Chemopreventive Agents For Colon Cancer Research Articles

Abstract B13: In vitro animal cancer model for assessment of sulfated polysaccharides extract of Ulva lactuca as colon cancer therapeutic and chemopreventive agents

Abstract Purpose: Sulfated polysaccharides extract of Ulva lactuca (ULSP) was in vitro assessed as probable chemopreventive and therapeutic agents for colon cancer (CC) comparing with currently used chemotherapy (5-fluorouracil “5-FU” and mitomycin-C “MMC”) using our newly developed in vitro transformed cancerous rat colon epithelial cells. Methods: The present study used an in vitro animal CC model as it resembles human multistage of chromosomal instability type of CC that is located mainly at distal colon. Hence, this model was used for evaluating ULSP by the detection of cytotoxicity against normal and cancerous colon cells and antimetastatic potency. Besides, ULSP efficacy as CC chemopreventive agent was assessed through the detection of CC progression markers; loss of heterozygosity of deleted in colorectal cancer (DCC) and p53, aldehyde dehydrogenase (ALDH) 1B1 expression, activities of ornithine decarboxylase (ODC), cyclooxygenase-2 (COX-2) and alkaline phosphatase (ALP), cellular redox state (Eh), doubling time, and mucin level. Results: The apoptosis-dependent anticancer activity of ULSP (4.9 µg/ml) was revealed at all stages of cancerous distal colon epithelial cells by the formation of apoptotic bodies and DNA fragmentation (above 43%) without affecting normal colon epithelial cells. Also, its inhibitory effect on the invasion of cancerous epithelial and Caco-2 cells (above 49%) was stronger than that of 5-FU+MMC (about 11%). Cells were treated with ULSP before exposure to CC inducers and they showed normal tumor suppressor genes (DCC and p53), normal expression of ALDH1B1, and normal activity of ODC (0.03±0.0004 U/μg) and null activity of COX-2. In addition, their normal pattern of proliferations (doubling time 3 days and Eh 252.7±2.12 mV) and differentiation was confirmed by the maintenance of ALP activity (0.46±0.01 mU/μg) and the secretion of mucin (29.2±0.2 μg/ml) compared to normal and cancerous colon epithelial cells. Conclusion: Our innovative in vitro model of colon cancer have proved that ULSP could have induced apoptosis at all stages of cancerous colon cells, inhibited cancerous cells to metastasize and blocked CC progression without effect on normal epithelial cells comparable to chemotherapy. Citation Format: Marwa Abu-Serie, Maha El Demellawy, Mohamed El-Sayed, Fatma El-Rashidy. In vitro animal cancer model for assessment of sulfated polysaccharides extract of Ulva lactuca as colon cancer therapeutic and chemopreventive agents. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B13.

The inhibition of Typhonium flagelliforme Lodd. Blume leaf extract on COX-2 expression of WiDr colon cancer cells

ObjectiveTo determine the inhibition activity of Typhonium flagelliforme Lodd. Blume (T. flagelliforme) leaf extract on cyclooxygenase 2 (COX-2) expression of colon cancer cells. MethodsT. flagelliforme leaf extract was prepared to macerate in ethyl acetate. In vitro anticancer activity was assayed by MTT method on WiDr colon cancer cells. This study applied apoptosis induction assay to investigate the mechanism of cell death using double staining method. COX-2 expression was stained by immunocytochemistry. ResultsT. flagelliforme showed anticancer activity and induced apoptosis on WiDr cells through inhibition of COX-2 expression with IC50 70 μg/mL. ConclusionsThis study showed that T. flagelliforme is a promising chemopreventive agent for colon cancer through COX-2 inhibition.

Anticancer Effects of Curcuma C20-Dialdehyde against Colon and Cervical Cancer Cell Lines.

