Study on anti-colon cancer effect and the underlying mechanisms of dehydroxyhispolon methylether

在台灣，大腸癌的發生率已經連續數年位居首位，致死率則排名第三，爰亟需有效之大腸癌治療藥物。先前研究已指出STAT3主導之促存活路徑在大腸癌細胞中普遍呈現持續性活化，並且STAT3路徑之持續性活化與大腸癌之不良預後呈正相關性，因此STAT3訊息路徑為開發大腸直腸癌治療藥物的關鍵標的。Hispolon為藥用真菌桑黃(Phellinus linteus)之藥用成份之一且具抗癌活性，但近年研究發現某些Hispolon類似物較Hispolon之抗癌效應更強，而Dehydroxyhispolon methylether為Hispolon類似物之一，惟目前缺乏Dehydroxyhispolon methylether之生物活性(包括抗癌活性)之研究。 在我們研究中發現Dehydroxyhispolon methylether確實具有明顯之抗大腸癌效應，並且可有效抑制STAT3之持續性活化。具體而言，我們發現：Dehydroxyhispolon methylether可有效抑制大腸癌細胞株之存活率與細胞群落生成能力，但對正常人類大腸上皮細胞則較無毒性；Dehydroxyhispolon methylether的抗大腸癌效應顯著地高於Hispolon；Dehydroxyhispolon methylether可以誘發大腸癌細胞株之細胞凋亡以毒殺細胞。信號轉導和轉錄激活因子3（STAT3）的組成型激活與結直腸癌腫瘤發生和預後不良高度相關，暗示STAT3是結直腸癌治療的有希望的藥物靶標。 我們發現Dehydroxyhispolon methylether顯著抑制大腸癌細胞株之STAT3持續性活化，當我們在HCT116、HCT-15及LoVo細胞株大量表達持續性活化STAT3能夠顯著拮抗Dehydroxyhispolon methylether抑制之細胞凋亡，證實抑制STAT3活化是Dehydroxyhispolon methylether誘導大腸癌細胞株細胞凋亡之必要機制。研究中發現Dehydroxyhispolon methylether對JAK2活性沒有影響；但我們證實Dehydroxyhispolon methylether能抑制大腸癌細胞株JAK1、SRC的活性，並在大量表現SRC活性之穩定細胞株中，Dehydroxyhispolon methylether抑制STAT3活性能力受到拮抗，證實Dehydroxyhispolon methylether乃透過抑制SRC活性以壓抑STAT3活性進而促進細胞凋亡。西方墨點法結果顯示Dehydroxyhispolon methylether能抑制STAT3下游之抗凋亡蛋白BCL-2蛋白表現量；此外，在大量表達持續性活化STAT3之HCT116、HCT-15及LoVo穩定細胞株中， BCL-2 無法被Dehydroxyhispolon methylether所抑制，顯示Dehydroxyhispolon methylether乃透過抑制STAT3活性以抑制BCL-2之轉錄。我們將BCL-2在HCT116、HCT-15及LoVo中大量表現，西方墨點法及Annexin V assay結果顯示大量表達BCL-2能拮抗Dehydroxyhispolon methylether誘導之大腸癌細胞之凋亡，證明Dehydroxyhispolon methylether抑制STAT3以調降BCL-2，進而誘發細胞凋亡。 總之，我們在此提供了支持抑制JAK1 / SRC-STAT3-BCL-2介導的存活途徑的第一證據，其解釋了DHME的抗結腸癌作用。Colorectal cancer is the most commonly diagnosed cancer and the third cause of cancer mortality in Taiwan. It is known that the signal transducer and activator of transcription 3 (STAT3) is a key signaling molecule implicated in the regulation of growth and malignant transformation, is aberrantly activated in colon cancer,implicating STAT3 as a promising drug target for colorectal cancer therapy. Hispolon, a polyphenolic compound, is one of the medicinal components of the medicinal fungus Phellinus linteus .Several studies have demonstrated the antioxidant,anti-inflammatory, anti-estrogenic activity and anti-cancer properties of hispolon. However, in recent years, some Hispolon analogues have been found to have stronger anticancer effects than Hispolon, and Dehydroxyhispolon methylether is one of Hispolon analogues.However, the underlying mechanisms of anti-colorectal cancer effect of Dehydroxyhispolon methylether are still poorly understood . Here, we reported that Dehydroxyhispolon methylether induce apoptosis and , less toxic to normal human large intestinal epithelial cells; the anti-colorectal cancer effect of Dehydroxyhispolon methylether is significantly higher than that of Hispolon ;markedly suppresse colony formation of colon cancer cell lines HCT116, HCT-15, and LoVo. Constitutive activation of signal transducer and activator of transcription 3 (STAT3) is highly correlated with colorectal cancer tumorigenesis and poor