Mo1162 Inflammation Increases Notch1 Activity in Colorectal Cancer Cells and Is Counteracted by...
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Mo1160 In Vitro and In Vivo Knock-Down of Smad7 Inhibits Growth and Promotes Apoptosis of Colon Cancer Cells Carmine Stolfi, Eleonora Franzè, Massimiliano Sarra, Vincenzo De Mare, Francesco Pallone, Giovanni Monteleone BACKGROUND AND AIM: In the early stages of tumorigenesis, transforming growth factor (TGF)-β1-driven signaling provides tumor-suppressive action. TGF- β1 activity is inhibited by SMAD7, a protein that binds to TGF- β receptor and prevents TGF- β1-induced signal transduction. Forced expression of SMAD7 in cancer cells differently regulates tumorigenesis depending on the cell context analyzed, and preliminary evidence suggests that amplification of SMAD7 associates with a significantly worse prognosis in patients with colorectal cancer (CRC). In this study we evaluated the role of SMAD7 in the process of colon carcinogenesis. METHODS: SMAD7 expression was evaluated in human CRC samples, CRC cell lines (i.e. HCT-116, DLD-1, HT-29 and HT-115) and normal colonic epithelial cells by immunohisto- chemistry and Western blotting. HCT-116 and DLD-1 cells were transfected with a specific oligonucleotide antisense (GED-0301) or control sense, and cell proliferation, cell cycle, caspase-3 activation and apoptosis were then evaluated by flow-cytometry. Expression of cell cycle-related proteins (e.g., CDK2, CDC25A, cyclin D1) was assessed by Western blotting. To evaluate the effect of SMAD7 knock-down on CRC growth in vivo, xenografts were induced in Rag1-deficient mice by subcutaneous injection of HCT-116 cells and mice were treated either with SMAD7 sense or GED-0301. RESULTS: High SMAD7 was seen in CRC samples as compared to matched normal, adjacent, colonic samples. Consistently, SMAD7 was highly expressed in several CRC cell lines but not in normal colonic epithelial cells. Transfection of HCT-116 and DLD-1 cells with GED-0301 down-regulated SMAD7 and inhibited cell growth. Cell cycle analysis revealed that silencing of SMAD7 with GED-0301 induced cells to accumulate in S phase and this effect was associated with a sustained phosphorylation of CDK2 and decreased expression of CDC25A and cyclin D1. Moreover, time-course studies revealed that GED-0301-mediated cell cycle arrest was followed by caspase-dependent induction of CRC cell apoptosis. Finally, in vivo studies showed that daily intra-peritoneal administration of GED-0301 markedly inhibited the development of HCT-116-derived xenografts. CONCLUSIONS: SMAD7 knock-down causes accumulation of CRC cells in S phase thereby promoting cell growth arrest and apoptosis. Data suggest that SMAD7 could be a promising molecular target for pharmacological intervention in CRC patients. Mo1161 Phase 2B Randomized Placebo-Controlled Spectral Markers As Biomarkers for Colonic Chemoprevention With Aspirin: Correlation With Rectal Apoptosis but Not Proliferation Hemant K. Roy, Andrew Radosevich, Ramesh K. Wali, Asad Umar, Gary Della'Zanna, Borko Janovic, Michael J. Goldberg, Laura K. Bianchi, David T. Rubin, L. M. Rodriguez, Tat-Kin Tsang, Vladimir Turzhitsky, Julia Shklovskaya, Silvia Skripkauskas, Irene Helenowski, Raymond Bergan, Vadim Backman Introduction: While the efficacy of aspirin against colonic neoplasia is unequivocal, only a subgroup of patients (30-50%) will derive a chemopreventive benefit. Finding a robust biomarker of chemopreventive efficacy would allow optimization of dose and selection of agent so as to maximize the anti-neoplastic effect. The attainment of this at the level of the individual would allow optimal tailoring of the risk-benefit ratio in a manner consistent with true personalized medicine. Conventional approaches include assaying for the reversal of decreased apoptosis/ increased proliferation that are hallmarks of colonic field carcinogenesis. However, the chemopreventive predictive value of apoptosis is modest and proliferation is marginal. Our group has pioneered optical detected micro-architectural manifestation of the field carcinogenesis for screening ( Clin Gastro Hep, 2012). Indeed, we reported that rectal spectral markers predicted colonic advanced adenomas with ~90% accuracy ( Cancer Res 2009,). We, therefore, wanted to assess whether these spectral marker would be normalized by aspirin and would correlate with cellular markers. Methods: Patients: This was a random- ized placebo-controlled