Combretastatin A-4 efficiently inhibits angiogenesis and induces neuronal apoptosis in zebrafish

Cis-stilbene combretastatin A-4 (CA-4) and a large group of its derivant compounds have been shown significant anti-angiogenesis activity. However the side effects even the toxicities of these chemicals were not evaluated adequately. The zebrafish model has become an important vertebrate model for evaluating drug effects. The testing of CA-4 on zebrafish is so far lacking and assessment of CA-4 on this model will provide with new insights of understanding the function of CA-4 on angiogenesis, the toxicities and side effects of CA-4. We discovered that 7–9 ng/ml CA-4 treatments resulted in developmental retardation and morphological malformation, and led to potent angiogenic defects in zebrafish embryos. Next, we demonstrated that intraperitoneal injection of 5, 10 and 20 mg/kg CA-4 obviously inhibited vessel plexus formation in regenerated pectoral fins of adult zebrafish. Interestingly, we proved that CA-4 treatment induced significant cell apoptosis in central nervous system of zebrafish embryos and adults. Furthermore, it was demonstrated that the neuronal apoptosis induced by CA-4 treatment was alleviated in p53 mutants. In addition, notch1a was up-regulated in CA-4 treated embryos, and inhibition of Notch signaling by DAPT partially rescued the apoptosis in zebrafish central nervous system caused by CA-4.

So far, the mechanism of anti-vascular of CA-4, with similar molecular skeleton of colchicine ( Figure S1), is well documented and concluded as disrupting mitotic spindle by binding to colchicine-binding site in the tubulin dimer 13 . Though great many of VDAs following the prototype CA-4 have been obtained [14][15][16] , the side effects even the toxicities of these compounds were not evaluated adequately.
The zebrafish model has been becoming an important vertebrate model for assessing drug effects 17 . This model fits into multiple stages of the drug discovery pipeline, from target identification to lead optimization of absorption, distribution, metabolism and excretion, and toxicity studies 18 . The testing of CA-4 on zebrafish model is so far lacking and assessing CA-4 on this model will provide with new insights of understanding the function of CA-4 on angiogenesis, the toxicities and side effects of CA-4.

Results
The effects of CA-4 treatment on zebrafish embryos. To investigate the suitable dosing time window, we treated zebrafish embryos with 10 ng/ml CA-4 solution at gastrula, segmentation and pharyngula period (Fig. 1A). At 6 hours post-fertilization (hpf) (early gastrula period), 10 ng/ml CA-4 treatment resulted in all embryos dead within 48 hpf. Then we dosed at later periods (from middle gastrula period 8 hpf to pharyngula period 30 hpf). As shown in Fig. 1A, dosing 10 ng/ml CA-4 at different time caused developmental retardation and malformation in different extent. Subsequently, we investigated the dosage of CA-4 between 1 ng/ml and 100 ng/ml, at which embryos were treated in triplicate from 8 hpf (Fig. 1B,C). CA-4 treatment at concentration higher than 40 ng/ml from 8 hpf to 36 hpf caused embryos dead or malformed (Fig. 1B). CA-4 treatment at concentration higher than 20 ng/ml from 8 hpf to 60 hpf resulted in all embryos dead (Fig. 1C). Part of the embryos with CA-4 treatment at concentration lower than 6 ng/ml appeared normal (Fig. 1C). Due to that the CA-4 treatment at the concentration higher than 10 ng/ml caused very severe phenotype, we selected 7, 8 and 9 ng/ml as the working dosages in the following research. The morphology of CA-4 treated embryos was shown in Figure S2.

CA-4 treatment blocked zebrafish embryonic angiogenesis.
