|Ahead of print publication
The effect of danusertib, an Aurora kinase inhibitor, onto the cytotoxicity, cell cycle and apoptosis in pancreatic ductal adenocarcinoma cells
Ismail Ayberk Kirbiyik, Ahmet Ata Ozcimen
Department of Biology, Faculty of Science and Letters, Mersin University Ciftlikkoy Campus, Mersin, Turkey
|Date of Submission||04-Oct-2019|
|Date of Decision||25-Nov-2019|
|Date of Acceptance||21-Jan-2020|
|Date of Web Publication||06-Oct-2020|
Ahmet Ata Ozcimen,
Department of Biology, Faculty of Science and Letters, Mersin University Ciftlikkoy Campus, 33343, Mersin
Source of Support: None, Conflict of Interest: None
Background: Pancreatic cancer is the second type of cancer that causes the most death among the digestive system cancers. Difficulties in early diagnosis and rapidly progressing to advanced stages are most common in high mortality rate of pancreatic carcinoma. The mutation of Bcr-Abl tyrosine kinase and mitotic kinases (such as Aurora kinases), which are involved in the cell cycle, plays an important role in the progression of cancer. Enzymes belonging to Aurora kinase family (-A, -B, -C) have been reported to play a major role in cancer progression, invasion and metastasis. Therefore, the purpose of this study, investigate of the effect of danusertib, an Aurora kinase inhibitor, onto cytotoxicity, apoptosis and cell cycle in human pancreatic carcinoma CFPAC-1 cells.
Materials and Methods: For determining the IC50 value, the 20,000 cells were seeded in E-plate 16 wells in a real-time cell analyzer and various concentrations of danusertib (1-10,000 nM) were applied onto CFPAC-1 cells incubated in IMDM medium. Cell index demonstrated that the proliferation of fraction cells was measured in real time. On the other hand, cell apoptosis and cell cycle arrest test were stained with Annexin V-APC/PI and DNA-cell cycle PI staining respectively by using flow cytometry.
Results: The IC50 value was found to be approximately 400 nM. Danusertib at this concentration induced apoptosis in CFPAC-1 cells (%14,8 at 24 hours; %21,3 at 48 hours). Furthermore, in the cells treated with danusertib, 31.77% and 11.05% were arrested in the S and G2 phases, respectively.
Conclusions: Aurora kinase inhibitor danusertib induced a significant effect of cytotoxic, apoptotic and cell cycle arrest in CFPAC-1 ductal adenocarcinoma cells. Therefore, it may be a potential alternative to the treatment of pancreatic cancers.
Keywords: Apoptosis, Aurora kinase, cell cycle, cystic fibrosis pancreatic adenocarcinoma cell line-1, danusertib
|How to cite this URL:|
Kirbiyik IA, Ozcimen AA. The effect of danusertib, an Aurora kinase inhibitor, onto the cytotoxicity, cell cycle and apoptosis in pancreatic ductal adenocarcinoma cells. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 6]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=297395
Pancreatic cancer has the lowest survival rate (9%) among all cancers. Statistical data shows that the treatment of pancreatic carcinoma is inadequate. However, searching new treatments focused on drugs which can act on several pathways in recent years. As it is well known, loss of cell cycle control generates tumors in a consequence of limitless cell division., Therefore, cancer drugs are primarily targets of arresting proliferative and metastatic cells. Studies on cell cycle inhibitors in cancer have dramatically increased. The most important inhibitors of cell cycle are Bcr-Abl tyrosine-kinase and Aurora kinase inhibitor. Bcr-Abl tyrosine-kinase is an active protein during the transition from the G1 phase to the S phase of the cell cycle. Aurora kinases are activated at the end of the G2 phase and are responsible for sequencing chromosomes, separation and maturation of the centrosome, and condensation of chromatins at poles and are finally effective in cytokinesis.,,,,, Although the role of Bcr-Abl tyrosine-kinase was first seen in leukemias, it was also found to be active in some solid tumors in later studies. The Aurora kinase family consists of three members such as -A, -B, and -C. Aurora-A is activated in the S phase and is responsible for the initiation of mitosis, separation of centrosomes, arrangement of spindle strands, alignment of chromosomes to metaphase plate, and termination of mitosis. Aurora-B is activated in the M phase and is responsible for the chromosomal passenger complex to the separation of chromosomes; regulation of cytokinesis, centromere, and kinetochore; correct attachment of microtubules to kinetochore; and regulation of mitotic control point. Aurora-C has similar tasks as Aurora-B. As a result of animal experiments, Aurora-C was found to be more active in germ cells, but it was found to be highly expressed in various human tumors.,,, As a result of the observations, overexpression of these proteins, which are active in the important stages of the cell cycle, gives the cells unlimited division characteristics and causes cancer.
