j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e1 0
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Kif20a inhibition reduces migration and invasion of pancreatic cancer cells Daniela Stangel, PhD,a Mert Erkan, MD,a,b,* Malte Buchholz, PhD,c Thomas Gress, MD,c Christoph Michalski, MD,a,d Susanne Raulefs, PhD,a Helmut Friess, MD,a and Jo¨rg Kleeff, MDa a
Department of Surgery, Klinikum Rechts der Isar, Technische Universita¨t Mu¨nchen, Munich, Germany Department of Surgery, Koc School of Medicine, Istanbul, Turkey c Department of Gastroenterology and Endocrinology, University Hospital, Philipps-University, Marburg, Germany d Department of Surgery, University of Heidelberg, Heidelberg, Germany b
article info
abstract
Article history:
Backround: The Translational Genome Research Network in Pancreatic Cancer performed a
Received 7 November 2014
meta-analysis of publicly available various high-throughput gene analysis panels to
Received in revised form
identify drugable targets. There, the most differentially expressed gene between normal
10 March 2015
and cancerous pancreas was Kif20a. The aim of the study was to verify this expression
Accepted 25 March 2015
pattern and further characterize Kif20a in pancreatic cancer.
Available online xxx
Materials and methods: Detailed expression analyses were carried out in pancreatic tissues and in a wide panel of pancreatic cells including ductal adenocarcinoma (PDAC) and
Keywords:
neuroendocrine-cancer cell lines as well as immortalized human pancreatic ductal
Motility
epithelial and primary stellate cells using quantitative real-time polymerase chain reac-
Chemoresistance
tion, immunohistochemistry, immunofluorescence, and immunoblot analyses. Effects on
Targeted therapy
proliferation, apoptosis, and cell cycle were assessed by MTT assays, caspase-cleavage assays, and fluorescence-activated cell sorting analysis after Kif20a silencing. Cell motility was assessed by migration and invasion assays as well as time-lapse microscopy. Results: Mean Kif20a messenger RNA expression was 18.4-fold upregulated in PDAC tissues compared with that in the normal pancreas. In line, neuroendocrine-cancer cell lines display a 1.6-fold increase and ductal adenocarcinoma cell lines a 11-fold increase of Kif20a messenger RNA (P ¼ 0.009) in comparison with primary stellate cells. A 7.3-fold overexpression was also found in immortalized pancreatic ductal epithelial cells. Kif20a silencing with small interfering RNA molecules resulted in an inhibition of proliferation, motility, and invasion of pancreatic cancer cell lines. Conclusions: Targeting Kif20a reduces proliferation, migration, and invasion of pancreatic cancer cells. Together with its significant overexpression in PDAC, this makes it a potential target for diagnostic and interventional purposes. ª 2015 Elsevier Inc. All rights reserved.
* Corresponding author. Department of Surgery, Koc University School of Medicine, Davutpasa Caddesi No: 4, Topkapi 34010, Istanbul, Turkey. Tel.: þ90 850 250 8 250; fax: þ90 212 311 34 10. E-mail address: [email protected] (M. Erkan). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2015.03.070
2
1.
