| 摘要: | 背景與目標 : 肝細胞癌是目前台灣最致命而且高發生率的主要癌症。因B型肝炎與C型肝炎的高盛行率，使慢性肝病成為台灣的國病。慢性肝病進一步發展成肝硬化及肝癌。肝癌容易造成遠端轉移以及產生抗藥性的特徵使得病人整體的存活率始終無法有效提昇。吸菸會導致惡性腫瘤發生，然而，煙草暴露如何促進肝臟腫瘤的發生或引起肝癌惡化作用所之甚少。煙草中含有不少的致癌劑與突變劑。香癮主成分，尼古丁對肺癌，胃癌，大腸癌，乳癌及胰臟癌更惡化的機轉已有文獻發表但對肝癌細胞之間的關係則尚未有任何完整的文獻報導。尼古丁會活化菸鹼型乙醯膽鹼受體，nAChR會增加組織的纖維化及發炎，因此也可能是會促進肝硬化及癌症，其中α7-膽鹼受體(α7-nAChR)已經是許多癌症研究的焦點。本論文主要是針對抽菸與肝癌病人術後的預後及尼古丁接收器,α7-nAChR在肝癌惡性進程之癌症基因分子機轉及治療上的角色。進一步針對分子機轉開發治療策略。
材料與方法 : 利用回朔性分析2010年1月1日至2015年12月31日在衛生福利部委託台北醫學大學辦理之雙和醫院所診斷出的肝癌患者與術後檢體與包含179位患者手術前之抽菸狀況記錄與病理資料。分析抽菸狀況與菸癮主要活性成分尼古丁對於肝癌復發、轉移及存活率的相關性，並比較危險或有益的因子。利用免疫組織染色與西方墨點法，分析α7-nAChR尼古丁受體在臨床肝癌組織表現對於病患預後的關係分析其與臨床病理特徵的相關性。藉由抑制α7-nAChR表現，探討α7-nAChR調節肝癌相關生長、轉移與幹細胞特性與調控相關基因表現。利用STRING分析與肝癌的基因微矩陣，建立α7-nAChR在誘發肝癌惡化之分子生物機轉與基因調控網絡對肝癌惡化的影響。進一步利用JAK抑制劑dehydrocrenatidine (DHCT)，治療肝癌細胞，證明DHCT對於nicotine誘發之肝癌細胞生長、轉移與癌症幹細胞特性。裸鼠的肝癌異體移植模式，研究DHCT對於腫瘤生長的抑制效果與促使腫瘤細胞凋亡，探討DHCT作為治療Nicotine誘發肝癌惡化的專一性藥物。
結果: 本研究顯示，肝癌病患抽菸的狀況與數量會降低肝癌病人的無病存活期與總體存活期。尼古丁受體α7-nAChR表現與肝癌病理分期成正相關與病人的無病存活期與總體存活期成負相關。抑制α7-nACh表現，能降低肝癌細胞生長、轉移、侵襲與幹細胞特性與相關基因表現。α7-nAChR對於肝癌惡化的分子機轉與JAK2與TRG5複合體形成有關。進一步也證明尼古丁暴露，會刺激尼古丁受體α7-nAChR表現與促進肝癌細胞生長、轉移、侵襲與幹細胞特性與相關基因表現，利用JAK抑制劑DHCT處理，可降低尼古丁誘導的肝癌惡化特性與相關基因表現。在異體肝癌模式中，DHCT治療亦會抑制肝癌細胞生長與促進肝癌細胞凋亡。
結論:本研究證明，肝癌患者的抽菸行為會縮短患者的無病存活期與總體存活期。尼古丁暴露會促進尼古丁受體α7-nAChR的表現與肝癌增生、轉移與幹細胞的特性，導致肝癌復發。α7-nAChR、JAK2會形成複合體活化JAK2/STAT3訊息路徑，造成肝癌的惡性進程。利用JAK抑制劑DHCT治療肝癌細胞，可降低肝癌細胞體外生長、轉移與幹細胞之相關特性與基因表現。同時也能抑制肝癌異體移植小鼠之腫瘤生長與促使細胞凋亡。本研究建立了，抽菸與尼古丁受體α7-nAChR在肝癌中的角色與相關分子機轉。更進一步地透過分子機轉，找到DHCT做為新的治療策略，能幫助臨床對於肝癌的藥物治療。
Introduction: Hepatocarcinoma (HCC) is one of Taiwan’s most significant lethal cancers. Due to the high prevalence of hepatitis B and C, chronic liver disease has become a national disease in Taiwan. Chronic liver disease further develops into liver cirrhosis and HCC. The prognosis of HCC remains poor according to the high recurrent rate. Even after curative liver resection, the intra-hepatic recurrent rate is 50-60% at 3yrs and up to 70-100% at 5ys. Besides the extrahepatic metastasis (EHM) rates is 15– 17%. Smoking contributes to the initiation of malignancies; however, little is known about how tobacco exposure contributes to HCC tumorigenesis. Tobacco combustion generates many carcinogens and mutagens. Nicotine, the critical addiction agent in smoking, promotes lung, breast, colon, gastric, and pancreatic cancer progression. However, there is no focusing Nicotine triggers nicotinic acetylcholine receptor (nAChR) activation and promotes tissue fibrosis and inflammation, and recent years the oncogenic role of alpha-7 nicotinic receptor (α7-nAChR) becomes more attracted. Here, we focus on the prognosis of smoking and HCC patients after surgery and the α7-nAChR function in HCC tumorigenesis and progression. Furthermore, we study the molecular mechanism and treatment niche in nicotine-induced HCC. Moreover, we also develop novel therapeutic strategies