Marion Ganslmayer

The histone-deacetylase inhibitor Trichostatin A blocks proliferation and triggers apoptotic programs in hepatoma cells

BACKGROUND/AIMS Effective treatment for hepatocellular carcinoma is urgently needed. The histone-deacetylase inhibitor Trichostatin A (TSA) was shown to induce apoptosis in non-hepatic cells at submicromolar concentrations. However, the effect of TSA on hepatoma cells is unknown. METHODS The hepatoma cells HepG2, MH1C1, Hepa1-6 and Hep1B as well as human fibroblasts (control cells) were exposed to TSA (10(-6) to 10(-9)M). Cell proliferation was assessed by measuring DNA-synthesis and cell numbers. Apoptosis was quantified by flow cytometry and by the TdT-mediated dUTP nick-end labeling method. Expression patterns of cell cycle- and/or apoptosis-associated p27, p21(cip/waf), bax, bcl-2, cyclin A and (pro)-caspase 3 were studied using quantitative Western blotting. Activation of caspase 3 was analyzed via a colorimetric assay. RESULTS 10(-6)M TSA inhibited DNA-synthesis by 46% (HepG2) to 64% (MH1C1) after 24h, inducing a G(2)/M-phase arrest and apoptosis. TSA increased activation of caspase 3 and expression of cyclin A, p2l(cip/waf), bax and (pro)-caspase 3, while bcl-2 was downregulated. Human fibroblasts remained unaffected. CONCLUSIONS TSA inhibits hepatoma cell growth in vitro, which are otherwise particularly resistant to chemotherapy. Its anti-proliferative activity is paralleled by a comparable rate of apoptosis. TSA may be a promising agent for treatment of hepatocellular carcinoma in vivo.

Inducibility of microsomal liver function may differentiate cirrhotic patients with maintained compared with severely compromised liver reserve.

Background and Aim: Quantitative tests of liver function (QTLF) can be modulated by enzyme‐inducing agents. The objective of the study was to examine changes in QTLF after treatment with Phenobarbital, a potent cytochrome P450‐inducing agent.

Can Quantitative Tests of Liver Function Discriminate Between Different Etiologies of Liver Cirrhosis

Studies comparing quantitative testing of liver function (QTLF) in large numbers of patients with defined etiology of cirrhosis are lacking. In all 316 patients with proven cirrhosis underwent QTLF, including aminopyrine breath test (ABT), galactose elimination capacity (GEC), sorbitol (SCI), and indocyanine green clearance (ICG). Values were correlated with the Child-Pugh classification (CP) and the etiology of liver cirrhosis. Fifty-five percent of the patients had alcoholic cirrhosis (ALC), 31% cirrhosis due to viral hepatitis (VIC), and 14% primary biliary cirrhosis (PBC). In all three groups there was a decrease of QTLF levels from CP grade A to C, which differed from normal values. QTLF was most compromised in patients with ALC and VIC compared to patients with PBC. In conclusion, QTLF in ALC and VIC patients was more reduced than in patients with PBC. This may be due to saturation of enzymes in ALC and ongoing inflammation in VIC.

The synthetic retinoid adapalene inhibits proliferation and induces apoptosis in colorectal cancer cells in vitro

