Cell membrane signaling as target in cancer therapy. II: Inhibitory effect of N,N,N-trimethylsphingosine on metastatic potential of murine B16 melanoma cell line through blocking of tumor cell-dependent platelet aggregation.

Two phenotypic parameters, aberrant expression of protein kinase C and tumor cell-induced platelet aggregation (PA), have been correlated with abnormal growth behavior and metastatic potential of tumor cells. We recently observed that N,N,N-trimethylsphingosine (TMS) and N,N-dimethylsphingosine (DMS), but not sphingosine (SPN), had an inhibitory effect (via blocking of transmembrane signaling) on the growth of various human tumor cell lines in vitro as well as in vivo in nu/nu mice (K. Endo et al., Cancer Res., 51: 1613-1618, 1991). We therefore investigated the effects of TMS, DMS, and SPN on (a) PA induced by ADP and thrombin; (b) PA induced by melanoma cell line B16/BL6; and (c) experimental lung colonization as well as spontaneous lung metastasis of BL6 cells in syngeneic C57BL/6 mice. In experiments on agonist-induced PA, TMS inhibited PA and ATP secretion 5-fold more strongly than DMS or SPN. This effect may be based on the inhibition of Mr 47,000 platelet protein phosphorylation and/or inhibition of phosphatidylinositol turnover as a transmembrane signaling pathway in platelets. Tumor cell (BL6 melanoma)-induced PA and ATP secretion were also strongly inhibited by TMS, but not by DMS or SPN. Unlike ADP- or thrombin-induced PA, BL6 cell-induced PA was not inhibited by Calphostin-C (a potent protein kinase C inhibitor) or cilostazol (a potent inhibitor of PA based on inhibition of cyclic AMP phosphodiesterase). Since many previous studies suggested that the ability of tumor cells to induce PA is related to the degree of malignancy (e.g., metastatic potential) of tumor cells, we studied the effect of TMS on lung metastatic potential. Three independent sets of experiments, as described below, all showed clear inhibition of lung metastasis by administration of TMS: (a) i.v. coinjection of BL6 melanoma cells and TMS; (b) i.v. injection of TMS and, 1 h later, BL6 cells; (c) spontaneous metastasis to lung from s.c. BL6 tumor (TMS administered after establishment of tumor, followed by resection of tumor). In comparison to tumor growth inhibition produced by TMS or DMS, inhibition of melanoma metastasis by TMS is obvious at lower doses.