Inhibition of DNA-PKcs enhances radiosensitivity and increases the levels of ATM and ATR in NSCLC cells exposed to carbon ion irradiation

Yang,Lina; Liu,Yuanyuan; Sun,Chao; Yang,Xinrui; Yang,Zhen; Ran,Juntao; Zhang,Qiuning; Zhang,Hong; Wang,Xinyu; Wang,Xiaohu

doi:10.3892/ol.2015.3730

Inhibition of DNA-PKcs enhances radiosensitivity and increases the levels of ATM and ATR in NSCLC cells exposed to carbon ion irradiation

Authors:
- Lina Yang
- Yuanyuan Liu
- Chao Sun
- Xinrui Yang
- Zhen Yang
- Juntao Ran
- Qiuning Zhang
- Hong Zhang
- Xinyu Wang
- Xiaohu Wang
View Affiliations

Affiliations: Institute of Cell Biology, School of Life Sciences, Lanzhou University, Lanzhou, Gansu 730000, P.R. China, Department of Heavy Ion Radiation Medicine, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, Gansu 730000, P.R. China, Department of Radiotherapy, Gansu Province Tumor Hospital, Lanzhou, Gansu 730005, P.R. China
Published online on: September 18, 2015 https://doi.org/10.3892/ol.2015.3730
Pages: 2856-2864
Copyright: © Yang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Non-small cell lung cancer (NSCLC) exhibits radioresistance to conventional rays, due to its DNA damage repair systems. NSCLC may potentially be sensitized to radiation treatment by reducing those factors that continuously enhance the repair of damaged DNA. In the present study, normal lung fibroblast MRC‑5 and lung cancer A549 cells were treated with NU7026 and CGK733, which are inhibitors of the DNA‑dependent protein kinase catalytic subunit (PKcs) and ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3‑related (ATR), respectively, followed by exposure to X‑rays and carbon ion irradiation. The cytotoxic activity, cell survival rate, DNA damage repair ability, cell cycle arrest and apoptosis rate of the treated cells were analyzed with MTT assay, colony formation assay, immunofluorescence and flow cytometry, respectively. The transcription and translation levels of the ATM, ATR and DNA‑PKcs genes were detected by reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. The results indicated that the radiosensitivity and DNA repair ability of A549 cells were reduced, and the percentages of apoptotic cells and those arrested at the G2/M phase of the cell cycle were significantly increased, following ionizing radiation with inhibitor‑pretreatment. The expression levels of ATM, ATR, DNA‑PKcs and phosphorylated histone H2AX, a biomarker for DNA double‑strand breaks, were all upregulated at the transcriptional or translational level in A549 cells treated with carbon ion irradiation, compared with the control and X‑rays‑treated cells. In addition, the treatment with 5‑50 µM NU7026 or CGK733 did not produce any obvious cytotoxicity in MRC‑5 cells, and the effect of the DNA‑PKcs‑inhibitor on enhancing the radiosensitivity of A549 cells was stronger than that observed for the ATM and ATR‑inhibitor. These findings demonstrated a minor role for ATM and ATR in radiation‑induced cell death, since the upregulation of ATM and ATR did not rescue the A549 cells subjected to ionizing irradiation. Therefore, future studies on DNA-PKcs, ATM and ATR may lead to novel specific therapies that supplement general radiotherapy for the treatment of lung cancer.

