Sensitization of colon cancer cell lines to butyrate-mediated proliferation inhibition by combined application of indomethacin and nordihydroguaiaretic acid

Cancer Detection and Prevention 29 (2005) 276–285 www.elsevier.com/locate/cdp

Sensitization of colon cancer cell lines to butyrate-mediated proliferation inhibition by combined application of indomethacin and nordihydroguaiaretic acid$ Peter Galfi DVM, PhDa, Zsuzsa Neogrady DVM, PhDa, Albert Amberger PhDb, Raimund Margreiter MDb, Adam Csordas PhDc,* a

Institute of Physiology and Biochemistry, Faculty of Veterinary Sciences, Szent-Istva´n University, Budapest, Hungary b Tyrolean Cancer Research Institute, Innsbruck, Austria c Division of Medical Biochemistry, Biocentre Innsbruck, Innsbruck Medical University, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria Accepted 1 December 2004

Abstract The aim of the study was to investigate the effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on histone deacetylase-mediated proliferation inhibition. In the colon cancer cell line HT29 butyrate-mediated proliferation inhibition was enhanced by the additional presence of indomethacin (IM) and/or nordihydroguaiaretic acid (NDGA). Sensitisation to butyrate-mediated proliferation inhibition was abolished by the general caspase inhibitor Z-VAD-fmk, however, only IM-induced cell detachment was prevented by the caspase inhibitor but not that induced by NDGA or NDGA plus IM. In contrast to the parental cell line HT29, in the methotrexate-resistant sub-lines HT29-12 and HT2921, IM counteracted butyrate-mediated proliferation inhibition, which was abrogated by NDGA. In all the investigated cell lines, proliferation inhibition was most effectively achieved under the combined application of butyrate with IM and NDGA, suggesting that inhibition of both cyclooxygenase (COX) and lipoxygenase (LOX) isoenzymes is needed for proliferation inhibition by NSAIDs in tumour cells. # 2004 International Society for Preventive Oncology. Published by Elsevier Ltd. All rights reserved. Keywords: Colon cancer cell lines; Proliferation inhibition; Butyrate; NSAIDs; Indomethacin; NDGA; Cyclooxygenases; Lipoxygenases

1. Introduction It has become increasingly clear that the cyclooxygenase (COX)/lipoxygenase (LOX) families of enzymes not only play a role in inflammation processes but also represent important regulators of cell proliferation. Since COX/LOX isoenzyme expression patterns are linked to the process of tumorigenesis in a highly cell type-specific manner, nonsteroidal anti-inflammatory drug (NSAID) inhibitors of these enzymes emerged as a new perspective in tumour therapy as well as cancer prevention [1–6]. Abbreviations: ASA, acetylsalicylic acid; COX, cyclooxygenase; IM, indomethacin; LOX, lipoxygenase; NDGA, nordihydroguaiaretic acid; NSAID, nonsteroidal anti-inflammatory drug; PBS, phosphate buffered saline $ This study was presented at the Seventh International Symposium of the ISPO Meeting, Nice, France, 7 February 2004. * Corresponding author. Tel.: +43 512 507 3504; fax: +43 512 507 2872. E-mail address: [email protected] (A. Csordas).

As related to cell proliferation and cancer, the interest in COX-2 was triggered by the observation that it was not expressed in normal quiescent intestinal epithelial cells, but was found to be expressed in colon carcinomas [7]. Moreover, in an in vitro study it was demonstrated that constitutive COX-2 expression made rat intestinal epithelial cells refractory to butyrate-induced apoptosis, but apoptosis sensitivity to butyrate was restored by inhibition of COX-2 [8]. In the course of further investigations, however, it was shown that NSAIDs induced apoptosis in spite of COX-2 expression in colon cancer cells [9]. Recent studies revealed that in addition to COXs also LOX isoenzymes play an important role in proliferation control. Pancreatic tumour cell lines have been found to be highly sensitive to various LOX-inhibitors [10]. It was recognized that LOX isoenzymes are functionally distinct, so that some are associated with promotion of proliferation [10], while others with induction of apoptosis [11,12].

0361-090X/$30.00 # 2004 International Society for Preventive Oncology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cdp.2004.12.001

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Butyrate, and histone deacetylase inhibitors generally, act at certain concentration ranges as proliferation inhibitors in animal cells. Butyrate, produced by bacterial fermentation of complex carbohydrates in the colon, is present at millimolar concentrations in stool ([13], for reviews see [14,15]). On the one hand, butyrate is the preferential energy source for colonocytes, and on the other, a proliferation inhibitor and apoptosis inducer. This ‘‘butyrate paradox’’ has not yet been elucidated. Thus, colonocytes grow, differentiate and die in the environment of the proliferation inhibitor butyrate, and therefore the question of how proliferation of normal colonic epithelial cells and colon cancer cells is influenced by various factors in the presence of butyrate is of special interest. A correlation between histone hyperacetylation and expression of 15-LOX-1 was reported in Caco-2 colon cancer [16] and A549 lung cancer cell lines [17]; furthermore, altered expression levels of 15LOX-1 have been observed in colorectal and oesophageal tumours [2,11,12,18]. Although histone deactelyase inhibitors as well as NSAIDs are emerging as novel and selective anti-tumour agents, there are only a few studies on their combined application [19,20]; in the present study, we raised the question whether and to what extent proliferation inhibition induced by the histone deacetylase inhibitor butyrate is influenced by the additional presence of the non-selective COX-1 inhibitors acetyl salicylic acid (ASA) and indomethacin (IM), the selective COX-2 inhibitor NS-398, and the general LOX inhibitor nordihydroguaiaretic acid (NDGA). We tested at the level of cell lines whether the anti-proliferative effects exerted by butyrate and NSAIDs are additive or synergistic and whether selective, cell typespecific proliferation inhibition can be accomplished by the combined application of histone deacetylase inhibitors with specific NSAIDs.

