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Angiotensin II controls fluid leak during inflammation due to upregulation and activation of the AT2 receptor
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Surgical Forum Abstracts
METHODS: 5 ⫻ 105 C57 murine dermal fibroblasts were transfected with either Smad3 siRNA or nonsense siRNA or left untreated, and cultured in floating type I collagen gels. Subsequently, they were irradiated with 20 Gy. The same treatment conditions were also applied to a nonirradiated group as controls. Gel area was measured for 24 hours using standardized digital photography and image analysis software. RESULTS: Inhibition of Smad3 expression using Smad3 siRNA resulted in decreased gel contraction after radiation compared with nonsense siRNA- and non-treated cells, 55.7% vs 71.3% and 73.9%, respectively. This difference was even more apparent in the nonirradiated group, where the Smad3 siRNA treated cells had a 37.7% contraction, compared with 70.7% and 69.6% in the nonsense siRNA- and non-treated cells, respectively. There was similar contraction between the irradiated and nonirradiated control cells, although the irradiated group contained 50% fewer viable cells. When controlled for cell number, the irradiated group demonstrated greater and more rapid gel contraction. CONCLUSIONS: These data demonstrate that Smad3 pathway interference can disrupt fibroblast-mediated collagen gel contraction. Since tissue contraction is a central endpoint of scar formation, SMAD3 pathway inhibition may mitigate scar formation in vivo and ameliorate radiation injury.
Resveratrol regulates the Notch2-mediated neuroendocrine phenotype in human carcinoid cancer cells Scott N Pinchot MD, Renata Jaskula-Sztul PhD, Pongthep Pisarnturakit MD, Muthusamy Kunnimalaiyaan PhD, Herbert Chen MD, FACS University of Wisconsin, Madison, WI INTRODUCTION: We have recently shown that activation of the Notch1 signaling pathway by resveratrol (RES), a nontoxic, plantderived compound found in grapes, markedly suppresses neuroendocrine (NE) tumor markers and inhibits cellular proliferation both in vitro and in vivo. However, the influence of resveratrol on the Notch2 isoform in carcinoid cells has not been described. METHODS: Human gastrointestinal carcinoid (BON) and pulmonary (H727) carcinoid cell lines were treated with 0 to 100 M RES for 2 days, and total mRNA was isolated to detect changes in Notch2 mRNA levels by quantitative polymerase chain reaction (qPCR) methods. To determine the effects of RES-induced Notch2 activation on NE tumor markers, transfection of Notch2-specific smallinterfering RNA was performed, and cell lysates were analyzed for the NE markers achaete-scute complex-like 1 (ASCL1) and chromogranin A (CgA) by Western blot. RESULTS: In RES-treated BON and H727 cell lines, quantification of Notch2 mRNA levels by qPCR demonstrated a dosedependent increase in transcript. Transfection of Notch2 siRNA into carcinoid tumor cells blocked the effects of RES on Notch2 activation, ASCL1 suppression, and CgA reduction.
J Am Coll Surg
CONCLUSIONS: These data suggest for the first time that RES is capable of activating multiple Notch isoforms. Furthermore, these findings confirm the important role of Notch2 in regulating the NE phenotype of carcinoid tumor cells. Based on these and previous data, RES warrants clinical investigation as a possible therapeutic and palliative strategy for patients with carcinoid disease.