Recent attention on chemotherapeutic intervention against cancer has been focused on discovering and developing phytochemicals as anticancer agents with improved efficacy, low drug resistance and toxicity, low cost and limited adverse side effects. In this study, we investigated the effects of Curcuma C20-dialdehyde on growth, apoptosis and cell cycle arrest in colon and cervical cancer cell lines. Antiproliferative, apoptosis induction, and cell cycle arrest activities of Curcuma C20-dialdehyde were determined by WST cell proliferation assay, flow cytometric Alexa fluor 488-annexin V/propidium iodide (PI) staining and PI staining, respectively. Curcuma C20 dialdehyde suppressed the proliferation of HCT116, HT29 and HeLa cells, with IC50 values of 65.4±1.74 μg/ml, 58.4±5.20 μg/ml and 72.0±0.03 μg/ml, respectively, with 72 h exposure. Flow cytometric analysis revealed that percentages of early apoptotic cells increased in a dose-dependent manner upon exposure to Curcuma C20-dialdehyde. Furthermore, exposure to lower concentrations of this compound significantly induced cell cycle arrest at G1 phase for both HCT116 and HT29 cells, while higher concentrations increased sub-G1 populations. However, the concentrations used in this study could not induce cell cycle arrest but rather induced apoptotic cell death in HeLa cells. Our findings suggest that the phytochemical Curcuma C20-dialdehyde may be a potential antineoplastic agent for colon and cervical cancer chemotherapy and/or chemoprevention. Further studies are needed to characterize the drug target or mode of action of the Curcuma C20-dialdehyde as an anticancer agent.

Nexrutine inhibits azoxymethane-induced colonic aberrant crypt formation in rat colon and induced apoptotic cell death in colon adenocarcinoma cells.

Colon cancer is the third most common cause of death in the United States. Therefore, new preventive strategies are warranted for preventing colon cancer. Nexrutine (NX), an herbal extract from Phellodendron amurense, has been shown to have anti-inflammatory, anti-microbial and anti-cancer activity for various tissue specific cancers, but its chemopreventive efficacy has not been evaluated against colon cancer. Here, we explored the mechanism of chemopreventive/chemotherapeutic efficacy of NX against colon cancer. We found that dietary exposure of NX significantly reduced the number of azoxymethane (AOM)-induced aberrant crypt foci (ACF) in rats. In addition, significant inhibition in AOM-induced cell proliferation and reduced expression of the inflammatory markers COX-2, iNOS as well as the proliferative markers PCNA and cyclin D1 were also seen. Moreover, NX exposure significantly enhanced apoptosis in the colon of AOM treated rats. Furthermore, in in vitro studies, NX (2.5, 5, 10 μg/ml, 48 h) decreased cell survival and colony formation while inducing G0/G1 cell cycle arrest and apoptosis in colon adenocarcinoma cells COLO205 and HCT-15. However, NX had minimal cytotoxic effect on IEC-6 normal rat intestinal cells, suggesting its high therapeutic index. NX treatment also modulates the level of Bax and Bcl-2 proteins along with cytochrome c release, cleavage and enhanced expression of poly (adenosine diphosphate-ribose) polymerase as well as the catalytic activity of caspase 3 and caspase 9 in both COLO205 and HCT-15 cells. Based on these in vivo and in vitro findings, we suggest that NX could be useful candidate agent for colon cancer chemoprevention and treatment. © 2015 Wiley Periodicals, Inc.

Abstract 4658: Resveratrol aspirinate derivatives as novel chemopreventive agents for colon cancer

Abstract In more than a century of use, the benefits of aspirin have been exploited in different therapeutic areas, from inflammation to cardiovascular diseases and cancer prevention. However, the major limitation to the use of aspirin is its side effect to cause ulceration and bleeding in the gastrointestinal tract. The present study found that resveratrol, a natural chemopreventive agent from grapes and berries, and its methylated derivatives can react with acetylsalicyloyl chloride to form resveratrol aspirinate (RAS) and related derivatives. Among all the synthesized derivatives, we found that RAS is the most active component and have much stronger growth inhibitory effect on human colon cancer cells (HCT-116 and HT-29) than resveratrol and aspirin along, as well as the combination of resveratrol and aspirin. In addition, we found that RAS can be hydrolyzed to regenerate resveratrol and aspirin in mice and in human colon cancer cells. Therefore they serve as prodrugs of aspirin and resveratrol and its derivatives. Compared to simply combining aspirin and resveratrol, RAS has not only greater growth inhibitory effects on human colon cancer cells, but also can prevent the damage of gastric epithelial cells caused by direct contact between aspirin and gastric mucosa. Further mechanistic studies found that RAS inhibited cell growth through regulating cell cycle arrest via down-regulation of cyclins D1 and E, and inducing cancer cell apoptosis via mitochondrial pathway. In addition, two stable RAS analogues, for the first time, were successfully synthesized to demonstrate the importance of the scaffold composed of resveratrol and aspirin in the anti-cancer activity of RAS. Our results demonstrated for the first time that resveratrol based aspirin derivatives represent a brand-new, safer and improved alternative to aspirin in anti-cancer and anti-inflammatory therapies. Note: This abstract was not presented at the meeting. Citation Format: Shengmin Sang, Yingdong Zhu, Junsheng Fu, Kelly Shurlknight. Resveratrol aspirinate derivatives as novel chemopreventive agents for colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4658. doi:10.1158/1538-7445.AM2015-4658