2. Materials and methods 2.1. Cell lines The cell lines investigated are listed in Table 1. Under standard culture conditions, the colon cancer cell line Caco-2 shows epithelial morphology and spontaneously highly differentiated phenotype (polarised). Two Caco-2 cell lines were investigated: Caco-2H (Dr. H. Hendriks, Department of Veterinary Pathology, Faculty of Veterinary Medicine, Utrecht University, The Netherlands) and Caco2P (Dr. G. Mo´ zsik, Department of Internal Medicine, Medical University Pe´ cs, Hungary). HT29 cells, which are undifferentiated under standard culture conditions, were from Dr. J. Rafter, Karolinska Institutet, Huddinge, Sweden. The HT29-12 and HT29-21 cell lines, which are sub-clones of HT29 resistant to high concentrations of methotrexate and 5-fluorouracil [21,22], were obtained from Dr. H. Hendriks. HT29-12 is of enterocytic phenotype, while HT29-21

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Table 1 Description of the investigated cell lines Cell type

Cell line

Tissue origin

Epithelial Epithelial Goblet Epithelial Epithelial Epithelial Epithelial Endothelial-like Epithelial

HT29 HT29-12 HT29-21 HT29cl.19a Caco-2H Caco-2P IEC-18 RCTC MMT-060562

Epithelial-like Fibroblast Epithelial-like Epithelial-like

J-111 MG-63 A549 CL

Human colon adenocarcinoma Human colon adenocarcinoma Human colon adenocarcinoma Human colon adenocarcinoma Human colon adenocarcinoma Human colon adenocarcinoma Rat ileum Ovine rumen-connective tissue Mouse (C57BlxAf)F1 mammary gland tumour Human monocytic leukemia Human osteosarcoma Human lung carcinoma Human liver (chang liver)

represents goblet cells [22]. A third sub-clone HT29cl.19a, which exhibits a highly differentiated phenotype (polarised) under standard culture conditions, was isolated after culturing HT29 in the presence of 5 mM sodium butyrate [23]. The HT29cl.19a cell line was from Dr. R. Busche, Institute of Physiological Chemistry, University of Veterinary Medicine, Hannover, Germany. The RCTC cell line was derived from primary culturing as described previously [24]. IEC-18, MMT, J-111, MG-63, A549 and CL cells were obtained from ICN Flow, England. 2.2. Culturing of cells The cell lines were routinely cultured in Dulbecco’s Modified Eagle’s Medium (Sigma) containing glucose (4500 mg/l for the colon cancer cell lines and IEC-18; 1000 mg/ml for the RCTC, MMT, J-111, MG-63, A549 and CL cell lines) supplemented with 10% heat-inactivated FCS, 4 mM glutamine, 25 mM HEPES, 1% of MEM nonessential amino acid solution 100, 100 mg/l kanamycin sulphate, 50 mg/l gentamicin sulphate, at 37 8C, saturated humidity, 5% CO2. FCS and supplements were from Sigma. For maintenance purposes, all the cell lines were passaged once or twice a week using 0.05% trypsin in 0.02% EDTA in HBSS. The medium was changed twice a week at all culture conditions. 2.3. Stock solutions of butyrate and NSAIDs Sodium-n-butyrate (BDH Chemicals Ltd., Poole, England) was dissolved in phosphate buffered saline (PBS) (Sigma) and sterilized by filtration, the concentration of the stock solution was 2 mol/l; ASA (Sigma) was dissolved in DMSO and kept in a stock solution of 2 mol/l; IM (Sigma) was dissolved in 0.1 M NaOH, sterilized by filtration and kept as a stock solution of 27.95 mmol/l; NS-398 (Sigma) and NDGA (Sigma) were dissolved in DMSO at the concentrations of 18 and 10 mmol/l, respectively. The

