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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Abstract:  Ionizing radiation can induce cell damage by generating reactive oxygen species (ROS). The present study was carried out to investigate the radio-protective effects of a flavonoid compound, morin (2′,3,4′,5,7-pentahydroxyflavone) and the underlying mechanisms. Morin was found to reduce the intracellular ROS generated by γ-irradiation. Moreover, morin protected cellular components against radiation-induced membrane lipid peroxidation and cellular DNA damage, which are the main 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 by γ-radiation. Irradiated cells with morin recovered the expression of Bcl-2 reduced by γ-radiation. Morin exerted anti-apoptotic effects via inhibition of mitogen-activated protein kinase kinase-4 (MKK4/SEK1)-c-Jun NH2-terminal kinase (JNK)-activator protein 1 (AP-1) cascades induced by γ-radiation. The results suggest that morin protects cells against oxidative stress induced by radiation via reduction of ROS and attenuation of SEK1-JNK-AP-1 pathway.

Gamma-ray radiation is known to induce oxidative stress by generating reactive oxygen species (ROS), including superoxide anion, hydroxyl radical, single oxygen and hydrogen peroxide in cells [1]. These ROS can lead to functional damage in lipid, proteins and DNA, which in turn can eventually result 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 treatment for various malignant diseases. However, the amounts of ionizing radiation that can be given to treat malignant tumours are often limited by toxicity in the surrounding normal tissues and organs [5,6]. Recently, synthetic agents such as WR2721 (amifostine), OK-432, and ethiofos were investigated for their efficacy in protecting cells against radiation-induced damage [5]. However, these agents have the potential to cause serious side effects, including decreased cellular function, nausea, hypotension and death [6]. Alternatively, natural plant extracts that can protect cells and tissues against ionizing radiation without overt side effects could be developed as adjuncts to radiotherapy. In fact, natural compounds with disparate structures were isolated from different natural plant species. Some of these compounds have shown to be toxic but other compounds apparently are devoid of adverse effects. Kähkönen et al. [7] have reported that natural plant extracts containing phenolic compounds exhibited antioxidant activities. To date, several phenolic compounds have been shown to protect cells against radiation-induced damage by virtue of their antioxidant properties [8,9].

Flavonoids are a family of phenol compound most commonly found in a variety of fruits, vegetables, juices and components of herbal-containing dietary supplements. The interests in the investigation of flavonoids stem from their biological properties, which include oxygen radical scavenging and antioxidant properties [10,11]. Morin (2′,3,4′,5,7-pentahydroxyflavone) is a member of the flavonoid family which consists of a yellowish pigment found in almond (Prunus dulcis), fig (Chlorophora tinctoria) and other moraceae used in food and herbal medicine [12]. Moreover, morin has been reported to possess a variety of biological properties against oxidative stress-induced damage. It protects cardiovascular cells, glomerular mesangial cells, hepatocytes, oligodendrocytes and neurons against damage by oxidative stress [13–16]. Parihar et al. [17] have reported that morin exhibited anti-clastogenic activity against γ-radiation in vivo system. Recently, we have reported morin’s cellular protective effect on hydrogen peroxide-induced oxidative stress [18]. Because irradiated cells generate ROS, we speculated that morin, with its ROS scavenging effect, may provide cytoprotective effects against γ-radiation-induced cell damage. Consequently, we elected to investigate the effects of morin on cell damage induced by γ-radiation, and the possible mechanism underlying this cytoprotective effect.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

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

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

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Cell culture and irradiation.  It has been reported that the lung is a radiosensitive organ [19,20]. To study the effect of morin on γ-radiation-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 a humidified atmosphere of 5% CO2, and cultured in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated foetal calf serum, streptomycin (100 μg/ml) and penicillin (100 units/ml). The cells were exposed to γ-ray radiation at 1.5 Gy/min. from a 60Co γ-ray source (MDS Nordion C-188 standard source; Jeju National University, Jeju, Korea).

