Junichi Shoda

Proton Beam Therapy for Hepatocellular Carcinoma: A Retrospective Review of 162 Patients

Purpose: We present results of patients with hepatocellular carcinoma (HCC) treated with proton beam therapy. Experimental Design: We reviewed 162 patients having 192 HCCs treated from November 1985 to July 1998 by proton beam therapy with or without transarterial embolization and percutaneous ethanol injection. The patients in the present series were considered unsuitable for surgery for various reasons, including hepatic dysfunction, multiple tumors, recurrence after surgical resection, and concomitant illnesses. The median total dose of proton irradiation was 72 Gy in 16 fractions over 29 days. Results: The overall survival rate for all of the 162 patients was 23.5% at 5 years. The local control rate at 5 years was 86.9% for all 192 tumors among the 162 patients. The degree of impairment of hepatic functions attributable to coexisting liver cirrhosis and the number of tumors in the liver significantly affected patient survival. For 50 patients having least impaired hepatic functions and a solitary tumor, the survival rate at 5 years was 53.5%. The patients had very few acute reactions to treatments and a few late sequelae during and after the treatments. Conclusions: Proton beam therapy for patients with HCC is effective, safe, well tolerable, and repeatable. It is the useful treatment mode for either cure or palliation for patients with HCC irrespective of tumor size, tumor location in the liver, insufficient feeding of the tumor with arteries, presence of vascular invasion, impaired hepatic functions, and coexisting intercurrent diseases.

A prospective study of hypofractionated proton beam therapy for patients with hepatocellular carcinoma.

PURPOSE To evaluate the efficacy and safety of hypofractionated proton beam therapy for patients with hepatocellular carcinoma (HCC). METHODS AND MATERIALS Between September 2001 and August 2004, 51 patients with HCC greater than 2 cm away from the porta hepatis or gastrointestinal tract were treated with proton beam therapy to 66 Gy-equivalents (GyE) in 10 fractions. RESULTS Overall survival rates were 49.2 and 38.7% at 3 and 5 years after treatment. Local control rates were 94.5 and 87.8% at 3 and 5 years after treatment. Posttreatment serum alpha-fetoprotein values were significantly reduced when compared with pretreatment values (p < 0.0001). Patients experienced only minor acute reactions of Grade 1 or less, and 3 patients experienced late sequelae of Grade 2 or higher. However, there were no treatment-related deaths. CONCLUSIONS Hypofractionated proton beam therapy is safe and well-tolerated by patients with HCC located greater than 2 cm away from the porta hepatis or gastrointestinal tract and may be effective alternative to other modalities.

Ursodeoxycholic acid stimulates Nrf2-mediated hepatocellular transport, detoxification, and antioxidative stress systems in mice

The protective action of ursodeoxycholic acid (UDCA) in cholestatic liver diseases may be mediated by choleresis, detoxification, and cytoprotection against oxidative stress. Nrf2, one transcription factor, serves as a cellular stress sensor and is a key regulator for hepatic induction of detoxifying enzymes, antioxidative stress genes, and numerous Mrp family members. We aimed to investigate whether UDCA induces hepatic Mrp expression along with that of detoxifying enzymes and antioxidative stress genes via the Nrf2 transcriptional pathway. The protein level, subcellular localization, and mRNA level of Mrp family members were assessed in livers of Keap1 gene-knockdown (Keap1-kd) mice and those of UDCA-fed wild-type (WT) and Nrf2 gene-null (Nrf2-null) mice. Nuclear levels of Nrf2 in livers of Keap1-kd mice markedly increased, resulting in constitutive activation of Nrf2. Keap1-kd mice have high-level expression of hepatic Mrp2, Mrp3, and Mrp4 relative to WT mice. UDCA potently increased nuclear Nrf2 expression level in livers of WT mice, and the treatment showed maximal hepatic induction of Mrp2, Mrp3, and Mrp4 in association with enhanced membranous localizations in an Nrf2-dependent manner. UDCA similarly increased nuclear Nrf2 expression level in rat hepatocytes. Chromatin immunoprecipitation assays using mouse hepatocytes revealed the binding of Nrf2 to antioxidant response elements in the promoter regions of Mrp2, Mrp3, and Mrp4. These findings demonstrate an important role of Nrf2 in the induction of Mrp family members in livers and suggest that a therapeutic mechanism of UDCA action is, via Nrf2 activation, a stimulation of detoxification and antioxidative stress systems, along with Mrp-mediated efflux transport.

