Abstract
Background: Osteosarcoma (OS) is known as the malignant tumors in the bone. Cyanidin 3-OGlucoside (C3G) has a potential to induce the apoptotic cell death in different cancer cells; however, the mechanisms of action for C3G have not been clarified yet.
Objective: In this study, the apoptotic effects of C3G on three different osteosarcoma cell lines including Saso-2, MG-63, and G-292 (clone A141B1) were investigated. Methodology: The 24-hr IC50 of C3G for Saso-2, G-292, and MG-63 cells was evaluated by the MTT assay. Apoptosis induction in these cell lines after treatment with the C3G was approved by the Annexin V/PI flow cytometry. Changes at the mRNA expression level of PPARγ, P21, Bax, and Bcl-xl genes were investigated by real-time Polymerase Chain Reaction (PCR) technique, and P21 expression was further confirmed by the western blotting. Results: The MTT assay results demonstrated that the 24-hr IC50 of C3G was equal to 110μg/ml for Saso-2 and G-292 cells while it was about 140μg/ml for the MG-63 cells. The results of real-time PCR clearly showed that treatment of the cells with 24hrs IC50 of C3G caused the upregulation of PPARγ, P21, and Bax genes. Moreover, western blot analysis confirmed that P21 protein overexpressed endogenously after treatment of the cells with the C3G, and it was more upregulated in the MG-63 cells compared to the other cell lines. Conclusion: According to the findings of the study, the C3G is a novel anti-osteosarcoma agent with the ability to induce the apoptosis in different osteosarcoma cells through upregulation of the PPARγ and P21 genes.Keywords: Osteosarcoma, cyanidin 3-O-glucoside, PPARγ, P21, apoptosis, MTT.
Graphical Abstract
[1]
Mesquita, L.; Mortier, J.; Ressel, L.; Finotello, R.; Silvestrini, P.; Piviani, M. Neoplastic pleural effusion and intrathoracic metastasis of a scapular osteosarcoma in a dog: A multidisciplinary integrated diagnostic approach. Vet. Clin. Pathol., 2017, 46(2), 337-343.
[http://dx.doi.org/10.1111/vcp.12474] [PMID: 28370186]
[http://dx.doi.org/10.1111/vcp.12474] [PMID: 28370186]
[2]
Quaye, A.A.; Raskin, K.A.; Ecker, J.L.; Leffert, L.R. Management of a parturient with high-grade osteosarcoma of the proximal femur: a multidisciplinary approach. Int. J. Obstet. Anesth., 2010, 19(3), 340-342.
[http://dx.doi.org/10.1016/j.ijoa.2010.03.013] [PMID: 20627530]
[http://dx.doi.org/10.1016/j.ijoa.2010.03.013] [PMID: 20627530]
[3]
Raymond, A.K.; Jaffe, N. Osteosarcoma multidisciplinary approach to the management from the pathologist’s perspective.Cancer
Treat. Res.,; , 2009, 152, pp. 63-84.
[http://dx.doi.org/10.1007/978-1-4419-0284-9_4] [PMID: 20213386]
[http://dx.doi.org/10.1007/978-1-4419-0284-9_4] [PMID: 20213386]
[4]
Wittig, J.C.; Bickels, J.; Priebat, D.; Jelinek, J.; Kellar-Graney, K.; Shmookler, B.; Malawer, M.M. Osteosarcoma: A multidisciplinary approach to diagnosis and treatment. Am. Fam. Physician, 2002, 65(6), 1123-1132.
[PMID: 11925089]
[PMID: 11925089]
[5]
Unni, K.K.; Dahlin, D.C. Osteosarcoma: Pathology and classification. Semin. Roentgenol., 1989, 24(3), 143-152.
[http://dx.doi.org/10.1016/0037-198X(89)90010-2] [PMID: 2772662]
[http://dx.doi.org/10.1016/0037-198X(89)90010-2] [PMID: 2772662]
[6]
Burns, B.S.; Edin, M.L.; Lester, G.E.; Tuttle, H.G.; Wall, M.E.; Wani, M.C.; Bos, G.D. Selective drug resistant human osteosarcoma cell lines. Clin. Orthop. Relat. Res., 2001, (383), 259-267.
[http://dx.doi.org/10.1097/00003086-200102000-00030] [PMID: 11210963]
[http://dx.doi.org/10.1097/00003086-200102000-00030] [PMID: 11210963]
[7]
Thomas, D.M.; Johnson, S.A.; Sims, N.A.; Trivett, M.K.; Slavin, J.L.; Rubin, B.P.; Waring, P.; McArthur, G.A.; Walkley, C.R.; Holloway, A.J.; Diyagama, D.; Grim, J.E.; Clurman, B.E.; Bowtell, D.D.; Lee, J.S.; Gutierrez, G.M.; Piscopo, D.M.; Carty, S.A.; Hinds, P.W. Terminal osteoblast differentiation, mediated by runx2 and p27KIP1, is disrupted in osteosarcoma. J. Cell Biol., 2004, 167(5), 925-934.
