Funda Yilmaz

Differential expression of Caveolin-1 in hepatocellular carcinoma: correlation with differentiation state, motility and invasion

BackgroundCaveolin-1 is the main component of caveolae membrane structures and has different roles during tumorigenesis in different cancer types with varying expression profiles, indicating that the role of caveolin-1 varies according to tumor type. In this study, we investigated the role and expression of caveolin-1 in hepatocellular carcinogenesis.MethodsWe analyzed the expression of Caveolin-1 in 96 hepatocellular carcinoma (HCC), 29 cirrhosis, 20 normal liver tissues and 9 HCC cell lines by immunostaining and western blotting, respectively. After caveolin-1 was stably transfected to HepG2 and Huh7 cells, the effects of Caveolin-1 on the cellular motility, matrix invasion and anchorage-independent growth were studied. Also, caveolae structure was disrupted in endogenously caveolin expressing cells, SNU 449 and SNU 475 by addition of methyl-β-cyclodextrin and analyzed cellular motility and invasion.ResultsIn HCC cell lines, Caveolin-1 expression is correlated to differentiation and basal motility status of these cells. The percentage of Caveolin-1 positivity was found extremely low in normal liver tissue (5%) while it was increased in cirrhosis (45%) and in HCC (66%) (p = 0.002 and p = 0.001 respectively). Cav-1 expression in poorly differentiated HCC samples has been found significantly higher than well differentiated ones (p = 0.001). The caveolin-1 expression was found significantly higher in tumor cells than its peritumoral cirrhotic tissues in HCC samples (p < 0.001). Additionally, the patients with positive staining for Caveolin-1 had significantly higher portal vein invasion than those with negative staining (p = 0.02). Caveolin-1 overexpression increased motility and invasion of HepG2 and Huh7 cells. And disruption of caveolae results in a dramatic decline in both motility and invasion abilities in SNU-449 and SNU-475 cells. Furthermore, caveolin-1 overexpression resulted in down-regulation of E-cadherin while up-regulation of Vimentin. Also, it increased secreted MMP-2 and expression levels of MMP-9 and MT1-MMP. There was no significant difference in colony formation in soft agar between stable clones and parental ones.ConclusionIn conclusion, stepwise increase in Cav-1 expression in neoplastic stage with respect to pre-neoplastic stage during hepatocellular carcinogenesis and its ability to stimulate HCC cell motility and invasiveness indicate that this protein plays a crucial role in tumor progression.

Efficacy of insulin-sensitizing agents in nonalcoholic fatty liver disease.

Aim To investigate the efficacy of insulin-sensitizing agents in nonalcoholic fatty liver disease (NAFLD) patients. Methods This is an open-label, randomized, a single-center study. Sixty-four patients, with impaired glucose metabolism and elevated alanine aminotransferase for at least 6 months before enrollment and NAFLD activity score at least 5 in liver biopsy, were randomized as group 1 and received metformin 1700 mg/day, group 2 received rosiglitazone 4 mg/day, and group 3 received a combination of metformin 1700 mg/day and rosiglitazone 4 mg/day for 12 months. Results Baseline demographic and laboratory findings were similar in all the three groups, except baseline insulin level that was significantly higher in group 1 and group 3 versus group 2 (P<0.05). Serum transaminase levels showed a significant decrease after treatment in both group 2 and group 3. Serum &ggr;-glutamyl transpeptidase levels decreased significantly only in the group 3. However, there was no significant change in liver tests of group 1. Postprandial glucose levels showed significant decrease in all of the three groups. Homeostasis model assessment-insulin resistance was reduced significantly in only group 2. NAFLD score was significantly decreased on follow-up biopsy of the patients in group 2 and group 3. Fibrosis did not change significantly after the treatment. Conclusion Rosiglitazone therapy seems to be more effective in metabolic control and histological improvement in NAFLD patients with impaired glucose metabolism.

Expression of matrix metalloproteinase‐9 in predicting prognosis of hepatocellular carcinoma after liver transplantation