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general caspase inhibitor Z-VAD-fmk was from Calbiochem (Merck KGaA, Darmstadt, Germany) and kept as 10 mmol/l stock solution in DMSO at 20 8C. For the inhibition experiments Z-VAD-fmk was applied together with butyrate and/or the NSAIDs at the beginning of the 72-h treatment periods, to yield final concentrations of 0.01 mmol/l. 2.4. Treatment procedures Solutions with the following concentrations in PBS were prepared from the stock solutions: 2 mol/l sodium butyrate, 0.66 mmol/l ASA, 27.95 mmol/l IM, 6 mmol/l NS-398, 3.33 mmol/l NDGA; 10 ml volumes of these solutions were placed into the first well of each of the series of eight. To each of the 10 ml solutions of the substances to be tested 90 ml of cell suspension was added, yielding total final volumes of 100 ml per well with the following concentrations: 200 mmol/l sodium butyrate, 66 mmol/1 ASA, 2.8 mmol/l IM, 600 mmol/1 NS-398, 333 mmol/l NDGA. Starting from these solutions, series of dilutions were made so that in rows of eight each of the subsequent solutions represented threefold dilution of the previous ones. 2.5. Proliferation assays Proliferation inhibition was determined with the MTSassay and/or Giemsa-staining of attached cells. 2.5.1. MTS-assay The MTS-assay kit ‘‘CellTiter 96 AQueousNon-Radioactive Cell Proliferation Assay’’ of Promega was used for determining the IC50-values. Exponentially growing cells (48–72 h cultures, 25 cm2 culture flasks, Corning Glass Works, New York, NY, USA) were trypsinized and passaged to 96-well plates, 5  103 cells/well, in 90 ml media. Cells were treated with 10 ml solutions of each of the NSAIDs under investigation. The microplates were incubated at 37 8C and cell growth was assessed after 72 h by adding 20 ml of MTS reagent to the 100 ml of solution present per well; incubation followed at 37 8C for a period of 1.5–2 h;

the absorbance of the colour which developed was determined with an ELISA reader at 492 nm. Non-treated media, media without cells and cultures, which were treated with the corresponding volumes of DMSO served as controls. The applied concentration ranges of DMSO and other solvents used did not have any detectable effect on cell proliferation. The IC50-values for butyrate, the NSAIDs and their various combinations were calculated by regression analysis from the absorbance data of dose-dependencies of proliferation inhibition. 2.5.2. Assessment of proliferation inhibition by Giemsastaining of attached cell colonies Cells cultivated on 96-well plates were fixed with 10% formalin in PBS for 10 min, and stained thereafter for 30 min with a 10% aqueous Giemsa solution. After one washing with distilled water and several hours of drying, the extent of proliferation inhibition/detachment was determined by measuring absorbance at 630 nm or viewing photographs of attached cell colonies. 2.6. Statistical analysis Each treatment of the cell lines was performed in triplicate. The three values were then averaged to obtain one value for each culture. From these data, the mean value  S.E.M. was calculated for each experimental condition. An analysis of variance was carried out on the data for testing the treatment effect. For comparison of two means we used Student’s t-test for unpaired samples. A difference was considered significant when the P-value was <0.05.

3. Results 3.1. IC50-values of NSAIDs for the investigated cell lines Table 2 shows the IC50-values of ASA, IM, NS-398 and NDGA for the investigated cell lines. The IC50-values of

Table 2 The IC50-values of ASA, IM, NS-398 and NDGA Cell lines

ASA IC50 (mM)

IM IC50 (mM)

NS-398 IC50 (mM)

NDGA IC50 (mM)

IEC-18 HT29-12 HT29-21 HT29cl.19a Caco-2P Caco-2H MMT J-111 MG-63 A549 CL RCTC

1.44  0.13 2.61  0.49 3.86  0.67 5.43  0.65 4.31  0.42 6.15  0.45 5.57  0.80 3.82  0.23 7.69  2.36 4.10  0.08 2.56  1.08 5.26  1.70

0.36  0.04 0.33  0.03 0.55  0.05 0.94  0.14 0.48  0.08 0.73  0.22 0.96  0.16 0.90  0.29 0.65  0.08 0.84  0.30 0.52  0.12 0.92  0.22

138.4  29.1 96.0  26.7 198.9  50.5 131.0  13.0 91.8  34.2 ND 98.3  16.1 80.9  8.3 150.1  20.7 143.0  27.3 42.1  5.85 ND

24.1  3.03 86.8  20.1 75.4  10.7 60.0  10.0 33.8a 15.8  2.4 13.3  3.2 57.9  21.9 13.2  2.5 128.6  40.1 65.8  20.8 81.5a

ND: not determined. a Made in duplicate.

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ASA, IM and NS-398 for all the investigated cell lines are higher than those reported for enzyme inhibition of COX-2 [25]. This may be due to up-regulation of compensatory LOX isoenzymes under COX-inhibition and/or COX-2 independent proliferation inhibition mechanisms. The nontransformed rat intestinal epithelial cell line IEC-18 appeared to be quite sensitive to ASA and IM (but not to NS-398 and NDGA) compared to the other cell lines, suggesting that under inhibition of COX-1, this cell line is unable to express COX-2 or proliferation-promoting LOXs. Three cell lines, namely Caco-2H, MMT and MG-63 showed higher sensitivities to NDGA, with IC50-values lower than those reported for enzyme inhibition of 12-LOX or 15-LOX but higher than required for enzyme inhibition of 5-LOX [25], pointing to a contribution of 5-LOX to proliferation. 3.2. Proliferation inhibition by butyrate and/or NSAIDs in HT29 colon cancer cells It is well known that butyrate – as other histone deacetylase inhibitors – is an inhibitor of proliferation. We raised the question how the sensitivity to butyrate-mediated proliferation inhibition is affected by the additional presence of IM and/or NDGA. Fig. 1 shows proliferation inhibition in HT29 cells by butyrate (7.4 mM), IM (0.31 mM), NDGA (37 mM) alone, their various combinations, in the absence or presence of the general caspase inhibitor Z-VAD-fmk. Proliferation inhibition was determined by the MTS assay (panel A) and Giemsa staining of attached cells (panel B). IM (0.31 mM) or NDGA (37 mM) inhibited proliferation only to a limited extent; inhibition was more pronounced under their combined application; the presence or absence of Z-VAD-fmk did not make much of a difference. Butyrate by itself (7.4 mM) was quite an effective inhibitor. Proliferation

