Article Cite This: Chem. Res. Toxicol. 2018, 31, 612−618
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Quinone-Based Antitumor Agent Sepantronium Bromide (YM155) Causes Oxygen-Independent Redox-Activated Oxidative DNA Damage Tasaduq H. Wani,†,§ Sreeraj Surendran,†,§ Anal Jana,‡ Anindita Chakrabarty,† and Goutam Chowdhury*,‡ Department of Life Sciences and ‡Department of Chemistry, Molecular Toxicology and Cancer Therapeutics Laboratories, School of Natural Sciences, Shiv Nadar University, NH-91, Tehsil Dadri, Gautam Buddha Nagar, Utter Pradesh 201314, India
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ABSTRACT: Sepantronium bromide (YM155) is a small molecule antitumor agent currently in phase II clinical trials. Although developed as survivin suppressor, YM155’s primary mode of action has recently been found to be DNA damage. However, the mechanism of DNA damage by YM155 is still unknown. Knowing the mechanism of action of an anticancer drug is necessary to formulate a rational drug combination and select a cancer type for achieving maximum clinical efficacy. Using cell-based assays, we showed that YM155 causes extensive DNA cleavage and reactive oxygen species generation. DNA cleavage by YM155 was found to be inhibited by radical scavengers and desferal. The reducing agent DTT and the cellular reducing system xanthine/xanthine oxidase were found to reductively activate YM155 and cause DNA cleavage. Unlike quinones, DNA cleavage by YM155 occurs in the presence of catalase and under hypoxic conditions, indicating that hydrogen peroxide and oxygen are not necessary. Although YM155 is a quinone, it does not follow a typical quinone mechanism. Consistent with these observations, a mechanism has been proposed that suggests that YM155 can cause oxidative DNA cleavage upon 2-electron reductive activation.
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INTRODUCTION
transported inside the cell by the cancer cell-specific solute carrier protein SLC35F2.9 Although, YM155 performed very well in preclinical and phase I studies, it did not provide any meaningful increase in survival over docetaxel in a randomized phase II clinical trial.16,17 Similar results were obtained for combination with rituximab.18 This is surprising because survivin is highly expressed in cancer cells.7 Because docetaxel is a chemotherapeutic agent, a possible explanation may be that YM155’s antitumor activity primarily stems from DNA damage. However, DNA damage being a nontargeted chemotherapeutic approach is generally associated with side effects and toxicity.19,20 The low dose efficacy and negligible side effects observed for YM155 is not consistent with a DNA damaging agent unless it involves a mechanism that is distinct. Using a YM155-resistant MCF7 cell line, we have shown elsewhere that DNA damage is its primary mode of action. Herein, we report the underlying mechanism of DNA damage by YM155. Elucidating the mechanism of action of a drug poses a formidable challenge. A thorough understanding of the mechanism of action of YM155 is necessary not only to have a
Sepantronium bromide, commonly known as YM155, is a small molecule suppressor of survivin currently in phase II clinical trials for the treatment of various cancers.1,2 Survivin is a nodal protein wired with multiple signaling circuitries and is the smallest member of the inhibitor of apoptosis protein family (IAP) that is preferentially expressed in cancer and stem cells.3−5 A study by the National Cancer Institute found that survivin is expressed in all 60 human tumor cell lines with highest levels being in breast and lung cancer cells.6 Considering survivin’s tumor specific expression and functional importance, it is a lucrative target for cancer therapy. YM155 inhibits the growth of 119 human cancer cell lines at mean GI (50) values of ∼15 nM.6 In the case of triple negative breast cancer (TNBC) cell lines, YM155 was shown to decrease proliferation and induce spontaneous apoptosis.7 In xenograft models, YM155 showed significant regression of tumor burden with negligible body weight loss.6 In normal cells, even 10 μM YM155 did not induce cell death.6 Although developed as a survivin suppressant, recently, Glaros and others have reported that YM155’s primary mode of action is DNA damage.8−13 Likewise, there are contradictory reports regarding DNA intercalation by YM155.9,14,15 YM155 is © 2018 American Chemical Society
Received: April 5, 2018 Published: June 13, 2018 612
DOI: 10.1021/acs.chemrestox.8b00094 Chem. Res. Toxicol. 2018, 31, 612−618
Article
Chemical Research in Toxicology
Figure 1. Detection of DNA damage by YM155 in various cell lines. (A) Comet assays using YM155 on various cell lines showing extensive DNA cleavage as evident from the intensity and length of the comet tail. (B) γH2AX assay to detect DNA double strand break by YM155 in various cell lines. unwinding in alkaline buffer (300 mM NaOH and 1 mM EDTA, pH >13) for 30 min. The slides were then electrophoresed in the same buffer at 21 V, 300 mA for 30 min. Cells were neutralized in 400 mM Tris-HCl, pH 7.5 and stained with ethidium bromide (2 μg/mL). Slides were visualized using a Leica DFC450C microscope (Wetzlar, Germany) and quantitated using ImageJ software with OpenComet plugin (Benjamin M. Gyori, 2014). Extent of DNA damage was converted as a percentage of DNA in tail. γH2AX Immunofluorescence. Cells were plated onto polylysinecoated coverslips and allowed to adhere for 16−24 h prior to drug treatment. After treatment, cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 20 min, blocked in 10% BSA for 60 min, and incubated with anti-γ-H2AX Alexa Fluor 488 antibody (1:100 dilution, BioLegend, CA, USA) for 60−120 min. This was followed by incubation with 100 μL (1 μg/mL) of 4′,6diamidino-2-phenylindole (DAPI, Sigma-Aldrich, MO, USA) for 20 min. All incubations were at room temperature. Imaging was done using a Nikon Ti fluorescence microscope (Tokyo, Japan). Plasmid DNA Cleavage Assay. In a punctured cap microfuge tube, 1 μg of pUC19 plasmid DNA, YM155, 5 mM DTT, 250 μg/mL of catalase, and 100−250 mM, pH 7.4 KPhos were incubated at 37 °C for 12 h. Some reactions were treated with 100 mM DMEDA at 37 °C for 2 h, quenched by adding 5 μL of glycerol loading buffer, and electrophoresed for 50 min at 80 V loaded onto a 1% agarose gel with 0.5 μg/mL of ethidium bromide. DNA was visualized and quantitated using an AlphaImager (ProteinSimple, CA) gel documentation system. Strand breaks per plasmid DNA molecule (S) was calculated using the equation S = −ln f1, where f1 is the fraction of plasmid present as form I. In some reactions, 1,3-propanedithiol (5 mM), 1,4butanedithiol (5 mM), glutathione, and β-ME (varying concentrations) were used instead of DTT. For assays with radical scavengers, methanol (0.1 M), ethanol (0.1 M), isopropanol (0.1 M), or DMSO (0.1−0.5 M) was used. For reactions indicating involvement of metals, desferal (1−10 mM) was used. In case of DNA binding competition assays, ethidium bromide (0−300 μM) or spermine (0−500 μM)) was added prior to adding YM155 and DTT. For assays involving the xanthine/xanthine oxidase (X/XO) system, xanthine (1 mM) and xanthine oxidase (2.5 unit) were used. For anaerobic assays, all reagents except for DNA and proteins were mixed in the required amount (as mentioned above) and freeze−pump−thaw degassed three times to remove all dissolved oxygen from the solutions. The degassing was carried in a roundbottom 50 mL flask having an airtight stopcock. The degassed
