Effects of a combination of docosahexaenoic acid and 1,4-phenylene bis(methylene) selenocyanate on cyclooxygenase 2, inducible nitric oxide synthase and ß-catenin pathways in colon cancer cells

Bhagavathi A. Narayanan, Narayanan K. Narayanan, Dhimant Desai, Brian Pittman and Bandaru S. Reddy1

Chemoprevention and Nutritional Carcinogenesis Program, Institute for Cancer Prevention Valhalla, NY 10595, USA

1 To whom correspondence should be addressed. Tel: +1 914 789 7149; Email: breddy{at}ifcp.us


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Epidemiological and preclinical studies suggest that diets that are rich in n-3 polyunsaturated fatty acids (PUFAs) and selenium (Se) reduce the risk of colon cancer. Studies conducted in our laboratory have indicated that synthetic organoselenium 1,4-phenylene bis(methylene) selenocyanate (p-XSC) is less toxic and more effective than inorganic Se and selenomethionine, the major Se compound in natural selenium yeast. Through cDNA microarray analysis, we have demonstrated earlier that the n-3 PUFA docosahexaenoic acid (DHA), modulated more than one signaling pathway by altering several genes involved in colon cancer growth. There is increasing interest in the use of combinations of low doses of chemopreventive agents that differ in their specific modes of action as this approach can minimize toxicity and increase efficacy in model assays. In the present study we assessed the efficacy of DHA and p-XSC individually and in combination at low doses in CaCo-2 colon cancer cells, using cell growth inhibition and apoptosis as measures of chemopreventive efficacy. On the basis of western blot and RT–PCR analysis, we also determined the effects of DHA and p-XSC on the levels of expression of cyclooxygenase-2, inducible nitric oxide synthase, cyclin D1, ß-catenin and nuclear factor {kappa}B, all of which presumably participate in colon carcinogenesis. A 48 h incubation of CaCo-2 cells with 5 µM each DHA or p-XSC induced cell growth inhibition and apoptosis and altered the expression of the above molecular parameters. Interestingly, the modulation of these cellular and molecular parameters was more pronounced in cells treated with low doses of DHA and p-XSC (2.5 µM each) in combination than in cells treated with these agents individually at higher concentrations (5.0 µM each). These findings are viewed as highly significant since they will provide the basis for the development of combinations of low dose regimens of DHA and p-XSC in preclinical models against colon carcinogenesis and, ultimately, in human clinical trials.

Abbreviations: COX-2, cyclooxygenase-2; DAPI, 4',6-diamidine phenylindole dihydrochloride; DHA, docosahexaenoic acid; FITC, fluorescein isothiocyanate; iNOS, inducible nitric oxide synthase; NF-{kappa}B, nuclear factor {kappa}B; PBS, phosphate-buffered saline; PUFAs, polyunsaturated fatty acids; p-XSC, 1,4-phenylene bis(methylene) selenocyanate


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Colon cancer, which is the fourth most common cancer in the world, is one of the leading causes of cancer death in both men and women in Western countries, including the USA (1,2). An expert panel assembled by the American Institute for Cancer Research/World Cancer Research Foundation came to a scientific consensus that there is evidence for a correlation between a high intake of saturated fats (and/or animal fat) and colon cancer risk (3). A recent ecological study suggests that mortality data for colorectal cancer in 22 European countries, the USA and Canada positively correlated with the consumption of animal fat and that mortality rates are inversely related to the consumption of fish and fish oil (4,5). There is also increasing evidence from preclinical efficacy studies that diets high in saturated fats and {Omega}-6 (n-6) polyunsaturated fatty acids (PUFAs) such as corn oil significantly increase chemically induced colon carcinogenesis, whereas dietary fish oil rich in n-3 PUFAs, including docosahexaenoic acid (DHA) and eicosapentaenoic acid, suppresses colon carcinogenesis (612). Although the mechanisms by which n-3 PUFAs inhibit the growth of colon tumor cells remain to be fully elucidated, existing evidence suggests that the inhibition of colon carcinogenesis by n-3 PUFAs is mediated, at least in part, through modulation of more than one signaling pathway, including down-regulation of ras p21, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and several other pro-inflammatory genes (10,1319). Importantly, our studies on global gene expression with cDNA microarrays indicate that treatment of human CaCo-2 colon cancer cells with DHA (Figure 1A) down-regulates the prostaglandin family of genes as well as COX-2, and iNOS expression (19). Also, treatment of CaCo-2 cells with DHA altered several cell cycle-related genes, while it up-regulated caspases 5, 8, 9 and 10, which are associated with apoptosis (18,19).



