天然物及合成化合物在人類荷爾蒙不依賴型前列腺癌 的抗癌機轉探討

Abstract

前列腺癌是男性罹患癌症致死的主因之一。目前前列腺癌的治療策略仍局限於手術、化學治療、冷凍切片、荷爾蒙治療及放射線治療。其中，對於荷爾蒙療法無效的荷爾蒙不依賴型前列腺癌仍是臨床治療上的一大困境。由於治療荷爾蒙不依賴型前列腺癌相當棘手，因此尋求有效的治療方式可說是備受矚目。在本篇論文中，將針對荷爾蒙不依賴型前列腺癌提出治療策略。第一部分與第二部分將著重於探討多種天然物及化學合成化合物的抗癌機轉，第三部分則是首次提出rottlerin與camptothecin合併療法的研究。 本論文的第一部分，我們將探討天然物costunolide及moniliformediquinone的抗癌機轉，且將著重於探討氧化性壓力、鈣離子失衡及DNA損傷的角色。Costunolide的結構為倍半萜內酯(sesquiterpene lactone)，本實驗室欲探討新的機轉來強調其對荷爾蒙不依賴型前列腺癌細胞具有抗癌效果，並使用多種實驗方法來釐清其抗癌作用機轉。首先使用SRB方法(sulforhodamine B assay)、clonogenic test、流式細胞儀及carboxyfluorescein succinimidyl ester (CFSE)染色來確定costunolide在荷爾蒙依賴型前列腺癌LNCaP細胞株及荷爾蒙不依賴型PC-3、DU145細胞株抑制生長的情況。接著發現costunolide在PC-3細胞株能快速造成細胞核中鈣離子上升，並引發DNA損傷與ATR磷酸化。同時造成細胞週期G1期停滯、p21蛋白表現增加、p21/cdk2/cyclinE複合物結合增加以及Rb蛋白去磷酸化。這些現象可被鈣離子螯合劑BAPTA-AM與抗氧化劑glutathione (GSH)及其前驅物N-acetylcysteine (NAC)所抑制，然而另一種抗氧化劑trolox卻無效。這表示調控細胞內鈣離子及thiol含量減少對於細胞凋亡的重要性，來達到抗前列腺癌的效果，而活性氧化物的角色較不重要。以上實驗結果顯示，costunolide會造成細胞內thiol含量減少及細胞核內鈣離子上升，造成DNA損傷及p21表現量增加，p21經由與cdk2/cyclin E複合物結合，而抑制cdk2的活性與Rb蛋白的磷酸化，最後造成細胞週期停留於G1期及細胞凋亡。 Moniliformediquinone屬於phenanthraquinone結構的化合物，是由石斛得到的天然物。其在高度轉移性荷爾蒙不依賴型前列腺癌細胞株PC-3中，展現抗細胞生長及促進細胞凋亡的能力。本研究中，使用了多種方法來釐清moniliformediquinone在PC-3細胞的抗癌機轉。細胞在moniliformediquinone的處理下， 分別使用monochlorobimane (mClB)及fluo-3-AM染劑偵測GSH及鈣離子，發現會快速地造成細胞內GSH含量下降以及細胞質內鈣離子濃度上升。此外，moniliformediquinone也會迅速引起DNA損傷反應，並且造成相關蛋白 ATM、CHK1、CHK2和RPA32的磷酸化。接著，moniliformediquinone會引起粒線體壓力，造成粒線體膜電位的下降、Mcl-1蛋白(Bcl-2家族蛋白中抗細胞凋亡蛋白)的斷裂以及cytochrome c由粒線體釋放到細胞質中。而且，經由偵測IκB-α的磷酸化與降解以及NF-κB冷光試驗，結果皆顯示moniliformediquinone會活化NF-κB。另外值得注意的是，由in vivo 微小管試驗中，處理moniliformediquinone也會造成微小管不穩定。若預先給予sulfhydryl化合物GSH及NAC，可完全抑制moniliformediquinone造成的細胞反應，顯示了細胞內GSH含量對moniliformediquinone抗癌機轉的重要性。由以上實驗結果得知，moniliformediquinone會造成細胞內GSH含量下降，使細胞內鈣離子濃度上升、引起 DNA 損傷反應與粒線體喪失功能，最後導致PC-3細胞的生長抑制及細胞凋亡。 第二部分將探討化學合成anthraquinone衍生物EJMC30的抗癌機轉。EJMC30對於PC-3及H9c2心肌母細胞株具有良好的選擇性，GI50的值分別為8.64及大於30 μM。細胞處理EJMC30會造成細胞週期停滯於G1期，伴隨著細胞週期調控蛋白cyclin D1與cyclin E的表現量減少，以及p21蛋白的表現量增加。EJMC30也經由抑制mTOR (Ser2448)、4E-BP1 (Thr37/Thr46/Thr70)與 p70S6K (Thr389)的磷酸化，造成mTOR主導的轉譯途徑受到抑制。進一步分析ribosomal profiling後發現EJMC30會抑制蛋白生成轉譯途徑的起始及延長階段。接著EJMC30能快速導致mTOR上游蛋白Liver kinase B1 (LKB1)- 5'-AMP-activated protein kinase (AMPK)途徑的活化，顯示LKB1-AMPK途徑對於EJMC30作用機轉的重要性。總結來說，EJMC30 會經由LKB1-AMPK-mTOR途徑而影響ribosomal profiling及蛋白質轉譯，導致細胞週期停滯於G1期而抑制癌細胞生長。 然而，對化學治療藥物產生抗藥性仍是前列腺癌進展過程的主要機轉之一。因此，針對藥物機轉研發化學治療藥物，找尋新方法來增強細胞死亡可視為治療前列腺癌的策略之一。合併療法即是對抗荷爾蒙不依賴型前列腺癌的方法，可增強藥物的細胞毒殺效果與減少抗藥性的產生。在本論文的第三部分，我們提出了合併使用rottlerin與camptothecin的可能性。Rottlerin為含多酚結構的天然物，本研究發現其與topoisomerase I inhibitor camptothecin對於荷爾蒙不依賴型前列腺癌細胞株PC-3的毒殺作用有協同作用。雖然rottlerin為已知的protein kinase C-δ (PKC-δ) 抑制劑，但使用siRNA技術降低PKC-δ 的表現量，結果顯示rottlerin造成的協同作用與PKC-δ無關。Rottlerin能增強camptothecin在S期引起的DNA斷裂以及ATM蛋白在Ser1981的磷酸化程度，且 ATM的磷酸化程度與細胞凋亡呈現正相關 (r2 = 0.9)。接著偵測更上游的訊號，camptothecin的作用能穩定topoisomerase與DNA的複合物，導致形成結合camptothecin的topoisomerase I-DNA斷裂複合物，而合併rottlerin能增加複合物的形成。接著為了偵測對DNA修復能力的影響，檢測了短暫處理camptothecin並移除後，γH2A.X在不同時間點的變化。結果顯示，camptothecin移除後γH2A.X在S期的表現量會隨時間而逐漸減少，但合併rottlerin會抑制此現象，表示rottlerin存在下會抑制DNA修復的能力。並且，合併處理camptothecin與rottlerin時會造成Bax構型的變化而活化Bax，同時形成Bad蛋白的降解，表示粒線體壓力對於細胞凋亡的貢獻。總結來說，rottlerin 增強camptothecin的細胞毒殺能力來自於增加TOP1cc的穩定而導致增強DNA損傷壓力，以及可能抑制了DNA修復的能力。接著，經由活化Bax及造成Bad的裂解而造成粒線體壓力且引發細胞凋亡。此研究的嶄新發現提供了rottlerin與camptothecin合併療法用於荷爾蒙不依賴型前列腺癌的可能性。

