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Objective: Leukemia is a group of hematologic malignancies in the bonemarrow that arise from the dysfunctional proliferation of developing leukocytes. It is classified as either acute or chronic based on the rapidity of proliferation and as myelocytic or lymphocytic based on the cell of origin. Newcastle Disease Virus (NDV) is an avian paramyxovirus, which has been demonstrated to possess significant oncolytic activity against mammalian cancers because its ability to kill tumor cells with limited toxicity to normal cells., Methods:  In this study, the morphophical changes and apoptosis induction of WEHI 3B leukemia cell line treated with NDV strain AF2240 were studied by scanning electron microscopes and  transmission electron microscopes techniques., Result: Electron microscopy indicated that NDV strain AF 2240 significantly altered cell morphology and reduced cell viability. Furthermore,  early apoptosis was observed 6 h post-inoculation by fluorescence microscope., Conclusion: Our results suggest that NDV has ability to induce significant apoptoic structural changes in WEHI 3B leukemia cell line. These findings provide new insights into the mechanism of action of NDV virotherapy and could lead to the development of more effective treatments for leukemia.

Aim: Newcastle disease virus (NDV) is a member of genus Avulavirus within the family Paramyxoviridae. Interest of using NDV as an anticancer agent has arisen from its ability to kill tumor cells with limited toxicity to normal cells. Methods: In this investigation, the proliferation of brain tumor cell line, glioblastoma multiform (DBTRG.05MG) induced by NDV strain AF2240 was evaluated in-vitro, by using MTT proliferation assay. Furthermore, Cytological observations were studied using fluorescence microscopy and transmission electron microscopy, DNA laddering in agarose gel electrophoresis assay used to detect the mode of cell death and analysis of the cellular DNA content by flowcytometery. Results: MTT proliferation assay, Cytological observations using fluorescence microscopy and transmission electron microscopy show the anti-proliferation effect and apoptogenic features of NDV on DBTRG.05MG. Furthermore, analysis of the cellular DNA content showed that there was a loss of treated cells in all cell cycle phases (G1, S and G2/M) accompanied with increasing in sub-G1 region (apoptosis peak). Conclusion: It could be concluded that NDV strain AF2240 is a potent antitumor agent that induce apoptosis and its cytotoxicity increasing while increasing of time and virus titer., (Creative Commons Attribution License)

