Canthin-6-One Inhibits Developmental and Tumour-Associated Angiogenesis in Zebrafish.

Tumour-associated angiogenesis play key roles in tumour growth and cancer metastasis. Consequently, several anti-angiogenic drugs such as sunitinib and axitinib have been approved for use as anti-cancer therapies. However, the majority of these drugs target the vascular endothelial growth factor A (VEGFA)/VEGF receptor 2 (VEGFR2) pathway and have shown mixed outcome, largely due to development of resistances and increased tumour aggressiveness. In this study, we used the zebrafish model to screen for novel anti-angiogenic molecules from a library of compounds derived from natural products. From this, we identified canthin-6-one, an indole alkaloid, which inhibited zebrafish intersegmental vessel (ISV) and sub-intestinal vessel development. Further characterisation revealed that treatment of canthin-6-one reduced ISV endothelial cell number and inhibited proliferation of human umbilical vein endothelial cells (HUVECs), suggesting that canthin-6-one inhibits endothelial cell proliferation. Of note, canthin-6-one did not inhibit VEGFA-induced phosphorylation of VEGFR2 in HUVECs and downstream phosphorylation of extracellular signal-regulated kinase (Erk) in leading ISV endothelial cells in zebrafish, suggesting that canthin-6-one inhibits angiogenesis independent of the VEGFA/VEGFR2 pathway. Importantly, we found that canthin-6-one impairs tumour-associated angiogenesis in a zebrafish B16F10 melanoma cell xenograft model and synergises with VEGFR inhibitor sunitinib malate to inhibit developmental angiogenesis. In summary, we showed that canthin-6-one exhibits anti-angiogenic properties in both developmental and pathological contexts in zebrafish, independent of the VEGFA/VEGFR2 pathway and demonstrate that canthin-6-one may hold value for further development as a novel anti-angiogenic drug.

Next-generation of melanoma researchers: Trainee perspectives from around the world.

The Society for Melanoma Research (SMR) was created 20 years ago and has unequivocally contributed to the vast progress of the field, particularly for the treatment of melanoma patients with metastatic disease by facilitating synergistic collaborations between clinicians, researchers at the bench, and industry. In commemoration of the 20th anniversary of the first SMR International Congress (held in 2003 in Philadelphia), we look to the future by highlighting the perspectives of the next generation of rising stars, medical, and graduate students across six continents.

Heterogeneity in Melanoma.

There is growing evidence that tumour heterogeneity has an imperative role in cancer development, evolution and resistance to therapy. Continuing advancements in biomedical research enable tumour heterogeneity to be observed and studied more critically. As one of the most heterogeneous human cancers, melanoma displays a high level of biological complexity during disease progression. However, much is still unknown regarding melanoma tumour heterogeneity, as well as the role it plays in disease progression and treatment response. This review aims to provide a concise summary of the importance of tumour heterogeneity in melanoma.

3,4-Difluorobenzocurcumin Inhibits Vegfc-Vegfr3-Erk Signalling to Block Developmental Lymphangiogenesis in Zebrafish.

Lymphangiogenesis, the formation of new lymphatic vessels from pre-existing vasculature, plays critical roles in disease, including in cancer metastasis and chronic inflammation. Preclinical and recent clinical studies have now demonstrated therapeutic utility for several anti-lymphangiogenic agents, but optimal agents and efficacy in different settings remain to be determined. We tested the anti-lymphangiogenic property of 3,4-Difluorobenzocurcumin (CDF), which has previously been implicated as an anti-cancer agent, using zebrafish embryos and cultured vascular endothelial cells. We used transgenic zebrafish labelling the lymphatic system and found that CDF potently inhibits lymphangiogenesis during embryonic development. We also found that the parent compound, Curcumin, does not inhibit lymphangiogenesis. CDF blocked lymphatic and venous sprouting, and lymphatic migration in the head and trunk of the embryo. Mechanistically, CDF impaired VEGFC-VEGFR3-ERK signalling in vitro and in vivo. In an in vivo pathological model of Vegfc-overexpression, treatment with CDF rescued endothelial cell hyperplasia. CDF did not inhibit the kinase activity of VEGFR3 yet displayed more prolonged activity in vivo than previously reported kinase inhibitors. These findings warrant further assessment of CDF and its mode of action as a candidate for use in metastasis and diseases of aberrant lymphangiogenesis.