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Fig. 1. Chemical structure. (A) Docosahexaenoic acid (DHA); (B) 1,4-phenylene bis(methylene) selenocyanate (p-XSC).

 
There is also evidence from epidemiological studies that insufficient intake of the trace element selenium is related to an increased risk for colon cancer (20). Dr Larry Clark's randomized double blind trial of selenium-enriched yeast in patients with non-melanoma skin cancer led to the unexpected discovery that selenium protects against cancer of the colon, lung and prostate (21). Preclinical investigations have provided evidence that at doses well above the RDA (required daily allowance) inorganic selenium protects against colon carcinogenesis (22). However, inorganic and some naturally occurring selenium-containing amino acids, such as selenomethionine and selenocysteine, have considerable toxicity (23). Preclinical studies in our laboratory have indicated that certain synthetic organoselenium compounds, including 1,4-phenylene bis(methylene) selenocyanate (p-XSC) (Figure 1B) are less toxic but even more efficacious against colon carcinogenesis in preclinical models than the historically used selenium compounds, such as sodium selenite and selenomethionine (24,25). We have also observed that colon tumor inhibition by p-XSC is associated with an increase in apoptosis (26).

Intervention with a single chemopreventive agent is unlikely to be effective for secondary prevention of colorectal cancer in high risk patients. It is reasonable to expect that appropriate combinations of chemopreventive agents at lower doses rather than administering a single agent at a higher dose can lead to increased efficacy and minimized toxicity. We have generated convincing evidence that the combination approach does indeed provide greater efficacy than the administration of individual agents at higher doses (27). The proven efficacy of dietary n-3 PUFAs and p-XSC against colon carcinogenesis makes a compelling case for the full evaluation of these agents for their efficacy and mechanisms of action when administered in combination at low dose levels. Mindful of the significance of DHA and of the organoselenium compound p-XSC in colon carcinogenesis, the present study was designed to assess the effect of each agent individually and that of a combination of both on (i) cell growth and apoptosis, (ii) expression levels of ß-catenin and cyclin D1 and (iii) pro-inflammatory genes such as nuclear factor {kappa}B (NF-{kappa}B), COX-2 and iNOS in CaCo-2 human colon cancer cells. The rationale for determining ß-catenin expression levels was based on the observation that aberrant expression of ß-catenin occurred in colonic tumors (28).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Cell growth treatment
CaCo-2 cells, obtained from the American Type Culture Collection (Rockville, MD), were grown under cell culture conditions and maintained in a humidified environment at 37°C with 5% CO2 and by serial passage in RMPI containing 10% fetal bovine serum. DHA was purchased from Cayman (Ann Arbor, MI), and the organoselenium compound p-XSC was synthesized according to methods described previously (29). For stimulation, DHA and p-XSC were dissolved in DMSO and CaCo-2 cells were treated with final concentrations of 5 µM and 2.5 µM each DHA and p-XSC in the cell culture medium for 48 h. Control cultures treated with DMSO alone were processed similarly. The dose levels of DHA used in this study were based on our previous study in which 2.5 and 5.0 µM DHA down-regulated iNOS expression in CaCo-2 cells (19). The dose levels of p-XSC used in this current study were based on our preclinical studies in colon cancer (24) and on in vitro studies with mammary cancer cells (30).

Cell viability assay
Cell viability with each individual agent at 5 µM and in combination at 2.5 µM each was determined by the trypan blue (0.2%) exclusion assay as described earlier (19). Briefly, CaCo-2 cells were treated with 5 µM DHA or p-XSC when applied alone and 2.5 µM each agent in the combination study for 48 h. Adherent and floating cells were harvested by trypsinization and recovered by centrifugation. Immediate staining of cells with trypan blue enables easy identification of dead cells, as these take up the dye and appear blue with uneven cell membranes. In contrast, living cells repel the dye and appear refractile and colorless. Total numbers of viable cells in each experiment were compared with those of the parallel control cell counts performed simultaneously in three independent experiments.

