Studies have reported that atorvastatin (ATO) may increase the radiosensitivity of malignant cells. However, the influence of ATO on reactive oxygen species (ROS) levels before and after irradiation has not been fully illustrated. In the present study, radiosensitivity was evaluated by a clonogenic assay and a cell survival curve and cell apoptosis was measured by flow cytometry. ROS were detected by a laser scanning confocal microscope and flow cytometry with a DCFH-DA probe. NADPH oxidases (NOXs) and superoxide dismutase (SOD) proteins were detected by immunoblotting, and total SOD activity was measured using an SOD kit. We also conducted transient transfection of
Radiotherapy is one of the main treatments used to deal with malignancy. More than 50% of patients with malignant tumors receive radiotherapy during their treatment (
Statin is a type of 3-hydroxy-3-methyl-glutaryl-CoA (HMGCoA) reductase inhibitor which has been used to lower plasma cholesterol in clinical practice (
ATO is a third generation statin. Studies have reported that ATO can influence ROS generation in vascular cells (
ATO was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Tempol was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA) and an apoptosis detection kit was purchased from BD Biosciences (Franklin Lakes, NJ, USA). An ROS assay kit and a SOD assay kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Hoechst 33342 staining solution was purchased from Bridgen Company (Beijing, China). A rabbit monoclonal anti-NOX2 antibody and a rabbit monoclonal anti-SOD1 antibody were purchased from Biosynthesis Biotechnology Corp. (Beijing, China) and a rabbit monoclonal anti-NOX4 antibody was obtained from Abcam (Cambridge, UK).
The PC-3 cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and the cells were maintained in RPMI-1640 medium that was supplemented with 10% (v/v) fetal bovine serum (FBS) and cultured at 37°C in an incubator with 5% CO2 and a humid atmosphere.
To evaluate the radiosensitization potential of ATO on PC-3 cells, clonogenic and apoptosis assays were first conducted. Next, we transiently transfected
Cells were seeded into seven 6-well plates at different quantities and cultured at 37°C in an incubator for >24 h. After being pretreated with the vehicle, ATO, tempol or transfected
A cell survival curve was plotted by GraphPad Prism 6 software according to survival fractions and was calculated as the clonogenic efficiency of the irradiated cells divided by that of the unirradiated cells. The clonogenic efficiency was calculated as the percentage of the clones of the treated cells divided by that of the control. To calculate the radiosensitivity parameters, including D0, Dq, N, SF2 and SERs (
After ATO treatment, the cells from the study group received a 1 Gy dose of irradiation using an Elekta linear accelerator (4 Gy/min at room temperature). Six hours later, all the cells were collected into a 15 ml centrifuge tube, digested with 0.25% trypsin (without 0.05% EDTA) and rinsed thrice with ice-cold phosphate-buffered saline (PBS). Subsequently, the cells were resuspended with 1X Annexin-binding buffer, and then 1×105 cells were transferred into a 1.5 ml Eppendorf tube and 5 µl Annexin-V FITC and 5 µl propidium iodide were added. After 15 min of lucifugal incubation at room temperature, cell apoptosis was detected by flow cytometry. The apoptosis rate was calculated as the early apoptosis rate plus the late apoptosis rate.
Total RNA was extracted by TRIzol method according to the manufacturers instructions (Invitrogen, Carlsbad, CA, USA), c-DNA was synthesized by Reverse transcription PCR kit (Tiangen Biotech Co., Ltd. (Beijing, China).
