- 學位論文
探討5-去甲基川陳皮素對benzo[a]pyrene/dextran sulfate sodium誘導結腸直腸癌進程之影響
The effects of 5-demethylnobiletin on benzo[a]pyrene/dextran sulfate sodium-induced colorectal carcinogenesis
摘要
根據世界衛生組織2018年統計資料顯示，全球結腸直腸癌 (colorectal cancer) 發生率排名第三，死亡率更高居第二位，由此可見預防或改善結腸直腸癌成為當前重要的研究議題。而患病率的提高與飲食習慣的改變密切相關，其中，由於食品加工過程會造成飲食中暴露於致癌物苯芘 (benzo[a]pyrene, B[a]P) 的機率增加。因此，本實驗利用benzo[a]pyrene/dextran sulfate sodium (B[a]P/DSS) 誘導腸癌模式作為模擬平台。而致癌過程 (carcinogenesis) 共可區分為三個階段，依序為起始期 (initiation stage)、促進期 (promotion stage) 及進展期 (progression stage)，若能抑制前兩階段的發展，便能防止腸癌的進程。近年來，許多研究證實柑橘類果皮含有特殊的類黃酮成分為多甲氧基黃酮 (polymethoxyflavones, PMFs)，具有抗氧化、抗發炎與抗癌等作用。其中含量最為豐富的PMF即為川陳皮素 (nobiletin)，而研究顯示，經酸水解後所形成的5-去甲基川陳皮素 (5-demethylnobiletin, DMNB)，更能有效發揮多種生物活性進而減少腸癌生成。然而，目前DMNB對於結腸直腸癌化過程中不同階段的影響尚未被探討。故本研究欲利用B[a]P/DSS誘導腸癌模式，探討DMNB在腸癌進程中所扮演的角色及其相關分子機制。由結果顯示，DMNB在40 mg/kg/day該劑量下於促進期介入能明顯減少腸道腫瘤生成及血漿中細胞激素interleukin 10 (IL-10) 及tumor necrosis factor alpha (TNF-α) 的含量。此外，於腸道組織分析結果也顯示，DMNB於促進期介入能夠抑制磷酸化extracellular signal-regulated kinase 1/2 (ERK1/2) 及其下游調控細胞週期蛋白Cyclin D1和抗凋亡蛋白B-cell lymphoma 2 (Bcl-2) 的表現。相反地，DMNB在相同劑量下於起始期介入無法減少腸道腫瘤生成及血漿中細胞激素interleukin 6 (IL-6)、IL-10和TNF-α的含量，而其腸道組織中磷酸化ERK1/2及下游調控之Cyclin D1和Bcl-2的蛋白表現量皆明顯提升，顯示DMNB於起始期介入無法藉由抑制細胞增生相關蛋白而減緩腸癌進程。另外，利用細胞試驗則發現，DMNB亦無法調節B[a]P代謝轉化路徑 aryl hydrocarbon receptor/cytochrome P450 (AhR/CYP450) 之相關蛋白表現，推測其不具抑制B[a]P經代謝活化生成高反應性致癌物之效用。綜合以上結果認為，DMNB在40 mg/kg/day該劑量下雖然不具阻止B[a]P生物轉化而產生致突變或致癌特性的能力，但可藉由抑制發炎反應及腫瘤增生而達到減緩腸癌進程，具有作為天然的癌症化學預防試劑之潛力。
並列摘要
According to the statistics from the World Health Organization (WHO) in 2018, the incidence and mortality of colorectal cancer are ranked separately the third and the second in all kinds of cancer. This shows that prevention or improvement of colorectal cancer has become an important issue. However, the increase in prevalence of colorectal cancer is closely related to the diet. Among them, some of the food processing contaminates the diet, then increase the risk of exposure to the carcinogen such as benzo[a]pyrene (B[a]P). Therefore, we used benzo[a]pyrene/dextran sulfate sodium (B[a]P/DSS)-induced colorectal cancer in mice to simulate the carcinogenesis in human body. In general, carcinogenesis can be divided into three stages, which are in the order of initiation, promotion and progression. If the initiation and promotion stage could be inhibited, it can prevent the development of carcinogenesis. In recent years, many studies have showed that polymethoxyflavones (PMFs), the specific flavonoids in citrus peel, exhibits anti-oxidant, anti-inflammatory and anti-cancer activity. Besides, nobiletin is the most abundant PMF in citrus peel. The previous studies showed that 5-demethylnobiletin (DMNB), the acid hydrolysis product of nobiletin, exhibits the stronger bioactivity of inhibiting the colorectal cancer than nobiletin. Nevertheless, it is still unclear that the effects of DMNB on different stages of colorectal carcinogenesis. In the present study, we investigated the mechanisms of DMNB involved in initiation and promotion stage of B[a]P/DSS-induced colorectal carcinogenesis. The results showed that intervention of DMNB with the dose of 40 mg/kg/day in promotion stage could reduce colorectal tumorigenesis and plasma levels of interleukin 10 (IL-10) and tumor necrosis factor alpha (TNF-α). Besides, post-treatment of DMNB could also decrease the protein levels of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), Cyclin D1 and B-cell lymphoma 2 (Bcl-2) in colorectal tissues. Conversely, intervention of DMNB with the same dose in initiation stage could not reduce colorectal tumorigenesis and plasma levels of interleukin 6 (IL-6), IL-10 and TNF-α. In addition, pre-treatment of DMNB increased the protein levels of p-ERK1/2, cyclin D1 and Bcl-2 in colorectal tissues. It revealed that pre-treatment of DMNB was unable to suppress the colorectal carcinogenesis via inhibition of cell proliferation. Furthermore, pre-treatment of DMNB failed to reduce the related protein levels of aryl hydrocarbon receptor/cytochrome P450 (AhR/CYP450) signaling pathway in B[a]P-induced HT-29 cell model. From the above, it was speculated that pre-treatment of DMNB was incapable of inhibiting the metabolic transformation of B[a]P into the DNA-reactive carcinogen. Overall, these results suggested that DMNB could not block the biotransformation of B[a]P with the dose of 40 mg/kg/day, but it may serve as a potential chemopreventive agent of colorectal cancer via inhibiting inflammation and alleviating tumorigenesis.
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