Radiation-induced DNA Double Strand Breaks and Their Modulations
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by Herbal Treatments: A Cancer Cell Culture Model
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Abstract: Gamma radiation brings deleterious effects upon human cells by inducing oxidative stress and 7
damages. Antioxidants have been shown to confer protective effects on irradiated normal cells. Moringa 8
oleifera Lam. is a widely used nutritional supplement with antioxidant activities. This report showed that 9
antioxidant-containing supplements, in addition to protecting normal cells, could protect cancer cells 10
against genotoxic effects of gamma radiation. -H2AX immunofluorescent foci were utilized as an indicator 11
of radiation-induced DNA double strand breaks. MCF-7 human breast adenocarcinoma cells were 12
irradiated with 2-8 Gy gamma radiation. A linear relationship between the formation of -H2AX foci and 13
radiation dose was observed with an average of 10 foci per cell per Gy. A 30-minute pretreatment of the 14
cells with either the aqueous or the ethanolic extract of M. oleifera leaves could partially protect the cells 15
from radiation-induced DNA double strand breaks. A pretreatment with 500 µg/mL aqueous extract reduced 16
the number of foci formed by 15% when assayed at 30 minutes post-irradiation. The ethanolic extract was 17
more effective; 500 µg/mL of its concentration reduced the number of foci among irradiated cells by 30%.
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Our results indicated that irradiated cancer cells responded similarly to nutritional supplements containing 19
antioxidants as irradiated normal cells. These natural antioxidants could confer protective effects upon 20
cancer cells against gamma radiation.
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Keywords: gamma radiation, MCF-7, DNA double strand breaks, -H2AX, antioxidant, Moringa oleifera 22
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Introduction 24
25
Radiation therapy has long been accepted as a conventional treatment of malignant tumors. Gamma 26
radiation is a low linear-energy-transfer radiation often used in radiation therapy. It principally acts via ionization of 27
cellular water molecules, generating DNA-damaging free radicals, especially superoxide and hydroxyl radicals, 28
resulting in oxidative DNA damage and double strand breaks, eventually leading to apoptosis (Meyn et al., 2009;
29
Kobayashi et al., 2008; Weterings & Chen., 2008). Typical therapeutic doses may range from 20 to 80 Gy, which are 30
given in fractions of 1.8 – 3 Gy each, depending on the type of cancer.
31
An early event in ionizing radiation induced DNA damage response is the rapid phosphorylation of histone 32
H2AX to become -H2AX, which often serves as an indicator of the presence of DNA double strand breaks (Costes 33
et al., 2007). Discernable foci of DNA damage response components can be observed, which colocalize with 34
phosphorylated H2AX (Paull et al., 2000). -H2AX foci are induced vary rapidly within a few minutes upon 35
irradiation; the number of foci reaches its maximum by 30 minutes to an hour. Then, the level of -H2AX declines 36
gradually over time together with the reduction in the number of its foci. This decline has been shown to correlate 37
with the activity of DNA damage repair components which are recruited to the double strand break sites. By 24 38
hours, -H2AX level will be reduced to almost its background level.
39
The observed damages brought about by gamma and other ionizing radiation are mainly caused by radiation- 40
induced reactive oxygen species and radicals within and outside the cells. Many antioxidants have been shown to 41
counter the effects of ionizing radiation including gamma rays. Antioxidant thiols confer protective effects on normal 42
human lymphocytes against gamma radiation (Tiwari et al., 2009). N-acetylcysteine, ascorbic acid, sodium ascorbate, 43
co-enzyme Q10, α-lipoic acid, L-selenomethionine, and vitamin E succinate and their combinations have been shown 44
to protect MCF10A normal human breast epithelial cells against many types of ionizing radiation including gamma 45
rays (Wan et al., 2006).
46
Due to this protective effect on normal cells, some patients with solid cancers requiring radiation treatments 47
opted to take nutritional supplements with antioxidant activities for protection purposes. However, within cancer 48
cells, radiation-induced oxidative stress is required to induce DNA strand breaks and eventual death. Therefore, 49
consumptions of nutritional supplements or herbal products with antioxidant activity might hamper the action of 50
ionizing radiation against cancer cells, especially if these cells respond similarly to the treatments of antioxidants as 51
normal cells do. To address this question, we set up an experimental system using MCF-7 human breast 52
adenocarcinoma cells as a cancer cell model and extracts of Moringa oleifera Lam. as antioxidants-containing 53
nutritional supplements. Leaf extracts of M. oleifera had been shown to contain antioxidant phenolics including 54
gallic acid, chlorogenic acid, ferulic acid, caffeic acid, and egallic acid (Verma et al., 2009). We asked whether the 55
-H2AX foci were utilized to monitor the extent of radiation-induced DNA damage.