[http://dx.doi.org/10.1083/jcb.200409187] [PMID: 15583032]
[http://dx.doi.org/10.1083/jcb.200409187] [PMID: 15583032]
[8]
Haydon, R.C.; Luu, H.H.; He, T.C. Osteosarcoma and osteoblastic differentiation: A new perspective on oncogenesis. Clin. Orthop. Relat. Res., 2007, 454(454), 237-246.
[http://dx.doi.org/10.1097/BLO.0b013e31802b683c] [PMID: 17075380]
[http://dx.doi.org/10.1097/BLO.0b013e31802b683c] [PMID: 17075380]
[9]
Carpio, L.; Gladu, J.; Goltzman, D.; Rabbani, S.A. Induction of osteoblast differentiation indexes by PTHrP in MG-63 cells involves multiple signaling pathways. Am. J. Physiol. Endocrinol. Metab., 2001, 281(3), E489-E499.
[http://dx.doi.org/10.1152/ajpendo.2001.281.3.E489] [PMID: 11500304]
[http://dx.doi.org/10.1152/ajpendo.2001.281.3.E489] [PMID: 11500304]
[10]
Nozaki, K.; Kadosawa, T.; Nishimura, R.; Mochizuki, M.; Takahashi, K.; Sasaki, N. 1,25-Dihydroxyvitamin D3, recombinant human transforming growth factor-beta 1, and recombinant human bone morphogenetic protein-2 induce in vitro differentiation of canine osteosarcoma cells. J. Vet. Med. Sci., 1999, 61(6), 649-656.
[http://dx.doi.org/10.1292/jvms.61.649] [PMID: 10423687]
[http://dx.doi.org/10.1292/jvms.61.649] [PMID: 10423687]
[11]
Torchia, J.; Glass, C.; Rosenfeld, M.G. Co-activators and co-repressors in the integration of transcriptional responses. Curr. Opin. Cell Biol., 1998, 10(3), 373-383.
[http://dx.doi.org/10.1016/S0955-0674(98)80014-8] [PMID: 9640539]
[http://dx.doi.org/10.1016/S0955-0674(98)80014-8] [PMID: 9640539]
[12]
Desvergne, B.; Wahli, W. Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocr. Rev., 1999, 20(5), 649-688.
[PMID: 10529898]
[PMID: 10529898]
[13]
Berger, J.; Leibowitz, M.D.; Doebber, T.W.; Elbrecht, A.; Zhang, B.; Zhou, G.; Biswas, C.; Cullinan, C.A.; Hayes, N.S.; Li, Y.; Tanen, M.; Ventre, J.; Wu, M.S.; Berger, G.D.; Mosley, R.; Marquis, R.; Santini, C.; Sahoo, S.P.; Tolman, R.L.; Smith, R.G.; Moller, D.E. Novel Peroxisome Proliferator-Activated Receptor (PPAR) gamma and PPARdelta ligands produce distinct biological effects. J. Biol. Chem., 1999, 274(10), 6718-6725.
[http://dx.doi.org/10.1074/jbc.274.10.6718] [PMID: 10037770]
[http://dx.doi.org/10.1074/jbc.274.10.6718] [PMID: 10037770]
[14]
Huang, Q.; Alvares, K.; Chu, R.; Bradfield, C.A.; Reddy, J.K. Association of peroxisome proliferator-activated receptor and Hsp72. J. Biol. Chem., 1994, 269(11), 8493-8497.
[PMID: 8132576]
[PMID: 8132576]
[15]
Yu, S.; Reddy, J.K. Transcription coactivators for peroxisome proliferator-activated receptors. Biochim. Biophys. Acta, 2007, 1771(8), 936-951.
