Expression and function of beta-glucuronidase in pancreatic cancer: potential role in drug targeting.

Improvement of non-surgical strategies is a pivotal task in the treatment of pancreatic cancer. Response to treatment with most anticancer agents has been very poor, probably due to insufficient drug concentration in tumor tissue. Increased response rates during chemotherapy might be achieved by dose escalation; however, this approach is often hampered by severe side effects. One strategy to overcome these adverse effects is application of nontoxic glucuronide prodrugs from which the active moiety is released by beta-glucuronidase within or near the tumor. The use of glucuronide prodrugs in pancreatic cancer requires increased expression of the enzyme in the diseased tissue, a problem that has not been addressed so far. We therefore investigated function and expression of beta-glucuronidase in tissue samples from human healthy pancreas (n=7) and pancreatic adenocarcinoma (n=8), respectively. Comparing the ability of tissue homogenates to cleave the standard substrate 4-methylumbelliferyl-beta-D-glucuronide, we found a significantly increased specific beta-glucuronidase activity (P<0.05) in pancreatic cancer (median: 133; 75% percentile: 286; 25% percentile: 111 nmol/mg per h) as compared to healthy pancreas (median: 74; 75% percentile: 113; 25% percentile: 71 nmol/mg per h). Enzyme kinetic experiments with the model prodrug N-[4-beta-glucuronyl-3-nitrobenzyloxycarbonyl] doxorubicin (HMR 1826) demonstrated bioactivation of HMR 1826 by pancreatic beta-glucuronidase. Enzymatic activity was found to be closely related to enzyme contents (r=0.87) as assessed by Western blot analysis. Our data indicate that increased beta-glucuronidase activity in pancreatic cancer seems to be due to an elevated steady-state level of the protein. This may be the basis for new therapeutic strategies in treatment of pancreatic carcinoma by using glucuronide prodrugs of anticancer agents.

Free radical scavenging and antioxidative properties of 'silibin' complexes on microsomal lipid peroxidation.

The antioxidant properties of silibin complexes, the water-soluble form silibin dihemisuccinate (SDH), and the lipid-soluble form, silibin phosphatidylcholine complex known as IdB 1016, were evaluated by studying their abilities to react with the superoxide radical anion (O2-.), and the hydroxyl radical (OH.). In addition, their effect on pulmonary and hepatic microsomal lipid peroxidation had been investigated. Superoxide radicals were generated by the PMS-NADH system and measured by their ability to reduce NBT. IC50 concentrations for the inhibition of the NBT reduction by SDH and IdB 1016 were found to be 25 microM and 316 microM respectively. Both silibin complexes had an inhibitory effect on xanthine oxidase activity. SDH reacted rapidly with OH radicals at approximately diffusion controlled rate and the rate constant was found to be (K = 8.2 x 10(9) M-1 s-1); it appeared to chelate Fe2+ in solution. In hepatic microsomes, when lipid peroxidation was induced by Fe2+, SDH inhibited by 39.5 per cent and IdB 1016 by 19.5 per cent, whereas when lipid peroxidation was induced by CuOOH, IdB 1016 exerted a better protective effect than SDH (29.4 per cent and 19.4 per cent inhibition, respectively). In both microsomal systems lipid peroxidation proceeded through a thiol depletion mechanism which could be restored in the presence of silibin complexes. Low levels of lipid peroxidation in pulmonary microsomes point out the differences between in-vitro lipid peroxidation occurring in microsomes of different tissues. The results support the free radical scavenger and antioxidative properties of silibin when it is complexed with a suitable molecule to increase its bioavailability.