A Dinucleotide Deletion in the CD24 Gene Is a Potential Risk Factor for Colorectal Cancer.

BACKGROUND: CD24 is a sialoglycoprotein anchored to the cell surface via glycosylphosphatidylinositol and is involved in intracellular signaling processes. It plays an important role in the early stages of the multistep process of colorectal carcinogenesis. Several single nucleotide polymorphisms in the CD24 gene are reported to exert a diverse effect on cancer risk. We aimed to elucidate whether CD24 TG/del genetic variants are associated with susceptibility to colorectal cancer (CRC). METHODS: The study included 179 subjects, 36 with CRC (prior to surgery) and 143 healthy control subjects. Deoxyribonucleic acid was purified from peripheral blood leukocytes, and by using restriction fragment length polymorphism analysis, the CD24 gene was genotyped for the specific genetic variant, TG deletion. Additionally, CD24 protein expression levels were determined by Western blotting analysis. RESULTS: The incidence of the TG/del was higher among the CRC patients compared with healthy controls, 14% and 10%, respectively (P = .54). CD24 protein levels were significantly higher among CRC patients. There were no significant differences in CD24 expression between CRC patients at different stages of the disease or between patients who carry the mutation and those who did not. CONCLUSIONS: CD24 genetic variant might be of clinical value for risk assessment as part of cancer prevention programs. Further study on larger populations is needed to validate the importance of this dinucleotide deletion in CRC development. Overexpression of CD24 protein occurs early along the multistep process of CRC carcinogenesis, and a simple blood sample based on CD24 expression on peripheral blood leukocytes can contribute to early diagnosis.

Selective eradication of cancer cells by delivery of adenovirus-based toxins.

BACKGROUND AND OBJECTIVE: KRAS mutation is an early event in colorectal cancer carcinogenesis. We previously reported that a recombinant adenovirus, carrying a pro-apoptotic gene (PUMA) under the regulation of Ets/AP1 (RAS-responsive elements) suppressed the growth of cancer cells harboring hyperactive KRAS. We propose to exploit the hyperactive RAS pathway, rather than to inhibit it as was previously tried and failed repeatedly. We aim to improve efficacy by substituting PUMA with a more potent toxin, the bacterial MazF-MazE toxin-antitoxin system, under a very tight regulation. RESULTS: A massive cell death, in a dose-dependent manner, reaching 73% at MOI 10 was seen in KRAS cells as compared to 22% in WT cells. Increase expression of MazE (the anti-toxin) protected normal cells from any possible internal or external leakage of the system and confirmed the selectivity, specificity and safety of the targeting system. Considerable tumor shrinkage (61%) was demonstrated in vivo following MazEF-encoding adenovirus treatment without any side effects. DESIGN: Efficient vectors for cancer-directed gene delivery were constructed; "pAdEasy-Py4-SV40mP-mCherry-MazF""pAdEasy-Py4-SV40mP-mCherry-MazF-IRES-TetR-CMVmp-MazE-IRES-EGFP","pAdEasy-ΔPy4-SV40mP-mCherry-MazF-IRES-TetR-CMVmp-MazE-IRES-EGFP "and "pAdEasy-mCherry". Virus particles were produced and their potency was tested. Cell death was measured qualitatively by using the fluorescent microscopy and colony formation assay, and was quantified by MTT. FACS analysis using annexin V and RedDot2 dyes was performed for measuring apoptotic and dead cells, respectively. In vivo tumor formation was measured in a xenograft model. CONCLUSIONS: A proof of concept for a novel cancer safe and effective gene therapy exploiting an aberrant hyperactive pathway is achievable.

Terpinen-4-ol: A Novel and Promising Therapeutic Agent for Human Gastrointestinal Cancers.

