The Natural Diterpenoid Isoforretin A Inhibits Thioredoxin-1 and … · HepG2, MCF7, MDA-MB-231, and Caco2 cells were cultured in MEM supplemented with 10% FBS. HeLa, A549, U2OS,

Therapeutics, Targets, and Chemical Biology

The Natural Diterpenoid Isoforretin A InhibitsThioredoxin-1 and Triggers Potent ROS-MediatedAntitumor EffectsXiaoyan Sun1,2,Weiguang Wang3, Jiao Chen1,2, Xueting Cai1,2, Jie Yang1,2, Yang Yang1,2,Huaijiang Yan1,2, Xiaolan Cheng1,2, Juan Ye1,2,Wuguang Lu1,2, Chunping Hu1,2,Handong Sun3, Jianxin Pu3, and Peng Cao1,2

Abstract

Aberrant expression of thioredoxin 1 (Trx1) plays an impor-tant role in cancer initiation and progression and has gainedattention as an anticancer drug target. Here we report that therecently discovered natural diterpenoid isoforretin A (IsoA)significantly inhibits Trx1 activity and mediates anticancereffects in multiple preclinical settings. The inhibitory effect ofIsoA was antagonized by free radical scavengers polyethyleneglycol-catalase, polyethylene glycol superoxide dismutase, thi-ol-based antioxidants N-acetylcysteine and glutathione. Massspectrometry analysis revealed that the mechanism of action

was based on direct conjugation of IsoA to the Cys32/Cys35residues of Trx1. This conjugation event attenuated reversiblethiol reduction of Trx1, leading to ROS accumulation and abroader degradation of thiol redox homeostasis in cancer cells.Extending these in vitro findings, we documented that IsoAadministration inhibited the growth of HepG2 tumors in amurine xenograft model of hepatocellular carcinoma. Takentogether, our findings highlight IsoA as a potent bioactiveinhibitor of Trx1 and a candidate anticancer natural product.Cancer Res; 77(4); 926–36. �2016 AACR.

IntroductionThioredoxin 1 (Trx1; approved symbol: TXN; synonyms: TRX)

is a critical antioxidant protein and participates in a wide range ofcellular processes, such as cell proliferation, apoptosis, and aging(1, 2). Trx1 contains the active site Cys-Gly-Pro-Cys, whichreduces the target proteins by cysteine thiol-disulfide exchange(3). Upon oxidation, the two conserved cysteine residues in theactive site form a disulfide bond, and the oxidized Trx (Trx-S2) isthen recycled to the reduced form Trx-(SH)2 through the action ofthioredoxin reductase and NADPH (4).

Accumulating evidence has suggested that Trx1 is extensivelyinvolved in tumor initiation and progression. Trx1 is overex-pressed in many malignant cell lines and in a wide variety ofhuman tumors compared with normal corresponding tissues

(5–12). Overexpression of Trx1 can promote tumor cell prolif-eration and help tumor cells evade apoptosis through binding toapoptosis signal regulating kinase-1 (ASK1; ref. 13) and the tumorsuppressor PTEN (14). Besides, Trx1 stimulates sustained angio-genesis in tumors by increasing hypoxia-inducible factor 1a leveland enhancing VEGF function (15, 16).Moreover, overexpressionof Trx1 promotes cancer metastasis, and clinically is correlated topoor prognosis (10–12, 17, 18). These actions evidently makeTrx1 a promising target for cancer therapy.

So far, several Trx1-specific inhibitors have been identified,such as semisynthetic unsymmetrical 2-imidazolyl disulfides,which interact with Trx1 as its substrates (3, 19). Of note,1-methylpropyl 2-imidazolyl disulfide (PX12) has been inclinical trials, but little success has been observed (20, 21).Currently, no natural compounds have shown specific inhib-itory effect on Trx1 by binding to Trx1. Therefore, it is of greatvalue to explore novel natural Trx1-specific inhibitors for effec-tive cancer therapy.

Isodon forrestii var. forrestii is a traditional Chinese medicinalherb of the Isodon species distributed in the Southwest China andused for centuries in China. The Isodon plants are rich in ent-kaurane diterpenoids, which attract considerable interest for theirwell-known anticancer activities (22–27). IsoA is a novel ent-kaurane constituent isolated from the leaves of I. forrestii var.forrestii. The anticancer activity of IsoA has not been fullycharacterized.

In this study, we report that IsoA inhibited the growth of cancercells both in vitro and in vivo. Mechanically, IsoA suppressed Trx1activity through covalently binding to the Cys32/Cys35 residuesof the activation sites of Trx1 and triggered ROS accumulation,resulting in DNA damage and apoptosis in cancer cells. Our worksuggested, for the first time, that IsoA, as a Trx1 inhibitor, is

1Key Laboratory of Drug Targets and Drug Leads for Degenerative Diseases,Affiliated Hospital of Integrated Traditional Chinese and Western Medicine,Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China. 2Laboratory ofCellular and Molecular Biology, Jiangsu Province Academy of Traditional Chi-nese Medicine, Nanjing, Jiangsu, China. 3State Key Laboratory of Phytochem-istry and Plant Resources in West China, Kunming Institute of Botany, ChineseAcademy of Sciences, Kunming, Yunnan, China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

X. Sun and W. Wang are the co-first authors of this article.

Corresponding Authors: Peng Cao, Nanjing University of Chinese Medicine, 100#,Shizi Street, Hongshang Road, Nanjing, Jiangsu, China. Phone/Fax: 8625-8560-8666; E-mail: [email protected]; and Jianxin Pu, [email protected]

doi: 10.1158/0008-5472.CAN-16-0987

�2016 American Association for Cancer Research.

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selectively cytotoxic to cancer cells and has potential to be a novelagent for cancer therapy.

