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Nanomedicines Eradicating Cancer Stem-like Cells in Vivo by pH-Triggered Intracellular Cooperative Action of Loaded Drugs Hiroaki Kinoh,† Yutaka Miura,‡ Tsukasa Chida,‡ Xueying Liu,‡ Kazue Mizuno,‡ Shigeto Fukushima,§ Yosuke Morodomi,∥ Nobuhiro Nishiyama,⊥ Horacio Cabral,*,# and Kazunori Kataoka*,†,‡,§,# †
Innovation Center of NanoMedicine, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki 210-0821, Japan Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan § Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan ∥ Department of Innovative Applied Oncology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan ⊥ Polymer Chemistry Division, Chemical Resources Laboratory, Tokyo Institute of Technology, R1-11, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan # Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan ‡
S Supporting Information *
ABSTRACT: Nanomedicines capable of control over drug functions have potential for developing resilient therapies, even against tumors harboring recalcitrant cancer stem cells (CSCs). By coordinating drug interactions within the confined inner compartment of core−shell nanomedicines, we conceived multicomponent nanomedicines directed to achieve synchronized and synergistic drug cooperation within tumor cells as a strategy for enhancing efficacy, overcoming drug resistance, and eradicating CSCs. The approach was validated by using polymeric micellar nanomedicines co-incorporating the pan-kinase inhibitor staurosporine (STS), which was identified as the most potent CSC inhibitor from a panel of signaling-pathway inhibitors, and the cytotoxic agent epirubicin (Epi), through rationally contriving the affinity between the drugs. The micelles released both drugs simultaneously, triggered by acidic endosomal pH, attaining concurrent intracellular delivery, with STS working as a companion for Epi, down-regulating efflux transporters and resistance mechanisms induced by Epi. These features prompted the nanomedicines to eradicate orthotopic xenografts of Epi-resistant mesothelioma bearing a CSC subpopulation. KEYWORDS: cancer stem-like cells, polymeric micelles, chemotherapy, mesothelioma, epirubicin, staurosporine and Bcl-2,6−10 which represent promising targets for the development of clinically useful CSC inhibitors.6,11−14 Besides, the crosstalk between these CSC-related signaling pathways and their influence on tumor progression offer extra opportunity for implementation of multitargeted therapies, in particular, for synergistic combination with cytotoxic treatments.15 However, as many of these pathways are found in
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ecalcitrant cancer stem cells (CSCs) have been identified in many cancer types1−3 and have been associated with metastasis, resistance to therapy, and the eventual relapse of the disease.4,5 With traditional chemotherapeutics being futile against the CSC population,5 the effective combination of CSC inhibitors with cytotoxic therapies emerges as crucial for achieving robust responses capable of long-term disease-free survival. Accordingly, CSCs have shown deregulation in several pathways involved in the control of self-renewal and differentiation, such as PI3K/Akt, Wnt/β-catenin, PTEN, NF-κB, JAK/STAT, hedgehog, Notch, © 2016 American Chemical Society
Received: February 4, 2016 Accepted: April 19, 2016 Published: April 19, 2016 5643
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Figure 1. ALDH-high subpopulation in MSTO-211H cells shows CSC features. (A) Percentage of ALDH-positive subpopulation from four kinds of mesothelioma cells after monolayer (white bars) and spheroid colony formation (black bars) culture. Data are expressed as the mean ± SD (n = 3). (B) FACS-based sorting of ALDH-low and ALDH-high (ALDH-positive) cells by MoFlo Astrios. Left panel: Representative FACS plot showing the percentage of ALDH-positive population from MSTO-211H cells. Right panel: FACS plot showing the negative control after adding DEAB. (C) Number of colony formations by FACS-sorted MSTO-211H cells after 1 week culture on nonadherent 6-well plates. White bar, nonsorted cells; red bar, ALDH-high fraction; black bar, ALDH-low fraction. Data are expressed as the mean ± SD (n = 3). (D) mRNA expression of CSC markers (Nanog, Sox2, and Oct4) in ALDH-high subpopulation (red bars) compared to ALDH-low subpopulation (black bars). Data are expressed as the mean ± SD (n = 3). (E) Tumorigenicity of ALDH-low and ALDH-high cells after inoculation on the right and left flanks of SCID mice (n = 3 or 4; 8 weeks after inoculation).
normal stem cells,6 the complete eradication of CSCs with such inhibitors compels a challenging task due to the underlying risk of damage to healthy tissues, and the toxicity of CSC inhibitors combined with other anticancer agents could be rendered intolerable. Therefore, effective strategies should be designed for sufficient selectivity of these inhibitors toward CSCs. Nanomedicine has demonstrated great potential for developing safe and targeted strategies against solid tumors.16,17 Thus, nanomedicine approaches involving the targeting of both CSC inhibitors and cytotoxic drugs to tumor tissues could serve as an effective way for controlling side effects while improving treatment outcomes, eventually against recalcitrant cancers. However, beyond the mere co-incorporation of drugs, such nanomedicines should precisely tailor the intracellular interplay of CSC inhibitors and cytotoxic agents to actually achieve synchronized activities and potentiate cooperative synergistic efficacies. For this purpose, drug-loaded polymeric micelles, that is, core−shell nanoassemblies, appear as a translationable nanomedicine modality with the capacity for effectively controlling the interaction of therapeutics through their sheltered core structure.18 Herein, by exploiting the molecular interaction between drug molecules, we co-loaded CSC inhibitors and cytotoxic drugs into polymeric micelles and profited from the compartmentalized architecture of the micelles for maintaining this interaction until reaching the intracellular space of cancer cells and CSCs in tumors, where the release of both drugs is triggered by endosomal pH to attain coordinated therapeutic effects. Staurosporine (STS), a broadly selective and potent protein kinase inhibitor, was selected as the CSC inhibitor in this study after being identified as the most potent agent against the CSC fraction from a panel of inhibitors of signaling pathways. The affinity of STS, as well as the synergistic efficacy, with the anthracycline epirubicin (Epi) was then exploited to encompass
STS into the core of Epi-loaded polymeric micelles (Epi/m), which are being studied in phase I clinical studies,19 for intracellular synchronization of therapeutic effects. Our results indicated that these multicomponent STS/Epi-loaded micelles (STS/Epi/m) eliminated orthotopic xenografts of Epi-resistant mesothelioma, a lethal cancer of the mesothelium, through facilitated co-delivery to tumor tissues, triggered and coordinated release of both drugs at endosomal pH, cooperative drug interactions, eradication of CSC subpopulation, and reversal of drug resistance through ABC transporter inhibition.