BACKGROUND: Terpinen-4-ol, a naturally occurring monoterpene is the main bioactive component of tea-tree oil and has been shown to have many biological activities. AIM: To study the antitumor effects of terpinen-4-ol and its mechanism of action in prostate and GI malignancies, alone and in combination with chemotherapeutic and biological agents. METHODS: Terpinen-4-ol was administrated alone or combined with standard chemotherapy (Oxaliplatin, Fluorouracil, Gemcitabine, Tarceva) and biological agent (Cetuximab). It was also combined with humanized anti-CD24 mAbs (was developed by us). Killing effects were measured qualitatively by light microscopy and quantitatively using the MTT and FACS analysis, following treatment of colorectal, pancreatic, gastric and prostate cancer cells. Terpinen-4-ol effect on tumor development was evaluated in xenograft model. RESULTS: Terpinen-4-ol induces a significant growth inhibition of colorectal, pancreatic, prostate and gastric cancer cells in a dose-dependent manner (10-90% in 0.005-0.1%). Terpinen-4-ol and various anti-cancer agents (0.2µM oxaliplatin and 0.5µM fluorouracil) demonstrated a synergistic inhibitory effect (83% and 91%, respectively) on cancer cell proliferation. In KRAS mutated colorectal cancer cells, which are resistant to anti-EGFR therapy, combining of terpinen-4-ol with cetuximab (1 µM) resulted in impressive efficacy of 80-90% growth inhibition. Sub-toxic concentrations of terpinen-4-ol potentiate anti-CD24 mAb (150µg/ml)-induced growth inhibition (90%). Considerable reduction in tumor volume was seen following terpinen-4-ol (0.2%) treatment alone and with cetuximab (10mg/kg) (40% and 63%, respectively) as compare to the control group. CONCLUSION: Terpinen-4-ol significantly enhances the effect of several chemotherapeutic and biological agents. The possible molecular mechanism for its activity involves induction of cell-death rendering this compound as a potential anti-cancer drug alone and in combination in the treatment of numerous malignancies. Terpinen-4-ol restores the activity of cetuximab in cancers with mutated KRAS.

The APC p.I1307K polymorphism is a significant risk factor for CRC in average risk Ashkenazi Jews.

BACKGROUND: The p.I1307K adenomatous polyposis coli (APC) gene variant, prevalent among Ashkenazi Jews, may increase the risk for colorectal neoplasia. We studied the clinical importance of screening for this polymorphism in 3305 Israelis undergoing colonoscopy. PATIENTS AND METHODS: Clinical data regarding potential risk factors for colorectal cancer (CRC) were collected from individuals undergoing colonoscopic examination at the Tel-Aviv medical center. The APC p.I1307K was detected using real-time PCR (polymerase chain reaction) from DNA extracted from peripheral mononuclear cells. RESULTS: The overall prevalence of the p.I1307K polymorphism was 8.0% (10.1% among Ashkenazi and 2.7% among Sephardic Jews, p<0.001). The overall adjusted odds ratio (OR) for colorectal neoplasia among carriers was 1.51 (95% confidence intervals (CI), 1.16-1.98). Among average risk Ashkenazi Jews, the adjusted OR was 1.75 (95% CI 1.26-2.45). A multiplicative interaction was identified between Ashkenazi ethnicity and APC p.I1307K carrier status (P(INTERACTION) = 0.055). The histopathological features of adenomas and carcinomas did not differ between carriers and non-carriers. CONCLUSIONS: The APC p.I1307K gene variant is an important risk factor for colorectal neoplasia in average risk Ashkenazi Jews. Carriers in this group should be considered for screening colonoscopy at the age of 40, to be repeated every 5 years, similar to recommendations in individuals with family history of colorectal cancer.

One stop screening for multiple cancers: the experience of an integrated cancer prevention center.

