Magnetic Tandem Apoptosis for Overcoming Multidrug-Resistant

Nov 28, 2016 - We further demonstrate that apoptosis of MDR cancer cells can be achieved in a spatially selective manner with single-cell level precis...
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Magnetic Tandem Apoptosis for Overcoming Multidrug-Resistant Cancer Mi Hyeon Cho,†,‡,§ Seulmi Kim,†,‡,§ Jae-Hyun Lee,†,‡,§ Tae-Hyun Shin,†,‡,§ Dongwon Yoo,†,‡ and Jinwoo Cheon*,†,‡,§ †

Center for Nanomedicine, Institute for Basic Science (IBS), Seoul 03722, Republic of Korea Yonsei-IBS Institute, Yonsei University, Seoul 03722, Republic of Korea § Department of Chemistry, Yonsei University, Seoul 03722, Republic of Korea ‡

S Supporting Information *

ABSTRACT: Multidrug resistance (MDR) is a leading cause of failure in current chemotherapy treatment and constitutes a formidable challenge in therapeutics. Here, we demonstrate that a nanoscale magnetic tandem apoptosis trigger (m-TAT), which consists of a magnetic nanoparticle and chemodrug (e.g., doxorubicin), can completely remove MDR cancer cells in both in vitro and in vivo systems. m-TAT simultaneously activates extrinsic and intrinsic apoptosis signals in a synergistic fashion and downregulates the drug efflux pump (e.g., Pglycoprotein) which is one of the main causes of MDR. The tandem apoptosis strategy uses low level of chemodrug (in the nanomolar (nM) range) to eliminate MDR cancer cells. We further demonstrate that apoptosis of MDR cancer cells can be achieved in a spatially selective manner with single-cell level precision. Our study indicates that nanoscale tandem activation of convergent signaling pathways is a new platform concept to overcome MDR with high efficacy and specificity. KEYWORDS: Magnetic nanoparticle, magnetic field, tandem activation, multidrug-resistant cancer, apoptosis

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inducing either extrinsic or intrinsic apoptosis have made substantial advances in the field, overcoming MDR remains a challenge. Here, we introduce a new strategy to induce complete death of MDR cancer cells via a magnetic tandem apoptosis trigger (m-TAT) (Figure 1a). The m-TAT is not a simple drug carrier but an apoptosis signal activator that can stimulate both intrinsic and extrinsic apoptosis signaling pathways in tandem. The m-TAT is composed of three functional subunits: a magnetic nanoparticle (MNP, a magnetic force generator), a death receptor 4 (DR4)-targeting monoclonal antibody (DR4 Ab), and doxorubicin (Dox, an anticancer drug) (Figure 1a). 15 nm zinc-doped iron oxide magnetic nanoparticles (Zn0.4Fe2.6O4) with high saturation magnetization (161 emu g−1)23 enable the facile assembly of extracellular receptors when exposed to a static magnetic field22,24 (Figure 1a(i)). Dox is attached to the MNP through a disulfide linker, which releases Dox only in response to intracellular physiological conditions, such as glutathione (GSH)25,26 (Figure 1a(iii)). Briefly, m-TAT is synthesized by coating the MNP with silica followed by covalent attachment of Dox and DR4 Ab. Dox and DR4 Ab are

nduction of apoptosis (i.e., programmed cell death) in cancer cells has been one of the main strategies to treat cancer owing to the advantageous features of apoptosis, which include the self-destructive nature and noninflammatory responses.1,2 Most anticancer drugs make use of the intrinsic pathway to induce apoptosis by artificially inducing cell stresses.3−5 Unfortunately, the effects of anticancer drugs are still limited for multidrug-resistant (MDR) cancer cells, in which the overexpression of the ATP-binding cassette (ABC) family such as P-glycoprotein (P-gp) removes drugs from the intracellular space to the extracellular region.6−8 To circumvent these transporter-dependent resistances, many approaches have been explored.9−18 For example, P-gp-specific peptides or antibodies can be used to inhibit MDR efflux pumps;9,10 however, the efficacy of the anti-MDR approach has been frequently compromised by its adverse interactions with cancer drugs.19,20 Another approach is the use of nanoparticles as drug carriers, which are less affected by the efflux effect and can codeliver multiple drugs.11−18,21 Alternatively, albeit less explored, extrinsic apoptosis activation via the natural ligand TRAIL4,5 and magnetic nanoparticle switches22 can be another approach to induce MDR cancer cell death by targeting extracellular death receptors. However, their efficacy has not been demonstrated to be sufficient to resolve the MDR of cancer. In short, even though new strategies and methodologies © XXXX American Chemical Society

