Xanthatin promotes apoptosis via inhibiting thioredoxin reductase and

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Xanthatin promotes apoptosis via inhibiting thioredoxin reductase and eliciting oxidative stress Ruijuan Liu, Danfeng Shi, Junmin Zhang, Xinming Li, Xiao Han, Xiaojun Yao, and Jianguo Fang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00338 • Publication Date (Web): 25 Jun 2018 Downloaded from http://pubs.acs.org on June 25, 2018

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Molecular Pharmaceutics

Xanthatin promotes apoptosis via inhibiting thioredoxin reductase and eliciting oxidative stress Ruijuan Liu a,b, Danfeng Shi c, Junmin Zhang b, Xinming Li a,c , Xiao Han a,c, Xiaojun Yao c and Jianguo Fang a,c* a

State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000,

China b

School of Pharmacy, Lanzhou University, Lanzhou 730000, China

c

College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China

*

Corresponding author, E-mail: [email protected] (J. Fang).

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ABSTRACT Xanthatin (XT), a naturally occurring sesquiterpene lactone presented in cocklebur (Xanthium strumarium L.), is under development as a potential anticancer agent. Despite of the promising anticancer effect of XT, the molecular mechanism underlying its cellular action has not been well elucidated. The mammalian thioredoxin reductase (TrxR) enzymes, essential seleno-flavoproteins containing a penultimate selenocysteine (Sec) residue at the C-terminus, represent a promising target for cancer chemotherapeutic agents. In this study, XT inhibits both the purified TrxR and the enzyme in cells. The possible binding mode of XT with the TrxR protein is predicted by the covalent docking method. Mechanism studies reveal that XT targets the Sec residue of TrxR and inhibits the enzyme activity irreversibly. Simultaneously, the inhibition of TrxR by XT promotes oxidative stress-mediated apoptosis of HeLa cells. Importantly, knockdown of the enzyme sensitizes the cells to XT treatment. Targeting TrxR thus discloses a novel molecular mechanism in accounting for the cellular action of XT, and provides insights into the development of XT as an anticancer agent. KEYWORDS: Xanthatin; Oxidative stress; Thioredoxin; Apoptosis; Molecular docking; Anticancer.


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Introduction Plant-derived natural products have historically proven to be a pivotal and unique source of therapeutic drugs against human diseases and still represent a significant pool for the discovery and exploitation of new drug leads at present. Sesquiterpene lactones, a family of many bioactive compounds in medicinal plants, exert multiple pharmacological properties1, 2. Xanthatin (XT), a natural sesquiterpene lactone isolated from the traditional Chinese herbal Xanthium strumarium L. (cocklebur), has been used for the treatment of diverse pathologies3, 4. XT has been reported to affect several therapeutic targets in multiple cell types5, 6, such as stimulating expression of DNA damage-inducible GADD45γ mRNA7-9, inhibiting vascular endothelial growth factor (VEGF)-stimulated angiogenesis10, blocking phosphorylation of NF-κB11,

12

,

inhibiting 5-lipoxygenase activity and activating of Wnt/β-catenin pathway13, 14, downregulating GSK3β and STAT3 activities15, 16, and inhibiting the degranulation of LAD2 cells17. Despite of the potent anticancer activity of XT, its molecular mechanism is still poorly defined, and the primary cellular target and mode of action of this molecule are still in debate. The thioredoxin system, which is composed of thioredoxin reductase (TrxR), thioredoxin (Trx) and NADPH, is a crucial antioxidant system in the maintenance of redox balance and regulation of redox-based signaling pathways18,

19

. Accumulating evidence suggests that this

system is a pivotal player closely related to many human diseases20, 21. The mammalian TrxR proteins, existing mainly as the cytosolic TrxR1 and mitochondrial TrxR218, 22, 23, are selenoflavoenzymes containing a penultimate C-terminal selenocysteine (Sec) residue24. TrxRs catalyze the transfer of electrons from NADPH to the active site of Trxs leading to the generation of reduced Trxs, and the reduced Trxs interact with downstream targets to regulate diverse redox-based intracellular responses involved in cell proliferation, differentiation and death25. The higher Trx/TrxR levels usually exist in malignant cells than in normal cells, and thus targeting the Trx/TrxR system is thought to be an effective approach to impede tumor progression and 3 ACS Paragon Plus Environment


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metastasis26. Therefore, the past years have witnessed continuing efforts on the development of small molecules targeting TrxR as potential therapeutic agents for cancer27-33. XT has been documented processing potent anticancer activity, and targeting TrxR inhibition is increasingly recognized as a promising approach for development of anticancer drugs. Thus, we hypothesize XT may be a novel inhibitor of TrxR. As part of our continuing efforts to discover small molecule modulators targeting the cellular redox system21, 31, 32, 34-36, we demonstrate in the present study that XT inhibits the TrxR efficiently, and the Sec residue of the enzyme appears to be the primary target for this inhibition. Furthermore, XT causes a strong TrxR inhibition in HeLa cells. The covalent docking approach supports that XT binds to the Sec498 of TrxR irreversibly and all the hydrogen bonding interactions between XT and TrxR facilitate the covalent bond formation. XT increases reactive oxygen species (ROS) accumulation and further promotes oxidative stress-mediated apoptosis of HeLa cells. The cytotoxicity of XT correlates well with its ability to inhibit the cellular TrxR activity. Furthermore, genetic knockdown of the enzyme augments the potency of XT, which suggests a physiological significance of targeting TrxR by XT. Taken together, our results account for the cellular action of XT via an unprecedented mechanism, and shed lights on understanding its anticancer effect and developing XT as a potential anticancer drug. Materials and methods Chemicals and enzymes XT (MW 246.3, 98% purity) was obtained from ChemFaces (Wuhan, China) and dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution (100 mM). Unless otherwise indicated, the final concentrations of DMSO in all experiments were no more than 0.1% (v/v). Nacetyl-Asp-Glu-Val-Asp-p-nitroanilide

