**3. Apoptosis-Inducing Effect**

There is a wealth of information pertaining to apoptosis in anti-cancer research, and the 

apoptosis-inducing properties of carotenoids may be an important approach to chemo-prevention and/or chemotherapy. Carotenoids from terrestrial plants, such as Ά-carotene and lycopene, have been extensively studied and implicated as cancer preventive agents. Carotenoids from marine sources have received much attention, as they are structurally different from those found in terrestrial sources. Recent studies have found that carotenoids from algae, including fucoxanthin and peridinin, induce apoptosis in cancer cells [6–9,20,21]. 

We investigated whether various carotenoids present in marine sources are potentially involved in cancer-preventing action in human leukemia HL-60 cells. We found that the 11 carotenoids investigated, siphonaxanthin most potently at inhibiting the viability of HL-60 cells [22]. Compared to fucoxanthin, siphonaxanthin, at a concentration of 20 ΐM, markedly reduced cell viability as early as 6 h after treatment. The cellular uptake of siphonaxanthin was 2-fold higher than fucoxanthin, demonstrating a positive correlation between cellular uptake and cell viability. The apoptotic activity of siphonaxanthin was characterized by increases in TUNEL-positive cells, and by increased chromatin condensation in the cells. This induction of apoptosis was accompanied by decreased expression of Bcl-2, and subsequent increase in the activation of caspase-3. These observations indicate that siphonaxanthin may be a more potent growth-inhibitor in cancer cells than fucoxanthin, possibly due to the differences in the structure of their different functional groups. The presence of hydroxyl group on the 19th carbon atom appeared to contribute to the strong apoptosis-inducing effect. This is further supported by the fact that siphonein (Figure 1C), an esterified form of siphonaxanthin that does not contain an additional hydroxyl group, had a reduced inhibitory effect on cell viability. 

Interestingly, siphonaxanthin has been reported to up-regulate the expression of death receptor 5 (DR5) [22]. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) promotes apoptosis selectively in tumor cells without much effect on normal cells by binding to the transmembrane receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 [23]. Thus, any chemotherapeutic agent that activates TRAIL-induced apoptosis in cancer cells may be an attractive strategy in anti-cancer research. Yoshida *et al.* reported that halocynthiaxanthin and peridinin sensitize cancer cells to TRAIL-induced apoptosis by up-regulating the expression of DR5 [21], which indicates that some carotenoids can induce apoptosis through this pathway. In our study, siphonaxanthin potently up-regulated the expression of DR5, but fucoxanthin did not [22]. Siphonaxanthin could be potentially useful as a chemopreventive and/or chemotherapeutic agent. 

## **4. Anti-Angiogenic Effect**

Angiogenesis is characterized by the growth and remodeling process of the primitive network of blood vessels into a complex network. In this process, endothelial cells secrete proteases, migrate through the extracellular matrix, proliferate, and differentiate [24]. Angiogenesis is involved in many physiological and pathological situations. In normal adults, most vasculature is quiescent, with only 0.01% of endothelial cells undergoing division [25]. However, angiogenesis is an essential process in the female reproductive cycle, along with the remodeling and regeneration of tissues [26]. Pathological angiogenesis is implicated in the pathogenesis of many diseases, including cancer, atherosclerosis, diabetic retinopathy, and rheumatoid arthritis [27]. The newly-formed blood vessels promote cancer growth by supplying nutrients and oxygen and by removing waste products. Metastasis also depends on angiogenesis, as tumor cells are shed from a primary tumor and grow in their target organs [28]. Angiogenesis is activated under other pathological conditions, such as ocular and inflammatory disorders [29]. Hence, prevention of angiogenesis under pathological conditions is a promising approach in the prevention of cancer and other pro-angiogenic diseases. It has been reported that some natural products, such as vitamin B6 [30], algal polysaccharides [31], and nasunin [32], suppress angiogenesis. Given this background, studies on natural bioactive molecules from marine algae have reported that fucoidans, polysaccharides from marine brown algae, and fucoxanthin and its deacetylated product, fucoxanthinol, exert anti-angiogenic properties [14,33]. 

To evaluate the anti-angiogenic effect of siphonaxanthin from green algae, we examined its 

anti-angiogenic effect in cell culture model systems and by employing *ex vivo* approaches using human umbilical vein endothelial cells (HUVECs) and rat aortic ring [34,35], respectively. Siphonaxanthin significantly suppressed HUVEC proliferation at relatively lower concentration of 2.5 ΐM, while its effect on chemotaxis was not significant [36]. In addition, siphonaxanthin exhibited a strong inhibitory effect on HUVEC tube formation in an *in vitro* angiogenesis model. Siphonaxanthin suppressed the tube length at a concentration of 10 ΐM, while no tube formation was observed at 25 ΐM, suggesting that this could be due to the suppression of angiogenic mediators. The *ex vivo* angiogenesis assay using rat aortic ring indicated reduced microvessel outgrowth in a dose-dependent manner, and the reduction was significant at concentrations of more than 2.5 ΐM. 

To elucidate the molecular mechanism underlying the anti-angiogenic activity of siphonaxanthin compared to fucoxanthin, we focused on the vascular specific proangiogenic factor, vascular endothelial growth factor (VEGF), and the other angiogenic factors, such as fibroblast growth factors (FGFs) [37,38]. It has been shown that both siphonaxanthin and fucoxanthin 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 [39]. These suppressive effects of siphonaxanthin were more effective than fucoxanthin. However, the mRNA expression of VEGFR-2, a potent signal transducer involved in the 

VEGF-mediated signaling pathway, was not significantly affected by these carotenoids. Furthermore, these two marine algal carotenoids can 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 these 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 effect of these two algal carotenoids and suggest that their anti-angiogenic effect is due to the down-regulation of signal transduction by 

FGFR-1 in vascular endothelial cells. Siphonaxanthin exhibited its anti-angiogenic effect at lower concentrations than fucoxanthin, indicating its potential as a strong angiogenesis inhibitor. 

These findings provide new insights into the novel bio-functional property of marine algal carotenoids, especially that of siphonaxanthin, which has the potential to enhance current anti-angiogenic therapies in the treatment of cancer and other pro-angiogenic diseases. 
