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

gRASping the redox lever to modulate cancer cell fate signaling

RAS proteins are critical regulators of signaling networks controlling diverse cellular functions such as cell proliferation and survival and its mutation are among the most powerful oncogenic drivers in human cancers. Despite intense efforts, direct RAS-targeting strategies remain elusive due to its “undruggable” nature. To that end, bulk of the research efforts has been directed towards targeting upstream and/or downstream of RAS signaling. However, the therapeutic efficacies of these treatments are limited in the long run due to the acquired drug resistance in RAS-driven cancers. Interestingly, recent studies have uncovered a potential role of RAS in redox-regulation as well as the interplay between ROS and RAS-associated signaling networks during process of cancer initiation and progression. More specifically, these studies provide ample evidence to implicate RAS as a redox-rheostat, manipulating ROS levels to provide a redox-milieu conducive for carcinogenesis. Importantly, the understanding of RAS-ROS interplay could provide us with novel targetable vulnerabilities for designing therapeutic strategies. In this review, we provide a brief summary of the advances in the field to illustrate the dual role of RAS in redox-regulation and its implications in RAS signaling outcomes and also emerging redox-based strategies to target RAS-driven cancers

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field

Development of secretory expression system using synthetic signal peptides spA and spD for protein production in Escherichia coli.

ecombinant protein expression is very important in biotechnology. Successful protein expression depends on the expression host, vector and target protein. Escherichia coli being a popular expression host, is still plagued by various problems in expression like formation of inclusion bodies, incorrect folding and low soluble protein yield. These problems can be circumvented by using different promoters, different host strains, co-expression of chaperones, reduction of culture temperature and secretion of proteins into periplasm and culture medium. In this study, periplasmic protein secretion was investigated by using novel synthetic signal peptides, spA and spD. The two signal peptides were designed based on signal peptides of Bacillus spp. for secretion of heterologous proteins. They were amplified and ligated to genes coding for the green fluorescent protein (GFP) and cyclodextrin lucanotransferase (CGTase) to construct secretion cassettes spA*GFP, spD*GFP and spA*CGT and spD*CGT. These secretion cassettes were first cloned into pCR®-Blunt II-TOPO cloning vector. The cassettes were then sub-cloned into pET-32b(+) expression vector to construct pAGFP, pDGFP, pACGT and transformed into competent E. coli BL21(DE3) and BL21(DE3)pLysS cells. The cloning of secretion cassette spD*CGT into pET-32b(+) was not successful. GFP without the signal peptide was cloned into similar pET-32b(+) as a control and the construct was named pGFP. SDS-PAGE and western blotting results for recombinant GFP clones showed successful expression and secretion into the periplasm. Fluorescence analysis for GFP clones showed that the secreted GFP was not fluorescent while cytoplasmically expressed GFP was fluorescent. Induction temperature also affected the secretion of recombinant GFP as better secretion was attained at 37°C. SDS-PAGE analysis for recombinant clone BLpACGT showed that CGTase was detected in both cytoplasm and periplasm but in Western blotting only cytoplasmic expression was detected. However, the positive control, (CGTase with native signal peptide) was detected in both cytoplasm and periplasm. Cell growth analysis for recombinant clones GFP and CGTase did not show any adverse effect to the secretion host. These results show that the synthetic signal peptides, spA and spD, could direct recombinant proteins to the periplasmic spac

Identification of interaction between the anti-apoptotic protein Bcl-2 and the small GTPase Rac1 and its functional relevance

Ph.DDOCTOR OF PHILOSOPH

Yes from Fas to invade

Cytometry Part A73

mitoEnergetics and cancer cell fate

10.1016/j.bbabio.2008.12.009Biochimica et Biophysica Acta - Bioenergetics17875462-46

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

Zebrafish embryonic development-interfering macrolides from Streptomyces californicus impact growth and mitochondrial function in human colorectal cancer cells

Microorganisms are now recognized as a keysource of medicine. In this study, we aim to identify and characterize target-specific inhibitors from actinobacteria using zebrafish embryo developmental defect as readouts. From our preliminary screen, we evaluated 176 Malaysian actinobacteria extracts and from these, 15 extracts (8.52%) were observed to be toxic to embryos at 20 μg/mL at 72-h post fertilization (hpf). Detailed characterization of these 15 toxic hits uncovered one extract derived from Streptomyces californicus TY004-069 that arrested zebrafish embryonic development at 4–6 hpf after 24 h treatment, a phenotype indicative potentially targeting mitochondrial activity. Subsequent validation on one of the three isolated compounds from this extract, dimeric dinactin (DD) revealed its ability to disrupt mitochondrial membrane potential (MMP) in human colorectal HCT116 and HT29 cancer cells in a dose-dependent manner and impacting cell proliferation through a cell cycle G block. Overall, using zebrafish phenotypic assay, we identified a potential mitochondrial function inhibitor from actinobacteria that warrants further detailed analysis and also demonstrated that macrolides may hold promise as therapeutic molecules for human diseases including cancer

Zebrafish phenotypic screen identifies novel Notch antagonists

The authors would like to note that in the original online first version of this article, the symbol ɣ is missing from the text where ɣ-secretase is stated. Also, labels to figures in the Results section should be “CYCLIN D1 levels were also reduced in a similar dose dependent manner while those of HES1 and NOTCH1 (shown by densitometry in Figure S6c) were marginally reduced and remained unaffected, respectively (Fig. 5d)” and “Also, we were able to show as mentioned earlier, that EDD3 treatment resulted in decreased CYCLIND1 levels and induction of p27 (Figure S8g)”. Subtitle in the Result section should be “Identification of 3 novel compounds with potential anti-Notch activity in zebrafish embryos” and ‘EDD3 inhibits proliferation and cell cycle progression of ORL-150 cells’. The original article was corrected