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Biomaterials
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Nanoparticle-directed sub-cellular localization of doxorubicin and the sensitization breast cancer cells by circumventing GST-Mediated drug resistance^
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Xianghui Zeng a, Ralf Morgenstern b, Andreas M. Nyström
a Karolinska Institutet, Institute of Environmental Medicine, Division of Molecular Toxicology, Nobels väg 13, SE-17177 Stockholm, Sweden b Karolinska Institutet, Institute of Environmental Medicine, Division of Biochemical Toxicology, Nobels väg 13, SE-17177 Stockholm, Sweden
ARTICLE INFO
ABSTRACT
Article history:
Received 25 September 2013 Accepted 12 October 2013 Available online 7 November 2013
Keywords:
Polymeric nanoparticle Drug resistance Glutathione transferase Endocytosis Doxorubicin Drug delivery
Resistance to single or multiple chemotherapeutic drugs is a major complication in clinical oncology and is one of the most common treatment limitations in patients with reoccurring cancers. Nanoparticle (NP)-based drug delivery systems (DDS's) have been shown to overcome drug resistance in cancer cells mainly by avoiding the activation of efflux pumps in these cells. We demonstrate in this work that polyester-based hyperbranched dendritic-linear (HBDL)-based NPs carrying doxorubicin (Dox) can effectively overcome microsomal glutathione transferase 1 (MGST1)-mediated drug resistance in breast cancer cells. Our DDS was much more effective at considerably lower intracellular Dox concentrations (IC50 6.3 |M vs. 36.3 |m) and achieved significantly greater reductions in viability and induced higher degrees of apoptosis (31% vs. 14%) compared to the free drug in the resistant cells. Dox-loaded HBDL NPs were found to translocate across the membranes of resistant cells via active endocytic pathways and to be transported to lysosomes, mitochondria, and the endoplasmic reticulum. A significantly lower amount of Dox accumulated in these cytoplasmic compartments in resistant cells treated with free Dox. Moreover, we found that Dox-HBDL significantly decreased the expression of MGST1 and enhanced mitochondria-mediated apoptotic cell death compared to free Dox. Dox-HBDL also markedly activated the JNK pathway that contributes to the apoptosis of drug-resistant cells. These results suggest that HBDL NPs can modulate subcellular drug distribution by specific endocytic and trafficking pathways and that this results in drug delivery that alters enzyme levels and cellular signaling pathways and, most importantly, increases the induction of apoptosis. Our findings suggest that by exploiting the cell transport machinery we can optimize the polymeric vehicles for controlled drug release to overcome drug resistance combat drug resistance with much higher efficacy.
© 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
1. Introduction
Chemotherapy is the most common anticancer therapy, after surgical resection, currently employed in the clinical management of cancer. Modern chemotherapeutic therapy has been a success story with increased survivability and positive therapeutic results for many of the most common cancers, including breast cancer [1].
Abbreviations: MGST1, microsomal glutathione transferase 1; Dox, doxorubicin; Dox-HBDL, hyperbranched dendritic-linear micelles encapsulating doxorubicin; JNK, c-Jun N-terminal kinase.
q This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike License, which permits noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. * Corresponding author. Tel.: +46 8 524 86942. E-mail address: andreas.nystrom@ki.se (A.M. Nystrom).
However, antitumor efficacy can be severely limited by the development of multi-drug resistance in cancer cells. Some cancer cells have intrinsic resistance before drug treatment while others develop resistance during the course of chemotherapy [2,3]. This acquired resistance frequently involves cross-resistance to other drugs and is known as multi-drug resistance [4]. Drug resistance is correlated with a variety of biochemical changes, including increased efflux of the cytotoxic drugs due to overexpression of the ATP-binding cassette transporters, DNA repair activation, and altered levels of apoptosis [5,6]. Enzymatic detoxification of cyto-static drugs also constitutes a major mechanism of tumor drug resistance [6], and it has been clearly shown that overexpression of the glutathione transferases (GSTs) in tumors are linked to the development of multi-drug resistance [7—9].
One important strategy for overcoming drug resistance is to restore drug sensitivity through the use of nanoscale DDS's [10,11]
0142-9612/$ — see front matter © 2013 The Authors. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.10.042
and liposomal [12-15] and/or polymer-based nanomedicines in particular [16-18]. Kabanov et al. have shown that Pluronic® block copolymer micellar types of carriers can avoid permeability glycoprotein (Pgp) activation in response to doxorubicin (Dox) treatment both in vitro and in vivo [19-21]. Other types of nanomedicines include poly(lactic-co-glycolic acid) (PLGA) NPs [22], poly(N-(2-hydroxypropyl)methacrylamide) (poly(HPMA))-Dox conjugates [23], and iron oxide based NPs [24] among several others. Another type of polymer-based NP that has been explored for DDS applications is dendrimers [25-28]. Dendrimers are highly branched single molecule entities that are being used either as single entities or as linear-dendritic hybrid materials [25-28]. Polyester-based dendritic materials [29], and especially aliphatic polyesters based on poly 2,2-bis (methylol) propionic acid (bis-MPA) building blocks exhibit promising characteristics for utilization as a polymer-based nanoscale DDS [30-35]. Bis-MPA materials have been shown to be non-toxic, non-immunogenic, and biodegradable in several publications as well as highly efficient in vivo DDS systems.
