Article Cite This: Chem. Res. Toxicol. 2018, 31, 945−953
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Vitamin E D‑α-Tocopheryl Polyethylene Glycol Succinate (TPGS) Provokes Cell Death in Human Neuroblastoma SK-N-SH Cells via a Pro-Oxidant Signaling Mechanism Cristian Ruiz-Moreno, Carlos Velez-Pardo,† and Marlene Jimenez-Del-Rio*,†
Chem. Res. Toxicol. 2018.31:945-953. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 09/23/18. For personal use only.
Neuroscience Research Group, Medical Research Institute, Faculty of Medicine, University of Antioquia (UdeA), Calle 70 No. 52-21 and Calle 62 No. 52-59, Building 1, Room 412, SIU Medellin 500001, Colombia ABSTRACT: Neuroblastoma (NB) is the most common neoplasm during infancy. Unfortunately, NB is still a lethal cancer. Therefore, innovative curative therapies are immediately required. In this study, we showed the prodeath activity of TPGS in human NB SK-N-SH cancer cells. NB cells were exposed to TPGS (10−80 μM). We report for the first time that TPGS induces cell death by apoptosis in NB cells via a pro-oxidant-mediated signaling pathway. Certainly, H2O2 directly oxidizes DJ-1 cysteine106-thiolate into DJ-1 cysteine106-sulfonate, indirectly activates the transcription factors NF-kappaB, p53, and c-JUN, induces the upregulation of mitochondria regulator proteins BAX/PUMA, and provokes the loss of mitochondrial membrane potential (ΔΨm) and the activation of caspase-3/AIF, leading to nuclear disintegration, demonstrated by cellular and biochemical techniques such as fluorescence microscopy, flow cytometry, and Western blot analysis. Since TPGS is a U.S. Food and Drug Administration (FDA)-approved pharmaceutical excipient, this molecule might be used in clinical trials for NB treatment.
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of poorly absorbed drugs.11 Notably, TPGS has also been shown to be noxious to malignant cells, such as lung adenocarcinoma and breast cancer,12,13 likely through the generation of ROS and mitochondria-associated apoptosis.14 In line with these observations, our research group has recently established that TPGS provokes apoptosis in acute lymphoblastic leukemia via OS involving a cell death signaling pathway.15 However, no data are available to determine whether TPGS might eliminate NB tumor cells. On the basis of the last consideration, we sought to determine whether TPGS triggers cell death via a ROS pathway in SK-N-SH cells, a model of human NB16 in vitro. We found that TPGS prompted apoptosis in SK-N-SH cells by H2O2 generation, reflected by the oxidation of the OS sensor protein DJ-1 into the DJ-1 Cys106-sulfonate derivate; activation of p53, which in turn caused cell cycle arrest; activation of the transcription factors NF-κB and c-JUN; upregulation of the proapoptotic proteins PUMA and BAX, resulting in the loss of mitochondrial membrane potential (ΔΨm); and activation of apoptosis-inducing factor (AIF) and protease caspase-3. In addition, SK-N-SH cells can be protected from TPGS by the antioxidant N-acetyl-L-cysteine (NAC) and by the pharmacological inhibition of NF-κB (PDTC), p53 (PFT), JNK (SP600123), and caspase-3 (NSCI). We conclude that TPGS-induced apoptosis in NB tumor cells through a pro-oxidant-mediated signaling
INTRODUCTION Neuroblastoma (NB) is the most common extracranial tumor in childhood and is derived from primitive neuroectodermal cells of the developing sympathoadrenal system.1 This malignant cancer affects approximately 800 children per year in the United States, and according to the Children’s Oncology Group, children in the high-risk group have a 5-year survival rate of around 40 to 50% (https://www.cancer.org/cancer/ neuroblastoma). Despite advances in the underlying biologic and genetic features of NB tumors,2 the prognosis is still unfavorable due to high resistance to the current therapy and the increasing number of refractory and relapsed patients. Therefore, innovative curative therapies are immediately needed for these patient groups. Although several therapeutic approaches have been implemented for its treatment,3 activating apoptosis, a regulated cell death process,4 by novel agents seems to be one of the most reliable methods to kill cancer cells (e.g., venetoclax).5 Since NB cells are known to exhibit increased intrinsic reactive oxygen species (ROS) and oxidative stress (OS) metabolism,6 ROS levels can be used as a thermostat to monitor the damage that cells can bear.7 Interestingly, increased OS by exogenous ROS generation might lead to mitochondria dysregulation and apoptosis.8 Hence, increasing OS in NB cells by ROSactivating anticancer molecules is a reasonable therapeutic approach.9,10 D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS) is a water-soluble derivative of natural vitamin E used as a vehicle for drug delivery systems to enhance the bioavailability © 2018 American Chemical Society