Materials and MethodsReagents

IsoA was isolated from the leaves of I. forrestii var. forrestiiaccording to Supplementary Methods. Its structure was char-acterized by one-dimensional nuclear magnetic resonance(NMR) spectrometer, 2D NMR analysis, and single-crystalX-ray diffraction experiment (Supplementary Materials). IsoA(99% or higher purity) was dissolved in dimethylsulfoxide as a40 mmol/L stock solution and stored at �20�C. Primaryantibodies against Trx1, gH2AX, 8-oxoguanine (8-oxoG),GAPDH, and nitrotyrosine were purchased from Santa CruzBiotechnology; goat anti-mouse IgG and goat anti-rabbitIgG antibodies from LI-COR Biosciences; benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Z-VAD-FMK) from Selleck;20,70-dichlorodihydrofluorescein diacetate (DCFH-DA) fromInvitrogen; 20,70-dichlorofluorescein (DCF), NAC, GSH, PEG-SOD, PEG-Catalase, and other chemical reagents from Sigma-Aldrich.

Cell lines and culturesHuman fetal lung fibroblasts HFL1, hepatocellular carcinoma

(HepG2, BEL7402, and QGY7701), breast cancer (MCF7 andMDA-MB-231), cervical carcinoma (HeLa), lung cancer (A549),and colon cancer (Caco2) cell lines were purchased from CellBank of Shanghai Institute of Biochemistry and Cell Biology inJune 2013. Human mammary epithelial cell line MCF 10A waskindly provided by Stem Cell Bank, Chinese Academy of Sciencesin November 2015. Human osteosarcoma (U2OS), melanoma(A375), and normal hepatic (LO2) cell lines were purchased fromCell Bank of Kunming Institute of Zoology, Chinese Academy ofSciences in June 2013. All the cell lines were kept within 10passages and preserved in liquid N2 after receipt. The used cellswere resuscitatedwithin1month.Cell lineswere authenticated bythe above cell banks through short tandem repeat (STR) analysis.HepG2, MCF7, MDA-MB-231, and Caco2 cells were cultured inMEM supplemented with 10% FBS. HeLa, A549, U2OS, A375,and LO2 cells were cultured in DMEM supplemented with 10%FBS. BEL7402 and QGY7701 cells were cultured in RPMI1640containing 10% FBS. HFL1 cells were grown in F12K mediumsupplemented with 10% FBS. MCF 10A cells were culturedwith MEGM Kit (Lonza/Clonetics, CC-3150) supplemented withcholera toxin (100 ng/mL) and 10% FBS.

Cell proliferation and apoptosis analysisCell proliferation and viability were determined by MTT assay

and Trypan blue exclusion. Apoptotic cells were stained withHoechst 33258, and also quantified using a FACScan laser flowcytometer (Guava easyCyteHT; Millipore) after staining withAnnexin V and 7-AAD.

ROS determinationThe level of cellular ROS was analyzed using DCFH-DA as a

fluorescent probe. Cells were incubated with 10 mmol/L DCFH-DA for 30minutes at 37�C in a 5%CO2 humidified environment.The labeled cells were washed with PBS for three times. Toquantify ROS, cells were harvested and the DCFH-DA fluores-cencewasmeasured using a FACScan laser flow cytometer (GuavaeasyCyteHT; Millipore).

Assessment of GSH levelsThe intracellular levels of GSH were measured using GSH and

GSSG Assay Kit (Beyotime). According to the manufacturer'sinstructions, the IsoA-treated cells were collected and homoge-nized. The protein concentrations were quantified using the BCAmethod. The total GSH levels were carefully measured by theenzymatic recycling method using glutathione reductase and 50,50-dithio-bis (2-nitrobenzoic acid). The sulfhydryl group of GSHreacts with DTNB and produces a yellow-colored 5-thio-2-nitro-benzoic acid, which has an absorbance at 405 to 414 nm.Oxidized glutathione (GSSG) levels were accomplished firstly byfirst derivatizing GSHwith 2-vinylpyridine. The concentrations ofreduced GSHwere calculated by subtracting the GSSG levels fromthe total GSH (GSH ¼ total GSH � 2 � GSSG). The intracellularlevels of GSH were determined on the basis of cellular proteinconcentrations.

Western blot analysisFor Western blot analysis, cells were lysed with RIPA to extract

total proteins. Equal amounts of proteins were separated by SDS-PAGE and transferred onto a PVDFmembrane (Millipore). Mem-branes were blocked with 5% nonfat milk at room temperature,and probed with primary antibodies at 4�C overnight, followedby IRDye-conjugated secondary antibodies. Themembranes werescanned with an Odyssey infrared fluorescent scanner (LI-CORBiosciences).

8-Oxoguanine quantification by flow cytometryThe IsoA-treated cells were fixed in 1% paraformaldehyde and

permeabilized by 0.5% Triton X-100 dissolved in PBS, followedby incubating with a FITC-conjugated 8-oxoguanine (8-oxoG)antibody at room temperature. Then, the cells were stained withpropidium iodide to determine DNA content. Fluorescence wasanalyzed using a FACScan laser flow cytometer (Guava easyCyteHT; Millipore).

Trx1 activity analysisFor in vivo Trx1 activity, cells or tissues were lysed in the lysis

buffer [20 mmol/L HEPES (pH 7.9), 300 mmol/L NaCl,100 mmol/L KCl, 10 mmol/L EDTA, and 0.1% Nonidet P-40]containing protease and phosphatase inhibitors cocktail (Pierce).The activity of Trx1 was measured according to the methoddescribed previously (28).