In this study, danusertib was used for inhibitory effect on Aurora kinase family. The most important feature of danusertib is that it has no effect on the kidneys. In other words, it does not have the same effect on healthy cells as it affects cancerous cells., Danusertib chemically formulated in N-[5- (2-metoksi-2-fenil-asetil)-1, 4, 5, 6-tetrahidro-pirrolo [3,4-c] pirazol-3yl]- 4-(4-metil-piperazin-1-yl)-benzamid [Figure 1],, and its half-maximal inhibitory concentration (IC50) values are measured as 13 nM for Aurora-A, 79 nM for Aurora-B, 61 nM for Aurora-C, and 25 nM for Abl kinase by using cell-free assays.,, Several studies have shown that Bcr-Abl tyrosine-kinase and Aurora kinase families are effective in pancreatic cancer., Therefore, the effects of danusertib on cystic fibrosis pancreatic adenocarcinoma cell line (CFPAC-1) have been shown in our study. The CFPAC-1 is a cell line of epithelial origin that metastasizes to the pancreas from the liver. A mutation in the cystic fibrosis transmembrane regulator gene of chromosome 7 in humans has caused severe cancer effect. This mutation has been found to disrupt the electrolyte transport system in cells. The doubling time of the cells is 32 h for CFPAC-1 cells.,,
The aim of this study was to demonstrate the cytotoxic, apoptotic, and cell cycle inhibitory effects of danusertib on pancreatic ductal adenocarcinoma, leading to further in-vivo studies.
| > Materials and Methods|| |
Danusertib (PHA-739358) (98% purity) was obtained from MyBioSource (San Diego, CA, USA). Fetal bovine serum (FBS), trypsin-ethylenediaminetetraacetic acid, penicillin-streptomycin, amphotericin B, Dulbecco's phosphate-buffered saline (DPBS), and L-glutamine were purchased from Biowest (Nuaillé, France). Iscove's Modified Dulbecco's Media (IMDM) were purchased from Lonza (Basel, Switzerland). CycleTEST Plus DNA Reagent Kit and Trypan Blue were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Annexin V-allophycocyanin (APC)/propidium iodide (PI) Apoptosis Detection Kit was purchased from eBioscience (San Diego, CA, USA). E-plate 16 system was purchased from ACEA Biosciences (San Diego, CA, USA).
The CFPAC-1 cells are characteristically derived from the liver metastatic cells. CFPAC-1 cell lines were cultured in the formulation of IMDM with 10% inactive FBS, 1% penicillin-streptomycin, and 0.5% amphotericin-B at the conditions of 37°C, 5% CO2, and 95% in humidity atmosphere. All experiments were performed with the same passage number cells and the same conditions.
Determination of cytotoxicity
The effects of danusertib on CFPAC-1 cells were identified by measuring the IC50 value of cells by adding different concentrations of danusertib. CFPAC-1 cell lines were cultured in IMDM medium at 37°C and 5% CO2 and 95% in humidity atmosphere. Then, the cells were adjusted to a concentration of 20.000 cells in each well (well volume 270 μl) and incubated with different concentrations of danusertib, which were applied in triplicate with 1 nm, 10 nm, 100 nm, 1 mM, and 10 mM in logarithmic concentrations for 24 h. The IC50 value was measured in E-plate 16 by using xCELLigence RTCA (ACEA Biosciences, California, USA) system.
