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European Journal of Pharmacology 771 (2016) 77–83
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Molecular and cellular pharmacology
Cordycepin increases radiosensitivity in cervical cancer cells by overriding or prolonging radiation-induced G2/M arrest Da Bin Seong a,1, Semie Hong b,1, Sridhar Muthusami a, Won-Dong Kim a, Jae-Ran Yu c, Woo-Yoon Park a,n a
Department of Radiation Oncology, Chungbuk National University, College of Medicine, Cheongju, Chungbuk 28644, Republic of Korea Department of Radiation Oncology, Konkuk University School of Medicine, Seoul 05029, Republic of Korea c Department of Environmental and Tropical Medicine, Konkuk University, College of Medicine, Chungju, Chungbuk 27478, Republic of Korea b
art ic l e i nf o
a b s t r a c t
Article history: Received 12 September 2015 Received in revised form 8 December 2015 Accepted 9 December 2015 Available online 10 December 2015
Cordycepin (3-deoxyadenosine) has many pharmacological activities. We studied the radiosensitising effect of cordycepin and the underlying mechanisms relating to cell cycle changes in two human uterine cervical cancer cell lines, ME180 and HeLa cells. Cordycepin produced concentration- and time-dependent reductions in cell viability with more pronounced effects in ME180 cells. Cells pre-treated with cordycepin showed lower cell survival than those exposed to irradiation only. Radiation-induced expression of the histone, γ-H2AX, and apoptosis were also increased following cordycepin pre-treatment. In ME180 cells, pre-treatment with cordycepin reduced radiation-induced G2/M arrest and this G2/M checkpoint override was sustained for longer than in HeLa cells, where G2/M arrest was observed earlier and more briefly, the number of HeLa cells in the G2/M phase was subsequently increased. Cordycepin produced different effects on the expression of p53 and cell cycle checkpoint proteins in these two cell lines. It can be assumed that the mechanism underlying cordycepin-mediated radiosensitisation involves multiple effects that are primarily based on the induction of p53-mediated apoptosis and modulation of the expression of cell cycle checkpoint molecules. & 2015 Elsevier B.V. All rights reserved.
Keywords: Cordycepin Radiation Cell cycle Cervical cancer Chemical compounds studied in this article: Cordycepin (PubChem CID 6303)
1. Introduction Cervical cancer is the third most common cancer in women worldwide (Jemal et al., 2011). Radiotherapy has been used as the major treatment modality for cervical cancer, and is commonly administered concurrently with platinum-based chemotherapy. Theoretically, chemotherapy can act synergistically with radiotherapy by inhibiting the repair of radiation-induced damage, promoting the synchronisation of cells into a radiation-sensitive phase of the cell cycle, and initiating proliferation of quiescent cells (Thomas, 1999). Despite recent improvements in outcome, more than quarter of the patients treated for locally advanced disease experience recurrence (Green et al., 2001), indicating that more effective strategies are needed. Cordycepin (3-deoxyadenosine) can be isolated from Cordyceps miltaris, and has been proposed to be the active constituent of traditional Chinese medications prepared from this parasitic fungus (Paterson, 2008; Yang and Li, 2008). Cordycepin has shown
various biological properties, including anti-tumour, anti-bacterial and anti-inflammatory effects (Wu et al., 2007; Lee et al., 2013a; Wang et al., 2014; Sugar and McCaffrey, 1998; Ahn et al., 2000; Kim et al., 2006; Jeong et al., 2010; Thomadaki et al., 2008). Cordycepin is an RNA antimetabolite that has been reported to increase radiosensitivity by inducing G2 cell cycle arrest (Tomasovic and Dewey, 1978; Rowley and Kort, 1988) and by inhibiting the repair of potentially lethal DNA damage (Hiraoka et al., 1988). However, the molecular mechanism underlying this radiosensitisation has not been completely elucidated. Recently, cordycepin was reported to reduce cell proliferation by inducing cell cycle arrest at the radiosensitive G2/M phases (Lee et al., 2009; Wu et al., 2007), which may sensitise cancer cells to radiation. Hence, the present study was designed to investigate the synergistic effects of cordycepin and radiation on cervical cancer cells.
2. Materials and methods 2.1. Cell lines and culture conditions
n
Corresponding author. E-mail address: [email protected] (W.-Y. Park). 1 These authors contributed equally for this work.
http://dx.doi.org/10.1016/j.ejphar.2015.12.022 0014-2999/& 2015 Elsevier B.V. All rights reserved.
