Antofine, a
phenanthroindolizidine alkaloid derived from Cryptocaryachinensis and Ficusseptica in the Asclepiadaceae milkweed family, is cytotoxic for various
cancer cell lines. In this study, we demonstrated that treatment of rat primary astrocytes with
antofine induced dose-dependent inhibition of gap junction intercellular communication (GJIC), as assessed by scrape-loading
6-carboxyfluorescein dye transfer. Levels of
Cx43 protein were also decreased in a dose- and time-dependent manner following
antofine treatment. Double-labeling immunofluorescence microscopy showed that
antofine (10ng/ml) induced endocytosis of surface gap junctions into the cytoplasm, where
Cx43 was co-localized with the early endosome marker EEA1. Inhibition of lysosomes or proteasomes by co-treatment with
antofine and their respective specific inhibitors, NH4Cl or
MG132, partially inhibited the
antofine-induced decrease in
Cx43 protein levels, but did not inhibit the
antofine-induced inhibition of GJIC. After 30min of treatment,
antofine induced a rapid increase in the intracellular Ca(2+) concentration and activation of
protein kinase C (PKC)α/βII, which was maintained for at least 6h. Co-treatment of astrocytes with
antofine and the intracellular Ca(2+)
chelator BAPTA-AM prevented downregulation of
Cx43 and inhibition of GJIC. Moreover, co-treatment with
antofine and a specific PKCβ inhibitor prevented endocytosis of gap junctions, downregulation of
Cx43, and inhibition of GJIC. Taken together, these findings indicate that
antofine induces
Cx43 gap junction disassembly by the PKCβ signaling pathway. Inhibition of GJIC by
antofine may undermine the
neuroprotective effect of astrocytes in CNS.