THE ANATOMICAL RECORD 298:1271–1281 (2015)
Effects of Antitumor Drug Sorafenib on Chick Embryo Development YI-SEN CHENG,1† XIAO-YU WANG,1† GUANG WANG,1 YAN LI,1 YUE-LEI CHEN,2 MAN-LI CHUAI,3 KENNETH KA HO LEE,4 XIAO-YAN DING,2* 1 AND XUE-SONG YANG * 1 Division of Histology and Embryology, Key Laboratory for Regenerative Medicine of the Ministry of Education, Medical College, Jinan University, Guangzhou 510632, China 2 The State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China 3 Division of Cell and Developmental Biology, University of Dundee, Dundee, Dd1 5EH, UK 4 Key Laboratory for Regenerative Medicine, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin, Hong Kong
ABSTRACT Sorafenib has been used as an oral anti-cancer drug because of its ability to inhibit tumor growth. However, the pharmacological effect of sorafenib is still the lack of in vivo experimental evidence. Tumor and embryonic cells share some similar features, so we investigated the effects of sorafenib on the development of gastrulating chick embryos. We found that sorafenib exposure was markedly attributed to the number of embryonic cell in proliferation and apoptosis. We also detected sorafenib significantly interfered with epithelial-mesenchymal transition (EMT). Furthermore, sorafenib treatment impaired the production and migration of neural crest cells. Anat Rec, 298:1271–1281, 2015. C 2015 Wiley Periodicals, Inc. V
Key words: sorafenib; chick embryos; EMT; gastrulation; neural crest cells
Sorafenib is the first orally used drug that shows significant inhibitory effects on tumor growth and EMT. It principally targets serine/threonine and receptor tyrosine kinases (Escudier et al., 2007; Llovet et al., 2008; Chen et al., 2011; Nagai et al., 2011; Zhang et al., 2013). In rat hepatocytes, the drug antagonizes TGF-b signaling and interferes with TGF-b1-induced EMT and apoptosis (Chen et al., 2011). These two processes play crucial roles in embryo development, especially during gastrulation when EMT is required for the generation of the three germ cell layers. Furthermore, apoptosis, cell migration, cell division, and differentiation are the processes that are tightly regulated to ensure that the early embryo develops normally. In tumor genesis, EMT is presented as a loss of epithelial features and an acquisition of migratory properties, as illustrated by a breakdown of epithelium homeostasis and progressive cancer aggression. Superficially, embryonic EMT is very similar to the transformation of cellular phenotypes that occur during carcinoma progression (Savagner). The similarC 2015 WILEY PERIODICALS, INC. V
Grant sponsor: “973 Project”; Grant number: 2010CB529703; Grant sponsor: NSFC; Grant numbers: 31071054, 30971493; Grant sponsor: Guangdong Natural Science Foundation; Grant numbers: S2011010001593, S2013010013392; Grant sponsor: Science and Technology Grant; Grant number: 2012DFH30060. † These authors contributed equally to this work. *Correspondence to: X. Yang, Division of Histology and Embryology, Key Laboratory for Regenerative Medicine of the Ministry of Education, Medical College, Jinan University, Guangzhou 510632, China. E-mail: [email protected] (or) X. Y. Ding, The State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. E-mail: [email protected] Received 9 June 2014; Revised 27 November 2014; Accepted 5 January 2015. DOI 10.1002/ar.23155 Published online 17 April 2015 in Wiley Online Library (wileyonlinelibrary.com).
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ities between cancer and embryonic cells are not only evident in EMT but also in other cellular behaviors. For an example, when cancer cells are injected into blastocysts, the cells were found to contribute to normal embryonic development (Pierce, 1983). This extraordinary phenomenon has been widely observed by several experimenters (Mintz and Cronmiller, 1978) and indicates that cancer cells share some similar characteristics with embryonic cells. Moreover, there are similarities between these cells during apoptosis and cell proliferation. In this context, it will possible to investigate the anti-tumor effects of sorafenib by using the developing chick embryo as a model. There are many advantages of using the chick embryo. It is highly accessible inside the egg for surgical manipulation, inexpensive and the developmental stages of the embryos are well characterized. The early chick embryo also lack an immune system, so tissues and cells could be transplanted into them and their developmental fate established without rejection. Because of these properties, it has been proposed that this model is useful for addressing problems associated with stem cell and cancer biology (Rashidi and Sottile, 2009). Moreover, extra information can be gleamed from the model, for example how sorafenib affects the proliferation and migration of neural crest cells in the embryo (Bronner-Fraser, 1994).
MATERIALS AND METHODS Chick Embryos and Tissue Transplantation Fertilized leghorn eggs were obtained from the Avian Farm of South China Agriculture University. The eggs were incubated in a humidified incubator (Yiheng Instruments, Shanghai, China) at 38 C until the chick embryos reached Hamburger and Hamilton (HH) stage 3 (Hamburger and Hamilton, 1992). Either the whole chick embryo or half-side of the embryos were exposed to 10 lM sorafenib (Nexavar, BAY 43-9006, Bayer Pharmaceuticals, USA) in early chick (EC) culture medium overnight (Chapman et al., 2001; Li et al., 2013). The transplantation of the primitive streak from donor to host embryo was performed as previously described (Yang et al., 2002). Briefly, a piece of GFP-expressing primitive streak, 200 mm wide was excised from a GFP-transfected donor embryo, and grafted into a similar position in an unlabeled host embryo at the same developmental stage. After transplantation, the embryos were incubated overnight to assess and compare the migratory patterns of GFP1 cells between the control and sorafenib-treated embryos.
Chick Embryo Electroporation EC culture (Chapman et al., 2001) was employed to maintain the embryos during and after gene electroporation. Briefly, 1 lL of 1.5 lg GFP expression plasmid (pEGFP-N1, Clontech) was microinjected into the space between the vitelline membrane and the epiblast prior to electroporation. Electroporation was performed on HH3 chick embryos using an electroporator (BTXECM399) and a pair of platinum electrodes arranged in a parallel fashion. Two pulses of 10 V were delivered between the electrodes as previously described (Yang
et al., 2002). The survival rate of the embryos after gene transfection was nearly 100%, dependent on the embryos used were in good condition. The transfected embryos were incubated at 37 C and 70% humidity until the desired developmental stage was reached. During incubation, the fluorescence of GFP was directly viewed under a stereo-fluorescence microscope (Olympus MVX10).
Whole-mount Embryo Immunohistochemistry Immunofluorescent staining were performed on whole-mount control and sorafenib-treated chick embryos against: E-Cadherin (BD, 610181), N-Cadherin (DSHB, 6B3), Laminin (DSHB, 3H11), phospho-histone H3 (pHIS3, SantaCruz, sc-12927-R), Cleaved-Caspase3 (c-Caspase3, Cell Signaling Technology, 9664p), CleavedPARP (c-PARP, Cell Signaling Technology, 5625p) and HNK1 (Sigma C0678). The embryos were fixed in 4% paraformaldehyde (PFA) at 4oC overnight, and then treated with 2% Bovine Serum Albumin (BSA) 1 1% Triton-X 1 1% Tween 20 in PBS for 2 hr to block unspecific immunoreactions. Next, unspecific binding were blocked using 2% Bovine Serum Albumin (BSA) 1 1% Triton-X 1 1% Tween 20 in PBS for 2 hr, at room temperature. The embryos were washed in PBS and then incubated in primary mouse monoclonal antibody against E-Cadherin (1:100), or N-Cadherin (1:100), or Laminin (1:100), or pHIS3 (1:400), or c-Caspase3 (1:100), or c-PARP (1:100) and HNK1 (1:100), overnight at 4oC on a shaker. The embryos were then extensive washed in PBS and incubated with alexa fluor 555 anti-mouse IgG secondary antibodies (1:1000, Invitrogen, USA) overnight at 4 C, to visualize the primary antibody bindings. Subsequently, the embryos were sectioned on a cryostat microtome (Leica CM1900). These sections were mounted with mounting solution (Mowiol 4-88, Sigma) onto glass slides and sealed with coverslips before photography. All experiments were repeated three times and around five to six embryos were used for each group.
RNA Isolation and RT-PCR Total RNA was isolated from the whole primary steak using a Trizol kit (Invitrogen, USA) according to the manufacturer’s instructions. First-strand cDNA was synthesized to a final volume of 25 lL using the SuperScript RIII First-Strand (Invitrogen, USA). Following reverse transcription, PCR amplification of the cDNA was performed as described previously (Larger et al., 2004; Bai et al., 2012) using specific primers as follows: N-cadherin 5’-AGATTCTGGAAATCCACATGC-3’; 5’-CTTCCTTCATAGTCAAAGACT-3’ Slug 5’-CCAATGACCTCTCTCCGCTTTCTG-3’, 5’-ATCGCTAATGGGACTTTCTGAACCG-3’; RhoA 5’-GCAGCCATTCGAAAAAGCT-3’; 5’-TTTATAAGAGAAGGCACCCG-3’ Reactions were performed in a Bio-Rad S1000TM Thermal cycler. (Bio-Rad, USA) The final reaction volume was 50 lL composing of 1 lL of first-strand cDNA, 25 lM forward primer, 25 lM reverse primer, 10 lL PrimeSTARTM Buffer (Mg21 plus), 4 lL dNTPs Mixture (TaKaRa, Japan), 0.5 lL PrimeSTARTM HS DNA Polymerase (2.5 U lL21 TaKaRa, Japan), and RNase-free water to 50 lL.
EFFECTS OF SORAFENIB ON CHICK EMBRYO DEVELOPMENT
The cDNAs were amplified for 30 cycles. One round of amplification was performed at 94 C for 30 sec, 30 sec at 58 C, and 30 sec at 72 C. The PCR products (20 ll) were resolved in 1% agarose gels (Biowest, Spain) in 13TAE buffer (0.04 M Trisacetate and 0.001 M EDTA), and 10,0003 GeneGreen Nucleic Acid Dye (TIANGEN, China) solution. The resolved products were visualized in a transilluminator (SYNGENE, UK) and photographs were captured using a computer-assisted gel documentation system (SYNGENE).