Two human cervical cancer cell lines (ME180 and HeLa) were purchased from the Korean Cell Line Bank (Seoul, Korea) and
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cultured in Roswell Park Memorial Institute (RPMI) 1640 or Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and antibiotics at 37 °C in an incubator containing 5% CO2. Cordycepin was purchased from Sigma-Aldrich. 2.2. Irradiation Cells were irradiated by a 6-MV photon beam using a linear accelerator (Mevatron, Siemens Medical System, Erlangen, Germany) at room temperature. Doses of 0, 2, 4 or 6 Gy were used, with a dose rate of 3 Gy/min. 2.3. Cytotoxicity assay
2.7. Confocal microscopy Cells were treated with cordycepin (0 or 25 mM) for 24 h, irradiated (0 or 4 Gy) and then incubated at 37 °C for 15 min, 1, 6 or 24 h. After incubation, the cells were washed twice with PBS, fixed in 4% paraformaldehyde for 20 min and treated with anti-phospho-histone H2AX (ser139) (Millipore Corp., Bedford, MA, USA) for 1–2 h. After washing with PBS, the cells were probed with Alexa Fluor488-labelled goat anti-rabbit IgG (A-11008; Invitrogen, Carlsbad, CA, USA) in PBS for 1 h. DNA was counterstained with DAPI. Slides were viewed on a Zeiss 510M confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany). 2.8. Western blotting
Cells were treated with various concentrations of cordycepin (0, 25, 50, 100 or 200 mM) for 4, 24 or 48 h. The cells were the harvested, fixed in 70% ethanol and stored at 20 °C overnight. The cells were then washed twice with PBS before incubation with RNase and the DNA-intercalating dye, propidium iodide (PI) for 30 min at 37 °C. Analysis of cell cycle distribution was performed using a flow cytometer (FACS Calibur, BD Bioscience, San Jose, CA, USA) and Modifit LT software.
Cells were treated with 0 or 25 mM cordycepin for 24 h and then irradiated (0 or 4 Gy). After 6 and 24 h, the cells were washed with ice-cold PBS and then treated with cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) for 15 min at 4 °C. Protein concentrations in the cell lysate were determined using protein assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts of sample protein from each group were separated using polyacrylamide gel electrophoresis. The proteins were then electrotransferred on to polyvinylidene difluoride membranes (Millipore Corp.). The blots were blocked with 5% skim milk in Tris-buffered saline containing Tween 20 (TBS-T) for 1 h prior to incubation with indicated antibody overnight at 4 °C. The primary antibodies for cyclinB1, Cdc2, CDC25c, p53 and ß-actin and the secondary antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). The antibodies for phospho-Cdc25C, phospho-Cdc2 and CHK1 were purchased from Cell Signaling Technology. The blots were then washed twice with TBS-T and incubated with the appropriate horseradish peroxidase conjugated secondary antibody for 1 h. The membrane was washed three times with TBS-T and the signals were detected using ECL reagent (West-Zol, iNtRON Biotechnology, Seongnam, Korea). The images of the resultant protein bands were captured using LAS-3000 (Fujifilm, Minato, Tokyo, Japan).
2.5. Clonogenic assay
2.9. Statistical analysis
Cells in the exponential growth phase were treated with cordycepin (0 or 25 mM) for 24 h then irradiated with 0, 2, 4 or 6 Gy. The cells were seeded out at appropriate dilutions to form colonies and incubated at 37 °C for 1–2 weeks. Colonies were then stained with 0.1% crystal violet solution, and the number of colonies containing at least 50 cells was counted under stereomicroscope. The surviving fraction (SF) was calculated as follows: SF¼ plating efficiency (PE) of treated cells/PE of control cells. PE (%) was obtained from (colonies counted/cell plated) 100. Survival curves were then plotted using the mean surviving fraction values.
All experiments were performed in triplicate. All results were expressed as the mean 7 standard deviation (S.D.). The statistical significance of differences between experimental groups was assessed using Student's t-test, which was performed using Microsoft Excel. A difference with Po 0.05 was considered statistically significant.
2.6. Assessment of apoptosis
Cells treated with cordycepin showed reduced viability, as assessed by the WST-1 assay. This effect was clearly concentrationand time-dependent in the ME180 cell lines. However, concentration- and time-dependence were less evident in the HeLa cell lines at concentrations up to 200 mM (Fig. 1A and B). After exposed to 25 mM cordycepin for 24 h, the number of cells in G2/M phase increased in a time-dependent manner. This effect continued beyond 24 h and was more evident in ME180 cells than in HeLa cells (Fig. 1C and D).
Cell viability was measured using a WST-1 tetrazolium salt [2(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] assay, in accordance with the manufacturer's instructions (EZ-CYTOX, Daeillab service, Seoul, Korea). The cells were grown exponentially in 96-well plates and treated with various concentrations of cordycepin (0, 25, 50, 100 or 200 mM) for 24, 48 or 72 h. WST-1 was then added to each well and incubated for 1 h at 37 °C. Optical density was measured spectophotometrically at 450 nm (Thermo Electric, Madison, WI, USA). All experiments were performed in triplicate. 2.4. Cell cycle analysis
Cells treated with cordycepin and/or radiation were analysed by flow cytometry following FITC-Annexin V and PI staining using the FITC Annexin V Apoptosis Detection Kit (BD Pharmingen™, San Jose, CA, USA). Briefly, cells were harvested and centrifuged for 10 min at room temperature at 1000g. Cells were washed with PBS and resuspended in binding buffer prior to adding 5 ml FITC-Annexin V (20 mg/ml) and 5 ml PI (50 mg/ml). After incubation in the dark for 15 min, the samples were analysed by flow cytometry using FACS Calibur (BD Bioscience). This enables classification of the cells as a live (Annexin V /PI ), early apoptotic (Annexin V þ / PI ), late apoptotic (Annexin V þ /PI þ ), or necrotic (Annexin V / PI þ ).
3. Results 3.1. Effects of cordycepin on cell viability and cell cycle distribution
3.2. Effect of cordycepin on radiosensitivity The survival curves of ME 180 and HeLa cells treated with cordycepin and radiation were compared with those of cells
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Fig. 1. A. Viability of human cervical cancer ME180 and HeLa cells exposed to the indicated concentrations of cordycepin for the indicated times, as determined by WST-1 assay. B. Proportion of ME180 and HeLa cells in G2/M arrest in the presence of 0, 25 μM cordycepin for the indicated times, as determined by flow cytometry. Data represent mean 7 S.D.