clear knowledge of its biochemistry but also to formulate a rational drug combination and selection of a cancer type for achieving maximum clinical efficacy. It is also almost impossible to anticipate or reduce any potential toxicity without knowing the mechanism of action of YM155. Moreover, the mechanism may also shade light on the poor performance of YM155 in phase II clinical trials and provide an opportunity to prevent another late stage attrition of a potential drug candidate.16,21,22
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EXPERIMENTAL PROCEDURES
Cell Lines and Materials. The MDA-MB-231, MDA-MB-453, and SW-480 cells were procured from the National Centre for Cell Science (Pune, India). The MCF-7 cell line was a generous gift from Dr. S. Sinha Roy (CSIR Institutes of Genomics and Integrative Biology, Delhi, India). Cells were maintained in DMEM supplemented with 10% FBS (ThermoFisher Scientific, MA) under standard culture conditions. The MCF10A cell line was generously provided by Dr. Carlos L. Arteaga (Vanderbilt University Medical Center, Nashville, TN, USA) and maintained according to a standard protocol.23 All cell lines were authenticated by Short Tandem Repeat DNA profiling from Life Code Technology, Delhi, India. All reagents, unless otherwise mentioned, were obtained from Sigma-Aldrich, MO, USA. Dithiothreitol (DTT) and N,N′-dimethylethylenediamine (DMEDA) were from Tokyo Chemical Industry. Trypan blue was from ThermoFisher Scientific, MA, USA. Catalase, ethanol, methanol, isopropanol, β-mercaptoethanol (β-ME), and DMSO were from HiMedia, Mumbai, India. YM155 and AZD7762 were procured from Selleckchem (TX, USA), and xanthine oxidase (XO) was from SRL Pvt. Ltd. Inhibition of Cell Growth. A cell monolayer, seeded at 2.5 × 104 cells/well in a 24-well plate, was grown in the presence or absence of inhibitors for 72 h. Adherent cells were detached by treating with 0.05% trypsin, and cell number was estimated using a hemocytometer slide. The vital dye trypan blue was used to differentiate live versus dead cells. Comet Assay. Comet assay was performed as described previously by Dhawan et al. Briefly, 1.5 × 104 cells pretreated with YM155 (5 and 25 nM) for 12 h were embedded in 0.5% low melting agarose (LMPA) and layered onto a 1% agarose-coated glass slide. Embedded cells were lysed in lysis buffer containing 2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCL, pH 10 and TritonX-100 followed by DNA 613
DOI: 10.1021/acs.chemrestox.8b00094 Chem. Res. Toxicol. 2018, 31, 612−618
Article
Chemical Research in Toxicology solution was transferred into an argon-filled glovebag, and individual reactions were set up adding DNA (1 μL, 1 μg) and catalase (1 μL, 250 μg/mL) that are not degassed. The reactions were incubated at 37 °C under argon inside the glovebag for 12 h. Detection of ROS. ROS was detected using CellROX deep red reagent (ThermoFisher Scientific, MA). Assays were performed by treating cells with 25 nM YM155 for 0, 3, and 6 h followed by two washes with PBS, 20 min staining with CellROX, and 10 min incubation with DAPI. Some assays were performed by pretreating cells with 10 μM dicoumarol (Sigma-Aldrich, MO) for 6 h or 1 mM buthionine sulfoximine (BSO) (ThermoFisher Scientific, MA, USA) for 15 h. For positive control, cells were treated with 100 μM menadione (Sigma-Aldrich, MO, USA) for 2 h. UV−Vis Assay. UV−vis assays were done using an Agilent Cary 8454 UV−vis diode array spectrophotometer. All absorptions were measured in aqueous media keeping the pH of the system 7.4 by using 100 mM potassium phosphate buffer solution. Absorptions were collected every 30 s for more than 30 min. Concentrations of YM155 (10 μM) and DTT (5 mM) were kept constant for every measurement. Concentrations of deferal and Fe (III) were kept at 1 mM and 10 μM, respectively. For the sake of the reactions, DTT was added last, and all the UV experiments were done using quartz cuvettes.
