- 學位論文
類黃酮素抑制人類直腸腫瘤增生之體內及體外活性研究
In Vitro and In Vivo Anti-Colorectal Carcinoma Activities of Flavonoids
摘要
癌症是目前導致人類死亡的主要原因之一，至今依然沒有有效之抗癌藥物。本研究中，選取了九種flavones 及八種flavanones的相關衍生物，探討其對於人類直腸及結腸癌細胞所具有之抗癌活性。本實驗使用三株含有不同p53基因表現型之人類直腸及結腸癌細胞，其中HT 29與COLO 320HSR是屬於p53突變型，COLO 205 則是屬於p53野生型。根據MTT分析結果顯示，在這些測試之類黃酮素化合物中， flavone 及2''-OH flavanone對於HT 29、COLO 320HSR及COLO 205這三株人類直腸癌細胞具有最顯著之細胞毒殺性，並且呈現出劑量及時間依循性的細胞凋亡特徵，包括DNA斷裂、細胞凋亡小體以及hypodiploid cells產生等現象。而投予flavone 及2''-OH flavanone後，HT29及COLO 205此兩株腸癌細胞之p21蛋白質皆被誘導大量表現; 然而p53蛋白質表現量並未隨著藥物的投與而產生變化。此外， flavone 及2''-OH flavanone能專一性誘導腸癌細胞HT29及COLO 205細胞內之caspase-3活化，並造成其受質PARP蛋白質的切割，而與活化caspase-1無關。 此外Flavone 與2''-OH flavanone所誘發之細胞凋亡現象亦可經由caspase-3之抑制劑z-DEVD-FMK前處理而降低，但不受caspase-1的抑制劑z-YVAD-FMK影響。利用DCHF-DA進一步分析細胞內自由基之變化，發現 flavone及2’-OH flavanone能誘導細胞內之活化態氧( ROS )大量增加，而經由事先投予tiron、catalase、superoxide dismutase及pyrrolidine dithiocarbamate等多種抗氧化劑，均可有效抑制其所誘導之活化態氧量產生與細胞凋亡，相反的NADPH oxidase的抑制劑diphenylene iodonium並無抑制效果。在Bcl-2群蛋白表現變化，抗細胞凋亡蛋白質Mcl-1及Bcl-XL的表現量會隨flavone 與2''-OH flavanone之投予而減少表現量，其他如Bcl-2、Bax及Bad蛋白質則沒有明顯變化。最後於裸鼠動物實驗中，flavone 與2''-OH flavanone亦能有效抑制COLO 205細胞轉植於裸鼠之腫瘤生長。此體內及體外的實驗結果證實， flavone 及2''-OH flavanone能經由誘導ROS的增加、p21蛋白質的大量表現及caspase-3的活化而抑制人類腸癌細胞。
並列摘要
Cancer is one of the major leading causes of human death, however no effective anti-cancer drug was developed. In this study, nine flavones and eight flavanones derivatives were used to examine their apoptotic activities and mechanism in three human colorectal carcinoma cells with different p53 status including HT29 and COLO 320HSR (p53 mut/mut) and COLO205 (p53 wt/wt). Among these tested compounds, flavone, and 2’-hydroxyflavanone (2’-OH flavanone) showed the significant cytotoxicity on cells by MTT assays, accompanied by a dose- and time- dependent occurrence of characteristics of apoptosis including appearances of DNA fragmentation, apoptotic bodies and hypodiploid cells. P21 protein were induced in flavone- and 2’-OH flavanone-treated cells, however p53 protein remained unchanged. Cleavage of caspase 3 substrates, poly-(ADP-ribose) polymerase (PARP), and a transient activation of caspase 3, but not caspase 1, activity appeared in flavone-, and 2’-OH flavanone-treated COLO205 and HT29 cells. Apoptosis induced by indicated flavonoids was also attenuated by caspase 3 inhibitor z-DEVD-FMK, but not caspase 1 inhibitor z-YVAD-FMK. An increase in endogenous ROS production was detected in flavone- or 2’-OH flavanone-treated colorectal carcinoma cells by DCHF-DA assay. Antioxidants such as tiron, catalase, superoxide dismutase and pyrrolidine dithiocarbamate, but not diphenylene iodonium, significantly inhibit flavone- or 2’-OH flavanone -induced apoptosis. Additionally, a decrease in anti-apoptotic protein Mcl-1 and Bcl-xL were found in flavone-, and 2’-OH flavanone-treated colorectal carcinoma cells, whereas Bcl-2, Bax, Bad and Bag proteins remained unchanged. In nude mice anti-tumoral animal model, flavone and 2’-OH flavanone exhibited the antitumor effects in nude mice with COLO 205 tumor xenografts. Results of the in vitro and in vivo study, apoptosis induced by flavone and 2’-OH flavanone in colon carcinoma cells were through ROS production, p21 and caspase 3 cascades are involved in the apoptotic mechanisms.
參考文獻
延伸閱讀
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