Morin (2′,3,4′,5,7-Pentahydroxyflavone) Protected Cells against γ-Radiation-Induced Oxidative Stress

Morin (2¢,3,4¢,5,7-Pentahydroxyflavone) Protected Cells againstc-Radiation-Induced Oxidative Stress

Rui Zhang1, Kyoung Ah Kang1, Sam Sik Kang2, Jae Woo Park3 and Jin Won Hyun1

1Department of Biochemistry, College of Medicine and Applied Radiological Science Research Institute, Jeju National University, Jeju-si,Korea, 2College of Pharmacy, Seoul National University, Seoul, Korea, and 3Department of Nuclear and Energy Engineering, Jeju National

University, Jeju-si, Korea

(Received 29 May 2010; Accepted 24 July 2010)

Abstract: Ionizing radiation can induce cell damage by generating reactive oxygen species (ROS). The present study was car-ried out to investigate the radio-protective effects of a flavonoid compound, morin (2¢,3,4¢,5,7-pentahydroxyflavone) and theunderlying mechanisms. Morin was found to reduce the intracellular ROS generated by c-irradiation. Moreover, morin pro-tected cellular components against radiation-induced membrane lipid peroxidation and cellular DNA damage, which are themain targets of radiation-induced cell damage. Morin recovered cell viability damaged by radiation via inhibition of apoptosis.Irradiated cells with morin treatment reduced Bax, phospho Bcl-2, active caspase 9 and caspase 3, which were induced byc-radiation. Irradiated cells with morin recovered the expression of Bcl-2 reduced by c-radiation. Morin exerted anti-apoptoticeffects via inhibition of mitogen-activated protein kinase kinase-4 (MKK4 ⁄ SEK1)-c-Jun NH2-terminal kinase (JNK)-activatorprotein 1 (AP-1) cascades induced by c-radiation. The results suggest that morin protects cells against oxidative stress inducedby radiation via reduction of ROS and attenuation of SEK1-JNK-AP-1 pathway.

Gamma-ray radiation is known to induce oxidative stress bygenerating reactive oxygen species (ROS), including superox-ide anion, hydroxyl radical, single oxygen and hydrogen per-oxide in cells [1]. These ROS can lead to functional damagein lipid, proteins and DNA, which in turn can eventuallyresult in cell death [2]. In many cases, ionizing radiation-induced cell death has been identified as apoptosis [3,4].Recently, radiation therapy is regarded as an important treat-ment for various malignant diseases. However, the amountsof ionizing radiation that can be given to treat malignanttumours are often limited by toxicity in the surrounding nor-mal tissues and organs [5,6]. Recently, synthetic agents suchas WR2721 (amifostine), OK-432, and ethiofos were investi-gated for their efficacy in protecting cells against radiation-induced damage [5]. However, these agents have the potentialto cause serious side effects, including decreased cellularfunction, nausea, hypotension and death [6]. Alternatively,natural plant extracts that can protect cells and tissuesagainst ionizing radiation without overt side effects could bedeveloped as adjuncts to radiotherapy. In fact, natural com-pounds with disparate structures were isolated from differentnatural plant species. Some of these compounds have shownto be toxic but other compounds apparently are devoid ofadverse effects. K�hkçnen et al. [7] have reported that natu-ral plant extracts containing phenolic compounds exhibitedantioxidant activities. To date, several phenolic compounds

have been shown to protect cells against radiation-induceddamage by virtue of their antioxidant properties [8,9].

Flavonoids are a family of phenol compound most com-monly found in a variety of fruits, vegetables, juices and com-ponents of herbal-containing dietary supplements. Theinterests in the investigation of flavonoids stem from theirbiological properties, which include oxygen radical scaveng-ing and antioxidant properties [10,11]. Morin (2¢,3,4¢,5,7-pentahydroxyflavone) is a member of the flavonoid familywhich consists of a yellowish pigment found in almond (Pru-nus dulcis), fig (Chlorophora tinctoria) and other moraceaeused in food and herbal medicine [12]. Moreover, morin hasbeen reported to possess a variety of biological propertiesagainst oxidative stress-induced damage. It protects cardio-vascular cells, glomerular mesangial cells, hepatocytes, oligo-dendrocytes and neurons against damage by oxidative stress[13–16]. Parihar et al. [17] have reported that morin exhibitedanti-clastogenic activity against c-radiation in vivo system.Recently, we have reported morin’s cellular protective effecton hydrogen peroxide-induced oxidative stress [18]. Becauseirradiated cells generate ROS, we speculated that morin, withits ROS scavenging effect, may provide cytoprotective effectsagainst c-radiation-induced cell damage. Consequently, weelected to investigate the effects of morin on cell damageinduced by c-radiation, and the possible mechanism underly-ing this cytoprotective effect.

