Salinomycin-loaded gold nanoparticles for treating cancer stem cells

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Salinomycin-loaded gold nanoparticles for treating cancer stem cells by ferroptosis-induced cell death Yongmei Zhao, Wei Zhao, Yi Chieh Lim, and Tianqing Liu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.9b00132 • Publication Date (Web): 22 Apr 2019 Downloaded from http://pubs.acs.org on April 23, 2019

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Molecular Pharmaceutics

Salinomycin-loaded gold nanoparticles for treating cancer stem cells by ferroptosis-induced cell death Yongmei Zhao1, Wei Zhao2, Yi Chieh Lim3, Tianqing Liu4*

1

School of Pharmacy, Nantong University, Nantong, China

2

School of Polymer Science and Engineering, Qingdao University of Science and

Technology, Qingdao, China 3Danish

4QIMR

Cancer Society Research Center, Copenhagen, Denmark Berghofer Medical Research Institute, Brisbane, QLD, Australia

Corresponding Authors * Email: [email protected]. QIMR Berghofer Medical Research Institute, 300 Herston Road, Brisbane, QLD 4006, Australia.

Conflict of Interest Statement All authors declare no conflicts of interest related to this study.

Acknowledgements Dr Tianqing Liu is supported by the National Health and Medical Research Council (NHMRC) Early Career Fellowship (Grant No. 1112258).

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Abstract: Cancer Stem Cells (CSCs) are a subpopulation of tumor cells which exhibits self-renewal, differentiation and tumorigenicity. CSCs are highly resistant to conventional cancer treatment and have been associated with metastasis. Several studies have been shown that salinomycin (Sal) has the potential to target cancer stem cells and evidenced by in vitro and in vivo tumor models. Here, salinomycin was conjugated with biocompatible gold nanoparticles (AuNPs) coated with poly(ethylene glycol) (PEG) to improve its specificity in targeting breast cancer stem cells (BCSC). BCSC derived from CD24low/CD44high subpopulation showed high sensitivity to Sal-AuNPs treatment. In-depth analysis on the mechanism of action of Sal-AuNPs indicated ferroptosis, an iron-dependent cell death was achieved as a result of iron accumulation and inhibition of antioxidant properties. This also led to the induction of oxidative stress, mitochondrial dysfunction, and lipid oxidation. Our findings suggest Sal-AuNPs is an efficient therapeutic avenue in eliminating cancer stem cells.

Key words: Gold nanoparticles, Salinomycin, cancer stem cells, ferroptosis, ROS

1. Introduction Cancer is a devastating disease of lethal consequence. Gain in oncogenic drivers mark the beginning of malignancy in which cancer cells are not only bestowed with the advantage of abnormal growth but also the ability to disseminate from the primary tumor. While surgical resection debulks the primary tumor with the intent to prolong patients’ survival, metastasis is largely incurable because tumour cells can disseminate to distant sites. This accounts for the majority of cancer death in patients.1,2 Globally, cancer is now the second leading cause of mortality.3 Disseminated tumour cells are increasingly recognized to contain pluripotent cells4 which can self-renew and initiate tumour growth upon settlement at distal sites away from the primary tumor.5,6,7 These breast cancer stems cells (BCSCs) exhibit high refractory towards conventional treatments such as chemo-8,9,10 and radiotherapies11.


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Hence, there is an urgent need to develop new treatment options that can specifically target BCSCs to improve patients’ survival, especially those with metastasis.