Antioxidant and antitumor effects of polysaccharides from the fungus Pleurotus abalonus

Dietary supplement of edible Pleurotus abalonus (P. abalonus) rich in fungal polysaccharides is associated with anticancer health benefit. We here isolated the polysaccharides (PAP) from the fruiting bodies of P. abalonus, and evaluated the antiproliferative activity of the polysaccharides in human colorectal carcinoma LoVo cells. HPLC analysis showed that PAP consisted of d-mannose, d-ribose, l-rhamnose, d-glucuronic acid, d-glucose and d-galactose, and their corresponding mole percentages were 3.4%, 1.1%, 1.9%, 1.4%, 87.9% and 4.4%, respectively. PAP was shown to exert a high antioxidant activity in vitro and a dose-dependent antiproliferative effect against LoVo cancer cells. Flow cytometry analysis demonstrated that PAP exhibited a stimulatory effect on apoptosis of LoVo cells, and induced the cell-cycle arrest at the S phase. We also found that PAP could increase the generation of intracellular ROS which was a critical mediator in PAP-induced cell growth inhibition. These findings suggest that PAP may serve as a potential novel dietary agent for human colon cancer chemoprevention.

In vitro animal cancer model for assessment of sulfated polysaccharides extract of Ulva lactuca as colon cancer therapeutic and chemopreventive agents

In vitro animal cancer model for assessment of sulfated polysaccharides extract of Ulva lactuca as colon cancer therapeutic and chemopreventive agents

Chemopreventive effects of North American cranberry (Vaccinium Macrocarpon) on colitis‐associated colon carcinogenesis in mice.

North American cranberry (Vaccinium Macrocarpon), has been reported to have beneficial biological activities. In this study, we investigated the chemopreventive effects of North American cranberry on colitis‐associated colon carcinogenesis in azoxymethane (AOM)/dextran sulfate sodium (DSS)‐treated mice. It was observed that dietary administration of whole cranberry powder (WCP) significantly decreased colon tumor incidence, multiplicity, average tumor size and tumor burden. qRT‐PCR and ELISA results showed that both gene and protein expression levels of proinflammatory cytokines IL‐1, IL‐6 and TNF‐α were inhibited by WCP treatment. Immunoblotting results showed that administration of WCP resulted in profound effects on multiple proteins related with cell proliferation, inflammation and apoptosis. For example, WCP treatment significantly decreased the expression levels of COX‐2, PI3K/Akt, MMP‐2, MMP‐9, VEGF, EGFR, cyclin D, CDK4, Rb, and increased the levels of p53, p21, p27, cleaved caspase‐3 and cleaved PARP in colonic mucosa of AOM/DSS‐treated mice. Moreover, we found that treatment with WCP significantly enhanced the expression levels of phase II antioxidant enzymes, such as HO‐1 and NQO1 in colonic mucosa. Overall, our results demonstrated that dietary administration of WCP significantly inhibited colitis‐associated colon carcinogenesis in mice, providing a solid scientific basis for using North American cranberry as a chemopreventive agent for colon cancer in human.