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DNA Cleavage by YM155 under Various Conditions. H2O2 is mitigated by various enzymes including catalase and peroxidases. Addition of 250 μg/mL of catalase inhibited the cleavage caused by DTT alone but not by YM155 and DTT together (Figure 2B). Increasing the catalase concentration to 750 μg/mL had a negligible effect on DNA cleavage (Figure 2C). This clearly indicated that DNA cleavage by YM155 in the presence of DTT is not mediated through the formation of H2O2, which is quite unusual for quinones.28 Because typically DNA cleavage by quinones occurs through Fenton chemistry involving Fe(II), we also looked into the effect of the prototypical metal chelator deferoxamine (desferal) on DNA cleavage by YM155.29,30 When desferal was used along with YM155 and DTT, DNA cleavage was completely inhibited, indicating the requirement for metals (Figure 2B). Together, these results clearly indicate that the typical quinone mechanism involving redox cycling, superoxide and hydrogen peroxide (H2O2) formation, and generation of hydroxyl radicals does not occur for YM155-mediated DNA cleavage. To confirm if DNA cleavage by YM155 follows a radicalmediated pathway, we performed the DNA cleavage assay in the presence of radical scavengers. Addition of various radical scavengers including DMSO, methanol, ethanol, and isoproponal abrogated DNA cleavage, indicating that DNA cleavage by YM155 is radical mediated (Figures 2C, D).31 In a typical quinone mechanism, oxygen is required for redox cycling of the drug and generation of H2O2.32 To confirm the involvement of oxygen, we performed the DNA cleavage assay under hypoxic conditions. Contrary to the expectation, we found an increase in DNA cleavage under hypoxic conditions (Figure 2E). Similar to the observations made under normoxic conditions, catalase had no effect, whereas radical scavengers and desferal inhibited DNA cleavage by YM155 under hypoxic conditions (Figure 2F). Together, these data clearly indicated that DNA cleavage by YM155 is radical mediated and does not result from a typical redox cycling pathway involving H2O2. Thus, to understand the unusual DNA damaging mechanism of YM155, we looked into the role of DTT. We examined various thiols including β-ME, GSH, 1,3-propanedithiol, and 1,4-butanedithol. YM155 was unable to cleave DNA in the presence of any of these thiols used here except DTT (Figure 2E). The standard state redox potential of GSH (EGSH°′) is approximately −260 mV,33−35 βME −260 mV,36 and DTT −330 mV.37 DTT being a better reducing agent was able to reduce YM155. However, because DTT is not a biologically relevant thiol, it was necessary to determine the cellular reducing system that can activate YM155 to cause DNA cleavage. The constitutively expressed reducing system X/XO was found to activate YM155 and cause DNA cleavage (Figure 3H). Similar to DTT, catalase has no effect, whereas the radical scavenger DMSO and the metal chelator desferal inhibited DNA cleavage by YM155 in the presence of X/XO. The results clearly indicated that the reductive activation of YM155 by X/ XO follow a similar pathway as DTT. Because desferal and not catalase inhibit DNA cleavage by YM155 following reductive activation, it is necessary to understand the role of metals. Dihydroquinones are efficiently oxidized to the semiquinone and quinone by metals including Fe(III). Accordingly, we followed the UV−vis spectrum of YM155 in the presence of DTT over time. A plot of absorbance at 253 nm over time clearly showed a biphasic curve with a lag and two sigmoidal increase in absorbance
RESULTS AND DISCUSSION
Inhibition of Cell Growth by YM155. Toward our goal to understand the mechanism of DNA damage by YM155, we decided to perform experiments both in a controlled environment of a test tube and in the complex environment of a cell. To this end, we used four cell lines: three breast cancer (MDA-MB-231, MDA-MB-453, and MCF-7) cell lines representing three breast cancer subtypes and a colon cancer cell line (SW-480). To confirm the activity of YM155 in these cell lines, we initially performed a cell survival assay. Cells were treated with 5 and 25 nM YM155, and their growth was followed for 72 h. YM155 was found to significantly inhibit growth of all four cell lines compared to that of the DMSO vehicle control. The effect was markedly pronounced in case of the triple negative