Salinomycin (Sal) is a polyether ionophore antibiotic with known efficacy as an anti-cancer drug.12 Pre-clinical data indicates this therapeutic agent has the potential to reduce tumorigenicity by targeting BCSCs and has been effective in several cancer types.13,14,15 As compared to standard chemotherapeutic agents, Sal is a potent drug with more than 100 fold efficacy in eliminating BCSCs.12,16 Currently, its molecular function is being elucidated. Several findings indicate Sal may play a role in dysregulating metal ions17 and inhibiting stem cell maintenance.18,19,20 However, therapeutic delivery of Sal to the site of tumor needs to be further studied due to the hydrophobicity and the cytotoxicity during systemic drug administration.12 Nanoparticle entrapment (nanocarrier) is a recent developed strategy to improve anti-cancer drug delivery. By increasing solubility as well as enabling enhanced permeability and retention effect (EPR), the system allows penetration across leaky vasculature and increase drug accumulation in the tumor.21,22 Therefore, nanocarriers are able to minimize free drug to non-target regions of the body while promoting better drug uptake by cancer cells.23 Drug nanoparticle formulations such as PLGA nanoparticles,24 lipid-polymer nanoparticles,25 and silver nanoparticles26 have been developed to improve the therapeutic efficacy of Sal. However, the underlying mechanisms of Sal-loaded nanoparticles to eradicate BCSCs are not well studied.

Gold nanoparticles (AuNPs) are excellent drug carriers due to their biocompatibility, easy synthesis and drug functionalization capability, and theranostic potentials.27,28,29 Several AuNPs-based therapies have been approved by U.S. Food and Drug Administration (FDA) for clinical trials.30 Taking into consideration the need for an efficacious anti-cancer drug while still able to reduce the potential damage to surrounding healthy tissues, we developed a biocompatible gold nanoparticles coated with poly(ethylene glycol) (PEG) which is conjugated to Sal. Our study indicates conjugation of Sal with AuNPs improves drug-induced cell death and BCSCs are



highly susceptible to this particular treatment. Hence, the use of Sal-AuNPs can improve the therapeutic efficacy of Sal against BCSCs and thus this strategy offers great potential in targeting BCSCs.

2. Materials and methods Materials: Chloroauric acid (HAuCl4·3H2O), sodium citrate, hydroquinone, Tween 20, N-hydroxysuccinimide (NHS), N-(3- dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), phosphate buffered saline (PBS), Salinomycin, Z-VAD-FMK, necrostatin-1, ferrostatin-1 were obtained from Sigma-Aldrich and used as received from the manufacturer without further purification. Water used in the experiments was purified with a Millipore water treatment system (organic content less than 5 ppb). Poly(ethylene glycol) thiol (HS-PEG-NH3) with an average molecular weight of 1000 g/mol was purchased from Rapp Polymer Gmbh.

Gold nanoparticles (AuNPs) synthesis and characterisation Monodisperse quasi-spherical AuNPs were synthesized using sodium citrate reduction methods.31 In brief, 1% gold chloride (HAuCl4) solution was heated to boiling, while 1% sodium citrate solution was added and reacted for 10 minutes. The color of the solution tunes to ruby red. After reaction, AuNPs were characterized using Varian Cary 5 UV-Vis-NIR spectrophotometer (UV-Vis), Zetasizer Nano ZS dynamic light scattering (DLS), and Philips CM100 transmission electron microscopy (TEM).

AuNPs surface modification AuNPs were first PEGylated by incubating with SH-PEG-NH3 under vortex overnight at 4 oC.31 After removal of the excess PEG molecules by centrifugation, the carboxylic acid groups of the prepared PEG-AuNPs were activated with EDC (15 mg/mL) and NHS (20mg/mL) for 5min. Salinomycin (0.1 mM) were mixed with 5 ml of AuNPs for 6 h and then sufficiently washed to remove excess Salinomycin before DLS measurements.


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Drug loading efficiency was measured by plate-reader After conjugation, Sal-AuNPs was separated from the solution by centrifugation and then salinomycin was dissolved in ethanol. Salinomycin loading efficiency was analysed by measuring UV-Vis-NIR spectrophotometer the following the published method.32 For the following cellular experiments, Sal and Sal-AuNPs were used at the same amount of salinomycin (Sal doses of 150 ng). DMSO used for preparing the drug stock solution was added drug vehicle control.