Cell viability.  To assess the cytoprotective effect of morin against γ-radiation, cells at 1 × 10cells/ml were treated with morin at 25 μM and 1 hr later, exposed to γ-ray at 5, 10, 15 and 20 Gy at a dose rate of 1.5 Gy/min. And to assess the cytoprotective effect of JNK inhibitor against γ-radiation, cells were treated with JNK inhibitor, SP600125, and 30 min. later, cells were treated with morin at 25 μM and 1 hr later, exposed to γ-ray at 10 Gy. After incubation for 48 hr, 50 μl of the MTT stock solution (2 mg/ml) was added to each well to reach a total reaction volume of 200 μl. After incubating for 4 hr, the plate was centrifuged at 800 × g for 5 min. followed by aspiration of the supernatants. Formazan crystals in each well were dissolved in 150 μl of dimethylsulfoxide and the A540 was read on a scanning multi-well spectrophotometer.

Intracellular reactive oxygen species measurement.  Cells were pre-treated with morin at 25 μM and exposed to γ-radiation 1 hr later or co-treated with morin and γ-radiation simultaneously. Cells were then incubated for an additional 24 hr at 37°C. After adding 25 μM of DCF-DA solution, fluorescent 2′,7′-dichlorofluorescein was detected using a Perkin Elmer LS-5B spectrofluorometer and a flow cytometer (Becton Dickinson, CA, USA) [21]. Image analysis for intracellular ROS generation was achieved by seeding the cells on cover slip-loaded six-well plate at 2 × 10cells/well. At 16 hr after plating, cells were treated with morin. After an hour, the plate was irradiated at 10 Gy. At 24 hr later, 100 μM of DCF-DA was added to each well and was incubated for an additional 30 min. at 37°C. The stained cells were mounted onto a microscope slide in mounting medium (DAKO, Carpinteria, CA, USA). Microscopic images were collected using Laser Scanning Microscope 5 PASCAL program (Carl Zeiss, Jena, Germany) on a confocal microscope.

Lipid peroxidation assay.  Lipid peroxidation was assayed by a thiobarbituric acid reaction to determine the contents of thiobarbituric acid reactive substances (TBARS). Cells were washed with cold PBS, scraped and homogenized in ice-cold 1.15% KCl. One hundred microlitres 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 added to the mixture to reach a final volume of 4 ml, followed by heating to 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 to each sample, followed by gentle shaking. After centrifuging the mixture at 1000 × g for 10 min., the supernatant fraction was isolated and 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 each well and was incubated for an additional 15 min. in the dark. DPPP fluorescence images were analysed by the Zeiss Axiovert 200 inverted microscope at an excitation wavelength of 351 nm and an emission wavelength of 380 nm.

Single-cell gel electrophoresis (Comet assay).  A comet assay was performed to determine the degree of oxidative DNA damage [23]. Cell suspension was mixed with 75 μl of 0.5% low-melting agarose (LMA) at 39°C, and spread on a fully frosted microscopic slide pre-coated with 200 μl of 1% normal melting agarose (NMA). After solidification of agarose, the slide was covered with another 75 μl of 0.5% 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 mM Na-EDTA (pH 13) for 40 min. to allow for DNA unwinding and expression of alkali labile damage. Next, an electrical field was applied (300 mA, 25 V) for 20 min. at 4°C to draw negatively charged DNA toward an anode. After electrophoresis, the slides were washed three times for 5 min. at 4°C in a neutralizing buffer (0.4 M Tris, pH 7.5), followed by staining with 75 μl of propidium iodide (20 μg/ml). The slides were observed with a fluorescence microscope and image analyser (Kinetic Imaging, Komet 5.5, UK). The percentage of total fluorescence in the tail, and tail length of the 50 cells per slide were recorded.

Nuclear staining with Hoechst 33342.  1.5 μl of Hoechst 33342 (stock 10 mg/ml), which is a DNA-specific fluorescent dye, was added to each well and incubated for 10 min. at 37°C. Stained cells were visualized under a fluorescent microscope, equipped with a CoolSNAP-Pro colour digital camera to examine the degree of nuclear condensation. The percentage of apoptotic cells was assessed by counting three random fields in triplicate wells.

DNA fragmentation.  Cellular DNA-fragmentation was assessed by analysing cytoplasmic histone-associated DNA fragmentation, using a kit from Roche Diagnostics (Hillsboro, OR, USA) according to the manufacturer’s instructions.

