The selective Aurora B kinase inhibitor AZD1152 as a novel treatment for hepatocellular carcinoma

"Clinical evidence exists indicating a significant relationship between Aurora B kinase expression and the aggressive progression of HCC [10], and our preclinical studies indicated that AZD1152, a specific inhibitor of Aurora B kinase, is a promising novel therapeutic approach for the treatment of human HCC. Urgent studies and clinical trials of AZD1152 will confirm its role in the treatment of HCC.".......AZD1152-HQPA showed potent antiproliferative activity in all HCC cell types.....These results indicate that inhibition of Aurora B kinase by AZD1152-HQPA can induce cell death in the SK-Hep1 and Hep3B cells in vitro.......As shown in Fig. 4A, significant regression of SK-Hep1 tumors was observed in the group of mice that received AZD1152 compared with control. The mean tumor volumes were substantially decreased by treatment with AZD1152 on day 14 following treatment.....These results indicate that inhibition of Aurora B kinase by AZD1152-HQPA can induce cell death in the SK-Hep1 and Hep3B cells in vitro......These results demonstrate that AZD1152 was able to significantly inhibit in vivo growth of a human HCC tumor in the liver microenvironment in mice"

The selective Aurora B kinase inhibitor AZD1152 as a novel treatment for hepatocellular carcinoma


Arihiro Aihara, Shinji Tanaka, Mahmut Yasen, Satoshi Matsumura, Yusuke Mitsunori, Ayano Murakata, Norio Noguchi, Atsushi Kudo, Noriaki Nakamura, Koji Ito, Shigeki Arii


Received 9 April 2009; received in revised form 2 July 2009; accepted 13 July 2009. published online 29 October 2009.

ABSTRACT

Background & Aims- We previously identified that high Aurora B expression was associated with hepatocellular carcinoma (HCC) recurrence due to tumor dissemination. In this preclinical study, a novel inhibitor of Aurora B kinase was evaluated as a treatment for human HCC.

Methods- AZD1152 is a selective inhibitor of Aurora B kinase. Twelve human HCC cell lines were analyzed for Aurora B kinase expression and the in vitro effects of AZD1152. The in vivo effects of AZD1152 were analyzed in a subcutaneous xenograft model and a novel orthotopic liver xenograft model.

Results- Aurora B kinase expression varied among the human HCC cell lines and was found to correlate with inhibition of cell proliferation, accumulation of 4N DNA, and the proportion of polyploid cells following administration of AZD1152-hydroxyquinazoline-pyrazol-anilide (AZD1152-HQPA). AZD1152-HQPA suppressed histone H3 phosphorylation and induced cell death in a dose-dependent manner. Growth of subcutaneous human HCC xenografts was inhibited by AZD1152 administration. In an orthotopic hepatoma model, treatment with AZD1152 significantly decelerated tumor growth and increased survival. Pharmacobiological analysis revealed that AZD1152 induced the rapid suppression of phosphohistone H3, followed by cellular apoptosis in the liver tumors but not in the normal tissues of the orthotopic models.

Conclusions- Our preclinical studies indicate that AZD1152 is a promising novel therapeutic approach for the treatment of HCC.


Introduction


Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide, accounting nearly for 1 million deaths per year [1], and the incidence is still increasing [2]. The primary curative treatment for HCC is surgical resection, and there has been limited improvement in the availability of alternative treatments in the last decade [3]. A major obstacle for the treatment of HCC is the high frequency of tumor recurrence after curative resection. In fact, it is the recurrence pattern, rather than the recurrence itself, that critically affects patient prognosis [4]. The systemic treatment of HCC using conventional anticancer agents has provided little clinical benefit or prolonged survival for patients with advanced HCC [5]. A recent clinical trial by Llovet et al. [6] revealed a molecular-targeted inhibitor, sorafenib, as the first agent that demonstrated an improved overall survival in patients with advanced HCC. The increased understanding of the molecular mechanisms regulating cancer progression has led to the development of novel targeted therapies [7], [8]. In order to fulfill this promise, there is an urgent need to identify the optimal targets for treatment.


