Seema R. Gandhi

Involvement of p21cip1/waf1 in the anti-proliferative effects of polyethylene glycol in colon carcinogenesis

Polyethylene glycol (PEG) is a safe and effective chemopreventive agent against colorectal carcinogenesis in cell culture, animal models and human subjects. Although the precise molecular mechanism is unclear, we previously reported that PEG suppresses colonic epithelial proliferation. As cellular proliferation is driven by complex G1-S phase transition, we now characterize the role of PEG on cell cycle regulation. We focused our attention on the effect of PEG on the CDK inhibitor p21cip1/waf1, which is implicated in early colon carcinogenesis and is upregulated by non-steroidal anti-inflammatory drugs. These studies were done in the azoxymethane-treated (AOM) rat model as well as in HT-29 colon cancer cells. Immunohistochemical analysis revealed that while AOM decreased the p21 expression (75%, p<0.01) in the premalignant colonic mucosa, PEG induced p21 levels back to normal. These findings paralleled a decreased BrdUrd incorporation (78%, p<0.001) and hypophosphorylated retinoblastoma protein (Rb; by 47%) signifying PEGs antiproliferative activity. Furthermore, in HT-29 cells, PEG decreased proliferation as measured by PCNA (68% reduction), increased p21 expression (2.3-fold), induced cell cycle arrest during G0/G1 phase (45% reduction in S phase cells) and inhibited the phosphorylation of Rb (by 52% compared to untreated). PEG caused greater than a 2-fold induction of protein and mRNA level of p21cip1/waf1 in HT-29 cells. These results demonstrate for the first time that PEG is involved in p21 regulation concomitant with G1S phase cell cycle arrest and it is through these effects that it can exert its anti-proliferative and hence chemopreventive role.

Association of stem-like cells in gender-specific chemoprevention against intestinal neoplasia in MIN mouse

This study was undertaken to examine the gender-sensitivity and chemopreventive responsiveness of celecoxib on intestinal stem-like cells as a biomarker of colon carcino-genesis, using the MIN mouse model. Male and female MIN mice (6-7-weeks old) were randomized to either control diet or to a diet supplemented with celecoxib (1,500 ppm). The animals were euthanized ten weeks later and the intestines were flushed and opened longitudinally to assess tumor count. Small intestinal segments were formalin-fixed and tissue sections were subjected to immunohistochemical evaluation of DCAMKL1, a known marker of stem-like cells. We found that in animals receiving control (AIN 76A diet) alone, female MIN mice had a higher polyp count than males (52.32 ± 13.89 vs. 35.43 ± 16.05; p<0.0005). However, compared to control diet groups, celecoxib supplementation caused a larger reduction in the number of polyps in females than their male cohorts (6.38 ± 1.43 vs. 12.83 ± 6.74; a reduction of 88% in females to 64% in males). Significant differences (p=0.013) were observed in the number of DCAMKL1-stained cells in the crypts of the wild-type (WT) (10.01 ± 1.07 stem cells per high powered field; HPF) compared to the MIN mice (24.15 ± 8.08 stem cells per HPF), illustrating increased stem-like cells in animals that are more prone to neoplasia. DCAMKL1 labeled stem-like cells were equal in number in the male and female groups receiving the control AIN 76A diet alone (females, 25.73 stem-like cells/HPF); males, 24.15 stem-like cells/HPF). However, females showed a greater reduction in the number of DCAMKL1-labeled stem-like cells with celecoxib supplementation than the respective males (16.63 ± 4.23 vs. 21.56 ± 9.06; a reduction of 35.4% in females to 10.7% in males). We conclude that a higher number of stem-like cells in the uninvolved mucosa paralleled tumorigenesis and mirrored greater chemopreventive responsiveness of female MIN mice compared to males.

