Polyunsaturated fatty acids sensitize human colon adenocarcinoma HT-29 cells to death receptor-mediated apoptosis

Cancer Letters 218 (2005) 33–41 www.elsevier.com/locate/canlet

Polyunsaturated fatty acids sensitize human colon adenocarcinoma HT-29 cells to death receptor-mediated apoptosis Jiøina Hofmanova´*, Alena Vaculova´, Alois Kozubı´k Laboratory of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kra´lovopolska´ 135, CZ-612 65 Brno, Czech Republic Received 15 June 2004; received in revised form 26 July 2004; accepted 29 July 2004

Abstract The proliferative and apoptotic response to TNF-a and anti-Fas antibody (CH-11) in human colon adenocarcinoma HT-29 cells was modulated by pretreatment with arachidonic (AA, 20:4, nK6) or docosahexaenoic (DHA, 22:6, nK3) fatty acids, which alone increased reactive oxygen species production and lipid peroxidation, and decreased the S-phase of the cell cycle. The higher amount of floating cells, subG0/G1 population and apoptotic cells detected in pre-treated cells was potentiated by cycloheximide. The effects of CH-11 were associated with activation of caspase-8, -9, and -3, cleavage of poly(ADPribose)polymerase-PARP, and decreased mitochondrial membrane potential (MMP), but these parameters were not significantly changed after PUFA pretreatment. q 2004 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Supplementation of cell cultures in vitro or feeding animals with nK3 or nK6 polyunsaturated fatty acids (PUFAs) led to an increase of these PUFAs in cell Abbreviations: AA, arachidonic acid; CHX, cycloheximide; DAPI, 4,6-diamidino-2-phenyl-indole; DHA, docosahexaenoic acid; FCM, flow cytometry; FCS, fetal calf serum; MMP, mitochondrial membrane potential; NaBt, sodium butyrate; PARP, poly(ADP-ribose) polymerase; PUFAs, polyunsaturated fatty acids; ROS, reactive oxygen species; TBARs, thiobarbituric acid reactive substances; TMRE, tetramethylrhodamine ethyl ester perchlorate. * Corresponding author. Tel.: C420 541517182; fax: C420 541211293. E-mail address: [email protected] (J. Hofmanova´).

membrane phospholipids [1,2] and may influence membrane properties [3]. Moreover, PUFAs and their metabolites, eicosanoids, are considered as important mediators and modulators of the intracellular network of signals [4], they change oxidative metabolism [5] and may have a direct effect on gene expression when activating the specific nuclear receptors and transcription factors [6,7]. Certain PUFAs (especially nK3 types) were reported to improve immunological response [8], prevent proliferation and initiate apoptosis [9], kill tumor cells in vitro [10], and inhibit tumor growth in experimental animals [11]. The interaction of PUFAs from dietary fat with naturally occurring endogenous factors regulating the cytokinetics is supposed particularly in the colon where epithelial cells are in direct contact with

0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.07.038

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the diet [12]. The important endogenous regulators are those of the TNF family (tumor necrosis factor-a— TNF-a and Fas ligand), whose precise role in regulating colon epithelial cell turnover is not fully understood [13]. Their effects are mediated through death receptors (DRs) which induce similar signaling pathways leading to apoptosis [14]. The TNF family members are considered as potential antitumor agents. However, many cancer cell types develop resistance to their effects, and the cause of this differential sensitivity remains unclear [15,16]. Our previous results showed that HT-29 human colon adenocarcinoma cells are relatively resistant to acute antiproliferative and cytotoxic effects of TNF-a [17,18] and show medium sensitivity to the Fas ligating antibody (CH-11) (our own observation) [19]. Suggesting that PUFAs could modulate colon cancer cell response to these apoptotic inducers, we studied parameters reflecting proliferation and apoptosis of HT-29 cells induced with TNF-a and CH-11 after their pretreatment with arachidonic (AA, 20:4, nK6) or docosahexaenoic (DHA, 22:6, nK3) acid.