Western Blot To detect the protein levels of MAPK pathway markers, chick embryo (HH4) treated as indicated were lysed with 70 mL of lysis buffer and subjected to western blot analysis. Approximately 30 mg of total protein was separated by 12% SDS-polyacrylamide gel, transferred to a PVDF membrane and incubated with the appropriate antibodies as indicated in the figure legends. The membrane was blocked with 5% fat-free milk. The primary antibody (1:1,000 dilution) incubation was performed overnight at 4 C and the secondary antibody incubation (1:3,000 dilution) was performed for 1 hr at room temperature. GAPDH was used as an internal control to normalize for loading equality. The protein bands were visualized using an enhanced chemiluminescence (ECL) detection kit (BIORAD). Primary antibodies against ERK1/2, ERK1/2 phosphorylation (1:1000, supplied by professor Yanhong Yu) and GAPDH (1:1,000, supplied by Yuhui Yan) and secondary goat anti-rabbit- HRP conjugated antibody were all obtained from CST.
Cell Culture Cranial neural crest cells were obtained from the chick embryo (HH9) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Biochrom, Berlin, Germany) at 37 C with 5% CO2 for 60 h.
Photography Following immunofluorescent staining, the whole mount embryos were photographed using a stereofluorescence microscope (Olympus MVX10) and the captured images analyzed using an Olympus software package Image-Pro Plus 7.0. The embryos were sectioned at 10–14 lm using a cryostat microtome (Leica CM1900). The slides were photographed and analyzed using an epi-fluorescence microscope (Olympus IX51, Leica DM 4000B) at 2003 and 4003 magnifications and associated software package Leica CW4000 FISH.
Statistical Analysis Data analysis and construction of charts were performed using a Graphpad Prism 5 software package (Graphpad Software, CA). The results were presented as mean value (x6SE). All data were analyzed using student t test or v2-test to determine statistical difference amongst the control and experimental groups. P < 0.05 was considered to be statistically significant.
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RESULTS Sorafenib Retards Chick Embryo Growth The effects of sorafenib on gastrulation in the chick embryo were investigated. Chick embryos (HH0) were randomly divided into two groups, with one group exposed to 10lM sorafenib solubilized in DMSO while another group exposed to DMSO only (serving as the control) for 28 hr. The morphology of the embryos was periodically photographed at 0 hr (Fig. 1A,D), 18-hr and 28-hr after incubation. We observed that the development of sorafenib-treated embryos (Fig. 1E) lagged behind the control embryos (Fig. 1B) at 18-hr incubation. The extent of this retardation was very obvious after 28-hr incubation (Fig. 1C,F). We examined a large cohort of embryos (20 embryos for each group) to avoid intragroup developmental differences. For control embryos incubated for 18-hr and 28-hr, we determined that the majority (92%) of the embryos were staged HH5-6. In contrast, only 40% of sorafenib-treated embryos could reach the HH5-6 stage (Fig. 1G). We recorded the length of the primitive streak, which make its appearance during gastrulation, as an indicator of embryonic development (Fig. 1H). We established that there was a significant difference in the length of the primitive streak between the control and sorafenib-treated embryos (Fig. 1I).
Effects of Sorafenib on Cell Death and Proliferation in the Gastrulating Chick Embryo We established that sorafenib retarded the growth of gastrulating embryos. Hence, we decided to investigate whether the retardation was attributed to altered cell proliferation and apoptosis. The extent of cell proliferation was determined by immunofluorescent staining with pHIS3 antibodies, which labels M-phase nuclei. We established that there were significant fewer pHIS31 cells in the sorafenib-treated embryos than control embryos (Fig. 2A–E)—suggesting that sorafenib inhibited cell proliferation. To determine the extent of cell death in our specimens, we performed immunofluorescent staining for c-Caspase3 and c-PARP. We established that sorafenib treatment significantly increased the number of c-Caspase31 and c-PARP1 in our gastrulating chick embryos (Fig. 2F–O). This implies that sorafenib increases the rate of apoptosis in the early embryo.
Sorafenib Impairs EMT During Gastrulation in the Chick Embryo We examined whether EMT was influenced by sorafenib treatment. We used E-cadherin, an epithelial cell marker that becomes indispensably down-regulated during EMT initiation, to track the EMT process in the embryo. Immunofluorescent staining revealed that ECadherin was normally expressed on the apical side of the epiblast (Fig. 3A). However, the intensity and extent of E-Cadherin staining increased dramatically when the embryos were treated with sorafenib (Fig. 3B), especially in the primitive streak (Fig. 3C,D). We also used NCadherin to identify the mesoderm which forms in the gastrulating embryos after EMT. N-Cadherin expression was found to be inhibited following sorafenib treatment (Fig. 3F–G) as compared with the control (Fig. 3E,F). Semi-quantitative RT-PCR analysis confirmed that NCadherin expression was reduced after sorafenib
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Fig. 1. Exposure to sorafenib retards development of the gastrulating chick embryo. HH0 chick embryos were treated with DMSO (control) or 10lM sorafenib for 18–28 hr. Representative appearance of control (A–C) and 10lM sorafenib-treated embryos (D–F) is presented at 0, 18, and 28 hr after incubation. (H) showing how the length of the primitive streak was measured after 28-hr incubation. (I) Bar chart revealing the average length of the primitive streaks between control
and sorafenib-treated embryos. (*: P < 0.05, significant difference between control and sorafenib-treated embryos). Table 1: The chart showing the developmental stage (HH) that the embryos managed to attain after 18-hr and 28-hr of DMSO and sorafenib treatment.Abbreviations: No, number; PS, primitive streak. Scale bar 5 500 lm in A–D and 1000 lm in H.
treatment (Fig. 3M,N). In addition, we also found Slug, a transcription factor which is vital for the downregulation of E-Cadherin and upregulation of NCadherin expression during EMT, expression was reduced by sorafenib (Fig. 3M,N). We also performed immunofluorescent staining for laminin in the gastrulating embryos. Laminin is a major glycoprotein component found in the basement membrane. Sorafenib treatment resulted in the laminin being chaotically distributed between the epiblast and mesoderm layers (Fig. 3K,L) in comparison with the control (Fig. 3I,J).
migration share some common characteristics. To study the effects of sorafenib on mesodermal cell migration, we transplanted a piece of GFP-labelled anterior primitive streak (containing progenitor cells for heart tube formation) (Yang et al., 2008; Yue et al., 2008) into corresponding sites in host chick embryos in the presence or absence of sorafenib (Fig. 4). In control embryos, we found the leading GFP1 mesodermal cells had reached to the site of heart tube formation after 14-hr incubation (Fig. 4A,B). In contrast, the leading GFP1 mesodermal cells in sorafenib-treated embryos lagged behind to those of control embryos (Fig. 4C–E). Moreover, the area occupied by fluorescent migratory GFP1 mesodermal cells in the sorafenib-treated embryos was also less than the control—as determined using an Image-Pro Plus software. RhoA (a member of small GTPase family) is an important player in cell migration—through its ability to regulate the actin cytoskeleton in the formation of
Sorafenib Represses Mesodermal Cell Migration in the Gastrulating Chick Embryo Mesodermal cell migration is a fundamental process in the formation of mesodermal structures during embryogenesis. Tumor cell invasion and mesoderm cell
EFFECTS OF SORAFENIB ON CHICK EMBRYO DEVELOPMENT
Fig. 2. Sorafenib inhibits cell proliferation and enhances apoptosis in gastrulating chick embryo. Transverse sections of control and 10lM sorafenib-treated embryos were immunofluorescently stained for pHIS3, c-Caspase3 and c-PARP. (A–D) showing the presence of pHIS31 (dividing) cells in control (A,B) and sorafenib-treated (C,D) embryos, in which DAPI staining was added in B and D. (F–I) showing the presence of c-Caspase3 (apoptotic) cells in control (F,G) and
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sorafenib-treated (H,I) embryos. (K–N) demonstrating the presence of c-PARP (apoptotic) cells in control (K–L) and sorafenib-treated (M,N) embryos. Bar charts show comparing the average percentage of pHIS31 (E), c-Caspase31 (J) and c-PARP (O) cells in control and sorafenib-treated embryos. (**: P < 0.01, significant difference between control and sorafenib-treated embryos in E, J and O). Scale bar 5 50 lm.
Fig. 3. Sorafenib increases E-cadherin and reduces N-Cadherin expressions in gastrulating chick embryos. Transverse sections of control and 10 lM sorafenib-treated embryos were immunofluorescence stained for E-Cadherin, N-Cadherin and laminin. A–D: showing the presence of E-Cadherin1 cells in control (A,B) and sorafenibtreated (C,D) embryos. A-a and C-b: E-Cadherin expression in the primitive groove was increased following sorafenib treatment. E–H: showing the presence of N-Cadherin1 cells in control (E,F) and sorafenib-treated (G,H) embryos. E-c and G-d: N-Cadherin expression in the mesodermal cells is more compared control group with sorafe-
nib group. I–L: revealing the presence of laminin in control (I,J) and sorafenib-treated (K,L) embryos. Semi-quantitative RT-PCR analysis showing Slug and N-Cadherin expressions were significantly repressed following sorafenib treatment. N: Bar chart showing Slug and N-Cadherin expressions in the presence and absence of sorafenib. O: Bar chart showing the number of mesoderm cells in control and sorafenib-treated embryos. *: P < 0.05-significant difference between control and sorafenib-treated embryos. Abbreviations: ECad, E-Cadherin; N-Cad, N-Cadherin. Scale bar 5 50 lm.