Inspired by the work described by Frechet et al. who used bis-MPA-based bow-tie dendron structures that exhibited unsurpassed results in vivo [25,32-34], our group previously developed hyperbranched dendritic-linear (HBDL)-based NPs made from poly(ethylene glycol) (PEG)-grafted from Boltorn® [36,37]. Hyperbranched polymers offer the high functionality seen with dendrimers. They are less well defined, but they are significantly easier to synthesize on a large scale and can be used to construct complex nanoparticle assemblies [30,31]. In our previous work, the anti-neoplastic drug Dox was successfully encapsulated within the HBDL NP assemblies, and the Dox-loaded NPs improved the antiproliferative efficacy of the drug over the non-formulated version of Dox in vitro [36,37]. In addition, our previous work demonstrated that breast cancer cells could efficiently take up the NPs via both macropinocytosis and clathrin-dependent endocy-tosis [36].
Based on the results of our previous studies, we hypothesized that the nanostructures might modulate the delivery of Dox to reverse drug resistance in breast cancer cells. Understanding the mechanisms of how the NPs interact with drug-resistant cancer cells would help lead to the improved design of NP-based DDS's as efficient and safe carriers for overcoming cancer drug resistance. In this study, we used the genetically engineered MCF-7/ADR cell line as a drug-resistant cell model that overexpresses microsomal gluta-thione transferase 1 (MGST1). MGST1 is up regulated in many different malignant tissues compared to normal tissues and has been linked to multidrug resistance, including resistance to Dox [38]. In this work, we have expanded the use of our DDS to the treatment of drug-resistant breast cancer cells and investigated the anti-proliferative effects, uptake profiles, endocytosis mechanisms, and subcellular drug delivery of this formulation as well as the altered cellular functions in drug-resistant cells. We show a dramatic reversal of the resistance to Dox, a significant reduction in cell viability, and an increase in apoptosis in the drug-resistant cells with our polyester-based nanomedicine system. Overcoming GST-mediated resistance represents a viable target for improving the chemotherapeutic treatment of drug-resistant cancers in the future.
2. Materials and methods
2.1. Preparation of HBDL, Dox-HBDL, and HBDL-FL nanoparticles
The Boltorn® H30-based hyperbranched dendritic-linear (HBDL) copolymers and fluorescein cadaverine-labeled (HBDL-FL) conjugates were synthesized and characterized as previously reported by our group [36,37]. In brief, the hyperbranched dendritic-linear (HBDL) polymers form micellar assemblies in the aqueous solutions due to its amphiphilic nature. The hydrophilic PEG segments served as a shell stabilizing the micelles, whereas H30 Boltorn constitutes the hydrophobic core. The micelles have on average 5 PEG chains (10 kDa) per hyperbranched core
molecule. BoltornH30-(PEG10k): MnNMR ~ 53,300 Da, MwGPC = 14,000 Da (polystyrene equivalent), Mn GPC = 11,400 Da (polystyrene equivalent), Mw/ Mn = 1.2.
The preparation of drug loaded Dox-HBDL nanoparticles was performed by employing similar methods as described earlier [37], and the formulation was measured by dynamic light scattering (DLS, Nano-ZS, Malvern). Diameter = 316 nm ± 158 nm (Z-average). Zeta potential = -1.8 mV ± 0.4 mV. The concentration of Dox and entrapment efficiency was assessed to be 0.097 mM and 30.6%, respectively, by UV spectrophotometry.
2.2. Cells and chemicals
The human breast carcinoma cell line MCF-7 with low endogenous expression of MGST1 was purchased from the American Type Culture Collection (ATCC; Manassas, VA) and maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin under 5% CO2 at 37 °C. MCF-7/ADR cells with overexpression of MGST1 were transfected with a vector containing rat MGST1 as described previously [39], and the cells were cultured in the DMEM medium supplemented with 1 mg/mL geneticin. MCF-7 cells are used as controls as no significant difference between these and antisense transfected MCF-7 was observed.
DAPI, Cholera toxin B subunit (CTB) Alexa 647, transferrin Alexa 647, lysosensor, ERtracker, Mitotracker, CellROX reagent, MitoSOX reagent, Thiol reagent, Bodipy probe, MitoDilC kit, JNK1&2(pTpY183'185), and secondary antibodies were obtained from Invitrogen (Eugene, OR). Specific antibodies against the early endosome marker (EEA1) and the Golgi marker (TGN46) were purchased from Abcam Inc. The polyclonal rabbit IgG against rat MGST1 was generated by the laboratory of Prof Ralf Morgenstern. Dextran-rhodamine was prepared by Jakob Regberg as previously described [36]. Water, dimethyl sulfoxide (DMSO), methanol, (3-(4,5-dimethylthiazol-2-yl))-2,5-diphenyl tetrazolium bromide (MTT), and other HPLC-grade solvents were from Sigma-Aldrich Company, Ltd. All pharmacological inhibitors and other reagents of general laboratory grade were purchased from Sigma (St. Louis, MO) unless otherwise stated. Stock solutions were prepared and stored at -80 °C in small aliquots as per manufacturer's recommendation.
2.3. Cytotoxicity studies
The level of MGST1 expression in MCF-7 and MCF-7/ADR cells was confirmed by immunostaining and confocal fluorescence microscopy. MCF-7 and MCF-7/ADR cells were seeded at 5 x 104 cells per well in a 96-well plate, pre-incubated for 24 h, then incubated with free Dox or Dox-HBDL for 48 h at Dox concentrations ranging from 0.001 mM to 10 mM. Untreated cells in medium were used as controls. The MTT assay was used to measure cytotoxicity [37]. All experiments were carried out with three replicates. The cytotoxicity of empty HBDL NPs was tested with the method above, but the concentration of empty micelles ranged from 50 mg/mL to 500 mg/mL.