To verify whether CA-4 blocks embryonic angiogenesis in zebrafish, we investigated the effects of CA-4 using transgenic line Tg(kdrl:EGFP::huc:mcherry), in which endothelial cells (ECs) are labeled in EGFP. It was shown that CA-4 treatment at 7, 8, and 9 ng/ml significantly inhibited ISV branching angiogenesis ( Fig. 2A-D,A′-D″,M-P). ISV length in the CA-4 treated group was obviously shorter than that of control group (Fig. 2U) and most of the ISV failed to form dorsal longitudinal anastomotic vessel (DLAV) until 55hpf ( Fig. 2A-D,A′-D″,M-P). The CA-4 treatment caused some ISVs absent especially in 9 ng/ml group (Fig. 2V). Additionally, we found that CA-4 treatment resulted in the brain vessels dorsal longitudinal vein (DLV), dorsal midline junction (DMJ), middle cerebral vein (MCeV), mesencephalic vein (MsV), central artery (CtA), basilar artery (BA), posterior communicating segment (PCS), primordial hindbrain channel (PHBC), and primary head sinus (PHS) partly absent ( Fig. 3A-D,A′-D′,M-P). The absence ratio was presented as means of ratio of the ten marked brain vessels in CA-4 treated embryos (Fig. 3A,Q). In order to study proliferation and migration of endothelial cells (ECs) in CA-4 treated embryos, the transgenic zebrafish line Tg(fli1a:nEGFP) was employed ( Fig. 2Q-T). In control embryos, there were 3-4 ECs in each ISV (Fig. 2Q), however, in the CA-4 treated embryonic zebrafish there were only 1-2 ECs in each ISV, suggesting that proliferation and migration of ECs were significantly inhibited ( Fig. 2R-T,X). In addition, we did not observe any significant change of lumen size in DAs or PCVs of CA-4 treated embryos ( Figure S3A,B). To examine the arterial-venous differentiation of early ECs in CA-4 treated embryos, we performed whole-mount in situ hybridization analysis by using flt4 and dll4 antisense probes. The expression of flt4 in PCV and dll4 in DA were not apparently changed in CA-4 treated embryos ( Figure S3C).

CA-4 treatment inhibited angiogenesis during fin regeneration in adult zebrafish. Furthermore,
we investigated whether the CA-4 treatment inhibits angiogenesis in adult zebrafish. We measured the length, width and area of regenerated vessel plexus in pectoral fins at 3 days post amputation (dpa) (Fig. 4A,B) and 9 dpa (Fig. 4A,C). It was demonstrated that injection of 5, 10 and 20 mg/kg CA-4 significantly reduced the size of regenerated vessel plexus ( Fig. 4B-F). We counted the number of branching points in regenerated vessels and defined the Number of branching points divided by Area of regenerated vessel plexus as regeneration score. It was shown that the number of branching points and the regeneration score of regenerated vessel plexus were significantly reduced in 5-20 mg/kg CA-4 injected zebrafish (Fig. 4G,H). Additionally, the regenerated vessel plexus were diagramed using Imaris software ( Next we investigated the effect of CA-4 treatment on motor neurons using transgenic line Tg(hb9:EGFP), in which motor neurons are labeled with green florescence. It was shown that CA-4 treatment at 7-9 ng/ml significantly reduced the ventral and dorsal axon length and branch points of motor neurons ( Fig. 5A-D,A′-D″,I,J), and CA-4 treatment at 8 and 9 ng/ml resulted in no apparent dorsal axon sprouting (Fig. 5C,D). The axons of motor neurons were diagrammed with Imaris software (Fig. 5E-H). Additionally, we found that CA-4 treatment also induced the absence of motor neurons (Fig. 5K). Through two-tailed Pearson correlation analysis, we found there were close correlations of axon length and ISV length, as well as absence ratio of motor neuron and absence ratio of ISV with Pearson correlation coefficient 0.895 and 0.903, respectively ( Figure S4C,D).

CA-4 treatment induced zebrafish neuronal apoptosis.