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Fig. 4. Sorafenib impairs mesodermal cell migration in gastrulating chick embryos. A piece of GFP1 anterior primitive streak tissue was transplanted into corresponding position in unlabeled HH3 host embryos. The manipulated embryos were then treated with DMSO (control) or 10 lM sorafenib for 14 hr. A,B: showing the appearance of control (A,B) and sorafenib-treated (C,D) embryos before and after 14hr incubation. Bar charts revealing the average distances migrated (E)
and area occupied (F) by the GFP1 mesodermal cells from the transplantation site after 14-hr incubation. G: Semi-quantitative RT-PCR analysis showing RhoA expression was significantly reduced by sorafenib treatment. **P < 0.01 and *P < 0.05, significant difference between control and sorafenib treated embryos. Scale bars 5 500 lm in A–C and 1000 lm in B and D.
pseudopodia. Semi-quantitative RT-PCR analysis revealed that RhoA expression was significantly inhibited following sorafenib exposure (Fig. 4G).
quantity HNK11 cells and the extent of their migration in the control (Fig. 5G) and sorafenib-treated embryos (Fig. 5I). The differences in phenotype were more obvious in transverse sections, which showed fewer HNK11 cells in sorafenib-treated embryos (Fig. 5J,K) than in control embryos (Fig. 5L,M). The reduction in cranial neural crest cells by sorafenib exposure further confirm the inhibitory effect of sorafenib on EMT and cell migration during morphogenesis.
Sorafenib Suppresses the Generation and Migration of Cranial Neural Crest Cells in Early Chick Embryos We observed the sorafenib exposure inhibited the generation of cranial neural crest cells as well (Fig. 5). Immunofluorescencet staining revealed that Pax7 was expressed in the generation of the neural crest cells of HH8-9 chick embryos. As shown in Fig. 5, it was noticeable that quantity Pax71 cells and the extent of their generation of the neural crest cell were affected because of the presence of sorafenib. Besides EMT and mesodermal cells, neural crest cells also migrate during in the early embryo. HNK1 is strongly expressed in migratory neural crest cells, so we used it as a marker to reveal the presence of neural crest cells. We examined the cranial region and discovered that sorafenib exposure inhibited the generation of cranial neural crest cells (Fig. 5C,D) in comparison with the control (Fig. 5A,B). This is also illustrated by the
Immunofluorescence Staining of the pHIS3, Hnk1, and F-Actin are Performed on the Neural Crest Cells We investigate whether the retardation was attributed to altered cell proliferation and apoptosis. The extent of cell proliferation was determined by immunofluorescence staining against pHIS3 antibodies, which labels M-phase nuclei. We established that there were significant fewer pHIS31 cells in the sorafenib-treated group than control group (Fig. 6 A,D)—suggesting that sorafenib inhibited cell proliferation. In addition, we used HNK1 as a marker to reveal the presence of neural crest cells since it is strongly
Fig. 5. Sorafenib inhibits generation and migration of cranial neural crest cells. Whole-mount (A–D) and transverse sections (E–H) of embryos were labeled for HNK1 (migrating neural crest cell maker) following sorafenib treatment. A,B: Pax7 immunofluorescence staining of the head section in control group. C,D: Pax7 immunofluorescence staining in sorafenib group. E: Bar chart: comparing the average percentage of Pax7 positive cells in control and sorafenib-treated embryos. (*: P < 0.05 significant difference between control and sorafenib-treated embryos in E). Whole-mount (F–I) and transverse sections (J–M) of embryos were immunofluorescently stained for
HNK1 (migrating neural crest cell maker) following sorafenib treatment. F: Representative bright-field image of a control HH10 embryo. G: Corresponding fluorescent image showing the extent of HNK11 neural crest cell migration and viewed in transverse section (J,K). H: Representative bright-field image of sorafenib-treated HH10 embryo. I: Corresponding fluorescent image of neural crest cell migration and viewed in transverse section (L,M). N: Bar chart showing the ratio of HNK11 cells present versus the area of the whole embryo. *: P < 0.05, significant difference between control and sorafenib-treated embryos. Scale bars 5 50lm in A–D, 250 lm in F–I, 100 lm in J–M.
EFFECTS OF SORAFENIB ON CHICK EMBRYO DEVELOPMENT
Fig. 6. Immunofluorescence staining of the pHIS3, HNK1 and Factin are performed on the head neural crest cells. A–C: showing immunofluorescence staining of pHIS3, HNK1, DAPI and F-actin in the cells of the control group. D–F: showing immunofluorescence staining of pHIS3, HNK1, DAPI and F-actin in the cells of the sorafenib-treated
expressed in migratory neural crest cells. And we discovered that sorafenib exposure inhibited the migration of cranial neural crest cells (Fig. 6E) in comparison with the control (Fig. 6B). This is also illustrated by the quantity HNK11 cells and the extent of their migration in the control (Fig. 5J) and sorafenib-treated embryos (Fig. 5L). Both cell migration and adhesion are closely associated with the regulation of cytoskeleton actin microfilament (Fei et al., 2010). To investigate whether sorafenib affects the cytoskeleton reorganization of neural crest cells, stereo-fluorescence microscope assays was applied using a probe for F-actin. Results of the cells in control and sorafenib-treated group were displayed representatively in reply Fig. 6C,F. In control cells F-actin dispersed throughout the whole cells uniformly. When incubated in the sorafenib-containing medium, the Factin underwent edge still uniform, and uniform F-actin signals were mainly localized in the cells membrane (Li et al., 2014). From the results in C and F, we cannot detect the changes of the F-actin signals in control and sorfenib group, that is to say, sorafenib did not affect the migration of neural crest cells.
Sorafenib Inhibited the Development of the Chick Embryo by the MAPK Pathway sorafenib inhibited multiple key enzymes in the MAPK signaling pathway (Fig. 7A). In comparison with DMSO control, sorafenib inhibited ERK1/2 phosphorylation by 55% (Fig. 7B). The finding indicates that sorafenib has a significant effect by preventing increased activity of non-targeted pathway in chick embryos. It is the same that already shown mechanism of action in the tumor.
DISCUSSIONS Sorafenib has been used as an oral anti-cancer drug against hepatocellular carcinoma (HCC). However, the efficacy and safety of sorafenib application in clinical practice has mainly been mainly addressed based on
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group. G: Bar chart showing the number of pHIS3 positive cells in control and sorafenib-treated cells. **: P < 0.01-significant difference between control and sorafenib-treated embryos. Scale bars 5 100lm in A–F.
Fig. 7. Sorafenib differentially inhibited phosphorylation of several key enzymes in MAPK and pathway. A: Western blot for p-ERK1/ 2(Thr202/204) level after different treatments. Also shown is endogenous ERK1/2. Phosphorylated kinase band density was assessed by Image-J software and normalized by GAPDH. B: A plot generated based on GAPDH normalized phosphorylated kinase band density in A. The GAPDH normalized density of each phosphorylated kinases is calculated in DMSO treatment and set at 100 arbitrary units and the density of phosphorylated kinases in the sorafenib treatment is as percentage of the DMSO treatment for easy comparison.
practical experiences (Di Marco et al.). To optimize the use of this small molecule, it is necessary to understand its exact pharmacological effects. Because tumor cells and embryonic cells share many identical features, we have presently employed the early chick embryo as an in vivo model to further investigate the anti-cancer effects of sorafenib. In the study, we found that exposure to sorafenib dramatically retarded the development of the gastrulating chick embryo. During gastrulation, the embryonic cells undergo extensive migration, proliferation and apoptosis which are highly coordinated and integrated. This morphogenetic process eventually generates the ectoderm, mesoderm and endoderm that make up the embryonic germ layers. These layers in turn produce all the organs and tissues found in adult. In this context, the ability of sorafenib to retard the development of the gastrulating chick embryo infers that it could interfere with apoptosis, cell proliferation, cell migration, and EMT. Interestingly, these are the biological processes that are inhibited by sorafenib in the
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growing tumor—implying that the impact of sorafenib on the developing embryo could mimic its anti-tumor effects. Embryonic cell proliferation and apoptosis are both relatively active during gastrulation. We estimated that the cell doubling time is 5–7 hr in the early embryo (unpublished observation). We investigated the effect of sorafenib on cell proliferation using pHIS3 antibody and found that it significantly inhibited cell division in vitro and in vivo. In addition, we also observed that sorafenib increased apoptosis as indicated by increased c-Caspase3 and c-PARP labeling of embryonic cells. These findings could explain how sorafenib was able to retard embryonic growth, by inhibiting cell proliferating and enhancing apoptosis. It is now well establish that EMT plays an important role in primary tumor’s progression to metastases. Similarly, EMT is indispensable for the conversion of the bilaminar epiblast germ disk into a trilaminar embryo. A proposal to classify EMTs into three subtypes based on the biological and biomarker context in which they occur was discussed at a 2007 meeting on EMT in Poland and at a subsequent conference in March 2008 at Cold Spring Harbor Laboratories. EMT I exists in embryo development, and EMT III is in cancer progression (Kalluri, 2009). Although type III EMT is far different from those observed in the other two types of EMT, the MT I and III have the association to some extent. Recent human studies found once the migratory cancer cells generated by type III EMT find themselves in distant tissue beds, and then they form secondary tumors exhibiting an epithelial phenotype (Brabletz et al., 2001; Rastaldi et al., 2002; Bataille et al., 2008; Reckamp et al., 2008; Trimboli et al., 2008). This suggests that the reversibility of EMT observed during embryonic development, when migratory mesenchyme gives rise to secondary epithelia, is also operational in the formation of secondary metastatic nodule. Additionally, there exist many similarity makers in EMT type 1 and 3, for example, slug, E-cadherin, N-cadherin and ZO-1 and so forth. Last but not the least, the environment in vivo of cancer progression is hardly copied completely. Just because of these reasons, we would like to use the embryo development, which may mimic the cancer progression in vivo to some extent. From these views, we can use the embryo development may act as the cancer progression models in vivo to some extent. What’s more, although much is known about the signaling pathways involved in type 1 and type 2 EMT (Acloque et al., 2009; Kalluri and Weinberg, 2009), it is still unclear what specific signals induce type 3 EMT in epithelial carcinoma cells. So we may use the embryo developmental model to detect the specific signals in 3 EMT in the future to some extent. Sorafenib’s use was approved because of its able to inhibit the EMT process through an epigenetic mechanism (Chen et al., 2011; Nagai et al., 2011; Zhang et al., 2013). We found that sorafenib treatment retarded the development of the gastrulating chick embryo, so it was reasonable to speculate that sorafenib affected EMT in the embryo. Our speculation was confirmed by sorafenib enhancing E-Cadherin expression while inhibiting NCadherin expression in the gastrulating embryos. Normally, these cell adhesion molecules are inversely expressed to permit proper EMT. Furthermore, Slug (transcriptional factor for EMT) and laminin expression
were also repressed by sorafenib. The abbreviated expression of EMT-related genes might account for the observed reduction in mesoderm cell numbers and retardation of embryo growth. Cell migration is another cellular process that occurs both in tumor metastasis and embryo gastrulation. Consequently, we investigated the effects of sorafenib on mesoderm cell migration during EMT. We transplanted a piece of GFP expressing primitive streak into corresponding site in unlabeled embryos to track cell migration during EMT in the absence or presence of sorafenib as described previously (Yang et al., 2002; Yang et al., 2008; Yue et al., 2008). We found sorafenib repressed mesoderm cell migration. In support, we also found sorafenib affected neural crest cell generation and migration. Our findings revealed the effect of sorafenib in inhibiting the generation of the neural crest cells but sorafenib did not affect the migration of cells itself, which demonstrate the decrease of HNK1 positive cells in sorafenibtreated group is due to the inhibition of the proliferation and the generation of neural crest cells not the migration action itself. These findings imply that sorafenib’s anti-cancer effect may be attributed to its ability to inhibit cancer cell invasion. It is noticeable that sorafenib is a non-selective multiple kinase inhibitor with proven anti-proliferative effects in thyroid, renal and hepatocellular carcinoma. Sorafenib acts on vascular endothelial growth factor (VEGF) and on platelet-derived growth factor (PDGF) related pathways. It also influences the rat sarcoma proto-oncogene/rat fibrosarcoma protein kinase/mitogen activated protein kinase (RAS/RAF/MAPK) pathway and blocks tumor growth factor beta-1 (TGF-b-1)-mediated epithelial-mesenchymal transition (EMT) (Gedaly et al., 2010; Chen et al., 2011; Smolle et al., 2014). Our findings show sorafenib inhibited multiple key enzymes in the MAPK signaling pathway in the chick embryo development like in the tumor. This result may be evidence that we can use the embryo development as the cancer progression, and metastasis in vivo model in the future. In sum, we first demonstrated that sorafenib retarded the development of the gastrulating chick embryos. This growth retardation was caused by a reduction in embryonic cell proliferation, an increase in apoptosis and disruption in EMT and mesoderm cell migration. The close similarity in cellular behavior between the gastrulating chick embryo and growing tumor makes the embryo a good model for testing out new anti-cancer drugs.