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e1 0
Introduction
Pancreatic cancer is the fourth most common cancer to cause death in Western societies [1]. The dismal prognosis is mostly due to the advanced tumor-stage at the time of diagnosis and poor response to current therapies. Because of an overall 5-y survival rate of approximately 5%, it is of utmost importance to develop novel therapeutic strategies [2,3]. To identify new candidate genes with functional significance in pancreatic cancer biology, a meta-analysis of publically available high-throughput gene expression data sets was performed within the framework of the German National Genome Research Network project “PaCa-Net” (www. ngfn-pacanet.de). Here, we focus on the kinesin family member 20a (Kif20a), which was consistently reported to be overexpressed in pancreatic cancer in several studies using different high-throughput expression profiling technologies [4,5]. From an oncological point of view, Kif20a has been reported to be overexpressed in various other cancers such as small cell lung cancer, bladder cancer, and breast cancer [4,6,7]. In normal tissues, Kif20a is abundantly expressed in fetal liver, adult bone marrow, and thymus, whereas low levels are found in the placenta and heart. Furthermore, Kif20a is barely detectable or absent in the normal spleen, lymph nodes, pancreas, lung, brain, liver, kidney, and skeletal muscle [8]. Kif20a belongs to the kinesin superfamily-6 and possesses a conserved motor domain. It binds to microtubules and couple adenosine triphosphate hydrolysis to generate mechanical force [9]. Kif20a was identified to localize in the Golgi apparatus where it interacts with guanosine triphosphate-bound forms of RAB6A/B and acts as a motor required for the retrograde RAB6-regulated transport of Golgi membranes and associated vesicles along microtubules [10]. In addition, Kif20a has a microtubule plus enddirected motility and thus, is involved in different cellular processes such as formation of the mitotic spindle and chromosome partitioning [11]. Taken together, Kif20a appears to be a suitable target in pancreatic ductal adenocarcinoma (PDAC) because of its significant overexpression and its vital cellular functions such as organelle transport [12], cell cycle [13,14], and cell motility [15].
2.
Materials and methods
2.1.
Cell lines
PDAC cell lines, Panc-1, SU86.86, and T3M4, were cultured as published previously [16]. Immortalized human pancreatic ductal epithelial (HPDE) cells were kindly provided by Dr MingSound Tsao and cultured in keratinocyte medium (Life Technologies, Carlsbad, CA) with supplements as published previously [17,18]. Neuroendocrine tumor cell lines (CM-1, BON, and QGP) were a kind gift of Dr Aldo Scarpa and were cultured as published previously [19]. Human stellate cell isolation and cultivation was performed using the outgrowth method as described previously [20].
2.2.
Tissue collection and protein expression analyses
After informed consent of patients and approval of the ethical committee (protocol number 5510/12), tissue collection was performed at Klinikum Rechts der Isar, Technical University of Munich, Germany, as published previously [21]. Kif20a protein expression was detected using a rabbit polyclonal antibody against Kif20a (A300-879A; Bethyl Laboratories, TX) at dilutions of 1:150 for immunohistochemistry and 1:100 for immunofluorescence analyses [20]. Quantification of immunostaining was done semiquantitatively as previously published [21]. Mouse antiea-tubulin (ab18251; Abcam, Cambridge, UK) antibody was used at a dilution of 1:100. For immunofluorescence analysis, secondary antibodies were Alexa594-conjugated anti-rabbit (A11012; Life Technologies, dilution 1:300) and Alexa488conjugated anti-mouse (A11001; Invitrogen, Karlsruhe, Germany, dilution 1:400). Slides were mounted with fluorescence mounting medium (DAKO, CA) containing 40 , 6-diamidino-2phenylindole and visualized with AxioVision scanning system (Zeiss, Oberkochen, Germany). For immunoblotting, antieKif20a rabbit polyclonal antibody was used at a dilution of 1:1000 using a previously published protocol [16]. Enhanced chemiluminescence antierabbit IgG was applied at a dilution of 1:2000 (GE Healthcare, Buckinghamshire, UK) for 45 min. Signal detection was made using the enhanced chemiluminescence system (Invitrogen). Antieglyceraldehyde-3-phosphate-dehydrogenase (sc365062; Santa Cruz, Dallas, TX, dilution 1:500) and/or a-tubulin (ab18251; Abcam, 1:1000) were used as internal loading controls. Immunoblots were scanned and quantified by densitometry using ImageJ software (NIH, Bedhesda, MD) as described previously [22].
2.3.