Objectives: Clinically approved iron chelators are effective in decreasing significant transfusional iron accumulation. Starch-Deferoxamine (S-DFO), a novel high molecular weight iron chelator, was produced to increase binding capacity to iron and reduce toxicity. Although its efficacy was established in one small cohort clinical trial, its potential adverse effect was not adequately addressed., Methods: We utilized zebrafish model to assess S-DFO toxicity using following assays: mortality, teratogenicity, hatching rate, tail flicking, Acridine Orange staining for apoptosis detection, o-dianisidine staining for hemoglobin synthesis, and the level of Hsp70 as a general stress indicator. Embryos were exposed to different concentrations of S-DFO, Zinc Oxide nanoparticle (ZnO) (positive control), along with untreated control (UC)., Results: S-DFO showed no significant mortality nor deformities at all tested concentrations (0.0-1000 μM). Thus, the LC50 is expected to >1000 μM. 100 μM S-DFO treatment did not affect embryo development (as judged by hatching rate); neuromuscular activity (as judged by tail flicking); and hemoglobin synthesis. Neither apoptosis, nor increase in Hsp70 level was noticed upon S-DFO treatment., Conclusion: Our assays demonstrate that S-DFO does not induce cellular or biochemical stress and has no adverse effect on organ development of zebrafish embryos, suggesting its safe use as an iron chelator., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background and Objectives: Behçet's disease (BD) is a multi-system inflammatory disorder that can cause vasculitis. Here we questioned whether Neutrophils in BD cause vasculitis via releasing Neutrophil Extracellular Traps (NETs), a process called NETosis., Methods: Circulating neutrophils were isolated from a cohort of Middle Eastern BD patients with an active disease and healthy volunteers. The percentage of NETs release was monitored in neutrophils stimulated or not with BD serum, and treated or not with Colchicine, Dexamethasone, Cl-amidine or N-Acetyl Cysteine (NAC). The mRNA expression levels of PAD4 (a key enzyme in NETosis) was also assessed. The effect of NETs on the proliferation and cell death of endothelial cells was investigated using an in vitro co-culture model. The presence of NETs in skin tissues of BD patients was examined using immunolabeling of NETs associated proteins., Results: Circulating Neutrophils from BD patients were more prone to release NETs in vitro and expressed higher levels of PAD4 compared to healthy volunteers. Spontaneous NETs formation in BD neutrophils was inhibited by Colchicine and Dexamethasone, two drugs used to treat BD. NETs formation was also inhibited by Cl-amidine, a specific PAD4 inhibitor, and by NAC, a ROS inhibitor. Interestingly, serum from BD patients stimulated circulating neutrophils from healthy volunteers to release more NETs and increased their mRNA PAD4 expression. Moreover, endothelial cells cultured in the presence of NETs from BD patients showed a decrease in proliferation and an increase in apoptosis and cell death. Finally, NETosis was predominantly identified around affected blood vessels in biopsies of vasculitis from BD patients., Conclusion: Our results provide evidence on the implication of NETosis in the pathophysiology of BD especially in inducing vasculitis., (Copyright © 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Ru(II)-polypyridyl complexes exhibit antitumor properties that can be systematically tailored by means of adjusting the ligand environment. In this work, the effect of incorporating π-extended moieties into anionic N ∧ O - based chelating ligands on the cytotoxic properties of Ru compounds is explored. Four new Ru(II) complexes, [Ru(bpy) 2 (dphol)][PF 6 ] (1; bpy = 2,2'-bipyridine, dphol = dibenzo[ a, c]phenazin-10-olate), [Ru(phen) 2 (dphol)][PF 6 ] (2; phen = 1,10-phenanthroline), [Ru(bpy) 2 (hbtz)][PF 6 ] (3; hbtz = 2-(benzo[ d]thiazol-2-yl)phenolate), and [Ru(phen) 2 (hbtz)][PF 6 ] (4) were synthesized and thoroughly characterized. In vitro cytotoxicity was investigated in human lung adenocarcinoma (A549) cells, which revealed that 4 is the most cytotoxic compound (IC 50 = 0.8 μM) in the series including a control compound [Ru(bpy) 2 (quo)][PF 6 ] (5; quo = 8-hydroxyquinolinate) and is nearly 8-fold more cytotoxic than cisplatin. An investigation of the mechanism of cell death led to the finding that compounds 1-4 disrupt the mitochondrial transmembrane potential (ΔΨ m ) in a concentration-dependent fashion, which is an event associated with the intrinsic pathway of apoptosis. Moreover, compound 4 triggers the activity of caspase-3/7, which eventually induces the apoptotic cellular death of A549 cells. Thus, increasing the overall lipophilicity of the Ru compounds by introducing π-extended moieties in the anionic N ∧ O - ligand is a successful strategy for realizing a new family of pro-apoptotic compounds with a [Ru II N 5 O] + coordination environment.