ACKNOWLEDGEMENTS The authors thank Wing Yan Wong for constructive help on the manuscript and Professor Yu Yanhong for kindly providing ERK antibody.
LITERATURE CITED Acloque H, Adams MS, Fishwick K, Bronner-Fraser M, Nieto MA. 2009. Epithelial-mesenchymal transitions: the importance of changing cell state in development and disease. J Clin Investig 119:1438–1449. Bai C, Hou L, Zhang M, Pu Y, Guan W, Ma Y. 2012. Characterization of vascular endothelial progenitor cells from chicken bone marrow. BMC Vet Res 8:54.
EFFECTS OF SORAFENIB ON CHICK EMBRYO DEVELOPMENT Bataille F, Rohrmeier C, Bates R, Weber A, Rieder F, Brenmoehl J, Strauch U, Farkas S, Furst A, Hofstadter F, Scholmerich J, Herfarth H, Rogler G. 2008. Evidence for a role of epithelial mesenchymal transition during pathogenesis of fistulae in Crohn’s disease. Inflamm Bowel Dis 14:1514–1527. Brabletz T, Jung A, Reu S, Porzner M, Hlubek F, Kunz-Schughart LA, Knuechel R, Kirchner T. 2001. Variable beta-catenin expression in colorectal cancers indicates tumor progression driven by the tumor environment. Proc Natl Acad Sci USA 98:10356– 10361. Bronner-Fraser M. 1994. Neural crest cell formation and migration in the developing embryo. FASEB J 8:699–706. Chapman SC, Collignon J, Schoenwolf GC, Lumsden A. 2001. Improved method for chick whole-embryo culture using a filter paper carrier. Dev Dyn 220:284–289. Chen YL, Lv J, Ye XL, Sun MY, Xu Q, Liu CH, Min LH, Li HP, Liu P, Ding X. 2011. Sorafenib inhibits transforming growth factor beta1-mediated epithelial-mesenchymal transition and apoptosis in mouse hepatocytes. Hepatology 53:1708–1718. Di Marco V, De Vita F, Koskinas J, Semela D, Toniutto P, Verslype C. 2013. Sorafenib: from literature to clinical practice. Ann Oncol 24 (Suppl 2):ii30–ii37. Escudier B, Eisen T, Stadler WM, Szczylik C, Oudard S, Siebels M, Negrier S, Chevreau C, Solska E, Desai AA, Rolland F, Demkow T, Hutson TE, Gore M, Freeman S, Schwartz B, Shan M, Simantov R, Bukowski RM. 2007. Sorafenib in advanced clear-cell renal-cell carcinoma. N Engl J Med 356:125–134. Fei F, Stoddart S, Muschen M, Kim YM, Groffen J, Heisterkamp N. 2010. Development of resistance to dasatinib in bcr/Abl-positive acute lymphoblastic leukemia. Leukemia 24:813–820. Gedaly R, Angulo P, Hundley J, Daily MF, Chen C, Koch A, Evers BM. 2010. PI-103 and sorafenib inhibit hepatocellular carcinoma cell proliferation by blocking ras/raf/MAPK and PI3K/AKT/mTOR pathways. Anticancer Res 30:4951–4958. Hamburger V, Hamilton HL. 1992. A series of normal stages in the development of the chick embryo. 1951. Dev Dyn 195:231–272. Kalluri R, Weinberg RA. 2009. The basics of epithelialmesenchymal transition. J Clin Investig 119:1420–1428. Larger E, Marre M, Corvol P, Gasc JM. 2004. Hyperglycemiainduced defects in angiogenesis in the chicken chorioallantoic membrane model. Diabetes 53:752–761. Li X, Guo H, Yang Y, Meng J, Liu J, Wang C, Xu H. 2014. A designed peptide targeting cxcr4 displays anti-acute myelocytic leukemia activity in vitro and in vivo. Sci Rep 4:6610. Li Y, Wang XY, Wu T, Chuai M, Lee KK, Wang LJ, Yang X. 2013. PTEN is involved in modulation of vasculogenesis in early chick embryos. Biol Open 2:587–595. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, de Oliveira AC, Santoro A, Raoul JL, Forner A, Schwartz M, Porta C, Zeuzem S, Bolondi L, Greten TF, Galle PR, Seitz JF, Borbath I,
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Haussinger D, Giannaris T, Shan M, Moscovici M, Voliotis D, Bruix J. 2008. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 359:378–390. Mintz B, Cronmiller C. 1978. Normal blood cells of anemic genotype in teratocarcinoma-derived mosaic mice. Proc Natl Acad Sci USA 75:6247–6251. Nagai T, Arao T, Furuta K, Sakai K, Kudo K, Kaneda H, Tamura D, Aomatsu K, Kimura H, Fujita Y, Matsumoto K, Saijo N, Kudo M, Nishio K. 2011. Sorafenib inhibits the hepatocyte growth factor-mediated epithelial mesenchymal transition in hepatocellular carcinoma. Mol Cancer Therap 10:169–177. Pierce GB. 1983. The cancer cell and its control by the embryo. Rous-whipple award lecture. Am J Pathol 113:117–124. Rashidi H, Sottile V. 2009. The chick embryo: hatching a model for contemporary biomedical research. Bioessays 31:459–465. Rastaldi MP, Ferrario F, Giardino L, Dell’Antonio G, Grillo C, Grillo P, Strutz F, Muller GA, Colasanti G, D’Amico G. 2002. Epithelialmesenchymal transition of tubular epithelial cells in human renal biopsies. Kid Int 62:137–146. Reckamp KL, Gardner BK, Figlin RA, Elashoff D, Krysan K, Dohadwala M, Mao J, Sharma S, Inge L, Rajasekaran A, Dubinett SM. 2008. Tumor response to combination celecoxib and erlotinib therapy in nonsmall cell lung cancer is associated with a low baseline matrix metalloproteinase-9 and a decline in serum-soluble E-cadherin. J Thorac Oncol Off Publ Int Assoc Study Lung Cancer 3:117–124. Savagner P. 2010. The epithelial-mesenchymal transition (EMT) phenomenon. Ann Oncol 21 (Suppl 7):vii89–vii92. Smolle E, Taucher V, Petru E, Haybaeck J. 2014. Targeted treatment of ovarian cancer—the multiple—kinase—inhibitor sorafenib as a potential option. Anticancer Res 34:1519–1530. Trimboli AJ, Fukino K, de Bruin A, Wei G, Shen L, Tanner SM, Creasap N, Rosol TJ, Robinson ML, Eng C, Ostrowski MC, Leone G. 2008. Direct evidence for epithelial-mesenchymal transitions in breast cancer. Cancer Res 68:937–945. Yang X, Chrisman H, Weijer CJ. 2008. PDGF signalling controls the migration of mesoderm cells during chick gastrulation by regulating N-cadherin expression. Development (Cambridge, England) 135:3521–3530. Yang X, Dormann D, Munsterberg AE, Weijer CJ. 2002. Cell movement patterns during gastrulation in the chick are controlled by positive and negative chemotaxis mediated by fgf4 and fgf8. Dev Cell 3:425–437. Yue Q, Wagstaff L, Yang X, Weijer C, Munsterberg A. 2008. Wnt3amediated chemorepulsion controls movement patterns of cardiac progenitors and requires RhoA function. Development (Cambridge, England) 135:1029–1037. Zhang J, Chen YL, Ji G, Fang W, Gao Z, Liu Y, Wang J, Ding X, Gao F. 2013. Sorafenib inhibits epithelial-mesenchymal transition through an epigenetic-based mechanism in human lung epithelial cells. PloS One 8:e64954.