Materials and Methods

Materials and reagents. Morin (2¢,3,4¢,5,7-pentahydroxyflavone, fig. 1)compound, 2¢,7¢-dichlorodihydrofluorescein diacetate (DCF-DA)and Hoechst 33342 were purchased from Sigma Chemical Company

Author for correspondence: Jin Won Hyun, Department of Bio-chemistry, College of Medicine, Jeju National University, Jeju-si 690-756, Korea (fax +82-64-702-2687, e-mail [email protected]).

Basic & Clinical Pharmacology & Toxicology, 108, 63–72 Doi: 10.1111/j.1742-7843.2010.00629.x

� 2010 The AuthorsBasic & Clinical Pharmacology & Toxicology � 2010 Nordic Pharmacological Society

(St Louis, MO, USA). Diphenyl-1-pyrenylphosphine (DPPP) waspurchased from Molecular Probes (Eugene, OR, USA), and5,5¢,6,6¢-tetrachloro-1,1¢3,3¢-tetraethyl-benzimidazolylcarbocyanineiodide (JC-1) was purchased from Invitrogen Corporation (Carlsbad,CA, USA) and thiobarbituric acid was purchased from BDH Labo-ratories (Poole, Dorset, UK). Primary anti-Bcl-2, -Bax, -phosphoBcl-2, -caspase 9, -caspase 3, -JNK, -phospho JNK, -SEK1 and -phospho SEK1 antibodies were purchased from Cell SignallingTechnology (Beverly, MA, USA). The plasmid containing the AP-1-binding site-luciferase construct was provided by professor YoungJoon Surh of Seoul National University (Korea).

Cell culture and irradiation. It has been reported that the lung is aradiosensitive organ [19,20]. To study the effect of morin on c-radia-tion-induced cell damage, Chinese hamster lung fibroblasts (V79-4)cells from the American Type Culture Collection (Rockville, MD,USA) were used and maintained at 37�C in an incubator with ahumidified atmosphere of 5% CO2, and cultured in Dulbecco’s mod-ified Eagle’s medium containing 10% heat-inactivated foetal calfserum, streptomycin (100 lg ⁄ ml) and penicillin (100 units ⁄ ml). Thecells were exposed to c-ray radiation at 1.5 Gy ⁄ min. from a 60Co c-ray source (MDS Nordion C-188 standard source; Jeju NationalUniversity, Jeju, Korea).

Cell viability. To assess the cytoprotective effect of morin againstc-radiation, cells at 1 · 104 cells ⁄ ml were treated with morin at25 lM and 1 hr later, exposed to c-ray at 5, 10, 15 and 20 Gy at adose rate of 1.5 Gy ⁄ min. And to assess the cytoprotective effect ofJNK inhibitor against c-radiation, cells were treated with JNK inhib-itor, SP600125, and 30 min. later, cells were treated with morin at25 lM and 1 hr later, exposed to c-ray at 10 Gy. After incubationfor 48 hr, 50 ll of the MTT stock solution (2 mg ⁄ ml) was added toeach well to reach a total reaction volume of 200 ll. After incubatingfor 4 hr, the plate was centrifuged at 800 · g for 5 min. followed byaspiration of the supernatants. Formazan crystals in each well weredissolved in 150 ll of dimethylsulfoxide and the A540 was read on ascanning multi-well spectrophotometer.

