娃兒藤生物鹼藉由c-Jun抗癌細胞生長分子機制之探討及其衍生物之抗冠狀病毒活性研究

Study on c-Jun mediated anticancer mechanisms of tylophorine and identification of tylophorine and its derivatives as novel potent anti-coronaviral agents

楊政偉(Cheng-Wei Yang)

指導教授 ： 李秀珠 ； 張晃猷

國立清華大學/生命科學院/分子醫學研究所/博士(2013年)

摘要

娃兒藤生物鹼(tylophorine)為菲駢類吲哚生物鹼(phenanthroindolizidine alkaloids)的一種，是印度娃兒藤草藥植物(Tylophora indica)裡主要的活性成分。先前已有許多研究證實它具抗發炎、抗病毒及抗癌之生物活性。然而娃兒藤生物鹼抗癌之作用機制仍需更進一步的探討。另外，過去也沒有報導它有抗冠狀病毒的生物活性。在第一部分我們探討娃兒藤生物鹼藉由c-Jun來調控癌細胞生長之分子機制。我們發現癌細胞在tylophorine處理後AP1轉錄因子中的c-Jun蛋白質會明顯增加，且in vitro 酵素活性分析(kinase assay)結果顯示c-Jun的磷酸化主要是經由活化之JNK來調控。此外我們利用流式細胞儀分析，證實在癌細胞裡異位表現(ectopically expressed)c-Jun-myc與tylophorine所導致癌細胞生長停留在細胞週期之G1 phase是有相關聯的。我們之前的研究指出tylophorine抑制癌細胞生長使之停滯在G1 Phase是透過抑制cyclin A2蛋白質的表現。所以我們利用ChIP及reporter assays方法來探討這個現象，證實tylophorine會藉由活化c-Jun去調降cyclin A2蛋白質的表現，而且是透過c-Jun結合至cyclin A2 啟動子(promoter)中調控因子結合位置上AP1和ATF的差異來抑制其活性。最後我們利用藥物抑制劑 (pharmacological inhibitors)和RNA干擾方法實驗證明tylophorine所誘發癌細胞內c-Jun蛋白質的累積與活性是透過二條訊息途徑來調控。其一，我們發現tylophorine會透過活化NF-B_PKC_(MKK4)這條訊息途徑影響JNK活化c-Jun之磷酸化，上調c-Jun的磷酸化和增加其蛋白質穩定性而減緩其降解。其二，是tylophorine透過PI3K_PDK1_PP2A_eEF2這條訊息途徑以維持eEF2活性，使得c-Jun蛋白質表達增加。在第二部分的研究結果，我們發現娃兒藤生物鹼，包含從植物萃取而來或經化學合成之菲駢類吲哚(phenanthroindo-
lizidines)和菲駢類喹喏(phenanthroquinolizidines)具有抑制豬傳染性胃腸炎病(TGEV) 和急性呼吸系統綜合症人類冠狀病毒之活性。利用免疫螢光分析這些具有潛力的化合物抑制TGEV的棘突蛋白(S)和核鞘蛋白(N)的表現量50%之有效抑制濃度(EC50)範圍在8 至 1468 nM，使用RT-PCR方法也發現娃兒藤生物鹼能有效的抑制病毒的產量。此外這些具有潛力之娃兒藤生物鹼化合物顯示出能抑制TGEV病毒的複製，所以皆能抑制病毒所造成細胞凋亡及其引起之細胞病變效應。藉由化學結構與活性關聯性分析結果顯示，這些娃兒藤生物鹼衍生物中最有活性的化合物是在類吲哚(indolizidine)C14位置或在架構菲(phenanthrene)C3位置具有氫氧基(hydroxyl group)，而且具類喹喏(quinolizidine)之娃兒藤生物鹼衍生物相較於其具(indolizidine)生物鹼活性更好。此外針對人類冠狀病毒(SARS-CoV)，這些娃兒藤生物鹼衍生物明顯減少病毒感染後引起的細胞病變效應，EC50落在小於5至 340 nM。此外藥物動力學實驗分析顯示7-methoxycryptopleurine 和tylophorine口服吸收率分別為52.7%及65.7%以上。因此我們的實驗結果顯示這類娃兒藤生物鹼化合物具有很大的潛力，可以進一步研發藥物來對抗TGEV或SARS CoV等冠狀病毒感染造成的威脅。本篇論文研究提供Tylophorine化合物之分子機制及生物活性的基礎訊息，以利其研發成治療藥物。

