Chemopreventive effects of Coltect, a novel dietary supplement, alone and in combination with 5-aminosalicylic acid in 1,2-dimethylhydrazine-induced colon cancer in rats

Abstract

Objectives: Coltect is a novel dietary supplement containing curcumin, green tea and selenomethionine. Previous reports have suggested that these agents can prevent colorectal cancer (CRC). The present study examined the chemopreventive effect of Coltect alone or combined with 5-aminosalicylic acid (5-ASA) using the 1,2-dimethylhydrazine (DMH) model in rats.

Methods: The effect of Coltect was examined on HT-29 CRC cells by growth inhibition assay. Apoptosis was determined by annexin V-FITC/PI staining. Male rats were injected with DMH in vivo and treated with Coltect 150 mg/kg, 5-ASA 50 mg/kg or their combination, by oral gavage. Aberrant crypt foci (ACF) were identified by methylene blue staining.

Results: HT-29 cells exhibited a dose-dependent response to Coltect. Part of the growth inhibition can be explained by the induction of mild-moderate apoptosis in cancer cells (28%) compared with the untreated cells (10%). In the in vivo model, the average number of ACF was divided into small (1–3 crypts) or large (≥4 crypts). The Coltect compound reduced the number of small and large ACF similarly to 5-ASA (40% reduction). This reduction was amplified by combining the two agents (70% reduction).

Conclusion: Coltect inhibits the growth of colon cancer cells, induces apoptosis and inhibits ACF development. Furthermore, it augments the growth inhibitory effect of 5-ASA in vivo. This may be clinically important since this safe dietary supplement-drug combination can be administered as a chemopreventive regimen for the treatment of CRC.

Introduction

Colorectal cancer (CRC) is rated third among cancers in the Western world for both men and women and second in the leading causes of death due to cancer, carrying a 9% lifetime risk [Jemal et al. 2009]. It is estimated that there will be over one million new cases of CRC worldwide in 2010, with an almost 50% death rate [Jemal et al. 2009]. In the past 20 years a new dogma of cancer prevention has arisen, which entails the use of specific natural or chemical compounds to prevent, inhibit or reverse the carcinogenetic process before its development [Greenwald and Kelloff, 1996]. It involves the long-term administration of a plethora of oral agents that can inhibit the multistep process of CRC carcinogenesis from normal-appearing mucosa to adenoma to carcinoma [Half and Arber, 2009].

Aberrant crypt foci (ACF), which were originally described by Bird and colleagues [McLellan and Bird, 1991; Bird, 1987] in unsectioned murine colon tissue exposed to azoxymethane (AOM), a colon-specific carcinogen, were recognized as early preneoplastic lesions. Subsequently, ACF induced by several kinds of chemical carcinogens have been widely used as a biomarker in short-term tests for the prediction of mucosal malignant transformation. The search for new chemopreventive compounds with minimal toxicity raised a special interest in phytochemicals. The Coltect compound, developed by BioPro Pharmaceuticals (Caesarea, Israel), contains potent anti-inflammatory and antineoplastic natural herbs (e.g. curcumin, green tea and selenomethionine).

Curcumin (diferuloylmethane) is a polyphenol derived from the plant Curcuma longa and is the active ingredient of the Indian curry spice that is used as a food additive. Curcumin is known for its antitumor, anti-oxidant, anti-amyloid and anti-inflammatory properties [Goel et al. 2008; Sharma et al. 2005; Gafner et al. 2004]. Its anticancer effects derive from its ability to induce apoptosis and cell-cycle arrest in CRC cells without cytotoxic effects on normal cells [Villegas et al. 2008]. Among the many mechanisms explaining its anticancer effects, curcumin was shown to interfere with the activity of the transcription factor NF-kB, suppress EGFR gene expression, and inhibit β-catenin-mediated transactivation and COX-2 expression [Anand et al. 2008; Singh and Khar, 2006; Goel et al. 2001]. Previous studies in our laboratory have demonstrated a synergistic effect between celecoxib (Celebrex, Pfizer, New York, USA), a COX-2 inhibitor, and curcumin, in the growth inhibition of CRC cells in vitro and in vivo [Shpitz et al. 2006; Lev-Ari et al. 2005a, 2005b], while others [Volate et al. 2005] showed that curcumin alone had an effect in vivo, and decreased the number of ACF in rats after injection of AOM. The ability of curcumin to modulate ACF correlates with its ability to induce apoptosis.

