Furanodiene (FUR) is a natural terpenoid isolated from Rhizoma Curcumae, a well-known Chinese medicinal herb that presents anti-proliferative activities in several cancer cell lines. In this study, we demonstrated that FUR concentration-dependently inhibited the cell proliferation of A549, NCI-H1299 and 95-D lung cancer cells with 50% concentrations of inhibition (IC50) of 157.9 μM, 126.1 μM and 118.3 μM after 48 h-treatment, respectively. β-Elemene, another terpenoid isolated from Rhizoma Curcumae, exhibited weaker anti-proliferative effects in A549 and NCI-H1299 cells (with IC50 of 347 μM and 184 μM, respectively after 48 h-treatment) and activities similar to FUR in 95-D cells. FUR significantly inhibited colony formation in A549 and 95-D cells and blocked their cell cycle progressions in G0/G1 phase through down-regulating the protein levels of cyclin D1 and CDK6, and up-regulating the protein expressions of p21 and p27. It affected the signaling molecules of apoptosis pathway in 95-D cells revealed by the down-regulation of the protein levels of PARP, caspase-7, Bcl-2 and survivin. FUR up-regulated the mRNA and protein expression levels of binding immunoglobulin protein (BIP) and C/EBP homologous protein (CHOP), and accumulated CHOP in the nucleus, indicating that endoplasmic reticulum stress (ERS) is induced. FUR also concentration-dependently enhanced the generation of reactive oxygen species (ROS). 80 μM FUR increased the ROS production to 2.4 folds of control after 24 h. ix As well as the induction of ERS, the significant increasing of ROS generation is likely to, at least in part, be responsible for the anti-proliferative activities of FUR. However, the adhesion ability of 95-D cells to matrigel and fibronectin had been rarely inhibited after FUR treatment for 24 h. According to the results of the transwell invasion assay and the wound healing assay, FUR also exhibited slight suppression on cell invasion and migration in 95-D cells. Furthermore, we investigated the effects of FUR in the combination with chemotherapy drugs. FUR plus doxorubicin (DOX) showed synergistic anti-cancer effects at high concentrations, whereas antagonism was observed at lower doses in all tested three cell lines according to the combination index (CI) after co-treatment. The combined treatment of FUR with paclitaxel (TAX) showed synergetic anti-proliferative activities in NCI-H1299 and 95-D cells, which was weaker in A549 cells. Using 95-D cells as a model to investigate the synergistic mechanisms, we demonstrated that 80 μM FUR reduced the cell numbers distributed in G2/M phase induced by 0.2 μM TAX (from 94.48% to 55.01% after 24 h-treatment) while increased those in G1 phase. The protein levels of cyclin D1, cyclin B1, CDK6 and c-Myc were all significantly down-regulated in the group of combined treatment. The co-treatment also induced more severe effects on the signaling molecules of apoptosis pathway revealed by decreasing the protein expressions of Bcl-2, caspase-3 and caspase-7. Though FUR alone obviously induced ERS, this signaling pathway may not contribute to the synergetic anti-proliferative effect as the protein expressions of CHOP and BIP in the combined treatment were similar to those in FUR alone group. The protein levels of integrin α5, integrin α6, integrin β1, integrin β3, integrin β4, focal adhesion kinase (FAK) and paxillin were detected to explore the effects of FUR plus TAX on integrins and its related signaling pathway. The dra- x matically down-regulated expression of integrin β4, FAK and paxillin might partially contribute the synergy effect. These results indicate that FUR, one of the main components of Rhizoma Curcumae, presents its anti-lung cancer activities mainly through the anti-proliferative effects on lung tumor cells. FUR also showed similar anti-cancer activity compared with that of β-Elemene, another anti-cancer component isolated from Rhizoma Curcumae. These findings suggest that FUR is not only one of the main pharmacologically active ingredients of Rhizoma Curcumae, but also a promising compound for anti-cancer agent development. Its synergetic activity with TAX further indicates its potential in the combined therapy.