Received: May 29, 2018 Published: August 9, 2018 945
DOI: 10.1021/acs.chemrestox.8b00138 Chem. Res. Toxicol. 2018, 31, 945−953
Article
Chemical Research in Toxicology
cat. no. sc-1694), rabbit anticaspase-3 (Santa Cruz, cat. no. 22171-R), rabbit anti-Bax (Santa Cruz, cat. no. sc-493), rabbit anti-oxidized DJ-1 (Abcam, cat. no. ab169520), and rabbit anti-PUMA (Abcam, cat. no. ab9643) antibodies and with the primary monoclonal antibody mouse anti-AIF (Santa Cruz, cat. no. sc-13116). All primary antibodies were prepared at a final concentration of 5−10 μg/mL. After several washes, cells were incubated with Alexa Fluor594 mouse antirabbit (cat. no. A21207) or Alexa Fluor488 donkey antimouse (cat. no. A-21202) IgG secondary antibodies according to the supplier’s protocol (Life Technologies, Eugene, Oregon, USA). Nucleus staining was carried out using Hoechst 33342 (Life Technologies). Protein Preparation and Western Blot. For total protein preparation, SK-N-SH cells (3 × 105 cells per well/mL) were left untreated or were treated with TPGS (60 μM) for 24 h and were then lysed in 50 nM Tris-HCl pH 8.0 with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.1% sodium dodecyl sulfate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 μg/mL leupeptin, 100 mM NaF, 1 nM PMSF, and a protease inhibitor cocktail (Sigma-Aldrich). Protein lysate (50 μg) was loaded and separated using 12% electrophoresis gels and transferred onto nitrocellulose membranes (Hybond-ECL, Amersham Biosciences) at 300 mA for 80 min by an electrophoretic transfer system (BIO-RAD). The membranes were incubated overnight at 4 °C with primary antibodies. The expression of the redox sensor protein was measured using the rabbit antioxidized DJ-1 (Abcam, cat. no. ab169520) antibody. The expression of pro-apoptotic transcription factors were determined using mouse anti-p53 (Thermo, cat. no. MA5-12453), rabbit anti-NF-κB (Thermo, cat. no. PA5-16545), and rabbit anti-c-Jun (Santa Cruz, cat. no. sc-1694) antibodies. Mitochondrial maintenance proteins were measured using rabbit-anti Bax (Santa Cruz, cat. no. sc-493) and rabbit-anti PUMA (Abcam, cat. no. ab9643). The expression of proapoptotic signaling proteins was determined using rabbit anticaspase-3 (Santa Cruz, cat. no. 22171-R) and mouse anti-AIF (Santa Cruz, cat. no. sc-13116). Normalization was performed with the mouse antiactin antibody (Thermo, cat. no. MA5-11869). IRDye 800CW/680CW goat antimouse or antirabbit and IRDye 800CW donkey antigoat (LI-COR Biosciences; 1:10 000) antibodies were used as the secondary probes. The blots were developed using the Odyssey infrared imaging system. The WB analysis was assessed three times in independent experiments. Photomicrography and Image Analysis. Fluorescent microphotographs were taken using a FLoid Cell Imaging Station (Life Technology). Single-cell image analysis was carried out using ImageJ software (http://imagej.nih.gov/ij/). The figures were transformed into 8-bit images, and the background was subtracted. The cellular measurement region of interest (ROI) was drawn around the cell, and the total fluorescence intensity was subsequently determined using the same threshold for controls and treatments. The mean fluorescence intensity (MFI) was obtained by normalizing the total fluorescence to the number of nuclei. Statistical Analysis. Statistical analyses were carried out using GraphPad Prism version 7.0 (GraphPad Software, Inc.). A comparison of means of two groups of data was made by using the unpaired and two-tailed Student t test. All data are reported as the mean ± standard deviation (SD) from at least three independent experiments. Statistical significance was considered at *P < 0.05 and **P < 0.01.
pathway. Understanding the pathways of TPGS-induced cell death may provide insight into more effective anti-NB cancer therapy. Our findings support the usefulness of TPGS as a drug for pediatric neuroblastoma cancer.