In our previous studies in HCC patients after curative resection, the aggressive recurrence exceeding Milan criteria showed extremely poor prognosis [9]; moreover, a genome wide microarray profiling analysis identified the over-expression of Aurora B kinase as the only independent factor predictive of the aggressive recurrence [10]. The Aurora kinase family of serine–threonine kinases control chromosome assembly and segregation during mitosis. Aberrant expression of the Aurora kinases has been reported in a variety of solid tumors including prostate [11], colon [12], pancreas [13], lung [14], breast [15], and thyroid [16]. These findings have led to an interest in these kinases as molecular targets for cancer treatment [17], [18]. Several small-molecule inhibitors of Aurora kinases have been developed as potential anticancer treatments. According to the recent review on Aurora inhibitors [19], ZM447439, Hesperadin, and MK0457/VX680 were the first to be described and to have similar potency versus Aurora A, Aurora B, and Aurora C. Currently, MLN8054 and MLN8237 are being developed as selective Aurora A kinase inhibitors. AZD1152 is a selective inhibitor of Aurora kinase activity with specificity for Aurora B kinase [20], [21]. AZD1152 is a prodrug that is rapidly converted to the active moiety AZD1152-hydroxyquinazoline-pyrazol-anilide (AZD1152-HQPA) in plasma. Thus, AZD1152 is used for in vivo studies, while AZD1152-HQPA is used for in vitro work.


The importance of the role of the organ microenvironment in cancer is being increasingly understood [22]. This is particularly true for HCC, an organotropic cancer in which the liver-specific microenvironment may play a critical role in HCC tumor development, cellular apoptosis, and drug sensitivity [23]. Additionally, hepatic tumors reside within the liver parenchyma, where drug metabolism and transformation occur. Thus, the pharmacodynamics of drug therapy for intrahepatic tumors may vary significantly from those drugs targeted at tumors in peripheral tissues. Several attempts have been made to generate a model of intrahepatic HCC via intraportal or intrahepatic injection of tumor cells in mice; however, frequent cancer dissemination makes it particularly difficult to generate a single quantitative tumor. A recent report describes development of a novel orthotopic liver tumor xenograft model that could be used in quantitative investigations of a single tumor within its native microenvironment [24]. This might provide a system in which the tumor’s biological response to therapeutic agents more closely mimics that observed in liver tumors in patients [25]. The in vivo efficacy of Aurora kinase inhibitors in orthotopic xenograft models of solid cancers has not been reported to date [20], [26], [27].


Outcome of HCC patients is determined by combination of two distinct types of HCC recurrence, and the aggressive recurrence is driven by malignant characteristics of the tumor [4], [9]. Because Aurora B kinase was found to be associated with the aggressive recurrence exceeding Milan criteria [10], it makes sense to target Aurora B kinase to treat the tumor. In this regard, the Aurora B kinase-specific inhibitor AZD1152 might be an attractive candidate for HCC therapy. This investigation evaluates the in vitro and in vivo effects and pharmacodynamics of AZD1152 in a number of preclinical liver tumor models, including an orthotopic model that more closely mimics the human disease.

Discussion


The Aurora family of serine–threonine kinases has recently emerged as a key mitotic regulator required for genome stability [38]. In mammals, the Aurora family consists of three members: Aurora A and B kinases and the less well-characterized Aurora C kinase. Aurora B kinase has been clearly shown to regulate kinetochore function, as it is required for correct chromosome alignment and segregation, spindle-checkpoint function, and cytokinesis. As Aurora kinases are frequently overexpressed in various tumors [39], they have received much attention as potential targets for novel anticancer therapeutics. Treatment with Aurora kinase inhibitors induces the accumulation of cells arrested in a pseudo-G1 state with >4N DNA content or the accumulation of cells with >4N DNA content, the latter population representing cells that exit mitosis and subsequently proceed through S phase in the absence of cell division [31]. Continued proliferation in the presence of aberrant mitosis and failed cytokinesis presumably gives rise to cells with higher DNA content due to an increase of the cell diameter, resulting in apoptosis [17], [18], [40], [41]. The defective cytokinesis, as well as the inhibition of PhH3 by Aurora kinase inhibitors, suggests that the cellular effects of Aurora kinase inhibitors might be largely mediated by the disruption of Aurora B kinase function [18]. AZD1152 is a selective inhibitor of Aurora kinase with specificity for Aurora B kinase. AZD1152 has the potential to be efficacious in multiple tumor types and is currently undergoing phase 1 clinical evaluation as a treatment for a range of malignancies [20], [21].


