Study On The Anticancer Effect Of Gekko Ethanol Extract(â…¡)

OBJECTIVE The animal Gekko (also called Shou Gong) is one of thetraditional Chinese medicines, which has been used to treat cancer for hundreds ofyears. In the last decades, a large number of researchers have proved that Gekko doreally have anticancer activity. However, there is still limited information about itsdefinite components and underlying mechanism. The aims of this study are: To extractand make study on the anti-cancer effect of Gekko active components both in vitro andin vivo; To supply experiment and theory foundation for the study on the anticancercomponents of Gekko and for its appliance in treating cancer in clinic.METHODS Mixture of100g dried Gekko power with400mL double distilledwater was made into homogenate. After centrifuged (5500rpm,5min), the collectedprecipitation was mixed with55%ethanol solution to400mL. Supernatant wascollected by the following centrifugation (5500rpm,5min), which was thencondensed in rotary evaporator.7.2g Gekko ethanol extraction (Ⅱ) powder (GEE Ⅱ)was ultimately obtained by the following Freeze-drying process.MTT assay was used to test the inhibitory effect of GEE (Ⅱ) on the proliferationof EC109, Hep2, HepG2, C6cells. Hoechst33258fluorescence staining was carriedout to investigate the changes in the nucleus of Hep2and HepG2cell lines exposed toGEE (Ⅱ). Mice subrenal capsule xenograft model (model1) and mice subcutaneousxenograft at the right armpit model (model2) of H22hepatocellular carcinoma wererespectively established to measure the inhibitory effect of GEE (Ⅱ) on the tumorgrowth in vivo. The expression levels of interleukin-6(IL-6) and tumor necrosisfactor-α (TNF-α) in both mice models were evaluated by ELISA assay. Western blotanalysis was performed to detect the protein expressions of VEGF and caspase-3inHep2cells and in xenograft tumor in model2after treated with GEE (Ⅱ).Gel filtration chromatography was used to isolate GEE (Ⅱ). Inhibitory effects ofeach peak components on the proliferation of Hep2and HepG2cells were evaluated by MTT assay.The most active component was analyzed by HPLC.RESULTS The yield rate of Gekko ethanol extract (Ⅱ) was7.2%. MTT assayresults indicated that GEE (Ⅱ) could significantly inhibit the proliferation of EC109,Hep2, HepG2, C6cell lines in a time-dose dependent manner. After exposed to GEE(Ⅱ) for48h, the IC50of GEE (Ⅱ) was211μg/mL,238μg/mL,170μg/mL,182μg/mL, respectively. By the results of Hoechst33258fluorescence staining, evenlystained with weak fluorescence, regular and round-shaped nucleus were observed inthe control groups. However, except the decreased cell number, cells treated with GEE(Ⅱ) also showed conspicuous morphological changes including cell volume shrinkage,brighter fluorescence, condensed or fragmented chromatin. GEE (Ⅱ) couldsignificantly inhibit the growth of xenograft tumors, and GEE (Ⅱ) lead to notablyreduced levels of IL-6protein and increased levels of TNF-α protein in the two micemodels. Western blot analysis demonstrated that VEGF expression was down-regulated while activation of caspase-3was increased by GEE (Ⅱ) treatment in atime-dose dependent manner both in Hep2cells and in model2.The first peak components (GEGA) obtained by gel filtration chromatographywas the most effect part of GEE (Ⅱ). After treated with GEGA for48h, the IC50ofGEGA was134μg/mL and132μg/mL in Hep2and HepG2cell lines, respectively.Compared with GEE (Ⅱ), IC50of GEGA was lower. HPLC spectrum analysis ofGEGA showed that GEGA still consisted of several kinds of components.CONLUSIONS A new method to extract the active anticancer components fromGekko is successfully established in this study. The yield rate of Gekko ethanol extract(Ⅱ) is7.2%. GEE (Ⅱ) may have anticancer effect both in vitro and in vivo byantiangiogenesis and apoptosis inducing properties. These properties may be involvedin increasing the activation of caspase-3, up-regulated expression of TNF-α anddecreasing the expression of VEGF, IL-6proteins. GEGA isolated by gel filtrationchromatography from GEE (Ⅱ) is more effective than GEE (Ⅱ). HPLC analysisindicated GEGA still consists of several constituents and further purification should bedone in the future work.