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

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.