Van-Gogh-like 2 antagonises the canonical WNT pathway and is methylated in colorectal cancers.

BACKGROUND: Aberrant activation of the canonical WNT signaling is a feature of colorectal cancer (CRC). Van-Gogh-like 2 (VANGL2) belongs to the non-canonical WNT pathway whose activation inhibits canonical WNT signaling. In this study, we investigated the role of VANGL2 and its epigenetic regulation in CRC. METHODS: Van-Gogh-like 2 expression and promoter methylation after 5-aza-2'-deoxycytidine (5-aza) treatment were evaluated in CRC cells. DNA samples from 418 sporadic CRCs were tested for VANGL2 promoter methylation and microsatellite instability (MSI). Proliferation, colony formation and activation of the WNT pathway were tested in cells after VANGL2 overexpression. RESULTS: Van-Gogh-like 2 mRNA was significantly higher in 5-aza-treated RKO, LOVO and SW48, whereas no differences were found in SW480. Van-Gogh-like 2 was fully methylated in RKO, SW48, HCT116, DLD1 and Caco2; partially methylated in LOVO, LS174T and SW837; and unmethylated in SW480, SW620 and HT29. Higher expression of VANGL2 mRNA was found in the unmethylated cell lines. In CRC specimens (8.93% MSI), methylated VANGL2 was associated with MSI, higher grade, proximal colon location and BRAF mutation. Van-Gogh-like 2 overexpression in SW480 significantly decreased proliferation, colony formation and ß-catenin levels. CONCLUSION: Van-Gogh-like 2 is frequently methylated in MSI-CRCs with BRAF mutation and may act as a tumour suppressor gene, counteracting WNT/ß-catenin signaling.

Efficacy of doxorubicin coupled to lactosaminated albumin on rat hepatocellular carcinomas evaluated by ultrasound imaging.

BACKGROUND/AIMS: Doxorubicin was conjugated with lactosaminated human albumin, a hepatotropic drug carrier, in order to increase its efficacy in the treatment of hepatocellular carcinoma. In rats bearing hepatocellular carcinomas induced by diethylnitrosamine, lactosaminated human albumin coupled doxorubicin enhanced the drug concentrations in the tumours and lowered those in extrahepatic tissues. The aim of the present study was to investigate the effects of lactosaminated human albumin coupled doxorubicin on the growth of established rat hepatocellular carcinomas induced by diethylnitrosamine. METHODS: Lactosaminated human albumin coupled doxorubicin and the free drug were i.v. administered to rats twice a week for 4 weeks at the single dose of 1 microg/g. Growth of individual tumours was followed through time by ultrasonography. RESULTS: In the control animals injected with saline the mean area of the tracked tumours significantly increased during the whole period of treatment. In the group of rats treated with lactosaminated human albumin coupled doxorubicin the mean area of the followed hepatocellular carcinomas remained practically unchanged. The free drug inhibited tumour growth only in the first period of drug administration. Lactosaminated human albumin coupled doxorubicin also hindered the development of new neoplastic nodules, which was unaffected by the free drug. CONCLUSIONS: The results support lactosaminated human albumin coupled doxorubicin as a promising agent for a systemic chemotherapy of hepatocellular carcinomas to treat noncurable patients.

Doxorubicin coupled to lactosaminated albumin: Effects on rats with liver fibrosis and cirrhosis.

BACKGROUND: The conjugate of doxorubicin with lactosaminated human albumin has the potential of increasing the doxorubicin efficacy in the treatment of hepatocellular carcinomas expressing the asialoglycoprotein receptor. However, coupled doxorubicin also accumulates in the liver, which might damage hepatocytes. AIMS: To verify whether coupled doxorubicin impairs liver function in rats with liver fibrosis and cirrhosis. METHODS: Coupled doxorubicin was administered using the same schedule which exerted an antineoplastic effect on rat hepatocellular carcinomas (4-weekly injections of doxorubicin at 1 microg/g). Liver fibrosis/cirrhosis was produced by carbon tetrachloride (CCl4) poisoning. Liver samples were studied histologically. Serum parameters of liver function and viability were determined. RESULTS: In normal rats, administration of coupled doxorubicin neither caused microscopic changes of hepatocytes nor modified serum liver parameters. In rats with fibrosis/cirrhosis, although a selective doxorubicin accumulation within the liver followed coupled doxorubicin administration, the drug did not have a detrimental effect on the histology of the liver and, among serum liver tests, only alanine aminotransferase and aspartate aminotransferase levels were moderately modified. CONCLUSIONS: Coupled doxorubicin can be administered to rats with liver fibrosis/cirrhosis without inducing a severe liver damage. If further studies will confirm the efficacy and safety of this compound, coupled doxorubicin therapy may open a new perspective in the treatment of hepatocellular carcinoma.