Intracellular reactive oxygen species measurement. Cells were pre-treated with morin at 25 lM and exposed to c-radiation 1 hr later orco-treated with morin and c-radiation simultaneously. Cells werethen incubated for an additional 24 hr at 37�C. After adding 25 lMof DCF-DA solution, fluorescent 2¢,7¢-dichlorofluorescein wasdetected using a Perkin Elmer LS-5B spectrofluorometer and a flowcytometer (Becton Dickinson, CA, USA) [21]. Image analysis forintracellular ROS generation was achieved by seeding the cells oncover slip-loaded six-well plate at 2 · 105 cells ⁄ well. At 16 hr afterplating, cells were treated with morin. After an hour, the plate wasirradiated at 10 Gy. At 24 hr later, 100 lM of DCF-DA was addedto each well and was incubated for an additional 30 min. at 37�C.The stained cells were mounted onto a microscope slide in mountingmedium (DAKO, Carpinteria, CA, USA). Microscopic images werecollected using Laser Scanning Microscope 5 PASCAL program(Carl Zeiss, Jena, Germany) on a confocal microscope.

Lipid peroxidation assay. Lipid peroxidation was assayed by a thio-barbituric acid reaction to determine the contents of thiobarbituricacid reactive substances (TBARS). Cells were washed with cold PBS,

scraped and homogenized in ice-cold 1.15% KCl. One hundredmicrolitres of the cell lysates was mixed with 0.2 ml of 8.1% SDS,1.5 ml of 20% acetic acid (adjusted to pH 3.5) and 1.5 ml of 0.8%thiobarbituric acid (TBA). Subsequently, distilled water was addedto the mixture to reach a final volume of 4 ml, followed by heatingto 95�C for 2 hr. After cooling the mixture to room temperature,5 ml of an n-butanol and pyridine mixture (15 : 1) was added toeach sample, followed by gentle shaking. After centrifuging the mix-ture at 1000 · g for 10 min., the supernatant fraction was isolatedand the absorbance was measured spectrophotometrically at 532 nm.Lipid peroxidation was also estimated using a fluorescent probe,diphenyl-1-pyrenylphosphine (DPPP) [22]. DPPP was added to eachwell and was incubated for an additional 15 min. in the dark. DPPPfluorescence images were analysed by the Zeiss Axiovert 200 invertedmicroscope at an excitation wavelength of 351 nm and an emissionwavelength of 380 nm.

Single-cell gel electrophoresis (Comet assay). A comet assay wasperformed to determine the degree of oxidative DNA damage [23].Cell suspension was mixed with 75 ll of 0.5% low-melting agarose(LMA) at 39�C, and spread on a fully frosted microscopic slide pre-coated with 200 ll of 1% normal melting agarose (NMA). Aftersolidification of agarose, the slide was covered with another 75 ll of0.5% LMA and then immersed in a lysis solution (2.5 M NaCl,100 mM Na–EDTA, 10 mM Tris, 1% Trion X-100, and 10%DMSO, pH 10) for 1 hr at 4�C. The slides were then placed in a gel-electrophoresis apparatus containing 300 mM NaOH and 10 mMNa-EDTA (pH 13) for 40 min. to allow for DNA unwinding andexpression of alkali labile damage. Next, an electrical field wasapplied (300 mA, 25 V) for 20 min. at 4�C to draw negativelycharged DNA toward an anode. After electrophoresis, the slides werewashed three times for 5 min. at 4�C in a neutralizing buffer (0.4 MTris, pH 7.5), followed by staining with 75 ll of propidium iodide(20 lg ⁄ ml). The slides were observed with a fluorescence microscopeand image analyser (Kinetic Imaging, Komet 5.5, UK). The percent-age of total fluorescence in the tail, and tail length of the 50 cells perslide were recorded.

Nuclear staining with Hoechst 33342. 1.5 ll of Hoechst 33342 (stock10 mg ⁄ ml), which is a DNA-specific fluorescent dye, was added toeach well and incubated for 10 min. at 37�C. Stained cells werevisualized under a fluorescent microscope, equipped with a Cool-SNAP-Pro colour digital camera to examine the degree of nuclearcondensation. The percentage of apoptotic cells was assessed bycounting three random fields in triplicate wells.

DNA fragmentation. Cellular DNA-fragmentation was assessed byanalysing cytoplasmic histone-associated DNA fragmentation, using

Fig. 1. Chemical structure of morin (2¢,3,4¢,5,7-pentahydroxyflavone).

