[A case of alpha-fetoprotein-producing fetal gut-like small intestinal cancer].

A 72-year-old Japanese woman was admitted because of vomiting and abdominal pain. An enhanced computed tomography scan showed a small intestinal obstruction due to ileal wall thickening and multiple liver metastases. Her serum alpha-fetoprotein (AFP) level was high at 1671.9ng/ml. An ileocecal resection was performed. The histological diagnosis was AFP-producing small intestinal cancer resembling the primitive gut epithelium of a fetus. The present case suggested that even intestinal cancer could produce AFP.

CD40 expression in HCV-associated chronic liver diseases.

CD40 is expressed primarily on B cells and plays an important role in antigen presentation, B cell proliferation, and T cell activation. It has been reported that the CD40 signal modulates apoptosis and has an anti-viral effect in certain cells. Therefore, we investigated the expression and the function of CD40 in HCV-associated chronic liver disease. The expression of CD40 on liver tissues was determined through immunohistochemistry on 50 liver specimens obtained from HCV-positive patients. The effect of CD40 signaling on apoptosis of HepG2 cells was assessed using the MTT assay. The effect of CD40 stimulation on NF-kappaB activation was determined in NF-kappaB reporter gene-transfected HepG2 cells with the Luciferase assay. CD40 positive hepatocytes were observed in both periportal and lobular areas, accompanied by inflammation. In both areas, CD40 staining intensity became significantly stronger, correlating with the histological grading. Similarly, it became stronger with the progression of the histological staging in F1, F2 and F3 cases; however, the expression level decreased in F4 cases. CD40 ligation induced apoptosis in HepG2 cells in the presence of 500 ng/ml of actinomycin D, while CD40 ligation alone could not. Anti-CD40 monoclonal antibody caused NF-kappaB activation in HepG2 cells in a dose-dependent manner. These results suggest that hepatocyte over-expression of CD40 might play an important role in regulating hepatocyte survival and death in HCV-associated chronic liver diseases.

Cellular apoptosis susceptibility protein and proliferation in human hepatocellular carcinoma.

The cellular apoptosis susceptibility protein (CAS) is the human homologue of the product of the essential yeast chromosome segregation gene, CSE1, and has important roles in tumor necrosis factor (TNF)-induced apoptosis and cell proliferation. In this study, we used immunoblotting and immunohistochemistry to look at CAS expression in human hepatocellular carcinoma (HCC) cells. We also studied the correlation between CAS expression and cell proliferation. To do this, we studied the expression of proliferating cell nuclear antigen (PCNA) by immunostaining and at apoptosis by in situ nick end-labeling (TUNEL), followed by calculation of the PCNA labeling index (PCNA LI) and TUNEL labeling index (TUNEL LI). CAS was constitutively expressed in human HCC cell lines and was primarily confined to the cytoplasm of the cells. PCNA LI and TUNEL LI were significantly higher in HCC than in non-tumor tissue (p<0.01); however, the ratio of TUNEL LI/PCNA LI in HCC was significantly lower than that of non-tumor tissue. Immunohistochemistry revealed that the staining intensity score of CAS in HCC was significantly higher than that of non-tumor tissue (p<0.05). These results indicate that there is an augmentation of pro-liferative activity and apoptosis in HCC tissue, as compared to non-tumor tissue. There was a significant positive correlation between CAS and PCNA LI (p<0.05). In addition, we observed an inverse relationship between CAS expression and TUNEL LI, although the correlation did not reach statistical significance. These results suggest that CAS is expressed at higher levels in human HCC tissue than in non-tumor tissue. CAS may be associated with cell proliferation rather than apoptosis, and further, CAS might play an important role in the development of human HCCs.

Different effects of bile acids, ursodeoxycholic acid and deoxycholic acid, on cell growth and cell death in human colonic adenocarcinoma cells.