TaqMan analysis of pancreatic tissues
RNA isolation was carried out using the peqGOLD Total RNA Kit (PEQLAB Biotechnologie GmbH, Erlangen, Germany) according to the manufacturers’ instructions. Concentrations were quantified with the NanoDrop ND-1000 (PEQLAB Biotechnology GmbH), followed by synthesis of complementary DNA (cDNA) from 1 mg of total RNA using the Omniscript Kit and protocol (Qiagen, Hilden, Germany). Quantitative real-time polymerase chain reaction (qRTPCR) was performed in a 7500 Fast Real-Time PCR System (Applied Biosystems, Warrington, UK) using gene-specific TaqMan primers and probes obtained from Applied Biosystems. For every tissue sample, expression of candidate genes was normalized to the average expression of seven “housekeeping” genes (HPRT1, GUSB, RPLP0, PPIA, RPL37A, RPL30, and RPS17).
2.4.
qRT-PCR analyses
Cancer cells, HPDE, and pancreatic stellate cells (PSC) were cultivated in T75 flasks until 80% confluency, and RNA extraction was carried out using the RNeasy Plus Mini Kit (Qiagen). One microgram of total RNA was reverse transcribed using the Revert Aid H Minus first Strand cDNA Synthesis Kit (Fermentas International Inc, SK, CAN) and 5 mL cDNA, corresponding to 10-ng reversed-transcribed RNA which was
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e1 0
analyzed using LightCycler480 with SYBR Green I Master (Roche Diagnostics, Rotkreuz, Risch, Switzerland). The primer sequence for Kif20a detection was 50 -CAAGGGCCTAACCCTCAA-30 and 50 -TCTGTCGTCTCTACCTCCCTAGA-30 Tm 65 C. For internal normalization, hypoxanthine-guanine phosphoribosyltransferase 50 -GCAGCCCTGGCGTCGTGATTAG -30 and 50 TCGAGCAAGACGTTCAGTCCTGT-30 Tm 60 C was used. The relative expression was normalized to human (reference gene) hypoxanthine-guanine phosphoribosyltransferase using LightCycler480 software release 1.5, version 1.05.0.39 (Roche).
2.5.
RNA interference
For transient messenger RNA (mRNA) silencing, 7 104 cells were seeded into 6-well plates and transfected with 20 mM of Kif20a small interfering RNA (siRNA; Hs_KIF20 A_5, sense: 50 GGCCAGGUUUCUGCCAAAATT-30 , antisense: 50 -UUUUGGCAGAAACCUGGCCTT-30 ) using Hiperfect transfection reagent (Qiagen) as published before [21]. As negative control, AllStars Negative Control siRNA (Qiagen) was used. Transfection efficiency was verified by qRT-PCR and immunoblot analysis. For functional experiments, cells were then transferred into appropriate chambers (i.e., invasion), and the timing of the experiments were adjusted accordingly so that the analyses were conducted 48e72 h after transfection for optimal effect of RNA-silencing at the protein level.
2.6.
Proliferation assay
Proliferation of transfected cells was determined using MTT (3-(4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, MO) colorimetric growth assay as published before [16]. Briefly, 5 103 cells were seeded into wells of a 96-well plate, 50 mg per well MTT-solution was added, and the samples were incubated for 4 h at 37 C. Cellular MTT was solubilized with isopropanol, and optical density was measured with an enzyme-linked immunosorbent assay reader at 570 nm. All experiments were performed three times in triplicates.
2.7. Cell-cycle analysis and evaluation of apoptosis using fluorescence-activated cell sorting Cancer cells were first synchronized in G1-phase using double thymidine block as published before [23]. Cells were seeded at a density of 40,000 cells in wells of 6-well plates. After incubation with 2.5 mM of thymidine (T9250; Sigma) for 20 h, cells were washed twice with phosphate-buffered saline and treated with normal medium, containing Kif20a siRNA. Afterward, cells were washed again twice with phosphatebuffered saline, and medium was changed to fresh medium containing 2.5 mM of thymidine. Cells were fixed in ethanol (48 h after siRNA transfection) stained with probidium iodide and analyzed using flow cytometry (Guava easyCyte Flow Cytometer; Millipore, MA). Apoptosis was additionally analyzed using immunofluorescence-based caspase assay as described previously [24]. Cells were first transfected with Kif20a siRNA and after 72 h transfection immunostained with rabbit polyclonal antibody against cleaved caspase 3 (Asp175; Cell Signaling, MA) at a concentration of 1:400. Samples were
3
then mounted with fluorescence mounting medium (DAKO) containing probidium iodide and visualized with AxioVision scanning system.