Western blot analysis.  Cells were then lysed on ice for 30 min. in 100 μl 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 supernatants were collected from the lysates and protein concentrations were determined. Aliquots of the lysates (40 μg of protein) were boiled for 5 min. and electrophoresed in 10% SDS–polyacrylamide gel. Blots in the gels were transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) and subsequently incubated with anti-primary antibodies. The membranes were further incubated with secondary anti-immunoglobulin-G-horseradish peroxidase conjugates (Pierce, Rockford, IL, USA), followed by exposure to X-ray film. Protein bands were detected using an enhanced chemiluminescence Western blotting detection kit (Amersham, Buckinghamshire, UK).

Mitochondrial membrane potential (Δψm) analysis.  Mitochondrial membrane potential (Δψm) analysis was determined by flow cytometer. Cells were suspended in PBS containing JC-1 (10 μg/ml). After incubation for 15 min. at 37°C, cells were analysed by a flow cytometer. In addition, for image analysis for mitochondrial membrane potential, JC-1 was added to each well and incubated for 30 min. at 37°C. The stained cells were mounted onto microscope slide in mounting medium (DAKO). Microscopic images were collected using the Laser Scanning Microscope 5 PASCAL program (Carl Zeiss) on confocal microscope [24].

Transient transfection and AP-1 luciferase assay.  Cells were transiently transfected with plasmid harbouring AP-1 promoter, using DOTAP as the transfection reagent according to the manufacturer’s instructions (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 with PBS and lysed with reporter lysis buffer (Promega, Madison, Wisconsin, USA). After vortex-mixing and centrifugation at 12,000 × g for 1 min. at 4°C, the supernatant was stored at −70°C for the luciferase assay. After mixing 20 μl of cell extract with 100 μl of the luciferase assay reagent at room temperature, the mixture was placed in an illuminometer to measure the light produced.

Statistical analysis.  All measurements were made in triplicate and all values were expressed as the mean ± standard error (SE). The results were subjected to an analysis of variance (anova) using the Tukey’s test to analyse the difference. A p-value of <0.05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Radioprotective effect of morin on γ-radiation.

In our previous study, we found that morin at 25 μM was an optimal concentration for determining the protective effect against oxidative stress-induced cellular damage [18]. In this study, we used 25 μM as optimal concentration of morin. We measured the cell viability at 48 hr after exposure of various radiation doses by MTT assay. As shown in 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 death induced by radiation until 15 Gy exposure. From these data, we chose 10 Gy as the optimal radiation dose to study the effect of morin against γ-radiation-induced oxidative stress.

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Figure 2.  Radio-protective effect of morin on γ-radiation. Cells were treated with morin at 25 μM, and 1 hr later, γ-radiation at indicated doses was exposed to cells. Cell viability was determined by MTT assay. *Significantly different from radiation-exposed cells (p < 0.05).

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Scavenging effect of morin on ROS generated by γ-irradiation.

Flow cytometric data showed that fluorescence intensity of ROS stained by DCF-DA dye was 191 value in morin-treated irradiated cells compared with 320 value of fluorescence intensity in irradiated cells (fig. 3A). Red fluorescence intensity of ROS detected using a confocal microscope was enhanced in γ-radiated cells; however, morin reduced red fluorescence intensity in γ-radiated cells (fig. 3B). Spectrofluorometric analysis revealed that pre-treatment or co-treatment with morin significantly decreased intracellular ROS levels in irradiated cells (fig. 3C), suggesting that the reduction of intracellular ROS levels is not totally caused by decreasing the initial levels of ROS by morin. Taken together, these results suggest that morin scavenges ROS generated by γ-ray radiation.

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Figure 3.  Effect of morin on scavenging intracellular ROS generated by γ-radiation. Cells were treated with morin at 25 μM, followed by γ-ray irradiation 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 μM and exposed to γ-ray irradiation simultaneously or an hour later. The intracellular ROS was detected with (C) a fluorescence spectrophotometer after DCF-DA staining. *,#Significantly different from control cells (p < 0.05); §,Δsignificantly different from 10 Gy-treated cells both of pre-treatment and co-treatment groups, respectively (p < 0.05).

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Effect of morin on lipid peroxidation and DNA damage induced by γ-radiation.