Cell culture and BCSC isolation MCF-7 cells were purchased from American Type Culture Collection (ATCC) and cultured in complete media consisting of DMEM supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 5% CO2. After serum-free treatment, BCSC sub-populations in the MCF-7 cells were isolated using MagCellect Human breast cancer stem cells isolation kit (R&D Systems) according to protocols from the manufacture. In brief, MCF-7 cells (1 x 107 cells/mL) were prepared in buffer and mixed with anti-human CD24 biotinylated antibody and then Streptavidin Ferrofluid for selection of CD24low cells. Those cells were subsequently incubated with a biotinylated anti-human CD44 antibody and Streptavidin Ferrofluid for selection of CD44high cells. After washing, magnetically tagged CD24low/CD44high BCSC cells were isolated through the magnet. The cells are directly treated with Sal, PEG-AuNPs, Sal-AuNPs, and control samples after isolation.

Cell proliferation assay Cells were seeded in a 96-well plate and incubated for 6 h to enable attachment. After adding control, Sal, PEG-AuNPs, or Sal-AuNPs samples, the cancer cell growth was measured every 6 h using real-time imaging with the Incucyte ZOOM. Cell confluency was determined using the Incucyte software.

Calcein/PI assay



BCSCs were treated with Sal, PEG-AuNPs, and Sal-AuNPs for 72h. To understand the cell death pathway, cells were also treated with or without apoptosis, necrosis, or ferroptosis inhibitors, which were Z-VAD-FMK (50uM), necrostatin-1 (20uM), or ferrostatin-1 (10uM), respectively. The concentrations used are based on earlier studies.19 After co-incubation, the medium were replaced with fresh cell culture medium. Cells were harvested and using Calcein/Propidium Iodide (PI) assay according to the manufacturer’s protocol (Life Technology). BCSC cell death was quantified using Canto II flow cytometer (BD Biosciences, San Jose, CA).

Analysis of ROS production Cellular ROS after the treatment was detected by the fluorescence change of an ROS sensitive dye, 2’,7’-dichlorofluorescin diacetate (DCFH-DA). After the treatment, the BCSC cells were incubated with cell culture medium with DCFH-DA (10 uM) for 30 min in dark. Cells were washed with PBS, harvested, and analyzed by FACS CANTO II (BD Biosciences) flow cytometer using 488nm laser for excitation.

Lipid ROS Lipid peroxide product malondialdehyde (MDA) was measured by MDA assay kit (Sigma-Aldrich) according to the manufacturer’s protocol. Briefly, isolated BCSC cells were treated, harvested, lysed, and reacted with thiobarbituric acid. MDA level was quantified by fluorescence signal at 532nm for excitation. Lipid ROS was also determined using C11-BODIPY dye. After the treatment, the BCSC cells were incubated with cell culture medium with C11-BODIPY (5 uM) for 20 min in dark. Cells were washed with PBS, harvested, and analysed by FACS CANTO II (BD Biosciences) flow cytometer using 488nm laser for excitation.

Mitochondria damage measurement Mitochondria superoxide was determined using MitoSOX dye. After the treatment, the BCSC cells were incubated with cell culture medium with MitoSOX (5 uM) for 20 min in dark. Cells were washed with PBS, harvested, and analysed by FACS


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CANTO II (BD Biosciences) flow cytometer. Mitochondria damage was evaluated with mitochondrial membrane potential dye Tetramethylrhodamine, methyl ester (TMRM). Cells were incubated with cell culture medium with TMRM (100 nM) and imaged using Zeiss 780-NLO scanning confocal microscope.

Intracellular glutathione (GSH) assay The intracellular GSH level was measured using a GSH commercial kit (Abcam) according to the manufacturer’s protocol. Briefly, isolated BCSC cells were treated, and then harvested and lysed using a homogenizer. The supernatant solution was then obtained by centrifugation at 10,000g. After mixing with reaction reagents, the GSH level was quantified by a colorimetric assay at the wavelength of 412 nm using a plate-reader.

