| Abstract: | 第一部份
近年來，許多研究趨向以天然物質來抑制或延遲癌症的發生，可食的植物被認為是抗癌藥物的來源之一。本研究室首先證實洛神花(Hibiscus Sabdariffa Linnaeus)具有抗氧化以及抗腫瘤促進作用之功效。更進一步針對洛神花萃取物成份之抗癌活性分析。探討洛神花多酚（H. polyphenol-rich extract，HPE）、水萃取物（H. sabdariffa extract，HSE）以及原兒茶酸（protocatechuic acid，PCA）對人類癌細胞的影響及其可能造成癌細胞死亡的機轉。以HPE進行篩選八種細胞株（NIH/3T3、Hep G2、MCF-7、KB、Caco-2、Hep 3B、HL-60和AGS）的存活測試。結果顯示：人類胃癌細胞（human gastric carcinoma，AGS）對於HPE抑制細胞存活的敏感度最高（0.95 mg/ml HPE即可達抑制細胞生長50%），而且呈現濃度依賴性。
接著以AGS細胞為實驗模式，各別處理HPE、HSE和PCA在不同劑量下（HPE：0.1、0.5、1.0和2.0 mg/ml；HSE：1.0、2.0和3.0 mg/ml；PCA：3.0、6.0、9.0 mM），經過24小時以後，觀察細胞凋亡的特徵。結果發現隨著處理HPE、HSE和PCA濃度增加對於造成AGS細胞凋亡的現象也愈顯著。包括細胞型態的改變、有效地增加DNA fragmentation的程度、以及流式細胞儀分析中，AGS處理HPE、HSE和PCA濃度分別在2.0 mg/ml、3.0 mg/ml和6.0 mM培養24小時後，其細胞週期subG1期出現明顯的apoptotic peak。並藉由p53蛋白磷酸化表現增加以及p38專一性的抑制劑或p38 dominant mutant證實HPE誘導AGS細胞凋亡是透過p53訊號傳遞以及p38 MAPK/FasL訊息路徑所調控。此外，HSE和PCA所傳遞的細胞凋亡路徑也與JNK/p38 MAPK的活化、增加FasL表現具有高度的相關性。由此我們推測洛神花具有治療胃癌的高度潛力，可能是藉由洛神花功能性成份-多酚，發揮其抗癌功效。
第二部分
洛神花多酚（H. polyphenol-rich extract，HPE）是從乾燥洛神花(Hibiscus Sabdariffa Linnaeus)花萼中萃取出來的天然抗氧化劑。近年來，有許多研究指出一些抗癌藥物可藉由引起癌細胞的程序式死亡或改變癌細胞的生長週期，來達到抑制癌細胞的增生與惡化。另一方面，了解抗癌藥物對細胞週期的調控作用有助於提高藥物之效率。本實驗室之前的研究指出，HPE具有促進人類胃癌細胞（AGS）細胞凋亡之功能（已發表論文HPE）。AGS細胞處理HPE在不同時間點（0、3、6、12、24和48小時）之下，藉由流式細胞儀測定顯示:HPE可促使AGS細胞週期停滯在G0/G1期。以西方點墨法和免疫沉澱法觀察其對癌細胞週期蛋白的影響，包括各種 cyclin-dependent kinase inhibitors（CKIs）、cyclin/cdk complexes，Rb phosphorylation及其與E2F之association，cell cycle蛋白分布等。結果發現HPE會增加p53蛋白和磷酸化表現，以及活化CKI（p21、p16、p27）；HPE並且抑制E2F和phospho-Rb的表現，以及cyclin E/cdk2和cyclin D1/cdk4的結合，導致Rb/E2F的結合增加，最後促使細胞週期停滯。進一步利用p53抑制劑（pifithrin-a）證實HPE誘導這些訊息傳遞因子之間的相關性。此研究將有助於洛神花抗癌機轉之釐清。
第三部份
原兒茶酸（protocatechuic acid，PCA）廣存於蔬菜、水果等植物中，是一結構簡單之多酚類化合物。已有報告指出原兒茶酸具有很強的抗氧化能力，可抑制化學致癌物誘導之癌化，並促進血癌以及胃癌細胞凋謝死亡。因此，本研究主要在於探討PCA之抑制癌細胞轉移侵襲作用及分子機制。首先，利用傷口癒合和Boyden chamber分析PCA對人類胃癌細胞株（AGS）移動和侵襲能力的影響，呈現濃度及時間的效應關係。我們再利用zymography的方法分析細胞移動侵襲相關蛋白表現，結果發現PCA對MMP-2、uPA的活性亦有明顯的抑制。為分析其中參與之分子機轉，我們藉由轉錄因子NF-kB表現及其與DNA結合能力、IkB ubiquitination、以及Ras/PI3K/Akt蛋白表現減少；而RhoB表現和RhoB/PKCe結合增加；以及PKCe專一性的抑制劑（GF 109203X）和基因轉殖K-ras，證實PCA抑制AGS細胞移動侵襲是透過阻斷Ras/PI3K/Akt訊息路徑，調控NF-kB/IkB機制以及活化RhoB/PKCe，抑制MMP-2之表現與活化。
後續我們利用癌症轉移之動物實驗模式，給予每隻小鼠5mg和10 mg的PCA作為治療組，結果顯示PCA可以有效抑制黑色素瘤細胞B16的轉移。從以上的結果，我們證實PCA具有抑制癌瘤細胞侵襲作用的能力。