For in vitro Trx1 activity, this assay was performed using amodified Thioredoxin Activity Fluorescent Assay Kit (CaymenChemical Company). The samples containing 0.02 mmol/L hTrx1(the final concentration in the assay) were prepared according tothe manufacturer's instructions. Next, 2 mL of IsoA at variousconcentrations (0.195–100 mmol/L) was added to each sample,followed by addition of 5 mL of b-NADPH to all samples andincubation at 37�C for 30 minutes. Finally, the fluorescent sub-strate was added, and the Trx1 activity was recorded as theemission at 518 nm after 488 nm excitation for 30 to 60minutes,with a Thermo Scientific Varioskan Flash fluorescent microplatereader.

Determination of glutaredoxin activity in vitroThe in vitro Grx activity assay was performed using a modified

Fluorescent Glutaredoxin Assay Kit (Caymen Chemical Compa-ny). The samples containing 1.5 nmol/L of hGrx-1 (the final

Isoforretin A, a Novel Diterpenoid, Targets Thioredoxin 1

www.aacrjournals.org Cancer Res; 77(4) February 15, 2017 927




concentration in the assay) were prepared according to the man-ufacturer's instructions. Next, 2 mL of IsoA at various concentra-tions (0.195–100mmol/L)was added to each sample, followedbyadding 10 mL of fluorescent substrate to each well, and recordingthe emission at 545 nm after excitation at 520 nm for 15 to 30minutes with a Thermo Scientific Varioskan Flash fluorescentmicroplate reader.

Covalent dockingThe crystal structure of human Trx1 was obtained from the

protein data bank (PDB ID: 4PUF_C). The protein structure wasprepared with Protein Preparation Tool (ProPrep) in the Schro-dinger 2014-4 suit software. Hydrogen atoms were added at pH7.0 by the PROPKA tool in Maestro with optimized hydrogen-bond network. OPLS_2005 force field was used for restrainedminimization with converge heavy atoms to 0.30 Å. The ligandIsoAwas sketched inMaestro and subjected to Ligand PreparationTool (LigPrep) usingOPLS_2005 at pH7.0 to generate low-energyconformation. Covalent docking was performed using GlideDocking module. The reactive residues Cys32 and Cys35 weresupposed to forma covalent bondwith the ketene of IsoA throughMichael addition reaction.

MS/MS analysisA stock solution (5 mg/mL) of human Trx1 protein (Sino

Biological Inc.) was prepared in HPLC-grade water. Next, 4 mLof IsoA (40mmol/L stock solution) was incubated with 140 mL ofhumanTrx1 stock solution in 25mmol/L Tris-HCl buffer solution(pH 7.4) for 2 hours at room temperature. In this reaction, thefinal concentrations of IsoA and Trx1 were about 80 mmol/L and291 mmol/L, respectively. The samples were reduced according tothe methods of Shimadzu Biotech Proteome Kit, trypsin digestedby MonoSpin Trypsin, and desalted using a MonoSpin C18column, before being analyzed usingMALDI 7090 (SHIMADZU).

RNA interferenceTrx1-specific siRNA and scramble siRNA duplexes were trans-

fected to HepG2 cells using Lipofectamine 2000 (Invitrogen).The siRNA sequences for Trx1 were SiTrx1-a# 50-CUGCAGGU-GAUAAACUUGUTT-30; SiTrx1-b# 50-GAUCAAGCCUUUCUUU-CAUTT-30. The scramble siRNA (negative control, NC) sequencewas 50-UUCUCCGAACGUGUCACGUTT-30. The knockdownefficiency was assessed by Western blotting, and the Trx1 activitywas determined by insulin reduction assay. Cell viability ofTrx1 knockdown cells after treating with IsoA was measured byMTT assay.

Murine modelsAll animal experiments were conducted under protocols

approved by the Animal Care and Use Committee of JiangsuProvince Academyof Traditional ChineseMedicine.Nude BALB/cmice (13 � 2 g) were raised in air-conditioned pathogen-freeenvironment. HepG2 cells (2 � 106) were injected subcutane-ously into the right flank of nude mice. When tumors becamepalpable, the mice were randomized into two groups (n ¼ 6, pergroup), and treated with IsoA (15mg/kg) or vehicle control for 14days. Thebodyweight and tumor sizeweremeasured every 3days.Tumor volumes were calculated according to following formula:a2� b� 0.4, where a is the smallest diameter and b is the diameterperpendicular to a. At the end of the experiments, all animals weresacrificed and the tumors were excised, weighed, snap-frozen in

liquid nitrogen, and stored at �80�C, or fixed in 4% paraformal-dehyde for further analysis.

To explore the toxicity of IsoA to main organs, nude BALB/cmice (13 � 2 g) were treated with IsoA (15 mg/kg) or vehiclecontrol for 14 days (n ¼ 5, per group). At the end of the experi-ments, all animals were anesthetized using isoflurane (3%) inha-lation, and blood samples were collected from the retro-orbitalplexus. Complete blood counts were done on Auto HematologyAnalyzer BC-5380 (Mindray) and plasma biochemical indiceswere analyzed on Cobas C311 (Roche Diagnostics).

Immunohistochemical staining and in situ terminaldeoxynucleotidyl transferase-mediated dUTP nick end labelingassay

The immunostaining assaywas performedon tumor specimensthat were fixed in 4% paraformaldehyde and embedded with

Figure 1.

Effects of IsoA on various cells. A, The chemical structure of IsoA. B, The IC50

values of IsoA for indicated cell lines. Cells were treated with IsoA at variousconcentrations for 24 hours and processed forMTT assay.C,Cytotoxicity of IsoAto hepatocellular carcinoma cell line HepG2 and normal human hepatic cell lineLO2. Cells were incubated with IsoA for 24 hours, and the viability wasdetermined by MTT assay. D, Selective inhibitory effects of IsoA on cancer cells.LO2 cells and HepG2 cells were treated with IsoA at indicated concentrations.Viable cell number was assessed by Trypan blue exclusion assay. E, HepG2cells were exposed to IsoA for 16 hours, and apoptosis was determined byHoechst 33258 staining analysis. F, HepG2 cells were pretreated with Z-VAD-FMK for 2 hours followed by treatment with IsoA for 16 hours, and the apoptoticcells were evaluated and quantified by Annexin V/7-AAD staining and flowcytometry. The mean � SD of three experiments is shown. ��, P < 0.001.

