-HPODE)-dependent formation of reactive oxygen species (ROS) in pheochromocytoma PC-12 cells in the presence of quercetin 3-O-beta-glucuronide (Q3GA) and related compounds. A 2',7'-dichlorofluorescin (DCFH) assay showed that Q3GA significantly suppressed the formation of ROS, when it was coincubated with 13-HPODE (coincubation system). However, it was less effective than quercetin aglycon in the concentration range from 0.5 to 10 microM. In an experiment in which the cells were incubated with the test compounds for 24 h before being exposed to 13-HPODE, Q3GA was also effective in suppressing the formation of ROS in spite that little Q3GA was taken up into the cells. These results suggest that antioxidative metabolites of quercetin are capable of protecting nerve cells from attack of lipid hydroperoxides.

Activation of AP-1 and increased synthesis of MMP-9 in the rabbit retina induced by lipid hydroperoxide.

PURPOSE: We identified the temporal expression of activator protein-1 (AP-1) and matrix metalloproteinases (MMPs) after linoleic acid hydroperoxide (LHP) induction of retinal neovascularization. METHODS: After injection of LHP into the vitreous of rabbits, samples were collected for AP-1 binding activity and mRNA for MMP-9 and MMPs activity. AP-1 binding activity was measured by electrophoretic mobility shift assay. MMP-9 activity was measured by zymography and mRNA by quantitative RT-PCR. RESULTS: AP-1 binding activity was increased at 1-3 hr. MMP-9 mRNA levels were increased at 3 hr in the neural retina and by 12 hr in the retinal pigment epithelium (RPE) layer. MMP-9 proteolytic activity was elevated within the neural retina and within the vitreous and in the RPE-interphotoreceptor matrix (IPM) at 12 hr and peaked at 24 hr or 4 days. CONCLUSIONS: LHP increases the transcription factor AP-1 which in turn may regulate retinal MMP-9 synthesis during neovascularization.

Effect of dietary fat on human breast cancer growth and lung metastasis in nude mice.

Results from epidemiological studies have generally indicated an association of dietary saturated animal fats with human breast cancer risk. Some studies, however, have suggested a similar association for some polyunsaturated vegetable fats shown to promote both rodent mammary carcinogenesis and metastasis. This study was performed to evaluate the effects of corn oil on growth and metastasis of MDA-MB-435 human breast cancer cells, which have a propensity for metastasis. Corn oil is rich in the omega-6 fatty acid linoleic acid. Fifty-eight female athymic nude mice (NCr-nu/nu) were fed a high-fat diet (23% wt/wt corn oil; 12% linoleic acid) or a low-fat diet (5% wt/wt corn oil; 2.7% linoleic acid). Seven days after diets were started, tumor cells (1 x 10(6) were injected into a mammary fat pad. The time to appearance of solid tumors and the tumor size were recorded. After 15 weeks, the study was terminated, and autopsies were performed to determine the weight of the primary tumor and the extent of metastasis. The latent interval for tumor appearance in the animals fed the high-fat diet was shorter than that in the low-fat diet group, and the tumor growth rate in the high-fat diet group showed a small but statistically significant increase compared with the low-fat diet group. Primary tumors developed in 27 of the 29 mice on the high-fat diet and in 21 of the 29 on the low-fat diet. Of the mice with palpable primary tumors, 18 of 27 in the high-fat diet group and eight of 21 in the low-fat diet group had macroscopic lung metastases. The extent of metastasis in the high-fat diet group was independent of the primary tumor weight, but only those in the low-fat diet group with primary tumors weighing more than 2 g developed metastases. These results suggest that a high-fat diet rich in omega-6 polyunsaturated fatty acid can enhance metastasis of human breast cancer cells in this mouse model. The findings support the need for further study of the relationship between dietary polyunsaturated fats and breast cancer risk and for experiments to determine the effect on metastasis of only a 50% difference in fat intake--the dietary goal of the proposed clinical trials of low-fat dietary intervention in breast cancer patients.