The amplified DNA was transfected into DH5α competent cells (GenStar, Biosolutions Co., Ltd., Beijing, China) and the plasmid was extracted using a plasmid extraction kit (Life Technologies, Grand Island, NY, USA). The plasmid and pcDNA3.1 vector (Invitrogen) were digested by the restriction enzymes
Before transfection, the PC-3 cells were seeded into 6-well plates at 2×105 cells/well and cultured in a 37°C incubator for 48 h. DNA and Lipo3000 transfection reagent (Invitrogen) were diluted using non-serum RPMI-1640 medium, mixed and maintained at room temperature for 15 min. Then, the cells were incubated with Lipo3000-DNA complexes in non-serum RPMI-1640 medium, cultured in a 37°C incubator for 6 h. Next, the old media was replaced with fresh RPMI-1640 (with 10% FBS) and cultured at 37°C for 24 h. Finally, the transfected cells were screened using G418 antibiotic (Amresco, LLC, Solon, OH, USA) in fresh RPMI-1640 for 14 days, and the clones which were enlarged in the culture medium were selected.
PC-3 cells for flow cytometry assay were seeded into 6-well plates. Before probe treatment, the cells were collected into 15 ml tubes digested with 0.25% trypsin (with 0.05% EDTA) digestion. The cells for laser scanning confocal microscopy assay were seeded into a confocal dish. During ROS detection, part of the cells were suspended in a positive control that was diluted with non-serum RPMI-1640 medium in 1:1,000 titer and cultured in a 37°C incubator. Fifteen minutes later, all the cells were rinsed thrice with non-serum RPMI-1640 medium, then suspended in DCFH-DA solution that was diluted with non-serum RPMI-1640 medium in 1:1,000 titer. The cells were cultured in a 37°C incubator for 20 min and then immediately detected by flow cytometry or observed under a laser scanning confocal microscope.
For the irradiation study, the cells received a 1 Gy dose of irradiation using an Elekta linear accelerator after ATO alone or combined treatment and then the cells were cultured in a 37°C incubator for 2 h before following the same steps as aforementioned.
The cells were rinsed twice with ice-cold PBS, and lysed with lysis buffer, which was supplemented with a protease inhibitor cocktail. Cell lysates were collected using a scraper and transferred into an Ependorf tube for high speed centrifugation (4°C, 12,000 rpm, 20 min), The liquid supernatant was divided into segments of protein samples and the concentrations were determined. The protein samples were diluted with 5X loading buffer and denaturated at 100°C for 5 min.
For immunoblotting, an equal amount of total proteins from each group was separated by SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes. Next, the antigens on the membranes were blocked with 5% dried skimmed milk/1X TBST for 1 h, and incubated overnight with corresponding primary antibodies against NOX2, NOX4, SOD1 and GAPDH, respectively. Subsequently, the membranes were washed thrice with 1X TBST and incubated with horseradish peroxidase-coupled secondary antibodies for 1 h. After washing thrice again with 1X TBST, blots on the membranes were finally detected by ECL western blotting detection reagent.
Total proteins of the PC-3 cells were extracted as aforementioned. When the protein concentration was determined, the assay was carried out according to the instructions from the total SOD assay kit with WST-8. Briefly, an equal amount of proteins was adjusted by SOD detection buffer and transferred into a 96-well plate. Then WST-8/enzyme operating solution and reaction activating solution were added. The reaction system was incubated for 30 min at 37°C and then the absorption was detected with photometry using a microplate reader at 459 nm.
Data are expressed as the mean ± SEM. Statistic software SPSS 13.0 and GraphPad Prism 6 were used to analyze data. ImageJ software was applied to analyze the relative intensity of the protein blots. A coupled t-test was used to contrast radiosensitivity parameters and a one-way ANOVA was used to evaluate the apoptosis rates, ROS levels, protein levels and SOD inhibition rates. P<0.05 was defined as statistically significant; P<0.01 was considered as a highly significant difference.
To evaluate the influence of ATO on the radiosensitivity of PC-3 cells, a clonogenic assay was first conducted and a cell survival curve was plotted. As shown in the survival curve (
Using GraphPad Prism 6 fitted cell survival curve with multi-target single hit equation, the results of the radiosensitivity parameters (
Apoptosis is the main form of cell death following irradiation (
In addition, the apoptosis rate of the irradiated cells was significantly increased when compared with this rate in the unirradiated ATO-treated group. However, there was no statistical significance in the apoptosis rate between the irradiated and unirradiated cells in the control group.