Secondary bile acids have been implicated as an important etiological factor in colorectal cancer. We investigated the effects of ursodeoxycholic acid (UDCA) and deoxycholic acid (DCA) on the growth and cytotoxicity in HT29 human colonic adenocarcinoma cells. Proliferation assay, cell cycle analysis and cell death characterization by bile acids were performed. Both UDCA and DCA reduced their proliferation rate of HT29 over 48 h in a concentration- and time-dependent manner compared with control cultures. In terms of cell cycle effects, however, UDCA induced G2/M arrest, while DCA induced G1 arrest in a concentration- and time-dependent manner. As for the effects of each bile acid on cell toxicity, UDCA induced early apoptosis and DCA induced both early apoptosis and necrosis. Bile acids play an important role in regulating cell survival and cell death in colon adenocarcinoma cells.

Acute pancreatitis in patients with ulcerative colitis.

Twenty-two patients (13 men and 9 women; median age, 34 years; range, 15-64 years) with ulcerative colitis (UC) were evaluated to determine the incidence of acute pancreatitis with UC at the First Department of Internal Medicine, Mie University School of Medicine, during 1989-2001. Among these, three patients (14%) were diagnosed as having had episodes of acute pancreatitis during the mean follow-up period of 6 years. One patient presented with acute pancreatitis and UC simultaneously. Two patients had drug-induced pancreatitis (one due to azathioprine and the other due to 5-ASA). In conclusion, acute pancreatitis is not a frequent, but an occasional extraintestinal manifestation of UC.

Expression of TNF-related apoptosis-inducing ligand in human hepatocellular carcinoma.

TNF-related apoptosis-inducing ligand (TRAIL), as well as Fas ligand, plays a pivotal role in lymphocyte cytotoxicity and the maintenance of immunological homeostasis in various tissues, but its physiological role in immune evasion of cancer cells remains unknown. We have previously shown strong resistance to TRAIL-induced cytotoxicity in human hepatocellular carcinomas (HCCs). The current study investigates the expression of TRAIL in HCCs. We found that three HCC cells, HepG2, Hep3B and Huh7 cells, constitutively express TRAIL mRNA and protein, as detected by reverse transcriptase PCR and Western blotting. Four of 10 human HCC tissues demonstrated positive staining for TRAIL, whereas non-tumor tissues showed little detectable staining. TRAIL expression on tumor cells was detected by flow cytometry and was dramatically induced after the addition of doxorubicin, a chemotherapeutic agent, or cytokine stimulation with TNF-alpha, IL-1beta or IL-18. This expression was induced principally via the NF-kappaB activation pathway, since IkappaB transfection significantly reduced TRAIL expression. In addition, the expressed TRAIL was functional. The TRAIL on HCC cells induced apoptosis in Jurkat cells that are sensitive to TRAIL-mediated apoptosis, and this process was specifically inhibited by recombinant TRAIL-receptors:Fc which binds to TRAIL. In conclusion, TRAIL expressed on the surface of HCC cells by cytokines or cytostatic drugs might contribute to an alternative mechanism that enables tumors to evade immune surveillance by inducing apoptosis of activated human lymphocytes.

Augmentation of tumor necrosis factor family-induced apoptosis by E3330 in human hepatocellular carcinoma cell lines via inhibition of NF kappa B.

AIM: To investigate the reduction of cell viability in human hepatocellular carcinoma (HCC) cell lines induced by inhibition of nuclear factor kappa B (NF kappa B). METHODS: HLE, SKHep1, and HepG2 were incubated and E3330 was used to compare the stimulation of some chemotherapeutic drugs with that of TNF family, Fas ligand, TNF alpha and TNF-related apoptosis-inducing ligand (TRAIL) at the point of the reduction of cell viability by inhibiting NF kappa B. RESULTS: E3330 decreased NF kappa B levels in HLE cells stimulated by TNF and TRAIL. The cytotoxicity of the combination of TRAIL, TNF alpha, Fas ligand, and E3330 increased synergistically in a dose-dependent manner compared to either E3330 alone in all HCC cell lines by MTT assay. However, the combination of some chemotherapeutic drugs and E3330 did not decrease the cell viability. CONCLUSION: Inhibition of NF kappa B sensitizes human HCC cell lines to TNF-mediated apoptosis including TRAIL, and TRAIL-based tumor therapy might be a powerful potential therapeutic tool in the treatment of human HCC.

