Resolvin E1 Reduces Tumor Growth in a Xenograft Model of Lung Cancer.

Inflammation plays a significant role in carcinogenesis and tumor growth. The current study was designed to test the hypothesis that resolvin E1 (RvE1) and overexpression of the receptor for RvE1 (ERV1) will prevent and/or reverse tumor generation in a gain-of-function mouse model of tumor seeding with lung cancer cells. To measure the impact of enhanced resolution of inflammation on cancer pathogenesis, ERV1-overexpressing transgenic (TG) and wild-type FVB mice were given an injection of 1 × 106 LA-P0297 cells subcutaneously and were treated with RvE1 (100 ng; intraperitoneally) or placebo. To assess the impact of RvE1 as an adjunct to chemotherapy, ERV1-TG and wild-type FVB mice were treated with cisplatin or cisplatin + RvE1. RvE1 significantly prevented tumor growth and reduced tumor size, cyclooxygenase-2, NF-κB, and proinflammatory cytokines in TG animals as compared to wild-type animals. A significant decrease in Ki-67, vascular endothelial growth factor, angiopoietin (Ang)-1, and Ang-2 was also observed in TG animals as compared to wild-type animals. Tumor-associated neutrophils and macrophages were significantly reduced by RvE1 in transgenics (P < 0.001). RvE1 administration with cisplatin led to a significant reduction of tumor volume and reduced cyclooxygenase-2, NF-κB, vascular endothelial growth factor-A, Ang-1, and Ang-2. These data suggest that RvE1 prevents inflammation and vascularization, reduces tumor seeding and tumor size, and, when used as an adjunct to chemotherapy, enhances tumor reduction at significantly lower doses of cisplatin.

Talin-Dependent Integrin Activation Regulates VE-Cadherin Localization and Endothelial Cell Barrier Function.

RATIONALE: Endothelial barrier function depends on the proper localization and function of the adherens junction protein VE (vascular endothelial)-cadherin. Previous studies have suggested a functional relationship between integrin-mediated adhesion complexes and VE-cadherin yet the underlying molecular links are unclear. Binding of the cytoskeletal adaptor protein talin to the ß-integrin cytoplasmic domain is a key final step in regulating the affinity of integrins for extracellular ligands (activation) but the role of integrin activation in VE-cadherin mediated endothelial barrier function is unknown. OBJECTIVE: To test the requirement of talin-dependent activation of ß1 integrin in VE-cadherin organization and endothelial cell (EC) barrier function. METHODS AND RESULTS: EC-specific deletion of talin in adult mice resulted in impaired stability of intestinal microvascular blood vessels, hemorrhage, and death. Talin-deficient endothelium showed altered VE-cadherin organization at EC junctions in vivo. shRNA (short hairpin RNA)-mediated knockdown of talin1 expression in cultured ECs led to increased radial actin stress fibers, increased adherens junction width and increased endothelial monolayer permeability measured by electrical cell-substrate impedance sensing. Restoring ß1-integrin activation in talin-deficient cells with a ß1-integrin activating antibody normalized both VE-cadherin organization and EC barrier function. In addition, VE-cadherin organization was normalized by reexpression of talin or integrin activating talin head domain but not a talin head domain mutant that is selectively deficient in activating integrins. CONCLUSIONS: Talin-dependent activation of EC ß1-integrin stabilizes VE-cadherin at endothelial junctions and promotes endothelial barrier function.

Strain-Specific Impact of Fusobacterium nucleatum on Neutrophil Function.