Effects of Antitumor Drug Sorafenib on Chick Embryo Development YI-SEN CHENG,1† XIAO-YU WANG,1† GUANG WANG,1 YAN LI,1 YUE-LEI CHEN,2 MAN-LI CHUAI,3 KENNETH KA HO LEE,4 XIAO-YAN DING,2* 1 AND XUE-SONG YANG * 1 Division of Histology and Embryology, Key Laboratory for Regenerative Medicine of the Ministry of Education, Medical College, Jinan University, Guangzhou 510632, China 2 The State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China 3 Division of Cell and Developmental Biology, University of Dundee, Dundee, Dd1 5EH, UK 4 Key Laboratory for Regenerative Medicine, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin, Hong Kong
ABSTRACT Sorafenib has been used as an oral anti-cancer drug because of its ability to inhibit tumor growth. However, the pharmacological effect of sorafenib is still the lack of in vivo experimental evidence. Tumor and embryonic cells share some similar features, so we investigated the effects of sorafenib on the development of gastrulating chick embryos. We found that sorafenib exposure was markedly attributed to the number of embryonic cell in proliferation and apoptosis. We also detected sorafenib significantly interfered with epithelial-mesenchymal transition (EMT). Furthermore, sorafenib treatment impaired the production and migration of neural crest cells. Anat Rec, 298:1271–1281, 2015. C 2015 Wiley Periodicals, Inc. V
Key words: sorafenib; chick embryos; EMT; gastrulation; neural crest cells
Sorafenib is the first orally used drug that shows significant inhibitory effects on tumor growth and EMT. It principally targets serine/threonine and receptor tyrosine kinases (Escudier et al., 2007; Llovet et al., 2008; Chen et al., 2011; Nagai et al., 2011; Zhang et al., 2013). In rat hepatocytes, the drug antagonizes TGF-b signaling and interferes with TGF-b1-induced EMT and apoptosis (Chen et al., 2011). These two processes play crucial roles in embryo development, especially during gastrulation when EMT is required for the generation of the three germ cell layers. Furthermore, apoptosis, cell migration, cell division, and differentiation are the processes that are tightly regulated to ensure that the early embryo develops normally. In tumor genesis, EMT is presented as a loss of epithelial features and an acquisition of migratory properties, as illustrated by a breakdown of epithelium homeostasis and progressive cancer aggression. Superficially, embryonic EMT is very similar to the transformation of cellular phenotypes that occur during carcinoma progression (Savagner). The similarC 2015 WILEY PERIODICALS, INC. V
Grant sponsor: “973 Project”; Grant number: 2010CB529703; Grant sponsor: NSFC; Grant numbers: 31071054, 30971493; Grant sponsor: Guangdong Natural Science Foundation; Grant numbers: S2011010001593, S2013010013392; Grant sponsor: Science and Technology Grant; Grant number: 2012DFH30060. † These authors contributed equally to this work. *Correspondence to: X. Yang, Division of Histology and Embryology, Key Laboratory for Regenerative Medicine of the Ministry of Education, Medical College, Jinan University, Guangzhou 510632, China. E-mail: [email protected] (or) X. Y. Ding, The State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. E-mail: [email protected] Received 9 June 2014; Revised 27 November 2014; Accepted 5 January 2015. DOI 10.1002/ar.23155 Published online 17 April 2015 in Wiley Online Library (wileyonlinelibrary.com).
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ities between cancer and embryonic cells are not only evident in EMT but also in other cellular behaviors. For an example, when cancer cells are injected into blastocysts, the cells were found to contribute to normal embryonic development (Pierce, 1983). This extraordinary phenomenon has been widely observed by several experimenters (Mintz and Cronmiller, 1978) and indicates that cancer cells share some similar characteristics with embryonic cells. Moreover, there are similarities between these cells during apoptosis and cell proliferation. In this context, it will possible to investigate the anti-tumor effects of sorafenib by using the developing chick embryo as a model. There are many advantages of using the chick embryo. It is highly accessible inside the egg for surgical manipulation, inexpensive and the developmental stages of the embryos are well characterized. The early chick embryo also lack an immune system, so tissues and cells could be transplanted into them and their developmental fate established without rejection. Because of these properties, it has been proposed that this model is useful for addressing problems associated with stem cell and cancer biology (Rashidi and Sottile, 2009). Moreover, extra information can be gleamed from the model, for example how sorafenib affects the proliferation and migration of neural crest cells in the embryo (Bronner-Fraser, 1994).
MATERIALS AND METHODS Chick Embryos and Tissue Transplantation Fertilized leghorn eggs were obtained from the Avian Farm of South China Agriculture University. The eggs were incubated in a humidified incubator (Yiheng Instruments, Shanghai, China) at 38 C until the chick embryos reached Hamburger and Hamilton (HH) stage 3 (Hamburger and Hamilton, 1992). Either the whole chick embryo or half-side of the embryos were exposed to 10 lM sorafenib (Nexavar, BAY 43-9006, Bayer Pharmaceuticals, USA) in early chick (EC) culture medium overnight (Chapman et al., 2001; Li et al., 2013). The transplantation of the primitive streak from donor to host embryo was performed as previously described (Yang et al., 2002). Briefly, a piece of GFP-expressing primitive streak, 200 mm wide was excised from a GFP-transfected donor embryo, and grafted into a similar position in an unlabeled host embryo at the same developmental stage. After transplantation, the embryos were incubated overnight to assess and compare the migratory patterns of GFP1 cells between the control and sorafenib-treated embryos.
Chick Embryo Electroporation EC culture (Chapman et al., 2001) was employed to maintain the embryos during and after gene electroporation. Briefly, 1 lL of 1.5 lg GFP expression plasmid (pEGFP-N1, Clontech) was microinjected into the space between the vitelline membrane and the epiblast prior to electroporation. Electroporation was performed on HH3 chick embryos using an electroporator (BTXECM399) and a pair of platinum electrodes arranged in a parallel fashion. Two pulses of 10 V were delivered between the electrodes as previously described (Yang
et al., 2002). The survival rate of the embryos after gene transfection was nearly 100%, dependent on the embryos used were in good condition. The transfected embryos were incubated at 37 C and 70% humidity until the desired developmental stage was reached. During incubation, the fluorescence of GFP was directly viewed under a stereo-fluorescence microscope (Olympus MVX10).
Whole-mount Embryo Immunohistochemistry Immunofluorescent staining were performed on whole-mount control and sorafenib-treated chick embryos against: E-Cadherin (BD, 610181), N-Cadherin (DSHB, 6B3), Laminin (DSHB, 3H11), phospho-histone H3 (pHIS3, SantaCruz, sc-12927-R), Cleaved-Caspase3 (c-Caspase3, Cell Signaling Technology, 9664p), CleavedPARP (c-PARP, Cell Signaling Technology, 5625p) and HNK1 (Sigma C0678). The embryos were fixed in 4% paraformaldehyde (PFA) at 4oC overnight, and then treated with 2% Bovine Serum Albumin (BSA) 1 1% Triton-X 1 1% Tween 20 in PBS for 2 hr to block unspecific immunoreactions. Next, unspecific binding were blocked using 2% Bovine Serum Albumin (BSA) 1 1% Triton-X 1 1% Tween 20 in PBS for 2 hr, at room temperature. The embryos were washed in PBS and then incubated in primary mouse monoclonal antibody against E-Cadherin (1:100), or N-Cadherin (1:100), or Laminin (1:100), or pHIS3 (1:400), or c-Caspase3 (1:100), or c-PARP (1:100) and HNK1 (1:100), overnight at 4oC on a shaker. The embryos were then extensive washed in PBS and incubated with alexa fluor 555 anti-mouse IgG secondary antibodies (1:1000, Invitrogen, USA) overnight at 4 C, to visualize the primary antibody bindings. Subsequently, the embryos were sectioned on a cryostat microtome (Leica CM1900). These sections were mounted with mounting solution (Mowiol 4-88, Sigma) onto glass slides and sealed with coverslips before photography. All experiments were repeated three times and around five to six embryos were used for each group.
RNA Isolation and RT-PCR Total RNA was isolated from the whole primary steak using a Trizol kit (Invitrogen, USA) according to the manufacturer’s instructions. First-strand cDNA was synthesized to a final volume of 25 lL using the SuperScript RIII First-Strand (Invitrogen, USA). Following reverse transcription, PCR amplification of the cDNA was performed as described previously (Larger et al., 2004; Bai et al., 2012) using specific primers as follows: N-cadherin 5’-AGATTCTGGAAATCCACATGC-3’; 5’-CTTCCTTCATAGTCAAAGACT-3’ Slug 5’-CCAATGACCTCTCTCCGCTTTCTG-3’, 5’-ATCGCTAATGGGACTTTCTGAACCG-3’; RhoA 5’-GCAGCCATTCGAAAAAGCT-3’; 5’-TTTATAAGAGAAGGCACCCG-3’ Reactions were performed in a Bio-Rad S1000TM Thermal cycler. (Bio-Rad, USA) The final reaction volume was 50 lL composing of 1 lL of first-strand cDNA, 25 lM forward primer, 25 lM reverse primer, 10 lL PrimeSTARTM Buffer (Mg21 plus), 4 lL dNTPs Mixture (TaKaRa, Japan), 0.5 lL PrimeSTARTM HS DNA Polymerase (2.5 U lL21 TaKaRa, Japan), and RNase-free water to 50 lL.
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The cDNAs were amplified for 30 cycles. One round of amplification was performed at 94 C for 30 sec, 30 sec at 58 C, and 30 sec at 72 C. The PCR products (20 ll) were resolved in 1% agarose gels (Biowest, Spain) in 13TAE buffer (0.04 M Trisacetate and 0.001 M EDTA), and 10,0003 GeneGreen Nucleic Acid Dye (TIANGEN, China) solution. The resolved products were visualized in a transilluminator (SYNGENE, UK) and photographs were captured using a computer-assisted gel documentation system (SYNGENE).
