Dose-dependent inhibitory effects of zoledronic acid on osteoblast viability and function in vitro

Huang,Xin; Huang,Shilong; Guo,Fengjin; Xu,Fei; Cheng,Peng; Ye,Yaping; Dong,Yonghui; Xiang,Wei; Chen,Anmin

doi:10.3892/mmr.2015.4627

Dose-dependent inhibitory effects of zoledronic acid on osteoblast viability and function in vitro

Authors:
- Xin Huang
- Shilong Huang
- Fengjin Guo
- Fei Xu
- Peng Cheng
- Yaping Ye
- Yonghui Dong
- Wei Xiang
- Anmin Chen
View Affiliations

Affiliations: Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China
Published online on: November 30, 2015 https://doi.org/10.3892/mmr.2015.4627
Pages: 613-622
Copyright: © Huang 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

Zoledronic acid (ZA), which is one of the most potent and efficacious bisphosphonates, has been commonly used in clinical practice for the treatment of various bone disorders. The extensive use of ZA has been associated with increasing occurrence of jaw complications, now known as bisphosphonate‑associated osteonecrosis of the jaw (BRONJ). However, the mechanism underlying BRONJ remains to be fully elucidated. The aim of the present study was to investigate the effects of different concentrations of ZA on the MC3T3‑E1 murine preosteoblast cell line cells and examine the possible pathogenesis of BRONJ. In the present study, the effect of ZA on the viability, apoptosis, differentiation and maturation of MC3T3‑E1 cells, as well as its relevant molecular mechanism, were examined The results of a Cell Counting Kit 8 assay, a flow cytometric Annexin‑V/propidium iodide assay and western blot analysis demonstrated that ZA exhibited a significant inhibition of cell viability and induction of apoptosis at concentrations >10 µM. Subsequently, the effect of ZA on cell differentiation at concentrations <1 µM were investigated. In this condition, ZA inhibited bone nodule formation and decreased the activity of alkaline phosphatase. The results of reverse transcription-quantitative polymerase chain reaction and western blot analyses indicated that ZA downregulated the expression levels of the marker genes and proteins associated with osteogenic differentiation. Further investigation revealed that the suppression of differentiation by ZA was associated with decreased expression of bone morphogenetic protein‑2 (BMP‑2) and downregulation of the phosphorylation levels in the downstream extracellular signal‑regulated kinase 1/2 and p38 pathways. These adverse effects of ZA were observed to be concentration‑dependent. The results from the present study suggested that ZA at higher concentrations induces cytotoxicity towards osteoblasts, and ZA at lower concentrations suppresses osteoblast differentiation by downregulation of BMP-2. These results assist in further understanding the mechanisms of BRONJ.

