Psoralen and Isopsoralen, Two Estrogen-Like Natural Products from Psoraleae Fructus, Induced Cholestasis via Activation of ERK1/2.

With the increasing use of oral contraceptives and estrogen replacement therapy, the incidence of estrogen-induced cholestasis (EC) has tended to rise. Psoralen (P) and isopsoralen (IP) are the major bioactive components in Psoraleae Fructus, and their estrogen-like activities have already been recognized. Recent studies have also reported that ERK1/2 plays a critical role in EC in mice. This study aimed to investigate whether P and IP induce EC and reveal specific mechanisms. It was found that P and IP increased the expression of esr1, cyp19a1b and the levels of E2 and VTG at 80 µM in zebrafish larvae. Exemestane (Exe), an aromatase antagonist, blocked estrogen-like activities of P and IP. At the same time, P and IP induced cholestatic hepatotoxicity in zebrafish larvae with increasing liver fluorescence areas and bile flow inhibition rates. Further mechanistic analysis revealed that P and IP significantly decreased the expression of bile acids (BAs) synthesis genes cyp7a1 and cyp8b1, BAs transport genes abcb11b and slc10a1, and BAs receptor genes nr1h4 and nr0b2a. In addition, P and IP caused EC by increasing the level of phosphorylation of ERK1/2. The ERK1/2 antagonists GDC0994 and Exe both showed significant rescue effects in terms of cholestatic liver injury. In conclusion, we comprehensively studied the specific mechanisms of P- and IP-induced EC and speculated that ERK1/2 may represent an important therapeutic target for EC induced by phytoestrogens.

Anti-inflammatory effects of 6S-5-methyltetrahydrofolate-calcium on RAW264.7 cells and zebrafish.

AIM: 6S-5-methyltetrahydrofolate is the predominant form of dietary folate in circulation and is used as a crystalline form of calcium salt (MTHF-Ca). Reports revealed that MTHF-Ca was more safe than folic acid, a synthetic and highly stable version of folate. Folic acid has been reported to have anti-inflammatory effects. The study's objective was to assess the anti-inflammatory effect of MTHF-Ca in vitro and in vivo. MAIN METHODS: In vitro, the ROS production was assessed by H2DCFDA, and nuclear translocation of NF-κB were evaluated by the NF-κB nuclear translocation assay kit. Interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and tumor necrosis factor-alpha (TNF-α) were assessed using ELISA. In vivo, ROS production was assessed by H2DCFDA, neutrophils and macrophages recruitment were evaluated in tail transection-induced and CuSO4-induced zebrafish inflammation models. Expression of inflammation related genes were also investigated based on CuSO4-induced zebrafish inflammation model. KEY FINDINGS: MTHF-Ca treatment decreased LPS-induced ROS production, inhibited nuclear translocation of NF-κB and decreased the levels of IL-6, IL-1ß and TNF-α in RAW264.7 cells. In addition, MTHF-Ca treatment inhibited ROS production, suppressed the recruitment of neutrophils and macrophages, and reduced the expression of inflammation related genes, including jnk, erk, nf-κb, myd88, p65, tnf-α, and il-1b in zebrafish larvae. SIGNIFICANCE: MTHF-Ca may play an anti-inflammatory role by reducing the recruitment of neutrophils and macrophages and keeping the low levels of proinflammatory mediators and cytokines. MTHF-Ca may have a potential role in the treatment of inflammatory diseases.

Efficacy and mechanism study of cordycepin against brain metastases of small cell lung cancer based on zebrafish.

