Inhibition of Aurora B by CCT137690 sensitizes colorectal cells to radiotherapy
- Xiaoyu Wu†1,
- Wentao Liu†2,
- Qinhong Cao1,
- Che Chen1,
- Zhiwei Chen1,
- Zhe Xu1,
- Weisu Li1,
- Fukun Liu1 and
- Xuequan Yao1Email author
© Wu et al.; licensee BioMed Central Ltd. 2014
Received: 16 October 2013
Accepted: 20 January 2014
Published: 29 January 2014
Colorectal cancer is the third most commonly diagnosed cancer worldwide. Although surgery remains the best treatment for this disease, adjuvant chemotherapy and radiotherapy are also very important in clinical practice. However, the notorious refractory lack of responses to radiochemotherapy greatly limits the application of radiochemotherapy in the context of colorectal cancer.
There is a growing interest in the role that Aurora B may play in colorectal cancer cell survival as well as other cancer subtypes. In the current study, we sought to ascertain whether blocking of Aurora B signaling machinery by a small molecule inhibitor, CCT137690, could synergize radiation-induced colorectal cancer cell death. Results showed that CCT137690 increases the sensitivity of SW620 cells to radiation. Mechanistic studies revealed that Aurora B-Survivin pathway may be involved in this synergistic effect.
Taken together, our results for the first time show that Aurora B inhibition and radiation exert a synergistic effect, resulting in enhanced colorectal cancer cell death. This synergistic effect is clinically relevant as lower doses of radiation could be used for cancer treatment, and could provide significant clinical benefits in terms of colorectal cancer management, while reducing unwanted side-effects.
KeywordsCCT137690 Aurora B Radiotherapy Colorectal cancer
Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide with over 1.4 million new cancer cases and 0.6 million estimated deaths every year [1–3]. The incidence of CRC has rapidly increased in China and other Asian countries over the last few decades [4–8], and identifying better ways of treating this cancer is paramount. Although surgery of CRC remains the best treatment, adjuvant chemotherapy and radiotherapy are also very important and beneficial treatments for patients [9, 10]. After surgery, chemotherapy and radiotherapy is used to target small cancerous tissues that may be missed during surgery and help to prevent cancer recurrence.
Aurora kinases (of which there are 3 isoforms: Aurora A, B and C) are the most important serine/threonine-protein kinases which regulate the function of centrosomes, spindles, and kinetochores for proper mitotic progression [9, 11]. Aurora A overexpression has been observed in various cancers including colorectal cancers. Baba et. al. reported overexpression of Aurora A protein in 19% of CRC by immunohistochemistry . High copy amplification of the Aurora A gene was found in colorectal tumors  and associated with chromosomal instability phenotypes . In another report, up-regulation of Aurora kinases were detected in 48.5% (97/200) of patients with colorectal carcinoma . Similarly, a previous study reported that the presence of nuclear Aurora B was strongly associated with lymph node metastasis in colorectal cancer . In metastatic colorectal cancer, patients with a high expression level of Aurora B lived significantly shorter compared with patients with a low expression level . Taken together, these studies highlight the association of altered aurora kinases and CRC.
As far as therapeutic options, 5-Fluorouracil (5-FU) remains the most commonly used chemotherapeutic agent for CRC. However, CRC tumors are highly refractory to chemotherapy and many patients eventually relapse. Because of the established roles of Aurora kinases in tumor initiation and progression, many inhibitors of Aurora kinases have been specifically tested for the treatment of colorectal cancers in combination with 5-FU, with some currently in clinical trials [18–22].
Recent studies showed that overexpression of Aurora kinases might have a role in chemo- and radiotherapy resistance of cancers [23, 24]. Consistent with this notion, inhibition of Aurora kinases can enhance radiation sensitivity of cancer cells [25, 26]. For example, inhibition of Aurora B sensitizes mesothelioma cells by enhancing mitotic arrests  and also potently suppresses repopulation during fractionated irradiation of human lung cancer cell lines .