View Figures

View References

1	Siegel R, DeSantis C, Virgo K, Stein K, Mariotto A, Smith T, Cooper D, Gansler T, Lerro C, Fedewa S, et al: Cancer treatment and survivorship statistics, 2012. CA Cancer J Clin. 62:220–241. 2012. View Article : Google Scholar : PubMed/NCBI
2	Jemal A, Siegel R, Xu J and Ward E: Cancer statistics, 2010. CA Cancer J Clin. 60:277–300. 2010. View Article : Google Scholar : PubMed/NCBI
3	Siegel R, Naishadham D and Jemal A: Cancer statistics, 2012. CA Cancer J Clin. 62:10–29. 2012. View Article : Google Scholar : PubMed/NCBI
4	Yang HJ, Kim N, Seong KM, Youn H and Youn B: Investigation of radiation-induced transcriptome profile of radioresistant non-small cell lung cancer A549 cells using RNA-seq. PLoS One. 8:e593192013. View Article : Google Scholar : PubMed/NCBI
5	Zhang W, Wang J, Tang M, Pan J, Bai P, Lin D, Qian F, Lin F, Yang X and Zhang S: Quantitative study of lung perfusion SPECT scanning and pulmonary function testing for early radiation-induced lung injury in patients with locally advanced non-small cell lung cancer. Exp Ther Med. 3:631–635. 2012.PubMed/NCBI
6	Hamada N, Imaoka T, Masunaga S, Ogata T, Okayasu R, Takahashi A, Kato TA, Kobayashi Y, Ohnishi T, Ono K, et al: Recent advances in the biology of heavy-ion cancer therapy. J Radiat Res (Tokyo). 51:365–383. 2010. View Article : Google Scholar
7	Janssens GO, Rademakers SE, Terhaard CH, Doornaert PA, Bijl HP, van den Ende P, Chin A, Marres HA, de Bree R, van der Kogel AJ, et al: Accelerated radiotherapy with carbogen and nicotinamide for laryngeal cancer: Results of a phase III randomized trial. J Clin Oncol. 30:1777–1783. 2012. View Article : Google Scholar : PubMed/NCBI
8	Bussink J, van der Kogel AJ and Kaanders JHAM: Activation of the PI3K/Akt pathway and implications for radioresistance mechanisms in head and neck cancer. Lancet Oncol. 9:288–296. 2008. View Article : Google Scholar : PubMed/NCBI
9	Nagelkerke A, Bussink J, van der Kogel AJ, Sweep FCGJ and Span PN: The PERK/ATF4/LAMP3-arm of the unfolded protein response affects radioresistance by interfering with the DNA damage response. Radiother Oncol. 108:415–421. 2013. View Article : Google Scholar : PubMed/NCBI
10	Serra V, Markman B, Scaltriti M, Eichhorn PJ, Valero V, Guzman M, Botero ML, Llonch E, Atzori F, Di Cosimo S, et al: NVP-BEZ235, a dual PI3K/mTOR inhibitor, prevents PI3K signaling and inhibits the growth of cancer cells with activating PI3K mutations. Cancer Res. 68:8022–8030. 2008. View Article : Google Scholar : PubMed/NCBI
11	Yajima H, Lee K-J, Zhang S, Kobayashi J and Chen BPC: DNA double-strand break formation upon UV-induced replication stress activates ATM and DNA-PKcs kinases. J Mol Biol. 385:800–810. 2009. View Article : Google Scholar : PubMed/NCBI
12	Zhuang W, Li B, Long L, Chen L, Huang Q and Liang Z: Induction of autophagy promotes differentiation of glioma-initiating cells and their radiosensitivity. Int J Cancer. 129:2720–2731. 2011. View Article : Google Scholar : PubMed/NCBI
13	Stiff T, ODriscoll M, Rief N, Iwabuchi K, Löbrich M and Jeggo PA: ATM and DNA-PK function redundantly to phosphorylate H2AX after exposure to ionizing radiation. Cancer Res. 64:2390–2396. 2004. View Article : Google Scholar : PubMed/NCBI
14	Ghosh S, Narang H, Sarma A and Krishna M: DNA damage response signaling in lung adenocarcinoma A549 cells following γ and carbon beam irradiation. Mutat Res. 716:10–19. 2011. View Article : Google Scholar : PubMed/NCBI
15	Okayasu R, Okada M, Okabe A, et al: Repair of DNA damage induced by accelerated heavy ions in mammalian cells proficient and deficient in the non-homologous end-joining pathway. Radiat Res. 165:59–67. 2006. View Article : Google Scholar : PubMed/NCBI
16	Vaucher RA, da Motta AS and Brandelli A: Evaluation of the in vitro cytotoxicity of the antimicrobial peptide P34. Cell Biol Int. 34:317–323. 2010. View Article : Google Scholar : PubMed/NCBI
17	Li Y, Song J, Yang P, Zou R, Fan X and Zhao Z: Establishment of a three-dimensional culture and mechanical loading system for skeletal myoblasts. Cell Biol Int. 33:192–198. 2009. View Article : Google Scholar : PubMed/NCBI
18	Shen H, Schultz M, Kruh GD and Tew KD: Increased expression of DNA-dependent protein kinase confers resistance to adriamycin. Biochim Biophys Acta. 1381:131–138. 1998. View Article : Google Scholar : PubMed/NCBI
19	Kantor AF, Li FP, Janov AJ, Tarbell NJ and Sallan SE: Hypertension in long-term survivors of childhood renal cancers. J Clin Oncol. 7:912–915. 1989.PubMed/NCBI
