Nereis active protease (NAP) is a novel fibrinolytic active serine protease from the polychaete Nereis virens. In this study, NAP was purified from Nereis virens and the effects of NAP on human lung cancer cells were investigated. Our results indicated that NAP inhibited the proliferation and induced apoptosis of H1299 cells in a time-and dose-dependent manner. The loss of mitochondrial membrane potential, the activation of Bax and cleaved-caspase 3/9, the release of cytochrome C, and the

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... ssion of Bcl-2 and poly-ADP ribose polymerase were observed in NAP-treated H1299 cells by flow cytometry and Western blotting. Moreover, the expression levels of Bax and Bcl-2 mRNA were determined by real-time quantitative polymerase chain reaction and the Bax/Bcl-2 expression ratio was increased in the NAP-treated cell lines. The results indicated that NAP-induced apoptosis may be related to mitochondria mediated apoptosis and occurs through caspase-dependent pathways. Then, the effects of NAP on tumor growth in animal models were observed, where 5 or 10 mg/kg of NAP noticeably reduced tumor volume and weight and increased apoptosis as determined by Western blotting when compared to the negative control group. Therefore, our findings suggest that NAP could be a hopeful anticancer medicine for its propensity to inhibit growth and induce of apoptosis in human lung cancer cells. or lead to treatment failures [15] . Apoptosis has become a preferable choice of cancer cell death for the treatment of cancers because of it does not trigger inflammatory or immune responses. Recently, several anticancer or anti-proliferative peptides have been purified from marine-derived protein hydrolysates to induce apoptosis. For example, Gln-Pro-Lys, a tripeptide from Sepia ink protein hydrolyates, was found to inhibit the proliferation of prostate cancer cells in a time-and dose-dependent manner [16] . Phe-Ile-Met-Gly-Pro-Tyr, a hexapeptide from protein hydrolysates of skate (Raja porosa) cartilage, was found to induce apoptosis by upregulating the Bax/Bcl-2 ratio and activating caspase-3 in HeLa cells [7] . Ala-Val-Leu-Val-Asp-Lys-Gln-Cys-Pro-Asp, a decapeptide purified from Ruditapes philippinarum protein hydrolysates, was found to have inhibitory effects on breast, prostate, and lung cancer cell proliferation [13] . However, there were no references in relation to anti-lung cancer protease extracted from marine sources. In this study, purified Nereis serine protease (NAP) was obtained from Nereis virens, according to the previous study [17] , and in vitro experiments carried out using lung cancer cells to determine the effectiveness of NAP in inducing apoptosis. Furthermore, in vivo experiments were also performed to determine whether NAP suppresses lung tumor growth. Our results indicated that NAP inhibited the proliferation of human lung cancer cells, especially H1299 cells, induced apoptosis and regulated protein expression related to apoptosis through mitochondrial dysfunction. Results Purification of NAP NAP was purified from Nereis virens through ammonium sulfate precipitation, anion exchange chromatography, and gel chromatography. Protease activity was used to monitor the purification. The results of the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis ( Figure 1 ) showed that purified NAP was successfully obtained and its molecular weight is estimated to be about 29 kDa, which is consistent with the previous study [18] . The total recovery of NAP from Nereis virens was approximately 35.6%.