Original Research Paper
Antitumour activity of AMG 900 alone or in combination with histone deacetylase inhibitor SaHa on medulloblastoma cell lines Lenisa Geron1,2, Kleiton Silva Borges1, Augusto Faria Andrade3, Veridiana Kill Suazo1,2, Carlos Alberto Scrideli1, Luiz Gonzaga Tone1,3 1
Department of Pediatrics, Ribeira˜o Preto Medical School, University of Sa˜o Paulo, Brazil, 2Department of Biology, Ribeira˜o Preto School of Philosophy, Sciences and Letters, University of Sa˜o Paulo, Brazil, 3 Department of Genetics, Ribeira˜o Preto Medical School, University of Sa˜o Paulo, Brazil Objectives: Medulloblastoma (MB) is the most common malignant childhood brain tumour. Aurora kinases are essential for cell division and are primarily active during mitosis. Recently, the combination of aurora kinases inhibitors (iAURK) and histone deacetylase inhibitors (iHDAC) has shown potential antitumour effects and had significant biological effects in preclinical cancer models. In this study, we analysed the effects of the pan-aurora kinases inhibitor AMG 900 alone or in combination with the iHDAC SaHa (Vorinostat) on paediatric MB cell lines (UW402, UW473 and ONS-76). Methods: Cell proliferation was measured by XTT assay, apoptosis was determined by flow cytometry and clonogenic capacity was studied. qRT-PCR assays were used to determine the mRNA expression in MB cell lines after treatment. Drug combination analyses were made based on Chou–Talalay method. Results: AMG 900 caused the inhibition of cell proliferation, diminution of clonogenic capacity and increased the apoptosis rate in cell lines (Pv0.05). A synergistic effect in the AMG900–SaHa combination was evidenced on the inhibition of cell proliferation in all cell lines, especially in sequential drug treatment. Moreover, the combination of these drugs reached 100% of the inhibition in colony formation (synergistic effect). The treatment with AMG 900 increased the p21 and GDF15 expression, but did not alter the TP53 in one of the cell lines. Conclusions: These results indicate that AMG 900 may be a promising drug for the adjuvant treatment of MB, mainly when combined with iHDAC. Keywords: Medulloblastoma, Aurora-kinase, AMG900, SaHa (Vorinostat), Combination
Introduction Medulloblastoma (MB) is the most frequent central nervous system (CNS) neoplasm in children. There are four molecularly defined subgroups for this tumour: Wnt, with a good prognosis, sonic-hedgehog (Shh), that presents intermediate prognosis and Groups 3 and 4, representing the worst prognosis subgroups.1–3 Despite aggressive multimodal therapy (surgery followed by local and craniospinal radiation and/or chemotherapy), the spread of the disease is common and a significant proportion of MBs have proved largely refractory to treatment, moreover, long-term side effects following treatment for MB have been consistently demonstrated.4–6
Correspondence to: Kleiton Silva Borges, Departamento de Puericultura e Pediatria, Faculdade de Medicina de Ribeira˜o Preto – USP, Hospital de Clinicas da FMRP/USP, Laborato´rio de Pediatria/Setor de Biologia Molecular, Av. Bandeirantes 3900/Bloco G, 1 andar, sala 218, Ribeira˜o Preto, Sa˜o Paulo, Brasil. Email: [email protected]
ß W. S. Maney & Son Ltd 2015
DOI 10.1179/1743132815Y.0000000048
Many advances have collaborated to the identification of new therapeutic targets for cancer treatment. These include the Aurora kinase family (Aurora-A, Aurora-B and Aurora-C), which are serine–threonine kinases that act in several cell cycle processes such as centrosome maturation, mitotic spindle assembly, chromosomal bi-orientation, cytokinesis and monitoring of the mitotic checkpoint.7–10 Alteration in the aurora kinases gene expression leads to disorders of the mitosis, causing aneuploidy and genomic instability.11,12 Several reports have shown the Aurora A and Aurora B overexpression in a variety of cancers such as breast,13,14 ovary, pancreas,15,16 gliomas17 and adrenocortical carcinoma.18 These data support the importance of these proteins in the tumourigenesis and indicate that such proteins may be interesting targets to cancer treatment. Following the identification of potential targets for cancer chemotherapy, many Aurora kinase inhibitors have been discovered and are currently
Neurological Research
2015
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37
NO .