(Ac-DEVD-pNA),

3-(3-cholamidopropyl)

dimethylammonio-1-propanesulfonate (CHAPS), L-buthionine-(S, R)-sulfoximine (BSO), DMSO, N-acetyl-L-cysteine (NAC), 2, 3-dimercapto-1-propanesulfonic acid (DMPS), bovine 4 ACS Paragon Plus Environment

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insulin, reduced and oxidized glutathione (GSH and GSSG), Tris (2-Carboxyethyl) phosphine (TCEP), 2’, 7’-dichlorfluorescein diacetate (DCFH-DA), Dulbecco's modified Eagle’s medium (DMEM) and Hoechst 33342 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-actin antibody (AA128-1), phenylmethylsulfonyl fluoride (PMSF), bovine serum albumin (BSA), trypan blue and sodium orthovanadate (V) (Na3VO4) were purchased from Beyotime (Nantong, China). Fetal bovine serum (FBS), as the growth supplement for cell culture, was a product of Sijiqing (Hangzhou, China). 5, 5’-Dithiobis-2-nitrobenzoic acid (DTNB) was purchased from J&K Scientific (Beijing, China). The fluorescein-5-isothiocyanateconjugated Annexin V (Annexin V-FITC) and propidium iodide (PI) apoptosis detection kit were purchased from Zoman Biotech (Beijing, China). 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT), streptomycin and penicillin were products of Amresco (Solon, OH, USA). NADPH and poly-vinylidene difluoride (PVDF) membrane were purchased from Roche (Mannheim, Germany) and Millipore (Billerica, MA), respectively. The anti-rabbit IgG-HRP (sc-2004), anti-mouse IgG-HRP (sc-2031), dihydroethidium (DHE) and TrxR1 primary antibody (sc-28321) were products of Santa Cruz Biotechnology (Santa Cruz, CA). The recombinant rat TrxR1 from Prof. Arne Holmgren (Karolinska Institute, Stockholm, Sweden) was essentially prepared as described and its activity was half of wild type TrxR137. The Escherichia coli Trx, PAO-sepharose and recombinant U498C TrxR1 mutant (Sec→Cys) were obtained by the method provided in our previous publication38. The recombinant rat TrxR1 and the mutant enzyme (U498C TrxR1) have an activity of ~666 and ~63 mol of NADPH oxidized/min/mol using the DTNB as a substrate, respectively. All other reagents were of analytical grade and used without further purification. Cell lines and culture conditions HeLa (cervical carcinoma), HepG 2 (hepatocellular carcinoma), A549 (lung carcinoma), HEK 293T (embryonic kidney–293T) and BEAS-2B (bronchial epithelial) cells were obtained 5 ACS Paragon Plus Environment


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from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Cells were cultured under the standard culture conditions (DMEM containing 100 units/ml penicillin/streptomycin, 2 mM glutamine and 10% FBS, 5% CO2, 37 °C). HeLa-shNT cells were used as described in our lab and maintained under the standard culture conditions supplemented with puromycin (1 µg/ml)39. Cell viability analysis MTT assay Following treatment of cells (1×104 cells/well in 96-well plates) with the tested compound in triplicate in a final volume of 100 µl for the indicated times, 10 µl/well of MTT (5 mg/ml) was added. After 4 h incubation at 37 °C, 100 µl of the extraction buffer containing 5% iso-butanol, 0.1% HCl and 10% SDS was added, and the cells were incubated overnight further. Controls were treated with 0.1% (v/v) DMSO alone. The absorbance (570 nm) of each well was detected using a microplate reader (Multiskan GO, Finland) and used to calculate the viability. Trypan blue exclusion assay HeLa cells (5×104 cells/well in 24-well plates) were treated with various concentrations of XT for 24 h. Controls were treated with DMSO only. The cell viability was measured by the trypan blue exclusion assay. After the treatment, the trypan blue (0.4%, w/v) was used to stain cells, and the number of viable (non-stained) cells was counted using a microscope. Molecular docking simulation In order to investigate the interaction between XT and TrxR, a covalent docking was performed in the program Schrödinger Suite 2015-1. A rat TrxR1 structure (PDB code 3EAN, Chain A and B) was obtained from the Protein Data Bank and further prepared in the Protein Preparation Wizard module. The residue Sec498 in the chain A was selected as the reactive 6 ACS Paragon Plus Environment