McCoy’s 5A medium supplemented with 1% ITS (insulin, transferrin, sodium selenite) without PUFAs and with or without TNF-a (30 ng/ml, Sigma), or antiFas monoclonal antibody (CH-11, 200 ng/ml, Immunotech). In the experiments using cycloheximide (CHX), the cells were treated with 5 mg/ml CHX added 3 h before the application of TNF-a or CH-11. 2.3. Analysis of fatty acid composition of cellular lipids After 48 h treatment with AA or DHA the cells were frozen (in cultivation medium containing 10% FCS and 5% DMSO in K80 8C). After thawing total lipids were extracted from the samples and the selected fatty acid content was determined after transmethylation using a GC-MS TurboMass instrument (Perkin-Elmer, Norwalk, USA). The derivatization procedure was performed as published previously [20]. The amount of fatty acids detected in the samples was expressed as nmol per 106 cells. 2.4. Floating cell quantification and viability assays

2. Material and methods 2.1. Cell culture The human colon adenocarcinoma HT-29 cells (ATCC, Rockville, MD, USA) were cultured in Mc Coy’s 5A medium (Sigma-Aldrich Corp., St Louis, MO, USA) with gentamycin (50 mg/l; Sigma) and 10% fetal calf serum (FCS; PAN Systems, Germany) at 37 8C in 5% CO2 and 95% humidity. 2.2. Experimental design The attached cells (24 h after seeding) were treated with AA or DHA (Sigma) freshly prepared from stock solutions (100 mM in 96% ethanol stored under nitrogen at K80 8C) and bound to fatty acid-free bovine serum albumin (BSA, SERVA Electrophoresis GmbH; Heidelberg, Germany) at a molar ratio of 1:2.5 in cultivation medium with 5% FCS. The control cells were treated with BSA and an appropriate concentration of ethanol, which did not influence any of the parameters tested. After 48 h cultivation of cells the medium was exchanged for a fresh serum-free

Floating and adherent cells were counted separately using a Coulter Counter (model ZM, Beckman-Coulter, Fullerton, CA, USA), and the amount of floating cells was expressed as a percentage of the total cell number. Cell viability was determined by eosin (0.15%) dye exclusion assay. 2.5. Flow cytometric (FCM) analysis of DNA Floating and adherent cells were harvested together, washed with PBS, and fixed in 70% ethanol at 4 8C. Low-molecular-weight fragments of DNA were extracted for 10 min in citrate buffer (Na2HPO4, C6H3O7, pH 7.8), RNA was removed by ribonuclease A (5 Kunitz U/ml), and DNA was stained with propidium iodide (PI; 20 mg/ml PBS) for 30 min in the dark. Fluorescence (2!104 cells per sample) was measured using a flow cytometer (FACSCalibur, Becton Dickinson, San Jose, CA) equipped with an argon laser at 488 nm wavelength for excitation. The ModFit 2.0 (Verity Software House, Topsham, ME) and CellQuest (Becton Dickinson) softwares were used to generate DNA content frequency histograms

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and quantify the amount of cells in the individual cell cycle phases including subG0/G1 population. 2.6. Fluorescence microscopy The cells were stained with a 4,6-diamidino-2phenyl-indol (DAPI, Fluka, Buche, Switzerland) solution (1 mg DAPI/ml ethanol) at room temperature in the dark for 30 min. They were then mounted in Mowiol, and the percentage of apoptotic cells (with chromatin condensation and fragmentation) was determined using a fluorescence microscope (Olympus IX-70, Prague, Czech Republic). 2.7. Production of reactive oxygen species (ROS) The intracellular production of ROS was detected by FCM analysis using dihydrorhodamine-123 (DHR123, Fluka, Switzerland), which reacts with intracellular hydrogen peroxide as described previously [21].

