| 摘要: | 為了尋找更有效、更有潛力的抗癌準新藥物質，仍有許多的研究和開發正不斷的進行。在本研究中，我們針對本實驗室中所合成具有不同骨架的數種衍生物進行體外癌細胞株之抗癌活性篩選。我們幸運的發現其中3個化合物(DBA、BTP-11以及YC-1)具有顯著的抗癌活性。三個化合物分屬不同化合物的骨架且各具有不同的抗癌活性機制。
在本論文的第一部份，我們將闡明化合物DBA及BTP-11對於人類前骨髓性白血病細胞株HL-60的抗癌機制。化合物DBA是以阿魏酸當起始物質所合成得到的ferulamide衍生物，阿魏酸是存在於天然界中最為普遍的酚類化學物質，其本身具有抗氧化及抗癌的活性。我們的結果顯示化合物DBA造成HL-60細胞生長的抑制具有濃度和時間的依存性，24小時的IC50值約為8.2 μM，而且對人類週邊血球沒有任何顯著的毒性出現。經細胞週期的分析後發現，化合物DBA造成HL-60細胞停滯於G2/M期，而且在顯微鏡觀察時發現具有細胞皺縮以及凋亡小體出現等細胞凋亡反應的表徵。在以反轉錄聚合酶連鎖反應檢測相關基因的表現結果中發現，化合物DBA顯著的增加p21 mRNA的表現；並抑制cyclin B1 mRNA的表現，這樣的結果於是造成細胞停滯於G2/M期，而且也發現到明顯降低Bcl-2 mRNA表現後造成細胞凋亡的發生。
全反式維生素甲酸是維生素A的衍生物，其透過誘發顯著的細胞分化能力對於急性骨髓性白血病的治療上展現極高的緩解率。而在我們過去研究中發現benzoditjiophenedione類衍生物對於HL-60細胞具有顯著的抗癌活性，於是我們進一步利用NBT還原法測試這一系列化合物對於HL-60細胞分化誘導的活性。有趣的是，我們發現化合物BTP-11具有明顯增強全反式維生素甲酸誘導人類前骨髓性白血病細胞株HL-60的分化活性，而且在併用0.1 μM的化合物BTP-11以及5 nM的全反式維生素甲酸情形下可達到近乎完全分化的能力。根據我們實驗的結果發現，化合物BTP-11也可增強全反式維生素甲酸促進HL-60細胞停滯於G0/G1期的效果，另一方面再以Liu’s染色判別細胞核型態與細胞表面抗原表現的結果證明，其與全反式維生素甲酸一樣造成細胞走向顆粒球分化成熟的途徑。進一步利用西方墨點法檢測調控G0/G1期與G1-S過渡期進程之相關蛋白質表現，結果發現，當併用化合物BTP-11與全反式維生素甲酸時對於cyclin D1、CDK4以及cyclin A有加成抑制其表現的效果，同時，不論是單獨使用化合物BTP-11或全反式維生素甲酸以及併用兩者時都有明顯增加骨髓性分化特異表現的轉錄因子C/EBPε的活性。以上結果可以讓我們進一步了解化合物DBA及化合物BTP-11的抗癌分子機制。
在本論文的第二部份中，吲唑類化合物YC-1經利用日本JFCR39癌細胞株之抗癌活性篩選後發現，YC-1對於人類肺鱗狀上皮細胞癌細胞NCI-H226以及人類腎癌細胞RXF-631L具有高度的抑制選擇性，於是我們選擇NCI-H226細胞株做為YC-1對抗肺癌之抗癌機制探討模式細胞。由實驗結果發現，YC-1造成NCI-H226細胞生長的抑制具有濃度和時間的依存性。YC-1會抑制cyclin D1、CDK2及cdc25A蛋白質表現；以及增加p16、p21和p53等蛋白質的表現，於是造成NCI-H226細胞大量停滯於細胞周期的G0/G1期。經長時間以YC-1處理後亦可誘發NCI-H226細胞進入經由粒線體啟動的細胞凋亡反應。另外，YC-1亦可以透過抑制基質金屬蛋白酶-2和-9的表現進而抑制NCI-H226細胞轉移的活性。根據上述結果指出，YC-1具有多重的抗癌機制表現並且有希望成為對抗肺癌的新化療藥物。
綜合本論文的研究成果，化合物DBA、化合物BTP-11以及YC-1均頗具潛力成為抗癌準新藥物質。
In order to seek and find a potential anti-cancer drug candidate, exploring and designing new cancer agents are still focus of cancer researches. In this study we used in vitro cancer screen to evaluate the anti-tumor activities of a part of compounds from our laboratory. Fortunately, we have found three compounds (DBA, BTP-11 and YC-1) which have significant anti-cancer activity. Each one belongs to different
chemical skeleton and exhibit different anti-cancer mechanism.
