research article gst-tat-sod: cell permeable bifunctional...

Research ArticleGST-TAT-SOD Cell Permeable Bifunctional AntioxidantEnzymemdashA Potential Selective Radioprotector

Jianru Pan1 Huocong He2 Ying Su2 Guangjin Zheng1

Junxin Wu2 Shutao Liu1 and Pingfan Rao13

1College of Biological Science and Engineering Fuzhou University No 2 Xue Yuan Road University Town FuzhouFujian 350108 China2Laboratory of Radiation Oncology and Radiobiology Fujian Provincial Cancer Hospital Teaching Hospital ofFujian Medical University Fujian Key Laboratory of Tumor Translational Cancer MedicineThe National Clinical Key Specialty Construction Program of China No 420 Fuma Road Fuzhou 350014 China3Food Nutrition Sciences Centre Zhejiang Gongshang University Room 407 No 1 Laboratory Building No 149 Jiaogong RoadXihu District Hangzhou 310012 China

Correspondence should be addressed to Jianru Pan panjrfzueducn and Pingfan Rao pingfanraogmailcom

Received 5 February 2016 Revised 28 April 2016 Accepted 8 May 2016

Academic Editor Janusz Gebicki

Copyright copy 2016 Jianru Pan et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Superoxide dismutase (SOD) fusion of TAT was proved to be radioprotective in our previous work On that basis a bifunctionalrecombinant protein which was the fusion of glutathione S-transferase (GST) SOD and TAT was constructed and named GST-TAT-SOD Herein we report the investigation of the cytotoxicity cell-penetrating activity and in vitro radioprotective effect ofGST-TAT-SOD compared with wild SOD single-function recombinant protein SOD-TAT and amifostine We demonstrated thatwild SOD had little radioprotective effect on irradiated L-02 and Hep G2 cells while amifostine was protective to both cell linesSOD-TAT or GST-TAT-SOD pretreatment 3 h prior to radiation protects irradiated normal liver cells against radiation damageby eliminating intracellular excrescent superoxide reducing cellular MDA level enhancing cellular antioxidant ability and colonyformation ability and reducing apoptosis rate Comparedwith SOD-TAT GST-TAT-SODwas proved to have better protective effecton irradiated normal liver cells and minimal effect on irradiated hepatoma cells Besides GST-TAT-SOD was safe for normal cellsand effectively transduced into different organs inmice including the brainThe characteristics of this protein suggest that it may bea potential radioprotective agent in cancer therapy better than amifostine Fusion of two antioxidant enzymes and cell-penetratingpeptides is potentially valuable in the development of radioprotective agent

1 Introduction

As a component of therapy for a wide range of malignantconditions radiotherapy is estimated to be used by halfof all cancer patients during the course of their treatmentfor cancer The absorption of ionizing radiation by livingcells can directly disrupt molecular structures producingchemical and biological changesThrough radiolysis of waterit can also act indirectly by generating reactive chemicalspecies thatmay damage nucleic acids proteins and lipids [1]The direct and indirect effects of ionizing radiation initiate

a series of biochemical and molecular signaling events [2]Irradiation of noncancerous ldquonormalrdquo tissues during thecourse of therapeutic radiation can result in a range of sideeffects including self-limited acute toxicities mild chronicsymptoms or severe organ dysfunction To protect organismsfrom radiation various agents called radioprotectors havebeen utilized Amifostine is the only clinical radioprotectorapproved by the Food and Drug Administration (FDA)for head and neck cancer patients [3] But it was provedto have low potency and poor bioavailability due to thestoichiometric nature of its action [4] What is more side

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 5935080 13 pageshttpdxdoiorg10115520165935080

2 Oxidative Medicine and Cellular Longevity

effects of amifostine such as fever rash severe nausea allergyand acute hypotension have prompted a continuing search forbetter radioprotector [5ndash7]

Superoxide radicals produced by ionizing radiation arehighly reactive and potentially damaging to cellsThe enzymesuperoxide dismutase (SOD) neutralizes superoxide radicalsby changing it into molecular oxygen and hydrogen perox-ide thereby preventing the formation of highly aggressivecompounds such as peroxynitrite Hydrogen peroxide isthen subsequently eliminated by catalase and glutathioneperoxidase [8 9] SOD is naturally present in human cellsand proved to play a key role in cellular defenses againstoxidative damage [1] But as a protein SOD is too large tofreely enter into cells Although the hypothesis that SOD isradioprotective has been supported by many studies throughtransgenic experiments [10ndash15] there were many limitationson its protecting against radiation-induced chronic injuryin humans SOD mimics are another way to overcomethe limitation of large molecular weight Some of themhave been proved to be radioprotective in various radiationinjury models [16] But their reaction efficiency of scaveng-ing superoxide anion is still inferior to wild SOD Theirmechanism selectivity and toxicity of mimics may varycompared with natural enzyme [16] In our previous workwe constructed a cell membrane permeable SOD by generecombinant technique to circumvent this limitation Therecombinant protein was the fusion of hCuZn-SOD (SOD1)and cell-penetrating peptide derived from HIV-1 TAT pro-tein transduction domain TAT (YGRKKRRQRRR) Proteintransduction domains are able to carry larger molecules suchas oligonucleotides peptides full-length proteins 40 nm ironnanoparticles bacteriophages and even 200 nm liposomesacross cellular membranes and have proven useful in deliv-ering biologically active cargoes in both in vitro and in vivomodels [17ndash22] The recombinant SOD had been purifiedcrystallized and proved to be effective in preventing andtreating the damage of guinea pigs skin caused by single doseUVB radiation [23ndash25] What is more it was proved to beeffective in preventing radiation-induced lung injury in mice[26]

Cell permeable SOD was confirmed to have remarkableradioprotective effects compared with wild SOD by aboveexperiments [23 24 26] However superoxide radicals werenot the only harmful reactive chemical species producedby ionizing radiation To find out whether a cell permeablerecombination of different antioxidase would encourage abetter outcome a bifunctional recombinant protein fusedwith glutathione S-transferase (GST) and cell permeable SODwas constructed firstly and named GST-TAT-SOD GST isan enzyme that aids in detoxification by speeding up thelinking of toxic compounds with glutathione (GSH) thusforming a less reactive substance Besides that fusional GSTcould enhance the expression quantity of soluble bifunctionalantioxidase and simplify its purification

Current study investigated the selective radioprotectiveeffects of this cell permeable bifunctional antioxidant enzymecompared with SOD-TAT and amifostine

2 Materials and Methods

21 Enzyme and Chemicals E coli strains with recombi-nant plasmid of GST-TAT-SOD containing GST TAT-PTDand human CuZn-SOD and recombinant protein SOD-TAT were obtained from Institute of Biotechnology FuzhouUniversity (Fujian China) Wild CuZn-SOD was purchasedfromDatian Huacan Biotechnology Co Ltd (Fujian China)GST affinity chromatography was purchased from Weishi-Bohui Chromatogram Technology Co Ltd (Beijing China)Amifostine was purchased fromMeiluo Yinhe Pharmacy CoLtd (Hunan China) Malondialdehyde (MDA) SOD cata-lase (CAT) total antioxidation capacity (T-AOC) and glu-tathione peroxidase (GSH-Px) reagent kits were purchasedfrom Nanjing Jiancheng Bioengineering Co Ltd (JiangsuChina) Micro-BCAtrade Protein Assay Kit was purchased fromThermo Scientific (USA) RPMI-1640 and fetal bovine serumwere purchased from HyClone and Gibco (USA) respec-tively All other chemicals were of analytical purity

22 Cell Cultures Normal human liver cell line L-02 cells andhuman hepatoma cell line Hep G2 cells are available fromShanghai Institute of Biochemistry andCell Biology (SIBCB)Cells were cultured in RPMI-1640 (HyClone) supplementedwith 10 fetal bovine serum (Gibco) 100UmL penicillinand 100mgmL streptomycin (Gibco) at 37∘C in a 5 CO

2

humidified chamber

23 Mice Male Kunming mice (Fujian Medical University)weighing 18ndash22 g each were used at 6ndash8 weeks of age forthese experiments All mice were housed in an animal roomat 22∘C in a 12 h light12 h dark cycle All mice were givena standard chow diet and water ad libitum Animal welfareand experimental procedures were carried out in accordancewith the Guide for the Care and Use of Laboratory Animals(Ministry of Science and Technology of China 2006) andwere approved by the Review Committee for the Use ofHuman or Animal Subjects of Institute of BiotechnologyFuzhou University

24 Preparation of Recombination Protein GST-TAT-SODBacteria were washed in PBS and lysed in buffer A (10mMNa2HPO4 18mM KH

2PO4 and 27mM KCl pH 73) using

a cell disrupter (Sonic SolutionsCo vc-750)The supernatant(extract) was collected after centrifugation of the lysatesfor 20min at 12000 rpm in a HITACHI RX series HimacCF15RX rotor at 4∘C The supernatant containing GST-TAT-SOD was precipitated by incubation with 561 g ammoniumsulfate per 100mL of culture medium at 4∘C For purificationof GST-TAT-SOD the precipitate was resuspended in 20mMPBS pH 74 and purified by GST affinity chromatogra-phy resin (Weishi-Bohui Chromtotech Co Beijing China)according to the manufacturerrsquos instructions After bindingand washing the bound protein was eluted with 50mMTris-HC1 containing 001M glutathione pH 80 The concentra-tion SOD activity and GST activity of the purified proteinwere determined by BCA Protein Assay Kit (Thermo USA)and SOD and GST reagent kits (Jiangsu China) respectivelyThe SOD and GST activity of purified GST-TAT-SOD were

