蒽醌衍生物在人類荷爾蒙不依賴型前列腺癌細胞之抗癌作用機轉探討

Abstract

為了發展治療人類荷爾蒙不依賴型前列腺癌之新藥，我們利用SRB assay篩選了大量的化合物，並發現一系列的蒽醌衍生物在PC-3和DU145兩種人類荷爾蒙不依賴型前列腺癌細胞株，有不錯的抑制生長效果。其中，FJ3化合物抑制細胞生長效果最為顯著，其IC50在PC-3細胞株中為0.5 μM，而在DU145細胞株中則為0.69 μM。藉由流式細胞儀也明顯地觀察到FJ3會造成細胞的凋亡作用(subG1的增加)。利用西方點墨法發現，細胞經過FJ3化合物處理三小時後，會造成PARP-1 cleavage及Chk1和Chek2的磷酸化作用，顯示了FJ3造成了DNA的損傷。進一步發現，FJ3作用三到九小時，造成了粒線體膜電位喪失；且FJ3也會導致GRP78的增加，說明了內質網壓力(Endoplasmic reticulum stress)可能與FJ3調控的細胞凋亡作用有關。另外，FJ3所引起的細胞凋亡作用，可以明顯地被兩種抗氧化劑N-acetyl cysteine與Trolox抑制，這個結果說明了氧化壓力(Oxidative stress)在細胞凋亡作用的貢獻。總括而論，在人類荷爾蒙不依賴型前列腺癌細胞中，FJ3造成了氧化壓力與DNA損傷，進而影響了粒線體與內質網，導致粒線體膜電位喪失與內質網壓力而造成細胞凋亡。

To develop anticancer agents against human hormone-refractory prostate cancers (HRPC), we did a large scale of screening test using sulforhodamine B assay and found a series of anthraquinone derivatives displaying antiproliferative activity in PC-3 and DU-145 cells (two HRPC cell lines). FJ3 stood out from the series compounds and showed a concentration-dependent inhibition of PC-3 cell growth with IC50 values of 0.5 and 0.69 μM in PC-3 and DU-145, respectively. The flow cytometric analysis of propidium iodide staining showed that FJ3 caused a significant increase of sub-G1 population (apoptosis) of the cell cycle. The Western blot analysis demonstrated that a three-hour exposure to FJ3 induced the phosphorylation and activation of Chk1 and Chek2 associated with the cleavage of PARP-1, indicating the induction of DNA damage response. Further identification showed that FJ3 caused a time-dependent (3 to 9 hours) loss of mitochondrial membrane potential. Moreover, the up-regulation of GRP78 was stimulated by FJ3, suggesting the involvement of endoplasmic reticulum stress (ER stress) in FJ3-mediated apoptotic response. Of note, the apoptosis induced by FJ3 was significantly inhibited by N-acetyl cysteine (NAC, a glutathione precursor and antioxidant) and trolox (an antioxidant). The data indicate that the oxidant stress may explain FJ3-induced effect. In summary, the data suggest that FJ3 effectively induces oxidant stress and DNA damage response, which in turn cause a crosstalk between mitochondria and ER, leading to the loss of mitochondrial membrane potential and ER stress response that ultimately result in apoptosis in HRPCs.