Chemotherapy of advanced stages of colorectal carcinoma is unsatisfactory. Retinoids inhibit cell growth and induce apoptosis in a variety of human malignancies. We compared the effect of the synthetic retinoid adapalene (ADA) and 9‐cis‐retinoic acid (CRA) on carcinoma cell lines in vitro. Colon carcinoma cell lines CC‐531, HT‐29 and LOVO as well as human foreskin fibroblasts were exposed to different concentrations of ADA and CRA for 3–72 hr. Proliferation was assessed by BrdU incorporation and apoptosis by FACS analysis. Breakdown of ΔΨm was determined by JC‐1 staining and activity of caspases 3 and 8, by a colorimetric assay. Quantitative Western blots were performed to detect changes in bax, bcl‐2 and caspase‐3. Both retinoic derivatives suppressed DNA synthesis and induced apoptosis in all tested cell lines time‐ and dose‐dependently. While the natural retinoid CRA showed moderate antiproliferative and proapoptotic effects only at the highest concentration (10–4 M), the synthetic retinoic ADA was significantly more effective, showing remarkable effects even at 10–5 M. ADA and CRA disrupt ΔΨm and induce caspase‐3 activity in responsive tumor cells. Quantitative Western blots showed a shift of the bax:bcl‐2 ratio toward proapoptotic bax in ADA‐treated cells. Our results clearly indicate the superiority of ADA compared to CRA. Therefore, we suggest that ADA may be far more suitable as an adjunctive therapeutic agent for treatment of colon cancer in vivo.

Peginterferon alfa-2a relapse rates depend on weight-based ribavirin dosage in HCV-infected patients with genotype 1: Results of a retrospective evaluation

Objective. The cumulative dosage of ribavirin per kilogram of body-weight prevents relapse and thus is a significant predictor of sustained virological response (SVR). Comparison of peginterferon (peg-IFN) alfa-2b/ribavirin and peg-IFN alfa-2a/ribavirin shows that the rates of SVR are similar, but the rates of relapse are significantly lower under the peg-IFN alfa-2b regimen. Depending on the weight-based ribavirin dose, patients with >105 kg reach a maximum of 13.2 mg/kg body-weight ribavirin in the peg-IFN alfa-2b regimen as opposed to only 11.3 mg/kg in the peg-IFN alfa-2a regimen. The aim of these investigations was to determine relapse rates in a retrospective analysis of 98 patients chronically infected with hepatitis C virus (HCV) genotype (GT) 1 in relation to the weight-based ribavirin dose. Material and methods. All patients completed treatment with peg-IFN alfa-2a/ribavirin (1000 mg/d or 1200 mg/d for patients weighing <75 kg or ≥75 kg) for 48 weeks. Classification of a low ribavirin dose with <13.2 mg/kg body-weight was used. Patients with a ribavirin dose ≥13.2 mg/kg were compared with those with a dose <13.2 mg/kg. Results. Patients with a ribavirin dose ≥ 13.2 mg/kg (n=84) showed a relapse rate of 19.0% in contrast to 71.4% in patients with a ribavirin dose of <13.2 mg/kg (n=14) (p=0.0013). The SVR rate was significantly higher in the ≥13.2 mg/kg ribavirin dosed group (59.5% versus 28.6%). Conclusions. Weight-adapted ribavirin dosing in combination with peg-IFN alfa-2a to avoid giving low doses of ribavirin should be evaluated. This will minimize relapse, especially in HCV GT 1 patients.

Antiphospholipid syndrome in combination with autoimmune hepatitis.

Autoimmune hepatitis is rarely described in combination with antiphospholipid syndrome. So far, only cases have been presented where the secondary antiphospholipid syndrome occurred as an effect of autoimmune hepatitis. We report on a 56-year-old Caucasian female with a history of thrombosis, thrombocytopenia and the detection of anti-cardiolipin antibodies. Interestingly, a few years later the patient developed liver failure with highly elevated liver enzymes. The immunological antibody pattern verified the diagnosis of type I autoimmune hepatitis, and immunosuppressive therapy led to the recovery of the patient. Thus, we present the first case of autoimmune hepatitis as a consequence of primary antiphospholipid syndrome.