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EXPERIMENTAL PROCEDURES
Cell Line and Reagents. SK-N-SH cells (catalog no. HTB-11; ATCC, Manassas, VA, USA) were cultured according to the supplier’s indications. Cells at passages 5−15 were plated at a density of 3 × 105 cells per well/mL in 24-well plates. TPGS was prepared according to ref. 15. Neuroblastoma cells were then exposed to increasing TPGS concentrations (10−80 μM), freshly prepared, and suspended in DMEM medium with high glucose (4500 mg/mL; Sigma-Aldrich, St Louis, MO, USA) and 20% FBS (Gibco/Invitrogen, Grand Island, NY, USA) in the absence or presence of different products of interest (e.g., antioxidant, inhibitors) for 24 h at 37 °C. All other reagents were from Sigma-Aldrich (St Louis, MO, USA). Determination of DNA Fragmentation and Cell Cycle by Flow Cytometry. DNA fragmentation was determined using a hypotonic solution of propidium iodide (PI) (BD Bioscience, San Jose, CA, USA) according to a previous report.17 Briefly, we used cells in the sub-G1 phase as markers of apoptosis. After detaching untreated and treated cells from well plates using 0.25% trypsin, neuroblastoma cells (1 × 105 cells per well/mL) were washed twice with PBS (pH 7.2) and stored in 95% ethanol overnight at −20 °C. Then, cells were washed and suspended in a 500 μL solution containing PI (50 μg/mL), RNase A (100 μg/mL), EDTA (50 mM), and Triton X-100 (0.2%) for 30 min at 37 °C. We analyzed the fluorescence of PI in the cell suspension using an Epics XL flow cytometer (Beckman Coulter, Miami, FL, USA). DNA fragmentation and cell cycles were assessed three times in independent experiments. Quantitative data and figures from the sub-G1 population were obtained using FlowJo X 10.0.7 data analysis software. Analysis of Mitochondrial Membrane Potential (ΔΨm) by Flow Cytometry. To test the ΔΨm, we incubated SK-N-SH cells (3× 105 cells per well/mL) for 20 min at RT in the dark with a cationic and lipophilic 3,3′-dihexyloxacarbocyanine iodide [DiOC6(3), 10 nM final concentration] compound (Calbiochem, Darmstadt, Germany; cat. no. D-273) and intercalating agent propidium iodide (PI, 12.5 ng/mL, final concentration) according to ref 17. Cells were then analyzed by using an Epics XL flow cytometer (Beckman Coulter). The combination of these two markers allowed for dividing the cells into two different populations. Cells with high fluorescence intensity for DiOC6(3) and that were impermeable to PI (DiOC6(3)high/PI−) were considered living cells (L). Cells with low fluorescence intensity for DiOC6(3) and impermeable to PI (DiOC6(3)low/PI−) and cells with low fluorescence intensity for DiOC6(3) and permeable to PI (DiOC6(3)low/PI+) were considered cells in apoptosis (A). The experiment was conducted three times, and 10 000 events were acquired for analysis. We used FlowJo X 10.0.7 software to obtain all quantitative data and figures. Protection Experiments. SK-N-SH cells (3 × 105 cells per well/mL) were left untreated or treated with TPGS (60 μM) alone or in the presence of antioxidant N-acetyl-L-cysteine (NAC, 1 mM) or specific inhibitor reagents ammonium pyrrolidinedithiocarbamate (PDTC, 10 nM), pifithrin-α (PFT, 50 nM), 1-(4-methoxybenzyl)5-[2-(pyridin-3-yl-oxymethyl)pyrrolidine-1-sulfonyl]-1H-indole-2,3dione (NSCI, 10 nM), and 1,9-pyrazoloanthrone (SP600125, 1 nM) according to ref 15 at 37 °C for 24 h. The concentrations of inhibitors were selected according to a previous report.15 Then, we evaluated cells for DNA fragmentation and ΔΨm by flow cytometry. The assessment was repeated three times in independent experiments. Immunofluorescence Analyses. For immunofluorescent staining, neuroblastoma cells were left untreated (control) or treated with TPGS (60 μM). Cells were fixed using 4% paraformaldehyde for 20 min at RT. After permeabilization (0.2% Triton X-100) and subsequent blockage (10% BSA), cells were incubated overnight at 4 °C with primary polyclonal rabbit anti-NF-κB (Thermo, cat. no. PA5-16545), rabbit antip53 (Santa Cruz, cat. no. sc-6243), rabbit anti-c-Jun (Santa Cruz,
