2,6雙取代醯胺基蒽醌衍生物之細胞毒殺活性篩選及相關抗癌分子機制之研究

Cytotoxic screening of 2,6-Disubstituted Amidoanthraquinones and Anticancer Molecular Mechanisms Studies.

許碩辰(Shuo-Chen Hsu)

指導教授 ： 邱慧芬

高雄醫學大學/醫學院/藥理學科研究所/碩士(2006年)

摘要

蒽醌衍生物(anthraquinones derivatives)近年來已被證實有許多生物活性，例如抗腫瘤、抗濾過性病毒、血管舒張、抑制腸臑動或輕瀉作用等藥理活性。目前我們的研究團隊已尋找出在1,4、1,5、1,8、2,6等雙取代位置之蒽醌衍生物中幾個化合物較具有明顯的抗癌藥理活性，而其結果亦已陸續發表於2001-2006年知名國際期刊。
今將對2,6雙對稱雙胺醯基蒽醌衍生物B7和B9兩者對各種癌細胞的初步毒殺作用和相關分子作用機轉做更進一步的探討，以了解其抗癌分子作用機轉。
不同濃度的2,6雙對稱雙胺醯基蒽醌衍生物(B7及B9)，對人類肝癌細胞(Hep G2、2.2.15)、大白鼠神經膠質瘤細胞(C6)和人類肺癌細胞(A549)之毒殺效果，以methylene blue方法測定其對細胞生長之調節或毒殺作用，發現B7及B9皆對癌細胞之生長有抑制作用。其中B7及B9對數種癌細胞(Hep G2、2.2.15、C6、A549)之50%抑制生長濃度(IC50)分別B7: 0.27, 0.41, 0.28, 0.47 μM；B9之IC50分別為:10.57, 10.28, 20.10, 12.30 μM。
B7和B9之抗癌活性與細胞週期之相關性測定則以流式細胞儀(flow cytometer)來測定，低濃度時B7 (0.05 μM)和B9 (1 μM)造成明顯抑制細胞週期作用在G1期的效果，其結果亦與天然蒽醌衍生物即大黃素emodin相似，無論在24或72小時均被觀察具有停滯週期在G1/G0期之作用，其作用機轉不同於mitoxantrone或adriamycin兩者之作用均造成停滯在G2/M期之現象。
在細胞凋亡之測定試驗中，以不同濃度(0.5-30 μM)之B7和B9作用於HepG2細胞後24及72小時，經由原位DNA片段測定法(TUNEL)分析，結果可發現細胞凋亡的數目隨著B7及B9兩者藥物濃度之增高呈現反應增加之現象。
B7和B9兩者對細胞膜上細胞凋亡因子TNF-R1之調節活性測定發現B7 (20 μM)、B9 (20 μM) 加入HepG2細胞作用72小時後，測定發現B7、B9對於人類肝癌細胞TNF-R1無明顯之作用。
一氧化氮(NO)與肝臟細胞之毒殺作用機轉可能密切相關，我們測定B7和B9 (0.1-10 M)作用24及72小時後，發現NO之含量兩者均有增加現象且B7較明顯，在西方點墨法(Western blot)測定中亦觀察到誘發型一氧化氮合成酶(iNOS)及上游之NFκB兩者之活性均有增加，證明NO 之增加可能增強B7和B9對肝癌細胞造成細胞凋亡之作用。
利用西方點墨法(Western blot)偵測促細胞凋亡相關因子如Fas、Fas Lignad、FADD、caspase-3，測定結果發現B7及B9會造成細胞凋亡各相關因子們之活性均明顯增加，而使抗細胞凋亡相關因子Bcl-2相對的有活性減少之現象。除此之外，為了確認B7、B9對細胞週期調節之作用機轉，我們亦利用西方點墨法(Western blot)偵測細胞週期素cyclin D1之表現及腫瘤抑制因子p53，我們發現B7、B9均會造成p53活性增加之作用，而以B7較為明顯，接著p53影響細胞週期素cyclin D1造成其活性下降，最後導致細胞週期停滯於G1/G0期。
本研究之結果顯示研究新型2,6雙對稱雙胺醯基蒽醌衍生物為一系列具有潛力之抗肝癌新藥，其B7及B9對細胞內NO濃度之調節、細胞凋亡因子之活化、細胞週期停滯帶動癌細胞生長停滯其為抗癌之相關分子作用機轉，而整體而言，B7之作用均較B9為顯著。

關鍵字

蒽醌衍生物 ； 細胞凋亡 ； 抗癌

並列摘要

In recent years, anthraquinones derivatives have been proved to possess various of biological activities including anticancer: antibacterial, anti-multiple sclerosis, vasodilatation and supperes intestine creeps effects.
Our research team has reported that hundreds synthesized small-molecule disubstituted anthraquinones have cytotoxic effects on various cancer cell lines. We found the different of anthraquinone derivatives (1,4 1,5 1,8 and 2,6 disubstituted derivatives) have demonstrated different cytotoxic potency (Huang et al.2001-2006).
For investigation the anticancer molecular mechanisms of the new 2,6 diamidoanthraquinone derivers, we focus on two compound, B7(CHH20060607) and B9(CHH20060609), study their molecular mechanisms of cytotoxic effects.
After cytotoxicity test, we found B7 & B9 possessed different inhibitory effect on various of tumor cell lines: human hepatoma cell(HepG2. 2.2.15), rat glioma cell(C6) and lung carcinoma cell(A549). Our results have indicated the IC50 of B7 on HepG2, 2.2.15, C6 and A549 cell are 0.27, 0.41, 0.28, 0.47 ?嵱, and the IC50 of B9 are 10.57, 10.28, 20.10, 12.30 ?嵱, respectively.
At low concentrations of B7 (0.05 ?嵱) or B9 (1 ?嵱) both caused cell cycle arrest at G0/G1 phase at 24hr or 72hr, this effect similar to the natural anthraquinone emodin. In contrast, the clinical anticancer drugs mitoxantrone and adriamycin both induced cell cycle arrest on G2/M phase which demonstrated different action mechanism.
The results of TUNEL assay have demonstrated that B7 and B9 both caused significant apoptosis change in HepG2 cells. B7(0.5-20 μM) and B9(1-30 μM) both shown a dose- response phenomenon.
Nitric oxide (NO) is a muti-regulated molecule in hepatocyte. B7 and B9 induced the increase of the intracellular “NO” release which might result in the formation of toxic reaction products, such as peroxynitrite that induced cell apoptosis. In our Western blot analysis, the “iNOS” expression and upstream “NFκB” activity both were found significent promotion.
With the Western bloting assessment, we detect other apoptosis signals: Fas, Fas Ligand, FADD, Caspase-3 and antiapoptosis signal:Bcl-2. Simutaneouly, Our results pointed out that B7 and B9 both can increase the activation of apoptoatic signal(Fas, Fas Ligand, FADD, Caspase-3, p53) and decreased the antiapoptosis Bcl-2 level. It were also indicated that B7 and B9 both could decrease the Cyclin D1 expression and change pERK and pAKT proliferative signal.
In conclusion, our data have shown the CHH20060607(B7) is a potential new anticancer drugs. Both B7 and B9 increase the release of NO synthesis, activating the apoptosis signal (Fas, Fas L, FADD, Caspase-3, p53, p27),and decreased both the antiapoptotic signal Bcl-2 protein expression and proliferative signal(pERK, pAKT).

並列關鍵字

amidoanthraquinones ； apoptosis ； anticancer

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