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Ghrelin promotes pancreatic adenocarcinoma cellular proliferation and invasiveness
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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS
Background: SRF is a transcription factor that controls mesodermal differentiation. To date, there has been no report demonstrating the role of SRF in endodermally derived tissue. Genetic ablation of SRF results in embryonic lethality precluding analysis of SRF in pancreatic development. We sought to determine the expression of SRF in the normal adult rodent pancreas and during pancreatic regeneration (PR). Methods: Pancreata from adult rodents were harvested and fixed in 4% paraformaldehyde. Sections were stained for SRF expression using a polyclonal antibody. 90% pancreatectomies were performed and regenerating pancreatic remnants harvested at 0, 24, 72 hrs and 7 days. Tissues were processed and stained as above. Sham laparotomies served as controls. Results: Nuclear staining for SRF was observed in normal pancreatic ductal, acinar, and islet cells. Staining was stronger in islet cells compared to ductal and acinar cells. Double staining for insulin demonstrated islet SRF staining was predominately within the beta cells. During PR, SRF expression was increased with maximal expression at 36 hours (Fig 1B). SRF expression continued to be increased compared to baseline and sham at 7 days. In contrast to normal pancreata, SRF expression in the regenerating pancreas was found predominately in the acinar cytoplasm (AC). Conclusion: For the first time, SRF expression is observed in all three cellular components of the pancreas. During PR, SRF expression is increased and redistributed to the acinar cytoplasm. The data suggests SRF may be an important regulator of pancreatic growth, replication, and differentiation.
Fig 1A: Sham acinar cells (36 hrs) no AC staining. Fig 1B: PR acinar cells (36hrs) SRF AC staining (arrow). 91. Ghrelin Promotes Pancreatic Adenocarcinoma Cellular Proliferation and Invasiveness. M. S. Duxbury, M.R.C.S., T. Waseem, M.D., H. Ito, M.D., M. J. Zinner, M.D., S. W. Ashley, M.D., E..E. Whang, M.D. Department of Surgery, Brigham & Women’s Hospital, Harvard Medical School, Boston, Massachusetts. Introduction: Ghrelin, a newly described 28 amino acid peptide hormone, has potent orexogenic properties that are of interest in developing novel therapies for cancer cachexia. We tested the hypothesis that pancreatic adenocarcinoma, commonly associated with marked cachexia, is a ghrelin-responsive malignancy. Methods: PANC1, MIAPaCa2, BxPC3 and Capan2 pancreatic adenocarcinoma cells were cultured in the presence of 1 to 100nM ghrelin or control vehicle (PBS). Proliferation was determined at 4 days by cell counting and MTT assay. Expression of ghrelin and the functional ghrelin 1a receptor was determined by RT-PCR and Western blot analysis. The effect of 10nM ghrelin on invasiveness was quantified in an 8m pore Matrigel Boyden chamber assay and normalized to proliferation. Phosphorylation of Akt, a signal transduction molecule associated with pancreatic cancer invasiveness, was assessed using phospho-Akt (pAKT)-specific immunoblotting, and normalized to total Akt. Results: All cell lines expressed ghrelin 1a receptor mRNA and protein but none expressed ghrelin. Ghrelin treatment increased cellular proliferation (see graph, mean ⫾ SD relative to control) and, at 10nM, increased cellular invasiveness (PANC1: 60 ⫾ 5%, MIAPaCa2: 35 ⫾ 3%, BxPC3: 30 ⫾ 3% Capan2: 15 ⫾ 3%. Mean % increase ⫾ SD. P ⬍ 0.05) relative to control. Activating phosphorylation of Akt was increased by 10nM ghrelin in all cell lines. Conclusion: Pancreatic adenocarcinoma is a ghrelin-responsive malig-
nancy. The use of ghrelin as a treatment for pancreatic cancer cachexia will require cautious evaluation.