BRN2 expression increases anoikis resistance in melanoma.

Melanoma tumors are highly heterogeneous, comprising of many cell populations that vary in their potential for growth and invasion. Differential transcription factor expression contributes to these phenotypic traits. BRN2, a member of the POU domain family of transcription factors is thought to play important roles in melanoma invasion and metastasis. However, the function of BRN2 during the metastatic process of melanoma remains largely unknown. We therefore investigated the effect of BRN2 expression in melanoma cells with no or low constitutive expression using a doxycycline-inducible system. Induction of BRN2 expression led to reduced proliferation and partial resistance to an inhibitor of mutated BRAF. Whole-genome profiling analysis revealed novel targets and signaling pathway changes related to prevention of cell death induced by detachment from the extracellular matrix, known as anoikis resistance. Further investigation confirmed increased survival of BRN2-expressing cell lines in non-adherent conditions. Functionally, expression of BRN2 promoted induction of c-MET levels as well as increased phosphorylation of STAT3. Treatment with crizotinib, a c-MET inhibitor, decreased cellular viability of BRN2-expressing cells under non-adherent conditions to death by anoikis. Alternative inhibitors of c-MET showed similar results. These results highlight the importance of a largely overlooked transcription factor in the progression and metastasis of melanoma, and may suggest a strategy to target BRN2-expressing cells resistant to therapy and cell death by anoikis.

Interference Potential of Tannins and Chlorophylls in Zebrafish Phenotypic-Based Assays.

Natural products are prolific producers of diverse chemical scaffolds, which have yielded several clinically useful drugs. However, the complex features of natural products present challenges for identifying bioactive molecules using high-throughput screens. For most assays, measured endpoints are either colorimetric or luminescence based. Thus, the presence of the major metabolites, tannins, and chlorophylls, in natural products could potentially interfere with these measurements to give either false-positive or false-negative hits. In this context, zebrafish phenotypic assays provide an alternative approach to bioprospect naturally occurring bioactive compounds. Whether tannins and/or chlorophylls interfere in zebrafish phenotypic assays, is unclear. In this study, we evaluated the interference potential of tannins and chlorophylls against efficacy of known small-molecule inhibitors that are known to cause phenotypic abnormalities in developing zebrafish embryos. First, we fractionated tannin-enriched fraction (TEF) and chlorophyll-enriched fraction (CEF) from Camellia sinensis and cotreated them with PD0325901 [mitogen-activated protein kinase-kinase (MEK) inhibitor] and sunitinib malate (SM; anti-[lymph]angiogenic drug). While TEF and CEF did not interfere with phenotypic or molecular endpoints of PD0325901, TEF at 100 µg/mL partially masked the antiangiogenic effect of SM. On the other hand, CEF (100 µg/mL) was toxic when treated up to 6 dpf. Furthermore, CEF at 100 µg/mL potentially enhanced the activity of γ-secretase inhibitors, resulting in toxicity of treated embryos. Our study provides evidence that the presence of tannin and/or chlorophyll in natural products do interfere with zebrafish phenotype assays used for identifying potential hits. However, this may be target/assay dependent and thus requiring additional optimization steps to assess interference potential of tannins and chlorophylls before performing any screening assay.

Zebrafish phenotypic screen identifies novel Notch antagonists.