RESULTS ALDH-High Subpopulation in Mesothelioma Cells Has a CSC Phenotype. The development of an aggressive phenotype in malignant mesothelioma is characterized by the development of CSC-like features and an enhancement of the invasive/metastatic potential, with widespread dissemination to almost all organs.20 The overall survival for mesothelioma patients having current frontline chemotherapy (i.e., pemetrexed and cisplatin) is only 12 months.21 Moreover, this therapeutic regime further enriches the CSC population,22 and responses to second- and third-line therapies are exceptional in patients failing first-line therapy. Several reports have shown various CSC markers in malignant mesothelioma, including ALDH-1, side population, CD44, CD24, and CD26,23−26 but their validity is still arguable. To search for a CSC inhibitor of mesothelioma, we first confirmed if ALDH-1 serves as a CSC marker. Accordingly, by using an Aldefluor fluorescent reagent, four mesothelioma cell lines were stained and the positive subpopulations were detected as follows: 6.2% in MSTO-211H cells, 3.1% in H226 cells, 1.5% in Meso-1 cells, and 1.1% in H2452 cells (Figure 1A; control). Through the spheroid colony formation assay, which is used to identify CSCs in their ability for self-renewal and differentiation, we demonstrated that the 5644
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Figure 2. Staurosporine depletes the CSC subpopulation in MSTO-211H mesothelioma cells. (A) Survival rate of total cells (blue bars) and Aldefluor-positive cells (red bars) after incubation with cisplatin, pemetrexed, STS, UCN-01, and enzasataurin. Data are expressed as the mean ± SD (n = 3). (B) Inhibitory effect of STS on the Aldefluor-positive subpopulation in other human mesothelioma cells. Data are expressed as the mean ± SD (n = 3).
subpopulation. Accordingly, STS showed an IC50 value of 9 nM against the total cell population and an EC50 value of 0.36 nM against the Aldefluor-positive fraction, resulting in a selectivity for CSCs 25-fold higher than that for differentiated cancer cells (Table S1). STS is a multikinase inhibitor, especially of protein kinase C (PKC), Akt-1, Cdk-1−4, and Chk-1 in the nanomolar order, suggesting its potential as an effective CSC inhibitor. It has been reported that PKC plays an essential role in the Wnt/JNK pathway by regulating the localization and activity of Dishevelled27 and in the downstream of the Notch pathway.28 Thus, we determined whether STS and its homologues UCN-01 and enzastaurin have an inhibitory effect on the Aldefluor-positive subpopulation, compared to the conventional drugs cisplatin and pemetrexed. While the percentage of Aldefluor-positive population is increased after exposure to cisplatin or pemetrexed, STS or UCN-01 decreased the proportion of the ALDH-high subpopulation (Figure 2A and Figure S2A), though enzastaurin was not effective for reducing the ALDHhigh fraction. Moreover, increasing the concentration of cisplatin or pemetrexed augmented the Aldefluor-positive fraction, whereas higher concentrations of STS or UCN-01 effectively inhibited this subpopulation, and STS even depleted the CSCs at 0.1 μM (Figure 2A). STS also reduced the Aldefluor-positive subpopulation in other human mesothelioma cells (Figure 2B). In addition, besides ALDH, other CSC markers for mesothelioma (i.e., side population and CD44/ CD44v10) were also evaluated. STS or UCN-01 treatment dramatically diminished the side population percentage from 4.8 to 0.8% at 0.01 μM STS and 1.0% at 0.1 μM UCN-01 in MSTO-211H cells (Figure S2B). It is worth noting that by STS treatment the CD44- and CD44v10-high subpopulations shifted to CD44/CD44v10-low subpopulations (Figure S2C). These findings indicate that STS and its homologue UCN-01 are effective inhibitors of CSCs in mesothelioma. STS Interaction with Epi Facilitates Its Loading in EpiLoaded Micelles for Synergistic Efficacy. STS should be used in combination with conventional anticancer agents to facilitate the eradication of both CSCs and their differentiated progeny. Because anthracyclines have been indicated as the most effective drugs among conventional chemotherapeutics against malignant mesothelioma cells,29 and Epi has demonstrated moderate activity as monotherapy in mesothelioma patients, with 15% of the patients showing partial response lasting for a median of 37 weeks, 40% of patients with stable disease, and a median survival of 40 weeks,30 we focused on the
four mesothelioma cell lines increased the number of colonies after 1 week incubation in assay medium, with a significant increase in the Aldefluor staining (Figure 1A; spheroid colony assay). To further verify that ALDH-high subpopulation behaves as CSCs, ALDH-high and ALDH-low populations in MSTO-211H cells were isolated by cell sorting (Figure 1B), and each population was characterized in detail. The ALDHhigh cells were 4-fold more effective than ALDH-low cells to form colonies (Figure 1C). Moreover, quantitative real-time polymerase chain reaction (PCR) of sorted ALDH-high population revealed high mRNA levels of CSC markers (Nanog, Sox2, Oct4) (Figure 1D). To assess the tumorforming potential of the ALDH-high subpopulation in MSTO211H cells, we performed a transplantation assay of the sorted ALDH-high subpopulation and ALDH-low cells in SCID mice (Figure 1E). The ALDH-high subpopulation could generate tumors even after injection of only 1000 cells, which was 10fold lower than that required for ALDH-low cells to effectively form tumors. CSCs are resistant to current treatments, such as chemotherapy and radiotherapy, as well as to hypoxic environment. Moreover, several reports indicate that the number of CSCs increases after these treatments.1,5,21 By continuously exposing MSTO-211H cells to low doses of cisplatin, pemetrexed, or Epi for more than 3 months, resistant sublines were derived from the original parental cell line. These drug-resistant sublines showed an increase of ALDH-positive population (Figure S1A). X-ray irradiation (2 Gy, 6 Gy, 6 Gy plus 6 Gy 1 week later) also increased the Aldefluor-positive percentage in MSTO-211H cells (Figure S1B). In addition, hypoxia promoted expansion of the ALDH-positive population, as the incubation of MSTO211H cells in 1% O2 for 72 h increased the population of Aldefluor-positive cells from 6 to 12% (Figure S1C). Together, these results indicate that ALDH is a valid marker for CSCs in MSTO-211H cells. STS Effectively Depletes the CSC Subpopulation in Malignant Mesothelioma MSTO-211H Cells. The inhibition of various signaling pathways, including Notch, Wnt, SHh, and PI3k/Akt, is a useful strategy for depleting CSC subpopulations. In this way, we assessed the activity of a panel of 22 small-molecule inhibitors against total MSTO211H cells, as well as against the Aldefluor-positive subpopulation. The results showed that nine compounds have IC50 against total MSTO-211H cells with nanomolar potency (Table S1). Among them, STS was the most effective compound against both the whole cell population and the CSC 5645
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Figure 3. Staurosporine interaction with epirubicin facilitates its loading in Epi-loaded micelles (Epi/m) for synergistic efficacy. (A) Effect of the addition of STS on the cytotoxicity of Epi against MSTO-211H cells. Data are expressed as the mean ± SD (n = 3); *p < 0.01. (B) Quenching of the fluorescence of STS in methanol by addition of Epi (brown line, without Epi; red line, with 0.5 mg/mL Epi). (C) Scheme of the preparation of STS/Epi-loaded micelles. (D) Size distribution of Epi/m and STS/Epi/m by volume determined by dynamic light scattering. Blue histogram, Epi/m; red histogram, STS/Epi/m.
