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[Cancer Research 65, 2899-2905, April 1, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

RNA Interference Targeting Aurora Kinase A Suppresses Tumor Growth and Enhances the Taxane Chemosensitivity in Human Pancreatic Cancer Cells

Tatsuo Hata1, Toru Furukawa1, Makoto Sunamura2, Shinichi Egawa2, Fuyuhiko Motoi2, Noriyuki Ohmura2, Tomotoshi Marumoto3, Hideyuki Saya3 and Akira Horii1

Departments of 1 Molecular Pathology and 2 Gastroenterological Surgery, Tohoku University School of Medicine, Sendai Miyagi, Japan and 3 Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan

Requests for reprints: Akira Horii, Department of Molecular Pathology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Japan. Phone: 81-22-717-8042; Fax: 81-22-717-8047; E-mail: horii{at}mail.tains.tohoku.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
AURKA/STK15/BTAK, the gene encoding Aurora A kinase that is involved in the regulation of centrosomes and segregation of chromosomes, is frequently amplified and overexpressed in various kinds of human cancers, including pancreatic cancer. To address its possibility as a therapeutic target for pancreatic cancer, we employed the RNA interference technique to knockdown AURKA expression and analyzed its phenotypes. We found that the specific knockdown of AURKA in cultured pancreatic cancer cells strongly suppressed in vitro cell growth and in vivo tumorigenicity. The knockdown induced the accumulation of cells in the G2-M phase and eventual apoptosis. Furthermore, we observed a synergistic enhancement of the cytotoxicity of taxanes, a group of chemotherapeutic agents impairing G2-M transition, by the RNA interference–mediated knockdown of AURKA. These results indicate that inhibition of AURKA expression can result in potent antitumor activity and chemosensitizing activity to taxanes in human pancreatic cancer.

Key Words: aurora A • pancreatic cancer • RNAi • taxane


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pancreatic cancer is one of the most common cancers with an extremely poor prognosis around the world because of its aggressive invasion, early metastasis, resistance to existing chemotherapeutic agents and radiation therapy, and lack of specific symptoms (1). To improve the horrible prognosis, we need to find novel approaches to both diagnosis and treatment that are far more efficient than currently available techniques. Molecular studies of cancers can lead us to find new drugs for molecular target therapy such as trastuzumab in breast cancer and gefitinib in lung cancer (2, 3). Pancreatic cancer involves very complicated molecular changes (4, 5); our previous comparative genomic hybridization analysis of the pancreatic cancer genome revealed intricate genomic alterations in multiple chromosome arms, including losses of 1p, 3p, 4q, 6q, 8p, 9p, 12q, 17p, 18q, and 21q and gains of 8q and 20q (6). The increase in copy number of 20q13 is especially prominent in pancreatic cancer (6, 7). Amplification of 20q13 is also found in several other types of human cancer such as colorectal cancer, breast cancer, bladder cancer, ovarian cancer, and hepatocellular cancer, suggesting the existence of an important oncogene(s) that plays a crucial role in a variety of human cancers in this area (8–12). AURKA was identified as one of the candidate oncogenes from the amplicon on 20q13 (13).

AURKA is one of three related genes (AURKA, AURKB, and AURKC) encoding AURORA kinases/serine-threonine kinases that play important roles in mitotic spindle formation and centrosome maturation and are physiologically essential for proper segregation of chromosomes into daughter cells (14). Since their discovery, the aurora kinases have been shown to be closely associated with carcinogenesis; an overexpression of AURKA transforms NIH3T3 cells and gives rise to aneuploid cells containing multiple centrosomes and multipolar spindles, indicating that AURKA is one of the fundamental cancer-associated genes and a potential target for diagnosis and treatment (14, 15). To further elucidate the possibility for utilization of AURKA in the treatment of human pancreatic cancer, we analyzed the phenotypes of cultured pancreatic cancer cells after RNA interference (RNAi)–mediated AURKA knockdown (16). Moreover, we tested the synergistic enhancement of the cytotoxicity of taxanes in pancreatic cancer cells by AURKA-RNAi.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Pancreatic cancer cell lines and cell culture. Three human pancreatic cancer cell lines, Panc-1, MIA PaCa-2, and SU.86.86, were purchased from American Type Culture Collection (Manassas, VA), and PK-1 was obtained from the original developer (17). All cells were maintained in RPMI 1640 containing 10% fetal bovine serum under atmosphere of 5% CO2 with humidity at 37°C.

