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Comparison of antiangiogenic therapy with thrombospondin type I repeats and gemcitabine in an orthotopic model of pancreatic cancer in SCID mice
Vol. 199, No. 3S, September 2004
Alimentary Tract I
S11
RNAi-mediated inhibition of cyclin D1 decreases colon cancer cell proliferation and MMP protein expression Carlos A Murillo MD, Piotr G. Rychahou MD, B Mark Evers, MD, FACS The University of Texas Medical Branch Galveston, TX INTRODUCTION: Cyclin D1, a downstream protein of the PI3K/GSK-3 pathway, regulates cell cycle progression and proliferation. Overexpression of cyclin D1 is associated with a poor prognosis in various human cancers (eg, colorectal cancers). The purpose of this study was to determine whether targeted inhibition of cyclin D1, using small interfering RNA (siRNA), alters proliferation and expression of the matrix metalloproteinase proteins (MMPs) in the colon cancer cell line KM20. METHODS: KM20 cells were treated with cyclin D1 siRNA or scrambled sequences (control); cell proliferation and viability was assessed. Proliferating cell nuclear antigen (PCNA) expression was measured by ELISA. Western blots were performed to determine expression of cyclin D1, PI3K subunits p85alpha and p110 and GSK-3alpha/beta (upstream regulators of cyclin D1), and MMP-2 and MMP-9 (tumor invasion proteins regulated by cyclin D1). RESULTS: Cyclin D1 expression was effectively blocked by siRNA treatment. Expression of MMP-2 and MMP-9 was decreased in KM20 cells by cyclin D1 inhibition; PI3K subunit and GSK-3 expression was not affected. Compared to control, decreased KM20 cell proliferation and PCNA translocation was noted with cyclin D1 siRNA treatment; no significant changes were noted in apoptosis. CONCLUSIONS: We show that selective inhibition of cyclin D1 inhibits MMP expression and KM20 cell proliferation, most likely due to cell cycle arrest. Selective targeting of cyclin D1 may enhance the effects of standard chemotherapeutic agents and provide novel adjunct treatment for selected colorectal cancers.
Transcriptomic fingerprints of Beta2mⴚ/Thy1ⴙ bone marrow stem cells derived from common bile ductligated rats Marjorie R. Chelly MD, Charles Wang MD, PhD, Thomas Hui MD, Ning-Ning Chai MD, PhD, Gulia Castiglione MD, Itzah Avital MD, Achilles A Demetriou MD, PhD, FACS Cedars Sinai Medical Center Los Angeles, CA INTRODUCTION: Previously our laboratory identified a population of cells within the bone marrow, known as Beta2m⫺/Thy1⫹ bone marrow derived stem cells (BMSC) that are able to differentiate into hepatocytes. The effect of liver injury on the genes expressed by Beta2m⫺/Thy1⫹ BMSC is not known. The objective of this study was to determine the gene expression profile of Beta2m⫺/Thy1⫹ BMSC in common bile duct-ligated (CBDL) rats using DNA microarray analysis. METHODS: Male Lewis rats underwent CBDL (n ⫽ 3)or a sham operation (n ⫽ 3). After 10 days, their bone marrow was harvested and Beta2m⫺/Thy1⫹ BMSC isolated using a two-step magnetic
bead cell sorting technique. The mRNA expression profile of primary hepatocytes harvested from sham-operated male Lewis rats (n ⫽ 3) was used for comparison. The gene expression profile of Beta2m⫺/Thy1⫹ BMSC was determined using Affymetrix rat genemone U34a array. RESULTS: In sham-operated rats, 24 common genes were found between Beta2m-/Thy1⫹ BMSC and normal hepatocytes. In the CBDL rats, 189 commonly expressed genes were found between the Beta2m-/ Thy1⫹ BMSC and normal hepatocytes. Several liver-specific genes were identified including HNF1, Complement component C3, cholesterol esterase, Cytochrome P50 3A2, D site albumin promoter binding protein and mitochondrial import stimulation factor. CONCLUSIONS: Our results demonstrate that liver injury by CBDL alters the gene expression profile of Beta2m-/Thy1⫹ BMSCs. Cluster analysis of hepatocyte genes revealed Beta2m-/Thy1⫹ BMSCs from CBDL rats shared more genes in common with primary hepatocytes than Beta2m-/ Thy1⫹ BMSCs from sham-operated rats.