Cells exposed to γ-radiation showed an increase in lipid peroxidation, which was substantiated by generation of TBARS. However, morin was found to prevent γ-radiation-induced lipid peroxidation (fig. 4A). The fluorescence intensity of DPPP, a specific fluorescent probe of lipid peroxidation, was enhanced in γ-radiated cells, however, morin reduced the fluorescence intensity of DPPP in γ-radiated cells (fig. 4B). In addition, cellular DNA damage induced by γ-radiation was detected by an alkaline comet assay. When cells were exposed to γ-radiation, the percentage of DNA in the tail increased to 26%; however, morin treatment resulted in a decrease to 11% as shown in fig. 5A and B.

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Figure 4.  Effect of morin on γ-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 from 10 Gy-treated cells (p < 0.05).

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

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Effect of morin against apoptosis induced by γ-radiation.

To study the cytoprotective effect of morin in terms of apoptosis, nuclei were stained with Hoechst 33342 for visualization by microscopy and quantitated. The microscopic pictures in fig. 6A demonstrated that the control cells had intact chromatin (apoptotic cells 4%), whereas radiation-exposed cells demonstrated significant chromatin condensation (apoptotic cells 32%), characteristic of apoptosis. However, cells with morin exhibited dramatic decrease in chromatin condensation (apoptotic cells 11%). In addition to morphological evaluation, it was also confirmed by ELISA, based on quantification of cytoplasmic histone-associated DNA fragmentation. As shown in fig. 6B, irradiated cells increased the levels of cytoplasmic histone-associated DNA fragmentations compared with control cells. However, morin treatment significantly decreased the level of DNA fragmentation.

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Figure 6.  Effect of morin on γ-radiation-induced apoptosis. (A) Apoptotic body formation was observed under a fluorescence microscope and quantitated 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 from control cells (p < 0.05) and #significantly different from 10 Gy-treated cells (p < 0.05).

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Effect of morin on the caspases-dependent pathway via mitochondria in apoptotic process induced by γ-radiation.

Bcl-2, an anti-apoptotic factor, is localized in the mitochondrial 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, and releases cytochrome c from mitochondria to cytosol, and induces apoptosis [26]. Morin-treated cells showed an increase in Bcl-2 expression, a decrease in phospho Bcl-2 and Bax expressions in γ-radiated cells (fig. 7A). During the apoptotic process, Bcl-2 prevented opening of the mitochondrial membrane pore, whereas Bax induced opening of membrane pore [27], and pore opening induces the loss of Δψm. In our system, the irradiated cells resulted in loss of Δψm, as substantiated by an increase in fluorescence (FL-1) with the JC-1 dye in flow cytometry analysis (fig. 7B). As shown in fig. 7C, the control cells and only morin-treated cells exhibited strong red fluorescence (polarized state of mitochondrial Δψ). The γ-radiation resulted in decreased red fluorescence and increased green fluorescence (depolarized state); however, morin blocked the loss of Δψm after radiation exposure. Caspase 9 is activated as a result of mitochondrial membrane disruption [28]. Morin inhibited the γ-radiation-induced active form of caspase 9 (39 kDa), and caspase 3 (17 kDa) (fig. 7A). These results suggest that morin protects cells from apoptosis by inhibiting the caspases-dependent pathway via mitochondria.

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Figure 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 (Δψm) was analysed with (B) flow cytometer and (C) confocal microscope after staining of JC-1.

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Effect of morin on SEK1-JNK-AP-1 signalling pathway.

Because the JNK signal pathway plays an important role in γ-radiation-induced apoptosis [29], we tested whether morin regulates this signalling pathway. SEK1 is known to be one of the upstream components in the JNK signalling pathway [30]. The γ-radiated cells markedly increased SEK1 phosphorylation (active form of SEK1) levels (fig. 8A). However, morin effectively inhibited γ-radiation-induced SEK1 phosphorylation. Furthermore, morin remarkably inhibited JNK activation (phosphorylated JNK) induced by γ-radiation (fig. 8A). In addition, treatment with SP600125, a JNK-specific inhibitor, increased the radio-protective effect of morin (fig. 8B). AP-1, a transcription factor, is a downstream target of the phospho JNK pathway, and activated AP-1 is involved in cell death including apoptosis [31]. The transcriptional activity of AP-1 was assessed using a promoter construct containing AP-1 binding DNA consensus sequences, which are linked to a luciferase reporter gene. As illustrated in fig. 8C, morin inhibited the transcriptional activity of AP-1 induced by γ-irradiation. These results suggest that morin inhibits γ-radiation-induced apoptosis through suppression of the SEK1-JNK-AP-1 pathway.