We found that CA-4 treatment caused spinal cord and brain region of zebrafish embryos become opaque ( Figure S2), suggesting CA-4 treatment induces zebrafish neuronal apoptosis. To verify this hypothesis we did the TUNEL staining analysis of central neural system (CNS) in zebrafish embryos and adults treated with CA-4. It was shown that CA-4 treatment at 7-9 ng/ml induced obvious cell apoptosis at concentration dependent manner (Fig. 6A,B). Moreover, we found that the significant cell apoptosis in brain of adult zebrafish with 5-20 mg/kg CA-4 I.P. injection (Fig. 6C,D). To confirm CA-4 treatment induces zebrafish neuronal apoptosis, we treated the p53 mutated zebrafish embryos with different concentration of CA-4. It was revealed that CA-4 treatment at concentrations higher than 40 ng/ml led to embryos dead and the p53 mutants treated with CA-4 at concentrations lower than 9 ng/ml showed no obvious developmental defects, whereas CA-4 treatment at 7-9 ng/ml resulted in embryos developmental retardation or malformation (Fig. 6E,F). In addition, we treated the p53 morpholino injected Tg(huc:EGFP) embryos with CA4 and found that p53 knockdown significantly reduced the apoptosis of EGFP positive cells (Fig. 7D). These results support that CA-4 treatment induces zebrafish neuronal apoptosis.
To investing the potential mechanism responsible for apoptosis, we examined the Notch and Wnt signaling in CA-4 treated zebrafish. A Wnt reporter Tg(7xTCF-Xla.Siam:GFP) ia4 , which is reliable and sensitive Wnt biosensors for in vivo studies 19,20 , was used to evaluate the involvement of Wnt signaling in CA-4 induced neuronal apoptosis. We did not observe the obvious alteration of EGFP expression in central nervous system (CNS) of CA-treated embyos compared with that of control ( Figure S5), indicating Wnt signaling is not responsible for the neuronal apoptosis. Then we examined the expression of Notch ligands and receptors using whole mount in situ hybridization analysis and found that notch1a was significantly up-regulated in CA-treated embyos (Figs 7A and S6A-F). This result was confirmed by real-time PCR and RT-PCR (Fig. 7B,C). It was reported that Notch activation induces apoptosis in neural progenitor cells through a p53-dependent pathway in mice 21 , which suggests that the elevation of notch1a expression linked to the neuronal apoptosis caused by CA-4 treatment. Inhibition of Notch signaling by DAPT partially rescued the apoptosis in zebrafish CNS caused by CA-4 (Fig. 7D), indicating that Notch signaling is involved in the neuronal apoptosis induced by CA-4. Additionally, we also examined the muscle structure in CA-4 treated embryos by Phalloidin staining, and did not detected apparent defects ( Figure S7).

Discussion
Humans are confronted with great stresses from environmental pollution, food safety, lifestyle shift and so on, resulting in a rising incidence of cancer diseases worldwide 22,23 . Based on the World Cancer Report 2014 of WHO, the worldwide burden of cancer is expected to rise to 22 million annually within the next two decades, and cancer deaths are predicted to rise from an estimated 8.2 million annually to 13 million per year 16 . The exploration of pathology mechanism of cancers and the development of therapeutic drugs are hot research topics in the field of medicine. Vigorous vasculature in tumor was commonly targeted in anti-tumor treatment. VDAs are the most important anti-tumor therapeutic agents. Among these agents, a large number of VDAs were designed and obtained based on the prototype compound CA-4. Thus, evaluating the efficacy and safety of CA-4 is necessary for the clinical application. In this study we firstly confirmed the anti-vascular effects of CA-4 using zebrafish model. Importantly, we found CA-4 displayed significant neuronal toxicity through inducing cell apoptosis in CNS.