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2.10. Immunoblotting The cells were lysed in sample buffer (100 mM Tris, pH 7.4; 1% sodium dodecyl sulfate; 10% glycerol), diluted to an equal concentration, mixed with bromphenol blue (0.01%) and mercaptoethanol (1%), and subjected (20 mg) to SDS-PAGE. The polyacrylamide gels were transferred to polyvinylidene difluoride membranes (Immobilon-P, Sigma) electrophoretically in a buffer containing 192 mM glycine, 25 mM Tris, and 15% methanol. The membranes were blocked overnight in 5% nonfat milk in wash buffer (0.05% Tween-20 in 20 mM Tris; pH 7.6; 100 mM NaCl), and then probed with rabbit anti-PARP (1:500, SC-7150, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The recognized proteins were detected using horseradish peroxidase-labeled rabbit anti-IgG (1:6000, #NA934, Amersham Biosciences) secondary antibody and an enhanced chemiluminescence kit (ECL, Amersham Biosciences). An equal loading was verified using b-actin (1:5000, A5441, Sigma) quantification. 2.11. Statistical analysis

2.8. Detection of mitochondrial membrane potential (MMP) The changes of MMP were analysed by FCM using tetramethylrhodamine ethyl ester perchlorate (TMRE; Molecular Probes, Eugene, OR, USA) as described previously [21].

The results of at least three independent experiments were expressed as the meansCSEM. Statistical significance (P!0.05) was determined by one-way ANOVA followed by Tukey or LSD tests.

3. Results 2.9. Caspase activities

3.1. Pretreatment of cells with PUFAs

The cells were lysed in lysis buffer (250 mM HEPES, 25 mM CHAPS, 25 mM DTT, 40 mM protease inhibitor cocktail, Sigma, Czech Republic) on ice for 20 min and then centrifuged at 15,000g, 15 min in 4 8C. The acquired proteins (equal concentrations) were incubated with caspase-3 (Ac-DMQDAMC, 50 mM, Alexis), caspase-8 (Ac-IETD-AMC, 50 mM, Sigma) or caspase-9 (Ac-LEHD-AMC, 50 mM, Alexis) substrates overnight in assay buffer (40 mM HEPES, 20% glycerol, 4 mM DTT). Fluorescence was measured (355/460 nm) using a Fluostar Galaxy fluorometer (BMG Labtechnologies GmbH, Offenburg, Germany).

HT-29 cells were treated with previously chosen equieffective doses of AA (50 mM) or DHA (20 mM) for 48 h. As we previously reported [21] after this time AA or DHA did not significantly influence the total cell number, but increased (in a concentrationdependent manner) the percentage of floating cells. However, the number of cells in subG0/G1 population and cell viability were not significantly affected. Compared to control, both PUFAs significantly decreased the amount of cells in the S phase and increased the amount of cells in either G2/M (AA) or G0/G1 (DHA). A significantly increased ROS production (200–300% of control value) and lipid

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peroxidation (measured by thiobarbituric acid assay) were observed. However, no changes of MMP compared to control were detected (not shown). An analysis of the fatty acid content in cellular lipids after 48 h of treatment showed a significant increase of the corresponding PUFA. Standardized as nmol per 106 cells, 56.6 nmol (after treatment with 20 mM DHA) vs. 2.6 nmol (control) of DHA and 34 nmol (after treatment with 50 mM AA) vs. 3.3 nmol (control) of AA were detected. 3.2. The effects of death inductors

3.4. Cell death Pretreatment of cells either with AA or DHA significantly potentiated the ability of TNF-a and particularly of CH-11 to induce cell death. It is documented primarily by a significantly increased percentage of floating cells and cells in subG0/G1 population (Fig. 2A, B), when compared both to untreated controls and to cells treated only with single compounds. These effects were more profound after treatment of cells with CH-11 and significantly more effective after inhibition of protein synthesis with

