Eiji Yamasaki

Effect of pravastatin on survival in patients with advanced hepatocellular carcinoma. A randomized controlled trial

Chemotherapy is not effective for hepatocellular carcinoma (HCC). HMG-CoA redutase inhibitors have cytostatic activity for cancer cells, but their clinical usefulness is unknown. To investigate whether pravastatin, a potent HMG-CoA reductase inhibitor, prolongs survival in patients with advanced HCC, this randomized controlled trial was conducted between February 1990 and February 1998 at Osaka University Hospital. 91 consecutive patients <71 years old (mean age 62) with unresectable HCC were enroled in this study. 8 patients were withdrawn because of progressive liver dysfunction; 83 patients were randomized to standard treatment with or without pravastatin. All patients underwent transcatheter arterial embolization (TAE) followed by oral 5-FU 200 mg–1d for 2 months. Patients were then randomly assigned to control (n = 42) and pravastatin (n = 41) groups. Pravastatin was administered at a daily dose of 40 mg. The effect of pravastatin on tumour growth was assessed by ultrasonography. Primary endpoint was death due to progression of HCC. The duration of pravastatin administration was 16.5 ± 9.8 months (mean ± SD). No patients in either group were lost to follow-up. Median survival was 18 months in the pravastatin group versus 9 months in controls (P = 0.006). The Cox proportional hazards model showed that pravastatin was a significant factor contributing to survival. Pravastatin prolonged the survival of patients with advanced HCC, suggesting its value for adjuvant treatment.

Modulation of the mevalonate pathway and cell growth by pravastatin and d -limonene in a human hepatoma cell line (Hep G2)

Modulation of cell growth by a combination of pravastatin [a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor] and d-limonene (an inhibitor of protein isoprenylation) was studied using Hep G2, a human hepatoma-derived cell line. Pravastatin, at 0.1 mM, produced 85% inhibition of cholesterol biosynthesis in Hep G2 cells. The combination of 0.1 mM pravastatin and 1.0 mM d-limonene had no further effect on the reduction seen with pravastatin alone. Addition of 0.1 mM pravastatin or 1.0 mM d-limonene did not significantly suppress DNA synthesis by the cells, whereas the combination suppressed it to 50% of the control level. Production of m-p21ras was markedly decreased to 35% of the control level by the combination of these two inhibitors. Both the reduction by pravastatin of farnesylpyrophosphate as substrate for protein:farnesyl transferase and inhibition of protein farnesylation by d-limonene seem to be responsible for the profound suppression of m-p21ras formation in the cells. However, dolichol synthesis was not suppressed by the combination of these inhibitors. In human fibroblasts, the combination suppressed m-p21ras production but not DNA synthesis. These findings suggest that the combination of pravastatin and d-limonene acts on cancer cell growth through inhibition of the post-translational processing of cellular proteins including p21ras, rather than through the suppression of cholesterol and dolichol biosynthesis. Thus, the combination of an HMG-CoA reductase inhibitor and an inhibitor of protein isoprenylation offers potential as a new approach for cancer therapy.

Inhibition of cell growth of human hepatoma cell line (HepG2) by a farnesyl protein transferase inhibitor: A preferential suppression of ras farnesylation

So far, treatment with anti‐cancer agents has failed to achieve satisfactory results in hepatocellular carcinoma. In the process of hepatocarcinogenesis, ras has been shown to play a role, ras requires a farnesyl moiety for activation. It has been found that UCFI‐C (manumycin), an antibiotic, inhibits farnesyl protein transferase, an enzyme that catalyzes farnesylation. Therefore, we investigated the effects of UCFI‐C on cell growth, prenylation of cellular proteins including ras and Rap I, MAP kinase activity, activities of 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase, and synthesis of cholesterol in a ras‐activated human hepatoma cell line, Hep G2. Treatment with varying concentrations of UCFI‐C (10–30 μM) for 24 and 72 hr resulted in a time· and dose‐dependent inhibition of cell numbers. 3H‐Thymidine incorporation was also inhibited in a dose‐dependent manner, with 50% inhibition after 44 hr being observed at a concentration of 17 μM. UCFI‐C dose‐dependently inhibited ras farnesylation and MAP kinase activity, but did not decrease Rap I geranylgeranylation or prenylation of 21‐ to 26‐kDa proteins. Neither the activities of 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase nor cholesterol synthesis were inhibited. These results suggest that UCFI‐C antagonizes the growth of Hep G2 via the suppression of ras farnesylation and could be a lead for the development of new anti‐cancer agents blocking the function of oncogenic ras associated with human cancer, including hepatocellular carcinoma.