2.8.
Migration, invasion, and motility assays
For migration studies, 24-Multiwell Insert System without Matrigel (BD Biosciences, Heidelberg, Germany) was used. Cancer cells were first transfected in 6-well plates with Kif20a siRNA or control siRNA. Forty-eight hours after transfection, cells were seeded in a density of 15 103 cells into the migration chambers and were incubated for another 24 h. Cells which passed the membrane were fixed with 4% paraformaldehyde and stained with crystal violet (Merck, Darmstadt, Germany). The migrated cells were counted under a light microscope (Zeiss). The assays were repeated three times and were performed in triplicates. Invasion studies were performed using 24-Multiwell Insert Systems with matrigel (BD Biosciences) as previously described [25]. Forty-eight hours after transfection, cells were seeded in a density of 7 103 cells per well into the top chamber and were incubated for 22 h. Invading cells were fixed with 4% paraformaldehyde and stained with crystal violet (Merck). Stained cells were counted under a light microscope (Zeiss). The assays were repeated three times and were performed in triplicates. Motility of cancer cells was analyzed using time-lapse microscopy as published before [26]. Fifty thousand transfected cells were seeded into wells of 6-well plates and 48 h later analyzed for 20 h using time-lapse microscopy. Manual cell tracking was performed using ImageJ software (NIH). Velocity, distance, and directionality were measured via the chemotaxis tool from Ibidi, Martinsried, Germany.
2.9.
Statistical analysis
Results were expressed as mean standard error of the mean. GraphPad Prism 5 software (GraphPad, San Diego, CA) was used to present graphs. Statistical analyses were performed using SPSS 20 for Windows (SPSS Inc, Chicago, IL). Statistical significance was set at a P value of
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Kif20a inhibition reduces migration and invasion of pancreatic cancer cells Daniela Stangel, PhD,a Mert Erkan, MD,a,b,* Malte Buchholz, PhD,c Thomas Gress, MD,c Christoph Michalski, MD,a,d Susanne Raulefs, PhD,a Helmut Friess, MD,a and Jo¨rg Kleeff, MDa a
Department of Surgery, Klinikum Rechts der Isar, Technische Universita¨t Mu¨nchen, Munich, Germany Department of Surgery, Koc School of Medicine, Istanbul, Turkey c Department of Gastroenterology and Endocrinology, University Hospital, Philipps-University, Marburg, Germany d Department of Surgery, University of Heidelberg, Heidelberg, Germany b
article info
abstract
Article history:
Backround: The Translational Genome Research Network in Pancreatic Cancer performed a
Received 7 November 2014
meta-analysis of publicly available various high-throughput gene analysis panels to
Received in revised form
identify drugable targets. There, the most differentially expressed gene between normal
10 March 2015
and cancerous pancreas was Kif20a. The aim of the study was to verify this expression
Accepted 25 March 2015
pattern and further characterize Kif20a in pancreatic cancer.
Available online xxx
Materials and methods: Detailed expression analyses were carried out in pancreatic tissues and in a wide panel of pancreatic cells including ductal adenocarcinoma (PDAC) and
Keywords:
neuroendocrine-cancer cell lines as well as immortalized human pancreatic ductal
Motility
epithelial and primary stellate cells using quantitative real-time polymerase chain reac-
Chemoresistance
tion, immunohistochemistry, immunofluorescence, and immunoblot analyses. Effects on
Targeted therapy
proliferation, apoptosis, and cell cycle were assessed by MTT assays, caspase-cleavage assays, and fluorescence-activated cell sorting analysis after Kif20a silencing. Cell motility was assessed by migration and invasion assays as well as time-lapse microscopy. Results: Mean Kif20a messenger RNA expression was 18.4-fold upregulated in PDAC tissues compared with that in the normal pancreas. In line, neuroendocrine-cancer cell lines display a 1.6-fold increase and ductal adenocarcinoma cell lines a 11-fold increase of Kif20a messenger RNA (P ¼ 0.009) in comparison with primary stellate cells. A 7.3-fold overexpression was also found in immortalized pancreatic ductal epithelial cells. Kif20a silencing with small interfering RNA molecules resulted in an inhibition of proliferation, motility, and invasion of pancreatic cancer cell lines. Conclusions: Targeting Kif20a reduces proliferation, migration, and invasion of pancreatic cancer cells. Together with its significant overexpression in PDAC, this makes it a potential target for diagnostic and interventional purposes. ª 2015 Elsevier Inc. All rights reserved.