breast cancer (TNBC) cell line MDA-MB231 (Figure S1). Detection of Cellular DNA Cleavage by YM155. To confirm if YM155 causes DNA damage in the cell lines used here,8 we performed comet assays following the standard protocol.24 Comets of varying tail length were observed for all cell lines used here with 5 and 25 nM YM155 (Figure 1A) except for 5 nM YM155-treated MCF-7 cells. The extent of DNA damage is represented by the length and intensity of the comet tail,25 which was significantly more for YM155-treated cells than DMSO control. Comets were observed in the order MDA-MB-231 > MDA-MB-453 > SW-480 > MCF-7. Consistent with the survival data, DNA damage caused by YM155 was high for the TNBC cells. Because YM155 is a quinone, for comparison, we performed a comet assay with 5 and 25 nM of the classical quinone redox cycling agent menadione (Figure S2).26 Interestingly, the DNA damage efficiency of YM155 was significantly higher than menadione. We also performed γ-H2AX assay. H2AX is a histone that is phosphorylated following DNA double strand break. The phosphorylated histone, commonly known as γ-H2AX, binds to double strand breaks in DNA and recruit other proteins to form a foci that can be detected using fluorescence-tagged γH2AX-specific antibodies.27 YM155 treatment resulted in γH2AX foci formation in all cell lines tested here (Figure 1B). The data revealed that YM155 does cause extensive DNA cleavage in a cell in lower nm concentration. 614
DOI: 10.1021/acs.chemrestox.8b00094 Chem. Res. Toxicol. 2018, 31, 612−618
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Fe(III) (10 μM) generated a biphasic curve with a very short lag between the phases, and the rate of the second phase is faster. On the basis of these observations, we concluded that YM155 is 2-electron reduced by DTT to dihydroquinone, which is back oxidized to the semiquinone by Fe(III). In the absence of Fe(III), there is no semiquinone formation, and thus, we see a monophasic curve with a steep slope. When Fe(III) is added, we get a similar curve as seen for DTT with the rates of each transition being faster. The UV−vis studies with X/XO cannot be performed because the spectra of uric acid, the oxidation product of xanthine, and YM155 overlap. Generation of ROS by YM155. Although the typical redox cycling pathway involving H2O2 may not be responsible for DNA cleavage here, it still may be a viable pathway that generates reactive oxygen species (ROS) in a cell. To confirm the presence of a redox cycling pathway generating ROS, we performed an imaging assay using the CellROX deep red reagent. CellROX is a cell-permeant dye that is nonfluorescent in the reduced state and exhibits bright red fluorescence upon oxidation by ROS (https://www.thermofisher.com/order/ catalog/product/C10422). We found that, following 25 nM YM155 treatment for 3 and 6 h, there is significant ROS generation (Figure 3C and Figure S3). Evidently, the ROS generated by 25 nM YM155 is more than that produced by 100 μM menadione. Effect of DNA Binding. There are contradictory literature reports that YM155 intercalates DNA.9,15 If YM155 does bind to DNA, it is conceivable that due to DNA binding by YM155 there is localized generation of H2O2 in the close vicinity of DNA that allows it to escape the mitigating effect of catalase and cause DNA cleavage. To eliminate this possibility, we performed the DNA cleavage assay with YM155 and DTT in the presence of varying concentrations of the well-known DNA intercalator ethidium bromide or the minor groove-binding agent spermine. The idea being, depending on the binding mode of YM155, addition of a molar excess amount of either of the binding agents should prevent YM155 from binding to DNA. Thus, if H2O2 is involved, we should be able to see the effect of catalase resulting in inhibition of DNA cleavage. We found that varying the concentration of ethidium bromide did not have any effect on DNA cleavage by YM155 (Figure 3D), whereas spermine caused a small increase. The increase in cleavage in the presence of spermine may be due to an increase in the effective free concentration of YM155 allowing its reduction by DTT, which probably does not happen in the DNA-bound state. Proposed Mechanism of DNA Cleavage by YM155. On the basis of the observations made here, a mechanism of DNA cleavage by YM155 was proposed (Figure 4). The proposed mechanism involves 2-electron reduction of the positively