Furanodiene Presents Synergistic Anti‐proliferative Activity With Paclitaxel Via Altering Cell Cycle and Integrin Signaling in 95‐D Lung Cancer Cells

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. Recently, we found that the combined treatment of FUR with paclitaxel (TAX) showed synergetic anti‐proliferative activities in 95‐D lung cancer cells. Herein, we showed that FUR reduced the cell numbers distributed in mitosis phase induced by TAX while increased those in G1 phase. The protein levels of cyclin D1, cyclin B1, CDK6 and c‐Myc were all down‐regulated in the group of combined treatment. The dramatically down‐regulated expression of integrin β4, focal adhesion kinase and paxillin might partially contribute to the synergic effect. Though FUR alone obviously induced endoplasmic reticulum stress, this signaling pathway may not contribute to the synergetic anti‐proliferative effect as the protein expression of CHOP and BIP was similar in FUR alone and combined treatment group. Copyright © 2013 John Wiley & Sons, Ltd.     

莪术油的HPLC指纹图谱研究

目的 建立莪术油的HPLC指纹图谱鉴别方法。方法 以呋喃二烯为参照物,采用HPLC检测方法;色谱条件：Agilent Extend C₁₈色谱柱（250 mm×4.6 mm,5 μm）,乙腈-水二元梯度洗脱,体积流量1.0 mL/min,检测波长216 nm,柱温30 ℃。结果 确定了8个共有峰,建立了莪术油的HPLC指纹图谱,可区别同属其他植物的挥发油。结论 本法操作简便,专属性强,重现性好,可有效控制莪术油的质量。     

呋喃二烯对肝脏细胞色素P450酶不同亚型的影响

目的 考察呋喃二烯(furanodiene,FDE)对肝脏微粒体主要的细胞色素P450(CYP)酶活性的影响。方法 采用探针底物法,考察FDE在体外孵育体系中对大鼠和人肝微粒体CYP1A、CYP2A、CYP3A、CYP2C、CYP2D、CYP2E1的抑制作用,并计算相应的IC50值;采用微粒体体外“鸡尾酒”孵育法(cocktail法),考察大鼠经低、高剂量FDE(40 mg·kg^-1和160 mg·kg^-1)连续灌胃7 d,其肝微粒体主要CYP酶活性的变化。结果 FDE对大鼠肝微粒体CYP2D1和CYP2C6/7有较弱的抑制作用,其IC50分别为15.8和23.8 μmol·L^-1;对人肝微粒体CYP2C9也有较弱的抑制作用,IC50为26.1 μmol·L^-1。与对照组比较,大鼠灌胃40 mg·kg^-1 FDE,肝微粒体主要CYP酶活性无显著变化;灌胃160 mg·kg^-1后,肝微粒体CYP2E1活性为对照组的164%。结论 FDE对大鼠和人肝微粒体CYP主要亚型的抑制作用较弱;40 mg·kg^-1 FDE对大鼠肝微粒体主要CYP酶未显示明显诱导作用,160 mg·kg^-1 FDE对肝微粒体CYP2E1有一定的诱导作用。     

CO2超临界流体色谱法同时测定莪术油中3个倍半萜类成分的含量

目的 建立CO₂超临界流体色谱法测定莪术油中呋喃二烯、牻牛儿酮和莪术二酮含量的方法。方法 采用ACQUITY UPC² HSS C₁₈ SB色谱柱（3.0 mm×150 mm,1.8 μm）,以CO₂-乙腈为流动相,梯度洗脱;流速为1.0 mL·min^-1;检测波长为216 nm,柱温为55℃,背压为2 000 psi。结果 呋喃二烯在2.67~1 337.26μg·mL^-1内线性关系良好（r=1.000）,加样回收率为97.94%（n=6,RSD=1.50%）。牻牛儿酮在2.77~1 386.00 μg·mL^-1内线性关系良好（r=1.000）,加样回收率为96.07%（n=6,RSD=1.68%）;莪术二酮在6.99~3 493.00 μg·mL^-1内线性关系良好（r=1.000）,加样回收率为99.33%（n=6,RSD=1.88%）。结论 本方法快捷准确、稳定且绿色环保,可用于莪术油中上述3个倍半萜类成分的质量控制。     

呋喃二烯抑制脑胶质瘤作用

目的观察呋喃二烯(furanodiene,FDE)纳米乳对颅内移植G422胶质母细胞瘤小鼠化疗后的生存期变化及病理改变,并初步探讨其作用机制,评估FDE纳米乳对胶质母细胞瘤的抑制作用。方法将G422胶质母细胞瘤细胞植入昆明小鼠脑白质中建模,考察FDE纳米乳及其与卡莫司汀联合组对荷瘤小鼠生存期的影响,观察各组瘤组织的病理结构变化。采用细胞增殖抑制法、Hoechst33258染色法和Annexin V-FITC/PI双染法流式细胞术检测细胞凋亡率等方法初步探索FDE对人胶质瘤U251细胞的作用机制。结果 FDE纳米乳(80 mg·kg^(-1))组治疗荷瘤小鼠生命延长率为28.1%,联合组生命延长率可达69.0%;组织病理学分析显示,FDE纳米乳组肿瘤细胞排列疏松,未见明显侵润;联合组肿瘤体积小,且与周围组织间边界清晰。细胞增殖抑制和Hoechst33258荧光染色实验均表明FDE对U251细胞增殖抑制具有浓度依赖性,流式细胞术结果分析当FDE浓度为18.5μmol·L(-1))组治疗荷瘤小鼠生命延长率为28.1%,联合组生命延长率可达69.0%;组织病理学分析显示,FDE纳米乳组肿瘤细胞排列疏松,未见明显侵润;联合组肿瘤体积小,且与周围组织间边界清晰。细胞增殖抑制和Hoechst33258荧光染色实验均表明FDE对U251细胞增殖抑制具有浓度依赖性,流式细胞术结果分析当FDE浓度为18.5μmol·L^(-1)时,U251细胞早期凋亡率可达71.8%。结论 FDE通过调节胶质母细胞的增殖、早期凋亡等机制,有效抑制小鼠胶质瘤的生长。     