Oxidative Medicine and Cellular Longevity 3

2476 and 766UmL respectively Purified protein was con-centrated and dialyzed for cellular experiments

25 Transduction of Recombination Protein GST-TAT-SOD InVitro L-02 cells plated in a 24-well plate were treated withGST-TAT-SOD or wild SOD then harvested washed andlysed using PBS containing 05 Triton X-100 Then the celllysate was removed and centrifuged at 4∘C at 13000 rpmfor 15min and the SOD activity and the protein content inthe supernatant were analyzed spectrophotometrically usingSODdiagnostic reagent kits (Nanjing Jiancheng Bioengineer-ing) and BCA Protein Assay Kit (Thermo USA) followingmanufacturerrsquos directions respectively

In addition the fusion protein GST-TAT-SOD is labeledusing fluorescein isothiocyanate (FITC) following standardmethods Briefly 2mgmL GST-TAT-SOD dialyzed againstsodium carbonate was incubated with 40120583gmL FITC for 1 hat room temperatureThen labeled proteins are purified fromfree dye using a gel filtration column

FITC-labeled GST-TAT-SOD internalized into culturecells was observed under fluorescence microscopy L-02 cellswere seeded into 24-well plates (Greiner Germany) at adensity of 4 times 105 cells per well and cultivated to semi-confluence in RPMI-1640 medium in a humid atmospheresupplemented with 5 CO

2for 24 h at 37∘C The culture

medium was removed and the cells were washed with PBStwice Appropriate amount of FITC-labeled GST-TAT-SODwasmixedwith freshmediumwithout serum and then addedto the cells and incubated for 3 h with 5 CO

2at 37∘C

The cells were further washed 5 times with PBS then thefluorescence was observed under fluorescence microscopy(ZEISS AXIOSKOP-50 Germany)

For quantification of the transduction of GST-TAT-SOD cells from the different groups were lysed using PBScontaining 05 Triton X-100 The cell lysate was removedand centrifuged at 4∘C at 13000 rpm for 15min and thefluorescence in the supernatant was read with a SynergyH4 Hybrid Multi-Mode Microplate Reader (BioTek Instru-ments Winooski VT) To normalize fluorescence per cellprotein was determined by BCA Protein Assay Kit (ThermoUSA) following manufacturerrsquos directions Fluorescence wascorrected for background signal and normalized for proteincontent and expressed as fluorescence120583g of protein

26 Transduction Experiment In Vivo Male Kunming micewere randomly divided into 3 groups (119899 = 5 mice pergroup) The first group (CON) was untreated The secondgroup (SOD) was injected with 05mL of FITC-labeledSOD (2 kUmL) intraperitoneally and the other group (GST-TAT-SOD) received intraperitoneal injection of 05mL ofthe FITC-labeled recombinant protein GST-TAT-SOD (2 kUmL) Three hours after injection all animals were sacrificedby cervical dislocation The liver spleen lung brain andkidney were weighed and 10 homogenates were preparedwith ice-cold saline using a homogenizer respectively Thefluorescence of tissue homogenates from organs was deter-mined with a Synergy H4 Hybrid Multi-Mode MicroplateReader (BioTek Instruments Winooski VT) To normalizefluorescence the protein contents of the 10 homogenates

were determined by BCA Protein Assay Kit (Thermo USA)following manufacturerrsquos directions Fluorescence was cor-rected for background signal and normalized for proteincontent and expressed as fluorescence120583g of protein

27 CytotoxicityMTT MTT a tetrazolium salt is reduced byviable mitochondria to formazan which causes a colorimet-ric change that can be measured at 570 nm Approximately1 times 105 cells were seeded per well in 96-well plates andincubated for 48 h in fresh medium along with the protein atthe final concentration indicated (three replicates each) Cellviability was estimated by a colorimetric assay using MTT(3-(45-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bro-mide) (Sigma Chemical Co)

28 Irradiation Cells were irradiated with X-ray generatedby a Linac (IEC 61217) with a nominal potential of 6 MV anda dose rate of 300UMmin at room temperature

29 Clonogenic Tests on Cells following X-Ray TreatmentConfluent 75 cm2 flasks of cells were treated with protein oramifostine and irradiated with 2Gy X-ray subsequently Thedose was confirmed to cause about half percent of survivingfraction of normal cell through an unpublished pretest onthe surviving fraction of cells at radiation dose range from 0to 8Gy After irradiation the cells were trypsinized countedwith a hemocytometer and diluted in complete media toobtain 100 cellsmL One milliliter of cell suspension wasplated in each well of a 6-well tissue culture plate to obtain100 cells per well For each cell line used experiments wereperformed in triplicate and clonogenic tests were performedon following different cells groups The first group (CON)did not receive any irradiation and was untreated the secondgroup (XRT) received irradiation only the third group(XRT+SOD) received irradiation pretreated with wild SOD(250ndash6000UmL) for 3 h the fourth group (XRT+AMFT)received irradiation pretreated with amifostine (4120583gmL)for 05 h the fifth group (XRT+GST-TAT-SOD) receivedirradiation pretreatedwithGST-TAT-SOD (250ndash6000UmL)for 3 h and the other groups (XRT+SOD-TAT) receivedirradiation pretreated with SOD-TAT (250ndash6000UmL) for3 hThe incubation time of proteinwas determined accordingto its transduction effect The incubation time of amifostinewas determined according to its medication guides Colonieswere stained with crystal violet after 14 days and thosecontaining at least 50 cells were counted as surviving coloniesThe plating efficiency (PE) and the survival fraction (SF) foreach cell line after each treatment were calculated accordingto themethod proposed by Franken et al [27] Protective rateof pretreated groups was calculated as the following formula

Protective rate () = SF (treatment) minus SF (XRT)100 minus SF (XRT)

times 100

(1)

210 Measurement of T-AOC SOD CAT MDA and GSH-PXActivity L-02 cells and Hep G2 cells in logarithmic growthphase were trypsinized and counted with a hemocytometer


Approximately 3 times 106 cells were seeded into 75 cm2 flasksFor each cell line used experiments were performed intriplicate and tests were performed on following differentcells groups The first group (CON) did not receive any irra-diation and was untreated the second group (XRT) received4Gy X-ray irradiation only the third group (XRT+SOD)received irradiation pretreated with wild SOD (6000UmL)for 3 h the fourth group (XRT+AMFT) received irradiationpretreated with amifostine (4 120583gmL) for 05 h the fifth group(XRT+GST-TAT-SOD) received irradiation pretreated withGST-TAT-SOD (2000UmL) for 3 h and the other groups(XRT+SOD-TAT) received irradiation pretreated with SOD-TAT (6000UmL) for 3 h

After irradiation cells were further cultured for 24 h at37∘C Then the cells were lysed using PBS containing 05Triton X-100 The cell lysate was removed and centrifuged at4∘C at 13000 rpm for 15min and the activities of SOD (Umgprotein) MDA (nmolmg protein) CAT (Umg protein)GSH-PX (Umg protein) GST (Umg protein) and T-AOC (Umg protein) in the supernatant were determinedspectrophotometrically using their corresponding diagnosticreagent kits (Nanjing Jiancheng Bioengineering) accordingto the manufacturerrsquos instructions The protein content ofthe lysate was determined by using BCA Protein Assay Kit(Thermo USA)

211 ROS Assays The production of endogenous oxidativestress by-product was assessed using the conversion of 2101584071015840-dichlorodihydrofluorescein diacetate (DCHF-DA Sigma)[28] Cells were plated in triplicate grouped as describedin Section 210 pretreated with protein or amifostine andincubated with 20120583MDCFH-DA for 1 h in PBS at 37∘C Afterincubation the cells were rinsed in PBS and irradiated at adose of 4GyThe fluorescence intensity of irradiated sampleswas observed within 10min using a fluorescent microscopy(ZEISS AXIOSKOP-50 Germany) equipped with a digitalcharge-coupled device camera and a PC for data acquisitionand analysis

212 Apoptosis Assays Apoptotic cells were detected using aFITC Annexin-V apoptosis detection kit 1 (BD PharmingenSan Diego CA USA) Briefly cells (grouped as describedin Section 210) pretreated with protein or amifostine wereirradiated at a dose of 4Gy and cultured for 24 h at 37∘CThen the cells were trypsinized and washed with PBS Thewashed cells were resuspended in Annexin-V binding buffercontaining 10mM HEPESNaOH pH 74 140mM NaCland 25mM CaCl

2according to the manufacturerrsquos protocol

The cells were stained simultaneously with FITC-conjugatedAnnexin-V andPI at room temperature for 15min in the darkprior to the addition of binding buffer The apoptotic cellswere measured using a FACScan flow cytometer The cellswere sorted into intact cells (Annexin-Vminus PIminus) early apop-totic cells (Annexin-V+ PIminus) late apoptotic cells (Annexin-V+PI+) and necrotic cells (Annexin-Vminus PI+)

