Hans Minderman

Cyclosporin A Is a Broad-Spectrum Multidrug Resistance Modulator

Purpose: Overexpression of the multidrug resistance proteins P-glycoprotein (Pgp), multidrug resistance protein (MRP-1), breast cancer resistance protein (BCRP), and lung resistance protein (LRP) is associated with treatment failure in acute myeloid leukemia (AML) and other malignancies. The Pgp modulator cyclosporin A has shown clinical efficacy in AML, whereas its analogue PSC-833 has not. Cyclosporin A is known to also modulate MRP-1, and we hypothesized that broad-spectrum multidrug resistance modulation might contribute to its clinical efficacy. Experimental Design: We studied the effects of cyclosporin A and PSC-833 on in vitro drug retention and cytotoxicity in resistant cell lines overexpressing Pgp, MRP-1, and BCRP and on nuclear-cytoplasmic drug distribution and cytotoxicity in cells overexpressing LRP. Cellular drug content was assessed by flow cytometry and nuclear-cytoplasmic drug distribution by confocal microscopy. Results: Cyclosporin A enhanced retention of the substrate drug mitoxantrone in cells overexpressing Pgp (HL60/VCR), MRP-1 (HL60/ADR), and BCRP (8226/MR20, HEK-293 482R) and increased cytotoxicity 6-, 4-, 4-, and 3-fold, respectively. Moreover, cyclosporin A enhanced nuclear distribution of doxorubicin in 8226/MR20 cells, which also express LRP, and increased doxorubicin cytotoxicity 12-fold without an effect on cellular doxorubicin content, consistent with expression of wild-type BCRP, which does not efflux doxorubicin. Cyclosporin A also enhanced nuclear doxorubicin distribution in a second cell line with LRP overexpression, HT1080/DR4. PSC-833 enhanced mitoxantrone retention and cytotoxicity in cells overexpressing Pgp, but had no effect in cells overexpressing MRP-1, BCRP, or LRP. Conclusions: Cyclosporin A modulates Pgp, MRP-1, BCRP, and LRP, and this broad-spectrum activity may contribute to its clinical efficacy.

VX-710 (Biricodar) Increases Drug Retention and Enhances Chemosensitivity in Resistant Cells Overexpressing P-Glycoprotein, Multidrug Resistance Protein, and Breast Cancer Resistance Protein

Purpose: The pipecolinate derivative VX-710 (biricodar; Incel) is a clinically applicable modulator of P-glycoprotein (Pgp) and multidrug resistance protein (MRP-1); we studied its activity against the third multidrug resistance (MDR)-associated drug efflux protein, breast cancer resistance protein (BCRP). Experimental Design: VX-710 modulation of uptake, retention, and cytotoxicity of mitoxantrone, daunorubicin, doxorubicin, topotecan, and SN38 was studied in cell lines overexpressing Pgp, MRP-1 and wild-type (BCRPR482) and mutant (BCRPR482T) BCRP. Results: In 8226/Dox6 cells (Pgp), VX-710 increased mitoxantrone and daunorubicin uptake by 55 and 100%, respectively, increased their retention by 100 and 60%, respectively, and increased their cytotoxicity 3.1- and 6.9-fold, respectively. In HL60/Adr cells (MRP-1), VX-710 increased mitoxantrone and daunorubicin uptake by 43 and 130%, increased their retention by 90 and 60%, and increased their cytotoxicity 2.4- and 3.3-fold. In 8226/MR20 cells (BCRPR482), VX-710 increased mitoxantrone uptake and retention by 60 and 40%, respectively, and increased cytotoxicity 2.4-fold. VX-710 increased daunorubicin uptake and retention by only 10% in 8226/MR20 cells, consistent with the fact that daunorubicin is not a substrate for BCRPR482, but, nevertheless, it increased daunorubicin cytotoxicity 3.6-fold, and this increase was not associated with intracellular drug redistribution. VX-710 had little effect on uptake, retention, or cytotoxicity of mitoxantrone, daunorubicin, doxorubicin, topotecan, or SN38 in MCF7 AdVP3000 cells (BCRPR482T). Conclusions: VX-710 modulates Pgp, MRP-1, and BCRPR482, and has potential as a clinical broad-spectrum MDR modulator in malignancies such as the acute leukemias in which these proteins are expressed.

In vitro and in vivo irinotecan-induced changes in expression profiles of cell cycle and apoptosis-associated genes in acute myeloid leukemia cells

Objective: To study irinotecan (CPT-11)–induced changes in expression profiles of genes associated with cell cycle control and apoptosis in myeloid leukemia cells in vitro and in vivo. Methods: HL60 cells were exposed to clinically achievable concentrations of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of CPT-11, and blood sampled from patients with acute myeloid leukemia and chronic myeloid leukemia in myeloid blast transformation treated with CPT-11. Gene expression changes were studied by cDNA microarray and correlated with biological responses by studying DNA distributions by flow cytometry. Results: cDNA microarray analysis showed down-regulation and up-regulation of specific cell cycle–associated genes, consistent with loss of S-phase cells and temporary delay of G1-S-phase transition seen by flow cytometry. Flow cytometry showed that cells in S phase during SN-38 exposure underwent apoptosis, whereas cells in G2-M and G1 were delayed in G1 and entered S phase only 6 to 8 hours after drug removal, consistent with the observed changes in gene expression. Proapoptotic changes in gene transcription included down-regulation of antiapoptotic genes and up-regulation of proapoptotic genes. Many gene expression changes observed following in vitro SN-38 exposure were also seen following in vivo administration of 10 or 15 mg/m2 CPT-11; notably, proapoptotic changes included reduced transcription of survivin pathway-associated genes and increased transcription of death receptor 5. Conclusion: CPT-11-induced changes in gene expression profiles in vitro and in vivo are consistent with temporary delay in G1-S transition and enhanced responsiveness to apoptosis, both of which may contribute to the synergistic interactions of this drug with antimetabolites.