Takao Iwasaki

Characterization of the epithelial cell adhesion molecule (EpCAM)+ cell population in hepatocellular carcinoma cell lines

Accumulating evidence suggests that cancer stem cells (CSC) play an important role in tumorigenicity. Epithelial cell adhesion molecule (EpCAM) is one of the markers that identifies tumor cells with high tumorigenicity. The expression of EpCAM in liver progenitor cells prompted us to investigate whether CSC could be identified in hepatocellular carcinoma (HCC) cell lines. The sorted EpCAM+ subpopulation from HCC cell lines showed a greater colony formation rate than the sorted EpCAM− subpopulation from the same cell lines, although cell proliferation was comparable between the two subpopulations. The in vivo evaluation of tumorigenicity, using supra‐immunodeficient NOD/scid/γcnull (NOG) mice, revealed that a smaller number of EpCAM+ cells (minimum 100) than EpCAM− cells was necessary for tumor formation. The bifurcated differentiation of EpCAM+ cell clones into both EpCAM+ and EpCAM− cells was obvious both in vitro and in vivo, but EpCAM− clones sustained their phenotype. These clonal analyses suggested that EpCAM+ cells may contain a multipotent cell population. Interestingly, the introduction of exogenous EpCAM into EpCAM+ clones, but not into EpCAM− clones, markedly enhanced their tumor‐forming ability, even though both transfectants expressed a similar level of EpCAM. Therefore, the difference in the tumor‐forming ability between EpCAM+ and EpCAM− cells is probably due to the intrinsic biological differences between them. Collectively, our results suggest that the EpCAM+ population is biologically quite different from the EpCAM− population in HCC cell lines, and preferentially contains a highly tumorigenic cell population with the characteristics of CSC. (Cancer Sci 2010)

Transient elastography for measurement of liver stiffness measurement can detect early significant hepatic fibrosis in Japanese patients with viral and nonviral liver diseases

BackgroundMany studies have reported the efficiency of transient elastography, a noninvasive, reproducible, and reliable method for predicting liver fibrosis, in patients with chronic hepatitis C (CHC) and B (CHB), but there are few reports about nonviral chronic liver disease (CLD) such as primary biliary cirrhosis (PBC), nonalcoholic steatohepatitis (NAFLD), and autoimmune hepatitis (AIH). We therefore compared the efficiency of transient elastography between CHC and nonviral CLD.MethodsWe assessed the accuracy of liver stiffness measurement (LSM) using Fibroscan, and compared these values with those of hyaluronic acid, type 4 collagen, platelet count, prothrombin index, and AST/platelet ratio index (APRI) as indices for the diagnosis of liver fibrosis in 114 patients with a variety of chronic liver diseases: CHC (n = 51), CHB (n = 11), NAFLD (n = 17), PBC (n = 20), and AIH (n = 15). The histology was assessed according to the METAVIR score by two pathologists.ResultsThe number of fibrosis stage (F0/1/2/3/4) with CHC was 9/15/12/6/10, and that with nonviral CLD was 10/21/11/4/6, respectively. The ability, assessed by area under receiver operating characteristic (AUROC) curve, to predict liver fibrosis F ≥ 2 for LSM, HA, type 4 collagen, platelet count, prothrombin index, and APRI, was 0.92, 0.81, 0.87, 0.85, 0.85, and 0.92 in CHC patients, respectively; and 0.88, 0.72, 0.81, 0.67, 0.81, and 0.77 in nonviral CLD patients, respectively.ConclusionsIn patients with nonviral CLD, LSM was most helpful in predicting significant fibrosis (F ≥ 2). Transient elastography is a reliable method for predicting significant liver fibrosis, not only in CHC patients but also in nonviral CLD patients.

Four‐year study of lamivudine and adefovir combination therapy in lamivudine‐resistant hepatitis B patients: influence of hepatitis B virus genotype and resistance mutation pattern