View Figures

View References

1	Bone HG, Hosking D, Devogelaer JP, Tucci JR, Emkey RD, Tonino RP, Rodriguez-Portales JA, Downs RW, Gupta J, Santora AC, et al: Ten years' experience with alendronate for osteoporosis in postmenopausal women. N Engl J Med. 350:1189–1199. 2004. View Article : Google Scholar : PubMed/NCBI
2	Aapro M, Abrahamsson PA, Body JJ, Coleman RE, Colomer R, Costa L, Crinò L, Dirix L, Gnant M, Gralow J, et al: Guidance on the use of bisphosphonates in solid tumours: recommendations of an international expert panel. Ann Oncol. 19:420–432. 2008. View Article : Google Scholar
3	Russell RG: Bisphosphonates: mode of action and pharmacology. Pediatrics. 119(Suppl 2): S150–S162. 2007. View Article : Google Scholar : PubMed/NCBI
4	Dunford JE, Rogers MJ, Ebetino FH, Phipps RJ and Coxon FP: Inhibition of protein prenylation by bisphosphonates causes sustained activation of Rac, Cdc42 and Rho GTPases. J Bone Miner Res. 21:684–694. 2006. View Article : Google Scholar : PubMed/NCBI
5	Lawson MA, Xia Z, Barnett BL, Triffitt JT, Phipps RJ, Dunford JE, Locklin RM, Ebetino FH and Russell RG: Differences between bisphosphonates in binding affinities for hydroxyapatite. J Biomed Mater Res B Appl Biomater. 92:149–155. 2010. View Article : Google Scholar
6	Vestergaard P, Schwartz K, Pinholt EM, Rejnmark L and Mosekilde L: Gastric and esophagus events before and during treatment of osteoporosis. Calcif Tissue Int. 86:110–115. 2010. View Article : Google Scholar
7	Coleman R, Burkinshaw R, Winter M, Neville-Webbe H, Lestera J, Woodward E and Brown J: Zoledronic acid. Expert Opin Drug Saf. 10:133–145. 2011. View Article : Google Scholar
8	Marx RE: Pamidronate (Aredia) and zoledronate (Zometa) induced avascular necrosis of the jaws: A growing epidemic. J Oral Maxillofac Surg. 61:1115–1117. 2003. View Article : Google Scholar : PubMed/NCBI
9	Ruggiero SL: Bisphosphonate-related osteonecrosis of the jaw: an overview. Ann N Y Acad Sci. 1218:38–46. 2011. View Article : Google Scholar
10	Ruggiero SL, Dodson TB, Assael LA, Landesberg R, Marx RE and Mehrotra B; American Association of Oral and Maxillofacial Surgeons: American Association of Oral and Maxillofacial Surgeons position paper on bisphosphonate-related osteonecrosis of the jaws - 2009 update. J Oral Maxillofac Surg. 67:2–12. 2009.PubMed/NCBI
11	Diel IJ, Fogelman I, Al-Nawas B, Hoffmeister B, Migliorati C, Gligorov J, Väänänen K, Pylkkänen L, Pecherstorfer M and Aapro MS: Pathophysiology, risk factors and management of bisphosphonate-associated osteonecrosis of the jaw: Is there a diverse relationship of amino- and non-aminobisphosphonates? Crit Rev Oncol Hematol. 64:198–207. 2007. View Article : Google Scholar : PubMed/NCBI
12	Huja SS, Fernandez SA, Phillips C and Li Y: Zoledronic acid decreases bone formation without causing osteocyte death in mice. Arch Oral Biol. 54:851–856. 2009. View Article : Google Scholar : PubMed/NCBI
13	Pozzi S, Vallet S, Mukherjee S, Cirstea D, Vaghela N, Santo L, Rosen E, Ikeda H, Okawa Y, Kiziltepe T and Schoonmaker J: High-dose zoledronic acid impacts bone remodeling with effects on osteoblastic lineage and bone mechanical properties. Clin Cancer Res. 15:5829–5839. 2009. View Article : Google Scholar : PubMed/NCBI
14	Bragdon B, Moseychuk O, Saldanha S, King D, Julian J and Nohe A: Bone morphogenetic proteins: a critical review. Cell Signal. 23:609–620. 2011. View Article : Google Scholar
15	Ten DP, Fu J, Schaap P and Roelen BA: Signal transduction of bone morphogenetic proteins in osteoblast differentiation. J Bone Joint Surg Am. 85-A(Suppl 3): 34–38. 2003.
16	Chen G, Deng C and Li YP: TGF-β and BMP signaling in osteoblast differentiation and bone formation. Int J Biol Sci. 8:272–288. 2012. View Article : Google Scholar
17	Hu Y, Chan E, Wang SX and Li B: Activation of p38 mitogen-activated protein kinase is required for osteoblast differentiation. Endocrinology. 144:2068–2074. 2003. View Article : Google Scholar : PubMed/NCBI
18	Guicheux J, Lemonnier J, Ghayor C, Suzuki A, Palmer G and Caverzasio J: Activation of p38 mitogen-activated protein kinase and c-Jun-NH2-terminal kinase by BMP-2 and their implication in the stimulation of osteoblastic cell differentiation. J Bone Miner Res. 18:2060–2068. 2003. View Article : Google Scholar : PubMed/NCBI