Sun et al.

Cancer Res; 77(4) February 15, 2017 Cancer Research928




paraffin. Tissue sections were deparaffinized and rehydrated,followed by antigen retrieval and endogenous peroxidase block-ing. Primary antibodies against 8-oxoG and nitrotyrosine wereadded and incubated overnight at room temperature. Immuno-signals were detected by the two-stage peroxidase-based EnVision(Dako) method. Apoptosis index in the tumor samples wasassessed by in situ terminal deoxynucleotidyl transferase-mediat-ed dUTP nick end labeling (TUNEL) analysis using the One StepTUNEL Apoptosis Assay Kit (Beyotime).

Statistical analysisThe difference between two different treatments was assessed

by unpaired Student t test using PRISM software. ANOVA wasused to comparemultiple treatment groups and the nontreatmentgroup. The intensity of the immune-reactive bands in Westernblots was quantified by ImageJ software (NIH, Bethesda, MD).P < 0.05 was considered as statistically significant.

ResultsIsoA preferably induced apoptosis in cancer cells

IsoA was obtained as colorless crystal needles. On the basis ofmeticulous one-dimensional (1D) NMR, 2D NMR analyses, andsingle-crystal X-ray diffraction using anomalous scattering ofCuKa radiation data (CCDC 1063025), the absolute configura-tion of IsoA was assigned and described according to the fol-lowing nomenclature: (2S, 3R, 5S, 6S, 8S, 9S, 10R, 11R, 12R, 13R)-6-hydroxy-2,3,11,12-tetraacethoxy-ent-kaur-16(17)-en-15-one(Fig. 1A). The detailed structural information of IsoA wasillustrated in the Supplementary Information (SupplementaryFigs. S1–S7; Supplementary Table S1).

To investigate the antitumor potential of IsoA, 10 humancancer cell lines, including hepatic, breast, lung, colon, and cervi-cal carcinoma, osteosarcoma, melanoma cells, along with non-malignant human hepatic cell line LO2, fetal lung fibroblastsHFL1, and mammary epithelial cell line MCF 10A were tested for

Figure 2.

IsoA induces ROS and DNA damage inhuman cancer cells. A, HepG2 cells weretreated with indicated concentrations ofIsoA for 2 hours, and the ROS level wasdetermined by flow cytometry (means� SD of three experiments). B, HepG2cells were treatedwith 15 mmol/L of IsoAfor the indicated time, and then ROSlevels were measured (means � SD ofthree experiments). C, BEL7402 cellswere treated with indicatedconcentrations of IsoA for 5 hours, andROS levels were measured (means� SDof three experiments). D, BEL7402 cellswere treated with 20 mmol/L of IsoA forthe indicated time, and ROS levels weremeasured (means � SD of threeexperiments). E, IsoA decreasedintracellular GSH levels. HepG2 cellswere treated with 15 mmol/L of IsoA forthe indicated time. The GSH levels weremeasured by GSH and GSSG Assay Kitand normalized to protein level (means� SD of three experiments; �� , P < 0.01).F, IsoA depleted GSH levels dose-dependently. HepG2 cells were treatedwith IsoA at indicated concentrations for24 hours. TheGSH levelsweremeasuredby GSH Detection Kit and normalized toprotein level. G, The cells wereincubated with 15 mmol/L of IsoA for 16hours. 8-oxoG DNA lesion was detectedand quantified by flow cytometry usinga FITC-conjugated antibody. H and I,Concentration- and time-dependentinduction of gH2AX in response to IsoA.HepG2 cells were treated with IsoA atindicated times or concentrations.gH2AX was detected by Western blotanalysis. GAPDH served as a loadingcontrol. The histograms representmeans � SD (of three experiments) ofWestern blot quantification of gH2AX/GAPDH.






the growth-inhibitory effect of IsoA. Compared with the nonma-lignant cells, IsoAdisplayed apreferential antiproliferative activityagainst cancer cells, with IC50 values of <30 mmol/L (Fig. 1B;Supplementary Table S2). By clonogenic assay, we further foundthat IsoA drastically inhibited the colony formation of all testedcancer cells at 10 mmol/L (Supplementary Fig. S8).

Of all the tested cancer cell lines, HepG2 cells were the mostsensitive to IsoA,with an IC50 value of 15.83� 1.10mmol/L. Thus,this cancer cell line was chosen as a model to further investigatethe anticancer activity and the underlying mechanisms of IsoA.MTT assay demonstrated that IsoA reduced the viability of HepG2cells in a concentration-dependentmanner, but did not obviouslyalter the viability of LO2 cells even at 40 mmol/L (Fig. 1C). Thiswas further verified by Trypan blue exclusion assay (Fig. 1D).Hoechst staining revealed that IsoA was able to induce chromatincondensation and fragmentation (Fig. 1E), suggesting inductionof apoptosis. Flow cytometry analysis further showed that thepan-caspase inhibitor Z-VAD-FMK significantly attenuated IsoA-induced apoptosis (Fig. 1F). These data indicated that the IsoA-induced apoptosiswas at least partiallymediatedby the activationof caspase cascade.