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inhibition in the presence of butyrate was further enhanced by the additional application of IM or NDGA, and was most effective under the combined application of both of them. Fig. 2 gives a closer view of the caspase-dependence of sensitisation to butyrate-mediated proliferation inhibition by IM and/or NDGA in HT29 cells. Panel A of Fig. 2 (MTSassay) shows that the sensitisation to butyrate-mediated proliferation inhibition by IM, NDGA or their combined application was abrogated in the presence of Z-VAD-fmk. Interestingly, Giemsa staining demonstrates that IM-induced cell detachment was prevented by Z-VAD-fmk when IM was applied in addition to butyrate (panel B of Fig. 2); cell detachment, however, was not prevented by Z-VAD-fmk when NDGA or IM plus NDGA was applied in addition to butyrate (Fig. 2, panel B). Thus, sensitisation of HT29 cells to butyrate-mediated proliferation inhibition by IM and/or NDGA is caspase-dependent (panel A) and occurs via enhanced cell detachment (panel B). Interestingly, IMinduced cell detachment was caspase-dependent, whereas that induced by NDGA or IM plus NDGA was caspaseindependent (i.e., was not abrogated by Z-VAD-fmk) (Fig. 2, panel B). It is also noteworthy that the detached cells under the given conditions (i.e., in the presence of Z-VAD-fmk, 72h treatment) retained their reducing capacity so that they appeared viable in the MTS-assay (Fig. 2, panel A). Fig. 3 shows photographs of Giemsa-stained attached HT29 cells after various treatments. Application of IM in addition to butyrate resulted in enhanced cell detachment, and this was prevented by Z-VAD-fmk. 3.3. Paradoxical modulation of butyrate-mediated proliferation inhibition by indomethacin Next we tested the cell type-specificity of sensitisation to butyrate-mediated proliferation inhibition by NSAIDs. In a

Fig. 1. Proliferation inhibition of HT29 cells determined under various treatment conditions. The drugs (butyrate (BU), 7.4 mM; IM, 0.31 mM; NDGA, 37 mM) were applied with (0.01 mM) or without the poly-caspase inhibitor Z-VAD-fmk. After 72-h treatment, proliferation inhibition was determined using the MTSassay (panel A). In separate experiments the density of attached cells was determined by measuring absorbance of Giemsa-stained cell colonies at 630 nm (panel B). *Differs significantly (P < 0.05) from the control.

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Fig. 2. Sensitisation of HT29 cells to butyrate-mediated proliferation inhibition by IM and/or NDGA. The drugs (butyrate (BU), 7.4 mM; IM, 0.31 mM; NDGA, 37 mM) were applied with (0.01 mM) or without the poly-caspase inhibitor Z-VAD-fmk. After 72-h treatment, proliferation inhibition was determined using the MTS-assay (panel A), or alternatively, by measuring the absorbance of Giemsa-stained attached cells at 630 nm (panel B). *Differs significantly (P < 0.05) from the control.

set of cell lines the IC50-value of butyrate alone was compared with that determined in the additional presence of ASA, IM, NS-398 or NDGA. Fig. 4 shows the IC50-values for butyrate-mediated proliferation (as controls) compared with those determined in the additional presence of different doses of the NSAIDs. ASA (7.4 mM) had a sensitising effect on butyrate-mediated proliferation inhibition, which was statistically significant in the non-tumour intestinal epithelial cell line IEC-18, the two Caco-2 cell lines and also in HT29-12, HT29cl.19a, J-111 and CL (panel A). When examining the sensitising effect of IM on butyrate-mediated proliferation inhibition, we made

the striking observation that in certain cell lines IM counteracted butyrate-mediated proliferation inhibition, i.e., the IC50-value was higher than that measured for butyrate alone. Although IM when applied alone was clearly a proliferation inhibitor for all cell lines investigated (Table 2), in the sub-lines HT29-12 and HT29-21 it not only failed to add to butyrate-mediated proliferation inhibition but even exerted a paradoxical proliferation promoting effect. A similar phenomenon of increased IC50-values compared to that measured with butyrate alone was also observed, even though to a lesser extent, in the additional

Fig. 3. Photographs of Giemsa-stained HT29 cell colonies. Treatment with the drugs (butyrate, 7.4 mM; IM, 0.31 mM) was for 72 h with (0.01 mM) or without Z-VAD-fmk. (a) Butyrate without Z-VAD-fmk. (b) Butyrate with Z-VAD-fmk. (c); Butyrate and IM without z-VAD-fmk. (d) Butyrate and IM with zVAD-fmk.