Resveratrol inhibits metastatic follicular thyroid cancer cell growth Susan Clare Pitt MD, Ruth J Davis, Scott N Pinchot MD, Herbert Chen MD, FACS University of Wisconsin, Madison, WI INTRODUCTION: Effective therapies for patients with invasive, metastatic follicular thyroid cancer (FTC) are lacking. In other aggressive thyroid cancer lines, we have previously shown that activation of Notch1 suppresses tumor cell proliferation. Therefore, the objective of this study was to investigate the effects of resveratrol, a naturally occurring Notch1 activator, on metastatic FTC cells. METHODS: FTC236 cells were treated in vitro with resveratrol (0 to 25 micromolar). Western blot analysis was performed on cell lysates isolated after 2 and 4 days. Cellular growth was measured by MTT assay over 6 days. RESULTS: Treatment of FTC236 cells with resveratrol caused a notable dose-dependent increase in the Notch1 intracellular domain (NICD), demonstrating successful activation of Notch1 signaling. Resveratrol treatment also significantly reduced FTC cell growth at all time points. The lowest dose of 5 micromolar decreased cellular proliferation 56% after 6 days (p⬍0.0001 vs control). This reduction in cellular proliferation was directly proportional to the degree of NICD expression. Western blot analysis also revealed increased expression of cleaved poly (ADP-ribose) polymerase (PARP) and cleaved caspase-3, with concurrent decreased expression of X-linked inhibitor of apoptosis (XIAP), suggesting that suppression of cell proliferation occurs via apoptosis. CONCLUSIONS: We demonstrate that treatment of metastatic FTC cells in vitro with resveratrol, at doses as low as 5 micromolar, inhibits tumor cell growth and upregulates Notch1 activity. Because 5 micromolar serum concentrations of resveratrol have been safely achieved in humans, this compound may be a potential therapy for patients with aggressive, metastatic FTC.
Angiotensin II controls fluid leak during inflammation due to upregulation and activation of the AT2 receptor Elizabeth L Cureton MD, Alexander Q Ereso MD, Kristopher D Dozier MD, Rita O Kwan MD, MPH, Javid Sadjadi MD, Brian Curran BS, Jane E Murphy PhD, Aditi Bhargava PhD, Gregory P Victorino MD, FACS University of California, San Francisco-East Bay, Oakland, CA INTRODUCTION: Angiotensin II (AngII) increases microvascular fluid leak during basal conditions and decreases leak during inflam-
Vol. 209, No. 3S, September 2009
mation. The opposing actions of AngII may be due to the differential activities of the AT1 and AT2 subtype receptors. Our hypothesis was that AngII decreases fluid leak during inflammation due to AT2 receptor activation. METHODS: Microvascular fluid leak (Lp) was measured in rat mesenteric postcapillary venules during inflammation induced by 10 mg/kg systemic LPS plus either: (1) AT1 agonism (10-4M [Sar1]AngII), (2) AT1 antagonism (3 ⫻ 10-5M ZD7155), (3) AT2 agonism (10-4M CGP42112), or (4) AT2 antagonism (3 ⫻ 10-5M PD123319). Additional rats underwent LPS administration and Western blots of small bowel used to quantitate AT1 and AT2 receptors (n ⫽ 3 for all groups). RESULTS: LPS increased Lp 2-fold (from 1.20 ⫾ 0.13 to 2.27 ⫾ 0.13; p⬍0.002). Compared with LPS alone, AT1 agonism increased Lp 2-fold (Lp ⫽ 5.51 ⫾ 0.65; p⬍0.01), AT1 antagonism decreased Lp 53% (Lp ⫽ 1.06 ⫾ 0.03; p⬍0.002), AT2 agonism decreased Lp 38% (Lp ⫽ 1.40 ⫾ 0.03; p⬍0.02), and AT2 antagonism increased Lp 3-fold (Lp ⫽ 6.62 ⫾ 0.59; p⬍0.001). Units for Lp are cm-sec/ cm-H2O ⫻ 10-7. There was no difference in AT1 expression after vehicle or LPS (p⫽0.1). The ratio of AT2 expression relative to actin was 0.98 ⫾ 0.01 after vehicle and increased to 1.2 ⫾ 0.08 after LPS (p⫽0.02). CONCLUSIONS: The AngII subtype receptors have opposing effects on microvascular fluid leak during inflammation, and AT2 expression is increased during inflammation. During inflammation, the AT2 receptor is upregulated and, when activated by AngII, decreases fluid loss. The opposing roles of the AngII subtype receptors may provide a therapeutic target during sepsis to help control volume loss.