We have previously identified Aurora B kinase as the only independent predictor for the aggressive recurrence of human HCC [10]. In our present study, AZD1152-HQPA substantially reduced in vitro proliferation in a variety of human HCC cell lines. The extent of proliferation inhibition was correlated with Aurora B kinase expression levels (Fig. 1). As shown in Fig. 1C, significant DNA fragmentation in the form of a sub-G1 peak could not be detected after 24h of treatment with AZD1152-HQPA, which is in line with data reported by Wilkinson et al. [20]. This inability to detect a sub-G1 population after AZD1152-HQPA treatment may result because inhibition of Aurora B kinase induces polyploidy before apoptosis, in which case DNA fragmentation will occur in the >4N population, making it difficult to detect a sub-G1 population.


Treatment with AZD1152-HQPA also led to inhibition of PhH3 as well as failure of tumor cell division, and ultimately induced death of human HCC cells (Fig. 2, Fig. 3). In vivo administration of AZD1152 suppressed the growth of human HCC tumors in established subcutaneous xenografts (Fig. 4). Although subcutaneous xenograft models have the benefits of easy visualization and monitoring of tumor growth, the biological response to therapeutic agents in the natural microenvironment of the tumor should be analyzed using orthotopic xenograft models [30], [42]. In this study, a novel model of intrahepatic inoculation with Matrigel was utilized to closely mimic HCC tumors in humans [24]. As shown in Fig. 5, AZD1152 inhibited in vivo growth of established liver tumors and increased survival in this model. Furthermore, pharmacobiological studies of AZD1152 confirmed in vivo suppression of PhH3 and induction of cellular apoptosis of human HCC (Fig. 6). AZD1152 was well tolerated at the dose required to elicit a potent and durable antitumor effect in mice. According to the previous report by Wilkinson et al. [20], mice were almost resistant to myelosuppression after AZD1152 treatment; the authors could not find any reductions in bone marrow nucleated cells at the end of the dosing period. In rats, there was a myelosuppressive effect of AZD1152 that was associated with a reduction in bone marrow nucleated cells to 34% of that seen in the controls at the end of the 48-h dosing period; however, the bone marrow nucleated cell content rapidly recovered such that it was 104.8% of control at the end of the study period. Although the phase 1 studies on the side effects of AZD1152 have not yet been reported in detail, humans might be more sensitive to the myelosuppressive effects compared to the experimental rodents. Further study should be required for clinical application to HCC patients, especially those with cirrhosis.


Clinical evidence exists indicating a significant relationship between Aurora B kinase expression and the aggressive progression of HCC [10], and our preclinical studies indicated that AZD1152, a specific inhibitor of Aurora B kinase, is a promising novel therapeutic approach for the treatment of human HCC. Urgent studies and clinical trials of AZD1152 will confirm its role in the treatment of HCC.

Results


Aurora B kinase expression and in vitro effects of AZD1152-HQPA in human hepatocellular carcinoma cells


Evaluation of Aurora B kinase protein in 12 human HCC cell lines revealed a variety of expression levels, as shown in Fig. 1A (Aurora B/tubulin expression ratio: JHH-1, 0.120; JHH-2, 0.039; JHH-4, 0.059; HuH-1, 0.078; HuH-6, 0.220; HuH-7, 0.243; HLE, 0.040; HLF, 0.032; PLC/PRF/5, 0.083; SK-Hep1, 0.107; Hep3B, 0.079; HepG2, 0.044). Expression of Aurora B kinase was approximately 7-fold higher in HuH-7 and HuH-6 cells than in JHH-2 and HLF cells. To evaluate the growth inhibitory effects of AZD1152-HQPA, cell proliferation assays were conducted in these HCC cell lines. AZD1152-HQPA showed potent antiproliferative activity in all HCC cell types with IC50 values (JHH-1, 17.4±1.0nM; JHH-2, 218.0±10.8nM; JHH-4, 155.6±16.8nM; HuH-1, 27.3±5.0nM; HuH-6, 3.7±0.6nM; HuH-7, 6.8±0.3nM; HLE, 45.9±6.4nM; HLF, 126.1±12.2nM; PLC/PRF/5, 76.9±9.9nM; SK-Hep1, 21.9±1.2nM; Hep3B, 7.6±1.2nM; HepG2, 14.7±1.7nM) (Fig. 1A). Fig. 1B demonstrates the relationship between Aurora B kinase expression and indexes of AZD1152-HQPA IC50 in the panel of cell lines tested (correlation coefficient: −0.72738; R2=0.529; p=0.0073).