20	Vandersickel V, Depuydt J, Van Bockstaele B, Perletti G, Philippe J, Thierens H and Vral A: Early increase of radiation-induced γH2AX foci in a human Ku70/80 knockdown cell line characterized by an enhanced radiosensitivity. J Radiat Res (Tokyo). 51:633–641. 2010. View Article : Google Scholar
21	Yang J, Xu ZP, Huang Y, Hamrick HE, Duerksen-Hughes PJ and Yu YN: ATM and ATR: Sensing DNA damage. World J Gastroenterol. 10:155–160. 2004.PubMed/NCBI
22	Hada M and Georgakilas AG: Formation of clustered DNA damage after high-LET irradiation: A review. J Radiat Res (Tokyo). 49:203–210. 2008. View Article : Google Scholar
23	Wang C and Lees-Miller SP: Detection and repair of ionizing radiation-induced DNA double strand breaks: New developments in nonhomologous end joining. Int J Radiat Oncol Biol Phys. 86:440–449. 2013. View Article : Google Scholar : PubMed/NCBI
24	Mukherjee B, Kessinger C, Kobayashi J, et al: DNA-PK phosphorylates histone H2AX during apoptotic DNA fragmentation in mammalian cells. DNA Repair (Amst). 5:575–590. 2006. View Article : Google Scholar : PubMed/NCBI
25	Durocher D and Jackson SP: DNA-PK, ATM and ATR as sensors of DNA damage: Variations on a theme? Curr Opin Cell Biol. 13:225–231. 2001. View Article : Google Scholar : PubMed/NCBI
26	Kim JS, Krasieva TB, Kurumizaka H, et al: Independent and sequential recruitment of NHEJ and HR factors to DNA damage sites in mammalian cells. J Cell Biol. 170:341–347. 2005. View Article : Google Scholar : PubMed/NCBI
27	Wang H, Wang M, Wang H, Böcker W and Iliakis G: Complex H2AX phosphorylation patterns by multiple kinases including ATM and DNA-PK in human cells exposed to ionizing radiation and treated with kinase inhibitors. J Cell Physiol. 202:492–502. 2005. View Article : Google Scholar : PubMed/NCBI
28	Yang J, Yu Y, Hamrick HE and Duerksen-Hughes PJ: ATM, ATR and DNA-PK: Initiators of the cellular genotoxic stress responses. Carcinogenesis. 24:1571–1580. 2003. View Article : Google Scholar : PubMed/NCBI
29	Löbrich M, Shibata A, Beucher A, et al: γH2AX foci analysis for monitoring DNA double-strand break repair: Strengths, limitations and optimization. Cell Cycle. 9:662–669. 2010. View Article : Google Scholar : PubMed/NCBI
30	Falck J, Coates J and Jackson SP: Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage. Nature. 434:605–611. 2005. View Article : Google Scholar : PubMed/NCBI
31	Takahashi A and Ohnishi T: Does γH2AX foci formation depend on the presence of DNA double strand breaks? Cancer Lett. 229:171–179. 2005. View Article : Google Scholar : PubMed/NCBI
32	Fernandez-Capetillo O, Chen HT, Celeste A, Ward I, Romanienko PJ, Morales JC, Naka K, Xia Z, Camerini-Otero RD, Motoyama N, et al: DNA damage-induced G2-M checkpoint activation by histone H2AX and 53BP1. Nat Cell Biol. 4:993–997. 2002. View Article : Google Scholar : PubMed/NCBI
33	Stucki M, Stagljar I, Jónsson ZO and Hübscher U: A coordinated interplay: Proteins with multiple functions in DNA replication, DNA repair, cell cycle/checkpoint control, and transcription. Prog Nucleic Acid Res Mol Biol. 65:261–298. 2001. View Article : Google Scholar : PubMed/NCBI
34	Ferguson DO, Sekiguchi JM, Frank KM, Gao Y, Sharpless NE, Gu Y, Manis J, DePinho RA and Alt FW: The interplay between nonhomologous end-joining and cell cycle checkpoint factors in development, genomic stability, and tumorigenesis. Cold Spring Harb Symp Quant Biol. 65:395–403. 2000. View Article : Google Scholar : PubMed/NCBI
35	Hong J, Zhang Z, Lv W, Zhang M, Chen C, Yang S, Li S, Zhang L, Han D and Zhang W: Icaritin synergistically enhances the radiosensitivity of 4T1 breast cancer cells. PLoS One. 8:e713472013. View Article : Google Scholar : PubMed/NCBI
36	Yang CJ, Wang CS, Hung JY, Huang HW, Chia YC, Wang PH, Weng CF and Huang MS: Pyrogallol induces G₂-M arrest in human lung cancer cells and inhibits tumor growth in an animal model. Lung Cancer. 66:162–168. 2009. View Article : Google Scholar : PubMed/NCBI
37	Sun Y, Sun D, Li F, Tian L, Li C, Li L, Lin R and Wang S: Downregulation of Sirt1 by antisense oligonucleotides induces apoptosis and enhances radiation sensitization in A549 lung cancer cells. Lung Cancer. 58:21–29. 2007. View Article : Google Scholar : PubMed/NCBI
38	Zangemeister-Wittke U and Hopkins-Donaldson S: Apoptosis regulation and drug resistance in malignant pleural mesothelioma. Lung Cancer. 49(Suppl 1): S105–S108. 2005. View Article : Google Scholar : PubMed/NCBI
39	Williams TM, Nyati S, Ross BD and Rehemtulla A: Molecular imaging of the ATM kinase activity. Int J Radiat Oncol Biol Phys. 86:969–977. 2013. View Article : Google Scholar : PubMed/NCBI