Cells either non-pretreated or pretreated for 48 h with PUFAs were washed and further incubated in PUFA-free medium with or without TNF-a (30 ng/ml) or CH-11 (200 ng/ml) as described in Section 2. TNF-a or CH-11 were also combined with CHX (5 mg/ml). After additional 24 h of cultivation, cell cycle parameters and induction of cell death including parameters reflecting some proposed mechanisms involved in the effects observed were investigated. 3.3. Cell cycle In the cells pretreated for 48 h with AA or DHA and then cultivated for 24 h in PUFA-free medium, a significantly decreased amount of cells in the S phase accompanied by an increased amount of cells in G2/M (for AA) or G0/G1 (for DHA) compared to control was still detected (Fig. 1A, B). Neither TNF-a nor CH-11 affected cell cycle parameters in PUFA-non-pretreated cells. In PUFA-pretreated cells, TNF-a did not significantly change the values detected in only PUFA-pretreated cells (except additionally decreased G2/M phase after DHA-pretreatment) and CH-11 shifted the values between those detected in the cells treated only with individual agents. Inhibition of protein synthesis by CHX slightly attenuated the effects of PUFA pretreatment, but did not significantly change the effects of TNF-a on both pretreated and non-pretreated cells. On the other hand, treatment of cells with CH-11 together with CHX led to a significant increase in S-phase accompanied by decreased G0/G1 and G2/M phases in both pretreated and non-pretreated cells.

Fig. 1. Cell cycle parameters of HT-29 cells non-pretreated or pretreated for 48 h with arachidonic (AA; panel A) or docosahexaenoic (DHA; panel B) acid and then incubated for 24 h in PUFAfree medium without or with TNF-a or anti-Fas. Experiments with cycloheximide (CHX) added to the cells for the last 27 h are presented in the right parts of panels. The values are meansCSEM; nZ4; statistical significance: P!0.05 (*) compared to appropriate non-treated control; (C) compared to TNF-a or anti-Fas and/or (!) to AA or DHA as single factors.

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CH-11 together with CHX. The viability of these cells was only 16–18% of the controls (data not shown). Although a lower concentration of DHA (20 mM) than of AA (50 mM) was used for cell pretreatment, it was even slightly more effective in potentiating the effects of both apoptotic inducers. 3.5. ROS production and membrane mitochondrial potential (MMP) Neither TNF-a nor CH-11 caused significant additional changes in ROS production in both PUFA-non-pretreated and PUFA-pretreated cells. Fig. 4A, B shows the results with DHA, which were similar to those with AA. Unlike no effects of TNF-a, CH-11 enhanced significantly the amount of cells with decreased MMP (40–50%). However, it was not significantly enhanced by PUFA pretreatment (not shown).

Fig. 2. The percentage of floating cells and subG0/G1 population of HT-29 cells non-pretreated or pretreated for 48 h with arachidonic (AA; panel A) or docosahexaenoic (DHA; panel B) acid and then incubated for 24 h in PUFA-free medium without or with TNF-a or anti-Fas. Experiments with cycloheximide (CHX) added to the cells for the last 27 h are presented in the right parts of panels. The values are meansCSEM; nZ4; statistical significance: P!0.05 (*) compared to non-treated control; (C) compared to TNF-a or antiFas and/or (!) to AA or DHA as single factors.

CHX (up to 77% of floating cells and 42% of cells in subG0/G1 population). A lesser efficiency of PUFA pretreatment on TNFa-induced apoptosis is also documented by morphologically detected apoptotic cells after DAPI staining (Fig. 3). While a significant increase of apoptotic cells after 24 h treatment with CH-11 in AA (16%) as well as DHA pretreated (20%) cells was detected in comparison with control as well as with PUFAs or CH-11 alone, no significant increase after TNF-a treatment was observed. In spite of detection of attributes of apoptosis, the viability of cells was not significantly decreased in comparison with control after 24 h treatment except for cells pretreated with PUFAs and then treated with

3.6. Activity of caspases and cleavage of PARP In cells treated with CH-11 the activities of caspase-8, -9, and -3 (Table 1), and cleavage of PARP (Fig. 5, results with DHA) were significantly increased compared to control. However, there

Fig. 3. The percentage of cells with apoptotic morphology (DAPI staining) in non-pretreated or pretreated for 48 h with 50 mM of arachidonic (AA) or 20 mM of docosahexaenoic (DHA) acid and then incubated for 24 h in PUFA-free medium without or with TNFa (30 ng/ml) or anti-Fas (200 ng/ml). P!0.05 (*) compared to nontreated control, (C) compared to TNF-a or anti-Fas and/or (!) to AA or DHA as single factors.