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RESULTS TPGS Induces DNA Fragmentation, Cell Cycle Arrest, and Loss of Mitochondrial Membrane Potential (ΔΨm) in Neuroblastoma SK-N-SH Cells. To assess whether TPGS was able to trigger apoptosis reflected as the fragmentation of DNA and arrest of the cell cycle in SK-N-SH cells, we first treated NB cells with increasing concentrations of TPGS (10 to 80 μM) for 24 h and then stained the untreated (control) or treated cells with propidium iodide (PI), a fluorescent probe which intercalated into the DNA strand and was easily detectable by flow cytometry. The sub-G1 population is therefore directly related to nuclear dismantling on cells 946
DOI: 10.1021/acs.chemrestox.8b00138 Chem. Res. Toxicol. 2018, 31, 945−953
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Chemical Research in Toxicology
Figure 1. TPGS induces nuclear, cell cycle, and mitochondrial damage in neuroblastoma cells. SK-N-SH cells (1 × 105 cells per well/mL) were left untreated or were treated with TPGS for 24 h at 37 °C. (A) Histogram showing the sub-G1 cell population indicative of DNA fragmentation (mean percentage ± 5% SD of three independent experiments). (B) Histogram of the cell cycle (mean percentage ± 5% SD of three independent experiments) and (C) ΔΨm assessed by flow cytometry according to the Experimental Procedures section
undergoing apoptosis. TPGS induced a concentration-dependent increase in the fragmentation of DNA in NB cells, and this effect was significant from 40 μM (Figure 1A). When we assessed the cell cycle of untreated cells and cells treated with TPGS, there was a clear cell cycle arrest of NB cells in the G1 phase starting from 20 μM (Figure 1B). In addition, the loss of Δψm, a well-known phenomenon present on apoptotic cells,18 can be detected through the use of lipophilic dyes that selectively stain the mitochondria of live cells. For this purpose, we used DiOC6(3) to assess Δψm in SK-N-SH cells under TPGS treatment. After 24 h of exposure, there was an evident increase in the number of cells that lost their Δψm (Figure 1C,A: % apoptotic cells, [pink background]). This change was notorious after 20 μM (P < 0.01) and was higher as the concentration increased. TPGS Generates H2O2, and the Latter Acts as a Trigger Molecule of Apoptosis in Neuroblastoma SK-N-SH Cells. Next, we assessed whether TPGS was able to produce ROS, specifically H2O2, in SK-N-SH cells. Therefore, the levels of oxidized DJ-1 protein were determined in untreated or treated cells. DJ-1 suffers oxidation at the cysteine residue at position 106 (Cys-106) under OS triggered by H2O2.15 Neuroblastoma cells under TPGS (40 μM) showed a significantly increased level of oxidized DJ-1 compared with control cells after
treatment assessed by immunofluorescence (Figure 2A) and Western blot (Figure 2B). Of note, cotreatment with N-acetylL-cysteine (NAC, 1 mM), an antioxidant compound, diminished the level of oxidized DJ-1 to nearly the basal level (Figure 2A,B). Furthermore, flow cytometry analysis revealed that NB cells exposed to TPGS (40 μM) and coincubated with NAC significantly increased both the number of cells in the G1/S/G2 phase (i.e., decreased the sub-G1 phase) and the percentage of cells with high ΔΨm compared to those of untreated cells (Figure 2C,D, respectively). TPGS Induces Activation of Several Pro-Apoptotic Proteins in Neuroblastoma SK-N-SH Cells. To further characterize the antitumor mechanism of TPGS in NB cells, we checked whether several proteins that have been