Green tea extract contains various polyphenols (epicatechin, epicatechin gallate, epigallocatechin and epigallocatechin gallate), and is widely consumed worldwide. Studies have shown that the green tea extract containing epigallocatechin-3-gallate (EGCG) inhibited the formation of ACF in vivo [Dashwood et al. 1999; Xu et al. 1996]. EGCG, the most abundant catechin in green tea, was found to possess many biological activities, including anti-oxidative [Yang and Wang, 1993], anti-inflammatory [August et al. 1999] and antimutagenic [Roy et al. 2003] effects, in a variety of experimental models. Chen and colleagues [Chen et al. 2003] demonstrated that EGCG triggers apoptotic cell death in HT-29 CRC cells by inducing caspase-3 and caspase-9 activities. Green tea extract also decreased the AOM-induced ACF number in rats by decreasing β-catenin and cyclin D1 levels [Xiao et al. 2008].

Selenium is an essential trace element for humans and animals. Its therapeutic role has been largely attributed to its presence in selenoproteins as the 21st amino acid, selenocysteine. Jariwalla and colleagues [Jariwalla et al. 2008] also showed that organic selenium derivatives, namely methyl-l-selenocysteine and selenomethionine, induced apoptosis in many cancer cell lines, including colorectal, breast and hepatic cancer cells. A diet enriched with selenium significantly suppressed ACF and cancer formation in experimental animals [Hu et al. 2008; Finley and Davis, 2001].

5-aminosalicylic acid (5-ASA), an anti-inflammatory drug, has been in the front line of inflammatory bowel disease (IBD) therapy for more than half a century. It has limited systemic adverse effects and gastrointestinal toxicity and is tolerated by most patients [Bjarnason and Macpherson, 1993]. Recent epidemiological, experimental and clinical investigations suggest that regular intake of 5-ASA may reduce the occurrence of CRC in patients with IBD as it is rapidly metabolized in the gut mucosa [Allgayer and Kruis, 2002; Moody et al. 1996]. To date, there is no consensus regarding the efficacy of 5-ASA in the prevention of sporadic CRC.

The present study aimed to examine the chemopreventive effects of Coltect alone or combined with 5-ASA in the 1,2-dimethylhydrazine (DMH)-induced model of CRC in rats. Our data demonstrate that Coltect is comparable to 5-ASA in inhibiting the formation of ACF, and that it exerts a synergistic effect when they are combined.

Materials and methods

Chemicals and reagents

The human HT-29 CRC cell line was obtained from the American Type Culture Collection. The cells were grown and maintained in Dulbecco’s Modified Eagle’s Medium (Biological Industries, Kibbutz Beit Haemek, Israel) supplemented with 5% fetal bovine serum, 1% penicillin and 1% streptomycin at 37°C, in an atmosphere of 95% oxygen and 5% carbon dioxide (complete medium). The Coltect compound was generously provided by BioPro Pharmaceuticals (Caesarea, Israel). The compound contains curcumin (Curcuma longa extract [95% curcuminoids] mixed with C. longa powder 1 : 1), green tea (Camellia sinensis extract [60% polyphenols and 25% EGCG] in a 2 : 1 ratio) and traces of L-selenomethionine (0.1 mg/ml).

DMH, 25 mg/kg stock and dimethyl sulfoxide (DMSO) were purchased from Sigma (Rehovot, Israel). DMH was dissolved in saline and titrated with sodium hydroxide to pH 7.4. 5-ASA (Pentasa, Ferring, Israel) was kindly provided by Ferring Pharmaceuticals.

Cell growth inhibition assay

HT-29 cells were plated in duplicates at a density of 3 × 10⁴ cells in 12-well plates containing 1 ml of complete medium. Coltect or 0.1% DMSO, the drug vehicle, was added to the culture medium at the selected concentrations 24 h after plating. The number of viable cells was determined in duplicates after 72 h using a Coulter counter.

Detection of apoptosis

HT-29 CRC cells were seeded in 10-cm plates (1 × 10⁵ cells/plate). Coltect (20 µM) was added for 72 h to the culture medium. Following incubation, the cells were stained for apoptosis using the MEBCYTO^® Apoptosis Kit (MBL International Corporation) according to the manufacturer’s instructions. Staining of annexin V-FITC/PI was detected by flow cytometry on a FACSCalibur (Becton Dickinson, San Jose, CA, USA). The results were analyzed using CELLQuest software (Becton Dickinson).