The expression levels of plasma membrane transporters in the cholestatic liver of patients undergoing biliary drainage and their association with the impairment of biliary secretory function

OBJECTIVES:Percutaneous transhepatic biliary drainage (PTBD) has been believed to reduce hyperbilirubinemia in patients with obstructive cholestasis and to lessen liver injury through bile acid retention. The efficacy may be closely related to the capability of cholestatic liver to produce and secrete bile, which in turn depends on the expressions and functional activities of plasma membrane transporters in the liver. The aim of the present study was to determine the expression levels of these transporters in the cholestatic liver of patients undergoing PTBD.METHODS:A total of 24 patients who had experienced obstructive cholestasis and had undergone preoperative PTBD were included in the study. Liver biopsy specimens were analyzed to determine the expression levels of the multidrug resistance-associated proteins (MRP) MRP2 and MRP3 and the canalicular bile salt export pump BSEP in the liver.RESULTS:The messenger RNA (mRNA) levels of MRP2, the canalicular bilirubin conjugate export pump, and bile salt export pump (BSEP) were unchanged in liver specimens from the 14 patients well drained by PTBD but were reduced in specimens from the 10 patients poorly drained, compared to the levels of control subjects. Immunostainings of MRP2 and BSEP outlined the canalicular membrane domain but seemed fuzzy to varying degrees in specimens obtained from cholestatic liver, especially in specimens from liver that had been poorly drained, in contrast to the linear and intense localization in the liver of control subjects, correlating with the impaired bilirubin conjugate and bile acid secretion. The mRNA of MRP3, functioning as an inducible export pump for bilirubin conjugate and bile acid, was expressed not only in the cholestatic liver but also in the liver of control subjects, and the mRNA level was increased in specimens from both the cholestatic liver that had been well drained and from the liver that had been poorly drained. Immunostaining of MRP3 was observed in the epithelia of intrahepatic bile ducts in the liver of both control subjects and cholestatic patients, and in the epithelia of proliferated bile ductules and the hepatocytes surrounding the portal tracts in the cholestatic liver.CONCLUSIONS:From the results of the present study, it is concluded that 1) the mRNA and immunohistochemical expression levels of MRP2 and BSEP may be altered in the cholestatic liver of patients undergoing PTBD; 2) both the decreased mRNA levels and the diminished canalicular membrane localization may be associated with the impairment of bile formation and secretion, i.e., the efficacy of PTBD; and 3) upregulated MRP3 in the cholangiocytes and hepatocytes may play a significant role in bile acid transport in the cholestatic hepatobiliary system.

Deletion of nuclear factor-E2-related factor-2 leads to rapid onset and progression of nutritional steatohepatitis in mice.