Smad3 is a regulator of MMP-2 Judy W Lee MD, John P Tutela MD, Phuong D Nguyen MD, Orlando Canizares MD, Cristian D Valenzuela BS, Gina K Paek BA, Jaimie P Levine MD, Stephen M Warren MD, Pierre B Saadeh MD, FACS New York University, New York, NY INTRODUCTION: Radiation injury is characterized by fibrosis. The Smad proteins play a critical role in transforming growth factor-beta (TGF-beta) signaling during fibrosis. Smads2 and 3 are receptormediated Smads, and upon injury, Smad3, and to a lesser extent Smad2, are activated by TGF-beta and translocate to the nucleus to regulate transcription of downstream target genes. MMP-2, or gelatinase, disrupts the basement membrane and is upregulated by TGFbeta activation, which is thought be controlled by a Smad2dependent mechanism. The effect of Smad3 on MMP-2 after irradiation injury is unknown. METHODS: C57 murine dermal fibroblasts were transfected with siRNA against Smad2 or Smad3, then irradiated with 20 Gy and allowed to incubate at 37° for 24 hours. Cells were harvested for
Surgical Forum Abstracts
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RNA isolation and semiquantitative real-time reverse transcription PCR (RT-PCR) to evaluate gene expression of Smad2, Smad3, and MMP-2. RESULTS: The fibroblasts treated with Smad2 siRNA showed an 89% reduction in Smad2 expression, while Smad3 expression was only decreased by 15%. Those cells treated with Smad3 siRNA had 31% knockdown of Smad2 expression, whereas Smad3 expression was reduced by 72%. MMP-2 expression was reduced by 44% in the Smad3 siRNA–treated cells compared with Smad2 siRNA–treated cells. CONCLUSIONS: These results indicate that Smad3 gene silencing has a greater effect on MMP-2 expression than Smad2 silencing. This novel association between MMP-2 and Smad3 extends the role played by Smad3 in fibrosis pathways.
Gallbladder cancer cells express 3 subtypes of somatostatin receptors and respond better to the treatment of cytotoxic drugs combined with somatostatin Wei Gong MD, MSc, Jiyu Li MD, Songgang Li MD, Zhiwei Quan MD, FACS Shanghai Jiaotong University School of Medicine, Shanghai, China INTRODUCTION: Gallbladder cancer is lethal digestive malignancy and nonsensitive to routine chemotherapy. We evaluated the role of somatostatin as an adjuvant chemotherapeutic agent in gallbladder cancer and the potential mechanism of its activity. METHODS: The reverse transcriptase–polymerase chain reaction (RT-PCR) and immunohistochemistry were used to detect the expression of somatostatin receptor (SSTR) subtypes in gallbladder cancer cells (GBC-SD). The cytotoxic effects of chemotherapeutic drugs combined with somatostatin were determined by MTT assay. Apoptosis and cell cycle changes were measured by flow cytometry. The effects of somatostatin on the expression of pRb, p53, and Bax were evaluated by Western blots. RESULTS: Three of the somatostatin receptors could be detected in GBC-SD cells. Somatostatin significantly enhanced the inhibitory effect of cytotoxic drug on GBC-SD (Pⱕ0.05 vs control). After 48-hour exposure to 75 g/mL somatostatin, flow cytometric analysis demonstrated an increased number of cells in S phase (Pⱕ0.05 vs control) associated with a decreased number of cells in G2/M and G0/G1 phase. No significant changes of apoptosis were seen in samples treated with somatostatin. The expression of pRB was promoted by somatostatin, while that of p53 and that of Bax were not changed. CONCLUSIONS: The results suggested that somatostatin enhanced the inhibitory effect of cytotoxic drug on gallbladder cells through S cell cycle arrest rather than through the process of apoptosis. These effects were partially mediated by enhancing the expression of pRB, whereas the amounts of p53 and Bax were not changed.
