植化素薑黃素藉由引發細胞凋亡促進新穎及傳統鉑金屬大腸直腸癌化療藥物之敏感性

Phytochemical curcumin enhances chemosensitivity of human colorectal cancer cells to novel and approved platinum-based anticancer drugs via induction of apoptosis

王盈堤(Ying-Ti Wang)

指導教授 ： 蘇純立

國立臺灣師範大學/教育學院/人類發展與家庭學系/碩士(2012年)

摘要

營養支持對於接受癌症治療的病人非常重要。透過營養素與臨床抗癌藥物的交互作用，營養素可能具有輔助或抑制藥物的效用，可能提高或降低癌症病人的治療效率，因此營養素的建議攝取對於藥物治療的效果有重要的角色。人類大腸直腸癌是目前第二大流行與致死率排行第三的癌症。然而在治療方法上除手術外，還有化學治療與放射線治療。化學治療藥物中包含鉑金屬化合物，如：cisplatin、carboplatin與oxaliplatin等。但化學治療藥物容易產生副作用，如：腎毒性、神經毒性、噁心嘔吐等，造成病患身體不適。為解決目前抗癌用藥抗藥性及副作用的困境，實驗想要藉由天然物薑黃素的輔助去降低新穎化合物或臨床藥物的使用劑量，進而解決困境。我們研究團隊從許多新穎合成的化合物中篩選出具有抗癌潛力的triazinone triepoxide TATT。藉由細胞毒殺試驗（MTT assay）得知triazinone triepoxide TATT比cisplatin、carboplatin或oxaliplatin對於人類大腸直腸癌細胞（HT-29）更具細胞毒殺或是抑制細胞生長的效用，對於人類正常臍靜脈內皮細胞（HUVEC），triazinone triepoxide TATT則比cisplatin、carboplatin或oxaliplatin有較低的毒性且較安全。進一步探討發現triazinone triepoxide TATT會引發HT-29之細胞凋亡（apoptosis）並對細胞週期造成影響。使用propidium iodide（PI）或annexin V染色，以流式細胞儀進行分析發現HT-29細胞經4 μM triazinone triepoxide TATT處理12小時後，細胞凋亡比例與G2/M期增加，處理24小時後，G2/M期減少而細胞凋亡比例持續增加。在同樣24小時4 μM條件之下，triazinone triepoxide TATT與cisplatin、carboplatin或oxaliplatin處理後的細胞相比，triazinone triepoxide TATT引發較多的細胞凋亡比例。進一步使用細胞凋亡機轉相關的抑制劑，如：caspase 8抑制劑Z-IETD-fmk及粒線體transition pore抑制劑cyclosporine A處理HT-29細胞，以annexin V染色進行流式細胞儀分析、西方點墨法、共軛焦顯微鏡與螢光顯微鏡分析，確認triazinone triepoxide TATT是透過caspase 8及粒線體相關蛋白質的內外路徑影響大腸直腸癌細胞產生細胞凋亡，並透過增加cyclin B1的表現與減少CDC25B與CDC25C的表現使G2/M期減少及sub-G1期的增加。進一步以薑黃素（30 μM）與triazinone triepoxide TATT（4 μM）處理HT-29細胞24小時後，以PI染劑或annexin V染色再以流式細胞儀進行分析，並透過Jin’s method公式計算出薑黃素與triazinone triepoxide TATT或oxaliplatin合併處理致使大腸直腸癌細胞產生凋亡具有加乘或協同的效果，但薑黃素與cisplatin或 carboplatin合併後效果為拮抗。本研究結果發現triazinone triepoxide TATT具有開發成為新一代的抗癌藥物的潛力，以提供癌症病患治療的不同選擇，期望能改善並提升醫療品質。在進行化療病人的飲食建議上，含薑黃素的食物，如：薑黃或咖哩等的攝取建議，可能會影響抗癌藥物的治療效果，值得再深入探討。其它天然植物與蔬果中之安全並有效的成分，如：苯甲基異硫、氰酸酯、木香烴內酯與酚類化合物白藜蘆醇等，對於藥物治療為輔助或抑制效果應有深入的了解。期望藉由飲食上增加補充或減少攝取天然物中的營養素，促使化療藥物發揮更好的治療效果。

關鍵字

Triazinone triepoxide TATT ； 薑黃素 ； 細胞凋亡 ； 訊息傳遞 ； 鉑金屬化合物 ； 人類大腸直腸癌

並列摘要

Nutritional support is very important for the patients undergoing cancer treatment. Through the interaction of nutrients with anti-cancer drugs, the efficacy of the treatment may be enhanced or inhibited. Therefore, dietary recommendation plays an vital role on the effects of the treatment. Human colorectal cancer is the second most popular and the third leading cause of death in cancers. Surgery, chemotherapy and radiation therapy are the common treatments, and platinum-based compounds such as: cisplatin, carboplatin and oxaliplatin are ordinary used. However, these chemotherapeutic drugs are likely to cause side effects and result in illness of the patients. In order to resolve the current plight of the anti-cancer drugs, a number of new synthetic compounds were screened and the anti-cancer potential of triazinone triepoxide TATT was discovered. Further study indicates that triazinone triepoxide TATT displays comparable anti-cancer effect on human colorectal cancer HT-29 cells. Nevertheless, triazinone triepoxide TATT displays relatively less cytotoxicity on normal human umbilical vein endothelial cells than cisplatin, carboplatin or oxaliplatin determined by the cytotoxicity MTT assay, suggesting that triazinone triepoxide TATT has lower toxicity and is more secure than the anti-cancer drugs. In addition, triazinone triepoxide TATT induced apoptosis and regulated cell cycle of HT-29 cells using propidium iodide (PI) or annexin V staining followed by flow cytometry. Of note is that when HT-29 cells were treated at 4 μM for 24 hours, triazinone triepoxide TATT exhibited greater effects than these anti-cancer drugs. Administration of caspase 8 inhibitor (Z-IETD-fmk) or mitochondrial transition pore inhibitor (cyclosporine A) followed by flow cytometry analysis, Western blotting, confocal microscopy and fluorescence microscopy confirmed that triazinone triepoxide TATT-induced apoptosis proceeded via caspase and mitochondrial pathways. triazinone triepoxide TATT-induced decrease in G2/M phase accompanied with the increase of cyclin B1. Addition of curcumin (30 μM) increased apoptosis of HT-29 cells and produced an additivity or synergistic effect on triazinone triepoxide TATT or oxaliplatin but resulted in an antagonistic effect on cisplatin or carboplatin using PI or annexin V staining followed by flow cytometry analysis and Jin's method to calculate the interaction of two agents. The results of this study indicates the anti-cancer potential of triazinone triepoxide TATT and triazinone triepoxide TATT may provide an alternative choice for treating cancer to improve the quality of medical care. Regarding to of the dietary recommendation for the patients with chemotherapy, suggestion of curcumin-rich foods, such as: turmeric or curry, etc., may affect the anti-cancer efficacy. Other phytochemicals, such as: benzene, methyl isothiocyanate, cyanate ester, costunolide and phenolic compounds resveratrol, etc., may also assist or inhibit the anti-cancer therapy. Further study is needed to understand the interactions of dietary supplements or the nutrients in natural products on chemotherapy to optimize the therapeutic effect.

並列關鍵字

Triazinone triepoxide TATT ； curcumin ； apoptosis ； signal transduction ； platinum-based compound ； human colorectal cancer

參考文獻

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