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ABSTRACT: p53 suppressor gene mutations are a well known step which occurs in the late stages of the complex tumourigenesis of colorectal cancer. A deregulation of p53 protein function may be associated with increased neovascularization and aggressive tumour growth. In vitro studies have shown that these genetic alterations cause a loss of wild-type p53-induced anti-angiogenetic control and could possibly induce expression of the neoangiogenic vascular endothelial growth factor (VEGF). Therefore, this in vivo study was performed to assess p53 mutations, i.e. hot spots in exons 4-9, in primary colorectal cancers and in corresponding liver metastases in order to test whether there is an association between p53 mutated tumours with increased microvessel density (MVD) and VEGF overexpression. Twenty-two tissue samples taken from primary colorectal cancers and the corresponding liver metastases were immediately snap-frozen in liquid nitrogen and fixed in formaldehyde. After DNA extraction exons 4-9 were amplified and directly sequenced. Cryostat sections were stained immunohistochemically using antibodies against VEGF, CD34, and p53 protein. A modified semiquantitative Weidner score and interactive computerized image analysis was used to assess MVD. Overexpression of immunohistochemically detected p53 protein was found in 7 of the 11 primary tumours and liver metastases (64%). Sequencing showed 3 out of 11 primary tumours (27%) and 5 out of 11 liver metastases (46%) to have p53 point or frameshift mutations; these samples tested immunohistochemically positive for p53 protein. Two p53 mutations in samples of liver metastases were not detectable in the corresponding primaries. We detected one frameshift mutation in exon 4 that has not yet been described in the literature. Tumour samples with p53 mutations and increased VEGF immunoreactivity were associated with higher MVD (p<0.01 and p<0.05, respectively). However, there was no association detected immunohistochemically between p53 and MVD as well as p53 mutations and VEGF overexpression. Our data demonstrate specific genetic alterations in the coding regions of p53 suppressor gene in both primary colorectal cancers and corresponding liver metastases, these alterations are associated with an increase in MVD, but not in VEGF overexpression. In addition, a novel frameshift mutation in both colorectal cancer and metastasis is described.
International Journal of Oncology 09/2002; 21(2):243-9. · 2.40 Impact Factor
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ABSTRACT: Thrombin, the central enzyme of the coagulation cascade, induces proliferation in different solid tumours. The effect is mainly mediated through the functional thrombin receptor PAR 1, a member of the G-protein coupled receptor family. The aim of this study was to assess the role of thrombin on adhesion of pancreatic cancer to extracellular matrix proteins and endothelial cells in vitro.
The human pancreatic adenocarcinoma cell line MIA PaCa-2 was treated with thrombin and the thrombin-receptor-activating peptide (TRAP), respectively. As a control the cells were pre-incubated with the thrombin-receptor-inhibiting peptide (T1). The cells were incubated on microtiter plates, which were pre-coated with extracellular matrix proteins (fibronectin, laminin, collagen IV) or human umbilical vein endothelial cells (HUVECs), for 30 and 60 min, respectively. The number of adherent cells were measured using the MTT method. ANOVA was used for statistical analysis.
Thrombin enhanced the adhesion of MIA PaCa-2 cells to extra-cellular matrix proteins and endothelial cells significantly (P< or =0.001). The effects of thrombin could be mimicked by TRAP. Pre-incubation with T1 inhibited the effect.
Thrombin enhances adhesion of pancreatic adenocarcinoma to extracellular matrix proteins and endothelial cells in vitro. The effect is mediated through the thrombin receptor PAR 1. The results emphasize the role of thrombin and PAR 1 in pancreatic tumour biology.
European Journal of Surgical Oncology 08/2001; 27(5):472-6. · 2.50 Impact Factor
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ABSTRACT: The liver represents the predominant site of cancer relapse after curative resection of hepatic metastases from colorectal carcinoma. Adjuvant intra-arterial chemotherapy was therefore considered a promising therapeutic approach in high-risk patients.
From July 1984 to December 1985, a total of 42 consecutive patients underwent R0 resection of colorectal liver metastases. Thirty patients with mesenteric lymph-node metastases (Dukes C) were randomised into two groups. In 14 group-A patients, a hepatic artery port catheter was placed during liver resection. Four courses of adjuvant chemotherapy were administered at 4-week intervals, consisting of mitomycin C (8 mg/m2, day 1) and 5-fluorouracil (800 mg/m2, days 1-5). Sixteen group-B patients served as controls. The 12 patients with no mesenteric lymph-node metastases (Dukes A/B) were included in the follow-up program.
After 5 years, 64% of Dukes A/B patients and 29% of Dukes C patients were alive (P<0.01). The probability of remaining free of recurrent disease after 5 years and 10 years was 55% and 18%, respectively (P<0.01). No significant difference in either 5-year survival (25% vs 31%) or long-term disease-free status (15% vs 23%) was detected between groups A and B. The initial tumour relapse was shifted towards extrahepatic sites in group-A patients, but no difference was obtained regarding the definite distribution of recurrent disease.
