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P.1.133: PONTIN52 IS A PPARGAMMA TARGET GENE AND ITS KNOCKDOWN REDUCES HEPATIC CANCER IN AN ORTHOTOPIC-SINGENIC MOUSE MODEL
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Abstracts of the XVII National Congress of Digestive Diseases / Digestive and Liver Disease 43S (2011) S115–S264
P.1.133 PONTIN52 IS A PPARGAMMA TARGET GENE AND ITS KNOCKDOWN REDUCES HEPATIC CANCER IN AN ORTHOTOPIC-SINGENIC MOUSE MODEL T. Mello ∗ ,1 , M. Tarocchi 1 , F. Buccoliero 1 , E. Ceni 1 , S. Polvani 2 , L. Cioni 1 , S. Milani 1 , A. Galli 1 1 University of Florence, Florence, Italy; 2 Fiorgen Foundation, Sesto F. No (FI), Italy
Background and aim: Pontin52 is an AAA+helicase involved in many cellular processes including cell growth and cancer. Pontin has been recently shown to be overexpressed in HCC and to be an negative prognostic factor. By proteomics approach, we identified Pontin as a gene downregulated by Rosiglitazone (RGZ) in a transgenic mice prone to HBV-related HCC (Galli et al., Hepatology 2010). Aim of this study is to asses the usefulness of Pontin inhibition in reducing HCC growth in vivo and to investigate the mechanisms its inhibition by RGZ. Material and methods: Mouse (Hepa1-6) and human (HepG2) liver cancer cells were used for in vitro assays. We developed a new orthotopic mice model that allows the growth of hepatic cancer cells directly injected in the liver. Pontin expression was evaluated by western-blot and qPCR. Pontin promoter activity was evaluated by luciferase reporter assay. Gene expression knockdown was performed by RNA interference. Results: RGZ down-regulation of Ruvbl-1 expression was confirmed both by qPCR, Western Blotting and Immunofluorescence analysis on Hepa1-6 (mouse) and HepG2 (human) liver cancer cells. After 48hs of RGZ treatment (20uM) Pontin expression was effectively reduced by a 50% at mRNA and protein levels. Surprisingly, PPARg silencing significantly reduces both Pontin mRNA levels and promoter activity. Over-expression of PPARg induces both Pontin promoter activity and mRNA levels. Moreover, the non-TZD PPARg agonist GW1929 induces Pontin expression, while the PPARg inhibitor GW9662 reduces it. On the other hand, RGZ treatment has negligible effects on Pontin promoter activity. Pontin down-regulation by either RNA interference or RGZ treatment significantly inhibits hepatoma cells proliferation in vitro. Pontin-silenced hepatoma cells form significantly less tumor foci than control cells in the orthotopic mice model. Conclusions: Pontin expression is induced by PPARg, but downregulated by RGZ most likely through PPARg-independent non-genomic mechanisms. Knockdown of Pontin effectively reduces the number of cancer lesion in a new orthotopic mice model as well as hepatoma cells proliferation in vitro. We suggest that to target Pontin expression could be of potential interest for developing novel anti-cancer strategies.