2.4. Cellular uptake of Dox-HBDL
MCF-7 and MCF-7/ADR cells were seeded onto 6-well culture plates (1 x 105 cells per well) and incubated for 24 h. The cells were treated with 1 mM Dox or Dox-HBDL (1.0 mM Dox-equivalent) for 1, 3, 6,12, or 24 h. The cells were then washed three times with PBS, trypsinized, and transferred to tubes and centrifuged. The pellets were re-suspended in 0.5 mL of PBS for immediate analysis by flow cytom-etry. Flow cytometry was performed on a FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA). Data from 1 x 104 cells were collected and analyzed using the CellQuest software (Becton Dickinson). The cellular uptake of Dox and Dox-HBDL was further confirmed by confocal laser scanning microscopy (CLSM, Olympus FV1000). Cells grown on coverslips in a 6-well tissue culture plate were cultured with Dox and Dox-HBDL NPs at a concentration of 1 mM. After incubation for 24 h, the cells were washed with PBS and fixed with 5% paraformaldehyde in PBS. Canadian Balsam was dropped on the slides to seal the cell samples after the cells were washed with PBS. The stained coverslips were imaged using CLSM.
2.5. Uptake mechanism studies
To study the endocytic pathways of the nanoparticles, MCF-7 and MCF-7/ADR cells were seeded as described above in 12-well plates. Cells were treated with 1 mM Dox or Dox-HBDL (1.0 mM Dox-equivalent) in medium and incubated for 3 h. Co-localization assays in living cells were performed to identify the endocytic vesicles involved in the nanoparticle internalization. Following the nanoparticle treatment, the cells were incubated with different dyes depending on the purpose of the staining: dextran-rhodamine conjugate to label macropinosomes, transferrin Alexa 647 conjugate to label clathrin, and CTB Alexa 647 conjugate to label caveolin. Cells were imaged with CLSM. ln the endocytic inhibition assays, cells were washed with warm (37 °C) PBS, and fresh medium was added containing the inhibitors of endocytic pathways 0.5 h prior to the addition of the Dox-HBDL suspensions. Next, the cells were treated with Dox-HBDL (1.0 mM Dox-equivalent) either in the presence or absence of the endocytosis inhibitors. Various well-characterized inhibiting drugs (filipin, nystatin, phenylarsine oxide, chlorpromazine, amiloride-HCl, and cytocha-lasin D) were selected for their ability to inhibit specific steps in the endocytic pathway, and the effects of low temperature (4 °C) on nanoparticle uptake were also
Fig. 1. Expression of MGST1 in cells and the cytotoxicity of doxorubicin (Dox). (A) Detection of the MGST1 expression in MCF-7/ADR cells or MCF-7 cells by confocal microscopy. Scale bar = 20 mm. Cytotoxicity of free Dox and Dox-HBDL NPs to (B) MCF-7 cells and (C) MCF-7/ADR cells as assessed by an MTT assay.
studied as described previously [36]. After a 0.5-h pre-incubation with the drugs at 37 °C, 1 тм of Dox and the Dox-HBDL nanoparticle (1.0 тм Dox-equivalent) solution was added to the cells for 3 h after which the cells were prepared for FACS analysis as described above.
2.6. Intracellular localization study
To study the subcellular localization of Dox, MCF-7 and MCF-7/ADR cells were seeded and treated as described above in 12-well plates. Immunostaining was used
to investigate intracellular drug localization. After 6 h incubation of Dox and Dox-HBDL NPs at a 5.0 mM Dox equivalent concentration, cells were fixed with 4% paraformaldehyde and labeled with specific antibodies for early endosomes (EEA1) or the Golgi network (TGN). Drug co-localization assays with Lysosensor, Mitotracker, and ERtracker were performed to label lysosomes, mitochondria, and endoplasmic reticulum, respectively. Overlap between drug and the FL-labeled HBDL carrier localization was also studied by incubation of Dox-encapsulated micelles in MCF-7/ ADR cells. Cells were imaged using CLSM, and the Olympus FV10-ASW and ImageJ
MCF-7/ADR
MCF-7/ADR
H9— MCF-7 A MCF-7
5 10 15 20 25 30 35 Distance (^m)
10' 102 10 Fluorescence intensity
Fig. 2. Cellular uptake of (A) free Dox and (B) Dox-HBDL NPs by MCF-7 and MCF-7/ADR cells. The concentration of Dox (free Dox and equivalent Dox in micelle form) in the cell culture was 1 |m. Flow cytometry was used to quantify the uptake of free Dox or Dox-HBDL NPs by the cells. Three independent experiments were performed. (C) Representative dot plots showing fluorescence channel analysis and (D) quantitative comparison of Dox accumulation in MCF-7 cells and MCF-7/ADR cells after incubation with free Dox or Dox-HBDL NPs for 24 h. (E) Confocal laser scanning microscopy images of cells treated with free Dox or Dox-HBDL NPs at 1 |m for 24 h. Nuclei are stained with DAPI (blue), and Dox is indicated by red fluorescence. Scale bar = 20 mm. The line-scanning profiles of fluorescence intensity of the cells are indicated by arrows. The blue line represents the intensity from DAPI and the red line represents the intensity from Dox. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
vl.47 software packages were used to analyze the co-localization and fluorescent topographic profiles.