8
703
Geron et al.
Antitumour activity of AMG 900 or with Vorinostat
under development.9,10 Among these, AMG 900 is an inhibitory/competitive adenosine triphosphate, highly potent and selective for Aurora A, B and C that is currently in phase I clinical trial. The AMG 900 has the ability to inhibit the autophosphorylation of Aurora kinases A and B, as well as the phosphorylation of histone H3, an Aurora B substrate.19,20 Aurora kinases inhibition may lead to the enhanced antitumour activity of the additional cancer chemotherapeutics agents.18,21,22 Previous studies have demonstrated that combination of histone deacetylases inhibitors (iHDACs) with aurora kinases inhibitors (iAURK) synergistically induced apoptosis and decreased cell proliferation as well as clonogenic survival in lymphoma,23 prostate cancer24 and BCR–ABLexpressing cells.25 Histone deacetylases (HDACs) have been widely studied since they are related to cancer pathogenesis and treatment with inhibitors of these enzymes shows promising results. Histone deacetylases inhibitors are known to bind to histone proteins and induce histone acetylation, interfering with its activities, which leads to significant biological effects in preclinical cancer models.26–28 Hence, we investigated the effects of AMG 900 alone or in combination with the iHDAC SaHa (Vorinostat) on MB paediatric cell lines. We found that inhibition of Aurora kinases, combined or not with iHDACs, may be a promising treatment for paediatric MB.
Materials and Methods Cell culture Paediatric MB cell lines UW402 and UW473 were kindly provided by Dr. Michael S. Bobola (Department of Neurological Surgery, University of Washington, Seattle, WA, USA) and ONS-76 cell line was obtained from the Rio de Janeiro Cell Bank (BCRJ, Federal University of Rio de Janeiro, Brazil). UW402 and UW473 were cultured in HAM-F10 medium and ONS-76 was cultured in RPMI medium both supplemented with 10% foetal bovine serum (FBS, Gibco BRL, Life Technologies, Carlsbad, CA, USA), penicillin (100 U/ml) and streptomycin (100 mg/ml) (SigmaH Chemical Co., St. Louis, MO, USA) at 37uuC in a humidified atmosphere of 5% CO2 incubator. Cell counts were performed using the test of Trypan blue exclusion. All assays were performed at least in triplicate at three different times. Culture medium, antibiotics and trypsin were purchased from Sigma (Sigma-Aldrich, Sa˜o Paulo, Brazil).
Drugs A stock solution of 1 mM Aurora kinase inhibitor AMG 900 (Selleckchem, Houston, TX, USA) and a stock solution of 1 mM iHDAC, SaHa (Vorinostat, ZolinzaH) were prepared in DMSO and stored in
704
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aliquots at {80 and {20uuC, respectively. Drugs were added in complete medium immediately before being applied to the cells. The final concentration of dimethyl sulfoxide (DMSO) in the mixture was 0.1%.
Cell cycle synchronisation assay To synchronise the cells at the mitotic phase, UW402 and UW473 cell lines were seeded a subconfluent density. On the following day, the cultures were rinsed with saline phosphate buffer (PBS) and cultured with serum free medium for 48 hours (serum starvation). After this time, cells were released into cell cycle by addition of medium supplemented with 10% serum. Twenty hours after the release, the cell lines were treated with colchicine (mitotic blocker) and with different AMG 900 concentrations (DMSO, 5 and 50 nmol/l) for 8 hours and then harvested and prepared for Western blot analysis.29
Western blotting Anti-AURKB (sc-25426, 1:200), anti-p-histone H3 (ser 10) (sc-8656, 1:200) and anti-GAPDH (sc-47724, 1:1000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Equal amounts of protein were size-fractionated by 16% SDS-PAGE, blotted onto a nitrocellulose membrane (Amersham Hybond2 ECL2, GE Healthcare) and incubated in Tris-buffered saline 0.1% Tween 20 (TBS-T) containing 5% (w/v) dried non-fat milk for 1 hour at room temperature. After blocking and washing in TBS-T with 0.1% Tween 20 for 30 minutes, each membrane was incubated with appropriately diluted primary antibodies overnight. Following membrane incubation, the membrane was washed three times in TPBS-T with 0.1% Tween 20 and bound to biotin-labelled horseradish peroxidase-conjugated species-specific secondary antibody (AbCam, CA, USA). The complexes were visualised using an enhanced chemiluminescence reagent (ECL; Amersham, Uppsala, Sweden).