Through investigation of dosing time window, we found CA-4 treatment from earlier stage caused zebrafish embryos more severe phenotype at the same dosage. And dosing from the same developmental stage, the embryos treated with CA-4 for longer duration displayed more severe phenotype. Dosing from 8 hpf and phenotyping at 36 hpf and 60 hpf, we demonstrated that CA-4 treatment at 4-10 ng/ml resulted in abnormal phenotype without death in wild type embryonic zebrafish. The previous studies reported 1 μ mol/l CA4P (equivalent to 316 ng/ml CA-4) for 15 min obviously inhibited human lens epithelial cells 24 , and IC 50 of 1.9-835 nmol/l CA-4 (equivalent to 0.6-263.84 ng/ml) showed against various human cancer cell lines and a MDR-resistant cancer cell line 6 . Compared with these reported dosages, our results showed the effective dosing range was relatively narrow, which might be attributed to the species diversity. In view of moderate efficacy, we selected 7-9 ng/ml CA-4 dosing at 8 hpf in the following work. 7-9 ng/ml CA-4 treatment induced ISVs and brain vessels inhibition or even absence in zebrafish embryos. Furthermore, we found CA-4 treatment inhibited ISV branching angiogenesis through retarding proliferation and migration of zebrafish ECs in dose-dependent manner. CA-4 and CA-4P were also reported to inhibit the proliferation and migration of epithelial cells in vitro 6,24 . Here we showed the potent anti-vascular effect of CA-4 via inhibiting proliferation and migration of developmental ECs in situ.
To investigate the anti-vascular effect of CA-4 in zebrafish adults, we conducted intraperitoneal injection of CA-4 into Tg(fli1a:EGFP) adult zebrafish. Thomas Nielsen et al. reported that 250 mg/kg CA-4P administered intraperitoneally significantly reduced tumor vessel volume and size distribution in mice 25 . It was also reported that rats dosed 30 and 100 mg/kg CA-4P showed tumor vascular shutdown 26 . 30 mg/kg CA-4P that Munich-Wistar rats received through intraperitoneal injection was considered to be a clinically relevant dose [27][28][29][30] . Based on these studies, we selected 5, 10 and 20 mg/kg as the treating dosages in the adult zebrafish experiments. Notably, intraperitoneal injection of 5 mg/kg CA-4, the lowest dosage we selected, resulted in potent anti-vascular effect in regenerated adult zebrafish pectoral fin. In conclusion, we firstly confirmed CA-4 possessed anti-vascular activity both in embryonic and adult zebrafish.
However, in the present study we reported firstly CA-4 exhibited potent negative effects in CNS of both embryonic and adult zebrafish. CA-4 treatment impairs zebrafish neural development and induces zebrafish neuronal apoptosis. CA-4 inducing apoptosis has been reported previously in several models in vitro 15,24,31 . The present data supports that CA-4 induces cell apoptosis in vivo. In addition, CA-4 has been reported to induce side effects such as anemia, dyspnoea, hypokalemia, headache, transient sensory neuropathy and renal impact 8,27,32 . In zebrafish embryos, CA-4 affected body formation, exerted potent anti-vascular effect, and resulted in abnormality in nervous system. These findings suggest that adequate evaluation on side effects and toxicity of CA-4 and its analogues is required in the future.

Materials and Methods
Ethics statement. All animal experimentation was carried out in accordance with the NIH Guidelines for the care and use of laboratory animals (http://oacu.od.nih.gov/regs/index.htm) and ethically approved by the Administration Committee of Experimental Animals, Jiangsu Province, China (Approval ID: SYXK(SU) 2007-0021).
Zebrafish, drug treatment and morpholino injection. The study was conducted conforming to the local institutional laws, and the Chinese law for the Protection of Animals. The embryos were obtained through natural mating. Zebrafish embryos and adults were raised and maintained on standard conditions in Zebrafish Center of Nantong University as we previously described 33,34 . The transgenic zebrafish lines Tg(kdrl:EGFP), Tg(kdrl:EGFP::huc:mCherry), Tg(fli1a:nEGFP), Tg(hb9:EGFP) and p53 mutants were used as described in previous work [34][35][36] . Developmental stages of embryonic zebrafish referred to the previously described by Kimmel et al. 37 . At 6 hpf, embryos were screened under anatomical microscope to remove the morphologically abnormal individuals. Around 10 healthy embryos were loaded into each well of 96-well plate in E3 solution. At the setting time, E3 solutions were replaced with CA-4 treatment solutions. The control and treated groups were analyzed at different intervals. At 55 hpf, zebrafish embryos were collected for imaging and fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for TUNEL staining.