Apoptotic analysis with Annexin V-APC/propidium iodide
Following cell culture, danusertib-treated (for 24 h and 48 h) CFPAC-1 cells (1 × 106 cells ml) were quantified by Annexin V-APC/PI Apoptosis Detection Kit by using flow cytometry according to the protocol provided. In short, CFPAC-1 cells were treated with danusertib for 24 h and 48 h and then washed with DPBS and resuspended in the binding buffer after being incubated with Annexin V-APC (5 μl) and PI (5 μl) mixture in the binding buffer (100 μL) for 15 min at 25°C in the dark atmosphere. Then, 400 μL of binding buffer was added to each tube. The cells were then immediately examined by a flow cytometer (BD FACSAria III). PI was excited using the 405 nm ultraviolet laser, and APC was excited using the 650 nm red laser. Apoptotic stages of the cells were analyzed by FACSDiva software BD Biosciences, Franklin Lakes, NJ, USA in the instrument of flow cytometry.
Cell cycle analysis by flow cytometry
To demonstrate the arresting of the cell cycle, the CFPAC-1 cells were then starved in serum-free medium for treatment with danusertib (400 nM) for 24 and 48 h. Following incubation, the cells were trypsinized and then washed twice with cold PBS. 1 × 106 cells were separated for each group and stained with PI according to the CycleTEST Plus DNA Reagent Kit protocol. After the dying stages, the cells were analyzed by ModFit software Verity Software House, Topsham, ME, USA in the flow cytometer.
The cytotoxicity results were evaluated by using xCELLigence RTCA software ACEA Biosciences, San Diego, CA, USA. The results of cell cycle analysis were converted to significant results by using ModFit software. The results were evaluated quantitatively.
| > Results|| |
The cytotoxic effect of danusertib onto the cystic fibrosis pancreatic adenocarcinoma cell line-1 cells
To quantify the cytotoxic effect of danusertib value were obtained from the xCELLigence RTCA system. The dose- and time-dependent effects of danusertib have been demonstrated on CFPAC-1 cells. Cytotoxic IC50 value was found as 400 nM at 64 h. The logarithmic analysis was performed considering CFPAC-1 cell-doubling time (32 h), as shown in [Figure 2]a. Dose–response curve was obtained as a result of the analysis [Figure 2]b.
|Figure 2: XCELLigence analysis result of danusertib effects on cystic fibrosis pancreatic adenocarcinoma cell line-1 cells. (a) Cells were incubated in the E-plate for 24 h. Danusertib applied at 24 h, and the cells were measured every 15 min during 96 h. (b) The IC50value of danusertib at 64 h demonstrated by using the dose–response curve. Value of R2 regression is “0.99”|
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The apoptotic effect of danusertib onto the cystic fibrosis pancreatic adenocarcinoma cell line-1 cells
To determine the apoptotic effect of danusertib on CFPAC-1 cells were measured by using flow cytometry. Danusertib-treated and nontreated CFPAC-1 cells were stained with Annexin V-APC/PI. As shown in [Figure 3] and [Figure 4], the early apoptotic cells are seen in the Q4 quadrant. While the early apoptotic effects were found for the control groups as 0.3% at 24 h and as 0.9% at 48 h, the corresponding effects of danusertib-treated (400 nmol/L) groups were found as 14.8% at 24 h and as 21.3% at 48 h [Table 1]. These values show that dose- and time-dependent effects of danusertib induce early apoptosis in CFPAC-1 cells.