Summary.  To investigate the efficacy of long‐term lamivudine (3TC) and adefovir dipivoxil (ADV) combination therapy in 3TC‐resistant chronic hepatitis B virus (HBV) infected patients, we analysed 28 3TC‐resistant patients treated with the combination therapy during 47 months (range, 9–75). At 12, 24, 36, and 48 months, the rates of virological response with undetectable HBV DNA (≤2.6 log copies/mL) were 56, 80, 86, and 92%, respectively. Among 17 hepatitis B e antigen (HBeAg)‐positive patients, HBeAg disappeared in 24% at 12 months, 25% at 24 months, 62% at 36 months, and 88% at 48 months. When HBV genotypes were compared, patients with genotype B achieved virological response significantly more rapidly than those with genotype C (P = 0.0496). One patient developed virological breakthrough after 54 months, and sequence analysis of HBV obtained from the patient was performed. An rtA200V mutation was present in the majority of HBV clones, in addition to the 3TC‐resistant mutations of rtL180M+M204V. The rtN236T ADV‐resistant mutation was observed in only 25% clones. In vitro analysis showed that the rtA200V mutation recovered the impaired replication capacity of the clone with the rtL180M+M204V mutations and induced resistance to ADV. Moreover, rtT184S and rtS202C, which are known entecavir‐resistant mutations, emerged in some rtL180M+M204V clones without rtA200V or rtN236T. In conclusion, 3TC+ADV combination therapy was effective for most 3TC‐resistant patients, especially with genotype B HBV, but the risk of emergence of multiple drug‐resistant strains with long‐term therapy should be considered. The mutation rtA200V with rtL180M+M204V may be sufficient for failure of 3TC+ADV therapy.

HEPATOCELLULAR CARCINOMAS SUPPLIED BY INFERIOR PHRENIC ARTERIES

Purpose: To assess the arterial supply to hepatocellular carcinomas (HCCs) by inferior phrenic arteries (IPA). Material and Methods: A total of 126 consecutive cases of HCC were studied by contrast-enhanced CT and conventional arteriography. Blood supply from an IPA was suspected when the size of the HCC mass as seen on contrast-enhanced CT did not match the size of the tumor mass as seen on hepatic arteriography. Inferior phrenic arteriography was employed to confirm these findings. HCCs fed by the IPA were analyzed in terms of size, location, and history of prior treatment. Results: In 14 (11%) of the 126 cases, the tumor was found to have a blood supply from an IPA. Eleven of these tumors were located in segments 2 and 7. Three tumors, which had not been treated previously, had a blood supply from an IPA. Six tumors were almost exclusively fed by an IPA and were located in segments 7, 1, and 4. Conclusion: HCCs located in segments which form the bare area of the liver (S1, S2, S7) can be supplied by an IPA. This should be suspected when a lesion or part of a lesion is identified on contrast-enhanced CT but not on hepatic arteriography.

The efficacy of the combination therapy of 5-fluorouracil, cisplatin and leucovorin for hepatocellular carcinoma and its predictable factors.

PurposeSeveral clinical trials in human neoplasms have demonstrated the effectiveness of combination therapy with 5-fluorouracil (FUra), cisplatin (CDDP), and leucovorin (LV). Thymidylate synthase (TS), the target enzyme of FUra, and dihydropyrimidine dehydrogenase (DPD), the rate-limiting catabolic enzyme of pyrimidines, have both been reported to be predictors of the response to FUra-based chemotherapies. Therefore, we aimed to clarify the effects of a combination of the three drugs against hepatoma cells and to determine the role of these two enzymes using in vitro models.MethodsFive human hepatoma cell lines (Hep3B, HepG2, HuH7, PLC/PRF/5 and Chang) were used. Cytotoxicity was determined after exposure to various concentrations and combinations of antitumor agents. The combination effects of FUra and CDDP in terms of synergy, additivity or antagonism were evaluated by median effect analysis. The mRNA levels of TS and DPD were measured by quantitative real-time PCR. Expression of TS and DPD proteins was also investigated.ResultsLV alone did not show any cytotoxicity, although it enhanced the cytotoxicity of FUra, but not that of CDDP. Synergistic enhancement was observed with the combination of FUra and CDDP against all cells. The median combination index at fraction 0.5 was 0.554 (range 0.273–0.616). All cells expressed TS and DPD with median relative quantities of mRNA normalized to that of HuH7 cells of 1.04 (range 1.00–1.32) and 1.18 (range 0.88–1.55), respectively. A strong correlation was found between the IC50 of FUra and the mRNA level of DPD (r=0.912, P=0.0295).ConclusionsLV and CDDP enhanced the cytotoxicity of FUra, which provided a rationale for the regimen combining the three drugs for the treatment of hepatocellular carcinoma. DPD plays an important role in the sensitivity to FUra, and the DPD mRNA expression level may be used to predict the response to FUra-based chemotherapy for HCC.

Rupture of rectal varices treated with endoscopic variceal ligation.