Clinical characteristics and prognostic indicators of drug-induced fulminant hepatic failure.

BACKGROUND/AIMS: In most cases of drug-induced liver injury, it is difficult to diagnose whether these cases would progress to fulminant hepatic failure. We investigated the characteristics of non-viral and suspiciously drug-induced fulminant hepatic failure by comparing clinical data between cases that progressed and those that did not progress to fulminant hepatic failure. METHODOLOGY: Ninety-five cases of suspicious drug-induced liver injury including 22 cases that had been treated at our hospital, and subsequently progressed to fulminant hepatic failure were involved in this study. We investigated the characteristics of drug-induced fulminant hepatic failure by a comparison of non-fulminant and fulminant cases, and simultaneously of survivors and fatal cases in the group of fulminant cases. RESULTS: Many of the clinical variables were significantly deteriorated in fulminant cases. The latent period, which means the duration of drug administration, correlated with the severity of drug-induced liver injury including fulminant hepatic failure. Suspicious cases of drug-induced liver injury where the bilirubin level at the time of definite diagnosis stayed over 13 mg/dL for more than one month were likely to progress to fulminant hepatic failure. CONCLUSIONS: Our results suggest that the latent period and the peak level of total bilirubin would be prognostic factors for the severity of drug-induced fulminant hepatic failure. Early preparation of liver transplantation should be recommended by referring these characteristics.

Overexpression of X-linked inhibitor of apoptosis in human hepatocellular carcinoma.

The X-linked inhibitor of apoptosis (XIAP) is a member of a novel family of inhibitors of apoptosis. Since suppression of apoptosis is fundamentally important for carcinogenesis and tumor growth, we investigated the expression and function of XIAP in human hepatocellular carcinomas (HCCs). XIAP was expressed constitutively in HCC cell lines. Fourteen out of 20 (70%) HCC tissues demonstrated moderate or strong cytoplasmic staining for XIAP, whereas non-tumor parts showed negative or weak staining for XIAP by immunohistochemistry. In addition, XIAP expression was inversely correlated with apoptosis, but not with proliferation in HCC tissues. These results indicated that XIAP is a principal inhibitor of apoptosis overexpressed in human HCCs and that XIAP may be a potential target for gene therapy of human HCCs.

Cellular FLICE/caspase-8-inhibitory protein as a principal regulator of cell death and survival in human hepatocellular carcinoma.

Human hepatocellular carcinomas (HCCs) show resistance to apoptosis mediated by several death receptors. Because cellular FLICE/caspase-8-inhibitory protein (cFLIP) is a recently identified intracellular inhibitor of caspase-8 activation that potently inhibits death signaling mediated by all known death receptors, including Fas, TNF-receptor (TNF-R), and TNF-related apoptosis-inducing ligand receptors (TRAIL-Rs), we investigated the expression and function of cFLIP in human HCCs. We found that cFLIP is constitutively expressed in all human HCC cell lines and is expressed more in human HCC tissues than in nontumor liver tissues. Metabolic inhibitors, actinomycin D (ActD) or cycloheximide (CHX), dramatically rendered HCC cells sensitive to Fas-mediated apoptosis. Neither caspase-8 nor caspase-3 was activated by agonistic anti-Fas antibody alone, but both caspases were activated by Fas stimulation in the presence of ActD or CHX, indicating the importance of caspase-8 inhibitors that are sensitive to metabolic inhibitors. Actually, cFLIP expression was decreased in ActD or CHX treatment. cFLIP down-regulation induced by cFLIP antisense oligodeoxynucleotides sensitized HLE cells to Fas, TNF-R, and TRAIL-R-mediated apoptosis. Furthermore, cFLIP over-expression activated nuclear factor (NF)-kappaB and cFLIP down-regulation attenuated NF-kappaB activation induced by TNF-alpha or TRAIL. Pretreatment with pan-caspase-inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD-fmk), restored NF-kappaB activity attenuated by cFLIP down-regulation. cFLIP expression was increased by TNF-alpha, TRAIL, or vascular endothelial growth factor but decreased by wortmannin, indicating that cFLIP expression is regulated by both the NF-kappaB and phosphatidylinostiol-3 kinase (PI-3)/Akt pathways. These results suggest that cFLIP plays an important role in cell survival not simply by inhibiting death-receptor-mediated apoptosis but also by regulating NF-kappaB activation in human HCCs.

