Mechanism of inhibition of tumor angiogenesis by beta-hydroxyisovalerylshikonin.

Shikonin and beta-hydroxyisovalerylshikonin (beta-HIVS) from Lithospermum erythrorhizon inhibit angiogenesis via inhibition of vascular endothelial growth factor receptors (VEGFR) in an adenosine triphosphate-non-competitive manner, although the underlying molecular mechanism has not been fully understood. In the present study, we found that beta-HIVS inhibited angiogenesis within chicken chorioallantoic membrane approximately threefold more efficiently than shikonin. beta-HIVS also significantly inhibited angiogenesis in two other assays, induced either by Lewis lung carcinoma cells implanted in mouse dorsal skin or by VEGF in s.c. implanted Matrigel plugs and metastasis of Lewis lung carcinoma cells to lung. Therefore, using beta-HIVS as a bioprobe, we investigated the molecular mechanism of shikonin's anti-angiogenic actions. beta-HIVS inhibited the phosphorylation and expression of VEGFR2 and Tie2 without affecting VEGFR1 and fibroblast growth factor receptor 1 levels. beta-HIVS suppressed the phosphorylation but not the expression of extracellular signal-regulated kinase, and an Sp1-dependent transactivation of the VEGFR2 and Tie2 promoters, thereby suppressing the proliferation of vascular endothelial and progenitor cells. This was mimicked by an Sp1 inhibitor mithramycin A and partially rescued by Sp1 overexpression. These results implicate potential use of shikonin and beta-HIVS as leading compounds for clinical application in the future by virtue of their unique properties including: (i) inhibition of VEGFR2 and Tie2 phosphorylation in an adenosine triphosphate-non-competitive manner; (ii) simultaneous inhibition of the phosphorylation and expression of VEGFR2 and Tie2; and (iii) bifunctional inhibition of the growth in endothelial cells and vascular remodeling.

Involvement of tumor necrosis factor receptor-associated protein 1 (TRAP1) in apoptosis induced by beta-hydroxyisovalerylshikonin.

beta-Hydroxyisovalerylshikonin (beta-HIVS), a compound isolated from the traditional oriental medicinal herb Lithospermum radix, is an ATP non-competitive inhibitor of protein-tyrosine kinases, such as v-Src and EGFR, and it induces apoptosis in various lines of human tumor cells. However, the way in which beta-HIVS induces apoptosis remains to be clarified. In this study, we performed cDNA array analysis and found that beta-HIVS suppressed the expression of the gene for tumor necrosis factor receptor-associated protein 1 (TRAP1), which is a member of the heat-shock family of proteins. When human leukemia HL60 cells and human lung cancer DMS114 cells were treated with beta-HIVS, the amount of TRAP1 in mitochondria decreased in a time-dependent manner during apoptosis. A similar reduction in the level of TRAP1 was also observed upon exposure of cells to VP16. Treatment of DMS114 cells with TRAP1-specific siRNA sensitized the cells to beta-HIVS-induced apoptosis. Moreover, the reduction in the level of expression of TRAP1 by TRAP1-specific siRNA enhanced the release of cytochrome c from mitochondria when DMS114 cells were treated with either beta-HIVS or VP16. The suppression of the level of TRAP1 by either beta-HIVS or VP16 was blocked by N-acetyl-cysteine, indicating the involvement of reactive oxygen species (ROS) in the regulation of the expression of TRAP1. These results suggest that suppression of the expression of TRAP1 in mitochondria might play an important role in the induction of apoptosis caused via formation of ROS.

Sophoranone, extracted from a traditional Chinese medicine Shan Dou Gen, induces apoptosis in human leukemia U937 cells via formation of reactive oxygen species and opening of mitochondrial permeability transition pores.

Screening of various natural products in a search for novel inducers of apoptosis in human leukemia cells led us to identify the strong apoptosis-inducing activity in a fraction extracted with methanol from the roots of Sophora subprostrata Chun et T. Chen. We purified the compound that induced apoptosis in human leukemia cells and identified it as sophoranone. Sophoranone inhibited cell growth and induced apoptosis in various lines of cells from human solid tumors, with 50% inhibition of growth of human stomach cancer MKN7 cells at 1.2 +/- 0.3 microM. The growth-inhibitory and apoptosis-inducing activities of sophoranone for leukemia U937 cells were very much stronger than those of other flavonoids, such as daidzein, genistein and quercetin. At the early stages of treatment of U937 cells with sophoranone, reactive oxygen species were formed, mitochondrial permeability pores were opened and cytochrome c was released from mitochondria. Cytochrome c was also released upon treatment of isolated mitochondria with sophoranone. Inhibitors of complexes III and IV, but not complexes I and II, of the mitochondrial respiratory chain prevented the release of cytochrome c from isolated mitochondria by sophoranone, as well as the induction of apoptosis in U937 cells in response to sophoranone. Our results indicate that sophoranone might be a unique apoptosis-inducing anticancer agent that targets mitochondria.