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residue involved in the Michael addition and also set as the centroid of the docking pocket. The docking simulation was performed with the default parameters. Purified TrxR activity assay The DTNB reduction assay is a convenient method to monitor the purified enzyme activity. Following treatment of the NADPH-reduced U498C TrxR (350 nM) or TrxR (85 nM) with the tested compound in a final volume of 50 µl for the indicated times, a master mixture (50 µl) with NADPH (200 µM) and DTNB (2 mM) in TE buffer (50 mM Tris-HCl, 1 mM EDTA, pH 7.5) were added. The relative enzyme activity was determined by recording the increasing optical density (412 nm). Using the DTNB as a substrate, the specific activities of the recombinant TrxR and U498C TrxR are ~666 and ~63 mol of NADPH oxidized/min/mol, respectively. For the convenience, the potency of the inhibition of recombinant and mutant enzymes by XT was presented as the percentage change from the corresponding control group. Imaging of TrxR activity TRFS-green, a cell membrane permeable dye developed in our laboratory40, is applied to visualize the activity of TrxR. Briefly, 5×105 HeLa cells were incubated in 12-well plates for 8 h with the indicated concentrations of XT, followed by additional incubation for 4 h with TRFSgreen (10 µM). The fluorescence images were acquired using an inverted Leica DMI4000 fluorescence microscope (Leica Microsystems GmbH, Germany). Determination of TrxR activity in cells After incubating with varying concentrations of XT for 24 h, HeLa cells (70-80% confluence) were harvested and washed twice by PBS. The total cellular protein was extracted by RIPA buffer (150 mM NaCl, 1% Triton X-100, 50 mM Tris-HCl, 2 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, pH 7.5, 0.1% SDS and 0.5% deoxycholate) for 30 min on ice, and

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quantified by the Bradford procedure. According to our published protocols39, the endpoint insulin reduction assay was adopted to measure the TrxR activity in cell lysates. Determination of Trx redox states The redox states of Trx were assayed according to the high affinity of phenylarsine oxide (PAO) with vicinal dithiols38, 41. After the reduced Trx was captured by PAO beads (PAOsepharose), which were obtained by coupling the PAO molecule to sepharose 4B38, the oxidized forms were reserved in the supernatant fraction. Then HeLa cells were treated with XT (20 µM) for 24 h. The total cellular protein was extracted by lysis of frozen samples with RIPA buffer and quantified using the Bradford protein assay. The control samples, i. e., the fully reduced Trx and fully oxidized Trx, were prepared by incubation of the non-treated cell lysates with TCEP (5 mM) and diamide (5 mM), respectively. The samples were loaded on PAO-sepharose and placed on a rotating shaker. After 30 min, the supernatant containing oxidized Trx was pooled. The sepharose beads were washed extensively with TE buffer and the reduced Trx was knocked out by DMPS (20 mM). The total cellular protein was fractionated by SDS-PAGE under reducing conditions and transferred to PVDF membranes for Western blot assay. Measurement of the intracellular ROS HeLa cells (5×105 cells/well) were incubated with XT in 12-well plates for 1 h. The ROS indicator DHE (10 µM) or DCFH-DA (10 µM) in fresh FBS-free medium was added to each well and allowed to incubate for 30 min at 37 °C. Images of sample were examined by an inverted fluorescence microscope. Assessment of intracellular thiols Following treatment with increasing concentrations of XT in 100 mm dishes for 24 h at room temperature, 2×106 HeLa cells were harvested and washed with PBS. The total cellular protein was extracted with RIPA buffer, and quantified using the Bradford assay. The 8 ACS Paragon Plus Environment

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concentration of total cellular thiols was measured using DTNB-titration method as described below. After adding cell lysate (10 µl) to wells with DTNB (Tris-HCl pH 8.0, 1 mM in 90 µl of 6 M guanidine hydrochloride), and incubating at room temperature for further 5 min, the absorbance at 412 nm was measured on a microplate reader. The total content of thiols was calculated according to a calibration curve using GSH as a standard. GSH and GSSG assay Total GSH and GSSG were measured and quantified by the enzymatic recycling method39, 42

. Following treatment of HeLa cells (2×106 cells/well in 100 mm dishes) for 24 h with the

indicated concentrations of XT, the cells were resuspended in ice-cold KPE buffer (0.6% sulfosalicyclic acid, 0.1% Triton X-100, 5 mM EDTA, pH 7.5, 0.1 M potassium phosphate buffer). After the suspension was sonicated in ice for 2-3 min with vortexing every 30 s, the solution was centrifuged at 3000g for additional 4 min at 4 °C, then the supernatant was immediately collected for measuring the total GSH. After GSH was derivatized by 2vinylpyridine, GSSG was measured. Briefly, the mixture of 2 µl of 2-vinylpyridine and 100 µl cell supernatant reacted for 1 h in a fume hood at room temperature, then triethanolamine (6 µl) was added to the supernatant and the solution was mixed. Assay of GSSG was performed using the procedure described above for the total GSH. Western blot analysis The same amount of lysate proteins from each fraction were isolated by SDS-PAGE (40 µg per lane) under reducing conditions and then electroblotted to PVDF membranes. After blocked with 5% non-fat milk in TBST for 2 h at room temperature, the membranes were probed with indicated primary antibodies at 4 °C overnight. After washed in TBST three times, the membranes were incubated with the peroxidase–conjugated secondary antibodies for 1 h at room temperature. Measurement of signal intensity on the membranes was performed with a chemiluminescence kit from GE Healthcare Life Science. 9 ACS Paragon Plus Environment