Figure 4. Staurosporine/epirubicin-loaded micelles inhibit the CSC subpopulation in resistant mesothelioma cells. (A) In vitro cytotoxicity of Epi, Epi/m, and STS/Epi/m against MSTO-211H and MSTO-211H Epi-R cells. Data are expressed as the mean ± SD (n = 4). (B) Quantification of the apoptosis induction by Epi, Epi plus STS, Epi/m, and STS/Epi/m determined by flow cytometry analysis with Annexin V. Data are expressed as the mean ± SD (n = 3); *p < 0.01. (C) ALDH-positive subpopulation in MSTO-211H Epi-R cells after treatment with Epi, STS plus Epi, Epi/m, and STS/Epi/m determined by flow cytometry. Top panels: Representative FACS plot after treatment with the drugs. Bottom panels: FACS plot showing the negative control after adding DEAB.
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Figure 5. Staurosporine/epirubicin-loaded micelles eradicate orthotopic mesothelioma tumors. (A) Antitumor effect against bioluminescent MSTO-211H-luc orthotopic tumors after intravenous injection with HEPES buffer, Epi/m (Epi 6 mg/kg), STS/Epi/m (STS 1.2 mg/kg, Epi 6 mg/kg), Epi (6 mg/kg), STS (1.2 mg/kg), STS plus Epi (STS 1.2 mg/kg and Epi 6 mg/mL), and Epi/m plus STS (Epi 4 mg/kg and STS 0.8 mg/kg). Arrows: injection points. Data are expressed as the mean ± SD (n = 10). (B) Representative IVIS images on day 25. (C) Body weight changes during the antitumor experiment. Data are expressed as the mean ± SD (n = 10). (D) Survival curves of mice; *p < 0.05 and **p < 0.001 by log-rank test.
(hAGP) in blood, and toxicity,31 STS/Epi/m could represent a safe and effective method for selectively delivering STS to tumor tissues. STS/Epi/m Effectively Inhibit CSCs in Resistant Mesothelioma Cells. As recurrent tumors have abundant drug-resistant and CSC subpopulations, we challenged the STS/Epi/m against Epi-resistant mesothelioma cells. The in vitro cytotoxicity of free Epi, Epi/m, and STS/Epi/m was studied against the original MSTO-211H cell line and the Epiresistant subline, that is, MSTO-211H Epi-R cells. While free Epi and Epi/m were less cytotoxic against MSTO-211H Epi-R cells than against the original MSTO-211H cells, STS/Epi/m with a 1:5 loading ratio of STS to Epi were more effective against MSTO-211H Epi-R cells than the original MSTO-211H cells (Figure 4A). Moreover, the induction of apoptosis was evaluated by Annexin V expression on the surface of the cells by flow cytometry after treatment with free Epi (0.05 μg/mL), free STS plus free Epi (0.01 and 0.05 μg/mL, respectively), Epi/m (0.05 μg/mL), and STS/Epi/m (0.01 μg/mL STS and 0.05 μg/ mL Epi). Representative data are shown in Figure S4A. The quantification of early plus late apoptosis indicated that free Epi was less efficient to induce apoptosis in MSTO-211H Epi-R cells than in MSTO-211H cells (Figure 4B), and Epi/m was more effective that free Epi for provoking apoptosis in MSTO211H Epi-R cells (Figure 4B). Remarkably, STS/Epi/m could induce more apoptosis than the combination of free STS plus free Epi (Figure 4B). To confirm whether STS/Epi/m are targeting CSCs in the MSTO-211H Epi-R cells, we analyzed their inhibitory effect on the ALDH-1-high subpopulation as well as the CD44v10positive subpopulation. ALDH-1-high subpopulation represented approximately 17.6% of the overall cellularity in MSTO-
combination of STS and Epi. The in vitro cytotoxicity studies against MSTO-211H cells revealed that the addition of only 25 ng/mL of STS to 125 ng/mL of Epi significantly decreased the surviving fraction of cells when compared to 125 ng/mL Epi or 125 ng/mL STS alone, supporting a synergistic effect between Epi and STS (Figure 3A). The cytotoxicity of the STS/Epi combination was further augmented at higher ratios of STS, and a minimal surviving fraction of cells was observed for 62.5 or 125 ng/mL STS plus 125 ng/mL Epi (Figure 3A), indicating the potential of the STS/Epi combination for eradicating whole tumor cells. Interestingly, we observed that STS and Epi interact with each other, as denoted by the quenching of the fluorescence signal of STS by the addition of Epi (Figure 3B). The interaction between the drugs was further confirmed by a Job plot from 1H NMR spectra (Figure S3). This affinity between the drugs can be advantageously exploited for incorporating STS molecules into the core of our previously developed Epi/ m, which are being evaluated in phase I clinical studies. These micelles are prepared from PEG-b-poly(aspartate) copolymers conjugating Epi through hydrazone links (Figure 3C), which allow controlled release of Epi at endosomal pH. Thus, STS was first mixed with the PEG-b-poly(aspartate-Epi) copolymers in methanol at a 1:5 weight ratio of STS to Epi, which showed synergistic activity in the in vitro cytotoxicity studies (Figure 3A). After the methanol was evaporated and 10 mM HEPES buffer was added (pH 7.4), micelles incorporating both STS and Epi in their core were obtained. The STS/Epi/m were approximately 50 nm in diameter, which was comparable to that of Epi/m (Figure 3D). As the translation of STS into clinical studies has been limited due to its poor solubility in water, rapid inactivation by human alpha-1 acid glycoprotein 5647
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Figure 6. Effect of the pH-sensitive drug release of STS/Epi/m on their activity. (A) Drug release rate from the micelles under pH conditions of the bloodstream (pH 7.4), intratumoral space (pH 6.5), and endosomes (pH 5.5). Top panel: pH-sensitive STS/Epi/m (with Epi conjugated via a hydrazone bond). Bottom panel: pH-insensitive STS/Epi/m (with Epi conjugated via and amide bond). (B) Antitumor activity against bioluminescent MSTO-211H-luc orthotopic tumors after intravenous injection of HEPES, pH-sensitive STS/EPI/m and pHinsensitive STS/EPI/m, Epi/m, and pH-insensitive STS/EPI/m plus STS. Data are expressed as the mean ± SD (n = 8).