Tu1279 Relationship Between Beliefs in Complementary and Alternative Medicine and Psychosocial Variables Among IBD Patients

Improving Case Definition of Crohns Disease and Ulcerative Colitis in Electronic Medical Records Using Natural Language Processing − a Novel Informatics Approach Ashwin N. Ananthakrishnan, Tianxi Cai, Su-Chun Cheng, Pei Jun Chen, Guergana Savova, Raul Guzman Perez, Vivian S. Gainer, Shawn N. Murphy, Peter Szolovits, Katherine Liao, Elizabeth W. Karlson, Susanne Churchill, Isaac Kohane, Robert M. Plenge

M1179 Targeting Epidermal Growth Factor Receptor With Polyethylene Glycol: Applications for Digestive Cancer Prevention

Introduction: H. pylori transactivates the epidermal growth factor (EGFR) and induces extracellular-signal related kinase (ERK) phosphorylation in gastric epithelial cells which may be important in the pathway to gastric carcinogenesis. The green tea catechin epigallocatechin-3-gallate (EGCG) has been reported in some studies to have chemoprotective properties against development of gastric cancer. The aim of this study was to investigate if H. pyloriinduced ERK phosphorylation in A431 epithelial cells was inhibited by EGCG.Methods: H. pylori (NCTC 11637, cag PAI+) were co-cultured with A431 epithelial cells for 2.5 hrs. EGF (100 ng/ml) was used as a positive control. “In-Cell Western” (ICW) assay, which is a rapid quantitative method for determining the inhibitory effects of chemicals of interest on H. pylori epithelial cell signalling responses (REF Du Y et al., 2007), was used to examine the effects of EGCG on ERK1/2 phosphorylation (pERK) in A431 epithelial cells. EGCG (1-200 μM) was pre-incubated with A431 cells for 1 hr prior to the co-culture experiments. Results: EGF significantly (p<0.001, n=4) increased pERK in A431 cells compared to unstimulated controls. EGCG at concentrations greater than 50 μM caused a small but significant (p< 0.01) increase in pERK in A431 cells. EGCG dose-dependently significantly inhibited EGF (100 ng/ml)-induced pERK at a concentration of 10 μM and above (p<0.01, n=4). H. pylori strain NCTC 11637 significantly (p<0.01, n=4) increased pERK in A431 cells compared to unstimulated controls. EGCG dose-dependently inhibited pERK induced by H. pylori. EGCG at 100 μM significantly inhibited (p<0.05, n=4) H. pylori-induced pERK 2.5 hrs postculture. Conclusions: H. pylori-induced ERK phosphorylation in A431 epithelial cells was significantly inhibited by green tea catechin EGCG in a dose-dependent manner. The use of EGCG as dietary phytochemical for H. pylori-induced gastric cancer chemoprevention is plausible, at least in a specific group of patients with higher risk of gastric cancer development but failure for H. pylori eradication.

M1176 MicroRNA 132 Regulation in Human Colon Carcinogenesis: Potential Role in NSAID Chemoprevention

