- Email: [email protected]
Mo1957 Identification of Small Molecules to Suppress Pancreatic Cancer Growth
group. For cytoskeletal markers, we investigated focal adhesion kinase (FAK) given evidence of its importance in a myriad of cancers, including pancreatic. Samples were then immediately fixed in formalin, processed for IHC, and stained for FAK. The lengths of microvilli and crypt lengths were calculated using the ImageJ software (NIH) by observer blinded to sample group. Results: In the LEBS analysis, there were profound alterations in several spectral markers. Indeed, there was a significant decrease in the E parameter and an increase in the m parameter (Table 1), suggesting that the contribution of larger length scales in the tissue increases. To investigate this, we rigorously measured crypt and microvillus length in the duodenum and found a significant increase in microvilli and crypt lengths (24% and 47%, respectively) in the patients with adenocarcinoma compared to control patients (P-values = 0.069 and 0.028, respectively). Additionally, we found FAK to be significantly upregulated in the cancer patients (130% increase, P < 0.001), demonstrating dysregulation of focal adhesion scaffolding and related pathways. Conclusions: We provide ultrastructural corroboration to the duodenal micro-architectural changes detected by LEBS in pancreatic cancer screening. Our findings show that both small (cytoskeletal) and large (crypt/villus) length scales are dysregulated. This work demonstrates the biological alterations in the extended pancreatic field carcinogenesis and may serve as minimally intrusive biomarkers for risk stratification of pancreatic cancer. LEBS analysis of the duodenal tissue in pancreatic cancer patients compared to control patients.
Rottlerin Promotes Apoptosis and Autophagy in Pancreatic Stellate Cells via AMPK Activation Aurelia Lugea, Payam Javaherizadeh, Hongxiang Hui, Richard T. Waldron, Stephen J. Pandol Background and Significance: Phytochemicals may play roles to reduce carcinogenesis by modulating central metabolic pathways in cancer and tumor stromal cells. AMP-activated kinases (AMPK) are key regulators of cellular metabolism, cell growth and proliferation. Activation of AMPK kinases by low energy status inhibits anabolic processes such as protein and fatty acid synthesis, activates autophagy and can lead to apoptosis. AMPK actions are mediated at least in part by inhibition of mTOR complex-1 (TORC1). Previous studies from our group showed that rottlerin, a phytochemical from the kamala tree, reduced pancreatic tumor volume in an orthotopic model of pancreatic cancer. Here we investigated the effects of rottlerin on AMPK/mTORC1 pathways in the pancreatic stellate cell (PaSC), a key cell type in the tumor microenvironment that promotes pancreatic carcinogenesis. Methods: Culture-activated primary mouse PaSC were incubated for up to 72 h in 10% FBS in the absence (control) or presence of rottlerin (0.5-10 μM). We then measured 1) activation of AMPK and Akt/mTORC1 pathways by Western blot analysis to assess phosphorylated states of intermediate proteins, 2) autophagy by Western blot and immunofluorescence to detect levels of and localize autophagic markers, 3) apoptosis by caspase-3 activity and ELISA to detect internucleosomal DNA fragmentation, and 4) cell viability/proliferation by MTT assay/ cell counting. Results: Rottlerin at concentrations as low as 0.5 μM induced rapid, sustained activation of AMPK, as determined by a significant increase in PaSC levels of phosphoAMPKα (Thr172). A marked inhibition of the Akt/mTOR pathway was indicated by reduced phosphorylation of Akt (Ser473) and the mTORC1 substrates p70 S6 kinase and 4EBP1. Further, mTORC1 inhibition blocked protein translation, and induced endoplasmic reticulum (ER) stress as indicated by phosphorylation of the translation initiator protein eiF2α (Ser51) and upregulation of the proapoptotic transcription factor CHOP. In addition, rottlerin treatment rapidly (within 15 min) induced LC3I-LC3II conversion that was sustained at least 48 h, and significantly increased p62/SQSTM1 protein expression. Together, these data suggest accumulation of autophagosomes as well as reduced autophagic flux in rottlerintreated PaSC. Interestingly, preincubation with the AMPK inhibitor compound C (20 μM) greatly reduced rottlerin-induced ER stress and autophagy. Furthermore, rottlerin dosedependently induced PaSC apoptosis, to a 6-fold increase over control at 1 μM and 36-fold at 10 μM after 72 h, and reduced cell viability by 35% at 0.5 μM, and by 70% at 10 μM. Conclusion: Our data elucidate the AMPK/mTOR pathways as a key target of rottlerin in PaSC and reinforce the notion that phytochemicals are therapeutically useful to reduce pancreatic cancer progression by eliminating pancreatic stellate cells.