|Figure 3: Q1 aggregate and dead cells, Q2 late apoptotic cells, Q3 live cells, and Q4 early apoptotic cells. (a) Cystic fibrosis pancreatic adenocarcinoma cell line-1 24 h control cells (Q1: 0.5%, Q2: 0.1%, Q3: 99.1%, and Q4: 0.3%). (b) Cells treated with danusertib for 24 h (Q1: 0.4%, Q2: 3.7%, Q3: 81%, and Q4: 14.8%)|
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|Figure 4: Q1 aggregate and dead cells, Q2 late apoptotic cells, Q3 live cells, and Q4 early apoptotic cells. (a) Cystic fibrosis pancreatic adenocarcinoma cell line-1 48 h control cells (Q1: 0.9%, Q2: 0.2%, Q3: 98%, and Q4: 0.9%). (b) Cells treated with danusertib for 48 h (Q1: 0.9%, Q2: 4.3%, Q3: 73.4%, and Q4: 21.3%)|
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|Table 1: Summary of apoptosis analysis of danusertib effect on cystic fibrosis pancreatic adenocarcinoma cell line-1 cells|
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The effects of cell cycle arrest onto the cystic fibrosis pancreatic adenocarcinoma cell line-1 cells
In this study, to determine whether danusertib regulates cell cycle progression in CFPAC-1 cells, the cells were treated with 400 nmol/L concentration of danusertib for 24 h and 48 h. The DNA was stained with PI. After desired incubation, the CFPAC-1 cells were analyzed by using MotFit software in flow cytometry. Comparing with the control, [Figure 5] and [Figure 6] show that the cells were accumulated at S and G2/M phases in a dose- and time-dependent manner of danusertib. Cells treated with 400 nmol/L danusertib for 24 h had a higher fraction of S-phase cells (31.77%) compared with that of cells treated for 48 h (22.99%). However, the arrest of cells in the G2/M phase increased at both 24 h and 48 h. In this study, dose- and time–dependent effects of danusertib arrested the CFPAC-1 cells in G2/M phase.
|Figure 5: Propidium iodide emission graphs of the 24-h groups. (a) Control group (G1, 74.36%; S, 20.65%; and G2, 4.99%). (b) Cells treated with danusertib (G1, 57.18%; S, 31.77%; and G2 11.05%)|
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|Figure 6: Propidium iodide emission graphs of the 48-h groups. (a) Control group (G1, 79.47%; S, 16.12%; and G2 4.41%). (b) Cells treated with danusertib (G1, 66.34%; S, 22.99%; and G2, 10.67%)|
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| > Discussion|| |
In recent years, solid tumor types have increased and have major deficiencies in their treatment. Although there are many reasons for an increase in cancer cases, one of the important reasons is Aurora kinase overexpression in solid tumors. Over the past decade, different inhibitor molecules of these kinase groups have been identified. Many kinase inhibitors have been revealed to have antitumor activity against several tumor types such as hepatocellular carcinoma Hep3B cells, mammary gland MDA-MB-231 cells, and mammary gland MCF-7 cells.,,,,,,, In this study, we used danusertib as an ABL and pan-Aurora kinase inhibitor. When we compared danusertib with other inhibitors such as apatinib (20 μM), BML-275 (10 μM), and gefitinib (20–40 μM), we observed that danusertib is more effective than other kinase inhibitors.,,,, Previous previous studies had shown that danusertib had significant antitumoral effects on several cancer types such as lymphomas, breast, pancreatic, and the many other solid tumors.,,
In the present study, danusertib exhibited to increase the cytotoxic effect onto the CFPAC-1 cells. Our results showed that danusertib is effective at 400 nmol/L on pancreatic adenocarcinoma CFPAC-1 cell line. When compared with other kinase inhibitors, the 400 nmol/L dose of danusertib has a better effect.,,, It may be presumed that the drugs having low-dose effects may have less side effects on the body. Thereafter, healthy cells will suffer less damage. As shown in previous studies, the effect of danusertib on the kidneys has only seen at high micromolar dosages (>1.5 μM)., In further studies, a synergistic effect may be created with danusertib and different kinase inhibitors or natural plant extracts. In addition, Liu et al. demonstrated that danusertib and BKM120 were able to produce a synergistic effect on the Namalwa B-lymphocyte cell line. Therefore, danusertib can be used for treating many types of cancer.