Combined inhibitors of angiogenesis and histone deacetylase: Efficacy in rat hepatoma

AIM To evaluate the antitumoral effect of combined inhibitors of angiogenesis and histone deacetylases in an experimental rat hepatoma model. METHODS MH7777A hepatoma cells were injected into the liver of male Buffalo rats. After 7 d treatment with the vascular endothelial growth factor receptor antagonist PTK787/ZK222584 (PTK/ZK), the histone deacetylase inhibitor MS-275, tamoxifen (TAM) and/or retinoic acid was initiated (n ≥ 8 animals/group). Natural tumor development was shown in untreated control groups (control 1 with n = 12, control 2 with n = 8). The control groups were initiated at different time points to demonstrate the stability of the hepatoma model. For documentation of possible side effects, we documented any change in body weight, loss of fur and diarrhea. After 21 d treatment, the rats were euthanized. Main target parameters were tumor size and metastasis rate. Additionally, immunohistochemistry for the proliferating cell nuclear antigen (PCNA) and TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay were performed. RESULTS The control groups developed large tumor nodules with extrahepatic tumor burden in the lung and abdominal organs (control 1: 6.18 cm(3) ± 4.14 cm(3) and control 2: 8.0 cm(3) ± 4.44 cm(3) 28 d after tumor cell injection). The tumor volume did not differ significantly in the control groups (P = 0.13). As single agents MS-275 and PTK/ZK reduced tumor volume by 58.6% ± 2.6% and 48.7% ± 3.2% vs control group 1, which was significant only for MS-275 (P = 0.025). The combination of MS-275 and PTK/ZK induced a nearly complete and highly significant tumor shrinkage by 90.3% ± 1% (P = 0.005). Addition of TAM showed no further efficacy, while quadruple therapy with retinoic acid increased antitumoral efficacy (tumor reduction by 93 ± 1%) and side effects. PCNA positive cells were not significantly reduced by the single agents, while dual therapy (MS-275 and PTK/ZK) and quadruple therapy reduced the PCNA-positive cell fraction significantly by 9.1 and 20.6% vs control 1 (P < 0.05). The number of TUNEL-positive cells, markers for ongoing apoptosis, was increased significantly by the single agents (control 1: 6.9%, PTK/ZK: 11.4%, MS-275: 12.2% with P < 0.05 vs control 1). The fraction of TUNEL-positive cells was upregulated highly significantly by dual therapy (18.4%) and quadruple therapy (24.8%, P < 0.01 vs control 1). For the proliferating (PCNA positive) and apoptotic cell fraction, quadruple therapy was significantly superior to dual therapy (P = 0.01). CONCLUSION Combined PTK/ZK and MS-275 were highly effective in this hepatoma model. Quadruple therapy enhanced the effects microscopically, but not macroscopically. These results should be investigated further.

A large cohort of patients with hepatocellular carcinoma in a single European centre: aetiology and prognosis now and in a historical cohort.

PRINCIPLES The incidence of hepatocellular carcinoma is rising. However, this is occurring not only in developing nations, but in industrial countries as well. Surveillance programmes, classification systems and therapeutic options have improved, but there is a lack of data regarding their impact on the prognosis of this difficult-to-treat cancer. MATERIALS AND METHODS We evaluated 484 patients and reported on disease stage, therapeutic procedures and survival time. Data were compared with a historical cohort treated in the same centre 10 years before. RESULTS In this cohort, the main reason for liver disease was alcoholism, although hepatitis B remains the leading cause of hepatocellular carcinoma worldwide. Now, most patients have compensated liver function and hepatocellular carcinoma is diagnosed in the early tumour stages (it was diagnosed in the advanced disease stages in the previous cohort). Overall, median survival time was 62.4 weeks, 1-year survival was 58.6% and 3-year survival was 23.2%. Survival time correlated with the stage of liver disease, tumour stage and with therapeutic options. CONCLUSION Surveillance programmes lead to diagnosis in earlier tumour stages. Differentiated classification systems allow individualised therapeutic approaches. Earlier cancer stage and compensated liver function allow combination or sequential therapy, which was nearly impossible some years ago but is an option for most now. Primary liver cancer remains a difficult-to-treat malignancy, but the prognosis has improved remarkably, at least in the western world.