P.1.134 CONTRAST-ENHANCED ULTRASONOGRAPHY IN EARLY ASSESSMENT OF THE EFFICACY OF CHEMOTERAPY IN PATIENTS WITH LIVER METATASIS: A PRELIMINARY REPORT A. Caruso ∗ , M. Cossignani, M. Cretella, M. Coppola, E. Mannisi, V. Formica, I. Portarena, I. Grenga, M. Roselli, F. Pallone, G. Del Vecchio Blanco Policlinico Tor Vergata, Roma, Italy Background and aim: Contrast-enhanced ultrasonography (CEUS) has been demonstrated to have high sensitivity and accuracy in the evaluation of the efficacy of the treatment in patients (pts) with hepatocarcinoma. Liver metastasis (LMs) can show different vascular patterns at CEUS, the major part of them are hypovascular, with the absence of arterial, portal and tardive enhancement. Hypervascular metastasis show arterial enhancement and quick wash out.Data about the role of CEUS in the evaluation of the efficacy of chemotherapy (CHT) in LMs are rare. We evaluated the possible role of CEUS in the early assessment of the efficacy of CHT in pts with LMs. Material and methods: We enrolled consecutive pts with CT diagnosed LMs. CEUS was performed at time 0, 2, 8 weeks (wks) after starting CHT. All
pts underwent a B-mode standard liver ultrasonography and CEUS using SonoVue (sulfur hexafluoride) agent contrast 5 mg e.v. in bolus followed by the injection of 10 ml of saline isotonic solution. CT was performed before and at 6 months after starting CHT. Results: We followed up 11 pts with LMs, 9 M, median age of 65 yrs, 8 with colorectal cancer, 2 with pancreatic cancer and 1 with metastatic melanoma. No side effects were reported during the contrast injection. 6/11 pts ended 6-months follow up; 5/11 pts died before the end of the study for primary disease.In 10/11 pts LMs were identified as hypovascular whereas in 1 pt as hypervascular. At 2 weeks-CEUS no modifications of LMs were identified, while 8 weeks-CEUS showed reduction in diameter in 2 pts with 1 and 2 LMs respectively and reduction in number in 4 pts with multiple LMs. The CT at 6 months confirmed the early results of CEUS in all pts. Conclusions: In our preliminary report CEUS performed after 8 weeks of CHT was able to prove early response to the therapy confirmed by CT at 6 months. Our data suggest that CEUS could be used as additional tool easy to perform and radiation-free in pts with LMs to assess early evaluation of the efficacy of CHT. Our study is actually ongoing to confirm the preliminary results in a larger cohort of pts with MLs.
P.1.135 ULTRASONOGRAPHIC FEATURES OF ACUTE INTESTINAL GRAFT-VERSUS-HOST DISEASE R. Ciccocioppo ∗ ,1 , G. Carnevale Maffè 1 , A. Gallia 1 , M.L. Russo 1 , V. Boccaccio 1 , V. Imbesi 1 , G. Zanellati 1 , F. Ripamonti 2 , E.P. Alessandrino 2 , G.R. Corazza 1 1 Clinica Medica I, Fondazione Irccs Policlinico San Matteo, Università degli Studi di Pavia, Pavia, Italy; 2 Clinica Ematologica, Fondazione Irccs Policlinico San Matteo, Università degli Studi di Pavia, Pavia, Italy
Background and aim: Acute intestinal graft-versus-host disease (I-GvHD) is a life-threatening complication of allogeneic haematopoietic stem cell transplantation (HSCT) which may involve any intestinal tract. An early diagnosis is crucial for improving clinical outcome, but symptoms become evident in an advanced phase and histological examination of perendoscopic mucosal biopsies may be inconclusive. Abdominal ultrasonography with colour Doppler (CD) allows evaluation of bowel wall layers and their blood flow. Up to now, only scanty information are available about the role of ecography in the diagnosis and staging of acute I-GVHD. We aimed, therefore, to characterize the sonographic findings of I-GvHD. Material and methods: Twenty patients (M/F ratio 11/9, mean age 45 years) who received allogeneic HSCT for haematological malignancies and suffering from gastrointestinal symptoms were enrolled. Ultrasonography was performed with both 3.5 and 7.5 Mhz probes, and the following parameters were evaluated: bowel loop transverse diameter (n.v.<15 mm), wall thickness (n.v.<3 mm), parietal stratification, intraparietal blood flow, free abdominal fluid, mesenteric inflammation and lymphnode size. Standard microbiology and laboratory tests were carried out on both blood and stool samples. Endoscopy was allowed in 11 out 20 cases due to critical conditions. Results: All patients showed an increase of both small intestinal loop transverse diameter with the proximal part more frequently involved, and bowel wall thickness of at least one tract, mainly the distal ileum (14/20) and sigmoid colon (10/20). An increased intraparietal blood flow was evident in 12/20 cases in the ileum and in 9/20 cases in the sigmoid colon. Mesenteric inflammation was found in 9/20 patients, whilst free abdominal fluid and increased lymphnode size were rarely observed. In 9 patient the diagnosis of acute I-GvHD was confirmed. In the other patients the clinical course was complicated by bacterial or viral infection, but one who developed radiation-induced enteritis and three whose gastrointestinal symptoms remained unexplained. Conclusions: Ultrasonography with CD is a feasible and safe method for the assessment of patients in whom I-GvHD is suspected and it should be included in the diagnostic algorithm. Even if not specific, the findings may help in both early detection and accurate staging of this condition.