BACKGROUND: Neutrophil function is critical for initiation and progression of infecto-inflammatory diseases. Key quorum-sensing plaque bacteria, such as Fusobacterium nucleatum, act as bridging species between early and late colonizer pathogens, such as Porphyromonas gingivalis, as the biofilm ages and periodontal inflammation increases. This study is designed to determine impact of different F. nucleatum strains on neutrophil function. METHODS: Cells of human promyelocytic leukemia cell line-60 were differentiated into neutrophil-like cells and cultured with F. nucleatum strains of subspecies (ssp.) nucleatum ATCC 25586, ssp. polymorphum ATCC 10953, and ssp. vincentii ATCC 49256. Neutrophil phagocytosis of F. nucleatum strains and neutrophil apoptosis were analyzed by flow cytometry. Superoxide generation was measured by cytochrome C reduction in the presence and absence of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) (1 µM) stimulation. Proinflammatory cytokine release was determined after 2, 6, and 24 hours of culture in the presence/absence of different F. nucleatum strains. Expression of Toll-like receptor (TLR)2, TLR4, and nuclear factor (NF)-kappa B mRNA levels were analyzed using real-time quantitative polymerase chain reaction. Each experiment was repeated at least three times in triplicate. Data were analyzed using analysis of variance followed by post hoc Bonferroni correction. RESULTS: All strains of F. nucleatum significantly increased phagocytic capacity of neutrophils. Neutrophil phagocytosis of F. nucleatum ssp. polymorphum was significantly greater than that of F. nucleatum ssp. vincentii and ssp. nucleatum (P <0.001). F. nucleatum ssp. nucleatum and ssp. polymorphum significantly blocked fMLP-induced superoxide generation (P <0.001). Although F. nucleatum vincentii also reduced superoxide generation (25%), the impact was not as strong as that of ssp. nucleatum (83%) and ssp. polymorphum (100%). All F. nucleatum strains stimulated significant increase in neutrophil apoptosis compared with control (P <0.001) and significantly increased expression of NF-κB mRNA in neutrophils (P <0.05). Levels of interleukin-8 and tumor necrosis factor-α produced by neutrophils were significantly increased in all F. nucleatum groups compared with control (P <0.001). CONCLUSIONS: These findings suggest that different strains of F. nucleatum impact neutrophil function in different ways. Two of three subspecies blocked neutrophil superoxide generation in response to a secondary stimulus, preventing oxidative killing by neutrophils. The direct role of bridging species in pathogenesis of periodontitis may be greater than previously suspected in which they create a favorable environment for pathogenic transition of the dental ecosystem.

Impact of resolvin E1 on murine neutrophil phagocytosis in type 2 diabetes.

Diabetic complications involve inflammation-mediated microvascular and macrovascular damage, disruption of lipid metabolism, glycosylation of proteins, and abnormalities of neutrophil-mediated events. Resolution of inflamed tissues to health and homeostasis is an active process mediated by endogenous lipid agonists, including lipoxins and resolvins. This proresolution system appears to be compromised in type 2 diabetes (T2D). The goal of this study was to investigate unresolved inflammation in T2D. Wild-type (WT) and genetically engineered mice, including T2D mice (db/db), transgenic mice overexpressing the human resolvin E1 (RvE1) receptor (ERV1), and a newly bred strain of db/ERV1 mice, were used to determine the impact of RvE1 on the phagocytosis of Porphyromonas gingivalis in T2D. Neutrophils were isolated and incubated with fluorescein isothiocyanate-labeled P. gingivalis, and phagocytosis was measured in a fluorochrome-based assay by flow cytometry. Mitogen-activated protein kinase (MAPK) (p42 and p44) and Akt (Thr308 and Ser473) phosphorylation was analyzed by Western blotting. The mouse dorsal air pouch model was used to evaluate the in vivo impact of RvE1. Results revealed that RvE1 increased the neutrophil phagocytosis of P. gingivalis in WT animals but had no impact in db/db animals. In ERV1-transgenic and ERV1-transgenic diabetic mice, phagocytosis was significantly increased. RvE1 decreased Akt and MAPK phosphorylation in the transgenic animals. In vivo dorsal air pouch studies revealed that RvE1 decreases neutrophil influx into the pouch and increases neutrophil phagocytosis of P. gingivalis in the transgenic animals; cutaneous fat deposition was reduced, as was macrophage infiltration. The results suggest that RvE1 rescues impaired neutrophil phagocytosis in obese T2D mice overexpressing ERV1.