Alterations in DNA ploidy in the human HCC cell lines were analyzed by flow cytometry (Fig. 1C). Accumulation of cells with >4N DNA content was observed in all of the cell lines following 24-h incubation with AZD1152-HQPA 100nM, with the exception of JHH-2 and HLF, which showed AZD1152 insensitivity with low expression levels of Aurora B kinase. As shown in Fig. 1D, the increasing rate of >4N DNA by AZD1152-HQPA (JHH-1, 40.5±3.6%; JHH-2, 24.2±0.2%; JHH-4, 2.3±0.1%; HuH-1, 32.0±0.1%; HuH-6, 31.5±0.8%; HuH-7, 53.7±2.9%; HLE, 24.9±1.8%; HLF, 8.4±1.7%; PLC/PRF/5, 37.3±2.7%; SK-Hep1, 22.0±2.5%; Hep3B, 45.4±1.9%; HepG2, 59.0±0.9%) was correlated with the indexes of IC50 values (correlation coefficient: −0.66534; R2=0.443; p=0.0129). The accumulation of polyploid cells is consistent with failed cytokinesis following inhibition of Aurora B kinase activity.


Previously, cellular apoptosis in response to the pan-Aurora kinase inhibitor VX680 was limited in cells expressing wild-type p53 but was enhanced in cells lacking p53 [31]. The p53 point mutations have been reported in four HCC cell lines (HuH-7 at codon 220 Tyr-to-Cys; HLF at codon 244 Gly-to-Ala; HLE and PLC/PRF/5 at codon 249 Arg-to-Ser), and null expression of p53 was reported due to the deletion in the Hep3B cell line, while SK-Hep1 and HepG2 have wild-type p53 [32], [33], [34]. There was no significant correlation between the efficacy of AZD1152-HQPA and the p53 status of each cell line in our experiments.

In vitro effects of AZD1152-HQPA on phosphorylation of histone H3 and cell death in human hepatocellular carcinoma cell lines


In the previous studies by Mortlock et al. [35], AZD1152-HQPA is a selective Aurora B kinase inhibitor with more than 1000- to 10,000-fold selectivity for Aurora A kinase and various tyrosine kinases including kinase insert domain receptor (KDR), the Abelson virus kinase (vABL), and epidermal growth factor receptor (EGFR). The inhibition of Aurora B kinase is determined by its specific cellular substrate histone H3 [36]. We investigated whether AZD1152-HQPA was able to inhibit PhH3 in the sensitive SK-Hep1 and Hep3B cells. As shown in Fig. 2, Fig. 3, AZD1152-HQPA 100nM yielded a substantial reduction in the level of PhH3. This inhibition of histone H3 phosphorylation was shown to be dose dependent in SK-Hep1 and Hep3B cells treated with AZD1152-HQPA 1–100nM (Fig. 3B). The cellular apoptosis was confirmed by analysis of Annexin-V binding (Fig. 3C). Cell death rates were measured and were also found be proportional to AZD1152-HQPA dose (Fig. 3D) [21]. These results indicate that inhibition of Aurora B kinase by AZD1152-HQPA can induce cell death in the SK-Hep1 and Hep3B cells in vitro. In contrast, the AZD1152-insensitive HLF cells with a low expression of Aurora B kinase (Fig. 1) showed no significant effects on PhH3 and apoptosis compared with SK-Hep1 and Hep3B cells (Fig. 2, Fig. 3).

In vivo effects of AZD1152 on subcutaneous xenografts of human hepatocellular carcinoma cells


The human HCC cell line SK-Hep1 (AZD1152 IC50: 21.9nM) is known to be aggressively tumorigenic in vivo [37]. To investigate in vivo antitumor activity, AZD1152 100mg/kg per day was administered to nude mice bearing established SK-Hep1 subcutaneous xenografts on 2 consecutive days per week for 2weeks (n=10). Tumor volumes were measured every other day. As shown in Fig. 4A, significant regression of SK-Hep1 tumors was observed in the group of mice that received AZD1152 compared with control. The mean tumor volumes were substantially decreased by treatment with AZD1152 on day 14 following treatment, and tumor volumes in treated mice were 15.5% of those in control mice (Fig. 4B). None of the AZD1152-treated mice showed signs of wasting or other toxicity relative to control mice. AZD1152 was tolerated at the dose at which antitumor efficacy was observed.

