There is a long history about the usage of traditional Chinese medicine (TCM), and the medicine is renowned in treating diseases. However, the correlations between active ingredients and efficacies of TCM are still poorly understood. To identify active compounds from TCM for drug development, a microarray-based drug screening platform, constructed by arraying HPLC fractions of herbal extracts onto a surface-activated polystyrene slide, has been developed. The compounds deriving from individual herbal fractions were chemically coupled to a chip, named as HerboChips®. The phytochemical-coated chip could be applied in drug screening. The biotinylated protein probes, e.g. growth factor, hormone and enzyme, could be used to screen thousands of HerboChips within a short period of ti...[ Read more ]

There is a long history about the usage of traditional Chinese medicine (TCM), and the medicine is renowned in treating diseases. However, the correlations between active ingredients and efficacies of TCM are still poorly understood. To identify active compounds from TCM for drug development, a microarray-based drug screening platform, constructed by arraying HPLC fractions of herbal extracts onto a surface-activated polystyrene slide, has been developed. The compounds deriving from individual herbal fractions were chemically coupled to a chip, named as HerboChips®. The phytochemical-coated chip could be applied in drug screening. The biotinylated protein probes, e.g. growth factor, hormone and enzyme, could be used to screen thousands of HerboChips within a short period of time. In view of this application, we aimed to identify effective compounds from TCM that could interact with vascular endothelial growth factor (VEGF) using HerboChips. The biotinylated-VEGF was hybridized with chips coated with different HPLC-separated fractions of herbal extracts. In the screening process, the Straptavidin-Cy5 was used to identify the VEGF-bound fractions. Over 500 TCM chips were screened, and several positive hits were identified, e.g. Ginkgo Folium, Polygoni Cuspidati Rhizoma et Radix, etc. The interaction of identified herbal extracts, or phytochemicals with VEGF, was further confirmed in cultured human umbilical endothelial cells (HUVECs). In HUVECs, the identified TCMs exerted potentiating or inhibitory effects on the VEGF-stimulated cell proliferation, cell migration, cell invasion and tube formation, as well as the VEGF-mediated downstream signallings.

By using biacore assay, immunoprecipitation assay and molecular docking, the identified chemicals, i.e. resveratrol and polydatin from Polygoni Cuspidati Rhizoma et Radix, ginkgetin and kaempferol from Ginkgo Folium, were further shown to bind with VEGF markedly. To demonstrate the angiogenic functions of identified compounds, the application of resveratrol, polydatin and ginkgetin suppressed VEGF-induced cell mobilities in cultured HUVECs. These compounds exerted inhibitory effects on the sub-intestinal vessel formation in zebrafish embryos in vivo, as well as microvascular sprouting in rat aortic ring ex vivo. In signalling cascade, the application of resveratrol/polydatin/ginkgetin attenuated VEGF-induced phosphorylations of VEGF receptor 2 and c-Jun N-terminal kinase (JNK), and the VEGF-induced phosphorylations of Akt, eNOS and Erk were significantly decreased after the treatment of these phytochemicals. In contrast, kaempferol potentiated the VEGF-induced cell motility in HUVECs, as well as the formation of sub-intestinal vessel in zebrafish embryos and microvascular sprouting in rat aortic ring. In line to these observations, the potentiation effect of kaempferol was revealed in VEGF-induced migration of skin cell and monocyte.

By auto-docking analysis, ginkgetin and resveratrol showed different binding sites to VEGF. The synergy of ginkgetin and resveratrol in suppressing the VEGF-induced endothelial cell proliferation, migration and tube formation was revealed; the calculated combination index was less than 1, indicating the synergistic effect in angiogenesis. The synergy of ginkgetin and resveratrol was further illustrated in HT-29 colon cancer xenograft nude mice. Ginkgetin and resveratrol, when applied together, exerted synergistic anti-tumour effect of 5-fluorouracil with decreasing microvessel density and suppression of CD31 expression in the tumours. In parallel, the combination of ginkgetin and resveratrol synergistically relieved 5-fluorouracil-induced inflammatory response by suppressing expressions of COX-2 and inflammatory cytokines.

Thus, our investigations supported applicability of HerboChips in screening VEGF binding components from TCM herbal extracts. The angiogenic roles of aforementioned identified TCMs and compounds, as well as their signalling mechanism in blocking the VEGF-mediated responses, provided support to possible development of herbals extract and/or compounds as potential therapeutic agents for prevention and treatment of angiogenesis-related diseases. For example, the anti-angiogenic roles of ginkgetin and resveratrol in combination could provide effective therapeutic strategy in cancer, similar to that of Avastin, in suppressing the VEGF-mediated angiogenesis during cancer development.