ONCOLOGY LETTERS 22: 610, 2021 Targeting glioma cells by antineoplastic activity of reversine CAMILA HIRAKATA1*, KELI LIMA1*, BRUNA OLIVEIRA DE ALMEIDA1, LÍVIA BASSANI LINS DE MIRANDA1, KATHARINE GURGEL DIAS FLORÊNCIO2, LUCIANA COSTA FURTADO1, LETICIA VERAS COSTA‑LOTUFO1 and JOÃO AGOSTINHO MACHADO‑NETO1 1Department of Pharmacology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, CEP 05508‑900; 2Department of Physiology and Pharmacology, Drug Research and Development Center, Federal University of Ceará, Fortaleza, Ceará, CEP 60440‑900, Brazil Received April 3, 2021; Accepted June 2, 2021 DOI: 10.3892/ol.2021.12871 Abstract. Gliomas are the most common type of primary DNA damage and may predict improved antiglioma responses central nervous system tumors and despite great advances to the drug. Reversine may be a potential novel drug in the in understanding the molecular basis of the disease very few antineoplastic arsenal against gliomas. new therapies have been developed. Reversine, a synthetic purine analog, is a multikinase inhibitor that targets aurora Introduction kinase A (AURKA) and aurora kinase B (AURKB). In gliomas, a high expression of AURKA or AURKB is associated with Gliomas are the most common primary central nervous a malignant phenotype and a poor prognosis. The present system (CNS) tumors, corresponding to ~80% of all malig‑ study investigated reversine‑related cellular and molecular nant primary brain tumors in adults (United States of antiglioma effects in HOG, T98G and U251MG cell lines. America, 2013‑2017) (1). According to the 2016 World Health Gene and protein expression were assessed by reverse tran‑ Organization (WHO) classification of CNS tumors, diffuse scription‑quantitative PCR and western blotting, respectively. gliomas are classified based on histological (astrocytoma, For functional assays, human glioma cell lines (HOG, T98G oligoastrocytoma, oligodendroglioma and glioblastoma) and U251MG) were exposed to increasing concentrations of and genetic features [isocitrate dehydrogenase mutations, reversine (0.4‑50 µM) and subjected to various cellular and alpha‑thalassemia/mental retardation, X‑linked (ATRX) molecular assays. Reversine reduced the viability and clonoge‑ loss, TP53 mutation and 1p/19q‑codeletion] (2). In addition, nicity in a dose‑ and/or time‑dependent manner in all glioma the WHO classified CNS tumors into 4 grades (I‑IV) that cells, with HOG (high AURKB‑expression) and T98G (high have distinct prognosis and require different therapeutic AURKA‑expression) cells being more sensitive compared decisions (3,4). Glioblastomas (grade IV), which correspond with U251MG cells (low AURKA‑ and AURKB‑expression). to ~50% of gliomas are highly malignant gliomas with a Notably, HOG cells presented higher levels of polyploidy, poor prognosis (5,6). The current therapeutic options for while T98G presented multiple mitotic spindles, which is these tumors include surgery, radiation, immunotherapy and consistent with the main regulatory functions of AURKB and chemotherapy with non‑specific and highly toxic agents (7‑9). AURKA, respectively. In molecular assays, reversine reduced Currently, alkylating agents (temozolomide, carmustine and AURKA and/or AURKB expression/activity and increased lomustine), mTOR inhibitors (everolimus) and monoclonal DNA damage and apoptosis markers, but autophagy‑related antibodies (bevacizumab and naxitamab‑gqgk) are approved proteins were not modulated. In conclusion, reversine potently by the Food and Drug Administration authority for the treat‑ induced mitotic catastrophe and apoptosis in glioma cells and ment of high‑grade gliomas (https://www.cancer.gov) (10). higher basal levels of aurora kinases and genes responsive to Inspite of considerable advances in the understanding of the molecular basis of gliomas (6,11), progress in improving the clinical outcomes of the disease is still slow and very few new therapies have been developed. Reversine [2‑(4‑morpholinoanilino)‑6‑cyclohexylamino‑ Correspondence to: Professor João Agostinho Machado‑Neto, purine] is an adenosine triphosphate analog that acts as a Department of Pharmacology, Institute of Biomedical Sciences, potent multi‑kinase inhibitor selective to aurora kinases, MPS1 University of São Paulo, 1524 Avenue Professor Lineu Prestes, (monopolar spindle 1) and JNK (12‑14). The antineoplastic São Paulo, CEP 05508‑900, Brazil E‑mail: jamachadoneto@usp.br activity of reversine has been demonstrated in hematological and solid tumors, in which it triggered cell cycle arrest, poly‑ *Contributed equally ploidy, apoptosis and autophagy (15). Aurora kinases, a family of serine/threonine kinases, Key words: gliomas, glioblastoma, aurora kinases, apoptosis, consisting of aurora kinase A (AURKA), aurora kinase