To explain the radiosensitization effect of ATO, the intracellular ROS level of PC-3 cells was then detected. ROS play an important role in the cell killing process resulting from irradiation. The ROS level in cells before irradiation corresponds to the endogenetic ROS level. Our results showed that the endogenetic ROS level of the study group was obviously decreased with ATO treatment. As the result from laser scanning confocal microscopy shows (
However, this trend was abrogated by
Next, the change in ROS level after irradiation was detected. Generally, radiation-induced ROS are only maintained ~10−9-10−7 sec in cells (
On the other hand, the DCF-tagged ROS fluorescence of the combined treatment groups, including ATO plus tempol or
NOX2 and NOX4 are two isoforms that are involved in prostate cancer progression and are two main sources of endogenous ROS generation (
Furthermore, our results showed that, if we transfected
The SOD family is able to eliminate redundant intracellular ROS in time. Based on the aforementioned results, the level of SOD was assessed. In our results, western blot analysis revealed little difference in the thickness and relative intensity of the SOD1 blots in all groups (
However, on the other hand, we found that the SOD activity decreased in the ATO-alone group. As our results showed (
The aforementioned results imply that a low level of endogenetic ROS and a high level of radiation-induced ROS are related to the radiosensitization effect of ATO, and a low expression of NOXs and decreased SOD activity contribute to these changes. Next, we combined ATO with
Our results indicate that combined treatment of ATO and
In the present study, we verified that ATO could increase the radiosensitivity of PC-3 cells. According to the cell survival curve, the D0, Dq, N, and SF2 values of the study group were all lower compared with the control. A low D0 value implies that the mean lethal dose is lower, in other words, the dose for reducing the survival fraction from 0.1–0.037 is lower than that of the control (
Similarly, the apoptosis assay also indicated that ATO increased the apoptosis rate of the irradiated PC-3 cells. In our study, the cells that underwent ATO pretreatment and 1 Gy irradiation showed a higher apoptosis rate than the other groups, especially the cells treated with ATO alone, which implies that ATO increases radiation-induced cell killing. Apoptosis is the main form of cell death during irradiation (
DNA damage is the key event following irradiation that causes cell death. Radiation-induced DNA damage can be defined as two types: direct damage and indirect damage. For indirect damage, its occurrence and degree are closely related to the ROS levels evoked by irradiation (
In addition, we also found that ATO significantly decreased endogenous ROS levels in the PC-3 cells. Normally, cells may generate endogenous ROS in metabolic processes, and these play an important role in signal transduction and ensuring cell growth (
This effect was confirmed by adding tempol and by
As a crucial factor in redox reactions, NOXs are essential for endogenous ROS generation. NOX1-NOX5 are five main isoforms of the NOX family (
The SOD family is the main scavenging system in cells for ROS elimination, especially superoxide anion (O2•−), a main form of endogenous ROS (
In contrast, a decrease in SOD activity may attenuate the ROS-eliminating capability during irradiation. Radiation-induced ROS is the product of radiolytic hydrolysis during irradiation, including superoxide anion (O2•−), hydroxy radical (OH•) and hydrogen peroxide (H2O2) (
This effect was finally verified by irradiative clonogenic assay. When we treated cells with ATO and tempol, the cell survival fraction was markedly increased compared with that of the cells treated with ATO alone, which proves that low SOD activity is responsible for the radiosensitization effect of ATO. However, when we treated cells with ATO and
In conclusion, our data indicate that ATO is effective in decreasing endogenous ROS levels through the reduction in NOX2 and NOX4 expression and in prolonging the lifespan of irradiation-induced ROS through the attenuation of SOD activity. These effects not only increased the DNA susceptibility to irradiation, but also increased the indirect DNA damage of irradiation-induced ROS (
We sincerely thank Professor De-Min Zhou for providing access to the laboratory equipment, facilities and reagents. Professor De-Min Zhou is the director of the State Key Laboratory of Natural and Biomimetic Drugs of Peking University (Beijing, China). This study was supported by the Department of Radiation Oncology, Peking University First Hospital, (Beijing, China).