Fig. 2. Cell survival curves of ME180 and HeLa cells treated with 0 or 25 μM cordycepin for 24 h and then irradiated (IR) with the indicated doses prior to determination of clonogenic cell survival. The data represent the mean 7 S.D. *; P o0.05 for the comparison with cells exposed of IR only.
treated with radiation alone. Pre-treatment with cordycepin enhanced radiation sensitivity in both cell lines (P o0.05), although this effect was not observed in HeLa cells exposed to 2 Gy irradiation (P ¼0.16). The radiosensitising effects of cordycepin were more evident in ME180 cells than in HeLa cells (Fig. 2).
3.3. Effects of cordycepin on radiation induced γ-H2AX expression The expression of γ-H2AX has been identified as a sensitive indicator of radiation-induced DNA double strand breaks (DSBs) (Rogakou et al., 1998). Western blot and immunohistochemical analyses using an anti-phospho-H2AX (ser139) antibody were performed to investigate the effects of cordycepin on radiationinduced DNA damage and repair. The γ-H2AX expression in cells
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Fig. 3. Expression of γ-H2AX in ME180 and HeLa cells. (A) Cells were treated with 0 or 25 mM cordycepin for 24 h, irradiated with 4 Gy, and γ-H2AX expression was measured by western blot at the indicated time points. ß-actin was used as a loading control. (B) ME180 and HeLa cells were treated with 0 or 25 mM cordycepin for 24 h and irradiated (4 Gy) 1 h prior to examination of γ-H2AX (green foci) and DAPI (blue) staining by confocal microscopy. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
treated with radiation alone was compared with that of those treated with cordycepin and irradiation. As shown in Fig. 3A, γH2AX expression was more prominent in cells pre-treated with cordycepin than in cells treated with radiation alone. Confocal microscopy identified more marked γ-H2AX foci after cordycepin pre-treatment followed by 4 Gy irradiation (Fig. 3B). Although γH2AX expression had decreased by 6 h in ME180 cells treated with cordycepin and irradiation, it was still visible at 24 h in HeLa cells.
3.4. Cordycepin pre-treatment enhances radiation-induced apoptosis In order to investigate the radiosensitising mechanism of cordycepin, apoptosis was evaluated using an Annexin V/PI staining assay. In ME180 cells, 16.6% of the control cells were apoptotic. After exposure to 25 mM cordycepin for 4 h, the proportion of apoptotic cells had increased to 47.3% (P o0.05). Exposure to cordycepin pre-treatment and irradiation (4 Gy), increased this
Fig. 4. Flow cytometry analysis of ME180 (upper row) and HeLa (lower row) cells stained with FITC-labeled annexin V and PI. Cells were treated with or without (control) 25 mM cordycepin for 24 h prior to irradiation (4 Gy) where indicated (IR). And were harvested after 24 h for analysis. (A, E) control, (B, F) cordycepin alone, (C, G) radiation (IR) alone, (D, H) cordycepin and radiation. The numbers indicate the percentage of cells in the indicated quadrant.*; P o0.05 for the indicated comparison.
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Fig. 5. Cell cycle distribution in ME180 and HeLa cells treated with 0 (control) or 25 mM cordycepin for 4 or 24 h prior to irradiation (IR) with 4 Gy, where indicated. Cell cycle distribution was measured by flow cytometry after 6 or 24 h. These graphs were produced using Modifit software.
proportion from 17.6% (irradiation alone) to 64.1% (Fig. 4, upper row, P o0.05). However, there was no significant difference between cells treated with cordycepin plus irradiation and those treated with cordycepin only (P¼ 0.80). In HeLa cells, treatment with 25 mM cordycepin alone did not increase the proportion of apoptotic cells. After irradiation (4 Gy), 14.4% of cells pre-treated with cordycepin were in apoptosis, as compared with 11.6% of cells exposed to irradiation alone (Fig. 4, lower row, P¼ 0.97).
3.5. Cordycepin alters cell cycle distribution and expression of G2/M checkpoint proteins in irradiated cells Cell cycle arrest to allow DNA damage repair is one of the most important cellular responses to radiation-induced DNA damage (Pellegata et al., 1996). Flow cytometry showed that in ME180 cells, G2/M phase prolongation was evident 24 h after cordycepin treatment alone (25 mM) and irradiation alone (4 Gy) (Fig. 5).
Fig. 6. Expression of G2/M cell cycle regulating proteins ME180 and HeLa cells were treated with or without 25 mM of cordycepin (as indicated) for 24 h prior to irradiation (0 or 4 Gy). Proteins were analysed by wetern blotting after 6 or 24 h, using β-actin as a loading control.
