探討Thapsigargin誘發肺癌細胞內質網壓力與端粒酶活性調控之機轉

The Mechanisms of Thapsigargin Induce Endoplasmic Reticulum Stress and Regulation of Telomerase Activity in Human Lung Cancer Cells

蕭月婕(Yueh-Chieh Hsiao)

指導教授 ： 柯俊良

中山醫學大學/醫學院/醫學研究所/碩士(2011年)

https://doi.org/10.6834/CSMU.2011.00002

摘要

Thapsigargin藥劑能誘發細胞內質網壓力，另外Thapsigargin也會抑制細胞端粒酶活性。本研究主要探討Thapsigargin是否透過誘發細胞內質網壓力來抑制端粒酶活性，並且透過其他因子表現影響細胞存活。首先，Thapsigargin刺激下能明顯促進A549肺癌細胞死亡，但對Calu-1肺癌細胞不具毒殺能力。並且以西方墨點法及RT-PCR方式觀察到A549細胞內質網壓力指標GADD153以及lipocalin2的表現量增加。以Reporter assay分析，NF-κB抑制劑Bay11-7082並不能降低Thapsigargin所誘發A549細胞中lipocalin2 啟動子活性。並透過核質分離技術分析，Thapsigargin無法活化NF-κB進入細胞核，但GADD153有明顯受活化。因此，我們認為Thapsigargin不是透過NF-κB調控lipocalin2表現，可能與GADD153的調控有關。利用VZV-G pseudotyped lentivirus-shRNA 系統抑制A549細胞GADD153表現確實會明顯減少lipocalin2的表現量。運用Q-ChIP結合實驗來分析， Thapsigargin能增強A549細胞中C/EBPβ及GADD153結合至lipocalin2啟動子能力。而抑制A549 細胞Lipocalin2表現，Thapsigargin明顯降低細胞端粒酶活性及減少細胞群落生成能力。綜合上述，本研究首度發現Thapsigargin會透過活化GADD153進而調控lipocalin2表現，並且lipocalin2的存在可能與細胞端粒酶活性及細胞死亡有關。

關鍵字

內質網壓力 ； 端粒酶活性 ； 中性粒细胞明膠酶相關脂質運載蛋白 ； 毒胡蘿蔔素

並列摘要

Thapsigargin is an ER stress inducer and represses telomerase activity. This study explored that the mechanisms of thapsigargin on telomerase activity suppression and cell death induction through ER stress. Thapsigargin promoted apoptosis in A549 lung cancer cells, but not in CaLu-1 lung cancer cells. Using western blot and RT-PCR analysis, thapsigargin increased expression of ER stress-associated protein GADD153 and lipocalin2 in A549 cells but not in Calu-1 cells. The reporter assay indicated that Bay11-7082, an NF-κB inhibitor, did not reduce thapsigargin-mediated lipocalin2 promoter activity in A549 cells. Using nuclear extraction, thapsigargin did not activate NF-κB. However, GADD153 was upregulated in the nuclear after thapsigargin treatment. Therefore, the results suggested that thapsigargin induced lipocalin2 expression by GADD153, but not via NF-κB activation. Using VZV-G pseudotyped lentivirus-shRNA system for GADD153 genes silencing, thapsigargin reduced lipocalin2 expression. It demonstrated that GADD153 and C/EBPβ did bind on lipocalin2 promoter using ChIP assay. In comparison with A549 shLuc cells, thapsigargin-suppressed cell telomerase activity and colony formation in A549 shLipocalin2 cells. This results suggested that thapsigargin induce lipocalin2 expression through GADD153 induction. And lipocalin2 may be related with telomerase activity and cell death.

並列關鍵字

ER stress ； Telomerase activity ； Lipocalin2(NGAL) ； Thapsigargin

參考文獻

Aktunc, E., Ozacmak, V.H., Ozacmak, H.S., Barut, F., Buyukates, M., Kandemir, O. and Demircan, N. (2011) N-acetyl cysteine promotes angiogenesis and clearance of free oxygen radicals, thus improving wound healing in an alloxan-induced diabetic mouse model of incisional wound. Clin Exp Dermatol, 35, 902-909.

Artandi, S.E. and DePinho, R.A. (2010) Telomeres and telomerase in cancer. Carcinogenesis, 31, 9-18.

Baehrecke, E.H. (2005) Autophagy: dual roles in life and death? Nat Rev Mol Cell Biol, 6, 505-510.

Bernales, S., Papa, F.R. and Walter, P. (2006) Intracellular signaling by the unfolded protein response. Annu Rev Cell Dev Biol, 22, 487-508.

Biessmann, H. and Mason, J.M. (1992) Genetics and molecular biology of telomeres. Adv Genet, 30, 185-249.