2.7. Assessment ofMGST1 expression, lipid peroxidation, and mitochondrial membrane potential
MCF-7 cells and MCF-7/ADR cells were plated in 6-well plates at a density of 1 x 105 cells per well and incubated at 37 °C in a 5% CO2 humidified atmosphere for 24 h. Cells were incubated with either free Dox or Dox-HBDL at a concentration of 5 mM for 24 h at 37 °C. After 24 h incubation, cells were collected, centrifuged at 5000 rpm for 5 min to pellet the cells, and washed with PBS. After incubation with the MGST1 antibody and washing, the cells were further incubated with the Alexa 647-labeled secondary antibody at room temperature. The cells were then washed and the MGST1 level was determined using the flow cytometer. To determine the intracellular lipid peroxidation and mitochondrial membrane potential, cells were treated with free Dox and Dox-HBDL using the same protocol described above. According to the manufacturer's protocol, cells were collected then resuspended in PBS with 50 nM MitoDilC (for assessment of mitochondrial membrane potential, Djm) or 5 mM C11-Bodipy581/591 (a marker of lipid peroxidation) and incubated at room temperature for 30 min. After washing with PBS, the cell samples were subjected to flow cytometer analysis.
2.8. Measurement of GSH, mitochondrial superoxide, and cellular ROS
lntracellular GSH, ROS, and mitochondrial superoxide levels were measured by assays as previously described [40]. Cells were exposed to free Dox (5 mM) and Dox-HBDL nanoparticles (5.0 mM Dox-equivalent) for 24 h. The following fluorescent probes were incubated with cells at 37 °C in the dark prior to harvest: (i) 20 mM Thioltracker dye (a marker of glutathione, GSH) for 30 min, (ii) 5 mM MitoSOX for 10 min (for assessment of mitochondria-generated superoxide), or (iii) 5 mM CellROX reagent for 30 min (for assessment of cellular ROS production). Cells were washed and the protein concentration was measured with the BCA protein assay (Sigma). Fluorescence of cell samples was measured using a spectrofluorimeter (BioTek Synergy MX, VT) according to the manufacturer's protocol, and fluorescence intensity was compared with equal amounts of protein.
2.9. Cell apoptosis and JNK activation analysis
MCF-7 cells and MCF-7/ADR cells were seeded onto 6-well culture plates at a concentration of 1 x 105 cells/well. The apoptosis of cells exposed to Dox and Dox-HBDL at a Dox concentration of 10 mM for 48 h was determined. After treatment, cells were trypsinized, centrifuged, washed with PBS, and stained with FlTC-labeled Annexin V and propidium iodide following the manufacturer's instructions. The numbers of cells undergoing necrosis (positive for propidium iodide), early apoptosis (positive for Annexin V), and late apoptosis (double-positive for Annexin V and propidium iodide) were quantified using flow cytometry. To study the induction of JNK phosphorylation, MCF-7 and MCF-7/ADR cells were seeded at semiconfluent concentrations on 12-mm coverslips in the wells of a 12-well plate. Cells were incubated with free Dox (5 mM) or Dox-HBDL nanoparticles (5.0 mM Dox-equivalent) for 24 h at 37 °C in 5% CO2. After incubation with primary antibody, Alexa 647-conjugated-secondary antibody was added. lmmunofluorescence was detected and analyzed using CLSM. All experiments were repeated three times and representative results are presented.
2.10. Statistical analysis
The data is presented as mean values with standard deviations (SD). Statistical analysis was performed with one-way ANOVA followed by Tukey's multiple comparison tests, and p < 0.05 was considered statistically significant. The data were processed and plotted in KaleidaGraph v4.1 (Synergy Software, Reading, PA).
3. Results and discussion
3.1. Uptake studies and cytotoxicity
Dox-encapsulated HBDL (Dox-HBDL) NPs were prepared by the mixed phase method as previously reported [37]. A multidrug resistant cancer cell line, MCF-7/ADR, which was engineered to overexpress MGST1, was selected to evaluate the ability of Dox-HBDL NPs to overcome drug resistance [39]. Immunofluorescent staining in Fig. 1A shows that MGST1 is expressed at much higher levels in the MCF-7/ADR cells compared to normal MCF-7 cells. The half maximal inhibitory concentration (lC50) of Dox was determined by MTT assays after incubation of free Dox or Dox-HBDL NPs at equivalent Dox concentrations with both MCF-7 and MCF-7/ADR cells for 72 h. Free Dox showed notable cytotoxicity to MCF-7 cells at low concentrations (lC50 7.5 mM ± 1.8 mM) but was much less
cytotoxic to MCF-7/ADR cells (lC50 36.2 mM ± 3.2 mM). This confirmed that MGST1 overexpression confers robust resistance to free Dox. Dox-HBDL NPs exhibited enhanced cytotoxicity compared to free Dox treatment under identical conditions with an lC50 of 4.8 mM ± 1.0 mM in MCF-7 cells and an lC50 of 6.3 mM ± 1.4 mM in MCF-7/ADR cells (Fig. 1B and C). As a control, empty HBDL NPs without Dox were not cytotoxic to either MCF-7 or MCF-7/ADR cells after 72 h incubation (Supplemental Information, Fig. S2) indicating that the cytotoxicity of Dox-HBDL is an effect of the nanoformulation of the chemotherapeutic agent within the HBDL carriers. These observations show that Dox-HBDL NPs effectively overcome MGST1-dependent drug resistance.