IsoA induced oxidative stress in cancer cellsInduction of ROShas been reported to preferably induce cancer

cell apoptosis (29, 30). Next, we therefore investigated the effectsof IsoA on the induction of ROS using a fluorescent probe DCFH-DA. As shown in Fig. 2A and B, the induction of ROSwas detectedin HepG2 cells after exposure to IsoA. Similar results wereobtained from another hepatocellular carcinoma cell line

BEL7402 (Fig. 2C andD). To rule out the changes in ester cleavage,uptake, or efflux of DCFH-DA, we further detected IsoA-inducedROS inHepG2 cells using the oxidation insensitive analogueDCFas a control. IsoA treatment caused a remarkable shift in thefluorescence signal in cells loaded with DCFH-DA, but not withDCF (Supplementary Fig. S9). These results suggested that induc-tion of ROS might play a critical role in inhibiting cancer cellgrowth.

Because GSH is an important antioxidant in cells to defendoxidant damage and regulate redox homeostasis (31), we nextexamined the association between intracellular GSH levels andthe IsoA-induced ROS elevation. As shown in Fig. 2E, IsoAtreatment of HepG2 cells resulted in a swift depletion of GSHcomparedwith that of the control cells. Furthermore, treatment ofHepG2 cells with IsoA for 24 hours also led to a dramaticdepletion of reduced GSH (Fig. 2F).

Themain cytotoxicity of excessive ROS is throughDNAdamageinduced by base oxidation and double-strand breaks (DSB).Using a fluorescent antibody that recognizes 8-oxoG, we charac-terized and quantified DNA damage by flow cytometry. As shownin Fig. 2G, a significant increase of 8-oxoG was detected in IsoA-treated HepG2 cells. To examine whether the DNA damageinvolves DSBs, we performed Western blot analysis for the phos-phorylated form of H2AX (gH2AX), a specific marker for DSBs(32). As shown in Fig. 2H and I, IsoA induced a dose- and time-dependent expression of gH2AX in HepG2 cells. Taken together,our results suggested that ROS accumulation might be a generalmechanism of IsoA in inhibiting cancer cell growth, and IsoAtriggered both DSBs and oxidative DNA lesions in cancer cells.

Figure 3.

ROS scavengers attenuate IsoA'sactivity in HepG2 cells. A–D, HepG2 cellswere pretreated with NAC (A), GSH (B),PEG-Catalase (C), and PEG-SOD (D) atindicated concentrations for 30minutes,and then incubated with indicatedconcentrations of IsoA for 24 hours. Thecell viability was determined by MTTassay. E, HepG2 cells were pretreatedwith 2 mmol/L of NAC for 30 minutesand then incubated with 15 mmol/L ofIsoA for 16 hours, followed by apoptosisassay using Annexin V staining and flowcytometry. F, Quantification of AnnexinV staining results. G, HepG2 cells werepretreated with 2 mmol/L of NAC for30 minutes and then incubated with 15mmol/L of IsoA for 1 hour. ROS levelswere measured by flow cytometry andquantified. H, HepG2 cells werepretreatedwith 1,000U of PEG-Catalasefor 30 minutes, followed by incubatingwith 15 mmol/L of IsoA for 16 hours, andcell apoptosis was analyzed by flowcytometry and quantified. I, HepG2 cellswere pretreated with 1,000 U of PEG-Catalase for 30 minutes, followed byincubating with 15 mmol/L of IsoA for1 hour, and ROS levels were analyzedby flow cytometry and quantified.

Sun et al.





Antioxidant alleviated IsoA-induced ROS accumulation andapoptosis

To further understand the contribution of ROS to IsoA-inducedcell growth inhibition and apoptosis, we investigated whether thethiol-based antioxidant agents NAC or GSH could antagonizeIsoA. For this, HepG2 cells were pretreated with NAC or GSHfor 30 minutes, and then exposed to IsoA (0–30 mmol/L) for24 hours, followed by MTT assay. NAC (2 mmol/L) or GSH(2 mmol/L) markedly attenuated the inhibitory effect of IsoA oncell viability (Fig. 3A and B). To further clarify whether ROS orsome other oxidants were induced, we evaluated the effect of thepowerful free radical scavenger PEG-Catalase or PEG-SOD onIsoA-induced cytotoxicity. The results showed that PEG-Catalaseor PEG-SOD also antagonized IsoA-induced cytotoxicity inHepG2 cells (Fig. 3C and D). Our additional experiments furtherdemonstrated that NAC and PEG-Catalase abrogated the IsoA-induced apoptosis and intracellular accumulation of ROS inHepG2 cells (Fig. 3E–H). Collectively, our results demonstrated

that the IsoA-induced apoptosis was primarily induced by oxi-dative stress.

Mitochondria are known to be the major place of intracellularROS generation during electron transport flow. To determinewhether IsoA induction of ROS was through disrupting theoxidative metabolism of mitochondria, we generated A549 Rho0cells, which lacked mitochondrial function due to depletingmitochondrial DNA. IsoA treatment induced similar levels ofROS in both normal A549 and A549 Rho0 cells (SupplementaryFig. S10A). In linewith this, IsoA treatment also resulted in similarreduction of cell viability in both A549 and A549 Rho0 cells(Supplementary Fig. S10B). These results suggested that mito-chondria should not be the main site of IsoA-induced ROSformation.

IsoA inhibited Trx1 activityChemical structure analysis revealed that IsoA contained an

a, b-unsaturated carbonyl group, which can readily interact

Figure 4.