Short interference RNA transfection. Oligonucleotides of short interference double-strand RNAs (siRNA) with two thymidine residues (dTdT) overhanging at the 3' end for knock down of the expressions of AURKA and the luciferase gene (GL2), including 5'-AUGCCCUGUCUUACUGUCA-3' in the sense strand corresponding to nucleotides 725 to 743 relative to its start codon for the former (18) and 5'-CGUACGCGGAAUACUUCGA-3' in the sense strand for the latter used as a control as described previously (19), were purchased from Japan Bioservice (Asaka, Japan). The siRNAs were dissolved into 5x annealing buffer [500 nmol/L potassium acetate, 150 nmol/L HEPES-KOH (pH 7.4), and 10 nmol/L magnesium acetate] to a final concentration of 20 µmol/L, boiled for 60 seconds, and gradually cooled down to 37°C for 60 minutes to anneal them. In vitro transfection was done using the Oligofectamine reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.

Immunoblotting. A total of 3 x 105 cells were plated in 6-well plates (35 mm in diameter) and allowed to adhere for 24 hours; the transfection of double-stranded siRNA oligonucleotides was done as described above. After 48 hours, cells were harvested, and protein concentrations in total cell lysates were measured using the detergent-compatible protein assay kit (Bio-Rad, Hercules, CA). A 50-µg aliquot of the protein was subjected to immunoblotting as described previously using a 10% to 20% polyacrylamide gradient gel (Bio-Rad; ref. 20). The antibodies used were anti-AURKA polyclonal antibody (Transgenic, Kumamoto, Japan), anti-ß actin monoclonal antibody (Sigma, St Louis, MO), and horseradish peroxidase–conjugated anti-mouse or anti-rabbit immunoglobulin antibodies (Amersham Biosciences Co., Piscataway, NJ). For blocking conditions and concentrations of antibodies, we followed the manufacturer's recommendations. Signals were visualized by reaction with enhanced chemiluminescence Detection Reagent (Amersham Biosiences) and digitally processed using LAS 1000 Plus with a Science Lab 99 Image Gauge (Fuji Photo Film, Minamiashigara, Japan).

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. A total of 5 x 103 cells in 100 µL of the medium were plated in 96-well plates, and the RNA oligonucleotides were transfected. Every 24 hours up to 7 days, the medium was replaced with 100 µL of 0.05% 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT)/PBS (–) and incubated for 1 hour. After the incubation, the MTT solution was removed, and the cells were suspended in 100% ethanol. Absorbance was measured at 590 nm using Versamax microplate reader (Amersham Biosiences).

Flowcytometry. Cells were harvested with trypsin-EDTA, washed with PBS (–), and fixed with 70% ethanol at –20°C for a few days. The fixed cells were pelleted, resuspended in 100 µL of hypotonic citric buffer (192 mmol/L Na2HPO4 and 4 mmol/L citric acid), and incubated for 30 minutes at room temperature. The cells were pelleted and suspended in PI/RNase/PBS (100 µg/mL propidium iodide and 10 µg/mL RNase A) overnight at 4°C. Analysis of DNA content was done on a FACSCalibur system (BD Immunocytometry Systems, San Jose, CA).