Abstracts of the XVII National Congress of Digestive Diseases / Digestive and Liver Disease 43S (2011) S115–S264
P.1.133 PONTIN52 IS A PPARGAMMA TARGET GENE AND ITS KNOCKDOWN REDUCES HEPATIC CANCER IN AN ORTHOTOPIC-SINGENIC MOUSE MODEL T. Mello ∗ ,1 , M. Tarocchi 1 , F. Buccoliero 1 , E. Ceni 1 , S. Polvani 2 , L. Cioni 1 , S. Milani 1 , A. Galli 1 1 University of Florence, Florence, Italy; 2 Fiorgen Foundation, Sesto F. No (FI), Italy
Background and aim: Pontin52 is an AAA+helicase involved in many cellular processes including cell growth and cancer. Pontin has been recently shown to be overexpressed in HCC and to be an negative prognostic factor. By proteomics approach, we identified Pontin as a gene downregulated by Rosiglitazone (RGZ) in a transgenic mice prone to HBV-related HCC (Galli et al., Hepatology 2010). Aim of this study is to asses the usefulness of Pontin inhibition in reducing HCC growth in vivo and to investigate the mechanisms its inhibition by RGZ. Material and methods: Mouse (Hepa1-6) and human (HepG2) liver cancer cells were used for in vitro assays. We developed a new orthotopic mice model that allows the growth of hepatic cancer cells directly injected in the liver. Pontin expression was evaluated by western-blot and qPCR. Pontin promoter activity was evaluated by luciferase reporter assay. Gene expression knockdown was performed by RNA interference. Results: RGZ down-regulation of Ruvbl-1 expression was confirmed both by qPCR, Western Blotting and Immunofluorescence analysis on Hepa1-6 (mouse) and HepG2 (human) liver cancer cells. After 48hs of RGZ treatment (20uM) Pontin expression was effectively reduced by a 50% at mRNA and protein levels. Surprisingly, PPARg silencing significantly reduces both Pontin mRNA levels and promoter activity. Over-expression of PPARg induces both Pontin promoter activity and mRNA levels. Moreover, the non-TZD PPARg agonist GW1929 induces Pontin expression, while the PPARg inhibitor GW9662 reduces it. On the other hand, RGZ treatment has negligible effects on Pontin promoter activity. Pontin down-regulation by either RNA interference or RGZ treatment significantly inhibits hepatoma cells proliferation in vitro. Pontin-silenced hepatoma cells form significantly less tumor foci than control cells in the orthotopic mice model. Conclusions: Pontin expression is induced by PPARg, but downregulated by RGZ most likely through PPARg-independent non-genomic mechanisms. Knockdown of Pontin effectively reduces the number of cancer lesion in a new orthotopic mice model as well as hepatoma cells proliferation in vitro. We suggest that to target Pontin expression could be of potential interest for developing novel anti-cancer strategies.