Animals

A total of 40 6–8-week-old male Wistar rats (Harlan, Rehovot, Israel) were bred and housed in the animal facility at Tel Aviv Sourasky Medical Center in plastic cages covered with metal grids (5 rats/cage), in a temperature-controlled room (21–25°C) with a 12-h light/12-h dark cycle. The rats were held in quarantine for 1 day prior to the initiation of the study and had free access to regular food and water. The study was approved by the institutional committee for animal welfare of the Tel Aviv Sourasky Medical Center (protocol number 24-7-08).

Induction of ACF and experimental design

A total of 15 rats were divided into one nontreated control and two treatment groups. Ten rats were subcutaneously injected with 0.2 ml of DMH (30 mg/kg body weight) twice a week for 2 weeks, after which they were randomly divided into two treatment groups, the drug vehicle DMSO alone and Coltect compound (150 mg/kg). The drugs were administered by oral gavage, using a 20G venflon’s polyethylene tube. Thirty days after receiving the DMH injection, the rats were sacrificed by carbon dioxide flow followed by cervical dislocation. Their colons were removed and flushed with an isotonic saline solution. They were then opened longitudinally, cut into three parts of equal length and labeled as the proximal, middle and distal segments. The colons were laid open on Whatman filter paper and fixed flat in 4% buffered formalin solution. The ACF were visualized and counted using a methylene blue 0.2% solution. The colons were placed into the staining solution for 10–15 min, after which excess dye was removed by flushing the mucosal surface with saline solution. The colons were placed on a Petri dish and transilluminated under a dissecting microscope (×40). The number and multiplicity of ACF for each colonic segment was evaluated and recorded by a single investigator (BS) who was blinded for all study procedures. The number and multiplicity of ACF were recorded separately for each segment. ACF were defined as a single aberrant crypt, as well as foci that contained more than one crypt per ACF. To determine multiplicity of ACF, the number of crypts in each focus was counted and recorded.

Additional 25 rats were divided into one nontreated control and four treatment groups. A total of 20 rats were subcutaneously injected with 0.2 ml of DMH (30 mg/kg body weight) twice a week for 2 weeks, after which they were randomly divided into four treatment groups: the drug vehicle DMSO alone; Coltect compound (150 mg/kg); 5-ASA (50 mg/kg); combined treatment of Coltect (150 mg/kg) and 5-ASA (50 mg/kg). Five rats were injected with 0.2 ml of saline, fed with the drug vehicle DMSO, and served as the nontreated control group. The drugs were administered by oral gavage, using a 20G venflon polyethelene tube. Sixty days after the injections the rats were sacrificed and the ACF visualized as described above.

Statistical analysis

The results are presented as mean ± standard deviation. The data were analyzed using Student’s t-test and two-way analysis of variance. A p-value of 0.05 or less was considered as statistically significant.

Results

Inhibition of cell growth by Coltect

We assessed the effect of the Coltect compound on the growth of HT-29 human colon carcinoma cells. Treatment with Coltect at concentrations of 5–25 µM demonstrated a significant dose-dependent inhibition of cell growth (Figure 1). The half maximal inhibitory concentration (IC50) was achieved at approximately 15 µM. Similar results were obtained in the rat epithelial cell line IEC18, and the pancreatic cancer cell lines, Panc-1, Colo357 and MiaPaCa. Coltect significantly inhibited the growth of these cells in a time- and dose-dependent manner (data not shown).

Figure 1.

Effect of the Coltect compound on the growth of colorectal cancer HT-29 cells. HT-29 cells were exposed for 72 h to different concentrations of Coltect as indicated. The data are mean ± standard deviation values from three individual experiments performed in duplicates. Differences in cell growth after exposure to Coltect compared with the negative control cells were determined by the Student’s t-test. *p < 0.05.

Induction of apoptosis by Coltect

We evaluated the ability of Coltect, at a concentration of 20 µM, to induce apoptosis in HT-29 cells. HT-29 cells were treated with Coltect for 72 h and stained for apoptosis using an annexin V-FITC/PI apoptosis assay. The results showed that Coltect was able to induce apoptosis in these cells compared with the untreated cells (Figure 2). Thus, the total percentage of apoptotic cells in the Coltect-treated group, including both early and late apoptosis, was 28% compared with 10% in the untreated group.