Oxidative stress is a critical mediator in liver injury of steatohepatitis. The transcription factor Nrf2 serves as a cellular stress sensor and is a key regulator for induction of hepatic detoxification and antioxidative stress systems. The involvement of Nrf2 in defense against the development of steatohepatitis remains unknown. We aimed to investigate the protective roles of Nrf2 in nutritional steatohepatitis using wild-type (WT) and Nrf2 gene-null (Nrf2-null) mice. WT and Nrf2-null mice were fed a methionine- and choline-deficient (MCD) diet for 3 and 6 wk, and the liver tissues were analyzed for pathology and for expression levels of detoxifying enzymes and antioxidative stress genes via the Nrf2 transcriptional pathway. In WT mice fed an MCD diet, Nrf2 was potently activated in the livers, and steatohepatitis did not develop over the observation periods. However, in Nrf2-null mice fed an MCD diet, the pathological state of the steatohepatitis was aggravated in terms of fatty changes, inflammation, fibrosis, and iron accumulation. In the livers of the Nrf2-null mice, oxidative stress was significantly increased compared with that of WT mice based on the increased levels of 4-hydroxy-2-nonenal and malondialdehyde. This change was associated with the decreased levels of glutathione, detoxifying enzymes, catalase, and superoxide dismutase activity. Correlating well with the liver pathology, the mRNA levels of factors involved in fatty acid metabolism, inflammatory cytokines, and fibrogenesis-related genes were significantly increased in the livers of the Nrf2-null mice. These findings demonstrate that Nrf2 deletion in mice leads to rapid onset and progression of nutritional steatohepatitis induced by an MCD diet. Activation of Nrf2 could be a promising target toward developing new options for prevention and treatment of steatohepatitis.

A new, effective and safe therapeutic option using proton irradiation for hepatocellular carcinoma

BACKGROUND/AIMS Conventional radiation is almost useless for hepatocellular carcinoma (HCC) because of the severe adverse effects of the irradiation to the accompanying liver cirrhosis. In contrast, the proton beam has Bragg peak, which limits distribution of the beam. The aim of this study was to prove the usefulness of proton irradiation for HCC. METHODS The proton irradiation was performed in 32 nodular lesions in 24 patients with HCC who had unresectable tumors or serious complications; the proton irradiation was performed either as monotherapy (15 lesions) or as combination therapy to insufficient Lipiodol-targeted chemotherapy (Kodama Co. Ltd., Tokyo, Japan) (17 lesions). The energy was 250 MeV, and 50-87 Gy (76.5 +/- 9.5, mean +/- SD) in total was irradiated for a time period of 17-69 days. RESULTS After 1 year, size reduction was seen in 12 out of 13 lesions (92%) in the monotherapy group and 9 out of 9 lesions (100%) in the combination therapy group; after 2 years, size reduction was seen 4 out of 5 lesions (80%) in the monotherapy group and 5 out of 5 lesions (100%) in the combination therapy group. Local tumor control has being assured for 2 years of the observation, which is continuing for another 2 years. None of the patients have experienced any serious adverse effects. CONCLUSIONS These results show that proton irradiation is a new, safe, and effective therapeutic option in cases of HCC, even in patients with unresectable tumors or those with serious complications.

Genipin enhances Mrp2 (Abcc2)-mediated bile formation and organic anion transport in rat liver

Inchin‐ko‐to (ICKT), an herbal medicine, and its ingredients exert potent choleretic effects by a “bile acid‐independent” mechanism. The current study was designed to determine whether ICKT or its ingredients potentiate multidrug resistance‐associated protein 2 (Mrp2; Abcc2)‐mediated choleresis in vivo. Biliary secretion of Mrp2 substrates and the protein mass, subcellular localization, and messenger RNA (mRNA) level of Mrp2 were assessed in rat liver after infusion of genipin, an intestinal bacterial metabolite of geniposide, a major ingredient of ICKT. The function of Mrp2 was also assessed by the adenosine triphosphate (ATP)‐dependent uptake of Mrp2‐specific substrates using canalicular membrane vesicles (CMVs) from the liver. Infusion of genipin increased bile flow by 230%. It also increased biliary secretion of bilirubin conjugates and reduced glutathione (GSH) by 513% and 336%, respectively, but did not increase bile acid secretion. The ATP‐dependent uptake of estradiol 17‐β‐D‐glucuronide (E217βG; by 265%), leukotriene C4 (LTC4; by 161%), taurolithocholate‐3‐sulfate (TLC‐3S; by 266%), and methotrexate (MTX; by 234%) was significantly stimulated in the CMVs from the liver. These effects were not observed in Mrp2‐deficient rats. Under these conditions, genipin treatment increased the protein mass of Mrp2 in the CMVs but not the mRNA level. In immunoelectron microscopic studies, a marked increase in Mrp2 density in the canalicular membrane (CM) and microvilli was observed in the genipin‐treated liver tissue sections when compared with the vehicle‐treated liver tissue sections. In conclusion, genipin may enhance the bile acid‐independent secretory capacity of hepatocytes, mainly by stimulation of exocytosis and insertion of Mrp2 in the bile canaliculi. ICKT may be a potent therapeutic agent for a number of cholestatic liver diseases. (HEPATOLOGY 2004;39:167–178.)

