Inhibition of Aurora kinases enhances chemosensitivity to ...

J Cancer Res Clin Oncol (2012) 138:405–414 DOI 10.1007/s00432-011-1111-0

ORIGINAL PAPER

Inhibition of Aurora kinases enhances chemosensitivity to temozolomide and causes radiosensitization in glioblastoma cells Kleiton Silva Borges · Angel Maurício Castro-Gamero · Daniel Antunes Moreno · Vanessa da Silva Silveira · Maria Sol Brassesco · Rosane Gomes de Paula Queiroz · Harley Francisco de Oliveira · Carlos Gilberto Carlotti Jr. · Carlos Alberto Scrideli · Luiz Gonzaga Tone

Received: 27 September 2011 / Accepted: 28 November 2011 / Published online: 9 December 2011 © Springer-Verlag 2011

Abstract Background Glioblastoma remains one of the most devastating human malignancies, and despite therapeutic advances, there are no drugs that signiWcantly improve the patient survival. Altered expression of the Aurora kinases was found in diVerent malignancies, and their inhibition has been studied in cancer therapy. In this study, we analyzed the expression of Aurora A and Aurora B in glioblastoma samples and also analyzed whether the eVects of Aurora kinase inhibition were associated with temozolomide or not on cell lines and primary cultures of glioblastoma. Materials and methods RT-PCR assays were used to determine the mRNA expression in glioblastoma tumor samples and in the cell lines. Cell proliferation was measured by XTT assay, and apoptosis was determined by Xow

cytometry. Drug combination analyses were made based in Chou-Talalay method. Gamma radiation for clonogenic survival used the doses of 2, 4 and 6 Gy. Changes in Aurora B level were assessed by Western blot analysis. Results Aurora A and B were expressed in glioblastoma samples as well as in the glioblastoma cell lines (n = 6). Moreover, ZM447439, a selective Aurora kinase inhibitor, decreased the proliferation separately and synergistically with temozolomide in primary cultures and cell lines of glioblastoma. ZM also enhanced the eVects of radiation on the two cell lines studied (U343 and U251), mainly when associated with TMZ in U343 cells. Treatment with ZM induced apoptotic cell death and diminished Aurora B protein level. Conclusions These data suggest that Aurora kinase inhibition may be a target for glioblastoma treatment and could be used as adjuvant to chemo- and radiotherapy.

K. S. Borges (&) · A. M. Castro-Gamero · D. A. Moreno · L. G. Tone Department of Genetics, School of Medicine of Ribeirão Preto, University of São Paulo (USP), Avenida Bandeirantes 3900, Ribeirão Preto, SP 14048-900, Brazil e-mail: [email protected]

Keywords Glioblastoma · Aurora A · Aurora B · ZM447439 · Temozolomide · Radiation

V. da Silva Silveira · M. S. Brassesco · R. G. de Paula Queiroz · C. A. Scrideli · L. G. Tone Department of Pediatrics, School of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, Brazil

Glioblastoma (GBM) is the most common type of primary malignant brain tumor in adults and is associated with a high mortality rate. Despite access to multimodality treatment (surgery, radiotherapy and chemotherapy with the alkylating agent temozolomide), patients diagnosed with GBM have a low survival rate (Khasraw and Lassman 2010; Ohgaki and Kleihues 2009). The Aurora kinase family is a collection of highly related and conserved serine– threonine kinases consisting of three members (A, B and C). These proteins are involved in the mitotic (M) phase of the cell cycle, playing essential roles in ensuring correct

H. F. de Oliveira Department of Clinical Medicine, School of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, Brazil C. G. Carlotti Jr. Department of Surgery and Anatomy, School of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, Brazil

Introduction

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J Cancer Res Clin Oncol (2012) 138:405–414

bipolar spindle formation, alignment of centrosomes on the mitotic spindle, centrosome separation, cytokinesis and monitoring of the mitotic checkpoint. Alterations in the expression of these genes cause disorders at diVerent stages of the mitotic phase and consequently the development of aneuploid cells with genomic instability and defects in the mitotic apparatus (Boss et al. 2009; Katayama and Sen 2010; Green et al. 2011). Overexpression of Aurora A caused the transformation of rodent Wbroblasts, further supporting the role of Aurora A as a potential oncogene (BischoV et al. 1998). In colorectal cell lines and in murine embryo Wbroblasts raised Aurora B protein was associated with aneuploidy and increased levels of phosphorylated H3 (Ser10). Moreover, these transformed Wbroblasts formed invasive tumors in nude mice (Ota et al. 2002). Several reports have shown the overexpression of Aurora A and Aurora B in a variety of cancers such as non-small-cell lung carcinoma (Zhang et al. 2008), breast cancer (Nadler et al. 2008; Tanaka et al. 1999) and head and neck carcinoma (Reiter et al. 2006), medulloblastoma (Neben et al. 2004) and neuroblastoma (Morozova et al. 2010). Taken together, these data support the importance of these proteins in the tumorigenesis process and indicate that inhibiting these kinases might be a powerful antitumor strategy. A number of small-molecule inhibitors of one or more Aurora kinases members have been developed to target these proteins and to investigate the antineoplastic eVects of Aurora kinase inhibition (Boss et al. 2009). ZM447439, AZD1152, VX680 and hesperadin are examples of Aurora kinase inhibitors that reduce histone H3 phosphorylation, endoreduplication and apoptosis (Green et al. 2011). ZM447439 is an ATP selective inhibitor of Aurora kinases A and B (DitchWeld et al. 2003). Previous studies have reported the antineoplastic eVects of ZM447439 in several cell lines from diVerent tumors (Georgieva et al. 2010; Long et al. 2008; Walsby et al. 2008). The present study shows the expression of Aurora A and Aurora B genes in the samples of patients diagnosed with glioblastoma as well as in a panel of glioblastoma cell lines. Furthermore, exposure of the cell lines and primary cultures of glioblastoma to ZM447439 induced growth inhibition and synergistic eVects when combined with TMZ. ZM also enhanced radiation eVects, alone or associated with TMZ, and caused apoptosis in glioblastoma cell lines.