BACKGROUND: Small cell lung cancer (SCLC) is an aggressive tumor with high brain metastasis (BM) potential. There has been no significant progress in the treatment of SCLC for more than 30 years. Cordycepin has shown the therapeutic potential for cancer by modulating multiple cellular signaling pathways. However, the effect and mechanism of cordycepin on anti-SCLC BM remain unknown. PURPOSE: In this study, we focused on the anti-SCLC BM effect of cordycepin in the zebrafish model and its potential mechanism. STUDY DESIGN AND METHODS: A SCLC xenograft model based on zebrafish embryos and in vitro cell migration assay were established. Cordycepin was administrated by soaking and microinjection in the zebrafish model. RNA-seq assay was performed to analyze transcriptomes of different groups. Geno Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were performed to reveal the underlying mechanism. Real-time qPCR was used to verify the effects of cordycepin on the key genes. RESULTS: Cordycepin showed lower cytotoxicity in vitro compared with cisplatin, anlotinib and etoposide, but showed comparable anti-proliferation and anti-BM effects in zebrafish SCLC xenograft model. Cordycepin showed significant anti-SCLC BM effects when administrated by both soaking and microinjection. RNA-seq demonstrated that cordycepin was involved in vitamin D metabolism, lipid transport, and proteolysis in cellular protein catabolic process pathways in SCLC BM microenvironment in zebrafish, and was involved in regulating the expressions of key genes such as cyp24a1, apoa1a, ctsl. The anti-BM effect of cordycepin in SCLC was mediated by reversing the expression of these genes. CONCLUSION: Our work is the first to describe the mechanism of cordycepin against SCLC BM from the perspective of regulating the brain microenvironment, providing new evidence for the anti-tumor effect of cordycepin.

Docetaxel and 5-FU enhanced the inhibitory effects of apatinib and ramucirumab on growth and migration of gastric cancer.

BACKGROUND: Gastric cancer (GC) is one of the most common malignant tumors in the world. The clinical benefit of anti-angiogenic strategy as a single drug is limited. Some studies showed that the combination of anti-angiogenic therapy and chemotherapy exhibited synergistic effect and reduced the side effects of chemotherapy drugs. We investigated the combined effects of these two types of drugs in gastric cancer cells in vitro and in vivo. METHODS: cell viability, migration, invasion, and apoptosis were evaluated by CCK-8, wound-healing, transwell, and Annexin V-FITC/PI assay, respectively. In vivo anti-cancer efficacy was tested for the cell proliferation and metastasis in cell line derived tumor xenograft (CDX) model and patient derived tumor xenografted (PDX) model based on Tg (fli-1: EGFP) zebrafish embryos; RESULTS: In the cell experiments, the combination of the two types of drugs could inhibit the proliferation and metastasis of gastric cancer cells and promote apoptosis through VEGFR-2/AKT/ERK1/2 signal. In the zebrafish CDX (zCDX) model and zebrafish PDX (zPDX) model, the combination of the two treatment also showed a synergistic effect in inhibiting gastric cancer cell metastasis and cell proliferation. CONCLUSIONS: Apatinib/ramucirumab targeted therapy combined with docetaxel or 5-fluorouracil (5-FU) may serve as an effective treatment strategy for patients with advanced gastric cancer.

Zebrafish xenograft model for studying mechanism and treatment of non-small cell lung cancer brain metastasis.

BACKGROUND: Brain metastasis (BM) is thought to be related to the mortality and poor prognosis of non-small cell lung cancer (NSCLC). Despite promising development of NSCLC treatment, the treatment of NSCLC BM is still not optimistic due to the existence of the blood-brain barrier (BBB) that prevent drug penetration, as well as the short median survival time of the patients left for treatment. In this context, further development of quick and effective pre-clinical models is needed in NSCLC BM treatment. Here, we report a model system using zebrafish to promote the development of drugs for patients with NSCLC BM. METHODS: Three different NSCLC cell lines (H1975, A549 and H1299) were used to establish zebrafish BM models. The embryo age and cell number for injection were first optimized. Metastatic cells were observed in the brain blood vessels of zebrafish and were verified by hematoxylin-eosin (HE) staining. Then, the metastasis potentials of H1975 and A549 with manipulated microRNA-330-3p (miR-330-3p) expression were also investigated. Finally, sensitivities of H1975 and A549 to osimertinib and gefitinib were tested. RESULTS: This zebrafish BM model could distinguish NSCLC cell lines with different BM potential. Over-expressed miR-330-p significantly improved the BM potential of the A549 cells while knockdown miR-330-p reduced the BM ability of the H1975 cells. Both osimertinib and gefitinib showed inhibition effect in zebrafish BM model with the inhibition rate higher than 50 %. H1975 cell showed much higher sensitivity to osimertinib rather than gefitinib both in vivo and in vitro. CONCLUSIONS: We established zebrafish brain metastasis model for studying mechanism and treatment of NSCLC BM. This study provided a useful model for NSCLC brain metastasis that could be used to study the mechanism that drive NSCLC cells to the brain as well as identify potential therapeutic options.

