類黃酮衍生物在人類雄性激素不依賴型前列腺癌細胞的抗癌作用機轉探討

Abstract

前列腺癌一直是西方國家高罹患率及高死亡率的癌症之一，而隨著生活習慣及飲食的西化，前列腺癌的罹患率及死亡率也在亞洲地區快速的攀升。造成前列腺癌高死亡率的主要原因來自於癌症的轉移及惡化，但目前並沒有有效治療的藥物，極需要相關研究的投入。本次研究之化合物WJ9708012 (6-(3-Hydroxy-3-methylbutyl)-2’-(7-hydroxy-3,7-dimethyloctyl)-3’,4’,5,7-Tetramethoxy -flavanone) 為類黃酮類結構，與一般類黃酮類化合物不同，其不具有強效的抗氧化能力，但對於人類賀爾蒙抗性前列腺癌細胞PC-3及DU-145，則展現抑制細胞生長及促進細胞凋亡之能力。於抑制細胞生長功能上，WJ9708012藉由抑制mTOR相關訊息傳遞路徑，使cyclin D1等G1細胞週期調控之重要分子表現量降低，造成G1細胞週期停滯現象。使用西方點墨法觀察發現，WJ9708012造成p-mTORser2448、p-P70S6KThr389及p-4E-BP1Thr37/46位置的磷酸化現象明顯減少，使eIF4E調控的7-methylguanosine 5’-triphosphate (m7GTP) cap-dependent translation無法進行，下游cyclin D1等G1細胞週期相關蛋白在mRNA沒有明顯減少的情況下蛋白表現量皆大幅降低，使細胞無法順利通過G1細胞週期，產生停滯生長現象。此現象可藉由給予胎牛血清使mTOR相關訊息傳遞路徑活化的方式降低，間接證明抑制mTOR相關訊息傳遞在此化合物抑制細胞生長之功能中扮演重要角色。在引起細胞凋亡方面，短時間處理WJ9708012可產生一個由細胞外進入胞內的鈣離子流，造成胞內鈣離子濃度迅速上升並活化鈣離子相關訊息分子，如：μ-calpain、PKC-α及calmodulin，使細胞內粒線體及內質網兩個調控鈣離子之重要胞器產生壓力或傷害等影響。粒線體部分，WJ9708012造成粒線體損傷，使膜電位流失及下游內生性細胞凋亡訊息途徑啟動。以西方點墨法觀察，可發現化合物造成粒線體凋亡因子釋出、caspase-9、-3、-7活化及晚期Bcl-2家族蛋白表現量改變及斷裂現象。另外，化合物也會造成細胞內能量恆定失調，使在此扮演細胞保護功能之AMPK蛋白活化，調控下游訊息分子。於內質網部分，則可觀察到內質網壓力產生，使GRP78、GADD153及Caspase12表現量增加或斷裂活化的現象。WJ9708012引起的粒線體損傷和內質網壓力在密切的訊息傳遞後，會造成細胞凋亡途徑的啟動。使用流式細胞儀可觀察到subG1細胞增加和具有PI-Annexin X雙重螢光訊號細胞增加的現象。此外，使用TUNEL assay亦可觀察到具有DNA斷點的螢光訊號之細胞增加情形。總體而言，WJ9708012引起人類賀爾蒙抗性前列腺癌細胞PC-3細胞G1細胞週期停滯及細胞凋亡反應，主要是藉由抑制mTOR相關訊息傳遞路徑及引起鈣離子流入細胞質，影響相關訊息傳遞分子之活性，並造成粒線體及內質網損傷，而啟動細胞凋亡途徑來達成。

Prostate cancer is one of the leading causes of cancer death among men in the United States and Western Europe. Following the transition of lifestyle to Western type, the mortality of prostate cancer is rapidly increased in Asian countries. Most prostate cancer-related deaths are due to advanced disease and, until recently, the hormone therapy was the only well-acceptable treatment approved for advanced prostate cancer. However, many patients were resistant to hormonal therapies after two to three years of treatment and no effective therapy currently exists for advanced prostate cancers that are resistant to hormonal therapy. In this thesis, we reported that a methoxyflavanone – WJ9708012 (6-(3-Hydroxy-3-methylbutyl)-2’-(7-hydroxy-3,7-dimethyloctyl)-3’,4’,5,7- Tetramethoxyflavanone) inhibited growth and produced apoptosis of human hormone-resistant prostate cancer PC-3 and DU-145 cells. WJ9708012 inhibited cell proliferation and induced G1-phase cell-cycle arrest. By using Western blotting analysis, we found that WJ9708012 induced a rapid and time-dependent decrease of phosphorylation level of mTORSer2448, P70S6KThr389 and 4E-BP1Thr37/Thr46, which would suppress 7-methylguanosine 5’-triphosphate (m7GTP) cap-dependent translation through inhibiting eIF4E release from the translational repressor 4E-BP1 and down-regulating the expression of cyclin D1. The stimulation of mTOR-related signaling pathway by fetal bovine serum could rescue the anti-proliferative effect of WJ9708012, indicating an important role of mTOR-related signaling pathway to WJ9708012 action. The treatment of PC-3 prostate cancer cells with WJ9708012 also induced a prompt calcium influx from the extracellular space. The increase in [Ca2+]i was associated with the activation of calcium-related signaling molecules, including calpain, PKC-α and calmodulin. Subsequently, these events caused mitochondria damage and resulted in the induction of cell apoptosis. By using Western blotting analysis, we found that WJ9708012 induced a mitochondria-regulated apoptosis, which involves the release of cytochrome c, activation of caspase-9, -3, and -7 and alteration of protein expression of Bcl-2 family members. Moreover, because of the damage of mitochondria function, WJ9708012 also caused the activation of AMPK, which served as a protective signal and regulated the downstream signal molecules. The endoplasmic reticulum (ER) stress was also induced by WJ9708012, which is associated with the cleavage of pro-caspase12 and the up-regulation of protein levels of GADD153 and GRP-78. A crosstalk between ER stress and mitochondrial insult was observed, leading to the apoptosis events in PC-3 cells. The apoptosis triggered by WJ9708012 in PC-3 cells was assessed using annexin V/ PI labeling and the TUNEL assay. Taken together, the data suggest that WJ9708012 causes a G1-phase cell cycle arrest by inhibiting mTOR-related pathways and induces the cell death through the activation of calcium influx from extracellular space matrix followed by ER stress and mitochondria damage, which would trigger the intrinsic apoptotic pathways.