19	Jadlowiec J, Koch H, Zhang X, Campbell PG, Seyedain M and Sfeir C: Phosphophoryn regulates the gene expression and differentiation of NIH3T3, MC3T3-E1 and human mesenchymal stem cells via the integrin/MAPK signaling pathway. J Biol Chem. 279:53323–53330. 2004. View Article : Google Scholar : PubMed/NCBI
20	Nohe A, Keating E, Knaus P and Petersen NO: Signal transduction of bone morphogenetic protein receptors. Cell Signal. 16:291–299. 2004. View Article : Google Scholar
21	Ducy P, Zhang R, Geoffroy V, Ridall AL and Karsenty G: Osf2/Cbfa1: A transcriptional activator of osteoblast differentiation. Cell. 89:747–754. 1997. View Article : Google Scholar : PubMed/NCBI
22	Scheper MA, Badros A, Salama AR, Warburton G, Cullen KJ, Weikel DS and Meiller TF: A novel bioassay model to determine clinically significant bisphosphonate levels. Support Care Cancer. 17:1553–1557. 2009. View Article : Google Scholar : PubMed/NCBI
23	von Knoch F, Jaquiery C, Kowalsky M, Schaeren S, Alabre C, Martin I, Rubash HE and Shanbhag AS: Effects of bisphos-phonates on proliferation and osteoblast differentiation of human bone marrow stromal cells. Biomaterials. 26:6941–6949. 2005. View Article : Google Scholar : PubMed/NCBI
24	Pan B, Farrugia AN, To LB, Findlay DM, Green J, Lynch K and Zannettino AC: The nitrogen-containing bisphosphonate, zoledronic acid, influences RANKL expression in human osteoblast-like cells by activating TNF-alpha converting enzyme (TACE). J Bone Miner Res. 19:147–154. 2004. View Article : Google Scholar : PubMed/NCBI
25	Schindeler A and Little DG: Osteoclasts but not osteoblasts are affected by a calcified surface treated with zoledronic acid. in vitro Biochem Biophys Res Commun. 338:710–716. 2005. View Article : Google Scholar
26	Sudo H, Kodama HA, Amagai Y, Yamamoto S and Kasai S: In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Biol. 96:191–198. 1983. View Article : Google Scholar : PubMed/NCBI
27	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
28	Ding WX, Ni HM, DiFrancesca D, Stolz DB and Yin XM: Bid-dependent generation of oxygen radicals promotes death receptor activation-induced apoptosis in murine hepatocytes. Hepatology. 40:403–413. 2004. View Article : Google Scholar : PubMed/NCBI
29	Niu YB, Li YH, Kong XH, Zhang R, Sun Y, Li Q, Li C, Liu L, Wang J and Mei QB: The beneficial effect of Radix Dipsaci total saponins on bone metabolism in vitro and in vivo and the possible mechanisms of action. Osteoporos Int. 23:2649–2660. 2012. View Article : Google Scholar : PubMed/NCBI
30	Allen MR and Burr DB: Mandible matrix necrosis in beagle dogs after 3 years of daily oral bisphosphonate treatment. J Oral Maxillofac Surg. 66:987–994. 2008. View Article : Google Scholar : PubMed/NCBI
31	Stresing V, Fournier PG, Bellahcene A, Benzaïd I, Mönkkönen H, Colombel M, Ebetino FH, Castronovo V and Clézardin P: Nitrogen-containing bisphosphonates can inhibit angiogenesis in vivo without the involvement of farnesyl pyrophosphate synthase. Bone. 48:259–266. 2011. View Article : Google Scholar
32	Naik NH and Russo TA: Bisphosphonate-related osteonecrosis of the jaw: the role of actinomyces. Clin Infect Dis. 49:1729–1732. 2009. View Article : Google Scholar : PubMed/NCBI
33	Kuhl S, Walter C, Acham S, Pfeffer R and Lambrecht JT: Bisphosphonate-related osteonecrosis of the jaws-a review. Oral Oncol. 48:938–947. 2012. View Article : Google Scholar
34	Hoff AO, Toth BB, Altundag K, Johnson MM, Warneke CL, Hu M, Nooka A, Sayegh G, Guarneri V, Desrouleaux K, et al: Frequency and risk factors associated with osteonecrosis of the jaw in cancer patients treated with intravenous bisphosphonates. J Bone Miner Res. 23:826–836. 2008. View Article : Google Scholar : PubMed/NCBI
35	Raje N, Woo SB, Hande K, Yap JT, Richardson PG, Vallet S, Treister N, Hideshima T, Sheehy N, Chhetri S, et al: Clinical, radiographic and biochemical characterization of multiple myeloma patients with osteonecrosis of the jaw. Clin Cancer Res. 14:2387–2395. 2008. View Article : Google Scholar : PubMed/NCBI