Cancer is one of the most common causes of death in the developed world, with one-third of people diagnosed with cancer during their lifetime. Oral cancer commonly occurs involving the buccal mucosa (cheeks), tongue, floor of the mouth and lip. It is one of the most devastating and disfiguring of malignancies. Morinda citrifolia L., commonly known as ‘noni’, belongs to the Rubiaceae family. It is native to the Pacific islands, Hawaii, Caribbean, Asia and Australia. The plant displays broad curative effects in pharmacological studies. Damnacanthal (DAM) and Nordamnacanthal (NDAM), anthraquinone compounds isolated from the roots of Morinda citrifolia L., has been used for the treatment of several chronic diseases including cancer. The objectives of this study were to evaluate cytotoxicity, morphological changes, cell death mode (apoptosis/necrosis), and cell migration induced by DAM and NDAM on the most common type of oral cancer, oral squamous cell carcinoma (OSCC)cells. Anti-proliferative effects of these compounds against OSCC cell lines were determined by MTT assay. The mode of cell death was analysed by phase contrast and fluorescent microscopy as well as flow cytometry. In addition, cell migration was assessed. The results showed that DAM and NDAM exerted cytotoxicity against OSCC cells with IC50 values of 1.9 to >30 μg/ml after 72 h treatment. Maximum growth inhibition among the tested cell lines for both compounds was observed in H400 cells, and thus it was selected for further study. The study demonstrated inhibition of H400 OSCC cell proliferation, marked apoptotic morphological changes, induction of early apoptosis, and inhibition of cell migration by DAM and NDAM. Therefore, this information suggests that these compounds from noni have potential for used as anti tumor agents for oral cancer therapy., (Creative Commons Attribution License)

Dracaena cinnabari Balf.f. is a red resin endemic to Socotra Island, Yemen. Although there have been several reports on its therapeutic properties, information on its cytotoxicity and anticancer effects is very limited. This study utilized a bioassay-guided fractionation approach to determine the cytotoxic and apoptosis-inducing effects of D. cinnabari on human oral squamous cell carcinoma (OSCC). The cytotoxic effects of D. cinnabari crude extract were observed in a panel of OSCC cell lines and were most pronounced in H400. Only fractions DCc and DCd were active on H400 cells; subfractions DCc15 and DCd16 exhibited the greatest cytotoxicity against H400 cells and D. cinnabari inhibited cells proliferation in a time-dependent manner. This was achieved primarily via apoptosis where externalization of phospholipid phosphatidylserine was observed using DAPI/Annexin V fluorescence double staining mechanism studied through mitochondrial membrane potential assay cytochrome c enzyme-linked immunosorbent and caspases activities revealed depolarization of mitochondrial membrane potential (MMP) and significant activation of caspases 9 and 3/7, concomitant with S phase arrest. Apoptotic proteins array suggested that MMP was regulated by Bcl-2 proteins family as results demonstrated an upregulation of Bax, Bad, and Bid as well as downregulation of Bcl-2. Hence, D. cinnabari has the potential to be developed as an anticancer agent.

Goniothalamin, a natural compound extracted from Goniothalamus sp. belonging to the Annonacae family, possesses anticancer properties towards several tumor cell lines. This study focused on apoptosis induction by goniothalamin (GTN) in the Hela cervical cancer cell line. Cell growth inhibition was measured by MTT assay and the IC50 value of goniothalamin was 3.2 ± 0.72 μg/ml. Morphological changes and biochemical processes associated with apoptosis were evident on phase contrast microscopy and fluorescence microscopy. DNA fragmentation, DNA damage, caspase-9 activation and a large increase in the sub-G1 and S cell cycle phases confirmed the occurrence of apoptosis in a time-dependent manner. It could be concluded that goniothalamin show a promising cytotoxicity effect against cervical cancer cells (Hela) and the cell death mode induced by goniothalamin was apoptosis.

Cancer is one of the major health problems worldwide and its current treatments have a number of undesired adverse side effects. Natural compounds may reduce these. Currently, a few plant products are being used to treat cancer. In this study, goniothalamin, a natural occurring styryl-lactone extracted from Goniothalamus macrophyllus, was investigated for cytotoxic properties against cervical cancer (HeLa), breast carcinoma (MCF-7) and colon cancer (HT29) cells as well as normal mouse fibroblast (3T3) using MTT assay. Fluorescence microscopy showed that GTN is able to induce apoptosis in HeLa cells in a time dependent manner. Flow cytometry further revealed HeLa cells treated with GTN to be arrested in the S phase. Phosphatidyl serine properties present during apoptosis enable early detection of the apoptosis in the cells. Using annexin V/PI double staining it could be shown that GTN induces early apoptosis on HeLa cells after 24, 48 and 72 h. It could be concluded that goniothalamin showing a promising cytotoxicity effect against several cancer cell lines including cervical cancer cells (HeLa) with apoptosis as the mode of cell death induced on HeLa cells by Goniothalamin was.