To investigate whether the effect of Dox-HBDL NPs was dependent on changes in intracellular drug accumulation, cellular uptake of free Dox or Dox-HBDL NPs in MCF-7 cells and MCF-7/ ADR cells was quantified at different time intervals from 1 h to 24 h. Free Dox or Dox-HBDL NPs in the culture medium was incubated with either MCF-7 or MCF-7/ADR cells at an equivalent dose of 1 mM Dox. The kinetics of drug accumulation was quantitatively determined by flow cytometry, and Fig. 2A illustrates the intracellular accumulation of free Dox in MCF-7 or MCF-7/ADR cells, respectively. The fluorescence intensity of free Dox in MCF-7/ADR cells (163 ± 5) was significantly lower than that seen in MCF-7 cells (212 ± 7) after incubation with free Dox for 24 h. The decrease in intracellular drug concentrations could be a result of the increased detoxification capacity of the cells due to the MGST1 overexpression that contributes to drug resistance [39]. However, when incubated with Dox-HBDL NPs the fluorescence intensity in MCF-7/ADR cells increased more rapidly than in MCF-7 cells (Fig. 2B). Much higher fluorescence intensities were observed in MCF-7/ADR cells (82 ± 4) compared to MCF-7 cells (61 ± 2) after incubation for the same period. These results indicate that Dox-HBDL NPs might not be detoxified as quickly as free Dox by MGST1, and the drug could be retained for a longer time in the resistant cells after internalization. The enhanced cellular uptake of Dox-HBDL NPs also contributes to the overcoming of the drug resistance.
The mean fluorescence intensities of MCF-7 cells and MCF-7/ ADR cells treated with free Dox were much higher than after incubation with an equivalent dose of Dox-HBDL NPs for the same period of time under the same conditions, and the trend increased with longer culturing time. As shown in Fig. 2C, the cellular fluorescence intensities of MCF-7 and MCF-7/ADR cells treated with free Dox were about 4 and 2 times higher, respectively, than the intensities after Dox-HBDL NP treatment. lt should be mentioned that the fluorescence intensity of Dox-HBDL NPs was not significantly different from that of free Dox at the same molar concentration. The cellular uptake of Dox (free Dox or its equivalent in nanoparticle form) was also investigated by confocal laser scanning microscopy. Compared with the cells treated with free Dox, the red fluorescence intensity was much weaker in cells treated with Dox-HBDL NPs (Fig. 2D). Furthermore, the red fluorescence intensity in the nuclei of the cells treated with Dox-HBDL NPs was also lower compared to the nuclei of cells treated with free Dox (Fig. 2E). These results indicate that cells apparently internalize the Dox-HBDL NPs more slowly than free Dox, and this might be due to the difference between endocytosis of the nanocarrier and the diffusion of a small-molecule drug [37,41].
The above results demonstrated that delivery of Dox using NPs could efficiently increase the cellular drug concentration in MCF-7/ADR cells compared with that in MCF-7 cells and could sensitize MCF-7/ADR cells to the drug and improve its cytotoxicity. However, it is interesting to note that the drug uptake of Dox-HBDL NPs was significantly lower than free drug in both sensitive cells and resistant cells under the same conditions, which means that the profiles of drug uptake are not fully
Fig. 3. (A) Effect of endocytic inhibitors or low temperature on accumulation of Dox-HBDL NPs in MCF-7/ADR cells. Cells were untreated (control) or pretreated with inhibitors for 30 min at 37 °C prior to addition of Dox-HBDL NPs (1 mM). The fluorescence signal was measured following incubation for 3 h at 37 °C in all cases except the one labeled 4 °C. Data represent the mean ± SD (n = 3). *p < 0.05 and **p < 0.01 compared to control. (B) The role of macropinosomes, clathrin-coated pits, and caveolae in endocytosis of Dox-HBDL NPs in MCF-7/ADR cells. Dox-HBDL micelle (red) co-localization with endocytic markers (green). High co-localization (yellow vesicles) was observed with dextran (macropinocytosis) and transferrin (clathrin pathway), but no correlation with Cholera toxin B subunit (CTB) signal (caveolin pathway) was observed. Scale bar = 20 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Intracellular accumulation of Dox in MCF-7/ADR cells after treatment with Dox-HBDL micelles (A) and free Dox (B), respectively. Co-localization of Dox-HBDL micelles (red) with lysosensor (lysosome marker, green), EEA1 (early endosome marker, green), ERtracker (endoplasmic reticulum marker, green), Mitotracker (mitochondria marker, green), and TGN46 (Golgi apparatus marker, green) 3 h after the nanoparticle treatment shows that the particles are mostly delivered to the lysosomes, mitochondria, and endoplasmic re-ticulum. Scale bar = 20 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
equivalent to the drug effects. This suggests that there are additional factors that contribute to the enhanced cytotoxicity of the NPs, for example, endocytosis, distribution, MGST1 inhibition, enhanced apoptosis, or altered molecular mechanisms of action.
3.2. Mechanisms of endocytosis and confocal studies
Resistant and non-resistant cells were treated with Dox-HBDL NPs to investigate the role of endocytosis in Dox uptake,
Table 1
Pearson's coefficients for Dox co-localization with markers of cytoplasmic compartments.
EEA1 Lysosensor Mitotracker ERtracker TGN
Free Dox Dox-HBDL -0.11 ± 0.21 0.13 ± 0.10 0.63 ± 0.14 0.76 ± 0.12 0.19 ± 0.16 0.71 ± 0.15 0.05 ± 0.19 0.63 ± 0.14 0.14 ± 0.13 0.19 ± 0.21
intracellular localization, and accumulation. Endocytosis is one of the most important entry mechanisms for various extracellular materials and is a primary mechanism for the uptake of NPs [4144]. Endocytosis is an energy-dependent mechanism and can be inhibited at low temperatures (e.g., 4 °C) [36,45]. As shown in Fig. 3A, incubation of MCF-7/ADR cells with Dox-HBDL NPs at 4 °C for 3 h resulted in a 32.5% decrease in Dox accumulation compared to cells incubated at 37 °C. Inhibition of macropinocytosis and the clathrin and caveolae pathways by exposing cells to their respective inhibitors cytochalasin D, amiloride, and phenylarsine oxide for 3 h resulted in significant inhibition of Dox-HBDL NP transmigration (Fig. 3A). Dox-HBDL NP (red) co-localization assays with endocytic
markers (green) were studied by confocal imaging in vitro, and a significant amount of co-localization (yellow vesicles) was observed with transferrin (a clathrin pathway marker), and dextran (a macropinocytosis marker) but no correlation with the caveolin signal was observed (Fig. 3B). We can conclude from these results that the processes involved in translocation of Dox-HBDL NPs across MCF-7/ADR cell membranes are energy-dependent and include macropinocytosis and clathrin-mediated endocytosis. This is in contrast to the diffusion-based translocation seen for free Dox across MCF-7 cell membranes.
