Inhibitory effects of autologous γ-irradiated cell conditioned medium on osteoblasts in vitro

Li,Xu‑Fang; Zhu,Guo‑Ying; Wang,Jian‑Ping; Wang,Yu

doi:10.3892/mmr.2015.3354

Inhibitory effects of autologous γ-irradiated cell conditioned medium on osteoblasts in vitro

Authors:
- Xu‑Fang Li
- Guo‑Ying Zhu
- Jian‑Ping Wang
- Yu Wang
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Affiliations: Department of Environmental Epidemiology and Bone Toxicology, Institute of Radiation Medicine, Fudan University, Shanghai 200032, P.R. China
Published online on: February 16, 2015 https://doi.org/10.3892/mmr.2015.3354
Pages: 273-280

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Abstract

Skeletal complications from radiation therapy have been reported in patients with breast, brain and pelvic cancer, and types of blood cancer. However, it remains to be elucidated whether localized radiotherapy may result in systemic adverse effects on the unirradiated skeleton through an abscopal mechanism. The present study investigated the abscopal effect of radiation on osteoblasts mediated by autologous γ‑irradiated cell conditioned medium. Osteoblasts obtained from calvarial bones were incubated with irradiated cell conditioned medium (ICCM) and changes in cell viability, alkaline phosphatase (ALP) activity, mineralization ability, cell apoptosis and the gene expression levels of ALP, osteocalcin (BGP), osteoprotegerin (OPG), receptor activator of nuclear factor‑κB ligand (RANKL) and caspase 3 were observed. Notably, ICCM regulated osteoblast function, inhibiting viability and differentiation, resulting in apoptosis or cell death. ICCM at 10 or 20%, from osteoblasts irradiated with 10 Gy γ‑rays, significantly inhibited the proliferation of osteoblastic cells (P<0.001). In addition, an increase in apoptosis was noted in the osteoblasts incubated with ICCM at 40% with increasing doses of radiation, accompanied by an upregulation in the mRNA expression of caspase 3. In addition, ICCM at 20% inhibited the ALP activity in the 5 and 10 Gy groups and osteoblast mineralization, particularly at 10 Gy ICCM. Additionally, the mRNA expression levels of ALP, BGP, OPG and RANKL of the cells treated with ICCM at 20% were downregulated significantly compared with those treated with medium from unirradiated cells. The present study provided novel evidence to elucidate radiation‑therapy‑associated side effects on the skeleton.