Surgical Forum Abstracts
METHODS: 5 ⫻ 105 C57 murine dermal fibroblasts were transfected with either Smad3 siRNA or nonsense siRNA or left untreated, and cultured in floating type I collagen gels. Subsequently, they were irradiated with 20 Gy. The same treatment conditions were also applied to a nonirradiated group as controls. Gel area was measured for 24 hours using standardized digital photography and image analysis software. RESULTS: Inhibition of Smad3 expression using Smad3 siRNA resulted in decreased gel contraction after radiation compared with nonsense siRNA- and non-treated cells, 55.7% vs 71.3% and 73.9%, respectively. This difference was even more apparent in the nonirradiated group, where the Smad3 siRNA treated cells had a 37.7% contraction, compared with 70.7% and 69.6% in the nonsense siRNA- and non-treated cells, respectively. There was similar contraction between the irradiated and nonirradiated control cells, although the irradiated group contained 50% fewer viable cells. When controlled for cell number, the irradiated group demonstrated greater and more rapid gel contraction. CONCLUSIONS: These data demonstrate that Smad3 pathway interference can disrupt fibroblast-mediated collagen gel contraction. Since tissue contraction is a central endpoint of scar formation, SMAD3 pathway inhibition may mitigate scar formation in vivo and ameliorate radiation injury.
Resveratrol regulates the Notch2-mediated neuroendocrine phenotype in human carcinoid cancer cells Scott N Pinchot MD, Renata Jaskula-Sztul PhD, Pongthep Pisarnturakit MD, Muthusamy Kunnimalaiyaan PhD, Herbert Chen MD, FACS University of Wisconsin, Madison, WI INTRODUCTION: We have recently shown that activation of the Notch1 signaling pathway by resveratrol (RES), a nontoxic, plantderived compound found in grapes, markedly suppresses neuroendocrine (NE) tumor markers and inhibits cellular proliferation both in vitro and in vivo. However, the influence of resveratrol on the Notch2 isoform in carcinoid cells has not been described. METHODS: Human gastrointestinal carcinoid (BON) and pulmonary (H727) carcinoid cell lines were treated with 0 to 100 M RES for 2 days, and total mRNA was isolated to detect changes in Notch2 mRNA levels by quantitative polymerase chain reaction (qPCR) methods. To determine the effects of RES-induced Notch2 activation on NE tumor markers, transfection of Notch2-specific smallinterfering RNA was performed, and cell lysates were analyzed for the NE markers achaete-scute complex-like 1 (ASCL1) and chromogranin A (CgA) by Western blot. RESULTS: In RES-treated BON and H727 cell lines, quantification of Notch2 mRNA levels by qPCR demonstrated a dosedependent increase in transcript. Transfection of Notch2 siRNA into carcinoid tumor cells blocked the effects of RES on Notch2 activation, ASCL1 suppression, and CgA reduction.
J Am Coll Surg
CONCLUSIONS: These data suggest for the first time that RES is capable of activating multiple Notch isoforms. Furthermore, these findings confirm the important role of Notch2 in regulating the NE phenotype of carcinoid tumor cells. Based on these and previous data, RES warrants clinical investigation as a possible therapeutic and palliative strategy for patients with carcinoid disease.