211H Epi-R (Figure 4C). After treatment with STS/Epi/m, the ALDH-1-high percentage was dramatically diminished to 4.45%. Moreover, STS/Epi/m significantly increased the CD44v10-negative population of MSTO-211H cells from 6.68 to 52.1% (Figure S4B). In addition, the ability of STS/ Epi/m to eradicate CSCs was also confirmed in Epi-resistant MCF-7 cells, MCF-7 Epi-R, which show approximately 25% of ALDH-high subpopulation (Figure S5A) and 35% of CD44positive/CD24-negative subpopulation (Figure S5B). Thus, after treatment with STS/Epi/m, the ALDH-high subpopulation was reduced to less than 1% (Figure S5C), while the CD44-positive/CD24-negative fraction was decreased to 2.4% (Figure S5D). These results indicate that STS/Epi/m could be a potent therapeutic strategy against recurrent tumors with increased CSCs. STS/Epi/m Eradicate Orthotopic Mesothelioma Tumors. The antitumor effect of STS/Epi/m was evaluated against an orthotopic dissemination model of bioluminescent mesothelioma (MSTO-211H-luc) cells (Figure 5). The tumor burden was followed through bioluminescence imaging by using an in vivo imaging system (IVIS) after the injection of 150 mg/kg of luciferin. Five days after tumor inoculation in the pleural cavity, mice were intravenously injected with Epi/m (6 mg/kg Epi), STS/Epi/m (1.2 mg/kg STS and 6 mg/kg Epi), free Epi (6 mg/kg), free STS (1.2 mg/kg), free STS plus free Epi (1.2 mg/kg and 6 mg/kg, respectively), and Epi/m plus free STS (6 mg/kg and 1.2 mg/kg, respectively) for nine times, initially every 4 days on days 5, 9, and 13 and then once every 10 days on days 23, 33, 43, 53, 63, and 73. By quantifying the bioluminescent signal from the orthotopic tumors, STS/Epi/m were found to reduce the tumor burden, as the bioluminescence of MSTO-211H-luc cells was effectively suppressed, whereas mice treated with HEPES, single drugs, or the combination of drugs failed to inhibit the growth of the tumors (Figure 5A). Representative bioluminescent images are shown
in Figure 5B, illustrating the strong and uniform suppression of the bioluminescent signal of MSTO-211H-luc tumors for mice treated with STS/Epi/m. Moreover, despite the repeated dosing of STS/Epi/m, mice did not show any body weight loss during the experiment (Figure 5C), indicating the safe profile of the micelles. Survival curves revealed that all mice injected with STS/Epi/m were alive for more than 3 months (Figure 5D). It is worth noting that the micelles were administered until day 73 and that the median survival of mice was extended for more than 1 year (Figure S6), which further supports the eradication of orthotopic MSTO-211-luc tumors by STS/Epi/ m. pH-Sensitive Drug Release of STS/Epi/m Is Essential for Activity. To confirm the role of the pH-sensitive design of the drug release from STS/Epi/m on their efficacy, a pHinsensitive version of the STS/Epi/m was constructed by using PEG-b-poly(aspartic acid) copolymers conjugating Epi via an amide bond (Figure S7A). The STS/Epi/m and the pHinsensitive version of the micelles were then incubated under pH conditions of the bloodstream (pH 7.4), intratumoral space (pH 6.5), and endosomes (pH 5.5), and the concentration of released drugs was measured by using high-performance liquid chromatography (HPLC). In agreement with their pH-sensitive design, the STS/Epi/m did not release the drug at pH 7.4, which serves to avoid drug leakage from the micelles during circulation in the body, while the Epi release was accelerated as the pH decreased (Figure 6A). Interestingly, STS followed the pH-sensitive release profiles of Epi from STS/Epi/m (Figure 6A), which may be associated with the aforementioned interaction between STS and Epi. Conversely, the pHinsensitive version of the micelles did not release the drug at any pH (Figure 6A). Thus, STS/Epi/m could synchronize the release of both drugs in intracellular acidic compartments, such as endosomes and lysosomes, allowing synergistic efficacy 5648
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Figure 7. Collaborative drug interactions in staurosporine/epirubicin-loaded micelles enable synergistic activity. (A) In vitro cytotoxicity of Epi, STS, STS plus Epi, Epi/m, and STS/Epi/m incubated with or without 0.5 mg/mL of human alpha-1 acid glycoprotein. Data are expressed as the mean ± SD (n = 3). (B) In vitro confocal laser scanning microscopy of MSTO-211H and MSTO-211H Epi-R cells incubated with Epi, Epi plus STS, Epi plus verapamil (VPM), Epi/m, and STS/Epi/m. Top panels: Intracellular fluorescence of Epi (red). Bottom panels: Intracellular fluorescence of eFluxx-IDH MDR (green). (C) Quantification of the fluorescence intensity of Epi or eFluxx-IDH MDR from the microscopies in (B) normalized by their fluorescence intensity in original MSTO-211H cells. Data are expressed as the mean ± SD (n = 3); *p < 0.01. (D) Western blot of Akt phosphorylation (p-Akt) after treatment with STS/Epi/m or Epi/m. GAPDH was used as internal reference. (E) Abrogation of cell cycle arrest by staurosporine/epirubicin-loaded micelles determined by flow cytometry. The cell cycle was analyzed by flow cytometry through quantitation of the DNA content with propidium iodide (PI) staining 24 h after the treatment with Epi/m and STS/ Epi/m. (F) Cell cycle distribution in MSTO-211H cells treated with Epi/m and STS/Epi/m assessed by PI staining and flow cytometry (n = 3). (G) Western blot of Chk-1 phosphorylation (p-Chk-1) after treatment with STS/Epi/m and Epi/m. G3PDH was used as an internal reference.
suppression of the tumor growth (Figure 6B), supporting their smart pH-sensitive design for synchronized synergistic therapy. Synergistic Antitumor Efficacy of STS/Epi/m Is Mediated by Collaborative Drug Interactions. The mechanisms for the enhanced efficacy of STS/Epi/m against CSCs and drug-resistant cells were thoroughly studied considering the micelle design and the cooperative behaviors of the drugs. First, as the access to the targeted kinases by STS, as well as its homologue UCN-01,31,32 is hindered by binding to hAGP in blood, we evaluated the ability of STS/Epi/m to prevent the inactivation of STS by hAGP. When the MSTO211H cells were treated with STS in medium without hAGP, free STS could display its antitumor effect, and the activity of the combination of free STS plus free Epi was comparable to
through coherent interactions between the drugs to affect interconnected signaling pathways within tumor cells. To prove that the pH-controlled drug release of STS/Epi/m is key in their biological performance, we first evaluated the in vitro cytotoxicity of the micelles against MSTO-211H cells. In accordance with the drug release rate, the pH-insensitive micelles demonstrated efficacy lower than that of pH-sensitive micelles (Figure S7B). Second, we studied the in vivo antitumor effect of the micelles against the orthotopic MSTO-211H-luc model by quantification of the bioluminescent signal (Figure 6B). Epi/m, STS/Epi/m, the pH-insensitive STS/Epi/m, and the pH-insensitive STS/Epi/m plus STS were injected three times on days 5, 9, and 12. Results indicated that only the administration of pH-sensitive STS/Epi/m resulted in the 5649
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Epi/m stopped the cell cycle of MSTO-211H cells at the G2/M period (Figure 7E; PI-A peak at 4N) depending on the concentration of Epi, with 0.5 μg/mL Epi arresting more cells than 0.05 μg/mL Epi. However, after exposure to STS/Epi/m, the amount of cells arrested at G2/M was diminished, and cells go through the G2 period (Figure 7E; PI-A peak at 4N). When the flow cytometry results were computed, approximately 50% of the cells treated with Epi/m (0.5 μg/mL Epi) were arrested at the G2/M phase, while less than 20% of the cells treated with STS/Epi/m remained in the G2/M period (Figure 7F). Western blotting showed that STS/Epi/m inhibited the phosphorylation of Chk-1 (Figure 7G). In addition, to distinguish between cells in different phases of the cell cycle, we used HeLa cells stably expressing fluorescent ubiquitinationbased cell cycle indicator proteins (HeLa-fucci) as cell cycle sensors.40 Fucci fusion protein effectively labels an individual G1 phase in red, the G2/M phase in green, and the S phase in yellow. We visualized the cell cycle of HeLa-fucci by time-lapse CLSM and counted the number of green, red, and yellow cells at indicated time points (Figure S11A and Movies S1−S6). When the cells were treated with free Epi, Epi/m, and free cisplatin, there was an increase of the cells in the G2/M phase and a decrease of the cells in the G1 phase in the 12−48 h period (Figure S11A). STS/Epi/m-treated cells showed a reduction of the G2/M cell fraction in the 12−48 h period, and the total number of cells decreased due to apoptosis (Figure S11A). Moreover, the pH-insensitive version of STS/Epi/m was not able to overcome the arrest in the G2/M period (Figure S11A and Movie S5). These results indicated that the STS/Epi/m overcame the cell cycle arrest in the G2/M phase induced by Epi, skipping the DNA repair checkpoint.