毛细管气相色谱法同时测定保妇康栓中冰片及3种倍半萜含量

目的建立GC-FID含量测定方法,同时测定保妇康栓中冰片、呋喃二烯、莪术醇和莪术二酮的含量。方法采用HP-5石英毛细管色谱柱(30 m×0.25 mm,0.25μm)程序升温,流速为2.5 mL.min-1,分流比为10∶1,进样量为1μL。结果冰片、呋喃二烯、莪术醇和莪术二酮质量浓度分别在28.0～224.0(r=0.999 8)、16.0～128.0(r=0.999 9)、2.8～22.4(r=0.999 8)、4.0～32.0 mg.L-1(r=0.999 9)内线性关系良好,平均回收率分别为103.7%、91.6%、102.5%、99.5%,RSD分别为4.7%、3.6%、3.9%、4.3%(n=9)。结论本方法可为保妇康栓的质量控制提供科学依据。     

Induction of apoptosis by furanodiene in HL60 leukemia cells through activation of TNFR1 signaling pathway

Objective Curcuma wenyujin,named Ezhu in Chinese,a traditional Chinese medicine,which has been shown to possess anticarcinogenic activity and used for the treatment of tumor in China,for example,hepatic cancer and leukemia.β-elemene,a component of Curcuma wenyujin,was largely reported as a main active component for anti-tumor effect of Curcuma wenyujin.However,furanodiene,another main component of Curcuma wenyujin,was seldom investigated for its anti-tumor activities.In the present study,we aim to investigate the effect of furanodiene on human leukemia HL60 cells and to study the accurate molecular mechanisms of its action.Methods Trypan blue exclusion experiment was used for cell growth inhibition assay.The apoptotic characterizations were assessed by flow cytometry analysis,AO/EB staining assay and agarose gel electrophoresis assay.The expression of apoptosis-related proteins and the release of cytochrome c from mitochondrial were detected by western blotting,and the mRNA levels of TNF-α and TNFR1 were probed by RT-PCR.Formation of TNFR1 complex was analyzed by using immunoprecipitation of TNFR1 with TRRF1/2 and RIP.Results Furanodiene(10-100 μM)treatment inhibited the growth of HL60 cells in a concentration-dependent manner.The effect of furanodiene on HL60 cells was associated with the induction of apoptosis,which was characterized by DNA fragmentation,cleavage of poly(ADP-ribose)polymerase(PARP),caspase-3,caspase-8 and caspase-9.In the Bcl-2 family proteins,Bid protein(a substrate of caspase-8)was activated by furanodiene,but Bcl-2,Bax and Bcl-xL proteins were not inuenced by furanodiene stimulation.Furanodiene elicited cytochrome c release from mitochondria into the cytosol.Moreover,furanodiene treatment caused the upregulation of TNFR1,the formation of TNFR1 complex and an obvious production of TNF-α in HL60 cells.The soluble TNFR1 receptor effectively inhibited furanodiene-induced apoptosis.Conclusions Furanodiene inhibited the growth of HL60 leukemia cells via induction of apoptosis.Furanodiene-induced apoptosis in HL60 cells is mediated by upregulation of TNF receptor 1 as well as induction of TNF-α production to activate TNFR1 signaling pathway.Our research provides insight into the molecular mechanisms on furanodiene-induced cell death,and may aid to the development of furanodiene as a new anti-tumor agent.     

复方莪术油栓中呋喃二烯及莪术醇的测定

目的:建立复方莪术油栓中呋喃二烯和莪术醇的含量测定方法。方法:采用《中国药典》2010年版复方莪术油栓〔含量测定〕项下色谱条件。结果:呋喃二烯在85.89～2579.4 ng范围内,莪术醇在16.272～488.16 ng范围内,线性关系良好,平均回收率分别为99%和97%,RSD分别为3.9%与4.0%(n=9)。结论:药典标准色谱条件可同时控制呋喃二烯和莪术醇的含量,为进一步完善复方莪术油栓质量标准提供了参考依据。     

莪术油的HPLC指纹图谱研究

目的建立莪术油的HPLC指纹图谱鉴别方法。方法以呋喃二烯为参照物,采用HPLC检测方法;色谱条件:Agilent Extend C18色谱柱(250 mm×4.6 mm,5μm),乙腈-水二元梯度洗脱,体积流量1.0 mL/min,检测波长216 nm,柱温30℃。结果确定了8个共有峰,建立了莪术油的HPLC指纹图谱,可区别同属其他植物的挥发油。结论本法操作简便,专属性强,重现性好,可有效控制莪术油的质量。