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

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Exposure of cells to ionizing radiation can lead to increased ROS generation including hydrogen peroxides (H2O2), hydroxyl radials (OH) and superoxide anions (O2). The damaging effects of free radicals by ionizing radiation are associated with an increased risk of various diseases [2]. As it is important to protect human beings from the detrimental effects of ionizing radiation, the screening of the compounds with the ability to scavenge these ROS generated by ionizing radiation is promising and can lead to the development of radio protectors [32]. We have previously shown that morin-protected cells against H2O2-induced cell damage via activation of cellular antioxidant [18]. However, the precise mechanism of morin to protect the cellular damage induced by oxidative stress is less obvious. In the present study, we examined whether morin can protect cells against γ-radiation-induced cell damage and the mechanisms involved. Morin is a member of the flavonoid family with a polyphenol structure. The existence of a phenolic group with an aromatic conjugation in the structure of morin contributes to the reduction of ROS generated by irradiation. Radiation-induced ROS attack vital cellular sites, such as cell membranes and DNA, which often result in lethal cellular damage. The formation of lipid peroxidation in cells exposed to γ-radiation is an important marker of cell membrane damage. Thus, inhibition of lipid peroxidation is a key target in developing successful radio-protection strategies [33].

Morin was found to protect cell membrane lipids from peroxidation damage induced by radiation. In addition, DNA damage is the main event in irradiated cells, inducing apoptosis as a nuclear mediator. Morin was found to inhibit DNA tail length induced by γ-radiation, indicating protection of cellular DNA by morin treatment. These inhibitory effects of morin against lipid and DNA damage resulted in protective effects against radiation-induced cell death. In many cases, γ-radiation- induced cell death has resulted in apoptosis [3,34]. Morin inhibited the γ-radiation-induced caspase-dependent apoptotic biochemical changes. The mitochondria act as an important apparatus for signals during apoptosis, and the loss of mitochondrial integrity can be prompted or inhibited by many regulators of apoptosis [25,35]. Morin inhibited γ-radiation-induced loss of mitochondrial Δψ. During the apoptotic process, Bcl-2 prevents the opening of mitochondrial membrane pores, whereas Bax induces the opening of membrane pores [27]. Therefore, blocked loss of Δψm by morin may be from the result of Bcl-2 up-regulation and Bax down-regulation. It has been reported that JNK translocates to mitochondria and then phosphorylates Bcl-2 and Bcl-XL, anti-apoptotic members of the Bcl-2 family, and presumably inactivate them [36]. In addition, JNK was found to induce mitochondrial pathway of apoptosis by activating Bim and Bax, proapoptotic members of the Bcl-2 family [37]. The JNK cascade is one of the signalling pathways that mediate γ-irradiation-induced apoptosis. The disruption of the JNK pathway by a dominant negative mutant abrogated radiation-induced apoptosis [38]. Therefore, activation of this pathway appeared to be essential in transducing apoptosis signals. Morin blocked γ-radiation-induced activation of the SEK1-JNK-AP-1 signalling pathway, resulted in protection from γ-radiation-induced apoptosis. It is known that the survival time for irradiated animals can be lengthened by various manipulations, such as inhibition of free radical generation or acceleration of the removal of free radicals, enhancement of DNA repair, replenishment of dead hematopoietic cells and stimulation of immune cell formation or activity [39]. Thus, the elimination of the free-radical species from the cell environment can inhibit the side effects induced by ionizing radiation. The potential applications of radio-protective compounds include their use in the event of a radiation accident or incident as well as in radiation therapy of cancer patients to protect normal cells. As we have manifested that morin exhibited radio-protection activity in vitro and our further study will be focused on exploring our findings by performing in vivo experiments.

In conclusion, morin exerted the cytoprotective effect against γ-radiation-induced cell death through scavenging of ROS, inhibition of the JNK pathway, and inhibition of mitochondria-involved caspase-dependent apoptosis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This research was performed under the programme of Basic Atomic Energy Research Institute (BAERI), which is part of the Nuclear R&D Programmes funded by the Ministry of Science & Technology of Korea (KOSEF).

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References