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, 2014, 819548

The Connection Between the Toxicity of Anthracyclines and Their Ability to Modulate the P-glycoprotein-mediated Transport in A549, HepG2, and MCF-7 Cells


The Connection Between the Toxicity of Anthracyclines and Their Ability to Modulate the P-glycoprotein-mediated Transport in A549, HepG2, and MCF-7 Cells

Aneta Rogalska et al. ScientificWorldJournal.


Multidrug resistance (MDR) is a major obstacle to the successful chemotherapy of solid tumors. We compared the resistance of the most popular solid tumors, breast adenocarcinoma (MCF-7 cell line) and nonsmall cell lung (A549 cell line) hepatocellular liver carcinoma (HepG2 cells), to aclarubicin (ACL) and doxorubicin (DOX). This research aimed at determining the relation between the toxicity of ACL and DOX, their cell accumulation, and then effect on P-glycoprotein functionality. ACL is more cytotoxic for tumor cells compared to DOX. The intracellular concentration of drugs in cancer cells was dependent on the dose of the drugs and the time of incubation. The P-gp inhibitor Verapamil (V) increased DOX accumulation in all tested cell lines. By contrast, the intracellular level of ACL was not affected by this modifying agent. The assessment of the uptake of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1) or Rhodamine 123 (R123) allows the evaluation of the different influence of drugs on P-gp activity which is in agreement with the estimation of expression measured by MDR-1 shift assay. These data suggest that ACL is less P-gp dependent than DOX and consequently may be used in a clinical setting to increase treatment efficacy in resistant human tumors.


Figure 1
Figure 1
Representative dose-response curves following exposure to DOX and ACL of A549, HepG2, and MCF-7 cells. The evaluation of cell survival was assessed by MTT assay. *P < 0.05, the effect of DOX and ACL on the viability of solid tumor cells. + P < 0.05, changes between the probes preincubated with Verapamil. The values are the mean ± SD of four independent experiments.
Figure 2
Figure 2
The effect of Verapamil on ACL and DOX accumulation in tested cells. The accumulation of ACL and DOX in A549, HepG2, and MCF-7 cell lines was examined after 2 h incubation with IC50 of the drugs and 24 h after incubation without drugs. The excitation and emission wavelengths were for ACL λ ex = 436 nm and λ ex = 585 nm and DOX λ ex = 488 nm and λ em = 566, respectively. *Values are statistically significant in comparison to the control, untreated cells, P < 0.05. +Values are statistically significant in comparison to the cells incubated without Verapamil, P < 0.05.
Figure 3
Figure 3
Intracellular DOX and ACL accumulation and their distribution in (a) A549, (b) HepG2, and (c) MCF-7 cell lines. The cells were incubated with IC50 of each anthracycline for 2 h, or, after 2 h incubation with the drugs, the medium was removed and the cells were cultured in fresh medium for another 24 h. In the experiments with Verapamil the cells were preincubated for 30 min at 37°C and then incubated with drugs. Fluorescence was monitored using an Olympus IX70, Japan, magnification 400x.
Figure 4
Figure 4
Uptake of R123 (right panel) or JC-1 (left panel) in the presence of Verapamil, DOX, and ACL by A549, HepG2, and MCF-7 cells in function of time. The values are the mean ± SD of four independent experiments.
Figure 5
Figure 5
Analysis of the P-gp expression in solid tumor cell lines using vinblastine as positive control (a) and comparison of the effect of ACL and DOX on P-gp expression (b) in drug treated cells. *P < 0.05, significant differences between drugs treated and control cells. **P < 0.05, significant differences between cells treated with ACL and DOX.

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