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Apoptosis assays Hoechst 33342 staining Following treatment of HeLa cells (5×105 cells/well in 12-well plates) with the indicated concentrations of XT for 24 h, Hoechst 33342 (5 µg/ml) was added and incubated for 15 min. Images of cells were captured under a Floid Cell Imaging Station (Life Technologies, USA). Determination of caspase 3 activity After incubation of 2×106 HeLa cells in 100 mm dishes for 24 h with different concentrations of XT, cells were washed twice with PBS and lysed on ice. The RIPA buffer and the Bradford procedure were used to extract and quantify the protein, respectively. According to the published protocols39, 43, the caspase 3 activity was determined by a colorimetric assay using Ac-DEVD-pNA as a substrate. Annexin V/PI staining Following incubation of 1×106 HeLa cells (in 6-well plates) for 24 h with the indicated concentrations of XT, the cells were rinsed twice with PBS. According to the manufacturer’s protocol, the Annexin V-FITC/PI double staining assay was used for the apoptosis analysis of necrotic cells, apoptotic cells and live cells. The stained cells were quantified by the FACSCantoTM flow cytometer (BD Biosciences, USA) with CellQuest software. Statistics All the values were means ± SE from at least 3 experiments. Student’s t-test was used to determine statistical significance of differences between groups. Multiple comparisons between control and treatment groups were made by one-way analysis of variance (ANOVA). Results were considered to be significant at p < 0.05.


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Results Inhibition of TrxR by XT The α-methylene-γ-lactone moiety, an electrophilic center, is a core structure of XT, and has been documented responsible for the observed biological functions of XT (Fig. 2)44-46. Several TrxR inhibitors, such as parthenolide32, alantolactone31 and elephantopus mollis 2347, also contain such functionality. Therefore, it is reasonable to speculate that XT is also an inhibitor of TrxR. Firstly, the inhibition of XT on purified TrxR was tested. Incubation of XT with the enzyme resulted in a dose-responsive inhibitory activity. The Sec residue is critical for the enzyme activity, and has been reported to be targeted by electrophilic inhibitors48. A survey of inhibition of U498C TrxR1 by XT, in which the Sec498 was replaced by Cys, was further determined. Due to the low activity of the U498C TrxR1, a high concentration was used in the assay. As shown in Fig. 1C, XT exhibited a trivial effect on U498C TrxR1. Selective inhibition of wild type (WT) TrxR1 over U498C TrxR1 indicated that XT specifically targeted the Sec residue in the active site of TrxR. It was worth nothing that the inhibitory efficacy of TrxR depended on the incubation time (Fig. 1A). Then, the TrxR inhibition by XT was determined in HeLa cells. The cellular TrxR activity was determined using TRFS-green, a green emission probe to detect TrxR developed by our group40. The obtained data (Fig. 1B) showed that the cells treated with XT had low fluorescence, supporting the inhibition of TrxR by XT. A classical Trxmediated insulin reduction assay was performed to verify the imaging results. Treatment of the cells with XT impaired the cellular TrxR activity dose-dependently (Fig. 1D), which was consistent with observations from live cell imaging assay. These results indicated that XT had a certain influence on enzyme activity, and also supported the involvement of Sec residue in XTinduced TrxR inhibition. Collectively, XT is a novel inhibitor of TrxR, and the Sec residue in TrxR is critical to the interaction between XT and the enzyme. Molecular docking simulation 11 ACS Paragon Plus Environment


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The effective inhibition of the purified TrxR and the enzyme in cells by XT was demonstrated, and then we predicted the possible binding mode for XT interacting with TrxR1 protein by the covalent docking method (Fig. 2). According to the Michael addition reaction mechanism between the electron-deficient double bond and the selenium atom of Sec498 in TrxR1, XT turns out to harbor two reactive sites (site 1 and site 2 in Fig. 2). The site 1 is a terminal α-methylene group connected to the γ-lactone moiety, and the site 2 contains a substituted α, β-unsaturated ketone structure. The binding affinities for both reactive sites were evaluated by three energy parameters in Schrödinger. As shown in Table 1, site 1 seemed to have greater binding affinity than site 2 with a lower docking score of -5.095 kcal/mol. The binding mode was further analyzed to recognize the key interactions between XT and TrxR1. The detailed interaction comparison between site 1 and site 2 was shown in Fig. 2. The binding mode showed that the binding pocket for XT was formed by the C-terminal redox active site of one monomer, including Gly470, Glu477, Thr481, Cys497, Sec498 and Gly499, and some hydrophobic residues of the other monomer, including Pro344, Ile347 and Gln348. The hydrogen bonding interactions were found between the carbonyl group of XT and the amino groups of Gly470 and Cys497, which were both located at the C-terminal redox active site and ensured the stability of XT binding. All these results above presented a clear view that XT reacted with Sec498 of TrxR1 irreversibly by forming a covalent bond at site 1 and all the hydrogen bonding interactions between XT and TrxR1 appeared to facilitate the covalent bond formation. Cytotoxicity of XT As described above, we have demonstrated that XT effectively inhibits the enzyme activity of TrxR. In order to extend this discovery, we adopted the MTT assay to detect the cytotoxicity of XT. The viability of HeLa cells after XT treatment was decreased in a dose-dependent manner (Fig. 3A), and the concentration causing half inhibition of the cell proliferation (IC50 value) could 12 ACS Paragon Plus Environment