double blinded trial which obtained biopsies of the endoscopically normal left colonic mucosa. Those with abnormal spectral slope (Spec) and fractal dimension (Frac) were randomized to aspirin (325 mg qd) or placebo and markers were reassessed by flexible sigmoidoscopy after 3 months. Biomarkers: Spec and Frac were calculated using low coherence enhanced backscattering spectroscopy (LEBS)( Gastro 2011) whereas apoptosis and proliferation was determined with immunohistochemistry (cleaved caspase 3 and Ki 67, respectively). Results: The 72 patients randomized to aspirin or placebo group were comparable with regards to age, gender, BMI etc. As can be seen, with regards to apoptosis (figure 1), patients manifested an anti-neoplastic benefit based on Frac (left panel) or Spec (right panel) also had the expected induction in apoptosis consistent with chemoprevention. However, regardless of spectral markers (figure 2) there was no suppression of proliferation consistent with a number of reports impugning the value of rectal proliferation as a chemo- preventive biomarker. Conclusions: Aspirin treatment resulted in trend of normalization of both spectral markers consonant with a decrease in risk of future neoplasia. When compared to conventional (albeit suboptimal) markers, spectral markers correlated with the more robust cellular marker, apoptosis but not proliferation. This proof of concept study demonstrating the promise of LEBS field carcinogenesis detection as an intermediate bio- marker for chemoprevention. Potential applications include personalizing therapy (adjusting dosage etc) and providing a platform for rapid clinical validation of novel agents. S-595 AGA Abstracts Figure 1: Correlation of aspirin induced alteration in spectral markers with that of apoptosis Figure 2: Correlation of aspirin induced alteration in spectral markers with that of prolif- eration Mo1162 Inflammation Increases Notch1 Activity in Colorectal Cancer Cells and Is Counteracted by Eicosapentaenoic Acid-Free Fatty Acid Chiara Fazio, Giulia Piazzi, Paola Vitaglione, Vincenzo Fogliano, Alessandra Munarini, Anna Prossomariti, Leonarda D'Angelo, Manuela Napolitano, Andrea Belluzzi, Franco Bazzoli, Luigi Ricciardiello Introduction/Objectives: The link between inflammation and colorectal cancer (CRC) is well established, and macrophages in the lamina propria are believed to be critical for colitis- associated CRC development. Notch signaling has a pivotal role in carcinogenesis and Notch1 can act as an oncogene by promoting cell survival and proliferation through its activation by the ligand Jagged1. A consumption of anti-inflammatory agents, such as ω-3 polyunsatu- rated fatty acids ( ω-3 PUFAs), has a protective role on colorectal carcinogenesis. The chemo- preventive activity of eicosapentaenoic acid free fatty acid (EPA-FFA), an ω-3 PUFA, has been demonstrated in preclinical models of CRC, but its activity in inflammatory-driven CRC remains to be elucidated. The aim of this study was to evaluate whether Notch1 can be activated by an inflammatory stimuli through STAT3-Jagged1 in CRC cells and whether this mechanism can be counteracted by treatment with EPA-FFA. Methods: The human monocyte cell line THP-1 was differentiated to macrophages using phorbol 12-myristate 13-acetate and stimulated with E.coli lipopolysaccharide to obtain a cytokine-enriched conditioned media (CM). Differentiation was evaluated by FACS analysis. Cytokines levels were analyzed by Luminex technology. The human CRC cell line HT29 was treated with different concentrations of EPA-FFA to test cell viability by MTT assay. Membrane incorpora- tion of EPA was evaluated using GC-MS. In order to explore the effect of the inflammatory microenvironment, the whole CM was applied to untreated and EPA-treated HT29 cells for different times. Protein levels of Notch1 intracellular Domain (NICD), STAT3 and its phosphorylated form (p-STAT3) were quantified by western blotting. Gene expression of the Notch1 ligand Jagged1 and Notch downstream targets NRARP and Hath1 were evaluated by Real-Time PCR. Results: THP1 differentiated macrophages produced a conditioned media with high concentrations of MIP-1, IL-8, TNF- α, MCP-1, IL-6 and IFN- γ. CM stimulation of HT29 induced a strong activation of pSTAT3, increased expressions of NICD and Jagged1. The activation of Notch1 caused a 3-fold up-regulation of NRARP and a 16-fold down- regulation of Hath1. Interestingly, pre-treatment with