Routine application of adjuvant regional chemotherapy after R0 liver resection is not warranted.
Langenbeck s Archives of Surgery 07/1999; 384(3):243-9. · 1.81 Impact Factor
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ABSTRACT: Portal vein thrombosis (PVT) was previously considered a contraindication to orthotopic liver transplantation (OLT) since adequate portal blood supply is mandatory for graft function and patient survival. Improvements in surgical technique, however, have meant that this problem now can be circumvented in most instances. Nevertheless portal vein thrombosis remains an obstacle in OLT and is associated with increased incidence of primary non-function and long-term liver failure.
A 55-yr-old patient underwent OLT for secondary biliary cirrhosis associated with hepatitis C infection and complicated by long standing PVT. Involvement of the portal, mesenteric, and splenic veins prevented standard portal venous reconstruction. Portal inflow was accomplished by a side-to-end anastomosis between the middle colic vein and the donor portal vein.
Hepatic reperfusion and subsequent liver function were excellent. Portal blood flow, as measured by color-enhanced Doppler ultrasound, was normal following surgery until discharge. The post-operative course was complicated by abdominal wound dehiscence and recurrent cytomegalovirus (CMV) infection. The patient was discharged in good clinical condition, with excellent liver function and patent portal vein 89 d after OLT.
The middle colic vein is a novel, not previously described, source of portal venous inflow for OLT complicated by extensive splanchnic venous inflow thrombosis.
Clinical Transplantation 01/1999; 12(6):538-42. · 1.67 Impact Factor
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ABSTRACT: In this article, the "tethered ligand" thrombin receptor was identified on human pancreatic tumor cells, MIA PaCa-2, using immunofluorescence studies with a monoclonal anti-thrombin receptor antibody. Pharmacological characterization, using 3H-labeled thrombin receptor activating peptide-6 (TRAP-6) as radioligand, demonstrated a single class of high-affinity binding sites (KD = 9.1+/-1.8 x 10(-7) M) and a binding capacity of 13.9+/-0.7 fmol/mg protein. These binding sites represent functional thrombin receptors, as shown by alpha-thrombin- and TRAP-6-induced mobilization of free intracellular calcium, protein kinase C translocation from cytosol to the cell membrane, and stimulation of DNA synthesis in MIA PaCa-2 cells. These results provide the first identification of tethered ligand thrombin receptor in human pancreatic cancer cells and suggest thrombin receptor involvement in mechanisms of human pancreatic tumor progression.
Pancreas 03/1998; 16(2):189-94. · 2.39 Impact Factor
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ABSTRACT: Cholecystokinin (CCK) is known to stimulate pancreatic cancer cell growth, but no detailed CCK receptor subtype characterization and investigation of CCK receptor-mediated cellular responses in human pancreatic cancer cells have been reported thus far. In this study, CCK binding sites were identified in human pancreatic cancer cells (MIA-PaCa-2) using radioligand binding studies. Pharmacological characterization demonstrated a single class of high-affinity CCK sites on MIA-PaCa-2 cells (326 +/- 18 pM, receptor density 16.9 +/- 2.3 fmol/mg protein). These CCK binding sites displayed a typical CCKB binding profile as shown in competition studies by using different CCK-related compounds and non-peptide CCK antagonists discriminating between CCKA and CCKB sites. CCKB receptor-connected effector systems have been characterized in MIA-PaCA-2 cells, and their involvement in CCK-8S-induced proliferative effects on MIA-PaCa-2 cells has been demonstrated.
Neuropeptides 01/1998; 31(6):573-83. · 1.55 Impact Factor
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ABSTRACT: Recently, the expression of the "tethered ligand" thrombin receptor in carcinosarcoma and melanoma cells has been shown. However, the role of the thrombin receptor in tumor cell metabolism still is undefined.
In this article, the "tethered ligand" thrombin receptor was identified on human epidermoid carcinoma cells (HEp-2g cell line) by using immunofluorescence studies with a monoclonal antithrombin receptor antibody and radioligand binding. Furthermore, the effects of alpha-thrombin and thrombin receptor activating peptides (TRAP)-6 on calcium mobilization, protein kinase C (PKC) translocation, and DNA synthesis were estimated.
Pharmacologic characterization using [3H]TRAP-6 as a radioligand demonstrated a single class of high affinity binding sites (dissociation constant [KD] = 7.2 +/- 2.2 x 10(-7) M) and a binding capacity of 27 +/- 3.4 fmol/mg protein. The function of these binding sites was demonstrated by alpha-thrombin- and TRAP-6-induced mobilization of free intracellular calcium, and translocation of PKC from cytosol to cell membrane. Moreover, alpha-thrombin and TRAP-6 induced an increase in [3H]thymidine incorporation in HEp-2g cells that could be blocked by the PKC inhibitor bisindolylmaleimide.
