Cytotoxic and apoptotic effects of zoledronic acid on D-17 canine osteosarcoma cell line

Document Type : Full paper (Original article)

Authors

Department of Biochemistry, Faculty of Veterinary Medicine, Aydın Adnan Menderes University, Aydın, Turkey

Abstract

Background: Pets are exposed to a multitude of carcinogenic substances in the modern world. Consequently, high rates of cancer are observed, particularly in middle-aged cats and dogs. Although bisphosphonates have been incorporated into the treatment of human cancers, there are few animal-specific studies. As the number of cancer cases in animals continues to rise, it becomes increasingly important to evaluate anticancer drugs on a species-specific basis. Aims: The present study aimed to examine the impact of zoledronic acid (ZA) on apoptotic pathways in canine osteosarcoma (OSA) cell lines. Methods: The apoptotic effects of ZA administration on D-17 canine OSA cells were analysed by determining apoptotic DNA breaks, caspase-3, -8 and -9 levels by ELISA method and Bax/Bcl-2 ratio by qRT-PCR. The effect of ZA on the colony formation capacity of cells was evaluated by crystal violet staining method. The mineral binding capacity of ZA application in cells was investigated by Alizarin Red S staining technique. The change in alkaline phosphatase enzyme activity in OSA cells due to ZA treatment was determined using the colorimetric method. The antimetastatic effect was determined using the wound healing method, which evaluated the migration potential of cells. Results: While ZA application did not show a significant cytotoxic effect in the cells in the first 24 h, a decrease observed in the viability of the cells depending on the increasing dose and time. Low dose ZA (1, 5, 7.5, 10 µM) concentrations increased mineral content and alkaline phosphatase enzyme activity. A significant decrease was found in the expression levels of survivin, which determines the cell survival, depending on the dose and time. Conclusion: It is expected that our obtained data will contribute to the more effective treatment of the disease by creating different treatment options for clinicians in the light of increasing knowledge in cancer cell biology.