Background: The combination of fish oil-derived docosahexaenoic acid (DHA) (22:6; omega 3 [n-3]) and butyrate (4:0), a fiber fermentation product, synergized to enhance colonocyte apoptosis by inducing a p53-independent, oxidation sensitive, mitochondrial Ca(2+) -dependent (intrinsic) pathway., Methods: In this study, the authors probed the specificity of n-6 and n-3 polyunsaturated fatty acid induction of Ca(2+) -dependent proapoptotic events in immortalized young adult mouse colonocytes and determined whether combinations of polyunsaturated fatty acid and butyrate could trigger endoplasmic reticulum (ER) stress conditions, thereby promoting mitochondrial Ca(2+) overload. Cultures were treated with 0 μM to 50 μM of DHA (22:6; n-3), EPA (20:5; n-3), arachidoinic acid (AA) (20:4; n-6), linoleic acid (18:2; n-6), or oleic acid (OA) (18:1; n-9) for a total of 72 hours with or without RU-360 (to inhibit the mitochondrial Ca(2+) uniporter) for 30 minutes before cotreatment with butyrate (0 mM or 5 mM)., Results: Combined DHA and butyrate maximally induced apoptosis and mitochondrial-to-cytosolic Ca(2+) levels. By comparison, EPA, a precursor to DHA, was minimally effective. Similarly, AA and OA in combination with butyrate had no effect on mitochondrial Ca(2+) or apoptosis compared with butyrate alone. DHA with or without butyrate cotreatment minimally altered the ER stress-regulated genes DNA damage-inducible transcript 3, the CCAAT enhancer binding protein (C/EBP) homologous protein (CHOP), and eukaryotic initiation factor 2α., Conclusions: The current data indicated that butyrate and DHA, but not EPA, worked in a coordinated fashion to trigger an ER-independent, Ca(2+) -dependent, intrinsic mitochondrial-mediated apoptotic pathway in colonocytes., (Copyright © 2011 American Cancer Society.)

Newcastle disease virus (NDV) is a member of genus Avulavirus within the family Paramyxoviridae. Interest of using NDV as an anticancer agent has arisen from its ability to kill tumor cells with limited toxicity to normal cells. In this investigation, the cytotolytic properties of NDV strain AF2240 were evaluated on brain tumor cell line, anaplastic astrocytoma (U-87MG), by using MTT assay. Cytological observations were studied using fluorescence microscopy and transmission electron microscopy to show the apoptogenic features of NDV on U-87MG. DNA laddering in agarose gel electrophoresis and terminal deoxyribonucleotide transferase-mediated dUTP-X nick end-labeling staining assay confirmed that the mode of cell death was by apoptosis. However, analysis of the cellular DNA content by flowcytometery showed that there was a loss of treated U-87MG cells in all cell cycle phases (G1, S and G2/M) accompanied with increasing in sub-G1 region (apoptosis peak). Early apoptosis was observed 6 h post-inoculation by annexin-V flow-cytometry method. It could be concluded that NDV strain AF2240 is a potent antitumor agent that induce apoptosis and its cytotoxicity increasing while increasing of time and virus titer.