Figure 2.

Effect of the Coltect compound on the induction of apoptosis in HT-29 cells. (a) Representative results of annexin-V-FITC/PI apoptosis assay analysis: untreated HT-29 cells (left); HT-29 treated with 20 µM Coltect for 72 h (right). Following treatment, the cells were harvested and double stained with annexin-V and PI (lower left, negative cells; lower right, early apoptosis; upper right, late apoptosis; upper left, necrosis). (b) Level of apoptosis in the untreated and Coltect-treated HT-29 cells. Values are mean percentages of total apoptotic cells, early and late apoptosis (error bars indicate standard deviations) obtained from three independent experiments of cells treated with Coltect compared with untreated cells (The numbers in the diagrams indicate the percentage of the cells).

Inhibition of ACF formation by Coltect

All rats survived to the end of the experiments. The animals’ weight in both experiments was comparable (data not shown). ACF were found only in DMH-treated rats and only in the middle and distal thirds of the colons. The data are therefore presented for these colonic sections. The ACF were considered as being either small (1–3 crypts/ACF) or large (≥4 crypts/ACF) (Figure 3).

Figure 3.

1,2-dimethylhydrazine-induced ACF in rats. (a) Methylene blue staining of normal appearing mucosa with no visible ACF. (b) Mucosa containing small ACF (1–3 crypts). (c) Mucosa containing large ACF (≥4 crypts). Crypts are indicated by arrows. ACF, aberrant crypt foci.

The first experiment was performed in order to ascertain the efficacy of the Coltect compound. Coltect indeed reduced the number of the DMH-induced total (p = 0.013), small (p = 0.023) and large ACF (p = 0.054) (Table 1). As DMH did not produce a large number of ACF, the treatment period was extended to 60 days.

Table 1.

Number of aberrant crypt foci per group.

Group	Total	1–3 crypts	≥4 crypts
Negative control	0	0	0
Positive control	30.75 ± 5.1	25.25 ± 5.6	5.5 ± 2.6
Coltect 150 mg/kg	18.8 ± 7.6*	16.4 ± 5.4*	2.4 ± 2.0

The average number ± standard deviation of total small (1–3 crypts) and large (≥4 crypts) aberrant crypt foci per group 30 days after 1,2-dimethylhydrazine injections. *p < 0.05, **p < 0.01 as determined by the Student’s t-test.

The second experiment compared Coltect with 5-ASA and examined their synergistic effect. The DMH induced many ACF, most of which were small (Table 2). There was a significant decrease in the number of small ACF in the Coltect and 5-ASA treatment when compared with the negative control group (p < 0.05). The protective effect of Coltect and 5-ASA was similar. Suppression of ACF formation was most evident in group 5. These rats were exposed to both agents, and their ACF was significantly lower than that in the rats who received monotherapy (for small and large ACF (p < 0.01) in comparison to the negative control). Total and small ACF in group 5 were significantly lower than the monotherapies (p < 0.01), indicating a synergistic inhibitory effect on ACF formation. The experiments were repeated twice and similar results were obtained (data not shown).

Table 2.

Number of aberrant crypt foci per group.

Group	Total	1–3 crypts	≥4 crypts
Negative control	0	0	0
Positive control	66.5 ± 8.2	47.5 ± 8.7	19 ± 5.3
Coltect 150 mg/kg	42 ± 10.1*	31 ± 5.9*	12 ± 5.4
5-ASA 50 mg/kg	37.2 ± 6.3*	28 ± 4.1*	11.6 ± 3.9
Coltect + 5-ASA	19.4 ± 3.4**	14 ± 6.3**	6 ± 1.1**

The average number ± standard deviation of total small (1–3 crypts) and large (≥4 crypts) aberrant crypt foci per group 60 days after 1,2-dimethylhydrazine injections. *p < 0.05, **p < 0.01 as determined by analysis of variance. 5-ASA, 5-aminosalicylic acid.

Discussion

The current study demonstrated that the combination of curcumin, green tea and selenomethionine can prevent malignant transformation in the colonic mucosa of rats. This effect is comparable to the preventive effect of 5-ASA. Combining the two agents prevented ACF formation in a synergistic manner.