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References

Adams, JM and Cory, S (1998). The Bcl-2 protein family: arbiters of cell survival. Science. 281: 1322-1326.
Ali, NN; Harrison, MA; Rowe, JN and Teich, NM (1993). Spectrum of osteoblastic differentiation in new cell lines derived from spontaneous murine osteosarcomas. Bone. 14: 847-858.
Al-Khan, AA; Nimmo, JS; Tayebi, M; Ryan, SD; Simcock, JO; Tarzi, R; Kuntz, CA; Saad, ES; Day, MJ; Richardson, SJ and Danks, JA (2020). Parathyroid hormone receptor 1 (PTHR1) is a prognostic indicator in canine osteosarcoma. Sci. Rep., 10: 1-10.
Basso, FG; Silveira Turrioni, AP; Hebling, J and De Souza Costa, CA (2013). Zoledronic acid inhibits human osteoblast activities. Gerontology. 59: 534-541.
Boerman, I; Selvarajah, GT; Nielen, M and Kirpensteijn, J (2012). Prognostic factors in canine appendicular osteosarcoma-a meta-analysis. BMC Vet. Res., 8: 56.
Bruzzese, F; Pucci, B; Milone, MR; Ciardiello, C; Franco, R; Chianese, MI; Rocco, M; Di Gennaro, E; Leone, A; Luciano, A; Arra, C; Santini, D; Caraglia, M and Budillon, A (2013). Panobinostat synergizes with zoledronic acid in prostate cancer and multiple myeloma models by increasing ROS and modulating mevalonate and p38-MAPK pathways. Cell Death Dis., 4: e878.
Caraglia, M; Marra, M; Naviglio, S; Botti, G; Addeo, R and Abbruzzese, A (2010). Zoledronic acid: an unending tale for an antiresorptive agent. Expert. Opin. Pharmaco., 11: 141-154.
Cheung, LC; Tickner, J; Hughes, AM; Skut, P; Howlett, M; Foley, B; Oommen, J; Wells, JE; He, B; Singh, S; Chua, GA; Ford, J; Mullighan, CG; Kotecha, RS and Kees, UR (2018). New therapeutic opportunities from dissecting the pre-B leukemia bone marrow micro-environment. Leukemia. 32: 2326-2338.
Christou, A; Ferreira, N and Sophocleous, A (2023). Effects of zoledronic acid on osteosarcoma progression and metastasis: systematic review and meta-analysis. Clin. Exp. Med., 7: 3041-3051.
Conry, RM; Rodriguez, MG and Pressey, JG (2016). Zoledronic acid in metastatic osteosarcoma: encouraging progression free survival in four consecutive patients. Clin. Sarcoma Res., 6: 1-7.
Evdokiou, A; Labrinidis, A; Bouralexis, S; Hay, S and Findlay, DM (2003). Induction of cell death of human osteogenic sarcoma cells by zoledronic acid resembles anoikis. Bone. 33: 216-228.
Farley, JR; Kyeyune-Nyombi, E; Tarbaux, NM; Hall, SL and Strong, DD (1989). Alkaline phosphatase activity from human osteosarcoma cell line SaOS-2: an isoenzyme standard for quantifying skeletal alkaline phosphatase activity in serum. Clin. Chem., 35: 223-229.
Forest, V; Peoc’h, M; Campos, L; Guyotat, D and Vergnon, JM (2005). Effects of cryotherapy or chemotherapy on apoptosis in a non-small-cell lung cancer xenografted into SCID mice. Cryobiology. 50: 29-37.
Fu, D; He, X; Yang, S; Xu, W; Lin, T and Feng, X (2011). Zoledronic acid inhibits vasculogenic mimicry in murine osteosarcoma cell line in vitro. BMC Musculoskelet. Disord., 12: 146.
Ge, XY; Yang, LQ; Jiang, Y; Yang, WW; Fu, J and Li, SL (2014). Reactive oxygen species and autophagy associated apoptosis and limitation of clonogenic survival induced by zoledronic acid in salivary adenoid cystic carcinoma cell line SACC-83. PLoS One. 9: e101207.
Gregory, CA; Gunn, WG; Peister, A and Prockop, DJ (2004). An alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Ana. Biochem., 329: 77-84.
Heymann, D; Ory, B; Blanchard, F; Heymann, MF; Coipeau, P; Charrier, C; Couillaud, S; Thiery, JP; Gouin, F and Redini, F (2005). Enhanced tumor regression and tissue repair when zoledronic acid is combined with ifosfamide in rat osteosarcoma. Bone. 37: 74-86.
Kavya, N; Rao, S; Sathyanarayana, ML; Narayanaswamy, HD; Byregowda, SM; Ranganath, L; Kamaran, A; Purushotham, KM and Kishore, TK (2017). Survivin expression in canine spontaneous cutaneous and subcutaneous tumors and its prognostic importance. Vet. World. 10: 1286-1291.
Koto, K; Murata, H; Kimura, S; Horie, N; Matsui, T; Nishigaki, Y; Ryu, K; Sakabe, T; Itoi, M; Ashihara, E; Maekawa, T; Fushiki, S and Kubo, T (2010). Zoledronic acid inhibits proliferation of human fibrosarcoma cells with induction of apoptosis, and shows combined effects with other anticancer agents. Oncol. Rep., 24: 233-239.
Liu, L; Geng, H; Mei, C and Chen, L (2021). Zoledronic acid enhanced the antitumor effect of cisplatin on orthotopic osteosarcoma by ROS-PI3K/AKT signaling and attenuated osteolysis. Oxid. Med. Cell. Longev., 2021: 6661534.