To the authors' knowledge, the results of this study demonstrate for the first time functional thrombin receptors in epidermoid carcinoma cells. The thrombin receptor appears to be involved in growth regulation in HEp-2g cells by a PKC-dependent mechanism.
Cancer 01/1998; 80(11):2068-74. · 4.77 Impact Factor
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Journal of Gastrointestinal Surgery 04/1997; 1(5):408-22. · 2.83 Impact Factor
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ABSTRACT: Cytological examination of peritoneal lavages is a useful predictor of peritoneal recurrence in gastrointestinal carcinoma patients. Nevertheless, it may be inadequate for those patients with lavages containing only few cancer cells. In the present study, sensitive detection of free cancer cells could be achieved through amplification of cytokeratin 19, carcinoembryonic antigen, alpha-fetoprotein mRNAs by means of multiplex reverse transcriptase polymerase chain reaction and nested polymerase chain reaction.
The multiplex reverse transcriptase polymerase chain reaction assay was used to examine lavage samples from 64 patients with various gastrointestinal malignant lesions (colorectal n = 27; duodenal carcinoma n = 1; gastric n = 7; pancreatic n = 4; hepatocellular carcinoma n = 2; gallbladder n = 1; cholangiocellular carcinoma n = 2 and 20 colorectal liver metastases. Specificity was assessed by examination of 15 donors without malignancies. In addition, nested polymerase chain reaction was used to improve the sensitivity of the assay for the detection of alpha-fetoprotein transcripts.
Peritoneal lavages from 12 of 64 gastrointestinal carcinoma patients were positive for carcinoembryonic antigen mRNA. Carcinoembryonic antigen proved a specific marker, as no false-positives were detected in any patients without gastrointestinal cancer. alpha-fetoprotein mRNA was detected exclusively in peritoneal lavages from tumor patients, i.e., in 16 of 27 colon cancer patients, 14 of 20 patients with colorectal liver metastasis, 2 of 7 patients with gastric cancer, two patients with hepatocellular carcinoma and 2 of 4 patients with pancreatic cancer. Cytokeratin 19 mRNA was not found a useful marker, since control patients without malignancies were also positive.
Our data suggest that carcinoembryonic antigen- and alpha-fetoprotein mRNA in peritoneal lavage are potentially useful specific markers for early diagnosis of metastasis of gastrointestinal cancer. It has been shown that alpha-fetoprotein-specific nested reverse transcriptase polymerase chain reaction can detect not only hepatocellular carcinoma cells, but also malignant cells from other gastrointestinal carcinomas. In contrast, cytokeratin 19 mRNA lacks specificity for gastrointestinal cancer.
Hepato-gastroenterology 48(42):1675-9. · 0.66 Impact Factor
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ABSTRACT: p53 suppressor gene mutations are a well known step which occurs in the late stages of the complex tumourigenesis of colorectal cancer. A deregulation of p53 protein function may be associated with increased neovascularization and aggressive tumour growth. In vitro studies have shown that these genetic alterations cause a loss of wild-type p53-induced anti-angiogenetic control and could possibly induce expression of the neoangiogenic vascular endothelial growth factor (VEGF). Therefore, this in vivo study was performed to assess p53 mutations, i.e. hot spots in exons 4-9, in primary colorectal cancers and in corresponding liver metastases in order to test whether there is an association between p53 mutated tumours with increased microvessel density (MVD) and VEGF overexpression. Twenty-two tissue samples taken from primary colorectal cancers and the corresponding liver metastases were immediately snap-frozen in liquid nitrogen and fixed in formaldehyde. After DNA extraction exons 4-9 were amplified and directly sequenced. Cryostat sections were stained immunohistochemically using antibodies against VEGF, CD34, and p53 protein. A modified semiquantitative Weidner score and interactive computerized image analysis was used to assess MVD. Overexpression of immunohistochemically detected p53 protein was found in 7 of the 11 primary tumours and liver metastases (64%). Sequencing showed 3 out of 11 primary tumours (27%) and 5 out of 11 liver metastases (46%) to have p53 point or frameshift mutations; these samples tested immunohistochemically positive for p53 protein. Two p53 mutations in samples of liver metastases were not detectable in the corresponding primaries. We detected one frameshift mutation in exon 4 that has not yet been described in the literature. Tumour samples with p53 mutations and increased VEGF immunoreactivity were associated with higher MVD (p<0.01 and p<0.05, respectively). However, there was no association detected immunohistochemically between p53 and MVD as well as p53 mutations and VEGF overexpression. Our data demonstrate specific genetic alterations in the coding regions of p53 suppressor gene in both primary colorectal cancers and corresponding liver metastases, these alterations are associated with an increase in MVD, but not in VEGF overexpression. In addition, a novel frameshift mutation in both colorectal cancer and metastasis is described
Int.J.Oncol. 21(2).