Received: July 27, 2016 Revised: October 22, 2016

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Figure 1. Magnetic tandem apoptosis trigger (m-TAT) and its tandem capability for magnetic clustering and Dox release. (a) Schematic illustration of m-TAT and its modes of action. Under a magnetic field, m-TAT synergistically induces apoptosis in MDR cancer cells via three modes of action: (i) magnetic assembly of DR4 to activate extrinsic death signal, (ii) drug efflux pump (i.e., P-gp) downregulation to enhance the efficacy of drugs, and (iii) intracellular drug release via cleavage of the disulfide linker to activate intrinsic death. (b) Scanning electron microscope (SEM) images showing m-TAT on the cell surface. m-TAT is dispersed throughout the cell surface (left panel), and magnetic nanoparticles are clustered upon magnetic field application (right panel). (c) Time-dependent cumulative profile of Dox release from m-TAT in the presence and absence of glutathione (GSH). The disulfide linkage is cleaved only in the presence of GSH (5 mM), whereas it is not responsive in the absence of GSH. (d) Fluorescence images of Dox (red) released from m-TAT in the in vitro cell test.

Figure 2. Behavior of m-TAT at the interface of MDR cancer cells. (a) Schematic illustrations showing the behavior of m-TAT upon treatment in MDR cancer cells. (i,ii) m-TAT first binds to DR4 and is subsequently clustered upon application of a magnetic field (MF) for extrinsic apoptosis signal initiation. (iii) The magnetically induced m-TAT clusters enter the cell via endocytosis and (iv) release Dox into the nucleus, which initiates the intrinsic apoptosis signal. (b) Transmission electron microscope (TEM) images showing the location of nanoparticles at the interface of the MDR cancer cells. Before MF application (0 h), nanoparticles are dispersed throughout the cell surface (yellow arrowheads). At 2 h post-MF application, nanoparticles are observed at the surface membrane of the cells (yellow arrowheads). At 6 h post-MF application, a large portion of the nanoparticles is endocytosed (red arrowheads), while some of the nanoparticles remain at the cell surface (yellow arrowheads). At 12 h post-MF application, all nanoparticles are observed at the endosomes (red arrowheads). (c) Fluorescence microscope images of m-TAT-treated MDR cancer cells. The blue (DAPI) and red (Dox) fluorescence, respectively, indicate the nucleus and released Dox. Red fluorescence is observed only in the images 6 and 12 h post-MF application.

We chose the DLD-1/ADR drug-resistant colon cancer cell line as an MDR cancer model. This cell line is highly resistant to doxorubicin and is cross-resistant to other types of chemodrugs; it was obtained by continuous culture of parental DLD-1 cells in doxorubicin with overexpressed DR4 and P-gp (Figure S2).

conjugated via a cleavable disulfide linker (3,3′-dithiodipropionic acid di(N-hydroxysuccinimide ester), DTSP) and protein A, respectively (Figure S1). Each m-TAT contains about ten Dox molecules and one DR4 Ab, which are quantified by photoluminescence of Dox and bicinchoninic acid assay of DR4 Ab. B

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Figure 3. m-TAT for the synergistic activation of two apoptosis signal pathways in MDR cancer cells. (a) Schematic of m-TAT-mediated tandem activation of apoptosis signaling pathways. m-TAT forms DISC and initiates the extrinsic apoptosis pathway, affording caspase-8. Then, the clustered m-TAT is endocytosed and releases Dox into the nucleus, which damages DNA to initiate the intrinsic apoptosis pathway, generating caspase-9 through the mitochondria. Both pathways induce apoptosis executor caspase-3, resulting in apoptotic cell death. (b) Pseudocolor-mapped confocal microscope images obtained at 2, 12, and 22 h post-MF application. Caspase-8, caspase-9, and caspase-3 are immunostained with FITC-labeled secondary antibodies and are pseudocolor-mapped with green, yellow, and red. Nuclei are stained with DAPI (blue). (c) Graph showing the relative caspase-3 expression level of DR4 Ab-MNP, Dox-MNP, and m-TAT, where m-TAT shows the highest caspase-3 expression level. (d) Plots of the concentration vs cell death. The viabilities of free Dox-, paclitaxel-, DR4 Ab-MNP-, Dox-MNP-, and m-TAT-treated cells are measured by CCK-8 assays. m-TAT induces 100% cell death. (e,f) Graphs showing the amount of (e) internalized Dox and (f) the expression of P-glycoprotein (P-gp) on the cell membrane. m-TAT shows the highest amount of internalized Dox and the lowest P-gp expression level.

