Cellular Uptake of Liposomal Daunorubicin and the Induction of Apoptosis

Abstract

Liposomal daunorubicin (L-DnR) has clinical activity against Kaposi's sarcoma, leukemias and lymphomas, but the uptake and cytotoxic mechanisms of its actions remain unclear. Uptake of LDnR and daunorubicin (DnR) by tumor cells and J774 (murine monocyte/macrophage) cells were compared and shown to occur both at 4 C and after formalin-fixation, suggesting that endocytosis is not the sole mechanism of liposome uptake even by phagocytic cells. L-DnR cytotoxicity in vitro was equivalent to that of DnR in standard, 48-hr assays, but substantial cytotoxicity (50% growth inhibition) also occurred following brief 1exposures to DnR and LDnR (5 and 30 min, respectively). Similar degrees of cytotoxicity were induced by DnR and L-DnR immobilized by adherence to tissue culture wells. As these data suggested that the induction of cytotoxicity was a rapid, early, and possibly cell-membrane-mediated event, we examined cell lysates for evidence of apoptosis. DNA fragmentation in J774 cells was demonstrable within 1-4 hr exposure to DnR and L-DnR by an ELISA method and by DNA laddering. Studies with human leukemia and lymphoma cell lines confirmed DnR- and L-DnR-induced apoptosis in HL60, JVM-2, and WSU-NHL cells. In studies of apoptosis-induction by the related anthracyclines doxorubicin (DoX) and liposomal doxorubicin (LPEG-DoX, polyethylene glycol-coated liposome), it was found that doxorubicin activity closely resembled that of L-DnR but LPEG-DoX showed no or little effect. These data show, for the first time, the induction of apoptosis by a conventional cytotoxic agent encapsulated in a liposome and suggest that the uptake and intracellular pharmacology may be similar to that of the free drug.