中藥溫莪術（Rhizoma Curcumae）具有較好的抗癌作用，呋喃二烯（furanodiene， FUR）是從中提取的倍半萜類化合物，約占溫莪術油的 20%-30%，是其主要成分之一。 我們的研究顯示，FUR 能劑量依賴性抑制 A549、NCI-H1299 和 95-D 三株肺癌細胞的 增殖，作用 48 h 後其半數抑制率（50% concentration of inhibition，IC50）分別為 157.9 μM、 126.1 μM、118.3 μM。與溫莪術油中的另一種廣泛用於各種腫瘤治療的倍半萜類化合物 β-欖香烯（β-elemene，β-ELE）相比，FUR 對 A549、NCI-H1299 細胞的抑制作用明顯 強於 β-ELE（作用 48 h，IC50 分別為 347 μM、184 μM），而對 95-D 細胞兩者的作用相 當。FUR 對 A549、95-D 細胞的克隆形成有顯著抑制作用，並阻滯腫瘤細胞於 G0/G1 期，下調細胞週期相關蛋白 cyclin D1、CDK6 表達，上調 p21、p27 表達。對於 95-D 細 胞 FUR 作用 24 h 後，細胞內 PARP、caspase-7、Bcl-2、survivin 等蛋白表達明顯下調， 說明 FUR 對於細胞凋亡通路信號分子有一定的作用。此外，FUR 顯著升高 A549、95-D 細胞中內質網應激（endoplasmic reticulum stress，ERS）分子標記物 BIP（binding immunoglobulin protein）、CHOP（C/EBP-homologous protein）的 mRNA 和蛋白表達以及 核內 CHOP 的聚集，說明 FUR 可誘導肺癌細胞產生 ERS。FUR 還可劑量依賴性誘導活 性氧（reactive oxygen species，ROS）生成，80 μM FUR 作用於 95-D 細胞 24 h 後其 ROS 生成量與對照組相比增加了 2.4 倍，這可能與其誘導的 ERS 作用一起貢獻於對肺癌細胞 的增殖抑制作用。FUR 作用於 95-D 細胞 24 h 後，與對照組相比，其對基質膠（matrigel， MA）和纖維粘連蛋白（fibronectin，FN）的粘附能力沒有明顯變化。通過 transwell 侵 vii 襲實驗和劃痕實驗證實 FUR 對 95-D 細胞侵襲轉移能力的抑制作用较弱。 本研究還對 FUR 與化療藥物的聯合作用進行了研究。通過計算聯合用藥指數 （comcination index，CI），我們發現，FUR 與阿黴素（doxorubicin，DOX）聯合用藥在 較高濃度時顯示出協同作用，低濃度則呈現拮抗作用。FUR 與紫杉醇（paclitaxel，TAX） 聯合用藥對 NCI-H1299 和 95-D 細胞有較明顯的協同作用，對 A549 細胞協同作用較弱。 以 95-D 細胞為模型，研究 FUR 與 TAX 的協同作用機制顯示，FUR 減少了 TAX 引起 的 G2/M 期阻滯作用，作用 24 h 後 G2/M 期細胞比例由 0.2 μM TAX 組的 94.48%降低至 聯合用藥組（80 μM FUR 加 0.2 μM TAX）的 55.01%，同時下調 cyclin D1、cyclin B1、 CDK6、c-Myc 等蛋白的表達。FUR 與 TAX 聯合使用後，其 95-D 細胞中 Bcl-2、caspase-3、 caspase-7 等蛋白的表達與其他組別相比也有明顯下調，說明 FUR 可增強 TAX 對細胞凋 亡通路信號分子的作用。雖然 FUR 有明顯 ERS 誘導作用，但我們的實驗條件下沒有觀 察到其與 TAX 聯合用藥後 BIP 和 CHOP 蛋白的表達變化，提示 ERS 通路可能並不參與 FUR 對 TAX 的協同作用。此外，通過檢測整合素（integrin）及其通路相關蛋白 integrin α5、integrin α6、integrin β1、integrin β3、integrin β4、focal adhesion kinase （FAK）和 paxillin 等的表達，我們發現聯合用藥組顯著下調 integrin β4、FAK 和 paxillin 的蛋白表 達，說明 FUR 對 integrin 及其通路的抑制可能是其協同作用機制之一。 綜上所述，中藥溫莪術中的主要成分之一 FUR 主要通過抑制腫瘤細胞的增殖來發 揮其抗肺癌作用，由於其與莪術中的抗腫瘤成分 β-欖香烯具有類似的作用效果，提示其 不僅是溫莪術中的主要藥理活性成分，同時也有可能進一步開發其成為新的抗腫瘤藥物。 其與 TAX 的協同作用更顯示了 FUR 今後可能用在聯合用藥方面的潛力。 關鍵字：呋喃二烯，β-欖香烯，紫杉醇，肺癌，內質網應激，協同作用，整合素