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be obtained as 4.92 µM following treatment of HeLa cells for 48 h. The cytotoxicity of XT to other cancer cell lines was also studied. As shown in Fig. 3C, XT exhibited significant efficacy against A549 and HepG 2 cells. Interestingly, XT showed less toxicity to two noncancerous cell lines, HEK 293T and BEAS-2B cells (Fig. 3D). To confirm the results from the MTT assay, the trypan blue exclusion assay was employed to verify the cell viability. After treatment for 24 h or 48 h with varying concentrations of XT, the dead cells were blue-stained, and the unstained live ones were counted (Fig. 3B). The trypan blue exclusion assay and the MTT assay yielded fairly consistent results (Fig. 3A), which further confirmed the observed time-dependent cytotoxicity of XT (Fig. 3B). In order to determine whether the toxicity of XT to the cells was related to the inhibition of TrxR activity, we also performed a correlation analysis between the two factors. The decreased TrxR activity in the XT-treated cells correlated well with the cytotoxicity of XT in both HeLa and A549 cells (Fig. 3E and 3F). The strong correlation between the cellular TrxR inhibition and cytotoxicity of XT implied that the inhibition of TrxR was important for the cytotoxicity of XT. Involvement of TrxR for cellular action of XT It has been demonstrated that XT is an efficient inhibitor of TrxR, and shows potent cytotoxic effects against multiple cell lines, especially cancer cells. To further elucidate the relationship between the observed cytotoxicity of XT and the interaction between TrxR and XT, we transfected short hairpin plasmids specifically targeting TrxR1 to generate HeLa cells stably knocking down the expression of TrxR1 (HeLa-shTrxR1). The control HeLa-shNT cells were also created by transfecting a non-targeting control plasmid. TrxR1 knockdown was adequately assessed by two different assays, i.e., the activity assessment (Fig. 4A) and expression of the protein (Fig. 4A inset). HeLa-shTrxR1 cells showed a >80% declines in TrxR1 expression compared with HeLa-shNT cells. The activity of total TrxR (including TrxR1 and TrxR2) decreased to ~50% in HeLa-shTrxR1 cells. Next, we determined the cytotoxicity of XT against 13 ACS Paragon Plus Environment


HeLa-shTrxR1 and HeLa-shNT cells. As shown in Fig. 4B, knockdown of TrxR was shown to sensitize cells to XT treatment. Less XT would be required to achieve inhibition of TrxR in cells with lower TrxR level, thereby enhancing the efficiency and effectiveness of XT. The results were consistent with many TrxR-targeting small molecules reported in other studies, such as parthenolide32 and alantolactone31. The findings indicated that TrxR was involved in the cytotoxicity induced by XT. Redox states of Trx in HeLa cells As TrxRs are the known enzymes to reduce oxidized Trx to the reduced state, we then determined the redox states of Trx after the cells were treated with XT. The PAO-sepharose pulldown assay was employed, and the assay principle was described previously38. To further verify this assay, the oxidized Trx and reduced Trx were produced in cell lysates treated with diamide and TCEP respectively. As illustrated in Fig. 5A, after the samples were loaded on PAO-sepharose, the oxidized Trx was contained in the supernatant only, while the reduced Trx was captured on the beads, which confirmed that the reduced Trx could be distinguished from the oxidized Trx by this assay. The greater part of Trx was presented in the reduced form in vehicle-treated cells, while the oxidized Trx significantly increased after treatment of the cells with XT. The bands intensity of the oxidized and reduced Trxs was quantified, and the reduced/oxidized Trx ratio was shown in Fig. 5B. Treatment with XT did not induce significant changes in total expression of Trx1 (Fig. 5C). Induction of oxidative stress Low level of ROS is essential for cellular signaling. However, the uncontrolled production of ROS is associated with oxidative stress49. The thioredoxin system keeps the intracellular redox balance to prevent excessive accumulation of ROS. Thus, a large number of TrxR inhibitors, such as alantolactone31 and plumbagin30, could lead to oxidative stress. In order to determine whether XT exhibited a similar effect, we then measured the ROS level in cells after XT 14 ACS Paragon Plus Environment

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treatment. Firstly, the total intracellular ROS content was determined by DCFH-DA, a general probe to detect the intracellular production of ROS. Treatment of HeLa cells with XT resulted in a remarkable increase in intracellular ROS level (Fig. 6A). As DCFH-DA-based ROS assay suffered from false positive results50, DHE was used to validate ROS production. Similarly, detectable fluorescence signals were induced in a dose-dependent manner (Fig. 6B) after treatment of the cells with XT. Both the DHE and DCFH-DA assays indicated that XT could elevate ROS production in HeLa cells. In general, cancer cells are more sensitive to oxidative stress than noncancerous cells as cancers cells usually harbor higher ROS level than noncancerous cells. This might account for the observed less cytotoxicity of XT to HEK 293T and BEAS-2B cells (Fig. 3D). The balance between disulfides and free sulfhydryl groups mainly dominated the cellular redox states41. To assess the overall redox states of HeLa cells treated with XT, the total thiol content was quantified by DTNB titration. Treatment of HeLa cells with low concentrations of XT (5 µM and 10 µM) caused a significant elevation of the total thiols (Fig. 6C). In mammalian cells, the thiol-dependent antioxidant system can be divided into two major systems, i.e., the thioredoxin system and the glutathione system. Furthermore, we determined the changes of glutathione homeostasis in HeLa cells treated with different concentrations of XT for 24 h. As shown in Fig. 6D and 6E, an increase of the total GSH and a higher or constant ratio of GSH/GSSG were presented in cells after XT treatment. The elevation of GSH is likely due to the activation of the Keap1-Nrf2 pathway as many small molecules containing the Michael acceptor scaffold are known activators of Nrf251, 52. The upregulation of GSH may contribute to the increase of total cellular thiols. The exposure of the cells to XT stimulated the production of GSH, indicating that the cells responded to oxidative stress by upregulation of cellular thiol pools. Promotion of cell death by GSH depletion and protection of cell death by NAC 15 ACS Paragon Plus Environment