concentration of GSH in the intracellular space is 5 mM,25,26 the m-TAT (20 nM) is incubated with 5 mM GSH in 37 °C. By measuring the recovery intensity of fluorescence of Dox, which is initially quenched upon conjugation with m-TAT, the amount of released Dox is quantified by measuring its fluorescence intensity (Figure 1c and Figure S4). According to Figure 1c, ca. 60% of the conjugated Dox bursts out from mTAT within 20 min post-incubation. Afterward, the remaining Dox is gradually released within 3 h of incubation, resulting in full release of Dox. By contrast, in the absence of GSH, no Dox release is observed (Figure 1c). The intracellular Dox release capability of m-TAT is also demonstrated in MDR cancer cells, where Dox internalized into the nuclei is observed in the red fluorescence image obtained at 12 h post-m-TAT treatment (Figure 1d). These data show that the Dox of m-TAT can be effectively released in the in vitro GSH conditions. The successful results of the magnetic clustering and the intracellular Dox release properties of m-TAT provide an ability to activate both the extrinsic and intrinsic apoptosis pathways. Prior to assessing the apoptosis signaling cascades, we monitor the time-dependent behaviors of m-TAT (e.g., receptor targeting, clustering, endocytosis, and Dox release) at the interface of MDR cancer cells (Figure 2a). In the transmission electron microscope (TEM) images obtained at 0 and 2 h postmagnetic field (MF) application, nanoparticles are located at the cell membrane (Figure 2b, yellow arrowheads). At 6 h postMF application, ca. 30% of nanoparticles are still observed at the cell membrane (yellow arrowheads), whereas the remainings are located at the endosomes (red arrowheads). At 12 h post-MF application, nanoparticles are endocytosed (red arrowheads), and no nanoparticles remain at the membrane. In the fluorescence microscope images (Figure

Electromagnetic coils that generate a static and low-gradient magnetic field of 0.5 T only when electricity is turned ON are utilized (Figure S3c). In our dual magnetic setup, clustering of nanoparticles is achieved through induced dipolar interparticle attraction and the magnetic field can reach deep inside the target tissue.22,27 Before application of the magnetic field, MDR cancer cells (1.5 × 104 cells/well) are treated with m-TAT (20 nM, metal basis) and incubated for 0.5 h to bind m-TAT to the DR4 on the cell surface. After removal of unbound m-TAT, the plate is placed in the middle of the electromagnetic coils, and a magnetic field of 0.5 T is applied for 2 h (Figure S3c). According to the scanning electron microscope (SEM) images obtained before application of the magnetic field, DR4-targeted m-TAT is dispersed throughout the cell surface and clusters of nanoparticles are observed after magnetic field application (Figure 1b). Under a static magnetic field of 0.5 T, clustering preferentially occurs on the two-dimensional (2D) surface membrane,22,28 whereas m-TAT is not assembled in threedimensional (3D) media (Figure S3). The preferred 2D clustering can be explained by the reduced degrees of freedom (i.e., from 3 in 3D to 2 in 2D), which can be a beneficial phenomenon of m-TAT for in vivo application because m-TAT is not assembled in 3D systems, such as plasma, before it binds to targets on the cell membrane. On average, the clusters are composed of approximately nine nanoparticles which is larger than minimally required number (ca. 3) of nanoparticle for extrinsic apoptosis activation1−5 (Supporting Information, methods section). The effective drug release capability (i.e., disulfide bond cleavage) of m-TAT is investigated by measuring the Dox release profile of m-TAT in test tubes mimicking the intracellular GSH environment (Figure 1c) and in vitro using an MDR cancer cell line (Figure 1d). Considering the average C

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Consequently, the use of m-TAT not only induces both extrinsic and intrinsic signals but also downregulates P-gp, finally resulting a synergistic anticancer effect against MDR cancer cells. As a note, unlike P-gp, other well-known efflux pumps such as multidrug resistance associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) are not overexpressed in the MDR cancer cells, and also the expression level does not change by m-TAT (Figure S6). This indicates that the downregulation of P-gp is the major contribution, while other known efflux transporters such as MRP1 and BCRP are negligible. Additionally, the anticancer effect is confirmed to be only attributed to force mediated tandem activation of apoptosis, not to the hyperthermic effect (Figure S7). Another advantage of m-TAT over conventional drugs is that m-TAT is capable of precision control of cell death at the single-cell level (Figure 4a, left panel). The magnetic field is