involved in the apoptosis signaling triggered by H2O2 are being activated by TPGS pro-oxidative stimuli.15 Therefore, NB cells were exposed to TPGS (60 μM) to evaluate changes in the expression/activation of different transcription factors (e.g., NF-κB, p53, and c-JUN), pro-apoptotic proteins (e.g., BAX and PUMA), and effector apoptotic proteins such as AIF and caspase-3. Immunocytochemistry staining analysis (Figure 3) showed the TPGS-induced activation of c-JUN (Figure 3B,C), NF-κB (Figure 3E,F), p53 (Figure 3H,I), PUMA (Figure 3K,L), BAX (Figure 3N,O), AIF (Figure 3Q,R), and caspase-3 947
DOI: 10.1021/acs.chemrestox.8b00138 Chem. Res. Toxicol. 2018, 31, 945−953
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Chemical Research in Toxicology
Figure 2. TPGS induces DJ-1 protein oxidation in neuroblastoma cells. SK-N-SH cells (3 × 105) were left untreated (−) or treated (+) with TPGS (40 μM) or N-acetyl-L-cysteine (NAC, 1 mM) for 24 h. (A) Cells were stained with Hoechst dye and anti-DJ-1 Cys106-sulfonate for IF analysis. Positive cells (green fluorescence) reflect oxidized DJ-1 protein. Fluorescence intensities (arbitrary units) were quantified and expressed as the mean ± SD from three independent experiments. Data are expressed as the mean ± SD. Intragroup statistical analysis was carried out using one-way ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001. (B) Protein extracts were blotted with anti-DJ-1 Cys106-sulfonate and antiactin antibodies. Relative fluorescence for DJ-1 was expressed as the fold increase relative to that in untreated cells and normalized to actin. Data are expressed as the mean ± SD. **P < 0.01. (C) Histogram showing the sub-G1 cell population indicative of DNA fragmentation (mean percentage ± 5% SD of three independent experiments). (D) Histogram of Δψm evaluated according to Experimental Procedures. Abbreviations: L, living cells; A, apoptotic cells. 948
DOI: 10.1021/acs.chemrestox.8b00138 Chem. Res. Toxicol. 2018, 31, 945−953
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Figure 3. TPGS induces differential activation of pro-apoptotic proteins in neuroblastoma cells (IF analysis). SK-N-SH cells (3 × 105) were left untreated or were treated with TPGS (60 μM) for 24 h. Cells were then stained with Hoechst dye and primary/secondary antibodies. Insets show merged images reflecting activation/nuclear translocation of the molecule (insets green/red fluorescence). Mean fluorescence intensities were quantified according to Experimental Procedures. Each fluorescent microphotograph is representative of three independent experiments. *P < 0.05 and **P < 0.001 versus control.
cells were exposed to TPGS (60 μM) alone or with/without JNK inhibitor SP600125, NF-κB inhibitor PDTC, p53 inhibitor PFT, and caspase-3 inhibitor NSCI. Cells displayed a significant reduction in the number of cells in the sub-G1
(Figure 3T,U) compared to that of unexposed cells (Figure 3A,D,G,J,M,P,S). These observations were confirmed by WB (Figure 4A,B). To further verify the involvement of TPGS-induced molecules in the apoptosis process, SK-N-SH 949
DOI: 10.1021/acs.chemrestox.8b00138 Chem. Res. Toxicol. 2018, 31, 945−953
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Chemical Research in Toxicology
Figure 4. TPGS induces differential activation of pro-apoptotic proteins in neuroblastoma cells: WB analysis. Cells (3 × 105) were treated with TPGS for 24 h as described in Figure 3. (A) Protein extracts were blotted with primary antibodies as listed. (B) Relative infrared fluorescence of antibodies was expressed as the fold increase relative to that in untreated cells and normalized to actin. Data are expressed as the mean ± SD *P < 0.001.
phase (Figure 5A) and the reduction of ΔΨm (Figure 5B) compared with cells treated with only TPGS.
