The fibrates are ligands for peroxisome proliferator-activated receptor (PPAR) α and used clinically as hypolipidemic drugs. The fibrates are known to cause peroxisome proliferation, enhance superoxide dismutase (SOD) expression and catalase activity. The antioxidant actions of the fibrates may modify radiation sensitivity. Here, we investigated the change of the radiation sensitivity in two cervix cancer cell lines in combination with fenofibrate (FF).
Activity and protein expression of SOD were measured according to the concentration of FF. The mRNA expressions were measured by using real time reverse-transcription polymerase chain reaction. Combined cytotoxic effect of FF and radiation was measured by using clonogenic assay.
In HeLa cells total SOD activity was increased with increasing FF doses up to 30 µM. In the other hand, the catalase activity was increased a little. As with activity the protein expression of SOD1 and SOD2 was increased with increasing doses of FF. The mRNAs of SOD1, SOD2, PPARα and PPARγ were increased with increasing doses of FF. The reactive oxygen species (ROS) produced by radiation was decreased by preincubation with FF. The surviving fractions (SF) by combining FF and radiation was higher than those of radiation alone. In Me180 cells SOD and catalase activity were not increased with FF. Also, the mRNAs of SOD1, SOD2, and PPARα were not increased with FF. However, the mRNA of PPARγ was increased with FF.
FF can reduce radiation sensitivity by ROS scavenging via SOD induction in HeLa. SOD induction by FF is related with PPARα.
The fibrates are ligands for peroxisome proliferator-activated receptor α (PPARα) and some of them are used clinically as hypolipidemic drugs to reduce blood triglyceride and cholesterol [
Radiation effects may occur as direct ionizations in an organic molecule or indirectly via free radical processes. In the indirect action, which is dominant in treatment with x-rays and γ-rays commonly used in clinical radiotherapy, radiation interacts with cellular molecules (mainly water) to produce free radicals, such as hydroxyl radicals (•OH) and aqueous electrons (e-aq). The radiation-induced reactive oxygen species (ROS) will undergo further reactions. The aqueous electron reacts with oxygen to produce the superoxide anion radicals (O2•-). The spontaneous or SOD-catalyzed dismutation of superoxide generates hydrogen peroxide (H2O2), which yields the highly toxic hydroxyl radicals through a Fenton reaction or a Haber-Weiss reaction [
The antioxidant actions of the fibrates may modify radiation sensitivity. If the patients taking the fibrates undergo radiation therapy the results may be changed. However, there is no report on this issue. Here, we investigated the change of the radiation sensitivity in cervix cancer cells combined with fenofibrate (FF), which is commonly used to reduce triglyceride.
The two cervix cancer cell lines (HeLa, Me180) were purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum supplemented with 100 IU/mL penicillin, 100 µg/mL streptomycin. The cells were kept in a humidified atmosphere containing 5% CO2 at 37℃. FF was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO) at a final concentration of less than 0.1% DMSO in the culture medium. All standard culture reagents were from Invitrogen (Carlsbad, CA, USA).
Cell proteins was obtained by rinsing the cells with phosphate-buffered saline (PBS) pH 7.2 three times, scraping the cells from the culture flasks with a rubber policeman, and centrifuging at 1,000 rpm for 10 minutes. The cells were washed with PBS two times and pellets were kept frozen at -80℃ until used. At the time of analysis, the cell pellets were resuspended in one volume of 50 mM potassium phosphate buffer (pH 7.85) and sonicated on ice 3 times for 10 seconds using a Vibra cell sonicator (Branson 1510R-DTH; Branson, Danbury, CT, USA). Protein concentration was determined using the Bradford method using bovine serum albumin as a standard [
Superoxide dismutase activity was assayed using an assay kit (Dojindo, Gaithersburg, MD, USA). Briefly, 1 × 104 cells were seeded in each well of 96-well micoplates and next day, treated with various concentration of FF for 48 hours. After adding a set of assay solutions as indicated in the manufacturer's protocol the absorbance at 450 nm was measured using a microplate reader and SOD activity were calculated.