第四部份
癌症預防已為一世界潮流，因此，尋找一化學預防物質（chemopreventive agent）為一重要工作。多酚（polyphenol）廣存於蔬菜、水果等植物中，已有許多報告證明多酚具抗氧化活性、降低血脂肪及防癌等作用。最近的發表也顯示此類洛神花多酚（H. polyphenol-rich extract，HPE）具有促進人類胃癌細胞（AGS）細胞凋謝死亡之功能（已發表論文HPE）。因此，本研究擬深入探討HPE之抗癌作用及其作用之分子機制。我們將利用多環芳香羥（B(a)P）致小白鼠（ICR mice）胃癌之動物模式，進行洛神花萃取物（H. sabdariffa extract，HSE以及HPE）抑制老鼠胃癌之研究。初步結果發現: B(a)P不只誘導一種形式的癌症產生，包含胃腺瘤、肺腺瘤、皮膚鱗狀上皮細胞癌和惡性肉瘤，造成多發性腫瘤(multiple tumor)之表徵。而 HSE或HPE對於B(a)P誘導之小鼠生理指標異常、腫瘤發生以及死亡確實有某個程度的抑制作用。因此，未來我們將進行其他的實驗，進一步驗證其功效。
Part 1
In view of the continuing need for effective anticancer agents, and the association of diet with reduced cancer risk, edible plants are increasingly being considered as sources of anticancer drugs. Hibiscus sabdariffa Linne (Malvaceae), an attractive plant believed to be native to Africa, is cultivated in Sudan and in Eastern Taiwan. Polyphenol had been demonstrated to possess antioxidative and antitumor promoting effects previously. In this study investigations were conducted to examine the mechanism of the anticancer activity of Hibiscus sabdariffa L., H. polyphenol-rich extracts (HPE), H. sabdariffa extract (HSE) and protocatechuic acid (PCA). Using HPLC assay, HPE was demonstrated to contain various polyphenolic components, including PCA, catechin, EGC, EGCG, caffeic acid and others. HPE induced cell death of 8 kinds of cell lines in a concentration-dependent manner. Among them human gastric carcinoma AGS cells were the most susceptible to HPE (0.95 mg/ml HPE inhibited its growth by 50%).
Our results revealed that AGS cells underwent DNA fragmentation, and had an increase in the distribution of hypodiploid phase (apoptotic peak, about 30-50%) after a 24-h treatment with HPE (2.0 mg/ml), HSE (3.0 mg/ml) and PCA (6.0 mM). This effect of HPE in AGS cells might be mediated via p53 signaling and p38 MAPK/FasL cascade pathway, as demonstrated by an increase in the phosphorylation of p53 and the usage of a specific p38 inhibitor, SB203580, or p38 dominant mutant. Thus, our data present the first evidence of HPE, HSE and PCA as an apoptosis inducer in AGS cells and these findings may open interesting perspectives to the strategy in human gastric cancer treatment.