Cell proliferation assay Cell proliferation was assessed using the XTT cell proliferation assay kit (XTT) as described previously.18 Cells were seeded at initial density of 2|103 in 96-well plates and maintained in culture conditions for 24 hours. After this period, the cells were treated with the inhibitor of Aurora kinases AMG 900 (5–50 nM) alone or in combination with the iHDAC [SaHa (50 nmol/l)] and incubated with the different treatments. After 48 hours treatment, cells were washed with PBS and medium without drug was added to culture. From this point onward, regarded as the time zero and cell proliferation was assessed at 24, 48 and 72 hours (48 hours for combination assays). After these treatment times, the formazan product was measured at
Geron et al.
450 nm using an iMark Microplate Reader (Bio-Rad Laboratories, Hercules, CA, USA). Drug-induced cytotoxic synergy was analysed using the CalcuSyn Software (Biosoft, Cambridge, UK).
Analysis of drug effects and interactions Cell lines were treated by both simultaneous and sequential drug exposure. On the basis of IC50 values after 48 hours of exposure to each drug alone, the doses used for drug combination assays were chosen. The simultaneous treatment regimen involved concomitant treatment of cells with AMG 900 and SaHa. For sequential drug exposure, cells were treated first with AMG 900, incubated for 48 hours and then incubated for an additional 48 hours with SaHa, or vice versa (SaHa first and then AMG 900). Analyses of the dose–effect relationships and the evaluation of drug interaction upon combination were carried out according to the median-effect method of Chou and Talalay30 using the CalcuSyn Software (Biosoft, Ferguson, MO, USA). A combination index (CI) of 1 indicates an additive drug interaction, whereas a CI of more than 1 is antagonistic and a score lower than 1 is synergistic.
Colony-forming cell assay Paediatric MB cell lines were plated in triplicate (500 cells/well), incubated with three different concentrations of AMG 900 (2.5, 5 and 50 mmol/l) for 48 hours, rinsed with PBS and cultured for 7–14 days. After that, colonies were then fixed with methanol and colonies with more than 50 cells were counted. Assays were performed in triplicate. In drug combination, cells were treated with AMG 900 (5 nM) and with IC30 values of SaHa (0.9 mM to UW402, 1.35 mM to UW473 and 1.00 mM to ONS-76) obtained by Calcusyn software (Biosoft, Ferguson, MO, USA) from proliferation assay with treatments of individual drugs. Cell lines were treated for both simultaneous and sequential exposure to the drug, conform previously mentioned.
Antitumour activity of AMG 900 or with Vorinostat
(CDKN1A) (Hs01034249) and TP53 (Hs00355782) were measured using TaqManH probes (PE Applied Biosystems, Foster City, CA, USA) in the ABI 7500 Real Time PCR System (PE Applied Biosystems). The relative expression was calculated using the 2{DDCT method31 with one internal control Gus (4326320/E). The expression levels in DMSO treated cells were used as calibrators.
Statistical analysis Statistical analysis was carried out using one- or twoway analysis of variance, followed by Bonferroni’s test, as appropriate, and Student’s t-test. A P value v0.05 was considered to be statistically significant. Data analysis was carried out using the SPSS 17.0 statistical software package (SPSS Inc., Chicago, IL, USA). Results are presented as means + SDs.
Results AMG 900 inhibits the phosphorylation of histone H3 at Ser 10 In order to analyse the effect of the drug AMG 900 (5–50 nmol/l) on the phosphorylation of histone H3, an Aurora B target, the cell synchronisation assay followed by Western blot analysis was performed. It was observed that histone H3 phosphorylation is low at concentrations of 5 and 50 nM tested and in UW402 and UW473 cell lines, confirming the ability of AMG 900 to inhibit the H3 phosphorylation (Fig. 1).
AMG 900 inhibits proliferation in a dosedependent manner and decreases clonogenic survival in paediatric MB cells The effects of AMG 900 (5–50 nM) on cell proliferation were assessed by XTT assay (Fig. 2A–C). Cell lines showed dose-dependent inhibition of proliferation after treatment (Pv0.05). There was a significant effect at 5 nM dose for ONS-76 cell line, which was not observed for UW402 and UW473 cell lines, and at 50 nM dose there was a significant decrease in cell proliferation in all paediatric MB cell lines (Pv0.05).
