Kinetics of Internalization and Cytotoxicity of Transferrin‐Neocarzinostatin Conjugate in Human Leukemia Cell Line, K562

Human serum transferrin was conjugated with an anticancer‐active polypeptide, neocarzinostatin, by using N‐succinimidy1‐3‐(2‐pyridyldithio)propionate. The conjugate consisted of 1.8 mol of neocarzinostatin per 1 mol of transferrin on average and retained cytotoxic activity against human tumor cells. This conjugate was capable of binding to the transferrin receptor of human myelogenous leukemia K562 cells and was internalized by endocytosis. The LD(50) values of the conjugate and neocarzinostatin alone in the presence of excess native bovine transferrin were 0.20 μ/ml and 1.80 μ/ml, respectively, suggesting that the effect of the conjugate was greater than that of neocarzinostatin alone. A pulse‐chase experiment using (125)I‐labeled conjugate revealed that 25% of the internalized conjugate was degraded in lysosomes and the rest was recycled back to the cell surface without degradation. About 75% of this conjugate recycled back to the cell surface in 18.3 min (3.4 min for receptor binding and 14.9 min for recycling to the cell surface through the acidosomes), while the rest was delivered from the cell surface to the lysosome in 19.6 min. This phenomenon was confirmed by chasing the radioactivity in subcellular fractions separated by Percoll density gradient centrifugation. Therefore, it was concluded that this conjugate is internalized specifically by transferrin receptors and is at least partly transferred to and accumulated in lysosomal compartments, resulting in the inhibition of cellular DNA synthesis.