CCT137690 is a newly synthesized compound which has been shown to inhibit the activities of Aurora kinases. IC50 values of CCT137690 are 15 and 25 nM for Aurora A and B, respectively. Although CCT137690 has shown promising therapeutic effects on different cancer cells (especially for colorectal cancer) [29–31], a narrow safety margin (due to its activity against hERG ion-channel) may limit its preclinical development .
The main cause of treatment failure and recurrence is resistance of cancer cells to radiation and drugs [32, 33]. Since inhibition of Aurora kinases can sensitize cancer cells to radiotherapy, it is expected that combining radiotherapy and Aurora inhibition for colorectal cancers may achieve a synergistic therapeutic effects. Concomitant inhibition of Aurora kinases and radiotherapy can also potentially decrease the dosages of either medicine or radiation, which in turns can reduce the side effects of the treatments. Therefore, in our current study we sought to explore whether the combination of radiotherapy with CCT137690 may prove efficacious in the treatment of colorectal cancer cell lines. In this way, optimized combinatorial treatment may lead to a decrease in the requirement of CCT137690 for therapeutic benefit.
Materials and methods
The authors declared that the current research has been approved by The Ethics Committee of Nanjing University of Traditional Chinese Medicine.
DMEM and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific at CHINA (Shanghai, China). 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazoliumbro-mide (MTT) was obtained from Sigma-Aldrich (Shanghai, China). Anti-Aurora B antibody and anti-Histone H3 (phospho S10) antibody were obtained from Abcam. Anti-Survivin antibody was purchased from Cell Signaling. Anti-Histone H3 and GAPDH antibody were obtained from Santa Cruz Biotechnology.
The human colorectal adenocarcinoma cell lines, SW48 and SW620, were obtained from the American Type Culture Collection. The cells were maintained in DMEM supplemented with 10% heat inactivated FBS at 37°C, 5% CO2, and 95% humidity.
Plasmids and transfection
The full-length cDNA sequence of survivin was amplified from total RNA of SW620 cells by using Reverse Transcription PCR. The fragment was inserted into pBABE-Puro vector. The control vector plasmid or the plasmid encoding survivin was transfected into Phoenix Retroviral Expression System. Virus was produced and applied onto target cells according to the standard protocol. The cells were subjected to drug-selection for 3 days (0.5 μg/ml of puromycin) to enrich for the desired cells.
Silencing of Aurora A and B in cells
1.5 × 105 cells were seeded in 60-mm plates and incubated for 24 h before transfection. The negative control siRNA or Aurora A or B siRNA was diluted in Opti-MEM I Reduced Serum Medium and mixed with Lipofectamine 2000 according to the manufacturer’s instructions. The mix of DNA and Lipofectamine was added to cells. After 72 hours post-transfection, expression levels of Aurora genes were determined by Real-time PCR and cells were used for different assays.
Cells were plated in dishes, and then irradiated with X-ray (104.93 cGy/min at 210 kV and 12 mA) by using an X-ray irradiator (MBR-1505R2; Hitachi Medico, Tokyo, Japan) for indicated dosages.
Determination of surviving fraction
2 × 105 cells were plated in a 60-mm dish. 24 hours later, the cells were exposed to different dosages of ionization radiation. After a 6-hour recovery, one percent of the cells were re-plated in a new dish. After 10 days the number of colonies formed were counted.
Combination effect of radiation and CCT137690
Cells were first treated with CCT137690 at different concentrations for 48 hours before they were exposed to different dosages of ionization radiation.
Cell cycle assay
Cells were collected by trypsinization and washed with PBS, centifuged and then resuspended in 0.4 ml of PBS and fixed by adding 1ml cold ethanol slowly. Cells were kept at 4°C overnight. For analysis, cell suspensions were centrifuged at 1500 rpm for 5 mins, washed with PBS and re-suspended in 500 μl staining solution (PBS with 50 μg/ml PI together with 50 μg/ml RNase A) at 37°C for 30 mins in the dark. Cells were analyzed by flow cytometry.
