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Mechanistic study of the anti-cancer effect of gynostemma pentaphyllum saponins in rat 6 fibroblast cell system

Authors Mo, Ziyao
Issue Date 2002
Summary The medicinal herb, Gynostemma pentaphyllum (Gp) has been shown to have anticancer effects. However, the precise mechanism is not known. The key active components of Gp are a group of saponins, structurally related to ginsenosides. In the present study, the anticancer effect of Gp saponins was assessed by testing their abilities to prevent the formation of foci induced by an activated c-Ha-ras oncogene in normal Rat 6 fibroblasts. The results indicated that the Gp saponins inhibited ras-induced foci in dosage and time dependent manners. To facilitate the investigation of the mode of inhibition of Gp in living cells, a green fluorescent protein-ras fusion construct (pGFP-ras) was used to substitute ras in the focus formation assay. Using this approach, the pGFP-ras transfected cells can be identified within 24 hours upon transfection under a fluorescent microscope. Cells acquired GFP-ras gene grew into green fluorescent foci with striking transforming morphology in the absence of Gp, whereas the GFP-ras transfected cell, in most of cases, remained as single green fluorescent cell with Gp saponins present in the medium. Based on cell proliferation, colony formation and cell cycle analysis, Gp saponins exhibited non-cytotoxic effect on either normal or the transformed R6 cells. However, Gp saponins posted a strong inhibition against the growth of the ras-transformed cells that were co-cultivated with normal R6 cells. Thus, the data strongly suggest that the inhibitory effect of Gp saponins is not due to a direct killing of transformed cells, rather, Gp might stimulate R6 cells to secret factor(s) that presumably has growth inhibitory effect against the transformed cells. Indeed, an active fraction against the transformed cells has been identified from the conditioned medium collected from Gp-treated R6 cultures. The bioactive component(s) seems to be heat-labile, with molecular weight larger than 14,000. Column purification of the active component(s) will be one of the immediate tasks. The potential gene targets of Gp were investigated. Based on the results obtained from the Western, Northern blot analysis and protein expression in situ, the addition of Gp does not affect the transforming ability, subcellular localization, or the level of expression of Ras protein. Instead, the level of Raf-1 protein was sharply down-regulated within 2 days of Gp treatment. Further investigation indicated that Gp treatment induced instability, instead of transcriptional inactivation of the Raf-1 expression. Besides Raf-1, the immediate downstream gene, Erk seemed to be dephosphorylated upon Gp treatment, while the total protein remained unchanged. To further understand the effect of Gp on R6 cells, a cDNA microarray containing 1176 rat cDNAs was used to examine the gene profilings between Gp-treated and -untreated R6 cells. The result showed that four genes: β2-microglobulin, GST7-7, gelatinase A and cathepsin L were up-regulated, while three genes: Erk-1, γIGFBP-6, and 14-3-3 zeta were down-regulated upon treatment with Gp saponins. The possible impact of the altered genes on ras-transformation is under evaluation. In order to identify the potential active glycosides in Gp, column chromatography, NMR and TLC were performed. Out of seventeen isolated saponins, five of which have retained the anti-cancer effect against ras-transformed cells. Ras proteins play a pivotal role in regulating cell growth and the development of cancer. The finding that an anti-cancer effect of a non-toxic drug may be mediated through the surrounding normal cells is conceptually novel and should have a broad implication in the future development of drugs or dietary supplements with cancer prevention function.
Note Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2002
Language English
Format Thesis
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