Western Blot To detect the protein levels of MAPK pathway markers, chick embryo (HH4) treated as indicated were lysed with 70 mL of lysis buffer and subjected to western blot analysis. Approximately 30 mg of total protein was separated by 12% SDS-polyacrylamide gel, transferred to a PVDF membrane and incubated with the appropriate antibodies as indicated in the figure legends. The membrane was blocked with 5% fat-free milk. The primary antibody (1:1,000 dilution) incubation was performed overnight at 4 C and the secondary antibody incubation (1:3,000 dilution) was performed for 1 hr at room temperature. GAPDH was used as an internal control to normalize for loading equality. The protein bands were visualized using an enhanced chemiluminescence (ECL) detection kit (BIORAD). Primary antibodies against ERK1/2, ERK1/2 phosphorylation (1:1000, supplied by professor Yanhong Yu) and GAPDH (1:1,000, supplied by Yuhui Yan) and secondary goat anti-rabbit- HRP conjugated antibody were all obtained from CST.
Cell Culture Cranial neural crest cells were obtained from the chick embryo (HH9) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Biochrom, Berlin, Germany) at 37 C with 5% CO2 for 60 h.
Photography Following immunofluorescent staining, the whole mount embryos were photographed using a stereofluorescence microscope (Olympus MVX10) and the captured images analyzed using an Olympus software package Image-Pro Plus 7.0. The embryos were sectioned at 10–14 lm using a cryostat microtome (Leica CM1900). The slides were photographed and analyzed using an epi-fluorescence microscope (Olympus IX51, Leica DM 4000B) at 2003 and 4003 magnifications and associated software package Leica CW4000 FISH.
Statistical Analysis Data analysis and construction of charts were performed using a Graphpad Prism 5 software package (Graphpad Software, CA). The results were presented as mean value (x6SE). All data were analyzed using student t test or v2-test to determine statistical difference amongst the control and experimental groups. P < 0.05 was considered to be statistically significant.
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RESULTS Sorafenib Retards Chick Embryo Growth The effects of sorafenib on gastrulation in the chick embryo were investigated. Chick embryos (HH0) were randomly divided into two groups, with one group exposed to 10lM sorafenib solubilized in DMSO while another group exposed to DMSO only (serving as the control) for 28 hr. The morphology of the embryos was periodically photographed at 0 hr (Fig. 1A,D), 18-hr and 28-hr after incubation. We observed that the development of sorafenib-treated embryos (Fig. 1E) lagged behind the control embryos (Fig. 1B) at 18-hr incubation. The extent of this retardation was very obvious after 28-hr incubation (Fig. 1C,F). We examined a large cohort of embryos (20 embryos for each group) to avoid intragroup developmental differences. For control embryos incubated for 18-hr and 28-hr, we determined that the majority (92%) of the embryos were staged HH5-6. In contrast, only 40% of sorafenib-treated embryos could reach the HH5-6 stage (Fig. 1G). We recorded the length of the primitive streak, which make its appearance during gastrulation, as an indicator of embryonic development (Fig. 1H). We established that there was a significant difference in the length of the primitive streak between the control and sorafenib-treated embryos (Fig. 1I).
Effects of Sorafenib on Cell Death and Proliferation in the Gastrulating Chick Embryo We established that sorafenib retarded the growth of gastrulating embryos. Hence, we decided to investigate whether the retardation was attributed to altered cell proliferation and apoptosis. The extent of cell proliferation was determined by immunofluorescent staining with pHIS3 antibodies, which labels M-phase nuclei. We established that there were significant fewer pHIS31 cells in the sorafenib-treated embryos than control embryos (Fig. 2A–E)—suggesting that sorafenib inhibited cell proliferation. To determine the extent of cell death in our specimens, we performed immunofluorescent staining for c-Caspase3 and c-PARP. We established that sorafenib treatment significantly increased the number of c-Caspase31 and c-PARP1 in our gastrulating chick embryos (Fig. 2F–O). This implies that sorafenib increases the rate of apoptosis in the early embryo.
Sorafenib Impairs EMT During Gastrulation in the Chick Embryo We examined whether EMT was influenced by sorafenib treatment. We used E-cadherin, an epithelial cell marker that becomes indispensably down-regulated during EMT initiation, to track the EMT process in the embryo. Immunofluorescent staining revealed that ECadherin was normally expressed on the apical side of the epiblast (Fig. 3A). However, the intensity and extent of E-Cadherin staining increased dramatically when the embryos were treated with sorafenib (Fig. 3B), especially in the primitive streak (Fig. 3C,D). We also used NCadherin to identify the mesoderm which forms in the gastrulating embryos after EMT. N-Cadherin expression was found to be inhibited following sorafenib treatment (Fig. 3F–G) as compared with the control (Fig. 3E,F). Semi-quantitative RT-PCR analysis confirmed that NCadherin expression was reduced after sorafenib
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Fig. 1. Exposure to sorafenib retards development of the gastrulating chick embryo. HH0 chick embryos were treated with DMSO (control) or 10lM sorafenib for 18–28 hr. Representative appearance of control (A–C) and 10lM sorafenib-treated embryos (D–F) is presented at 0, 18, and 28 hr after incubation. (H) showing how the length of the primitive streak was measured after 28-hr incubation. (I) Bar chart revealing the average length of the primitive streaks between control
and sorafenib-treated embryos. (*: P < 0.05, significant difference between control and sorafenib-treated embryos). Table 1: The chart showing the developmental stage (HH) that the embryos managed to attain after 18-hr and 28-hr of DMSO and sorafenib treatment.Abbreviations: No, number; PS, primitive streak. Scale bar 5 500 lm in A–D and 1000 lm in H.
treatment (Fig. 3M,N). In addition, we also found Slug, a transcription factor which is vital for the downregulation of E-Cadherin and upregulation of NCadherin expression during EMT, expression was reduced by sorafenib (Fig. 3M,N). We also performed immunofluorescent staining for laminin in the gastrulating embryos. Laminin is a major glycoprotein component found in the basement membrane. Sorafenib treatment resulted in the laminin being chaotically distributed between the epiblast and mesoderm layers (Fig. 3K,L) in comparison with the control (Fig. 3I,J).
migration share some common characteristics. To study the effects of sorafenib on mesodermal cell migration, we transplanted a piece of GFP-labelled anterior primitive streak (containing progenitor cells for heart tube formation) (Yang et al., 2008; Yue et al., 2008) into corresponding sites in host chick embryos in the presence or absence of sorafenib (Fig. 4). In control embryos, we found the leading GFP1 mesodermal cells had reached to the site of heart tube formation after 14-hr incubation (Fig. 4A,B). In contrast, the leading GFP1 mesodermal cells in sorafenib-treated embryos lagged behind to those of control embryos (Fig. 4C–E). Moreover, the area occupied by fluorescent migratory GFP1 mesodermal cells in the sorafenib-treated embryos was also less than the control—as determined using an Image-Pro Plus software. RhoA (a member of small GTPase family) is an important player in cell migration—through its ability to regulate the actin cytoskeleton in the formation of
Sorafenib Represses Mesodermal Cell Migration in the Gastrulating Chick Embryo Mesodermal cell migration is a fundamental process in the formation of mesodermal structures during embryogenesis. Tumor cell invasion and mesoderm cell
EFFECTS OF SORAFENIB ON CHICK EMBRYO DEVELOPMENT
Fig. 2. Sorafenib inhibits cell proliferation and enhances apoptosis in gastrulating chick embryo. Transverse sections of control and 10lM sorafenib-treated embryos were immunofluorescently stained for pHIS3, c-Caspase3 and c-PARP. (A–D) showing the presence of pHIS31 (dividing) cells in control (A,B) and sorafenib-treated (C,D) embryos, in which DAPI staining was added in B and D. (F–I) showing the presence of c-Caspase3 (apoptotic) cells in control (F,G) and
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sorafenib-treated (H,I) embryos. (K–N) demonstrating the presence of c-PARP (apoptotic) cells in control (K–L) and sorafenib-treated (M,N) embryos. Bar charts show comparing the average percentage of pHIS31 (E), c-Caspase31 (J) and c-PARP (O) cells in control and sorafenib-treated embryos. (**: P < 0.01, significant difference between control and sorafenib-treated embryos in E, J and O). Scale bar 5 50 lm.
Fig. 3. Sorafenib increases E-cadherin and reduces N-Cadherin expressions in gastrulating chick embryos. Transverse sections of control and 10 lM sorafenib-treated embryos were immunofluorescence stained for E-Cadherin, N-Cadherin and laminin. A–D: showing the presence of E-Cadherin1 cells in control (A,B) and sorafenibtreated (C,D) embryos. A-a and C-b: E-Cadherin expression in the primitive groove was increased following sorafenib treatment. E–H: showing the presence of N-Cadherin1 cells in control (E,F) and sorafenib-treated (G,H) embryos. E-c and G-d: N-Cadherin expression in the mesodermal cells is more compared control group with sorafe-
nib group. I–L: revealing the presence of laminin in control (I,J) and sorafenib-treated (K,L) embryos. Semi-quantitative RT-PCR analysis showing Slug and N-Cadherin expressions were significantly repressed following sorafenib treatment. N: Bar chart showing Slug and N-Cadherin expressions in the presence and absence of sorafenib. O: Bar chart showing the number of mesoderm cells in control and sorafenib-treated embryos. *: P < 0.05-significant difference between control and sorafenib-treated embryos. Abbreviations: ECad, E-Cadherin; N-Cad, N-Cadherin. Scale bar 5 50 lm.