Western blot The total cellular proteins were extracted by ice cold lysis buffer (Cell Signalling Technology) and quantified using the bicinchoninic acid protein assay. Protein samples were diluted and 50 μg of protein was loaded to SDS-polyacrylamide gel, separated by electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes. Proteins were probed with Glutathione Peroxidase 4 (GPX-4) (1:5000, Abcam), ferritin (1:1000, Abcam), and beta-actin antibodies (1:10000, Abcam). After sufficient washing, the membranes were incubated with near-infrared fluorescent secondary antibodies (Odyssey) in dark. The expression of the protein of interest was detected with Odyssey CLx system.

Tumorsphere formation MCF-7 cells were seeded into 24-well ultra-low attachment multiwell plates at density of 1000 cells/well. Tumorsphere formation was monitored using an inverted light microscope for 7 days. Tumorspheres larger than 50 μm in diameter were counted as positive.33,34,35 Tumorsphere formation efficiency was calculated as the tumorsphere number divided by the initial single cell number plated.



Statistical analysis All the experiments are performed at least three times. All data are presented as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA. *P-values < 0.05, **P-values < 0.01 and *** P-values < 0.001 were considered statistically significant. 3. Result and Discussion 3.1 Nanoparticle preparation and salinomycin loading AuNPs was first synthesized based on a well-established sodium citrate reduction method.31 It has been reported that smaller AuNPs (~20 nm) can be rapidly uptake by cells, making it an ideal delivery system for this study.36, 37 To reduce the aggregation of AuNPs, thiol-terminated polyethylene glycol (SH-PEG-NH3) was then conjugated to yield a high affinity Au-S-bond.38,39,40,41 The prepared PEGylated AuNPs with particle size of ~20 nm were observed using TEM (Figure 1A) that AuNPs showed uniform spherical shape. We confirmed the characteristic plasmon resonance absorption peak of the PEGylated AuNPs by using UV-Vis (Figure 1B). The particle size, zeta potential and drug loading coefficient of all three AuNPs (AuNPs, PEG-AuNPs and Sal-AuNPs) was then measured using TEM, DLS and UV-Vis, respectively (Figure 1C). The hydrodynamic size of the nanoparticles increased from 20.1 ± 0.5 nm to 20.9 ± 1.1 nm after conjugation with salinomycin. The surface charge also increased after drug loading, which confirmed the configuration of Sal-AuNPs. Approximately, 63.2 ± 4.7 % of salinomycin was loaded in Sal-AuNPs, characterized by measuring UV-Vis and free salinomycin were separated from the solution and subsequently removed by centrifugation.


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Figure 1. Characterization of gold nanoparticles: (A) TEM imaging of synthesized PEG-AuNPs; (B) UV–visible absorption of as-synthesized PEG-AuNPs; and (C) physiochemical properties of AuNPs, PEG-AuNPs and Sal-AuNPs. 3.2 In vitro cell viability With the success of establishing the Sal-AuNPs, we investigated if this nanocarrier can target breast cancer cells (BCCs) specifically for the cellular inhibition of breast cancer stem cells (BCSCs). In this study, BCCs is defined as CD24low / CD44low and BCSCs as CD24low / CD44high. As shown in Figure 2A and B, Sal-AuNPs under the same corresponding drug concentration showed cellular growth inhibition when compared to PEGylated AuNPs without Sal conjugation in BCCs and BCSCs. Moreover, BCCs and BCSCs were more sensitive to Sal-AuNPs treatment as compared to other treatment. This indicated the efficacy of Sal can be greatly improved by conjugating with AuNPs. The sensitivity and therapeutic efficacy of Sal-AuNPs was significantly improved. Cells treated with PEG-AuNPs showed reduction of cell proliferation as compared to control group, indicating good biocompatibility of the nanoparticles without drug loading. To better understand the activation of cellular induced-death by Sal-AuNPs, cells were treated with or without inhibitors of apoptosis, necrosis, and ferroptosis respectively as shown in Figure 2C. Cell death induced by Sal or Sal-AuNPs groups could be efficiently prevented by the ferroptosis inhibitor ferrostatin-1, whereas the apoptosis inhibitor Z-VAD-FMK and



necrosis inhibitor necrostatin-1, had no little effect on the prevention of cell death. These results are consistent with previous data showing Sal activates ferroptosis.19 Our findings suggested that ferroptosis is the dominant cell death pathway induced by both Sal-AuNPs and Sal, while Sal-AuNPs can enhance ferroptotic BCSC cell death.