Mo1957 Identification of Small Molecules to Suppress Pancreatic Cancer Growth David Liu, Xiangxun Zhao, David Ostrov, Chen Liu, Roniel Cabrera Background: Pancreatic cancer is the fourth leading cause of cancer death in the United States. The current treatment is not effective and the 5-year survival rate is less than 5%. Survivin is a cellular protein that is overexpressed in many types of cancer cells, including pancreatic cancer cells. Thus, it may be an ideal target for pancreatic cancer therapy. Since survivin functions as a dimer, we hypothesize that small molecules that can interfere the dimerization of survivin would be viable candidates for cancer therapy. Aim: The objective of our study is to identify small molecules that interfere with survivin dimerization through in silico molecular docking. These compounds will then be tested in pancreatic cell lines. Materials and Methods: We utilized the chemical and geometric characteristics of survivin dimerization sites by conducting dynamic molecular docking simulations. We used a docking program with more than 140,000 small molecules (available through the NCI/DTP) that tested their interactions with active sites on a survivin protein using the atomic coordinates provided by the crystal structures. The best-scored compounds were identified and identified as“lead” compounds. The lead compounds were tested for their ability to block dimerization of survivin in In Vitro assays using two pancreatic cancer cell lines, Panc-1 and MIA PaCa2. Cell growth was determined by an MTT assay, and the induction of apoptosis was determined by detection of caspase cleavage by Western blot. Results: Using the molecular docking approach, we identified 28 lead compounds. Three of these compounds showed a signficant ability to interfere with survivin dimerization. Varying concentrations of these compounds were then tested in Panc-1 and MIA PaCa-2 pancreatic cancer cell lines. Two compounds showed inhibitory effects on these cell lines. Western blot analysis showed the presence of caspase 3 cleavage, suggesting that these two compounds induced apoptosis in pancreatic cancer cells. Conclusion: We have demonstrated the feasibility of the molecular docking approach for the identification of survivin inhibitors. Two small molecular compounds were shown to inhibit survivin activity and induce apoptosis, resulting in suppression of pancreatic cancer cell growth.
Mo1955 Selective Eradication of Cancer Cells by Adenovirus-Based Delivery of Cytotoxic Agents: an Alternative Method for Targeting Pancreatic Cancer Shiran Shapira, Sarah Kraus, Diana Kazanov, Nadir Arber Background: Pancreatic cancer (PC) is a highly lethal disease. Mutation of the K-Ras gene is an early event that plays a direct causal role in PC carcinogenesis. The mutated K-Ras is a prime target for therapy, however most/all Ras inhibitors had failed. Adenovirus-based vectors are promising tools for cancer-directed cytotoxic gene transfer. Controlled transgeneexpression can improve their efficiency and safety. Aim: To evaluate a novel adenovirusbased strategy to eradicate PC-harboring mutated K-Ras. Methods: To achieve efficient DNA delivery into mammalian cells; the ribonuclease MazF-mCherry fusion gene, truncated derivative of Pseudomonas exotoxin (PE38), the full regulated-cassette encoding for the MazF-MazE (MazEF unique toxin-antitoxin (TA) system), and the corresponding control vectors were cloned into a “first generation” ΔE1/ΔE3 human type-5 adenoviral-vector under the control of the RRE or SV40 minimal promoter (Fig. 1). Virus particles were propagated in HEK293 toxin-resistant packaging-cells that were established in our lab. Cells harboring WT-K-Ras and mutated K-Ras were used as well as K-Ras-transformed enterocytes (R1 cells) (Arber et al., Gastroenterology 1996). Inhibition of proliferation, cell viability and toxinexpression were determined in-vitro. Results: Toxin-resistant stable cells and efficient toxic vectors for cancer-directed gene delivery were established and produced. Massive cell death (>50%) at low MOIs was seen in cells exhibiting activated K-Ras following exposure to the adenoviral vectors compared to WT-K-Ras-expressing cells. Conclusions: These novel adenoviral vectors carrying either, PE38, MazF or MazEF genes that target the K-Ras pathway can improve the outcome in this devastating disease.