Fig. 2. Radio-protective effect of morin on c-radiation. Cells weretreated with morin at 25 lM, and 1 hr later, c-radiation at indicateddoses was exposed to cells. Cell viability was determined by MTTassay. *Significantly different from radiation-exposed cells (p < 0.05).

64 RUI ZHANG ET AL.

� 2010 The AuthorsBasic & Clinical Pharmacology & Toxicology � 2010 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 108, 63–72

a kit from Roche Diagnostics (Hillsboro, OR, USA) according tothe manufacturer’s instructions.

Western blot analysis. Cells were then lysed on ice for 30 min. in100 ll of a lysis buffer [120 mM NaCl, 40 mM Tris (pH 8), 0.1%NP 40] and centrifuged at 13,000 · g for 15 min. The supernatantswere collected from the lysates and protein concentrations weredetermined. Aliquots of the lysates (40 lg of protein) were boiled for5 min. and electrophoresed in 10% SDS–polyacrylamide gel. Blots inthe gels were transferred onto nitrocellulose membranes (Bio-Rad,

Hercules, CA, USA) and subsequently incubated with anti-primaryantibodies. The membranes were further incubated with secondaryanti-immunoglobulin-G-horseradish peroxidase conjugates (Pierce,Rockford, IL, USA), followed by exposure to X-ray film. Proteinbands were detected using an enhanced chemiluminescence Westernblotting detection kit (Amersham, Buckinghamshire, UK).

Mitochondrial membrane potential (Dwm) analysis. Mitochondrialmembrane potential (Dwm) analysis was determined by flow cytome-ter. Cells were suspended in PBS containing JC-1 (10 lg ⁄ ml). After

A

B

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Fig. 3. Effect of morin on scavenging intracellular ROS generated by c-radiation. Cells were treated with morin at 25 lM, followed by c-rayirradiation at 10 Gy an hour later. After cells were incubated for 24 hr, the intracellular ROS was detected using (A) flow cytometer and (B)confocal microscope. FI indicates the fluorescence intensity of 2¢,7¢-dichlorofluorescein. Cells were treated with morin at 25 lM and exposed toc-ray irradiation simultaneously or an hour later. The intracellular ROS was detected with (C) a fluorescence spectrophotometer after DCF-DAstaining. *,#Significantly different from control cells (p < 0.05); §,Dsignificantly different from 10 Gy-treated cells both of pre-treatment and co-treatment groups, respectively (p < 0.05).

RADIO-PROTECTIVE EFFECTS OF MORIN 65


incubation for 15 min. at 37�C, cells were analysed by a flow cytome-ter. In addition, for image analysis for mitochondrial membranepotential, JC-1 was added to each well and incubated for 30 min. at37�C. The stained cells were mounted onto microscope slide inmounting medium (DAKO). Microscopic images were collectedusing the Laser Scanning Microscope 5 PASCAL program (Carl Ze-iss) on confocal microscope [24].

Transient transfection and AP-1 luciferase assay. Cells were tran-siently transfected with plasmid harbouring AP-1 promoter, usingDOTAP as the transfection reagent according to the manufacturer’sinstructions (Roche Diagnostics). After an overnight transfection,cells were treated with morin. After additional incubation for 1 hr,cells were irradiated. After 3 hr, cells were then washed twice withPBS and lysed with reporter lysis buffer (Promega, Madison, Wis-consin, USA). After vortex-mixing and centrifugation at 12,000 · gfor 1 min. at 4�C, the supernatant was stored at )70�C for the lucif-erase assay. After mixing 20 ll of cell extract with 100 ll of the lucif-erase assay reagent at room temperature, the mixture was placed inan illuminometer to measure the light produced.

Statistical analysis. All measurements were made in triplicate and allvalues were expressed as the mean € standard error (SE). The resultswere subjected to an analysis of variance (ANOVA) using the Tukey’stest to analyse the difference. A p-value of <0.05 were considered sta-tistically significant.