Apoptosis assessment by annexin V/propidium iodide staining Apoptotic cell death was determined after 48 hours of AMG 900 exposure, as described above in the cell proliferation, by labelling with annexin V fluorescein isothiocyanate (BD Biosciences Pharmigen, San Jose, CA, USA), as described previously.18
RNA isolation and reverse transcription-PCR Total RNA was extracted from each cell line using the Trizol reagent (Gibco BRL, Life Technologies, Carlsbad, CA, USA). cDNA was obtained using the High Capacity Kit (PdE Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. cDNA levels of genes GDF15 (Hs0017132), p21
Figure 1 Effects of AMG 900 on p-histone H3 Ser10 level. Western blotting of cell lysates from UW402 and UW473 cells synchronised in mitosis and treated with DMSO and AMG 900 for 8 hours at the indicated concentrations. Inhibition of histone H3 phosphorylation was verified using the anti-phosphorylated histone H3 antibody. The effects of AMG 900 on Histone H3 phosphorylation are independent of Aurora-B protein level. GAPDH was used as loading control
Neurological Research
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Antitumour activity of AMG 900 or with Vorinostat
AMG 900 also inhibited significantly colony formation of cell lines. ONS-76 cells were more sensitive to AMG 900 than UW402 and UW473 cells. AMG 900 reduced the capacity of colony formation by 92, 97 and 100% in UW402, UW473 and ONS-76 cells, respectively, at the dose of 50 nmol/l (Fig. 2D). These data is in agreement with Payton et al.19 that reported that AMG 900 induced a significant decrease in proliferative and clonogenic survival capacity in other tumours.
AMG 900 induces and enhances apoptotic cell death in MB cell lines The ability of AMG 900 to induce apoptosis was assessed by measuring annexin V/PI staining in UW402, UW473 and ONS-76 cells treated with the Aurora kinases inhibitor AMG 900 for 48 hours. Exposure of these cells to AMG 900 (5–50 nM) induced apoptosis in a dose-dependent manner (Fig. 3). AMG 900 induced 30% of apoptosis in UW402 and ONS-76 cells and 35% in UW473 cells after of 50 nM treatment.
AMG 900 causes increased expression of p21 and GDF15 genes in MB cell lines To evaluate the expression pattern of GDF15, P21 and TP53 genes after treatment with AMG 900,
both cell lines were treated with doses of 2.5 and 50.0 nM and incubated for 48 hours (Fig. 4). The GUS gene was used as endogenous control. At the dose of 50 nM, it was observed an increase in gene expression of 2.5-, 11.7- and 1.34-fold for the p21, GDF15 and TP53 genes, respectively for UW402 cell line (Fig. 4). For UW473 cell line, there was a ninefold increase in GDF15 gene expression, compared with the control (Fig. 4C). On the other hand, there was only a small increase in the p21 genes with 1.56-fold (Fig. 4A), while the TP53 gene did not show a significant change in its expression at both concentrations tested in this cell line (Fig. 4B).
