Astragalus membranaceus has been used to ameliorate the side effects of
antineoplastic drugs because of its immunomodulating nature. We had recently demonstrated that total Astragalus
saponins (AST) possess anticarcinogenic and proapoptotic properties in human
colon cancer cells and
tumor xenograft. In this study, we identified
NSAID-activated gene (NAG-1) as a potential molecular target of AST. The growth-inhibitory and proapoptotic effects of AST were assessed in a panel of human
cancer cell lines.
Hoechst 33342 nuclear staining,
Annexin V-FITC/
propidium iodide staining, Western immunoblotting, real-time PCR,
luciferase reporter assay and electrophoretic mobility shift assay were conducted to determine the association of NAG-1 and related
transcription factors with AST during its regulation of apoptotic activities. Moreover, the combined proapoptotic and NAG-1 promoting activities of AST and/or inhibitors of the PI3K-Akt pathway were also examined. AST caused overexpression of NAG-1, leading to PARP cleavage and apoptosis. The induction of NAG-1 promoter activity by the
drug was associated with increased gene expression, in addition to prior increase in Egr-1 expression and
DNA binding activity. AST-induced NAG-1 activation was intensified when PI3K inhibitor
LY294002 or Akt inhibitor was co-treated and reversed by NAG-1
siRNA transfection. Nevertheless, the extent of NAG-1 induction could not be altered by the ERK inhibitor
PD98059. Our results indicate that NAG-1 is a potential molecular target of AST in its antitumorigenic and proapoptotic actions, which would have additive effects when used along with PI3K-Akt inhibitors. The information obtained could facilitate future development of a novel target-specific chemotherapeutic agent with known molecular pathway.