Results

Radioprotective effect of morin on c-radiation.In our previous study, we found that morin at 25 lM wasan optimal concentration for determining the protective

effect against oxidative stress-induced cellular damage [18].In this study, we used 25 lM as optimal concentration ofmorin. We measured the cell viability at 48 hr after expo-sure of various radiation doses by MTT assay. As shownin fig. 2, cell viability was 77% at 5 Gy, 61% at 10 Gy,54% at 15 Gy and 45% at 20 Gy in only irradiated cells;morin significantly prevented the decrease in cell deathinduced by radiation until 15 Gy exposure. From thesedata, we chose 10 Gy as the optimal radiation dose tostudy the effect of morin against c-radiation-induced oxida-tive stress.

Scavenging effect of morin on ROS generated by c-irradiation.Flow cytometric data showed that fluorescence intensity ofROS stained by DCF-DA dye was 191 value in morin-trea-ted irradiated cells compared with 320 value of fluorescenceintensity in irradiated cells (fig. 3A). Red fluorescence inten-sity of ROS detected using a confocal microscope wasenhanced in c-radiated cells; however, morin reduced red flu-orescence intensity in c-radiated cells (fig. 3B). Spectrofluo-rometric analysis revealed that pre-treatment or co-treatmentwith morin significantly decreased intracellular ROS levels inirradiated cells (fig. 3C), suggesting that the reduction ofintracellular ROS levels is not totally caused by decreasingthe initial levels of ROS by morin. Taken together, theseresults suggest that morin scavenges ROS generated by c-rayradiation.

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B

Fig. 4. Effect of morin on c-radiation-induced lipid membrane. Lipid peroxidation was detected (A) by measuring the amount of TBARS and(B) by observing confocal microscope after DPPP staining. *Significantly different from control cells (p < 0.05); #significantly different from10 Gy-treated cells (p < 0.05).

66 RUI ZHANG ET AL.


Effect of morin on lipid peroxidation and DNA damageinduced by c-radiation.Cells exposed to c-radiation showed an increase in lipid per-oxidation, which was substantiated by generation of TBARS.However, morin was found to prevent c-radiation-inducedlipid peroxidation (fig. 4A). The fluorescence intensity ofDPPP, a specific fluorescent probe of lipid peroxidation, wasenhanced in c-radiated cells, however, morin reduced the flu-orescence intensity of DPPP in c-radiated cells (fig. 4B). Inaddition, cellular DNA damage induced by c-radiation wasdetected by an alkaline comet assay. When cells were exposedto c-radiation, the percentage of DNA in the tail increasedto 26%; however, morin treatment resulted in a decrease to11% as shown in fig. 5A and B.

Effect of morin against apoptosis induced by c-radiation.To study the cytoprotective effect of morin in terms of apop-tosis, nuclei were stained with Hoechst 33342 for visualiza-tion by microscopy and quantitated. The microscopicpictures in fig. 6A demonstrated that the control cells hadintact chromatin (apoptotic cells 4%), whereas radiation-exposed cells demonstrated significant chromatin condensa-tion (apoptotic cells 32%), characteristic of apoptosis. How-ever, cells with morin exhibited dramatic decrease inchromatin condensation (apoptotic cells 11%). In addition tomorphological evaluation, it was also confirmed by ELISA,based on quantification of cytoplasmic histone-associated

DNA fragmentation. As shown in fig. 6B, irradiated cellsincreased the levels of cytoplasmic histone-associated DNAfragmentations compared with control cells. However, morintreatment significantly decreased the level of DNA fragmen-tation.

Effect of morin on the caspases-dependent pathway viamitochondria in apoptotic process induced by c-radiation.Bcl-2, an anti-apoptotic factor, is localized in the mitochon-drial inner membrane, and its levels regulated apoptosis;phosphorylated Bcl-2 fails to inhibit cell apoptosis [25]. Bax,a pro-apoptotic factor, is localized in cytosol as a monomer,but during apoptosis, it translocates to mitochondria, andreleases cytochrome c from mitochondria to cytosol, andinduces apoptosis [26]. Morin-treated cells showed anincrease in Bcl-2 expression, a decrease in phospho Bcl-2and Bax expressions in c-radiated cells (fig. 7A). During theapoptotic process, Bcl-2 prevented opening of the mitochon-drial membrane pore, whereas Bax induced opening of mem-brane pore [27], and pore opening induces the loss of Dwm.In our system, the irradiated cells resulted in loss of Dwm, assubstantiated by an increase in fluorescence (FL-1) with theJC-1 dye in flow cytometry analysis (fig. 7B). As shown infig. 7C, the control cells and only morin-treated cells exhib-ited strong red fluorescence (polarized state of mitochondrialDw). The c-radiation resulted in decreased red fluorescenceand increased green fluorescence (depolarized state);