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Irradiation following cordycepin pre-treatment, resulted in a reduced G2/M phase prolongation. In HeLa cells, G2/M phase prolongation was observed 24 h after cordycepin pre-treatment, although this was not a prominent as the effect observed in ME180 cells. Radiation-induced G2/M phase prolongation was observed earlier in HeLa cells (6 h after irradiation) than in ME180 cells. Pretreatment with cordycepin attenuated the radiation-induced G2/ M prolongation 6 h after irradiation. However, the number of cells in G2/M phase was increased and this effect was sustained 24 h after irradiation (Fig. 5). Western blot analyses were performed to investigate expression of G2/M regulating molecules (Fig. 6). In ME180 cells, cordycepin pretreatment prevented radiation-mediated induction of cyclin B1, CDC2, p-CDC2, CDC25C and p-CDC25C expression. P53 was increased by cordycepin pretreatment and irradiation. These features were more evident at 24 h than at 6 h. In HeLa cells, the levels of cyclin B1, p-CDC25C and CHK1 proteins, which are the key molecules controlling G2/M progression, were increased by cordycepin pre-treatment and irradiation, as compared with cordycepin or irradiation alone. But p53 expression was not increased in HeLa cells (Fig. 6). These results indicated that different cell type showed different responses to cordycepin and radiation.
4. Discussion Multiple pathways are involved in maintaining the genetic integrity of cells after their exposure to ionising radiation. Cell cycle regulation is perhaps the most important determinant of sensitivity to ionising radiation. One of the key checkpoint pathway proteins is the tumour suppressor, p53, which coordinates DNA repair with cell cycle progression and apoptosis (Pawlik and Keyomarsi, 2004). In this study, cordycepin produced a radiosensitising effect in both of the cervical cancer cell lines examined. Interestingly, we found different modes of action in these cell lines. In ME180 cells, cordycepin decreased cell viability and induced apoptosis in a time- and concentration-dependent manner. Cordycepin pretreatment enhanced the radiation sensitivity of these cells and also promoted apoptotic cell death. This may be mediated by an increase in DSBs, as evidenced by elevated expression levels of γH2AX, a DSB indicator. The increased apoptosis was associated with increased expression levels of p53. ME180 cells that were pre-treated with cordycepin and then irradiated showed G2/M checkpoint abrogation and suppressed level of proteins involved in cell cycle control. Cordycepin was previously shown to induce apoptosis in human colorectal cancer cells through p38 signaling (He et al., 2010). In addition, Lu et al. (2015) reported that ruthenium(II) complex coordinated by cordycepin induced apoptosis by suppressing bcl-2 expression and stimulating p53 expression. In contrast, HeLa cells treated with cordycepin did not show clear changes in cell viability or levels of apoptosis. Cordycepin pre-treatment and irradiation produced less effect on the proportion of apoptotic HeLa cells that it did in ME180 cells. In HeLa cells radiation-induced G2/M arrest was evident 6 h after irradiation and declined at 24 h. Following pre-treatment with cordycepin and irradiation, the number of cells in G2/M phase was reduced at 6 h and increased at 24 h, consistent with levels of cyclin B1, p-CDC25c and CHK1 proteins in these cells. The G2/M transition in the cell cycle is positively regulated by a complex composed of Cdc2 (CDK1) and B-type cyclin (Nigg, 1995). Cdc25 phosphatases control cell-cycle progression by dephosphorylation and activation of CDK at positions Thr14 and Tyr15. Dephosphorylation of Cdc2 by Cdc25c activates the kinase activity of the Cdc2/cyclin B1 complex, thereby permitting cell entry into mitosis (Gautier et al., 1991). The cellular response to DNA damage
is mediated by evolutionarily conserved Ser/Thr kinases, phosphorylation of Cdc25 phosphatases, binding to 14-3-3 proteins, and exit from the cell cycle. MAPKAP kinase-2 undergoes initial activation in the nucleus with subsequent export of the active kinase to the cytoplasm (Ben-Levy et al., 1998). Thus, MAPKAP kinase-2 is well positioned to function as both a nuclear initiator of Cdc25B/C phosphorylation in response to DNA damage and as a maintenance kinase, which keeps Cdc25B/C inhibited in the cytoplasm (Manke et al., 2005). The present study found that an increase in the phosphorylation of Cdc25C in HeLa cells following irradiation in cordycepin pretreated cells could produce inhibitory effects on MAPKAP kinase-2. CHK1 and CHK2 phosphorylate and inactivate the dual specific phosphatase Cdc25C, which maintains the progression of the G2 phase by dephosphorylating and activating the cyclin-dependent kinase Cdc2 resulting in increased G2/ M arrest (Bernhard et al., 1995). Lee et al. (2010) reported that cordycepin activated p21/WAF1 expression, inducing G2/M phase arrest via a p53-independent pathway. In this report, cordycepin decreased the expression of cyclin B1, Cdc25c, and Cdc2, and increased the protein levels of p21/WAF1. The association of p21/WAF1 and Cdc2 also increased in a cordycepin-treated human colorectal cell line. However, cordycepin had no effect on the expression of either p27 or p53. Several studies have demonstrated the anti-cancer effects of cordycepin in different cell lines (Lee et al., 2009; Wu et al., 2007; Lee et al., 2013b). The mechanism underlying cordycepin-medicated cell death differs according to the cell type. Our findings indicated that the radiosensitising effect of cordycepin on two cervical cancer cell lines was not mediated by a single effect, but reflected of various activities that were primarily based on the induction of p53-mediated apoptosis and modulation of cell cycle checkpoint protein expression. Although future studies are warranted to elucidate the mechanisms underlying these different effects, the present results suggest that cordycepin may be a candidate radiosensitising agent in cervical cancer treatment.
Acknowledgement This work was supported by the National Research Foundation of Korea (NRF), Republic of Korea (2009–0075292 and 2012R1A1A2043237).