213 Statistical Analyses Statistical analysis of all data wasperformed using Excel The results are reported as means plusmnSE or SEM The 119875 values were determined using the Student

two-tailed 119905-test and 119875 lt 005 or 119875 lt 001 was consideredstatistically significant

3 Result

31 Transduction of Recombination Protein GST-TAT-SOD InVitro and In Vivo The restoration of enzymatic activities oftransduced fusion protein into cells is critical for the appli-cation of protein transduction technology or therapeutic useTherefore we determined the SOD activities of cells trans-duced with GST-TAT-SOD firstly As shown in Figure 1(a)GST-TAT-SOD was successfully delivered into cells whereaswild SODwas notThe enzyme activity of SOD in transducedcells increased rapidly in 2 h and basically levelled off inthe next 10 hours (Figure 1(a)) Besides that the amount oftransduced fusion protein also increased in a dose-dependentmanner (Figure 1(b)) To visualize the transduction of GST-TAT-SOD fusion protein was labeled by FITC internalizedinto culture cells observed under fluorescence microscopyand quantified by fluorescence Microplate Reader As shownin Figures 1(c) and 1(d) compared with wild SOD GST-TAT-SOD (2 kUmL) was efficiently transduced into cells afterincubation for 3 h (119875 lt 001) In vivo GST-TAT-SOD exhib-ited excellent transduced ability as was shown in Figure 1(e)The fluorescence intensity in liver lung spleen kidney andbrain tissues of mice almost had no difference between CONgroup and wild SOD group while the fluorescence intensityin those tissues of mice treated with GST-TAT-SOD wasconsiderably increased compared to other two groups (119875 lt005 119875 lt 001) This result implicates that GST-TAT-SODcould be effectively transduced into different organs in vivo

32 In Vitro Cytotoxicity The MTT assay was used to assessthe effect of GST-TAT-SOD concentration on cell viabilityin L-02 and Hep G2 cells as shown in Figure 2 GST-TAT-SOD (500ndash2000UmL) had little cytotoxicity on both cellsHigher concentration GST-TAT-SOD (6000UmL) resultedin inhibition of L-02 cell proliferation about 2125 (Fig-ure 2(a)) A dose-dependent (2000ndash6000UmL) reduction incell viability of GST-TAT-SOD was observed in Hep G2 celllines up to about 3339 (Figure 2(b)) SOD-TAT and wildSOD both were nontoxic to two cell lines (Figure 2)

33 Clonogenic Tests on Cells following X-Ray TreatmentColony-forming assays were used to determine the radiopro-tective effect of cell permeable antioxidant enzyme (Figure 3)As shown in Figure 3(a) surviving fraction of GST-TAT-SOD on L-02 cells colonies tended to follow a bell curve overprotein concentration The effect of SOD-TAT represented alittle bell curve in low concentration (lt2000UmL) whilehigher concentration showed upward trend in concentrationCompared with two cell permeable SOD wild SOD showeda most slowly rising trend in concentration Otherwise all ofthree proteins in low concentration (lt500UmL) seemed toenhance the surviving fraction of Hep G2 cells to a certaindegree In higher concentration GST-TAT-SOD showed acontinuous downward trend while SOD-TAT and wild SODtend to level off (Figure 3(b))When protective effects on L-02andHepG2 cells are considered together the optimal doses of


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Figure 1 Transduction of GST-TAT-SOD in vitro and in vivoThe transduction activity of the protein was analyzed by measuring the level ofthe transduced proteins in the cells by SOD activity (a and b) or visualization and quantification of the protein by FITC-labeled (c d and e)(a) Protein was added to culture media and incubated for various time intervals (b) Various concentrations of protein were added to culturemedia and incubated for 3 h (c) Visualization of GST-TAT-SOD transduced into cells L-02 cells were treated with 2 kUmL FITC-labeledGST-TAT-SOD fusion proteins and control FITC-labeled SOD for 3 h and the transduced proteinswere identified by fluorescencemicroscopy(d) Quantification of the transduction of GST-TAT-SOD into cells L-02 cells plated in a 24-well plate were treated with FITC-labeled proteinthen harvested and washedThe transduction activity of each protein was analyzed by measuring fluorescence intensity in the cells The barsindicate the means plusmn SD (119899 = 3 compared with control group 119875 lt 001 compared with wild SOD group lowastlowast119875 lt 001) (e) Transduction ofGST-TAT-SOD in vivo FITC-labeled GST-TAT-SOD or SOD (05mL 2 kUmL) was injected intraperitoneally into mice The homogenatesof the liver spleen lung brain and kidney were prepared and transduction efficiencies were analyzed by measuring fluorescence intensityof tissue homogenates from organs after 3 h The bars indicate the means plusmn SD (119899 = 5 compared with control group 119875 lt 005 119875 lt 001compared with wild SOD group lowast119875 lt 005 lowastlowast119875 lt 001)


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Figure 2 Cytotoxic effects of GST-TAT-SOD on cells Approximately 1 times 105 cells were seeded per well in 96-well plates and incubated for48 h in fresh medium along with the protein at the final concentration indicated (three replicates each) The bars indicate the means plusmn SD(119899 = 3) (a) L-02 cells (b) Hep G2 cells

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Figure 3 Clonogenic tests on L-02 (a) and Hep G2 (b) cells following X-ray treatmentThe cell strains were treated in the presence of proteinor amifostine and irradiated with 2Gy X-ray subsequently After irradiation the cells were detached with trypsin-EDTA and plated at aconcentration of 100 cellsdish in drug-free medium Fourteen days later the colonies were fixed and counted by using a crystal violet stainingto evaluate the number of colonies present in the dishes (c) Comparison of protective rate between three proteins and amifostine The barsindicate the means plusmn SD (119899 = 3 compared with group received irradiation only lowast119875 lt 005 lowastlowast119875 lt 001)


GST-TAT-SOD SOD-TAT and wild SOD were 2000UmL6000UmL and 6000UmL respectively The optimal doseof radioprotector used in clinic amifostine was proved tobe 4 120583gmL (unpublished) As was shown in Figure 3(c)amifostine SOD-TAT and GST-TAT-SOD all had significantprotective rate on irradiated L-02 cells (119875 lt 005 119875 lt 001)GST-TAT-SOD exhibited the optimal effect among abovethree pretreated groups Wild SOD have a little protectiveeffect but have no significance All of them have a certaindegree of protective effect on Hep G2 cells but no significantenhancement was observed

34 Measurement of SOD CAT T-AOC GSH-PX and MDAActivity A significant reduction in the activity of antioxi-dant enzymes (SOD CAT and GSH-PX) and a remarkableincrease in the level of MDA were observed in both L-02 andHep G2 XRT groups 24 h after irradiation (Figure 4(a) 119875 lt005 119875 lt 001) T-AOC of both cells was also significantlydecreased after irradiation (Figure 4(b)119875 lt 001) Comparedwith XRT group pretreatment of amifostine significantly(119875 lt 005 119875 lt 001) decreased the level of MDA inboth two cell lines but failed to keep the T-AOC activityof cells (Figures 4(a) and 4(b)) This treatment seemed toenhance the SOD activity GSH-Px activity and CAT activityof irradiated cells to some extent but those indexes werenot significant in comparison with XRT group (Figures 4(c)ndash4(e)) Wild SOD pretreatment failed to significantly improveall antioxidant indexes in L-02 cells but it essentially increasedthe CAT activity GSH-PX activity andMDA level in Hep G2cells (Figures 4(c)ndash4(e) 119875 lt 005) SOD-TAT pretreatmentgreatly increased the SOD GSH-Px and T-AOC activity anddecreased the MDA level in L-02 cells (Figure 4 119875 lt 005)It had no obvious effect on antioxidant index in Hep G2cells expect CAT activity and T-AOC activity (Figures 4(e)and 4(b) 119875 lt 005) GST-TAT-SOD treatment which wasthe best one among four pretreatment groups remarkablykept all antioxidant indexes in L-02 especially CAT activityand MDA level which reached or were higher than thoseof the control group (Figures 4(e) and 4(a) 119875 lt 005)Furthermore GST-TAT-SOD treatment had no noteworthyprotective effect on those indexes in Hep G2 cells (Figure 4)

35 ROS Assay ROS production in both two cells inducedby X-ray radiation was evaluated using a ROS-dependentoxidation of DCFH-DA A remarkable increase in ROSproduction was observed in both two cells after irradiation(Figures 5(a)-(B) and 5(b)-(B)) Pretreatment of L-02 cellswith amifostine SOD-TAT or GST-TAT-SOD especiallyGST-TAT-SOD significantly suppressed the elevation of ROSproduction (Figures 5(a)-(D) 5(a)-(E) and 5(a)-(F)) Asother cancer cells Hep G2 cells have higher levels of ROSthan normal cells (Figure 5(b)-(A)) After irradiation higherlevels of ROS were induced in Hep G2 cells than that ofL-02 cells (Figure 5(b)-(B)) Pretreatment with SOD-TATor GST-TAT-SOD slightly decreased the elevation of ROSproduction (Figures 5(b)-(E) and 5(b)-(F)) while amifostinepretreatment significantly suppressed that (Figure 5(b)-(D))By contrast wild SOD could not prevent the production of

intracellular ROS induced by irradiation (Figures 5(a)-(C)and 5(b)-(C))

