第二型前列腺素還原酶在胃癌中的角色:功能與機制之探討

Role of prostaglandin reductase 2 in gastric cancer: functional and mechanistic studies

張芸嘉(Emily Yun-Chia Chang)

指導教授 ： 莊立民

國立臺灣大學/醫學院/分子醫學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.03207

摘要

前列腺素還原酶PTGR2是一種可將15-keto-PGE2代謝成為13,14-dihydro-15- keto-PGE2的酵素。在我們之前已發表的研究中發現15-keto-PGE2是PTGR2的受質，且也是PPARγ的天然配體，而PPARγ本身可受配體調控進而影響脂肪的代謝與細胞分化。目前除已知PTGR2可抑制PPARγ活性與脂肪細胞分化外，對PTGR2的生化功能了解極為有限。目前許多研究證實前列腺素與其相關合成酵素與胃癌腫瘤的發育和轉移有關。而前列腺素與其代謝產物皆是PPARγ的配體且經由其交互作用可在腫瘤形成與轉移扮演一定角色。因此是否PTGR2也與癌症有關是一個待研究的有趣課題。
本論文中我們針對PTGR2在胃癌中的功能特性與機制做探討。透過慢病毒感染方式，我們建立了PTGR2剃除與PTGR2過度表現之胃癌細胞株AGS與SNU-16。 體外分析發現PTGR2剃除之胃癌細胞株其貼附性與非貼附性細胞生長以及貼附性細胞在半固狀的凝膠中懸浮生長能力皆受到抑制，而體內腫瘤異種移植老鼠研究中也顯示腫瘤生長較慢。 在機制方面，PTGR2剃除之胃癌細胞株可改變線粒體的功能，並產生了較高的活性氧自由基（ROS），因而導致ERK1/2和caspase3的活化與增加Bcl-2和抑制Bax的表現量，因而誘導細胞死亡，和PTGR2過度表現之胃癌細胞株呈現相反的對比。用PTGR2抑制劑以及冷凍乾燥後微脂粒重組的PTGR2代謝產物13,14-dihydro-15-keto-PGE2，我們發現PTGR2的致癌潛能是依賴它酵素本身的催化活性。為了尋找PTGR2其他的致癌途徑及受它調控的分子, 微陣列分析中也顯示PTGR2在對於腫瘤細胞遷移的功能也有潛在作用。最後，臨床免疫病理染色也發現PTGR2在胃癌檢體腫瘤區中相較鄰近非腫瘤區域有顯著性表現，且其和腸道型胃癌患者存活率亦有相關性。此外，剃除PTGR2之胃癌細胞株對於胃癌化學療法常用藥物cisplatin和5-fluorouracil更為敏感。基於以上的結果，我們的研究不僅提供PTGR2在胃癌中扮演的功能和機制的證據，臨床觀察也肯定PTGR2對胃癌病變有重要角色，所以設立PTGR2為未來治療目標是值得進一步評估。

關鍵字

前列腺素還原酶PTGR2 ； 前列腺素新陳代謝 ； 胃癌 ； 線粒體 ； 細胞計畫性的死亡

並列摘要

Prostaglandin reductase 2 (PTGR2) is an enzyme that catalyses the NADPH- dependent reduction of 15-keto-PGE2 into the downstream metabolite 13,14-dihydro- 15-keto-PGE2. Previously, 15-keto-PGE2 was demonstrated to act as a natural ligand for peroxisome proliferator-activated receptor γ (PPARγ), which plays a distinct role in mediating differentiation of adipocytes and regulating lipid metabolism. The suppressive effect of PTGR2 on PPARγ activity and adipocyte differentiation were characterized. However, there is still very little knowledge on the biological role of PTGR2. It is well established that different prostanoids & their synthases play an important role in gastric cancer. Many of these prostaglandin metabolites also serve as natural ligands for PPARγ. Thus, whether PTGR2 plays a distinct role in cancer biology is of interest.
In this thesis, functional and mechanistic analyses on the impact of PTGR2 in gastric cancer are presented. Downregulation or overexpression of PTGR2 was performed by lentiviral technology in AGS and SNU-16 gastric cancer cell lines. In contrast to overexpressing PTGR2, in vitro analysis showed that PTGR2 knockdown resulted in decreased proliferation rate and colony formation, and in vivo xenograft models showed slower growth of tumors. Mechanistically, PTGR2 knockdown induced cell death, altered mitochondrial function, and produced higher reactive oxygen species (ROS), which lead to the activation of ERK1/2 and caspase 3, with increased Bcl-2 and suppressed Bax expressions. Furthermore, treatment of AGS cells with synthetic PTGR2 inhibitors as well as lyophilized lipids reconstituted with 13,14-dihydro-15- keto-PGE2 showed the dependency of PTGR2 on its catalytic activity in its oncogenic potency. To investigate additional oncogenic pathways and molecules in which PTGR2 act on, microarray was also performed and identified potential role of PTGR2 in cancer cell migration. Lastly, clinical immunopathological staining showed strong PTGR2 expression in the gastric tumor portion relative to nearby non-tumor areas, and its expression negatively correlated with the survival of patients with intestinal type gastric cancer. Furthermore, PTGR2-knockdown cells were more sensitive to cisplatin and 5-fluorouracil. Taken together, our findings not only provide functional and mechanistic evidence of the involvement of PTGR2 in gastric cancer, but also clinical observations affirm the significance of PTGR2 in gastric cancer and suggest PTGR2-target based therapy is worth further evaluation.

並列關鍵字

PTGR2 ； prostaglandin metabolism ； gastric cancer ； mitochondria ； apoptosis

參考文獻

1. Stoicov, C., et al., Molecular biology of gastric cancer: Helicobacter infection and gastric adenocarcinoma: bacterial and host factors responsible for altered growth signaling. Gene, 2004. 341: p. 1-17.

2. Fuchs, C.S. and R.J. Mayer, Gastric carcinoma. N Engl J Med, 1995. 333(1): p. 32-41.

4. Takiuchi, H., Second-line chemotherapy for gastric cancer: a new issue lies ahead in global trials. Gastric Cancer, 2011. 14(3): p. 206-11.

5. Matsuzaki, T., et al., Synergistic antiproliferative effect of mTOR inhibitors in combination with 5-fluorouracil in scirrhous gastric cancer. Cancer Sci, 2009. 100(12): p. 2402-10.

6. Wu, B., et al., Anti-proliferative and chemosensitizing effects of luteolin on human gastric cancer AGS cell line. Mol Cell Biochem, 2008. 313(1-2): p. 125-32.