Pharmacokinetic analysis of protein-conjugated doxorubicin (DXR) and its degraded adducts in DXR-sensitive and -resistant rat hepatoma cells.
After treatment of AH66DR cells with the multidrug resistance (MDR) phenotype with bovine serum albumin (BSA)-conjugated [14C]doxorubicin (DXR), accumulation of the drug in the secondary lysosomal fraction increased as a function of time up to 24 h without any significant increase of the drug in other organellae. By contrast, AH66P cells showed a marked increase in accumulation of the drug in the mitochondrial fraction, and a moderate increase in the lysosomal and nuclear fractions. The intracellular degradation of the internalized conjugate was assessed by HPLC gel filtration as molecular change of the drug. The initial molecular mass (M(r)) of BSA-conjugated [14C]DXR was estimated to be 70 kDa; however, the secondary lysosomal fraction contained mainly three peaks of [14C]compounds ranging from 3 to 70 kDa. The [14C]compound extracted from the nuclear and mitochondrial fractions showed only one peak, which was estimated to be smaller than 2 kDa. By contrast, the cytosolic fraction contained mainly two peaks of [14C]compounds, which were smaller than 2 kDa and larger than 500 kDa. These results indicated that the intracellular distribution of the administered drug, based probably on the drug-traffic mechanism in the cells, was quite different between the two cell lines, but some of the biochemical characteristics of the degraded compounds from each subcellular fraction were similar because the degradation processes in each fraction might be almost identical. The possibility of lysosomal degradation of the protein-conjugated DXR leading to expression of cytotoxicity was also confirmed by the fact that only lysosomal digestable poly-L-lysine-conjugated DXR exhibited dose-dependent cytotoxicity against both cell lines in marked contrast to the cells treated with poly-D-lysine-conjugated DXR. It was concluded that lysosomal breakdown of protein-conjugated DXR, which had been taken up by endocytosis, and the liberation of the degraded active adducts of the conjugate without efflux by the MDR pump mechanism must be an essential stage in the development of the cytotoxicity against tumor cells with or without the MDR phenotype.