* Corresponding author. Department of Surgery, Koc University School of Medicine, Davutpasa Caddesi No: 4, Topkapi 34010, Istanbul, Turkey. Tel.: þ90 850 250 8 250; fax: þ90 212 311 34 10. E-mail address: [email protected] (M. Erkan). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2015.03.070
2
1.
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e1 0
Introduction
Pancreatic cancer is the fourth most common cancer to cause death in Western societies [1]. The dismal prognosis is mostly due to the advanced tumor-stage at the time of diagnosis and poor response to current therapies. Because of an overall 5-y survival rate of approximately 5%, it is of utmost importance to develop novel therapeutic strategies [2,3]. To identify new candidate genes with functional significance in pancreatic cancer biology, a meta-analysis of publically available high-throughput gene expression data sets was performed within the framework of the German National Genome Research Network project “PaCa-Net” (www. ngfn-pacanet.de). Here, we focus on the kinesin family member 20a (Kif20a), which was consistently reported to be overexpressed in pancreatic cancer in several studies using different high-throughput expression profiling technologies [4,5]. From an oncological point of view, Kif20a has been reported to be overexpressed in various other cancers such as small cell lung cancer, bladder cancer, and breast cancer [4,6,7]. In normal tissues, Kif20a is abundantly expressed in fetal liver, adult bone marrow, and thymus, whereas low levels are found in the placenta and heart. Furthermore, Kif20a is barely detectable or absent in the normal spleen, lymph nodes, pancreas, lung, brain, liver, kidney, and skeletal muscle [8]. Kif20a belongs to the kinesin superfamily-6 and possesses a conserved motor domain. It binds to microtubules and couple adenosine triphosphate hydrolysis to generate mechanical force [9]. Kif20a was identified to localize in the Golgi apparatus where it interacts with guanosine triphosphate-bound forms of RAB6A/B and acts as a motor required for the retrograde RAB6-regulated transport of Golgi membranes and associated vesicles along microtubules [10]. In addition, Kif20a has a microtubule plus enddirected motility and thus, is involved in different cellular processes such as formation of the mitotic spindle and chromosome partitioning [11]. Taken together, Kif20a appears to be a suitable target in pancreatic ductal adenocarcinoma (PDAC) because of its significant overexpression and its vital cellular functions such as organelle transport [12], cell cycle [13,14], and cell motility [15].
2.
Materials and methods
2.1.
Cell lines
PDAC cell lines, Panc-1, SU86.86, and T3M4, were cultured as published previously [16]. Immortalized human pancreatic ductal epithelial (HPDE) cells were kindly provided by Dr MingSound Tsao and cultured in keratinocyte medium (Life Technologies, Carlsbad, CA) with supplements as published previously [17,18]. Neuroendocrine tumor cell lines (CM-1, BON, and QGP) were a kind gift of Dr Aldo Scarpa and were cultured as published previously [19]. Human stellate cell isolation and cultivation was performed using the outgrowth method as described previously [20].
2.2.