Combination of AMG 900 with SaHa decrease proliferation in MB cell lines In order to investigate the effects of drug combination, the IC50 values of SaHa alone were determined. SaHa-IC50 value for UW402 was 7.5 mM, for UW473 was 5.9 mM, whereas for ONS-76 cells it was 4.2 mM. Two types of combinations were performed, simultaneous and sequential. For the former, cells were treated concomitantly with AMG 900 and with
Figure 2 Effects of AMG 900 on proliferation and clonogenic survival of medulloblastoma (MB) cell lines. AMG 900 caused inhibition of proliferation in a dose-dependent manner in (A) UW402, (B) UW473 and (C) ONS-76 cell lines. (D) Treatment with AMG 900 inhibits colony formation in MB cells in a dose-dependent manner. For clonogenic assay, cells were treated with ranging doses of AMG 900 for 48 hours. Untreated cells served as a control. After 10 days, colonics greater than 50 cells were counted. Data represent the mean 6 SD of three independent experiments each conducted in triplicate. *P
Antitumour activity of AMG 900 alone or in combination with histone deacetylase inhibitor SaHa on medulloblastoma cell lines Lenisa Geron1,2, Kleiton Silva Borges1, Augusto Faria Andrade3, Veridiana Kill Suazo1,2, Carlos Alberto Scrideli1, Luiz Gonzaga Tone1,3 1
Department of Pediatrics, Ribeira˜o Preto Medical School, University of Sa˜o Paulo, Brazil, 2Department of Biology, Ribeira˜o Preto School of Philosophy, Sciences and Letters, University of Sa˜o Paulo, Brazil, 3 Department of Genetics, Ribeira˜o Preto Medical School, University of Sa˜o Paulo, Brazil Objectives: Medulloblastoma (MB) is the most common malignant childhood brain tumour. Aurora kinases are essential for cell division and are primarily active during mitosis. Recently, the combination of aurora kinases inhibitors (iAURK) and histone deacetylase inhibitors (iHDAC) has shown potential antitumour effects and had significant biological effects in preclinical cancer models. In this study, we analysed the effects of the pan-aurora kinases inhibitor AMG 900 alone or in combination with the iHDAC SaHa (Vorinostat) on paediatric MB cell lines (UW402, UW473 and ONS-76). Methods: Cell proliferation was measured by XTT assay, apoptosis was determined by flow cytometry and clonogenic capacity was studied. qRT-PCR assays were used to determine the mRNA expression in MB cell lines after treatment. Drug combination analyses were made based on Chou–Talalay method. Results: AMG 900 caused the inhibition of cell proliferation, diminution of clonogenic capacity and increased the apoptosis rate in cell lines (Pv0.05). A synergistic effect in the AMG900–SaHa combination was evidenced on the inhibition of cell proliferation in all cell lines, especially in sequential drug treatment. Moreover, the combination of these drugs reached 100% of the inhibition in colony formation (synergistic effect). The treatment with AMG 900 increased the p21 and GDF15 expression, but did not alter the TP53 in one of the cell lines. Conclusions: These results indicate that AMG 900 may be a promising drug for the adjuvant treatment of MB, mainly when combined with iHDAC. Keywords: Medulloblastoma, Aurora-kinase, AMG900, SaHa (Vorinostat), Combination
Introduction Medulloblastoma (MB) is the most frequent central nervous system (CNS) neoplasm in children. There are four molecularly defined subgroups for this tumour: Wnt, with a good prognosis, sonic-hedgehog (Shh), that presents intermediate prognosis and Groups 3 and 4, representing the worst prognosis subgroups.1–3 Despite aggressive multimodal therapy (surgery followed by local and craniospinal radiation and/or chemotherapy), the spread of the disease is common and a significant proportion of MBs have proved largely refractory to treatment, moreover, long-term side effects following treatment for MB have been consistently demonstrated.4–6
Correspondence to: Kleiton Silva Borges, Departamento de Puericultura e Pediatria, Faculdade de Medicina de Ribeira˜o Preto – USP, Hospital de Clinicas da FMRP/USP, Laborato´rio de Pediatria/Setor de Biologia Molecular, Av. Bandeirantes 3900/Bloco G, 1 andar, sala 218, Ribeira˜o Preto, Sa˜o Paulo, Brasil. Email: [email protected]
ß W. S. Maney & Son Ltd 2015
DOI 10.1179/1743132815Y.0000000048
Many advances have collaborated to the identification of new therapeutic targets for cancer treatment. These include the Aurora kinase family (Aurora-A, Aurora-B and Aurora-C), which are serine–threonine kinases that act in several cell cycle processes such as centrosome maturation, mitotic spindle assembly, chromosomal bi-orientation, cytokinesis and monitoring of the mitotic checkpoint.7–10 Alteration in the aurora kinases gene expression leads to disorders of the mitosis, causing aneuploidy and genomic instability.11,12 Several reports have shown the Aurora A and Aurora B overexpression in a variety of cancers such as breast,13,14 ovary, pancreas,15,16 gliomas17 and adrenocortical carcinoma.18 These data support the importance of these proteins in the tumourigenesis and indicate that such proteins may be interesting targets to cancer treatment. Following the identification of potential targets for cancer chemotherapy, many Aurora kinase inhibitors have been discovered and are currently
Neurological Research
2015
VOL .