prognosis, implicating STAT3 as a promising drug target for colorectal cancer therapy. We found that Dehydroxyhispolon methylether suppresse constitutive activation of STAT3, as evidenced by reduced levels of phosphorylated tyrosine 705 of STAT3 (activated STAT3). Notably, overexpression of constitutively active STAT3 mutant (STAT3-CA) reversed apoptosis of Dehydroxyhispolon methylether -treated cells. It was found that Dehydroxyhispolon methylether had no effect on JAK2 activity; however, we confirmed that Dehydroxyhispolon methylether inhibited the activity of colorectal cancer cell lines JAK1 and SRC, and that the ability of Dehydroxyhispolon methylether to inhibit STAT3 activity was antagonized in a large number of stable cell lines expressing SRC activity. Dehydroxyhispolon methylether promotes apoptosis by inhibiting SRC activity by suppressing STAT3 activity. Western blotting results showed that Dehydroxyhispolon methylether inhibited the expression of anti-apoptotic protein BCL-2 protein downstream of STAT3; in addition, BCL-2 could not be expressed in HCT116, HCT-15 and LoVo stable cell lines expressing a large number of persistently activated STAT3 Inhibition by Dehydroxyhispolon methylether showed that Dehydroxyhispolon methylether inhibited the transcription of BCL-2 by inhibiting STAT3 activity. We showed a large number of BCL-2 in HCT116, HCT-15 and LoVo. Western blotting and Annexin V assay showed that BCL-2 could antagonize the apoptosis of colorectal cancer cells induced by Dehydroxyhispolon methylether, demonstrating that Dehydroxyhispolon methylether inhibits STAT3. To reduce BCL-2, and then induce apoptosis. In conclusion, we herein provide the first evidence supporting that suppression of JAK1/SRC- STAT3-BCL-2-mediated survival pathway accounts for the anti-colon cancer effect of Dehydroxyhispolon methylether.目次 中文摘要………………………………………………………………………………i Abstract………………………………………………………………………………iii 前言（Introduction）………………………………………………………………..1 一、大腸癌…………………………………………………………………………1 二、桑黃衍生物 ( Dehydroxyhispolon methylether )………………………… ..10 三、STAT3 (Signaling transducer and activator of transcription 3)……………11 四、STAT3與大腸癌……………………………………………………………...13 五、JAK(Janus kinase)……………………………………………………………14 六、Src（Proto-oncogene tyrosine-protein kinase Src）………………………….16 七、細胞凋亡（Apoptosis）……………………………………………………….17 八、Bcl-2家族……………………………………………………………………19 研究目的（Aim）…………………………………………………………………..21 實驗材料及試劑配方 (Materials)………………………………………………….22 一、藥物……………………………………………………………………………22 二、試劑 (Buffer) …………………………………………………………22 三、質體建構（Plasmid construction）…………………………………………..24 四、抗體配製………………………………………………………………………25 實驗方法（Methods）……………………………………………….……………..27 一、細胞培養 （Cell lines and cell culture）……………………..…………….27 二、細胞冷凍保存與解凍……………………………………..…………………28 三、細胞存活率測試（Cell viability assay）………...…………………………28 四、細胞群落形成能力檢測（Colony formation assay）…………….…..…….29 五、細胞凋亡試驗 （Apoptosis assay）………………………………..………29 六、細胞總量蛋白萃取（Whole protein extraction）……………….….………29 七、蛋白濃度定量分析（Protein quantification）………………...……………30 八、西方墨點法（Western blotting）……………………..……………….