Resveratrol inhibits metastatic follicular thyroid cancer cell growth Susan Clare Pitt MD, Ruth J Davis, Scott N Pinchot MD, Herbert Chen MD, FACS University of Wisconsin, Madison, WI INTRODUCTION: Effective therapies for patients with invasive, metastatic follicular thyroid cancer (FTC) are lacking. In other aggressive thyroid cancer lines, we have previously shown that activation of Notch1 suppresses tumor cell proliferation. Therefore, the objective of this study was to investigate the effects of resveratrol, a naturally occurring Notch1 activator, on metastatic FTC cells. METHODS: FTC236 cells were treated in vitro with resveratrol (0 to 25 micromolar). Western blot analysis was performed on cell lysates isolated after 2 and 4 days. Cellular growth was measured by MTT assay over 6 days. RESULTS: Treatment of FTC236 cells with resveratrol caused a notable dose-dependent increase in the Notch1 intracellular domain (NICD), demonstrating successful activation of Notch1 signaling. Resveratrol treatment also significantly reduced FTC cell growth at all time points. The lowest dose of 5 micromolar decreased cellular proliferation 56% after 6 days (p⬍0.0001 vs control). This reduction in cellular proliferation was directly proportional to the degree of NICD expression. Western blot analysis also revealed increased expression of cleaved poly (ADP-ribose) polymerase (PARP) and cleaved caspase-3, with concurrent decreased expression of X-linked inhibitor of apoptosis (XIAP), suggesting that suppression of cell proliferation occurs via apoptosis. CONCLUSIONS: We demonstrate that treatment of metastatic FTC cells in vitro with resveratrol, at doses as low as 5 micromolar, inhibits tumor cell growth and upregulates Notch1 activity. Because 5 micromolar serum concentrations of resveratrol have been safely achieved in humans, this compound may be a potential therapy for patients with aggressive, metastatic FTC.
Angiotensin II controls fluid leak during inflammation due to upregulation and activation of the AT2 receptor Elizabeth L Cureton MD, Alexander Q Ereso MD, Kristopher D Dozier MD, Rita O Kwan MD, MPH, Javid Sadjadi MD, Brian Curran BS, Jane E Murphy PhD, Aditi Bhargava PhD, Gregory P Victorino MD, FACS University of California, San Francisco-East Bay, Oakland, CA INTRODUCTION: Angiotensin II (AngII) increases microvascular fluid leak during basal conditions and decreases leak during inflam-
Vol. 209, No. 3S, September 2009
mation. The opposing actions of AngII may be due to the differential activities of the AT1 and AT2 subtype receptors. Our hypothesis was that AngII decreases fluid leak during inflammation due to AT2 receptor activation. METHODS: Microvascular fluid leak (Lp) was measured in rat mesenteric postcapillary venules during inflammation induced by 10 mg/kg systemic LPS plus either: (1) AT1 agonism (10-4M [Sar1]AngII), (2) AT1 antagonism (3 ⫻ 10-5M ZD7155), (3) AT2 agonism (10-4M CGP42112), or (4) AT2 antagonism (3 ⫻ 10-5M PD123319). Additional rats underwent LPS administration and Western blots of small bowel used to quantitate AT1 and AT2 receptors (n ⫽ 3 for all groups). RESULTS: LPS increased Lp 2-fold (from 1.20 ⫾ 0.13 to 2.27 ⫾ 0.13; p⬍0.002). Compared with LPS alone, AT1 agonism increased Lp 2-fold (Lp ⫽ 5.51 ⫾ 0.65; p⬍0.01), AT1 antagonism decreased Lp 53% (Lp ⫽ 1.06 ⫾ 0.03; p⬍0.002), AT2 agonism decreased Lp 38% (Lp ⫽ 1.40 ⫾ 0.03; p⬍0.02), and AT2 antagonism increased Lp 3-fold (Lp ⫽ 6.62 ⫾ 0.59; p⬍0.001). Units for Lp are cm-sec/ cm-H2O ⫻ 10-7. There was no difference in AT1 expression after vehicle or LPS (p⫽0.1). The ratio of AT2 expression relative to actin was 0.98 ⫾ 0.01 after vehicle and increased to 1.2 ⫾ 0.08 after LPS (p⫽0.02). CONCLUSIONS: The AngII subtype receptors have opposing effects on microvascular fluid leak during inflammation, and AT2 expression is increased during inflammation. During inflammation, the AT2 receptor is upregulated and, when activated by AngII, decreases fluid loss. The opposing roles of the AngII subtype receptors may provide a therapeutic target during sepsis to help control volume loss.