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Fig. 4. Sorafenib impairs mesodermal cell migration in gastrulating chick embryos. A piece of GFP1 anterior primitive streak tissue was transplanted into corresponding position in unlabeled HH3 host embryos. The manipulated embryos were then treated with DMSO (control) or 10 lM sorafenib for 14 hr. A,B: showing the appearance of control (A,B) and sorafenib-treated (C,D) embryos before and after 14hr incubation. Bar charts revealing the average distances migrated (E)
and area occupied (F) by the GFP1 mesodermal cells from the transplantation site after 14-hr incubation. G: Semi-quantitative RT-PCR analysis showing RhoA expression was significantly reduced by sorafenib treatment. **P < 0.01 and *P < 0.05, significant difference between control and sorafenib treated embryos. Scale bars 5 500 lm in A–C and 1000 lm in B and D.
pseudopodia. Semi-quantitative RT-PCR analysis revealed that RhoA expression was significantly inhibited following sorafenib exposure (Fig. 4G).
quantity HNK11 cells and the extent of their migration in the control (Fig. 5G) and sorafenib-treated embryos (Fig. 5I). The differences in phenotype were more obvious in transverse sections, which showed fewer HNK11 cells in sorafenib-treated embryos (Fig. 5J,K) than in control embryos (Fig. 5L,M). The reduction in cranial neural crest cells by sorafenib exposure further confirm the inhibitory effect of sorafenib on EMT and cell migration during morphogenesis.
Sorafenib Suppresses the Generation and Migration of Cranial Neural Crest Cells in Early Chick Embryos We observed the sorafenib exposure inhibited the generation of cranial neural crest cells as well (Fig. 5). Immunofluorescencet staining revealed that Pax7 was expressed in the generation of the neural crest cells of HH8-9 chick embryos. As shown in Fig. 5, it was noticeable that quantity Pax71 cells and the extent of their generation of the neural crest cell were affected because of the presence of sorafenib. Besides EMT and mesodermal cells, neural crest cells also migrate during in the early embryo. HNK1 is strongly expressed in migratory neural crest cells, so we used it as a marker to reveal the presence of neural crest cells. We examined the cranial region and discovered that sorafenib exposure inhibited the generation of cranial neural crest cells (Fig. 5C,D) in comparison with the control (Fig. 5A,B). This is also illustrated by the
Immunofluorescence Staining of the pHIS3, Hnk1, and F-Actin are Performed on the Neural Crest Cells We investigate whether the retardation was attributed to altered cell proliferation and apoptosis. The extent of cell proliferation was determined by immunofluorescence staining against pHIS3 antibodies, which labels M-phase nuclei. We established that there were significant fewer pHIS31 cells in the sorafenib-treated group than control group (Fig. 6 A,D)—suggesting that sorafenib inhibited cell proliferation. In addition, we used HNK1 as a marker to reveal the presence of neural crest cells since it is strongly
Fig. 5. Sorafenib inhibits generation and migration of cranial neural crest cells. Whole-mount (A–D) and transverse sections (E–H) of embryos were labeled for HNK1 (migrating neural crest cell maker) following sorafenib treatment. A,B: Pax7 immunofluorescence staining of the head section in control group. C,D: Pax7 immunofluorescence staining in sorafenib group. E: Bar chart: comparing the average percentage of Pax7 positive cells in control and sorafenib-treated embryos. (*: P < 0.05 significant difference between control and sorafenib-treated embryos in E). Whole-mount (F–I) and transverse sections (J–M) of embryos were immunofluorescently stained for
HNK1 (migrating neural crest cell maker) following sorafenib treatment. F: Representative bright-field image of a control HH10 embryo. G: Corresponding fluorescent image showing the extent of HNK11 neural crest cell migration and viewed in transverse section (J,K). H: Representative bright-field image of sorafenib-treated HH10 embryo. I: Corresponding fluorescent image of neural crest cell migration and viewed in transverse section (L,M). N: Bar chart showing the ratio of HNK11 cells present versus the area of the whole embryo. *: P < 0.05, significant difference between control and sorafenib-treated embryos. Scale bars 5 50lm in A–D, 250 lm in F–I, 100 lm in J–M.
EFFECTS OF SORAFENIB ON CHICK EMBRYO DEVELOPMENT
Fig. 6. Immunofluorescence staining of the pHIS3, HNK1 and Factin are performed on the head neural crest cells. A–C: showing immunofluorescence staining of pHIS3, HNK1, DAPI and F-actin in the cells of the control group. D–F: showing immunofluorescence staining of pHIS3, HNK1, DAPI and F-actin in the cells of the sorafenib-treated
expressed in migratory neural crest cells. And we discovered that sorafenib exposure inhibited the migration of cranial neural crest cells (Fig. 6E) in comparison with the control (Fig. 6B). This is also illustrated by the quantity HNK11 cells and the extent of their migration in the control (Fig. 5J) and sorafenib-treated embryos (Fig. 5L). Both cell migration and adhesion are closely associated with the regulation of cytoskeleton actin microfilament (Fei et al., 2010). To investigate whether sorafenib affects the cytoskeleton reorganization of neural crest cells, stereo-fluorescence microscope assays was applied using a probe for F-actin. Results of the cells in control and sorafenib-treated group were displayed representatively in reply Fig. 6C,F. In control cells F-actin dispersed throughout the whole cells uniformly. When incubated in the sorafenib-containing medium, the Factin underwent edge still uniform, and uniform F-actin signals were mainly localized in the cells membrane (Li et al., 2014). From the results in C and F, we cannot detect the changes of the F-actin signals in control and sorfenib group, that is to say, sorafenib did not affect the migration of neural crest cells.
Sorafenib Inhibited the Development of the Chick Embryo by the MAPK Pathway sorafenib inhibited multiple key enzymes in the MAPK signaling pathway (Fig. 7A). In comparison with DMSO control, sorafenib inhibited ERK1/2 phosphorylation by 55% (Fig. 7B). The finding indicates that sorafenib has a significant effect by preventing increased activity of non-targeted pathway in chick embryos. It is the same that already shown mechanism of action in the tumor.
DISCUSSIONS Sorafenib has been used as an oral anti-cancer drug against hepatocellular carcinoma (HCC). However, the efficacy and safety of sorafenib application in clinical practice has mainly been mainly addressed based on
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group. G: Bar chart showing the number of pHIS3 positive cells in control and sorafenib-treated cells. **: P < 0.01-significant difference between control and sorafenib-treated embryos. Scale bars 5 100lm in A–F.
Fig. 7. Sorafenib differentially inhibited phosphorylation of several key enzymes in MAPK and pathway. A: Western blot for p-ERK1/ 2(Thr202/204) level after different treatments. Also shown is endogenous ERK1/2. Phosphorylated kinase band density was assessed by Image-J software and normalized by GAPDH. B: A plot generated based on GAPDH normalized phosphorylated kinase band density in A. The GAPDH normalized density of each phosphorylated kinases is calculated in DMSO treatment and set at 100 arbitrary units and the density of phosphorylated kinases in the sorafenib treatment is as percentage of the DMSO treatment for easy comparison.
practical experiences (Di Marco et al.). To optimize the use of this small molecule, it is necessary to understand its exact pharmacological effects. Because tumor cells and embryonic cells share many identical features, we have presently employed the early chick embryo as an in vivo model to further investigate the anti-cancer effects of sorafenib. In the study, we found that exposure to sorafenib dramatically retarded the development of the gastrulating chick embryo. During gastrulation, the embryonic cells undergo extensive migration, proliferation and apoptosis which are highly coordinated and integrated. This morphogenetic process eventually generates the ectoderm, mesoderm and endoderm that make up the embryonic germ layers. These layers in turn produce all the organs and tissues found in adult. In this context, the ability of sorafenib to retard the development of the gastrulating chick embryo infers that it could interfere with apoptosis, cell proliferation, cell migration, and EMT. Interestingly, these are the biological processes that are inhibited by sorafenib in the
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growing tumor—implying that the impact of sorafenib on the developing embryo could mimic its anti-tumor effects. Embryonic cell proliferation and apoptosis are both relatively active during gastrulation. We estimated that the cell doubling time is 5–7 hr in the early embryo (unpublished observation). We investigated the effect of sorafenib on cell proliferation using pHIS3 antibody and found that it significantly inhibited cell division in vitro and in vivo. In addition, we also observed that sorafenib increased apoptosis as indicated by increased c-Caspase3 and c-PARP labeling of embryonic cells. These findings could explain how sorafenib was able to retard embryonic growth, by inhibiting cell proliferating and enhancing apoptosis. It is now well establish that EMT plays an important role in primary tumor’s progression to metastases. Similarly, EMT is indispensable for the conversion of the bilaminar epiblast germ disk into a trilaminar embryo. A proposal to classify EMTs into three subtypes based on the biological and biomarker context in which they occur was discussed at a 2007 meeting on EMT in Poland and at a subsequent conference in March 2008 at Cold Spring Harbor Laboratories. EMT I exists in embryo development, and EMT III is in cancer progression (Kalluri, 2009). Although type III EMT is far different from those observed in the other two types of EMT, the MT I and III have the association to some extent. Recent human studies found once the migratory cancer cells generated by type III EMT find themselves in distant tissue beds, and then they form secondary tumors exhibiting an epithelial phenotype (Brabletz et al., 2001; Rastaldi et al., 2002; Bataille et al., 2008; Reckamp et al., 2008; Trimboli et al., 2008). This suggests that the reversibility of EMT observed during embryonic development, when migratory mesenchyme gives rise to secondary epithelia, is also operational in the formation of secondary metastatic nodule. Additionally, there exist many similarity makers in EMT type 1 and 3, for example, slug, E-cadherin, N-cadherin and ZO-1 and so forth. Last but not the least, the environment in vivo of cancer progression is hardly copied completely. Just because of these reasons, we would like to use the embryo development, which may mimic the cancer progression in vivo to some extent. From these views, we can use the embryo development may act as the cancer progression models in vivo to some extent. What’s more, although much is known about the signaling pathways involved in type 1 and type 2 EMT (Acloque et al., 2009; Kalluri and Weinberg, 2009), it is still unclear what specific signals induce type 3 EMT in epithelial carcinoma cells. So we may use the embryo developmental model to detect the specific signals in 3 EMT in the future to some extent. Sorafenib’s use was approved because of its able to inhibit the EMT process through an epigenetic mechanism (Chen et al., 2011; Nagai et al., 2011; Zhang et al., 2013). We found that sorafenib treatment retarded the development of the gastrulating chick embryo, so it was reasonable to speculate that sorafenib affected EMT in the embryo. Our speculation was confirmed by sorafenib enhancing E-Cadherin expression while inhibiting NCadherin expression in the gastrulating embryos. Normally, these cell adhesion molecules are inversely expressed to permit proper EMT. Furthermore, Slug (transcriptional factor for EMT) and laminin expression
were also repressed by sorafenib. The abbreviated expression of EMT-related genes might account for the observed reduction in mesoderm cell numbers and retardation of embryo growth. Cell migration is another cellular process that occurs both in tumor metastasis and embryo gastrulation. Consequently, we investigated the effects of sorafenib on mesoderm cell migration during EMT. We transplanted a piece of GFP expressing primitive streak into corresponding site in unlabeled embryos to track cell migration during EMT in the absence or presence of sorafenib as described previously (Yang et al., 2002; Yang et al., 2008; Yue et al., 2008). We found sorafenib repressed mesoderm cell migration. In support, we also found sorafenib affected neural crest cell generation and migration. Our findings revealed the effect of sorafenib in inhibiting the generation of the neural crest cells but sorafenib did not affect the migration of cells itself, which demonstrate the decrease of HNK1 positive cells in sorafenibtreated group is due to the inhibition of the proliferation and the generation of neural crest cells not the migration action itself. These findings imply that sorafenib’s anti-cancer effect may be attributed to its ability to inhibit cancer cell invasion. It is noticeable that sorafenib is a non-selective multiple kinase inhibitor with proven anti-proliferative effects in thyroid, renal and hepatocellular carcinoma. Sorafenib acts on vascular endothelial growth factor (VEGF) and on platelet-derived growth factor (PDGF) related pathways. It also influences the rat sarcoma proto-oncogene/rat fibrosarcoma protein kinase/mitogen activated protein kinase (RAS/RAF/MAPK) pathway and blocks tumor growth factor beta-1 (TGF-b-1)-mediated epithelial-mesenchymal transition (EMT) (Gedaly et al., 2010; Chen et al., 2011; Smolle et al., 2014). Our findings show sorafenib inhibited multiple key enzymes in the MAPK signaling pathway in the chick embryo development like in the tumor. This result may be evidence that we can use the embryo development as the cancer progression, and metastasis in vivo model in the future. In sum, we first demonstrated that sorafenib retarded the development of the gastrulating chick embryos. This growth retardation was caused by a reduction in embryonic cell proliferation, an increase in apoptosis and disruption in EMT and mesoderm cell migration. The close similarity in cellular behavior between the gastrulating chick embryo and growing tumor makes the embryo a good model for testing out new anti-cancer drugs.