BACKGROUND: Cancer is a leading cause of mortality worldwide. Screening is a key strategy for reducing cancer morbidity and mortality. METHODS: We aimed to describe the experience of an integrated cancer prevention center in screening an asymptomatic population for the presence of neoplasia. One-thousand consecutive asymptomatic, apparently healthy adults, aged 20-80 years, were screened for early detection of 11 common cancers that account for 70-80% of cancer mortality. RESULTS: Malignant and benign lesions were found in 2.4% and 7.1% of the screenees, respectively. The most common malignant lesions were in the gastrointestinal tract and breast followed by gynecological and skin. The compliance rate for the different screening procedures was considerably higher than the actual screening rate in the general Israeli population - 78% compared to 60% for mammography (p<0.001) and 39% compared to 16% for colonoscopy (p<0.001). Advanced age, family history of cancer and certain lifestyle parameters were associated with increased risk. Moreover, polymorphisms in the APC and CD24 genes indicated high cancer risk. When two of the polymorphisms existed in an individual, the risk for a neoplastic lesion was extremely high (OR 2.3 [95% CI 0.94-5.9]). CONCLUSIONS: One stop shop screening for 11 common cancers in the setting of a multidisciplinary outpatient clinic is feasible and can detect cancer at an early stage.

Gene expression following exposure to celecoxib in humans: pathways of inflammation and carcinogenesis are activated in tumors but not normal tissues.

BACKGROUND: The Cox-2 inhibitor, celecoxib (Pfizer Inc., N.Y., USA), is a promising chemopreventive agent [Arber et al.: N Engl J Med 2006;355:885-895; Bertagnolli et al.: N Engl J Med 2006;355:873-884]. This study aims to explore its mechanism by defining changes in gene expression between neoplastic and normal tissue samples before and after treatment. METHODS: Patients with documented colorectal neoplasia in screening colonoscopy, destined to undergo surgical colectomy, were randomized for treatment with celecoxib (n = 11; 400 mg/day) or placebo (n = 3) for 30 days. Tissue samples were taken from the tumor and from normal adjacent mucosa during both colonoscopy and surgery. RNA was extracted and analyzed using Affymetrix Genechip®. RESULTS: 687 genes differentiated tumor samples before and after treatment, among which 310 genes did not show the same differential expression in the placebo group or normal samples. These genes were significantly related to pathways of cell cycle regulation and inflammation, and of note was the TGF-ß pathway, which held a strong association with the list of genes formerly found to be associated with the colorectal cancer expression profile in microarray analyses, as summarized in a meta-analysis by Cardoso et al. [Biochim Biophys Acta 2007;1775:103-137]. CONCLUSIONS: Celecoxib selectively affects genes and pathways involved in inflammation and malignant transformation in tumor but not normal tissues, this may assist in the development of safer and more effective chemopreventive agents.

First report of screening an asymptomatic population for cancer: the yield of an integrated cancer prevention center.

BACKGROUND: Cancer is a leading cause of mortality worldwide. The most effective way to combat cancer is by prevention and early detection. OBJECTIVES: To evaluate the outcome of screening an asymptomatic population for the presence of benign and neoplastic lesions. METHODS: Routine screening tests for prevention and/or early detection of 11 common cancers were conducted in 300 consecutive asymptomatic apparently healthy adults aged 25-77 years. Other tests were performed as indicated. RESULTS: Malignant and benign lesions were found in 3.3% and 5% of the screenees, respectively, compared to 1.7% in the general population. The most common lesions were in the gastrointestinal tract followed by skin, urogenital tract and breast. Advanced age and a family history of a malignancy were associated with increased risk for cancer with an odds ratio of 9 and 3.5, respectively (95% confidence interval 1.1-71 and 0.9-13, respectively). Moreover, high serum C-reactive protein levels and polymorphisms in the APC and CD24 genes indicated high cancer risk. When two of the polymorphisms existed in an individual, the risk for a malignant lesion was extremely high (23.1%; OR 14, 95% CI 2.5-78). CONCLUSIONS: Screening asymptomatic subjects identifies a significant number of neoplastic lesions at an early stage. Incorporating data on genetic polymorphisms in the APC and CD24 genes can further identify individuals who are at increased risk for cancer. Cancer can be prevented and/or diagnosed at an early stage using the screening facilities of a multidisciplinary outpatient clinic.