Introduction: H. pylori transactivates the epidermal growth factor (EGFR) and induces extracellular-signal related kinase (ERK) phosphorylation in gastric epithelial cells which may be important in the pathway to gastric carcinogenesis. The green tea catechin epigallocatechin-3-gallate (EGCG) has been reported in some studies to have chemoprotective properties against development of gastric cancer. The aim of this study was to investigate if H. pyloriinduced ERK phosphorylation in A431 epithelial cells was inhibited by EGCG.Methods: H. pylori (NCTC 11637, cag PAI+) were co-cultured with A431 epithelial cells for 2.5 hrs. EGF (100 ng/ml) was used as a positive control. “In-Cell Western” (ICW) assay, which is a rapid quantitative method for determining the inhibitory effects of chemicals of interest on H. pylori epithelial cell signalling responses (REF Du Y et al., 2007), was used to examine the effects of EGCG on ERK1/2 phosphorylation (pERK) in A431 epithelial cells. EGCG (1-200 μM) was pre-incubated with A431 cells for 1 hr prior to the co-culture experiments. Results: EGF significantly (p<0.001, n=4) increased pERK in A431 cells compared to unstimulated controls. EGCG at concentrations greater than 50 μM caused a small but significant (p< 0.01) increase in pERK in A431 cells. EGCG dose-dependently significantly inhibited EGF (100 ng/ml)-induced pERK at a concentration of 10 μM and above (p<0.01, n=4). H. pylori strain NCTC 11637 significantly (p<0.01, n=4) increased pERK in A431 cells compared to unstimulated controls. EGCG dose-dependently inhibited pERK induced by H. pylori. EGCG at 100 μM significantly inhibited (p<0.05, n=4) H. pylori-induced pERK 2.5 hrs postculture. Conclusions: H. pylori-induced ERK phosphorylation in A431 epithelial cells was significantly inhibited by green tea catechin EGCG in a dose-dependent manner. The use of EGCG as dietary phytochemical for H. pylori-induced gastric cancer chemoprevention is plausible, at least in a specific group of patients with higher risk of gastric cancer development but failure for H. pylori eradication.

T1192 The Role of Transketolase-Like Proteins During Early Stages of Colorectal Carcinogenesis

G A A b st ra ct s this Snail phosphorylation and nuclear localization. In addition, we show that a chemical inhibitor of iNOS as well as siRNA iNOS knockdown prevent activation of Snail by alcohol. Finally, we show that at rest, Snail is associated with Flotillin-2 membrane rafts while iNOS is associated with Caveolin-1 rafts. PAK1 is not associated with the membrane rafts. Upon stimulation PAK1 is translocated to Flotillin-2 rafts along with iNOS and Snail and activated Snail is translocated to the nucleus. Conclusions. Our data support a mechanism is which Flotillin-2 membrane rafts form a signaling platform for alcohol-induced activation of Snail via iNOS and PAK1.These are the first data to show iNOS activation of PAK1 and Snail and Snail association with membrane rafts by any stimulus and provide new insight into mechanisms regulating EMT as well as mechanisms through which alcohol may promote colon cancer progression through EMT that could be targeted for therapy.

T1190 MicroRNA Modulation is Involved in the Progression Phase of Colon Carcinogenesis in the Azoxymethane-Treated Rat Model: Implications for NSAID Chemoprevention

G A A b st ra ct s this Snail phosphorylation and nuclear localization. In addition, we show that a chemical inhibitor of iNOS as well as siRNA iNOS knockdown prevent activation of Snail by alcohol. Finally, we show that at rest, Snail is associated with Flotillin-2 membrane rafts while iNOS is associated with Caveolin-1 rafts. PAK1 is not associated with the membrane rafts. Upon stimulation PAK1 is translocated to Flotillin-2 rafts along with iNOS and Snail and activated Snail is translocated to the nucleus. Conclusions. Our data support a mechanism is which Flotillin-2 membrane rafts form a signaling platform for alcohol-induced activation of Snail via iNOS and PAK1.These are the first data to show iNOS activation of PAK1 and Snail and Snail association with membrane rafts by any stimulus and provide new insight into mechanisms regulating EMT as well as mechanisms through which alcohol may promote colon cancer progression through EMT that could be targeted for therapy.

M1180 Gender Predilection for Stem Cell Targeting by Celecoxib: Implications for Chemoprevention

1.00±0.76, p<0.049). Epithelial IHC staining revealed decreased PCNA staining (proliferation) in the initiation and post-initiation groups and an increased activated caspase-3 staining (apoptosis) in the post-initiation group. Conclusion: Topical PEG application resulted in EGFR downregulation with a concomitant decrease in proliferation and induction of apoptosis. This resulted in a robust chemopreventive benefit in oral cancer consistent with our previous data in colorectal neoplasia (currently in phase 2 clinical trials). This suggests a wider application for GI malignancies where PEG can be applied topically (esophageal, gastric, anal etc).