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Fig. 4. Production of reactive oxygen species (ROS) in HT-29 cells non-pretreated or pretreated for 48 h with 20 mM of docosahexaenoic (DHA) acid and then incubated for 24 h in PUFA-free medium without or with TNF-a (30 ng/ml, panel A) or anti-Fas (200 ng/ml, panel B). ROS were measured by FCM as dihydrorhodamine-123 (DHR-123) fluorescence (FL1-H). The figure is a representative result of three independent experiments.

were no significant differences between PUFA-nonpretreated and pretreated cells.

4. Discussion Our results showed that PUFAs, which comprise a part of dietary fat, induce processes promoting the sensitivity of colon cancer cells to endogenous apoptotic regulators. In agreement with other authors [2,5,22,23] we observed that the treatment of cells with both AA and DHA led to their incorporation into cellular lipids, enhanced lipid peroxidation and ROS production, and changed cell cycle parameters [21]. The cell response mentioned above outlasted even after washing and incubation of cells in PUFA-free medium for 24 h. In PUFA-pretreated cells, apoptotic parameters were not significantly different from the control, but these cells became more sensitive to

the effects of TNF-a and particularly CH-11 than nonpretreated cells. In contrast to our results, inhibition of Fas-mediated apoptosis in a different type of the colon cancer cell line by linoleic acid and no effect of DHA were reported [24]. In monocytic U937 cells DHA and vitamin E reduced TNF-a-induced apoptosis [25]. Our previous results and data from other laboratories showed relative resistance of HT-29 cells to TNF-a (up to 100 ng/ml) as well as to anti-Fas antibody, which can be overcome by combination with IFN-g or CHX [16,18,26]. In our experiments, pretreatment with both types of PUFAs was more effective in CH-11 than TNF-a-induced effects. In addition to a higher concentration of CH-11 used in our experiments the difference between them can be associated with the reported divergent signaling pathways of TNF-a and Fas receptors and slower kinetics of TNF-a [27–29]. As a consequence of PUFA pretreatment we detected a primarily increased percentage of floating cells after TNF-a or CH-11 treatment. These cells were primed for apoptosis as demonstrated by the increased amount of cells in subG0/G1 population and cells with apoptotic morphology. After CH-11 treatment, detachment from the substratum involved activation of caspase-8, -3 and -9, a marked collapse of MMP, and cleavage of PARP. Our results are in agreement with the statement that one consequence of Fas (CD95) signaling is loss of adhesion, a phenomenon termed ‘active disintegration’, during which apoptosis is initiated [13,14]. However, there were no significant differences in the above-mentioned parameters between PUFA-pretreated and non-pretreated cells. Table 1 Activity of caspase-3, -9, and -8 of HT-29 cells non-pre-treated or pre-treated for 48 h with 50 mM of arachidonic (AA) or 20 mM of docosahexaenoic (DHA) acid and then incubated for 24 h in PUFAfree medium without or with anti-Fas (200 ng/ml) Caspase activity (% of control) Control Anti-Fas AA AA-anti-Fas DHA DHA-anti-Fas

Caspase-3

Caspase-9

Caspase-8

100.0G0.0 175.6G16.8* 128.4G14.4 167.5G13.0* x 100.8G10.0 173.5G14.4* x

100.0G0.0 186.3G33.2 120.3G34.3 154.7G26.9 96.5G35.3 178.5G49.4

100.0G0.0 139.5G9.4* 110.8G5.6 124.9G8.0* 96.1G3.3 138.1G8.1* x

Values are meansCSEM; nZ3. P!0.05, (*) vs. control, (x) vs. PUFA, Tukey or LSD test.

