The bryostatins inhibit growth of B16/F10 melanoma cells in vitro through a protein kinase C-independent mechanism: Dissociation of activities using 26-epi-bryostatin

Bryostatin 1 is a potential cancer chemotherapeutic agent in Phase II clinical trials, with positive responses observed for malignant melanoma, among other tumors. The bryostatins are known to be potent ligands for protein kinase C (PKC), functioning as partial antagonists. In the present study, we explore the mechanism by which the bryostatins inhibit growth of B16/F10 mouse melanoma cells in vitro. Three experimental approaches suggest that the growth inhibition is independent of PKC. First, we characterized in detail the translocation and down-regulation of the PKC isozymes α, δ, and ε in response to phorbol ester and bryostatin 1 in these cells. Although the dose-response curves obtained for the translocation-activation of PKC isozymes showed good correlation with the growth-enhancing activity of phorbol 12-myristate 13-acetate, for no PKC isozyme was there a good correlation with the growth-inhibitory activity of bryostatin 1. Second, inhibition of PKC enzymatic activity by the specific PKC inhibitor bisindolyl-maleimide I did not block the inhibition of thymidine incorporation induced by bryostatin 1. Finally, 26-epi-bryostatin 1, a stereoisomer of the naturally occurring bryostatin 1 designed to have markedly reduced affinity for PKC, inhibited the growth of the B16/F10 melanoma cell lines with potency similar to that of bryostatin 1. We confirmed here that 26-epi-bryostatin 1 showed 60-fold reduced affinity for PKC and 30-60-fold reduced potency to translocate and down-regulate PKC isozymes compared with bryostatin 1. We presume that the principal toxicity of bryostatin 1 reflects its interaction with PKC, and we would thus predict that epi-bryostatin 1 would be less toxic. Indeed, we found at least 10-fold reduced toxicity of 26-epi-bryostatin 1 in C57BL/6 mice compared with bryostatin 1. We conclude that the growth inhibition of the bryostatins, at least in this system, does not result from interaction with PKC. As exemplified by 26-epi-bryostatin 1, this insight permits the design of analogues with comparable growth inhibition to bryostatin 1 but with reduced toxicity.