[http://dx.doi.org/10.1016/j.bbalip.2007.01.008] [PMID: 17306620]
[http://dx.doi.org/10.1016/j.bbalip.2007.01.008] [PMID: 17306620]
[16]
Wagner, E.R.; He, B.C.; Chen, L.; Zuo, G.W.; Zhang, W.; Shi, Q.; Luo, Q.; Luo, X.; Liu, B.; Luo, J.; Rastegar, F.; He, C.J.; Hu, Y.; Boody, B.; Luu, H.H.; He, T.C.; Deng, Z.L.; Haydon, R.C. Therapeutic implications of PPARgamma in human osteosarcoma. PPAR Res., 2010, 2010, 956427
[http://dx.doi.org/10.1155/2010/956427] [PMID: 20182546]
[http://dx.doi.org/10.1155/2010/956427] [PMID: 20182546]
[17]
Mirmalek, S.A.; Azizi, M.A.; Jangholi, E.; Yadollah-Damavandi, S.; Javidi, M.A.; Parsa, Y.; Parsa, T.; Salimi-Tabatabaee, S.A.; Ghasemzadeh Kolagar, H.; Alizadeh-Navaei, R. Cytotoxic and apoptogenic effect of hypericin, the bioactive component of Hypericum perforatum on the MCF-7 human breast cancer cell line. Cancer Cell Int., 2016, 16, 3.
[http://dx.doi.org/10.1186/s12935-016-0279-4] [PMID: 26865836]
[http://dx.doi.org/10.1186/s12935-016-0279-4] [PMID: 26865836]
[18]
Javidi, M.A.; Zolghadr, F.; Babashah, S.; Sadeghizadeh, M. Introducing dendrosomal nanocurcumin as a compound capable of in vitro eliminating undifferentiated stem cells in cell therapy practices. Exp. Clin. Endocrinol. Diabetes, 2015, 123(10), 632-636.
[http://dx.doi.org/10.1055/s-0035-1555775] [PMID: 26179929]
[http://dx.doi.org/10.1055/s-0035-1555775] [PMID: 26179929]
[19]
Mirmalek, S.A.; Jangholi, E.; Jafari, M.; Yadollah-Damavandi, S.; Javidi, M.A.; Parsa, Y.; Parsa, T.; Salimi-Tabatabaee, S.A.; Ghasemzadeh Kolagar, H.; Khazaei Jalil, S.; Alizadeh-Navaei, R. Comparison of in vitro cytotoxicity and apoptogenic activity of magnesium chloride and cisplatin as conventional chemotherapeutic agents in the MCF-7 cell line. Asian Pac. J. Cancer Prev., 2016, 17(S3), 131-134.
[http://dx.doi.org/10.7314/APJCP.2016.17.S3.131] [PMID: 27165250]
[http://dx.doi.org/10.7314/APJCP.2016.17.S3.131] [PMID: 27165250]
[20]
Olivas-Aguirre, F.J.; Rodrigo-García, J.; Martínez-Ruiz, N.D.; Cárdenas-Robles, A.I.; Mendoza-Díaz, S.O.; Álvarez-Parrilla, E.; González-Aguilar, G.A.; de la Rosa, L.A.; Ramos-Jiménez, A.; Wall-Medrano, A. Cyanidin-3-O-glucoside: Physical-chemistry, foodomics and health effects. Molecules, 2016, 21(9), E1264
[http://dx.doi.org/10.3390/molecules21091264] [PMID: 27657039]
[http://dx.doi.org/10.3390/molecules21091264] [PMID: 27657039]
[21]
Courts, C.; Pfaffl, M.W.; Sauer, E.; Parson, W. Pleading for adherence to the MIQE-Guidelines when reporting quantitative PCR data in forensic genetic research. Forensic Sci. Int. Genet., 2019, 42, e21-e24.
[http://dx.doi.org/10.1016/j.fsigen.2019.06.021] [PMID: 31270013]
[http://dx.doi.org/10.1016/j.fsigen.2019.06.021] [PMID: 31270013]
[22]
Bustin, S.A.; Benes, V.; Garson, J.A.; Hellemans, J.; Huggett, J.; Kubista, M.; Mueller, R.; Nolan, T.; Pfaffl, M.W.; Shipley, G.L.; Vandesompele, J.; Wittwer, C.T. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin. Chem., 2009, 55(4), 611-622.
[http://dx.doi.org/10.1373/clinchem.2008.112797] [PMID: 19246619]
[http://dx.doi.org/10.1373/clinchem.2008.112797] [PMID: 19246619]
[23]
Liu, Z.Q.; Mahmood, T.; Yang, P.C. Western blot: Technique, theory and trouble shooting. N. Am. J. Med. Sci., 2014, 6(3), 160.
[http://dx.doi.org/10.4103/1947-2714.128482] [PMID: 24741558]
[http://dx.doi.org/10.4103/1947-2714.128482] [PMID: 24741558]
[24]
Mahmood, T.; Yang, P.C. Western blot: Technique, theory, and trouble shooting. N. Am. J. Med. Sci., 2012, 4(9), 429-434.