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Fig. 5. Cleavage of poly(ADP-ribose) polymerase (PARP) to its 89 kDa fragment detected by Western blotting. An equal was verified using b-actin (40 kDa) antibody. HT-29 cells were nonpretreated or pretreated for 48 h with 20 mM of docosahexaenoic (DHA) acid and then incubated for 24 h in PUFA-free medium without or with anti-Fas (200 ng/ml). The figure is a representative result of three independent experiments.

In addition to the relatively strong ‘background effects’ of CH-11 alone, other sites and mechanisms of interaction could be considered. PUFA pretreatment can change cell adherence properties, and thus prime the cells to be more effectively shed to the media and to undergo apoptosis [30]. Thus, mechanisms associated with cell adhesion and subsequent events such as activation of specific types of kinases [7,31,32] can be supposed. In other cell types it was also reported that accumulation of triacylglycerol droplets in the cytoplasm after PUFA treatment can be associated with apoptosis, and that AA-induced apoptosis can be independent on caspase-3 activity [33]. Moreover, changes in cell cycle parameters observed after PUFA treatment and after inhibition of protein synthesis by CHX imply participation of some cell cycle regulatory molecules. In our recently published results we proved participation of p27Kip1 in the potentiation effects of PUFA pretreatment on the effects of sodium butyrate on HT-29 cells [21]. A substantial increase in apoptotic effects caused by CHX implies participation of some newly synthesized proteins playing a role in apoptosis blockage [14,16, 34]. The possibilities mentioned above are under investigation in our laboratory. It was reported that Fas-induced apoptosis was preceded by an increase of ROS [35]. Regardless of

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the fact that we did not detect ROS production after CH-11 treatment, elevated ROS production and lipid peroxidation in PUFA-pretreated cells can predispose them for more effective or more rapid apoptosis [5]. A similar effect was reported for HT-29 pretreated with NSAIDs or butyrate, where a potential role for NF-kB is supposed [19]. As in this report, we did not detect any changes in the expression of plasma membrane receptor Fas (CD95, APO-1). We can summarize that dietary PUFAs of both nK6 and nK3 series significantly alter the response of HT-29 colon cancer cells so that they become more sensitive to apoptosis induced by CH-11 and TNF-a. We conclude that although lower concentrations of AA or DHA alone were not capable of inducing apoptosis in colonic cells, our results imply that they can, through changes of membrane properties, oxidative metabolism [5] and other mechanisms [36,37] start a cascade of processes which prepare permissive environment for a more effective action of apoptotic inducers. This suggests that the composition of dietary fat should be included in strategies for colon cancer prevention and therapy.

Acknowledgements This work was supported by the Grant Agency of the Czech Republic (No. 524/04/0895). The authors thank Dr R. Hysˇpler, Dr J. Netı´kova´ and Mrs Lenka Vystrcˇilova´ for their willing technical assistance.

References [1] A.P. Simopoulos, Omega-3 fatty acids in health and disease and in growth and development, Am. J. Clin. Nutr. 54 (1991) 438–463. [2] A.B. Awad, A. Kamei, P.J. Horvath, C.S. Fink, Prostaglandin synthesis in human cancer cells: influence of fatty acids and butyrate, Prostaglandins Leukot. Essent. Fatty Acids 53 (1995) 87–93. [3] J.E. Kinsella, Lipids, membrane receptors, and enzymes: effects of dietary fatty acids, J. Parenter. Enteral Nutr. 14 (1990) 200S–217S. [4] V. DiMarzo, Arachidonic acid and eicosanoids as targets and effectors in second messenger interactions, Prostaglandinds Leukot. Essent. Fatty Acids 53 (1995) 239–254.