Tissue collection and protein expression analyses
After informed consent of patients and approval of the ethical committee (protocol number 5510/12), tissue collection was performed at Klinikum Rechts der Isar, Technical University of Munich, Germany, as published previously [21]. Kif20a protein expression was detected using a rabbit polyclonal antibody against Kif20a (A300-879A; Bethyl Laboratories, TX) at dilutions of 1:150 for immunohistochemistry and 1:100 for immunofluorescence analyses [20]. Quantification of immunostaining was done semiquantitatively as previously published [21]. Mouse antiea-tubulin (ab18251; Abcam, Cambridge, UK) antibody was used at a dilution of 1:100. For immunofluorescence analysis, secondary antibodies were Alexa594-conjugated anti-rabbit (A11012; Life Technologies, dilution 1:300) and Alexa488conjugated anti-mouse (A11001; Invitrogen, Karlsruhe, Germany, dilution 1:400). Slides were mounted with fluorescence mounting medium (DAKO, CA) containing 40 , 6-diamidino-2phenylindole and visualized with AxioVision scanning system (Zeiss, Oberkochen, Germany). For immunoblotting, antieKif20a rabbit polyclonal antibody was used at a dilution of 1:1000 using a previously published protocol [16]. Enhanced chemiluminescence antierabbit IgG was applied at a dilution of 1:2000 (GE Healthcare, Buckinghamshire, UK) for 45 min. Signal detection was made using the enhanced chemiluminescence system (Invitrogen). Antieglyceraldehyde-3-phosphate-dehydrogenase (sc365062; Santa Cruz, Dallas, TX, dilution 1:500) and/or a-tubulin (ab18251; Abcam, 1:1000) were used as internal loading controls. Immunoblots were scanned and quantified by densitometry using ImageJ software (NIH, Bedhesda, MD) as described previously [22].
2.3.
TaqMan analysis of pancreatic tissues
RNA isolation was carried out using the peqGOLD Total RNA Kit (PEQLAB Biotechnologie GmbH, Erlangen, Germany) according to the manufacturers’ instructions. Concentrations were quantified with the NanoDrop ND-1000 (PEQLAB Biotechnology GmbH), followed by synthesis of complementary DNA (cDNA) from 1 mg of total RNA using the Omniscript Kit and protocol (Qiagen, Hilden, Germany). Quantitative real-time polymerase chain reaction (qRTPCR) was performed in a 7500 Fast Real-Time PCR System (Applied Biosystems, Warrington, UK) using gene-specific TaqMan primers and probes obtained from Applied Biosystems. For every tissue sample, expression of candidate genes was normalized to the average expression of seven “housekeeping” genes (HPRT1, GUSB, RPLP0, PPIA, RPL37A, RPL30, and RPS17).
2.4.
qRT-PCR analyses
Cancer cells, HPDE, and pancreatic stellate cells (PSC) were cultivated in T75 flasks until 80% confluency, and RNA extraction was carried out using the RNeasy Plus Mini Kit (Qiagen). One microgram of total RNA was reverse transcribed using the Revert Aid H Minus first Strand cDNA Synthesis Kit (Fermentas International Inc, SK, CAN) and 5 mL cDNA, corresponding to 10-ng reversed-transcribed RNA which was
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e1 0
analyzed using LightCycler480 with SYBR Green I Master (Roche Diagnostics, Rotkreuz, Risch, Switzerland). The primer sequence for Kif20a detection was 50 -CAAGGGCCTAACCCTCAA-30 and 50 -TCTGTCGTCTCTACCTCCCTAGA-30 Tm 65 C. For internal normalization, hypoxanthine-guanine phosphoribosyltransferase 50 -GCAGCCCTGGCGTCGTGATTAG -30 and 50 TCGAGCAAGACGTTCAGTCCTGT-30 Tm 60 C was used. The relative expression was normalized to human (reference gene) hypoxanthine-guanine phosphoribosyltransferase using LightCycler480 software release 1.5, version 1.05.0.39 (Roche).
2.5.
