三唑為基礎及芳烴釕為基礎化學合成物在人類荷爾蒙不依賴型轉移性前列腺癌細胞之抗癌作用機轉研究

Abstract

賀爾蒙難治性轉移性前列腺癌（Hormone-refractory metastatic prostate cancer，HRMPC）是一種難以治癒的惡性癌症。由於其未滿足的醫療需求，故開發治療HRMPC的藥物非常重要。本論文中三唑類及芳烴釕類化合物皆顯示抗癌活性。論文的第一部分顯示，以三唑為基礎的FTY720類似物SPS-7使用sulforhodamine B（SRB）assay測定HRMPC細胞株PC-3和DU-145引發抗增生作用，並使用CFSE測定雙重證實。高濃度胸腺嘧啶阻止DNA複製，使細胞滯留於晚期G1/S phase，當從高濃度胸腺嘧啶釋放後，進行細胞週期，但SPS-7引發細胞停滯於G0/G1 phase且增加細胞凋亡。SPS-7引發細胞週期調控蛋白c-Myc、cyclin D1和phospho-RbSer807/811蛋白表現量顯著減少，與G0/G1 phase停滯作用有高度相關性。若長時間處理SPS-7將引發LC3-I轉化為LC3-II顯著增加、P62蛋白表現量顯著減少皆驗證自噬作用。Chloroquine（CQ）是自噬抑制劑會協同SPS-7引發的粒線體依賴性細胞凋亡，代表自噬扮演促進生存的角色。SPS-7顯著抑制Akt-mTOR-p70S6K-4EBP1訊息傳遞。轉染myristylated-Akt（持續Akt活化）將逆轉SPS-7造成的c-Myc、P62和Akt/mTOR蛋白表現量減少，代表Akt應作為重要的上游調節角色。值得注意的是，於動物實驗顯示相較SPS-7，FTY720顯著降低周邊血的淋巴細胞數、耗竭脾邊緣區域的淋巴球及減少鞘氨醇-1-磷酸受體蛋白表現量，代表FTY720而非SPS-7具有顯著免疫抑制性。論文第二部分顯示，有機金屬化合物CA219透過SRB及CFSE染色法證實對PC-3和DU-145細胞有抗癌活性。CA219引發顯著細胞凋亡是透過線粒體膜電位喪失，隨後Bak和Bax活化並形成多聚體膜孔，導致cytochrome c釋放和caspase活化。進一步證實，CA219快速引發活性氧化物釋放，造成DNA damage及Chk2的活化。此外，CA219引發LC3-I轉化LC3-II。已證明自噬可能作為自我保護性角色用以抵抗凋亡。使用自噬抑制劑CQ無法改變CA219引發細胞凋亡，代表自噬與細胞凋亡無關。抗氧化劑NAC可完全但NADPH氧化酶抑製劑（DPI）只能部分逆轉CA219引發的細胞凋亡和DNA損傷。綜合上述的數據顯示，SPS-7和CA219均顯示抗HRMPC的活性。 SPS-7透過抑制Akt-mTOR-p70S6K-4EBP1訊息途徑並減少c-Myc和cyclin D1蛋白表現量，導致G1 phase停滯，最終導致HRMPC細胞抑制增生和細胞凋亡。相比之下，CA219透過引起ROS伴隨粒線體依賴性凋亡來抑制HRMPC活性。

Hormone-refractory metastatic prostate cancer (HRMPC) is a type of incurable progressive cancer. Development of anti-HRMPC drugs is crucial due to its unmet medical need. Triazole-based and arene-ruthenium-based synthetic compounds in this thesis have shown anticancer activities. The first part of the thesis showed that SPS-7, a triazole-based FTY720 analogue, induced anti-proliferative effects against HRMPC cells lines, PC-3 and DU-145, using sulforhodamine B (SRB) assay. The effect was confirmed by CFSE assay. High concentrations of thymidine overload blocked DNA replication, synchronizing cells mainly at late G1/S phase. After release from thymidine block, cell cycle progression was triggered afterwards; however, SPS-7 induced profound G0/G1 arrest of cell cycle and an increase of cell apoptosis. Detection of cell cycle regulators revealed that SPS-7 induced dramatic down-regulation of protein levels of c-Myc, cyclin D1 and phospho-RbSer807/811, which were well correlated with the induction of G1 phase arrest. The conversion from LC3-I to LC3-II, an indicator of autophagy, was significantly increased in a time-dependent exposure of SPS-7. The decreased p62 protein expression validated the autophagic effect. Chloroquine, an inhibitor of autophagy, synergized mitochondria-dependent apoptosis induced by SPS-7 that indicates a pro-survival role of autophagy. SPS-7 significantly inhibited Akt-mTOR-p70S6K-4EBP1 axis signaling pathway. Transient transfection of myristylated-Akt (constitutive Akt activation) rescued SPS-7-mediated inhibition of c-Myc, p62 and Akt/mTOR signaling related protein expressions, suggesting the key role of Akt as an upstream regulator. Notably, the in vivo study showed that FTY720- but not SPS7 significantly decreased the lymphocyte counts in blood, depleted the marginal zone B cells and decreased the sphingosine-1-phosphate receptors, indicating the immunosuppressive effect of FTY720 but not SPS-7. The second part of the thesis have shown that CA219, an organometallic compound exhibits anticancer activity against PC-3 and DU-145 cells by SRB assay and CFSE staining. CA219 triggered profound apoptosis through the loss of mitochondrial membrane potential (ΔΨm), accompanied by activation of Bak and Bax in forming oligomeric pore, release of cytochrome C and caspase activation. Further examination demonstrated that CA219 induced rapid production of reactive oxygen species (ROS), DNA damage response and activation of Chk2. Furthermore, the conversion of LC3 type-I to type-II was quickly induced to CA219 action. It has been documented that autophagy may play a cytoprotective role to counteract apoptosis. The inhibition of autophagy using CQ did not modify CA219-induced apoptosis suggesting that the autophagy was independent of apoptosis. The antioxidant N-acetylcysteine (NAC) completely but NADPH oxidase inhibitor (DPI) partly rescued CA219-induced apoptosis and DNA damage. In conclusion, the data suggest that both SPS-7 and CA219 display anti-HRMPC activity. SPS-7 inhibits Akt-mTOR-p70S6K-4EBP1 axis signaling pathway and down-regulates c-Myc and cyclin D1, leading to G1 phase arrest which ultimately causes anti-proliferative effect and apoptosis in HRMPC cells. In contrast, ROS mediated and mitochondria-dependent apoptotic signaling explains CA219-mediated HRMPC activity.