According to flow cytometric analysis, the concentration of 400 nmol/L danusertib arrests the cell cycle in S (31.77% at 24 h and 22.99% at 48 h) and G2/M phases (11.05% at 24 h and 10.67% at 48 h) and induces apoptosis (14.8% at 24 h and 21.3% at 48 h). When we look at other studies on danusertib, it appears to be effective only in the G2/M phase of other tumor cell types (Hep3B, MCF7, AGS, and NCI-N78 cell lines).,, Therefore, danusertib may be more effective in pancreatic tumor types. However, danusertib also arrests the cell cycle in the G2/M phase. Xie et al. showed cell cycle arrest in the G2/M phase on other pancreatic cancer cell lines. The apoptotic effect of danusertib with a lower dose when compared to the effects of other kinase inhibitors (apatinib) on CFPAC-1 cells was found to be same. Apoptosis plays a major role in the elimination of mutations, which is especially important for the rapid recovery of early diagnosed cancer patients.
In this study, the antiproliferative effect of danusertib was demonstrated on CFPAC-1 cells. It is also estimated that fast-progressive pancreatic cancers can be treated rapidly with the ability of danusertib to induce apoptosis and the arresting cell cycle on S and G2/M phases. However, the effects of danusertib should be supported by gene expression assays. Thus, this preliminary study showed that we could focus on further investigation such as the in-vivo model.
The CFPAC-1 cells used in this study were donated by Prof. Dr. Güneş Esendagli. Our deepest appreciations go to him for the kind donation. This study was supported by Mersin University's BAP Unit with project number TP2-2017-2252.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin 2019;69:7-34.
Hartwell LH, Kastan MB. Cell cycle control and cancer. Science 1994;266:1821-8.
Cahill DP, Lengauer C, Yu J, Riggins GJ, Willson JK, Markowitz SD, et al
. Mutations of mitotic checkpoint genes in human cancers. Nature 1998;392:300-3.
Nigg EA. Mitotic kinases as regulators of cell division and its checkpoints. Nat Rev Mol Cell Biol 2001;2:21-32.
Zhu Q, Yu X, Zhou ZW, Luo M, Zhou C, He ZX, et al
. A quantitative proteomic response of hepatocellular carcinoma Hep3B cells to danusertib, a pan-aurora kinase inhibitor. J Cancer 2018;9:2061-71.
Weimer AK, Demidov D, Lermontova I, Beeckman T, Van Damme D. Aurora kinases throughout plant development. Trends Plant Sci 2016;21:69-79.
Willems E, Dedobbeleer M, Digregorio M, Lombard A, Lumapat PN, Rogister B. The functional diversity of aurora kinases: A comprehensive review. Cell Div 2018;13:7.
Li JP, Yang YX, Liu QL, Zhou ZW, Pan ST, He ZX, et al
. The pan-inhibitor of aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells. Drug Des Devel Ther 2015;9:1027-62.
Bischoff JR, Anderson L, Zhu Y, Mossie K, Ng L, Souza B, et al
. A homologue of Drosophila
aurora kinase is oncogenic and amplified in human colorectal cancers. EMBO J 1998;17:3052-65.
Greuber EK, Smith-Pearson P, Wang J, Pendergast AM. Role of ABL family kinases in cancer: From leukaemia to solid tumours. Nat Rev Cancer 2013;13:559-71.
Marumoto T, Hirota T, Morisaki T, Kunitoku N, Zhang D, Ichikawa Y, Sasayama T, Kuninaka S, Mimori T, Tamaki N, Kimura M, Okano Y, Saya H. Roles of aurora-A kinase in mitotic entry and G2 checkpoint in mammalian cells. Genes To Cells 2002;7:1173-82
Tang CJ, Lin CY, Tang TK. Dynamic localization and functional implications of aurora-C kinase during male mouse meiosis. Dev Biol 2006;290:398-410.
Sasai K, Katayama H, Stenoien DL, Fujii S, Honda R, Kimura M, et al
. Aurora-C kinase is a novel chromosomal passenger protein that can complement aurora-B kinase function in mitotic cells. Cell Motil Cytoskeleton 2004;59:249-63.
Carvajal RD, Tse A, Schwartz GK. Aurora kinases: New targets for cancer therapy. Clin Cancer Res 2006;12:6869-75.