The effect of endoscopic injection sclerotherapy on portal blood flow and liver function.

BACKGROUND/AIMS: It is still controversial whether endoscopic injection sclerotherapy can affect portal blood flow and liver function. To clarify this issue, we investigated the change in portal blood flow velocity and the relationship between liver function and portal blood flow after sclerotherapy. METHODOLOGY: Ten liver cirrhosis patients with F2 esophageal varices were enrolled in this study. All patients underwent sclerotherapy until all varices were eradicated. Portal blood flow velocity was measured using Doppler ultrasonography. The changes in laboratory parameters and the portal flow velocity were analyzed in each patient. RESULTS: Total mean portal blood flow velocity was changed from 7.5 +/- 5.3 cm/s to 10.3 +/- 3.5 cm/s by sclerotherapy. Six of ten cases had increase in portal blood flow velocity. Among them, 5 cases demonstrated improvement in serum albumin and total cholesterol level 3 months after sclerotherapy. CONCLUSIONS: Prophylactic endoscopic injection sclerotherapy among patients with early liver cirrhosis can improve liver function by increasing the portal blood flow.

Long-term follow-up of chronic hepatitis C in Japan.

BACKGROUND/AIMS: Chronic hepatitis C which exhibits a varied natural course, is becoming a major problem worldwide. METHODOLOGY: In this study, we investigated 161 patients with chronic hepatitis C by repeated liver biopsies. From initial biopsies, we diagnosed 56 patients with chronic persistent hepatitis, 74 with chronic active hepatitis 2A, and 31 with chronic active hepatitis 2B. RESULTS: During the follow-up period, a progression from chronic hepatitis to liver cirrhosis was recognized among all stages, however the rate of progression to liver cirrhosis was less in chronic persistent hepatitis than in chronic active hepatitis 2A and chronic active hepatitis 2B. Hepatocellular carcinoma was detected in chronic active hepatitis 2A and chronic active hepatitis 2B at the initial stage, however, no tumors developed in chronic persistent hepatitis at the initial stage. Most hepatocellular carcinomas were concomitant with liver cirrhosis. CONCLUSIONS: We suggest a close follow-up of patients with chronic hepatitis C, especially those patients with chronic active hepatitis 2A or 2B and exhibiting successive active inflammation of liver.

Transcatheter arterial embolization and extrabeam radiation therapy for subcutaneous seeding of hepatocellular carcinoma by percutaneous ethanol injection.

A 74-year-old man with a hepatocellular carcinoma received percutaneous ethanol injection twice following the needle biopsy of the tumor. Two years and 6 months after percutaneous ethanol injection, a subcutaneous tumor, which appeared to be a needle tract seeding by percutaneous ethanol injection, was recognized in the right lower anterior chest wall. A curative surgical resection was impossible because of the patient's decreased coagulopathy and severe liver dysfunction. The disseminated tumor was treated with extrabeam radiotherapy (20 fractions; total dose of 50 grays) followed by transcatheter arterial embolization by means of superselective catherization. The size of the subcutaneous tumor was decreased to about 15 mm in diameter. Contrast medium enhanced computed tomography demonstrated no enhancement in the tumor. The patient is currently doing well without further recurrence of hepatocellular carcinoma and without enlargement of the subcutaneous tumor after extrabeam radiation therapy and transcatheter arterial embolization.