Comparison of antiangiogenic therapy with thrombospondin type I repeats and gemcitabine in an orthotopic model of pancreatic cancer in SCID mice Xue Feng Zhang MD, PhD, Xue Zhang MD, PhD, Eric Galardi BA, Mark Duquette BA, Jack Lawler PhD, Sareh Parangi MD, FACS Beth Israel Deaconess Medical Center Boston, MA INTRODUCTION: Pancreatic cancer is a devastating disease. Efficacy of type I repeats of thrombospondin (TSP-1) in mice with orthotopic pancreatic cancer was tested and compared to gemcitabine. METHODS: SCID mice treated with an intrapancreatic injection of a human pancreatic cancer cell line (ASPC-1) developed large ortho-
S12
Alimentary Tract I
topic tumors with local invasion, peritoneal and and mesenteric implants. Recombinant Thrombospondin Type I repeats (TSR), one of the antiangiogenic domains of TSP, was cloned and purified. One week after tumor implantation, mice were treated with 3TSR (n ⫽ 9, 0.3 mg/kg/day), gemcitabine (n ⫽ 6, 15 mg/kg, twice weekly) or the combination of the two drugs (n ⫽ 11) for 3 weeks. Subsequently, mouse weight, tumor size, number of metasteses were compared. Tumor volume was calculated using the formula 0.52 (width) 2 (length). Student’s t test (Mann-Whitney) was used for statistical comparison. RESULTS: Tumor bearing mice treated with TSR had a 65% reduction in tumor volume (371 ⫾ 97 vs 1064 ⫾ 375 mm3, p ⬍ 0.0001). Tumor volume was reduced 84% with gemcitabine alone (167 ⫾ 67 mm3 ) and 79% with gemcitabine plus TSR treatment (219.39 ⫾ 78.37 mm3 ) p ⬍ 0.001 when compared to control but p ⬎ 0.05 when compared to eachother. Mean vessel density was lowest in the TSR treated animals (3.74 ⫾ 1.2) and highest in the control group (7.96 ⫾ 6.7), p ⬍ 0.05. Proliferation and apoptosis rates were compared. CONCLUSIONS: Antiangiogenic therapy using TSR reduces tumor volume and vascular density in mice with orthotopic pancreatic tumors and can be used safely alone or in combination with gemcitabine.
Aerosolization of heparin and hyaluronan reduce peritoneal loco-regional tumour growth Nawar A Alkhamesi MD, FRCS, FRCSEd, David H Peck PhD, Ara W Darzi MD, FRCS, FRCSI, FACS Imperial College London London, United Kingdom INTRODUCTION: Loco-regional intraperitoneal recurrence remains a critical issue in the management of many abdominal malignancies and is associated with poor prognosis. Around 25% of patients that undergo curative surgery will develop peritoneal recurrence at some stage. Recently, we have shown that this can be inhibited successfully using both heparin and hyaluronan both in vitro and in vivo. The former via an ICAM-1 dependent mechanism while the latter functioned as a barrier. We used an aerosolization device to deliver our therapeutics during laparoscopic procedures. The design of the machine and the catheter enable the operator to cover the whole surface area of the peritoneum with the correct therapeutics’ dose and volume. METHODS: Thirty male WAG rats were divided into 3 groups of 10. After the introduction of 1x105 CC5135, synergistic rat colorectal cancer cells, either 100U/ml heparin, 0.25% hyaluronan or PBS (control) was introduced into the peritoneum using the peritoneal aerosilization technique. After a recovery period of 2 weeks, the animals were sacrificed and the tumor growth was assessed macroscopically and microscopically
J Am Coll Surg
RESULTS: Heparin completely blocked tumor growth with no obvious macroscopic or microscopic invasion detected (p ⬍ 0.05). Hyaluronic acid reduced tumor number and size in comparison to control but at a lower level then heparin CONCLUSIONS: Both modalities were capable of inhibiting tumor spread in vivo. The ability of heparin to abolish tumor development with no local or systemic complications indicates a possible therapeutic to prevent loco-regional recurrence and shows the efficacy of aerosolizarion within the peritoneum.