Pro-Apoptotic Activities of Polyphenolics From Açai (Euterpe oleracea Martius) in Human SW-480 Colon Cancer Cells

This study aimed to evaluate the cell growth inhibition activity of açai (Euterpe oleracea Mart.) polyphenolic extract against colon cancer HT-29 and SW-480 cells and the nonmalignant CCD-18Co colon fibroblast cells. Results showed that açai polyphenolic extract (5–20 mg/L) inhibited preferentially the growth of SW-480 cells with no toxicity in CCD-18Co cells, and this was accompanied by reduction of H2O2-induced reactive oxygen species (ROS) generation. The mechanisms involved in SW-480 cell growth-inhibition by açai polyphenolic extract included the downregulation of NF-κB proinflammatory transcription factor and the nuclear factor-kappa B targets intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Furthermore, prooncogenic specificity proteins (Sp) were downregulated as well as Sp-targets Bcl-2, vascular endothelial growth factor, and survivin. This was accompanied by activation of mitochondrial proapoptotic pathway involving increase of cytochrome c, cleavage of caspase-3, and decrease of PARP-1. Results strongly suggest that açai polyphenolic extract has antiinflammatory and cytotoxic activities in colon cancer cells and can be effective as natural colon cancer chemopreventive agents.

Abstract 2513: NOSH-aspirin alone or in combination with 5-fluorouracil induces tumor regression in a xenograft model of estrogen receptor positive breast cancer

Abstract Introduction: In the US, breast cancer is the second leading cause of cancer deaths in women. Estrogen receptor positive [ER(+)] breast cancers represent 70-75% of human breast cancers. Selective estrogen receptor modulators such as tamoxifen, have a well-established role in preventing ER (+) breast cancer. However, these agents have limitations due to development of tamoxifen resistance. It is now well accepted that non-steroidal anti-inflammatory drugs (NSAIDs) reduce the risk of and mortality from colon cancer and constitute the prototypical colon cancer chemopreventive agents. Recent exhaustive meta-analysis also provides evidence that NSAID also reduce the risk for ER(+) breast cancer. However, regular NSAID use may lead to life threatening side effects. To overcome these limitations, we developed NOSH-aspirin, a nitric oxide-, and hydrogen sulfide-releasing hybrid that is very potent yet devoid of any cyto-toxicity. Here we report on the effects of NOSH-aspirin alone or in combination with 5-fluorouracil (5-FU) on treatment of established tumors in a xenograft model of human ER(+) breast cancer. Methods: NOSH-aspirin was synthesized and purified by us. Cell line: breast MCF-7 (ER+), Xenografts: Female athymic nude mice (N=25) were implanted s.c. in the right flank with MCF-7 cells (2 x 106), when the tumor volumes reached ∼100 mm3, the animals were randomly divided into 5 groups (N=5/gp) and treated with NOSH-aspirin (po,50 and 100 mg/kg body wt/day), 5-FU (ip, 10 mg/kg x 2/week), high dose NOSH-aspirin+5-FU as above, or vehicle (po). Tumor volume and animal weight were recorded every 3 days. After 3 weeks of treatment, mice were sacrificed, tumors excised, weighed, and fixed in 10% buffered formalin for IHC studies. We also weighed the livers and the kidneys. Results: NOSH-aspirin dose-dependently reduced tumor volume as a function of time. 5-FU also significantly reduced tumor volume, however, there was a synergistic effect in the combination group. Tumor mass at sacrifice in each group was, vehicle (2.35±0.22g), NOSH-aspirin 50 mg/kg (0.80±0.28g, 66% reduction, P<0.01), NOSH-aspirin 100 mg/kg (0.17±0.08g, 93% reduction, P<0.001), 5-FU (0.065±0.024g, 97% reduction, P<0.001), high dose NOSH-aspirin+5-FU (0.018±0.007g 99% reduction P<0.001). The tumor regression was significantly more in the combination group compared to NOSH-aspirin or 5-FU alone. NOSH-aspirin had no effect on the weight of the mice whether alone or in combination with 5-FU, there were no overt signs of toxicity. There was a 10% reduction in the weight of animals in the 5-FU group; however, this difference was not significant. There were no differences in liver or kidney masses amongst the groups. Conclusions: NOSH-aspirin has potent anti-cancer properties and demonstrates synergistic properties with 5-FU. It merits further evaluation as a chemotherapeutic agent. Citation Format: Sarin A. Soyemi, Mitali Chattopadhyay, Khosrow Kashfi. NOSH-aspirin alone or in combination with 5-fluorouracil induces tumor regression in a xenograft model of estrogen receptor positive breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2513. doi:10.1158/1538-7445.AM2014-2513

4′-Geranyloxyferulic acid: an overview of its potentialities as an anti-cancer and anti-inflammatory agent

