20-O-(beta-D-glucopyranosyl)-20(S)-protopanaxadiol (
IH901), an intestinal bacterial metabolite of ginseng
saponin formed from
ginsenosides Rb1, Rb2, and Rc, is suggested to be a potential chemopreventive agent. Here, we show that
IH901 induces apoptosis in human
hepatoblastoma HepG2 cells.
IH901 led to an early activation of
procaspase-3 (12 h posttreatment), and the activation of
caspase-8 became evident only later (18 h posttreatment).
Caspase activation was a necessary requirement for apoptosis because
caspase inhibitors significantly inhibited cell death by
IH901. Treatment of HepG2 cells with
IH901 also induced the cleavage of cytosolic factors such as Bid and Bax and translocation of truncated Bid (tBid) to mitochondria. A time-dependent release of
cytochrome c from mitochondria was observed, which was accompanied by activation of
caspase-9. A broad-spectrum
caspase inhibitor, N-
benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (
zVAD-fmk), and a specific inhibitor for
caspase-8, N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone (zIETD-fmk), abrogated Bid processing and translocation, and
caspase-3 activation.
Cytochrome c release was inhibited by
zVAD-fmk, however, the inhibition by zIETD-fmk was not complete. The activation of
caspase-8 was inhibited not only by zIETD-fmk but also by
zVAD-fmk. The results, together with the kinetic change of
caspase activation, indicate that activation of
caspase-8 occurred downstream of
caspase-3 and -9. Our data suggest that the activation of
caspase-8 after early
caspase-3 activation might act as an amplification loop necessary for successful apoptosis. Primary hepatocytes isolated from normal Sprague-Dawley rats were not affected by
IH901 (0-60 microM). The very low toxicity in normal hepatocytes and high activity in
hepatoblastoma HepG2 cells suggest that
IH901 is a promising experimental
cancer chemopreventive agent.