that of the STS/Epi/m (Figure 7A). However, the addition of 0.5 mg/mL of hAGP to the cell culture media extremely decreased the antitumor effect of free STS against MSTO211H, and the addition of free STS to free Epi did not improve the cytotoxicity (Figure 7A). On the other hand, the cytotoxicity of STS/Epi/m was not affected by addition of hAGP (Figure 7A), indicating that the stable loading of STS in the micelles and the selective intracellular release at acidic endosomal pH can avoid the binding of STS to hAGP and may permit preserving the effectiveness of STS in the in vivo setting. The STS/Epi/m could overcome Epi resistance in mesothelioma cells, which can be associated with the increase of ABC transporters that efflux anticancer agents from cells.33 This resistance to Epi in MSTO-211H Epi-R occurs by the induction of a particular ABC transporter (i.e., MDR-1), as confirmed by flow cytometry studies, but not the ABC transporters BCRP or MRP-2 (Figure S8). Because STS and its homologues can inhibit ABC transporters,34,35 we studied the ability of STS/Epi/m to suppress the efflux of Epi from MSTO-211H Epi-R cells by using confocal laser scanning microscopy (CLSM). The fluorescent signal of free Epi was observed inside the original MSTO-211H cells after 1 h incubation (Figure 7B(i), red). The same cells incubated with the ABC transporter indicator, eFluxx-ID-GFP, also showed high fluorescent levels (Figure 7B(ii), green). However, in MSTO-211H Epi-R cells, the intracellular fluorescence of free Epi and eFluxx-ID-GFP was severely reduced (Figure 7B(i,ii)). Quantification of the fluorescent signals of Epi and eFluxx-IDGFP confirmed the significant depletion of the drugs (Figure 7C(i,ii)). The addition of free STS significantly suppressed the outflow of free Epi and eFluxx-ID-GFP from MSTO-211H EpiR, and the fluorescent levels were comparable to that of the cells treated with the ABC transporter inhibitor, verapamil (Figure 7B(i,ii),C(i,ii)). MSTO-211H Epi-R also expelled Epi/ m micelles (Figure 7B(iii),C(iii)), which is in agreement with our recent observations showing that doxorubicin-conjugated block copolymers are removed by ABC transporters.36 In contrast, STS/Epi/m successfully maintained high fluorescent levels for both Epi and eFluxx-ID-GFP (Figure 7B(iii,iv),Cii,iv),C(iii,iv)), indicating the effective suppression of the efflux by ABC transporters. The levels of eFluxx-ID-GFP in the same cells were also analyzed by flow cytometry (Figure S9). The ability of the STS/Epi/m to reduce drug efflux in resistant cells was also verified in the Epi-resistant MCF-7 cells (MCF-7 EpiR) (Figure S10). The results further confirmed the inhibition of the drug efflux by the STS/Epi/m. Several reports have also shown that Akt, which promotes cell survival, is transiently elevated in various cancer cell lines as a result of the exposure to doxorubicin, conferring drug resistance.37 Here, we found that Epi/m increased phosphorylated Akt (p-Akt) in MSTO-211H cells, whereas STS/Epi/m remarkably down-regulated p-Akt (Figure 7D), further supporting the cooperative interaction for intracellular drug release of STS/Epi/m. STS/Epi/m Abrogate the Cell Cycle Arrest. Another mechanism of chemoresistance in cancer cells and CSCs is the repair of DNA damage.5 After the damage, the cell cycle stops at the G2 checkpoint and DNA is repaired. In this process, Chk-1 is a key enzyme for arresting the cycle for DNA damage inspection.38 As STS has been identified as a potent inhibitor of Chk-1, which can abrogate cell cycle arrest at the G2/M period and recovery of DNA damage,39 we evaluated if STS/Epi/m could prevent cells from arresting in response to DNA damage.
DISCUSSION Effective therapies against CSCs, in addition to conventional chemotherapy, appear to be essential for achieving long-term patient survival and avoiding relapse of the disease. Herein, we found that the pan-kinase inhibitor STS and its homologues can effectively eliminate the CSC subpopulation in mesothelioma cells at picomolar concentrations. The incorporation of STS into EPI/m not only overcame the inherent issues of STS for clinical translation, such as poor tumor selectivity, low solubility in water, and inactivation by hAGP, but also emerged as a safe and potent therapeutic entity cooperatively synchronizing the therapeutic effects of STS and EPI at the intracellular level. Thus, the resulting STS/EPI/m eradicated orthotopic xenografts of Epi-resistant mesothelioma by down-regulating drug resistance mechanisms and depleting the CSC in the tumors to extend the median survival of mice for more than 1 year. These findings indicate the high potential of targeted therapies of STS for treating recurrent and recalcitrant tumors and the ability of polymeric micelles to evolve into multicomponent nanoformulations rationally devised for achieving synchronized and cooperative drug interactions with synergistic therapeutic effects. Nanomedicine has demonstrated advantages for combining drugs within single platforms for regulated pharmacokinetics and concentration of drugs in tumors.41−43 Our study innovates the strategy by evolving multicomponent polymeric micelles designed to achieve synchronized synergistic drug cooperation from a molecular viewpoint, which affect interconnected signaling pathways in cancer cells and CSCs. Thus, the multicomponent STS/Epi/m rationally considered the interaction of STS and Epi molecules for incorporation in the core 5650
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controlling the drug release mechanisms of nanomedicines allows circumventing the leakage of the loaded drugs during circulation and specifically releasing the cargo based on endogenous triggers, such as endosomal pH, which can avoid toxicity and improve efficacy. Indeed, polymeric micelles designed for selectively releasing Pt drugs in the late endosomes/lysosomes after internalization by cancer cells showed enhanced antitumor activity and were able to overcome drug resistance by evading detoxification mechanisms present in the cytoplasm.55 However, even though Epi/m cells used in current study are designed for specific drug release at endosomal pH, they may not overcome Epi resistance, as doxorubicin-loaded micelles with comparable structure are recognized by ABC transporters and excreted from doxorubicin-resistant cancer cells.41 Therefore, the convenient coincorporation of STS, which readily down-regulates the ABC transporter, inside the multicomponent STS/Epi/m and the endosomal pH-mediated co-release of both STS and Epi from the micelles conform a rational configuration for overcoming Epi resistance, while destroying differentiated cancer cells and CSCs. STS/Epi/m were found to be even more effective than the combination of free Epi and free STS both in vitro and in vivo, most likely due to the protection of STS inside the core of the micelles from the inactivation by hAGP, the effective intracellular drug release mediated by the pH of endosomes, and the synchronized intracellular action of the drugs. Moreover, while most CSC inhibitors in clinical stages present low potency and should be administered repeatedly and at high concentration, the strong inhibitory activity of STS against CSCs allows using low amounts of STS inside STS/Epi/m, which was precisely tailored to 1:5 weight ratio of STS to Epi, and still obtains synergistic efficacy. Besides, the stable loading of STS and Epi inside the micelles at pH 7.4, which avoided drug release during circulation in the bloodstream, was also essential for the safety of STS/Epi/m. Thus, the weekly administration of STS/Epi/m could eradicate orthotopic mesothelioma without any body weight loss.