37
NO .
8
703
Geron et al.
Antitumour activity of AMG 900 or with Vorinostat
under development.9,10 Among these, AMG 900 is an inhibitory/competitive adenosine triphosphate, highly potent and selective for Aurora A, B and C that is currently in phase I clinical trial. The AMG 900 has the ability to inhibit the autophosphorylation of Aurora kinases A and B, as well as the phosphorylation of histone H3, an Aurora B substrate.19,20 Aurora kinases inhibition may lead to the enhanced antitumour activity of the additional cancer chemotherapeutics agents.18,21,22 Previous studies have demonstrated that combination of histone deacetylases inhibitors (iHDACs) with aurora kinases inhibitors (iAURK) synergistically induced apoptosis and decreased cell proliferation as well as clonogenic survival in lymphoma,23 prostate cancer24 and BCR–ABLexpressing cells.25 Histone deacetylases (HDACs) have been widely studied since they are related to cancer pathogenesis and treatment with inhibitors of these enzymes shows promising results. Histone deacetylases inhibitors are known to bind to histone proteins and induce histone acetylation, interfering with its activities, which leads to significant biological effects in preclinical cancer models.26–28 Hence, we investigated the effects of AMG 900 alone or in combination with the iHDAC SaHa (Vorinostat) on MB paediatric cell lines. We found that inhibition of Aurora kinases, combined or not with iHDACs, may be a promising treatment for paediatric MB.
Materials and Methods Cell culture Paediatric MB cell lines UW402 and UW473 were kindly provided by Dr. Michael S. Bobola (Department of Neurological Surgery, University of Washington, Seattle, WA, USA) and ONS-76 cell line was obtained from the Rio de Janeiro Cell Bank (BCRJ, Federal University of Rio de Janeiro, Brazil). UW402 and UW473 were cultured in HAM-F10 medium and ONS-76 was cultured in RPMI medium both supplemented with 10% foetal bovine serum (FBS, Gibco BRL, Life Technologies, Carlsbad, CA, USA), penicillin (100 U/ml) and streptomycin (100 mg/ml) (SigmaH Chemical Co., St. Louis, MO, USA) at 37uuC in a humidified atmosphere of 5% CO2 incubator. Cell counts were performed using the test of Trypan blue exclusion. All assays were performed at least in triplicate at three different times. Culture medium, antibiotics and trypsin were purchased from Sigma (Sigma-Aldrich, Sa˜o Paulo, Brazil).
Drugs A stock solution of 1 mM Aurora kinase inhibitor AMG 900 (Selleckchem, Houston, TX, USA) and a stock solution of 1 mM iHDAC, SaHa (Vorinostat, ZolinzaH) were prepared in DMSO and stored in
704
Neurological Research
2015
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8
aliquots at {80 and {20uuC, respectively. Drugs were added in complete medium immediately before being applied to the cells. The final concentration of dimethyl sulfoxide (DMSO) in the mixture was 0.1%.
Cell cycle synchronisation assay To synchronise the cells at the mitotic phase, UW402 and UW473 cell lines were seeded a subconfluent density. On the following day, the cultures were rinsed with saline phosphate buffer (PBS) and cultured with serum free medium for 48 hours (serum starvation). After this time, cells were released into cell cycle by addition of medium supplemented with 10% serum. Twenty hours after the release, the cell lines were treated with colchicine (mitotic blocker) and with different AMG 900 concentrations (DMSO, 5 and 50 nmol/l) for 8 hours and then harvested and prepared for Western blot analysis.29
Western blotting Anti-AURKB (sc-25426, 1:200), anti-p-histone H3 (ser 10) (sc-8656, 1:200) and anti-GAPDH (sc-47724, 1:1000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Equal amounts of protein were size-fractionated by 16% SDS-PAGE, blotted onto a nitrocellulose membrane (Amersham Hybond2 ECL2, GE Healthcare) and incubated in Tris-buffered saline 0.1% Tween 20 (TBS-T) containing 5% (w/v) dried non-fat milk for 1 hour at room temperature. After blocking and washing in TBS-T with 0.1% Tween 20 for 30 minutes, each membrane was incubated with appropriately diluted primary antibodies overnight. Following membrane incubation, the membrane was washed three times in TPBS-T with 0.1% Tween 20 and bound to biotin-labelled horseradish peroxidase-conjugated species-specific secondary antibody (AbCam, CA, USA). The complexes were visualised using an enhanced chemiluminescence reagent (ECL; Amersham, Uppsala, Sweden).