40

J. Hofmanova´ et al. / Cancer Letters 218 (2005) 33–41

[5] D.G. Tang, E. La, J. Kern, J.P. Kehrer, Fatty acid oxidation and signaling in apoptosis, Biol. Chem. 383 (2002) 425–442. [6] S.A. Kliewer, S.S. Sundseth, S.A. Jones, P.J. Brown, G.B. Wisely, C.S. Koble, et al., Fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferator-activated receptors alpha and gamma, Proc. Natl. Acad. Sci. USA 94 (1997) 4318–4323. [7] Q.N. Diep, R.M. Touyz, E.L. Schiffrin, Docosahexaenoic acid, a peroxisome proliferator-activated receptor-alpha ligand, induces apoptosis in vascular smooth muscle cells by stimulation of p38 mitogen-activated protein kinase, Hypertension 36 (2000) 851–855. [8] P.C. Calder, Polyunsaturated fatty acids, inflammation, and immunity, Lipids 36 (2001) 1007–1024. [9] I.L. Rudolph, D.S. Kelley, K.C. Klasing, K.L. Erickson, Regulation of cellular differentiation and apoptosis by fatty acids and their metabolites, Nutr. Res. 21 (2001) 381–393. [10] S.I. Grammatikos, P.V. Subbaiah, T.A. Victor, W.M. Miller, Diverse effects of essential (n-6 and n-3) fatty acids on cultured cells, Cytotechnology 15 (1994) 31–50. [11] G. Calviello, P. Palozza, N. Maggiano, E. Piccioni, P. Franceschelli, A. Frattucci, et al., Cell proliferation, differentiation, and apoptosis are modified by n-3 polyunsaturated fatty acids in normal colonic mucosa, Lipids 34 (1999) 599–604. [12] R.S. Chapkin, J.R. Lupton, Colonic cell proliferation and apoptosis in rodent species, Modulation by diet, Adv. Exp. Med. Biol. 470 (1999) 105–118. [13] J. O’Connell, M.W. Bennett, K. Nally, G.C. O’Sullivan, J.K. Collins, F. Shanahan, Interferon-gamma sensitizes colonic epithelial cell lines to physiological and therapeutic inducers of colonocyte apoptosis, J. Cell Physiol. 185 (2000) 331–338. [14] J. Strater, P. Moller, Expression and function of death receptors and their natural ligands in the intestine, Ann. NY Acad. Sci. 915 (2000) 162–170. [15] P.H. Krammer, P.R. Galle, P. Moller, K.M. Debatin, CD95(APO-1/Fas)-mediated apoptosis in normal and malignant liver, colon, and hematopoietic cells, Adv. Cancer Res. 75 (1998) 251–273. [16] U. von Reyher, J. Strater, W. Kittstein, M. Gschwendt, P.H. Krammer, P. Moller, Colon carcinoma cells use different mechanisms to escape CD95-mediated apoptosis, Cancer Res. 58 (1998) 526–534. [17] M. Kovarikova, J. Pachernik, J. Hofmanova, Z. Zadak, A. Kozubik, TNF-alpha modulates the differentiation induced by butyrate in the HT-29 human colon adenocarcinoma cell line, Eur. J. Cancer 36 (2000) 1844–1852. [18] A. Vaculova, J. Hofmanova, K. Soucek, M. Kovarikova, A. Kozubik, Tumor necrosis factor-alpha induces apoptosis associated with poly(ADP-ribose) polymerase cleavage in HT-29 colon cancer cells, Anticancer Res. 22 (2002) 1635–1639. [19] C. Giardina, H. Boulares, M.S. Inan, NSAIDs and butyrate sensitize a human colorectal cancer cell line to TNF-alpha