Plasmid constructions and colony formation assay. pSUPER-retro.neo+GFP (pSR) vectors (ref. 21; Oligoengine, Seattle, WA) harboring gatccccATGCCCTGTCTTACTGTCAttcaagagaTGACAGTAAGACAGGGCATttttta and gatccccCGTACGCGGAATACTTCGAttcaagagaTCGAAGTATTCCGCGTACGttttta at its BglII/HindIII sites were prepared for expressing short hairpin RNAs (shRNA), as indicated in upper cases, specific for interfering expressions of AURKA (pSR-shAURKA) and luciferase (pSR-shGL2), respectively. The fidelity of the inserts was confirmed by sequencing both strands with primers of 5'-CGATCCTCCCTTTATCCAGC-3' for the sense strand and 5'-CAGAACACATAGCGACATGC-3' for the antisense strand using an ABI PRIZM BigDye Terminator Cycle Sequencing FS Ready Reaction Kit and an ABI PRIZM 310 DNA Analyzer according to the manufacturer's instructions (Applied Biosystems, Foster City, CA). For colony formation assays, 1 x 106 cells were plated in 10-cm culture dishes and transfected with 4 µg of either pSR-shAURKA, pSR-shGL2, or pSR using LipofectAMINE PLUS reagent (Invitrogen) according to the manufacturer's protocol. After 24 hours, transfected cells were passaged and cultured in the appropriate culture medium containing G418 at 500 µg/mL in concentration. After 14 days, cells were fixed with methanol and stained with 0.1% crystal violet. Visible colonies were manually counted.

Tumorigenicity in mice xenograft model. Four-week-old female Crj:CD-1(ICR)-nu mice were obtained from Charles River Japan, Inc. (Yokohama, Japan) and maintained under pathogen-free conditions. Each aliquot of 2 x 106 cells of MIA PaCa-2 stably trasfected with either pSR-shAURKA or pSR-shGL2 was suspended into 100 µL of PBS (–) containing 20% of Matrigel Growth Factor Reduced (Becton Dickinson Labware, Flanklin, NJ). These two sets of cells were s.c. injected into both flanks of mice. The inoculations were done in six mice. Tumor diameters were measured every 3 days, and each tumor volume in mm3 was calculated by the following formula: V = 0.4 x D x d2 (V, volume; D, longitudinal diameter; d, latitudinal diameter). Animal experiments in this study were done in compliance with Tohoku University School of Medicine institutional guidelines.

Statistical analysis. All experiments were done in duplicate or triplicate. A two-tailed Student's t test was used for statistical analysis of comparative data using Microsoft Excel software (Microsoft Co., Tokyo, Japan). Values of P < 0.05 were considered as significant and indicated by asterisks in the figures.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Specific knockdown of AURKA in pancreatic cancer cell lines. To address the question of whether AURKA could serve as a therapeutic target for pancreatic cancer, we employed the siRNA method in an attempt to deplete the expression of AURKA in cultured pancreatic cancer cells. We prepared 21-mer oligoribonucleotides targeting AURKA and Photinus pyralis luciferase (GL2) based on information described elsewhere (18, 19). The oligoribonucleotides were annealed to give a double-strand siRNA and were transfected at 200 nmol/L into pancreatic cancer cells, MIA PaCa-2, Panc-1, PK-1, and SU.86.86, using the Oligofectamine reagent. After 48 hours, the cells were harvested, and their total lysates were analyzed by immunoblotting to see the effects of the siRNA on AURKA protein levels. As shown in Fig. 1A, dramatic suppression of AURKA expression was observed in all four cell lines by the siRNA targeting AURKA but not GL2. The siRNA oligonucleotides did not cause a nonspecific inhibition of gene expression, as shown by expressions of ß-actin. Furthermore, the suppression of AURKA protein levels was achieved in a dose-dependent manner as shown in Fig. 1B; partial to complete suppressions were observed along with increasing concentrations of the siRNA oligonucleotides.



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  		Figure 1. siRNA directed against AURKA specifically inhibits its expression. A, expression of AURKA 48 hours after the transfection of siRNA at 200 nmol/L directed against AURKA (AU) and luciferase (GL2) in pancreatic cancer cell lines, MIA PaCa-2, Panc-1, PK-1, and SU.86.86, detected by immunoblotting. NO, no transfection. B, a dose-dependent knockdown of AURKA expression by siRNA. Panc-1 and MIA PaCa-2 cells were transfected with the siRNA targeting AURKA at various concentrations ranging from 0 to 200 nmol/L. Expression of AURKA 48 hours after the transfection was detected by immunoblotting. ß-Actin expression was monitored as the control. The ratio of AURKA/ß-actin was calculated by using densitometry, and values were normalized by dividing by the ratio at no treatment (0 nmol/L).