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Fig. 4. The IC50-values of butyrate (BU) as affected by the additional presence of ASA, IM, NS-398 or NDGA. The IC50-value of butyrate alone (control) is compared with those measured in the additional presence of different concentrations of ASA (panel A), IM (panel B), NS-398 (panel C) or NDGA (panel D). After 72-h treatment, the MTS-assay was used for determining the IC50-values. *Differs significantly (P < 0.05) from the control.

presence of NS-398 in the sub-lines HT29-12, HT29-21 and also in the leukaemia cell line J-111 (Fig. 4, panel C). Panel D of Fig. 4 shows that in the cell lines Caco-2H, MG-63 and MMT, NDGA (12.3 mM) exerted a significant and dose-dependent sensitisation to butyrate-mediated proliferation inhibition. These additional anti-proliferative effects were observed at NDGA concentrations above that reported for enzyme inhibition of 5-LOX but below those reported for 12-LOX and 15-LOX enzyme activities [25]. These three cell lines also exhibited a higher sensitivity to NDGA, when NDGA was applied alone (without butyrate) (see the IC50-values in Table 2). The apparently paradoxical increase of the IC50-values (compared to that measured for butyrate alone) in the additional presence of IM or NS-398 (i.e., under COX-2 inhibition) may have been due to up-regulation/activation of proliferation promoting LOX isoenzyme(s) triggered by COX-2 inhibition. If this supposition is correct, a combined application of NDGA with IM can be expected to abrogate the paradoxical IM effect. Fig. 5 shows these experiments with the cell lines HT29-12 and HT29-21. The IC50-value of

butyrate alone (control) is compared with those under the combined applications with IM or IM plus NDGA. The additional presence of IM, at the concentrations 0.31 and 0.93 mM, caused in both cell lines HT29-12 (Fig. 5a) and HT29-21 (Fig. 5b) a marked dose-dependent increase of the IC50-values compared to the control (IC50-value of butyrate alone), i.e., an increased resistance to proliferation inhibition by butyrate. When, however, IM was applied together with NDGA, the paradoxical IM-effect counteracting butyratemediated proliferation inhibition was abrogated. These observations are consistent with the working hypothesis that in the presence of the histone deacetylase inhibitor butyrate, the inhibition of COXs may trigger a compensatory upregulation of LOX isoenzyme(s), which were inhibited by NDGA. 3.4. Assessment of proliferation inhibition by cell colony staining with Giemsa For calculation of the IC50-values depicted in the Figs. 4 and 5, proliferation inhibition was measured with the MTSassay. In the following, we investigated the effects of IM

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Fig. 5. Abrogation of the paradoxical proliferation-promoting effect of IM by NDGA in the methotrexate-resistant sub-lines HT29-12 (panel A) and HT29-21 (panel B). IC50-values for butyrate alone (control) are compared with those in the additional presence of IM, NDGA or IM plus NDGA. The IC50-values were determined using the MTS-assay (after 72-h treatment). Dose-dependent desensitisation to butyrate-mediated proliferation inhibition by IM (increased IC50values) is abrogated by NDGA (37 mmol/1).

and/or NDGA on butyrate-mediated proliferation inhibition by Giemsa staining of the attached cell colonies in the wells of 96-well plates. Assessment of proliferation inhibition was by inspection of their photographs. In order to explore further the cell type-specificity of the effects of IM and NDGA on butyrate-mediated proliferation inhibition, we compared the extent of proliferation inhibition/detachment induced by butyrate, IM or NDGA, when applied alone and under various combined applications employing the parental cell line HT29, its three sub-lines (HT29-12, HT29-21 and HT29cl.19a) and the Caco-2H and Caco-2P cell lines; the Caco-2 cell lines were obtained from two different laboratories (see Materials and methods) and were found to be identical according to 15 different criteria of STRgenotyping (Dr. A. Amberger, Tyrolean Cancer Research Institute, Innsbruck, Austria); we observed, however, differences between the two Caco-2 cell lines under certain treatment conditions (for instance, see Table 2). Fig. 6 shows photographs of the Giemsa-stained attached cell colonies after the various treatment procedures. IM or NDGA applied alone caused some proliferation inhibition (compare with Table 2) with the most visible effects in HT29, HT29cl.19a and Caco-2H. The combined application of IM and NDGA resulted in marked proliferation inhibition in almost all the cell lines investigated. When butyrate (7.4 mM) was applied alone, the density of attached cell colonies was quite reduced compared to the untreated control, except in Caco-2P, where the effect was marginal. Although in the parental HT29 cell line co-application of IM with butyrate led to a further sensitisation to proliferation inhibition compared to that with butyrate alone, in the three sub-lines of HT29-12, HT29-21, HT29cl.19a and in Caco2H, under the same conditions, the additional presence of IM resulted in a paradoxical proliferation promoting effect, which was abrogated by NDGA. When NDGA was applied together with butyrate, the number/size of attached colonies was clearly reduced compared to that with butyrate alone in all the investigated cell lines. A further sensitisation to proliferation inhibition could be achieved under combined application of IM and NDGA together with butyrate. Thus,

the most efficient proliferation inhibition of the colon cancer cell lines was achieved when the histone deacetylase inhibitor butyrate was applied together with the nonselective COX inhibitor IM and the general LOX inhibitor NDGA.

