2-Phenyl-4-Quinolone在人類雄激素不依賴型前列腺癌細胞的抗癌作用機轉探討

Abstract

經由干擾微小管功能的化療藥物，已廣泛地使用在對抗腫瘤。在本論文的實驗，我們探討了2-phenyl-4-quinolone化合物(2P4Q)，其在人類荷爾蒙不依賴型前列腺癌細胞的抗癌作用機轉。經由SRB細胞生長測定方法發現，2P4Q對人類荷爾蒙不依賴型前列腺癌細胞PC-3有抑制生長的作用(IC50=1.3 μM)，且其作用濃度與抑制腫瘤細胞生長的比例呈正向關係。此外，對於人類多重抗藥性乳癌細胞NCI/ADR-RES (P-glycoprotein-rich的細胞株)也有類似的生長抑制效果 (IC50=1.8 μM)，顯示2P4Q沒有微小管藥物常見的抗藥性問題。以流氏細胞儀觀察到，2P4Q會先造成細胞週期中G2/M期細胞的累積，而之後subG1期 (細胞凋亡)的增加；且由TUNEL測定法發現，2P4Q確實會引起細胞凋亡。由共軛焦螢光顯微鏡觀察到，2P4Q對好幾種tubulin isotypes (α、βI、βII、βIII、βIV)會造成聚集現象，且以monopolar、bipolar、multipolar各種方式呈現；此外，使用胞內微小管聚合實驗也發現，2P4Q有促進α、βI、βII、βIII、βIV-tubulin的聚合作用，此與共軛焦顯微鏡所觀察到的現象符合。接著我們以西方墨點法觀察各種與細胞凋亡相關的蛋白質表現，發現2P4Q會造成Bcl-2和Bcl-xL磷酸化現象、Mcl-1表現量下降及Bad的裂解現象；而且，由共軛焦螢光顯微鏡可觀測到cytochrome c及AIF有釋放的情形；此外，2P4Q也會造成多種caspase的活化，包括有：caspase-2、caspase-8、caspase-3、caspase-7，但caspase-9並沒有活化現象發生，且Apaf-1及pro-caspase-9間交互作用並無改變。我們也觀察到survivin會分佈到細胞核內且有表現量增加的情形，這現象與調控細胞週期及對抗細胞凋亡的功能有關，這也可以解釋caspase-9無法活化的現象。由於2P4Q會造成G2/M週期停滯的現象，我們也觀察了一些調控G2/M週期的蛋白表現，結果發現在2P4Q作用下，在Cdk1的Tyr-15位置有去磷酸化作用，而Cdc25C在Ser-216的位置也有去磷酸化情形發生；此外，也偵測到大量的MPM2產生。由這些情形推知，在2P4Q作用之下，Cdk1是呈現活化狀態且細胞會被停滯在有絲分裂週期 (mitosis)。綜合上述結果推論，2P4Q的作用機轉為：經由影響與tubulin之間的作用而干擾紡錘體 (mitotic spindle)的正常功能，導致細胞停滯在有絲分裂週期，進而啟動細胞凋亡的一連串訊息傳遞。

Cancer chemotherapeutic agents that interfere with tubulin/microtubule function are in extensive use against both hematological malignancies and solid tumors. In this study, we have identified the anti-tumor mechanisms of 2-phenyl-4-quinolone (2P4Q) in androgen-independent prostate cancer cell line. By sulforhodamine B (SRB) assays, we found that 2P4Q induced an anti-proliferative effect in human hormone-resistant prostate cancer PC-3 cells in a dose-dependent manner with an IC50 of 1.3 μM. 2P4Q also displayed a similar anti-tumor effect in human breast cancer NCI/ADR-RES cells (P-glycoprotein-rich) with an IC50 of 1.8 μM, revealing that 2P4Q was not the substrate of P-glycoprotein. By FACScan flow cytometric analysis, 2P4Q caused an arrest of the cell cycle in G2/M phase, and a subsequent increase in hypodiploid phase (apoptosis). The apoptotic cell death induced by 2P4Q was also identified by TUNEL reaction technique. The immuno-histochemical examination by confocal microscopy showed that 2P4Q induced the assembly of several tubulin isotypes, including α, βI, βII, βIII, and βIV tubulin, with characteristics of monopolar, bipolar, and multipolar microtubule assembly. Using in vivo tubulin polymerization assay, we found that 2P4Q promoted the polymerization of α, βI, βII, βIII, and βIV tubulin, which is corresponsive with the confocal data. We used Western blot analysis to detect the expression of some apoptosis-related proteins and found that 2P4Q was able to induce phosphorylation of Bcl-2 and Bcl-xL, downregulation of Mcl-1, and proteolysis of Bad. The immuno-histochemical detection by confocal microscopic examination showed that 2P4Q induced release reaction of cytochrome c and AIF. Additionally, 2P4Q also induced the activation of several caspases, including caspase-2, -8, -3 and -7. However, neither the association of pro-caspase-9 with Apaf-1 was monitored, nor the caspase-9 was activated by 2P4Q. Our data also showed that survivin was significantly up-regulated and was translocated into the nucleus in cells responsive to 2P4Q, revealing that survivin played a role in the regulation of cell cycle progression and the anti-apoptotic function. This effect also explained the inability of caspase-9 activation in 2P4Q-treated cells. Since 2P4Q induced G2/M arrest of the cell cycle, a further experiment on the relevant proteins was conducted. The data showed that a 24-h treatment with 2P4Q caused the dephosphorylation on inhibitory Tyr-15 of Cdk1 and Ser-216 of Cdc25c. The data together with a significant induction of MPM2 expression suggested that Cdk1 was activated and the cells were arrested in the mitotic phase after an insult by 2P4Q. In summary, it is suggested that the mechanism of action of 2P4Q involves an interaction with tubulin resulting in disturbance of regular function of mitotic spindles, leading to mitotic arrest and activation of apoptotic signaling cascades.