3-(4′-Geranyloxy-3′-methoxyphenyl)-2-trans propenoic acid (4′-geranyloxyferulic acid, GOFA) 1 is an oxyprenylated ferulic acid derivative that has been extracted for the first time in 1966 from the root and bark extracts of Acronychia baueri Schott (Fam. Rutaceae). In the last decade the pharmacological properties of the title compound began to be characterized, revealing its valuable potentialities as an anti-inflammatory and anti-tumor agent. In this context the aim of this review article is to report the insofar described biological effects of 4′-geranyloxyferulic acid. Data collected during the recent years indicates that 1 is an effective dietary feeding colon cancer chemopreventive agent in vivo. When administered to animals, in which colon adenoma and adenocarcinoma have been induced by the administration of azoxymethane in the diet, as a large bowel delivered prodrug linked to dipeptides, or as an inclusion complex in cyclodextrins, or coupled to known anti-inflammatory agents like L-NAME, a protective effect on colon cancer growth and development have been recorded. Recent studies on the natural occurrence of 4′-geranyloxyferulic acid, showed the presence of this oxyprenylated secondary metabolite in widely consumed edible fruits and vegetables like citrus fruits and their phytopreparations.

Kynurenic acid inhibits colon cancer proliferation in vitro: effects on signaling pathways.

Kynurenic acid (KYNA), a tryptophan metabolite, inhibits proliferation of several cancer cell lines including colon cancer, renal cancer and glioblastoma cells. Previous studies reported that inhibitory properties of KYNA may be related to interactions of KYNA with cell cycle regulators and signaling proteins. However, the exact molecular interaction of KYNA with signaling pathways in colon cancer cells has not been studied to date. The molecular mechanism of KYNA activity towards colon cancer cells may be of great importance taking into consideration that KYNA is present in several tissues and physiological fluids, including gastrointestinal tract, and it is also present in various products of human diet. In this study, the inhibitory effect of KYNA on activation of phosphoinositide 3-kinase/Akt (PI3K/Akt) and mitogen-activated protein kinase (MAPK) signaling pathways in colon adenocarcinoma HT-29 cells was revealed. KYNA decreased phosphorylation of Akt, ERK 1/2 and p38 kinases in HT-29 cells. Interestingly, the study revealed also unexpected effect of KYNA on Wnt pathway in HT-29 cells. KYNA in millimolar concentrations increased protein expression of β-catenin. However, the nuclear translocation of β-catenin in HT-29 cells exposed to KYNA was not observed. Moreover, KYNA 1 mM increased antiproliferative properties of inhibitors of signaling pathways: wortmannin, PD98059, SB202190 and IWR-1. Taking into consideration these results, KYNA may be seen as a potential chemopreventive agent in colon cancer or supportive agent in standard cancer chemotherapy. However, the interactions between KYNA, Wnt signaling pathway and β-catenin need further studies to exclude potential effect of KYNA on colon carcinogenesis.

Cytotoxic and apoptotic activities of Amorphophallus campanulatus (Roxb.) Bl. tuber extracts against human colon carcinoma cell line HCT-15

Colorectal cancer is one of the leading causes of cancer death worldwide and is the third most common form of malignancy in both men and women. Several possible colon cancer chemopreventive agents are found in edible plants. Amorphophallus campanulatus (Roxb.) Blume (family: Araceae) is a tuber crop, largely cultivated throughout the plains of India for using its corm as food. This tuber has also been traditionally used for the treatment of abdominal tumors, liver diseases, piles etc. The aim of this study was to evaluate the dose-dependent cytotoxic and apoptosis inducing effects of the sub fractions of A. campanulatus tuber methanolic extract (ACME) viz. petroleum ether fraction (PEF), chloroform fraction (CHF), ethyl acetate fraction (EAF) and methanolic fraction (MEF) on the colon cancer cell line, HCT-15. Antiproliferative effects of the sub fractions of ACME were studied by MTT assay. Apoptotic activity was assessed by DAPI, Annexin V-FITC and JC-1 fluorescent staining. The chemotherapeutic drug, 5-flurouracil (5-FU) was used as positive drug control. The sub fractions of ACME significantly inhibited the proliferation of HCT-15 cells in a dose-dependent manner. In addition, the extracts were found to induce apoptosis and were confirmed by DAPI, Annexin V-FITC and JC-1 fluorescent staining. A pronounced results of cytotoxic and apoptotic activities were observed in the cells treated with 5-FU and CHF, whereas, EAF and MEF treated cells exhibited a moderate result and the least effect was observed in PEF treated cells. Our results suggested that, among the sub fractions of ACME, CHF had potent cytotoxic and apoptotic activity and thus it could be explored as a novel target for anticancer drug development. Furthermore, these findings confirm that the sub fractions of ACME dose-dependently suppress the proliferation of HCT-15 cells by inducing apoptosis.

