Genetically encoded dihydroxyphenylalanine coupled with tyrosinase for strain promoted labeling.

Protein modifications through genetic code engineering have a remarkable impact on macromolecule engineering, protein translocation, protein-protein interaction, and cell biology. We used the newly developed molecular biology approach, genetic code engineering, for fine-tuning of proteins for biological availability. Here, we have introduced 3, 4-dihydroxy-l-phenylalanine in recombinant proteins by selective pressure incorporation method for protein-based cell labeling applications. The congener proteins treated with tyrosinase convert 3, 4-dihydroxy-l-phenylalanine to dopaquinone for strain-promoted click chemistry. Initially, the single-step Strain-Promoted Oxidation-Controlled Cyclooctyne-1,2-quinone Cycloaddition was studied using tyrosinase catalyzed congener protein and optimized the temporally controlled conjugation with (1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethanol. Then, the feasibility of tyrosinase-treated congener annexin A5 with easily reactive quinone functional moiety was conjugated with fluorescent tag dibenzocyclooctyne-PEG4-TAMRA for labeling of apoptotic cells. Thus, the congener proteins-based products demonstrate selective cell labeling and apoptosis detection in EA.hy926 cells even after the protein modifications. Hence, genetic code engineering can be coupled with click chemistry to develop various protein-based fluorescent labels.

Siphonaxanthin, a green algal carotenoid, as a novel functional compound.

Siphonaxanthin is a specific keto-carotenoid in green algae whose bio-functional properties are yet to be identified. This review focuses on siphonaxanthin as a bioactive compound and outlines the evidence associated with functionality. Siphonaxanthin has been reported to potently inhibit the viability of human leukemia HL-60 cells via induction of apoptosis. In comparison with fucoxanthin, siphonaxanthin markedly reduced cell viability as early as 6 h after treatment. The cellular uptake of siphonaxanthin was 2-fold higher than fucoxanthin. It has been proposed that siphonaxanthin possesses significant anti-angiogenic activity in studies using human umbilical vein endothelial cells and rat aortic ring. The results of these studies suggested that the anti-angiogenic effect of siphonaxanthin is due to the down-regulation of signal transduction by fibroblast growth factor receptor-1 in vascular endothelial cells. Siphonaxanthin also exhibited inhibitory effects on antigen-induced degranulation of mast cells. These findings open up new avenues for future research on siphonaxanthin as a bioactive compound, and additional investigation, especially in vivo studies, are required to validate these findings. In addition, further studies are needed to determine its bioavailability and metabolic fate.

Marine algal carotenoids inhibit angiogenesis by down-regulating FGF-2-mediated intracellular signals in vascular endothelial cells.

Discovery of natural compounds as effective angiogenesis inhibitors has become an important approach in the prevention of cancer. We previously demonstrated the anti-angiogenic potential of two marine algal carotenoids, fucoxanthin and siphonaxanthin. In this study, we evaluated the molecular mechanisms of the anti-angiogenic activity of those two carotenoids using human umbilical vein endothelial cells. This study showed that both fucoxanthin and siphonaxanthin suppress the mRNA expression of fibroblast growth factor 2 (FGF-2) and its receptor (FGFR-1) as well as their trans-activation factor, EGR-1. But, the mRNA expression of VEGFR-2 did not show significant effect by those two carotenoids. Further, those two marine algal carotenoids down-regulate the phosphorylation of FGF-2-mediated intracellular signaling proteins such as ERK1/2 and Akt. Inhibition of FGF-2-mediated intracellular signaling proteins by those carotenoids represses the migration of endothelial cells as well as their differentiation into tube-like structures on Matrigel. These results demonstrate for the first time the possible molecular mechanism underlying the anti-angiogenic effects of fucoxanthin and siphonaxanthin and suggest that these effects are due to the down-regulation of signal transduction by FGFR-1. Our findings imply a new insight into the novel bio-functional property of marine algal carotenoids which should improve current anti-angiogenic therapies in the treatment of cancer and other pro-angiogenic diseases.

Siphonaxanthin, a marine carotenoid from green algae, effectively induces apoptosis in human leukemia (HL-60) cells.

BACKGROUND: Bioactive marine molecules have recently received considerable attention for their nutraceutical characteristics. Considering the ever-increasing demand of nutraceuticals for anti-cancer therapy, we investigated the apoptosis-inducing effects of marine carotenoids, including siphonaxanthin, on human leukemia (HL-60) cells. METHODS: Apoptotic effects were evaluated by cell viability assay, TUNEL assay, and caspase-3 activity. The expression of apoptosis-inducing death receptor-5 (DR5), Bcl-2 and Bax were assayed by Western blot analysis, and mRNA expression of GADD45α was assayed by quantitative RT-PCR analysis. RESULTS: Siphonaxanthin potently inhibited the viability of HL-60 cells compared with the other carotenoids evaluated. In comparison with fucoxanthin, siphonaxanthin at a concentration of 20µM markedly reduced cell viability (p<0.05) as early as within 6h of treatment. The effective apoptotic activity of siphonaxanthin was observed by increases in TUNEL-positive cells, and by increased chromatin condensation in HL-60 cells. This induction of apoptosis was associated with the decreased expression of Bcl-2, and the subsequently increased activation of caspase-3. In addition, siphonaxanthin up-regulated the expression of GADD45α and DR5. CONCLUSIONS: These data suggest that the dietary carotenoid siphonaxanthin could be potentially useful as a chemo-preventive and/or chemotherapeutic agent. GENERAL SIGNIFICANCE: Our findings demonstrate for the first time the novel functional property of siphonaxanthin as a potent inducer of apoptosis in HL-60 cells.

Anti-angiogenic effect of siphonaxanthin from green alga, Codium fragile.

Since anti-angiogenic therapy has becoming a promising approach in the prevention of cancer and related diseases, the present study was aimed to examine the anti-angiogenic effect of siphonaxanthin from green alga (Codium fragile) in cell culture model systems and ex vivo approaches using human umbilical vein endothelial cells (HUVECs) and rat aortic ring, respectively. Siphonaxanthin significantly suppressed HUVEC proliferation (p<0.05) at the concentration of 2.5 µM (50% as compared with control) and above, while the effect on chemotaxis was not significant. Siphonaxanthin exhibited strong inhibitory effect on HUVEC tube formation. It suppressed the formation of tube length by 44% at the concentration of 10 µM, while no tube formation was observed at 25 µM, suggesting that it could be due to the suppression of angiogenic mediators. The ex vivo angiogenesis assay exhibited reduced microvessel outgrowth in a dose dependent manner and the reduction was significant at more than 2.5 µM. Our results imply a new insight on the novel function of siphonaxanthin in preventing angiogenesis related diseases.