To confirm that the red fluorescence in Fig. 3B represents not just that of released Dox from the HBDL NPs, a sample of the cells was dosed with Dox-loaded and fluorescently labeled (Dox-HBDL-FL) nanocarriers that were synthesized as previously reported [36,37]. Confocal microscopy showed the co-localization of the FL and Dox channels (green and red) depicting the cellular internali-zation of Dox-HBDL-FL NPs as a nanocarrier (Supplemental Information, Fig. S4). These observations agree with our previous study that showed that endocytic internalization was the main
Fig. 5. MGST1 expression and intracellular glutathione (GSH) levels in MCF-7 cells and MCF-7/ADR cells treated with Dox and Dox-HBDL nanoparticles at a Dox concentration of 5 mM for 24 h. (A) Representative flow cytometric analyses of MGST1 expression. (B) Quantification of the flow cytometry results. (C) A thiol assay was used to determine GSH levels. *p < 0.01, **p < 0.01, and ***p < 0.001 compared to control.
Fig. 6. Mitochondrial function, oxidative stress, and lipid peroxidation of MCF-7 cells and MCF-7/ADR cells treated Dox and Dox-HBDL at a Dox concentration of 5 mM for 24 h. (A) The mitochondrial membrane potential (Ajm) of the cells was measured by flow cytometry using the MitoDilC kit. (B) Mitochondria-generated superoxide in the cells was measured using the MitoSox assay. (C) Cellular ROS production was measured using the CellROX assay. (D) Lipid peroxidation in the cells was measured by the Bodipy assay. **p < 0.01 and ***p < 0.001 compared to control.
mechanism for uptake of these HBDL NPs [36,37]. Also, Dox-HBDL NPs have diameters of around 320 nm (Supplemental lnformation) and are within the favorable size range for macropinocytosis and clathrin-mediated endocytosis [36,37].
The energy dependence and uptake pathways of HBDL-DOX transport in MCF-7 cells were also studied, and clear reductions in the intracellular accumulation of Dox were observed at 4 °C and with inhibitors of macropinocytosis compared with the control group (Supplemental Information, Fig. S5). However, inhibitors of the clathrin pathway did not reduce the uptake of Dox-HBDL significantly. Furthermore, less transferrin signal was detected in MCF-7 cells compared with resistant cells, and a faint signal of co-localization of Dox-HBDL and transferrin was observed in MCF-7 cells by confocal imaging (Supplemental ln-formation, Fig. S6). This is in contrast to the substantial amount
of co-localization seen in MCF-7/ADR cells (Fig. 3B). We interpret this to be due to the MGST1 overexpression. Clathrin binding by fusion to GST has been reported to be associated with the assembly of clathrin coats [46], and the higher levels of MGST1 in MCF-7/ADR cells is suggested to promote assembly of clathrin coats and to enhance the uptake of Dox-HBDL NPs. The data indicate that not only the physicochemical properties of the NPs but also cellular factors influenced the rate of Dox-HBDL uptake across the plasma membrane.
3.3. Intracellular localization of Dox
To identify the intracellular distribution of Dox (red channel) delivered by HBDL NPs, we performed co-localization assays with EEA1, lysotracker, ERtracker, Mitotracker, and TGN as known
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markers (green channel) for early endosomes, lysosomes, the endoplasmic reticulum, mitochondria, and Golgi apparatus, respectively. As shown in Fig. 4, high co-localization (yellow) of red fluorescence with lysosensor, ERtracker, and Mitotracker demonstrated that Dox-HBDL and its released Dox are primarily transported to lysosomes, endoplasmic reticulum, and mitochondria following endocytosis of the carrier in MCF-7/ADR cells. Conversely, a low co-localization signal was observed with EEA1 and TGN, and this indicates that the drug is not typically localized within early endosomes or the Golgi network (Fig. S4 in the Supplemental Information shows localization of free Dox after 6 h of cell incubation). Table 1 provides Pearson's coefficients for each co-localization. These results show that free Dox significantly co-localized with lysosensor, but no Dox signal was observed in early endosomes or the Golgi network and there was no strong correlation with the endoplasmic reticulum or mitochondria. Free drug in the cytosol of the cells can be degraded by lysosomes, but the encapsulated drugs can be slowly released through either passive diffusion or degradation of the biodegradable polymers [47]. The sustained drug release from our nanocarriers in cytoplasmic depots might be a contributive factor to the improvement of chemotherapy outcome and sensitization of resistant cells to drugs. However, further studies are needed to elucidate the details of cellular degradation of the HBDL NPs and its kinetics.