Figure 2. The therapeutic impact of salinomycin nanocarrier on BCCs and BCSCs. (A) and (B) Cell proliferation study of Sal, PEG-AuNPs, and Sal-AuNPs against MCF-7 BCCs and BCSCs, respectively; and (C) cell viability of BCSCs were treated with Sal, PEG-AuNPs, and Sal-AuNPs for 72h in the presence or absence of the indicated cell death pathway inhibitors. 3.3 In vitro reactive oxygen species (ROS) study Oxidative stress plays a critical role in regulating ferroptosis of cancer cells.42,43 Abnormal accumulation of ROS leads to dysfunction of antioxidant systems in the cells, which can induce cell damage in DNA, protein and lipids, and consequently


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causing cell death. To investigate the mechanisms of cell death caused by Sal and AuNPs, BCSCs were treated with Sal, PEG-AuNPs and Sal-AuNPs. Reactive oxygen species (ROS) production was determined by the increase of 2’,7’-dichlorofluorescin diacetate (DCFH-DA) fluorescent intensity. As shown in Figure 3A, both Sal, and Sal-AuNPs increased ROS. Additionally, Sal-AuNPs treatment induced more ROS generation compared to Sal treatment. The molecular mechanism of Sal-induced cancer cell death has generally associated with interference of mitochondrial function.44,45 Excessive superoxide which entered the mitochondrial matrix will elicit oxidative stress and be determined by MitoSOX dye.

46-47

The quantitative results showed significant generation of mitochondrial

superoxide in Sal-AuNPs treated cells as compared to other treatments. Using an independent observation, we monitored cellular mitochondria damage by determining mitochondrial membrane potential, TMRM. We observed a reduction in membrane potential in both Sal and Sal-AuNPs group (Figure 3B). Moreover, Sal-AuNPs treatment had a significant loss of mitochondrial membrane potential compared to free drug Sal. Previously it has been reported that Sal treatment to cancer cells can increase accumulation of dysfunctional mitochondria with increased ROS.45,48 Consistent with previous results, we also observed Sal increased ROS generation in breast cancer cells. We found Sal-AuNPs can further promote mitochondrial superoxide generation and mitochondrial damage resulting in cell death to a greater extent in BCSCs. To determine whether Sal-AuNPs affect lipid oxidation which correlates with ferroptosis sensitivity, cellular toxic lipid ROS accumulation was firstly monitored using C11-BODIPY (581/591), a lipid ROS fluorescent probe.49 BCSCs treated with either Sal or Sal-AuNPs resulted in a higher level of lipid ROS accumulation as shown in Figure 4B. We then investigated the lipid peroxide product MDA. A similar response was observed when Sal-AuNPs resulted in the high levels of lipid oxidation as shown in Figure 4C. GSH is actively involved in removing cellular hydroperoxides and preventing oxidative stress during ferroptosis.50 Therefore it is important to monitor cellular GSH level to understand cellular antioxidant capability. Figure 4D