non-cytotoxic concentration of EPA- FFA, corresponding to an incorporation of approximately 20% of EPA in the cellular mem- brane, counteracted the CM-driven activation of NICD and pSTAT3. Importantly, Jagged1 induction returned to basal levels after EPA-FFA treatment. Finally, EPA-FFA effects on Notch1 were supported by a concordant modulation of its downstream targets NRARP and HATH1. Conclusion: Inflammation is a strong activator of Notch1 signaling in CRC cells and this activation is reduced by EPA-FFA treatment. Our data suggest a possible new mechanism of action of eicosapentaenoic acid as chemopreventive agent in inflammatory driven-CRC. Mo1163 Carcinogenic Effects of Benzo[a]Pyrene (Cyp 450 Activation) on HET-1A and Bar-T Cell Line Could Be Reversed by Curcumin Carlos D. Minacapelli, Manisha Bajpai, Xin Geng, Kiron M. Das Introduction: Smoking and history of gastroesophageal reflux disease (GERD), are associated risk factors for development of esophageal cancers. Benzo[a]pyrene (B[a]P) is a prototype of poly aromatic hydrocarbons found in cigarette smoke, charbroiled or grilled foods, and has been implicated in several human malignancies including cancers of aero-digestive tract. B[a]P, a promutagen, follows the aromatic hydrocarbon receptor (AhR) Pathway, which results in up-regulation of cytocrome P450 (CYP) genes. These CYP genes metabolize B[a]P to potent carcinogens like B[a]P diol epoxide. The oxidation reaction that most efficiently generates B[a]P-dependent mutagenesis is catalyzed by CYP1A1 and CYP1B1 genes. Cur- cumin has been shown to have chemopreventive effects in cell line models derived from some of the common cancer like those of colon and breast. One of the mechanisms of action AGA Abstracts
Transcript of Mo1162 Inflammation Increases Notch1 Activity in Colorectal Cancer Cells and Is Counteracted by...
Mo1160
In Vitro and In Vivo Knock-Down of Smad7 Inhibits Growth and PromotesApoptosis of Colon Cancer CellsCarmine Stolfi, Eleonora Franzè, Massimiliano Sarra, Vincenzo De Mare, FrancescoPallone, Giovanni Monteleone
BACKGROUND AND AIM: In the early stages of tumorigenesis, transforming growth factor(TGF)-β1-driven signaling provides tumor-suppressive action. TGF-β1 activity is inhibitedby SMAD7, a protein that binds to TGF- β receptor and prevents TGF-β1-induced signaltransduction. Forced expression of SMAD7 in cancer cells differently regulates tumorigenesisdepending on the cell context analyzed, and preliminary evidence suggests that amplificationof SMAD7 associates with a significantly worse prognosis in patients with colorectal cancer(CRC). In this study we evaluated the role of SMAD7 in the process of colon carcinogenesis.METHODS: SMAD7 expression was evaluated in human CRC samples, CRC cell lines (i.e.HCT-116, DLD-1, HT-29 and HT-115) and normal colonic epithelial cells by immunohisto-chemistry and Western blotting. HCT-116 and DLD-1 cells were transfected with a specificoligonucleotide antisense (GED-0301) or control sense, and cell proliferation, cell cycle,caspase-3 activation and apoptosis were then evaluated by flow-cytometry. Expression ofcell cycle-related proteins (e.g., CDK2, CDC25A, cyclin D1) was assessed byWestern blotting.To evaluate the effect of SMAD7 knock-down on CRC growth in vivo, xenografts wereinduced in Rag1-deficient mice by subcutaneous injection of HCT-116 cells and mice weretreated either with SMAD7 sense or GED-0301. RESULTS: High SMAD7 was seen in CRCsamples as compared to matched normal, adjacent, colonic samples. Consistently, SMAD7was highly expressed in several CRC cell lines but not in normal colonic epithelial cells.Transfection of HCT-116 and DLD-1 cells with GED-0301 down-regulated SMAD7 andinhibited cell growth. Cell cycle analysis revealed that silencing of SMAD7 with GED-0301induced cells to accumulate in S phase and this effect was associated with a sustainedphosphorylation of CDK2 and decreased expression of CDC25A and cyclin D1. Moreover,time-course studies revealed that GED-0301-mediated cell cycle arrest was followed bycaspase-dependent induction of CRC cell apoptosis. Finally, in vivo studies showed thatdaily intra-peritoneal administration of GED-0301 markedly inhibited the development ofHCT-116-derived xenografts. CONCLUSIONS: SMAD7 knock-down causes accumulationof CRC cells in S phase thereby promoting cell growth arrest and apoptosis. Data suggestthat SMAD7 could be a promising molecular target for pharmacological intervention inCRC patients.