of micelles and simultaneous release inside the cells, which allows concurrent delivery of both drugs, with STS working as a companion for Epi, where the ABC transporters and Akt induced by Epi37 as well as the inherent Epi-resistance mechanisms in CSCs are down-regulated by STS. In addition, STS/Epi/m build on the standpoints of practicality and safety, indicating that this approach has the potential to launch translationable therapies with remarkable efficacy for efficient eradication of tumors and circumvention of recurrence by eliciting synergistic functions through coherent synchronization of drug actions within tumor cells. STS/EPI/m effectively eliminated the CSC fraction in MSTO-211H cells. This potent efficacy can be correlated with the strong inhibitory effects of STS on PKC, Akt, and Chk-1 in the nanomolar order.44 Moreover, as CSCs are maintained by PKC/Akt signaling in various tumors, such as colon cancer, glioma, or pancreatic cancer,6,7,45 STS may serve as a broad and effective CSC inhibitor for other malignancies besides mesothelioma. STS can also inhibit PKC and focal adhesion kinase (FAK) signaling,44,46 which is involved in selfrenewal, maintenance, and tumorigenicity of CSCs in various cancers. Interestingly, even though the CSCs of the MSTO211H cells used in this study were found to be particularly insensitive to FAK inhibition due to the high expression of Merlin protein,23 STS could effectively eradicate them probably due to its pan-kinase inhibitory activity. In addition, an inhibitory activity in the micromolar order of STS against sonic hedgehog (SHh) signaling has been reported.47 SHh signaling has been described in several human cancers and plays a significant role in self-renewal and survival in CSCs,6,10 and Vismodegib, that is, the first clinically approved SHh inhibitor,48 has confirmed the practicality of the SHh inhibition strategy in patients of various cancers, including basal cell carcinoma,49 medulloblastoma,50 and pancreatic cancer.51 Yet, Vismodegib and most drugs targeting the Hh pathway inhibit SHh through the smoothened receptor (SMO), which may not affect tumors having mutations in SMO or molecular dysregulations downstream of SMO.52 Since the inhibition of SHh by STS is related to GLI1 inactivation, STS could overcome issues regarding SMO targeting. In addition, STS is a potent inducer of apoptosis through the formation of reactive oxygen species, generation of cytochrome c, and inhibition of topoisomerase, which can further facilitate the removal of cancer cells and CSCs.53 While the most effective method of cancer treatment by using molecular inhibitors is to adjust the therapy that specifically targets altered epigenetics on an individual basis, the extent of distress in the signaling pathways is usually variable, and hitting only one pathway is unlikely to eradicate cancer. Thus, the multitargeting capability of STS offers an efficient therapeutic approach against CSCs, concurrently bypassing the development of drug resistance, though such strategy could also lead to undesired effects, as the targeted signaling pathways are also found in normal stem cells. In this way, nanomedicine holds great promise for developing safe and effective CSC directed therapies by selectively delivering otherwise toxic multitargeted inhibitors, such as STS, to their site of action within tumors. Nanomedicines capable of broad distribution in tumor tissues could further ease reaching CSCs. In this way, the 50 nm diameter of STS/EPi/m is a substantial advantage for achieving effective targeting, as we have recently demonstrated that the size of polymeric micelles is essential for their extravasation and deep penetration within tumors.54 Moreover,
CONCLUSIONS STS/Epi/m nanomedicines eradicated orthotopic mesothelioma xenografts bearing recalcitrant CSC subpopulation through the facilitated co-delivery of STS and Epi to tumor tissues and triggered and coordinated release of both drugs at endosomal pH, which allowed the reversal of drug resistance through inhibition of the ABC transporter, abrogation of the cell cycle arrest, and effective elimination of CSC subpopulation. Our study demonstrates the potential of manipulating drug interactions within nanomedicine compartments as an effective modality for designing therapies capable of coordinating synergistic effects for eliminating both cancer cells and recalcitrant CSC subpopulations in tumors. Such nanomedicines could allow achieving the long-sought disease-free survival, majorly impacting cancer treatment. METHODS Materials. The chemicals, cells lines, and animals used in this study are listed in Supporting Information and Methods. All the experiments were conducted under the ethical guidelines of The University of Tokyo. Aldefluor Activity Assay and Sorting ALDH-High Population by FACS. The Aldefluor kit (StemCell Technologies, Durham, NC, USA) was used to isolate the population with a high ALDH enzymatic activity, according to the manufacturer’s instructions. Cells were 5651
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fluorescence spectrometry by using a Jasco FP-6500 spectrometer (Jasco, Tokyo, Japan). Then, increasing concentrations of Epi were added to 0.03 mg/mL STS, and the drop in the fluorescence intensity of STS was followed. Second, the interaction of STS and Epi was confirmed by 1H NMR. The drugs were dissolved in methanol-d4 and tetrahydrofuran-d8 (1:1 v/v ) and mixed at 10:0, 8:2, 6:4, 5:5, 4:6, 2:8, and 0:10 ratios. The shift of the peaks of STS and Epi was measured by 1H NMR (Varian UNITY INOVA 500 MHz, CA, USA). Preparation of pH-Sensitive and pH-Insensitive STS/Epi/m. The pH-sensitive Epi/m were prepared from PEG-b-poly(aspartatehydrazide-epirubicin) copolymer (100 mg) (Mw of PEG = 12 000 Da; poly(aspartate) units = 40; Epi units = 8), while the pH-sensitive STS/ Epi/m were prepared with the same block copolymer plus STS (5 mg). The block copolymer, or the block copolymer plus STS, was dissolved in methanol (50 mL), protected from light, and stirred for 30 min at room temperature. Then, the solutions were evaporated using a rotatory evaporator, followed by addition of 50 mL of HEPES buffer (10 mM, pH 7.4) into the flask containing the dried sample, and the mixture was sonicated for 30 min. The pH-insensitive STS/Epi/m were prepared in a similar manner from PEG-b-poly(aspartate-amideepirubicin) copolymer (100 mg) (Mw of PEG = 12 000 Da; poly(aspartate) units = 40; Epi units = 8) plus STS (5 mg). All micelles were then purified by ultrafiltration (molecular weight cutoff (MWCO) = 30 000 Da) and concentrated to a total volume of 15 mL. Finally, the micelles were filtered by using a PES filter (0.22 μm). The size of the micelles was measured by dynamic light scattering by using a