Matrix metalloproteinases (MMPs) are known to play an important role in cell migration during cancer invasion by degrading extracellular matrix proteins. This study aimed to determine the role of MMP‐9 in hepatocellular carcinoma (HCC) carcinogenesis. Eighty‐nine cases who underwent liver transplantation for HCC in cirrhotic liver were selected for this study. The tumor characteristics such as nodule number, maximal diameter, portal vein invasion, and the preoperative alpha‐fetoprotein levels were reviewed. The intensity of immunostaining and the percentage of immunoreactive cells with MMP‐9 were evaluated. All patients were evaluated for HCC recurrence and/or death, and cause of death was noted. There was a lower survival and more recurrence risk among participants with 4 or more nodules exceeding 3 cm in diameter, with poorly differentiated tumor, and with large‐vessel involvement. Eleven patients developed recurrent HCC (12.4%). Twelve patients died as a result of HCC (13.5%). Among 89 HCCs, the incidences of a weak (+) and moderate (++) expression of MMP‐9 in carcinoma cells were 30.3% (23/89) and 43.8% (39/89), respectively. Increased expression and intensity of MMP‐9 were found to be inversely associated with poor tumor differentiation (P = 0.016, P = 0.009, respectively). A significant correlation between expression and intensity of MMP‐9 and large vascular invasion (P = 0.01, and P = 0.03) was also observed. As far as prognosis is concerned, increased immunoreactivity and intensity of MMP‐9 were found to exert an unfavorable impact on overall survival rates (P < 0.01, P = 0.01, respectively) and recurrences (P = 0.001, P = 0.02). Multivariate analyses revealed that MMP‐9 staining percentage (P = 0.007) and portal vein invasion (P = 0.002) were independent predictors of survival, whereas the only independent predictor of recurrences was portal vein invasion (P = 0.007). In this study, our results indicate a positive association between MMP‐9 expression and histopathologic parameters that indicate poor prognosis. We conclude that together, MMP‐9 staining percentage and portal vein invasion in HCC may aid to predict poor outcome. Nevertheless MMP‐9 staining percentage is expected to be a potential predictive marker on survival and needs to be studied more in detail. Liver Transpl 16:621‐630, 2010.

Liver transplantation for progressive familial intrahepatic cholestasis: Clinical and histopathological findings, outcome and impact on growth

Abstract:  In this study, we analyze the demographic features, clinical and histopathological findings in patients who underwent liver transplantation for progressive familial intrahepatic cholestasis. We also analyze outcome and impact of liver transplantation on growth and bone mineral content. Most of the patients were presented with jaundice mainly beginning within the first six months. At the time of initial admission; eight patients had short stature (height SD score <2), and four patients had weight SD score <2. Liver transplantation were performed at the age of 43.2 ± 27 months (range 9 to 96 mfonths), 6.5 ± 3.5 months later after the first admission. Infection, surgical complications and osmotic diarrhea associated with severe metabolic acidosis were noted in 41.4%, 16.6% and 33.3%, respectively. One patient developed posttransplant lymphoproliferative disorder. Overall; 1 year graft and patient survival was 69.2% and 75%, respectively. At the end of the 1st year only 2 patients had height SD score <2. Linear regression of height gain against increase in total body BMD measured at the time of transplantation and 1 year after liver transplantation gave a coefficient r = 0.588 (p = 0.074). No correlation was found between the height gain and age and PELD score at time of transplantation, and no difference was noted between the sexes and donor type. Liver transplantation is effective treatment modality with good outcome and little morbidity, and increases the growth acceleration in patients with PFIC associated with cirrhosis.

Intracranial meningeal tumours in childhood: a clinicopathologic study including MIB-1 immunohistochemistry.