In the first part of our studies, we elucidate the anti-cancer mechanisms of DBA and BTP-11 in the human acute promyelocytic leukemia cell line HL-60 cells. (2E)-N,N-dibutyl-3-(4-hydroxy-3- methoxy-phenyl)acrylamide (DBA), a ferulamide derivative was synthesized from the starting material “ferulic acid” in our laboratory. Ferulic acid is one of the most ubiquitous phenolic compounds in nature, which has antioxidant and anticancer activity. Our data demonstrate that DBA inhibited cell growth in a concentration- and time-dependent manner, with IC50 value being to 8.2 μM (24 h) and without any detectable effects on PBMC cells. The cell cycle analysis indicated that DBA-treated cells were arrested in G2/M phase. Microscopic examination showed that treatment with DBA displayed typical morphological features of apoptotic cells, with cell shrinking and formation of apoptotic bodies. Reverse transcription polymerase chain reaction (RT-PCR) analysis showed a dramatic induction of CDK inhibitor p21 to inhibit the expression of cyclin B1, thereby resulting in G2/M phase arrest. After G2/M phase arrest, cells underwent apoptosis via significant down-regulation of Bcl-2 expression level.
The vitamin A derivative, all-trans retinoic acid (ATRA), has the remarkable ability to induce differentiation of leukemic promyelocytes in patients with APL, resulting in high CR rates. In our prior work, we have found that some benzodithiophenedione derivatives showed potent cytotoxicity against HL-60 cells. We therefore analyzed the induction of leukemic cell differentiation potential of these series compounds as determined by the nitroblue tetrazolium (NBT) assay in human HL-60 cells. Interestingly, 2-(1-hydroxylethyl)-4,8-dihydrobenzo[1,2-b:5,4-b''] dithiophene-4,8-dione (BTP-11) showed the greatest enhancement of ATRA-induced differentiation in HL-60 cells, inducing nearly complete differentiation at combination of 5 nM ATRA and 0.1 μM BTP-11. In this study, we also reported that BTP-11 could enhance ATRA-induced G0/G1 cell-cycle arrest in HL-60 cells. On the other hand, Liu’s staining, phagocytic activity and expression of cell surface markers (CD11b and CD14) confirmed that the inhibition of proliferation is associated with differentiation especially toward granulocytic differentiation pathway. Furthermore, we found a synergistic reduction of the G0/G1 and G1-S phase regulators, cyclin D1, CDK4 and cyclin A in response to the combination of BTP-11 and ATRA. Additionally, expression of the novel myeloid specific transcription factor C/EBPε was increased by BTP-11 or ATRA used alone and together. These results provide important information towards the understanding of the action mechanisms by DBA
or BTP-11 mediated anticancer effects.
In the second part of our research, YC-1, an indazole derivative, displayed impressive selective toxicity against the human lung squamous carcinoma cell line NCI-H226 and human kidney tumor cell lines RXF-631L by a panel of 39 human cancer cell lines (JFCR39) screening. Therefore, the molecular mechanism by which YC-1 affects NCI-H226 cell growth was studied. YC-1 inhibited NCI-H226 cell growth in a time- and a concentration-dependent manner. The data showed that YC-1 suppress the protein levels of cyclin D1, CDK2 and cdc25A, up-regulate p16, p21 and p53, increase the number of NCI-H226 cells in the G0/G1 phase of the cell cycle. Long exposure to YC-1 induced apoptosis by mitochondrial-dependent pathway. In addition, YC-1 inhibited matrix metalloproteinases-2 (MMP-2) and MMP-9 protein activities to abolish tumor cells metastasis. These findings suggest that YC-1 exhibit multiple anti-cancer mechanisms and indicate that YC-1 may be a promising chemotherapy agent against lung cancer.
In conclusion, our data demonstrate that DBA, BTP-11 and YC-1 are identified in this study as new anti-cancer drug candidates. |