Alterations in lipid mediated signaling and Wnt/ ß -catenin signaling in DMH induced colon cancer on supplementation of fish oil.

Ceramide mediates inhibition of cyclooxygenase-2 (COX-2) which catalyzes formation of prostaglandin further activating peroxisome proliferator-activated receptor γ (PPAR γ ) and Wnt/ ß -catenin pathway; and hence plays a critical role in cancer. Therefore, in current study, ceramide, COX-2, 15-deoxy prostaglandin J2(15-deoxy PGJ2), PPAR γ , and ß -catenin were estimated to evaluate the effect of fish oil on lipid mediated and Wnt/ ß -catenin signaling in colon carcinoma. Male Wistar rats in Group I received purified diet while Groups II and III received modified diet supplemented with FO : CO(1 : 1) and FO : CO(2.5 : 1), respectively. These were further subdivided into controls receiving ethylenediaminetetraacetic acid and treated groups receiving dimethylhydrazine dihydrochloride (DMH)/week for 4 weeks. Animals sacrificed 48 hours after last injection constituted initiation phase and those sacrificed after 16 weeks constituted postinitiation phase. Decreased ceramide and increased PPAR γ were observed in postinitiation phase only. On receiving FO+CO(1 : 1)+DMH and FO+CO(2.5 : 1)+DMH in both phases, ceramide was augmented whereas COX-2, 15-deoxy PGJ2, and nuclear translocation of ß -catenin were reduced with respect to cancerous animals. Decrease was more significant in postinitiation phase with FO+CO(2.5 : 1)+DMH. Treatment with oils increased PPAR γ in initiation phase but decreased it in postinitiation phase. Hence, fish oil altered lipid mediated signalling in a dose and time dependent manner so as to inhibit progression of colon cancer.

Fish oil suppresses cell growth and metastatic potential by regulating PTEN and NF-κB signaling in colorectal cancer.

Homeostasis in eukaryotic tissues is tightly regulated by an intricate balance of the prosurvival and antisurvival signals. The tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10), a dual-specificity phosphatase, plays a functional role in cell cycle arrest and apoptosis. NF-κB and its downstream regulators (such as VEGF) play a central role in prevention of apoptosis, promotion of inflammation and tumor growth. Therefore, we thought to estimate the expression of PTEN, Poly-ADP-ribose polymerase (PARP), NF-κBp50, NF-κBp65 and VEGF to evaluate the effect of supplementation of fish oil on apoptotic and inflammatory signaling in colon carcinoma. Male wistar rats in Group I received purified diet while Group II and III received modified diet supplemented with FO∶CO(1∶1)&FO∶CO(2.5∶1) respectively. These were further subdivided into controls receiving ethylenediamine-tetra acetic-acid and treated groups received dimethylhydrazine-dihydrochloride (DMH)/week for 4 weeks. Animals sacrificed 48 hours after last injection constituted initiation phase and that sacrificed after 16 weeks constituted post-initiation phase. We have analysed expression of PTEN, NF-κBp50, NF-κBp65 by flowcytometer and nuclear localization of NF-κB by immunofluorescence. PARP and VEGF were assessed by immunohistochemistry. In the initiation phase, animals receiving DMH have shown increased % of apoptotic cells, PTEN, PARP, NF-κBp50, NF-κBp65 and VEGF however in post-initiation phase no significant alteration in apoptosis with decreased PTEN and increased PARP, NF-κBp50, NF-κBp65 and VEGF were observed as compared to control animals. On treatment with both ratios of fish oil in both the phases, augmentation in % of apoptotic cells, decreased PTEN, PARP, NF-κBp50, NF-κBp65 and VEGF were documented with respect to DMH treated animals with effect being more exerted with higher ration in post-initiation phase. Hence, fish oil activates apoptosis, diminishes DNA damage and inhibits inflammatory signalling in a dose and time dependent manner so as to inhibit progression of colon cancer.