Expression of survivin during liver regeneration.

Survivin functions to suppress cell death and regulate cell division, and is observed uniquely in tumor cells and developmental cells. However, the expression and regulation of survivin in non-transformed cells are not well elucidated. Therefore, we investigated the expression of survivin in a murine liver regeneration model after partial hepatectomy and intraperitoneal carbon tetrachloride (CCl(4)) injection. We found that the expression of survivin transcript and protein were markedly elevated with the onset of DNA synthesis and remained elevated during G2 and M phases during liver regeneration. In a normal mouse liver cell line, over-expression of survivin resulted in a decrease in the G0/G1 phase and an increase in the S and G2/M phases, resulting in Rb phosphorylation. These findings suggest that survivin is dramatically expressed in a cell cycle-dependent manner during liver regeneration and provide a new insight into the regulation of cell proliferation and differentiation.

Functional expression of tumor necrosis factor-related apoptosis-inducing ligand in human colonic adenocarcinoma cells.

TNF-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in various transformed cell lines. Therefore, we investigated TRAIL sensitivity, TRAIL-induced nuclear factor-kappaB (NF-kappaB) activation, and expression of TRAIL in human colonic adenocarcinoma cell lines (HT-29, LS180, SK-CO-1). All four TRAIL receptors (TRAIL-R1 through TRAIL-R4) are expressed in these cell lines. TRAIL sensitivity was assessed by assay of cell viability. Cancer cell viabilities were 83 +/- 3.1% (HT-29), 90 +/- 4.3% (LS180), and 88 +/- 6.3% (SK-CO-1) at 24 hours after the addition of 100 ng/ml TRAIL, indicating that these cell lines were relatively resistant to TRAIL. Activation of NF-kappaB was variably influenced by TRAIL administration, with no consistent tendency among the cell lines, indicating that TRAIL-induced NF-kappaB activation might be cell-type dependent. In contrast, TRAIL was expressed in the human colonic adenocarcinoma cell lines by Western blotting and RT-PCR. Increased expression of TRAIL on tumor cells was observed by flow cytometry after cytokine stimulation (IFN-gamma, TNF-alpha) or the addition of chemotherapeutic agents (camptothecin, doxolubicin hydrochloride). TRAIL on HT-29 cells was functional and able to induce apoptosis in Jurkat cells. Jurkat cell viability was increased by the addition of TRAILR1-R4-Fc. In the presence of various cytokines or chemotherapeutic agents, functional TRAIL is expressed on the surface of tumor cells, and this expressed TRAIL might contribute to tumor immune privilege by inducing apoptosis of activated human lymphocytes.

Detection of precore-mutant hepatitis B virus genome in patients with acute and fulminant hepatitis using mutation site-specific assay (MSSA).

BACKGROUND/AIMS: Precore mutation of hepatitis B virus was recently been suggested to be involved in the pathogenesis of fulminant hepatitis. In this study, we analyzed the occurrence of precore mutants in patients with acute and fulminant hepatitis B using new simple rapid and sensitive MSSA (mutation site-specific assay) and evaluated this method for predicting prognosis. METHODOLOGY: We analyzed HBV-DNA of 10 patients with fulminant hepatitis B, 15 patients with acute self-limited hepatitis, and 4 patients with acute severe hepatitis using MSSA. Precore point mutation (G to A; 83rd base of precore region) was examined using a mutation-trapped oligonucleotide primer, which would yield a polymerase chain reaction amplification product only with precore mutants. RESULTS: We distinguished precore mutants from wild type according to the presence or absence of the band at 203 bp, which was amplified in only precore mutants by polymerase chain reaction. Mutation of the precore region was observed in all 10 patients with fulminant hepatitis, in 3 of the 4 patients with severe hepatitis, and 11 of 15 patients with self-limited hepatitis. Negative pre-C mutants in patients with HBeAg indicates good prognosis of hepatitis. CONCLUSIONS: Precore mutant strains of HBV-DNA play an important role but are not specific for fulminant hepatitis, and the mere presence of precore mutants may not directly lead to fulminant hepatitis or severe hepatitis. However, this method is useful for predicting outcome of patients with acute HBV hepatitis, especially in HBeAg-positive state.