charged YM155 to a dihydroquinone by DTT. In presence of metals, the dihydroquinone is oxidized back to the semiquinone radical. The semiquinone radical can either abstract a H atom from DNA and form dihydroquinone or it can be oxidized to the quinone in the presence of oxygen. Abstraction of the H atom from DNA by the semiquinone radical results in DNA cleavage, whereas oxidation to the quinone causes superoxide and peroxide formation. The presence of metals generates a cyclic pathway that produces a stoichiometric excess of semiquinone radicals and H atom abstraction. This explains the unusually high DNA cleavage observed in the presence of low nM YM155 concentration. In the absence of metals under the conditions used here, a
Figure 2. Plasmid DNA cleavage assays with YM155 in the presence of various agents. Supercoiled plasmid DNA (1 μg) was incubated with YM155 (10 μM), DTT (5 mM), catalase (250 μg/mL), potassium phosphate buffer (100−250 mM, pH 7.4), and various agents at 37 °C for 12 h followed by agarose gel electrophoretic analysis. Strand breaks per plasmid DNA molecule (S) was calculated using the equation S = −ln f1, where f1 is the fraction of plasmid present as form I: (A) in the presence and absence of DTT (5 mM), (B) in the presence and absence of catalase (250 μg/mL) or desferal (1 mM), (C) in the presence and absence of varying concentrations of catalase (250−750 μg/mL) or DMSO (0.1−0.5 M), (D) in the presence of various radical scavengers (0.1 M each), (E) under hypoxic and normoxic conditions, (F) effect of radical scavengers under hypoxic conditions, (G) with the use of various thiols with YM155, and (H) DNA cleavage in the presence of the X/XO reducing system (1 mM xanthine, 2.5 units xanthine oxidase, 100 mM DMSO, and 10 mM desferal).
(Figure 3A). We found that in the presence of DTT over a period of 30 min there are two distinct types of spectra (Figure 3B). Interestingly, in the presence of desferal (1 mM), a monophasic curve was obtained (Figure 3A). Addition of 615
DOI: 10.1021/acs.chemrestox.8b00094 Chem. Res. Toxicol. 2018, 31, 612−618
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Chemical Research in Toxicology
Figure 3. (A) Change in absorbance at 253 nm vs time following treatment with DTT (5 mM), DTT + desferal (1 mM), and/or DTT + Fe(III) (10 μM). (B) Change in UV−vis spectrum of YM155 vs time following treatment with DTT (5 mM). (C) Microscopic detection of ROS in YM155-treated MDA-MB-231 cells using the CellROX reagent as depicted by the formation of red fluorescence. MDA-MB-231 cells in culture were treated with 25 nM YM155 for 0−6 h. (D) Effect of ethidium bromide or spermine on DNA cleavage by YM155 + DTT.
Figure 4. Proposed mechanism of activation and DNA damage by YM155.
semiquinone radical is not formed. Hence, we do not see any DNA cleavage when desferal is present in the reaction mixture. DNA cleavage resulting from drug radical-mediated H abstraction is well-documented and observed for various molecules including neocarzinostatin, bleomycin, and calicheamicin.38 Similar to DTT, when the X/XO system was used as a reducing system, desferal and DMSO inhibited DNA cleavage by YM155. This observation can be explained if the X/XO system also reduces YM155 by 2 electrons, resulting in direct formation of the dihydroquinone. A similar observation was made for the hypoxia-selective antitumor agent 3-amino 2quinoxalinecarbonitrile 1,4 di-N-oxide.31
Semiquinones in the presence of O2 can be oxidized to YM155 along with the formation of superoxide radical. However, because YM155 causes DNA cleavage in the presence of catalase, this pathway, although operational, is not responsible for DNA cleavage observed here in the presence of catalase. However, it might be the cause of generation of ROS in the cell. Semiquinone radicals are generally anionic at physiological pH. In principle, for neutral quinone molecules, abstraction of H atoms by the corresponding anionic semiquinone radical from negatively charged DNA is not favorable. However, for positively charged YM155, the 616
DOI: 10.1021/acs.chemrestox.8b00094 Chem. Res. Toxicol. 2018, 31, 612−618
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Chemical Research in Toxicology
TNBC, triple negative breast cancer; X/XO, xanthine/xanthine oxidase
semiquinone radical is neutral, and YM155 has an affinity for DNA, which may facilitate the H atom abstraction process.