36 Apoptotic Index To further confirm the effect of GST-TAT-SOD on irradiation-induced apoptosis in both L-02 andHep G2 cells Annexin-V-FITCPI staining experiment wasperformed (Figure 6) The number of Annexin-V-positivecells increased in both L-02 and Hep G2 cells after irra-diation treatment the number of total apoptotic indexeswas 1494 and 610 respectively Amifostine pretreatmentcould reduce the apoptotic index of both L-02 and Hep G2cells to 951 and 316 respectively Pretreatment with wildSOD failed to lower the apoptotic index of L-02 cells whilethe apoptotic index of Hep G2 cells with same treatmentwas decreased to 445 respectively SOD-TAT pretreatmentcould lessen the apoptotic index of both L-02 and Hep G2cells to 1369 and 505 respectively Pretreatment withGST-TAT-SOD could go down the apoptotic index of two celllines to 1179 and 549 respectively

4 Discussion

In this study a bifunctional protein GST-TAT-SOD arecombinant fusion of GST SOD and TAT from E coliwas prepared Firstly GST-TAT-SOD was proved to be cellpermeable and safe to normal cellsThen the radioprotectiveeffect of bifunctional GST-TAT-SOD on radiation-inducedcellular damage was tested in comparison with monofunc-tional SOD-TAT and amifostine

Our results show that intracellular ROS were induced inboth human normal liver cells and human hepatoma cellsby ionizing radiation (Figure 5) which caused a significantreduction of antioxidant system (Figure 4) decline in colony-forming ability and apoptosis of cells (Figures 3 and 6)Amifostine could remove intracellular ROS in irradiatednormal liver cells effectively (Figure 5) and reduce subsequentradiation injury (Figures 3 4 and 6)

SOD enzymes are indispensable and ubiquitous antiox-idant defenses protecting oxygen-utilizing cells from thetoxicity of the ROS produced by irradiation But our resultsshowed that wild SOD had little protective effect for itsinefficient delivery across the biological membranes due tothe large molecular size (Figures 3 4 5 and 6)

Intramuscular injection of liposomal CuZn-SOD intra-tracheal injections of Mn-SOD plasmid-liposome or Mn-SOD adenovirus gene therapy and overexpression of EC-SOD in transgenic mice prior to irradiation were effectivein attenuating radiation damage [10 14 29ndash31] which furtherindicated the importance of intracellular delivery of SOD forthe radioprotective effect But these applications in humanare far away from practical due to the apparent technicaldifficulties

SOD mimics provide a novel potential developmentpattern of SOD Mn porphyrins are most valid SOD mimicsup to now and have been proven to be anticancer andradioprotective [16] But it still needs further improvement tomatch the catalytic efficiency of natural enzyme Otherwise


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mg

pro)

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

lowastlowast

(c)

75

125

175

225

GSH

-Px

activ

ity (U

mg

pro)

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

lowastlowast lowast

(d)

000510152025

CAT

activ

ity (U

mg

pro)

lowast

lowast

lowast

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

(e)

Figure 4 Biochemical estimations of both cells (a) MDA level (b) T-AOC activity (c) SOD activity (d) GSH-PX activity (e) CAT activityCells were pretreated with protein or amifostine and irradiated with 4Gy X-ray subsequently After irradiation the cells were further culturedfor 24 h at 37∘C Then the cells were lysed and centrifuged and the antioxidant activities in the supernatant were determined subsequentlyThe bars indicate the means plusmn SD (119899 = 3)The XRT group was compared with the control group (119875 lt 005 119875 lt 001)The pretreated groupwas compared with the XRT group (lowast119875 lt 005 lowastlowast119875 lt 001)

their actionmechanism is varied due to vastly different sterics[16]

SOD fused with cell-penetrating TAT-PTD overcamethe above-mentioned shortcoming Both monofunction andbifunctional cell membrane permeable SOD SOD-TAT andGST-TAT-SOD can enter into irradiated normal cells (Fig-ure 1) remarkably clear up intracellular redundant ROS

(Figure 5) maintain antioxidant system (Figure 4) enhancecolony-forming ability (Figure 3) and suppress apoptosis(Figure 6)

Bifunctional GST-TAT-SOD was superior to monofunc-tional SOD-TAT in many ways such as higher antioxidantcapacity colony-forming ability and lower apoptosis index ofirradiated normal cells which may be due to the additional


(A) (B) (C)

(D) (E) (F)

100120583m 100120583m 100120583m

100120583m 100120583m 100120583m

(a)

(A) (B) (C)

(D) (E) (F)

100120583m 100120583m 100120583m

100120583m 100120583m 100120583m

(b)

Figure 5 DCFH-DA detection of ROS in L-02 (a) andHep G2 (b) cells treated with X-ray radiation Cells were plated in triplicate pretreatedwith protein or amifostine and incubated with DCFH-DA for 1 h in PBS at 37∘C After incubation the cells were rinsed in PBS and irradiatedat a dose of 4Gy The fluorescence intensity of irradiated samples was observed within 10min using a fluorescent microscopy (A) CON (B)XRT (C) XRT+SOD (D) XRT+AMFT (E) XRT+GST-TAT-SOD (F) XRT+SOD-TAT

fusion of GST Via a sulfhydryl group GSTs catalyze theconjugation of reduced glutathione (GSH) to electrophiliccentres on a wide variety of substrates [32] This activitydetoxifies endogenous compounds such as peroxidised lipids[33] Although GST activity of irradiated cells would not be

impacted with low irradiation dose [34] increasing intracel-lular GST activity did help to enhance the ability to removefree radicals and antioxidant capacity of GST-TAT-SODcompared to that of SOD-TAT (Figures 5 and 4) As such theradioprotective effect of bifunctional antioxidant enzymes in


PIPE

Annexin-V FITCAnnexin-V FITCAnnexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

100 101 102 103 104 100 101 102 103 104100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104PI

PE

100

101

102

103

104

PIPE

100

101

102

103

104

(A) (B) (C)

(D) (E) (F)

882

612

869

593

524

427

712

467

782

587

(a)

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

(A) (B) (C)

(D) (E) (F)

340

270

264

181

137

179

301

248

244

261

(b)

Figure 6 Continued


0

4

8

12

16

20

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

Tota

l apo

ptot

ic in

dex

()

L-02Hep G2

(c)

Figure 6Annexin-VPI analysis of apoptosis in L-02 (a) andHepG2 (b) cells at 24 h after treatmentwith 4Gyof irradiation Cellswere stainedwith Annexin-V FITC and PI and detected by flow cytometryThe lower right quadrant (Annexin-V+ PIminus) represents early apoptosis whereasupper right quadrant (Annexin-V+ PI+) represents late apoptosis ((A) CON (B) XRT (C) XRT+SOD (D) XRT+AMFT (E) XRT+GST-TAT-SOD (F) XRT+SOD-TAT) (c) Total apoptotic index of cells with different treatment

low dose (2000UmL) was better than that of single-functionantioxidant enzymes in higher dose (6000UmL)

One of the major concerns with the use of radioprotectorduring the course of radiotherapy is the possibility of tumorprotection Many antioxidants such as alpha tocopherol andbeta carotene used during the course of radiotherapy wereassociated with evidence of poorer tumor control in random-ized trials [35 36] although they could reduce normal tissuetoxicity in many instances with promising results

Amifostine has been shown to concentrate more rapidlyin normal tissues than in tumor tissues in studies of tumor-bearing animals [37] But concerns about tumor protectionand toxicity of this agent have led to controversy regardingthe appropriate setting for its use [38] Our results showedthat although amifostinewas a good radioprotector of normalhepatocytes it showed some degree of protective effectson hepatoma cells at the same time (Figures 3 4 5 and6) Instead both SOD-TAT and GST-TAT-SOD seemed toeliminate free radical cell selectively (Figure 5(b)) whichmay be due to their selective cell-penetrating capabilityon cell lines [39] Particularly the latter seemed to haveno significant effect on antioxidant system (Figure 4) SF(Figure 3(c)) and apoptotic index of irradiated hepatomacells (Figure 6) Otherwise intraperitoneal injection of GST-TAT-SOD was proved to be successfully transduced intodifferent organs inmice including the brainwhere amifostinecould not reach (Figure 1(e)) What is more protein fusionwith TAT could be delivered by a variety of routes includingoral administration [40] and parenteral administration suchas transdermal administration [23 24] and intraperitonealinjection [26 41] All these showed that bifunctional GST-TAT-SOD has the potential to be a safer and more efficientradioprotector for clinical applications in radiotherapy orintentional exposures to ionizing radiation More detailedselectively protective mechanism and protective effect in vivoof GST-TAT-SOD need to be studied in the future for acomprehensive assessment of its potential However fusion

of two antioxidant enzymes and cell-penetrating peptides ispotentially valuable in the development of radioprotectiveagent

5 Conclusions

The present study has not yet provided various evidencesfor the selectively radioprotective effect of bifunctional GST-TAT-SOD on irradiated cells Nonetheless it confirms thatGST-TAT-SOD pretreatment is safe and better than amifos-tine or SOD-TAT pretreatment to selectively scavenge intra-cellular free radical maintain antioxidant system enhancecolony-forming ability and suppress apoptosis