Mo1958 Nutritional Management of Pediatric Acute Pancreatitis Soma Kumar, Cheryl E. Gariepy, Wallace Crandall Background: The incidence of acute pancreatitis (AP) is increasing in the pediatric population. There is a paucity of literature on the management of AP in children, especially with regards to nutrition. In adult severe AP, enteral nutrition (EN) is associated with less infectious complications, surgical interventions and mortality when compared to parenteral nutrition (PN). Recent studies have also shown the added protective effect of EN that is initiated early (within 24-48 hours). The aim of this study was to describe the nutritional management of pediatric patients with AP and identify factors associated with the initiation of PN. Methods: We performed a retrospective chart review of 188 patients seen between Jan 2009 to Sept 2010 with a primary/secondary diagnosis of pancreatitis. The diagnosis and severity of AP was determined using the Atlanta criteria. Demographic, laboratory and radiographic data was collected along with data regarding nutritional management. Univariate analysis was used to determine factors associated with PN use, any significant factors were then combined in a multivariate logistic regression model. Results: 110 patients (99 mild, 11 severe) met the definition of AP and were included. The average patient age was 11.6 yrs. Lipase and amylase were >3x upper limit of normal in 96% and 33%, respectively. Pancreatic imaging was abnormal in 28%. 98% were initially fasted and the average fast time for mild and severe patients was 57 hours and 183 hours, respectively (p=0.0006). Once a diet was resumed, 73% were given clear liquids, 10% were allowed a solid diet and 17% were started on tube feeds. 29% of patients were considered intolerant of their initial diet most commonly due to abdominal pain (31%) and increasing lipase levels (28%). This resulted in withdrawal of enteral nutrition in 67% and the initiation of PN in 27%. By univariate analyses, four variables were significant (p<0.05) with regards to requiring PN; severe AP, younger age (<=3 years), BMI < 85% and discharge from a surgical service. When combined in a multivariable logistic regression model, only BMI, severe AP, and service were significant
Mo1956 Ultrastructural Alterations in Microscopically Normal Duodenal Mucosa as a Biomarker for Pancreatic Cancer Field Carcinogenesis Yolanda E. Stypula, Andrew Radosevich, Dhananjay Kunte, Nikhil N. Mutyal, Mart DeLaCruz, Tina P. Gibson, Ramesh K. Wali, Wentao Qi, Hemant K. Roy, Vadim Backman Introduction: Our group has developed a novel optical technology termed low-coherence enhanced backscattering (LEBS) spectroscopy, capable of detecting microarchitectural alterations of field carcinogenesis and hence enabling cancer risk stratification (reviewed in Gastro, 2011). For pancreatic cancer, emerging evidence suggests that the duodenum may be a surrogate site, potentially due to the impact of pancreatic juices. Indeed, we have reported that LEBS interrogation of the peri-ampullary duodenal tissue was accurate at discriminating patients with pancreatic cancers from controls (Clin Cancer Res, 2007; Dis Markers, 2008). However, the biological underpinnings remain unclear. In the colon, we have noted that alterations in crypt length and colonocytes cytoskeletal perturbations appear central (Biophysics J, 2010). We therefore, wanted to investigate these parameters in the duodenum of pancreatic cancer patients versus controls. Methods: The instrument setup used to collect LEBS signals was similar to as previously described (Opt Lett, 2005). Measurements were taken on duodenal samples from control patients (n=22) or patients harboring an adenocarcinoma in the pancreas (n=6). LEBS markers were calculated by observer blinded to sample
S-707
AGA Abstracts
AGA Abstracts
Mo1954
Rottlerin Promotes Apoptosis and Autophagy in Pancreatic Stellate Cells via AMPK Activation Aurelia Lugea, Payam Javaherizadeh, Hongxiang Hui, Richard T. Waldron, Stephen J. Pandol Background and Significance: Phytochemicals may play roles to reduce carcinogenesis by modulating central metabolic pathways in cancer and tumor stromal cells. AMP-activated kinases (AMPK) are key regulators of cellular metabolism, cell growth and proliferation. Activation of AMPK kinases by low energy status inhibits anabolic processes such as protein and fatty acid synthesis, activates autophagy and can lead to apoptosis. AMPK actions are mediated at least in part by inhibition of mTOR complex-1 (TORC1). Previous studies from our group showed that rottlerin, a phytochemical from the kamala tree, reduced pancreatic tumor volume