The traditional medicinal herb Andrographis paniculata has known anti-tumor activity, and the potential active compound is andrographolide (ANGL), a diterpenoid lactone isolated from its leaves.ANGL inhibited tumor growth of hepatoma cancer cells (Hep3B) in a xenograft mouse tumor model, concomitant with a reduction in tumor vessel counts.No adverse effects such as reduced body weight were observed in vivo.Further, via targeting inhibiting angiogenesis and inducing apoptotic cytotoxicity, the involved mechanism was investigated.ANGL inhibited VEGF-induced phosphorylation of VEGFR2, with a consequent inhibition of the phosphorylated activation of downstream signals such as c-Jun N-terminal kinase (JNK), p38 and extracellular regulated kinase (ERK).ANGL interfered with the binding of VEGF to VEGFR2, but had no effect on VEGFR2 kinase activity in vitro.Our findings indicate that ANGL has anti-angiogenic activity which is mediated by preventing the binding of VEGF to VEGFR2, and downstream signaling through the VEGF-VEGFR2 cascade.Buthionine sulfoximine (BSO), an inhibitor of cellular GSH biosynthesis,significantly augmented ANGL-induced cytotoxicity in hepatoma Hep3B and HepG2 cells.BSO depleted cellular GSH, and augmented ANGL-induced apoptosis, inhibition of colony formation and JNK activation in Hep3B cells.BSO also increased ANGL-induced activation of apoptosis signal-regulating kinase 1 (ASK1), mitogen-activated protein kinase kinase-4 (MKK4) and c-Jun, which are all up-stream or down-stream signals of JNK.JNK inhibitor SP600125 and 420116 both reversed ANDRO-induced cytotoxicity.Our results demonstrate that there is a crosstalk between JNK activation and cellular GSH homeostasis, and ANDRO targets this to induce cytotoxicity in hepatoma cells.