Efflux Kinetics and Intracellular Distribution of Daunorubicin Are Not Affected by Major Vault Protein/Lung Resistance-Related Protein (Vault) Expression

Abstract

Vaults may contribute to multidrug resistance by transporting drugs away from their subcellular targets. To study the involvement of vaults in the extrusion of anthracyclines from the nucleus, we investigated the handling of daunorubicin by drug-sensitive and drug-resistant non-small lung cancer cells, including a green fluorescent protein (GFP)-tagged major vault protein (MVP)-overexpressing transfectant (SW1573/MVP-GFP). Cells were exposed to 1 M daunorubicin for 60 min, after which the cells were allowed to efflux the accumulated drug. No significant differences in daunorubicin efflux kinetics were observed between the sensitive SW1573 and SW1573/MVP-GFP transfectant, whereas the drugresistant SW1573/2R120 cells clearly demonstrated an increased efflux rate. It was noted that the redistribution of daunorubicin from the nucleus into distinct vesicular structures in the cytoplasm was not accompanied by changes in the intracellular localization of vaults. Similar experiments were performed using mouse embryonic fibroblasts derived from wildtype and MVP knockout mice, which were previously shown to be devoid of vault particles. Both cell lines showed comparable drug efflux rates, and the intracellular distribution of daunorubicin in time was identical. Reintroduction of a human MVP tagged with GFP in the MVP Ϫ/Ϫ cells results in the formation of vault particles but did not give rise an altered daunorubicin handling compared with MVP Ϫ/Ϫ cells expressing GFP. Our results indicate that vaults are not directly involved in the sequestration of anthracyclines in vesicles nor in their efflux from the nucleus.

Reversal of multidrug resistance by cisplatin-loaded magnetic Fe3O4 nanoparticles in A549/DDP lung cancer cells in vitro and in vivo

“…34,35 Accumulating evidence indicates that NSCLC with high expression of LRP is resistant to cisplatin. 36,37 In our present study, LRP expression in the Fe 3 O 4 -MNP-DDP group was less than that in the DDP group, whereas Fe 3 O 4 -MNP alone did not decrease LRP expression in DDP-resistant A549 cells. These results suggest that the regulatory mechanism of both LRP and MRP1 expression is associated with the multidrug resistance phenotype.…”

Depletion of major vault protein increases doxorubicin sensitivity and nuclear accumulation and disrupts its sequestration in lysosomes

“…Control transfections were carried out using siRNA-targeting luciferase. MVP-GFP that has previously been rigorously evaluated (29) was mutated in four silent positions to destroy the siRNA target site using the QuikChange Site-Directed Mutagenesis kit from Stratagene. The following primers were used: MVP3MUTF, 5 ¶-CTCAGCCCGCATCATACGGACAGCAGTCTTTGGC-TTTGAGACC-3 ¶ and MVP3MUTR, 5 ¶-GGTCTCAAAGC-CAAAGACTGCTGTCCGTATGATGCGGGCTGAG-3 ¶.…”

Major Vault Protein, in Concert with Constitutively Photomorphogenic 1, Negatively Regulates c-Jun–Mediated Activator Protein 1 Transcription in Mammalian Cells

“…Purified COP1 and MVP polyclonal antibodies were previously described (9,20). Anti-Flag (Sigma, St. Louis, MO), anti-MVP (LRP, BD Transduction Laboratories, San Diego, CA), anti-MVP (LRP56, Kamiya, Seattle, WA), anti-MVP (LRP56, Calbiochem, La Jolla, CA), anti-P-Tyr (Sigma), anti-c-Jun (SC-45, Santa Cruz, Biotech, Inc., Santa Cruz, CA), anti-P-c-Jun (S63, Cell Signaling, Beverly, MA), anti-tubulin (Sigma), and anti-Hsp70 (BD Transduction Laboratories) antibodies were used according to the recommendations of the manufacturers.…”