Effect of some fatty acid methyl esters on gastrointestinal carcinogenesis by N-methyl-N-nitro-N-nitrosoguanidine in rats.

Methyl linoleate hydroperoxide (MLHP) and native methyl linoleate (ML) were tested for carcinogenicity toward the gastrointestinal (GI) tract in male specific-pathogen-free outbred Wistar rats. N-Methyl-N-nitro-N-nitrosoguanidine (MNNG) was given in the drinking water in a dose of 20 mg/liter when cocarcinogenic properties of the test substances were to be tested. MLHP and ML were fed by stomach tube and had no effect as complete carcinogens. Given concomitantly with MNNG, ML did not enhance carcinogenesis. MLHP in conjunction with MNNG was the only treatment which, as treatment with MNNG in a dose of 83 mg/liter, led to an increase of GI cancers in animals that died before day 354. Cumulative results after a maximum of 612 days showed a distribution of GI cancers in favor of the glandular stomach only after MLHP was given with MNNG.

Spontaneous tumor incidence in female brown Norway rats after lifelong diets high and low in linoleic acid.

High linoleic acid (C18:2) (group I; 17.7 cal%) and low C18:2 (group II; 3.3 cal%) diets were given to groups of inbred Brown Norway virgin female rats (100 animals/group), during their whole life-span. A total of 140 tumors were found in group I and 123 tumors in group II; the median survival times of the 2 groups were 124.2 and 118.5 weeks, respectively. Total spontaneous tumor incidence and median survival times were not significantly different. However, significant differences were found in the incidences of some specific tumors: The numbers of reticuloendothelial tumors and adrenocortical carcinomas were significantly higher in the group of animals receiving the low-C18:2 diet. A high incidence of tumor multiplicity, however, resulted in a significantly greater number of mammary tumors in the high-C18:2 diet group.

Requirement of essential fatty acid for mammary tumorigenesis in the rat.

In an attempt to determine the requirement of essential fatty acid for dimethylbenz(a)anthracene-induced mammary tumorigenesis, rats were fed diets containing different levels of linoleate: 0.5, 1.1, 1.7, 2.2, 3.5, 4.4, 8.5, or 11.5%. Each diet contained 20% of fat by weight, with varying amounts of coconut oil and corn oil added to achieve the desired levels of linoleate. Mammary tumorigenesis was very sensitive to linoleate intake and increased proportionately in the range of 0.5 to 4.4% of dietary linoleate. Regression analysis indicated that a breakpoint occurred at 4.4%, beyond which there was a very poor linear relationship, suggesting the possibility of a plateau. From the intersection of the regression lines in both the upper and lower ranges, the level of linoleate required to elicit the maximal tumorigenic response was estimated to be around 4%. The differences in tumor yield could not be correlated with changes in prostaglandin E concentration in the mammary fat pads of normal animals maintained on similar diets, suggesting that linoleate may act by some other mechanism to stimulate mammary tumorigenesis.

Effects of dietary linoleic acid on pancreatic carcinogenesis in rats and hamsters.