Primary tumours of the meninges with a relatively high tendency for malignant behaviour are uncommon in childhood. This study concerns 18 cases of meningeal tumours in children under the age of 16, of which 13 were meningiomas and five were other tumours arising in the meninges. Meningiomas showed a preponderance in females as in adult series, and the majority were supratentorial in localisation. The percentage of meningeal tumours and meningiomas among all brain tumours in our centre were 3.72% and 2.69%, respectively. Four out of 13 meningiomas were fibroblastic, four were transitional, one was meningothelial, two were psammomatous and two were papillary meningiomas. Seven (38.8%) out of 18 tumours showed anaplastic features, including two papillary meningiomas, two hemangiopericytomas, one mesenchymal chondrosarcoma, one pleomorphic sarcoma and one anaplastic meningeal tumour. Papillary meningiomas with hemangiopericytoma-like solid areas were seen frequently in our cases (15.3%). Meningoangiomatosis was associated with two meningeal tumours. MIB1 (Ki-67) labelling indices (LIs) ranged between 0% and 13.6% (mean 1.83%) in benign, and between 1% and 20% (mean 7.2%) in malignant tumour, including papillary meningiomas. Mean MIB-1 LIs were 5.61% and 1.14% in non-recurrent and recurrent cases, respectively. MIB-1 LIs showed significant differences between benign and malignant meningeal tumours but no significant correlation either with prognosis or recurrence. Despite the fact that brain tumours are among the most common neoplasms of childhood, meningeal tumours are rare lesions, accounting for less than 2% of published series of intracranial neoplasms in childhood [5, 8, 18, 24, 30, 32]. It has been suggested that the clinical and pathological characteristics of meningiomas in this age group differ from those of adults [14, 18, 24, 45]. Besides meningiomas, there are a few reports of other meningeal tumours in childhood and difficulties in differential diagnosis may arise within this group, especially in anaplastic tumours [11, 13, 32, 44, 46]. One of the major problems in meningiomas and some tumours arising in the meninges is the discordance that arises between the histologic appearance of the tumour and behaviour [4]. Several studies have attempted to determine the proliferation potential of meningiomas, including immunohistochemical labelling with monoclonal antibodies to Ki-67, proliferating cell nuclear antigen (PCNA), and bromodeoxyuridine (BUdR); flow cytometric DNA analysis; or argyrophilic nucleolar organizer regions (AgNORs) counting [9, 10, 15, 19, 22, 26, 31, 35, 53]. The studies concerning proliferation markers have contradictory results [9, 10, 15, 26, 31, 42, 53]. MIB-1 detects the same or a similar epitope as the original antibody Ki-67 and reacts with a proliferation associated antigen expressed in all active parts of the cell cycle, G1, S, G2 and M (mitosis), but not in the G0 or quiescent phases [7]. In this study we examined the clinicopathological characteristics and MIB1 values of 18 meningeal tumours in children under the age of 16 years within the last 25 years (from 1970 to 1995).

Association between hepatitis B and hepatocellular carcinoma recurrence in patients undergoing liver transplantation.

BACKGROUND/AIMS Hepatitis B virus (HBV) and hepatocellular carcinoma (HCC) recurrences affect both patient and graft survivals post-orthotopic liver transplantation (OLT) in HBV patients with HCC. We analyzed the relationship between HBV and HCC recurrence in a large cohort of HBV-OLT patients with versus without HCC. METHODS Two hundred eighty-seven HBV patients with OLT (72 also with HCC) were included in the study. Mean follow-up in the post-OLT period was 31.7 +/- 24.7 (range, 3-119) months. RESULTS Post-OLT HBV recurrence observed in 10.1% of patients was more prevalent among the HCC group; 23.6% versus 5.5% in patients with and without HCC, respectively. The mean interval for the development of HBV recurrence was 39.5 +/- 28.5 (range, 2-99) months. Among 72 HCC patients, 8 patients (11.1%) had recurrent HCC, and 7 of them also had HBV recurrence. The mean interval for the development of HCC recurrence was 11.2 +/- 7.85 (range, 2-23) months after OLT. OLT patients with HCC with tumors exceeding the Milan criteria had worse 1-, 3-, and 5-year survival rates than patients with HCC meeting the Milan criteria. HBV and HCC recurrence-free survivals were significantly lower in patients with HCC and HBV recurrence, respectively. In the 7 patients with both HCC and HBV recurrence, mean HBV recurrence time was 9.42 +/- 6.75 months and mean HCC recurrence time was 9.57 +/- 6.75 months. There was a strong correlation between HBV and HCC recurrence times. Cox proportional hazards regression analysis showed that only HCC recurrence was a significant independent predictor of HBV recurrence (P < .001; hazard ratio [HR] = 26.94; 95% confidence interval [CI] = 10.81-67.11). On the other hand, HBV recurrence (P = .013; HR = 5.80; 95% CI = 1.45-23.17) and nodule count (P = .014; HR = 13.08; 95% CI = 1.70-100.83) were significant predictors of HCC recurrence. CONCLUSIONS HBV and HCC recurrences demonstrate a close relationship in patients with OLT.

Reciprocal Activating Crosstalk between c-Met and Caveolin 1 Promotes Invasive Phenotype in Hepatocellular Carcinoma

c-Met, the receptor for Hepatocyte Growth Factor (HGF), overexpressed and deregulated in Hepatocellular Carcinoma (HCC). Caveolin 1 (CAV1), a plasma membrane protein that modulates signal transduction molecules, is also overexpressed in HCC. The aim of this study was to investigate biological and clinical significance of co-expression and activation of c-Met and CAV1 in HCC. We showed that c-Met and CAV1 were co-localized in HCC cells and HGF treatment increased this association. HGF-triggered c-Met activation caused a concurrent rise in both phosphorylation and expression of CAV1. Ectopic expression of CAV1 accelerated c-Met signaling, resulted in enhanced migration, invasion, and branching-morphogenesis. Silencing of CAV1 downregulated c-Met signaling, and decreased migratory/invasive capability of cells and attenuated branching morphogenesis. In addition, activation and co-localization of c-Met and CAV1 were elevated during hepatocarcinogenesis. In conclusion reciprocal activating crosstalk between c-Met and CAV1 promoted oncogenic signaling of c-Met contributed to the initiation and progression of HCC.