Hepatic angiosarcoma mimicking cavernous hemangioma on angiography.

A 60-year-old woman was admitted to our department for evaluation of a hepatic mass. The mass was diagnosed as a hemangioma of the liver by abdominal angiography because of typical cotton wool appearance and stretched arterial vessels and no peripheral staining. However, one month later, the mass was surgically removed because of extravasation. Histological findings of a specimen of the mass revealed that it entirely contained abundant necrotic tissue, and a small residual part after transcatheter arterial embolization was consistent with hemangioma. However, she complained of hemoptysis and thigh pain after several weeks. Computed tomography revealed multiple lung masses and a mass of right musculus gluteus medius. Reexamined histological findings of the liver tumor showed hemangiosarcoma. We should pay attention to the fact that it is sometimes difficult to differentiate cavernous hemangioma from angiosarcoma by angiography.

Endoscopic sclerotherapy (ethanolamine oleate injection) for acute rectal varices bleeding in a patient with liver cirrhosis.

Treatment for acute rectal bleeding from rectal varices in a patient with liver cirrhosis is often difficult. Herein, we report an elderly male with cirrhosis who successfully underwent endoscopic sclerotherapy for rectal bleeding from rectal varices. He had a history of esophageal varices, which were treated by endoscopic sclerotherapy. Three years after the treatment of esophageal varices, he developed massive bright red rectal bleeding. Taking into consideration the risk of treating rectal varices by surgery, we decided to try endoscopic sclerotherapy of the rectal varices. In this case, we injected 16.4 mL of 5% ethanolamine oleate with iopamidole to the rectal varices for the purpose of confirmation of the feeding vein for the varices. After six months, the rectal varices had disappeared. We believe endoscopic sclerotherapy might be an effective therapeutic modality for rectal varices with cirrhosis.

High expression of p21WAF1/CIP1 is correlated with human hepatocellular carcinoma in patients with hepatitis C virus-associated chronic liver diseases.

p21(WAF1/CIP1) (p21) protein is a universal inhibitor of cyclin-dependent kinases and is regulated transcriptionally by p53, which is activated by DNA stress. Hepatocytes in chronic hepatitis receive several DNA stresses by lymphocytes and Kupffer cells. Therefore, we analyzed p21 expression of hepatocytes in hepatitis C virus (HCV)-associated chronic liver diseases and investigated the possible involvement of p21 in hepatocarcinogenesis. We examined p21 expression in 35 cases of HCV-associated chronic hepatitis and 25 cases of HCV-associated liver cirrhosis by immunohistochemical analysis. The p21 labeling index (LI) was calculated as the ratio of positive cells to total cells. p21-positive hepatocytes were more numerous in areas of intense inflammation and spotty necrosis and areas close to fibrosis, and were increased according to the degrees of grading and staging. The p21 LI with liver cirrhosis was significantly higher than that with chronic hepatitis (14.4 +/- 5.9 versus 11.1 +/- 4.2, P = 0.014). The cumulative incidence of hepatocellular carcinoma (HCC) was significantly higher in the p21 LI >or=14% group than in the p21 LI <14% group (P = 0.0079). Multivariate analysis demonstrated that p21 expression can be recognized as an independent significant factor for HCC development (relative risk 5.00, P = 0.039). p21 LI decreased significantly after interferon therapy. These results suggested that p21 is up-regulated by the stress of inflammation and fibrosis in HCV-associated chronic liver diseases and that high p21 expression might be related to hepatocarcinogenesis in cirrhotic patients.