ACKNOWLEDGEMENTS The authors thank Wing Yan Wong for constructive help on the manuscript and Professor Yu Yanhong for kindly providing ERK antibody.
LITERATURE CITED Acloque H, Adams MS, Fishwick K, Bronner-Fraser M, Nieto MA. 2009. Epithelial-mesenchymal transitions: the importance of changing cell state in development and disease. J Clin Investig 119:1438–1449. Bai C, Hou L, Zhang M, Pu Y, Guan W, Ma Y. 2012. Characterization of vascular endothelial progenitor cells from chicken bone marrow. BMC Vet Res 8:54.
EFFECTS OF SORAFENIB ON CHICK EMBRYO DEVELOPMENT Bataille F, Rohrmeier C, Bates R, Weber A, Rieder F, Brenmoehl J, Strauch U, Farkas S, Furst A, Hofstadter F, Scholmerich J, Herfarth H, Rogler G. 2008. Evidence for a role of epithelial mesenchymal transition during pathogenesis of fistulae in Crohn’s disease. Inflamm Bowel Dis 14:1514–1527. Brabletz T, Jung A, Reu S, Porzner M, Hlubek F, Kunz-Schughart LA, Knuechel R, Kirchner T. 2001. Variable beta-catenin expression in colorectal cancers indicates tumor progression driven by the tumor environment. Proc Natl Acad Sci USA 98:10356– 10361. Bronner-Fraser M. 1994. Neural crest cell formation and migration in the developing embryo. FASEB J 8:699–706. Chapman SC, Collignon J, Schoenwolf GC, Lumsden A. 2001. Improved method for chick whole-embryo culture using a filter paper carrier. Dev Dyn 220:284–289. Chen YL, Lv J, Ye XL, Sun MY, Xu Q, Liu CH, Min LH, Li HP, Liu P, Ding X. 2011. Sorafenib inhibits transforming growth factor beta1-mediated epithelial-mesenchymal transition and apoptosis in mouse hepatocytes. Hepatology 53:1708–1718. Di Marco V, De Vita F, Koskinas J, Semela D, Toniutto P, Verslype C. 2013. Sorafenib: from literature to clinical practice. Ann Oncol 24 (Suppl 2):ii30–ii37. Escudier B, Eisen T, Stadler WM, Szczylik C, Oudard S, Siebels M, Negrier S, Chevreau C, Solska E, Desai AA, Rolland F, Demkow T, Hutson TE, Gore M, Freeman S, Schwartz B, Shan M, Simantov R, Bukowski RM. 2007. Sorafenib in advanced clear-cell renal-cell carcinoma. N Engl J Med 356:125–134. Fei F, Stoddart S, Muschen M, Kim YM, Groffen J, Heisterkamp N. 2010. Development of resistance to dasatinib in bcr/Abl-positive acute lymphoblastic leukemia. Leukemia 24:813–820. Gedaly R, Angulo P, Hundley J, Daily MF, Chen C, Koch A, Evers BM. 2010. PI-103 and sorafenib inhibit hepatocellular carcinoma cell proliferation by blocking ras/raf/MAPK and PI3K/AKT/mTOR pathways. Anticancer Res 30:4951–4958. Hamburger V, Hamilton HL. 1992. A series of normal stages in the development of the chick embryo. 1951. Dev Dyn 195:231–272. Kalluri R, Weinberg RA. 2009. The basics of epithelialmesenchymal transition. J Clin Investig 119:1420–1428. Larger E, Marre M, Corvol P, Gasc JM. 2004. Hyperglycemiainduced defects in angiogenesis in the chicken chorioallantoic membrane model. Diabetes 53:752–761. Li X, Guo H, Yang Y, Meng J, Liu J, Wang C, Xu H. 2014. A designed peptide targeting cxcr4 displays anti-acute myelocytic leukemia activity in vitro and in vivo. Sci Rep 4:6610. Li Y, Wang XY, Wu T, Chuai M, Lee KK, Wang LJ, Yang X. 2013. PTEN is involved in modulation of vasculogenesis in early chick embryos. Biol Open 2:587–595. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, de Oliveira AC, Santoro A, Raoul JL, Forner A, Schwartz M, Porta C, Zeuzem S, Bolondi L, Greten TF, Galle PR, Seitz JF, Borbath I,
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Haussinger D, Giannaris T, Shan M, Moscovici M, Voliotis D, Bruix J. 2008. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 359:378–390. Mintz B, Cronmiller C. 1978. Normal blood cells of anemic genotype in teratocarcinoma-derived mosaic mice. Proc Natl Acad Sci USA 75:6247–6251. Nagai T, Arao T, Furuta K, Sakai K, Kudo K, Kaneda H, Tamura D, Aomatsu K, Kimura H, Fujita Y, Matsumoto K, Saijo N, Kudo M, Nishio K. 2011. Sorafenib inhibits the hepatocyte growth factor-mediated epithelial mesenchymal transition in hepatocellular carcinoma. Mol Cancer Therap 10:169–177. Pierce GB. 1983. The cancer cell and its control by the embryo. Rous-whipple award lecture. Am J Pathol 113:117–124. Rashidi H, Sottile V. 2009. The chick embryo: hatching a model for contemporary biomedical research. Bioessays 31:459–465. Rastaldi MP, Ferrario F, Giardino L, Dell’Antonio G, Grillo C, Grillo P, Strutz F, Muller GA, Colasanti G, D’Amico G. 2002. Epithelialmesenchymal transition of tubular epithelial cells in human renal biopsies. Kid Int 62:137–146. Reckamp KL, Gardner BK, Figlin RA, Elashoff D, Krysan K, Dohadwala M, Mao J, Sharma S, Inge L, Rajasekaran A, Dubinett SM. 2008. Tumor response to combination celecoxib and erlotinib therapy in nonsmall cell lung cancer is associated with a low baseline matrix metalloproteinase-9 and a decline in serum-soluble E-cadherin. J Thorac Oncol Off Publ Int Assoc Study Lung Cancer 3:117–124. Savagner P. 2010. The epithelial-mesenchymal transition (EMT) phenomenon. Ann Oncol 21 (Suppl 7):vii89–vii92. Smolle E, Taucher V, Petru E, Haybaeck J. 2014. Targeted treatment of ovarian cancer—the multiple—kinase—inhibitor sorafenib as a potential option. Anticancer Res 34:1519–1530. Trimboli AJ, Fukino K, de Bruin A, Wei G, Shen L, Tanner SM, Creasap N, Rosol TJ, Robinson ML, Eng C, Ostrowski MC, Leone G. 2008. Direct evidence for epithelial-mesenchymal transitions in breast cancer. Cancer Res 68:937–945. Yang X, Chrisman H, Weijer CJ. 2008. PDGF signalling controls the migration of mesoderm cells during chick gastrulation by regulating N-cadherin expression. Development (Cambridge, England) 135:3521–3530. Yang X, Dormann D, Munsterberg AE, Weijer CJ. 2002. Cell movement patterns during gastrulation in the chick are controlled by positive and negative chemotaxis mediated by fgf4 and fgf8. Dev Cell 3:425–437. Yue Q, Wagstaff L, Yang X, Weijer C, Munsterberg A. 2008. Wnt3amediated chemorepulsion controls movement patterns of cardiac progenitors and requires RhoA function. Development (Cambridge, England) 135:1029–1037. Zhang J, Chen YL, Ji G, Fang W, Gao Z, Liu Y, Wang J, Ding X, Gao F. 2013. Sorafenib inhibits epithelial-mesenchymal transition through an epigenetic-based mechanism in human lung epithelial cells. PloS One 8:e64954.