Analysis of the effect of ATO on the survival fraction and apoptosis rate of irradiated PC-3 cells. (A) The survival curve of PC-3 cells under different irradiation doses. Survival fractions are presented as a napierian logarithm (ln). (B) The scatterplot of flow cytometry on cell apoptosis. (C) The apoptosis rate histogram of PC-3 cells. The ATO-treated PC-3 cells received 1 Gy irradiation or were not irradiated. The data are analyzed from 4 independent experiments and expressed as the mean ± SEM, *P<0.05. ATO, atorvastatin; IR, irradiation.
Effect of ATO on intracellular ROS levels. (A) Laser scanning confocal microscopy results of endogenous ROS levels. Green fluorescence indicated DCF-tagged ROS, blue fluorescence indicated nuclear staining by Hoechst. (B) Histogram of endogenous ROS levels by flow cytometry detection. (C) Laser scanning confocal microscopy results of intracellular ROS levels after the irradiation had been terminated for 2 h. (D) Histogram of intracellular ROS levels by flow cytometry detection after the irradiation had been terminated for 2 h. The data were analyzed from 3 independent experiments and are expressed as the mean ± SEM, **P<0.01. ATO, atorvastatin; ROS, reactive oxygen species.
Effect of ATO on the levels of NOX2, NOX4 and SOD1 proteins and total SOD activity. (A) Immunoblotting results of NOX2, NOX4 and SOD1 protein levels. (B) Histogram of NOX2 and NOX4 relative expression. (C) Histogram of SOD1 relative expression. (D) Histogram of SOD inhibition rates. The data are analyzed from 3 independent experiments and are expressed as the mean ± SEM, *P<0.05, **P<0.01. ATO, atorvastatin; SOD, superoxide dismutase.
Changes in the survival fraction of irradiated PC-3 cells with NOX and SOD alteration. (A) The survival curve of irradiated PC-3 cells with ATO and tempol combined treatment. (B) The survival curve of irradiated PC-3 cells with combined treatment of ATO and
Model dispalying the influence of ATO on intracellular ROS and the irradiation-induced cell killing effect. i) Atorvastatin decreases endogenous ROS levels through the reduction in NOX expression, thus compensating the attenuation of SOD activity. ii) Attenuated SOD activity fails to eliminate radiation-induced ROS effectively and iii) causes accumulation of radiation-induced ROS in the cytoplasm, so that the indirect DNA damage is increased. ATO, atorvastatin; ROS, reactive oxygen species; SOD, superoxide dismutase; NOXs, NADPH oxidases.
Parameters of the cell survival curves fitted by the multi-target single hit equation.
Parameters | n | Control | ATO group | t-stat | P-value |
---|---|---|---|---|---|
D0 (Gy) | 3 | 0.710±0.021 | 0.585±0.017 |
8.013 | 0.001 |
Dq (Gy) | 3 | 1.430±0.130 | 1.006±0.132 |
3.964 | 0.008 |
N | 3 | 7.495±1.770 | 5.582±1.502 | 1.427 | 0.113 |
SF2 | 3 | 0.301±0.020 | 0.161±0.008 |
8.774 | 0.013 |
SERD0 | 3 | 1 | 1.277±0.065 |
−7.381 | 0.001 |
SERDq | 3 | 1 | 1.356±0.196 |
−3.146 | 0.017 |
SERSF2 | 3 | 1 | 1.739±0.222 |
−5.766 | 0.002 |
Data are expressed as mean ± SEM for three independent experiments. In the ATO study group, radiosensitivity parameters (D0, Dq, N and SF2) were universally lower than that of the control. SERs were all >1.2. Coupled t-test displayed a statistically significant difference in parameters between the two groups (P<0.05), except N-value (P>0.05).
P<0.05 vs. control
P<0.01 vs. control. SF2, survival fraction at 2 Gy; SER, sensitivity enhancement ratio; ATO, atorvastatin.