showed Sal-AuNPs depleted the intracellular GSH level. Our results are consistent with other studies which reported that Sal caused BCSC cell death by increasing lipid ROS production.19,26 Collectively, we found that combination of Sal with AuNPs can significantly increase ROS-dependent cell death via suppression of mitochondrial damage, GSH, and accumulation of lipid ROS. During ferroptosis, iron-storage protein ferritin is degraded by ferritinophagy to release labile iron, leading to an increase of oxidative stress.51 Glutathione peroxidase 4 (GPX-4) however is charged with the removal of lipid hydroperoxides to promote cellular antioxidant properties.52 Downregulation of GPX-4 resulted in the intracellular accumulation of lipid peroxides, which is the main determinant for ferroptosis. Hence, we investigated the expression levels of two ferroptotic markers, ferritin and GPX-4 in BCSC after treatment (Figure 5). Both Sal and Sal-AuNPs showed reduced expression of GPX-4 and ferritin in Sal and Sal-AuNPs groups, while Sal-AuNPs showed greatly decrease in the expression of these proteins among all the groups. This finding agrees with previous published data, showing as the response to the increased accumulation iron in lysosomes after Sal treatment, cancer stem cells trigger the degradation of ferritin which leads to ROS and ferroptosis.19 These results indicated that Sal-AuNPs have higher ability to increase iron accumulation, which cause the increasing of ROS generation and dramatically consuming GSH and GPX-4, thus leading to ferroptosis.


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Figure 3 (A) Fluorescence microscopy images showing the mitochondria damage of Sal, PEG-AuNPs, and Sal-AuNPs in cells treated as indicated. (B) BCSCs were treated as indicated in the figure and the levels of mitochondrial superoxide was quantified using MitoSox dye incubated with BCSCs and analyzed by flow cytometer. Scale bar, 20 μm.



Figure 4 In vitro reactive oxygen species (ROS) levels analyzed by flow cytometer: (A) DCF fluorescence, (B) C11-BODIPY, (C) Lipid peroxide product MDA, and (D) the intracellular GSH level.

Figure 5 (A) Immunoblot shows level of actin as control, ferritin and GPX-4 in cells treated with Sal, PEG-AuNPs, and Sal-AuNPs as indicated. (B) Data analysis of GPX-4 determined by western blot and quantified against loading control of actin. (C) Data analysis of ferritin determined by western blot and quantified against loading control of actin.


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Figure 6 MCF-7 cells were seeded for the tumorsphere formation and tumorspheres larger than 50 μm in diameter were counted as positive. (A) Light microscopic imaging of the tumorsphere formation of control, Sal, PEG-AuNPs and Sal-AuNPs; and (B) tumorsphere formation efficiency of Sal, PEG-AuNPs and Sal-AuNPs (v.s. control) respectively. Scale bar, 200 μm.

3.5 In vitro tumorsphere formation study Tumorsphere formation investigates the self-renewal capacity of BCSCs and is a useful tool for anti-cancer drug screening.13 The tumorsphere formation of BCSCs treated with Sal, PEG-AuNPs and Sal-AuNPs was imaged and quantified as presented in Figure 6. The tumorsphere formation efficiency of Sal and Sal-AuNPs were significantly decreased compared to either PEG-AuNPs or control, which agrees with the anti-CSC activity of Sal as reported.13,53 Interestingly, Sal-AuNPs treatment exhibited even fewer tumorspheres compared with Sal. Both PEG-AuNPs and control treated BCSCs can still form solid cellular cluster as shown in Figure 6B, suggesting their self-renewal capability was not influenced. Our results indicated that Sal-AuNPs can efficiently prevented BCSCs from developing tumourspheres. Thus, these data provided solid evidence that Sal-AuNPs can enhance therapeutic efficacy towards



BCSCs in vitro. 4. Conclusion In summary, Sal is an efficacy anti-cancer drug which can be further improved by conjugating with AuNPs. BCSCs showed high sensitivity to Sal-AuNPs with better efficacy of cell-death. Our study revealed the mechanism of Sal-AuNPs of inducing ferroptosis in BCSCs which includes eliciting of ROS, mitochondrial dysfunction, and lipid oxidation. As an iron-dependent cell death pathway, ferroptosis is tightly controlled by iron storage protein ferritin and antioxidant GPX-4. Both higher iron accumulation and GPX-4 inactivation indicates Sal-AuNPs exhibit higher ability to induce ferroptosis during BCSC treatment. Collectively, our findings suggest Sal-AuNPs could be an efficient therapeutic strategy for cancer stem cells, and provide insight into their mechanism of action of ferroptosis.

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Salinomycin-loaded gold nanoparticles for treating cancer stem cells

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