Ovarian cancer remains the most lethal gynecological malignant tumor. High ratio of metastasis in malignant stages makes it difficult to eradicate the lesions by surgical therapy, thus chemotherapy is required to eliminate the inoperable lesions. Meanwhile, platinum-taxane doublet remains to be the first-line chemotherapy among the past 20 years. To overcome the risks of recurrence and drug-resistance caused by the homogenized chemo strategy, developing new kind of drugs is imperative as always. Garcinone E (GE) is a xanthone isolated from the fruit hull of mangosteen. It has been reported that GE exhibits cytotoxicity in several kinds of tumor cell lines, while indepth mechanism hasn't been well studied. This research is to study on the antitumor activity and mechanism of GE, and to find out a new impetus of ovarian cancer treatment. GE inhibited cell viabilities of ovarian cancer cell lines HEY, A2780, and A2780/Taxol in concentration- and time- dependent manners. The IC50s were: HEY: 7.79±1.12 µM (24 h)，3.55±0.35 µM (48 h); A2780: 3.09±0.81 µM (24 h), 2.91±0.50 µM (48 h); A2780/Taxol: 4.68±0.61 µM (24 h), 3.25±0.13 µM (48 h). And the resistance index of A2780/Taxol on GE were 1.51 (24 h) and 1.12 (48 h), respectively, indicating that GE presents anti-drug resistant potential. High concentration of GE caused LDH leakage of HEY cells, suggested it excert cytotoxicity. It caused decline of mitochondrial membrane potential, shrinkage of nuclei, and apoptosis. GE downregulated Bcl-xL and activated caspase signaling pathway, which led to caspaseddependent apoptosis. Meanwhile, GE induced endoplasmic reticulum stress in HEY and A2780 cells and thus activated IRE-1α signaling pathway. When IRE-1α was knocked down by siRNA, caspase cascade was further activated and more cell death was caused by GE. On the other hand, the anti-migration and anti-invasion effects of GE on HEY cells were tested by wound-healing assay as well as transwell assay. GE eliminated migrative ability of HEY cells by reducing the expression of Ras homolog gene family, member A (RhoA) and Ras-related C3 botulinum toxin substrate (Rac), and it vi blockaded invation by down-regulating the protein level of matrix metalloproteinase (MMP) -9 and MMP-2, and up-regulating the protein level of tissue inhibitors of metalloproteinase (TIMP) -1 and TIMP-2, thus reduced the activities of MMP-9 and MMP-2 confirmed by gelatin zymography assay. Moreover, GE could modulate the progress of autophagic flux of ovarian cancer cells. On the initial stage of autophagy, GE enhanced the genesis of microtubule-associated protein 1 light chain 3 (LC3) II and p62, and induced the occurrence of autophagy. While on the late stage, GE might inhibit the function of lysosome, and prevent the fusion with autophagosome by down-regulating the protein level of transcript factor EB (TFEB), and blockading the activation of cathepsin L (CTSL), which leads to homeostasis damage and cell death. In summary, we found out that GE exerted promising antitumor potential on antiproliferation, anti-migration, anti-invasion, and autophagic-modulation, suggesting its prospects as an anti-ovarian cancer candidate.