Cell proliferation assay Cell proliferation was assessed using the XTT cell proliferation assay kit (XTT) as described previously.18 Cells were seeded at initial density of 2|103 in 96-well plates and maintained in culture conditions for 24 hours. After this period, the cells were treated with the inhibitor of Aurora kinases AMG 900 (5–50 nM) alone or in combination with the iHDAC [SaHa (50 nmol/l)] and incubated with the different treatments. After 48 hours treatment, cells were washed with PBS and medium without drug was added to culture. From this point onward, regarded as the time zero and cell proliferation was assessed at 24, 48 and 72 hours (48 hours for combination assays). After these treatment times, the formazan product was measured at
Geron et al.
450 nm using an iMark Microplate Reader (Bio-Rad Laboratories, Hercules, CA, USA). Drug-induced cytotoxic synergy was analysed using the CalcuSyn Software (Biosoft, Cambridge, UK).
Analysis of drug effects and interactions Cell lines were treated by both simultaneous and sequential drug exposure. On the basis of IC50 values after 48 hours of exposure to each drug alone, the doses used for drug combination assays were chosen. The simultaneous treatment regimen involved concomitant treatment of cells with AMG 900 and SaHa. For sequential drug exposure, cells were treated first with AMG 900, incubated for 48 hours and then incubated for an additional 48 hours with SaHa, or vice versa (SaHa first and then AMG 900). Analyses of the dose–effect relationships and the evaluation of drug interaction upon combination were carried out according to the median-effect method of Chou and Talalay30 using the CalcuSyn Software (Biosoft, Ferguson, MO, USA). A combination index (CI) of 1 indicates an additive drug interaction, whereas a CI of more than 1 is antagonistic and a score lower than 1 is synergistic.
Colony-forming cell assay Paediatric MB cell lines were plated in triplicate (500 cells/well), incubated with three different concentrations of AMG 900 (2.5, 5 and 50 mmol/l) for 48 hours, rinsed with PBS and cultured for 7–14 days. After that, colonies were then fixed with methanol and colonies with more than 50 cells were counted. Assays were performed in triplicate. In drug combination, cells were treated with AMG 900 (5 nM) and with IC30 values of SaHa (0.9 mM to UW402, 1.35 mM to UW473 and 1.00 mM to ONS-76) obtained by Calcusyn software (Biosoft, Ferguson, MO, USA) from proliferation assay with treatments of individual drugs. Cell lines were treated for both simultaneous and sequential exposure to the drug, conform previously mentioned.
Antitumour activity of AMG 900 or with Vorinostat
(CDKN1A) (Hs01034249) and TP53 (Hs00355782) were measured using TaqManH probes (PE Applied Biosystems, Foster City, CA, USA) in the ABI 7500 Real Time PCR System (PE Applied Biosystems). The relative expression was calculated using the 2{DDCT method31 with one internal control Gus (4326320/E). The expression levels in DMSO treated cells were used as calibrators.
Statistical analysis Statistical analysis was carried out using one- or twoway analysis of variance, followed by Bonferroni’s test, as appropriate, and Student’s t-test. A P value v0.05 was considered to be statistically significant. Data analysis was carried out using the SPSS 17.0 statistical software package (SPSS Inc., Chicago, IL, USA). Results are presented as means + SDs.
Results AMG 900 inhibits the phosphorylation of histone H3 at Ser 10 In order to analyse the effect of the drug AMG 900 (5–50 nmol/l) on the phosphorylation of histone H3, an Aurora B target, the cell synchronisation assay followed by Western blot analysis was performed. It was observed that histone H3 phosphorylation is low at concentrations of 5 and 50 nM tested and in UW402 and UW473 cell lines, confirming the ability of AMG 900 to inhibit the H3 phosphorylation (Fig. 1).
AMG 900 inhibits proliferation in a dosedependent manner and decreases clonogenic survival in paediatric MB cells The effects of AMG 900 (5–50 nM) on cell proliferation were assessed by XTT assay (Fig. 2A–C). Cell lines showed dose-dependent inhibition of proliferation after treatment (Pv0.05). There was a significant effect at 5 nM dose for ONS-76 cell line, which was not observed for UW402 and UW473 cell lines, and at 50 nM dose there was a significant decrease in cell proliferation in all paediatric MB cell lines (Pv0.05).