Portal vein arterialization for the treatment of post resection acute liver failure in the rat.

INTRODUCTION: Hyperoxygenation of the liver has been suggested to improve its regenerative capacity. Thus, this study sought to determine whether an additional supply of oxygenated blood delivered by portal vein arterialization (PVA) was protective against acute liver failure induced by hepatectomy. METHODS: Sprague-Dawley rats (six per each group) were divided to either undergo PVA or be untreated after extended hepatectomy. Liver injury was evaluated by the serum alanine aminotransferase (ALT) levels. Hepatocyte regeneration was assessed by calculating the mitotic index and bromodeoxyuridine staining. The 10-day survival was assessed in separate experimental groups. RESULTS: The pO(2) in portal blood increased significantly following PVA. Serum ALT levels were significantly reduced in arterialized versus nonarterialized rats. PVA promotes liver regeneration. Finally, PVA significantly improved host survival compared to the controls: 90% versus 30%, respectively. CONCLUSION: These data suggested that an additional supply of arterial oxygenated blood through PVA promoted a rapid regeneration, leading to a faster restoration of liver mass after partial hepatectomy in rats. Thus, PVA may represent a novel tool to optimize hepatocyte regeneration.

Successful treatment of CCL4-induced acute liver failure with portal vein arterialization in the rat.

INTRODUCTION: Optimization of the conditions for regeneration of the native diseased liver is a major goal in patients with acute liver failure. This study sought to determine whether portal vein arterialization (PVA), which increases the oxygen supply to the liver, was protective in a rat model of liver failure. METHODS: At 24 hours after CCl(4) intoxication, Sprague-Dawley rats (six per group) were assigned to receive PVA or as controls. We determined blood tests, histology, and 10-day survivals. Hepatocyte regeneration was assessed by the mitotic index and bromodeoxyuridine (BrdU) incorporation. RESULTS: Serum transaminases were significantly lower in PVA-treated rats than in control animals: liver necrosis resolved rapidly after PVA. The BrdU staining and mitotic index were severalfold higher among PVA-treated than in untreated rats. Survival was 100% among rats with PVA and 40% in untreated animals (P < .01). CONCLUSIONS: PVA led to resolution of CCl(4)-induced massive liver necrosis in the rat. This effect was probably mediated by activation of rapid and extensive hepatocyte regeneration. PVA might provide a novel, alternative approach to treat acute liver failure.

Technical aspects of portal vein arterialization for acute liver failure: from rat lab to man.

Survival rates of patients with acute liver failure (ALF) without transplantation are poor. However, many of them die awaiting a transplant because of the donor organ shortage. Supporting these patients until an organ becomes available or until their own liver is able to regenerate itself thus avoiding transplantation is a major goal in their multidisciplinary treatment. Animal experimental studies have shown that portal vein arterialization (PVA) enhances the regenerative capacity of hepatocytes by increasing the oxygen supply to the liver after extended hepatectomy or in toxin-induced ALF models. Furthermore, we have reported the application of PVA in patients with ALF. We herein have described the technical aspects of the PVA procedure both in preclinical models and in man.

Low p27 expression is an independent predictor of survival for patients with either hilar or peripheral intrahepatic cholangiocarcinoma.