Mo1161
Phase 2B Randomized Placebo-Controlled Spectral Markers As Biomarkers forColonic Chemoprevention With Aspirin: Correlation With Rectal Apoptosisbut Not ProliferationHemant K. Roy, Andrew Radosevich, Ramesh K. Wali, Asad Umar, Gary Della'Zanna,Borko Janovic, Michael J. Goldberg, Laura K. Bianchi, David T. Rubin, L. M. Rodriguez,Tat-Kin Tsang, Vladimir Turzhitsky, Julia Shklovskaya, Silvia Skripkauskas, IreneHelenowski, Raymond Bergan, Vadim Backman
Introduction: While the efficacy of aspirin against colonic neoplasia is unequivocal, only asubgroup of patients (30-50%) will derive a chemopreventive benefit. Finding a robustbiomarker of chemopreventive efficacy would allow optimization of dose and selection ofagent so as to maximize the anti-neoplastic effect. The attainment of this at the level of theindividual would allow optimal tailoring of the risk-benefit ratio in a manner consistentwith true personalizedmedicine. Conventional approaches include assaying for the reversal ofdecreased apoptosis/ increased proliferation that are hallmarks of colonic field carcinogenesis.However, the chemopreventive predictive value of apoptosis is modest and proliferation ismarginal. Our group has pioneered optical detected micro-architectural manifestation of thefield carcinogenesis for screening (Clin Gastro Hep, 2012). Indeed, we reported that rectalspectral markers predicted colonic advanced adenomas with ~90% accuracy ( Cancer Res2009,). We, therefore, wanted to assess whether these spectral marker would be normalizedby aspirin and would correlate with cellular markers. Methods: Patients: This was a random-ized placebo-controlled double blinded trial which obtained biopsies of the endoscopicallynormal left colonic mucosa. Those with abnormal spectral slope (Spec) and fractal dimension(Frac) were randomized to aspirin (325 mg qd) or placebo and markers were reassessed byflexible sigmoidoscopy after 3 months. Biomarkers: Spec and Frac were calculated usinglow coherence enhanced backscattering spectroscopy (LEBS)(Gastro 2011) whereas apoptosisand proliferation was determined with immunohistochemistry (cleaved caspase 3 and Ki67, respectively). Results: The 72 patients randomized to aspirin or placebo group werecomparable with regards to age, gender, BMI etc. As can be seen, with regards to apoptosis(figure 1), patients manifested an anti-neoplastic benefit based on Frac (left panel) or Spec(right panel) also had the expected induction in apoptosis consistent with chemoprevention.However, regardless of spectral markers (figure 2) there was no suppression of proliferationconsistent with a number of reports impugning the value of rectal proliferation as a chemo-preventive biomarker. Conclusions: Aspirin treatment resulted in trend of normalizationof both spectral markers consonant with a decrease in risk of future neoplasia. Whencompared to conventional (albeit suboptimal) markers, spectral markers correlated with themore robust cellular marker, apoptosis but not proliferation. This proof of concept studydemonstrating the promise of LEBS field carcinogenesis detection as an intermediate bio-marker for chemoprevention. Potential applications include personalizing therapy (adjustingdosage etc) and providing a platform for rapid clinical validation of novel agents.