4. Discussion In the present study we investigated the effects of the COX-inhibitors ASA, IM, NS-398 and the general LOX inhibitor NDGA on proliferation in the cancer cell lines HT29, Caco-2, the three HT29 sub-lines HT29-12, HT2921, HT29cl.19a and several other cell lines in order to determine whether and to what extent sensitisation to butyrate-mediated proliferation inhibition can be achieved. We made the observation that the combined application of the COX inhibitor IM and the LOX inhibitor NDGA with the histone deacetylase inhibitor butyrate represents the most efficient proliferation inhibition for all the cell types investigated. We made, however, also the observation that the additional presence of an NSAID does not necessarily result in sensitisation to butyrate-mediated proliferation inhibition, but can even effectively counteract it. Although in HT29 butyrate-mediated proliferation inhibition and cell detachment was enhanced by the additional presence of IM, in the sub-lines HT29-12, HT29-21, the presence of IM resulted in a decrease of proliferation inhibition compared to that exerted by butyrate alone. Thus, it depends on the cell type whether the application a COX inhibitor will result in sensitisation or desensitisation to histone deacetylasemediated proliferation inhibition. A plausible explanation for these observations appears to be that in the presence of butyrate, the inhibition of certain COX isoenzyme(s) may trigger the up-regulation of proliferation promoting COX or LOX isoenzyme(s). It should be pointed out that the paradoxical proliferation promoting effect of IM was observed only in the presence of butyrate, i.e., in the presence of a histone deacetylase inhibitor, a condition which leads to chromatin hyperacetylation and consequently

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Fig. 6. Photographs of the Giemsa-stained attached cell colonies. Comparison of proliferation inhibition/detachment after various treatments (72 h) of the cell lines HT29, HT29-12, HT29-21, HT29cl.19a, Caco-2H and Caco-2P. Treatment with IM (0.31 mmol/l), NDGA or butyrate (BU) (7.4 mmol/l) alone and their various combinations. NDGA concentration for the HT29 cell lines was 37 mmol/1, for the Caco-2 cell lines 12 mmol/l.

to modulation of gene expression [26]. The cell typedependence of this phenomenon might be explained by the differing COX/LOX expression patterns of cell types, due to different organization of the COX/LOX genes within the chromatin structure and thus differing abilities to induce compensatory proliferation-promoting COX/LOX isoenzyme(s). Abrogation of the proliferation promoting effects of IM by the LOX inhibitor NDGA can be considered as an indirect proof of our hypothesis. Thus, combined application of COX and LOX inhibitors may be needed for effective tumour therapy. Indeed dual inhibitors, i.e., inhibitors of COX-2 and LOX are being developed and investigated [27,28]. There are an increasing number of studies reporting on some kind of interrelationship in the expression of various COX and LOX isoenzymes [28]. COX-1 up-regulation was causally associated with up-regulation of COX-2 in HeLa cells [29], and in colorectal and oesophageal tumour cells COX-2 inhibition resulted in up-regulation of 15-LOX-1 [2,11,12], which via its metabolite 13-S-HODE, induced

apoptosis. Furthermore, 15-LOX-1 expression was reported to be linked to histone acetylation [16,17], and the degree of histone acetylation [16] as well as the expression level of 15LOX-1 was found to be different when colorectal [11,16] or oesophageal tumour cells [12] were compared with the adjacent normal cells. Butyrate caused hyperacetylation and up-regulation of 15-LOX-1 in Caco-2 colon cancer cells [16]; in A549 lung epithelial cells it was shown that 15LOX-1 is upregulated by IL-4 or butyrate; furthermore, histone acetylation and acetylation of STAT6 was required for activation of the 15-LOX-1 gene [17]. Interestingly, in the methotrexate-resistant sub-lines HT29-12, HT29-21, and in HT29cl.19a, which was isolated in the presence of 5 mM butyrate, the additional presence of the COX-2 inhibitor IM counteracted butyrate-mediated proliferation inhibition, while in the parental cell line HT29 the presence of IM resulted in enhanced proliferation inhibition (Fig. 6). The phenomenon of a proliferation promoting action of an NSAID in the presence of a histone deacetylase inhibitor might possibly be encountered in other