B reversine (AURKB) and aurora kinase C (AURKC) serve an essential role in cell cycle progression and cellular division (16). High 2 HIRAKATA et al: ANTINEOPLASTIC EFFECTS OF REVERSINE IN GLIOMAS expression of AURKA, AURKB and AURKC has observed Inc.) for the expression of aurora kinase A (AURKA), aurora in several types of cancer, including gliomas and is associ‑ kinase B (AURKB), BCL2 apoptosis regulator (BCL2), BCL2 ated with a poor prognosis (17‑19). AURKA and AURKB are like (BCL2L1 1), baculoviral IAP repeat containing 5 (BIRC5), members with the most detailed characterizations: AURKA BCL2 interacting protein 3 (BNIP3), BCL2 interacting serves a role in the formation of the mitotic spindle during protein 3 like (BNIP3L), BCL2 associated agonist of cell death mitosis, while AURKB serves a key role in the organization of (BAD), BCL2 associated X, apoptosis regulator (BAX), BCL2 the centromere‑kinetochore region, microtubule‑kinetochore binding component 3 (BBC3), phorbol‑12‑myristate‑13‑ace‑ attachments and cytokinesis (16,20,21). Due to the differential tate‑induced protein 1 (PMAIP1), cyclin dependent kinase expression and biological functions related to the malignant inhibitor 1A (CDKN1A), cyclin dependent kinase inhibitor 1B phenotype attributed to these proteins, aurora kinases have (CDKN1B) and growth arrest and DNA damage inducible been proposed as potential targets for pharmacological inter‑ alpha (GADD45A) genes (Table SI) according to the manu‑ vention, particularly in cancer (22‑24). facturers' protocol. The thermocycling conditions included an In gliomas, high AURKA and AURKB expression is initial denaturation at 95˚C for 10 min, followed by 40 cycles associated with a poor prognosis and chemoresistance (18,25), of denaturation (95˚C for 15 sec), annealing (60˚C for 30 sec) which led to the study of the reversine effects in these disease and extension (60˚C for 30 sec). Hypoxanthine phosphoribo‑ models. Currently, reversine is in the preclinical phase of syltransferase 1 (HPRT1) and β‑actin were used as reference study in oncology, but the data accumulated thus far has genes. Relative quantification values were calculated using the indicated interesting antineoplastic activity (15). Hence, the 2‑ΔΔCt equation (26). A negative ‘No Template Control’ was identification of the most sensitive type of tumors to reversine, included for each primer pair as control for contamination including predictors of response for this drug, may identify a or unspecific amplification. Data were visualized using the profile of cancer patients eligible for clinical trials. The present multiple experiment viewer (MeV) v.4.9.0 software (27). study aimed to target aurora kinases using reversine and to identify the cellular and molecular mechanisms underlying its Western blotting. Total protein extraction was performed using anti‑glioma effects. a buffer containing 100 mM Tris (pH 7.6), 1% Triton X‑100, 150 mM NaCl, 2 mM PMSF, 10 mM Na3VO4, 100 mM NaF, Materials and methods 10 mM Na4P2O7, and 4 mM EDTA from untreated U87MG, HOG, T98G and U251MG cells, or HOG, T98G and U251MG Cell culture, reagents and chemicals. Oligodendroglioma cells treated with vehicle or reversine (1.6, 3.2 or 6.4 µM) (HOG) and glioblastoma (T98G, U251MG and U87MG) cell for 24 h or for cells treated with 1.6 µM reversine for 24, 48 lines were kindly provided by Professor Regina Pekelmann and 72 h. Extracted proteins were quantified by the Bradford Markus (University of São Paulo, São Paulo, Brazil). The method. Equal amounts of protein (30 µg) were separated by authenticity of the cells was determined by Short Tandem 8‑15% SDS‑PAGE (28) and transferred onto nitrocellulose Repeat (STR). The STR data from U87MG cells used in the membranes (GE Healthcare). Antibodies against AURKA present study is compatible with a glioblastoma of unknown (cat. no. sc‑25425), AURKB (cat. no. sc‑25426), phos‑ origin (https://web.expasy.org/cellosaurus/CVCL_0022). phorylated (p)‑histone H3S10 (sc‑8659‑R) and p‑histone H2A. All cell lines were cultured in the Roswell Park Memorial XS139 (γH2AX; sc‑51748) were purchased from Santa Cruz Institute medium (RPMI)‑1640 and supplemented with 10% Biotechnology, Inc. Antibodies against PARP1 (cat. no. 9542), fetal bovine serum (FBS), glutamine, 100 U/ml penicillin, SQSTM1/p62 (cat. no. 88588), LC3BI/II (cat. no. 2775), and 100 µg/ml streptomycin. The cells were maintained at β‑actin (cat. no. 4970) and α‑tubulin (cat. no. 2144) were 37˚C in a 5% CO2 humidified incubator and regularly split to obtained from Cell Signaling Technology, Inc. All primary maintain their exponential growth state (up to six passages). antibodies were used at 1:1,000 dilution and incubated for 16 h Reversine [2‑(4‑morpholinoanilino)‑6‑cyclohexylaminopu‑