Apoptosis detection
CaCo-2 cells that were untreated (control) or treated with DHA or p-XSC individually at 5.0 µM each or with a combination of both at 2.5 µM each for 48 h were harvested and stained with 4',6-diamidine phenylindole dihydrochloride (DAPI) to detect apoptotic cells. Briefly, after fixing the cells with 10% formalin for 15 min and washing with phosphate-buffered saline (PBS), the cells were then incubated with DAPI for 30 min. After washing with PBS, the stained cells were examined for changes indicating convoluted budding and blebbing of the membrane, chromatin aggregation and nuclear and cytoplasmic condensation pertaining to apoptosis using an epifluorescence microscope (AX-70; Olympus, Tokyo, Japan) as described earlier (19). In addition, Annexin V [fluorescein isothiocyanate (FITC)-conjugated] staining of the membrane for phosphatidylserine externalization in the apoptotic cells was performed in the control and in cells treated with DHA in combination with p-XSC. Annexin V-positive cells with the characteristic morphological changes of apoptosis were identified with an epifluorescence microscope. Necrotic cell death was assessed on the basis of (i) morphological evaluation by light microscopy of the integrity and nature of the cell membrane and the release of the cellular contents, which is different from apoptosis, showing condensation of the nuclear material associated with blebbing of the membrane indicative of apoptosis, and (ii) appearance of a DNA smear in an agarose gel instead of DNA ladder fragmentation, which is characteristic of apoptosis.

Cellular localization of ß-catenin
CaCo-2 cells that were untreated (control) or treated with DHA or p-XSC individually (5.0 µM each) or with a combination of DHA and p-XSC (2.5 µM each) for 48 h were fixed in 10% formalin, then treated with 0.1% Triton X-100 and 2 N HCl at 37°C for 10 min each and, finally, with 0.1 M sodium borate for 5 min and washed with PBS three times. Immunofluorescence detection of ß-catenin-positive cells was with anti-ß-catenin antibody (Santa Cruz Biotechnology, Santa Cruz, CA), followed by incubation with FITC-conjugated goat anti-mouse IgG for 30 min. An epifluorescence microscope (AX-70; Olympus, Japan) was used to detect the green fluorescence in ß-catenin-positive cells (nuclear and cytoplasmic). The positively stained cells were quantified with Image Pro plus software (Media Cybernetics, Silverspring, MD).

Western blot analyses for ß-catenin, COX-2, iNOS, NF-{kappa}Bp65 and cyclin D1 expressions
CaCo-2 cells treated with DHA or p-XSC individually (5 µM) or with a combination of both agents (2.5 µM each) for 48 h were harvested by trypsinization. Total protein was isolated with protein extraction buffer containing 150 mM NaCl, 10 mM Tris (pH 7.2), 5 mM EDTA, 0.1% Triton X-100, 5% glycerol and 2% SDS in addition to a cocktail of protease inhibitors (Boehringer Mannheim, Mannheim, Germany). Aliquots of protein (20 µg/lane) were fractionated on 10% SDS–PAGE gels and transferred to PVDF membranes. The western blot procedure was carried out as described earlier (19) with iNOS polyclonal antiserum and COX-2 antibody as primary antibodies (Cayman, Ann Arbor, MI). Antibodies against ß-catenin, cyclin D1 and NF-{kappa}Bp65 were purchased from Santa Cruz Biotechnology. Horseradish peroxidase-conjugated IgG was used as the secondary antibody and the reactive protein band was detected with chemiluminescence reagents (ECL; Amersham). Densitometric analysis of the protein bands was performed with Gel-Pro Analyzer software (Media Cybernetics).