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1	Bentzen SM: Preventing or reducing late side effects of radiation therapy: radiobiology meets molecular pathology. Nat Rev Cancer. 6:702–713. 2006. View Article : Google Scholar : PubMed/NCBI
2	Robbins ME and Zhao W: Chronic oxidative stress and radiation-induced late normal tissue injury: a review. Int J Radiat Biol. 80:251–259. 2004. View Article : Google Scholar : PubMed/NCBI
3	Baxter NN, Habermann EB, Tepper JE, Durham SB and Virnig BA: Risk of pelvic fractures in older women following pelvic irradiation. JAMA. 294:2587–2593. 2005. View Article : Google Scholar : PubMed/NCBI
4	Abe H, Nakamura M, Takahashi S, Maruoka S, Ogawa Y and Sakamoto K: Radiation-induced insufficiency fractures of the pelvis: evaluation with 99mTc-methylene diphosphonate scintigraphy. AJR Am J Roentgenol. 158:599–602. 1992. View Article : Google Scholar : PubMed/NCBI
5	Blomlie V, Rofstad EK, Talle K, Sundfør K, Winderen M and Lien HH: Incidence of radiation-induced insufficiency fractures of the female pelvis: evaluation with MR imaging. AJR Am J Roentgenol. 167:1205–1210. 1996. View Article : Google Scholar : PubMed/NCBI
6	Van Staa TP, Leufkens HG, Abenhaim L, Zhang B and Cooper C: Use of oral corticosteroids and risk of fractures. June, 2000. J Bone Miner Res. 20:1487–1494. 2005. View Article : Google Scholar : PubMed/NCBI
7	Nelson HD, Helfand M, Woolf SH and Allan JD: Screening for postmenopausal osteoporosis: a review of the evidence for the U.S. Preventive Services Task Force. Ann Intern Med. 137:529–541. 2002. View Article : Google Scholar : PubMed/NCBI
8	Zuppinger A and Minder W: Calcium uptake in irradiated bone. United Nations International Conference on the Peaceful Uses of Atomic Energy; United Nations, Geneva, Switzerland. 22. pp. p2471959
9	Pappas AM and Cohen J: The abscopal effect of X-irradiation on bone growth in rats. J Bone Joint Surg. 45A:765–772. 1963.
10	Jia D, Gaddy D, Suva LJ and Corry PM: Rapid loss of bone mass and strength in mice after abdominal irradiation. Radiat Res. 176:624–635. 2011. View Article : Google Scholar : PubMed/NCBI
11	Chen Z, Maricic M, Aragaki AK, et al: Fracture risk increases after diagnosis of breast or other cancers in postmenopausal women: results from the Women’s Health Initiative. Osteoporos Int. 20:527–536. 2009. View Article : Google Scholar
12	Hinoi E, Fujimori S, Takemori A and Yoneda Y: Cell death by pyruvate deficiency in proliferative cultured calvarial osteoblasts. Biochem Biophys Res Commun. 294:1177–1183. 2002. View Article : Google Scholar : PubMed/NCBI
13	Hansen MB, Nielsen SE and Berg K: Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J Immunol Methods. 119:203–210. 1989. View Article : Google Scholar : PubMed/NCBI
14	Kirsch T, Nah HD, Shapiro IM and Pacifici M: Regulated production of mineralization-competent matrix vesicles in hypertrophic chondrocytes. J Cell Biol. 137:1149–1160. 1997. View Article : Google Scholar : PubMed/NCBI
15	Schmittgen TD and Livak KJ: Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 3:1101–1108. 2008. View Article : Google Scholar : PubMed/NCBI
16	Dare A, Hachisu R, Yamaguchi A, Yokose S, Yoshiki S and Okano T: Effects of ionizing radiation on proliferation and differentiation of osteoblast-like cells. J Dent Res. 76:658–664. 1997. View Article : Google Scholar : PubMed/NCBI
17	Aksnes LH and Bruland ØS: Some musculo-skeletal sequelae in cancer survivors. Acta Oncol. 46:490–496. 2007. View Article : Google Scholar : PubMed/NCBI
18	Jia D, Koonce NA, Griffin RJ, Jackson C and Corry PM: Prevention and mitigation of acute death of mice after abdominal irradiation by the antioxidant N-acetyl-cysteine (NAC). Radiat Res. 173:579–589. 2010. View Article : Google Scholar : PubMed/NCBI
19	Travis EL: The sequence of histological changes in mouse lungs after single doses of x-rays. Int J Radiat Oncol Biol Phys. 6:345–347. 1980. View Article : Google Scholar : PubMed/NCBI
20	Hopewell JW: Radiation-therapy effects on bone density. Med Pediatr Oncol. 41:208–211. 2003. View Article : Google Scholar : PubMed/NCBI
21	Zhou G, Gu G, Li Y, et al: Effects of cerium oxide nanoparticles on the proliferation, differentiation, and mineralization function of primary osteoblasts in vitro. Biol Trace Elem Res. 153:411–418. 2013. View Article : Google Scholar : PubMed/NCBI
22	Lyng FM, Seymour CB and Mothersill C: Early events in the apoptotic cascade initiated in cells treated with medium from the progeny of irradiated cells. Radiat Prot Dosimetry. 99:169–172. 2002. View Article : Google Scholar : PubMed/NCBI
23	Lehnert BE, Goodwin EH and Deshpande A: Extracellular factor(s) following exposure to alpha particles can cause sister chromatid exchanges in normal human cells. Cancer Res. 57:2164–2171. 1997.PubMed/NCBI
