| Abstract: | Part I Eesculetin( 6,7-dihydroxycoumarin )是香豆精( coumarin )的衍生物之一，常見於藥用植物如菊科的茵陳蒿、芸香科的橙柚、玄參科的毛地黃、大戢科的續隨、茄科的顛茄、蔓陀羅、茛宕等，結構是一多酚類化合物。過去本研究在抗氧化天然物開發中發現其具有不錯的抗氧化和抗發炎之作用，而近年來有文獻報告香豆精衍生物( 如：1,2-benzopyrone、7-hydroxycoumarin )對人類惡性細胞株皆具有抑制生長的作用，但是作用的分子之機制仍不清楚。
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。在本實驗中，觀察到esculetin可以抑制人類肝癌細胞株( H3B )的生長，而由流速細胞儀測定顯示esculetin可以使H3B細胞週期停滯在G1 phase，以esculetin分別處理H3B細胞0、12、24、36、48小時後由西方墨點法發現，esculetin可以使cyclin D及cyclin E表現量減少，尤其對cyclin D1的影響較明顯；但是並不影響CDK-2、CDK-4的蛋白表現。而cyclin dependent kinase inhibitor ( CKI / CDKI ) p27、p21、p16的表現量都有增加， p21在24小時達最高量，p27則在24小時開始上升。另外esculetin處理H3B細胞後增加了transcription factor E2F-1蛋白的表現量; 但是另一個transcription factor DP-1則不受影響，同時也跟兩者的association無關。p38MAPK、phospho-MAPK、phospho-JNK的磷酸化會增加，尤其以phospho-MAPK、phospho-JNK的磷酸化較明顯，在24小時達最高峰，對於p38、JNK蛋白的表現量並沒有影響。但是增加了ERK-1、ERK-2蛋白的表現量;尤其是ERK-1蛋白，在24小時達到最大量。
Part II
咖啡酸(caffeic acid [ CA / 3,4-dihydroxy-cinnamic acid ])及其衍生物caffeic acid phenethyl ester [ CAPE / 3-( 3,4-dihydroxy-phenyl ) acrylic acid phenethyl ester]和caffeic acid ethyl ester [ EC / 3-( 3,4-dihydroxy-phenyl ) acrylic acid ethyl ester]都是峰膠的主要成分之一。咖啡酸(Caffeic acid [ CA / 3,4-dihydroxycinnamic acid ])廣泛分布於蔬菜、水果、全榖類、和蜂蜜等物中。CA及其衍生物CAPE和EC結構上均屬於多酚類化合物，由於近年來對於蜂蜜的相關研究漸趨白熱，發現其許多治療預防的特性。例如: 抗氧化、抗突變、抗癌、和抗免疫發炎反應等等功能( 1-5 )。在本篇實驗中由MTT的結果知道蜂膠中的主要活性成分CAPE對於腦神經膠瘤細胞株C6 glioma cell及人類肝癌細胞株H3B hepatocyte的作用高於CA; 且CAPE的作用發生較EC早，0.05 mM CAPE在12 hr即可降低50 % 細胞存活。而C6 glioma cell受CAPE，EC的影響較人類肝癌細胞株H3B hepatocyte明顯。同時觀察加藥處理24小時後的細胞型態，發現細胞的mitosis降低，且伴隨著mophology的變化。自流式細胞分析儀分析資料顯示CAPE可使細胞週期停滯在G1/S期; 接著西方點墨法的分析CAPE促使C6 glioma cell 生長週期停滯是透過p53活化下游的p21，和cyclin dependent protein kinase ( CDKI / CDI ) — p27; 而非透過另一tumor suppressor — RB來執行調控細胞週期的功能。
Part I Esculetin, a phenolic compound, is a coumarin derivative containing in many plants such as Atemisiae capillaris Flos ( Compositae ), leaves of Citrus limonia ( Rutaceae ), Digitalis purpurea L. ( Scrophulariaceae ), Euphorbia lathyris L. ( Euphobiaceae ), Atropa belladonna ( Solanaceae ), Datura stramonium L. ( Solanceae ) Hyoscyamus niger L.( Solanceae ). Previously, we found that esculetin exhibited antioxidant and anti-inflammatory bioactivities. It is reported that coumarin derivatives can inhibit the proliferation of human malignant cell lines. However, the molecular mechanism of anti-proliferation of these compounds are remain to be defined.
Recently, some studies have suggest that anticancer drugs may induce cancer cell apoptosis ( programmed cell death ) or alter cell cycle to prevent prolifaration of the cancer cell. Although, the mechanisms about apoptosis were unclear, is supposed generally that a promising chemotherapeutic agent will have to promote the cell entering these two pathway. In this study, we found esculetin presents the ability of inhibiting cell growth in H3B hepatocytes. The flow cytometry analysis showed the esculetin arrest cell cycle of H3B cells in G1 phase. In addition cell cycle analysis, we also found esculetin treatd H3B hepatocytes reduced the expression of cyclin D and cyclin E, especially in cyclin D1; but has no effect on CDK-2、CDK-4. Cyclin dependent kinase inhibitors( CKI / CDKI )--p27、p21、p16 were increase after esculetin treatment, transcrption factor--E2F-1 content was decrease, DP-1 expression and the dimerization of E2F-1-DP-1 had no relationship to esculetin induced cell cycle arrest. Finally we observe the phosphorylation of MAPK siganling cascades members--38MAPK、phospho-MAPK、phospho-JNK and the protein expression of ERK-1、ERK-2 were increased, but do not effect expression of p38MAPK、JNK proteins.
Part II
Caffeic acid [ CA / 3,4-dihydroxycinnamic acid ]and caffeic acid esters: including caffeic acid phenethyl ester [ CAPE / 3-( 3,4-dihydroxy-phenyl ) acrylic acid phenethyl ester] and caffeic acid ethyl ester [ EC / 3-( 3,4-dihydroxy-phenyl ) acrylic acid ethyl ester] were phonolic components of propolis . Propolis , a honey bee hive product , exihibits a specturm of medical appication . For example : anti-oxidant , tumor growth inhibition , anti-mutagenic , anti-cancer , and anti-flammatory . Recently several investigate suggest that some of the observed biological effects may be due to caffeic acid esters which were present in propolis . some scholar bring an idea that anticancer drugs may induced cancer apoptosis or alter cell cycle to provent the cancer cell proliferation . In this experiment , we found the ability of CAPE ( the active compoment of propolis extract ) to inhibit cell growth also execute a better action to C6 glioma cells than EC and CA . And C6 was more sensitive to CAPE、EC treatment than H3B hepatocytes . Flowcytometry analysis show CAPE arrest the cell cycle of C6 cells at G1/S phase . Following by morphology / nuclear staining , CAPE reduced mitosis and change morphology of C6 cells . Furthermore analysis the proteins association with cell cycle . We found C6 cells treatment with CAPE 12 hr would increase tumor suppressor — p53 , p53 target protein — p21 , and cyclin dependent kinase inhibitor ( CDKI / CKI ) — p27 expression , but has no relationship with anthor tumor suppressor — Rb . The results sggest that CAPE may as a powerful chemotherapy agent for some neoplastic cells . |
