The study of tumor growth inhibiting activity purified from conditioned medium of human malignant melanoma cells

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In the present study, we purified the growth-inhibitory factors released by cultured MMN9 melanoma cells and identified one factor to be β_2-microglobulin (β_2M). Exogenous b2M induced apoposis in all melanoma cell lines tested but not normal fibroblasts. Thus, β_2M is a pro-apoptotic factor. The general caspase inhibitor Z-VAD-fmk and the caspase-3/-7 inhibitor DEVD-CHO did not block β_2M-induced apoptosis, indicating β_2M-induced apoposis does not involve the caspase-dependent pathway that characterizes the classical Fas-mediated apoptosis. Furthermore, bcl-2 expression did not alter during β_2M-induced apoptosis and the cleaved bcl-2 band that appears during Fas-mediated apoptosis of MMN9 cells was never observed. However, expression of the pro-apoptotic bax and bak molecules was upregulated later in β_2M-induced apoptosis. Thus, β_2M-induced apoptosis may involve bak- and bax-dependent apoptotic pathways. The morphological features of β_2M-induced apoptosis are similar to those observed in apoposis induced by the antibody ligation of MHC I molecules. In addition, like MHC I ligation-induced apoptosis, PI-3 kinase was activated at an early stage of β_2M-induced apoptosis and the PI-3 kinase inhibitor wortmannin blocked this apoptosis. Thus, β_2M-induced apoptosis of MMN9 melanoma cells may involve a signal sent through the MHC I molecule that activates PI-3 kinase, which in turn activates the bcl-2 oncoprotein family at a later stage of apoptosis. Understanding the apoptotic pathway induced by β_2M may suggest useful therapeutic strategies that aim to eliminate melanoma cells.