[Locally ablative therapies of hepatocellular carcinoma]. / Lokal ablative Therapien des hepatozellulären Karzinoms.

Locally Ablative Therapies of Hepatocellular Carcinoma Delayed diagnosis of hepatocellular carcinomas (HCC) leads to a poor prognosis with a median survival time of less than 10 months. Surgical resection of small HCCs is the treatment of choice in patients with good residual liver function. The recurrence-free 5-year survival rate after curative resection is 33%. Resectability of HCC is often limited by the low hepatic functional reserve. Only 20% of all HCC are resectable in spite of novel diagnostic tools, an intensified screening, and advances in surgical technique. Local methods for tumor ablation are promising extensions of tumor therapy, especially in patients with limited liver function, nonresectable tumors, or multifocal tumors. Vis-à-vis a change of therapeutic options, local methods of tumor ablation in combination with tumor resection promise a yet unknown improvement of the prognosis for patients with HCC. Controlled randomized studies comparing and validating these methods of local tumor ablation are eagerly awaited. In the following article different methods of tumor ablation are described. Percutaneous interventions can be distinguished into vascular regional (TAE = transarterial embolization and TACE = transarterial catheter embolization) and local ablative approaches like PEI (percutaneous ethanol instillation), LITT (laser-induced thermotherapy), Cryo (cryotherapy), and RFA (radio frequency ablation).

Microinjected glutathione reductase crystals as indicators of the redox status in living cells.

The flavoenzyme glutathione reductase catalyses electron transfer reactions between two major intracellular redox buffers, namely the NADPH/NADP+ couple and the 2 glutathione/glutathione disulfide couple. On this account, microcrystals of the enzyme were tested as redox probes of intracellular compartments. For introducing protein crystals into human fibroblasts, different methods (microinjection, particle bombardment and optical tweezers) were explored and compared. When glutathione reductase crystals are present in a cytosolic environment, the transition of the yellow Eox form to the orange-red 2-electron reduced charge transfer form, EH2, is observed. Taking into account the midpoint potential of the Eox/EH2 couple, the redox potential of the cytosol was found to be < -270 mV at pH 7.4 and 37 degrees C. As a general conclusion, competent proteins in crystalline--that is signal-amplifying--form are promising probes for studying intracellular events.

Dinitrosyl-dithiol-iron complexes, nitric oxide (NO) carriers in vivo, as potent inhibitors of human glutathione reductase and glutathione-S-transferase.

Human glutathione reductase (GR) and rat liver glutathione-S-transferases (GSTs) had been shown to be inhibited by the nitric oxide (NO) carrier S-nitroso-glutathione (GSNO). We have now extended these studies by measuring the effects of dinitrosyl-iron complexed thiols (DNIC-[RSH]2) on human GR, GST and glutathione peroxidase. DNIC-[RSH]2 represent important transport forms of NO but also of iron ions and glutathione in vivo. Human GR was found to be inhibited by dinitrosyl-iron-di-glutathione (DNIC-[GSH]2) and dinitrosyl-iron-di-L-cysteine (DNIC-Cys2) in two ways: both compounds were competitive with glutathione disulfide (GSSG), the inhibition constant (Ki) for reversible competition of DNIC-[GSH]2 with GSSG being approximately 5 microM; preincubating GR for 10 min with 4 microM DNIC-[GSH]2 and 40 microM DNIC-Cys2, respectively, led to 50% irreversible enzyme inactivation. More than 95% GR inactivation was achieved by incubation with 36 microM DNIC-[GSH]2 for 30 min. This inhibition depended on the presence of NADPH. Absorption spectra of inhibited GR showed that the charge-transfer interaction between the isoalloxazine moiety of the prosthetic group flavin adenine dinucleotide (FAD) and the active site thiol Cys63 is disturbed by the modification. Cys2 and FAD could be ruled out as sites of the modification. Isolated human placenta glutathione-S-transferase and GST activity measured in hemolysates were also inhibited by DNIC-[GSH]2. This inhibition, however, was reversible and competitive with reduced glutathione, the Ki being 20 nM. The inhibition of GST induced by GSNO was competitive with reduced glutathione (GSH) (Ki = 180 microM) and with the second substrate of the reaction, 1-chloro-2,4,-dinitrobenzene (Ki = 170 microM). An inhibition of human glutathione peroxidase by GSNO or DNIC-[RSH]2 was not detectable. Inactivation of GR by DNIC-[GSH]2 is by two orders of magnitude more effective than modification by GSNO; this result and the very efficient inhibition of GST point to a role of DNIC-[RSH]2 in glutathione metabolism.

Inhibition of glutathione reductase by dinitrosyl-iron-dithiolate complex.

The biological signal molecule nitric oxide (NO) exists in a free and carrier-bound form. Since the structure of the carrier is likely to influence the interaction of NO with macromolecular targets, we assessed the interaction of a dinitrosyl-iron-dithiolate complex carrying different thiol ligands with glutathione reductase. The enzyme was irreversibly inhibited by dinitrosyl-iron-di-L-cysteine and dinitrosyl-iron-di-glutathione in a concentration- and time-dependent manner (IC50 30 and 3 microM, respectively). Evaluation of the inhibition kinetics according to Kitz-Wilson yielded a Ki of 14 microM, and a k3 of 1.3 x 10(-3) s-1. A participation of catalytic site thiols in the inhibitory mechanism was indicated by the findings that only the NADPH-reduced enzyme was inhibited by dinitrosyl-iron complex and that blockade of these thiols by Hg2+ afforded protection against irreversible inhibition. This inhibition was not accompanied by formation of a protein-bound dinitrosyl-iron complex and/or S-nitrosation of active site thiols (Cys-58 and Cys-63). However, one NO moiety exhibiting an acid lability similar to a secondary N-nitrosamine was present per mol of inhibited monomeric enzyme. These findings suggest specifically N-nitrosation of glutathione reductase as a likely mechanism of inhibition elicited by dinitrosyl-iron complex and demonstrate in general that structural resemblance of an NO carrier with a natural ligand enhances NO+ transfer to the ligand-binding protein.