PURPOSE AND EXPERIMENTAL DESIGN: The prognosis for intrahepatic cholangiocarcinoma (ICC) depends mainly on the feasibility of complete surgical resection. In the absence of demonstrated biological predictors of survival, we evaluated the prognostic value of the cyclin-dependent kinase inhibitor p27 by immunohistochemistry in a series of routine specimens from 47 ICC patients, 22 with the hilar and 25 with the peripheral subtype. Proliferation rate was also evaluated in the same cases by the MIB1 index. Tumors were scored as high, low, and negative p27 expressers (> or =50%, <50%, and no positive nuclei, respectively). RESULTS: High, low, and negative p27 expression was recorded in 18 (38%), 17 (36%), and 12 (26%) cases, respectively. No significant correlation was found between p27 expression and gender, age, tumor grade, tumor location, vascular or perineural invasion, or proliferative index. Tumors with low or absent p27 expression were associated with poor survival compared with the high-expresser group. Kaplan-Meier curves comparing different p27 expression levels with survival showed highly significant separation (P < 0.0001, log-rank test). With univariate Cox proportional hazard regression, only p27 score among all of the parameters was found to influence survival (P = 0.0003). CONCLUSION: We conclude that in ICC, low or absent p27 expression can predict poor survival, regardless of tumor location, pathological features, and tumor proliferation. Immunohistochemical detection of p27 on routine sections may provide the first biological prognostic marker for ICC, thus influencing the therapeutic strategies for these patients.

Aberrant fragile histidine triad gene transcripts in primary hepatocellular carcinoma and liver cirrhosis.

To determine whether transcriptional alterations of the fragile histidine triad (FHIT) gene play a role in the development and progression of human hepatocellular carcinoma (HCC) we used reverse transcription-PCR to examine mRNA FHIT expression in 28 paired samples of HCC (24 in cirrhotic and 4 in noncirrhotic livers) and matched noncancerous tissue and in 10 normal livers. We also assessed loss of heterozygosity of the polymorphic D3S1300 microsatellite marker in the intron between exons 5 and 6 of the FHIT gene. Abnormal FHIT transcripts were detected in 13 cases (46.4%): 10 in the cancerous tissue only, 1 with the same pattern in both cancerous and matched noncancerous tissue, and 2 in the noncancerous tissue only. The four HCCs that arose in noncirrhotic liver all showed abnormal FHIT transcripts. No alterations were found in normal livers. Sequence analysis of abnormally sized transcripts revealed that they were generated by the fusion of exons 3 or 4 with exons 8 or 9. Among the cancerous specimens, one case showed only an abnormal sized transcript derived from the fusion of exons 4 and 9 in the absence of any normal-sized transcript, and another case showed deletion of a sequence comprised between nucleotides -35 and 399 resulting in an exon 4-9 fusion not respecting the exons' bounds. Loss of heterozygosity was found in two cases with abnormal FHIT transcripts and in only one case with normal transcript. Patients with aberrant FHIT transcripts showed a significantly higher relapse rate and shorter recurrence time (P = 0.001). This could be related to a primary genomic instability affecting particularly susceptible regions like FRA3B and could be associated with an increasing risk of recurrence without involving a causative role.

Use of low temperatures for glutathione histochemical stain.

We performed glutathione (GSH) staining at a low temperature to prevent GSH release from the section and, hence, improve morphology. Fresh frozen sections of liver, lung, kidney, heart, and stomach were incubated for GSH activity in an ice bath (2-5 degrees C) for 5-10 min. Low temperature incubation prevented GSH diffusion out of cells and minimized migration of granules into vessels and outside of tissue. Incubation at low temperature generally reduced the intensity of the stain compared to the standard method. We conclude that low temperature incubation improves GSH localization in cells, probably by regulating the rate, formation, and the size of GSH-mercury orange complexes.

Localization of high benzaldehyde dehydrogenase activity in rat upper gastrointestinal tract mucosa: a quantitative histochemical study.

