Regulation of epidermal growth factor receptor activity by crotoxin, a snake venom phospholipase A(2) toxin - A novel growth inhibitory mechanism

Crotoxin (CT), a phospholipase A(2) (PLA(2)) derived from the venom of Crotalus durissus terrificus, is a heterodimeric protein composed of subunit B with enzymatic activity and a binding regulatory subunit (A) without enzyme activity. Although the PLA(2) activity of CT may be important in its anti-proliferative activity, its cytostatic mechanism is unknown. In this study, we examined the cytostatic effect of PLA(2)-associated CT activity on squamous carcinoma cells expressing distinct levels of epidermal growth factor receptor (EGFr). CT was most effective in suppressing growth on cells expressing high intrinsic levels of EGFr. Cardiotoxin, another membrane active toxin with no intrinsic PLA(2) activity, had no differential anti proliferative activity on cells expressing high EGFr levels, suggesting a correlation between EGFr expression and CT-directed antiproliferative activity. Both chemically modified CT (MCT) devoid of PLA(2) activity and covalently cross linked CT (CCT), which is functionally unable to utilize cellular membranes as PLA(2) substrate, were also without growth inhibitory activity. No evidence for direct binding of CT to EGFr was found, although pretreatment with EGF was able to partially suppress the anti-proliferative activity of CT. Tyrosine phosphorylation of EGFr, however, was stimulated by CT in intact A431 cells. Tyrosine phosphorylation of EGFr was concentration-dependently stimulated (3- to 8-fold) in cellular membranes of A431 cells treated in vitro with CT but not with anti-proliferatively inactive MCT or CCT. The data provide evidence for transmembrane receptors involved in growth signaling (namely EGFr) as cellular targets and potential effectors of PLA(2)-mediated anti-proliferative activity of snake venom.