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CONCLUSIONS YM155 is a first in its class antitumor agent that suppresses the expression of survivin. However, recent work by others and us reveal that YM155 is a DNA damaging agent. Although it is quite evident that DNA damage plays a significant role in its pharmacology, the mechanism of DNA damage remains elusive. In this study, we have clearly shown that DNA damage by YM155 is primarily responsible for its pharmacology. We have presented convincing evidence that DNA damage by YM155 is distinct from that of typical quinones. According to our proposed mechanism, YM155 requires reductive activation by 2 electrons and the presence of metals to cause DNA damage. The cellular reducing system consisting of X/XO can activate YM155 to cause DNA cleavage. Although YM155 does produce ROS in a cell, catalase has no effect on DNA cleavage by YM155, and oxygen is not necessary. DNA cleavage by YM155 occurs not through the generation of HO• but by direct abstraction of H atoms from DNA by the semiquinone drug radical. The results suggest that YM155 has the potential to be a good chemotherapeutic drug if used with the right combination against the right cancer subtype.
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(1) Yamanaka, K., Nakata, M., Kaneko, N., Fushiki, H., Kita, A., Nakahara, T., Koutoku, H., and Sasamata, M. (2011) YM155, a selective survivin suppressant, inhibits tumor spread and prolongs survival in a spontaneous metastatic model of human triple negative breast cancer. Int. J. Oncol. 39, 569−575. (2) Altieri, D. C. (2008) Survivin, cancer networks and pathwaydirected drug discovery. Nat. Rev. Cancer 8, 61−70. (3) Coumar, M. S., Tsai, F. Y., Kanwar, J. R., Sarvagalla, S., and Cheung, C. H. (2013) Treat cancers by targeting survivin: just a dream or future reality? Cancer Treat. Rev. 39, 802−811. (4) Altieri, D. C. (2013) Targeting survivin in cancer. Cancer Lett. 332, 225−228. (5) Santa Cruz Guindalini, R., Mathias Machado, M. C., and Garicochea, B. (2013) Monitoring survivin expression in cancer: implications for prognosis and therapy. Mol. Diagn. Ther. 17, 331− 342. (6) Nakahara, T., Kita, A., Yamanaka, K., Mori, M., Amino, N., Takeuchi, M., Tominaga, F., Kinoyama, I., Matsuhisa, A., Kudou, M., and Sasamata, M. (2011) Broad spectrum and potent antitumor activities of YM155, a novel small-molecule survivin suppressant, in a wide variety of human cancer cell lines and xenograft models. Cancer Sci. 102, 614−621. (7) Yamanaka, K., Nakata, M., Kaneko, N., Fushiki, H., Kita, A., Nakahara, T., Koutoku, H., and Sasamata, M. (2011) YM155, a selective survivin suppressant, inhibits tumor spread and prolongs survival in a spontaneous metastatic model of human triple negative breast cancer. Int. J. Oncol. 39, 569−575. (8) Glaros, T. G., Stockwin, L. H., Mullendore, M. E., Smith, B., Morrison, B. L., and Newton, D. L. (2012) The ″survivin suppressants″ NSC 80467 and YM155 induce a DNA damage response. Cancer Chemother. Pharmacol. 