Prostate cancer is a major cause of cancer mortality in males. Prostate cancer treatment options are now limited to surgery, chemotherapy, cryotherapy, hormonal therapy, and radiation. Hormone-refractory prostate cancer (HRPC), resistant to hormone therapy, is a major obstacle in clinical treatment. Owning to the intractable treatment of HRPC, the development of efficient approaches is highly demanded. In this thesis, we have identified the mechniasms of several nature products and synthetic compounds against HRPC on the part-I and part-II. We provided a cancer therapeutic strategy of combinatory use between rottlerin and camptothecin for the effective treatment of HRPC on the part-III. On the Part-I, we have identified the anti-cancer mechanism of natural products, costunolide and moniliformediquinone. The roles of oxidative stress, calcium imbalance and DNA damage were focused on this part. Several pharmacological and biochemical assays were used to characterize the apoptotic signaling pathways of costunolide, a natural sesquiterpene lactone, in prostate cancer cells. Costunolide showed effective antiproliferative activity against hormone dependent (LNCaP) and independent (PC-3 and DU-145) prostate cancer cells by sulforhodamine B assay, clonogenic test and flow cytometric analysis of carboxyfluorescein succinimidyl ester labeling. The data showed that costunolide induced a rapid overload of nuclear Ca2+, DNA damage response and ATR phosphorylation in PC-3 cells. Costunolide induced G1-phase cell cycle arrest, which was supported by p21 up-regulation and its association with the cyclin dependent kinase 2/cyclin E complex. The association resulted in inhibition of the complex activity and inhibition of Rb phosphorylation. Costunolide mediated effects were substantially inhibited by glutathione, the reactive oxygen species scavenger and glutathione precursor N-acetylcysteine, and the Ca2+ chelator BAPTA-AM other than the reactive oxygen species scavenger Trolox. This indicated the crucial role of intracellular Ca2+ mobilization and thiol depletion but not of reactive oxygen species production in apoptotic signaling. Data suggest that costunolide induces the depletion of intracellular thiols and overload of nuclear Ca2+ that cause DNA damage and p21 up-regulation. The association of p21 with the cyclin dependent kinase 2/cyclin E complex blocks cyclin dependent kinase 2 activity and inhibits Rb phosphorylation, leading to G1 arrest of the cell cycle and subsequent apoptotic cell death in human prostate cancer cells. Moniliformediquinone, a nature phenanthraquinone compound obtained from Dendrobium moniliforme, displayed antiproliferative and apoptotic activity in PC-3 cells, a highly metastatic HRPC. In the present work, several approaches were used to clarify the anticancer mechanism of Moniliformediquinone in PC-3 cells. Moniliformediquinone treatment rapidly caused the depletion of the intracellular glutathione (GSH) and an increase of cytosolic calcium concentration by using mClB and fluo-3-AM staining, respectively. Furthermore, Moniliformediquinone promptly induced DNA damage response (DDR) and phosphorylation of DDR-related protein, such as ATM, CHK1, CHK2 and RPA32. The mitochondrial stress was subsequently induced by Moniliformediquinone through the identification of the loss of mitochondrial membrane potential (ΔΨm), cleavage of Mcl-1 (an anti-apoptotic Bcl-2 family member) and the release of cytochrome c from mitochondria to cytosol. Moreover, the data by the detection of phosphorylation and degradation of IκB-α and NF-κB luciferase assay showed that NF-κB was markedly activated. Of note, cytoskeleton instability was observed after Moniliformediquinone treatment by in vivo tubulin assay. Pretreatment with sulfhydryl compounds, GSH and N-acetylcysteine (NAC), completely blocked the Moniliformediquinone-mediated cellular effect, highlighting the critical role of the intracellular GSH level to Moniliformediquinone