Alterations resulting in enhanced epidermal growth factor receptor (EGFR) expression or function have been documented in a variety of tumors. Therefore, EGFR‐tyrosine kinase is a promising therapeutic target. Although in vitro and in vivo studies have shown the anti‐tumor activity of EGFR‐tyrosine kinase inhibitors against various tumor types, little is known about the mechanism by which such inhibitors effect their anti‐tumor action. AG1478 is known to selectively inhibit EGFR‐tyrosine kinase. In this study, we showed that AG1478 caused apoptosis and apoptosis‐related reactions such as the activation of caspase 3 in human non‐small cell lung cancer cell line PC‐9. To investigate the signaling route by which AG1478 induced apoptosis, we examined the activation of c‐Jun N‐terminal kinase (JNK) and mitogen‐activated protein kinase p38 in AG1478‐treated PC‐9 cells. JNK, but not p38, was significantly activated by AG1478 as determined by both immunoblot analysis for levels of phosphorylated JNK and an in vitro activity assay. Various types of stimuli activated JNK through phosphorylation by the dual‐specificity JNK kinases, but the dual‐specificity JNK kinases MKK4 and MKK7 were not activated by AG1478 treatment. However, JNK phosphatase, i.e. mitogen‐activated protein kinase phosphatase‐1 (MKP‐1), was constitutively expressed in the PC‐9 cells, and its expression level was reduced by AG1478. The inhibition of JNK activation by ectopic expression of MKP‐1 or a dominant‐negative form of JNK strongly suppressed AG1478‐induced apoptosis. These results reveal that JNK, which is activated through the decrease in the MKP‐1 level, is critical for EGFR‐tyrosine kinase inhibitor‐induced apoptosis.

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Abstract. Angiostatin (AS), a proteolytic fragment of plasminogen, is a potent antiangiogenic factor. It was reported that AS attenuates the vasodilatory response to vascular endothelial growth factor (VEGF) in isolated interventricular arterioles. Here, we investigated the effect of AS on nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs). AS inhibited VEGF-stimulated NO production in a dose-dependent manner, whereas AS alone did not affect basal NO production. Disruption of kringle structures by reduction of disulfide bonds resulted in the loss of the inhibitory effect of AS on VEGF-stimulated NO production. To elucidate how AS might impair VEGF activation of endothelial NO synthase (eNOS), we further examined whether AS would affect Ca 2+ -dependent and -independent pathways of eNOS activation. AS had no effect on the transient increase in cytosolic Ca 2+ levels elicited by VEGF. In contrast, AS prevented VEGF-potentiated eNOS phosphorylation at Ser1177. These results clearly indicate that AS inhibits VEGF-stimulated NO production in HUVECs without affecting basal NO production. The kringle structures of AS are required for this effect, and impairment of Ser1177 phosphorylation of eNOS might be involved in the inhibition of VEGF-stimulated NO production by AS.

Hit, Lead & Candidate Discovery Antiangiogenesis therapy is a promising way for treatment of solid cancers, and many angiogenesis inhibitors that target vascular endothelial growth factor (VEGF) or its receptors have been developed. We explored novel antiangiogenic compounds other than anti‐VEGF drugs by screening our synthetic compound library and found that 6‐thiophen‐3‐yl‐2‐methoxy‐1,4‐naphthoquinone (6‐TMNQ) had potential as a novel angiogenesis inhibitor. This paper describes the effects of 6‐TMNQ on angiogenesis and tumor growth in vitro and in vivo. 6‐TMNQ inhibited serum‐, VEGF‐, and basic fibroblast growth factor (bFGF)‐stimulated proliferation of endothelial cells in a concentration‐dependent manner, but had no effect on the proliferation of fibroblasts. VEGF‐induced activation of VEGF receptor‐2 in endothelial cells was not affected by the compound. 6‐TMNQ markedly abrogated both migration and tube formation of endothelial cells. Orally administered 6‐TMNQ inhibited angiogenesis in response to VEGF or bFGF in mice in a dose‐dependent manner. Furthermore, when tumor‐bearing mice were treated with 6‐TMNQ, increase in tumor size was significantly prevented due to inhibition of angiogenesis in the tumor tissues. These results demonstrate that 6‐TMNQ is an orally available compound that selectively inhibits endothelial cell proliferation and migration, and abrogates angiogenesis, resulting in the prevention of tumor growth. The mechanism of 6‐TMNQ action is different from that of conventional anti‐VEGF drugs.