Alimentary leptin receptors are most abundant in the gallbladder Deborah A Swartz-Basile PhD, Carol Svatek BS, Shannon Graewin MD, James Kiely MD, Henry Pitt MD, FACS Medical College of Wisconsin Milwaukee, WI INTRODUCTION: We have demonstrated that leptin deficiency and leptin resistance are associated with abnormal motility in the gallbladder. In addition, we have shown that serum glucose and insulin are inversely correlated with biliary motility and that administration of leptin to deficient mice normalizes gallbladder function. Thus, leptin administration may be therapeutic for biliary dyskinesia. However, no data are available on biliary leptin receptor levels. Therefore, we hypothesized that leptin receptors would be abundant in the gallbladder. METHODS: 11 week old female lean control (C57Bl/6J, n ⫽ 12), leptin-deficient obese/diabetic (Lepob, n ⫽ 8), leptin-resistant obese/diabetic (Lepdb, n ⫽ 8) and non-obese, diabetic (NOD/LtJ, n ⫽ 8) mice were fasted overnight. The next day alimentary tissues including stomach, duodenum/jejunum, ileum, colon, gallbladder, liver and spleen were removed, and leptin receptor (Ob-Rb, long form) protein levels were determined by ELISA. Data were analyzed by Two-way and One-way ANOVA and by Student-Newman-Keuls for pair-wise multiple comparisons. RESULTS: Leptin receptors were similarly abundant in the gallbladder of each strain (p ⬍ 0.001) compared to spleen (6.8%), stomach (4.4%), jejunum (3.5%), colon (3.3%), ileum (2.8%), and liver (⬍1%). Leptin receptors (ng/mg protein) in the gallbladder by strain are presented in the table below. C57BI/6J
Lepob
Lepdb
NOD
Leptin receptors 22.6 ⫾ 4.9 23.8 ⫾ 3.5 24.6 ⫾ 4.0 35.5 ⫾ 8.3* *p ⬍ 0.001 vs other strains.
CONCLUSIONS: These data suggest that leptin receptors 1) are found most commonly in the gallbladder and 2) are increased in the gallbladder of non-obese diabetic mice. We conclude that alimentary leptin receptors are most abundant in the gallbladder and that leptin provides a link among obesity, diabetes, and gallstone formation.
Alimentary Tract I
S11
RNAi-mediated inhibition of cyclin D1 decreases colon cancer cell proliferation and MMP protein expression Carlos A Murillo MD, Piotr G. Rychahou MD, B Mark Evers, MD, FACS The University of Texas Medical Branch Galveston, TX INTRODUCTION: Cyclin D1, a downstream protein of the PI3K/GSK-3 pathway, regulates cell cycle progression and proliferation. Overexpression of cyclin D1 is associated with a poor prognosis in various human cancers (eg, colorectal cancers). The purpose of this study was to determine whether targeted inhibition of cyclin D1, using small interfering RNA (siRNA), alters proliferation and expression of the matrix metalloproteinase proteins (MMPs) in the colon cancer cell line KM20. METHODS: KM20 cells were treated with cyclin D1 siRNA or scrambled sequences (control); cell proliferation and viability was assessed. Proliferating cell nuclear antigen (PCNA) expression was measured by ELISA. Western blots were performed to determine expression of cyclin D1, PI3K subunits p85alpha and p110 and GSK-3alpha/beta (upstream regulators of cyclin D1), and MMP-2 and MMP-9 (tumor invasion proteins regulated by cyclin D1). RESULTS: Cyclin D1 expression was effectively blocked by siRNA treatment. Expression of MMP-2 and MMP-9 was decreased in KM20 cells by cyclin D1 inhibition; PI3K subunit and GSK-3 expression was not affected. Compared to control, decreased KM20 cell proliferation and PCNA translocation was noted with cyclin D1 siRNA treatment; no significant changes were noted in apoptosis. CONCLUSIONS: We show that selective inhibition of cyclin D1 inhibits MMP expression and KM20 cell proliferation, most likely due to cell cycle arrest. Selective targeting of cyclin D1 may enhance the effects of standard chemotherapeutic agents and provide novel adjunct treatment for selected colorectal cancers.