IsoA inhibits Trx1 activity. A, In vitro assays for Trx1 and Grx activity. For Trx1 acticity assay, 0.02 mmol/L of hTrx1 protein (the final concentration) was subjected tothe assay in the presence of indicated concentrations of IsoA. For Grx activity assay, 1.5 nmol/L of hGrx-1 (the final concentration) was subjected to the assayin the presence of indicated concentrations of IsoA (means � SD of three experiments). B, HepG2 cells were treated with IsoA at the indicated concentrationsfor 8 hours, and Trx1 activity in cell lysates was determined by insulin endpoint assay (means � SD of three experiments). C, BEL7402 cells were treated with20 mmol/L of IsoA for 8 hours, and Trx1 activity in cell lysates was measured by insulin endpoint assay (means � SD of three experiments). D, HepG2 cells wereincubated with 15 mmol/L of IsoA for the indicated time, and Trx1 and thioredoxin reductase levels were detected by Western blotting. GAPDH served as aloading control. Bar graphs represent the quantified results of protein levels (Trx1 and TrxR), which were normalized to corresponding GAPDH protein leveland expressed as fold of control (mean fold of control � SD of three experiments). E, HepG2 cells transfected with the scrambled siRNA (control) and Trx1-specific siRNA for 48hours, and the knockdownefficiency of Trx1 levelswas analyzed byWestern blotting. The histogramshowedmeans�SD (of three experiments)of Western blot quantification of Trx1/GAPDH. �� , P < 0.001. F, The Trx1 activity in Trx1-silenced HepG2 cells was determined by insulin endpoint assay.G, Trx1-silenced HepG2 cells and control cells were treated with IsoA for 24 hours, and cell viability was measured by MTT assay (means � SD of threeexperiments).






with biological molecules by forming covalent bonds with freethiol of cysteine or by acting as an electrophilic center in redoxreactions. Hence, we speculated that IsoA might be a novelinhibitor of Trx1. For this, the levels of Trx1 in all cell lines weredetected by Western blotting. As shown in SupplementaryFig. S11, Trx1 was extensively overexpressed in cancer cellsrelative to nonmalignant cells, which was consistent withprevious reports (5–10). Next, we investigated the inhibitionpotency of IsoA toward Trx1 using an in vitro Trx1 activityfluorescent assay. Our results demonstrated that IsoA effectivelyinhibited Trx1 activity with an IC50 of approximately 5.177mmol/L, whereas it had little inhibitory effect on Grx (Fig. 4A).To investigate the specificity of IsoA for Trx1, we analyzed the invitro–inhibitory effects of IsoA on a spectrum of thiol-contain-ing enzymes including glutathione reductase, thioredoxinreductase, glutathione-S-transferase, and protein disulfide iso-merases. The results showed that IsoA inhibited thioredoxinreductase activity with an IC50 of approximately 130.1 mmol/L,approximately 25 times of IC50 for Trx1 (SupplementaryFig. S12A). In addition, 100 mmol/L of IsoA inhibitedthe activity of protein disulfide isomerases only by 10% (Sup-plementary Fig. S12B), and almost had no inhibitory effects on

glutathione reductase and glutathione-S-transferase (Supple-mentary Fig. S12C and S12D). Using the insulin reductionassay, we found that IsoA suppressed Trx1 activity in cancercells in a dose-dependent manner (Fig. 4B and C), despite noeffects on the protein levels of Trx1 (Fig. 4D). These resultsindicate that IsoA is a potent inhibitor of Trx1.

Trx1 interacts with ASK1, and inhibits its kinase activity andASK1-mediated apoptosis (13). ROS releases Trx1 from ASK1partly through oxidizing Trx1 and activates ASK1/JNK deathsignaling cascade (33). To determine whether IsoA-mediatedcell apoptosis is associated with the activation of ASK1 sig-naling, we examined the effect of IsoA on the phosphorylationlevels of ASK1 and JNK. The Western blot analysis resultsshowed that IsoA indeed increased the phosphorylation levelsof ASK1 and JNK (Supplementary Fig. S13), suggesting that theactivation of ASK1/JNK may contribute to IsoA-induced cellapoptosis.

To assess whether the effects of IsoA are Trx1 dependent, weused siRNAs to knockdown Trx1 expression. Trx1 protein levelwas reduced in the Trx1 knockdown cells (Fig. 4E and F), whichrendered cancer cells more sensitive to IsoA (Fig. 4G). Theseresults indicated that Trx1 levels influence cell sensitivity to IsoA.

Figure 5.

IsoA covalently binds with Cys32 and Cys35 in Trx1.A andB,Molecular docking for bindingmodels of IsoA in Cys32 and Cys35 residues of Trx1. C, IsoA (200 mmol/L)was incubated with Trx1 protein (70 mg) at 37�C for 2 hours, and the adduct forms were assayed by MS. The molecular weight of peptide P1 was 1624.85,representing 22 to 36 amino acids of Trx1, and themolecular weight of the adduct product between peptide P1 and two IsoA, whichwas the detected peptide P2, was2694.14. C� represents the cysteine binding with IsoA. D, The identified peptide P2 was further analyzed by MS/MS. b1, b2, b3, b4, b5, b6, and b7 representthe dissociated fragments of peptide P2. C� represents the cysteine binding with IsoA. b2, b4, and b5 represent the fragments conjugated with IsoA.

Sun et al.





IsoA covalently conjugated to Trx1 at Cys32 and Cys35residues

To investigate whether IsoA directly interacts with the catalyticsite of Trx1, we predicted the binding mode between IsoA andTrx1 through molecular docking. As shown in Fig. 5A and B, thedocking poses of IsoAwere almost identical to each other. The freesulfhydryl of reactive residues Cys32 and Cys35 acted as a nucle-ophile to attack a, b-unsaturated carbonyl group of IsoA, formingstrong covalent bondbetween the receptor and the ligand throughMichael addition in each complex. Meanwhile, the hydrogenbond formed between the acetyl of IsoA and Met74 also contrib-uted to the stable binding interaction in both complexes.