Part 2
H. ployphenol-rich extract (HPE) was isolated from the dried flower of Hibiscus sabdariffa L. (Malvaceae). Previously, we have reported that HPE induced apoptosis in gastric carcinoma cells (Lin and Wang et al., Mol Carcinog. 43, 86-99, 2005). Besides, HPE through apoptosis and cell cycle arrest at G0/G1 may be associated with the induction of p53. In this report we further investigate the mechanism of cell cycle arrest induced by HPE in AGS cells. HPE treatment of AGS cells resulted in G0/G1 arrest in cell cycle progression. HPE increased the content of the cyclin-dependent kinase inhibitors (CKIs) p21, p16 and p27 protein and this effect correlated with the elevation in p53 levels. Further, we used immunoprecipitation and found that cyclin E/cdk2 and cyclin D1/cdk4 complexes reduction, which prevent the expression of Rb phosphorylation, so that Rb/E2F complex increased, resulted in inhibiting cell cycle entry response to HPE induction. These results illustrated that cell death is caused by HPE through cell cycle arrest at G0/G1 phase via induction of p53/p21, leading to reduction of cyclin E/cdk2 and cyclin D1/cdk 4 activity and decrease phosphorylation of Rb, as demonstrated by the usage of a specific p53 inhibitor, pifithrin-a.
Part 3
Protocatechuic acid (PCA), a phenolic compound ubiquitous in vegetable and fruit, had been demonstrated to possess antioxidative and antitumor promoting effects, and induce apoptosis in HL-60 and AGS cells previously. In this study investigations were conducted to examine the mechanism of the anti-invasive potentials of PCA. First, PCA (0.1, 0.5, 1.0 and 2.0 mM) dose-dependently inhibited AGS cell migration and invasion by wound-healing assay and in vitro transwell assay. Second, the decreasing activities of metalloproteinase were observed by zymographic and urokinasetype plasminogen activator (uPA) assay. The result revealed that the treatment of PCA could diminish the activities of MMP-2 more than MMP-9 and uPA. Next, our results showed that PCA exerted an induction effect on the amount of RhoB, a suppressor of transformation, invasion and metastasis, while no effects on Rho A and Cdc42 belong to small GTPase family. The PCA-mediated inhibition of MMP-2 and induction of RhoB appear to occur via NF-kB because their DNA binding activity was suppressed by PCA. Furthermore, PCA significantly inhibited the expression of Ras, PI3K, Akt and NF-kB in a dose- and time-dependent manner. The pretreatment of an inhibitor specific for RhoB/PKCe to AGS cells could result in reverse the PCA-mediated inhibition of MMP-2 with a marked inhibition on cell migration and invasion, respectively. Finally, PCA inhibited melanoma metastasis to the liver in a mouse model.
Taken together, these results indicate that PCA targets the activation of RhoB as a mechanism by blocking the Ras/PI3K/Akt and NF-kB/IkB pathway, which in turn led to the reduced MMP-mediated cellular events in cancer cells and provides a new mechanism for its anticancer activity.
Part 4
Considerable attention has been focused on chemopreventive agents that occur naturally or are added to food and beverages for human consumption. Polyphenol is ubiquitous in vegetable and has antioxidative properties. H. ployphenol-rich extract (HPE) was isolated from the dried flower of Hibiscus sabdariffa L. (Malvaceae). Recently, our data also showed that H. sabdariffa extract (HSE) and HPE possess antitumor potentials in gastric carcinoma cells (Lin and Wang et al., Mol Carcinog. 43, 86-99, 2005; Chem Biol Interact. 165, 59-75, 2007.). Further study is needed to assess more accurately the mechanisms of HSE and HPE on the antitumor effect in vivo.
The results of these studies may be used to (i) demonstrate the antitumor effect of HSE and HPE on benzo(a)pyrene-induced gastric carcinogenesis and to (ii) provide some momentums for developing HSE and HPE as new chemopreventive agents. |