et al. 2007), from subjects admitted for diagnosis and treatment to the University Hospital of the School of Medicine of Ribeirão Preto, SP, Brazil. Five samples of microdissected non-neoplastic brain tissue (white matter) were obtained from patients who had undergone surgery for the treatment of epilepsy. The average age of the patients was 44.3 years, and eleven subjects were men and nine were women. The study was approved by the Research Ethics Committee of the University Hospital, School of Medicine of Ribeirão Preto, University of São Paulo (protocol number 8273/2008).

Materials and methods

RNA isolation and reverse transcription-PCR

Patients analyzed

Total RNA was extracted from each cell line using the Trizol® reagent (Gibco BRL, Life technologies®, Carlsbad, CA, USA). Complementary DNA (cDNA) was obtained with the High Capacity® kit (Applied Biosystems®, Foster City, CA, USA) according to the manufacturers’ instructions. cDNA

For this study, 20 fresh-frozen microdissected tumor samples were obtained from gross total surgical resection of glioblastomas according to the WHO classiWcation (Louis

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Cell lines and primary culture The human adult glioblastoma cell lines U251, U343, T98G and U87 were purchased from the American Type Culture Collection, and cell line LN319 was kindly provided by Dr. Frank Furnari (Ludwig Institute for Cancer Research, La Jolla, CA). Pediatric glioblastoma cell line SF188 was kindly provided by Dr Michael S. Bobola (Department of Neurological Surgery, University of Washington, Seattle, WA). Cells were cultured in HAM F10 (Gibco BRL, Life Technologies®, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, penicillin (100 U/mL) and streptomycin (100 g/mL) (Sigma® Chemical Co., St. Louis, MO, USA) at 37°C in a humidiWed 5% CO2 incubator. The primary culture was derived from the biopsy specimens of glioblastoma patients under a protocol approved by the institutional review board at the University Hospital of the School of Medicine of Ribeirão Preto, SP, Brazil. Cultures were obtained according to Brassesco et al. (2009) and maintained at low passage numbers (p2–p5) under standard culture conditions. Drugs ZM447439 (ZM) was obtained from Tocris Cookson Inc. (Ellisville, MO). The compound was diluted to 10 mmol/L using 99% DMSO (Mallinckrodt. Chemical Works, St. Louis, Mo) and stored at ¡20°C. Temozolomide was obtained from Sigma (Sigma® Chemical Co., St. Louis, MO, USA) and Schering-Plough Brazil. The former was diluted to 200 mM using DMSO and stored at ¡20°C, and the latter was prepared as previously described (Borges et al. 2011) and used only for the radiation assay.


levels of genes Aurora A and Aurora B were measured using an ABI 7500 Real Time PCR System (PE Applied Biosystems). AmpliWcations were obtained using on-demand TaqMan® probes: AURKA (Hs00269212_m1), AURKB (Hs00177782_m1), TBP (4326322E0811008), HPRT (431 0809E0502006) and GAPDH (4326317-E 0905031) (Applied Biosystems). Blank and standard controls (calibrators) were run in parallel to verify the ampliWcation eYciency within each experiment. The level of internal control genes, human GAPDH, TBP and HPRT, were used to normalize the cDNA levels of the Aurora genes. The material was prepared and stored according to the manufacturer’s instructions, and the Wnal volume of each reaction was optimized to 12 L (6.0 L of TaqMan PCR Master Mix, 0.6 L of TaqMan probe and 5.4 L of cDNA—diluted 1:20). Real-time PCR was performed in duplicate, and a standard deviation (SD) of 1 is antagonistic and a score lower than 1 is synergistic.