Statistical analysis
All analyses were carried out in triplicate as parallel but independent experiments. Expression of COX-2 and associated genes was compared between the control and treatment groups using analysis of variance (ANOVA), followed by Tukey's multiple comparisons test (32).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Effect of DHA and p-XSC on cell viability
In this study we have determined the effects of 2.5 and 5.0 µM DHA and p-XSC individually and of a combination of 2.5 µM each agent on the viability of CaCo-2 colon cancer cells. DHA and p-XSC individually at 2.5 and 5.0 µM induced significant cell growth inhibition by reducing the number of viable cells compared with the control. A greater extent of cell death along with a few necrotic cells was observed at the 5 µM concentration of p-XSC alone after 48 h. Interestingly, an additive effect on cell viability was observed when cells were exposed to 2.5 µM DHA together with 2.5 µM p-XSC (P < 0.001) (Figure 2). Cell growth in terms of percentage viable cells was 58% with 2.5 µM DHA, 42% with 2.5 µM p-XSC and 26% with the combination of DHA and p-XSC (2.5 µM each) (Figure 2), i.e. cell growth inhibition with DHA and p-XSC (2.5 µM each) was 42 and 58%, respectively, and with the combination of DHA and p-XSC (2.5 µM of each) was 74%.



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Fig. 2. Cell growth inhibition. Cell viability was determined by the trypan blue (0.2%) exclusion assay as described in Materials and methods. Viability of CaCo-2 cells was analyzed after these cells were treated with 5 µM DHA or pXSC individually or a combination of 2.5 µM of each agent for 48 h. Each bar represents the mean percent viable cells measured in three parallel but independent experiments.

 
Induction of apoptosis
Apoptosis induced by DHA or p-XSC (individually and in combination) was measured by identifying cells that showed typical changes in the nuclear material. Apoptotic cells were visualized in terms of characteristic morphological changes determined by DAPI staining, namely blebbing of the membrane, chromatin aggregation and nuclear and cytoplasmic condensation (Figure 3A–D) and by localizing Annexin V-positive cells (Figure 3E and F). It is noteworthy that when the cells were exposed to a combination of DHA and p-XSC (2.5 µM each) greater apoptotic cell death was evident compared with the control.



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Fig. 3. Apoptosis. Individual and combined effects of DHA and pXSC on apoptosis in colon cancer cells were detected using DAPI staining as described in Materials and methods. CaCo-2 cells were treated with DHA or p-XSC for 48 h. (A) Control; (B) DHA (5 µM); (C) p-XSC (5 µM); (D) combined application of DHA (2.5 µM) with p-XSC (2.5 µM); (E) Annexin V staining, control; (F) Annexin V-positive apoptotic cells (arrow) with combined application of DHA (2.5 µM) with p-XSC (2.5 µM). See online supplementary material for a colour version of this figure.

 
A quantitative analysis of apoptotic cell death is summarized in Figure 4. In comparison with the untreated control, which shows 6% apoptosis, DHA and p-XSC at 5.0 µM effectively induced apoptosis in ~28 and 24% of CaCo-2 cells, respectively (P < 0.001). Thus, at high concentrations both agents induced apoptosis, however, treatment of the cells with the lower concentrations of DHA and p-XSC (2.5 µM each) in combination synergistically induced apoptosis more effectively (>50%, P < 0.001).



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Fig. 4. Quantitative analysis of apoptotic cells. Apoptotic cells determined with DAPI/Annexin V staining as described in Materials and methods. Each bar represents the percentage of apoptotic cells induced by DHA or p-XSC for 48 h.

 
Effect of DHA and p-XSC on ß-catenin expression
Immunofluorescence detection of ß-catenin in CaCo-2 cells treated with DHA or p-XSC, individually or in combination, is shown in Figure 5. We observed that treatment of CaCo-2 cells with DHA or p-XSC decreased the levels of ß-catenin expression in CaCo-2 cells compared with the untreated control. Green fluorescence detection at the cellular level indicated down-regulation of cytoplasmic and nuclear accumulation of ß-catenin, with very few cells showing a signal indicative of ß-catenin at the membrane level (Figure 5A–C). Although down-regulation of ß-catenin was observed upon treatment with the individual agents, it is noteworthy that a more significant decrease (>5-fold decrease) in expression of ß-catenin was observed in CaCo-2 cells treated with a combination of p-XSC and DHA at the lower dose levels (Figure 5D).



