ABSTRACT
Aim To investigate the proliferative effect and the apoptosis of rat epididymal epithelial cells induced by podophyllotoxin and its underlying mechanisms.Methods Primary epididymal epithelial cells were cultured in vitro.CCK-8 assay was used to analyze proliferation of epididymal epithelial cells induced by podophyllotoxin on 24, 48 and 72 h.The ultra structural changes of the epididymal epithelial cells were observed by transmission electron microscope.AnnexinV-FITC/PI staining was used to quantify the percentages of apoptosis in the total cell population.The TdTmediated dUTP nick end labeling(TUNEL) technique was applied to observe the morphological changes of apoptotic cells.The expression of tumor necrosis factor alpha(TNF-α) mRNA was investigated by real-time RT-PCR.The level of TNF-α in cell culture supernatant was measured by enzyme-linked immunosorbent assay(ELISA) technology.Western blot was per-formed to determine the protein expression of cytochrome C, caspase-3, caspase-8, caspase-9.Results Podophyllotoxin significantly inhibited the activity of proliferation and induced apoptosis of epididymal epithelial cells in a dose-and time-dependent manner(P<0.05), with a 50％ inhibitory concentration(IC50) value and corresponding 95％ confidence intervals(CI) of 59.36(15.50～227.41), 0.37(0.080～1.70), 0.077(0.017～0.35) μmol·L-1 at 24, 48 and 72 h, respectively.Podophyllotoxin induced cell volume turned round and cell nuclear fragmentation and mitochondrial vacuolation.RT-PCR and ELISA results showed that podophyllotoxin improved the expression of TNF-α mRNA and protein.Western blot results demonstrated that podophyllotoxin activated the protein expression of cytochrome C, caspase-3, caspase-8 and caspase-9.Conclusion Podophyllotoxin can induce rat epididymal epithelial cell apoptosis through both the mitochondria-regulated intrinsic pathway and the TNF receptor-mediated extrinsic pathway.
ABSTRACT
Aim Toinvestigatetheeffectsoftriptonot-erpene methyl ether ( TME ) , a diterpene derived from the medicinal plant Triptergium wilfordii, on human gastric cancer AGS cell proliferation inhibition and ap-optosisinducedinvitro.Methods MTTassaywas used for screening tumor spectrum and detecting the vi-ability of AGS cells and normal human gastric epitheli-al cells GES-1 . Cell morphology was observed by light microscopy and AO / EB staining. Flow cytometry was used to detect cell apoptotic rate and cell cycle. JC-1 staining and fluorescence probe DCFH-DA were em-ployed to detect the changes of mitochondrial mem-brane potential and reactive oxygen species ( ROS ) . The effect of inhibiting AGS clonogenic survival was as-sayed by the method of plate clone formation. Western blot was used to analyse the expression of caspase-3 , caspase-8,Bcl-2andBax.Results MTTresults showed that TME exhibited significantly higher cytotox-icity to gastric cancer AGS cell line than to noncancer-ous cell line GES-1. IC50 for AGS of 48 h treatment was 23 . 85 μmol · L-1 . TME significantly inhibited colony formation and caused morphological changes in AGS cells. Annexin V-FITC / PI double staining showed the apoptotic rate increased. DCFH-DA stai-ning showed TME resulted in an increase in intracellu-lar ROS levels. Mitochondrial membrane potential de-creased after TME treatment. Western blot results showed that TME increased the proportion of Bax /Bcl-2 , with the activation of caspase-8 and caspase-3 . The broad-spectrum caspase inhibitor z-VAD-fmk pre-treatment reduced the expression of caspase-8 and caspase-3. TME enabled AGS cell cycle arrest in G0/G1phase.Conclusion TMEpossessespotenttumor selected toxicity and can induce apoptosis of AGS cells through cell cycle arrest, which is associated with Bcl-2 protein family.
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Aim To study the mechanism of andrographolide(AD) on the proliferation and apoptosis induction in human esophageal cancer Ec9706 cells.Methods The spectrometry was used to detect the activity of caspase-3 in human esophageal cancer Ec9706 cells treated with or without AD for 6 h,12 h and 18 h,and to detect the activity of caspase-8 and caspase-9 in human esophageal cancer Ec9706 cells treated with or without AD for 6 h.The influence of AD on the proliferation of Ec9706 cells after treatment with or without Z-VAD-FMK(a broad-spectrum caspase inhibitor) was determined by MTT method and the result was compared.The changes of gene expression levels of bcl-2 were determined by immunohistochemical method.Results The expression level of bcl-2 gene was obviously lower in the cells treated with AD(30 mg?L-1,P