Sparstolonin B (SsnB) is a novel bioactive compound isolated from Sparganium stoloniferum, an herb historically used in Traditional Chinese Medicine as an anti-tumor agent. Angiogenesis, the process of new capillary formation from existing blood vessels, is dysregulated in many pathological disorders, including diabetic retinopathy, tumor growth, and atherosclerosis. In functional assays, SsnB inhibited endothelial cell tube formation (Matrigel method) and cell migration (Transwell method) in a dose-dependent manner. Microarray experiments with human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (HCAECs) demonstrated differential expression of several hundred genes in response to SsnB exposure (916 and 356 genes, respectively, with fold change ≥2, p<0.05, unpaired t-test). Microarray data from both cell types showed significant overlap, including genes associated with cell proliferation and cell cycle. Flow cytometric cell cycle analysis of HUVECs treated with SsnB showed an increase of cells in the G1 phase and a decrease of cells in the S phase. Cyclin E2 (CCNE2) and Cell division cycle 6 (CDC6) are regulatory proteins that control cell cycle progression through the G1/S checkpoint. Both CCNE2 and CDC6 were downregulated in the microarray data. Real Time quantitative PCR confirmed that gene expression of CCNE2 and CDC6 in HUVECs was downregulated after SsnB exposure, to 64% and 35% of controls, respectively. The data suggest that SsnB may exert its anti-angiogenic properties in part by downregulating CCNE2 and CDC6, halting progression through the G1/S checkpoint. In the chick chorioallantoic membrane (CAM) assay, SsnB caused significant reduction in capillary length and branching number relative to the vehicle control group. Overall, SsnB caused a significant reduction in angiogenesis (ANOVA, p<0.05), demonstrating its ex vivo efficacy.
References
[1]
Qiu C, Xu X, Zhu H (2008) The studies on the anti-inflammatory and analgesia effects of SLW. Pharmacology and Clinics of Chinese Materia Medica 14: 7–10.
[2]
Xiong Y, Deng KZ, Guo YQ, Gao WY, Zhang TJ (2009) New chemical constituents from the rhizomes of Sparganium stoloniferum. Arch Pharm Res 32: 717–720.
[3]
Liang Q, Wu Q, Jiang J, Duan J, Wang C, et al. (2011) Characterization of sparstolonin B, a Chinese herb-derived compound, as a selective Toll-like receptor antagonist with potent anti-inflammatory properties. J Biol Chem 286: 26470–26479.
[4]
Folkman J (1971) Tumor angiogenesis: therapeutic implications. N Engl J Med 285: 1182–1186.
[5]
Risau W (1997) Mechanisms of angiogenesis. Nature 386: 671–674.
[6]
Staton CA, Reed MW, Brown NJ (2009) A critical analysis of current in vitro and in vivo angiogenesis assays. Int J Exp Pathol 90: 195–221.
[7]
Bussolino F, Mantovani A, Persico G (1997) Molecular mechanisms of blood vessel formation. Trends Biochem Sci 22: 251–256.
[8]
Carmeliet P (2003) Angiogenesis in health and disease. Nat Med 9: 653–660.
[9]
Liekens S, De Clercq E, Neyts J (2001) Angiogenesis: regulators and clinical applications. Biochem Pharmacol 61: 253–270.
[10]
Pandya NM, Dhalla NS, Santani DD (2006) Angiogenesis–a new target for future therapy. Vascul Pharmacol 44: 265–274.
[11]
Wu Z, Cho H, Hampton GM, Theodorescu D (2009) Cdc6 and cyclin E2 are PTEN-regulated genes associated with human prostate cancer metastasis. Neoplasia 11: 66–76.
[12]
Borlado LR, Mendez J (2008) CDC6: from DNA replication to cell cycle checkpoints and oncogenesis. Carcinogenesis 29: 237–243.
[13]
Nigg EA (2001) Mitotic kinases as regulators of cell division and its checkpoints. Nat Rev Mol Cell Biol 2: 21–32.
[14]
Sullivan M, Morgan DO (2007) Finishing mitosis, one step at a time. Nat Rev Mol Cell Biol 8: 894–903.
[15]
Tassan JP, Schultz SJ, Bartek J, Nigg EA (1994) Cell cycle analysis of the activity, subcellular localization, and subunit composition of human CAK (CDK-activating kinase). J Cell Biol 127: 467–478.
[16]
O'Farrell PH (2001) Triggering the all-or-nothing switch into mitosis. Trends Cell Biol 11: 512–519.
[17]
Maxwell PH, Wiesener MS, Chang GW, Clifford SC, Vaux EC, et al. (1999) The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis [see comments]. Nature 399: 271–275.
[18]
Meng H, Xing G, Sun B, Zhao F, Lei H, et al. (2010) Potent angiogenesis inhibition by the particulate form of fullerene derivatives. ACS Nano 4: 2773–2783.
[19]
Rohde LE, Lee RT (2003) Pathophysiology of atherosclerotic plaque development and rupture: an overview. Semin Vasc Med 3: 347–354.
[20]
Scoditti E, Calabriso N, Massaro M, Pellegrino M, Storelli C, et al. (2012) Mediterranean diet polyphenols reduce inflammatory angiogenesis through MMP-9 and COX-2 inhibition in human vascular endothelial cells: a potentially protective mechanism in atherosclerotic vascular disease and cancer. Arch Biochem Biophys 527: 81–89.
[21]
Kwak HJ, Park MJ, Park CM, Moon SI, Yoo DH, et al. (2006) Emodin inhibits vascular endothelial growth factor-A-induced angiogenesis by blocking receptor-2 (KDR/Flk-1) phosphorylation. Int J Cancer 118: 2711–2720.
[22]
Wang XH, Wu SY, Zhen YS (2004) Inhibitory effects of emodin on angiogenesis. Yao Xue Xue Bao 39: 254–258.
[23]
Hsu WH, Lee BH, Pan TM (2010) Red mold dioscorea-induced G2/M arrest and apoptosis in human oral cancer cells. J Sci Food Agric 90: 2709–2715.
[24]
Cude K, Wang Y, Choi HJ, Hsuan SL, Zhang H, et al. (2007) Regulation of the G2-M cell cycle progression by the ERK5-NFkappaB signaling pathway. J Cell Biol 177: 253–264.
[25]
Liu F, Li X, Wang C, Cai X, Du Z, et al. (2009) Downregulation of p21-activated kinase-1 inhibits the growth of gastric cancer cells involving cyclin B1. Int J Cancer 125: 2511–2519.
[26]
Wang Q, Zou L, Liu W, Hao W, Tashiro S, et al. (2011) Inhibiting NF-kappaB activation and ROS production are involved in the mechanism of silibinin's protection against D-galactose-induced senescence. Pharmacol Biochem Behav 98: 140–149.
[27]
Meteoglu I, Erdogdu IH, Meydan N, Erkus M, Barutca S (2008) NF-KappaB expression correlates with apoptosis and angiogenesis in clear cell renal cell carcinoma tissues. J Exp Clin Cancer Res 27: 53.
[28]
Johnson C, Sung HJ, Lessner SM, Fini ME, Galis ZS (2004) Matrix metalloproteinase-9 is required for adequate angiogenic revascularization of ischemic tissues: potential role in capillary branching. Circ Res 94: 262–268.
[29]
Huang B, Zhao J, Unkeless JC, Feng ZH, Xiong H (2008) TLR signaling by tumor and immune cells: a double-edged sword. Oncogene 27: 218–224.