The mode of nanoparticle trafficking through various subcellu-lar compartments usually determines the path of drug distribution [47], and our current results are in agreement with our previous studies. This HBDL NP carrier has been shown to bypass early endosomes and the Golgi network and to be routed to the lyso-somal compartments following its internalization [36], and Dox-HBDL NP-treated cultures showed diffuse fluorescence throughout the cytoplasm [37]. This is contrast to the free Dox-treated cultures that exhibited high drug accumulation in the nuclei of the cells (Fig. 4). The present study suggests that Dox-HBDL NPs have a strong affinity for specific cellular organelles, and the unique capacity for these carriers to facilitate sustained release of the drug payload from the specific compartments could be highly advantageous for specific drug delivery applications. The endoplasmic reticulum and mitochondria are the major sites of MGST1 expression [48] and are also the main cellular localization sites of the Dox delivered by HBDL carriers (Fig. 4).
3.4. Evaluation ofMGSTl expression, glutathione levels, and oxidative stress
To explore the interaction of Dox-HBDL and MGST1, the protein expression level of MGST1 in the cells was determined by flow cytometry after treatment with Dox-HBDL. Fig. 5A shows that Dox-HBDL clearly leads to decreased MGST1 levels, and the decline is greater in MCF-7/ADR cells compared to MCF-7 cells that have low endogenous levels of MGST1. We have further shown that free Dox had less effect on MGST1 expression in both cell lines relative to Dox-HBDL, and the lower activity of free Dox most likely stems from the low levels of free Dox located in the endoplasmic reticulum and the mitochondria. The inhibition of
GST enzymes has been reported to result in the overcoming of the drug resistance [48,49], and these observations suggest that inhibition of MGST1 expression could account for the enhanced drug activity in MCF-7/ADR cells treated with Dox-HBDL NPs. GST enzymes are known to catalyze the conjugation of gluta-thione (GSH) with xenobiotics, and GSH plays an important role in protecting cells against damage from free radicals, oxidants, and electrophiles [50]. The effects of Dox-HBDL NPs on GSH levels were examined using the ThioGlo-1 assay. As shown in Fig. 5C, the GSH level in MCF-7/ADR cells decreased after a 24-h treatment with Dox-HBDL presumably because more GSH is consumed as more free radicals, lipid peroxides, and oxidants are formed. Lower GSH levels are expected during cell toxicity (for example by mitochondrial damage caused by Dox-HBDL), and not from alteration of MGST1 per se, as shown by Johansson et al. [39].
We further assessed mitochondrial function, the production of reactive oxygen species (ROS), and lipid peroxidation to investigate the cytotoxic effects of free Dox and Dox-HBDL NPs. A MitoDilc assay [40] showed that Dox-HBDL NPs induced significant dissipation of the mitochondrial membrane potential in both cell lines, but free Dox only triggered mitochondrial dysfunction in MCF-7 cells (Fig. 6A). Moreover, Dox-HBDL treatment resulted in significant production of mitochondrial ROS, cellular ROS, and lipid peroxides as shown in Fig. 6B, C, and D, respectively. ln contrast, free Dox treatment induced comparable effects on cellular ROS and lipid peroxides only in MCF-7 cells and not in MCF-7/ADR cells. Mitochondria are the major source of ROS generation in the cell [51]. Overproduction ROS and the accumulation of ROS in cells causes damage to lipids, proteins, and DNA and leads to mitochondrial membrane permeabilization that further initiates apoptosis [51,52]. The present study shows that Dox-HBDL increases ROS production and lipid peroxidation as well as induces dissipation of the mitochondrial membrane potential in MCF-7/ADR cells more efficiently than free Dox. ln contrast to free Dox that mainly accumulates in the nuclei, we show here that the mitochondria is the major target and plays a central role in how Dox-HBDL is able to overcome drug resistance. As MGST1 is known to protect cells from oxidative stress induced cell death [53], the lowered protection against Dox-HBDL is somewhat counterintuitive. We suggest that the lowering of MGST1 expression together with the more severe oxidative stress induced overcomes the protective ability.
3.5. Induction of apoptosis and JNK activation
Because Dox-HBDL NPs induced the dissipation of the mito-chondrial membrane potential, we further studied the apoptosis and necrosis that is induced by Dox-HBDL NPs. lt is well known that mitochondria are involved in the regulation of apoptosis [51], and the loss of the mitochondrial membrane potential can trigger the release of apoptogenic factors from mitochondria into the cytosol that further initiates the sequential death of the cell [52] Fig. 7 shows that MCF-7 cells treated with Dox suffered significant levels of apoptosis and necrosis. Dox-HBDL NPs enhance the susceptibility of both cancer cell lines to Dox and induce significantly
Fig. 7. Evaluation of the apoptosis mechanisms of breast cancer cells treated with PBS (control), free Dox, and Dox-HBDL micelles. (A) Representative dot plots showing fluorescence channel analyses of MCF-7 and MCF-7/ADR cells after dual staining with FlTC-conjugated annexin V and propidium iodide. The cells were treated with free Dox or Dox-HBDL NPs for 48 h and stained with FlTC-conjugated Annexin V (horizontal axis) and propidium iodide (vertical axis) before being analyzed using flow cytometry and fluorescence-activated cell sorting protocols. (B) Comparison of the cell death rates of MCF-7 and MCF-7/ADR cells exposed to free Dox and Dox-HBDL NPs for 48 h. The concentration of Dox (free or equivalent) in the cell culture was 10 mM in all experiments. Data are shown as the mean ± SD (n = 3). (C) Dox-HBDL micelles induced significant activation of JNK compared to free Dox in MCF-7/ADR cells. Cells were incubated with the equivalent of 5 mM free Dox or Dox-HBDL NPs for 24 h. lmmunofluorescence of phosphorylated JNK (p-JNK) was detected using a confocal laser microscope system. The nuclei were stained with DAPl (blue). Scale bar = 20 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
more apoptosis in the cells. Apoptosis was induced in 27.1% and 43.4% of the MCF-7 cells after treatment with Dox and Dox-HBDL NPs, respectively. Treatment with Dox had a slight influence on the MCF-7/ADR cells and induced apoptosis in 14% of the cells. Treatment of MCF-7/ADR cells with Dox-HBDL NPs induced necrosis and apoptosis in 8.4% and 31.3% of the cells, respectively. The enhanced apoptotic effects of Dox-HBDL NPs correlated linearly with the effects of the HBDL NP on the mitochondrial function in resistant cells shown in Fig. 6. This implies that mitochondria-mediated apoptosis plays a central role in the cell death induced by Dox-HBDL NPs.