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

Acknowledgments

The financial support was provided by grants from theNational Natural Science Foundation of China (8147290730800285) and Joint Research Project of Fujian ProvinceEducational Committee and Health Department (WKJ2008-2-47)

References

[1] P A Riley ldquoFree radicals in biology oxidative stress and theeffects of ionizing radiationrdquo International Journal of RadiationBiology vol 65 no 1 pp 27ndash33 1994

[2] D R Spitz E I Azzam J J Li and D Gius ldquoMetabolicoxidationreduction reactions and cellular responses to ionizingradiation a unifying concept in stress response biologyrdquoCancerand Metastasis Reviews vol 23 no 3-4 pp 311ndash322 2004


[3] J Gu S Zhu X LiHWu Y Li and FHua ldquoEffect of amifostinein head and neck cancer patients treated with radiotherapya systematic review and meta-analysis based on randomizedcontrolled trialsrdquo PLoS ONE vol 9 no 5 Article ID e959682014

[4] MMabro S Faivre and E Raymond ldquoA risk benefit assessmentof amifostine in cytoprotectionrdquo Drug Safety vol 21 no 5 pp367ndash387 1999

[5] M I Koukourakis D Pitsiava A Giatromanolaki G Kam-bouromiti E Sivridis and G Kartalis ldquoAmifostine-relatedfever-rash during fractionated radiotherapy diagnostic andpredictive role of C-reactive proteinrdquo American Journal ofClinical Oncology Cancer Clinical Trials vol 34 no 3 pp 281ndash285 2011

[6] VMDest ldquoRadioprotectants adding quality of life to survivor-shiprdquo Seminars in Oncology Nursing vol 22 no 4 pp 249ndash2562006

[7] D Rades F Fehlauer A Bajrovic B Mahlmann E Richterand W Alberti ldquoSerious adverse effects of amifostine duringradiotherapy in head and neck cancer patientsrdquo Radiotherapyand Oncology vol 70 no 3 pp 261ndash264 2004

[8] T Quinlan S Spivack and B T Mossman ldquoRegulation ofantioxidant enzymes in lung after oxidant injuryrdquo Environmen-tal Health Perspectives vol 102 supplement 2 pp 79ndash87 1994

[9] M-F Tsan ldquoSuperoxide dismutase and pulmonary oxygentoxicityrdquo Proceedings of the Society for Experimental Biology andMedicine vol 214 no 2 pp 107ndash113 1997

[10] Z N Rabbani M S Anscher R J Folz et al ldquoOverexpressionof extracellular superoxide dismutase reduces acute radiationinduced lung toxicityrdquoBMCCancer vol 5 no 1 article 59 2005

[11] S K Kang Z N Rabbani R J Folz et al ldquoOverexpres-sion of extracellular superoxide dismutase protects mice fromradiation-induced lung injuryrdquo International Journal of Radi-ation Oncology Biology Physics vol 57 no 4 pp 1056ndash10662003

[12] M W Epperly V E Kagan C A Sikora et al ldquoManganesesuperoxide dismutase-plasmidliposome (MnSOD-PL) admin-istration protects mice from esophagitis associated with frac-tionated radiationrdquo International Journal of Cancer vol 96 no4 pp 221ndash231 2001

[13] MW Epperly J A Bray S Krager et al ldquoIntratracheal injectionof adenovirus containing the human MnSOD transgene pro-tects athymic nude mice from irradiation-induced organizingalveolitisrdquo International Journal of Radiation Oncology BiologyPhysics vol 43 no 1 pp 169ndash181 1999

[14] M W Epperly S Defilippi C Sikora J Gretton A Kalendand J S Greenberger ldquoIntratracheal injection of manganesesuperoxide dismutase (MnSOD) plasmidliposomes protectsnormal lung but not orthotopic tumors from irradiationrdquo GeneTherapy vol 7 no 12 pp 1011ndash1018 2000

[15] M Epperly J Bray S Kraeger et al ldquoPrevention of late effectsof irradiation lung damage bymanganese superoxide dismutasegene therapyrdquo Gene Therapy vol 5 no 2 pp 196ndash208 1998

[16] I Batinic-Haberle A Tovmasyan E R H Roberts ZVujaskovic K W Leong and I Spasojevic ldquoSOD therapeu-tics latest insights into their structure-activity relationshipsand impact on the cellular redox-based signaling pathwaysrdquoAntioxidants amp Redox Signaling vol 20 no 15 pp 2372ndash24152014

[17] A Eguchi T Akuta H Okuyama et al ldquoProtein transductiondomain of HIV-1 tat protein promotes efficient delivery of DNA

into mammalian cellsrdquoThe Journal of Biological Chemistry vol276 no 28 pp 26204ndash26210 2001

[18] L Josephson C-H Tung A Moore and R Weissleder ldquoHigh-efficiency intracellular magnetic labeling with novel superpar-amagnetic-tat peptide conjugatesrdquo Bioconjugate Chemistry vol10 no 2 pp 186ndash191 1999

[19] M Lewin N Carlesso C-H Tung et al ldquoTat peptide-derivatized magnetic nanoparticles allow in vivo tracking andrecovery of progenitor cellsrdquo Nature Biotechnology vol 18 no4 pp 410ndash414 2000

[20] J B Rothbard S Garlington Q Lin et al ldquoConjugation ofarginine oligomers to cyclosporin A facilitates topical deliveryand inhibition of inflammationrdquo Nature Medicine vol 6 no 11pp 1253ndash1257 2000

[21] V P Torchilin R Rammohan V Weissig and T S LevchenkoldquoTAT peptide on the surface of liposomes affords their efficientintracellular delivery even at low temperature and in thepresence of metabolic inhibitorsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 98 no15 pp 8786ndash8791 2001

[22] J S Wadia and S F Dowdy ldquoProtein transduction technologyrdquoCurrent Opinion in Biotechnology vol 13 no 1 pp 52ndash56 2002

[23] J Pan S Peng J Zhou et al ldquoProtective effects of recombinantprotein PTD-SOD on guinea pigs damaged by ultraviolet BrdquoRadiation Protection vol 30 no 2 pp 209ndash213 2010

[24] J Pan J Zhou S Peng H He S Liu and P Rao ldquoThepreventing effects of recombinant protein PTD-SOD on guineapigs damaged by Ultraviolet Brdquo Journal of Radiation Researchand Radiation Processing vol 27 no 5 pp 297ndash302 2009

[25] J Pan K Zhou G Zheng S Liu and P Rao ldquoCrystallizationand preliminary X-ray diffraction analysis of the SOD-TATfusion proteinrdquo Acta Crystallographica Section F StructuralBiology and Crystallization Communications vol 68 no 5 pp543ndash546 2012

[26] J Pan Y Su X Hou et al ldquoProtective effect of recombinantprotein SOD-TAT on radiation-induced lung injury in micerdquoLife Sciences vol 91 no 3-4 pp 89ndash93 2012

[27] N A P Franken H M Rodermond J Stap J Haveman and Cvan Bree ldquoClonogenic assay of cells in vitrordquo Nature Protocolsvol 1 no 5 pp 2315ndash2319 2006

[28] O Myhre J M Andersen H Aarnes and F Fonnum ldquoEvalu-ation of the probes 2rsquo7rsquo-dichlorofluorescin diacetate luminoland lucigenin as indicators of reactive species formationrdquoBiochemical Pharmacology vol 65 no 10 pp 1575ndash1582 2003

[29] S Delanian F Baillet J Huart J-L Lefaix C Maulard andM Housset ldquoSuccessful treatment of radiation-induced fibrosisusing liposomal CuZn superoxide dismutase clinical trialrdquoRadiotherapy and Oncology vol 32 no 1 pp 12ndash20 1994

[30] S K Kang Z N Rabbani R J Folz et al ldquoOverexpres-sion of extracellular superoxide dismutase protects mice fromradiation-induced lung injuryrdquo International Journal of Radia-tionOncology Biology Physics vol 57 no 4 pp 1056ndash1066 2003

[31] J-L Lefaix S Delanian J-J Leplat et al ldquoSuccessful treat-ment of radiation-induced fibrosis using CuZn-SOD and Mn-SOD an experimental studyrdquo International Journal of RadiationOncology Biology Physics vol 35 no 2 pp 305ndash312 1996

[32] K T Douglas ldquoMechanism of action of glutathione-dependentenzymesrdquo Advances in Enzymology and Related Areas of Molec-ular Biology vol 59 pp 103ndash167 1987

[33] M J Leaver and S G George ldquoA piscine glutathione S-transferase which efficiently conjugates the end-products of


lipid peroxidationrdquoMarine Environmental Research vol 46 no1ndash5 pp 71ndash74 1998

[34] K-S Nam M-K Kim and Y-H Shon ldquoCancer chemopre-ventive enzymes of human colorectal adenocarcinoma cellsirradiated with proton beamsrdquo Journal of the Korean PhysicalSociety vol 52 no 3 pp 945ndash948 2008

[35] I Bairati F Meyer M Gelinas et al ldquoRandomized trialof antioxidant vitamins to prevent acute adverse effects ofradiation therapy in head and neck cancer patientsrdquo Journal ofClinical Oncology vol 23 no 24 pp 5805ndash5813 2005