A novel β1,3-N-acetylglucosaminyltransferase (β3Gn-T8), which synthesizes poly-N-acetyllactosamine, is dramatically upregulated in colon cancer

A new member of the UDP‐N‐acetylglucosamine: β‐galactose β1,3‐N‐acetylglucosaminyltransferase (β3Gn‐T) family having the β3‐glycosyltransferase motifs was identified using an in silico method. This novel β3Gn‐T was cloned from a human colon cancer cell line and named β3Gn‐T8 based on its position in a phylogenetic tree and enzymatic activity. β3Gn‐T8 transfers GlcNAc to the non‐reducing terminus of the Galβ1–4GlcNAc of tetraantennary N‐glycan in vitro. HCT15 cells transfected with β3Gn‐T8 cDNA showed an increase in reactivity to both LEA and PHA‐L4 in a flow cytometric analysis. These results indicated that β3Gn‐T8 is involved in the biosynthesis of poly‐N‐acetyllactosamine chains on tetraantennary (β1,6‐branched) N‐glycan. In most of the colorectal cancer tissues examined, the level of β3Gn‐T8 transcript was significantly higher than in normal tissue. β3Gn‐T8 could be an enzyme involved in the synthesis of poly‐N‐acetyllactosamine on β1–6 branched N‐glycans in colon cancer.

Proton beam therapy for hepatocellular carcinoma: the University of Tsukuba experience.

The authors have published a series of studies evaluating the safety and efficacy of proton beam therapy for the treatment of hepatocellular carcinoma in a variety of clinical settings. In the current study, they retrospectively reviewed their entire experience treating hepatocellular carcinoma patients with proton beam therapy at their hospital‐based facility at the University of Tsukuba.

Multivariate analysis of risk factors for hepatocellular carcinoma in patients with hepatitis C virus-related liver cirrhosis.

To elucidate the risk factors for hepatocellular carcinoma (HCC) in hepatitis C virus (HCV)-related liver cirrhosis (LC), we examined 204 cirrhotic patients negative for hepatitis B surface antigen and positive for HCV antibodies. The independent influence of various clinical characteristics in these patients was analyzed by multiple logistic regression, and the risk factors for HCC were identified. Multiple logistic regression analysis identified and ranked the following four risk factors: male sex (P<0.001), habitual heavy drinking (P<0.005), hepatitis B virus antibody positivity (anti-HBs and/or anti-HBc,P<0.05), and age greater than 60 years (P<0.05). The odds ratio of HCC was 4.20 (95% confidence interval; CI, 1.80–9.78) in male patients, 3.27 (95% CI, 1.46–7.30) in habitual heavy drinkers, 2.01 (95% CI, 1.01–3.99) in patients positive for hepatitis B virus antibodies, and 2.06 (95% CI, 1.00–4.23) in patients older than 60 years. The cumulative occurrence rates of HCC after blood transfusion were significantly higher in habitual heavy drinkers (4.8%, 49.4%, and 74.7% at 10, 20, and 30 years, respectively) than in non-drinkers (0%, 21.0%, and 23.3% at 10, 20, and 30 years, respectively,P<0.0003). The mean interval for progression to LC after blood transfusion was significantly shorter in the habitual heavy drinkers than in the non-drinkers (22.4±4.4 years vs 28.4±3.9 years;P<0.0003). This multivariate analysis revealed that habitual heavy drinking and hepatitis B virus antibody positivity are significant risk factors for HCC in HCV-related liver cirrhosis.