In vivo effects of AZD1152 on orthotopic liver xenografts of human hepatocellular carcinoma cells


A novel orthotopic xenograft model of liver tumors with Matrigel was utilized to explore tumor growth inhibition in situ [25] (Fig. 5A). AZD1152 100mg/kg was administered to mice bearing SK-Hep1 orthotopic xenografts on 2 consecutive days per week for 2weeks (n=5). Histological analysis of the liver tumors was conducted within 4weeks after treatment. Growth of liver tumors was found to be suppressed in all of the mice that had been treated with AZD1152 (Fig. 5B). After drug administration, the mean liver tumor weight in those animals that had received AZD1152 was 10% of that in the control mice (Fig. 5C). Similar growth inhibition was observed in Hep3B orthotopic xenografts by administration of AZD1152 (Fig. 5B and C). In the orthotopic model, mouse survival was significantly enhanced by AZD1152 treatment in comparison with the control (p<0.005; Fig. 5D). These results demonstrate that AZD1152 was able to significantly inhibit in vivo growth of a human HCC tumor in the liver microenvironment in mice. All of the host tissues examined, including liver, bone marrow, kidney, intestine, and lung, were histologically normal in all experiments.

Pharmacobiological effects of AZD1152 on orthotopic liver xenografts of human hepatocellular carcinoma cells


The liver xenograft model described above was subjected to histological analysis by immunostaining to investigate the pharmacobiological effects of AZD1152 in the hepatic microenvironment (Fig. 6). Three days after treatment with AZD1152, there was a substantial decrease in PhH3 (Fig. 6E and H) compared with the control (Fig. 6D and G), although after 5days, PhH3 had recovered (Fig. 6F and I). Staining of tumor samples for apoptotic marker cCasp-3 showed gradually elevating levels following AZD1152 treatment (Fig. 6J–O). The hepatocytes from the host liver were histologically normal at all points following AZD1152 administration (Fig. 6A–C).

Materials and methods


Reagents


AZD1152-HQPA and its prodrug AZD1152 were provided by AstraZeneca Pharmaceuticals (Macclesfield, UK).

Cell culture


The human HCC cell lines SK-Hep1, Hep3B, and PLC/PRF/5 were obtained from the American Type Culture Collection (Manassas, VA, USA). Other human HCC cell lines—JHH-1, JHH-2, JHH-4, HuH-1, HuH-6, HuH-7, HLE, HLF, and HepG2—were obtained from the Human Science Research Resources Bank (Osaka, Japan). Culture media were RPMI-1640 (SK-Hep1, Hep3B, HuH-7, and HepG2), Dulbecco’s modified Eagle’s medium (PLC/PRF/5, HuH-1, HuH-6, HLE, and HLF), and William’s E medium (JHH-1, JHH-2, and JHH-4), supplemented with 5% fetal bovine serum (FBS) for HLF cells or 10% FBS for the remaining cell lines. All media supplemented 100U/mL of penicillin and 100μg/mL of streptomycin; all cell lines were cultivated in a humidified incubator at 37°C in 5% carbon dioxide and harvested with 0.25% trypsin–0.03% EDTA.

Analysis of cell proliferation and cell viability


All cell lines were cultured in logarithmic growth phase in the presence of various concentrations of AZD1152-HQPA (0.3–1000nM) for 72h. Cells were seeded at 4×104 cells in six-well plates with the appropriate control medium. After 24h, plates were treated with compound and incubated for 72h at 37°C. At the end of the incubation time, cells were detached from each plate, and viable cells were counted using a hemocytometer. Half-maximal inhibitory concentration (IC50) values were calculated with BioDataFit v.1.02 software using the four-parameter logistic model. The mean values and standard deviations of IC50 were calculated in triplicate for each cell line. To investigate cell viability, triplicate samples of SK-Hep1, Hep3B, and HLF cells were cultured in the presence of various concentrations of AZD1152-HQPA (1–100nM) for 72h. The number of nonviable cells was assessed using a hemocytometer and trypan blue dye exclusion.

Western blotting


Total protein was extracted from each cell line, as described previously [28]. Protein levels of Aurora B kinase, phosphohistone H3 (PhH3), and alpha-tubulin (control) were detected using standard western blot analysis on 8–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Blots were incubated overnight at 4°C with the primary antibody antihuman Aurora B (1:1000; Abcam, Cambridge, UK; Catalog No. ab2254) or antihuman PhH3 (1:200; Santa Cruz, CA, USA; Catalog No. sc-8656-R), then at room temperature for 1h with anti-alpha-tubulin (1:5000; Sigma–Aldrich, St. Louis, MO, USA; Catalog No. T9026). Appropriate secondary antibodies were added for 2h, and protein expression was visualized with enhanced chemiluminescence by the ECL western blotting detection system (GE Healthcare, Buckinghamshire, UK). The expression ratio of Aurora B kinase to the control was analyzed using Multi-Gage software (FUJIFILM, Tokyo, Japan).