[36] F Meyer I Bairati A Fortin et al ldquoInteraction betweenantioxidant vitamin supplementation and cigarette smokingduring radiation therapy in relation to long-term effects onrecurrence and mortality a randomized trial among head andneck cancer patientsrdquo International Journal of Cancer vol 122no 7 pp 1679ndash1683 2008

[37] J M Yuhas ldquoActive versus passive absorption kinetics asthe basis for selective protection of normal tissues by S-2-(3-aminopropylamino)-ethylphosphorothioic acidrdquo CancerResearch vol 40 no 5 pp 1519ndash1524 1980

[38] D M Brizel and J Overgaard ldquoDoes amifostine have a role inchemoradiation treatmentrdquoThe Lancet Oncology vol 4 no 6pp 378ndash381 2003

[39] C Zhang X ChenW Ding et al ldquoDifferent protective effect ofPTD-SOD on UVB-irradiated L-O2 and HepG2 cellsrdquo ChineseJournal of Radiological Medicine and Protection vol 28 no 4pp 424ndash425 2008

[40] N Ye Y Lin S Liu et al ldquoProtection of fusion protein PTD-SOD by oral on rats of focal cerebral ischemiareperfusioninjuryrdquo Chinese Journal of Modern Applied Pharmacy vol 28no 7 pp 602ndash606 2011

[41] N Ye S Liu Y Lin and P Rao ldquoProtective effects of intraperi-toneal injection of TAT-SOD against focal cerebral ischemiareperfusion injury in ratsrdquo Life Sciences vol 89 no 23-24 pp868ndash874 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


effects of amifostine such as fever rash severe nausea allergyand acute hypotension have prompted a continuing search forbetter radioprotector [5ndash7]

Superoxide radicals produced by ionizing radiation arehighly reactive and potentially damaging to cellsThe enzymesuperoxide dismutase (SOD) neutralizes superoxide radicalsby changing it into molecular oxygen and hydrogen perox-ide thereby preventing the formation of highly aggressivecompounds such as peroxynitrite Hydrogen peroxide isthen subsequently eliminated by catalase and glutathioneperoxidase [8 9] SOD is naturally present in human cellsand proved to play a key role in cellular defenses againstoxidative damage [1] But as a protein SOD is too large tofreely enter into cells Although the hypothesis that SOD isradioprotective has been supported by many studies throughtransgenic experiments [10ndash15] there were many limitationson its protecting against radiation-induced chronic injuryin humans SOD mimics are another way to overcomethe limitation of large molecular weight Some of themhave been proved to be radioprotective in various radiationinjury models [16] But their reaction efficiency of scaveng-ing superoxide anion is still inferior to wild SOD Theirmechanism selectivity and toxicity of mimics may varycompared with natural enzyme [16] In our previous workwe constructed a cell membrane permeable SOD by generecombinant technique to circumvent this limitation Therecombinant protein was the fusion of hCuZn-SOD (SOD1)and cell-penetrating peptide derived from HIV-1 TAT pro-tein transduction domain TAT (YGRKKRRQRRR) Proteintransduction domains are able to carry larger molecules suchas oligonucleotides peptides full-length proteins 40 nm ironnanoparticles bacteriophages and even 200 nm liposomesacross cellular membranes and have proven useful in deliv-ering biologically active cargoes in both in vitro and in vivomodels [17ndash22] The recombinant SOD had been purifiedcrystallized and proved to be effective in preventing andtreating the damage of guinea pigs skin caused by single doseUVB radiation [23ndash25] What is more it was proved to beeffective in preventing radiation-induced lung injury in mice[26]

Cell permeable SOD was confirmed to have remarkableradioprotective effects compared with wild SOD by aboveexperiments [23 24 26] However superoxide radicals werenot the only harmful reactive chemical species producedby ionizing radiation To find out whether a cell permeablerecombination of different antioxidase would encourage abetter outcome a bifunctional recombinant protein fusedwith glutathione S-transferase (GST) and cell permeable SODwas constructed firstly and named GST-TAT-SOD GST isan enzyme that aids in detoxification by speeding up thelinking of toxic compounds with glutathione (GSH) thusforming a less reactive substance Besides that fusional GSTcould enhance the expression quantity of soluble bifunctionalantioxidase and simplify its purification

Current study investigated the selective radioprotectiveeffects of this cell permeable bifunctional antioxidant enzymecompared with SOD-TAT and amifostine

2 Materials and Methods

21 Enzyme and Chemicals E coli strains with recombi-nant plasmid of GST-TAT-SOD containing GST TAT-PTDand human CuZn-SOD and recombinant protein SOD-TAT were obtained from Institute of Biotechnology FuzhouUniversity (Fujian China) Wild CuZn-SOD was purchasedfromDatian Huacan Biotechnology Co Ltd (Fujian China)GST affinity chromatography was purchased from Weishi-Bohui Chromatogram Technology Co Ltd (Beijing China)Amifostine was purchased fromMeiluo Yinhe Pharmacy CoLtd (Hunan China) Malondialdehyde (MDA) SOD cata-lase (CAT) total antioxidation capacity (T-AOC) and glu-tathione peroxidase (GSH-Px) reagent kits were purchasedfrom Nanjing Jiancheng Bioengineering Co Ltd (JiangsuChina) Micro-BCAtrade Protein Assay Kit was purchased fromThermo Scientific (USA) RPMI-1640 and fetal bovine serumwere purchased from HyClone and Gibco (USA) respec-tively All other chemicals were of analytical purity

22 Cell Cultures Normal human liver cell line L-02 cells andhuman hepatoma cell line Hep G2 cells are available fromShanghai Institute of Biochemistry andCell Biology (SIBCB)Cells were cultured in RPMI-1640 (HyClone) supplementedwith 10 fetal bovine serum (Gibco) 100UmL penicillinand 100mgmL streptomycin (Gibco) at 37∘C in a 5 CO

2

humidified chamber

23 Mice Male Kunming mice (Fujian Medical University)weighing 18ndash22 g each were used at 6ndash8 weeks of age forthese experiments All mice were housed in an animal roomat 22∘C in a 12 h light12 h dark cycle All mice were givena standard chow diet and water ad libitum Animal welfareand experimental procedures were carried out in accordancewith the Guide for the Care and Use of Laboratory Animals(Ministry of Science and Technology of China 2006) andwere approved by the Review Committee for the Use ofHuman or Animal Subjects of Institute of BiotechnologyFuzhou University

24 Preparation of Recombination Protein GST-TAT-SODBacteria were washed in PBS and lysed in buffer A (10mMNa2HPO4 18mM KH

2PO4 and 27mM KCl pH 73) using

a cell disrupter (Sonic SolutionsCo vc-750)The supernatant(extract) was collected after centrifugation of the lysatesfor 20min at 12000 rpm in a HITACHI RX series HimacCF15RX rotor at 4∘C The supernatant containing GST-TAT-SOD was precipitated by incubation with 561 g ammoniumsulfate per 100mL of culture medium at 4∘C For purificationof GST-TAT-SOD the precipitate was resuspended in 20mMPBS pH 74 and purified by GST affinity chromatogra-phy resin (Weishi-Bohui Chromtotech Co Beijing China)according to the manufacturerrsquos instructions After bindingand washing the bound protein was eluted with 50mMTris-HC1 containing 001M glutathione pH 80 The concentra-tion SOD activity and GST activity of the purified proteinwere determined by BCA Protein Assay Kit (Thermo USA)and SOD and GST reagent kits (Jiangsu China) respectivelyThe SOD and GST activity of purified GST-TAT-SOD were








2at 37∘C









times 100

(1)











3 Result





30

60

90

120

150

0 2 4 6 8 10 12

SOD

activ

ity (U

mg

pro)

t (h)

SODGST-TAT-SODCON

(a)

50

70

90

110

0 1000 2000 3000 4000

SOD

activ

ity (U

mg

pro)

SOD activity (UmL)

SODGST-TAT-SOD

(b)

GST-TAT-SODCON SOD

(c)

0

15

30

45

60

75

90

CON SOD GST-TAT-SOD

Fluo

resc

ence

inte

nsity

mg

pro lowastlowast

(d)

Fluo

resc

ence

inte

nsity

mg

pro

0

200

400

600

800

1000

1200


SODGST-TAT-SOD

CON

lowastlowast

lowastlowast

lowast

lowast

lowast

(e)



0

20

40

60

80

100

0 1500 3000 4500 6000SOD activity (UmL)

Inhi

bitio

n ra

tio (

)

SOD-TATGST-TAT-SOD

SOD

(a)

0

20

40

60

80

100

0 1500 3000 4500 6000

Inhi

bitio

n ra

tio (

)

SOD activity (UmL)

SOD-TATGST-TAT-SOD

SOD

(b)


25

50

75

100

0 1000 2000 3000 4000 5000 6000

Surv

ivin

g fr

actio

n (

)

SOD activity (UmL)

SOD-TATGST-TAT-SOD

SOD

(a)

Surv

ivin

g fr

actio

n (

)

SOD activity (UmL)

25

50

75

100

0 1000 2000 3000 4000 5000 6000

SOD-TATGST-TAT-SOD

SOD

(b)