Mechanism of hepatotoxicity of first-line tyrosine kinase inhibitors: Gefitinib and afatinib.

AIMS: Both gefitinib and afatinib are epidermal growth factor tyrosine kinase inhibitors (EGFR-TKI) in the treatment of non-small cell lung cancer (NSCLC). It has been reported that gefitinib and afatinib could cause hepatotoxicity during the clinic treatment, therefore it is critical to investigate their hepatotoxicity systematically. In this study, zebrafish (Danio rerio) were used as model animals to compare the hepatotoxicity and their toxic mechanism. MAIN METHODS: The zebrafish transgenic line [Tg (fabp10a: dsRed; ela3l:EGFP) was used in this study. After larvae developed at 3 days post fertilization (dpf), they were put into different concentrations of gefitinib and afatinib. At 6 dpf, the viability, liver area, fluorescence intensity, histopathology, apoptosis, transaminase reflecting liver function, the absorption of yolk sac, and the expression of relative genes were observed and analyzed respectively. KEY FINDINGS: Both gefitinib and afatinib could induce the larvae hepatotoxicity dose-dependently. Based on the liver morphology, histopathology, apoptosis and function assessments, gefitinib showed higher toxicity, causing more serious liver damage. Both gefitinib and afatinib caused abnormal expressions of genes related to endoplasmic reticulum stress (ERS) pathway and apoptosis. For example, jnk, perk, bip, chop, ire1, bid, caspase3 and caspase9 were up-regulated, while xbp1s, grp78, bcl-2/bax, and caspase8 were down-regulated. The hepatotoxicity difference of gefitinib and afatinib might be due to the different expression level of related genes.

Isoliquiritin promote angiogenesis by recruiting macrophages to improve the healing of zebrafish wounds.

Licorice is a widely used herbal medicine for the treatment of various diseases in southern Europe and parts of Asia. It has been reported that the isoliquiritin (ISL) from Glycyrrhiza root has the activity of promoting angiogenesis. The purpose of this study was to investigate the effect of ISL on the wound healing activity of zebrafish and its mechanism. 6-month-old zebrafish were injured in the skin (2 mm in diameter) and then treated with ISL. By measuring wound size and by histological examination, we found that ISL improved wound healing. In addition, 4-day-old zebrafish embryos of double transgenic line [Tg(fli-1:EGFP)]/[Tg(mpeg:mCherry)] were suffered from tissue traumas and then treated with ISL. Through fluorescent microscopy, we found that ISL promoted macrophage recruitment and angiogenesis in the wound area. Through qPCR analysis, we found that ISL up-regulated the expression of genes related to inflammation and angiogenesis in zebrafish embryos. These results showed that ISL could promote inflammatory response and angiogenesis, which played key roles in promoting wound healing. Therefore, ISL can be used as a promising candidate to promote wound healing.

Pro-angiogenic activity of Tongnao decoction on HUVECs in vitro and zebrafish in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Tongnao Decoction (TND) is a Chinese decoction approved and used in Jiangsu Province Hospital for the treatment of ischemic stroke. It shows conclusive efficiency in the improvement of neurologic impairment and activities of daily living of the patients. AIM OF THE STUDY: Recently, angiogenesis has been recognized as a potential therapeutic strategy for treating cerebral ischemia. This study was aimed to provide comprehensive evidence for the pro-angiogenic effect of TND and characterize the underlying mechanism. MATERIALS AND METHODS: We firstly established the chemical fingerprinting of TND. Then, the in vitro pro-angiogenic activities of TND were tested on human umbilical vein endothelial cells (HUVECs) through cell viability, wound healing and tube formation assays. The in vivo pro-angiogenic effects were evaluated on transgenic zebrafish embryos [Tg (fli-1: EGFP)] through the formation of intersegmental vessels (ISVs), subintestinal vessels (SIVs) and central arteries (CtAs). Lastly, the potential mechanisms of TND were analyzed by a blocking assay with eight pathways-specific kinase inhibitors. RESULTS: TND promoted the proliferation, migration and tube formation of HUVECs. TND also rescued the impairment of ISVs, SIVs and CtAs caused by VRI in a dose-dependent manner in zebrafish embryos. TND could activate vascular endothelial growth factor receptor-2 (VEGFR-2), phosphoinositide 3-kinase (PI3K) - protein kinase B (Akt) and Raf - mitogen-activated protein kinase1/2 (MEK1/2) - extracellular regulated kinase 1/2 (ERK1/2) signaling pathways. CONCLUSION: Our study firstly demonstrated the pro-angiogenic activities of TND. Our work provided evidences for the clinical usage of TND in restoring neurovascular function through promoting angiogenesis in the ischemic cerebral microvascular.