Macrophage migration inhibitory factor expression and MIF gene -173 G/C polymorphism in nonalcoholic fatty liver disease.

Aim To investigate the macrophage migration inhibitory factor (MIF) expression and −173 G/C polymorphism of the MIF gene in nonalcoholic fatty liver disease (NAFLD). Method Ninety-one patients with diagnosis of NAFLD and 104 healthy controls were included in the study. MIF −173 G/C polymorphism was detected using the PCR–restriction fragment length polymorphism based method. NAFLD was stratified as nonalcoholic steatohepatitis (NASH), probable NASH and steatosis, respectively in groups 1, 2 and 3, according to NAFLD Activity Score. MIF expression was detected by immunohistochemistry staining. Results Mean age of the patients was 50.1±9.6 years, and 54 of them were male. Serum alanine aminotransferase and aspartate aminotransferase were 50/83, 42/63 and 31/32, respectively in groups 1, 2 and 3, (P<0.05). Both the MIF expression of hepatocytes and mononuclear cells were more prominent in groups 1 and 2 than group 3. There was no correlation between MIF expression of hepatocytes and fibrosis stage. However, MIF expression of mononuclear cells significantly increased according to fibrosis stage (P<0.05, R : 0.2). There was no significant correlation between MIF genotype and MIF expression in the liver. Conclusion MIF expression is significantly increased especially by mononuclear cells in liver tissue of patients with NASH secondary to inflammation. Thus, it should be considered as a consequence not a causal factor.

Active form of AKT controls cell proliferation and response to apoptosis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related mortality worldwide. Deregulation of the AKT signaling pathway has been found in HCC. However, the effect of AKT activation on the proliferation and apoptosis in HCC is not clear. Herein, expression of phosphorylated form of AKT (Ser 473) was investigated in HCC tumor (n=73), cirrhosis (n=17), normal liver (n=22) samples and in HCC cell lines (n=8). The results showed that expression of p-AKT was higher in tumor (53%) than in cirrhotic tissues (12%) while it was absent in normal liver (p<0.0001). p-AKT expression was also associated with number of tumor nodules and differentiation status (p<0.05). LY294002 induced cell cycle arrest at G0/G1 in SNU-449 and Mahlavu cells by decreasing expression of CDK2, CDK4, CycD1, CycD3, CycE, CycA and increasing expression of p21 and p27 as well; it also caused a decrease in the E2F1 transcriptional activity through declining phosphorylated Rb. LY294002 did not affect the basal level of apoptosis; however, it amplified cisplatin-induced apoptosis in SNU-449 cells. When the p-AKT level was decreased specifically after transfection with the DN-AKT plasmid, SNU-449 cells became more sensitive to cisplatin-induced apoptosis. HuH-7 cells with no basal p-AKT, were markedly affected by the treatment of doxorubicin. Thus, Akt signaling controls growth and chemical-induced apoptosis in HCC and p-AKT may be a potential target for therapeutic interventions in HCC patients.

Biliary intraductal papillary mucinous neoplasia: three case reports

Intrahepatic cholangiocarcinoma is subdivided as mass-forming, periductal-infiltrating, and intraductal-growing types. Intraductal-growing type is an entity described in recent years as mucin-producing intrahepatic cholangiocarcinoma or intrahepatic (biliary) intraductal papillary mucinous neoplasia (b-IPMN). b-IPMN is classified as adenoma, borderline tumor, carcinoma in situ, and carcinoma, from benign to malignant. Using a different classification, b-IPMNs are subdivided into intestinal, pancreatobiliary, gastric, or oncocytic based on morphology of the cells forming the lesion and expression of MUC1, MUC2, and MUC5 gene proteins in the mucin family. The clinical and histopathological features of b-IPMN diagnosed in three cases are presented herein. Case 1 was classified as borderline. Case 2 was diagnosed as carcinoma in situ. Case 3 had large invasive areas, and was diagnosed as carcinoma. In all three cases, immunohistochemical investigation revealed MUC1 and MUC5AC to be positive, and MUC2 to be negative. We present herein three cases diagnosed with the clinical and pathological findings of a new entity in the literature, b-IPMN, and we discuss the macroscopic, histological, and immunohistochemical features.