Curcumin potentiates the pro-apoptotic effects of sulindac sulfone in colorectal cancer.

OBJECTIVE: The use of sulindac sulfone (SFN) for colorectal cancer (CRC) therapy is limited due to its toxicity. The present study was carried out to examine whether curcumin, a novel chemopreventive agent, can potentiate the effects of low dosages of SFN in CRC treatment. METHODS: HT-29 CRC cells were exposed to SFN (200 - 400 microM), curcumin (5 - 10 microM) or their combination. The cytotoxic effects of the drugs were evaluated using growth inhibition assays. Annexin V/PI and cell cycle analysis were employed to study the mechanism of action of the drugs. The therapeutic efficacy of the drugs in vivo was examined using the aberrant crypt foci (ACF) model. The treatment groups included eight rats/group. RESULTS: Treatment of cells with curcumin and SFN resulted in a synergistic inhibitory effect of 50 - 90% (p < 0.005) on cell growth. Growth inhibition was associated with inhibition of proliferation, G2/M arrest and induction of apoptosis. Administration of curcumin (0.6%) and SFN (0.06%) to 1, 2-dimethylhydrazine treated rats significantly reduced (by 75%, p < 0.01) the number of ACF. CONCLUSIONS: Curcumin augments the therapeutic effects of SFN. This may be clinically important since the addition of curcumin to low dosages of SFN may encourage a safer and potent combinatorial treatment regimen for CRC.

Malignant transformation of normal enterocytes following downregulation of Bak expression.

Bak is a pro-apoptotic gene, which plays an important role in the multi-step process of gastrointestinal tumorigenesis. We hypothesized that downregulation of Bak expression in normal enterocytes will result in a transformed phenotype. The nontumorigenic intestinal epithelial cell line (IEC18) was transfected with the vector pMV12-AS-bak (encoding anti-sense bak). Three clones, with Bak protein levels similar to those seen in colon cancer cell lines and significantly lower than those found in the parental cells, were further evaluated. The three clones proliferated faster, demonstrated anchorage-independent growth in soft agar and a higher saturation density and plating efficiency. Furthermore, when injected into nude mice, these cells generated tumors after approximately 2-3 weeks. The cells were more resistant to the induction of apoptosis by sulindac sulfide and sulindac sulfone but more sensitive to COX 2 inhibitors (celecoxib and nimesulide). The levels of p16, cyclin D1 and COX 2 were higher in the three transformed clones. In summary,downregulation of Bak expression in normal enterocytes contributes to abnormal growth and tumorigenesis. COX 2 inhibitors may serve as important agents in the prevention and treatment of CRC as they only inhibit the growth of malignant cells.

Gene expression analysis proposes alternative pathways for the mechanism by which celecoxib selectively inhibits the growth of transformed but not normal enterocytes.

PURPOSE: Cyclooxygenase-2 inhibitor (celecoxib, Pfizer) is a promising chemopreventive agent, yet its long-term use may be limited due to increased cardiovascular toxicity. This study was aimed to identify genes and pathways involved in colorectal tumorigenesis and affected by celecoxib. EXPERIMENTAL DESIGN: Normal rat enterocytes (IEC18 cells) and their Ras-transformed derivatives (R1) were exposed for 72 h or over 6 months to celecoxib and analyzed for gene expression pattern using Genechip (RG-U34). Cluster and pathway analyses were done using GeneSpring software and Gene Ontology database. Cyclin D1 was overexpressed in IEC18 cells using stable transfection; cell cycle and prostaglandin synthesis were assessed. RESULTS: Five hundred thirty-eight genes were differentially expressed after transformation, and 70 and 126 genes, respectively, were affected by short and long treatments with celecoxib. Clusters of expression showed different expression in the transformed cells that revert to normal after treatment; they included Ras/Erk/Ral-B, Jagged2/Notch, calcineurin, lysyl-oxidase, etc. Cyclin D1 is up-regulated under the Ras pathway and is down-regulated by celecoxib. Thus, we showed that cyclin D1-transformed cells are resistant to inhibition by celecoxib. Celecoxib was also shown to work via cyclooxygenase-2 inhibition in transformed cells. CONCLUSIONS: Celecoxib selectively affects transformed and not normal enterocytes by targeting genes and pathways that are involved in the transformation. Thus, an alternative mechanism is proposed for the cancer-preventive role of celecoxib other than the classic mechanism of inhibiting prostaglandin synthesis, stressing mainly the role of cyclin D1. These data may help in the development of safer and more effective preventive drugs.