action. Taken together, the data suggest that Moniliformediquinone may deplete intracellular GSH content which, in turn, causes an increase of cytosolic calcium level and induce DNA damage response and mitochondrial dysfunction, leading to antiproliferative effect and apoptosis in PC-3 cells. On the Part-II, we have identified the anti-cancer mechanism of an anthraquinone derivative, EJMC30. EJMC30 presented a remarkable selectivity between PC-3 and H9c2 cardiomyoblasts with GI50 values of 8.64 and more than 30 μM, respectiviely. EJMC30 treatment caused G1 arrest of cell cyle associated with down-regulation of cell cycle related proteins, such as cyclinD1 and cyclin E, and up-regulattion of p21 expression. EJMC30 also inhibited mTOR-dependent translational pathways through inhibition of mTOR (Ser2448), 4E-BP1 (Thr37/Thr46/Thr70) and p70S6K (Thr389). Further analysis showed that EJCM30 inhibited the initiation and elongation of protein translation by ribosomal profiling analysis. Moreover, EJMC30 quickly induced the activation of LKB1-AMPK, the upstream kinase of mTOR pathway, suggesting the central role of LKB1-AMPK to EJMC30 action. Taken together, the data suggest EJMC30 affects ribosomal profiling and protein translation that may be through LKB1-AMPK-mTOR dependent pathway, leading to G1 arrest of the cell cycle and growth inhibition. However, resistance to chemotherapeutic agents is a major mechanism in prostate cancer progression. With the mechanism-based development of chemotherapy, discovery of new approaches to enhance cell death signals may be an attractive strategy in prostate cancer treatment. Combination therapy, which can optimize killing activity to cancers and minimize drug resistance, is a mainstream therapy against HRPCs. On the Part-III, we provided an anticancer strategy of combinatory use between rottlerin and camptothecin. Rottlerin, a natural polyphenolic component, synergistically increased PC-3 apoptosis induced by camptothecin (a topoisomerase I inhibitor). Using siRNA technique to knockdown protein kinase C-δ (PKC-δ), the data showed that rottlerin-mediated synergistic effect was PKC-δ-independent, although rottlerin has been used as a PKC-δ inhibitor. Rottlerin potentiated camptothecin-induced DNA fragmentation at S phase and ATM phosphorylation at Ser1981. The effect was correlated to apoptosis (r2 = 0.9). To detect upstream signals, the data showed that camptothecin acted on and stabilized topoisomerase I-DNA complex, leading to the formation of camptothecin-trapped cleavage complexes. The effect was potentiated by rottlerin. To determine DNA repair capability, the time-related γH2A.X formation was examined after camptothecin removal. Consequently, rottlerin significantly inhibited camptothecin removal-mediated decline of γH2A.X formation at S phase, indicating the impairment of DNA repair activity in the presence of rottlerin. The combinatory treatment of camptothecin and rottlerin induced conformational change and activation of Bax and formation of truncated Bad, suggesting the contribution of mitochondria stress to apoptosis. In summary, the data suggest that rottlerin-mediated camptothecin sensitization is through the augmented stabilization of topoisomerase I-DNA cleavage complex (TOP1cc), leading to an increase of DNA damage stress and, possibly, an impairment of DNA repair capability. Subsequently, mitochondria-involved apoptosis is triggered through Bax activation and truncated Bad formation. The novel discovery may provide an anticancer approach of combinatory use between rottlerin and camptothecin for the treatment of HRPCs.