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Cordycepin increases radiosensitivity in cervical cancer cells by overriding or prolonging radiation-induced G2/M arrest Da Bin Seong a,1, Semie Hong b,1, Sridhar Muthusami a, Won-Dong Kim a, Jae-Ran Yu c, Woo-Yoon Park a,n a
Department of Radiation Oncology, Chungbuk National University, College of Medicine, Cheongju, Chungbuk 28644, Republic of Korea Department of Radiation Oncology, Konkuk University School of Medicine, Seoul 05029, Republic of Korea c Department of Environmental and Tropical Medicine, Konkuk University, College of Medicine, Chungju, Chungbuk 27478, Republic of Korea b
art ic l e i nf o
a b s t r a c t
Article history: Received 12 September 2015 Received in revised form 8 December 2015 Accepted 9 December 2015 Available online 10 December 2015
Cordycepin (3-deoxyadenosine) has many pharmacological activities. We studied the radiosensitising effect of cordycepin and the underlying mechanisms relating to cell cycle changes in two human uterine cervical cancer cell lines, ME180 and HeLa cells. Cordycepin produced concentration- and time-dependent reductions in cell viability with more pronounced effects in ME180 cells. Cells pre-treated with cordycepin showed lower cell survival than those exposed to irradiation only. Radiation-induced expression of the histone, γ-H2AX, and apoptosis were also increased following cordycepin pre-treatment. In ME180 cells, pre-treatment with cordycepin reduced radiation-induced G2/M arrest and this G2/M checkpoint override was sustained for longer than in HeLa cells, where G2/M arrest was observed earlier and more briefly, the number of HeLa cells in the G2/M phase was subsequently increased. Cordycepin produced different effects on the expression of p53 and cell cycle checkpoint proteins in these two cell lines. It can be assumed that the mechanism underlying cordycepin-mediated radiosensitisation involves multiple effects that are primarily based on the induction of p53-mediated apoptosis and modulation of the expression of cell cycle checkpoint molecules. & 2015 Elsevier B.V. All rights reserved.
Keywords: Cordycepin Radiation Cell cycle Cervical cancer Chemical compounds studied in this article: Cordycepin (PubChem CID 6303)
1. Introduction Cervical cancer is the third most common cancer in women worldwide (Jemal et al., 2011). Radiotherapy has been used as the major treatment modality for cervical cancer, and is commonly administered concurrently with platinum-based chemotherapy. Theoretically, chemotherapy can act synergistically with radiotherapy by inhibiting the repair of radiation-induced damage, promoting the synchronisation of cells into a radiation-sensitive phase of the cell cycle, and initiating proliferation of quiescent cells (Thomas, 1999). Despite recent improvements in outcome, more than quarter of the patients treated for locally advanced disease experience recurrence (Green et al., 2001), indicating that more effective strategies are needed. Cordycepin (3-deoxyadenosine) can be isolated from Cordyceps miltaris, and has been proposed to be the active constituent of traditional Chinese medications prepared from this parasitic fungus (Paterson, 2008; Yang and Li, 2008). Cordycepin has shown
various biological properties, including anti-tumour, anti-bacterial and anti-inflammatory effects (Wu et al., 2007; Lee et al., 2013a; Wang et al., 2014; Sugar and McCaffrey, 1998; Ahn et al., 2000; Kim et al., 2006; Jeong et al., 2010; Thomadaki et al., 2008). Cordycepin is an RNA antimetabolite that has been reported to increase radiosensitivity by inducing G2 cell cycle arrest (Tomasovic and Dewey, 1978; Rowley and Kort, 1988) and by inhibiting the repair of potentially lethal DNA damage (Hiraoka et al., 1988). However, the molecular mechanism underlying this radiosensitisation has not been completely elucidated. Recently, cordycepin was reported to reduce cell proliferation by inducing cell cycle arrest at the radiosensitive G2/M phases (Lee et al., 2009; Wu et al., 2007), which may sensitise cancer cells to radiation. Hence, the present study was designed to investigate the synergistic effects of cordycepin and radiation on cervical cancer cells.
2. Materials and methods 2.1. Cell lines and culture conditions
n
Corresponding author. E-mail address: [email protected] (W.-Y. Park). 1 These authors contributed equally for this work.
http://dx.doi.org/10.1016/j.ejphar.2015.12.022 0014-2999/& 2015 Elsevier B.V. All rights reserved.
Two human cervical cancer cell lines (ME180 and HeLa) were purchased from the Korean Cell Line Bank (Seoul, Korea) and
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cultured in Roswell Park Memorial Institute (RPMI) 1640 or Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and antibiotics at 37 °C in an incubator containing 5% CO2. Cordycepin was purchased from Sigma-Aldrich. 2.2. Irradiation Cells were irradiated by a 6-MV photon beam using a linear accelerator (Mevatron, Siemens Medical System, Erlangen, Germany) at room temperature. Doses of 0, 2, 4 or 6 Gy were used, with a dose rate of 3 Gy/min. 2.3. Cytotoxicity assay
2.7. Confocal microscopy Cells were treated with cordycepin (0 or 25 mM) for 24 h, irradiated (0 or 4 Gy) and then incubated at 37 °C for 15 min, 1, 6 or 24 h. After incubation, the cells were washed twice with PBS, fixed in 4% paraformaldehyde for 20 min and treated with anti-phospho-histone H2AX (ser139) (Millipore Corp., Bedford, MA, USA) for 1–2 h. After washing with PBS, the cells were probed with Alexa Fluor488-labelled goat anti-rabbit IgG (A-11008; Invitrogen, Carlsbad, CA, USA) in PBS for 1 h. DNA was counterstained with DAPI. Slides were viewed on a Zeiss 510M confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany). 2.8. Western blotting
Cells were treated with various concentrations of cordycepin (0, 25, 50, 100 or 200 mM) for 4, 24 or 48 h. The cells were the harvested, fixed in 70% ethanol and stored at 20 °C overnight. The cells were then washed twice with PBS before incubation with RNase and the DNA-intercalating dye, propidium iodide (PI) for 30 min at 37 °C. Analysis of cell cycle distribution was performed using a flow cytometer (FACS Calibur, BD Bioscience, San Jose, CA, USA) and Modifit LT software.