0

30

60

90


Prot

ectiv

e rat

e (

)

L-02Hep G2

lowastlowast

lowast

lowast

(c)








4 Discussion







468

101214161820

MD

A le

vel (

nmol

mg

pro)

L-02Hep G2

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

lowastlowast

lowast

lowastlowast lowast

(a)

020

025

030

035

T-AO

C ac

tivity

(Um

g pr

o)

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

lowastlowast

lowastlowast

L-02Hep G2

(b)

8

10

12

14

16

18

SOD

activ

ity (U

mg

pro)

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

lowastlowast

(c)

75

125

175

225

GSH

-Px

activ

ity (U

mg

pro)

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

lowastlowast lowast

(d)

000510152025

CAT

activ

ity (U

mg

pro)

lowast

lowast

lowast

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

(e)







(A) (B) (C)

(D) (E) (F)

100120583m 100120583m 100120583m

100120583m 100120583m 100120583m

(a)

(A) (B) (C)

(D) (E) (F)

100120583m 100120583m 100120583m

100120583m 100120583m 100120583m

(b)





PIPE


Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

100 101 102 103 104 100 101 102 103 104100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104PI

PE

100

101

102

103

104

PIPE

100

101

102

103

104

(A) (B) (C)

(D) (E) (F)

882

612

869

593

524

427

712

467

782

587

(a)

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

(A) (B) (C)

(D) (E) (F)

340

270

264

181

137

179

301

248

244

261

(b)

Figure 6 Continued


0

4

8

12

16

20

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

Tota

l apo

ptot

ic in

dex

()

L-02Hep G2

(c)






5 Conclusions


Competing Interests


Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























2at 37∘C









times 100

(1)











3 Result





30

60

90

120

150

0 2 4 6 8 10 12

SOD

activ

ity (U

mg

pro)

t (h)

SODGST-TAT-SODCON

(a)

50

70

90

110

0 1000 2000 3000 4000

SOD

activ

ity (U

mg

pro)

SOD activity (UmL)

SODGST-TAT-SOD

(b)

GST-TAT-SODCON SOD

(c)

0

15

30

45

60

75

90

CON SOD GST-TAT-SOD

Fluo

resc

ence

inte

nsity

mg

pro lowastlowast

(d)

Fluo

resc

ence

inte

nsity

mg

pro

0

200

400

600

800

1000

1200


SODGST-TAT-SOD

CON

lowastlowast

lowastlowast

lowast

lowast

lowast

(e)



0

20

40

60

80

100

0 1500 3000 4500 6000SOD activity (UmL)

Inhi

bitio

n ra

tio (

)

SOD-TATGST-TAT-SOD

SOD

(a)

0

20

40

60

80

100

0 1500 3000 4500 6000

Inhi

bitio

n ra

tio (

)

SOD activity (UmL)

SOD-TATGST-TAT-SOD

SOD

(b)


25

50

75

100

0 1000 2000 3000 4000 5000 6000

Surv

ivin

g fr

actio

n (

)

SOD activity (UmL)

SOD-TATGST-TAT-SOD

SOD

(a)

Surv

ivin

g fr

actio

n (

)

SOD activity (UmL)

25

50

75

100

0 1000 2000 3000 4000 5000 6000

SOD-TATGST-TAT-SOD

SOD

(b)

0

30

60

90


Prot

ectiv

e rat

e (

)

L-02Hep G2

lowastlowast

lowast

lowast

(c)








4 Discussion







468

101214161820

MD

A le

vel (

nmol

mg

pro)

L-02Hep G2

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

lowastlowast

lowast

lowastlowast lowast

(a)

020

025

030

035

T-AO

C ac

tivity

(Um

g pr

o)

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

lowastlowast

lowastlowast

L-02Hep G2

(b)

8

10

12

14

16

18

SOD

activ

ity (U

mg

pro)

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

lowastlowast

(c)

75

125

175

225

GSH

-Px

activ

ity (U

mg

pro)

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

lowastlowast lowast

(d)

000510152025

CAT

activ

ity (U

mg

pro)

lowast

lowast

lowast

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

(e)







(A) (B) (C)

(D) (E) (F)

100120583m 100120583m 100120583m

100120583m 100120583m 100120583m

(a)

(A) (B) (C)

(D) (E) (F)

100120583m 100120583m 100120583m

100120583m 100120583m 100120583m

(b)





PIPE


Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

100 101 102 103 104 100 101 102 103 104100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104PI

PE

100

101

102

103

104

PIPE

100

101

102

103

104

(A) (B) (C)

(D) (E) (F)

882

612

869

593

524

427

712

467

782

587

(a)

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

(A) (B) (C)

(D) (E) (F)

340

270

264

181

137

179

301

248

244

261

(b)

Figure 6 Continued


0

4

8

12

16

20

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

Tota

l apo

ptot

ic in

dex

()

L-02Hep G2

(c)






5 Conclusions


Competing Interests


Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























3 Result





30

60

90

120

150

0 2 4 6 8 10 12

SOD

activ

ity (U

mg

pro)

t (h)

SODGST-TAT-SODCON

(a)

50

70

90

110

0 1000 2000 3000 4000

SOD

activ

ity (U

mg

pro)

SOD activity (UmL)

SODGST-TAT-SOD

(b)

GST-TAT-SODCON SOD

(c)

0

15

30

45

60

75

90

CON SOD GST-TAT-SOD

Fluo

resc

ence

inte

nsity

mg

pro lowastlowast

(d)

Fluo

resc

ence

inte

nsity

mg

pro

0

200

400

600

800

1000

1200


SODGST-TAT-SOD

CON

lowastlowast

lowastlowast

lowast

lowast

lowast

(e)



0

20

40

60

80

100

0 1500 3000 4500 6000SOD activity (UmL)

Inhi

bitio

n ra

tio (

)

SOD-TATGST-TAT-SOD

SOD

(a)

0

20

40

60

80

100

0 1500 3000 4500 6000

Inhi

bitio

n ra

tio (

)

SOD activity (UmL)

SOD-TATGST-TAT-SOD

SOD

(b)


25

50

75

100

0 1000 2000 3000 4000 5000 6000

Surv

ivin

g fr

actio

n (

)

SOD activity (UmL)

SOD-TATGST-TAT-SOD

SOD

(a)

Surv

ivin

g fr

actio

n (

)

SOD activity (UmL)

25

50

75

100

0 1000 2000 3000 4000 5000 6000

SOD-TATGST-TAT-SOD

SOD

(b)

0

30

60

90


Prot

ectiv

e rat

e (

)

L-02Hep G2

lowastlowast

lowast

lowast

(c)








4 Discussion







468

101214161820

MD

A le

vel (

nmol

mg

pro)

L-02Hep G2

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

lowastlowast

lowast

lowastlowast lowast

(a)

020

025

030

035

T-AO

C ac

tivity

(Um

g pr

o)

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

lowastlowast

lowastlowast

L-02Hep G2

(b)

8

10

12

14

16

18

SOD

activ

ity (U

mg

pro)

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

lowastlowast

(c)

75

125

175

225

GSH

-Px

activ

ity (U

mg

pro)

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

lowastlowast lowast

(d)

000510152025

CAT

activ

ity (U

mg

pro)

lowast

lowast

lowast

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

(e)







(A) (B) (C)

(D) (E) (F)

100120583m 100120583m 100120583m

100120583m 100120583m 100120583m

(a)

(A) (B) (C)

(D) (E) (F)

100120583m 100120583m 100120583m

100120583m 100120583m 100120583m

(b)





PIPE


Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

100 101 102 103 104 100 101 102 103 104100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104PI

PE

100

101

102

103

104

PIPE

100

101

102

103

104

(A) (B) (C)

(D) (E) (F)

882

612

869

593

524

427

712

467

782

587

(a)

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

(A) (B) (C)

(D) (E) (F)

340

270

264

181

137

179

301

248

244

261

(b)

Figure 6 Continued


0

4

8

12

16

20

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

Tota

l apo

ptot

ic in

dex

()

L-02Hep G2

(c)






5 Conclusions


Competing Interests


Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















30

60

90

120

150

0 2 4 6 8 10 12

SOD

activ

ity (U

mg

pro)

t (h)

SODGST-TAT-SODCON

(a)

50

70

90

110

0 1000 2000 3000 4000

SOD

activ

ity (U

mg

pro)

SOD activity (UmL)

SODGST-TAT-SOD

(b)

GST-TAT-SODCON SOD

(c)

0

15

30

45

60

75

90

CON SOD GST-TAT-SOD

Fluo

resc

ence

inte

nsity

mg

pro lowastlowast

(d)

Fluo

resc

ence

inte

nsity

mg

pro

0

200

400

600

800

1000

1200


SODGST-TAT-SOD

CON

lowastlowast

lowastlowast

lowast

lowast

lowast

(e)



0

20

40

60

80

100

0 1500 3000 4500 6000SOD activity (UmL)

Inhi

bitio

n ra

tio (

)

SOD-TATGST-TAT-SOD

SOD

(a)

0

20

40

60

80

100

0 1500 3000 4500 6000

Inhi

bitio

n ra

tio (

)

SOD activity (UmL)

SOD-TATGST-TAT-SOD

SOD

(b)