P.1.134 CONTRAST-ENHANCED ULTRASONOGRAPHY IN EARLY ASSESSMENT OF THE EFFICACY OF CHEMOTERAPY IN PATIENTS WITH LIVER METATASIS: A PRELIMINARY REPORT A. Caruso ∗ , M. Cossignani, M. Cretella, M. Coppola, E. Mannisi, V. Formica, I. Portarena, I. Grenga, M. Roselli, F. Pallone, G. Del Vecchio Blanco Policlinico Tor Vergata, Roma, Italy Background and aim: Contrast-enhanced ultrasonography (CEUS) has been demonstrated to have high sensitivity and accuracy in the evaluation of the efficacy of the treatment in patients (pts) with hepatocarcinoma. Liver metastasis (LMs) can show different vascular patterns at CEUS, the major part of them are hypovascular, with the absence of arterial, portal and tardive enhancement. Hypervascular metastasis show arterial enhancement and quick wash out.Data about the role of CEUS in the evaluation of the efficacy of chemotherapy (CHT) in LMs are rare. We evaluated the possible role of CEUS in the early assessment of the efficacy of CHT in pts with LMs. Material and methods: We enrolled consecutive pts with CT diagnosed LMs. CEUS was performed at time 0, 2, 8 weeks (wks) after starting CHT. All
pts underwent a B-mode standard liver ultrasonography and CEUS using SonoVue (sulfur hexafluoride) agent contrast 5 mg e.v. in bolus followed by the injection of 10 ml of saline isotonic solution. CT was performed before and at 6 months after starting CHT. Results: We followed up 11 pts with LMs, 9 M, median age of 65 yrs, 8 with colorectal cancer, 2 with pancreatic cancer and 1 with metastatic melanoma. No side effects were reported during the contrast injection. 6/11 pts ended 6-months follow up; 5/11 pts died before the end of the study for primary disease.In 10/11 pts LMs were identified as hypovascular whereas in 1 pt as hypervascular. At 2 weeks-CEUS no modifications of LMs were identified, while 8 weeks-CEUS showed reduction in diameter in 2 pts with 1 and 2 LMs respectively and reduction in number in 4 pts with multiple LMs. The CT at 6 months confirmed the early results of CEUS in all pts. Conclusions: In our preliminary report CEUS performed after 8 weeks of CHT was able to prove early response to the therapy confirmed by CT at 6 months. Our data suggest that CEUS could be used as additional tool easy to perform and radiation-free in pts with LMs to assess early evaluation of the efficacy of CHT. Our study is actually ongoing to confirm the preliminary results in a larger cohort of pts with MLs.
P.1.135 ULTRASONOGRAPHIC FEATURES OF ACUTE INTESTINAL GRAFT-VERSUS-HOST DISEASE R. Ciccocioppo ∗ ,1 , G. Carnevale Maffè 1 , A. Gallia 1 , M.L. Russo 1 , V. Boccaccio 1 , V. Imbesi 1 , G. Zanellati 1 , F. Ripamonti 2 , E.P. Alessandrino 2 , G.R. Corazza 1 1 Clinica Medica I, Fondazione Irccs Policlinico San Matteo, Università degli Studi di Pavia, Pavia, Italy; 2 Clinica Ematologica, Fondazione Irccs Policlinico San Matteo, Università degli Studi di Pavia, Pavia, Italy
Background and aim: Acute intestinal graft-versus-host disease (I-GvHD) is a life-threatening complication of allogeneic haematopoietic stem cell transplantation (HSCT) which may involve any intestinal tract. An early diagnosis is crucial for improving clinical outcome, but symptoms become evident in an advanced phase and histological examination of perendoscopic mucosal biopsies may be inconclusive. Abdominal ultrasonography with colour Doppler (CD) allows evaluation of bowel wall layers and their blood flow. Up to now, only scanty information are available about the role of ecography in the diagnosis and staging of acute I-GVHD. We aimed, therefore, to characterize the sonographic findings of I-GvHD. Material and methods: Twenty patients (M/F ratio 11/9, mean age 45 years) who received allogeneic HSCT for haematological malignancies and suffering from gastrointestinal symptoms were enrolled. Ultrasonography was performed with both 3.5 and 7.5 Mhz probes, and the following parameters were evaluated: bowel loop transverse diameter (n.v.<15 mm), wall thickness (n.v.<3 mm), parietal stratification, intraparietal blood flow, free abdominal fluid, mesenteric inflammation and lymphnode size. Standard microbiology and laboratory tests were carried out on both blood and stool samples. Endoscopy was allowed in 11 out 20 cases due to critical conditions. Results: All patients showed an increase of both small intestinal loop transverse diameter with the proximal part more frequently involved, and bowel wall thickness of at least one tract, mainly the distal ileum (14/20) and sigmoid colon (10/20). An increased intraparietal blood flow was evident in 12/20 cases in the ileum and in 9/20 cases in the sigmoid colon. Mesenteric inflammation was found in 9/20 patients, whilst free abdominal fluid and increased lymphnode size were rarely observed. In 9 patient the diagnosis of acute I-GvHD was confirmed. In the other patients the clinical course was complicated by bacterial or viral infection, but one who developed radiation-induced enteritis and three whose gastrointestinal symptoms remained unexplained. Conclusions: Ultrasonography with CD is a feasible and safe method for the assessment of patients in whom I-GvHD is suspected and it should be included in the diagnostic algorithm. Even if not specific, the findings may help in both early detection and accurate staging of this condition.