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Materials and Methods 59
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Cell Culture 61
MCF7 human breast adenocarcinoma cells (American Type Culture Collection, USA) were cultured in 62
MEM medium (Invitrogen, USA) supplemented with 110 mg/L sodium pyruvate, non-essential amino acids, 10%
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fetal bovine serum, 100 µg/L streptomycin and 100 U/L penicillin in a 5% CO2 incubator at 37oC (Binder, Bohemia, 64
NY, USA). The culture medium was replaced every 3 days. Cells were removed by treatment with 0.25% trypsin 65
and 0.1% EDTA.
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Preparation of Moringa oleifera Lam. Leaf Extracts 68
Powdered dried leaves of Moringa oleifera Lam. were obtained from a commercial source. The aqueous 69
extract was prepared 1:10 (w/v) in boiling distilled water for 10 min and left to cool down at room temperature for 1 70
h, filtered and lyophilized in a ScanVac freeze dryer (Labogene, Denmark). The ethanolic extract was prepared 1:10 71
(w/v) in 95% ethanol by shaking at room temperature for 24 h, filtered and evaporated in a Hei-Vac rotary evaporator 72
(Heidolph Instruments, Germany). The extracts were both stored at -20°C until use.
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M. oleifera leaf Extract Treatments 75
MCF-7 cells were seeded in 60 mm culture dishes. After 1 - 2 days of growth, the culture was replenished 76
with fresh medium. To test the effects of MO leaf extracts on MCF-7 cells, the aqueous and the ethanolic extracts of 77
MO were dissolved in phosphate buffered saline (PBS) and dimethyl sulfoxide (DMSO), respectively. They were 78
filter-sterile and added directly to the culture medium at the concentration of 500 µg/mL for 30 minutes before 79
subsequent irradiation. The concentration of DMSO was kept at 1% or less in the treated medium.
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Radiation Treatments 82
Cells were seeded and grown for two days to sub-confluence. On the day of radiation treatment, the growth 83
medium was replaced with a fresh batch prior to and after irradiation. Cells were irradiated with gamma radiation 84
from a 60Co source in the culture medium at 0.50 – 0.70 Gy/min (Gems Irradiation Center, Thailand Institute of 85
Nuclear Technology). Radiation absorbed doses were monitored using optically-stimulated luminescence dosimeters 86
provided by Nuclear Technology Service Center, Thailand Institute of Nuclear Technology.
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Immunofluorescence staining 89
Treated cells were fixed 30 min post-irradiation with cold 3.7% formaldehyde in phosphate-buffered saline 90
for 10 min, blocked with 3% bovine serum albumin and 0.1% Triton X-100, immunostained with 2µg/mL mouse anti- 91
phospho-histone H2A.X (Ser139) (clone JBW301) (EMD Millipore, Billerica, MA, USA) for 1 h at room 92
temperature, blocked and incubated with Alexa 488 goat anti-mouse IgG1(1) secondary antibodies (Life 93
Technologies Corporation, Carlsbad, CA, USA) at 1:500 dilution, and counterstained with 60 µg/mL bisbenzimide.
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The samples were washed at least three times with Tris-buffered saline between each step. For microscopic 95
observation, they were mounted in ProLong Gold (Life Technologies Corporation, USA). Gamma H2AX foci were 96
observed with fluorescence imaging using an Olympus IX71 inverted microscope (Olympus, USA). Images were 97
captured with Olympus DP70 camera (Olympus, USA).
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Results 100
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Effects of Gamma Radiation 102
Cultured MCF-7 cells responded to gamma radiation similarly to other cell types. Upon irradiation with 2 – 103
8 Gy gamma rays, certain morphological changes could be observed at 30 minutes post-irradiation. Enlargement of 104
cell and nuclear volume was specifically noted, which became more pronounced at higher radiation dose (Figure 1A – 105
1D). Cells also appeared more rounded with increasing radiation dose. These observations were similar to what had 106
been previously reported on irradiated MCF-7 and normal breast epithelial cells (Somosy, 2003; Botlagunta et al., 107
2010). Alteration in plasma membrane morphology and reorganization of actin filament close to the plasma 108
membrane had also been reported.
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Figure 1. MCF-7 breast adenocarcinoma cells 30 min post-irradiation, showing cell morphology (A – D) and - 111
H2AX foci (E – H), which represent sites of DNA double strand breaks. Cells were irradiated with gamma radiation 112
at 0 (A, E), 2 (B, F), 4 (C, G) and 8 (D, H) Gy. Images on the top and bottom panels are of the same sets of cells.