25

50

75

100

0 1000 2000 3000 4000 5000 6000

Surv

ivin

g fr

actio

n (

)

SOD activity (UmL)

SOD-TATGST-TAT-SOD

SOD

(a)

Surv

ivin

g fr

actio

n (

)

SOD activity (UmL)

25

50

75

100

0 1000 2000 3000 4000 5000 6000

SOD-TATGST-TAT-SOD

SOD

(b)

0

30

60

90


Prot

ectiv

e rat

e (

)

L-02Hep G2

lowastlowast

lowast

lowast

(c)








4 Discussion







468

101214161820

MD

A le

vel (

nmol

mg

pro)

L-02Hep G2

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

lowastlowast

lowast

lowastlowast lowast

(a)

020

025

030

035

T-AO

C ac

tivity

(Um

g pr

o)

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

lowastlowast

lowastlowast

L-02Hep G2

(b)

8

10

12

14

16

18

SOD

activ

ity (U

mg

pro)

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

lowastlowast

(c)

75

125

175

225

GSH

-Px

activ

ity (U

mg

pro)

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

lowastlowast lowast

(d)

000510152025

CAT

activ

ity (U

mg

pro)

lowast

lowast

lowast

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

(e)







(A) (B) (C)

(D) (E) (F)

100120583m 100120583m 100120583m

100120583m 100120583m 100120583m

(a)

(A) (B) (C)

(D) (E) (F)

100120583m 100120583m 100120583m

100120583m 100120583m 100120583m

(b)





PIPE


Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

100 101 102 103 104 100 101 102 103 104100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104PI

PE

100

101

102

103

104

PIPE

100

101

102

103

104

(A) (B) (C)

(D) (E) (F)

882

612

869

593

524

427

712

467

782

587

(a)

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

(A) (B) (C)

(D) (E) (F)

340

270

264

181

137

179

301

248

244

261

(b)

Figure 6 Continued


0

4

8

12

16

20

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

Tota

l apo

ptot

ic in

dex

()

L-02Hep G2

(c)






5 Conclusions


Competing Interests


Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

0 1500 3000 4500 6000SOD activity (UmL)

Inhi

bitio

n ra

tio (

)

SOD-TATGST-TAT-SOD

SOD

(a)

0

20

40

60

80

100

0 1500 3000 4500 6000

Inhi

bitio

n ra

tio (

)

SOD activity (UmL)

SOD-TATGST-TAT-SOD

SOD

(b)


25

50

75

100

0 1000 2000 3000 4000 5000 6000

Surv

ivin

g fr

actio

n (

)

SOD activity (UmL)

SOD-TATGST-TAT-SOD

SOD

(a)

Surv

ivin

g fr

actio

n (

)

SOD activity (UmL)

25

50

75

100

0 1000 2000 3000 4000 5000 6000

SOD-TATGST-TAT-SOD

SOD

(b)

0

30

60

90


Prot

ectiv

e rat

e (

)

L-02Hep G2

lowastlowast

lowast

lowast

(c)








4 Discussion







468

101214161820

MD

A le

vel (

nmol

mg

pro)

L-02Hep G2

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

lowastlowast

lowast

lowastlowast lowast

(a)

020

025

030

035

T-AO

C ac

tivity

(Um

g pr

o)

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

lowastlowast

lowastlowast

L-02Hep G2

(b)

8

10

12

14

16

18

SOD

activ

ity (U

mg

pro)

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

lowastlowast

(c)

75

125

175

225

GSH

-Px

activ

ity (U

mg

pro)

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

lowastlowast lowast

(d)

000510152025

CAT

activ

ity (U

mg

pro)

lowast

lowast

lowast

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

(e)







(A) (B) (C)

(D) (E) (F)

100120583m 100120583m 100120583m

100120583m 100120583m 100120583m

(a)

(A) (B) (C)

(D) (E) (F)

100120583m 100120583m 100120583m

100120583m 100120583m 100120583m

(b)





PIPE


Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

100 101 102 103 104 100 101 102 103 104100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104PI

PE

100

101

102

103

104

PIPE

100

101

102

103

104

(A) (B) (C)

(D) (E) (F)

882

612

869

593

524

427

712

467

782

587

(a)

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

(A) (B) (C)

(D) (E) (F)

340

270

264

181

137

179

301

248

244

261

(b)

Figure 6 Continued


0

4

8

12

16

20

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

Tota

l apo

ptot

ic in

dex

()

L-02Hep G2

(c)






5 Conclusions


Competing Interests


Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















4 Discussion







468

101214161820

MD

A le

vel (

nmol

mg

pro)

L-02Hep G2

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

lowastlowast

lowast

lowastlowast lowast

(a)

020

025

030

035

T-AO

C ac

tivity

(Um

g pr

o)

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

lowastlowast

lowastlowast

L-02Hep G2

(b)

8

10

12

14

16

18

SOD

activ

ity (U

mg

pro)

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

lowastlowast

(c)

75

125

175

225

GSH

-Px

activ

ity (U

mg

pro)

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

lowastlowast lowast

(d)

000510152025

CAT

activ

ity (U

mg

pro)

lowast

lowast

lowast

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

(e)







(A) (B) (C)

(D) (E) (F)

100120583m 100120583m 100120583m

100120583m 100120583m 100120583m

(a)

(A) (B) (C)

(D) (E) (F)

100120583m 100120583m 100120583m

100120583m 100120583m 100120583m

(b)





PIPE


Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

100 101 102 103 104 100 101 102 103 104100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104PI

PE

100

101

102

103

104

PIPE

100

101

102

103

104

(A) (B) (C)

(D) (E) (F)

882

612

869

593

524

427

712

467

782

587

(a)

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

(A) (B) (C)

(D) (E) (F)

340

270

264

181

137

179

301

248

244

261

(b)

Figure 6 Continued


0

4

8

12

16

20

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

Tota

l apo

ptot

ic in

dex

()

L-02Hep G2

(c)






5 Conclusions


Competing Interests


Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















468

101214161820

MD

A le

vel (

nmol

mg

pro)

L-02Hep G2

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

lowastlowast

lowast

lowastlowast lowast

(a)

020

025

030

035

T-AO

C ac

tivity

(Um

g pr

o)

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

lowastlowast

lowastlowast

L-02Hep G2

(b)

8

10

12

14

16

18

SOD

activ

ity (U

mg

pro)

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

lowastlowast

(c)

75

125

175

225

GSH

-Px

activ

ity (U

mg

pro)

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

lowastlowast lowast

(d)

000510152025

CAT

activ

ity (U

mg

pro)

lowast

lowast

lowast

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

L-02Hep G2

(e)







(A) (B) (C)

(D) (E) (F)

100120583m 100120583m 100120583m

100120583m 100120583m 100120583m

(a)

(A) (B) (C)

(D) (E) (F)

100120583m 100120583m 100120583m

100120583m 100120583m 100120583m

(b)





PIPE


Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

100 101 102 103 104 100 101 102 103 104100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104PI

PE

100

101

102

103

104

PIPE

100

101

102

103

104

(A) (B) (C)

(D) (E) (F)

882

612

869

593

524

427

712

467

782

587

(a)

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

(A) (B) (C)

(D) (E) (F)

340

270

264

181

137

179

301

248

244

261

(b)

Figure 6 Continued


0

4

8

12

16

20

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

Tota

l apo

ptot

ic in

dex

()

L-02Hep G2

(c)






5 Conclusions


Competing Interests


Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(A) (B) (C)

(D) (E) (F)

100120583m 100120583m 100120583m

100120583m 100120583m 100120583m

(a)

(A) (B) (C)

(D) (E) (F)

100120583m 100120583m 100120583m

100120583m 100120583m 100120583m

(b)





PIPE


Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

100 101 102 103 104 100 101 102 103 104100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104PI

PE

100

101

102

103

104

PIPE

100

101

102

103

104

(A) (B) (C)

(D) (E) (F)

882

612

869

593

524

427

712

467

782

587

(a)

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

(A) (B) (C)

(D) (E) (F)

340

270

264

181

137

179

301

248

244

261

(b)

Figure 6 Continued


0

4

8

12

16

20

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

Tota

l apo

ptot

ic in

dex

()

L-02Hep G2

(c)






5 Conclusions


Competing Interests


Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















PIPE


Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

100 101 102 103 104 100 101 102 103 104100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104PI

PE

100

101

102

103

104

PIPE

100

101

102

103

104

(A) (B) (C)

(D) (E) (F)

882

612

869

593

524

427

712

467

782

587

(a)

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

Annexin-V FITC100 101 102 103 104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

PIPE

100

101

102

103

104

(A) (B) (C)

(D) (E) (F)

340

270

264

181

137

179

301

248

244

261

(b)

Figure 6 Continued


0

4

8

12

16

20

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

Tota

l apo

ptot

ic in

dex

()

L-02Hep G2

(c)






5 Conclusions


Competing Interests


Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

4

8

12

16

20

CON

XRT

XRT+

SOD

XRT+

SOD

-TA

T

XRT+

GST

-TA

T-SO

D

XRT+

AM

FT

Tota

l apo

ptot

ic in

dex

()

L-02Hep G2

(c)






5 Conclusions


Competing Interests


Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article gst-tat-sod: cell permeable bifunctional...

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