in an orthotopic model of pancreatic cancer. Here we investigated the effects of rottlerin on AMPK/mTORC1 pathways in the pancreatic stellate cell (PaSC), a key cell type in the tumor microenvironment that promotes pancreatic carcinogenesis. Methods: Culture-activated primary mouse PaSC were incubated for up to 72 h in 10% FBS in the absence (control) or presence of rottlerin (0.5-10 μM). We then measured 1) activation of AMPK and Akt/mTORC1 pathways by Western blot analysis to assess phosphorylated states of intermediate proteins, 2) autophagy by Western blot and immunofluorescence to detect levels of and localize autophagic markers, 3) apoptosis by caspase-3 activity and ELISA to detect internucleosomal DNA fragmentation, and 4) cell viability/proliferation by MTT assay/ cell counting. Results: Rottlerin at concentrations as low as 0.5 μM induced rapid, sustained activation of AMPK, as determined by a significant increase in PaSC levels of phosphoAMPKα (Thr172). A marked inhibition of the Akt/mTOR pathway was indicated by reduced phosphorylation of Akt (Ser473) and the mTORC1 substrates p70 S6 kinase and 4EBP1. Further, mTORC1 inhibition blocked protein translation, and induced endoplasmic reticulum (ER) stress as indicated by phosphorylation of the translation initiator protein eiF2α (Ser51) and upregulation of the proapoptotic transcription factor CHOP. In addition, rottlerin treatment rapidly (within 15 min) induced LC3I-LC3II conversion that was sustained at least 48 h, and significantly increased p62/SQSTM1 protein expression. Together, these data suggest accumulation of autophagosomes as well as reduced autophagic flux in rottlerintreated PaSC. Interestingly, preincubation with the AMPK inhibitor compound C (20 μM) greatly reduced rottlerin-induced ER stress and autophagy. Furthermore, rottlerin dosedependently induced PaSC apoptosis, to a 6-fold increase over control at 1 μM and 36-fold at 10 μM after 72 h, and reduced cell viability by 35% at 0.5 μM, and by 70% at 10 μM. Conclusion: Our data elucidate the AMPK/mTOR pathways as a key target of rottlerin in PaSC and reinforce the notion that phytochemicals are therapeutically useful to reduce pancreatic cancer progression by eliminating pancreatic stellate cells.
Mo1957 Identification of Small Molecules to Suppress Pancreatic Cancer Growth David Liu, Xiangxun Zhao, David Ostrov, Chen Liu, Roniel Cabrera Background: Pancreatic cancer is the fourth leading cause of cancer death in the United States. The current treatment is not effective and the 5-year survival rate is less than 5%. Survivin is a cellular protein that is overexpressed in many types of cancer cells, including pancreatic cancer cells. Thus, it may be an ideal target for pancreatic cancer therapy. Since survivin functions as a dimer, we hypothesize that small molecules that can interfere the dimerization of survivin would be viable candidates for cancer therapy. Aim: The objective of our study is to identify small molecules that interfere with survivin dimerization through in silico molecular docking. These compounds will then be tested in pancreatic cell lines. Materials and Methods: We utilized the chemical and geometric characteristics of survivin dimerization sites by conducting dynamic molecular docking simulations. We used a docking program with more than 140,000 small molecules (available through the NCI/DTP) that tested their interactions with active sites on a survivin protein using the atomic coordinates provided by the crystal structures. The best-scored compounds were identified and identified as“lead” compounds. The lead compounds were tested for their ability to block dimerization of survivin in In Vitro assays using two pancreatic cancer cell lines, Panc-1 and MIA PaCa2. Cell growth was determined by an MTT assay, and the induction of apoptosis was determined by detection of caspase cleavage by Western blot. Results: Using the molecular docking approach, we identified 28 lead compounds. Three of these compounds showed a signficant ability to interfere with survivin dimerization. Varying concentrations of these compounds were then tested in Panc-1 and MIA PaCa-2 pancreatic cancer cell lines. Two compounds showed inhibitory effects on these cell lines. Western blot analysis showed the presence of caspase 3 cleavage, suggesting that these two compounds induced apoptosis in pancreatic cancer cells. Conclusion: We have demonstrated the feasibility of the molecular docking approach for the identification of survivin inhibitors. Two small molecular compounds were shown to inhibit survivin activity and induce apoptosis, resulting in suppression of pancreatic cancer cell growth.