……30 九、大腸桿菌質體轉型作用（Transformation）………..……………………...32 十、病毒製備與病毒感染（Retrovirus and Lentivirus production and infection） ………………………………………..…………………………………….…..32 結果 (Results)…………………………………………………………………….. 34 一、DHME有效抑制大腸癌細胞株之存活率………………............34 二、DHME能有效抑制人類大腸癌細胞株群落生成能力………………………34 三、DHME誘發大腸癌細胞株細胞凋亡以毒殺細胞……………….34 四、DHME抑制大腸癌細胞株內持續性之STAT3活化…………………35 五、DHME可抑制IL-6誘導之STAT3活化……………………………………..35 六、抑制STAT3活化為DHME誘發人類大腸癌細胞凋亡之必要機制……………………………………………………………………….…………36 七、DHME透過抑制JAK1、SRC以抑制STAT3活化……………………36 八、DHME透過SRC-STAT3路徑促使大腸癌細胞之細胞凋亡………………37 九、DHME抑制STAT3轉錄其下游基因BCL-2…………………37 十、DHME抑制STAT3以調降BCL-2以誘發大腸癌細胞之凋亡……………………………………………..……………………………….38 討論（Discussion）…………………………………………………………….…..39 一、本論文首度發現之結果………………………………………………………..39 二、探討Dehydroxyhispolon methylether (DHME)之抗大腸癌潛力…………39 三、探討DHME與 Hispolon對大腸癌細胞存活率之影響………………………40 四、抑制轉錄因子STAT3活性在DHME誘導之人類大腸癌細胞凋亡之意義…………………………………………………………………………………..40 五、DHME透過抑制SRC、JAK1以壓抑STAT3活化促使細胞凋亡…………………………………………………………………………………41 六、DHME抑制STAT3轉錄下游標的基因BCL-2，誘發大腸癌細胞凋亡………………………………………………………………………………..42 七、其餘可能機制之探討……………………………………………………………43 結論 (Conclusion) ………………………………………………………………44 實驗結果圖表 (Results and Figures) ……………………………………………..45 圖一(A)、Dehydroxyhispolon methylether ( DHME )有效抑制大腸癌細胞株 之存活率…………………………………………………………………45 圖一(B)、Hispolon 與Dehydroxyhispolon methylether ( DHME )毒殺大腸癌 細胞株的毒性相比有較差…………………………………………….46 圖二、Dehydroxyhispolon methylether( DHME )有效抑制人類大腸癌細胞群落 生成能力……………………………………………………………………47 圖三、Dehydroxyhispolon methylether促使PARP截切活化影響細胞生存….48 圖四、Dehydroxyhispolon methylether處理促進人類大腸癌細胞株細胞凋亡….49 圖五、Dehydroxyhispolon methylether抑制多株人類大腸癌細胞STAT3持續性 活化………………………………………………………………………..50 圖六、Dehydroxyhispolon methylether抑制人類大腸癌細胞株持續性STAT3活 化…………………………………………………………………………….51 圖七、Dehydroxyhispolon methylether有效抑制誘導性STAT3活化…….52 圖八、抑制STAT3之持續性活化為Dehydroxyhispolon methylether誘導大腸癌 細胞細胞凋亡之必要機制…………………………………………………53 圖九、抑制STAT3之持續性活化為Dehydroxyhispolon methylether抑制大腸癌 細胞存活率之必要機制……………………………………………………54 圖十、抑制STAT3之持續性活化為Dehydroxyhispolon methylether抑制大腸癌 細胞存活率之必要機制…………………………………………………..55 圖十一、抑制STAT3之持續性活化為Dehydroxyhispolon methylether誘導大腸 癌細胞細胞凋亡之必要機制………………………………………….56 圖十二、Dehydroxyhispolon methylether並非透過JAK2路徑影響人類大腸癌細 胞株中STAT3活性………………………………………………………57 圖十三 、Dehydroxyhispolon methylether抑制STAT3上游蛋白JAK1磷酸 化使人類大腸癌細胞株STAT3活性下降………………………….58 圖十四、Dehydroxyhispolon methylether抑制STAT3上游蛋白SRC磷酸化 使人類大腸癌細胞株STAT3活性下降…………………………………59 圖十五、Dehydroxyhispolon methylether透過SRC-STAT3路徑促使大腸癌細胞 細胞凋亡…………………………………………………………………60 圖十六、抑制SRC之持續性活化為Dehydroxyhispolon methylether抑制大腸 癌細胞存活率之必要機制………………………………………………61 圖十七、Dehydroxyhispolon methylether抑制STAT3下游人類大腸癌細胞株之 抗凋亡蛋白 BCL-2蛋白表現量…………………………………………62 圖十八、在STAT3持續性活化之人類大腸癌細胞株HCT-15、HCT 116及LoVo中Dehydroxyhispolon methylether抗凋亡蛋白BCL-2蛋白表現量……..63 圖十九、在大量表現constitutively active BCL-2之人類大腸癌細胞中， Dehydroxyhispolon methylether所誘導之細胞凋亡受到抑制……..64 圖二十、在大量表現constitutively active BCL-2之人類大腸癌細胞中，BCL-2 之持續性活化為Dehydroxyhispolon methylether抑制大腸癌細胞存活 率之必要機制…………………………………………………………….65 圖二十一、在大量表現constitutively active BCL-2之人類大腸癌細胞中， Dehydroxyhispolon methylether所誘導之細胞凋亡受到抑制…………66 參考文獻（References）…………………………………………………………….6