70, 207−212. (9) Winter, G. E., Radic, B., Mayor-Ruiz, C., Blomen, V. A., Trefzer, C., Kandasamy, R. K., Huber, K. V., Gridling, M., Chen, D., Klampfl, T., Kralovics, R., Kubicek, S., Fernandez-Capetillo, O., Brummelkamp, T. R., and Superti-Furga, G. (2014) The solute carrier SLC35F2 enables YM155-mediated DNA damage toxicity. Nat. Chem. Biol. 10, 768−773. (10) Kasap, C., Elemento, O., and Kapoor, T. M. (2014) DrugTargetSeqR: a genomics- and CRISPR-Cas9-based method to analyze drug targets. Nat. Chem. Biol. 10, 626−628. (11) Hu, S., Fu, S., Xu, X., Chen, L., Xu, J., Li, B., Qu, Y., Yu, H., Lu, S., and Li, W. (2015) The mechanism of radiosensitization by YM155, a novel small molecule inhibitor of survivin expression, is associated with DNA damage repair. Cell. Physiol. Biochem. 37, 1219−1230. (12) Chang, B. H., Johnson, K., LaTocha, D., Rowley, J. S., Bryant, J., Burke, R., Smith, R. L., Loriaux, M., Muschen, M., Mullighan, C., Druker, B. J., and Tyner, J. W. (2015) YM155 potently kills acute lymphoblastic leukemia cells through activation of the DNA damage pathway. J. Hematol. Oncol. 8, 39. (13) Holmes, D. (2012) Cancer drug’s survivin suppression called into question. Nat. Med. 18, 842−843. (14) Ho, S. H., Sim, M. Y., Yee, W. L., Yang, T., Yuen, S. P., and Go, M. L. (2015) Antiproliferative, DNA intercalation and redox cycling activities of dioxonaphtho[2,3-d]imidazolium analogs of YM155: A structure-activity relationship study. Eur. J. Med. Chem. 104, 42−56. (15) Hong, M., Ren, M. Q., Silva, J., Paul, A., Wilson, W. D., Schroeder, C., Weinberger, P., Janik, J., and Hao, Z. (2017) YM155 inhibits topoisomerase function. Anti-Cancer Drugs 28, 142−152. (16) Clemens, M. R., Gladkov, O. A., Gartner, E., Vladimirov, V., Crown, J., Steinberg, J., Jie, F., and Keating, A. (2015) Phase II, multicenter, open-label, randomized study of YM155 plus docetaxel as first-line treatment in patients with HER2-negative metastatic breast cancer. Breast Cancer Res. Treat. 149, 171−179. (17) Satoh, T., Okamoto, I., Miyazaki, M., Morinaga, R., Tsuya, A., Hasegawa, Y., Terashima, M., Ueda, S., Fukuoka, M., Ariyoshi, Y.,
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.8b00094. Cell survival assays, comet assay, and ROS detection (PDF)
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*Tel: 91-9821131095; e-mail:
[email protected]. ORCID
Goutam Chowdhury: 0000-0003-1397-8684 Author Contributions
G.C. and A.C. designed the experiments and analyzed the data; T.H.W., S.S., and A.J. performed the experiments, and G.C. wrote the manuscript. Author Contributions §
T.H.W. and S.S. contributed equally to this work.
Funding
This work was supported in part by the Department of Biotechnology, Ramalingaswami Re-entry fellowships [BT/ HRD/35/02/2006 to G.C. and 102/IFD/SAN/1576/2014-15 to A.C.], SERB, Department of Science and Technology [ECR/2015/000197 to G.C. and A.C., ECR/2015/000198 to A.C.] and Shiv Nadar University. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS BSO, buthionine sulfoxamine; βME, beta mercaptoethanol; DTT, dithiothreitol; DMSO, dimethyl sulfoxide; DMEDE, N,N′-dimethylethylenediamine; EDTA, ethylenediaminetetraacetic acid; GSH, glutathione; H2O2, hydrogen peroxide; NaOH, sodium hydroxide; ROS, reactive oxygen species; 617
DOI: 10.1021/acs.chemrestox.8b00094 Chem. Res. Toxicol. 2018, 31, 612−618
Article
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DOI: 10.1021/acs.chemrestox.8b00094 Chem. Res. Toxicol. 2018, 31, 612−618