卵巢癌在婦科惡性腫瘤中的致死率高居首位，至今紫杉醇/卡鉑聯合化療仍 是晚期卵巢癌術後的標准化療法之一，相對單一的治療方案使不少患者面臨化 療耐藥和腫瘤復發的風險，因此新型藥物與治療方案亟待開發。 藤黃酮 E（garcinone E, GE）是從山竹果殼中提取分離出的氧雜蒽酮類化合 物，目前有報道它在肝癌等腫瘤細胞上具有細胞毒性，但尚未有文獻系統地對 其藥理作用尤其是抗腫瘤機制進行深入探討。為進一步探究 GE 的抗腫瘤藥理 活性，為卵巢癌的治療提供新選擇，本課題首次從抗增殖、抗轉移等多角度對 GE 進行評價和機制研究，發現其在卵巢癌細胞上具有良好的抗腫瘤效應。 本課題研究結果顯示，GE 能呈劑量、時間依賴性地抑制卵巢癌細胞 HEY、A2780 和 A2780/Taxol 的增殖，其半數抑制濃度（half-maximal inhibitory concentration，IC50）分別為：HEY：7.79±1.12 µM（24 h），3.55±0.35 µM（48 h）；A2780：3.09±0.81 µM（24 h），2.91±0.50 µM（48 h）；A2780/Taxol： 4.68±0.61 µM（24 h），3.25±0.13 µM（48 h）。相對於親本細胞株 A2780， A2780/Taxol 对 GE 的耐藥指數分別是 1.51（24 h）和 1.12（48 h），說明 GE 具 有顯著的抗耐藥作用。LDH 釋放實驗表明，10 µM 以上的 GE 可直接損傷細胞 膜，造成明顯的細胞毒性。而 Hoechst 33342 染色、annexin V/PI 雙染、JC-1 染 色、western blot 等實驗表明，GE 可誘導細胞發生凋亡，造成細胞膜通透性上 升，線粒體膜電位下降、細胞核皺縮、染色質凝聚斷裂、形成凋亡小體。在卵 巢癌 HEY 和 A2780 細胞上，GE 下調 Bcl-2 類似蛋白 1（B-cell lumphoma-2 like 1，Bcl-xL），激活 caspase 通路，導致多聚二磷酸腺苷-核糖聚合酶（poly (ADP-ribose) polymerase ，PARP）的失活，最終誘導細胞發生 caspase 依賴性的 凋亡。同時，GE 介導細胞發生內質網應激，從而激活未折疊蛋白反應中的肌醇 需求激酶-1α（inositol-requiring kinase-1α，IRE-1α）信號通路，致使 IRE-1α、 免疫球蛋白結合蛋白（immunoglobulin-binding protein，BiP）和 C/EBP 同源蛋 白（C/EBP homologous protein，CHOP）表達上調。當採用 siRNA 沉默 IRE-1α 後，HEY 細胞的 IRE-1α 存活通路受到抑制，死亡細胞數目進一步上升。 iv 在抑制細胞增殖的同時，GE 還能抑制卵巢癌 HEY 細胞的遷移和侵襲。劃 痕實驗和 transwell 遷移實驗表明 GE 可顯著限制 HEY 細胞的運動遷移能力，而 transwell 侵襲實驗表明 GE 可顯著抑制 HEY 細胞的侵襲作用。通過 western blot 實驗考察相關蛋白水平的變化，發現 GE 可能通過下調 Rho GTPase 複合體中 Ras 同源物家族成員 A（Ras homolog gene family, member A，RhoA）和 Ras 相 關 C3 肉毒素底物（Ras-related C3 botulinum toxin substrate，Rac）的表達，抑 制 HEY 細胞的運動遷移能力，同時一方面降低 MMP-9 和 MMP-2 的蛋白水 平 ， 另 一 方 面 提 高 金 屬 蛋 白 酶 組 織 抑 制 劑 （ tissue inhibitors of metalloproteinase，TIMP）-1 和 TIMP-2 的蛋白水平。明膠酶譜實驗驗證，GE 處理後，MMP-9 和 MMP-2 位置的酶解條帶均相應減弱，表明 GE 抑制了 HEY 細胞釋放到細胞外環境的 MMPs 的活性，使 MMPs 降解基質的能力下降，從而 抑制了 HEY 細胞的侵襲能力。 此外，GE 還能調控卵巢癌細胞的自噬過程。在自噬的初始階段，GE 促進 了微管相關蛋白 1 輕鏈 3（microtubule-associated protein 1 light chain 3，LC3）I 到 LC3II 的轉變和 p62 的轉錄翻譯，誘導 HEY 細胞自噬的發生，而在自噬的晚 期可能通過下調轉錄因子 TFEB（transcript factor EB，TFEB）的蛋白水平和組 織蛋白酶 L（cathepsin L，CTSL）的活化，抑制溶酶體功能及其與自噬體融合 的過程，導致兩者的降解進程受到阻斷而發生積聚，破壞細胞內穩態和能量循 環體系，從而對細胞的死亡過程起到一定促進作用。 綜上所述，本課題首次對 GE 抑制卵巢癌細胞增殖的機制進行了較深入的 探討，揭示了 GE 誘導的內質網應激在其發揮增殖抑制作用的過程中所扮演的 角色，還發現 GE 可通過抑制 HEY 細胞的自噬過程造成細胞內穩態失衡和物 質-能量代謝異常而促使細胞死亡，并通過抑制 HEY 細胞遷移和侵襲等關鍵環 節防止腫瘤轉移的發生。以上研究結果為進一步開發以 GE 為代表的山竹來源 氧雜蒽酮化合物作為卵巢癌及其他癌種新型藥物提供了理論基礎。