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

and Fas ligation: the role of reactive oxygen species, Biochim. Biophys. Acta 1448 (1999) 425–438. H.M. Liebich, C. Wirth, B. Jakober, Analysis of polyunsaturated fatty acids in blood serum after fish oil administration, J. Chromatogr. 572 (1991) 1–9. J. Hofmanova, A. Vaculova´, A. Lojek, A. Kozubı´k, Interaction of polyunsaturated fatty acids and sodium butyrate during apoptosis in HT-29 human colon adenocarcinoma cells, Eur. J. Nutr. 10 (2004) 1–12. R.S. Chapkin, M.Y. Hong, Y.Y. Fan, L.A. Davidson, L.M. Sanders, C.E. Henderson, et al., Dietary n-3 PUFA alter colonocyte mitochondrial membrane composition and function, Lipids 37 (2002) 193–199. U.N. Das, Tumoridical action of cis-unsaturated fatty acids and their relationship to fre radicals and lipid peroxidation, Cancer Lett. 56 (1991) 235–243. S. Meterissian, M. Kontogiannea, H. Murty, A. Gupta, Omega-6 fatty acids can inhibit Fas-mediated apoptosis in a human colorectal carcinoma cell line: a potential mechanism for escape from immune surveillance, Int. J. Surg. Investig. 2 (2000) 253–257. M. Yano, E. Kishida, M. Iwasaki, S. Kojo, Y. Masuzawa, Docosahexaenoic acid and vitamin E can reduce human monocytic U937 cell apoptosis induced by tumor necrosis factor, J. Nutr. 130 (2000) 1095–1101. N.K. Ossina, A. Cannas, V.C. Powers, P.A. Fitzpatrick, J.D. Knight, J.R. Gilbert, et al., Interferon-gamma modulates a p53-independent apoptotic pathway and apoptosisrelated gene expression, J. Biol. Chem. 272 (1997) 16351–16357. M.T. Abreu-Martin, A. Vidrich, D.H. Lynch, S.R. Targan, Divergent induction of apoptosis and IL-8 secretion in HT-29 cells in response to TNF-alpha and ligation of Fas antigen, J. Immunol. 155 (1995) 4147–4154. M. Enari, H. Hug, M. Hayakawa, F. Ito, Y. Nishimura, S. Nagata, Different apoptotic pathways mediated by Fas and the tumor-necrosis-factor receptor. Cytosolic phospholipase A2 is not involved in Fas-mediated apoptosis, Eur. J. Biochem. 236 (1996) 533–538. D. De Valck, D. Vercammen, W. Fiers, R. Beyaert, Differential activation of phospholipases during necrosis or apoptosis: a comparative study using tumor necrosis factor and anti-Fas antibodies, J. Cell Biochem. 71 (1998) 392–399. P. Latham, E.K. Lund, J.C. Brown, I.T. Johnson, Effects of cellular redox balance on induction of apoptosis by eicosapentaenoic acid in HT29 colorectal adenocarcinoma cells and rat colon in vivo, Gut 49 (2001) 97–105. A.J. Bretland, J. Lawry, R.M. Sharrard, A study of death by anoikis in cultured epithelial cells, Cell Prolif. 34 (2001) 199–210. T.P. Dalton, H.G. Shertzer, A. Puga, Regulation of gene expression by reactive oxygen, Annu. Rev. Pharmacol. Toxicol. 39 (1999) 67–101. M.A. Puertollano, M.A. de Pablo, G. Alvarez de Cienfuegos, Polyunsaturated fatty acids induce cell death in YAC-1

J. Hofmanova´ et al. / Cancer Letters 218 (2005) 33–41 lymphoma by a caspase-3-independent mechanism, Anticancer Res. 23 (2003) 3905–3910. [34] A. Srinivasan, F. Li, A. Wong, L. Kodandapani, R. Smidt Jr., J.F. Krebs, et al., Bcl-xL functions downstream of caspase-8 to inhibit Fas- and tumor necrosis factor receptor 1-induced apoptosis of MCF7 breast carcinoma cells, J. Biol. Chem. 273 (1998) 4523–4529. [35] K. Banki, E. Hutter, N.J. Gonchoroff, A. Perl, Elevation of mitochondrial transmembrane potential and reactive oxygen intermediate levels are early events and occur independently

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from activation of caspases in Fas signaling, J. Immunol. 162 (1999) 1466–1479. [36] B.A. Narayanan, N.K. Narayanan, B.S. Reddy, Docosahexaenoic acid regulated genes and transcription factors inducing apoptosis in human colon cancer cells, Int. J. Oncol. 19 (2001) 1255–1262. [37] C. Pompeia, T. Lima, R. Curi, Arachidonic acid cytotoxicity: can arachidonic acid be a physiological mediator of cell death?, Cell. Biochem. Funct. 21 (2003) 97–104.