ONCOLOGY PARALLEL SESSION II 92. Upregulation of the Arginine Transporter Atb(0,ⴙ) in Colorectal Cancer. N. Gupta, M.B.B.S., M.S., S. Miyauchi, Ph.D., R. G. Martindale, M.D., Ph.D., A. Herdman, M.D., H. Hu, M.D., R. Podolsky, Ph.D., S. Mager, Ph.D., V. Ganapathy, Ph.D. Medical College of Georgia Department of Surgery, Augusta, GA. Introduction: Excess production of nitric oxide (NO) from arginine by the enzyme iNOS plays a crucial role in colorectal cancer. Recently, a highly efficient arginine transporter ATB(0,⫹) has been described in human tissues. We hypothesize that ATB(0,⫹) is upregulated in colorectal cancer as a means to deliver arginine to iNOS for excess NO production. Methods: Total RNA was extracted from paired normal and cancer tissue harvested from colectomy specimens of 10 enrolled patients. Expression of ATB(0,⫹) mRNA was determined by semi-quantitative RT-PCR using 18S as an internal control, and confirmed by Northern blot (2 transcripts, 4.5 and 2 kb), with -actin as an internal control. ATB(0,⫹) and iNOS protein expression was determined by immunohistochemistry (IHC). Excess NO was detected by IHC using antibodies against nitrosylated tyrosine residues. Results: ATB(0,⫹) mRNA showed an 11-fold increase in colorectal cancer by RT-PCR (the 95% lower confidence limit for relative expression was a 6.55 fold increase) and a greater than 10-fold increase by Northern blot. ATB(0,⫹) protein expression showed a corresponding increase by IHC. ATB(0,⫹) and iNOS proteins co-localized in the cell. Cancer tissue showed increased NO levels. Conclusions: ATB(0,⫹) is significantly up-regulated in colorectal cancer and co-localizes with iNOS. We propose that upregulation of ATB(0,⫹) underlies the increased production of NO in colorectal cancer. Since ATB(0,⫹) has recently been shown to efficiently transport inhibitors of iNOS, this could provide a novel therapeutic strategy in colorectal cancer.
ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS
Background: SRF is a transcription factor that controls mesodermal differentiation. To date, there has been no report demonstrating the role of SRF in endodermally derived tissue. Genetic ablation of SRF results in embryonic lethality precluding analysis of SRF in pancreatic development. We sought to determine the expression of SRF in the normal adult rodent pancreas and during pancreatic regeneration (PR). Methods: Pancreata from adult rodents were harvested and fixed in 4% paraformaldehyde. Sections were stained for SRF expression using a polyclonal antibody. 90% pancreatectomies were performed and regenerating pancreatic remnants harvested at 0, 24, 72 hrs and 7 days. Tissues were processed and stained as above. Sham laparotomies served as controls. Results: Nuclear staining for SRF was observed in normal pancreatic ductal, acinar, and islet cells. Staining was stronger in islet cells compared to ductal and acinar cells. Double staining for insulin demonstrated islet SRF staining was predominately within the beta cells. During PR, SRF expression was increased with maximal expression at 36 hours (Fig 1B). SRF expression continued to be increased compared to baseline and sham at 7 days. In contrast to normal pancreata, SRF expression in the regenerating pancreas was found predominately in the acinar cytoplasm (AC). Conclusion: For the first time, SRF expression is observed in all three cellular components of the pancreas. During PR, SRF expression is increased and redistributed to the acinar cytoplasm. The data suggests SRF may be an important regulator of pancreatic growth, replication, and differentiation.
