The synthetic derivative of
ascochlorin, 4-O-carboxymethyl
ascochlorin (AS-6) is an agonist of the
nuclear hormone receptor PPARĪ³ and has been shown to induce differentiation in mouse pre-adipocytes and to ameliorate type II diabetes in a murine model.
AS-6 was cytotoxic when added at micromolar concentrations to cultures of three different human
cancer cell lines. We used gel electrophoresis and mass spectrometry to identify
proteins with altered expression in human hepatocarcinoma cells (HepG2) cells after 12 h in the presence of
AS-6 and found 58
proteins that were differentially expressed. Many of the
proteins showing increased expression in cells treated with
AS-6 are involved in
protein quality control, including
glucose-regulated
protein 78 (
GRP78/BiP), a regulator of ER stress responses, and the transcriptional regulator CHOP, which mediates ER stress-induced apoptosis. Cells treated with
AS-6 undergo an autophagic response accompanied by increased expression of
beclin1, ATG5, and LC3-II and autophagosome formation marked by the appearance of large vesicles containing LC3-II.
Grp78 induction was inhibited when the PPARĪ³ antagonist,
GW9662, was added together with
AS-6, and autophagy and cell death were partially blocked. 3-methyl-adenine (3-MA), an inhibitor of
phosphatidyl inositol 3-kinase (PI3-kinase) prevented induction of ATG5 and activation of LC3-II and blocked autophagosome formation. 3-MA also blocked induction of
GRP78 and CHOP, suggesting that
PI3-kinase, which is known to mediate ER stress-induced autophagy, also plays a role in initiating apoptosis in response to ER stress. Together these data establish that the cytotoxicity of
AS-6 operates by a mechanism dependent on ER stress-induced autophagy and apoptosis.