Apoptosis-inducing protein, AIP, from parasite-infected fish induces apoptosis in mammalian cells by two different molecular mechanisms.

AlP (apoptosis-inducing protein) is a protein purified and cloned from Chub mackerel infected with the larval nematode, Anisakis simplex, which induces apoptosis in various mammalian cells including human tumor cell lines. AlP has shown structural and functional homology to Lamino acid oxidase (LAO) which oxidizes several L-amino acids including L-lysine and AlP-induced apoptosis has been suggested to be mediated by H[sub 2]O[sub 2] generated by LAO activity of AlP. In this study, we confirmed that recombinant AlP generated enough H[sub 2]O[sub 2] in culture medium to induce rapid apoptosis in cells and this apoptosis was clearly inhibited by co-cultivation with antioxidants such as catalase and Nacetyl-cysteine. Surprisingly, however, we found that AlP still could induce H[sub 2]O[sub 2]-independent apoptosis more slowly than H[sub 2]O[sub 2]-dependent one in HL-60 cells even in the presence of antioxidants. In addition, the HL-60-derived cell line HP100-1, which is a H[sub 2]O[sub 2]-resistant variant, underwent apoptosis on treatment with AlP with a similar delayed time course. The latter apoptosis was completely blocked by addition of L-lysine to the culture medium, which is the best substrate of AlP as LAO, indicating that decreased concentration of L-lysine in the culture medium by AlP treatment induced apoptosis. We also showed that the both apoptosis by AlP were associated with the release of cytochrome c from mitochondria and activation of caspase9, and overexpressed Bcl-2 could inhibit both of the AlPinduced apoptosis. These results indicate that AlP induces apoptosis in cells by two distinct mechanisms; one rapid and mediated by H[sub 2]O[sub 2], the other delayed and mediated by deprivation of L-lysine, both of which utilize caspase-9/ cytochrome c system. [ABSTRACT FROM AUTHOR]