TPGS/ROS/H2O2 as a primary trigger of apoptotic cell death in SK-N-SH cells. We demonstrated, for the first time, the TPGS-induced activation of transcription factors (c-JUN, NF-κB, and c-JUN), upregulation of pro-apoptotic proteins (PUMA and BAX), and activation of dismantling nuclear proteins (AIF and caspase-3) in NB cells. Indeed, elevating ROS/H2O2 to high intracellular toxic levels activates various ROS-induced cell death pathways25 involving the mitogen-activated protein kinase (MAPK) signaling pathways.26 In our study, we detected an increase in the expression of the c-JUN protein, a transcription factor that might be activated through the ASK1/MKK4 (MAKK7)/JNK signaling pathway.27 Interestingly, the activation of ASK1 is mediated by the H2O2 oxidation of TRX1 and the subsequent dissociation of ASK1.28 In agreement with others,29 we found that pro-apoptotic protein PUMA may function as a downstream effector of the JNK/c-JUN in NB cells under TPGS exposure.15 Likewise, we found the levels of expression of NF-κB to be significantly increased in NB treated with TPGS compared to in untreated cells. This observation suggests that H2O2 might activate NF-κB, probably through Syk proteintyrosine kinase.30 Once NF-κB was activated, it enhanced gene expression of a subset of NF-κB-dependent pro-apoptotic genes (NF-kB target genes, https://www.bu.edu/nf-kb/generesources/target-genes/) such as p53.31 As guardian of the human genome, p53 plays an essential role in regulating the cell cycle and cell death in response to DNA damage. Functionally active p53 transactivates a set of target genes to induce cell cycle arrest and/or apoptosis. Indeed, the detected increase in p53 levels by IF and WB suggests that it might be involved in the arrest in the G1 cell cycle phase of NB cells treated with TPGS, possibly through the upregulation of the cell cycle inhibitor p21WAF1 gene.32 Furthermore, high p53 levels found in NB cells under TPGS
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DISCUSSION Exploring established noncancer drugs or pro-oxidant molecules for anticancer activity against neuroblastoma tumors offers a new approach to advancing curative strategies into clinical trials.2 In the present investigation, we demonstrate for the first time that TPGS induces apoptosis in NB cancer cells in vitro through an ROS/H2O2-induced signaling mechanism. This finding is supported by several observations. TPGS produces H2O2 as revealed by the specific oxidation of DJ-1 Cys106-thiolate (DJ-1-Cys106SO−) to DJ-1 Cys106-sulfonate (DJ-1-Cys106SO3−),19 which is a biomarker associated with high OS.20 This observation complies with the notion that TPGS is capable of generating H2O2 and may be linked to the loss of the DJ-1 antioxidant function.21 Although the underlying mechanisms by which TPGS generates H2O2 remain to be elucidated in depth, it has been suggested that once TPGS enters the cell by passing easily through the plasma membrane it reaches the cytoplasm,22 where it might be hydrolyzed into two main products: the hydrophilic head (polyethylene glycol chain) and the lipophilic tail (tocopheryl group). The latter has shown a powerful capacity to induce ROS production either by interacting with mitochondrial respiratory complex II on its ubiquinone-binding site23 or by the inhibition of mitochondrial complex I.24 Whatever the mechanism of generation might be, we demonstrated that H2O2 is a critical molecule in TPGSinduced apoptosis. This last finding was further correlated with the ability of NAC, a well-known antioxidant compound, to reduce the levels of oxidized protein DJ-1-Cys106SO3− assessed by IF and WB and to prevent the changes related to apoptosis cells such as an increase in the sub-G1 population and a loss of ΔΨm. Taken together, our results show clearly the key role of 950
DOI: 10.1021/acs.chemrestox.8b00138 Chem. Res. Toxicol. 2018, 31, 945−953
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Chemical Research in Toxicology
Figure 5. TPGS-induced cell death signaling in neuroblastomas (pharmacological inhibition). Cells (3 × 105) were pretreated with the indicated inhibitor for 30 min and then treated with TPGS for 24 h as described in Figure 3. (A) Histogram showing the sub-G1 cell population indicative of DNA fragmentation (mean percentage ± 5% SD of three independent experiments). (B) Histogram of Δψm evaluated according to Experimental Procedures. Abbreviations: L, living cells; A, apoptotic cells. Each panel is representative of three independent experiments.