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Fig. 5. Effect of morin on c-radiation-induced DNA damages. (A) Representative image and (B) percentage of cellular DNA damage weredetected by an alkaline comet assay. *Significantly different from control cells (p < 0.05); #significantly different from 10 Gy-treated cells(p < 0.05).



however, morin blocked the loss of Dwm after radiationexposure. Caspase 9 is activated as a result of mitochondrialmembrane disruption [28]. Morin inhibited the c-radiation-induced active form of caspase 9 (39 kDa), and caspase 3(17 kDa) (fig. 7A). These results suggest that morin protectscells from apoptosis by inhibiting the caspases-dependentpathway via mitochondria.

Effect of morin on SEK1-JNK-AP-1 signalling pathway.Because the JNK signal pathway plays an important role inc-radiation-induced apoptosis [29], we tested whether morin

regulates this signalling pathway. SEK1 is known to be oneof the upstream components in the JNK signalling pathway[30]. The c-radiated cells markedly increased SEK1 phos-phorylation (active form of SEK1) levels (fig. 8A). However,morin effectively inhibited c-radiation-induced SEK1 phos-phorylation. Furthermore, morin remarkably inhibited JNKactivation (phosphorylated JNK) induced by c-radiation(fig. 8A). In addition, treatment with SP600125, a JNK-spe-cific inhibitor, increased the radio-protective effect of morin(fig. 8B). AP-1, a transcription factor, is a downstream targetof the phospho JNK pathway, and activated AP-1 is

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Fig. 6. Effect of morin on c-radiation-induced apoptosis. (A) Apoptotic body formation was observed under a fluorescence microscope andquantitated after Hoechst 33342 staining and apoptotic bodies are indicated by arrows. *Significantly different from control cells (p < 0.05);#significantly different from 10 Gy-treated cells (p < 0.05). (B) DNA fragmentation was quantified by ELISA kit. *Significantly different fromcontrol cells (p < 0.05) and #significantly different from 10 Gy-treated cells (p < 0.05).

68 RUI ZHANG ET AL.


B

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A

Fig. 7. Effects of morin on apoptosis regulatory proteins and mitochondrial function. (A) Cell lysates were electrophoresed and Bcl-2, Bax,phospho-Bcl-2, active caspase 9, and caspase 3 proteins were detected by their specific antibodies. The mitochondrial membrane potential(Dwm) was analysed with (B) flow cytometer and (C) confocal microscope after staining of JC-1.



involved in cell death including apoptosis [31]. The transcrip-tional activity of AP-1 was assessed using a promoter con-struct containing AP-1 binding DNA consensus sequences,which are linked to a luciferase reporter gene. As illustratedin fig. 8C, morin inhibited the transcriptional activity of AP-1 induced by c-irradiation. These results suggest that morininhibits c-radiation-induced apoptosis through suppressionof the SEK1-JNK-AP-1 pathway.

Discussion

Exposure of cells to ionizing radiation can lead to increasedROS generation including hydrogen peroxides (H2O2),hydroxyl radials (OH) and superoxide anions (O2

)). Thedamaging effects of free radicals by ionizing radiation areassociated with an increased risk of various diseases [2]. As itis important to protect human beings from the detrimentaleffects of ionizing radiation, the screening of the compoundswith the ability to scavenge these ROS generated by ionizingradiation is promising and can lead to the development ofradio protectors [32]. We have previously shown that morin-protected cells against H2O2-induced cell damage via activa-tion of cellular antioxidant [18]. However, the precise mecha-nism of morin to protect the cellular damage induced byoxidative stress is less obvious. In the present study, weexamined whether morin can protect cells against c-radia-tion-induced cell damage and the mechanisms involved.Morin is a member of the flavonoid family with a polyphe-nol structure. The existence of a phenolic group with an aro-matic conjugation in the structure of morin contributes tothe reduction of ROS generated by irradiation. Radiation-induced ROS attack vital cellular sites, such as cellmembranes and DNA, which often result in lethal cellulardamage. The formation of lipid peroxidation in cells exposedto c-radiation is an important marker of cell membranedamage. Thus, inhibition of lipid peroxidation is a key targetin developing successful radio-protection strategies [33].