RNA interference
For transient messenger RNA (mRNA) silencing, 7 104 cells were seeded into 6-well plates and transfected with 20 mM of Kif20a small interfering RNA (siRNA; Hs_KIF20 A_5, sense: 50 GGCCAGGUUUCUGCCAAAATT-30 , antisense: 50 -UUUUGGCAGAAACCUGGCCTT-30 ) using Hiperfect transfection reagent (Qiagen) as published before [21]. As negative control, AllStars Negative Control siRNA (Qiagen) was used. Transfection efficiency was verified by qRT-PCR and immunoblot analysis. For functional experiments, cells were then transferred into appropriate chambers (i.e., invasion), and the timing of the experiments were adjusted accordingly so that the analyses were conducted 48e72 h after transfection for optimal effect of RNA-silencing at the protein level.
2.6.
Proliferation assay
Proliferation of transfected cells was determined using MTT (3-(4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, MO) colorimetric growth assay as published before [16]. Briefly, 5 103 cells were seeded into wells of a 96-well plate, 50 mg per well MTT-solution was added, and the samples were incubated for 4 h at 37 C. Cellular MTT was solubilized with isopropanol, and optical density was measured with an enzyme-linked immunosorbent assay reader at 570 nm. All experiments were performed three times in triplicates.
2.7. Cell-cycle analysis and evaluation of apoptosis using fluorescence-activated cell sorting Cancer cells were first synchronized in G1-phase using double thymidine block as published before [23]. Cells were seeded at a density of 40,000 cells in wells of 6-well plates. After incubation with 2.5 mM of thymidine (T9250; Sigma) for 20 h, cells were washed twice with phosphate-buffered saline and treated with normal medium, containing Kif20a siRNA. Afterward, cells were washed again twice with phosphatebuffered saline, and medium was changed to fresh medium containing 2.5 mM of thymidine. Cells were fixed in ethanol (48 h after siRNA transfection) stained with probidium iodide and analyzed using flow cytometry (Guava easyCyte Flow Cytometer; Millipore, MA). Apoptosis was additionally analyzed using immunofluorescence-based caspase assay as described previously [24]. Cells were first transfected with Kif20a siRNA and after 72 h transfection immunostained with rabbit polyclonal antibody against cleaved caspase 3 (Asp175; Cell Signaling, MA) at a concentration of 1:400. Samples were
3
then mounted with fluorescence mounting medium (DAKO) containing probidium iodide and visualized with AxioVision scanning system.
2.8.
Migration, invasion, and motility assays
For migration studies, 24-Multiwell Insert System without Matrigel (BD Biosciences, Heidelberg, Germany) was used. Cancer cells were first transfected in 6-well plates with Kif20a siRNA or control siRNA. Forty-eight hours after transfection, cells were seeded in a density of 15 103 cells into the migration chambers and were incubated for another 24 h. Cells which passed the membrane were fixed with 4% paraformaldehyde and stained with crystal violet (Merck, Darmstadt, Germany). The migrated cells were counted under a light microscope (Zeiss). The assays were repeated three times and were performed in triplicates. Invasion studies were performed using 24-Multiwell Insert Systems with matrigel (BD Biosciences) as previously described [25]. Forty-eight hours after transfection, cells were seeded in a density of 7 103 cells per well into the top chamber and were incubated for 22 h. Invading cells were fixed with 4% paraformaldehyde and stained with crystal violet (Merck). Stained cells were counted under a light microscope (Zeiss). The assays were repeated three times and were performed in triplicates. Motility of cancer cells was analyzed using time-lapse microscopy as published before [26]. Fifty thousand transfected cells were seeded into wells of 6-well plates and 48 h later analyzed for 20 h using time-lapse microscopy. Manual cell tracking was performed using ImageJ software (NIH). Velocity, distance, and directionality were measured via the chemotaxis tool from Ibidi, Martinsried, Germany.
2.9.
Statistical analysis
Results were expressed as mean standard error of the mean. GraphPad Prism 5 software (GraphPad, San Diego, CA) was used to present graphs. Statistical analyses were performed using SPSS 20 for Windows (SPSS Inc, Chicago, IL). Statistical significance was set at a P value of