24	Lyng FM, Seymour CB and Mothersill C: Production of a signal by irradiated cells which leads to a response in unirradiated cells characteristic of initiation of apoptosis. Br J Cancer. 83:1223–1230. 2000. View Article : Google Scholar : PubMed/NCBI
25	Li P, Nijhawan D, Budihardjo I, et al: Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 91:479–489. 1997. View Article : Google Scholar : PubMed/NCBI
26	Collett WK, Watson JA and Wald N: Abscopal and direct effects on calcium mobilization, alkaline phosphatase levels, and dentin formation following x-irradiation of either the rat incisor or the thyroid-parathyroid region. J Dent Res. 45:1529–1538. 1966. View Article : Google Scholar : PubMed/NCBI
27	Montour JL: Abscopal radiation damage to chick thymus and bursa of Fabricius. Acta Radiol Ther Phys Biol. 10:150–160. 1971. View Article : Google Scholar : PubMed/NCBI
28	Nobler MP: The abscopal effect in malignant lymphoma and its relationship to lymphocyte circulation. Radiology. 93:410–412. 1969. View Article : Google Scholar : PubMed/NCBI
29	Van der Meeren A, Monti P, Vandamme M, Squiban C, Wysocki J and Griffiths N: Abdominal radiation exposure elicits inflammatory responses and abscopal effects in the lungs of mice. Radiat Res. 163:144–152. 2005. View Article : Google Scholar : PubMed/NCBI
30	Kurnick NB and Nokay N: Abscopal effects and bone-marrow repop-ulation in man and mouse. Ann N Y Acad Sci. 114:528–537. 1964. View Article : Google Scholar : PubMed/NCBI
31	Conard RA: Indirect Effect of X-radiation on bone growth in rats. Ann N Y Acad Sci. 114:335–338. 1964. View Article : Google Scholar : PubMed/NCBI
32	Mothersill C, Rea D, Wright EG, et al: Individual variation in the production of a ‘bystander signal’ following irradiation of primary cultures of normal human urothelium. Carcinogenesis. 22:1465–1471. 2001. View Article : Google Scholar : PubMed/NCBI
33	Azzam EI, de Toledo SM, Gooding T and Little JB: Intercellular communication is involved in the bystander regulation of gene expression in human cells exposed to very low fluences of alpha particles. Radiat Res. 150:497–504. 1998. View Article : Google Scholar : PubMed/NCBI
34	Azzam EI, de Toledo SM, Waker AJ and Little JB: High and low fluences of alpha-particles induce a G1 checkpoint in human diploid fibroblasts. Cancer Res. 60:2623–2631. 2000.PubMed/NCBI
35	Ghandhi SA, Yaghoubian B and Amundson SA: Global gene expression analyses of bystander and alpha particle irradiated normal human lung fibroblasts: synchronous and differential responses. BMC Med Genomics. 1:632008. View Article : Google Scholar : PubMed/NCBI
36	Belyakov OV, Folkard M, Mothersill C, Prise KM and Michael BD: A proliferation-dependent bystander effect in primary porcine and human urothelial explants in response to targeted irradiation. Br J Cancer. 88:767–774. 2003. View Article : Google Scholar : PubMed/NCBI
37	Lyng FM, Howe OL and McClean B: Reactive oxygen species-induced release of signalling factors in irradiated cells triggers membrane signalling and calcium influx in bystander cells. Int J Radiat Biol. 87:683–695. 2011. View Article : Google Scholar : PubMed/NCBI
38	Lyng FM, Maguire P, McClean B, Seymour C and Mothersill C: The involvement of calcium and MAP kinase signaling pathways in the production of radiation-induced bystander effects. Radiat Res. 165:400–409. 2006. View Article : Google Scholar : PubMed/NCBI
39	Shao C, Folkard M and Prise KM: Role of TGF-beta1 and nitric oxide in the bystander response of irradiated glioma cells. Oncogene. 27:434–440. 2008. View Article : Google Scholar
40	Facoetti A, Ballarini F, Cherubini R, et al: Gamma ray-induced bystander effect in tumour glioblastoma cells: a specific study on cell survival, cytokine release and cytokine receptors. Radiat Prot Dosimetry. 122:271–274. 2006. View Article : Google Scholar
41	Facoetti A, Mariotti L, Ballarini F, et al: Experimental and theoretical analysis of cytokine release for the study of radiation-induced bystander effect. Int J Radiat Biol. 85:690–699. 2009. View Article : Google Scholar : PubMed/NCBI
42	Mothersill C and Seymour CB: Cell-cell contact during gamma irradiation is not required to induce a bystander effect in normal human keratinocytes: evidence for release during irradiation of a signal controlling survival into the medium. Radiat Res. 149:256–262. 1998. View Article : Google Scholar : PubMed/NCBI
43	Mothersill C, Saroya R, Smith RW, Singh H and Seymour CB: Serum serotonin levels determine the magnitude and type of bystander effects in medium transfer experiments. Radiat Res. 174:119–123. 2010. View Article : Google Scholar : PubMed/NCBI