 
Knockdown of AURKA inhibits in vitro growth and colony formation. In phenotypic analyses, we first investigated effects of AURKA siRNA on the in vitro growth of pancreatic cancer cells. The siRNA transfection was done at 200 nmol/L to achieve complete suppression of AURKA expression, and cellular proliferations were monitored by MTT assay daily for 7 days. As shown in Fig. 2A, cell proliferation was significantly suppressed by AURKA-siRNA in all four pancreatic cancer cell lines as compared with GL2-siRNA. To observe the stable phenotypic consequences of siRNA-mediated knockdown in the cells, the 19-mer target sequences bridged by a 9-mer spacer were introduced into the pSUPER.retro.neo+GFP (pSR) vector to generate a short hairpin RNA targeting AURKA (pSR-shAURKA) or luciferase (pSR-shGL2), as described in Materials and Methods. For the colony formation assay using these vectors, MIA PaCa-2 and Panc-1 cells were transfected with either pSR-shAURKA, pSR-shGL2, or pSR empty vector and maintained in the selection medium containing G418 for 2 weeks. As expected from the MTT assay done in the siRNA experiment, the numbers of colonies were significantly decreased in pSR-shAURKA transfectants compared with the controls in both cell lines (see Fig. 2B). These results indicated that the RNAi-mediated specific knockdown of AURKA induced strong inhibition of pancreatic cancer cell growth in vitro.



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  		Figure 2. siRNA directed against AURKA suppresses in vitro growth of the pancreatic cancer cells. A, MTT assay of in vitro proliferation of the cells. These experiments were performed for four times. B, colony formation assay of G418-resistant colonies of the cells transfected either with pSUPER.retro.neo+GFP vector (pSR), pSR expressing short hairpin RNA directed against AURKA (pSR-shAURKA) or that directed against luciferase (pSR-shGL2). These experiments were performed for four times. *, P < 0.05.

 
Knockdown of AURKA suppresses tumorigenicity in vivo. We wondered whether the down-regulation of AURKA expression in pancreatic cancer cells would affect their ability to form tumors in nude mice. To address this question, we established stable transfectants of MIA PaCa-2 cells with treatment by either pSR-shAURKA or pSR-shGL2. These cells had modestly reduced expression of the AURKA protein (Fig. 3A). We then tested the in vitro growth of these cells and found that they showed rational growth retardation but not complete suppression, probably because of their modest level of knockdown of AURKA expression (Fig. 3B). Next, we injected the aliquot of 2 x 106 cells s.c. into six athymic nude mice and monitored their tumor growth. As shown in Fig. 3C, the pSR-shGL2 transfected cells gave rise to tumors within 4 weeks in all six mice, whereas the pSR-shAURKA transfected cells did not develop tumors in any of them. These results indicated that RNAi-mediated knockdown of AURKA exerted a strong antitumorigenic effect in vivo on pancreatic cancer cells.



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  		Figure 3. Stable suppression of AURKA inhibits in vivo tumorigenicity. A, AURKA expressions in MIA PaCa-2 cells stably transfected with pSR-shAURKA or pSR-GL2. Mock, no transfection. B, MTT assay of in vitro growth of the stable clones. These experiments were performed for four times. *, P < 0.05 (significant differences between mock and pSR-shAURKA and between pSR-shGL2 and pSR-shAURKA). C, tumorigenicity of the stable clones in the mouse-xenografted model. The stable clones were inoculated s.c. into both flanks of six nude mice. Sizes of the tumors generated were measured at 4 weeks after the inoculation. D, representative features of tumors in a mouse 4 weeks after the inoculation.

