TGF-β在細菌影響胰臟癌惡化所扮演的角色

Role of TGF-beta signaling pathway in bacteria-affected pancreatic cancer progression

陳宥任(Yu-Jen Chen)

指導教授 ： 詹明修

中山醫學大學/醫學院/生化微生物免疫研究所/碩士(2017年)

摘要

胰臟癌是惡性腫瘤之一，五年內存活率只有5-6%，目前缺乏有效的治療。乙型轉化生長因子 (Transforming growth factor-β; TGF-β)為多功能性細胞激素。當TGF-β功能喪失或訊息傳遞異常突變，將導致細胞異常增生、誘導上皮間質轉化 (epithelial-mesenchymal transition; EMT) 及轉移。由流行病學、微生物相及本實驗室動物實驗的研究，牙周致病菌牙齦卟啉菌 (Porphyromonas gingivalis; P.gingivalis) 為胰臟癌的致病因子之一。在動物模式中，我們發現益生菌可抑制小鼠之胰臟癌惡化。本研究主要探討TGF-β訊息路徑是否參與益生菌減少胰臟癌惡化的機制。人類胰臟癌細胞株BxPC-3與P. gingivalis培養液共同作用，我們發現TGF-β訊息路徑相關分子包括TGF-β1, Smad2, Smad3, and Smad4顯著增加，其磷酸化的Smad2和Smad3也增加。而Smad7蛋白顯著降低。為了瞭解TGF-β訊息路徑在P. gingivalis促進胰臟癌進展所扮演的角色，利用LSL-KrasG12D; Pdx-1-Cre (KC) 基因轉殖胰臟癌小鼠口腔塗抹P. gingivalis。一個月後，我們發現TGF-β及其下游訊息分子Smad2，Smad3，Smad4在胰腺細胞的細胞質中明顯增加。另外也發現TGF-β訊息路徑活化之phospho-Smad2和phospho-Smad3則有入細胞核的情況，則Smad7的表現量降低。給予益生菌治療抑制P. gingivalis處理之胰腺細胞TGF-β訊息路徑的活化。已知Smad3會調控PD-1的表現，結果顯示口腔塗抹P. gingivalis使PD-1和PD-L1表現增加，而給予益生菌則抑制PD-1和PD-L1的表現。在BxPC-3與P. gingivalis培養液共同作用，也發現PD-1與PD-L1 (及SHP-2) 蛋白表現增加。綜合以上結果，我們在活體外及活體內的實驗均證實給予P. gingivalis活化TGF-β 與PD-1/PD-L1訊息路徑與胰臟癌的惡化程度呈正相關性。重要的是，益生菌會抑制胰臟癌進展之TGF-β訊息路徑的活化。未來我們將更進一步來了解P. gingivalis和益生菌透過何種機制來調控TGF-β/Smad與PD-1/PD-L1訊息路徑。

關鍵字

牙周致病菌 ； 胰臟癌 ； 轉化生長因子-β

並列摘要

Pancreatic cancer is one of the malignant tumors which survival rate is only 5-6% in 5 years, and has no effective therapeutic strategy. Transforming growth factor-β(TGF-β) is a multifunctional cytokine that can inhibit cell growth. Loss of function or mutation of TGF-βwill cause the abnormal proliferation of cells. According the findings through epidemiological studies, microbiota analysis and our animal studies, the periodontitis pathogen Porphyromonas gingivalis (P. gingivalis) may act as a risk factor of pancreatic cancer. We also found that oral administration of probiotics could inhibit pancreatic cancer progression in vivo. In this study, we analyzed whether downregulation of TGF-βsignal pathway play a role of probiotics in the pancreatic cancer suppression. We found that TGF-βsignaling pathway-associated molecules including TGF-β1, Smad2, Smad3, and Smad4 proteins were significantly increased, and phosphorylation levels of Smad2 and Smad3 were increased too. In addition, protein level of Smad7 protein decreased in P. gingivalis culture broth-treated human pancreatic cancer BxPC-3 cells than non-treated cells in vitro. To understand whether the TGF-βsignaling pathway play role in the pathogenesis of P. gingivalis–promoted pancreatic cancer progression, LSL-KrasG12D;Pdx-1-Cre (KC) transgenic pancreatic cancer mice were oral smear periodontal pathogens P. gingivalis. After one month, we found that TGF-βand its downstream signaling molecules Smad2, Smad3, Smad4 were significantly increased in the cytoplasm of pancreatic cells. In addition, nucleus translocation of phospho-Smad2 and phospho-Smad3 through TGF-βsignaling pathway activation were found too. Smad7 protein expression were decreased in pancreatic tissues from P. gingivalis-treated KC mice. On the other hand, probiotic treatment cold reverse the activation of TGF-βpathway by P. gingivalis-treatment. It has been known that Smad3 control immune check point PD-1 expression. Our results indicated that P. gingivalis treatment increased, but probiotics treatment suppressed, PD-1 and PD-L1 protein expressions. The enhancement of protein levels of PD-1 and PD-L1 (also SHP-2) by P. gingivalis treatment were found in P. gingivalis culture broth-treated BxPC-3 cells too. In conclusion, TGF-βand PD-1/PD-L1 pathway activation were observed after P. gingivalis-treatment in vivo and in vitro and it was correlated with the pancreatic cancer progression. More importantly, probiotics administration may reverse the activation of TGF-βpathway in the development process of pancreatic cancer. What are the mechanisms of P. gingivalis and probiotics for regulating TGF-βand PD-1/PD-L1 signal pathway should be clarified in more detail in the future.

並列關鍵字

Porphyromonas gingivalis ； Pancreatic cancer ； Transforming growth factor β

參考文獻

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