Mo1955 Selective Eradication of Cancer Cells by Adenovirus-Based Delivery of Cytotoxic Agents: an Alternative Method for Targeting Pancreatic Cancer Shiran Shapira, Sarah Kraus, Diana Kazanov, Nadir Arber Background: Pancreatic cancer (PC) is a highly lethal disease. Mutation of the K-Ras gene is an early event that plays a direct causal role in PC carcinogenesis. The mutated K-Ras is a prime target for therapy, however most/all Ras inhibitors had failed. Adenovirus-based vectors are promising tools for cancer-directed cytotoxic gene transfer. Controlled transgeneexpression can improve their efficiency and safety. Aim: To evaluate a novel adenovirusbased strategy to eradicate PC-harboring mutated K-Ras. Methods: To achieve efficient DNA delivery into mammalian cells; the ribonuclease MazF-mCherry fusion gene, truncated derivative of Pseudomonas exotoxin (PE38), the full regulated-cassette encoding for the MazF-MazE (MazEF unique toxin-antitoxin (TA) system), and the corresponding control vectors were cloned into a “first generation” ΔE1/ΔE3 human type-5 adenoviral-vector under the control of the RRE or SV40 minimal promoter (Fig. 1). Virus particles were propagated in HEK293 toxin-resistant packaging-cells that were established in our lab. Cells harboring WT-K-Ras and mutated K-Ras were used as well as K-Ras-transformed enterocytes (R1 cells) (Arber et al., Gastroenterology 1996). Inhibition of proliferation, cell viability and toxinexpression were determined in-vitro. Results: Toxin-resistant stable cells and efficient toxic vectors for cancer-directed gene delivery were established and produced. Massive cell death (>50%) at low MOIs was seen in cells exhibiting activated K-Ras following exposure to the adenoviral vectors compared to WT-K-Ras-expressing cells. Conclusions: These novel adenoviral vectors carrying either, PE38, MazF or MazEF genes that target the K-Ras pathway can improve the outcome in this devastating disease.
Mo1958 Nutritional Management of Pediatric Acute Pancreatitis Soma Kumar, Cheryl E. Gariepy, Wallace Crandall Background: The incidence of acute pancreatitis (AP) is increasing in the pediatric population. There is a paucity of literature on the management of AP in children, especially with regards to nutrition. In adult severe AP, enteral nutrition (EN) is associated with less infectious complications, surgical interventions and mortality when compared to parenteral nutrition (PN). Recent studies have also shown the added protective effect of EN that is initiated early (within 24-48 hours). The aim of this study was to describe the nutritional management of pediatric patients with AP and identify factors associated with the initiation of PN. Methods: We performed a retrospective chart review of 188 patients seen between Jan 2009 to Sept 2010 with a primary/secondary diagnosis of pancreatitis. The diagnosis and severity of AP was determined using the Atlanta criteria. Demographic, laboratory and radiographic data was collected along with data regarding nutritional management. Univariate analysis was used to determine factors associated with PN use, any significant factors were then combined in a multivariate logistic regression model. Results: 110 patients (99 mild, 11 severe) met the definition of AP and were included. The average patient age was 11.6 yrs. Lipase and amylase were >3x upper limit of normal in 96% and 33%, respectively. Pancreatic imaging was abnormal in 28%. 98% were initially fasted and the average fast time for mild and severe patients was 57 hours and 183 hours, respectively (p=0.0006). Once a diet was resumed, 73% were given clear liquids, 10% were allowed a solid diet and 17% were started on tube feeds. 29% of patients were considered intolerant of their initial diet most commonly due to abdominal pain (31%) and increasing lipase levels (28%). This resulted in withdrawal of enteral nutrition in 67% and the initiation of PN in 27%. By univariate analyses, four variables were significant (p<0.05) with regards to requiring PN; severe AP, younger age (<=3 years), BMI < 85% and discharge from a surgical service. When combined in a multivariable logistic regression model, only BMI, severe AP, and service were significant
Mo1956 Ultrastructural Alterations in Microscopically Normal Duodenal Mucosa as a Biomarker for Pancreatic Cancer Field Carcinogenesis Yolanda E. Stypula, Andrew Radosevich, Dhananjay Kunte, Nikhil N. Mutyal, Mart DeLaCruz, Tina P. Gibson, Ramesh K. Wali, Wentao Qi, Hemant K. Roy, Vadim Backman Introduction: Our group has developed a novel optical technology termed low-coherence enhanced backscattering (LEBS) spectroscopy, capable of detecting microarchitectural alterations of field carcinogenesis and hence enabling cancer risk stratification (reviewed in Gastro, 2011). For pancreatic cancer, emerging evidence suggests that the duodenum may be a surrogate site, potentially due to the impact of pancreatic juices. Indeed, we have reported that LEBS interrogation of the peri-ampullary duodenal tissue was accurate at discriminating patients with pancreatic cancers from controls (Clin Cancer Res, 2007; Dis Markers, 2008). However, the biological underpinnings remain unclear. In the colon, we have noted that alterations in crypt length and colonocytes cytoskeletal perturbations appear central (Biophysics J, 2010). We therefore, wanted to investigate these parameters in the duodenum of pancreatic cancer patients versus controls. Methods: The instrument setup used to collect LEBS signals was similar to as previously described (Opt Lett, 2005). Measurements were taken on duodenal samples from control patients (n=22) or patients harboring an adenocarcinoma in the pancreas (n=6). LEBS markers were calculated by observer blinded to sample
S-707
AGA Abstracts
AGA Abstracts
Mo1954