Oral supplementation with troxerutin (trihydroxyethylrutin), modulates lipid peroxidation and antioxidant status in 1,2-dimethylhydrazine-induced rat colon carcinogenesis

The present study was aimed to investigate the chemopreventive potential of troxerutin on 1,2-dimethylhydrazine (DMH) induced rat colon carcinogenesis by evaluating the antioxidant and lipid peroxidation (LPO) status. Rats were randomly divided into six groups. Group I rats served as control. Group II rats received troxerutin (50mg/kgb.w., p.o.) for 16 weeks. Groups III–VI rats received subcutaneous injections of DMH (20mg/kgb.w., s.c.) once a week, for the first 4 weeks. In addition to DMH, groups IV–VI rats received troxerutin at the doses of 12.5, 25 and 50mg/kgb.w., respectively. In DMH treated rats, our results showed decreased activities of antioxidants and increased levels of LPO in the liver. Moreover, LPO and antioxidants in the colon were found to be significantly diminished in DMH the treated rats. Furthermore, enhanced activity of colonic vitamin C and vitamin E levels were observed in DMH alone treated rats (group III), which was significantly reversed on troxerutin supplementation. Troxerutin at the dose of 25mg/kgb.w. had shown profound beneficial effects by exhibiting near normal biochemical profile and well-preserved colon histology as compared to the other two tested doses (12.5 and 50mg/kgb.w.). These findings suggest that troxerutin could serve as a novel agent for colon cancer chemoprevention.

Multitargeted Low-Dose GLAD Combination Chemoprevention: A Novel and Promising Approach to Combat Colon Carcinogenesis

Preclinical studies have shown that gefitinib, licofelone, atorvastatin, and α-difluoromethylornithine (GLAD) are promising colon cancer chemopreventive agents. Because low-dose combination regimens can offer potential additive or synergistic effects without toxicity, GLAD combination was tested for toxicity and chemopreventive efficacy for suppression of intestinal tumorigenesis in adenomatous polyposis coli (APC)Min/+ mice. Six-week-old wild-type and APCMin/+ mice were fed modified American Institute of Nutrition 76A diets with or without GLAD (25 + 50 + 50 + 500 ppm) for 14 weeks. Dietary GLAD caused no signs of toxicity based on organ pathology and liver enzyme profiles. GLAD feeding strongly inhibited (80–83%, P < .0001) total intestinal tumor multiplicity and size in APCMin/+ mice (means ± SEM tumors for control vs GLAD were 67.1 ± 5.4 vs 11.3 ± 1.1 in males and 72.3 ± 8.9 vs 14.5 ± 2.8 in females). Mice fed GLAD had >95% fewer polyps with sizes of >2 mm compared with control mice and showed 75% and 85% inhibition of colonic tumors in males and females, respectively. Molecular analyses of polyps suggested that GLAD exerts efficacy by inhibiting cell proliferation, inducing apoptosis, decreasing β-catenin and caveolin-1 levels, increasing caspase-3 cleavage and p21, and modulating expression profile of inflammatory cytokines. These observations demonstrate that GLAD, a novel cocktail of chemopreventive agents at very low doses, suppresses intestinal tumorigenesis in APCMin/+ mice with no toxicity. This novel strategy to prevent colorectal cancer is an important step in developing agents with high efficacy without unwanted side effects.