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Fig. 5. Immunofluorescence detection of ß-catenin. For the detection of ß-catenin CaCo-2 cells treated with DHA and p-XSC individually or in combination for 48 h were labeled with FITC-conjugated anti-ß-catenin antibody and ß-catenin-positive cells were detected as described in Materials and methods. (A) Control; (B) DHA (5 µM); (C) p-XSC (5 µM); (D) DHA (2.5 µM) + p-XSC (2.5 µM). See online supplementary material for a colour version of this figure.

 
Quantification of the ß-catenin signal intensities clearly showed significant differences in the expression levels between the control and individual agents (Figure 6, P < 0.01). It is noteworthy that the combination of DHA and p-XSC (2.5 µM each) reduced expression of ß-catenin >2-fold compared with the effect with each single agent at the higher dose (P < 0.01). However, western blot analysis for the expression of ß-catenin protein in total cell lysate indicated a significant (50%) down-regulation with DHA alone, in comparison with 90% inhibition when the cells were treated with a combination of DHA and p-XSC (2.5 µM each) for 48 h (Figure 7A).



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Fig. 6. Quantitative analysis of ß-catenin-positive cells. Each bar represents the mean percentage of ß-catenin-positive cells counted in three similar and independent experiments.

 


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Fig. 7. Western blots for molecular targets. Individual and combined effect of DHA and p-XSC on the expression of ß-catenin (A), COX-2 (B), iNOS (C), NF-{kappa}Bp65 (D) and cyclin D1 (E) proteins. Reactive protein bands were analyzed using ECL detection with specific antibodies as described in Materials and methods. Expressed proteins were quantified as a ratio to the control ß-actin. Each bar represents the approximate amount of protein corresponding to the banding pattern measured by densitometric analysis performed with the software Gel-Pro Analyzer (Media Cybernetics, Silver Spring, MD). The significant difference between treatment and control was determined from pair-wise comparisons and a P value <0.001 indicates a statistical difference, as explained under Results.

 
Effect of DHA and p-XSC on the expression of ß-catenin, cyclin D1, COX-2, iNOS and NF-{kappa}Bp65
To gain further insight into the molecular parameters involved in the progression of colon carcinogenesis we analyzed total cell lysate protein to detect changes in the expression levels of COX-2, iNOS, NF-{kappa}B p65, ß-catenin and cyclin D1 using western blot analysis (Figure 7). Our results indicate that treatment of CaCo-2 cells with the higher dose of 5 µM DHA alone moderately suppressed expression of ß-catenin. However, exposure to a combination of 2.5 µM each DHA and p-XSC remarkably reduced ß-catenin expression >2-fold in comparison with the control, suggesting a synergistic effect.

The level of expression of COX-2 protein was effectively reduced by DHA individually at the higher dose (P < 0.01), however, the degree of inhibition was even more pronounced, by >2-fold (P < 0.01) (Figure 7B), when low doses of DHA or p-XSC were tested. At higher doses iNOS expression was reduced by DHA alone, while DHA and p-XSC in combination at lower doses (2.5 µM each) produced a more effective reduction. In addition, the inhibition of iNOS expression by DHA alone and in combination with p-XSC is highly significant when compared with control cells (P < 0.01) (Figure 7C). The level of NF-{kappa}B p65 expression was also significantly reduced by a combination of DHA and p-XSC. Interestingly, although DHA alone at the higher dose of 5 µM reduced the level of expression of NF-{kappa}B, the low dose combination regimen enhanced the effect of p-XSC by altering the nuclear factor protein (Figure 7D). As indicated in Figure 7E, the level of cyclin D1 expression was effectively reduced by DHA, both individually at the high dose level and in combination with p-XSC at low dose when compared with the control (P < 0.01). Overall, these findings indicate that at high concentrations both agents modulated several molecular and cellular parameters relevant to colon carcinogenesis. A combination of these agents at low concentrations modulated pro-inflammatory, cell cycle regulatory and apoptosis-inducing proteins through a possible additive effect and thus induced protective anticancer effects.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
The present study is part of a large-scale investigation into the development of novel strategies for colon cancer prevention by means of combinations of low doses of potential chemopreventive agents. The rationale for using a combination of agents is to increase chemopreventive efficacy and to minimize side-effects associated with long-term administration of such agents. We have shown in our earlier studies that a combination of agents such as piroxicam, a non-steroidal anti-inflammatory drug, and difluoromethylornithine, an ornithine decarboxylase inhibitor, or the HMG-CoA reductase inhibitor lovastatin with the non-steroidal anti-inflammatory drug sulindac (27,29) produces significant synergistic efficacy against colon cancer development. In the present study we have examined the effects of low doses of DHA and p-XSC in combination for their efficacy against colon carcinogenesis, specifically targeting key molecular parameters encompassing cell adhesion and pro-inflammatory processes. This study demonstrates for the first time that in cell culture a combination of low doses of DHA and p-XSC suppresses colon cancer cell growth and induces apoptosis more effectively than high doses of the individual agents.