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It has been indicated that XT causes the elevation of ROS levels and cellular thiol pools, especially the boost of cellular GSH. In order to verify the role of GSH during XT treatment, the functions of NAC supplement and GSH depletion were examined. NAC acts as a precursor for biosynthesis of cellular GSH and thiol antioxidant. The cytotoxicity of XT was attenuated after pretreatment of HeLa cells with NAC, and the HeLa cell death induced by XT was blocked entirely with a high concentration of NAC (Fig. 7A). In contrast to the NAC-dependent protection, the cytotoxicity of XT was elevated significantly via GSH depletion in cells pretreated with BSO (Fig. 7B). After treatment of HeLa cells with BSO at a concentration of 50 µM for 24 h, the intracellular level of GSH was downregulated to < 20% of that in the control group under our experimental conditions. GSH is a crucial component of the glutathione system, another redox regulation network besides the thioredoxin system in cells. Our results, protecting and sensitizing cells to XT treatment by the supplement of NAC and depletion of GSH respectively, support the involvement of oxidative stress in the pharmacological effects of XT. Apoptosis induction in HeLa cells Apoptosis, a precisely regulated process under both physiological and pathological conditions, is frequently avoided by cancer cells, which arise from a complex interplay of genetic aberrations and misregulated death pathways53. In this study, we identified that HeLa cells were killed by XT mainly through the apoptosis (Fig. 8). The apoptosis was demonstrated using different assays. Firstly, Hoechst 33342, a membrane permeable dye was used to characterize morphological changes of the nuclei. The untreated control cells showed regular circular nuclei with weak and uniform fluorescence, as shown in Fig. 8A. However, the cells treated with XT displayed

condensed

and

highly

fluorescence, the

morphological hallmark

of

the

apoptotic nuclei. Secondly, the apoptotic cells were quantified by flow cytometer using the Annexin V-FITC/PI double staining assay. The scattergrams and quantification results were shown in Fig. 8B and 8D, respectively. Upon treatment of the cells with 5 µM, 10 µM and 20 16 ACS Paragon Plus Environment

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µM XT for 24 h, the cell apoptosis was ~22%, ~31% and ~53%, respectively. Finally, after treatment of the cells with XT, the activation of caspase 3, a key mediator during the apoptosis process, was measured. As shown in Fig. 8C, XT activated caspase 3 in HeLa cells. Combined with the above results, we concluded that XT predominantly induced apoptosis in HeLa cells. Discussion Recently, increasing studies have shown that the role of TrxR in a variety of cellular processes is closely related to the development of tumors21. Overexpression of the thioredoxin system was observed in tumor cells, such as colorectal cancer54, hepatocellular cancer55, lymphoblastic leukemia56, gastric cancer57, lung cancer54 and breast cancer58. Furthermore, the αmethylene-γ-lactone moiety was reported to be responsible for the inhibition of TrxR by many natural products, such as parthenolide32, alantolactone31 and elephantopus mollis 2347. XT also contains this moiety, thus we speculated XT might also be an inhibitor of TrxR. Firstly, whether XT could inhibit TrxR was examined. XT efficiently inhibits both the purified TrxR and the enzyme in cellular environments (Fig. 1). The more potent inhibition of WT TrxR1 than the mutant enzyme U498C TrxR1 suggested that targeting the Sec residue by XT was critically involved in the inhibition process. The molecular docking results further supported that XT covalently bound to the Sec498 of TrxR1 (Fig. 2). All the hydrogen bonding interactions between XT and TrxR1 are favorable for the covalent bond formation. Then, evidences were supplied to support that TrxR played a critical role for the cytotoxicity of XT. XT exhibited significant cytotoxicity against different human cancer cells (Fig. 3), and the inhibition of cellular TrxR by XT showed a strong correlation with the decreased cell survival rate (Fig. 3), which implied that the inhibition of TrxR contributed to the observed cytotoxicity of XT. More importantly, the potency of XT was enhanced through genetic knockdown of the enzyme, which further supported TrxR was critically involved in the biological functions of XT. Furthermore, XT-treatment caused the oxidized Trx accumulation in cells, which also suggested the inhibition 17 ACS Paragon Plus Environment