Figure 4. Single-cell level apoptosis induction by m-TAT. (a) Schematic illustrations of the magnetic setup for the site-specific apoptosis activation study. Micrometer-sized magnetic probe tips exerting a 0.8 T magnetic field (yellow region outlined with gray dashed line) are located to pinpoint a single cell. The magnetic field generated from the magnetic setup is simulated by FEMM software (right panel). (b) (i) Optical microscope images showing m-TATtreated MDR cancer cells. Two target cells “A” and “B” are outlined with a white dotted line. (ii) The magnetic tips are first focused on target cell “A”, and a 0.8 T magnetic field is applied for 1 h. (iii) The magnetic tips are repositioned at target cell “B”. Green fluorescence signal (FITC-annexin V) indicates the induction of apoptosis.

focused on a single MDR cancer cell of micron size for 1 h using two magnetic probe tips, where the distance between the magnetic probe tips is 15 μm (Figure 4a, right panel). According to the simulated magnetic field strength map, the dual magnetic probe tips generate a ca. 0.8 T magnetic field (Figure 4a (right panel), yellow region outlined with gray dashed line). Upon application of the magnetic field on target cell “A” for 1 h, a green fluorescence signal (FITC-annexin V, an early apoptosis marker) is selectively observed (i.e., target cell “A”) (Figure 4b(i) and (ii)). Then, the magnetic tips are repositioned at target cell “B” to obtain an activated apoptosis signal (Figure 4b(iii)). Based on these successful in vitro results, we further examine the in vivo feasibility of m-TAT in an MDR tumor xenograft mouse model. Figure 5a shows the experimental setup. MDR cancer cells (DLD-1/ADR cell line) are xenografted to the right thigh of eight nude mice, which is confirmed to be a sufficient D

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Figure 5. In vivo efficacy of m-TAT-mediated apoptosis induction in MDR tumors. (a) Schematic of the in vivo magnetic apoptosis experiments. mTAT (500 μg) is directly injected into the xenograft tumor (DLD-1/ADR, 30 mm3) of a mouse and exposed to a static magnetic field of 0.5 T. (b) Photographs of xenograft tumor-bearing mice obtained before (day 0) and after (day 14) treatment with various anticancer agents, including vehicle (nontreated), free Dox (1 nmol), paclitaxel (1 nmol), Dox-MNP (0.1 nmol), DR4 Ab-MNP (0.1 nmol), and m-TAT (0.1 nmol). The insets show tumors isolated at day 14. (c) A plot of the relative tumor volume vs time after treatment. The tumor sizes drastically increase in the nontreated (black line), free Dox (blue line), paclitaxel (purple line), Dox-MNP (orange line), and DR4 Ab-MNP (green line) groups. Only m-TAT (red line) treatment completely eliminates the tumor (n = 8, **P < 0.001). (d) A graph of the average weight of tumors (black square) and the tumor inhibitory rate (TIR, green square). Data are represented as the average ± standard deviation (n = 8). (e) Immunohistochemistry (IHC) images of the cross-sectioned xenograft tumor lesion obtained after anticancer treatments. In the images, xenograft human tumor tissue and mouse skin are shown in brown and blue, respectively. Brown-colored tumor tissue is observed in all control groups, except the m-TAT-treated group.

show strong brown color, indicating the presence of MDR cancer cells (Figure 5e). These in vivo results are in line with the in vitro results, which indicate that m-TAT is also effective in an in vivo mouse model of MDR cancer cells. In this study, we demonstrate that one of the major hurdles for overcoming MDR cancer can be resolved by a new methodology, m-TAT-mediated apoptosis induction, which facilitates two convergent apoptosis signal activations in tandem. This approach is potent in in vitro and in vivo, even with only a small amount of chemodrugs at the nanomolar range. Our tandem activation strategy has diverse applicability for important biological systems because convergent signal pathways are frequently found in various biological processes. In addition, the use of noninvasive external stimuli (e.g., magnetic field) can provide additional advantages of precision therapeutics with space and time selectivity with reduced offtarget side effects.