Zetasizer Nano ZS (Malvern, UK), and the concentration of the drugs inside the micelles was determined by HPLC (column: Tosoh with TSK gel 80-TM; temperature = 40 °C; flow rate = 0.8 mL/min; mobile phase = 2:3 vol/vol mixture of 1 mM formic acid and methanol) by using a UV detector at 254 nm for STS and 290 nm for Epi. Drug Release Rate. The pH-sensitive or the pH-insensitive STS/ Epi/m (0.3 mL; 1 mg/mL Epi; 0.2 mg/mL STS) were loaded in dialysis bags (MWCO = 3500 Da) and dialyzed against HEPES buffer (30 mL) at various pH values (5.5, 6.5, and 7.4). The concentration of the drugs in the dialysate was determined at defined time points by HPLC by using above-mentioned conditions. Antitumor Activity against an Orthotopic Mesothelioma Model. Orthotopic xenograft models of malignant mesothelioma were prepared as previously described.56 Four week old male Balb/c nu/nu mice were anesthetized, and 2 × 106 MSTO-211H-luc cells suspended in 100 μL PBS solution were injected into their left pleural cavity. The day that the cells were injected into the mice was defined as day 0. Five days later, when the tumor nodules were established, mice (n = 10) were randomized, and drugs or micelles were administered by tail vein injection. Epi/m (6 mg/kg Epi), STS/Epi/m (1.2 mg/kg STS and 6 mg/kg Epi), free Epi (6 mg/kg), free STS (1.2 mg/kg), free STS plus free Epi (1.2 mg/kg and 6 mg/kg, respectively), and Epi/m plus free STS (6 mg/kg and 1.2 mg/kg, respectively) were intravenously injected nine times, initially every 4 days on days 5, 9, and 13 and then once every 10 days on days 23, 33, 43, 53, 63, and 73. The tumor luminescence signal from the MSTO-211H-luc tumors was followed twice a week by using an in vivo imaging system after the injection of 150 mg/kg of luciferin. The body weight of the mice was also measured and taken as a parameter of treatment toxicity. The statistical significance was assessed by the Mann−Whitney U-test. The survival of mice was followed for more than 1 year, and the significance of the extension was determined by a log-rank test. Apoptosis Analysis with Annexin V Staining. Epi, STS, Epi plus STS, Epi/m, Epi/m plus STS, or STS/Epi/m with various concentrations was added to the cells and harvested for 24 h. The cells were then stained with Annexin V and DAPI, and 1 × 106 cells were counted to identify living (Annexin V-negative, DAPI-negative), apoptotic (Annexin V-positive, DAPI-positive or -negative), and necrotic cells (Annexin V-negative, DAPI-positive) by a FlowJo software. Data were analyzed by ANOVA with the Student− Newman−Keuls method multiple comparison test using Prism 5 software.
suspended in Aldefluor assay buffer containing ALDH substrate (BODIPY-aminoacetaldehyde) and incubated for 30 min at 37 °C. As the negative control, an aliquot of each cell was treated with 50 mM diethylaminobenzaldehyde (DEAB) inhibitor. The sorting gates were established using, as negative controls, the cells stained with DAPI (4′,6-diamidino-2-phenylindole) only and, for viability, the Aldefluorstained cells treated with DEAB and the staining with secondary antibody alone. Sorting of MSTO-211H-luc cell subpopulations was performed by using a MoFlo Astrios FACS instrument (Beckman Coulter, Brea, CA, USA). Spheroid Colony Formation Culture. Cells from a MoFlo Astrios FACS (Beckman Coulter, Brea, CA, USA) sorting were immediately plated in ultralow attachment plates (Corning Life Sciences, Acton, MA, USA) at a density of 2000 viable cells per milliliter in serum-free DMEM/F12 medium containing 5 μg/mL insulin (Sigma-Aldrich, St. Louis, MO, USA), 0.4% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA), 10 ng/mL basic fibroblast growth factor (Invitrogen, Waltham, MA, USA), and 20 ng/ mL human recombinant epidermal growth factor (Invitrogen, Waltham, MA, USA) until the growth of spheres. Single cell suspensions were prepared using Accutase (Millipore, Billerica, MA, USA) after 7 days. The number of colonies was counted in 6-well plates. Real-Time PCR. Real-PCR experiments for Nanog, Sox2, and Oct4 are described in Supporting Information and Methods. SCID Mouse Implantation. ALDH-high and ALDH-low cells were collected, mixed with Matrigel (BD Bioscience, Franklin Lakes, NJ, USA), and implanted subcutaneously into the left (ALDH-high) and right (ALDH-low) flanks of SCID mice (n = 3 or 4) at 1 × 103, 1 × 104, or 1 × 105 cells per mouse. The size of the tumors was assessed twice a week to monitor the tumor growth. Effect of Radiation and Hypoxia on the CSC Subpopulation of MSTO-211H Cells. The assessment of the Aldefluor-positive subpopulation after various X-ray irradiations is described in Supporting Information and Methods. The effect of incubating MSTO-211H cells under hypoxic conditions on the Aldefluor-positive subpopulation is also depicted in Supporting Information and Methods. Screening of CSC Inhibitors. MSTO-211H cells were seeded at 1 × 106 cells in 75 cm2 dishes with 10 mL of medium. Twenty-four hours later, the medium was changed. The CSC inhibitors were dissolved in DMSO, and serial concentrations of the drugs (final concentrations of 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, and 10 μM) were added to 10 mL of medium (n = 3). After 72 h, the surviving cells (Trypan blue negative cell) were counted by a Countess automated cell counter (Invitrogen, Carlsbad, CA, USA), and 1 × 106 living cells were stained by the Aldefluor assay kit and analyzed by a BD LSR II flow cytometer (BD Bioscience, Franklin Lakes, NJ, USA). Data analysis was performed using FlowJo software (Tree Star, Inc., Ashland, OR, USA). IC50 and EC50 values were measured against DMSO-treated cells using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA). Analysis of Side Population. The side population in MSTO211H cells after treatment with different drugs is shown in Supporting Information and Methods. Staining of CSC Marker Phenotype (FCM) and ABC Transporters. To identify the CSC markers (CD44, CD24, or CD44v10) and ABC transporter (MDR-1, BCRP, or MRP-2), the cells were removed from the culture dish with Accutase, washed with phosphate-buffered saline (PBS), suspended at 1 × 107 cell/ml in 100 μL of PBS, and labeled with antibodies. The cells were marked with anti-CD44-APC, anti-CD24-FITC (fluorescein isothiocyanate), antiCD44v10-PE-Cy5, anti-MDR-1-PE, anti-BCRP-APC, and anti-MRP2-FITC antibodies. Thirty minutes after being mixed at 4−8 °C, the cells were washed twice with PBS and analyzed by the BD LSR II flow cytometer. Interaction between STS and Epi. The interaction between STS and Epi was first studied by the fluorescence quenching of STS after the addition of Epi in methanol. For this, STS and Epi were dissolved in methanol and the fluorescence spectra of each drug obtained by 5652