Morin was found to protect cell membrane lipids fromperoxidation damage induced by radiation. In addition,DNA damage is the main event in irradiated cells, inducingapoptosis as a nuclear mediator. Morin was found to inhibitDNA tail length induced by c-radiation, indicating protec-tion of cellular DNA by morin treatment. These inhibitoryeffects of morin against lipid and DNA damage resulted inprotective effects against radiation-induced cell death. Inmany cases, c-radiation- induced cell death has resulted inapoptosis [3,34]. Morin inhibited the c-radiation-inducedcaspase-dependent apoptotic biochemical changes. The mito-chondria act as an important apparatus for signals duringapoptosis, and the loss of mitochondrial integrity can beprompted or inhibited by many regulators of apoptosis[25,35]. Morin inhibited c-radiation-induced loss of mito-chondrial Dw. During the apoptotic process, Bcl-2 preventsthe opening of mitochondrial membrane pores, whereas Baxinduces the opening of membrane pores [27]. Therefore,blocked loss of Dwm by morin may be from the result of Bcl-2 up-regulation and Bax down-regulation. It has beenreported that JNK translocates to mitochondria and thenphosphorylates Bcl-2 and Bcl-XL, anti-apoptotic membersof the Bcl-2 family, and presumably inactivate them [36]. Inaddition, JNK was found to induce mitochondrial pathwayof apoptosis by activating Bim and Bax, proapoptotic mem-bers of the Bcl-2 family [37]. The JNK cascade is one of thesignalling pathways that mediate c-irradiation-induced apop-tosis. The disruption of the JNK pathway by a dominantnegative mutant abrogated radiation-induced apoptosis [38].

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Fig. 8. Effects of morin on c-radiation-induced SEK1-JNK-AP-1activation. (A) Cell lysates were electrophoresed and the cell lysateswere immunoblotted using anti-phospho SEK1, -SEK1, -JNK and -phospho JNK antibodies. (B) After treatment with SP600125, morinand radiation, cell viability was assessed by MTT assay. *Signifi-cantly different from control cells (p < 0.05); #significantly differentfrom 10 Gy-treated cells (p < 0.05); §significantly different frommorin plus 10 Gy-treated cells (p < 0.05). (C) The transcriptionalactivity of AP-1 was assessed using the plasmid containing the AP-1-binding site-luciferase construct. *Significantly different from controlcells (p < 0.05); #significantly different from 10 Gy-treated cells(p < 0.05).

70 RUI ZHANG ET AL.


Therefore, activation of this pathway appeared to be essen-tial in transducing apoptosis signals. Morin blocked c-radia-tion-induced activation of the SEK1-JNK-AP-1 signallingpathway, resulted in protection from c-radiation-inducedapoptosis. It is known that the survival time for irradiatedanimals can be lengthened by various manipulations, such asinhibition of free radical generation or acceleration of theremoval of free radicals, enhancement of DNA repair,replenishment of dead hematopoietic cells and stimulation ofimmune cell formation or activity [39]. Thus, the eliminationof the free-radical species from the cell environment can inhi-bit the side effects induced by ionizing radiation. The poten-tial applications of radio-protective compounds include theiruse in the event of a radiation accident or incident as well asin radiation therapy of cancer patients to protect normalcells. As we have manifested that morin exhibited radio-pro-tection activity in vitro and our further study will be focusedon exploring our findings by performing in vivo experiments.

In conclusion, morin exerted the cytoprotective effectagainst c-radiation-induced cell death through scavenging ofROS, inhibition of the JNK pathway, and inhibition of mito-chondria-involved caspase-dependent apoptosis.

AcknowledgementsThis research was performed under the programme of

Basic Atomic Energy Research Institute (BAERI), which ispart of the Nuclear R&D Programmes funded by the Minis-try of Science & Technology of Korea (KOSEF).

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Morin (2′,3,4′,5,7-Pentahydroxyflavone) Protected Cells against γ-Radiation-Induced Oxidative Stress