113 114
Irradiated MCF-7 cells were assayed for DNA double strand breaks by immunofluorescence staining with 115
anti--H2AX antibodies at 30 minutes post-irradiation. Without radiation treatment, no -H2AX foci were observed 116
indicating that the background level of DNA double strand breaks was almost undiscernible. Upon gamma 117
irradiation, foci of DNA double strand breaks were detected within the cell nuclei, appearing as bright spots when 118
observed under the microscope (Figures 1E – 1H). The number of foci increased linearly with increasing radiation 119
dose from 2 to 8 Gy, yielding 10 -H2AX foci per cell per Gy on average. Our observation confirmed the linear 120
correlation between the number of DNA double strand breaks and radiation dose, which had been reported for other 121
cell types (Ivashkevich et al., 2011). The average number of foci was also similar to what was observed for irradiated 122
normal human lymphocytes (Valente et al., 2011).
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Effects of Moringa oleifera Lam. Extracts 125
Moringa oleifera Lam. is a widely-used nutritional supplement, which has been shown to possess both 126
antioxidant and anticancer activities including activities against breast cancer (Anusarnsunthorn, -; Khalafalla et al., 127
2010; Verma et al., 2009). Aqueous and ethanolic extracts of M. oleifera were prepared and supplemented to MCF-7 128
cells for 24 hours. At 500 µg/mL, the aqueous extract appeared relatively non-toxic. No significant morphological 129
changes were observed, and the cells remained attached to the substratum. On the other hand, the ethanolic extracts 130
appeared toxic at 500 µg/mL. Treated cells were rounded and most of the cells became detached (data not shown).
131
Therefore, for our purposes, cells were treated with M. oleifera extracts for only 30 minutes. With this treatment 132
duration, no major changes in cellular morphology could be observed (Figures 2A – 2C). When assayed for -H2AX 133
foci, no significant changes in the foci number were detected (Figures 2D – 2F), indicating that the aqueous and the 134
ethanolic extracts of M. oleifera did not induce significant numbers of DNA double strand breaks in MCF-7 breast 135
cancer cells.
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µg/mL (A and D), 500 µg/mL aqueous extract (B and E) and 500 µg/mL ethanolic extract (C and F). The top panels 139
(A – C) show cell morphology and the bottom panels (D – F) show -H2AX foci of the same sets of cells.
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Effects of Treatment Combinations 142
Aqueous and ethanolic extracts of M. oleifera were supplemented to MCF-7 breast cancer cells 30 min prior 143
to and during gamma irradiation of 0, 2, 4, and 8 Gy. Cells were assayed for DNA double strand breaks at 30 minutes 144
post-irradiation by immunofluorescence staining for -H2AX foci. As previously discussed, minimal DNA breaks 145
were observed in untreated cells, and the number of -H2AX foci per cell increased with increasing radiation dose.
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Not much differences in cellular and nuclear morphology were seen when cells were treated with M. oleifera extracts.
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However, the number of -H2AX foci per cell decreased when cells were treated with either the aqueous or the 148
ethanolic extracts prior to and during irradiation, comparing to irradiated cells without treatments with either of the 149
extracts. This observation held true for all irradiation doses from 2 to 8 Gy (Figure 3). The ethanolic extract 150
appeared more effective as the number of foci in pre-treated cells (Figures 3F, 3L & 3R) was more reduced than cells 151
pre-treated with the aqueous extract (Figures 3E, 3K & 3Q). Treatments with the ethanolic extract caused 30%
152
reduction in the number of foci or events of DNA double strand breaks among irradiated cells, while treatments with 153
the aqueous extract only brought about 15% reduction in DNA double strand break events.
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Discussion 156
DNA double strand breaks are a type of gamma radiation-induced damage which is especially important.
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First, it is the major type of damage found resulting from gamma radiation. Experiments by Roots et al. (1985) 158
showed that the ratio of DNA double strand breaks to single strand breaks induced by 1 Gy gamma radiation could be 159
as high as 110 in mammalian cells. Second, DNA double strand breaks are potentially lethal if left unrepaired, due to 160
destabilization of the genome. It was estimated that natural causes resulted in 10 DNA double strand breaks per cell 161
per day in normal human cells. -H2AX foci have been widely used to track DNA double strand break events in 162
various cell types due to their close correlation with the number of DNA double strand breaks as assayed by the decay 163
of incorporated 125I (Sedelnikova et al., 2002). Here, we have shown that gamma radiation additionally induced 164
approximately 10 DNA double strand breaks per cell per Gy in MCF-7 human breast adenocarcinoma cells, 165
visualized as -H2AX foci. This number of breaks was comparable to the numbers reported for gamma-irradiated 166
human lymphocytes (Valente et al., 2011), but very low comparing to the numbers reported for irradiated human lung 167
fibroblasts where as high as 75 breaks were reported per cell after 2 Gy X-irradiation (Rothkamm & Löbrich, 2003).
