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Title: | 探討 Benzimidazole 衍生物在人類荷爾蒙不依賴型前列腺癌細胞
的抗癌作用機轉 Investigation of the Anti-Tumor Mechanism of Benzimidazole Derivative in Human Androgen-Independent Prostate Cancer Cells |
Authors: | Wei-Ling Chang 張瑋凌 |
Advisor: | 顧記華 |
Keyword: | 細胞凋亡,細胞週期停止於G2/M時期,抗微管蛋白作用,JNK抑制劑, apoptosis,G2/M arrest,anti-microtubule,JNK inhibitor, |
Publication Year : | 2008 |
Degree: | 碩士 |
Abstract: | RFLC021是benzimidazole的衍生物,可抑制多種癌細胞的生長活性,包括攝護腺癌細胞(PC-3, DU145, LNCaP)、肝癌細胞(Hep3B, HepG2)、非小細胞肺癌(A549),與表現大量p-glycoprotein的乳癌細胞 (NCI/ADR-RES),其IC50濃度大約介於1.7到3.7 μM。由於RFLC021也抑制NCI/ADR-RES細胞的生長,因此,此化合物可能非p-glycoprotein的受質。RFLC021隨著濃度與時間的增加,造成PC-3、DU-145與LNCaP細胞的細胞週期停止在G2/M時期;在長時間的作用下,RFLC021引起hypodiploid之DNA增加,表示細胞凋亡的發生。由in vitro 微小管試驗與免疫螢光試驗可知,RFLC021於PC-3細胞中會抑制微小管聚合,並造成不正常的有絲分裂紡錘體。此化合物會造成cyclin B1與MPM-2的表現量增加、使CDK1的Thr161與Tyr15位置磷酸化。這些結果顯示RFLC021使細胞停止在G2/M phase。此外,粒線體相關的訊息傳遞也受到RFLC021的影響,包括Mcl-1減少與Bad的斷裂、Bcl-2與Bcl-xL的磷酸化、caspase-9與-3的活化以及poly(ADP-ribose) polymerase (PARP)的斷裂。此外,RFLC021也造成DNA 損傷。有趣的是,JNK抑制劑 SP600125可顯著的降低由RFLC021引起的細胞凋亡,且降低Bcl-2、Bcl-xL磷酸化的表現及caspase-3, -9和PARP的活化,同時也減少了型態異常的紡錘絲形成。另外,SP600125也影響了RFLC021對細胞週期的調控,包括減少MPM-2與cyclin B的表現量,增加cyclin E與p27表現量,使細胞通過Mitotic phase,以2N或4N的形式回到G1 phase。總言之,這些實驗結果顯示出由RFLC021引發之抗生長活性與細胞凋亡作用是透過抑制微小管聚合、使得細胞週期停止在G2/M時期,引發JNK相關的細胞凋亡。 RFLC021, a benzimidazole derivative, inhibited cell growth in various cancer cell lines, including prostate cancer PC-3, DU-145 and LNCaP, hepatocellular carcinoma Hep3B and HepG2, non-small cell lung cancer A549, chronic myeloid leukemia K562, acute promyelocytic leukemia HL-60 and P-glycoprotein-rich NCI/ADR-RES cells with IC50 values ranging from 1.7 to 3.7 μM. NCI/ADR-RES cells were susceptible to RFLC021-mediated anticancer effect suggesting that this compound is not a substrate for P-glycoprotein transporter. In flow cytometry analysis, PC-3, DU-145 and LNCaP cells were arrested at G2/M phases in a time- and concentration-dependent manner, followed by a sequential increase of hypodiploid phase (apoptosis). In vitro tubulin turbidity and immunofluorescence assays showed that RFLC021 inhibited tubulin polymerization and caused abnormal mitotic spindles in PC-3 cells. This compound also activated the up-regulation of cyclin B1 expression, Cdk1 phosphorylation at Thr161 as well as Cdc25C phosphorylation and elevation of MPM-2 levels. The data indicate the occurrence of mitotic arrest to RFLC021 action. Moreover, several mitochondria-related signals were modified by RFLC021, including the cleavage of Mcl-1 and Bad, phosphorylation of Bcl-2 and Bcl-xL, activation of caspase-9 and -3, and cleavage of poly(ADP-ribose) polymerase (PARP). Interestingly, the JNK inhibitor SP600125 significantly blunted RFLC021-induced apoptotic cell death associated with an inhibition Bcl-2 and Bcl-xL phosphorylation, caspase-3 activity and PARP cleavage, as well as blocking the formation of abnormal mitotic spindle. In addition, SP600125 also inhibited RFLC021-induced G2/M arrest through the regulation of various cell cycle regulators, including the decrease of MPM-2 and cyclin B levels, increase of cyclin E and p27 levels. It suggests that cells escape from mitotic arrest and re-enter the cell cycle with 2N or 4N (hyperdiploid) DNA content. In summary, the data suggest that RFLC021 induces the anti-proliferative and apoptotic effects through the inhibition of tubulin polymerization, induction of mitotic arrest and triggering of JNK-dependent apoptotic cell death. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/26717 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 藥學系 |
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