Adult Tg(fli1a:EGFP) zebrafish line at 6 months post fertilization were randomly divided into control group (n = 10, intraperitoneal injection normal saline), 5, 10 and 20 mg/kg groups (n = 10, intraperitoneal injection CA-4 solution). Zebrafish was anesthetized with 0.16-mg/ml tricaine and one of the pectoral fins was amputated at the first injection. Confocal imaging analysis of pectoral fins was carried out at 3 and 9 days post amputation (dpa) respectively. After intraperitoneal injection daily for 10 days, all the zebrafish were sacrificed and the brain tissues were collected for TUNEL staining. The DAPT treatment and morpholino injection was carried out as previously described 38,39 . Cryostat section and TUNEL staining. Zebrafish embryos or tissues were fixed with 4% PFA overnight at 4 °C. Then samples were washed for 3 × 5 min with PBS and immersed with the melted agarose-sucrose (1.5% agarose, 5% sucrose). The solidified agarose block was trimmed and incubated in 30% sucrose overnight at 4 °C. After being embedded with OCT compound (Tissue Tek), the block was fixed on the platform and equilibrated in the chamber of cryostats at least 30 min, and then sectioned at 10 μ m according to the manufacture's instruction. We mounted the sections on slides and dried the slides in the air for 4-5 hours. The sections were re-fixed on the slides for 20-30 min with 4% PFA, washed with PBS 3 × 5 min and incubated in permeabilisation solution (0.1% Triton X-100, 0.1% sodium citrate) for 2 min on ice. The TUNEL reaction solution was prepared according to the manufacture's instruction (Roche). The sections were labeled with TUNEL reaction solution strictly following the manufacture's protocol (Roche).
RNA extraction, reverse transcription, and PCR. Tissue was homogenized and frozen in Trizol reagent (Invitrogen) and stored at − 80 °C. Total RNA was extracted following the manufacturer's instructions. 1 μ g of RNA was reverse transcribed into cDNA using Transcriptor First Strand cDNA Synthesis Kit (Roche) according to the manufacturer's instructions. Synthesized cDNA was stored at − 20 °C. The Left primer for RT-PCR is 5′ -ATGACATCACCCTTCCAGCA-3′ ; Right primer is 5′ -GGTGATTGGGTGTGTTGTCC-3′ . PCR amplifications were carried out in a total volume of 50 μ l using specific primers and Advantage2 Polymerase Kit (Clontech). The Left primer for Real-time PCR is 5′ -ATTGATGAGTGTGTGAGCGC-3′ ; Right primer is 5′ -CAGTTGATGCCACTGAAGCC-3′ . The Real-time PCR was carried out as previously described 38 .
Riboprobe synthesis and whole-mount in situ hybridization. The coding sequence for zebrafish Notch signaling genes were amplified by PCR using the primers as previously described 40 . Digoxigenin (DIG)-labeled RNA sense and antisense probes were made from the linearized plasmids according to the manufacturer's protocol using the DIG RNA Labeling Kit (SP6/T7) (Roche). Whole-mount in situ hybridization was carried out as we previously described 35,41 . Imaging. At 55 hpf, for confocal imaging embryos were anesthetized with E3/0.16 mg/mL tricaine/1% 1-phenyl-2-thiourea (Sigma) and embedded in 0.8% low melt agarose. Images were acquired with an Olympus DP71 camera on an Olympus stereomicroscope. Confocal imaging was performed with a Leica TCS-SP5 LSM. Analysis was performed using Imaris software. The final figure processing was performed with Adobe Photoshop and Illustrator CS6.
Statistics. ISV length, size of regenerated pectoral fin vascular plexus, mCherry positive cell number, motor neuron axon length, and branch points were measured with Imaris (version 7.2.3). These data were statistically analyzed with GraphPad Prism 5. All data were expressed as mean ± S.E.M. Statistical analysis were performed using a one-way analysis of variance (ANOVA) (P < 0.05). The correlations between the changes of quantifications of blood vessels and those of nervous systems were analyzed by SPSS software (version 13.0).