Fig 1A: Sham acinar cells (36 hrs) no AC staining. Fig 1B: PR acinar cells (36hrs) SRF AC staining (arrow). 91. Ghrelin Promotes Pancreatic Adenocarcinoma Cellular Proliferation and Invasiveness. M. S. Duxbury, M.R.C.S., T. Waseem, M.D., H. Ito, M.D., M. J. Zinner, M.D., S. W. Ashley, M.D., E..E. Whang, M.D. Department of Surgery, Brigham & Women’s Hospital, Harvard Medical School, Boston, Massachusetts. Introduction: Ghrelin, a newly described 28 amino acid peptide hormone, has potent orexogenic properties that are of interest in developing novel therapies for cancer cachexia. We tested the hypothesis that pancreatic adenocarcinoma, commonly associated with marked cachexia, is a ghrelin-responsive malignancy. Methods: PANC1, MIAPaCa2, BxPC3 and Capan2 pancreatic adenocarcinoma cells were cultured in the presence of 1 to 100nM ghrelin or control vehicle (PBS). Proliferation was determined at 4 days by cell counting and MTT assay. Expression of ghrelin and the functional ghrelin 1a receptor was determined by RT-PCR and Western blot analysis. The effect of 10nM ghrelin on invasiveness was quantified in an 8m pore Matrigel Boyden chamber assay and normalized to proliferation. Phosphorylation of Akt, a signal transduction molecule associated with pancreatic cancer invasiveness, was assessed using phospho-Akt (pAKT)-specific immunoblotting, and normalized to total Akt. Results: All cell lines expressed ghrelin 1a receptor mRNA and protein but none expressed ghrelin. Ghrelin treatment increased cellular proliferation (see graph, mean ⫾ SD relative to control) and, at 10nM, increased cellular invasiveness (PANC1: 60 ⫾ 5%, MIAPaCa2: 35 ⫾ 3%, BxPC3: 30 ⫾ 3% Capan2: 15 ⫾ 3%. Mean % increase ⫾ SD. P ⬍ 0.05) relative to control. Activating phosphorylation of Akt was increased by 10nM ghrelin in all cell lines. Conclusion: Pancreatic adenocarcinoma is a ghrelin-responsive malig-
nancy. The use of ghrelin as a treatment for pancreatic cancer cachexia will require cautious evaluation.
ONCOLOGY PARALLEL SESSION II 92. Upregulation of the Arginine Transporter Atb(0,ⴙ) in Colorectal Cancer. N. Gupta, M.B.B.S., M.S., S. Miyauchi, Ph.D., R. G. Martindale, M.D., Ph.D., A. Herdman, M.D., H. Hu, M.D., R. Podolsky, Ph.D., S. Mager, Ph.D., V. Ganapathy, Ph.D. Medical College of Georgia Department of Surgery, Augusta, GA. Introduction: Excess production of nitric oxide (NO) from arginine by the enzyme iNOS plays a crucial role in colorectal cancer. Recently, a highly efficient arginine transporter ATB(0,⫹) has been described in human tissues. We hypothesize that ATB(0,⫹) is upregulated in colorectal cancer as a means to deliver arginine to iNOS for excess NO production. Methods: Total RNA was extracted from paired normal and cancer tissue harvested from colectomy specimens of 10 enrolled patients. Expression of ATB(0,⫹) mRNA was determined by semi-quantitative RT-PCR using 18S as an internal control, and confirmed by Northern blot (2 transcripts, 4.5 and 2 kb), with -actin as an internal control. ATB(0,⫹) and iNOS protein expression was determined by immunohistochemistry (IHC). Excess NO was detected by IHC using antibodies against nitrosylated tyrosine residues. Results: ATB(0,⫹) mRNA showed an 11-fold increase in colorectal cancer by RT-PCR (the 95% lower confidence limit for relative expression was a 6.55 fold increase) and a greater than 10-fold increase by Northern blot. ATB(0,⫹) protein expression showed a corresponding increase by IHC. ATB(0,⫹) and iNOS proteins co-localized in the cell. Cancer tissue showed increased NO levels. Conclusions: ATB(0,⫹) is significantly up-regulated in colorectal cancer and co-localizes with iNOS. We propose that upregulation of ATB(0,⫹) underlies the increased production of NO in colorectal cancer. Since ATB(0,⫹) has recently been shown to efficiently transport inhibitors of iNOS, this could provide a novel therapeutic strategy in colorectal cancer.