Catalase activity was quantitated spectrophotometrically following the decomposition of H2O2 at 240 nm [
Proteins was denatured in 1 volume of sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% sodium dodecyl sulfate (SDS), 5% β-mercaptoethanol (v/v) and 2-3 drops of saturated bromophenol blue solution at 100℃ for 3 min. The proteins were separated in a 12.5% denaturing polyacrylamide gel by electrophoresis and then transferred onto nitrocellulose membranes at 100 V for 1 hour at 4℃ The blots were then blocked in 4% dry milk in TTBS (0.02 M Tris buffer [pH 7.0] and 0.5% Tween 20) at room temperature for 2 hours and incubated with primary antibody (1:1,000) in TTBS at 4℃ overnight. After washing three times with TTBS, 5 minutes each, the blots were incubated with secondary antibody (1:10,000). After washing three times, the blots were visualized using chemiluminescence. The antibodies for CuZnSOD (SOD1), MnSOD (SOD2) and catalase were purchased from AbFrontier (Seoul, Korea).
RNA was isolated from the cells using TRIzol reagent according to the manufacturer's protocol (Invitrogen). cDNA was synthesized using iScript reverse transcriptase reagent (Bio-Rad, Hercules, CA, USA) from 1 µg of RNA. The following primers were used for the real-time quantitative reversetranscription polymerase chain reaction (RT-PCR) with SOD1 (forward, 5'-CTCTCAGGAGACCATTGCATCAT-3'; reverse, 5'-CCAATTACACCACAAGCCAAACG-3'), SOD2 (forward 5'-TCAATAAGGAACGGGGACAC-3'; reverse 5'-GTAGTAAGCGTGCTCCCACAC-3'), and PPARα (forward 5'-GGCGAATATGCTCAATGGT-3'; reverse 5'-GGCCACGAATTTTGAGGTTA-3'). The RT-PCR was performed with 20 µL of reaction solution containing diluted cDNA, 20 pmol of each primer, and 10 µL of two-step SYBR Green PCR Master Mix (Invitrogen). The thermal cycling parameters were 95℃ for 10 minutes for Taq polymerase activation followed by 40 cycles of 95℃ for 15 seconds and 60℃ for 1 minute. The reactions were performed in triplicate in each experiment. The threshold number of cycles (Ct) was recorded from each sample for both the target gene and the reference gene (GAPDH). Melting curve analysis was performed for each run. The relative expression of the target gene was calculated as ΔΔCt, determined by subtracting ΔCt of the reference gene from ΔCt of the target gene. Each experiment was performed independently three times.
Intracellular production of ROS was measured using the fluorescent dye, 2',7'-dichlorodihydrofluorescein-diacetate (H2DCFDA; Sigma, St. Louis, MO, USA) as described by Wan et al. [
After treated with FF for defined duration the cells were irradiated. Irradiation was done using a linear accelerator (Mevatron; Siemens) with a dose rate of 3 Gy/min at room temperature. The cells were trypsinized and varying number of cells for optimal colony counting was seeded in 60 mm dishes. After 6-7 days the cells were stained with 0.1% crystal violet solution and the number of colonies, composed of at least 50 cells, was counted. Surviving fraction (SF) was calculated as follows: SF = plating efficiency (PE) of treated cells / PE of control cells. PE (%) was obtained from (colonies counted / cells plated) × 100. Cell survival curves were plotted by using linear regression model.
