Confocal Scanning Microspectrofluorometry Reveals Specific Anthracyline Accumulation in Cytoplasmic Organelles of Multidrug-resistant Cancer Cells

Abstract

We used confocal microspectrofluorometry to investigate intracellular distribution of pirarubicin or THP-DOX in parental K562, CEM, and LR73 tumor cells and their corresponding multidrug-resistant (MDR) strains. Each spectrum of a recorded image was considered as a combination of cell autofluorescence and fluorescence of the drug. In the cytoplasm of parental K562, CEM, and LR73 cells, THP-DOX fluorescence emission profile was similar to that of free drug in aqueous buffer. The (I550nm/I600nm) ratio was 0. 50 +/- 0.1. However, in the cytoplasm of resistant cells the 550-nm band profile was modified. The I550nm/I600nm ratio was 0.85 +/- 0.2 in MDR K562 cells, which is significantly different from the ratio in sensitive cells (p<0.01). This appeared first to correspond to accumulation and self-oligomerization of THP-DOX in cytoplasmic organelles of MDR cells. Transfection of LR73 cells with the mdr1 gene conferred this characteristic on the resistant LR73R cells. Bodipy-ceramide, a trans-Golgi probe, was co-localized with the typical fluorescence emission peak at 550 nm observed in the cytoplasm of MDR cells. This organelle has been shown to be more acidic in MDR cells. Moreover, this specific pattern was similar to that observed when anthracycline is complexed with sphingomyelin. The typical fluorescence emission peak at 550 nm decreased in MDR cells incubated simultaneously in the presence of the drug and quinine, verapamil, or S9788. Growth inhibitory effect and nuclear accumulation of THP-DOX data obtained on LR73R and LR73D cell lines showed that only during reversion of resistance by verapamil and S9788 was an increase of nuclear THP-DOX accumulation observed. Our data suggest that characteristics of molecular environment, such as higher pH gradient or lipid structures, would be potential mechanisms of multidrug-resistance via the sequestration of anthracyclines.

Therapeutic efficacy of targeting chemotherapy using local hyperthermia and thermosensitive liposome: evaluation of drug distribution in a rat glioma model

“…Applying these advantages, some groups have examined the sub-cellular distribution of anthracycline derivatives, such as ADR (DOX) and MX2, in vitro and/or in vivo using CLSM [28][29][30] . Kabuto et al 31 examined the sub-cellular distribution of MX2 in a rat glioma model and reported that CLSM was useful for the study of the cellular pharmacokinetics of anti-tumour agents.…”

Considerations for Treatment Development in Rhabdomyosarcoma: In Vitro Assessment of Novel DNA Binding Drugs

“…In another study it was shown, using microspectrofluorometry, that the anthracycline antibiotic pirarubicin accumulates in the Golgi body of drug resistant K562, CEM and LR73 cells. It was proposed that this accumulation occurred as a result of the Golgi body being more acidic in MDR cells and hence possessing a stronger pH gradient across its membrane that influenced the passive diffusion of the compound (Belhoussine et al, 1998). The importance of pH gradients in the vesicular sequestration of the anthracycline antibiotics was again reported in 2003 by Ouar et al In this study pH gradients were abolished in resistant cells using concanamycin A, an inhibitor of the vacuolar proton pumps that are responsible for the maintenance of pH.…”

“…BFA, a potent disrupter of Golgi, functions due to its inhibition of Arf1, which is vital for formation of transport vesicles originating at the TGN (Klausner et al, 1992). It has previously been shown that small molecules may be taken up into Golgi apparatus (Belhoussine et al, 1998) and it is well established that nuclear waste is often removed via the ER and sorted in the TGN before being transported to vesicles of the membrane trafficking system for degradation. With this in mind Golgi function was disrupted by BFA in order to establish the influence this organelle plays in AS-DACA's sequestration by the membrane trafficking system.…”

A fluorescence-based assay for monitoring clinical drug resistance

“…Indeed, 30–40% of patients with breast or urothelial cancer treated with either doxorubicin or a combination of methotrexate, vinblastine, doxorubicin and cisplatin (MVAC) develop MDR 17. Furthermore fluorescence properties of anthracycline-like molecules such as doxorubicin have been used to study cellular uptake and distribution in sensitive and resistant living cancer cell lines including lung,18–20 melanoma,21 breast,22 urothelial,23–28 leukemia29 30 and colon 31…”