An unusual aldehyde dehydrogenase (AlDH) phenotype, histochemically similar to the "tumor-associated" AlDH appearing during rat hepatocarcinogenesis, was detected in normal rat upper gastrointestinal tract tissues. This phenotype is characterized by high activities with aromatic substrates, i.e., benzaldehyde (Bz) and NADP. Frozen sections of GI tract tissue from normal rats and from liver nodules induced by a Solt-Farber protocol were evaluated for AlDH activity. A sensitive, high-resolution procedure was used in which sections are pre-incubated in nitroblue tetrazolium and then incubated at 20 degrees C in a viscous polyvinyl alcohol medium containing buffer, phenazine methosulfate, sodium azide, substrate, co-enzyme, and nitroblue tetrazolium. Incubation at a suboptimal pH of 7.0 was found to improve retention of the final reaction product and the linearity with time. Activity was quantitated by computer-assisted microscopic photometry. Intense BzDH-NADP activity was localized in the squamous epithelium of the tongue, esophagus, and fore-stomach, and in the glandular pit cells of the glandular stomach; this activity was not evident in the submucosa, muscle walls, and vessels. Little if any BzDH-NADP activity was observed in the small or large intestine, pancreas, and liver. AlDH in upper GI tissues and in liver nodules shared three characteristics: a sharp localization; a preference for Bz and NADP compared to the aliphatic substrate acetaldehyde and NAD; and a high co-enzyme-independent activity in the presence of Bz.

Quantitative histochemistry of benzaldehyde dehydrogenase in hepatocellular carcinomas of vinyl chloride-treated rats.

Hepatocarcinogenesis in rats treated with several chemicals is associated with changes in aldehyde dehydrogenase (AlDH) activity, particularly heterogeneous expression of a "tumor specific" phenotype that is very active with aromatic aldehydes, e.g., benzaldehyde (Bz). Objectives of this study were first, to determine if liver cancers in vinyl chloride-treated rats also expressed this AlDH phenotype, and second, to quantitate the NAD- and NADP-dependent AlDH activity for the substrates Bz and acetaldehyde (Ac) in the cancers and surrounding tissue. Small cubes of tissue containing well-differentiated hepatocellular carcinoma were obtained from five Sprague-Dawley rats exposed to 2500 ppm vinyl chloride for 55 weeks. An optimized procedure was developed for AlDH histochemistry. Frozen sections were preincubated in nitroblue tetrazolium/acetone and then incubated at 20 degrees C in viscous polyvinyl alcohol media containing buffer, phenazine methosulfate, sodium azide, substrate, coenzyme, and nitroblue tetrazolium. Background activity was evaluated by omission of substrate. Activity was quantitated by computer-assisted microscopic photometry. All five carcinomas had heterogeneous staining of NADP- and NAD-dependent BzDH and AcDH activity, with clusters of very high-activity cells. The magnitude of staining in the high-activity neoplastic cells was at least tenfold greater for BzDH-NADP and about twofold greater for BzDH-NAD, AcDH-NADP, and AcDH-NAD than the staining in other liver cells. More neoplastic cells had high BzDH than high AcDH activity. Only BzDH-NADP was localized predominantly to the carcinoma.

1,1-Dichloroethylene: an apoptotic hepatotoxin?

Within 2 hr after 1,1-dichloroethylene administration, the following phenomena occur in livers of fasted rats: dilation and disruption of bile canaliculi, plasma membrane invagination and loss of microvilli, cytoplasmic vacuolation, and loss of density in mitochondrial matrices. Early, selective loss of enzyme activities was localized by histochemical staining to bile canalicular, and inner and outer mitochondrial membranes. Biliary permeability to inulin increased, a change suggestive of the breakdown of junctions between hepatocytes. Endoplasmic reticulum and lysosomes appeared spared. In addition, scattered, individual hepatocytes exhibited changes characteristic of apoptosis by 2 hr: chromatin aggregation and margination, nucleolar coarse granulation and enlargement, rounded blebs and proturberances on cell surfaces, and the separation of these cells from surrounding parenchyma. In contrast, evidence of plasma membrane leakiness to K+, Ca2+ and soluble cytoplasmic enzymes was not detected until after 2 hr. Based on these observations, we propose that 1,1-dichloroethylene may initiate apoptosis-like cell degradation in selected parenchymal cells prior to or coincident with centrolobular necrosis.

Retinoblastoma (RB1) gene product expression in breast carcinoma. Correlation with Ki-67 growth fraction and biopathological profile.