Transcriptomic fingerprints of Beta2mⴚ/Thy1ⴙ bone marrow stem cells derived from common bile ductligated rats Marjorie R. Chelly MD, Charles Wang MD, PhD, Thomas Hui MD, Ning-Ning Chai MD, PhD, Gulia Castiglione MD, Itzah Avital MD, Achilles A Demetriou MD, PhD, FACS Cedars Sinai Medical Center Los Angeles, CA INTRODUCTION: Previously our laboratory identified a population of cells within the bone marrow, known as Beta2m⫺/Thy1⫹ bone marrow derived stem cells (BMSC) that are able to differentiate into hepatocytes. The effect of liver injury on the genes expressed by Beta2m⫺/Thy1⫹ BMSC is not known. The objective of this study was to determine the gene expression profile of Beta2m⫺/Thy1⫹ BMSC in common bile duct-ligated (CBDL) rats using DNA microarray analysis. METHODS: Male Lewis rats underwent CBDL (n ⫽ 3)or a sham operation (n ⫽ 3). After 10 days, their bone marrow was harvested and Beta2m⫺/Thy1⫹ BMSC isolated using a two-step magnetic
bead cell sorting technique. The mRNA expression profile of primary hepatocytes harvested from sham-operated male Lewis rats (n ⫽ 3) was used for comparison. The gene expression profile of Beta2m⫺/Thy1⫹ BMSC was determined using Affymetrix rat genemone U34a array. RESULTS: In sham-operated rats, 24 common genes were found between Beta2m-/Thy1⫹ BMSC and normal hepatocytes. In the CBDL rats, 189 commonly expressed genes were found between the Beta2m-/ Thy1⫹ BMSC and normal hepatocytes. Several liver-specific genes were identified including HNF1, Complement component C3, cholesterol esterase, Cytochrome P50 3A2, D site albumin promoter binding protein and mitochondrial import stimulation factor. CONCLUSIONS: Our results demonstrate that liver injury by CBDL alters the gene expression profile of Beta2m-/Thy1⫹ BMSCs. Cluster analysis of hepatocyte genes revealed Beta2m-/Thy1⫹ BMSCs from CBDL rats shared more genes in common with primary hepatocytes than Beta2m-/ Thy1⫹ BMSCs from sham-operated rats.
Comparison of antiangiogenic therapy with thrombospondin type I repeats and gemcitabine in an orthotopic model of pancreatic cancer in SCID mice Xue Feng Zhang MD, PhD, Xue Zhang MD, PhD, Eric Galardi BA, Mark Duquette BA, Jack Lawler PhD, Sareh Parangi MD, FACS Beth Israel Deaconess Medical Center Boston, MA INTRODUCTION: Pancreatic cancer is a devastating disease. Efficacy of type I repeats of thrombospondin (TSP-1) in mice with orthotopic pancreatic cancer was tested and compared to gemcitabine. METHODS: SCID mice treated with an intrapancreatic injection of a human pancreatic cancer cell line (ASPC-1) developed large ortho-
S12
Alimentary Tract I
topic tumors with local invasion, peritoneal and and mesenteric implants. Recombinant Thrombospondin Type I repeats (TSR), one of the antiangiogenic domains of TSP, was cloned and purified. One week after tumor implantation, mice were treated with 3TSR (n ⫽ 9, 0.3 mg/kg/day), gemcitabine (n ⫽ 6, 15 mg/kg, twice weekly) or the combination of the two drugs (n ⫽ 11) for 3 weeks. Subsequently, mouse weight, tumor size, number of metasteses were compared. Tumor volume was calculated using the formula 0.52 (width) 2 (length). Student’s t test (Mann-Whitney) was used for statistical comparison. RESULTS: Tumor bearing mice treated with TSR had a 65% reduction in tumor volume (371 ⫾ 97 vs 1064 ⫾ 375 mm3, p ⬍ 0.0001). Tumor volume was reduced 84% with gemcitabine alone (167 ⫾ 67 mm3 ) and 79% with gemcitabine plus TSR treatment (219.39 ⫾ 78.37 mm3 ) p ⬍ 0.001 when compared to control but p ⬎ 0.05 when compared to eachother. Mean vessel density was lowest in the TSR treated animals (3.74 ⫾ 1.2) and highest in the control group (7.96 ⫾ 6.7), p ⬍ 0.05. Proliferation and apoptosis rates were compared. CONCLUSIONS: Antiangiogenic therapy using TSR reduces tumor volume and vascular density in mice with orthotopic pancreatic tumors and can be used safely alone or in combination with gemcitabine.