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tumour cell types, too. In previous studies it was reported that NS-398 and IM suppressed proliferation of two cell lines highly expressing COX-2 (Caco-2 and MKN45); however, the same inhibitors exerted only marginal effects on proliferation inhibition in cell lines which expressed significantly lower levels of COX-2 [30]. Butyrate led to upregulation of COX-2 protein in HT29 carcinoma and PC/ AA/C1 adenoma cells [19]. When applied alone, 10 mM NS398 did not result in detectable growth inhibition; in combination with butyrate, however, NS-398 sensitised HT29 cells to the growth-inhibiting effect exerted by butyrate, and this sensitisation was observed only in the COX-2 expressing HT29 cells and not in the COX-2 nonexpressing S/KS cells [19]. When measuring proliferation inhibition with the MTSassay, we observed that sensitisation of HT29 cells to butyrate-mediated proliferation inhibition by IM and/or NDGA was a caspase-dependent process since it could almost completely be prevented by Z-VAD-fmk (Figs. 1A and 2A). When; however, proliferation inhibition was measured by Giemsa-staining of attached cells, we noted that IM-induced cell detachment was prevented by Z-VADfmk but not that induced by NDGA or IM plus NDGA (Figs. 1B and 2B). Although NDGA-induced proliferation inhibition was caspase-dependent, NDGA-induced cell detachment proved to be clearly caspase-independent with the detached cells remaining viable according to the criteria of the MTS-assay under the conditions of the 72-h treatment (Figs. 1B and 2B). Thus, the process of anoikis (detachmentinduced apoptosis) may be more complex than anticipated. It remains to be investigated whether detached cells always have the same fate or whether they embark with different kinetics on different modes of cell death programmes. Little is known about the COX/LOX isoenzyme expression patterns of the sub-lines HT29-12 and HT2921. The enterocyte model HT29cl.19a, which was isolated as a clone resistant to 5 mM sodium butyrate [23], was reported to express 5-LOX, 15-LOX, leukotriene A4 hydrolase activities and COX-1 and COX-2 mRNA [30]. Despite the lack of five-lipoxygenase-activating protein (FLAP) expression, 5-LOX expression and metabolism was observed in this cell line [31]. The ‘‘constitutive’’ COX-2 expression in HT29cl.19a was found to be ‘‘inducible’’ i.e., markedly upregulated in the total absence of fetal bovine serum or by exogenous inducers [32]. Interestingly, FLAP transfection in HT29cl.19a resulted in up-regulation of COX-2, which appears to be a further piece of evidence for the interrelatedness of expression levels of COX-2 and LOX-5 [33]. If cell types, including tumour cell types, differ in their COX/LOX isoenzyme expression patterns and their ability to induce these isoenzymes under various treatment conditions, or if such distinctions are provoked by histone deacetylase inhibitors, appropriate combinations of histone deacetylase inhibitors with NSAIDs would represent a new perspective in tumour-selective chemotherapy. The paradoxical proliferation-promoting effect of the NSAID IM in

the presence of the histone deacetylase inhibitor butyrate, and its abrogation by the general LOX inhibitor NDGA (Fig. 5) highlight the impact of specific COX/LOX expression patterns on proliferation control. A better understanding of the inter-relatedness of COX and LOX isoenzyme expression patterns should be helpful in cell type-specific targeting of tumour cells. It should be also kept in mind that in the colon, millimolar concentrations of butyrate are present, a condition causing hyperacetylation and proliferation inhibition in colon cancer cell lines. For future studies the HT29 parental cell line and its sub-lines represent a model to learn more about the underlying mechanism of the observed phenomena. The potential contribution of NSAIDs to cancer prevention should not be underestimated considering that plant-derived nutritional components have been reported to act as COX or LOX inhibitors, and NSAIDs are in frequent use by patients with chronic inflammatory diseases.

Acknowledgements This work was supported by grants from the Hungarian Research Fund (OTKA, T 037401), the Austro-Hungarian Scientific and Technical Cooperation Programme (A-18/01 and A7-2004) and the Austrian Cancer Society/Tyrol. We are indebted to Ms. Rajam Csordas-Iyer for critical reading and editorial assistance.

References [1] DeWitt D, Smith WL. Yes, but do they still get headaches. Cell 1995;83:345–8. [2] Shureiqi I, Chen D, Lee JJ, et al. 15-LOX-1: a novel molecular target of nonsteroidal anti-inflammatory drug-induced apoptosis in colorectal cancer cells. J Nat Cancer Inst 2000;92:1136–42. [3] Shiff SJ, Koutsos MI, Qiao L, Rigas B. Nonsteroisdal antiinflammatory drugs inhibit the proliferation of colon adenocarcinoma cells: effects on cell cycle and apoptosis. Exp Cell Res 1996; 222:179–88. [4] Cuendet M, Pezzuto JM. The role of cyclooxygenase and lipoxygenase in cancer chemoprevention. Drug Metabol Drug Interact 2000;17:109–57. [5] Kawai N, Tsujii M, Tsuji S. Cyclooxygenases and colon cancer. Prostaglandins Other Lipid Mediat 2002;68–69:187–96. [6] Chan TA. Nonsteroidal anti-inflammatory drugs, apoptosis, and coloncancer prevention. Lancet Oncol 2002;3:166–74. [7] Sano H, Kawahito Y, Wilder RL, et al. Expression of cyclooxygenase1 and -2 in human colorectal cancer. Cancer Res 1995;55:3785–9. [8] Tsujii M, DuBois RN. Alteration in cellular adesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell 1995;83:493–501. [9] Shureiqi I, Chen D, Lotan R, et al. 15-Lipoxygenase-1 mediates nonsteroidal anti-inflammatory drug-induced apoptosis independently of cyclooxygenase in colon cancer cells. Cancer Res 2000;60: 6846–50. [10] Ding X-Z, Iversen P, Cluck MW, Knezetic JA, Adrian TE. Lipoxygenase inhibitors abolish proliferation of human pancreatic cancer cells. Biochem Biophys Res Commun 1999;261:218–23.