It has been suggested that linoleic acid (LA) is responsible for the promoting effect of dietary polyunsaturated fat on pancreatic carcinogenesis via an accelerated prostaglandin synthesis, caused by metabolism of LA-derived arachidonic acid in (pre)neoplastic tissue. The purpose of the present study was to investigate whether dietary LA is the cause of pancreatic tumor promotion by a high fat diet. Five groups of 30 azaserine-treated rats and 5 groups of 30 N-nitrosobis(2-oxopropyl)amine-treated hamsters were maintained for 6 months (rats) and 12 months (hamsters) on high fat (25 weight %) AIN diets containing 2, 4, 6, 10, or 15 weight % LA. The results indicated that the strongest enhancing effect on the growth of pancreatic (pre)neoplastic lesions in rats and hamsters was obtained with 4 and 2 weight % of dietary LA, respectively. At higher LA levels the tumor response seemed to decrease rather than increase. In both rats and hamsters the fatty acid profiles of blood plasma and pancreas showed an accurate reflection of the dietary fatty acid profiles: a proportional increase in LA levels was observed in plasma and pancreas with increasing dietary LA. In both species plasma and pancreatic AA levels remained constant, except for arachidonic acid levels in rat plasma, which significantly increased with increasing dietary LA levels. Fatty acid profiles in hamster pancreatic tumors did not differ from fatty acid profiles in nontumorous pancreatic tissue from hamsters fed the same diet. Prostaglandin (PG) E2, 6-keto-PGF1 alpha, PGF2 alpha, and thromboxane B2-concentrations in nontumorous pancreatic tissue were similar among the diet groups. Ductular adenocarcinomas from hamster pancreas showed significantly higher levels of 6-keto-PGF1 alpha, PGF2 alpha, and thromboxane B2, but not of PGE2 in comparison with nontumorous pancreas. It is concluded that the strongest pancreatic tumor promotion by dietary LA is 4 weight % in rats and 2 weight % or less in hamsters, and that PGs may be involved in the development of ductular adenocarcinomas induced in hamster pancreas by N-nitrosobis(2-oxopropyl)amine.

Effects of conjugated linoleic acid on prostaglandins produced by cells isolated from maternal intercotyledonary endometrium, fetal allantochorion and amnion in late pregnant ewes.

The anticarcinogenic properties of conjugated linoleic acid (CLA) are, at least partially, attributed to its ability to interrupt the n-6 polyunsaturated fatty acid (PUFA) metabolic pathway for the biosynthesis of eicosanoids, including prostaglandins (PG). Both PGE(2) and PGF(2alpha) play key roles in parturition. In the present study, we compared the effects of CLA (a mixture of cis- and trans-9, 11- and -10, 12-octadecadienoic acid) and linoleic acid (LA) on PG production by cells isolated from maternal intercotyledonary endometrium, fetal allantochorion and amnion from late pregnant ewes. The results demonstrated that supplementation of LA and CLA significantly affected both the proportions and the amounts of PGs produced by all three tissue types. The ability of the uterus and placenta to respond to oxytocin (OT, endometrium only) and lipopolysaccharide (LPS) was also affected. LA inhibited PGE(2) and PGF(2alpha) production in the absence or presence of either oxytocin or LPS. In endometrial cells with or without oxytocin or LPS, CLA dose-dependently suppressed PGF(2alpha) generation, whereas low doses of CLA (20 microM) increased PGE(2) generation. Supplementation with CLA therefore increased the PGE(2)/PGF(2alpha) ratio in the endometrial cells. These results suggest that dietary supplementation of LA or CLA may affect both the initiation and progression of parturition.

Conjugated linoleic acid supplementation reduces adipose tissue by apoptosis and develops lipodystrophy in mice.

Conjugated linoleic acid (CLA) is a naturally occurring group of dienoic derivatives of linoleic acid found in beef and dairy products. CLA has been reported to reduce body fat. To examine the mechanism(s) of CLA reduction of fat mass, female C57BL/6J mice were fed standard semipurified diets (10% fat of total energy) with or without CLA (1% wt/wt). Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick endlabeling (TUNEL) and DNA fragmentation analysis revealed that fat-mass decrease by CLA was mainly due to apoptosis. Tumor necrosis factor (TNF)-alpha and uncoupling protein (UCP)-2 mRNA levels increased 12- and 6-fold, respectively, in isolated adipocytes from CLA-fed mice compared with control mice. Because it is known that TNF-alpha induces apoptosis of adipocytes and upregulates UCP2 mRNA, a marked increase of TNF-alpha mRNA with an increase of UCP2 in adipocytes caused CLA-induced apoptosis. However, with a decrease of fat mass, CLA supplementation resulted in a state resembling lipoatrophic diabetes: ablation of brown adipose tissue, a marked reduction of white adipose tissue, marked hepatomegaly, and marked insulin resistance. CLA supplementation decreased blood leptin levels, but continuous leptin infusion reversed hyperinsulinemia, indicating that leptin depletion contributes to the development of insulin resistance. These results demonstrate that intake of CLA reduces adipose tissue by apoptosis and results in lipodystrophy, but hyperinsulinemia by CLA can be normalized by leptin administration.