Aerosolization of heparin and hyaluronan reduce peritoneal loco-regional tumour growth Nawar A Alkhamesi MD, FRCS, FRCSEd, David H Peck PhD, Ara W Darzi MD, FRCS, FRCSI, FACS Imperial College London London, United Kingdom INTRODUCTION: Loco-regional intraperitoneal recurrence remains a critical issue in the management of many abdominal malignancies and is associated with poor prognosis. Around 25% of patients that undergo curative surgery will develop peritoneal recurrence at some stage. Recently, we have shown that this can be inhibited successfully using both heparin and hyaluronan both in vitro and in vivo. The former via an ICAM-1 dependent mechanism while the latter functioned as a barrier. We used an aerosolization device to deliver our therapeutics during laparoscopic procedures. The design of the machine and the catheter enable the operator to cover the whole surface area of the peritoneum with the correct therapeutics’ dose and volume. METHODS: Thirty male WAG rats were divided into 3 groups of 10. After the introduction of 1x105 CC5135, synergistic rat colorectal cancer cells, either 100U/ml heparin, 0.25% hyaluronan or PBS (control) was introduced into the peritoneum using the peritoneal aerosilization technique. After a recovery period of 2 weeks, the animals were sacrificed and the tumor growth was assessed macroscopically and microscopically
J Am Coll Surg
RESULTS: Heparin completely blocked tumor growth with no obvious macroscopic or microscopic invasion detected (p ⬍ 0.05). Hyaluronic acid reduced tumor number and size in comparison to control but at a lower level then heparin CONCLUSIONS: Both modalities were capable of inhibiting tumor spread in vivo. The ability of heparin to abolish tumor development with no local or systemic complications indicates a possible therapeutic to prevent loco-regional recurrence and shows the efficacy of aerosolizarion within the peritoneum.
Alimentary leptin receptors are most abundant in the gallbladder Deborah A Swartz-Basile PhD, Carol Svatek BS, Shannon Graewin MD, James Kiely MD, Henry Pitt MD, FACS Medical College of Wisconsin Milwaukee, WI INTRODUCTION: We have demonstrated that leptin deficiency and leptin resistance are associated with abnormal motility in the gallbladder. In addition, we have shown that serum glucose and insulin are inversely correlated with biliary motility and that administration of leptin to deficient mice normalizes gallbladder function. Thus, leptin administration may be therapeutic for biliary dyskinesia. However, no data are available on biliary leptin receptor levels. Therefore, we hypothesized that leptin receptors would be abundant in the gallbladder. METHODS: 11 week old female lean control (C57Bl/6J, n ⫽ 12), leptin-deficient obese/diabetic (Lepob, n ⫽ 8), leptin-resistant obese/diabetic (Lepdb, n ⫽ 8) and non-obese, diabetic (NOD/LtJ, n ⫽ 8) mice were fasted overnight. The next day alimentary tissues including stomach, duodenum/jejunum, ileum, colon, gallbladder, liver and spleen were removed, and leptin receptor (Ob-Rb, long form) protein levels were determined by ELISA. Data were analyzed by Two-way and One-way ANOVA and by Student-Newman-Keuls for pair-wise multiple comparisons. RESULTS: Leptin receptors were similarly abundant in the gallbladder of each strain (p ⬍ 0.001) compared to spleen (6.8%), stomach (4.4%), jejunum (3.5%), colon (3.3%), ileum (2.8%), and liver (⬍1%). Leptin receptors (ng/mg protein) in the gallbladder by strain are presented in the table below. C57BI/6J
Lepob
Lepdb
NOD
Leptin receptors 22.6 ⫾ 4.9 23.8 ⫾ 3.5 24.6 ⫾ 4.0 35.5 ⫾ 8.3* *p ⬍ 0.001 vs other strains.
CONCLUSIONS: These data suggest that leptin receptors 1) are found most commonly in the gallbladder and 2) are increased in the gallbladder of non-obese diabetic mice. We conclude that alimentary leptin receptors are most abundant in the gallbladder and that leptin provides a link among obesity, diabetes, and gallstone formation.