 
Knockdown of AURKA induces G2-M accumulation and apoptosis. AURKA is an important regulator of bipolar spindle formation and therefore essential for accurate chromosome segregation. We hypothesized that the growth suppression of the pancreatic cancer cells we observed by the RNAi-mediated knockdown of AURKA was caused by disruption of cell cycle transition with delay in mitotic entry, which has been shown in other kinds of mammalian cells (19, 22). To determine this possibility, we analyzed the DNA contents of cell populations reflecting the cell cycle distribution after knockdown of AURKA mediated by transfection of the siRNA in 200 nmol/L in MIA PaCa-2 and Panc-1 cells. As shown in Fig. 4A, an increase in the G2-M population with a concomitant decrease in the G0-G1 population was observed after AURKA-siRNA treatment in both cells. Moreover, as we observed the changes in the DNA content during the time course after the transfection, we found obvious and significant increases in the sub-G1 populations after 72 hours in MIA PaCa-2 cells and after 96 hours in Panc-1 cells (see Fig. 4B). These results indicate that the siRNA-mediated knockdown of AURKA led the pancreatic cancer cells to abnormal accumulation in the G2-M phase and to eventual apoptosis.



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  		Figure 4. siRNA directed against AURKA induces G2/M accumulation and eventual apoptosis. A, cells were collected 72 hours after siRNA transfection at 200 nmol/L and subsequently assayed for their DNA content by flow cytometry. These experiments were performed for four times. Representative results are shown. B, time course quantification of sub-G1 population after siRNA transfection at 200 nmol/L. These experiments were performed for four times. *, P < 0.05.

 
Knockdown of AURKA significantly enhances cytotoxicities of taxanes. Taxanes, chemotherapeutic agents impairing the disassembly of microtubules that is crucial for the proper segregation of chromosomes during mitosis of eukaryotic cells, may synergistically enhance the effect of RNAi-mediated knockdown of AURKA, because it can accumulate cells in the G2-M phase where AURKA plays essential roles. To determine this possibility, we investigated the synergistic effects of AURKA siRNA and taxanes in MIA PaCa-2 and Panc-1 cells. First, we searched for the best concentration of siRNAs in this experiment because siRNA treatment itself showed some cytotoxicities. The concentration at 10 nmol/L seemed to be the best because no significant difference in cell proliferation between AURKA-siRNA and GL2-siRNA treatments was found (data not shown). The concentration for paclitaxel and docetaxel were set by IC50 previously determined by MTT assay (data not shown). Then we tested the potential enhancement of the cytotoxic effect of taxanes by AURKA-siRNA by treating cells either with AURKA or GL2-siRNA at 10 nmol/L in concentration followed by addition of either 10 nmol/L paclitaxel or 5 nmol/L docetaxel 4 hours later. After 72 hours of incubation, the viabilities of the cells were measured by MTT assay. Although the modest AURKA-siRNA at 10 nmol/L alone did not show any difference in cytotoxic effect when compared with the control treatment withGL2-siRNA, it enhanced the cytotoxic effects induced by taxanes significantly more strongly than the control treatment (Fig. 5A). A reciprocal set of experiments showed that taxanes can enhance the cytotoxic effect of AURKA-siRNA, as shown in Fig. 5B; the synergistic enhancement of the cytotoxic effect of AURKA-siRNA by taxanes were obvious from the treatment at 10 nmol/L siRNA and accelerated with increasing doses. These results indicate that the RNAi-mediated knockdown of AURKA can synergistically enhance the chemosensitivities of these pancreatic cancer cells to taxanes.