Grape seed proanthocyanidins inhibit colon cancer-induced angiogenesis through suppressing the expression of VEGF and Angl

Tumor cells trigger angiogenesis through overexpression of various angiogenic factors including vascular endothelial growth factor (VEGF) and angiopoietin 1 (Ang1). Therefore, inhibition of the expression of both VEGF and Ang1, the initial step of tumor angiogenesis, is a promising strategy for cancer chemoprevention and therapy. Grape seed proanthocyanidins (GSPs) are widely consumed dietary supplements that have antitumor activity. Due to their polymeric structure, GSPs are poorly absorbed along the gastrointestinal tract and can reach the colon at high concentrations, allowing these chemicals to act as chemopreventive agents for colon cancer. In the present study, we found that GSPs inhibited colon tumor-induced angiogenesis and, thus, the growth of colon tumor xenografts on the chick chorioallantoic membranes. The mechanisms of their action were related to inhibiting the expression of both VEGF and Ang1 through scavenging reactive oxygen species. Previous studies have demonstrated that the chemopreventive effects of GSPs on colon cancer are associated with their growth inhibitory and apoptosis-inducing effects. Our results demonstrate another mechanism by which GSPs inhibit colon tumor growth, which will be helpful for developing GSPs as a pharmacologically safe angiopreventive agent against colorectal cancer.

Two novel inclusion complexes of colon cancer chemopreventers, Auraptene and 4'-Geranyloxyferulic acid

In recent years we have demonstrated that two prenyloxyphenylpropanoids, namely the geranyloxycoumarin auraptene, widespread in edible fruits of plants belonging to the genus Citrus, and 4'-geranyloxyferulic acid, extracted from the Australian shrub Acronychia baueri Schott (Rutaceae), exert valuable effects as dietary feeding colon cancer chemopreventive agents.1 The same was seen for the well known iNOS inhibitor N-nitro-L-arginine methyl ester (L-NAME).2 As a continuation of our studies about physico-chemical and pharmacological properties of cyclodextrin-inclusion compounds of prenyloxyphenylpropanoids, we describe herein the synthesis and structural characterization of complexes between auraptene, 4'-geranyloxyferulic acid, each coupled to L-NAME, and β-cyclodextrin to be used as novel prodrugs of the title cancer chemopreventers.

Silibinin, a natural flavonoid, modulates the early expression of chemoprevention biomarkers in a preclinical model of colon carcinogenesis

The flavonolignan silibinin, the major biologically active compound of the milk thistle (Silybum marianum), has been shown to possess anticancer properties in a variety of epithelial cancers. The present study investigated the potential of silibinin as a chemopreventive agent in colon carcinogenesis. The rat azoxymethane (AOM)-induced colon carcinogenesis model was used because of its molecular and clinical similarities to sporadic human colorectal cancer. One week after AOM injection (post-initiation), Wistar rats received daily intragastric feeding of 300 mg silibinin/kg body weight per day until their sacrifice after 7 weeks of treatment. Silibinin-treated rats exhibited a 2-fold reduction in the number of AOM-induced hyperproliferative crypts and aberrant crypt foci in the colon compared to AOM-injected control rats receiving the vehicle. Silibinin-induced apoptosis in the colon mucosal cells was demonstrated by flow cytometry after propodium iodide staining and by colorimetric measurement of caspase-3 activity. Mechanisms involved in silibinin-induced apoptosis included the downregulation of the anti-apoptotic protein Bcl-2 and upregulation of the pro-apoptotic protein Bax, inverting the Bcl-2/Bax ratio to <1. This modulation already takes place at the mRNA expression level as shown by real-time RT-PCR. Furthermore, silibinin treatment significantly (P<0.01) decreased the genetic expression of biomarkers of the inflammatory response such as IL1β, TNFα and their downstream target MMP7, all of them shown to be upregulated during colon carcinogenesis. The downregulation of MMP7 protein was confirmed by western blot analysis. The present findings show the ability of silibinin to shift the disturbed balance between cell renewal and cell death in colon carcinogenesis in rats previously injected with the carcinogen AOM. Silibinin administered via intragastric feeding exhibited potent pro-apoptotic, anti-inflammatory and multi-targeted effects at the molecular level. The effective reduction of preneoplastic lesions by silibinin supports its use as a natural agent for colon cancer chemoprevention.