Total SOD and catalase activity was measured in the cells treated with 0, 3, 10, 30, 60 µM of FF for 48 hours. Total SOD activity was increased with increasing FF doses and it was maximal at 30 µM of FF: about two times higher than control in HeLa cells (
To investigate whether the SOD and catalase activity is related with their protein expression, Western blot was done for SOD1, SOD2 and catalase in HeLa cells treated with various doses of FF for 48 hours. As with the activity the protein expression of SOD1 and SOD2 were increased with increasing doses of FF (up to 60 µM) and that of catalase was decreased with increasing doses (30 and 60 µM) of FF (
To investigate whether protein expression of SOD1 and SOD2 is related with their mRNA expression, cells were treated with varying doses of FF for 48 hours and real time RT-PCR was done. Both mRNA of SOD1 and SOD2 were increased with increasing doses of FF in HeLa cells, but they were not increased in Me180 cells (
To investigate whether protein expression of SOD1 and SOD2 is related with PPARα and PPARγ mRNA expression, cells were treated with varying doses of FF for 48 hours and real time RT-PCR was done. PPARγ mRNA was increased with increasing doses of FF in both cell lines. However, PPARα mRNA was increased with increasing doses of FF in only HeLa cells (
Next, we were interested in whether FF affects on radiation sensitivity. Cells were treated with 10 µM of FF for 48 hours and irradiated. The survival curves showed radioprotective effect of FF in HeLa cells. However, such an effect was not observed in Me180 cells (
Finally, we were interested in whether FF modifies radiation sensitivity by scavenging ROS. To investigate whether SOD induced by FF contributes to scavenge ROS, HeLa cells were treated with 10 µM of FF for 48 hours and irradiated with 4 Gy. ROS produced by 4 Gy was decreased in the cells treated with FF (
Peroxisomes are found in virtually all eukaryotic cells. A major function of the peroxisome is the breakdown of fatty acid molecules, in a process called β-oxidation. Peroxisomes contain oxidative enzymes for certain oxidative reactions, like the β-oxidation of fatty acids. The β-oxidation of fatty acids are the main processes that generate ROS in peroxisomes, in particular H2O2. In several animal species, activators of PPARα, like fibrates increase the number and volume of peroxisomes and this peroxisomal proliferation increases the expression of peroxisomal oxidases that produce H2O2, such as acyl-CoA oxidase (ACO) [
More importantly, the increased activities of antioxidants, including the SODs, in cancer cells and tissues might reduce the cytotoxic effects of ROS-generating chemotherapy drugs and radiation, although the final effect may also depend on several factors, such as cell type, environmental factors, and the balance of antioxidant enzymes in the cell. However, the published studies about the relation of SODs to radiation sensitivity show some conflicting results depending on the increased levels of SOD activities. In the MnSOD-transfected HeLa cervical carcinoma cells, MnSOD overexpression protected the cells from growth suppression by serum deprivation, which is associated with increase in ROS generation [
There is an intense argument on the concurrent use of antioxidants with chemotherapy and/or radiotherapy. Although considerable existing data support that dietary antioxidants do not interfere with these therapeutic modalities for cancer [
This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund; KRF-2007-313-E00370).
No potential conflict of interest relevant to this article was reported.
Effect of fenofibrate (FF) on total superoxide dismutase (SOD) and catalase activity in HeLa and Me180 cells. Cells were treated with the indicated concentrations of FF for 48 hours. Dose-dependent effect of FF treatment on total SOD and catalase activity determined by SOD and catalase activity assay.
Effect of fenofibrate (FF) on superoxide dismutases (SODs) and catalase protein expression in HeLa cells. Cells were treated with 0-60 µM concentration of FF for 48 hours. Equal amounts of lysates were subjected to electrophoresis and analyzed by Western blotting for SOD1, SOD2 and catalase as described under Materials and Methods.
Effect of fenofibrate (FF) on the mRNA expression of superoxide dismutase 1 (SOD1), SOD2, peroxisome proliferator-activated receptor α (PPARα), and PPARγ in HeLa and Me180 cells. Cells were treated with the indicated concentrations of FF for 48 hours and RNA was isolated for reverse-transcription polymerase chain reaction analysis of PPARα gene expression as explained in Material and Methods. Data are presented as fold changes compared to sham-treated cells.
The change of radiation sensitivity in fenofibrate (FF) pretreated HeLa and Me180 cells. Cells were treated with 10 µM FF (FF10) for 48 hours and irradiated with 0-8 Gy X-rays. Following irradiation, cells were incubated at 37℃/5% CO2 for 6-7 days and then colonies containing ≥50 cells were scored. SF, surviving fraction; IR, ionizing radiation.
reactive oxygen species (ROS) changes by fenofibrate (FF) treatment. HeLa cells were treated with 10 µM of FF for 48 hours and irradiated with 4 Gy. ROS produced by 4 Gy was decreased in the cells treated with FF.