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We extracted marine low-temperature lysozyme (MLTL), a novel lysozyme, from a marine microorganism through fermentation. Our previous study suggested that a low molecular weight (16 kDa) may exert anti-tumor activity through antiangiogenesis. In this study, we extracted a high weight (39 kDa) and investigated its antiangiogenic activity in vivo and in vitro. Using zebrafish embryos as an in vivo study model, we found that treatment with MLTL significantly inhibited the growth of subintestinal vessels (SIVs) in a dose-dependent manner and that 400 µg/ml MLTL was sufficient to block the growth of SIVs. An in vitro study conducted using human umbilical vein endothelial cells (HUVECs) revealed that MLTL suppressed the proliferation, migration and tube formation of HUVECs in a dose-dependent manner. Interestingly, assays by flow cytometry and DNA electrophoresis indicated that MLTL was able to induce apoptosis of HUVECs. Moreover, further study demonstrated that the disruption of intracellular Ca2+ homeostasis may play an important role in MLTL induced apoptosis of HUVECs. Taken together, the results of this study demonstrate for the first time that MLTL inhibits angiogenesis through its pleiotropic effects on vascular endothelial cells and induces apoptosis through regulation of cellular Ca2+ levels. The results of this study also revealed a possible mechanism underlying the antiangiogenic effect of MLTL and suggested that MLTL may be a promising new antiangiogenic agent for use in cancer therapy.

Polypeptide from Chlamys farreri (PCF) is a novel marine bioactive product that was isolated from the gonochoric Chinese scallop Chlamys farreri, and was found to be an effective antioxidant in our recent studies. In this study, we investigated the effect of PCF on ultraviolet B (UVB)-induced apoptosis of HaCaT cells and the intracellular signaling pathways involved. Pretreatment with the inducible nitric oxide synthase (iNOS) inhibitor S-methylisothiourea sulfate inhibited UVB-induced apoptosis, indicating that iNOS and NO play important roles in apoptosis. On the other hand, the inhibition of UVB-induced apoptosis in the immortalized keratinocyte (HaCaT) cells by PCF was estimated using a DNA ladder. PCF treatment inhibited UVB-induced iNOS activation, as determined by RT-PCR, NO production, as determined by ESR, and up-regulated heat shock protein (HSP) 90 activation, as determined by Western blotting. Our results indicate that iNOS and NO are involved in UVB-induced apoptosis of HaCaT cells and the protective effect of PCF against UVB irradiation is exerted by suppressing the expression of iNOS, followed by inhibition of NO release and enhanced activation of HSP90.

A novel marine active polypeptide (PCF), isolated from the gonochoric Chinese scallop, Chlamys farreri, has potential antioxidant and anti-apoptotic activity against ultraviolet irradiation. We investigated whether UVB-induced HaCaT cell apoptosis occurs via the mitochondrial pathways Apaf-1/caspase-9 and Smac/XIAP/caspase-3. We then investigated the molecular mechanisms controlling the anti-apoptotic effect of PCF. Pre-treatment with PCF and caspase-9 inhibitor significantly inhibited UVB-induced apoptosis in HaCaT cells based on a DNA fragmentation assay and Hoechst 33258 staining. The expression of Apaf-1 and the cleavage of procaspase-9 were dose-dependently reduced by 1.42–5.96 mmol/L PCF pretreatment in UVB-irradiated HaCaT cells. This was followed by inhibition of cleavage of procaspase-3, whose activation induced cell apoptosis. Meanwhile, PCF significantly and dose-dependently enhanced the activation of ATPase. Furthermore, we demonstrated that PCF strongly inhibited the release of Smac from the mitochondria to cytosol by reducing the degradation of XIAP dose-dependently. We conclude that the protective effect of PCF against UVB irradiation in HaCaT cells may be attributed to the inhibition of the Apaf-1/caspase-9 and Smac/XIAP/caspase-3 apoptotic signaling pathways.