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  		Figure 5. Synergistic enhancement of cytotoxicity between siRNA directed against AURKA and taxanes. A, survival cells quantitated by MTT assay after siRNA transfection targeting AURKA or luciferase (GL2) at 10 nmol/L only or subsequent addition of 10 nmol/L paclitaxel or 5 nmol/L docetaxel. These experiments were performed for four times. *, P < 0.05. N.S., not significantly different. B, survival cells quantitated by MTT assay after various doses of siRNA transfection targeting AURKA or luciferase (GL2) only or subsequent addition of 10 nmol/L paclitaxel or 5 nmol/L docetaxel. Values were normalized by dividing them by control values of siRNA-GL2 at each concentration. These experiments were performed for four times. *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
AURKA is a commonly amplified and overexpressed gene in various types of cancers, including pancreatic cancer. In attempting to determine the possibility of AURKA as a therapeutic target, we employed the RNAi technique for knockdown of its expression and analyzed its phenotype. We found that a transient knockdown of AURKA strikingly inhibited growth and colony formation of pancreatic cancer cells in vitro. Stable suppression of AURKA in pancreatic cancer cells revealed an almost complete abrogation of their tumorigenicity in a mouse xenograft model. The knockdown induced accumulation of the cells in the G2-M phase and eventual apoptosis. Rojanala et al. (23) recently reported that antisense oligonucleotide mediated transient suppression of AURKA resulted in growth suppression, G2-M arrest, and eventual apoptosis in vitro. Our results are in good agreement with theirs. We further showed that the knockdown of AURKA significantly enhanced the cytotoxic effect of taxanes. Our findings indicate that AURKA is an attractive candidate for a therapeutic target, because it can regress tumorigenicity and enhance chemosensitivity to taxanes in pancreatic cancer.

We were able to achieve almost complete suppression of AURKA expression by using our siRNA treatment strategy in pancreatic cancer cells. The knockdown of AURKA induced the strong suppression of growth, accumulation in G2-M phase, and eventual apoptosis of the cells. This result suggests that AURKA is an essential molecule for proliferation of cancer cells and a good target for halting proliferation and triggering apoptosis; this can be explained by its key roles in mitosis, as we expected. More strikingly, the knockdown of AURKA completely inhibited tumorigenesis in vivo, even in the modest suppression of its expression achieved by our stable vector-mediated shRNAi strategy. This result suggests that overexpression of AURKA is strongly associated with the in vivo tumorigenic ability of pancreatic cancer cells, leading us to an interpretation of the frequent overexpression of AURKA in primary pancreatic cancer tissues and to an expectation that the knockdown strategy will be practical in stopping the progression of the cancer in vivo.

We found that the RNAi-mediated knockdown of AURKA synergistically enhanced the cytotoxicity of taxanes. Taxanes bind to free tubulin and promote the assembly of tubulin into stable microtubules by interfering with their disassembly. They inhibit cell cycle progression by accumulating cells in M phase at the metaphase-anaphase transition and subsequently lead them to apoptosis. Knockdown of AURKA also induced accumulation of cells in the G2-M phase and led to eventual apoptosis. As we noted, AURKA is essential for the proper arrangement of centrosomes and microtubules. Our results suggest that the combination of AURKA knockdown and taxanes results in strong impairment of M phase progression and the synergistic induction of apoptosis. This is consistent with the recent report indicating that HeLa cells with overexpression of AURKA gained a resistance to paclitaxel by decreasing spindle checkpoint activity (24). In that report, Anand et al. noted that overexpression of AURKA may decrease spindle checkpoint activity. In our experiments, AURKA knockdown may have recovered spindle checkpoint activity and thus increased the sensitivity of taxanes. The mechanism that triggers apoptosis by AURKA knockdown remains to be clarified. Taxanes have cytotoxic activity against various types of cancers including pancreatic cancer. Docetaxel is used for pancreatic cancers as first-line chemotherapy or a second-line combination with gemcitabine in phase II clinical trials (25, 26). Paclitaxel has been used as a radiation sensitizer (27). These taxane-mediated chemotherapies could be more effective in combination with knockdown of AURKA.

RNAi is becoming a conventional application for in vivo cancer therapy (28, 29). An efficient delivery system of siRNA into solid tumors has been developed (30). Our results suggest that RNAi-mediated knockdown of AURKA can be used as a specific gene-targeting therapy to suppress progression of pancreatic cancer. Interestingly, in a recent report, Harrington et al. developed a selective small-molecule inhibitor of Aurora kinases, VX-680, and showed a potent antitumor activity (31). We can assume that this different type of approach is also promising for in vivo abrogation of progression in pancreatic cancer.


   Acknowledgments
 
Grant support: Ministry of Education, Culture, Science, Sports, and Technology of Japan.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Barbara Lee Smith Pierce (a professor with the University of Maryland University College) for editorial work in the preparation of this article.

Received 11/ 5/04. Revised 1/ 5/05. Accepted 1/25/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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