Zinc protoporphyrin binding to telomerase complexes and inhibition of telomerase activity.

Zinc protoporphyrin (ZnPP), a naturally occurring metalloprotoporphyrin (MPP), is currently under development as a chemotherapeutic agent although its mechanism is unclear. When tested against other MPPs, ZnPP was the most effective DNA synthesis and cellular proliferation inhibitor while promoting apoptosis in telomerase positive but not telomerase negative cells. Concurrently, ZnPP down-regulated telomerase expression and was the best overall inhibitor of telomerase activity in intact cells and cellular extracts with IC50 and EC50  values of ca 2.5 and 6 µM, respectively. The natural fluorescence properties of ZnPP enabled direct imaging in cellular fractions using non-denaturing agarose gel electrophoresis, western blots, and confocal fluorescence microscopy. ZnPP localized to large cellular complexes (>600 kD) that contained telomerase and dysskerin as confirmed with immunocomplex mobility shift, immunoprecipitation, and immunoblot analyses. Confocal fluorescence studies showed that ZnPP co-localized with telomerase reverse transcriptase (TERT) and telomeres in the nucleus of synchronized S-phase cells. ZnPP also co-localized with TERT in the perinuclear regions of log phase cells but did not co-localize with telomeres on the ends of metaphase chromosomes, a site known to be devoid of telomerase complexes. Overall, these results suggest that ZnPP does not bind to telomeric sequences per se, but alternatively, interacts with other structural components of the telomerase complex to inhibit telomerase activity. In conclusion, ZnPP actively interferes with telomerase activity in neoplastic cells, thus promoting pro-apoptotic and anti-proliferative properties. These data support further development of natural or synthetic protoporphyrins for use as chemotherapeutic agents to augment current treatment protocols for neoplastic disease.

Metalloprotoporphyrin Inhibition of HCV NS3-4A Protease: Structure-Activity Relationships.

BACKGROUND: Antiviral actions of tetrapyrroles have been described in a number of systems. Our goal was to evaluate antagonism of the HCV NS3-4A protease by a variety of common porphyrins and characterize structure-activity relationships that may be useful for future drug design of HCV and related Flaviviruses. METHODS: Using fluorometric assays, common metalloprotoporphyrins (MPP) all inhibited NS3-4A protease with IC50 values in low micromolar ranges [CoPP (1.4 µM) < ZnPP = MnPP = SnPP < CuPP < FePP (6.5 µM) = protoporphyrin]. RESULTS: Lineweaver-Burk plots confirmed that MPP: NS3 inhibition was basically competitive. All tested MPPs inhibited HCV genotype 1A, 1B, 2A and 3A recombinant proteases with the same fidelity suggesting wide antagonistic capabilities. However, when the MPPs were tested in cellular incubations with HCV replicons only Zn, Fe and free-base protoporphyrin showed comparable EC50 and IC50 values suggesting that there may be critical differences in MPP uptake and intracellular availability. Meso, deutero, and isohematoporphyrin derivatives, with or without metal substitution, all showed less anti-protease and antiviral activities as compared to protoporphyrins, suggesting that the planar, vinyl side chains are important for protease active site binding. MPPs were also active against three common protease mutants (T54A, A156T, and V36M) with equivalent or better IC50 values as compared to wild type enzyme. CONCLUSION: These findings document the versatility of MPPs as antiviral agents with an expanded sensitivity for HCV genotypes and resistance to some common viral mutations. The results also suggest that further study of MPP structure and function will be useful for the development of new antiviral agents.

Reduced heme oxygenase-1 expression in steatotic livers infected with hepatitis C virus.

UNLABELLED: Hepatic nonalcoholic fatty liver disease (NAFLD) is known to exacerbate liver injury due to chronic hepatitis C infection. Heme oxygenase-1 (HO-1) is an important protective antioxidative defense enzyme that is known to be induced in response to NAFLD and other liver injuries. The aim of this study was to evaluate HO-1 expression in HCV infected human livers with concomitant NAFLD. METHODS: We compared levels of HO-1 in NAFLD liver biopsies from patients with or without chronic HCV infection using immunohistochemistry, immunoblots and real time RT-PCR. We also evaluated frozen sections of liver with dihydroethidium (DHE) or dichlorofluorescein (DCF) fluorescence staining to evaluate O(2)(-) and peroxide production respectively. RESULTS: HO-1 expression was only increased in NAFLD livers without HCV infection, while HCV infected livers showed reduced HO-1 levels, regardless whether NAFLD was present. In uninfected livers with NAFLD, HO-1 expression was primarily localized in hepatocytes containing fat and areas of injury around the central vein. However, both NAFLD with and without concomitant HCV infection showed high levels of O(2)(-) or peroxide production compared to normal human liver control samples. CONCLUSIONS: These findings support the hypothesis that NAFLD is an important process for hepatocyte oxidative stress and injury in liver diseases. They also suggest that HCV can repress HO-1 induction in vivo even when other inducers of HO-1 are present.

Increased plasma nitric oxide, L-arginine, and arginase-1 in cirrhotic patients with progressive renal dysfunction.

BACKGROUND AND AIMS: Increased levels of nitric oxide (NO) are hypothesized to contribute to renal dysfunction in patients with decompensated cirrhosis. In this study, we examined whether splanchnic and/or peripheral NO levels and L-arginine (L-Arg) correlate with progressive renal dysfunction in cirrhotics. METHODS: Serum NO metabolites (NOx) and L-Arg were measured in: controls (n = 10); organ donors (n = 12); compensated cirrhotics (n = 17), cirrhotics with ascites (n = 25), refractory ascites (n = 11) or hepatorenal syndrome type II (HRS) (n = 11) and chronic renal failure patients (n = 18). RESULTS: Plasma NOx and L-Arg levels rose progressively with worsening renal function in decompensated cirrhotics. Both NOx and L-Arg levels were highest in patients with HRS (P < 0.001 and P < 0.025, respectively). While there were no differences in NOx levels related to the site of sampling, L-Arg levels were lowest in hepatic venous blood. There were significant relationships of NOx and L-Arg with Model for End-Stage Liver Disease score and Child-Pugh scores (P < 0.04 and P < 0.01, respectively). Multivariate analysis showed a significant relationship between NOx, L-Arg and HRS. CONCLUSION: Worsening renal function in decompensated cirrhosis is accompanied by progressive elevation in plasma NOx and L-Arg. These findings support the hypothesis that NO-mediated vasodilation is probably linked with the mechanism of progressive renal failure in decompensated cirrhotics.