Gene therapy approach in prostate cancer cells using an active Wnt signal.

BACKGROUND: Functional activation of beta-catenin/T-cell factor (Tcf) signaling plays an important role in the early events of carcinogenesis. In past recent years accumulated evidence has demonstrated a significant role for the Wnt pathway in the development and progression of human prostate cancer. The objective of the current study was to use a gene-targeting approach to selectively kill human prostate cancer cells with activated beta-catenin/Tcf signaling. METHODS: A recombinant adenovirus that carries a lethal gene (PUMA) under the control of a beta-catenin/T-cell factor (Tcf)-responsive promoter (Ad-TOP-PUMA), was used to selectively target human prostate cancer cells (PC-3) in which the beta-catenin/Tcf pathway is activated, and compared its killing efficiency in cancer cells in which this pathway is inactive (DU145 cells). Ad-FOP-PUMA, carrying a mutant Tcf binding site, was used as a control virus. Cell viability was measured by methylene blue assay, and the level of beta-catenin/Tcf activity was measured by luciferase assay. RESULTS: The Ad-TOP-PUMA adenovirus inhibited PC-3 cell growth in a dose and time-dependent fashion, but did not had any effect on DU145 cell growth. CONCLUSIONS: Selective targeting of prostate cancer cells with the activated beta-catenin pathway may be a novel and effective therapy in prostate cancer.

Suppression of gastric cancer cell growth by targeting the beta-catenin/T-cell factor pathway.

BACKGROUND: Functional activation of beta-catenin/T-cell factor (Tcf) signaling plays an important role in the early events of carcinogenesis. Recently, it was demonstrated that adenomatous polyposis coli or beta-catenin genes are mutated frequently in gastric cancer cells. The objective of the current study was to use a gene-targeting approach to kill human gastric cancer cells selectively with activated beta-catenin/Tcf signaling. METHODS: A recombinant adenovirus that carries a lethal gene (p53 up-regulated modulator of apoptosis [PUMA]) under the control of a beta-catenin/Tcf-responsive promoter (AdTOP-PUMA) was used selectively to target gastric cancer cells (AGS) that posses an active beta-catenin/Tcf pathway. The combined effect of AdTOP-PUMA and several chemotherapeutic agents (5-florouracil, doxorubicin, paclitaxel) also was evaluated. Cell viability was measured by methylene blue assay, protein expression was measured by Western blot analysis, and cell cycle and apoptosis were evaluated by fluorescent-activated cell sorter analysis. RESULTS.: The TOP-PUMA adenovirus inhibited AGS cell growth in a dose- and time-dependent fashion. Growth inhibition was associated with the up-regulation of PUMA expression and the induction of apoptosis. Chemotherapy synergistically enhanced the killing effect of AdTOP-PUMA. CONCLUSIONS: Selective targeting of gastric cancer cells with the activated beta-catenin pathway may be a novel and effective therapy in gastric cancer. Combination of this gene-therapy approach with standard therapy may improve efficacy and reduce toxicity.

Targeting the active beta-catenin pathway to treat cancer cells.