One of the lessons learned from cancer research in recent years is that combination strategies in cancer therapy can provide dramatic improvement in safety and efficacy over monotherapy regimens, especially if the drugs differ in their mode of action. Several combinations of nonsteroidal anti-inflammatory drugs with other chemopreventive drugs have previously been investigated [Torrance et al. 2000; Agarwal et al. 1999]. Among these are celecoxib and curcumin, which in combination demonstrated a synergistic effect in inhibiting the growth of CRC cells in vitro and in vivo [Shpitz et al. 2006; Lev-Ari et al. 2005a, 2005,b]. Other studies have shown that curcumin and green tea synergistically inhibit cell proliferation and induce apoptosis in the ACF rat model and that a combination of celecoxib and difluoromethylornithine can reverse DNA hypomethylation on CRC in rats [Xu et al. 2010; Pereira et al. 2004]. Amantana and colleagues [Amantana et al. 2002] demonstrated that supplementing green tea with selenium increased the green tea’s antimutagenic effects. Therefore, the Coltect compound described herein is a novel mix of anticancer agents that seems to incorporate all the beneficial traits of these natural medicines in the prevention of ACF formation.

5-ASA, an aspirin derivative, is an anti-inflammatory drug that has been extensively used in the treatment of IBD. Our results show that it decreased the number of both small and large ACF (40% and 39%, respectively). These results coincide with those of other studies. MacGregor and colleagues [MacGregor et al. 2000] showed that the 5-ASA derivative balsalazide disodium, which is a colon-specific prodrug of 5-ASA, and 5-ASA itself induced a decrease in ACF formation in an AOM-induced ACF formation model in the rat colon by means of a mechanism involving inhibition of proliferation and induction of apoptosis. Stolfi and colleagues [Stolfi et al. 2008] used the HT-29 colon cancer cell line as a model for a COX-2-dependent inhibition pathway and DLD-1, a COX-deficient CRC cell line, as a model for a COX-2-independent inhibition pathway and reported that 5-ASA causes both a COX-2-dependent and COX-2-independent inhibition of CRC cell growth.

This research introduces the novel compound Coltect and its synergistic effect when combined with 5-ASA. As mentioned above, the Coltect compound consists of curcumin, green tea and selenomethionine. Our previous studies have shown that curcumin treatment alone reduced the number of ACF by 39% compared with an untreated control group [Shpitz et al. 2006]. Curcumin (0.6%) was administered in the animal chow and contained a 2.4-fold higher concentration than the one used herein. Thus, we were able to demonstrate comparable results induced by Coltect with a lower concentration of curcumin, implying a higher efficacy of the compound. Understanding the mechanism/s of action of Coltect and its individual components can aid in achieving improved performance. Further in vitro analysis is required to investigate the effects of Coltect on different cancer cell lines. We are aware of the limitations of the growth inhibition assay and we intend to investigate further the effects of Coltect using cell-cycle analysis. Our data indicate that Coltect induces mild to moderate apoptosis in CRC cells. Thus, additional in vivo studies can also be expanded and include further evaluation of the antitumor and pro-apoptotic activity of Coltect. This novel compound may be effective for the chemoprevention of other cancers as suggested by our preliminary data on pancreatic carcinoma cells.

Combination therapy can serve as an effective weapon against colon cancer. Our results showed a 70% decrease in both small and large ACF after treatment with both agents, thus clearly exhibiting a synergistic effect. In a clinical randomized, multicenter, double-blind, placebo-controlled trial that used curcumin and 5-ASA in ulcerative colitis, Hanai and colleagues [Hanai et al. 2006] showed that curcumin improved the life span and disease remission in patients taking curcumin and 5-ASA compared with patients taking 5-ASA and placebo, thus further supporting our current results.

In summary, the present study demonstrated that administration of the Coltect compound (150 mg/kg) or 5-ASA (50 mg/kg) decreases the number of ACF, while administration of both agents synergistically significantly suppresses the number of small (1–3 crypts/ACF) and large (≥4 crypts/ACF) ACF. This synergistic effect is clinically important because it can be an important tool in the prevention and treatment of CRC. This study has shown the therapeutic effect of Coltect and its synergistic effect with 5-ASA on ACF formation in a rat model. The data have demonstrated the effects of the different treatments mainly in the therapeutic aspect and have not specified the molecular mechanisms of the various treatments. We intend to investigate, with an assortment of methods, the signal transduction pathways affected by the treatments at the molecular level.

Funding

This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.

Conflicts of interest statement

None declared.