Uptake of radiolabeled morphiceptin and its analogs by experimental mammary adenocarcinoma: in vitro and in vivo studies.

Morphiceptin (Tyr-Pro-Phe-Pro-NH(2)) and its analogs modified at position 3: [D-Phe(3)]morphiceptin, [D-ClPhe(3)]morphiceptin and [D-Cl(2)Phe(3)]morphiceptin were synthesized and labeled with [(125)I] or [(131)I]. Their binding to membranes isolated from experimental adenocarcinoma was examined in vitro with the use of a cross-linking assay followed by the Western blot technique. The radioactive complex had molecular weight of about 65 kDa and was detectable by anti-mu-opioid receptor polyclonal antibody. Expression of the mu-opioid receptor in mouse mammary adenocarcinoma was confirmed by reverse transcriptase-polymerase chain reaction. The binding studies showed the highest affinity and capacity for [D-Phe(3)]morphiceptin (K(d) 0.39 and B(max) 1112) and [D-ClPhe(3)]morphiceptin (K(d) 1.8 and B(max) 220). Morphiceptin and its D-Cl(2)Phe analog had significantly lower B(max) values (131 and 83, respectively). Biodistribution experiments in tumor-bearing C3H/Bi mice with the use of the (131)I-labeled peptides confirmed the results of our in vitro studies. The highest accumulation of radioactive peptides in the tumor tissue was also found for peptides with D-Phe and D-ClPhe.

Synthesis of novel morphiceptin analogues modified in position 3 and their binding to mu-opioid receptors in experimental mammary adenocarcinoma.

Binding of the (125)I-labeled mu-opioid receptor selective ligands, morphiceptin (Tyr-Pro-Phe-Pro-NH(2)) and [D-Phe(3)]morphiceptin, to membranes isolated from experimental mouse mammary adenocarcinoma was examined in vitro using a cross-linking assay followed by a Western blot technique. The radioactive complex had a molecular weight of about 65 kDa and was detectable by anti-mu-opioid receptor antibody, indicating the presence of mu-opioid receptors in tumor membranes. A series of new morphiceptin analogues, modified at the pharmacophoric position 3, was synthesized in order to find the correlation between the lipophilicity, electronic and steric properties of the amino acid in this position and the in vitro affinity of new analogues for mu-opioid receptors on mouse brain and tumor membranes. In in vivo studies the uptake of (131)I-labeled analogues by experimental mammary adenocarcinoma was estimated. The highest affinity for mu-opioid receptors in both, in vitro and in vivo experiments was observed for [D-Phe(3)]morphiceptin and [D-ClPhe(3)]-morphiceptin.

Uptake of radiolabelled alpha-fetoprotein by experimental mammary adenocarcinoma and adenoma: in vivo and in vitro studies.

The biodistribution of iodine-labelled alpha-fetoprotein ( I-AFP) in experimental mammary tumours was studied. C3H mice with subcutaneously transplanted mammary adenocarcinoma and Sprague-Dawley rats treated with -methyl- -nitrosourea for mammary adenoma induction were used as animal models. The accumulation of labelled I-AFP in mouse mammary adenocarcinoma was significantly higher than that in rat mammary adenoma. The tumour/muscle radioactivity ratios increased with time and, 48 h after intravenous injection, were estimated as 23.4 and 6.7, respectively. For experiments, extracts from both mammary tumours were prepared. The extracts were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes and incubated with I-AFP. A single major AFP-binding protein with a molecular weight of about 30 kDa was detected in both extracts. The amount of AFP-binding protein was clearly higher for adenocarcinoma than for adenoma. In the presence of cross-linking reagent, I-AFP formed a complex (about 100 kDa) with adenocarcinoma proteins.