Cells were treated with 0 or 25 mM cordycepin for 24 h and then irradiated (0 or 4 Gy). After 6 and 24 h, the cells were washed with ice-cold PBS and then treated with cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) for 15 min at 4 °C. Protein concentrations in the cell lysate were determined using protein assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts of sample protein from each group were separated using polyacrylamide gel electrophoresis. The proteins were then electrotransferred on to polyvinylidene difluoride membranes (Millipore Corp.). The blots were blocked with 5% skim milk in Tris-buffered saline containing Tween 20 (TBS-T) for 1 h prior to incubation with indicated antibody overnight at 4 °C. The primary antibodies for cyclinB1, Cdc2, CDC25c, p53 and ß-actin and the secondary antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). The antibodies for phospho-Cdc25C, phospho-Cdc2 and CHK1 were purchased from Cell Signaling Technology. The blots were then washed twice with TBS-T and incubated with the appropriate horseradish peroxidase conjugated secondary antibody for 1 h. The membrane was washed three times with TBS-T and the signals were detected using ECL reagent (West-Zol, iNtRON Biotechnology, Seongnam, Korea). The images of the resultant protein bands were captured using LAS-3000 (Fujifilm, Minato, Tokyo, Japan).
2.5. Clonogenic assay
2.9. Statistical analysis
Cells in the exponential growth phase were treated with cordycepin (0 or 25 mM) for 24 h then irradiated with 0, 2, 4 or 6 Gy. The cells were seeded out at appropriate dilutions to form colonies and incubated at 37 °C for 1–2 weeks. Colonies were then stained with 0.1% crystal violet solution, and the number of colonies containing at least 50 cells was counted under stereomicroscope. The surviving fraction (SF) was calculated as follows: SF¼ plating efficiency (PE) of treated cells/PE of control cells. PE (%) was obtained from (colonies counted/cell plated) 100. Survival curves were then plotted using the mean surviving fraction values.
All experiments were performed in triplicate. All results were expressed as the mean 7 standard deviation (S.D.). The statistical significance of differences between experimental groups was assessed using Student's t-test, which was performed using Microsoft Excel. A difference with Po 0.05 was considered statistically significant.
2.6. Assessment of apoptosis
Cells treated with cordycepin showed reduced viability, as assessed by the WST-1 assay. This effect was clearly concentrationand time-dependent in the ME180 cell lines. However, concentration- and time-dependence were less evident in the HeLa cell lines at concentrations up to 200 mM (Fig. 1A and B). After exposed to 25 mM cordycepin for 24 h, the number of cells in G2/M phase increased in a time-dependent manner. This effect continued beyond 24 h and was more evident in ME180 cells than in HeLa cells (Fig. 1C and D).
Cell viability was measured using a WST-1 tetrazolium salt [2(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] assay, in accordance with the manufacturer's instructions (EZ-CYTOX, Daeillab service, Seoul, Korea). The cells were grown exponentially in 96-well plates and treated with various concentrations of cordycepin (0, 25, 50, 100 or 200 mM) for 24, 48 or 72 h. WST-1 was then added to each well and incubated for 1 h at 37 °C. Optical density was measured spectophotometrically at 450 nm (Thermo Electric, Madison, WI, USA). All experiments were performed in triplicate. 2.4. Cell cycle analysis
Cells treated with cordycepin and/or radiation were analysed by flow cytometry following FITC-Annexin V and PI staining using the FITC Annexin V Apoptosis Detection Kit (BD Pharmingen™, San Jose, CA, USA). Briefly, cells were harvested and centrifuged for 10 min at room temperature at 1000g. Cells were washed with PBS and resuspended in binding buffer prior to adding 5 ml FITC-Annexin V (20 mg/ml) and 5 ml PI (50 mg/ml). After incubation in the dark for 15 min, the samples were analysed by flow cytometry using FACS Calibur (BD Bioscience). This enables classification of the cells as a live (Annexin V /PI ), early apoptotic (Annexin V þ / PI ), late apoptotic (Annexin V þ /PI þ ), or necrotic (Annexin V / PI þ ).
3. Results 3.1. Effects of cordycepin on cell viability and cell cycle distribution
3.2. Effect of cordycepin on radiosensitivity The survival curves of ME 180 and HeLa cells treated with cordycepin and radiation were compared with those of cells
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Fig. 1. A. Viability of human cervical cancer ME180 and HeLa cells exposed to the indicated concentrations of cordycepin for the indicated times, as determined by WST-1 assay. B. Proportion of ME180 and HeLa cells in G2/M arrest in the presence of 0, 25 μM cordycepin for the indicated times, as determined by flow cytometry. Data represent mean 7 S.D.