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ACS Nano Evaluation of Drug Efflux by the ABC Transporter. MSTO211H Epi-R cells were incubated with free Epi, free Epi plus STS, free Epi plus verapamil, Epi/m, and STS/Epi/m for 1 h. A LSM780 confocal laser scanning microscope (Zeiss, Germany) equipped with He/Ne and argon lasers and filters set at 480 nm excitation/585 nm emission or 530 nm emission was used. The excretion activity of the transporters was confirmed by following the fluorescence signal of eFluxx-ID green dye (ENZO Life Sciences, Inc., Farmingdale, NY, USA). The efflux of the drugs was further studied by using a BD LSR II flow cytometer equipped with a blue (488 nm) laser, and the signals were registered in the FITC (530/30 filter) channels. Data analysis was performed using FlowJo software (Tree Star, Inc., Ashland, OR, USA). Cytotoxicity Assay and Effect of Human Alpha-1 Glycoprotein on Cytotoxicity with STS. The cytotoxicity of the drugs against MSTO-211H cells was compared with or without the addition of 0.5 mg/mL human alpha-1 glycoprotein (Sigma, St. Louis, MO, USA). The cells (3 × 105) were seeded in 96-well plates with 100 μL medium containing the drugs. At each time point, the cytotoxicity was measured using a cell counting kit 8 (Funakoshi, Tokyo, Japan). Western Blot Analysis. The Western blot study of the phosphorylation of Akt and Chk-1 is described in Supporting Information and Methods. Effect of Drugs on the Cell Cycle of MSTO-211H. MSTO211H cells were plated at 2 × 105 cells/75 cm2, which provided 10− 20% confluence 24 h later. Cells were then synchronized in the S phase by adding 2 mM thymidine for 18 h, released for 8 h by three washes, and treated again with 2 mM thymidine. Following the second release from thymidine, 95% of cells were in the S phase. Synchronized cells were then returned to complete medium containing 0.5 μg/mL Epi/m, which was found to be sufficient to arrest cells in early G2 phase without inducing apoptosis, with or without the combination with STS, Epi/m, or STS/Epi/m for up to 18 h. Cell cycle profiles were performed with a BD LSR II flow cytometer using a procedure for Hoechst 33342 staining of nuclei and analyzed by FlowJo software. Time-Lapse Microscopy of the Effect of Drugs on the Cell Cycle of HeLa-Fucci Cells. The progression of the cell cycle during drug exposure was also followed in human cervical cancer HeLa cells using the fluorescent FUCCI system.40 The procedure is explained in Supporting Information and Methods.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS The authors thank Q. Bou and K. Date for their technical assistance. This research was financially supported by Center of Innovation Program (COI stream) from Japan Science and Technology Agency (JST) and Funding Program for WorldLeading Innovative R&D on Science and Technology (FIRST) from the Japan Society for the Promotion of Science (JSPS). A part of the data were originally obtained through the project “DDS” (led by Prof. Kataoka) in the Project for Development of Innovative Research on Cancer Therapeutics (P-DIRECT)/ Ministry of Education, Culture, Sports, Science and Technology of Japan and was provided through the website of the National Bioscience Database Center (NBDC)/the Japan Science and Technology Agency (JST). REFERENCES (1) Visvader, J. E.; Lindeman, G. J. Cancer Stem Cells in Solid Tumours: Accumulating Evidence and Unresolved Questions. Nat. Rev. Cancer 2008, 8, 755−768. (2) Magee, J. A.; Piskounova, E.; Morrison, S. J. Cancer Stem Cells: Impact, Heterogeneity, and Uncertainty. Cancer Cell 2012, 21, 283− 296. (3) Medema, J. P. Cancer Stem Cells: The Challenges Ahead. Nat. Cell Biol. 2013, 15, 338−344. (4) Diehn, M.; Majeti, R. Metastatic Cancer Stem Cells: An Opportunity for Improving Cancer Treatment? Cell Stem Cell 2010, 6, 502−503. (5) Dean, M.; Fojo, T.; Bates, S. Tumour Stem Cells and Drug Resistance. Nat. Rev. Cancer 2005, 4, 275−284. (6) Dreesen, O.; Brivanlou, A. H. Signaling Pathways in Cancer and Embryonic Stem Cells. Stem Cell Rev. 2007, 3, 7−17. (7) Dubrovska, A.; Kim, S.; Salamone, R. J.; Walker, J. R.; Maira, S.M.; García-Echeverría, C.; Schultz, P. G.; Reddy, V. A. The Role of PTEN/Akt/PI3K Signaling in the Maintenance and Viability of Prostate Cancer Stem-like Cell Populations. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 268−273. (8) Reya, T.; Clevers, H. Wnt Signalling in Stem Cells and Cancer. Nature 2005, 434, 843−850. (9) Hoffmeyer, K.; Raggioli, A.; Rudloff, S.; Anton, R.; Hierholzer, A.; Del Valle, I.; Hein, K.; Vogt, R.; Kemler, R. Wnt/Beta-Catenin Signaling Regulates Telomerase in Stem Cells and Cancer Cells. Science 2012, 336, 1549−1554. (10) Zhou, J.; Wulfkuhle, J.; Zhang, H.; Gu, P.; Yang, Y.; Deng, J.; Margolick, J. B.; Liotta, L. A.; Petricoin, E., 3rd; Zhang, Y. Activation of the PTEN/Mtor/STAT3 Pathway in Breast Cancer Stem-Like Cells is Required for Viability and Maintenance. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 16158−16163. (11) Takebe, N.; Harris, P. J.; Warren, R. Q.; Ivy, S. P. Targeting Cancer Stem Cells by Inhibiting Wnt, Notch, and Hedgehog Pathways. Nat. Rev. Clin. Oncol. 2011, 8, 97−106. (12) Grinshtein, N.; Datti, A.; Fujitani, M.; Uehling, D.; Prakesch, M.; Isaac, M.; Irwin, M. S.; Wrana, J. L.; Al-Awar, R.; Kaplan, D. R. Small Molecule Kinase Inhibitor Screen Identifies Polo-Like Kinase 1 as a Target for Neuroblastoma Tumor-Initiating Cells. Cancer Res. 2011, 71, 1385−1395. (13) Gupta, P. B.; Onder, T. T.; Jiang, G.; Tao, K.; Kuperwasser, C.; Weinberg, R. A.; Lander, E. S. Identification of Selective Inhibitors of Cancer Stem Cells by High-Throughput Screening. Cell 2009, 138, 645−659. (14) Sachlos, E.; Risueño, R. M.; Laronde, S.; Shapovalova, Z.; Lee, J. H.; Russell, J.; Malig, M.; McNicol, J. D.; Fiebig-Comyn, A.; Graham, M.; Levadoux-Martin, M.; Lee, J. B.; Giacomelli, A. O.; Hassell, J. A.; Fischer-Russell, D.; Trus, M. R.; Foley, R.; Leber, B.; Xenocostas, A.; Brown, E. D.; et al. Identification of Drugs Including a Dopamine
ASSOCIATED CONTENT S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b00900. Characterization of the CSC subpopulation in mesothelioma cells, the in vitro evaluation of a panel of signaling-pathway inhibitors, activity of STS and its homologues against CSCs, inhibition of CSCs in mesothelioma and Epi-resistant breast cancer by STS/ Epi-loaded micelles, preparation and characterization of pH-insensitive micelles for control, a 1 year follow up of the mice survival, study of the ability of STS/Epi-loaded micelles to overcome drug resistance mechanisms (PDF) Movie S1 (MPG) Movie S2 (MPG) Movie S3 (MPG) Movie S4 (MPG) Movie S5 (MPG) Movie S6 (MPG)
AUTHOR INFORMATION Corresponding Authors
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