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Radiation clonogenic survival assay A clonogenic assay was performed to study the eVects of radiation as described previously (Tao et al. 2007). U343 and U251 cells were plated into six-well plates with 300 cells per dish. After 24 h, cells were treated with ZM (400 nM), TMZ (1 M) or ZM + TMZ for 48 h. Cells were then irradiated with 2, 4 and 6 Gy of X-ray radiation (irradiation rate of 3.20 Gy/min) using a Gammatron S-80 device equipped with a Cobalt-60 source (Siemens Medical Systems, Inc., South Iselin, USA). The irradiated cells were then cultured in a 5% CO2 incubator at 37°C for an additional 10–14 days. Individual colonies (>50 cells per colony) were Wxed with methanol, stained with crystal violet and subsequently counted. Plating eYciency (PE) represents the percentage of cells seeded that grow into colonies under a speciWc culture condition of a given cell line. The survival fraction, expressed as a function of irradiation, was calculated as follows: survival fraction (SF) at 2 Gy (SF) = colonies counted of 2 Gy/(cells seeded of 2 Gy*PE/ 100). In the clonogenic survival curve, we normalized the diVerent conditions according to the control. The radiation dose enhancement ratio (DER) of the diVerent treatments was calculated using the following formula: DER = (SF at an indicated dose of radiation alone)/(SF at an indicated dose of radiation + treatment). Radiosensitization is the term used when the treatment increases the sensitivity of cells to radiation. This is calculated with the formula listed above and represented in the form of DER. Thus, DER is deWned as the ratio of surviving cells with radiation alone compared with a combination of radiation and diVerent drug exposures. A dose enhancement ratio = 1 suggests an additive radiation eVect and DER >1, a supra-additive eVect as opposed to a sub-additive eVect in the case of DER 0.005 (d). Radiosensitization was observed only at the dose of 2 Gy for U251, DER: 3.01

Table 3 EVects of treatment on the dose enhancement ratio (DER) of cell lines U343 and U251 U343

U251

2 Gy

4 Gy

6 Gy

2 Gy

4 Gy

6 Gy

TMZ

1.71

1.37

2.80

1.06

1.28

2.18

ZM

2.70

4.30

5.44

1.76

2.23

2.50

ZM + TMZ

3.83

5.81

13.32

3.01

1.78

2.36

DER = 1 additive radiation eVect, DER > 1, a supra-additive eVect and DER < 1 sub-additive eVect

the capacity to enhance the radiosensitivity of cancer cells (Niermann et al. 2011; Moretti et al. 2011), but this is the Wrst description of the radiosensitization caused by the combination of an Aurora kinase inhibitor and a chemotherapeutic agent. This evidence conWrms the capacity of Aurora kinase inhibition to sensitize cancer cells to available therapy modalities. This might be helpful in clinical

Fig. 7 EVects of ZM447439 on Aurora kinase B protein level. U251 (a) and U343 (b) cells were treated with increasing concentrations of ZM447439 (0–20 M) for 48 h. Changes in the expression of Aurora kinase B were analyzed by Western blotting. The experiments were

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practice, increasing the therapeutic eVects and facilitating the reduction of TMZ doses, with a consequent reduction of side eVects and treatment costs. Distinct mechanisms involved in the inhibition of Aurora kinase activity are being reported, such as the reduction in phosphorylation of histone H3 at serine 10 after Aurora B inhibition (DitchWeld et al. 2003; Long et al. 2008). Recently, it was described that, in addition to inhibiting Aurora B kinase activity, AZD1152-HQPA and ZM also decreased Aurora B protein levels in a dose-dependent manner (Georgieva et al. 2010; Gully et al. 2010). Gully et al. (2010) showed that AZD1152-HQPA increased the turnover rate of Aurora B by increasing polyubiquitination and proteasomal degradation. In our study, we found similar results, with ZM treatment aVecting Aurora B levels in the glioblastoma cell lines studied. This is an interesting molecular result found after treatment with an Aurora kinase inhibitor, and further studies are needed to investigate its importance regarding the eVects of Aurora kinase inhibition.

described in more detail, including the reagents origin. The reason for the cells choice was tumor aggressiveness and the unavailability of eVective treatment, as described in the text


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References

Fig. 8 ZM induces apoptosis in a dose-dependent manner in glioblastoma cell lines. Cell apoptosis was analyzed by Xow cytometry. Cells were incubated with increasing amounts of ZM (2–20 M) and control (0.1% DMSO) for 48 h before being collected, and subjected to Xow cytometry analysis. a U343. b U251. The graph shows three independent experiments; Columns, mean; bars, SD; *P < 0.05

In the current study, there are potential results for clinical translation. We have shown that ZM is eVective against cell lines and primary culture of glioblastoma. Moreover, we found that the combination of ZM and current standard therapies for glioblastoma is eVective. Altogether, these data prompted further investigation into the antineoplastic activity of ZM in an in vivo model, and these preclinical Wndings suggest that the inhibition of Aurora kinases is a potentially useful therapeutic strategy for glioblastoma. Acknowledgments We would like to thank Patrícia Vianna Bonini Palma, Camila Cristina de Oliveira Menezes Bonaldo and Daiane Fernanda dos Santos, Hemocentro-FMRP-USP, Ribeirão Preto, Brazil, for their assistance with the Xow cytometry. Financial Support from Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP, process number 2009/50118-2), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior and Fundação de Apoio ao Ensino, Pesquisa e Assistência do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo is acknowledged. ConXict of interest interest.

The authors declare that they have no conXict of

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