168
The observed differences indicated that the extent of radiation-induced damage to the DNA is cell-type dependent.
169
Rothkamm & Löbrich (2003) have also shown that significantly different numbers of DNA breaks were induced in 170
different fibroblast cell lines irradiated with the same radiation dose.
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The ability of radiation to produce adverse effects on cancer cells constitutes the basis for the use of 172
radiotherapeutic approach to cure cancer. Ionizing radiation induced oxidative stress among irradiated cells leading 173
to an increased level of intracellular reactive oxygen species, lipid peroxidation and oxidative damage to the DNA 174
(Azzam et al., 2012; Widel et al., 2012; Morales et al., 1998; Leach et al., 2001) as we have observed in irradiated 175
MCF-7 cancer cells. Moringa oleifera Lam. is one of many plants which have been shown to possess antioxidant 176
activities and suspected to have ameliorative effects against ionizing radiation. The leaf extracts exhibited 1,1- 177
diphenyl-2-picrylhydrazyl, superoxide and hydroxyl radical scavenging activities, inhibitory effects on lipid 178
peroxidation, and protective effects against oxidative damage to naked DNA in vitro (Iqbal and Bhanger, 2006;
179
Chumark et al., 2008; Verma et al., 2009; Singh et al., 2009; Atawodi et al., 2010; Santos et al., 2012). We assayed 180
the ability of the aqueous and the ethanolic extracts of M. oleifera leaves to protect MCF-7 cells from the genotoxic 181
effect of acute gamma radiation from 2 to 8 Gy. Interestingly, both types of extracts were able to protect the cells 182
from radiation-induced DNA double strand breaks as evidenced by the reduction of -H2AX foci upon pre-treatments 183
with either of the extracts.
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Figure 3. Treatment combinations of gamma radiation and leaf extracts of Moringa olerfera Lam. MCF-7 cells were 187
pre-treated for 30 minutes without the extracts (A, D, G, J, M, P) or with 500 µg/mL of either the aqueous (B, E, H, 188
K, N, Q) or the ethanolic extract (C, F, I, L, O. R). Radiation doses were indicated on the left of the panels. Bright- 189
field images (A – C, G – I, M – O) showed cell morphology. Fluorescence images (D – F, J – L, P – R) showed - 190
H2AX foci, which indicated the sites of DNA double strand breaks.
191
Many herbal extracts with antioxidant activities have been shown to exhibit protective effects upon 192
irradiated normal cells. These extracts must be administered prior to radiation treatment. The extract of Zingerone, a 193
phenolic alkanone from ginger, was shown to exert radioprotective effects upon Chinese hamster fibroblast cells 194
(V79) when given to the cells 1 h prior to radiation exposure (Rao & Rao, 2010). Studies have been conducted to 195
investigate M. oleifera’s protective effects against radiation-induced oxidative stress. Swiss albino mice were 196
injected with a total dose of 150 mg/kg body weight of 50% methanolic extract of M. oleifera leaves as a single or 197
five daily doses prior to a whole-body gamma irradiation at 4 Gy. Supplementation of the extract was able to reduce 198
2001), indicating the radioprotection efficacy of M. oleifera. Although not a scientific study, in Thailand, there has 200
been a report that the aqueous extract of M. oleifera could alleviate radiation toxicity in cancer patients receiving 201
radiation therapy (Anusarnsunthorn, -), another support for its radioprotective ability.
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Conclusion 204
The above studies showed the radio-protective effects of antioxidant herbal extracts upon normal cells. Our 205
study is one of a few that investigated these effects on irradiated cancer cells. The results of our investigation 206
indicated that a 30-minute pre-treatment with M. oleifera leaf extracts yielded 15 – 30% reduction in radiation- 207
induced DNA double strand breaks in MCF-7 human breast cancer cells. This observation suggested that irradiated 208
cancer cells respond similarly to natural antioxidants as irradiated normal cells. Radiation protection that was to be 209
conferred upon normal cells would also be perceived by cancer cells. Additional investigation into the effects of 210
antioxidant pretreatments on the eventual survival or death of irradiated cancer cells would be an important next step 211
in understanding the implications of the radio-protective effects of antioxidant supplements on cancer patients.
212 213
Acknowledgements 214
215
This research was funded by Thailand Research Fund Grant no. DBG5380043 to K. Boonsirichai. We 216
thanked S. Pongpat and P. Uttayarat for technical helps.
217 218
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