Livak, KJ and Schmittgen, TD (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods. 25: 402-408.
Nash, LA; Sullivan, PJ; Peters, SJ and Ward, WE (2015). Rooibos flavonoids, orientin and luteolin, stimulate mineralization in human osteoblasts through the Wnt pathway. Mol. Nutr. Food Res., 59: 443-453.
Orriss, IR; Key, ML; Colston, KW and Arnett, TR (2009). Inhibition of osteoblast function in vitro by amino-bisphosphonates. J. Cell Biochem., 106: 109-118.
Ory, B; Rogers, M and Heymann, D (2008). Farnesyl diphosphate synthase is involved in the resistance to zoledronic acid of osteosarcoma cells. J. Cell Mol. Med., 12: 928-941.
Patntirapong, S; Singhatanadgit, W; Chanruangvanit, C; Lavanrattanakul, K and Satravaha, Y (2012). Zoledronic acid suppresses mineralization through direct cytotoxicity and osteoblast differentiation inhibition. J. Oral Pathol. Med., 41: 713-720.
Pautke, C; Schieker, M; Tischer, T; Kolk, A; Neth, P; Mutschler, W and Milz, S (2004). Characterization of osteosarcoma cell lines MG-63, Saos-2 and U-2 OS in comparison to human osteoblasts. Anticancer Res., 24: 3743-3748.
Poirier, VJ; Huelsmeyer, MK; Kurzman, ID; Thamm, DH and Vail, DM (2003). The bisphosphonates alendronate and zoledronate are inhibitors of canine and human osteosarcoma cell growth in vitro. Vet. Comp. Oncol., 1: 207-215.
Poradowski, D; Janus, I; Chrószcz, A and Obmińska-Mrukowicz, B (2021). In vitro studies on the influence of meloxicam on cytotoxic activity induced by risedronate sodium in canine (D-17) and human (U-2 OS) osteo-sarcoma cell lines. Animals. 11: 3135.
Ren, HY; Sun, LL; Li, HY and Ye, ZM (2015). Prognostic significance of serum alkaline phosphatase level in osteosarcoma: A meta-analysis of published data. Biomed.
Res. Int. 2015: 160835.
Rodriguez, LG; Wu, X and Guan, JL (2005). Wound-healing assay. Methods Mol. Biol., 294: 23-29.
Ryu, K; Murata, H; Koto, K; Horie, N; Matsui, T; Nishigaki, Y; Sakabe, T; Takeshita, H; Itoi, M; Kimura, S; Ashihara, E; Maekawa, T; Fushiki, S and Kubo, T (2010). Combined effects of bisphosphonate and radiation on osteosarcoma cells. Anticancer Res., 30: 2713-2720.
Shoeneman, JK; Ehrhart, EJ; Eickhoff, JC; Charles, JB; Powers, BE and Thamm, DH (2012). Expression and function of survivin in canine osteosarcoma. Cancer Res., 72: 249-259.
Smith, AA; Lindley, SE; Almond, GT; Bergman, NS; Matz, BM and Smith, AN (2023). Evaluation of zoledronate for the treatment of canine stage III osteosarcoma: A phase II study. Vet. Med. Sci., 9: 59-67.
Suva, LJ; Cooper, A; Watts, AE; Ebetino, FH; Price, J and Gaddy, D (2021). Bisphosphonates in veterinary medicine: The new horizon for use. Bone. 142: 115711.
Szewczyk, M; Lechowski, R and Zabielska, K (2015). What do we know about canine osteosarcoma treatment? Review. Vet. Res. Commun., 39: 61-67.
Tassone, P; Tagliaferri, P; Viscomi, C; Palmieri, C; Caraglia, M; D’Alessandro, A; Galea, E; Goel, A; Abbruzzese, A; Boland, CR and Venuta, S (2003). Zoledronic acid induces antiproliferative and apoptotic effects in human pancreatic cancer cells in vitro. Br. J. Cancer. 88: 1971-1978.
Tietz, NW; Burtis, CA; Duncan, P; Ervin, K; Petitclerc, CJ; Rinker, AD; Shuey, D and Zygowicz, ER (1983). A reference method for measurement of alkaline phosphatase activity in human serum. Clin. Chem., 29: 751-761.
Tobias, JH; Chow, JWM and Chambers, TJ (1993). 3-Amino-1-hydroxypropylidine-1-bisphosphonate (AHPrBP) suppresses not only the induction of new, but also the persistence of existing bone-forming surfaces in rat cancellous bone. Bone. 14: 619-623.
Uchide, T; Takatsu, N; Fujimori, Y; Fukushima, U and Itoh, H (2005). Expression of survivin mRNA in dog tumors. DNA Seq., 16: 329-334.
Ullén, A; Schwarz, S; Lennartsson, L; Kälkner, KM; Sandström, P; Costa, F; Lennerna, B; Linder, S and Nilsson, S (2009). Zoledronic acid induces caspase-dependent apoptosis in renal cancer cell lines. Scand. J. Urol. Nephrol., 43: 98-103.
Wilson, H; Huelsmeyer, M; Chun, R; Young, KM; Friedrichs, K and Argyle, DJ (2008). Isolation and characterisation of cancer stem cells from canine osteosarcoma. Vet. J., 175: 69-75.
Yu, JQ; Bao, W and Lei, JC (2013). Emodin regulates apoptotic pathway in human liver cancer cells. Phyther. Res., 27: 251-257.
Zhang, R; Thamm, DH and Misra, V (2015). The effect of zhangfei/CREBZF on cell growth, differentiation, apoptosis, migration, and the unfolded protein response in several canine osteosarcoma cell lines. BMC Vet. Res., 11: 1-11.

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