(∼50%) the percentage of cells in the sub-G1 phase and the loss of ΔΨm. Besides the oxidation of DJ-1-Cys106SO−, our data suggest that TPGS induces at least two independent but complementary signaling mechanisms in NB cells: (i) TPGS > H2O2 > ASK1 > MEKK4 > JNK > c-JUN > PUMA and (ii) TPGS > H2O2 > Syk > IKK > NF-κB > p53 > PUMA, BAX. Interestingly, TPGS-induced signaling converged on the loss of
exposure might be linked to the induction of apoptosis due to the fact that p53 upregulated the pro-apoptotic proteins BAX33 and PUMA,34,35 which are tightly associated with the mitochondrial outer membrane permeabilization (MOMP).36 Remarkably, the involvement of the JNK and NF-κB/p53 pathways were further confirmed through the use of specific pharmacological inhibitors, where each one was able to significantly reduce 951
DOI: 10.1021/acs.chemrestox.8b00138 Chem. Res. Toxicol. 2018, 31, 945−953
Article
Chemical Research in Toxicology ΔΨm. Noticeably, TPGS provokes the depolarization of mitochondria, most likely as a consequence of its interaction with mitochondrial complex I/II, as mentioned above.23 This observation supports the notion that TPGS may act as a mitocan class V molecule, as defined in ref 37. Finally, in this study, we found high levels of the caspase-3 and AIF proteins concomitantly with the dissipation of ΔΨm and an increased sub-G1 population in NB treated with TPGS, and the inhibition of caspase-3 by specific inhibitor NSCI dramatically reduced TPGS-induced apoptosis. These results suggest that TPGS may be used as a specific mitochondria-targeted agent against NB.38 In conclusion, we provide a mechanistic explanation for our findings consistent with all of the data demonstrating that TPGS induces apoptosis in SK-N-SH cells through oxidative stress mediated by H2O2 signaling. Since TPGS is a U.S. Food and Drug Administration (FDA)-approved pharmaceutical excipient, this molecule might be used in clinical trials for NB treatment.
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Annicchiarico-Petruzzelli, M., Antonov, A. V., Arama, E., Baehrecke, E. H., Barlev, N. A., Bazan, N. G., Bernassola, F., Bertrand, M. J. M., Bianchi, K., Blagosklonny, M. V., Blomgren, K., Borner, C., Boya, P., Brenner, C., Campanella, M., Candi, E., Carmona-Gutierrez, D., Cecconi, F., Chan, F. K., Chandel, N. S., Cheng, E. H., Chipuk, J. E., Cidlowski, J. A., Ciechanover, A., Cohen, G. M., Conrad, M., Cubillos-Ruiz, J. R., Czabotar, P. E., D’Angiolella, V., Dawson, T. M., Dawson, V. L., De Laurenzi, V., De Maria, R., Debatin, K. M., DeBerardinis, R. J., Deshmukh, M., Di Daniele, N., Di Virgilio, F., Dixit, V. M., Dixon, S. J., Duckett, C. S., Dynlacht, B. D., El-Deiry, W. S., Elrod, J. W., Fimia, G. M., Fulda, S., Garcia-Saez, A. J., Garg, A. D., Garrido, C., Gavathiotis, E., Golstein, P., Gottlieb, E., Green, D. R., Greene, L. A., Gronemeyer, H., Gross, A., Hajnoczky, G., Hardwick, J. M., Harris, I. S., Hengartner, M. O., Hetz, C., Ichijo, H., Jaattela, M., Joseph, B., Jost, P. J., Juin, P. P., Kaiser, W. J., Karin, M., Kaufmann, T., Kepp, O., Kimchi, A., Kitsis, R. N., Klionsky, D. J., Knight, R. A., Kumar, S., Lee, S. W., Lemasters, J. J., Levine, B., Linkermann, A., Lipton, S. A., Lockshin, R. A., Lopez-Otin, C., Lowe, S. W., Luedde, T., Lugli, E., MacFarlane, M., Madeo, F., Malewicz, M., Malorni, W., Manic, G., Marine, J. C., Martin, S. J., Martinou, J. C., Medema, J. P., Mehlen, P., Meier, P., Melino, S., Miao, E. A., Molkentin, J. D., Moll, U. M., Munoz-Pinedo, C., Nagata, S., Nunez, G., Oberst, A., Oren, M., Overholtzer, M., Pagano, M., Panaretakis, T., Pasparakis, M., Penninger, J. M., Pereira, D. M., Pervaiz, S., Peter, M. E., Piacentini, M., Pinton, P., Prehn, J. H. M., Puthalakath, H., Rabinovich, G. A., Rehm, M., Rizzuto, R., Rodrigues, C. M. P., Rubinsztein, D. C., Rudel, T., Ryan, K. M., Sayan, E., Scorrano, L., Shao, F., Shi, Y., Silke, J., Simon, H. U., Sistigu, A., Stockwell, B. R., Strasser, A., Szabadkai, G., Tait, S. W. G., Tang, D., Tavernarakis, N., Thorburn, A., Tsujimoto, Y., Turk, B., Vanden