Smad3 is a regulator of MMP-2 Judy W Lee MD, John P Tutela MD, Phuong D Nguyen MD, Orlando Canizares MD, Cristian D Valenzuela BS, Gina K Paek BA, Jaimie P Levine MD, Stephen M Warren MD, Pierre B Saadeh MD, FACS New York University, New York, NY INTRODUCTION: Radiation injury is characterized by fibrosis. The Smad proteins play a critical role in transforming growth factor-beta (TGF-beta) signaling during fibrosis. Smads2 and 3 are receptormediated Smads, and upon injury, Smad3, and to a lesser extent Smad2, are activated by TGF-beta and translocate to the nucleus to regulate transcription of downstream target genes. MMP-2, or gelatinase, disrupts the basement membrane and is upregulated by TGFbeta activation, which is thought be controlled by a Smad2dependent mechanism. The effect of Smad3 on MMP-2 after irradiation injury is unknown. METHODS: C57 murine dermal fibroblasts were transfected with siRNA against Smad2 or Smad3, then irradiated with 20 Gy and allowed to incubate at 37° for 24 hours. Cells were harvested for
Surgical Forum Abstracts
S127
RNA isolation and semiquantitative real-time reverse transcription PCR (RT-PCR) to evaluate gene expression of Smad2, Smad3, and MMP-2. RESULTS: The fibroblasts treated with Smad2 siRNA showed an 89% reduction in Smad2 expression, while Smad3 expression was only decreased by 15%. Those cells treated with Smad3 siRNA had 31% knockdown of Smad2 expression, whereas Smad3 expression was reduced by 72%. MMP-2 expression was reduced by 44% in the Smad3 siRNA–treated cells compared with Smad2 siRNA–treated cells. CONCLUSIONS: These results indicate that Smad3 gene silencing has a greater effect on MMP-2 expression than Smad2 silencing. This novel association between MMP-2 and Smad3 extends the role played by Smad3 in fibrosis pathways.
Gallbladder cancer cells express 3 subtypes of somatostatin receptors and respond better to the treatment of cytotoxic drugs combined with somatostatin Wei Gong MD, MSc, Jiyu Li MD, Songgang Li MD, Zhiwei Quan MD, FACS Shanghai Jiaotong University School of Medicine, Shanghai, China INTRODUCTION: Gallbladder cancer is lethal digestive malignancy and nonsensitive to routine chemotherapy. We evaluated the role of somatostatin as an adjuvant chemotherapeutic agent in gallbladder cancer and the potential mechanism of its activity. METHODS: The reverse transcriptase–polymerase chain reaction (RT-PCR) and immunohistochemistry were used to detect the expression of somatostatin receptor (SSTR) subtypes in gallbladder cancer cells (GBC-SD). The cytotoxic effects of chemotherapeutic drugs combined with somatostatin were determined by MTT assay. Apoptosis and cell cycle changes were measured by flow cytometry. The effects of somatostatin on the expression of pRb, p53, and Bax were evaluated by Western blots. RESULTS: Three of the somatostatin receptors could be detected in GBC-SD cells. Somatostatin significantly enhanced the inhibitory effect of cytotoxic drug on GBC-SD (Pⱕ0.05 vs control). After 48-hour exposure to 75 g/mL somatostatin, flow cytometric analysis demonstrated an increased number of cells in S phase (Pⱕ0.05 vs control) associated with a decreased number of cells in G2/M and G0/G1 phase. No significant changes of apoptosis were seen in samples treated with somatostatin. The expression of pRB was promoted by somatostatin, while that of p53 and that of Bax were not changed. CONCLUSIONS: The results suggested that somatostatin enhanced the inhibitory effect of cytotoxic drug on gallbladder cells through S cell cycle arrest rather than through the process of apoptosis. These effects were partially mediated by enhancing the expression of pRB, whereas the amounts of p53 and Bax were not changed.