類黃酮衍生物對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

前列腺癌為致命且廣泛存在於全世界的癌症之一，容易由前列腺擴散至骨頭和淋巴結。阻斷二氫睪酮素常用於治療前列腺癌，可以阻止癌細胞生長並使其縮小。然而，前列腺癌常於荷爾蒙阻斷治療一至兩年後產生抗性。因此，找尋新穎且有效的治療藥物，來對抗荷爾蒙不依賴型的前列腺癌是有意義的。類黃酮衍生物 WJ-970808-10，發現可有效抑制荷爾蒙不依賴型前列腺癌細胞 PC-3 和DU-145 的生長，其 GI50 分別為 5.86 和 9.21 μM。使用流式細胞儀分析發現， WJ-970808-10 可以引起 PC-3 癌細胞週期 SubG1 期的增加和 G0/G1 期的停滯，其效果和處理時間成正相關。此外還觀測到一些粒線體訊息，包括：粒線體膜電位去極化、Mcl-1、 Bak、Bad、Bim、Bid 的裂解、凋亡物質 cytochrome c 和 AIF 的釋出、ROS 的生成、survivin 的減少和 caspase cascade 的活化，至於死亡受器之相關訊息則無顯著變化。另外，處理藥物短時間可使細胞週期調控者 Cyclin D1、Cdk4、Cyclin E、Cdk2、Cdc25A 和 E2F 表現量減少與 p21 和 p27 表現量增加及 Rb 磷酸化減少。推測 WJ-970808-10 藉由造成細胞週期調控者Cyclin D1、Cdk4、Cyclin E、cdk2 的表現量減少，使細胞週期停滯在 G0/G1 期。使用 RT-PCR 證實 WJ-970808-10 處理 6 小時後，會減少 Cyclin D 和 Cyclin E 的 mRNA 表現，但對 cdk4、cdk2 的 mRNA 表現量則無顯著影響。此外 mTOR 下游 p70S6K (T389/T421/S424)、4E-BP-1 (T37/T46) 和 eIF4E (S209) 的磷酸化於 WJ-970808-10 處理 3小時後明顯受到抑制。以上結果得知，WJ-970808-10 在荷爾蒙不依賴型前列腺癌細胞 PC-3 中會藉由 mTOR 的抑制、造成粒線體損傷與 caspase cascade 的活化，引發細胞週期停滯和後期的細胞凋亡。

Prostate cancer is one of the most malignant and prevalent cancer types worldwide and easily spread from prostate to bone and lymph nodes. Blocking dihydrotestosterone (DHT) often causes prostate cancer to stop growing and even shrink. However, cancer cells which initially respond to hormonal therapy usually become chemo-resistant after one- to two-year therapy. Therefore, it is important to discover effective chemotherapeutic agents for treatment of hormone-independent prostate cancer. WJ-970808-10, a flavonoid derivative, suppressed cell growth in hormone-independent prostate cancer cell lines, including PC-3 and DU-145, with GI50 values of 5.86 and 9.21 μM respectively. In flow cytometry analysis, WJ-970808-10 induced an increase of cell population at SubG1 and G0/G1 phase of the cell-cycle in PC-3 cells in a time-dependent manner. The detection of cellular events, including the loss of mitochondria membrane potential, cleavage of Mcl-1, Bak, Bad, Bim and Bid, release of cytochrome c and AIF, production of ROS, down-regulation of survivin and activation of caspase cascade, suggested that mitochondria-related singnals were involved in WJ-970808-10-induced cell apoptosis. However, WJ-970808-10 had little effect on death receptor-related signals. The data also demonstrated that several cell-cycle regulators, including Cyclin D1, Cdk4, Cyclin E, Cdk2, Cdc25A, Rb, E2F, p21 and p27, were affectd by WJ-970808-10. The data suggested that WJ-970808-10 induced down-regulation of Cyclin D1, cdk4, Cyclin E and cdk2, leading to G0/G1 arrest of the cell-cycle. The mRNA level of cell cycle regulators was also examined by RT-PCR and the data showed that the mRNA levels of Cyclin D and Cyclin E, but not cdk4 and cdk2, were down-regulated after a 6-hour treatment of WJ-970808-10. Moreover, downstream factors of mTOR signaling, including the phosphorylation of p70S6K (T389/T421/S424) and 4E-BP-1 (T37/T46), were significantly inhibited after a 3-hour treatment of WJ-970808-10, suggesting the translation-inhibitory activity of this compound. Taken together, WJ-970808-10 induces G1 arrest of the cell-cycle and subsequent apoptosis of human prostate cancer cells through inhibition of mTOR-related pathways, induction of mitochondrial stress and activation of caspases.