Synergistic vascular toxicity and fatty acid anilides in the toxic oil syndrome.

The underlying etiology of the toxic oil syndrome may be related to any of several toxic contaminants. The hypothesis is made that two or more toxic compounds may act synergistically to cause vascular damage in the toxic oil syndrome. To support this hypothesis, previous studies are reviewed concerning the remarkable synergistic toxic action of allylamine and beta-aminopropionitrile on the media of blood vessels. Although these toxins are not directly related to the toxic oil syndrome, this previous experimental work emphasizes the possibility that unexplored synergistic actions may be important. Furthermore, the hypothesis that contaminating fatty acid anilides in toxic oil undergo alterations during cooking is supported by high pressure liquid chromatographic analysis. The theoretic metabolism of fatty acid anilides is discussed. Recent data concerning the toxic actions of the anilides of oleic and linoleic acid are given. These data suggest that these anilides induce immunologic alterations that may be similar to those seen in the toxic oil syndrome. In addition, the heated anilides appear to have increased toxicity, supporting the concept that the use of toxic oil in cooking may increase its toxicity.

Suppression of tumour-promoting factors in fat-induced colon carcinogenesis by the antioxidants caroverine and ubiquinone.

Fatty acid hydroperoxides are produced from unsaturated fatty acids in the presence of oxygen at elevated temperatures during food processing. Their effects on gene expression in colorectal tumour cells were studied using linoleic acid hydroperoxide (LOOH) as a model compound. Addition of LOOH to the medium of LT97 adenoma and SW480 carcinoma cells enhanced the production of hydrogen peroxide. Both cell lines were observed to increase VEGF factors based on mRNA. High consumption of dietary fat promotes colon carcinogenesis in the long-term. While this effect is well known, the underlying mechanisms are not understood. An approach was made starting from the assumption that LOOH is present in dietary fats as a result of heating. LOOH undergoes homolytic cleavage in the presence of iron. Various radicals are formed on mixing LT97 or SW480 cells with LOOH. The expression of tumour-promoting factors was inhibited by caroverine and ubiquinone, which may be justified as active chemopreventive agents.

Protection of linoleic acid hydroperoxide-induced cytotoxicity by phenolic antioxidants.

The protective effects of phenolic antioxidants on linoleic acid hydroperoxide (LOOH)-induced toxicity to cultured human umbilical vein endothelial cells were examined. Our previous results were confirmed that for tocopherol homologs, lipophilicity and the presence of a phenolic hydroxyl group and two alkyl groups at its ortho positions are critical for protection against LOOH-induced cytotoxicity. Probucol and butylated hydroxytoluene (BHT) were more effective than other simple alkylated phenols. It was found that the protective effects of alkylated phenols were depended on by the presence of two alkyl groups; in particular, two tert-butyl groups, at positions ortho to a hydroxyl group and an alkyl group at the para position. Among alpha-tocopherol, 2,2,5,7,8-pentamethylchroman-6-ol, and BHT, the relative effectiveness of protection against the cytotoxicity (1.0:0.86:0.58, respectively) was inconsistent with the previously reported, relative antioxidant activity in homogeneous solution (1.0:1.2:0.004, respectively). Probably, the effectiveness of protection by phenolic antioxidants against the cytotoxicity depend primarily on their incorporation rate into cells due to their lipophilicity, secondly on their antioxidant activity, and thirdly on their orientation in biomembranes.

Linoleic acid hydroperoxide-induced peroxidation of endothelial cell phospholipids and cytotoxicity.