A systematical comparison of anti-angiogenesis and anti-cancer efficacy of ramucirumab, apatinib, regorafenib and cabozantinib in zebrafish model.

AIMS: Gastric cancer (GC) is one of the most common malignant tumors in the world. Anti-angiogenic therapy is a useful strategy for the treatment of advanced GC. This study was aimed to systemically compare the anti-angiogenesis, anti-cancer efficacy, as well as the safety of four known anti-angiogenic drugs, namely ramucirumab, apatinib, regorafenib and cabozantinib. MAIN METHODS: Anti-angiogenic effect was evaluated for the intersegmental vessels (ISVs) and subintestinal veins (SIVs) formation in the Tg (fli-1: EGFP) zebrafish embryos. Anti-cancer efficacy was tested for the in vivo cell proliferation in cell line derived tumor xenograft (CDX) model based on Tg (fli-1: EGFP) zebrafish embryos. KEY FINDINGS: All four drugs exhibited anti-angiogenic abilities and tumor inhibition effects in fli-1: EGFP transgenic zebrafish. Using zebrafish xenografted model, we found that effectiveness of ramucirumab in anti-GC-proliferation is better than apatinib, regorafenib and cabozantinib. The combination of anti-angiogenic drugs and cisplatin showed no significant benefit in tumors. Meanwhile, toxicity assay showed that all tested anti-angiogenic drugs could cause cardiovascular-related side effects. The therapeutic index (LD50/ED50) of cabozantinib is higher than apatinib and regorafenib, suggesting a potential as an anti-GC drug. SIGNIFICANCE: The comparison of GC-related anti-angiogenic drugs was first reported. It was found that cabozantinib had a potential as an anti-GC drug. Zebrafish model was an ideal animal model for the research of anti-angiogenic behaviors.

Cancer-associated fibroblast regulate proliferation and migration of prostate cancer cells through TGF-ß signaling pathway.

AIMS: Prostate cancer (PCa) incidence rates are rising in China currently. Cancer-associated fibroblasts (CAFs), as a major component of tumor microenvironment, are crucial for tumor progression. This study was aimed to explore the promotion effect of patient-derived CAFs on the proliferation and migration of prostate cancer cells. MAIN METHODS: CAFs were isolated from tumor tissues of PCa patients. The promotion effect of CAFs on the proliferation and migration of PC-3 and LNCaP cells were evaluated in vitro and in vivo. The concentration of TGF-ß1 was measured by Luminex assay. The blocking activity of LY2109761 on the promotion effect of CAFs was also evaluated. KEY FINDINGS: CAFs could significantly promote the proliferation and migration of PC-3 and LNCaP cells both in vitro and in vivo. TGF-ß1 was identified as a highly increased factor in CAFs-CM compared with the normal culture medium of these two cancer cell lines. TGF-ß receptor inhibitor LY2109761 could suppress the CAFs-induced cellular proliferation and migration of PC-3 cells but not LNCaP cells. SIGNIFICANCE: Our study suggested a crucial role for CAFs and TGF-ß signaling in the progression of PCa. Zebrafish xenograft model was an ideal animal model for the study of CAFs and cancer cell interaction.

Zebrafish Xenograft Model of Human Lung Cancer for Evaluating Osimertinib Resistance.