To further confirm whether IsoA binds to Trx1 directly, weincubated IsoA with Trx1 protein in vitro, and examined theproducts by MS. We detected two major components at m/z1624.79 and m/z 2694.12, respectively. The molecular weight ofIsoA was 534.6. The component at m/z 1624.79 represented the

peptide LVVVDFSATWCGPCK, corresponding to the 22 to 36residues of Trx1. The molecular weight of this peptide plus twoIsoA molecules was 2694.12, equal to the molecular weight ofanother component at m/z 2694.12, indicating that a covalentreaction occurred between IsoA and Trx1 at the ratio of 2:1(Fig. 5C). Further MS/MS analysis demonstrated that IsoA cova-lently conjugated to both Cys32 and Cys35 residues of Trx1together (Fig. 5D).

In vivo antitumor efficacy of IsoA in a xenograft mousemodel

We next evaluated the in vivo antitumor efficacy of IsoA. In thexenograft model, HepG2 cells were inoculated subcutaneouslyinto the nudemice. Themice were then treated by intraperitonealinjectionwith vehicle or IsoA (15mg/kg/d) for 14 days.We foundthat treatment with IsoA did not show significant toxicity on thebasis of stable body weights (Fig. 6A). However, treatment

Figure 6.

IsoA treatment suppresses tumorgrowth in vivo. A–C,Mice implanted withHepG2 cells were administered IsoA orthe vehicle control for 14 days. Bodyweights (A), tumor volume (B), andtumor mass (C) of IsoA-treated orvehicle-treated animals were monitoredas described in Materials and Methods.Means� SD; �� , P <0.001; �� , P <0.01.D,The DNA oxidative damage marker 8-oxoG and protein oxidative markernitrotyrosine were evaluated byimmunostaining with anti-8-oxoG andanti-nitrotyrosine antibodies. E,Apoptosiswas examined using the in situTUNEL assay. F, Reduced GSH levelswere evaluated by the GSH DetectionKit (means � SD of three experiments;�� , P < 0.001). Trx1 activity in thexenograft was assessed using aThioredoxin Activity Fluorescent AssayKit (means � SD of three experiments;� , P < 0.05).






with IsoA significantly inhibited the growth of HepG2 xenograft(Fig. 6B) and reduced the weights of tumors compared with thevehicle-treated group (Fig. 6C). The DNA damagemarker 8-oxoGand protein oxidative marker nitrotyrosine (Fig. 6D) were obvi-ously increased in IsoA-treated tumors, suggesting that IsoAinduced oxidative stress in the xenografts. Further analysisrevealed that there was a significant increase of TUNEL-positivecells in the xenografts treated with IsoA, compared with thevehicle control (Fig. 6E). Besides, IsoA significantly suppressedthe activity of Trx1 anddepleted theGSH levels in the tumors (Fig.6F), consistent with our in vitro observations. These results dem-onstrated that IsoA exhibits potent antitumor activity in vivo.

To explore the safety of IsoA further, we screened its toxicity tobone marrow (blood counts), liver (aspartate aminotransferase,AST; alanine aminotransferase, ALT; alkaline phosphatase, ALP),and kidney (creatinine, and blood urea nitrogen, BUN) in mice.All the indices of routine blood test including redblood cell count,white blood cell (WBC) count, lymphocyte count, platelet count,and hemoglobin level remained in the normal ranges after IsoAtreatment (Fig. 7A). There were no significant differences in bloodbiochemical parameters (ALT, AST, ALP, BUN, and creatinine)between IsoA-treated and the control groups (Fig. 7B–F). Inaddition, after collecting blood samples for hematology, the vitalorgans (liver, spleen, and kidney) were collected, fixed in forma-lin, and processed for hematoxylin and eosin (H&E) staining.Histopathologic evaluation did not reveal any significant differ-ences between the vehicle and IsoA-treated groups (Supplemen-tary Fig. S14).

DiscussionIn this study, we evaluated anticancer potentials of IsoA, a key

component and a novel ent-kaurane diterpenoid from I. forrestiivar. forrestii. Our data established IsoA as a Trx1 inhibitor, whichinhibited tumor cell growth both in vitro and in vivo.

The inhibitory effect of IsoA on Trx1 in cancer cells wasdemonstrated with the following lines of evidence. First, IsoAinduced a rapid increase of ROS in cancer cells, and the collapse ofredox homeostasis may be responsible for IsoA's anticanceractivity. This result suggested that IsoA regulated cellular anti-oxidant systems. Second, IsoA suppressed in vitro and in vivoactivity of Trx1. In the in vitro reaction, IsoA inhibited Trx1 activitywith the IC50 of approximately 5.177 mmol/L, but had little effectson several thiol-containing enzymes, such as glutaredoxin, glu-tathione reductase, thioredoxin reductase, glutathione-S-transfer-ase, and protein disulfide isomerases (Fig. 4A; SupplementaryFig. S12). At the in vivo level, IsoA showed an inhibitory effect onTrx1 both in tumor cells and xenograft tumors (Figs. 5B and Cand6F). Third, downregulationof Trx1 in cancer cells significantlyenhanced IsoA-induced growth-inhibitory effects. Importantly,IsoA–Trx1 adducts were detected byMS/MS. Two residues, Cys32andCys35, at the catalytic sites of Trx1were identified to be criticalamino acids for the IsoA–Trx1 interaction, probably through aMichael addition reaction. Taken together, these results indicatedthat IsoA directly targeted the Cys32/Cys35 amino acids to form acovalent complex to inhibit Trx1 activity. This oxidative-likemodification inhibited the reversible reduction of Trx1 by TrxRand NADPH. Consequently, cellular redox balance was disruptedand ROS-mediated cell oxidative damage and apoptosis ensued.