P. Galfi et al. / Cancer Detection and Prevention 29 (2005) 276–285 [11] Shureiqi I, Wojno KJ, Poore JA, et al. Decreased 13-S-hydroxyoctadecadienoic acid levels and 15-lipoxygenase-1 expression in human colon cancers. Carcinogenesis 1999;20:1985–95. [12] Shureiqi I, Xu X, Chen D, et al. Nonsteroidal anti-inflammatory drugs induce apoptosis in esophageal cancer cells by restoring 15-lipoxygenase-1 expression. Cancer Res 2001;61:4879–84. [13] Cummings JH. Short-chain fatty acids in the human colon. Gut 1981;22:763–79. [14] Csordas A. Toxicology of butyrate and short-chain fatty acids. In: Hill MJ, editor. Role of gut bacteria in human toxicology and pharmacology. London: Taylor & Francis; 1995. p. 105–27. [15] Kruh J, Defer N, Tichonicky L. Effects of butyrate on cell proliferation and gene expression. In: Cummings JH, Rombeau JL, Sakata T, editors. Physiological and clinical aspects of short-chain fatty acids. Cambridge: Cambridge University Press; 1995. p. 275–88. [16] Kamitani H, Taniura S, Ikawa H, Watanabe T, Kalevkar US, Eling TE. Expression of 15-lipoxygenase-1 is regulated by histone acetylation in human colorectal carcinoma. Carcinogenesis 2001;22:187–91. [17] Shankaranarayanan P, Chaitidis P, Ku¨ hn H, Nigam S. Acetylation by histone acetyltransferase CREB-binding protein/p300 of STAT6 is required for transcriptional activation of the 15-lipoxygenase-1 gene. J Biol Chem 2001;276:42753–60. [18] Ikawa H, Kamitani H, Calvo BF, Foley JF, Eiling TE. Expression of 15-lipoxygenase-1 in human colorectal cancer. Cancer Res 1999; 59:360–6. [19] Crew TE, Elder DJE, Paraskeva Ch. A cyclooxygenase-2 (COX-2) selective non-steroidal antiinflammatory drug enhances the growth inhibitory effect of butyrate in colorectal carcinoma cells expressing COX-2 protein: regulation of COX-2 by butyrate. Carcinogenesis 2000;21:69–77. [20] Menzel T, Schauber J, Kreth F, et al. Butyrate and aspirin in combination have an enhanced effect on apoptosis in human colorectal cancer cells. Eur J Cancer Prev 2002;11:271–81. [21] Galfi P, Neogrady Zs, Csordas A. Apoptosis sensitivity is not correlated with sensitivity to proliferation inhibition by the histone deacetylase inhibitors butyrate and TSA. Cancer Lett 2002;188:141–52. [22] Lesuffleur T, Barbat A, Dussaulx E, Zweibaum A. Growth adaptation to methotrexate of HT-29 human colon carcinoma cells is associated

[23]

[24]

[25]

[26]

[27] [28]

[29]

[30]

[31]

[32]

[33]

285

with their ability to differentiate into columnar absorptive and mucussecreting cells. Cancer Res 1990;50:6334–43. Lesuffleur T, Violette S, Vasile-Pandrea I, et al. Resistance to high concentrations of methotrexate and 5-fluorouracil of differentiated HT-29 colon-cancer cells is restricted to cells of enterocytic phenotype. Int J Cancer 1998;76:383–92. Augeron C, Laboisse CL. Emergence of permanently differentiated cell clones in a human colonic cancer cell line in culture after treatment with sodium butyrate. Cancer Res 1984;44:3961–9. Hong SH, Avis I, Vos MD, Martı´nez A, Treston AM, Mulshine JL. Relationship of arachidonic acid metabolizing enzyme expression in epithelial cancer cell lines to the growth effect of selective inhibitors. Cancer Res 1999;59:2223–8. Marks P, Richon VM, Rifkind RA. Histone deacetylase inhibitors: inducers of differentiation or apoptosis of transformed cells. J Nat Cancer Inst 2000;92:1210–6. Leval X, Julemont F, Delarge J, Pirotte B, Dogne JM. New trends in dual 5-LOX/COX inhibition. Curr Med Chem 2002;9:941–62. Romano M, Cla`ria C. Cyclooxygenase-2 and 5-lipoxygenase converging functions on cell proliferation and tumor angiogenesis: implications for cancer therapy. FASEB J 2003;17:1986–95. Sales KJ, Katz AA, Howard B, Soeters RP, Millar RP, Jabbour HN. Cyclooxygenase-1 is up-regulated in cervical carcinomas: autocrine/ paracrine regulation of cyclooxygenase-2, prostaglandin E receptors, and angiogenic factors by cyclooxygenase-1. Cancer Res 2002; 62:424–32. Tsuji S, Kawano S, Sawaoka H, et al. Evidences for involvement of cyclooxygenase-2 in proliferation of two gastrointestinal cancer cell lines. Prostaglandins Leukot Essent Fatty Acids 1996;55:179–83. Battu S, Clement G, Heyman M, et al. Production of arachidonic acid metabolites by the colon adenocarcinoma cell line HT29cl.19a and their effect on chloride secretion. Cancer Lett 1997;116:213–23. Battu S, Chable-Rabinovitch H, Rigaud M, Beneytout JL. Cyclooxygenase-2 expression in human adenocarcinoma cell line HT29cl.19a. Anticancer Res 1998;18:2397–404. Battu S, Beneytout JL, Pairet M, Rigaud M. Cyclooxygenase-2 upregulation after FLAP tranfection in human adenocarcinoma cell line HT29cl.19A. FEBS Lett 1998;437:49–55.