Peroxidation of endothelial cell phospholipids was examined following treatments with linoleic acid hydroperoxide. The treatment effects were analyzed over a range of toxicities and exposure intervals as determined by cell plating efficiencies and survival. Over the concentration ranges where lipid peroxidation was evident (20-40 microM treatments in complete medium), significant cytotoxicity was apparent after 1 h of exposure. The extent of toxicity was dependent on the time interval between the end of peroxide treatment and replating of cells. Maximum toxicity was found when cells were replated 1-3 h after treatment. When cells were replated 4 h after treatment a linear increase in cell survival was found as a function of replating time following peroxide exposure. Analysis of cell phospholipids by HPLC after 1 h of exposure to linoleic acid hydroperoxide revealed that peroxidation (evidenced by conjugated diene content) had taken place among a number of phospholipid species with the most marked increases in phosphatidylcholine. Analysis of the fatty acyl composition of phospholipids also showed that the proportions of polyunsaturated fatty acids were reduced relative to saturated fatty acids, indicating peroxidative damage to phospholipids. Pretreatment of cells with vitamin E prevented the peroxidation of all phospholipids and blocked the cytotoxic action of linoleic acid hydroperoxide. These findings indicate that an immediate cytotoxic action of lipid hydroperoxide is associated with peroxidation of membrane phospholipids. This cytotoxicity is a transient effect, and cells surviving the acute injury display a time-dependent increase in plating efficiency representing a period of repair.

Newly recognized cytotoxic effect of conjugated trienoic fatty acids on cultured human tumor cells.

We investigated the cytotoxic effect of conjugated trienoic fatty acids on various human tumor cell lines: DLD- 1, colorectal; HepG2, hepatoma; A549, lung; MCF-7, breast; and MKN-7, stomach. Conjugated linoleic acid (CLA) and conjugated linolenic acid were prepared from linoleic acid (18:2, n-6) and alpha-linolenic acid (18:3, n-3), respectively, by treatment with 6.6% or 21% potassium hydroxide. Spectrophotometric readings at 235 nm for the conjugated diene formation, and at 268 nm for the conjugated triene, were confirmed for the respective conjugated fatty acids. In addition, tung oil (Aleurites fordii) fatty acids consisting principally of a conjugated triene (eleostearic acid, approximately 80% of total fatty acids) were prepared using an alkaline saponification procedure. All tumor cells were incubated for 24 h with 5-100 microM of the conjugated fatty acids, and MTT dye reduction was measured to verify the cell viability. Among the conjugated fatty acids examined, conjugated linolenic acid and tung oil fatty acids exhibited the most intense cytotoxic effects on DLD-1, HepG2, A549, MCF-7 and MKN-7 cells, while CLA was not cytotoxic to the tumor cells. These results demonstrate that conjugated trienoic fatty acids are more cytotoxic to human tumor cells than the conjugated dienoic fatty acid, CLA.

Effects of fatty acids on invasion through reconstituted basement membrane ('Matrigel') by a human breast cancer cell line.

An in vitro invasion assay system was used to examine the effects of linoleic acid, an omega-6 fatty acid, and two omega-3 fatty acids, eicosapentaenoic acid and docosahexaenoic acid, on the invasive capacity of MDA-MB-435 human breast cancer cells. Linoleic acid stimulated, and the omega-3 fatty acids inhibited, tumor cell invasion at concentrations of 0.25 and 0.5 microgram/ml. Indomethacin, 20 micrograms/ml, completely suppressed the stimulatory activity of linoleic acid, suggesting that these fatty acid effects are mediated via eicosanoid biosynthesis.

Lipids alter the level and distribution of mouse mammary tumor virus gp52 in vitro.

Linoleic acid, cholesterol, dexamethasone and progesterone were tested by immunocytochemistry and immunoprecipitation for their single and combined effects in vitro on mouse mammary tumor virus (MMTV) gp52 distribution among three compartments: cell-associated antigen, extracellular virus particles and extracellular shed antigen unassociated with virus particles. Results indicated that all additives significantly increased total MMTV gp52 levels and altered the distribution. Linoleic acid and dexamethasone induced the greatest relative proportion of extracellular gp52, whereas cholesterol and progesterone induced the greatest proportion of cell-associated gp52. The implications of these findings for the immune response to mammary tumors is discussed.