The combination of low doses of DHA and p-XSC also modulates several molecular parameters, including ß-catenin and cyclin D1, which have been associated with colon cancer progression. ß-Catenin is known to be a key component of the cadherin-mediated cell–cell adhesion system and an important molecule in the Wnt-APC signal transduction pathways (34). Accumulation of cytoplasmic and nuclear ß-catenin is associated with colon carcinogenesis (35). Recently, Yamada et al. (36) reported that ß-catenin accumulation results in the transcriptional activation of genes involved in proliferation and related oncogenic pathways (37). ß-Catenin gene mutations are associated with K-ras mutations and aberrant crypt foci in the colon, thus indicating a potential role in progression to malignant lesions (3840). These results of the present study with regard to alterations in the expression of cyclin D1 further support the concept that DHA and p-XSC in combination down-regulate the levels of cyclins involved in cell cycle regulatory pathways. A considerable body of data suggests that cyclin D1 mediates the effects of ß-catenin in mammary neoplasia (41). It can be expected, therefore, that transcriptionally active ß-catenin and its target gene cyclin D1, modulated by the combination of DHA and p-XSC, prevent a cascade of molecular changes involved in colon carcinogenesis.

Several pro-inflammatory pathway genes, including COX-2, NF-{kappa}B and iNOS, are known to be involved in the progression of colon carcinogenesis (18,42). In our earlier studies we demonstrated an effect of DHA on COX-2, iNOS and RXR-related genes that regulate colon cancer growth, development, differentiation and apoptosis (19). Preclinical studies clearly demonstrate that diets rich in {Omega}-3 PUFAs, including DHA, induce apoptosis or inhibit COX-2 and iNOS activity in colon tumors (10,4346). In the present study we have documented for the first time that co-treatment with a combination of low doses of DHA and p-XSC caused colon cancer cell growth inhibition and induced apoptosis and that these events are mediated through down-regulation of several molecular targets involved in the inflammation cascade and cell adhesion molecules.

In conclusion, we report here that in an in vitro system using colon cancer cell line CaCo-2 the efficacy of DHA and p-XSC in combination is mediated through a cascade of events in which the primary inhibitory effect may be on expression of ß-catenin, followed by reduced expression of the nuclear transcription factor NF-{kappa}B and COX-2 and iNOS. These events eventually impair the process of DNA synthesis and cell cycle regulatory events, as indicated by a reduced level of cyclin D1 expression. The above cellular effects make the cells susceptible to apoptosis and result in cell growth inhibition. The mode of action of these two agents in combination at low doses gives an insight into the changes occurring at both the metabolic and cellular levels. Our findings in the present study in conjunction with our earlier results (19) indicate a potential role for DHA in combination with p-XSC at low doses compared with the effect induced by DHA or p-XSC alone at higher doses. Combination studies of potential chemopreventive agents tested in in vitro cell culture models provide a lead to test such agents and dose regimens in preclinical models. In vivo studies in preclinical models using low doses of p-XSC in combination with DHA appear to be a highly promising approach that may evolve into a realistic chemopreventive strategy for colon cancer. This approach can be applied to other cancers.


    Supplementary material
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Supplementary material is available online at http://www.carcin.oupjournals.org/.


    Acknowledgments
 
We thank Dominic Nargi for his technical assistance, Ilse Hoffmann for editing and Laura McDermott for preparation of the manuscript. This work was supported in part by USPHS grants CA-37663, and CA-17613 from the National Cancer Institute.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 

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Received May 11, 2004; revised June 30, 2004; accepted July 25, 2004.