Fig. 2. Cell survival curves of ME180 and HeLa cells treated with 0 or 25 μM cordycepin for 24 h and then irradiated (IR) with the indicated doses prior to determination of clonogenic cell survival. The data represent the mean 7 S.D. *; P o0.05 for the comparison with cells exposed of IR only.
treated with radiation alone. Pre-treatment with cordycepin enhanced radiation sensitivity in both cell lines (P o0.05), although this effect was not observed in HeLa cells exposed to 2 Gy irradiation (P ¼0.16). The radiosensitising effects of cordycepin were more evident in ME180 cells than in HeLa cells (Fig. 2).
3.3. Effects of cordycepin on radiation induced γ-H2AX expression The expression of γ-H2AX has been identified as a sensitive indicator of radiation-induced DNA double strand breaks (DSBs) (Rogakou et al., 1998). Western blot and immunohistochemical analyses using an anti-phospho-H2AX (ser139) antibody were performed to investigate the effects of cordycepin on radiationinduced DNA damage and repair. The γ-H2AX expression in cells
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Fig. 3. Expression of γ-H2AX in ME180 and HeLa cells. (A) Cells were treated with 0 or 25 mM cordycepin for 24 h, irradiated with 4 Gy, and γ-H2AX expression was measured by western blot at the indicated time points. ß-actin was used as a loading control. (B) ME180 and HeLa cells were treated with 0 or 25 mM cordycepin for 24 h and irradiated (4 Gy) 1 h prior to examination of γ-H2AX (green foci) and DAPI (blue) staining by confocal microscopy. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
treated with radiation alone was compared with that of those treated with cordycepin and irradiation. As shown in Fig. 3A, γH2AX expression was more prominent in cells pre-treated with cordycepin than in cells treated with radiation alone. Confocal microscopy identified more marked γ-H2AX foci after cordycepin pre-treatment followed by 4 Gy irradiation (Fig. 3B). Although γH2AX expression had decreased by 6 h in ME180 cells treated with cordycepin and irradiation, it was still visible at 24 h in HeLa cells.
3.4. Cordycepin pre-treatment enhances radiation-induced apoptosis In order to investigate the radiosensitising mechanism of cordycepin, apoptosis was evaluated using an Annexin V/PI staining assay. In ME180 cells, 16.6% of the control cells were apoptotic. After exposure to 25 mM cordycepin for 4 h, the proportion of apoptotic cells had increased to 47.3% (P o0.05). Exposure to cordycepin pre-treatment and irradiation (4 Gy), increased this
Fig. 4. Flow cytometry analysis of ME180 (upper row) and HeLa (lower row) cells stained with FITC-labeled annexin V and PI. Cells were treated with or without (control) 25 mM cordycepin for 24 h prior to irradiation (4 Gy) where indicated (IR). And were harvested after 24 h for analysis. (A, E) control, (B, F) cordycepin alone, (C, G) radiation (IR) alone, (D, H) cordycepin and radiation. The numbers indicate the percentage of cells in the indicated quadrant.*; P o0.05 for the indicated comparison.
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Fig. 5. Cell cycle distribution in ME180 and HeLa cells treated with 0 (control) or 25 mM cordycepin for 4 or 24 h prior to irradiation (IR) with 4 Gy, where indicated. Cell cycle distribution was measured by flow cytometry after 6 or 24 h. These graphs were produced using Modifit software.
proportion from 17.6% (irradiation alone) to 64.1% (Fig. 4, upper row, P o0.05). However, there was no significant difference between cells treated with cordycepin plus irradiation and those treated with cordycepin only (P¼ 0.80). In HeLa cells, treatment with 25 mM cordycepin alone did not increase the proportion of apoptotic cells. After irradiation (4 Gy), 14.4% of cells pre-treated with cordycepin were in apoptosis, as compared with 11.6% of cells exposed to irradiation alone (Fig. 4, lower row, P¼ 0.97).
3.5. Cordycepin alters cell cycle distribution and expression of G2/M checkpoint proteins in irradiated cells Cell cycle arrest to allow DNA damage repair is one of the most important cellular responses to radiation-induced DNA damage (Pellegata et al., 1996). Flow cytometry showed that in ME180 cells, G2/M phase prolongation was evident 24 h after cordycepin treatment alone (25 mM) and irradiation alone (4 Gy) (Fig. 5).
Fig. 6. Expression of G2/M cell cycle regulating proteins ME180 and HeLa cells were treated with or without 25 mM of cordycepin (as indicated) for 24 h prior to irradiation (0 or 4 Gy). Proteins were analysed by wetern blotting after 6 or 24 h, using β-actin as a loading control.