Abstract 532: Nod1 functions in T cells to suppress inflammation-associated colorectal cancer

Abstract A major risk factor for the development of colorectal cancer is the presence of chronic inflammation within the colon. Innate immune receptors have emerged as key players in regulating intestinal homeostasis and the development of colorectal cancer in mouse models. There are two major classes of innate immune receptors, the Toll-like receptors and the Nod-like receptors (NLRs), which are important in pathogen recognition and eradication, but are also capable of sensing commensal bacteria within the gut. We have previously demonstrated that the ubiquitously expressed NLR family member, Nod1, acts as a tumor suppressor in the setting of chronic inflammation. The purpose of the current study is to better understand how Nod1 suppresses tumor development, which would be important in developing a chemoprevention strategy for inflammation-associated colon carcinogenesis using available Nod1 ligands. Using a mouse model of colitis-associated colon cancer in which mice are given a single intraperitoneal injection of the carcinogen, azoxymethane, followed by multiple rounds of water containing dextran sulfate sodium to induce chronic colitis, we generated bone marrow chimeras to determine whether Nod1 functions to suppress inflammation-related tumors in the intestinal epithelial versus hematopoietic compartment. We also performed adoptive transfer experiments to specifically determine the role of Nod1 in T cells in tumor suppression. Finally, Nod1 mice were crossed with T-cell deficient, TCRβ-/- mice, to further determine T-cell intrinsic properties of Nod1 in limiting tumor potential. Real-time PCR analysis and cultures of colon lamina propria cells from wildtype and Nod1-deficient mice were performed to determine recruitment of and production of cytokines by T cells within the colon. Our studies suggest that Nod1 functions in the hematopoietic cell compartment to protect against inflammation-induced tumorigenesis. More specifically, adoptive transfer of wildtype T cells was capable of rescuing Nod1-deficient mice from developing significant numbers of tumors, suggesting that Nod1 signaling in T cells is important for tumor suppression. Consistent with a T cell-specific function for Nod1 in tumor suppression, adoptive transfer of Nod1-deficient T cells in either TCRβ-deficient or TCRβ/Nod1 doubly-deficient mice resulted in significant numbers of tumors in contrast to adoptive transfer of wildtype T cells, which resulted in fewer tumors. Cytokine expression analysis of colon tissue as well as colon lamina propria cells demonstrated that Nod1-deficient mice exhibit an impairment in IFN-γ production during inflammation. These studies suggest a T-cell intrinsic role for Nod1 in protecting against inflammation-associated colon neoplasia, which may be related to a defect in immune surveillance. These observations set the stage for development of Nod1 agonists as colon cancer chemopreventive agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 532. doi:1538-7445.AM2012-532

Epigenetic inactivation of calcium-sensing receptor in colorectal carcinogenesis

Ca2+ is a chemopreventive agent for colon cancer. Ion transport systems are often altered in human cancer. The aim of this study was to clarify the alterations of calcium-sensing receptor (CASR), a member of the G protein-coupled receptor family, in colorectal carcinogenesis. We analyzed the expression of CASR in colorectal cancer cell lines and in cancer and adenoma tissues by RT-PCR and immunostaining. In addition, we analyzed methylation of the CASR promoter by using bisulfite sequence analysis and methylation-specific PCR. CASR mRNA and protein expression was significantly downregulated in most of the cancer cell lines. CpG islands were densely methylated in cancer cell lines with reduced CASR mRNA expression. Treatment with a demethylating agent, 5-aza-2′-deoxycytidine, and/or a histone deacetylase inhibitor, trichostatin A, restored CASR expression in the cancer cell lines. Disruption of CASR expression in CASR-unmethylated HCT-8 cells blocked the enhancing effect of Ca2+ on the cytotoxic response to 5-fluorouracil. CASR expression was observed in normal colonic epithelial cells and was retained in most adenoma tissues. CASR mRNA and protein expression was significantly downregulated in cancer tissues. There was an inverse relationship between CASR expression and degree of differentiation. Immunohistochemical CASR staining was reduced more predominantly in less-differentiated cancer tissues and/or in cancer cells at the invasive front, where nuclear/cytoplasmic β-catenin was often localized. CASR methylation was detected in 69% of colorectal cancer tissues and 90% of lymph node metastatic tissues and was significantly correlated with reduced CASR expression. CASR methylation was also detected in 32% of advanced adenoma tissues but was detected in only 9% of adenoma tissues and was not detected in hyperplastic polyp tissues. CASR methylation seems to occur at an early stage and progress in colorectal carcinogenesis. The results suggest that epigenetic inactivation of CASR has an important role in colorectal carcinogenesis.