The adenomatous polyposis coli or beta-catenin genes are frequently mutated in colorectal cancer cells, resulting in oncogenic activation of beta-catenin signaling. We tried to establish in vitro and in vivo models for selectively killing human cancer cells with an activated beta-catenin/T-cell factor (Tcf) pathway. We used a recombinant adenovirus that carries a lethal gene [p53-up-regulated modulator of apoptosis (PUMA)] under the control of a beta-catenin/Tcf-responsive promoter (AdTOP-PUMA) to selectively target human colorectal cancer cells (SW480, HCT116, DLD-1, and LS174T), hepatocellular carcinoma (HepG2), and gastric cancer cells (AGS) in which the beta-catenin/Tcf pathway is activated, and compared its efficiency in killing cancer cells in which this pathway is inactive or only weakly active. AdFOP-PUMA, carrying a mutant Tcf-binding site, was used as control virus. The combined effect of AdTOP-PUMA with several chemotherapeutic agents (5-florouracil, doxorubicin, and paclitaxel) was also evaluated. The effect of AdTOP-PUMA on colorectal cancer cells was also examined in nude mice: SW480 cells were infected with the AdTOP-PUMA and AdFOP-PUMA, and then inoculated s.c. into nude mice. The TOP-PUMA adenovirus inhibited cell growth in a dose-dependent fashion, depending on the signaling activity of beta-catenin. The growth of cells displaying high levels of active beta-catenin/Tcf signaling was inhibited after infection with AdTOP-PUMA, whereas that of cells with low levels of beta-catenin signaling was not. Growth inhibition was associated with induction of apoptosis. Chemotherapy synergistically enhanced the effect of AdTOP-PUMA. A combination of the adenovirus system with standard therapy may improve the efficacy and reduce the toxicity of therapy in humans.

CD24 is a new oncogene, early at the multistep process of colorectal cancer carcinogenesis.

BACKGROUND & AIMS: The aim of this study was to identify genes that play a role in colorectal cancer (CRC) carcinogenesis by analysis of differential gene expression of normal and transformed CRC cell lines. METHODS: Gene expression array analysis ([RG-U34] GeneChip) was performed in normal and transformed rat intestinal epithelial cells before and after exposures to celecoxib. In particular, we were looking for (1) altered gene expression in the transformed cells that reverts to normal following exposure to a selective cyclooxygenase-2 inhibitor, (2) novel genes, and (3) genes encoding membrane receptors or ligands. As a validation of the results and for human patients, immunohistochemistry was performed on 398 biological samples from the gastrointestinal tract (normal, polyps, and adenocarcinomas). Human cancer cell lines were tested for their response to anti-CD24 monoclonal antibodies. RESULTS: A total of 1081 genes were differently expressed following malignant transformation; 71 genes showed altered expression that reverted to normal following treatment with celecoxib, including the CD24 gene. Immunohistochemistry confirmed that increased expression of CD24 is an early event in CRC carcinogenesis. It was expressed in 90.7% of adenomas and 86.3% of CRCs. Very low expression was seen in normal epithelium (16.6%). Human cancer cell lines showed growth inhibition in response to the antibodies, according to their expression levels of CD24 and in dose- and time-dependent manners. These results were repetitive for 3 different antibodies. CONCLUSIONS: CD24 is overexpressed in the colonic mucosa, already at an early stage of carcinogenesis. It may be a useful target for early detection and in CRC therapy.

Celecoxib leads to G2/M arrest by induction of p21 and down-regulation of cyclin B1 expression in a p53-independent manner.

A unique in vitro system has been developed in our lab that consists of normal enterocytes derived from the rat ileum (IEC-18 cells) and their transformed derivatives with c-K-ras (R1 cells), anti-sense bak (B3 cells) and cyclin D1 (D1 cells). R1 and B3 cells express high level of COX-2 protein and PGE2. IEC 18 and D1 cells express negligible amount of COX-2, and produce very low level of PGE2. A relatively low dose of celecoxib (5-10 microM) induced G2/M arrest, followed by induction of apoptosis in the transformed but not in the normal cells. Down-regulation of cyclin B1 and up-regulation of p21 expressions independent of p53 might have cause this cell cycle block. Growth inhibition was related to COX-2 function with 90-95% reduction in PGE2 production. These findings may be of clinical importance, since low concentration of celecoxib can be achieved in human serum following standard anti-inflammatory (100-200 mg bid) regime.