Zebrafish represents a powerful in vivo model for phenotype-based drug discovery to identify clinically relevant small molecules. By utilizing this model, we evaluated natural product derived compounds that could potentially modulate Notch signaling that is important in both zebrafish embryogenesis and pathogenic in human cancers. A total of 234 compounds were screened using zebrafish embryos and 3 were identified to be conferring phenotypic alterations similar to embryos treated with known Notch inhibitors. Subsequent secondary screens using HEK293T cells overexpressing truncated Notch1 (HEK293TΔE) identified 2 compounds, EDD3 and 3H4MB, to be potential Notch antagonists. Both compounds reduced protein expression of NOTCH1, Notch intracellular domain (NICD) and hairy and enhancer of split-1 (HES1) in HEK293TΔE and downregulated Notch target genes. Importantly, EDD3 treatment of human oral cancer cell lines demonstrated reduction of Notch target proteins and genes. EDD3 also inhibited proliferation and induced G0/G1 cell cycle arrest of ORL-150 cells through inducing p27KIP1. Our data demonstrates the utility of the zebrafish phenotypic screen and identifying EDD3 as a promising Notch antagonist for further development as a novel therapeutic agent.

Canthin-6-one Isolated from Bruceajavanica Root Blocks Cancer Cells in the G2/M phase and Synergizes with Cisplatin.

Poor prognosis of most cancer patients is in part, due to limited therapeutic options. Furthermore, as chemotherapy remains the standard-of-care for several cancers, partial or lack of response remains a concern and compounding this are the adverse side effects of the treatment that severely impacts the quality of life and survival. In pursuit of improving treatment options, we have opted to investigate the unique chemical skeleton of natural compounds as anticancer therapies. In this study, from an initial screen of 31 crude methanol extracts from -15 plant species using HL60 cells, the root extract of Bruceajavanica (L.) Merr indicated the presence of bioactive compounds. Subsequent bioassay-guided purification on the root extract yielded two alkaloids canthin-6-one (1) and bruceolline J (2), which were further investigated for their bioactivity in representative human cancer lines and normal phenotypic counterparts. MTT assay demonstrated ED50 values from 34.7-72.9 gM for 1 and 16.0-54.0 gM for 2 for the cancer cell lines panel. NP69 cells also demonstrated sensitivity to. both compounds (9.3 piM and 4.5 pM). As amount of 2 isolated were limiting, we focused on 1 to further identify novel anticancer properties in PC3 and HeLa cancer lines. We observed at 30 gM, I induced a G2/M phase arrest coinciding with decreased cell proliferation. Furthermore, I was able to synergize the cytotoxic effect of cisplatin when used in combination, suggesting the potential of combination therapy for those less responsive lesions to standard chemotherapy.

Utilizing Zebrafish to Identify Anti-(Lymph)Angiogenic Compounds for Cancer Treatment: Promise and Future Challenges.

Cancer metastasis which predominantly occurs through blood and lymphatic vessels, is the leading cause of death in cancer patients. Consequently, several anti-angiogenic agents have been approved as therapeutic agents for human cancers such as metastatic renal cell carcinoma. Also, anti-lymphangiogenic drugs such as monoclonal antibodies VGX-100 and IMC-3C5 have undergone phase I clinical trials for advanced and metastatic solid tumors. Although anti-tumor-associated angiogenesis has proven to be a promising therapeutic strategy for human cancers, this approach is fraught with toxicities and development of drug resistance. This emphasizes the need for alternative anti-(lymph)angiogenic drugs. The use of zebrafish has become accepted as an established model for high-throughput screening, vascular biology, and cancer research. Importantly, various zebrafish transgenic lines have now been generated that can readily discriminate different vascular compartments. This now enables detailed in vivo studies that are relevant to both human physiological and tumor (lymph)angiogenesis to be conducted in zebrafish. This review highlights recent advancements in the zebrafish anti-vascular screening platform and showcases promising new anti-(lymph)angiogenic compounds that have been derived from this model. In addition, this review discusses the promises and challenges of the zebrafish model in the context of anti-(lymph)angiogenic compound discovery for cancer treatment.