Manumycin and gliotoxin derivative KT7595 block Ras farnesylation and cell growth but do not disturb lamin farnesylation and localization in human tumour cells.

Recently, many inhibitors of farnesyl protein transferase (FPTase) have been identified. Some of them interrupt cell growth in addition to Ras and nuclear lamin processing of Ras-transformed cells. We have tested the effect of the FPTase inhibitors manumycin, an analogue of farnesyl diphosphate, and KT7595, a gliotoxin derivative, on Ras farnesylation, DNA synthesis and the anchorage-dependent and -independent growth of human colon carcinoma (LoVo), hepatoma (Mahlavu and PLC/PRF/5) and gastric carcinoma (KATO III). Both drugs severely inhibited DNA synthesis, cellular proliferation and Ras farnesylation in LoVo and moderately reduced them in Mahlavu and PLC/PRF/5 but not in KATO III. Complete sequencing of ras genes, however, revealed that LoVo and KATO III have activated Ki-ras and activated N-ras, respectively, whereas Mahlavu and PLC/PRF/5 have no activated ras. We next checked whether the inhibition of the cellular proliferation is due to the blocking of nuclear lamin function. Neither drug disturbed lamin farnesylation and localization, as demonstrated using metabolic labelling, immunoblotting and indirect immunofluorescence. These results indicate that manumycin and KT7595 can inhibit Ras farnesylation and cell growth without disturbing the farnesylation and localization of the lamins on human tumour cell lines.

Reduced nitric oxide production by L-arginine deficiency in lysinuric protein intolerance exacerbates intravascular coagulation

Lysinuric protein intolerance (LPI) results in low serum L-arginine, hyperammonemia, mental retardation, thrombocytopenia, and an increased frequency of bowel movements. Our objective was to evaluate the effects of low serum L-arginine, the essential substrate for reactions catalyzed by nitric oxide synthetase (NOS), on the serum nitric oxide (NO) level and coagulation activity in a patient with LPI. A 37-year-old Japanese man who presented with abdominal pain and subnormal fasting levels of serum L-arginine and L-lysine was found to have LPI. The result of oral administration of diamino acids was an increased in urine and a decrease in serum, thus confirming the diagnosis. A decrease in the platelet count and an increase in the plasma levels of thrombin-antithrombin III complex (TAT) and fibrin degradation products (FDPs) indicated the presence of subclinical intravascular coagulation. Serum levels of NO derivatives and L-arginine were determined after intravenous administration of L-arginine. The effects of intravenous L-arginine or transdermal nitroglycerin on the plasma level of TAT were also investigated. Serum levels of NO derivatives were significantly reduced in the LPI patient versus the healthy control group (n = 5). Intravenous administration of L-arginine increased the serum level of NO derivatives and the platelet count and reduced plasma TAT and FDP levels. The plasma level of TAT was also reduced by transdermal nitroglycerin. A decrease in the serum level of L-arginine in patients with LPI appears to result in a decrease in NO production. The improvement in plasma TAT levels produced by administration of intravenous L-arginine or transdermal nitroglycerin suggests that intravascular coagulation is exacerbated by the decrease of NO production in patients with LPI.

Interleukin‐6 in transcatheter arterial embolization for patients with hepatocellular carcinoma. Effects of serine protease inhibitor

Background. Modulation of serum levels of circulating cytokines and inflammatory responses with a serine protease inhibitor was studied in 34 patients with hepatocellular carcinoma (HCC) after transcatheter arterial embolization (TAE).