Berghe, T., Vandenabeele, P., Vander Heiden, M. G., Villunger, A., Virgin, H. W., Vousden, K. H., Vucic, D., Wagner, E. F., Walczak, H., Wallach, D., Wang, Y., Wells, J. A., Wood, W., Yuan, J., Zakeri, Z., Zhivotovsky, B., Zitvogel, L., Melino, G., and Kroemer, G. (2018) Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Differ. 25, 486−541. (5) Mihalyova, J., Jelinek, T., Growkova, K., Hrdinka, M., Simicek, M., and Hajek, R. (2018) Venetoclax: A new wave in hematooncology. Exp. Hematol. 61, 10−25. (6) Novotny, N. M., Grosfeld, J. L., Turner, K. E., Rescorla, F. J., Pu, X., Klaunig, J. E., Hickey, R. J., Malkas, L. H., and Sandoval, J. A. (2008) Oxidative status in neuroblastoma: a source of stress? J. Pediatr. Surg. 43, 330−334. (7) Idelchik, M., Begley, U., Begley, T. J., and Melendez, J. A. (2017) Mitochondrial ROS control of cancer. Semin. Cancer Biol. 47, 57−66. (8) Liu, J. H., and Wang, Z. C. (2015) Increased Oxidative Stress as a Selective Anticancer Therapy. Oxid. Med. Cell. Longevity 2015, 1. (9) Gorrini, C., Harris, I. S., and Mak, T. W. (2013) Modulation of oxidative stress as an anticancer strategy. Nat. Rev. Drug Discovery 12, 931−947. (10) Galadari, S., Rahman, A., Pallichankandy, S., and Thayyullathil, F. (2017) Reactive oxygen species and cancer paradox: To promote or to suppress? Free Radical Biol. Med. 104, 144−164. (11) Yang, C., Wu, T., Qi, Y., and Zhang, Z. (2018) Recent Advances in the Application of Vitamin E TPGS for Drug Delivery. Theranostics 8, 464−485. (12) Constantinou, C., Neophytou, C. M., Vraka, P., Hyatt, J. A., Papas, K. A., and Constantinou, A. I. (2012) Induction of DNA Damage and Caspase-Independent Programmed Cell Death by Vitamin E. Nutr. Cancer 64, 136−152. (13) Neophytou, C. M., Constantinou, C., Papageorgis, P., and Constantinou, A. I. (2014) D-alpha-tocopheryl polyethylene glycol succinate (TPGS) induces cell cycle arrest and apoptosis selectively in Survivin-overexpressing breast cancer cells. Biochem. Pharmacol. 89, 31−42. (14) Cheng, G., Zielonka, J., McAllister, D. M., Mackinnon, A. C., Jr., Joseph, J., Dwinell, M. B., and Kalyanaraman, B. (2013) Mitochondria-targeted vitamin E analogs inhibit breast cancer cell energy metabolism and promote cell death. BMC Cancer 13, 285.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Marlene Jimenez-Del-Rio: 0000-0003-3477-2386 Author Contributions †
These authors share senior authorship.
Author Contributions
M.J.-D.-R. and C.V.-P. conceived and designed the experiments. C.R.-M. performed the experiments. C.R.-M., C.V.-P., and M.J.-D.-R. analyzed the data. M.J.-D.-R. contributed reagents, materials, and analysis tools. C.R.-M. wrote an original draft. C.R.-M., C.V.-P., and M.J.-D.-R. wrote and approved the article. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Committee for Development and Research (Comité para el Desarrollo y la InvestigaciónCODI, Universidad de Antioquia-UdeA) (grant no. 2014935). C.R.-M. is a researcher from the Young Researcher and Innovator Program (Programa Jovenes Investigadores e Innovadores) Colciencias 2016-761. The authors thank the Flow Cytometry Unit (GICIG-SIU-UdeA) and Material Science Group (CIENMAT) for the use of the instruments and technical assistance. The authors also thank the Neuroscience Research Group at the UdeA for the use of the Odyssey Infrared Imaging System.
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