Curcumin, a phenol in turmeric (Curcuma longa), has been studied for the last decade as a potential anticancer drug. It has been shown to reduce viability of the highly malignant, metastatic rat mammary gland cell line ENU1564 in culture and reduce metastasis of these cells injected into nude mice. The purpose of this study was to identify the mechanisms by which curcumin induces apoptosis in these ENU1564 cells in vitro, and to examine its effects on mitochondrial membrane potential and mitochondrial Ca(2+) homeostasis. The results show that curcumin induced apoptosis in ENU1564 cells through the intrinsic pathway of apoptosis, as evident by an increase in mitochondrial Ca(2+) accumulation and a decrease in mitochondrial membrane potential. However, treatment of the ENU1564 cells with the mitochondrial uniporter inhibitor RU-360 prior to curcumin treatment partially inhibited the curcumin effects. SKF-96365, a store-operated Ca(2+) channel blocker, suppressed the curcumin effect on mitochondrial Ca(2+). In addition, curcumin down-regulated the expressions of Bcl-2 and procaspase-3 and increased the production of reactive oxygen species in ENU1564 cells. These data suggest that the mitochondrial Ca(2+) is the leading factor by which curcumin induced apoptosis in ENU1564 cells, followed by reactive oxygen species production and inhibition of Bcl-2 oncoprotein., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Newcastle disease virus (NDV) is used as an antineoplastic agent in clinical tumor therapy. It has prompted much interest as an anticancer agent because it can replicate up to 10,000 times better in human cancer cells than in most normal cells. This study was carried out to determine the oncolytic potential of NDV strain AF2240 and V4-UPM on WEHI-3B leukemia cell line. Results from MTT cytotoxicity assay showed that the CD(50) values for both strains were 2 and 8 HAU for AF2240 and V4-UPM, respectively. In addition, bromodeoxyuridine (BrdU) and trypan blue dye exclusion assays showed inhibition in cell proliferation after different periods. Increase in the cellular level of caspase-3 and detection of DNA laddering using agarose gel electrophoresis on treated cells with NDV confirmed that the mode of cell death was apoptosis. In addition, flow-cytometry analysis of cellular DNA content showed that the virus caused an increase in the sub-G1 region (apoptosis peaks). In conclusion, NDV strains AF2240 and V4-UPM caused cytolytic effects against WEHI-3B leukemic cell line.

Butyrate, a short-chain fatty acid fiber fermentation product, induces colonocyte apoptosis in part via a Fas-mediated (extrinsic) pathway. In previous studies, we demonstrated that docosahexaenoic acid (DHA, 22:6(Delta4,7,10,13,16,19)) enhances the effect of butyrate by increasing mitochondrial lipid oxidation and mitochondrial Ca(2+)-dependent apoptosis in the colon. In this study, we further examined the mechanism of DHA-butyrate synergism in 1) human colon tumor (HCT-116 isogenic p53+/+ vs. p53-/-) cells and 2) primary cultures of rat colonic crypts. Herein, we show that DHA and butyrate promote apoptosis by enhancing mitochondrial Ca(2+) accumulation in both isogenic cell lines. Ca(2+) accumulation and apoptosis were inhibited by blockade of mitochondrial uniporter-mediated Ca(2+) uptake. In addition, Mito-Q, a mitochondria-targeted antioxidant, also blocked apoptosis induced by DHA and butyrate. In complementary experiments, rats were fed diets supplemented with either corn oil (control, contains no DHA) or fish oil (contains DHA). Colonic crypts were isolated and incubated with or without butyrate, after which the mitochondria-to-cytosol Ca(2+) ratio and crypt viability were measured. No significant difference (P > 0.05) in basal mitochondrial Ca(2+) levels was observed between fish oil- or corn oil-fed animals. In contrast, when fish oil was the dietary lipid source, crypts incubated with butyrate exhibited a significant increase (3.6-fold, P < 0.001) in mitochondrial Ca(2+) compared with corn oil plus butyrate treatment. On the basis of these data, we propose that the combination of DHA and butyrate compared with butyrate alone further enhances colonocyte apoptosis by inducing a p53-independent, oxidation-sensitive, mitochondrial Ca(2+) -dependent (intrinsic) pathway.