Apoptosis assessment by annexin V/propidium iodide staining Apoptotic cell death was determined after 48 hours of AMG 900 exposure, as described above in the cell proliferation, by labelling with annexin V fluorescein isothiocyanate (BD Biosciences Pharmigen, San Jose, CA, USA), as described previously.18
RNA isolation and reverse transcription-PCR Total RNA was extracted from each cell line using the Trizol reagent (Gibco BRL, Life Technologies, Carlsbad, CA, USA). cDNA was obtained using the High Capacity Kit (PdE Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. cDNA levels of genes GDF15 (Hs0017132), p21
Figure 1 Effects of AMG 900 on p-histone H3 Ser10 level. Western blotting of cell lysates from UW402 and UW473 cells synchronised in mitosis and treated with DMSO and AMG 900 for 8 hours at the indicated concentrations. Inhibition of histone H3 phosphorylation was verified using the anti-phosphorylated histone H3 antibody. The effects of AMG 900 on Histone H3 phosphorylation are independent of Aurora-B protein level. GAPDH was used as loading control
Neurological Research
2015
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NO .
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Geron et al.
Antitumour activity of AMG 900 or with Vorinostat
AMG 900 also inhibited significantly colony formation of cell lines. ONS-76 cells were more sensitive to AMG 900 than UW402 and UW473 cells. AMG 900 reduced the capacity of colony formation by 92, 97 and 100% in UW402, UW473 and ONS-76 cells, respectively, at the dose of 50 nmol/l (Fig. 2D). These data is in agreement with Payton et al.19 that reported that AMG 900 induced a significant decrease in proliferative and clonogenic survival capacity in other tumours.
AMG 900 induces and enhances apoptotic cell death in MB cell lines The ability of AMG 900 to induce apoptosis was assessed by measuring annexin V/PI staining in UW402, UW473 and ONS-76 cells treated with the Aurora kinases inhibitor AMG 900 for 48 hours. Exposure of these cells to AMG 900 (5–50 nM) induced apoptosis in a dose-dependent manner (Fig. 3). AMG 900 induced 30% of apoptosis in UW402 and ONS-76 cells and 35% in UW473 cells after of 50 nM treatment.
AMG 900 causes increased expression of p21 and GDF15 genes in MB cell lines To evaluate the expression pattern of GDF15, P21 and TP53 genes after treatment with AMG 900,
both cell lines were treated with doses of 2.5 and 50.0 nM and incubated for 48 hours (Fig. 4). The GUS gene was used as endogenous control. At the dose of 50 nM, it was observed an increase in gene expression of 2.5-, 11.7- and 1.34-fold for the p21, GDF15 and TP53 genes, respectively for UW402 cell line (Fig. 4). For UW473 cell line, there was a ninefold increase in GDF15 gene expression, compared with the control (Fig. 4C). On the other hand, there was only a small increase in the p21 genes with 1.56-fold (Fig. 4A), while the TP53 gene did not show a significant change in its expression at both concentrations tested in this cell line (Fig. 4B).
Combination of AMG 900 with SaHa decrease proliferation in MB cell lines In order to investigate the effects of drug combination, the IC50 values of SaHa alone were determined. SaHa-IC50 value for UW402 was 7.5 mM, for UW473 was 5.9 mM, whereas for ONS-76 cells it was 4.2 mM. Two types of combinations were performed, simultaneous and sequential. For the former, cells were treated concomitantly with AMG 900 and with
Figure 2 Effects of AMG 900 on proliferation and clonogenic survival of medulloblastoma (MB) cell lines. AMG 900 caused inhibition of proliferation in a dose-dependent manner in (A) UW402, (B) UW473 and (C) ONS-76 cell lines. (D) Treatment with AMG 900 inhibits colony formation in MB cells in a dose-dependent manner. For clonogenic assay, cells were treated with ranging doses of AMG 900 for 48 hours. Untreated cells served as a control. After 10 days, colonics greater than 50 cells were counted. Data represent the mean 6 SD of three independent experiments each conducted in triplicate. *P
