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Targeted inhibition of ATF5 function of paclitaxel -induced apoptosis of SW1990

Author: HuMing
Tutor: WangBin
School: Qingdao University
Course: Pathogen Biology
Keywords: ATF5 dominant-negative paclitaxel apoptosis pancreatic cancer
CLC: R735.9
Type: Master's thesis
Year: 2011
Downloads: 4
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Abstract

Objective:To investigate the effect and mechanism about apoptosis in paclitaxel-treated pancreatic cancer cell SW1990 after destructing the function of ATF5 by dominant-negative.Methods:1. The expressions of ATF5 in pancreatic cancer cells Capan-2、SW1990 and ASPC-1 were detected by RT-PCR and Western-blot.2. SW1990 cells were treated with paclitaxel at different concentrations(0、6.25、12.5、25、50、100、200nM) for different hours(0、6、12、24、48h) respectively. Viabilities were determined by MTT assay.3. SW1990 cells were treated with 100nM paclitaxel at different hours(0、12、24、48h). Morphologic changes were observed by inverted microscope. The cell apoptosis was observed by flow cytometry with AnnexinV/7-AAD double staining and SR-VAD-FMK/7-AAD double staining. The expression of ATF5 mRNA and BCL-2 mRNA were monitored by RT-PCR at the same time.4. The function of ATF5 was destructed by dominant-negative in SW1990 cells. Then the cells were treated with 100nM paclitaxel at different hours(12h、24h、48h). The sensitivity of paclitaxel-induced apoptosis after losing ATF5 function achieved with a dominant-negative (d/n) form of ATF5 was observed by flow cytometry to.5. Detect the expression of BCL-2 protein after losing ATF5 function achieved with a dominant-negative (d/n) form of ATF5 to study the mechanism between ATF5 and sensitivity of paclitaxel-induced apoptosis.Results:1. The expressions of ATF5 were detected by RT-PCR and Western-blot in pancreatic cancer cells Capan-2、SW1990 and ASPC-1. The percentage of mRNA ATF5/β-actin was 0.2±0.06、0.26±0.04、0.36±0.05 respectively; and the percentage of protein ATF5/p-actin was 0.33±0.09、0.35±0.08、0.60±.010 respectively. Expression level of ATF5 both mRNA and protein in SW1990 was highest. Then SW1990 was chosen for coming research.2. The MTT assay indicated proliferation of SW1990 cells can be arrested by paclitaxel. The IC50 was 115nM and the 100nM was chosen for coming research. There was a significant increased amount of floating cellular debris in the cultures at 12h after treated with 100nM paclitaxel. At 48h almost all cells were dead. The Annexin V/7-AAD double staining technique results shown that the ratio of cell apoptosis was 13.2%、19%、31.8% at 12h、24h、48h after treated with 100nM paclitaxel, while control group was 7.4%、9.9%、9.6% respectively(p<0.05). And the SR-VAD-FMK/7-AAD double staining technique results shown that the ratio of cell apoptosis was 13.1%、16.%、39.9%, compared to control group 9.0%、10.1%、11.0%(p<0.05).3. The expression of ATF5 and BCL-2 mRNA in SW1990 declined with paclitaxel-induced apoptosis. The percentage of mRNA ATF5/β-actin dropped 26.1% while BCl-2/β-actin dropped 12.5% at 48h after treated with 100nM paclitaxel.4. The ratio of cell apoptosis increased 13.6% at 48h after losing ATF5 function achieved with a dominant-negative (d/n) form of ATF5, compared to vector group 5%(p<0.05). The percentage of protein BCl-2/β-actin declined 39%, compared to vector group from 3%(p<0.05) at the same time. The ratio of apoptosis at 48h after d/n ATF5 combined with paclitaxel increased 25.7% compared to paclitaxel group18.6%(p<0.05),and enhanced the sensitivity of paclitaxel-induced apoptosis.Conclusions:1. The declining of ATF5 and BCl-2 expression was involved in the apoptosis of pancreatic cancer cells SW1990 induced by paclitaxel.2. A dominant-negative (d/n) form of ATF5 could enhance the sensitivity of pancreatic cancer cells SW1990 to paclitaxel-induced apoptosis.

Targeted inhibition of ATF5 function of paclitaxel -induced apoptosis of SW1990

Abstract

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