Effect of Pravastatin, a Potent 3-Hydroxy-3-methylgliitaryl-coenzyme A Reductase Inhibitor, on Survival of AH130 Hepatoma-bearing Rats

3‐Hydroxy‐3‐methylglutaryl‐coenzyme A (HMG‐CoA) reductase inhibitor is known to have an inhibitory effect on cell growth in addition to a cholesterol‐lowering effect. This study examined the effect of pravastatin, a potent inhibitor of HMG‐CoA reductase, on the survival of AH130 hepatoma‐bearing rats. Pravastatin (1, 2, or 8 mg/kg body weight) was intraperitoneally injected once a day into tumor‐bearing rats. The difference in the survival curves was significant between the controls and the rats treated with S mg/kg of pravastatin (P < 0.019 by logrank test) but not between the controls and the rats treated with 1 or 2 mg/kg of the inhibitor. The tumor volume was significantly decreased in the rats treated with 8 mg/kg of pravastatin (P < 0.05). These observations showed that intraperitoneal injection of pravastatin could improve the survival of AH130 hepatoma‐bearing rats and had an inhibitory effect on the growth of the ascites form tumor.

Effect of farnesyltransferase overexpression on cell growth and transformation.

A series of studies using farnesyltransferase (FTase) inhibitors that the inhibition of FTase function suppresses the growth of ras-transformed cells in vitro and in vivo. However, whether FTase is directly involved in the regulation of cell proliferation remains to be demonstrated. To investigate whether overexpression of FTase results in altered cell growth and transformation, we thus used NIH3T3 cells transfected with cDNA constructs of both alpha and beta subunits of human FTase. FTase-overexpressing cells resulted in a 3- to 13-fold increase in the expression of the alpha and beta subunit protein of FTase and a 1.5- to 3-fold increase in the level of the enzyme activity compared with untransfected NIH3T3 cells or vector-transfected cells. Further investigations using metabolic labeling indicated that farnesylation of Ras was enhanced in FTase-overexpressing cells. Insulin-like growth factor-I, platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF) more potently enhanced DNA synthesis and anchorage-dependent growth in FTase-overexpressing cells than in control cells, in a dose-dependent manner. In particular, PDGF and bFGF also induced dose-dependently enhanced colony formation in soft agar in FTase-overexpressing cells. Furthermore, in FTase-transfectants, bFGF stimulated high activation of mitogen-activated protein kinase. Interestingly, FTase-transfectants developed progressive tumors in nude mice. Light and electron microscopy showed that the tumors were characteristic of fibrosarcoma, which were distinct from v-ras-induced tumors. Overexpression of FTase in NIH3T3 cells thus amplifies growth-factor-mediated cell growth and transformation, and FTase-overexpressing cells form tumors in nude mice.

Efficacy of combination therapy of interferon-α with ursodeoxycholic acid in chronic hepatitis C: A randomized controlled clinical trial

The efficacy of interferon-α therapy in the treatment of chronic hepatitis C is still limited. A combination therapy of interferon-α with ursodeoxycholic acid (UDCA) was tested for its efficacy in the treatment of chronic hepatitis C by a randomized controlled study. Eighty consecutive Japanese patients with chronic hepatitis C were randomly divided into two groups: one group was treated with interferon-α (group A,n=40) and the other with a combination of interferon-α and UDCA (group B,n=40). In both groups, human interferon-α (6 million units per day) was intramuscularly injected daily for 2 weeks and then three times a week for 22 weeks: this 24-week period was followed by 24 weeks of observation. In group B, UDCA was also administered, daily at a dose of 600mg orally, from the beginning of the interferon therapy and administration was continued for 48 weeks. The rates for ALT normalization and clearance of hepatitis C virus (HCV) viremia at the end of the 24-week interferon therapy were similar for groups A and B (58% vs 60% and 55% vs 48%, respectively). At the end of the 24-week follow-up, the sustained normalization rates for ALT levels for the two groups were not different (35% vs 43%), while the rate of clearance was higher in group B (40%) than in group A (23%), but the difference was not significant (P=0.14). The sustained complete response, i.e., HCV RNA negativity at the end of the follow-up, as well as the maintenance of ALT normalization during the follow-up period, was more frequent in group B (38%) than in group A (18%) although the difference was not significantP=0.08). The rate of HCV reactivation after interferon was discontinued was significantly lower in group B (16%) than in group A (59%) (P<0.01). Although this combination therapy did not lead to a sufficiently sustained complete response, it could serve as adjuvant antiviral therapy when a suitable dosage and administration period are determined.