CYTOSTATIC AND CYTOTOXIC PROPERTIES OF THE MARINE PRODUCT BISTRATENE-A AND ANALYSIS OF THE ROLE OF PROTEIN-KINASE-C IN ITS MODE OF ACTION

Bistratene A is a polyether which was isolated from the marine ascidia n Lissoclinum bistratum Sluiter. The hypothesis has been tested that t he cytostatic effect of bistratene A is mediated by modulation of prot ein kinase C (PKC). Human-derived A549 lung and MCF-7 breast adenocarc inoma cells are extremely sensitive to growth inhibition induced by ac tivators of PKC. Therefore, the effect of bistratene A on these cell l ines was compared with that of the known PKC activator 12-O-tetradecan oylphorbol-13-acetate (TPA). The ability of bistratene A to modulate P KC activity in cellular cytosol was assessed to determine the involvem ent of PKC in the induction of cytostasis. Bistratene A inhibited the growth of both cell lines and initial seeding density determined its c ytostatic potency. IC50 values were between 1.0 and 2.9 nM. Bistratene A also had a profound effect on the colony forming ability of A549 ce lls, preventing clonal growth at 5 nM. Using the incorporation of [H-3 ]thymidine into cells to assess DNA synthetic activity and the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay to define cytotoxicity, the compound was found to have both cytostatic and cyto toxic properties. bistratene A decomposed by 50% after only 2.8 hr in cell culture medium. TPA induced rapid motility and the formation of a network of branched colonies in both cell lines grown on Matrigel, wh ereas bistratene A did not cause the same effect. Cell cytosol was ana lysed for phorbol ester binding sites after treatment with bistratene A or TPA. Incubation with TPA (10 nM) caused a reduction in binding si tes to 57% of binding in control cells after 30 min and to 35% after 2 4 hr. Bistratene A did not cause a significant change in binding sites . Assays of PKC activity in cellular cytosol revealed that bistratene A was unable to activate or inhibit the enzyme at concentrations of up to 10 muM. The results suggest that bistratene A is an exquisitely po tent cytostatic agent in the two cell lines studied, but modulation of PKC is not involved in the mode of action by which it elicits this ef fect.