S-595 AGA Abstracts
Figure 1: Correlation of aspirin induced alteration in spectral markers with that ofapoptosis
Figure 2: Correlation of aspirin induced alteration in spectralmarkers with that of prolif-eration
Mo1162
Inflammation Increases Notch1 Activity in Colorectal Cancer Cells and IsCounteracted by Eicosapentaenoic Acid-Free Fatty AcidChiara Fazio, Giulia Piazzi, Paola Vitaglione, Vincenzo Fogliano, Alessandra Munarini,Anna Prossomariti, Leonarda D'Angelo, Manuela Napolitano, Andrea Belluzzi, FrancoBazzoli, Luigi Ricciardiello
Introduction/Objectives: The link between inflammation and colorectal cancer (CRC) is wellestablished, and macrophages in the lamina propria are believed to be critical for colitis-associated CRC development. Notch signaling has a pivotal role in carcinogenesis and Notch1can act as an oncogene by promoting cell survival and proliferation through its activationby the ligand Jagged1. A consumption of anti-inflammatory agents, such as ω-3 polyunsatu-rated fatty acids (ω-3 PUFAs), has a protective role on colorectal carcinogenesis. The chemo-preventive activity of eicosapentaenoic acid free fatty acid (EPA-FFA), an ω-3 PUFA, hasbeen demonstrated in preclinical models of CRC, but its activity in inflammatory-drivenCRC remains to be elucidated. The aim of this study was to evaluate whether Notch1 canbe activated by an inflammatory stimuli through STAT3-Jagged1 in CRC cells and whetherthis mechanism can be counteracted by treatment with EPA-FFA. Methods: The humanmonocyte cell line THP-1 was differentiated to macrophages using phorbol 12-myristate13-acetate and stimulated with E.coli lipopolysaccharide to obtain a cytokine-enrichedconditioned media (CM). Differentiation was evaluated by FACS analysis. Cytokines levelswere analyzed by Luminex technology. The human CRC cell line HT29 was treated withdifferent concentrations of EPA-FFA to test cell viability by MTT assay. Membrane incorpora-tion of EPA was evaluated using GC-MS. In order to explore the effect of the inflammatorymicroenvironment, the whole CM was applied to untreated and EPA-treated HT29 cellsfor different times. Protein levels of Notch1 intracellular Domain (NICD), STAT3 and itsphosphorylated form (p-STAT3) were quantified by western blotting. Gene expression ofthe Notch1 ligand Jagged1 and Notch downstream targets NRARP and Hath1 were evaluatedby Real-Time PCR. Results: THP1 differentiated macrophages produced a conditioned mediawith high concentrations of MIP-1, IL-8, TNF-α, MCP-1, IL-6 and IFN-γ. CM stimulationof HT29 induced a strong activation of pSTAT3, increased expressions of NICD and Jagged1.The activation of Notch1 caused a 3-fold up-regulation of NRARP and a 16-fold down-regulation of Hath1. Interestingly, pre-treatment with non-cytotoxic concentration of EPA-FFA, corresponding to an incorporation of approximately 20% of EPA in the cellular mem-brane, counteracted the CM-driven activation of NICD and pSTAT3. Importantly, Jagged1induction returned to basal levels after EPA-FFA treatment. Finally, EPA-FFA effects onNotch1 were supported by a concordant modulation of its downstream targets NRARP andHATH1. Conclusion: Inflammation is a strong activator of Notch1 signaling in CRC cellsand this activation is reduced by EPA-FFA treatment. Our data suggest a possible newmechanism of action of eicosapentaenoic acid as chemopreventive agent in inflammatorydriven-CRC.
Mo1163
Carcinogenic Effects of Benzo[a]Pyrene (Cyp 450 Activation) on HET-1A andBar-T Cell Line Could Be Reversed by CurcuminCarlos D. Minacapelli, Manisha Bajpai, Xin Geng, Kiron M. Das
Introduction: Smoking and history of gastroesophageal reflux disease (GERD), are associatedrisk factors for development of esophageal cancers. Benzo[a]pyrene (B[a]P) is a prototypeof poly aromatic hydrocarbons found in cigarette smoke, charbroiled or grilled foods, andhas been implicated in several human malignancies including cancers of aero-digestive tract.B[a]P, a promutagen, follows the aromatic hydrocarbon receptor (AhR) Pathway, whichresults in up-regulation of cytocrome P450 (CYP) genes. These CYP genes metabolize B[a]Pto potent carcinogens like B[a]P diol epoxide. The oxidation reaction that most efficientlygenerates B[a]P-dependent mutagenesis is catalyzed by CYP1A1 and CYP1B1 genes. Cur-cumin has been shown to have chemopreventive effects in cell line models derived fromsome of the common cancer like those of colon and breast. One of the mechanisms of action
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