關鍵字

菲并吲哚啶生物鹼 ； 印度娃兒藤 ； 冠狀病毒 ； 人類急性嚴重呼吸道症候群 ； 娃兒藤生物鹼

並列摘要

Tylophorine, a phenanthroindolizidine alkaloid, is the major medicinal constituent of herb Tylophora indica. It exhibits anti-inflammatory, antiviral and anti-cancerous growth activities. However, the underlying mechanisms of its anticancer activity have not been elucidated and its anti-coronavirus activities not reported before. At the first part we examined the c-Jun mediated molecular mechanism of anticancer tylophorine. Tylophorine treatment increased the accumulation of c-Jun protein, a component of activator protein 1 (AP1), in carcinoma cells. An in vitro kinase assay revealed that the resultant c-Jun phosphorylation was primarily mediated via activated c-Jun N-terminal protein kinase (JNK). Moreover, flow cytometry indicated that ectopically overexpressed c-Jun in conjunction with tylophorine significantly increased the number of carcinoma cells that were arrested at the G1 phase. The tylophorine-mediated downregulation of cyclin A2 protein levels is known to be involved in the primary G1 arrest. Chromatin immunoprecipitation and reporter assays revealed that tylophorine enhanced the c-Jun downregulation of the cyclin A2 promoter activity upon increased binding of c-Jun to the deregulation AP1 site and decreased binding to the upregulation activating transcription factor (ATF) site in the cyclin A2 promoter, thereby reducing cyclin A2 expression. Further, biochemical studies using pharmacological inhibitors and RNA silencing approaches demonstrated that tylophorine-mediated elevation of the c-Jun protein level occurs primarily via two discrete prolonged signaling pathways: (i) the NF-κB/PKCδ_(MKK4)_JNK cascade, which phosphorylates c-Jun and increases its stability by slowing its ubiquitination, and (ii) the PI3K_PDK1_PP2A_eEF2 cascade, which sustains eukaryotic elongation factor 2 (eEF2) activity and thus c-Jun protein translation. At the second part, we identified tylophorine compounds, including naturally occurring and synthetic phenanthroindolizidines and phenanthroquinolizidines, as potent in vitro inhibitors of enteropathogenic coronavirus transmissible gastroenteritis virus (TGEV) and human severe acute respiratory syndrome coronavirus. The potent compounds showed 50% maximal effective concentration (EC50) values ranging from 8 to 1468 nM as determined by immunofluorescent assay of the expression of TGEV N and S proteins and by real time-quantitative PCR analysis of viral yields. Furthermore, the potent tylophorine compounds exerted profound anti-TGEV replication activity and thereby blocked the TGEV-induced apoptosis and subsequent cytopathic effect in ST cells. Analysis of the structure–activity relations indicated that the most active tylophorine analogues were compounds with a hydroxyl group at the C14 position of the indolizidine moiety or at the C3 position of the phenanthrene moiety and that the quinolizidine counterparts were more potent than indolizidines. In addition, tylophorine compounds strongly reduced cytopathic effect in Vero 76 cells induced by human severe acute respiratory syndrome coronavirus (SARS CoV), with EC50 values ranging from less than 5 to 340 nM. Moreover, a pharmacokinetic study demonstrated high and comparable oral bioavailabilities of 7-methoxycryptopleurine (52.7%) and the naturally occurring tylophorine (65.7%) in rats. Thus, our results suggest that tylophorine compounds are novel and potent anti-coronavirus agents that may be developed into therapeutic agents for treating TGEV or SARS CoV infection. These studies provide fundamental information regarding mechanisms of activities and biological activities for tylophorine compounds to be developed into therapeutic drugs.

並列關鍵字

phenanthroindolizidine alkaloids ； T. indica ； Coronavirus ； Human severe acute respiratory syndrome ； Tylophorine ； c-Jun

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