Jun-terminal kinase (JNK), which is responsible for the stress response through apoptosis, has been reported to be maintained at a low level because GST functions as an endogenous negative regulatory switch for this kinase [54,55]. Based on the MCF-7/ADR cell model in which MGST1 is overexpressed and Dox-HBDL NPs decrease the levels of MGST1, we hypothesized that the JNK-mediated pathway of apoptosis would contribute to the cytotox-icity of Dox-HBDL NPs in MCF-7/ADR cells. JNK activation is mediated by phosphorylation at threonine 183 and tyrosine 185 in the conserved TPY tripeptide motif in the kinase domain. Therefore, the levels of phosphorylated JNK at threonine 183 and tyrosine 185 were analyzed after treatment of cells with Dox and Dox-HBDL NPs. Confocal microscopy showed that JNK had translocated into the nucleus, which is characteristic for JNK activation. As shown in Fig. 7C, much stronger JNK fluorescence was detected in the nuclei of MCF-7/ADR cells treated with Dox-HBDL NPs compared to free Dox suggesting that Dox-HBDL NPs could activate the JNK pathway to a higher degree. This in turn contributes to the enhancement of apoptosis of MCF-7/ADR cells and the ability to overcome drug resistance and suggests that JNK might be employed as a potential therapeutic target for the use of nanocarriers against drug resistance.
Both free Dox and Dox-HBDL NPs activated JNK in MCF-7 cells because translocation of phosphorylated JNK to the nuclei could be observed within 24 h at equivalent Dox concentrations of 5 p,M (Supplemental Information, Fig. S7). Therefore, it is concluded that JNK activation contributes to the apoptosis of MCF-7 cells treated by both free drug and Dox-HBDL, and the enhanced apoptosis of the MCF-7/ADR cells by Dox-HBDL is most probably due to the highly activated JNK. In control experiments, empty HBDL NPs without Dox did not have any significant effect on the cell functions studied in either MCF-7 or MCF-7/ADR cells. This indicates that the effects of Dox-HBDL NPs are not due to the presence of the polymer-based carriers.
4. Conclusions
Drug resistance remains one of the most challenging problems to overcome in cancer treatment. The use of nanotechnology for drug delivery is a promising strategy for combating drug resistance, but understanding how the nanocarriers affect drug delivery remains a major obstacle to their potential applications. Here, we demonstrate that drug resistance in an MGST1-overexpressing human breast cancer cell line can be overcome by treatment with Dox encapsulated within our polyester-based DDS. Although Dox-HBDL NPs achieved enhanced drug accumulation in drug-resistant cancer cells compared to drug-sensitive cells, much less drug from the NPs was delivered in both the resistant and sensitive cells compared to treatment with free drug. Our data show that the uptake of Dox-HBDL is via an active endocytic transport mechanism involving macropinocytosis and clathrin-mediated pathways and the triggering of multistep processes that are distinct from the uptake of free Dox. This is strongly supported by our results showing 1) the accumulation of Dox-HBDL NPs in lysosomes,
endoplasmic reticulum, and mitochondria and a lack of accumulation in nuclei; 2) inhibition of the GST detoxification system that increases the amount of the drug available to react with its target molecules; 3) dissipation of the mitochondrial membrane potential and an increase in ROS levels in the cytoplasm; 4) activation of pro-apoptotic JNK signaling in drug-resistant cells; 5) a decrease in drug degradation within lysosome vesicles; and 6) an overall increase in apoptosis and cell death. In summary, our findings strongly suggest that drug resistance in cancer cells can be significantly circumvented through the use of HBDL NPs that can deliver drugs to specific organelles while minimizing the intracellular concentrations of the drugs. This work is significant in the field of nano-medicine research because it provides understanding of the nanomaterial's modulation of chemotherapeutic mechanisms of action and has important implications for rational design of DDS's to improve the efficacy of nuclear-acting chemotherapeutics against drug-resistant cancers.
Acknowledgments
Funding support to A.M.N. by the Royal Swedish Academy of Sciences, the Falk Foundation, and the Swedish Research Council (VR) under grants 2011-3720 and 2009-3259 is gratefully acknowledged. A.M.N. is the recipient of an assistant professorship from Carl Bennet AB, Karolinska Institutet, and Vinnova. Grants to X.H. Z. by Karolinska Institutets Research Funds (2012FoBi34650), the National Natural Science Foundation of China (81101688), and the Specialized Research Fund for the Doctoral Program of Higher Education (20105301120002) are acknowledged. R.M. was supported by the Swedish Research Council, the Swedish Foundation for Strategic Research, VINNOVA and funds from Karolinska Insti-tutet. Katarina Johansson is acknowledged for help with setting up the cells. The authors declare the following competing financial interest: Andreas M. Nystrom is the CEO of Polymer Factory Sweden, which commercializes bis-MPA-based dendrimers.
Appendix A. Supplementary data
Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.biomaterials.2013.10.042.
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