Alteration in apoptosis and cell cycle by celecoxib and/or fish oil in 7,12-dimethyl benzene (α) anthracene-induced mammary carcinogenesis.

Cyclooxygenase (COX)-2 inhibition by nonsteroidal anti-inflammatory drugs is a useful approach for cancer prevention but has several side effects. A novel approach combining these chemopreventive agents at low doses with dietary elements has been suggested to augment their effects and reduce side effects. Dietary fats, particularly, n-3 polyunsaturated fatty acids (PUFA) also exert cancer chemopreventive effect mediated through COX-2 inhibition. Therefore, the present study was designed to investigate the effect of combined dosage of celecoxib and n-3 PUFA-rich fish oil in experimental mammary carcinogenesis. Female Wistar rats were distributed into control and DMBA-treated groups. The groups were further subdivided based on pretreatment with celecoxib and/or fish oil. The animals were maintained for 90 days before sacrifice. To analyze the role of redox signaling, the two mediators, reactive oxygen species and calcium, and their effects on c-myc expression were evaluated. The chemopreventive effect was assessed by measurement of cell proliferation, apoptosis, and p53 in isolated mammary epithelial cells. Increased redox signaling with enhanced c-myc, p53 expression, and augmented apoptotic and proliferative rate were observed in carcinogen-treated animals. Pretreatment of carcinogen-treated animals with celecoxib and/or fish oil altered redox signaling with reduced c-myc, p53 expression, apoptosis, and proliferation. However, a combination dosage of celecoxib and fish oil had a better chemopreventive effect. The results suggest that a combination of celecoxib and fish oil is more effective in the chemoprevention of experimental mammary carcinogenesis, and this effect can be attributed to the modification of redox signaling.

Alterations in mitochondrial membrane in chemopreventive action of fish oil.

Mitochondria are major regulators of pathways related to tumorigenesis; therefore, mitochondrial membrane characteristics and associated cell signaling events were evaluated with different ratios of fish oil (FO) and corn oil (CO) in experimental colon carcinogenesis. Treatment with carcinogen 1,2-dimethylhydrazine (DMH) altered reactive oxygen species (ROS), Ca(2+), and membrane characteristics, which resulted in an elevation in apoptosis in initiation phase and reduction in post-initiation phase. FO+CO(2.5:1)+DMH treatment, however, altered mitochondrial membrane parameters, ROS, and Ca(2+) to increase apoptosis in both phases, whereas FO+CO(1:1)+DMH treatment enhanced apoptosis only in post-initiation phase suggesting that FO supplementation in higher ratio has better chemopreventive efficacy.

Chemopreventive effect of different ratios of fish oil and corn oil on prognostic markers, DNA damage and cell cycle in colon carcinogenesis.

Fish oil (FO) rich in n-3 polyunsaturated fatty acids (PUFAs) have a protective role in autoimmune disorders, type 2 diabetes, rheumatoid arthritis, and cancer, whereas corn oil (CO) rich in n-6 PUFAs has a proinflammatory and procarcinogenic effect. A balanced n-3/n-6 PUFA ratio in diet rather than absolute intake of either may be responsible for decreasing cancer incidence. This study was designed to evaluate the chemopreventive effect of different ratios of FO and CO on prognostic markers, DNA damage, and cell cycle distribution in colon carcinogenesis. Male Wistar rats were divided into control, N,N'-dimethylhydrazine dihydrochloride (DMH) treated, FO+CO(1 : 1)+DMH, and FO+CO(2.5 : 1)+DMH. All the groups, except control, received a weekly injection of DMH for 4 weeks. The animals were given modified AIN-76A diets and killed either 48 h later (initiation phase) or kept for 16 weeks (postinitiation phase). The animals treated with DMH in both the phases showed an increase in multiple plaque lesions, total sialic acid, lipid associated sialic acid, DNA damage and cell proliferation. However, levels of p53 in the postinitiation and cyclin D1 in both the phases were significantly elevated. FO+CO(2.5 : 1)+DMH treatment in both the phases led to a decrease in multiple plaque lesions, DNA damage, total sialic acid, lipid associated sialic acid as compared with the DMH treated group. There was a G1 arrest with a decrease in p53 and cyclin D1 levels in FO+CO(2.5 : 1) in both the phases whereas treatment with FO+CO(1 : 1)+DMH led to same results in the postinitiation phase only. This study suggests that FO+CO(2.5 : 1) is more effective in chemoprevention of experimental colon carcinogenesis.