MF tricyclic and sulindac retard tumor formation in an animal model.

New selective cyclooxygenase-2 inhibitors offer the benefit of cancer protection with less gastrointestinal toxicity associated with nonselective nonsteroidal anti-inflammatory drugs (NSAIDs). We hypothesize that MF tricyclic and sulindac can retard all stages of tumor formation in nude mice. In a blinded placebo controlled study, 3 types of experiments were performed: 1) 2.5 x 10(6) cells were injected into 2 flanks of nude mice subcutaneously, as a model for in situ cancer (n = 192); 2) 1 x 10(6) cells were injected into the cecum of mice as a model for in situ colorectal cancer (n = 78) and 3) 0.5 x 10(6) cells were implanted into the splenic subcapsule to establish a colorectal cancer liver metastasis model (n = 78). The animals were fed with standard chow containing either placebo, MF tricyclic (67 mg/kg of chow) or sulindac (150 mg/kg of chow). Mice that were given MF tricyclic or sulindac, at clinical anti-inflammatory plasma concentrations, were significantly more tumor free and had significantly smaller primary tumors and fewer metastases, as compared to mice that consumed placebo. The mortality and the latency period were significantly better in the treatment groups. These findings suggest that selective COX-2 inhibitors may serve as an adjunct to standard therapy in colorectal cancer.

Celecoxib and curcumin additively inhibit the growth of colorectal cancer in a rat model.

BACKGROUND: Multiple studies have indicated that specific COX-2 inhibitors may prevent CRC. However, the long-term use of COX-2 inhibitors is not toxicity-free and may be limited due to its cardiovascular side effects. The present study was carried out to examine the chemopreventive effects of celecoxib and curcumin alone and in combination using the 1,2-dimethylhydrazine (DMH) rat model. METHODS: Male rats were injected with DMH and randomly divided into four groups that consumed one of the following diets: (a) AIN-076 control diet; (b) AIN-076/curcumin (0.6%); (c) AIN-076/celecoxib (0.16%), or (d) AIN-076/celecoxib (0.16%) and curcumin (0.6%). Aberrant crypt foci (ACF) were identified by intensive staining with methylene blue in comparison to the surrounding normal crypts. RESULTS: The average number of ACF per rat colon was 64.2 +/- 3 in the control group, 39 +/- 5 and 47 +/- 10 for the curcumin- and celecoxib-treated group, respectively, and 24.5 +/- 6 in the group that had received both agents. CONCLUSIONS: In vivo, curcumin augments the growth inhibitory effect of celecoxib. This may be clinically important as this dose of celecoxib can be achieved in human serum following standard anti-inflammatory dosing of 100 mg.

Celecoxib and curcumin synergistically inhibit the growth of colorectal cancer cells.

