Eliezer Liberman

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 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 μm) and rofecoxib (0–20 μm). 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 μm, 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.

Cerebrospinal fluid leukocyte aggregation in meningitis.

OBJECTIVE To evaluate whether the difference in aggregation of cerebrospinal fluid cells from patients with bacterial, viral, aseptic and partially treated meningitis can be used for diagnostic purposes. METHODS Cerebrospinal fluid samples of 100 patients with meningitis (15 bacterial, 13 partially treated, 10 viral and 62 aseptic) were compared on the basis of the predefined leukocyte aggregation score (LAS). RESULTS Mean LAS was 56% in the bacterial meningitis group (range, 15 to 90%), 5.8% in the partially treated meningitis group (range, 0 to 27%), 2% in the proven viral meningitis group (range, 0 to 5%) and 2% in the aseptic meningitis group (range, 0 to 15%). All patients with bacterial meningitis had a LAS of > 15%, whereas all those with viral or aseptic meningitis had a score of < 15%. Although most patients with partially treated meningitis had a low LAS, several had higher scores, which may indicate bacterial infection. There was no statistical correlation between number of cells, type of cells (mononuclear or polymorphonuclear) or cerebrospinal fluid protein and glucose concentration and degree of leukocyte aggregation for the different groups. CONCLUSION Measurement of the LAS may contribute to the immediate differential diagnosis of bacterial or viral meningitis, especially in patients with very high pleocytosis, as sometimes seen in enteroviral meningitis. It may also serve as a guide for the likelihood of bacterial infection in cases of partially treated meningitis. Additional studies are needed to confirm these observations.

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

Functional activation of β‐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 β‐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 β‐catenin/Tcf signaling.

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 β-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.

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.

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.

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 Bakexpression 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 Bakprotein 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.

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 × 106 cells were injected into 2 flanks of nude mice subcutaneously, as a model for in situ cancer (n=192); 2) 1 × 106 cells were injected into the cecum of mice as a model for in situ colorectal cancer (n=78) and 3) 0.5 × 106 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.

Increased leukocyte adhesiveness/ aggregation in patients with inflammatory bowel disease during remission

PURPOSE: We have used a novel leukocyte adhesiveness/ aggregation test (LAAT) to show that many patients with inflammatory bowel disease (TOD) in clinical remission have a subclinical low grade inflammation. METHODS: Included in the study are 500 controls, 96 patients with IBD in remission, and 106 patients in relapse. RESULTS: The percent of aggregated white blood cells detected in the peripheral blood was 5.9±3.9, 9.1±5.9, and 18.8±9.4, respectively. The difference between each group and any other was significant atP<0.0001. Similar results were obtained when other acute phase reactants like the erythrocyte sedimentation rate, white blood cell count, differential count, and C-reactive protein level were examined. However, in a linear regression analysis, LAAT was the only significant (P<0.0006) variable that could classify correctly each subject to the appropriate category of control and IBD in remission or relapse. CONCLUSIONS: Identification of patients with IBD in clinical remission who have ongoing inflammation may be of clinical-therapeutic relevance. The LAAT is a simple, rapid, and convenient test. The present study indicates that it is also very sensitive.