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of TrxR by XT. All these findings demonstrated the crucial involvement of TrxR in the cellular effects induced by XT. ROS are produced from many metabolic pathways and play a critical role in regulating diverse cellular processes. However, an excessive production of ROS causes oxidative stress and is harmful to cells. Cancer cells usually harbor elevated ROS level due to their high metabolic rate. Therefore, one attractive strategy for cancer treatment is to use ROS or oxidative stressinduced agents to kill cancer cells59, 60. In our study, stimulation of HeLa cells with XT resulted in a remarkable increase in the intracellular ROS level (Fig. 6A). In addition, inhibition of TrxR limits the availability of the reduced Trx, an electron donor for many antioxidant enzymes24, 61 and an inhibitor of many apoptosis-relevant enzymes62, 63, which may result in oxidative stressmediated apoptosis. It should be noted that we observed the accumulation of total cellular thiols (Fig. 6C) and increased glutathione (Fig. 6D) after treatment of the cells with XT. The glutathione and thioredoxin systems are two main networks to maintain the cellular redox homeostasis64, 65. We speculated the elevation of GSH may contribute to the increase of total cellular thiols. At the same time, we observed that the upregulation of GSH by NAC alleviated the cytotoxicity of XT (Fig. 7A), while the depletion of GSH by BSO enhanced the cytotoxicity of XT (Fig. 7B). In short, the exposure of the cells to XT stimulated the production of GSH, indicating the cells responded to oxidative stress by upregulation of cellular thiol pools. Taken together, we proposed that the ROS accumulation and inhibition of TrxR contributed to inducing apoptosis of HeLa cells. Summary In conclusion, XT acts as a potent inhibitor of TrxR to induce apoptosis of cancer cells through a previously unrecognized mechanism. Discovery of XT-TrxR interaction in cells helps understanding how this sesquiterpene lactone acts in cellular environments, while the novel


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TrxR-target molecular mechanism would also provide insights for further development of XT as an anticancer agent.

Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (21572093 & 21778028) and the Fundamental Research Funds for the Central Universities (lzujbky-2017-ot02). The authors also appreciate Prof. Constantinos Koumenis (University of Pennsylvania) for shRNA plasmids and cells, and Prof. Arne Holmgren (Karolinska Institute, Sweden) for the recombinant rat TrxR1. Conflicts of interest The authors declare no conflicts of interest.



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Table 1. The covalent binding affinity of XT with Sec498 of TrxR1 Reactive site

Docking score

MM/GBSA

Docking affinity

Site1

-5.095

-49.631

-4.633

Site2

-4.179

-30.505

-3.330

The unit of the energy values is in kcal/mol.


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Figure captions Figure 1. Inhibition of TrxR by XT. (A) Time-dependent inhibition of TrxR by XT. After incubation of the NADPH-reduced recombinant TrxR with XT (20 µM) at room temperature for the indicated times, DTNB reduction assay was performed to measure the enzyme activity. (B) Inhibition of TrxR in cells determined by TRFS-green. After treatment of HeLa cells with XT for 12 h, fluorescence changes of TRFS-green (reflecting the TrxR activity) in live HeLa cells were acquired. Scale bar: 20 µm. (C) Effects of XT on the recombinant TrxR and the mutant enzyme U498C TrxR1. The NADPH-reduced enzymes were incubated with XT for 2 h, and their activity was determined by the DTNB reduction assay. (D) Inhibition of TrxR in cells determined by the endpoint insulin assay. Following treatment of the cells with XT for 24 h at the indicated concentrations, the cellular TrxR activity was determined by the endpoint insulin assay. Data in (A), (C) and (D) are from three independent experiments and expressed as mean ± SE, and all the activity was expressed as the percentage of the control at P < 0.01(**), P < 0.05(*).

Figure 2. Molecular docking of XT with TrxR1. The docking experiments were carried out using the covalent docking protocol in the program Schrödinger Suite 2015-1. The covalent reactive sites on XT were labeled by red asterisks and the binding mode for two sites was depicted in (A) and (B), respectively. Two monomers of TrxR1 were represented by green and cyan cartoons. XT and the interactive residues in active site were shown in orange and green/cyan sticks.


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Figure 3. Involvement of TrxR in the cellular actions of XT. (A) Cytotoxicity of XT to HeLa cells. After treatment of the cells with XT for 48 h at different concentrations, the MTT assay was performed to determine the viability. (B) Trypan blue exclusion test for the cytotoxicity of XT. Following treatment of the cells for 24 or 48 h with various concentrations of XT, the trypan blue staining was used to count the viable and dead cells. (C) Cytotoxicity of XT to HeLa, A549 and HepG 2 cells. Cell viability was assessed using the MTT assay after incubation of the cells with the indicated concentrations of XT for 48 h. (D) Cytotoxicity of XT towards HeLa, BEAS2B and HEK 293T cells. Cell viability was assessed using the MTT assay after incubation of the cells with the indicated concentrations of XT for 48 h. (E), (F) Correlation of the cellular TrxR inhibition and the cell viability. After the HeLa cells (E) or A549 cells (F) were treated with varying concentrations of XT, the cellular TrxR activity was determined by the endpoint insulin reduction assay and the cell viability was determined by the MTT assay. The graphs show the percent of cell survivals and the TrxR activity in cells (mean ± SD). Pearson correlation tests (95% confidence interval) on each data set indicate a significant correlation between declines in TrxR activity and decreased cell survival: A549 cells (r=0.976, P=0.001, 2-tailed), HeLa cells (r=0.959, P=0.003, 2-tailed). Data in (A), (B), (C) and (D) are from three independent experiments and expressed as mean ± SE. P < 0.01(**) vs. the control groups in (A) and (B); P < 0.01(**) vs. the HeLa group in (D).