PURPOSE: Multiple studies have indicated that cyclooxygenase-2 (COX-2) inhibitors may prevent colon cancer, which is one of the leading causes of cancer death in the western world. Recent studies, however, showed that their long-term use may be limited due to cardiovascular toxicity. This study aims to investigate whether curcumin potentiates the growth inhibitory effect of celecoxib, a specific COX-2 inhibitor, in human colon cancer cells. EXPERIMENTAL DESIGN: HT-29 and IEC-18-K-ras (expressing high levels of COX-2), Caco-2 (expressing low level of COX-2), and SW-480 (no expression of COX-2) cell lines were exposed to different concentrations of celecoxib (0-50 micromol/L), curcumin (0-20 micromol/L), and their combination. COX-2 activity was assessed by measuring prostaglandin E(2) production by enzyme-linked immunoassay. COX-2 mRNA levels were assessed by reverse transcription-PCR. RESULTS: Exposure to curcumin (10-15 micromol/L) and physiologic doses of celecoxib (5 micromol/L) resulted in a synergistic inhibitory effect on cell growth. Growth inhibition was associated with inhibition of proliferation and induction of apoptosis. Curcumin augmented celecoxib inhibition of prostaglandin E(2) synthesis. The drugs synergistically down-regulated COX-2 mRNA expression. Western blot analysis showed that the level of COX-1 was not altered by treatment with celecoxib, curcumin, or their combination. CONCLUSIONS: Curcumin potentiates the growth inhibitory effect of celecoxib by shifting the dose-response curve to the left. The synergistic growth inhibitory effect was mediated through a mechanism that probably involves inhibition of the COX-2 pathway and may involve other non-COX-2 pathways. This synergistic effect is clinically important because it can be achieved in the serum of patients receiving standard anti-inflammatory or antineoplastic dosages of celecoxib.

Celecoxib but not rofecoxib inhibits the growth of transformed cells in vitro.

PURPOSE: Nonsteroidal anti-inflammatory drugs reduce the risk of colorectal cancer. The cyclooxygenase (COX) pathway of arachidonic acid metabolism is an important target for nonsteroidal anti-inflammatory drugs. Increased expression of COX-2 was recently shown to be an important step in the multistep process of colorectal cancer carcinogenesis. The new COX-2-specific inhibitors offer the benefit of cancer protection without the gastrointestinal toxicity reported for the old drugs. The purpose of this study was to compare the growth effects of two specific COX-2 inhibitors, celecoxib (Pfizer, Inc., New York, NY), and rofecoxib (Merck, White House Station, NJ) in normal and transformed enterocytes. EXPERIMENTAL DESIGN: Cultures of normal rat intestinal epithelial cell line, IEC-18, vector control cells, c-K-ras, c-K-ras-bak, and antisense-bak derivatives were treated with different dosages of celecoxib (0-60 micro M) and rofecoxib (0-20 micro M). Cell cycle analysis and apoptosis were assessed by fluorescence-activated cell sorting analysis. Protein expression was assessed by Western blot analysis and caspases 3 and 8 activities by ELISA. RESULTS: Celecoxib inhibited cell growth and induced apoptosis in a time- and dose-dependent manner. IEC18 parental cells were two to four times more resistant to celecoxib than ras, ras-bak, and antisense bak transformed cells that overexpress the COX-2 protein. The induction of apoptosis by celecoxib involved the caspase pathways. Rofecoxib, up to its maximal concentration of 20 micro M, did not inhibit cell growth or induce apoptosis. CONCLUSIONS: Celecoxib may prove to be a very efficient component in the prevention and treatment of gastrointestinal tumors because it inhibits the growth of cancerous cells without affecting the growth of normal cells.

Oncogenic transformation of normal enterocytes by overexpression of cyclin D1.

Cyclin D1 plays an important role in the multi-step process of gastrointestinal tumorigenesis. We hypothesize that normal enterocytes over-expressing cyclin D1 will demonstrate a transformed phenotype. The nontumorigenic intestinal epithelial cell line, IEC-18, was transfected with the vector pMV7-CCND1, encoding cyclin D1. Three clones, with cyclin D1 levels similar to those seen in colon cancer cell lines, were further evaluated in comparison to the vector control cells. They proliferated faster and demonstrated anchorage-independent growth in soft agar, higher saturation density, and higher plating efficiency. When injected into nude mice, tumors were generated after 6-8 weeks. On the other hand these cells were more sensitive to induction of apoptosis. There was no change in the level of beta-catenin protein. In conclusion, cyclin D1 can act as an oncogene in vitro and in vivo, when produced in immortalized normal intestinal epithelial cells. This model may be useful for understanding the role and interrelationships of cyclin D1 in colorectal tumorigenesis.