About half of NSCLC patients with EGFR mutation had secondary mutation T790M after treatment with a first-generation tyrosine kinase inhibitor (TKI), Gefitinib. The third-generation of EGFR-TKI Osimertinib is suitable for patients with EGFR mutation and T790M mutation. However, drug screening for NSCLC patients after the emergence of acquired resistance has become a difficult problem for clinicians. In this study, we established drug-resistant cell lines of Gefitinib and Osimertinib to evaluate cell proliferation in vitro. And we investigated the inhibitory effect of different drug concentration gradients on cancer cells. Zebrafish with high homology to human genes were selected as xenotransplantation models to compare the effects of different concentrations of Osimertinib on the proliferation and angiogenesis of zebrafish tumors after transplantation of different lung cancer cell lines. It was confirmed that Osimertinib could inhibit the proliferation of tumor cells with EGFR mutation and T790M resistance mutation in zebrafish, which was consistent with the clinical research conclusion.

Pro-angiogenic activity of isoliquiritin on HUVECs in vitro and zebrafish in vivo through Raf/MEK signaling pathway.

AIMS: Liquorice is a widely used herbal medicine for treating various diseases native to southern Europe and parts of Asia. Isoliquiritin (ISL), a licorice root-derived flavonoid, has been reported to exhibit antioxidant, anti-inflammatory, anti-genotoxic activity and anti-depression activities. This study was aimed to explore the pro-angiogenic activity of ISL and explicate the underlying mechanism. MAIN METHODS: In vitro, ISL-treated human umbilical vein endothelial cells (HUVECs) were analyzed for cell viability, cell migration and tube formation. In vivo, pro-angiogenic effects were evaluated for the intersegmental vessels (ISVs) formation in transgenic zebrafish embryos [Tg(fli-1: EGFP)]. Furthermore, a blocking assay with eight pathways-specific kinase inhibitors were also used to determine the potential pro-angiogenic mechanism of ISL. KEY FINDINGS: ISL counteracted tyrosine kinase inhibitor II (VRI)-induced endothelial cell apoptosis and promoted cell migration and tube formation in HUVECs. ISL markedly rescued ISVs loss induced by VRI in zebrafish embryos, probably by activating vascular endothelial growth factor receptor-2 (VEGFR-2), phosphoinositide 3-kinase (PI3K), Raf and mitogen-activated protein kinase (MEK)-dependent signaling pathways. SIGNIFICANCE: Our study first discovered and confirmed the pro-angiogenic activity of ISL both in HUVECs and zebrafish. Thus, ISL could be developed as a potential therapeutic agent by the role of pro-angiogenic activity for the treatment of cardiovascular diseases, cerebrovascular diseases and other vascular diseases.

Anti-angiogenic activity of para-coumaric acid methyl ester on HUVECs in vitro and zebrafish in vivo.

BACKGROUND: Para-coumaric acid methyl ester (pCAME) is one of the bioactive components of Costus speciosus (Koen) Sm. (Zingiberaceae). This plant is traditionally used in Asia to treat catarrhal fevers, worms, dyspepsia, and skin diseases. PURPOSE: To investigate the anti-angiogenic activity of pCAME and its molecular mechanism of action. STUDY DESIGN: We investigated the anti-angiogenic activity of pCAME on human umbilical vein endothelial cells (HUVECs) in vitro and zebrafish (Danio rerio) in vivo. METHODS: In vitro cell proliferation, would healing, migration and tube formation assays were used, along with in vivo physiological angiogenic vessel formation, tumor-induced angiogenic vessel formation assays on zebrafish model. qRT-PCR and RNA-seq were also used for the target investigation. RESULTS: pCAME could inhibit the proliferation, would healing, migration and tube formation of HUVECs, disrupt the physiological formation of intersegmental vessels (ISVs) and the subintestinal vessels (SIVs) of zebrafish embryos, and inhibit tumor angiogenesis in the zebrafish cell-line derived xenograft (zCDX) model of SGC-7901 in a dose-dependent manner. Mechanistic studies revealed that pCAME inhibited vegf/vegfr2 and ang/tie signaling pathways in zebrafish by quantitative RT-PCR analysis, and regulated multi-signaling pathways involving immune, inflammation and angiogenesis in SGC-7901 zCDX model by RNA-seq analysis. CONCLUSION: pCAME may be a multi-target anti-angiogenic drug candidate and hold great potential for developing novel therapeutic strategy for cancer treatment.

1-Methoxycarbony-ß-carboline from Picrasma quassioides exerts anti-angiogenic properties in HUVECs in vitro and zebrafish embryos in vivo.

