Synergistic effects of A-B-C-type amphiphilic copolymer on reversal of drug resistance in MCF-7/ADR breast carcinoma

Abstract

P-glycoprotein (P-gp) overexpression has become the most common cause of occurrence of multidrug resistance in clinical settings. We aimed to construct a micellar polymer carrier to sensitize drug-resistant tumors to doxorubicin (DOX). This A-B-C-type amphiphilic copolymer was prepared by the sequential linkage of β-cyclodextrin, hydrophobic poly(d,l-lactide), and hydrophilic poly(ethylene glycol). Upon incubation of the DOX-loaded micelles with DOX-resistant human breast carcinoma MCF-7/ADR cells, significantly enhanced cytotoxicity and apoptosis were achieved. A series of studies on the action mechanism showed that the polymer components such as β-cyclodextrin, hydrophobic poly(d,l-lactide) segment, and poly(ethylene glycol) coordinatively contributed to the improved intracellular ATP depletion and ATPase activity, increased intracellular uptake of P-gp substrates via competitive binding to P-gp, and decreased P-gp expression in MCF-7/ADR cells. More interestingly, a similar phenomenon was observed in the zebrafish xenograft model, resulting in ~64% inhibition of MCF-7/ADR tumor growth. These results implied that the polymeric micelles displayed great potentials as P-gp modulators to reverse DOX resistance in MCF-7/ADR breast carcinoma.

Keywords: P-glycoprotein; doxorubicin; micelle; multidrug resistance; zebrafish.

Figure 1

¹H NMR spectra of different compounds. Notes: (A) β-CD in DMSO-d6. (B) PEG5000-CD in DMSO-d6. (C) PELA54 in CDCl₃. (D) PELA54-CD in DMSO-d6. Abbreviations: NMR, nuclear magnetic resonance; CD, cyclodextrin; DMSO, dimethyl sulfoxide; mPEG5000, poly(ethylene glycol); PELA54, 5:4 monomethoxy poly(ethylene glycol):d,l-lactide; PLA, poly(d,l-lactide); β-CD, beta-cyclodextrin.

Figure 2

Cytotoxicity and apoptosis studies against MCF-7/ADR cells. Notes: (A) Cytotoxicity of different formulations containing DOX against MCF-7/ADR cells for 48 hours. (B) Percentage of DOX-induced apoptosis in MCF-7/ADR cells detected by flow cytometry after 48 hours. *P<0.01 and ^#P<0.05, compared with DOX·HCl. The results are represented as mean ± SD (n=3). Abbreviations: DOX, doxorubicin; SD, standard deviation; PELA54, 5:4 monomethoxy poly(ethylene glycol):d,l-lactide; CD, cyclodextrin; mPEG5000, poly(ethylene glycol); β-CD, beta-cyclodextrin.

Figure 3

The cellular accumulation of Rh123 in MCF-7/ADR cells in the presence of 0.1 mg/mL PELA54-CD, PELA54, PEG5000-CD, and β-CD. Notes: The results are represented as mean ± SD (n=3). Statistical significance (*P<0.01) was compared with control group. Abbreviations: Rh123, Rhodamine 123; PELA54, 5:4 monomethoxy poly(ethylene glycol):d,l-lactide; CD, cyclodextrin; mPEG5000, poly(ethylene glycol); SD, standard deviation; β-CD, beta-cyclodextrin.

Figure 4

Effects of micelles on the intracellular ATP level. Notes: Cells were incubated with different polymers at the concentration of 0.1 or 0.5 mg/mL for 2 hours. The data are presented as percent of the untreated control. The results are represented as mean ± SD from three independent experiments (n=3). Statistical significance (^#P<0.05) was compared with control group. Abbreviations: SD, standard deviation; PELA54, 5:4 monomethoxy poly(ethylene glycol):d,l-lactide; CD, cyclodextrin; mPEG5000, poly(ethylene glycol); β-CD, beta-cyclodextrin.

Figure 5

Stimulation of P-gp ATPase activity by polymers as compared with verapamil and DOX·HCl. Notes: Data were analyzed in terms of ATPase activity by comparison to the ATP standard curve. The results are represented as mean ± SD (n=3). Statistical significance (^#P<0.05 and *P<0.01) was compared with untreated samples (NT) group. Abbreviations: P-gp, p-glycoprotein; DOX, doxorubicin; SD, standard deviation; PELA54, 5:4 monomethoxy poly(ethylene glycol):d,l-lactide; CD, cyclodextrin; mPEG5000, poly(ethylene glycol); β-CD, beta-cyclodextrin.

Figure 6

P-gp expression in the surface of MCF-7/ADR cells after exposing to different polymers (PELA54-CD, PELA54, PEG5000-CD, β-CD) at 50 μg/mL for 24 hours. Notes: The data measured by FCM are presented as percent of the untreated MCF-7/ADR control. The results are represented as mean ± SD (n=3). Statistical significance (*P<0.01) was compared with control group. Abbreviations: P-gp, p-glycoprotein; PELA54, 5:4 monomethoxy poly(ethylene glycol):d,l-lactide; CD, cyclodextrin; mPEG5000, poly(ethylene glycol); FCM, flow cytometry; SD, standard deviation; β-CD, beta-cyclodextrin.

Figure 7

Rhodamine B based P-gp activity assay and P-gp expression in zebrafish. Notes: (A) Rhodamine B accumulation in zebrafish co-treated with a series of concentrations of PELA54-CD. Images under a fluorescence microscope after being co-treated with PELA54-CD and 6 μM Rhodamine B for 20 hours (positive, 1,000 μg/mL CsA). PELA54-CD concentrations (in μg/mL): a, 0.1; b, 1; c, 10; d, 100; e, 250; f, 500; and g, 1,000. Randomly selected ten zebrafish from nine groups were imaged under a stereo fluorescent microscope. Scale bar =100 μm. (B) The inhibition rate of P-gp after co-treated with a series of concentrations of PELA54-CD (positive, 1,000 μg/mL CsA). The results are represented as mean ± SE (n=10). Statistical significance (**P<0.001) was analyzed comparing with control. (C) Twenty-four-hour Western blotting of P-gp. β-Actin was used as internal control, while 20 μM CsA acted as positive control. The concentration of PELA54-CD micelles was 200, 666, and 2,000 μg/mL. (D) Densitometry analysis in AB strain wild-type zebrafish. Western blotting was analyzed by ImageJ. The results are represented as mean ± SD (n=3). Statistical significance (^#P<0.05) was analyzed comparing with control. Abbreviations: PELA54, 5:4 monomethoxy poly(ethylene glycol):d,l-lactide; CD, cyclodextrin; CsA, cyclosporine A; P-gp, p-glycoprotein; SE, standard error; SD, standard deviation.

Figure 8

Inhibitory effect on MCF-7/ADR cells and cardiac toxicity of DOX·HCl or PELA54-CD/DOX micelle in zebrafish xenografts. Notes: (A–C) Inhibitory effect on MCF-7/ADR cells and (D) cardiac toxicity of DOX·HCl or PELA54-CD/DOX micelle in zebrafish xenografts. The results in (B) and (C) are represented as mean ± SE (n=10). Statistical significance (^##P<0.0001) was analyzed comparing with control. (A and D) Scale bar =100 μm. Pericardium of zebrafish shown by red dotted lines. Abbreviations: DOX, doxorubicin; PELA54, 5:4 monomethoxy poly(ethylene glycol):d,l-lactide; CD, cyclodextrin; SE, standard error.