Flow cytometry


Samples of all cell lines in logarithmic growth phase were exposed to AZD1152-HQPA 100nM for 24h, and then fixed in 70% ethanol at −20°C overnight. Cells were rehydrated in phosphate-buffered saline (PBS), and then resuspended in PBS containing RNase 100μg/mL (Sigma) and propidium iodide 10μg/mL. Cellular DNA content was analyzed on a FACS Caliber flow cytometer (Becton & Dickinson Biosciences, San Jose, CA, USA). For detection of apoptosis, cells were labeled with the Annexin V-FITC Kit (Miltenyi Biotec, Bergisch Gladbach, Germany; Catalog No. 130-092-052) at room temperature for 15min, followed by analysis on a FACS Caliber flow cytometer.

Immunocytochemistry and immunohistochemistry


SK-Hep1, Hep3B, and HLF cells were cultured on glass slides coated with silane in the presence of various concentrations of AZD1152-HQPA (1–100nM) for 4h. They were then fixed using 3.7% formalin for 10min and permeabilized using 100% methanol for 20min for immunocytochemical detection of PhH3.


Xenograft tumor tissue was harvested, formalin fixed, and paraffin embedded. The primary antibodies, PhH3 (Upstate Cell Signaling Solution, Danvers, MA, USA; Catalog No. 9701) and anti-cleaved caspase-3 (anti-cCasp-3; Upstate Cell Signaling Solution; Catalog No. 9661), were used at 1:100 and 1:400 dilution, respectively, in PBS containing 1% bovine serum albumin. The tissue sections and slides were stained with an automated immunostainer (BenchMark XT; Ventana Medical Systems, Tucson, AZ, USA) using heat-induced epitope retrieval and a standard diaminobenzidine detection kit (Ventana).

In vivo studies in a subcutaneous tumor xenograft model


A subcutaneous tumor model was used to analyze the in vivo activity of AZD1152, as described previously [29]. Five-week-old female nude mice (nu/nu) were purchased from Japan SLC (Shizuoka, Japan) and kept under pathogen-free conditions, fed standard food, and given free access to sterilized water. In all experiments, mice were anesthetized by 100mg/kg Nembutal intraperitoneal injection. Subcutaneous xenografts were established by inoculating 1×107 SK-Hep1 cells into the right dorsal flank. Palpable tumors were confirmed on day 5 following inoculation, and mice were randomized into treatment groups to receive AZD1152 or the control Tris-buffered saline. AZD1152 was prepared in Tris-buffered saline (pH 9) and administered by intraperitoneal injection. Tumor size was measured using calipers as frequently as every other day for 2weeks, and tumor volumes were calculated as AB2×0.5 (A, length; B, width). The Animal Care Committee of Tokyo Medical and Dental University School of Medicine approved the experimental protocols in accordance with its institutional guidelines.

In vivo studies in a novel orthotopic xenograft model


An orthotopic xenograft model was created by direct intrahepatic inoculation of SK-Hep1 and Hep3B cells, as described by Lu et al. [25]. With the mice fully anesthetized, a small transverse incision was made below the sternum to expose the liver. Then, 2.5×106 cells suspended in 25μL of RPMI-1640 and 25μL of Matrigel (Becton & Dickinson Biosciences) were slowly injected at a 30° angle into the upper left lobe of the liver using a 28-gauge needle. After injection, a small piece of sterile gauze was placed on the injection site, and light pressure was applied for 1min to prevent bleeding. The abdomen was then closed with a 6–0 silk suture. Pilot studies confirmed development of liver tumors in 6 of 6 mice at 14days after inoculation. AZD1152 (100mg/kg) or the control Tris buffer was administered to mice by intraperitoneal injection on 2 consecutive days per week for 2weeks starting on day 14 after inoculation. In both cell lines at 4weeks after initiation of treatment, mice were sacrificed to assess the antitumor effects of AZD1152. The survival end points were defined as ascites formation in the hepatoma-bearing mice [30]. Animal survival data were entered in the Kaplan–Meier Life Table format and presented as the cumulative survival plot. Statistical differences were analyzed by Mantel–Cox log-rank test. All in vivo procedures were approved by the Animal Care Committee of Tokyo Medical and Dental University (Permission No. 090235).


The pharmacobiological effects of AZD1152 treatment in the orthotopic liver tumors were assessed by immunohistochemical analysis of PhH3 and cCasp-3 expression in control tumors and in those harvested 3 and 5days after initiation of AZD1152 treatment.