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Modulation of Subcellular Distribution of Doxorubicin in Multidrug-resistant P388/ADR Mouse Leukemia Cells by the Chemosensitizer {{2-Isopropyl-1-{4-[3-N-methyl-N-(3,4-dimethoxy-β-phenethyl)amino]propyloxy}-benzenesulfonyl}}indolizine¹

Laboratoire de Pharmacologie et de Toxicologie Fondamentales, Centre Nationale de la Recherche Scientifique [J. P. J. G. L.], Laboratoire de Biochimie Médicale, Centre Hospitalier Universitaire Rangueil [T. L.], and Service d'Hematologie, Centre Hospitalier Universitaire Purpan [G. L.], Toulouse, France, and Sanofi-Labaz Research Center, Brussels, Belgium [P. C.]

The impact of the novel chemosensitizer {{2-isopropyl-1-{4-[3-N-methyl-N-(3,4-dimethoxy-β-phenethyl)amino]propyloxy}benzenesulfonyl}}indolizine (SR33557) on the intracellular distribution of doxorubicin (DOX) within the multidrug-resistant murine P388/ADR leukemia cell line was studied by fluorescence microscopy. We found that under conditions which modulated multidrug-resistant (30 µm SR33557 for 1 h), P388/ADR cells presented an original sequestration of DOX in large intracellular vesicles, where SR33557 is itself sequestered, as seen by colocalization studies. Colocalization experiments with lysosomal and mitochondrial probes suggest that these vesicles are neither mitochondrial in nature nor functional lysosomes.

To investigate the biochemical basis for this effect, we studied the impact of SR33557 on the sphingolipid metabolism of P388/ADR cells. We observed that although P388/ADR cells normally catabolized exogenous [³H]sphingomyelin, when pretreated with SR33557 they showed almost complete inhibition of sphingomyelin breakdown. Finally, in order to demonstrate that the inability of P388/ADR cells to degrade sphingomyelin in the presence of SR33557 (which is a potent inhibitor of acid lysosomal sphingomyelinase) leads to phospholipid accumulation, we performed electron microscopy where we observed laminated inclusions. These morphological modifications are similar to those observed in Niemann-Pick disease lymphoblastoid cell lines which are inherently deficient in acid sphingomyelinase activity. The observation that, in the absence of SR33557, these Niemann-Pick disease cell lines presented similar DOX sequestration to that of SR33557-treated P388/ADR cells strongly suggests that DOX accumulates in SR33557-induced myeloid bodies. The redistribution of DOX within these vesicles, perhaps by preventing its expulsion by P-glycoprotein, may be a key in discovering the mechanism of action of SR33557.

¹ This work was supported in part by Grants ARC 6891 and 6249 from l'Association pour la Recherche sur le Cancer, Sanofi, and l'Université Paul Sabatier of Toulouse, France.

² Supported by a grant from La Ligue Nationale Contre le Cancer and in part by the Philippe Foundation, Inc. To whom requests for reprints should be addressed. Present address: Oncology Division, Room M-211, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305-5306.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6/12/92. Accepted 9/18/92.

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