Mitoxantrone衍生物在人類去勢抗性轉移性前列腺癌之抗癌機轉探討

Abstract

前列腺惡性腫瘤在男性中，是目前最頻繁被診斷出的癌症。一般而言，早期階段的病人經過適當治療後可延長五年壽命的比率為百分之百。但是當癌症發展到了晚期腫瘤開始轉移時，此比率將急劇下降至百分之三十以下。目前對於轉移性前列腺惡性腫瘤的治療方式主要為「男性荷爾蒙去勢治療法」，但是經過18個月至24個月許多病患會持續惡化發展成轉移性去勢抗性前列腺癌（Castration-Resistant Metastatic Prostate Cancer），發展對於轉移性去勢抗性前列腺癌的治療實為當務之急。蒽醌（Anthracenedione）為抗癌藥物的重要結構，此結構的許多藥物例如mitoxantrone （MTX）和ametantrone都已應用於像是乳癌、前列腺癌、急性骨髓性白血病和非霍奇金淋巴瘤，但是現行蒽醌類藥物有較劇烈的副作用，例如噁心、嘔吐、腹瀉、低血壓和心毒性，因此我們的合作團隊合成了一系列的蒽醌類衍生物，以sulforhodamine B（SRB）assay 檢測，結果顯示一系列衍生物中編號YYC-577的合成物顯著地抑制去勢抗性前列腺癌細胞株 PC-3 （GI50 = 21.82 nM）和DU-145 （GI50 = 26.13 nM）之細胞增生作用，相較mitoxantrone的GI50 為364 nM ，由此可見YYC-577具有較好的抗癌能力。以carboxyfluorescein succinimidyl ester （CFSE）staining與流式細胞儀分析，證實YYC-577抑制細胞增生，並造成細胞停滯於 G2/M期。YYC-577會增加cleaved caspase-3和cleaved PARP，經由粒線體膜電位的測量也發現細胞的粒線體膜電位會被YYC-577破壞，進而造成前列腺癌細胞凋亡。YYC-577引起細胞週期延遲的作用與週期調控蛋白 cyclin D1和cyclin E有關。另外也觀察到和DNA損傷相關蛋白，例如p-chk2、Rad51、γH2AX和RPA32的表現量有顯著增加，免疫螢光染色影像及流式細胞儀定量顯示在YYC-577的作用下，細胞核內的γH2AX表現量顯著增加。Comet assay進一步證實YYC-577會對PC-3和DU-145 細胞株造成DNA的受損。另外透過in vivo complex of enzyme (ICE) assay分析細胞內拓撲異構酶（topoisomerase）和DNA的共價複合物，結果顯示YYC-577影響DNA受損主要干擾拓撲異構酶和DNA的作用。總結來說，YYC-577相較mitoxantrone 對於去勢抗性前列腺癌細胞有更好的抗癌能力，機轉方面主要是透過影響拓撲異構酶的功能造成DNA受損來造成細胞週期停滯和凋亡。

Carcinoma of the prostate is one of the most frequently diagnosed cancers in men. With adequate treatment, the 5-year relative survival rate is 100%; however, it drops to less than 30% if a distant metastasis occurs at the time of diagnosis. Androgen-deprivation therapy has been the mainstay of therapy for advanced metastatic prostate cancer. However, the growth of castration-resistant prostate cancer (CRPC) occurs ultimately after an 18- to 24-month treatment. Due to the widespread incidence and unmet medical need, prostate cancer is one of the important targets in both biomedical and clinical research. Anthracenedione is a leading structure in several cancer chemotherapeutic agents, such as mitoxantrone (MTX) and ametantrone used in several types of cancers including metastatic breast cancer, prostate cancer, acute myeloid leukemia, and non-Hodgkin’s lymphoma. Therefore, it is a crucial structure lead in the design of anticancer compound derivatives. However, existing anthracenedione agents show adverse side effects such as nausea, vomiting, diarrhea, low blood pressure and heart damage. Hence, our colleagues have synthesized a number of anthracenedione derivatives and after a screening test in CRPC PC-3 and DU-145 cells based on sulforhodamine B assay, we have found that YYC-577 shows a better anticancer efficacy than mitoxantrone with an GI50 of 21.82 nM in PC-3 cells and an GI50 of 26.13 nM in DU-145 cells (mitoxantrone, 364 nM). Both flow cytometric analysis of CFSE staining and colony assay have confirmed that YYC-577 displays a potent anti-proliferative activity. YYC-577 induces cell cycle arrest at G2/M phase and increases apoptosis-related signals such as cleaved PARA and caspase-3. YYC-577 also induces the loss of mitochondrial membrane potential using cytofluorometric analysis of JC-1 staining. YYC-577 exposure also results in increased expressions of DNA-damage-related signals, including p-chk2, Rad51, γH2AX and RPA32. Immunofluorescence imaging and cytofluorometric analysis show that the expression of γH2AX in nucleus is significantly increased by YYC-577. Comet assay further validates YYC-577-induced DNA damage. Moreover, in vivo complex of enzyme (ICE) assay that measures endogenous topoisomerase DNA covalent complexes demonstrates that YYC-577 preferentially targets topoisomerase II for the DNA damage effect. In summary, the data suggest that YYC-577 is a potential anthracenedione derivative that targets topoisomerase II, leading to G2/M arrest of the cell cycle and subsequent cell death in CRPCs.