Accumulating evidence has indicated that thioredoxin proteinsare potential diagnostic and prognostic markers of cancer(34, 35). Decreasing expression levels of Trx1 or altering its redoxstate from the reduced to the oxidized state increases ROS level,leading to apoptosis in hepatocellular carcinoma cells (36, 37).Downregulating Trx1 also triggers intrinsic apoptosis in MCF-7human breast cancer cells (38). These findings make Trx1 anactionable target for cancer treatment. Indeed, several specific Trx1inhibitors have been reported to possess potent anticancer activ-ity, such as PX-12 series and quinol compounds (3, 19, 39, 40).These reports, together with our results, suggest that covalent

Figure 7.

IsoA shows no toxicity to main organs.Nude BALB/c mice (13 � 2 g) weretreated with IsoA (15 mg/kg) or vehiclecontrol by intraperitoneal injection onceevery day for 14 days (5 mice/group). Allthe animals were sacrificed aftercollecting blood samples from the retro-orbital plexus. A, Complete blood countswere done onAutoHematologyAnalyzerBC-5380 (Mindray). Normal referenceranges for the tested parameters: WBC(2.6–12 � 103/mL), lymphocyte (1.3–9 �103/mL), hemoglobin (10.1–16.1 g/dL),platelets (5.92–29.72 � 105/mL), and redblood cells (6.5–10.1 � 106/mL). B–F,Results of plasma biochemical tests forALT, AST, ALP, BUN, and creatinine invehicle or IsoA-treated mice. ns,nonsignificant.

Sun et al.





modification of cysteine residues of Trx1 may be a direct andeffective way to suppress its activation.

As IsoA showed stronger suppression effect against Trx1 thanseveral other thiol-containing enzymes (glutaredoxin, glutathionereductase, thioredoxin reductase, glutathione-S-transferase, andprotein disulfide isomerases) tested (Fig. 4A; SupplementaryFig. S12), we propose that IsoA is a relatively specific inhibitor ofTrx1. The results that IsoA triggered ROS and activated ASK1/JNKcascade associated with apoptosis (Supplementary Fig. S13) andknockdownof Trx1 sensitized the cytotoxicity of IsoA (Fig. 4F) alsodemonstrated a critical role of Trx1 in IsoA-mediated apoptosis ofcancer cells. Currently, we could not rule out the possibility thatIsoAmay interactwith other thiol-containing enzymes or proteins.Nevertheless, at least, Trx1 is one of the important targets for IsoA.Accumulating evidence clearly showed that the reactivity of thiol-containing proteins was not determined solely by their –CXXC-motif, but also by several residues spatially close to the active sites(41, 42). The formation of a hydrogen bond or a salt bridge in theactive site has a dramatic effect on the function of the protein. Inour study, besides the Cys32/35 in the active sites, moleculardocking showed that IsoA also formed a hydrogen bond withMet74, which also contributed to the stable binding interaction inboth complexes (Fig. 5A and B). Consequently, we reasoned thatthe relatively specific inhibitory effect of IsoA on Trx1 mightattribute to the extended active-sitemotif aswell as residues distantin sequence but spatially close to the active site (Cys-Gly-Pro-Cys)of Trx1. Further research is needed to explore the mechanism bywhich IsoA had a relatively higher affinity with Trx1.

Recently, numerous ROS-inducing agents have been describedto selectively kill cancer cells but spare normal cells (29–31, 43–45). In this work, IsoA induced an intracellular burst of ROS incancer cells (Fig. 2A–D). Antioxidants NAC, GSH, PEG-SOD, orPEG-Catalase markedly abolished IsoA-induced growth inhibi-tion, ROS generation, and apoptosis (Fig. 3). Therefore, ROSelevation may play a central role in mediating antitumor activityof IsoA through blocking Trx1. Consistent with the ROS thresholdtheory, our findings suggest that IsoA may be a novel Trx1inhibitor with minimal toxicity to normal cells.

In conclusion, herewepresent evidence that IsoA is a novel Trx1inhibitor through covalently binding to its catalytic sites Cys32

and Cys35 and inhibits tumor cell growth both in vitro and in vivo.The selective cytotoxic activity of IsoA to cancer cells can beexplored further to develop novel antitumor agents.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y. Yang, H. Sun, J. Pu, P. CaoDevelopment of methodology: W. LuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): X. Sun, W. Wang, X. Cai, J. Yang, J. Ye, C. HuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): X. Sun, W. Wang, J. Chen, Y. Yang, W. Lu, J. PuWriting, review, and/or revision of the manuscript: X. Sun, J. Chen, J. Pu,P. CaoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): X. ChengStudy supervision: J. Pu, P. Cao

AcknowledgmentsWe thank Zhimin Yin (NanjingNormal University) andQiang Yu (Shanghai

Institute of Materia Medica, Chinese Academy of Sciences) for helpful adviceand critical reading of the manuscript. We thank Nanjing Newtop Biotechnol-ogy Company (China) for their help with MS/MS analysis.

Grant SupportP. Cao received the Priority Academic Program Development of Jiangsu

Higher Education Institutions (Integration of Chinese and Western Medicine)grant, National Natural Science Foundation of China (nos. 81622048 and81274150) grants, and Jiangsu Province Funds for Distinguished Young Scien-tists (BK20140049) grant. X.Y. Sun received National Natural Science Founda-tion of China (no. 81202576) grant and Natural Science Foundation of JiangsuProvince Grant (BK20131038). J.X. Pu received National Natural ScienceFoundation of China (no. 21322204) grant and NSFC-Joint Foundation ofYunnan Province (U1302223) grant.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 10, 2016; revised November 9, 2016; accepted November 16,2016; published OnlineFirst December 23, 2016.

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Sun et al.




2017;77:926-936. Published OnlineFirst December 23, 2016.Cancer Res Xiaoyan Sun, Weiguang Wang, Jiao Chen, et al. Triggers Potent ROS-Mediated Antitumor EffectsThe Natural Diterpenoid Isoforretin A Inhibits Thioredoxin-1 and

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