Evaluation of the role of oxidative stress in chemopreventive action of fish oil and celecoxib in the initiation phase of 7,12-dimethyl benz(α)anthracene-induced mammary carcinogenesis.

Cyclooxygenase-2 (COX-2) enzyme plays an important role in cancer development. COX-2 inhibition by non-steroidal anti-inflammatory drugs is a useful approach for cancer prevention, but its usage has been associated with side effects. n-3 polyunsaturated fatty acids also exhibit a chemopreventive effect mediated by COX-2 inhibition. Therefore, the present study was designed to evaluate the effect of combined dosage of celecoxib and fish oil in experimental mammary carcinogenesis. Female Wistar rats were distributed as control, 7,12-dimethyl benz(α)anthracene (DMBA) treated, celecoxib + fish oil (20 mg/kg b.w. + 0.5 ml), celecoxib + fish oil (30 mg/kg b.w. + 0.25 ml), and their corresponding controls treated with fish oil or celecoxib only. The treatment was given for 7 days, and on the 8th day animals of all the groups except the control group received DMBA orally and sacrificed after 90 days. The histopathology, DNA fragmentation, total sialic acid (TSA), lipid-associated sialic acid (LASA), and oxidative stress were measured in mammary tissue and liver mitochondrial fraction. The results showed ductal hyperplasia and an increase in TSA, LASA, lipid peroxidation, and nitrite levels with a decrease in the antioxidants on DMBA treatment. Pretreatment with celecoxib and fish oil in DMBA-treated animals led to normal histology, increase in DNA fragmentation, and decrease in TSA and LASA levels with reduced oxidative stress, and the effect was more pronounced than animals pretreated with either celecoxib/fish oil alone suggesting a synergistic effect of the two regimens. To conclude, a combination of celecoxib and fish oil is a better strategy for cancer chemoprevention than celecoxib/fish oil alone.

Chemopreventive effect of different ratios of fish oil and corn oil in experimental colon carcinogenesis.

n-3 Polyunsaturated fatty acids (PUFA) have a chemopreventive effect while n-6 PUFA promote carcinogenesis. The effect of these essential fatty acids may be related to oxidative stress. Therefore, the study was designed to evaluate the effect of different ratios of fish oil (FO) and corn oil (CO) in the prevention of colon cancer. Male Wistar rats were divided into control, dimethylhydrazine dihydrochloride (DMH) treated, FO + CO (1:1) and FO + CO (2.5:1). All the groups, except the control received a weekly injection of DMH for 4 weeks. The animals were sacrificed either 48 h later (initiation phase) or kept for 16 weeks (post initiation phase). DMH treatment in the initiation phase animals showed mild to moderate inflammation, decreased ROS and TrxR activity, increased antioxidants, apoptosis and ACF multiplicity. The post initiation study showed severe inflammation with hyperplasia, increased ACF multiplicity and ROS levels, a decrease in antioxidants and apoptosis. The FO + CO (1:1) treated animals showed severe inflammation, a decrease in ROS, an increase in antioxidants and apoptosis in the initiation phase. FO + CO (1:1) in the post initiation phase and FO + CO (2.5:1) in the initiation showed mild inflammation, increased ROS, apoptosis and decreased antioxidants. There was a decrease in ACF multiplicity and ROS levels, increased antioxidants and apoptosis in the post initiation phase study. The present study suggests that FO has a dose- and time-dependent chemopreventive effect in colon cancer mediated through oxidative stress and apoptosis.