| OBJECTIVE: The present study is to extract GPM from GEE, investigate theantitumor effect of GPM in vitro and the molecular mechanisms involved.METHODS: GEE was mixed with distilled water and made the concentration ofthe solution was50mg/mL, the solution was isolated by Sephadex G-25(Gel filtrationchromatography), the components were collected and the residue freeze-dried byfreeze-drying. The protein concentration of GPM was determined by Bradford assay.MTT method was used to determine the effect of Gecko Peptides Mixture onproliferation of EC109and HepG2cell line in vitro. Morphological changes in EC109and HepG2cells treated with Gecko Peptides Mixture (0-0.3mg/mL) after24h wereobserved by inverted microscope. Hoechst33258staining was used to investigate themorphological changes of EC109and HepG2cells. The expression of Bcl-2andcaspase-3in EC109cells were detected by using immunohistochemical method.Apoptosis was detected by caspase-3and caspase-9activity analysis after HepG2cellsexposed to GPM. The influence of GPM on the caspase-3, caspase-9, Cyt c and AIFprotein expressions of HepG2cells was performed by Western blotting.RESULTS: The first peak components which can significantly inhibit the growthof EC109and HepG2cells were the GPM. Bradford assay results indicated that theprotein concentration of GPM was43.9%.MTT assay results showed that EC109and HepG2cells significantly inhibited byGPM in dose-and time-dependent manners. After treated with different concentration(0.1mg/mL,0.15mg/mL,0.2mg/mL,0.25mg/mL,0.3mg/mL) of GPM for24h,48h,72h，the IC50of EC109is0.317mg/mL,0.227mg/mL,0.183mg/mL, respectively.the IC50of HepG2is0.154mg/mL,0.133mg/mL,0.051mg/mL, respectively. GPM- treated HepG2cells became small, exhibited a rounded morphology, shrinkage andattachment loss. Hoechst33258staining demonstrated that GPM induce typicalapoptotic morphological changes in EC109and HepG2cells, such as nuclearcondensation and brighter fluorescence. Immunocytochemistry indicated thatexpression of Bcl-2protein decreased while caspase-3expression increased comparedwith control group. GPM treatment caused a significant dose-dependent increase incaspase-3and Caspase-9activities. Western blotting analysis revealed that after24hof treatment with60,80μmol/L GPM, caspase-3, caspase-9, Cytochrome c and AIFprotein expression in HepG2cells increased.CONCLUSIONS: GPM exert anti-cancer effects in vitro and induce apoptosisthrough activating the mitochondrial pathway. |