targeting nicotine-induced HCC. Material and Methods: The retrospective analysis of HCC patients’ specimens who were diagnosed at Shuang Ho Hospital, Taipei Medical University from January 1, 2010, to Dec 31, 2015, and including 179 patients with smoking status records and pathological data before surgery. To analyze the correlation between smoking status and nicotine amount, the main active ingredient of smoking addiction, on the HCC recurrence, metastasis, and survival rate, and compare the risk or beneficial factors. Using IHC and Western blotting, we analyzed the HCC prognosis value of α7-nAChR, the primary nicotine receptor, and its expression elevation in tumor stages. To elucidate α7-nAChR, we used sh-α7-nAChR to silence α7-nAChR expression in HCC cells and examined the HCC proliferation, metastasis, and cancer-stemness properties. To clarify the α7-nAChR regulation network in HCC, we used STRING analysis and HCC microarray from the GEO database. Moreover, we used JAK inhibitor dehydrocrenatidine (DHCT) to treat nicotine-stimulated HCC for in vitro proliferation, migration, cancer stemness, and suppressed EMT and CSC gene expression of HCC progression. Moreover, we used the nude mice HCC xenograft model to study the in vivo DHCT tumor suppression and pro-apoptotic functions.
Results: Our results demonstrated that cigarette smoking status reduced overall and disease-free survival (DFS) in HCC patients. Expression of α7-nAChR positively correlated to the HCC stage and negative to OS and DFS. Silence of α7-nAChR reduced in vitro HCC proliferation, migration, invasion and cancer stemness abilities, and gene expression. α7-nAChR, JAK2, and TGR5 complex promoted JAK2 activation. Furthermore, HCC nicotine exposure promoted α7-nAChR expression and in vitro HCC proliferation, migration, invasion, cancer stemness abilities, and relative gene expression. DHCT treatment abolished nicotine-induced HCC proliferation, migration, invasion and cancer stemness abilities, and relative EMT and CSC gene expression. Moreover, DHCT treatment suppressed in vivo HCC tumor growth and promoted HCC apoptosis.
Conclusion: Our study proved that smoking decreased OS and DFS in HCC patients. Nicotine exposure promoted the expression of nicotine receptor α7-nAChR and the in vitro HCC proliferation, metastasis, and stem cells’ oncogenic properties, leading to HCC recurrence. α7-nAChR and JAK2 complex activated the JAK2/STAT3 signaling pathway to promote HCC malignant progression. DHCT treatment could reduce in vitro HCC proliferation, metastasis, and cancer stemness. Moreover, DHCT also suppressed the in vivo HCC growth and promoted apoptosis in the nude mice xenograft models. Our study elucidated the smoking-promoting HCC progression mechanism and found the novel therapy agent, DHCT, in nicotine-promoted HCC. |