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Figure 4. Protection against cell death by TrxR. (A) Quantification of the cellular total TrxR activity in HeLa-shNT and HeLa-shTrxR1 cells. Cell extracts were prepared and the endpoint insulin reduction assay was performed to determine the enzyme activity. The inset shows the TrxR1 protein expression in HeLa-shNT and HeLa-shTrxR1 cells by Western blotting. P < 0.01(**) vs. HeLa-shNT cells. (B) Cytotoxic effects of XT on HeLa-shNT and HeLa-shTrxR1 cells. After the cells were treated with varying concentrations of XT for 48 h, the viability was determined by the the MTT assay. Data are expressed as mean ± SE from three independent experiments. P < 0.01(**) vs. HeLa-shTrxR1 cells.

Figure 5. Accumulation of oxidized Trx in cells. (A) Measurement of the oxidized Trx1 and reduced Trx1 by the PAO-sepharose assay. After treatment of HeLa cells with vehicle or XT (20 µM) for 24 h, the redox states of Trx1 were determined. S: samples in the supernatant; P: samples eluted from the PAO-sepharose beads; R: reduced form; O: oxidized form. (B) The ratio of reduced/oxidized Trx1 by evaluating the band intensity using ImageJ. (C) There was no significant change of Trx1 protein expression after XT treatment. After treatment of HeLa cells with XT for 24 h, Western blot was performed to detect the expression of Trx1 protein. P < 0.01(**) vs. the control groups.


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Figure 6. Induction of oxidative stress by XT. (A) DCFH-DA or (B) DHE staining. Following HeLa cells treatment for 1 h with varying concentrations of XT, the DCFH-DA (10 µM) or DHE (10 µM) was incubated for 30 min. The fluorescence pictures (bottom panel) and bright filed images (top panel) were acquired. Scale bar: 20 µm. (C) Alteration of cellular total thiol levels after XT treatment. After 24 h treatment of HeLa cells with 5, 10 and 20 µM XT, the DTNB titration was performed to determine the cellular total thiols. (D) Effect of XT on the intracellular GSH levels in HeLa cells. After treatment of the cells for 24 h with XT at varying concentrations, the cellular GSH levels were detected according to the procedure as described under materials and methods. (E) Alteration of GSH/GSSG ratio in cells. After 24 h treatment of HeLa cells with XT, the enzymatic method was performed to measure the intracellular GSH and GSSG levels, and then the GSH/GSSG ratio was presented. Data in (C), (D) and (E) are expressed as mean ± SE from three independent experiments. P < 0.05(*) and P < 0.01(**) vs. the control groups.

Figure 7. Effect of NAC supplement and GSH depletion on the cytotoxicity of XT. (A) Attenuation of the cytotoxicity of XT by NAC. After 24 h treatment of HeLa cells with the indicated concentrations of XT and NAC, the MTT assay was performed to determine the cell viability. (B) Enhancement of the cytotoxicity of XT by GSH depletion. After 24 h treatment of HeLa cells with 50 µM BSO, the intracellular GSH level was reduced. After treatment for further 48 h, the MTT assay was performed to determine the cell viability. Data are from three independent experiments and expressed as mean ± SE. P < 0.01(**) vs. the groups without NAC in (A); P < 0.01(**) vs. the groups without BSO in (B).


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Figure 8. Induction of apoptosis of HeLa cells. (A) Morphological changes of nuclei by treatment with XT. After treatment of the cells with XT at various concentrations for 24 h, the nuclei were stained by Hoechst 33342. The bright filed photographs and blue channel fluorescence images were shown on the top and bottom panel respectively. The scale bar represents 20 µM. (B) Analysis of apoptosis with FITC-Annexin V/PI double staining test. After 24 h incubation of HeLa cells with XT at various concentrations to induce apoptosis, the representative FACS analysis scatter-gating for 10000 cells were presented. (C) Quantification of the population of live cells, apoptotic cells, and necrotic cells from (B). (D) Activation of caspase 3 by XT. After treatment of the cells with XT at various concentrations for 24 h, the proteolytic activity of caspase 3 in cell lysates was detected using the standard chromogenic assay as described in the method section. Data in (C) and (D) are from three independent experiments and expressed as mean ± SE. P < 0.05(*) and P < 0.01(**) vs. the vehicle groups.


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Figure 1. Inhibition of TrxR by XT.


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Figure 2. Molecular docking of XT with TrxR1.



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Figure 3. Involvement of TrxR in the cellular actions of XT.


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Figure 4. Protection against cell death by TrxR.



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Figure 5. Accumulation of oxidized Trx in cells.


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Figure 6. Induction of oxidative stress by XT.



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Figure 7. Effect of NAC supplement and GSH depletion on the cytotoxicity of XT.


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Figure 8. Induction of apoptosis of HeLa cells.



Xanthatin promotes apoptosis via inhibiting thioredoxin reductase and eliciting oxidative stress Ruijuan Liu a,b, Danfeng Shi c, Junmin Zhang b, Xinming Li a,c , Xiao Han a,c, Xiaojun Yao c and Jianguo Fang a,c* a

State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou

730000, China b

School of Pharmacy, Lanzhou University, Lanzhou 730000, China

c

College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou

730000, China

*

Corresponding author, E-mail: [email protected] (J. Fang).


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Xanthatin promotes apoptosis via inhibiting thioredoxin reductase and

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