[http://dx.doi.org/10.4103/1947-2714.100998] [PMID: 23050259]
[http://dx.doi.org/10.4103/1947-2714.100998] [PMID: 23050259]
[25]
Hosseini, M.M.; Karimi, A.; Behroozaghdam, M.; Javidi, M.A.; Ghiasvand, S.; Bereimipour, A.; Aryan, H.; Nassiri, F.; Jangholi, E. Cytotoxic and apoptogenic effects of cyanidin-3-glucoside on the glioblastoma cell line. World Neurosurg., 2017, 108, 94-100.
[http://dx.doi.org/10.1016/j.wneu.2017.08.133] [PMID: 28867321]
[http://dx.doi.org/10.1016/j.wneu.2017.08.133] [PMID: 28867321]
[26]
Lietti, A.; Forni, G. Studies on Vaccinium myrtillus anthocyanosides. II. Aspects of anthocyanins pharmacokinetics in the rat. Arzneimittelforschung, 1976, 26(5), 832-835.
[PMID: 989354]
[PMID: 989354]
[27]
Sousa, A.; Araújo, P.; Azevedo, J.; Cruz, L.; Fernandes, I.; Mateus, N.; de Freitas, V. Antioxidant and antiproliferative properties of 3-deoxyanthocyanidins. Food Chem., 2016, 192, 142-148.
[http://dx.doi.org/10.1016/j.foodchem.2015.06.108] [PMID: 26304331]
[http://dx.doi.org/10.1016/j.foodchem.2015.06.108] [PMID: 26304331]
[28]
Pratheeshkumar, P.; Son, Y.O.; Wang, X.; Divya, S.P.; Joseph, B.; Hitron, J.A.; Wang, L.; Kim, D.; Yin, Y.; Roy, R.V.; Lu, J.; Zhang, Z.; Wang, Y.; Shi, X. Cyanidin-3-glucoside inhibits UVB-induced oxidative damage and inflammation by regulating MAP kinase and NF-κB signaling pathways in SKH-1 hairless mice skin. Toxicol. Appl. Pharmacol., 2014, 280(1), 127-137.
[http://dx.doi.org/10.1016/j.taap.2014.06.028] [PMID: 25062774]
[http://dx.doi.org/10.1016/j.taap.2014.06.028] [PMID: 25062774]
[29]
Tan, J.; Li, Y.; Hou, D.X.; Wu, S. The effects and mechanisms of cyanidin-3-glucoside and its phenolic metabolites in maintaining intestinal integrity. Antioxidants, 2019, 8(10), 479.
[http://dx.doi.org/10.3390/antiox8100479] [PMID: 31614770]
[http://dx.doi.org/10.3390/antiox8100479] [PMID: 31614770]
[30]
Hainaut, P.; Plymoth, A. Targeting the hallmarks of cancer: Towards a rational approach to next-generation cancer therapy. Curr. Opin. Oncol., 2013, 25(1), 50-51.
[http://dx.doi.org/10.1097/CCO.0b013e32835b651e] [PMID: 23150341]
[http://dx.doi.org/10.1097/CCO.0b013e32835b651e] [PMID: 23150341]
[31]
Hanahan, D.; Weinberg, R.A. Hallmarks of cancer: The next generation. Cell, 2011, 144(5), 646-674.
[http://dx.doi.org/10.1016/j.cell.2011.02.013] [PMID: 21376230]
[http://dx.doi.org/10.1016/j.cell.2011.02.013] [PMID: 21376230]
[32]
Shen, Z.N.; Nishida, K.; Doi, H.; Oohashi, T.; Hirohata, S.; Ozaki, T.; Yoshida, A.; Ninomiya, Y.; Inoue, H. Suppression of chondrosarcoma cells by 15-deoxy-Delta 12,14-prostaglandin J2 is associated with altered expression of Bax/Bcl-xL and p21. Biochem. Biophys. Res. Commun., 2005, 328(2), 375-382.
[http://dx.doi.org/10.1016/j.bbrc.2004.12.186] [PMID: 15694358]
[http://dx.doi.org/10.1016/j.bbrc.2004.12.186] [PMID: 15694358]
[33]
Lu, Y.; Sun, Y.; Zhu, J.; Yu, L.; Jiang, X.; Zhang, J.; Dong, X.; Ma, B.; Zhang, Q. Oridonin exerts anticancer effect on osteosarcoma by activating PPAR-γ and inhibiting Nrf2 pathway. Cell Death Dis., 2018, 9(1), 15.
[http://dx.doi.org/10.1038/s41419-017-0031-6] [PMID: 29323103]
[http://dx.doi.org/10.1038/s41419-017-0031-6] [PMID: 29323103]