Angiogenesis is a crucial process in the development of inflammatory diseases, including cancer, psoriasis and rheumatoid arthritis. Recently, several alkaloids from Picrasma quassioides had been screened for angiogenic activity in the zebrafish model, and the results indicated that 1-methoxycarbony-ß-carboline (MCC) could effectively inhibit blood vessel formation. In this study, we further confirmed that MCC can inhibit, in a concentration-dependent manner, the viability, migration, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro, as well as the regenerative vascular outgrowth of zebrafish caudal fin in vivo. In the zebrafish xenograft assay, MCC inhibited the growth of tumor masses and the metastatic transplanted DU145 tumor cells. The proteome profile array of the MCC-treated HUVECs showed that MCC could down-regulate several angiogenesis-related self-secreted proteins, including ANG, EGF, bFGF, GRO, IGF-1, PLG and MMP-1. In addition, the expression of two key membrane receptor proteins in angiogenesis, TIE-2 and uPAR, were also down-regulated after MCC treatment. Taken together, these results shed light on the potential therapeutic application of MCC as a potent natural angiogenesis inhibitor via multiple molecular targets.

Patient-derived xenograft in zebrafish embryos: a new platform for translational research in gastric cancer.

BACKGROUND: Gastric cancer (GC) is among the most commonly cancer occurred in Asian, especially in China. With its high heterogeneity and few of validated drug targets, GC remains to be one of the most under explored areas of precision medicine. In this study, we aimed to establish an in vivo patient-derived xenograft (PDX) model based on zebrafish (Danio rerio) embryos, allowing for a rapid analysis of the angiogenic and invasive potentials, as well as a fast drug sensitivity testing. METHODS: Two human gastric cancer cell lines (AGS and SGC-7901) were xenografted into zebrafish embryos, their sensitivity to 5-FU were tested both in vitro and in vivo. Fourteen human primary cells from gastric cancer tissue were xenografted into zebrafish embryos, their proliferating, angiogenic and metastatic activities were evaluated in vivo. Sensitivity to 5-FU, docetaxel, and apatinib were also tested on primary samples from four patients. RESULTS: SGC-7901 showed higher sensitivity to 5-FU than AGS both in vitro (6.3 ± 0.9 µM vs.10.5 ± 1.8 µM) and in vivo. Nine out of fourteen patient samples were successfully transplanted in zebrafish embryos and all showed proliferating, angiogenic and metastatic potentials in the living embryos. Four cases showed varied sensitivity to the selected three chemotherapeutic drugs. CONCLUSIONS: Our zebrafish PDX (zPDX) model is a preclinically reliable in vivo model for GC. The zPDX model is also a promising platform for the translational research and personalized treatment on GC.

Developmental toxicity of auranofin in zebrafish embryos.

Auranofin (AF) is used in clinic for the treatment of rheumatoid arthritis, repurposing of AF as an anticancer drug has just finished a phase I/II clinical trial, but the developmental toxicity of AF remains obscure. This study focused on its developmental toxicity by using zebrafish embryos. Zebrafish embryos were exposed to different concentrations (1, 2.5, 5, 10 µm) of AF from 2 h post-fertilization (hpf) to 72 hpf. At 72 hpf, two major developmental defects caused by AF were found, namely severe pericardial edema and hypopigmentation, when embryos were exposed to concentrations higher than 2.5 µm. Biochemical detection of oxidative stress enzyme combined with expressions of a series of genes related to oxidative stress, cardiac, metal stress and pigment formation were subsequently tested. The superoxide dismutase activity was decreased while malondialdehyde content was accumulated by AF treatment. The expression of oxidative stress-related genes (sod1, gpx1a, gst), pigment-related genes (mitfb, trp-1a) and one metal stress-related gene ctr1 were all decreased by AF exposure. The expressions of cardiac-related genes (amhc, vmhc) and one metal-related gene hsp70 were found to be significantly upregulated by AF exposure. These findings indicated the potential developmental toxicity of AF on zebrafish early development. Copyright © 2016 John Wiley & Sons, Ltd.

Gambogic acid causes fin developmental defect in zebrafish embryo partially via retinoic acid signaling.