number to demonstrate statistically meaningful in vivo results based on power analysis.32 Then, m-TAT (500 μg, 0.1 nmol, metal basis) possessing Dox (0.58 μg, 1 nmol) is dispersed in normal saline (100 μL) and directly injected into the tumor when the tumor volume reaches ca. 30 mm3. The in vivo dosage of m-TAT is determined by dosage-dependent antitumor effect study (Figure S8). The mouse is placed in the magnet system, which generates a static magnetic field of 0.5 T for 3 h (Figure 5a). The in vivo apoptosis efficacy of mTAT is assessed by plotting the relative tumor volume vs time (Figure 5b and c) along with five control groups (i.e., nontreated, free Dox, paclitaxel, Dox-MNP, and DR4 AbMNP groups). After treatment, the changes in tumor volume are monitored for 14 days. For the nontreated control group, the tumor size increases by approximately 6.7-fold compared to the initial volume (Figure 5b and c). In the free Dox-treated and paclitaxel-treated groups, the tumor grows without meaningful inhibition due to the MDR characteristics of the DLD-1/ADR tumor model. Although the Dox-MNP- and DR4 Ab-MNP-treated groups show a slight suppression of the tumor growth rate, the final volume of the tumor is ca. 4.6-fold and 2.5-fold larger than the initial volume, respectively. By contrast, the tumor is completely removed in the m-TAT-treated group within 10 days. Based on the weight of tumors isolated at day 14 post-treatment, the m-TAT-treated group has a dominant effect on MDR tumor removal compared with the other control groups (Figure 5b insets and d). The tumor inhibitory rate (TIR) is further calculated from the weight of the isolated tumors (Figure 5d, green square). Compared with the TIR of the nontreated group, the TIR of m-TAT is 100%, which is significantly higher than those of the control groups (free Dox, ca. 1%; paclitaxel, ca. 5%; Dox-MNP, ca. 28%; DR4 Ab-MNP, ca. 69%) (Figure 5d, green square). Immunohistochemistry (IHC) using human mitochondria antibody confirms that there are no human MDR cancer cells residing in the subcutaneous region of the m-TAT-treated group, whereas the control groups

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.6b03122. Detailed methods and supplementary figures: m-TAT synthesis, drug release profile from m-TAT, hydrodynamic size of m-TAT, size analysis of magnetically clustered m-TAT, aggregation test of m-TAT on a 2D and 3D systems, single-cell precision control of apoptosis, m-TAT in vivo dosage dependence and in vivo mouse experiments, expression of death receptors and drug efflux pumps (i.e., P-gp, MRP1, and BCRP), dose-response of free Dox, temperature monitoring profile (PDF) E

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(23) Jang, J. -t.; Nah, H.; Lee, J.-H.; Moon, S. H.; Kim, M. G.; Cheon, J. Angew. Chem., Int. Ed. 2009, 48, 1234−1238. (24) Lee, J.-H.; Kim, E. S.; Cho, M. H.; Son, M.; Yeon, S.-I.; Shin, J.S.; Cheon, J. Angew. Chem., Int. Ed. 2010, 49, 5698−5702. (25) Ducry, L.; Stump, B. Bioconjugate Chem. 2010, 21, 5−13. (26) Santra, S.; Kaittanis, C.; Santiesteban, O. J.; Perez, J. M. J. Am. Chem. Soc. 2011, 133, 16680−16688. (27) Dobson, J. Nat. Mater. 2012, 11, 1006−1008. (28) Mannix, R. J.; Kumar, S.; Cassiola, F.; Montoya-Zavala, M. M.; Feinstein, E.; Prentiss, M.; Ingber, D. E. Nat. Nanotechnol. 2008, 3, 36−40. (29) Seo, S.; Hur, J.-G.; Kim, M.-J.; Lee, J.-W.; Kim, H.-B.; Bae, J.-H.; Kim, D.-W.; Kang, C.-D.; Kim, S.-H. Mol. Cancer 2010, 9, 199−213. (30) Kim, D.-Y.; Kim, M.-J.; Kim, H.-B.; Lee, J.-W.; Bae, J.-H.; Kim, D.-W.; Kang, C.-D.; Kim, S.-H. Biochim. Biophys. Acta, Mol. Basis Dis. 2011, 1812, 796−805. (31) Mantovani, I.; Cappellini, A.; Tazzari, P. L.; Papa, V.; Cocco, L.; Martelli, A. M. J. Cell. Physiol. 2006, 207, 836−844. (32) NCSS Statistical Software Home Page. PASS14 (Power Analysis and Sample Size) Software. https://www.ncss.com/software/pass/ (accessed Oct. 6, 2016).

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jae-Hyun Lee: 0000-0002-9236-157X Author Contributions

M.H.C., J.-H.L., D.Y., and J.C. designed the experiments. M.H.C., S.K., and D.Y. performed the experiments. M.H.C., S.K., J.-H.L., T.-H.S., D.Y., and J.C. wrote the manuscript. All of the authors discussed the results and commented on the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Institute for Basic Science (IBS-R026-D1). The authors thank Prof. J.-S. Shin (College of Medicine, Yonsei University) for providing the MDR cancer cell line.

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