We report here 3 cases of rectal varices treated with endoscopic variceal ligation and discuss the pathogenesis, treatment, and prognosis of rectal varices with referring to previous reports. Of the 3 patients, 2 had been diagnosed as liver cirrhosis and 1 as extrahepatic portal hypertension. All of the 3 patients had previously undergone treatment of esophagogastric varices. The rupture of rectal varices appeared to have some relationship with the treatment of esophageal varices. In previous reports, 73% of patients with ruptured rectal varices treated with endoscopic injection sclerotherapy or endoscopic variceal ligation had undergone treatments of esophageal varices. The endoscopic treatments resulted in a favorable prognosis in 2 patients. Although no fatality from endoscopic injection sclerotherapy or endoscopic variceal ligation has been reported, 1 of the present 3 cases died of liver failure.

Effects of collateral vessel occlusion on oral glucose tolerance test in liver cirrhosis.

Alterations in carbohydrate metabolism associated with liver cirrhosis are characterized by a high serum insulin level and prolonged hyperglycemia on oral glucose tolerance test (OGTT). We measured plasma glucose, immunoreactive insulin (IRI), and C-peptide immunoreactivity (CPR) levels during a 75-g OGTT before and after varices obliteration in 10 cirrhotic patients with gastric varices. After obliteration, the indocyanine green retention rate was decreased and the portal flow velocity was increased. A significant decline in plasma glucose and IRI levels was also noted on OGTT. Moreover, the plasma glucose and IRI levels declined at 90 and 120 min in OGTT while they increased progressively by 120 min before obliteration. The levels of CPR were similar before and after treatment. These results indicate that decreased portal flow due to extrahepatic shunt and consequent impairment of insulin metabolism play a role in glucose intolerance observed in cirrhotic patients and that shunt occlusion improves glucose metabolism.

Correlation between p21waf1 and p16INK4a expression in hepatocellular carcinoma

Abstract Background: The cyclin dependent kinase p21waf1 plays a crucial role in the regulation of cell cycle. The family of p53 proteins has the ability to induce p21waf1, whereas p16INK4a modulates post-transcriptionally the expression of p21waf1. Methods: Total 36 hepatocellular carcinomas (HCCs) and 24 paired adjacent liver tissues were evaluated for the following: (1) expression of p21waf1 and p16INK4a; (2) that of p21waf1, p73 and p63 mRNAs; (3) genomic mutations and the loss of heterozygosity of p73 and p53; and (4) frequency of methylation in the 5′CpG promoter region of p16INK4a. Results: In HCCs compared with the adjacent non-cancerous liver tissues, the expression of p21waf1 and p16INK4a was reduced. Indeed, p21waf1 was not detected in 36% (8/22) of HCCs in spite of the presence of p21waf1 mRNA: among them, mutations of p53 gene were found in 50%, whereas a lack of p16INK4a expression in all of them. p21waf1 and p16INK4a were reduced in proportion to the degree of methylation in p16INK4a gene. p73 did not mutated, and p63 did not expressed in HCCs. Conclusion: Methylation status of p16INK4a gene will play a part for reducing constitutive expression of p16INK4a and of p21waf1 coordinately in HCCs.

A novel third generation bisphosphonate, minodronate (YM529), prevented proliferation and migration of hepatocellular carcinoma cells through inhibition of mevalonate pathway

Aim:  Skeletal metastases and bone metasitasis are a common occurrence in patients with advanced hepatocellular carcinoma (HCC). Bisphosphonates (BPs), which are used for the treatment of osteoporosis and tumor‐associated hypercalcemia, have recently been reported to decrease skeletal morbidity in patients with metastatic bone disease. Several studies revealed that nitrogen‐containing BPs (N‐BPs) could inhibit tumor growth and migration, indicating the possibility that N‐BPs have direct inhibitory effects. We aimed to determine the effects of novel a N‐BP (YM529) on human HCC cells in vitro.

Injection of ethanolamine oleate into a segmental portal branch for pharmacologic hepatic segmentectomy in dogs

RATIONALE AND OBJECTIVES Currently available treatments for hepatocellular carcinoma are not satisfactory in terms of recurrence rates. In this study, we injected ethanolamine oleate (EO) into a portal branch in an attempt to cause necrosis of a liver segment in which hepatocellular carcinoma might be located. METHODS Nine dogs received EO injections via a balloon catheter into a segmental portal branch of the liver. RESULTS Immediately after injection, 80-100% of the liver cells in the EO-injected segment underwent coagulative necrosis. After 1 week, the EO-injected segment had become completely necrotic in two dogs. Only a few viable hepatocytes were still observed around the arteries and beneath the liver capsule in another dog. No pathologic changes were observed in the lungs, kidneys, or heart of any dog. There was a correlation between the EO dosages and the volume of the EO-injected liver tissue. CONCLUSION EO injection into a portal branch results in the pharmacologic destruction of the corresponding liver segment. This procedure may be beneficial in the treatment of hepatic malignancies.