We have previously shown that butyrate, a short-chain fatty acid fiber fermentation product, induces colonocyte apoptosis via a nonmitochondrial, Fas-mediated, extrinsic pathway. Interestingly, fermentable fiber when combined with fish oil containing docosahexaenoic acid (DHA, 22:6n-3) exhibits an enhanced ability to induce apoptosis and protect against colon tumorigenesis. To determine the molecular mechanism of action, the effect of DHA and butyrate cotreatment on intracellular Ca2+ homeostasis was examined. Mouse colonocytes were treated with 50 micromol/L DHA or linoleic acid (LA) for 72 h +/- butyrate (0-10 mmol/L) for the final 24 h. Cytosolic and mitochondrial Ca2+ levels were measured using Fluo-4 and Rhod-2. DHA did not alter basal Ca2+ or the intracellular inositol trisphosphate (IP3) pool after 6 h butyrate cotreatment. In contrast, at 12 and 24 h, DHA- and butyrate-treated cultures exhibited a 25% and 38% decrease in cytosolic Ca2+ compared with LA and butyrate. Chelation of extracellular Ca2+ abolished the effect of thapsigargin on the IP3-releasable Ca2+ pool. DHA and butyrate cotreatment compared with untreated cells increased the mitochondrial-to-cytosolic Ca2+ ratio at 6, 12, and 24 h by 73%, 18%, and 37%, respectively. The accumulation of mitochondrial Ca2+ preceded the onset of apoptosis. RU-360, a mitochondrial-uniporter inhibitor, abrogated mitochondrial Ca2+ accumulation and also partially blocked apoptosis in DHA and butyrate cotreated cells. Collectively, these data show that the combination of DHA and butyrate, compared with butyrate alone, further enhances apoptosis by additionally recruiting a Ca2+ -mediated intrinsic mitochondrial pathway.

1,1-Bis(3'-indolyl)methane (DIM) and the 5,5'-dibromo ring substituted DIM (5,5'-diBrDIM) inhibited growth of MCF-7 and MDA-MB-231 breast cancer cells, and IC50 values were 10-20 and 1-5 microM, respectively, in both cell lines. DIM and 5,5'-diBrDIM did not induce p21 or p27 protein levels or alter expression of Sp1 or Sp3 proteins in either cell line. In contrast, 10 microM 5,5'-diBrDIM downregulated cyclin D1 protein in MCF-7 and MDA-MB-231 cells 12 and 24 h after treatment. DIM (20 microM) also decreased cyclin D1 in MCF-7 (24 h) and MDA-MB-231 (12 h), and the DIM/5,5'-diBrDIM-induced degradation of cyclin D1 was blocked by the proteasome inhibitor MG132. Both DIM and 5,5'-diBrDIM induced apoptosis in MCF-7 cells and this was accompanied by decreased Bcl-2, release of mitochondrial cytochrome c, and decreased mitochondrial membrane potential as determined by the red/green fluorescence of JC-1. DIM and 5,5'-diBrDIM induced extensive necrosis in MDA-MB-231 cells; however, this was accompanied by decreased mitochondrial membrane potential primarily in cells treated with 5,5'-diBrDIM but not DIM. Thus, DIM and 5,5'-diBrDIM induce cell death in MCF-7 and MDA-MB-231 cells by overlapping and different pathways, and the ring-substituted DIM represents a novel class of uncharged mitochondrial poisons that inhibit breast cancer cell and tumor growth.