Gambogic acid (GA), the major active ingredient of gamboge, has been approved by the Chinese Food and Drug Administration for clinical trials in cancer patients due to its strong anticancer activity. However, our previous research showed that GA was teratogenic against zebrafish fin development. To explore the teratogenicity and the underlying mechanisms, zebrafish (Danio rerio) embryos were used. The morphological observations revealed that GA caused fin defects in zebrafish embryos in a concentration-dependent manner. The critical exposure time of GA to reveal teratogenicity was before 8 hpf (hours post fertilization). LC/MS/MS analysis revealed that a maximum bioconcentration of GA was occurred at 4 hpf. Q-PCR data showed that GA treatment resulted in significant inactivation of RA signaling which could be partially rescued by the exogenous supply of RA. These results indicate the potential teratogenicity of GA and provide evidence for a caution in its future clinic use.

Docosahexaenoic Acid Modulates Invasion and Metastasis of Human Ovarian Cancer via Multiple Molecular Pathways.

OBJECTIVE: We investigated the effect of docosahexaenoic acid (DHA) on the invasion and metastasis of ovarian cancer cells (A2780, HO8910, and SKOV-3). METHODS: Cytotoxicity assay was performed to determine the optimal doses of DHA in this experiment. The effects of DHA on invasion ability were assessed by invasion assay. The expressions of messenger RNA and/or proteins associated with invasion or metastasis were detected by quantitative Real Time-Polymerase Chain Reaction or Western blot. The effect of DHA on cell metastasis was assessed in xenograft model of zebrafish. RESULTS: Docosahexaenoic acid and α-linolenic acid could reduce the cell vitalities in dose-dependent manner. However, DHA inhibited the invasion and metastasis of ovarian cancer cells, but α-linolenic acid did not (**P < 0.01). Docosahexaenoic acid could downregulate the expressions of WAVE3, vascular endothelial cell growth factor, and MMP-9, and upregulate KISS-1, TIMP-1, and PPAR-γ, which negatively correlated with cell invasion and metastasis (*P < 0.05). Docosahexaenoic acid restrained the development of subintestinal vessels and cancer cell metastasis in xenograft model of zebrafish (**P < 0.01). CONCLUSIONS: Docosahexaenoic acid inhibited the invasion and metastasis of ovarian cancer cells in vitro and in vivo through the modulation of NF-κB signaling pathway, suggesting that DHA is a promising candidate for ovarian cancer therapy.

Anti-angiogenic effect of auranofin on HUVECs in vitro and zebrafish in vivo.

Angiogenesis plays an essential role in many physiological and pathological processes. Auranofin (Ridaura®), an important gold(I) complex, is used to treat rheumatoid arthritis. However, the effect of auranofin on blood vessel formation is still unclear. In this study, we investigated the anti-angiogenic activity of auranofin on human umbilical vein endothelial cells (HUVEC) in vitro and zebrafish in vivo. Our results showed that auranofin could inhibit the proliferation, migration and tube formation of HUVECs and disrupted the formation of intersegmental vessels and the subintestinal vessels of zebrafish embryos. Auranofin inhibited the phosphorylation of vascular endothelial growth factor 2 (p-VEGFR2) on HUVECs and suppressed the vascular endothelial growth factor (VEGF) signaling pathway (vegfa, flt-1, kdr) but not thioredoxin reductase (TrxR) on zebrafish. Our study suggested that auranofin might serve as a potential anti-angiogenic compound candidate.

Toxicity assessment of 7 anticancer compounds in zebrafish.

Toxicity is one of the major reasons for failure in drug development. Zebrafish, as an ideal vertebrate model, could also be used to evaluate drug toxicity. In this study, we aimed to show the predictability and highlight novel findings of toxicity in zebrafish model. Seven anticancer compounds, including triptolide (TP), gambogic acid (GA), mycophenolic acid (MPA), curcumin, auranofin, thalidomide, and taxol, were assessed in zebrafish for their toxicity. Three compounds (GA, TP, and taxol) showed highest acute lethality, with 50% lethal concentration ≈ 1 µmol/L. Missing tails, severe pericardial edema, and enlarged yolk sacs were observed in MPA-treated embryos. The development of pectoral fins was severely disturbed in thalidomide-, GA-, and TP-treated embryos. Bradycardia was observed in MPA- and thalidomide-treated groups. Our findings suggested that the zebrafish are a good model for toxicity assessment of anticancer compounds.