Gontarewicz A, Brümmendorf TH. Danusertib (formerly PHA-739358) - A novel combined pan-Aurora kinases and third generation Bcr-Abl tyrosine kinase inhibitor. Recent Results Cancer Res 2010;184:199-214
Cohen RB, Jones SF, Aggarwal C, von Mehren M, Cheng J, Spigel DR, et al
. A phase I dose-escalation study of danusertib (PHA-739358) administered as a 24-hour infusion with and without granulocyte colony-stimulating factor in a 14-day cycle in patients with advanced solid tumors. Clin Cancer Res 2009;15:6694-701.
Fancelli D, Moll J, Varasi M, Bravo R, Artico R, Berta D, et al
. 1, 4, 5, 6-tetrahydropyrrolo [3, 4-c] pyrazoles: İdentification of a potent aurora kinase inhibitor with a favorable antitumor kinase inhibition profile. J Med Chem 2006;49:7247-51.
Kollareddy M, Zheleva D, Dzubak P, Brahmkshatriya PS, Lepsik M, Hajduch M. Aurora kinase inhibitors: Progress towards the clinic. Invest New Drugs 2012;30:2411-32.
Yuan CX, Zhou ZW, Yang YX, He ZX, Zhang X, Wang D, et al
. Danusertib, a potent pan-aurora kinase and ABL kinase inhibitor, induces cell cycle arrest and programmed cell death and inhibits epithelial to mesenchymal transition involving the PI3K/Akt/mTOR-mediated signaling pathway in human gastric cancer AGS and NCI-N78 cells. Drug Des Devel Ther 2015;9:1293-318.
Hata T, Furukawa T, Sunamura M, Egawa S, Motoi F, Ohmura N, et al
. RNA interference targeting aurora kinase a suppresses tumor growth and enhances the taxane chemosensitivity in human pancreatic cancer cells. Cancer Res 2005;65:2899-905.
Li J, Kleeff J, Guo J, Fischer L, Giese N, Büchler MW, et al
. Effects of STI571 (Gleevec) on pancreatic cancer cell growth. Mol Cancer 2003;2:32.
Schoumacher RA, Ram J, Iannuzzi MC, Bradbury NA, Wallace RW, Hon CT, et al
. A cystic fibrosis pancreatic adenocarcinoma cell line. Proc Natl Acad Sci U S A 1990;87:4012-6.
Duong HQ, Hwang JS, Kim HJ, Seong YS, Bae I. BML-275, an AMPK inhibitor, induces DNA damage, G2/M arrest and apoptosis in human pancreatic cancer cells. Int J Oncol 2012;41:2227-36.
Xia Y, Lei Q, Zhu Y, Ye T, Wang N, Li G, et al
. SKLB316, a novel small-molecule inhibitor of cell-cycle progression, induces G2/M phase arrest and apoptosisin vitro
and inhibits tumor growth in vivo
. Cancer Lett 2014;355:297-309.
He K, Wu L, Ding Q, Haider F, Yu H, Wang H, et al
. Apatinib promotes apoptosis of pancreatic cancer cells through downregulation of hypoxia-ınducible factor-1α and ıncreased levels of reactive oxygen species. Oxid Med Cell Longev 2019;2019:5152072.
Green MR, Woolery JE, Mahadevan D. Update on aurora kinase targeted therapeutics in oncology. Expert Opin Drug Discov 2011;6:291-307.
Zhou X, Zheng M, Chen F, Zhu Y, Yong W, Lin H, et al
. Gefitinib inhibits the proliferation of pancreatic cancer cells via cell cycle arrest. Anat Rec (Hoboken) 2009;292:1122-7.
Liu J, Hong J, Ahn KS, Go J, Han H, Park J, et al
. ERK-dependent IL-6 positive feedback loop mediates resistance against a combined treatment using danusertib and BKM120 in Burkitt lymphoma cell lines. Leuk Lymphoma 2019;60:2532-40.
Xie L, Kassner M, Munoz RM, Que QQ, Kiefer J, Zhao Y, et al
. Kinome-wide siRNA screening identifies molecular targets mediating the sensitivity of pancreatic cancer cells to aurora kinase inhibitors. Biochem Pharmacol 2012;83:452-61.
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