AIMS: To investigate the expression of retinoblastoma protein (pRb) in invasive breast tumours and compare its expression with the major biopathological prognostic indicators to identify more aggressive subgroups. MATERIAL: Archival paraffin embedded tissues from 153 consecutive primary breast carcinomas. METHODS: pRb, Ki-67, and oestrogen receptor/progesterone receptor proteins were identified by immunohistochemistry and score values were recorded by image cytometric analysis; p53 and EGFr expression was also evaluated. RESULTS: pRb scores correlated strongly with proliferation activity as determined by Ki-67 staining. Positive relations were also observed between pRb scores, tumour size, nuclear and histological grade, and oestrogen receptor/progesterone receptor content, while abnormal p53 accumulation was not associated with pRb expression. Among the high proliferating carcinomas it was possible to identify 13 cases with loss of pRb expression. CONCLUSIONS: pRb expression paralleled proliferative activity in the majority of breast carcinomas examined, suggesting that in these cases the protein behaves normally in regulating the cell cycle. Conversely in cases with loss of pRb immunostaining, the combined expression of specific highly aggressive factors (EGFr and p53 expression, oestrogen receptor/progesterone receptor negative status, and high K67) seems to characterise a more aggressive phenotype showing growth advantage and cellular "progression" rather than significant nodal involvement.

Liver ischemia for hepatic resection: where is the limit?

BACKGROUND: A consecutive series of 50 patients who submitted to 53 hepatic resections with use of continuous normothermic liver ischemia is reported. METHODS: Portal triad clamping has been used in 28 cases, with associated inferior vena caval clamping above and below the liver (hepatic vascular exclusion) in 25 patients. The size of the tumor required major hepatic resection in 38 cases (71.7%). Malignant tumors (83%) were the most common indication for liver resection. Patients were placed in three groups according to the duration of liver ischemia: group A, less than 30 minutes (9 patients); group B, 30 to 60 minutes (29 patients); and group C, 60 or more (15 patients). RESULTS: No differences in mortality rates (5.7% in the entire series and 0% in group C) and morbidity rate could be shown. No significant difference was found in postoperative liver test results, and no persistent alteration remained thereafter. Liver biopsy at 6 and 12 months after operation did not reveal any chronic damage. Liver capability to regenerate was maintained as documented by postoperative computerized tomography scan or magnetic resonance imaging. CONCLUSIONS: Because interruption of hepatic blood flow in normothermia is safe for at least 60 minutes (up to 85 minutes in this study), vascular clamping is recommended for hazardous liver resections to minimize blood loss, which appears to be the main factor of death and morbidity.

Technique of hepatic vascular exclusion for extensive liver resection.

Hepatic vascular exclusion, which includes clamping of the portal pedicle along with the inferior vena cava below and above the liver, may be a useful procedure for resection of liver tumors close to the hepatic veins or the vena cava that are usually considered unresectable by conventional techniques. Since complete caval exclusion is the key to good hemodynamic tolerance and a bloodless transection of the liver parenchyma, several technical aspects of the procedure must be accomplished and are detailed.

AgNORs in breast tumours.

There is evidence that the quantitative distribution of AgNOR proteins is a proliferation-related parameter that can be used as a prognostic index in tumour pathology. In breast cancer, some authors found a significant prognostic correlation of AgNOR protein quantity, whereas other did not. However, in all the reports dealing with AgNOR area (as opposed to count) this parameter was always turned out to be an independent prognostic indicator. The present study tests the significance of AgNOR proteins in a large series of primary breast carcinomas, exploring the associations between the AgNOR protein amount, as evaluated by image cytometry, and the other well-established prognostic markers commonly considered for breast cancer, along with patients' survival. Our results demonstrated a highly significant association between AgNOR protein quantity and tumour prognosis. Moreover, when the AgNOR area values were entered in multivariate analysis together with the other predictive parameters commonly considered in breast carcinomas, they showed an independent prognostic value together with Ki67-labelling index (LI), N-status and tumour size. Considering node-negative and -positive cases separately, the AgNOR protein area and Ki67-LI both come out as a independent predictors only in the latter group: the short follow-up time of our series (36 months median) could be responsible for this discrepancy.