MTT assay for cell viability
104 cells were seeded into 96-well plates and were treated to either vehicle (DMSO) or different concentrations of CCT137690 for 48 hours. Cell viability was determined and quantified by using MTT assay.
Guava Nexin assay
The Guava Nexin assay was performed following manufactory protocol (Millipore). Briefly, attached and suspended cells were all collected. Cells were resuspended in 100 μL of medium and incubated together with 100 μL of Guava Nexin Reagent for 20 minutes at room temperature in the dark. Samples then were measured on a Guava System (Millipore). The data were analyzed by using the software provided by the company.
Radiotherapy stands a major adjunctive therapeutic option for colorectal cancer management. Although there have been intensive investigations on the optimal regimen of radiotherapy for this lethal disease, very limited success have been made during the past several decades.
CRC is notorious for being refractory to both chemotherapy and radiotherapy. Thus investigators are particularly interested in characterizing novel molecule targets which exert regulatory effects on sensitivity to radiochemotherapy in CRC patients. Positive results from these studies might be clinically important since untoward side effects from radiotherapy or chemotherapy stands as major concerns for clinicians in tumor management and sensitizers of radiochemotherapy may help to reduce dosage load and associated toxic side effects.
In light of this notion, we started to search for potential sensitization targets for radiotherapy of CRC subjects and we found that there is a recent growing interest in the role of Aurora B and cancer biology.
In terms of synergistic effect of Aurora B inhibition and radiotherapy sensitivity, a previous study has shown that Aurora B inactivation sensitizes mesothelioma cells . In addition, Aurora B inhibition also potently suppresses repopulation during fractionated irradiation of human lung cancer cells .In the current study, we first show that SW-620 colorectal cancer cells are relatively resistant to Aurora B inhibition by CCT137690 and to radiation (Figures 1 and 2). However, we found that the combination of Aurora B inhibition and radiation exerts synergistic effects on cancer cell growth inhibition. Our results showed that low-dose radiation (1 Gy) greatly exaggerates the growth inhibitory effect of CCT137690 on SW-620 cells (Figure 3A), as well as a low-dose of CCT137690 dramatically increases the sensitivity of cells to radiation (Figure 4A).
Our observations provide a good proof of concept that both chemotherapy and radiotherapy doses could be greatly lowered by taking the advantages of synergistic effects of those two interventions. This could be translated into the clinic where the expectation is that there would be less adverse side-effects and better patient tolerance at lower doses. These findings are especially important given that CT137690 has a narrow safety margin.
In terms of understanding of the mechanism by which inhibiting Aurora B increases radiosensitivity of CRC cells, we found that Aurora B-survivin pathway may be involved (Figure 5). These findings are consistent with several reports showing the association of Aurora B and survivin in context of CRC. For example, Tuncel et al. reported that nuclear Aurora B and cytoplasmic survivin expression is involved in lymph node metastasis of colorectal cancer . Moreover, Aurora-survivin signaling machinery has been implicated in other cancers such as myelodysplasia , chronic lymphocytic leukemia , head and neck squamous cell cancer . In this regard, we observed that forced-expression of survivin dramatically ameliorates Aurora B-inhibition induced CRC cell death in the context of radiation (Figure 5I).
Taken together, our results for the first time showed that Aurora B inhibition, via CCT137690, and radiation exposure may play synergistic effects in colorectal cancer death. Taking advantage of this synergistic effect, a lower dose of radiation exposure and/or chemical exposure is required for cancer cell death induction, which may have significant clinical implications for CRC management.
Funding disclosure statement
No current external funding sources for this study, all fundings for the current study are from intramural funding program to Liu FK and Yao XQ.
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