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Irradiation following cordycepin pre-treatment, resulted in a reduced G2/M phase prolongation. In HeLa cells, G2/M phase prolongation was observed 24 h after cordycepin pre-treatment, although this was not a prominent as the effect observed in ME180 cells. Radiation-induced G2/M phase prolongation was observed earlier in HeLa cells (6 h after irradiation) than in ME180 cells. Pretreatment with cordycepin attenuated the radiation-induced G2/ M prolongation 6 h after irradiation. However, the number of cells in G2/M phase was increased and this effect was sustained 24 h after irradiation (Fig. 5). Western blot analyses were performed to investigate expression of G2/M regulating molecules (Fig. 6). In ME180 cells, cordycepin pretreatment prevented radiation-mediated induction of cyclin B1, CDC2, p-CDC2, CDC25C and p-CDC25C expression. P53 was increased by cordycepin pretreatment and irradiation. These features were more evident at 24 h than at 6 h. In HeLa cells, the levels of cyclin B1, p-CDC25C and CHK1 proteins, which are the key molecules controlling G2/M progression, were increased by cordycepin pre-treatment and irradiation, as compared with cordycepin or irradiation alone. But p53 expression was not increased in HeLa cells (Fig. 6). These results indicated that different cell type showed different responses to cordycepin and radiation.
4. Discussion Multiple pathways are involved in maintaining the genetic integrity of cells after their exposure to ionising radiation. Cell cycle regulation is perhaps the most important determinant of sensitivity to ionising radiation. One of the key checkpoint pathway proteins is the tumour suppressor, p53, which coordinates DNA repair with cell cycle progression and apoptosis (Pawlik and Keyomarsi, 2004). In this study, cordycepin produced a radiosensitising effect in both of the cervical cancer cell lines examined. Interestingly, we found different modes of action in these cell lines. In ME180 cells, cordycepin decreased cell viability and induced apoptosis in a time- and concentration-dependent manner. Cordycepin pretreatment enhanced the radiation sensitivity of these cells and also promoted apoptotic cell death. This may be mediated by an increase in DSBs, as evidenced by elevated expression levels of γH2AX, a DSB indicator. The increased apoptosis was associated with increased expression levels of p53. ME180 cells that were pre-treated with cordycepin and then irradiated showed G2/M checkpoint abrogation and suppressed level of proteins involved in cell cycle control. Cordycepin was previously shown to induce apoptosis in human colorectal cancer cells through p38 signaling (He et al., 2010). In addition, Lu et al. (2015) reported that ruthenium(II) complex coordinated by cordycepin induced apoptosis by suppressing bcl-2 expression and stimulating p53 expression. In contrast, HeLa cells treated with cordycepin did not show clear changes in cell viability or levels of apoptosis. Cordycepin pre-treatment and irradiation produced less effect on the proportion of apoptotic HeLa cells that it did in ME180 cells. In HeLa cells radiation-induced G2/M arrest was evident 6 h after irradiation and declined at 24 h. Following pre-treatment with cordycepin and irradiation, the number of cells in G2/M phase was reduced at 6 h and increased at 24 h, consistent with levels of cyclin B1, p-CDC25c and CHK1 proteins in these cells. The G2/M transition in the cell cycle is positively regulated by a complex composed of Cdc2 (CDK1) and B-type cyclin (Nigg, 1995). Cdc25 phosphatases control cell-cycle progression by dephosphorylation and activation of CDK at positions Thr14 and Tyr15. Dephosphorylation of Cdc2 by Cdc25c activates the kinase activity of the Cdc2/cyclin B1 complex, thereby permitting cell entry into mitosis (Gautier et al., 1991). The cellular response to DNA damage
is mediated by evolutionarily conserved Ser/Thr kinases, phosphorylation of Cdc25 phosphatases, binding to 14-3-3 proteins, and exit from the cell cycle. MAPKAP kinase-2 undergoes initial activation in the nucleus with subsequent export of the active kinase to the cytoplasm (Ben-Levy et al., 1998). Thus, MAPKAP kinase-2 is well positioned to function as both a nuclear initiator of Cdc25B/C phosphorylation in response to DNA damage and as a maintenance kinase, which keeps Cdc25B/C inhibited in the cytoplasm (Manke et al., 2005). The present study found that an increase in the phosphorylation of Cdc25C in HeLa cells following irradiation in cordycepin pretreated cells could produce inhibitory effects on MAPKAP kinase-2. CHK1 and CHK2 phosphorylate and inactivate the dual specific phosphatase Cdc25C, which maintains the progression of the G2 phase by dephosphorylating and activating the cyclin-dependent kinase Cdc2 resulting in increased G2/ M arrest (Bernhard et al., 1995). Lee et al. (2010) reported that cordycepin activated p21/WAF1 expression, inducing G2/M phase arrest via a p53-independent pathway. In this report, cordycepin decreased the expression of cyclin B1, Cdc25c, and Cdc2, and increased the protein levels of p21/WAF1. The association of p21/WAF1 and Cdc2 also increased in a cordycepin-treated human colorectal cell line. However, cordycepin had no effect on the expression of either p27 or p53. Several studies have demonstrated the anti-cancer effects of cordycepin in different cell lines (Lee et al., 2009; Wu et al., 2007; Lee et al., 2013b). The mechanism underlying cordycepin-medicated cell death differs according to the cell type. Our findings indicated that the radiosensitising effect of cordycepin on two cervical cancer cell lines was not mediated by a single effect, but reflected of various activities that were primarily based on the induction of p53-mediated apoptosis and modulation of cell cycle checkpoint protein expression. Although future studies are warranted to elucidate the mechanisms underlying these different effects, the present results suggest that cordycepin may be a candidate radiosensitising agent in cervical cancer treatment.
Acknowledgement This work was supported by the National Research Foundation of Korea (NRF), Republic of Korea (2009–0075292 and 2012R1A1A2043237).
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