36	Recker RR, Delmas PD, Halse J, Reid IR, Boonen S, García-Hernandez PA, Supronik J, Lewiecki EM, Ochoa L, Miller P, et al: Effects of intravenous zoledronic acid once yearly on bone remodeling and bone structure. J Bone Miner Res. 23:6–16. 2008. View Article : Google Scholar
37	Peter B, Zambelli PY, Guicheux J and Pioletti DP: The effect of bisphosphonates and titanium particles on osteoblasts: An in vitro study. J Bone Joint Surg Br. 87:1157–1163. 2005. View Article : Google Scholar : PubMed/NCBI
38	Orriss IR, Key ML, Colston KW and Arnett TR: Inhibition of osteoblast function in vitro by aminobisphosphonates. J Cell Biochem. 106:109–118. 2009. View Article : Google Scholar
39	Bellido T and Plotkin LI: Novel actions of bisphosphonates in bone: Preservation of osteoblast and osteocyte viability. Bone. 49:50–55. 2011. View Article : Google Scholar
40	Im GI, Qureshi SA, Kenney J, Rubash HE and Shanbhag AS: Osteoblast proliferation and maturation by bisphosphonates. Biomaterials. 25:4105–4115. 2004. View Article : Google Scholar : PubMed/NCBI
41	van der Rest M and Garrone R: Collagen family of proteins. FASEB J. 5:2814–2823. 1991.PubMed/NCBI
42	Owen TA, Holthuis J, Markose E, van Wijnen AJ, Wolfe SA, Grimes SR, Lian JB and Stein GS: Modifications of protein-DNA interactions in the proximal promoter of a cell-growth-regulated histone gene during onset and progression of osteoblast differentiation. Proc Natl Acad Sci USA. 87:5129–5133. 1990. View Article : Google Scholar : PubMed/NCBI
43	Delany AM, Amling M, Priemel M, Howe C, Baron R and Canalis E: Osteopenia and decreased bone formation in osteo-nectin-deficient mice. J Clin Invest. 105:13252000. View Article : Google Scholar
44	Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao YH, Inada M, et al: Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell. 89:755–764. 1997. View Article : Google Scholar : PubMed/NCBI
45	Idris AI, Rojas J, Greig IR, Van't Hof RJ and Ralston SH: Aminobisphosphonates cause osteoblast apoptosis and inhibit bone nodule formation in vitro. Calcif Tissue Int. 82:191–201. 2008. View Article : Google Scholar : PubMed/NCBI
46	Reinholz GG, Getz B, Pederson L, Sanders ES, Subramaniam M, Ingle JN and Spelsberg TC: Bisphosphonates directly regulate cell proliferation, differentiation and gene expression in human osteoblasts. Cancer Res. 60:6001–6007. 2000.PubMed/NCBI
47	Kellinsalmi M, Mönkkönen H, Mönkkönen J, Leskelä HV, Parikka V, Hämäläinen M and Lehenkari P: In vitro comparison of clodronate, pamidronate and zoledronic acid effects on rat osteoclasts and human stem cell-derived osteoblasts. Basic Clin Pharmacol Toxicol. 97:382–391. 2005. View Article : Google Scholar : PubMed/NCBI
48	Sykaras N and Opperman LA: Bone morphogenetic proteins (BMPs): how do they function and what can they offer the clinician? J Oral Sci. 45:57–73. 2003. View Article : Google Scholar : PubMed/NCBI
49	Xiao Y, Haase H, Young WG and Bartold PM: Development and transplantation of a mineralized matrix formed by osteo-blasts in vitro for bone regeneration. Cell Transplant. 13:15–25. 2004. View Article : Google Scholar
50	Zhao M, Harris SE, Horn D, Geng Z, Nishimura R, Mundy GR and Chen D: Bone morphogenetic protein receptor signaling is necessary for normal murine postnatal bone formation. J Cell Biol. 157:1049–1060. 2002. View Article : Google Scholar : PubMed/NCBI
51	Jaiswal RK, Jaiswal N, Bruder SP, Mbalaviele G, Marshak DR and Pittenger MF: Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase. J Biol Chem. 275:9645–9652. 2000. View Article : Google Scholar : PubMed/NCBI
52	Cortizo AM, Lettieri MG, Barrio DA, Mercer N, Etcheverry SB and McCarthy AD: Advanced glycation end-products (AGEs) induce concerted changes in the osteoblastic expression of their receptor RAGE and in the activation of extracellular signal-regulated kinases (ERK). Mol Cell Biochem. 250:1–10. 2003. View Article : Google Scholar : PubMed/NCBI
53	Gallea S, Lallemand F, Atfi A, Rawadi G, Ramez V, Spinella-Jaegle S, Kawai S, Faucheu C, Huet L, Baron R, et al: Activation of mitogen-activated protein kinase cascades is involved in regulation of bone morphogenetic protein-2-induced osteoblast differentiation in pluripotent C2C12 cells. Bone. 28:491–498. 2001. View Article : Google Scholar : PubMed/NCBI