Docosahexaenoic acid (DHA, 22:6 n-3) from fish oil, and butyrate, a fiber fermentation product, work coordinately to protect against colon tumorigenesis in part by inducing apoptosis. We have recently demonstrated that dietary DHA is incorporated into mitochondrial membrane phospholipids, thereby enhancing oxidative stress induced by butyrate metabolism. In order to elucidate the subcellular origin of oxidation induced by DHA and butyrate, immortalized young adult mouse colonocytes were treated with 0-200 microM DHA or linoleic acid (LA, 18:2 n-6; control) for 72 h with or without 5 mM butyrate for the final 24 h. Cytosolic reactive oxygen species, membrane lipid oxidation, and mitochondrial membrane potential (MP), were measured by live-cell fluorescence microscopy. After 24 h of butyrate treatment, DHA primed cells exhibited a 151% increase in lipid oxidation (P < 0.01), compared with no butyrate treatment, which could be blocked by a mitochondria-specific antioxidant, 10-(6'-ubiquinoyl) decyltriphenylphosphonium bromide (MitoQ) (P < 0.05). Butyrate treatment of LA pretreated cells did not show any significant effect. In the absence of butyrate, DHA treatment, compared with LA, increased resting MP by 120% (P < 0.01). In addition, butyrate-induced mitochondrial membrane potential (MP), dissipation was 21% greater in DHA primed cells as compared with LA at 6 h. This effect was reversed by preincubation with inhibitors of the mitochondrial permeability transition pore, cyclosporin A or bongkrekic acid (1 microM). The functional importance of these events is supported by the demonstration that DHA and butyrate-induced apoptosis is blocked by MitoQ. These data indicate that DHA and butyrate potentiate mitochondrial lipid oxidation and the dissipation of MP which contribute to the induction of apoptosis.

We showed previously that the dietary combination of fish oil, rich in (n-3) fatty acids, and the fermentable fiber pectin enhances colonocyte apoptosis in a rat model of experimentally induced colon cancer. In this study, we propose that the mechanism by which this dietary combination heightens apoptosis is via modulation of the colonocyte redox environment. Male Sprague-Dawley rats (n = 60) were fed 1 of 2 fats (corn oil or fish oil) and 1 of 2 fibers (cellulose or pectin) for 2 wk before determination of reactive oxygen species (ROS), oxidative DNA damage, antioxidant enzyme activity [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx)] and apoptosis in isolated colonocytes. Fish oil enhanced ROS, whereas the combination of fish oil and pectin suppressed SOD and CAT and enhanced the SOD/CAT ratio compared with a corn oil and cellulose diet. Despite this modulation to a seemingly prooxidant environment, oxidative DNA damage was inversely related to ROS in the fish oil and pectin diet, and apoptosis was enhanced relative to other diets. Furthermore, apoptosis increased exponentially as ROS increased. These results suggest that the enhancement of apoptosis associated with fish oil and pectin feeding may be due to a modulation of the redox environment that promotes ROS-mediated apoptosis.

We have shown that a combination of fish oil (high in n-3 fatty acids) with the butyrate-producing fiber pectin, upregulates apoptosis in colon cells exposed to the carcinogen azoxymethane, protecting against colon tumor development. We now hypothesize that n-3 fatty acids prime the colonocytes such that butyrate can initiate apoptosis. To test this, 30 Sprague-Dawley rats were provided with diets differing in the fatty acid composition (corn oil, fish oil or a purified fatty acid ethyl ester diet). Intact colon crypts were exposed ex vivo to butyrate, and analyzed for reactive oxygen species (ROS), mitochondrial membrane potential (MMP), translocation of cytochrome C to the cytosol, and caspase-3 activity (early events in apoptosis). The fatty acid composition of the three major mitochondrial phospholipids was also determined, and an unsaturation index calculated. The unsaturation index in cardiolipin was correlated with ROS levels (R = 0.99; P = 0.02). When colon crypts from fish oil and FAEE-fed rats were exposed to butyrate, MMP decreased (P = 0.041); and translocation of cytochrome C to the cytosol (P = 0.037) and caspase-3 activation increased (P = 0.032). The data suggest that fish oil may prime the colonocytes for butyrate-induced apoptosis by enhancing the unsaturation of mitochondrial phospholipids, especially cardiolipin, resulting in an increase in ROS and initiating apoptotic cascade.