从白屈菜乙醇提取物中分离出的白屈菜碱通过p38-p53和PI3K/AKT信号转导通路促进 HeLa细胞凋亡(英文)

目的:研究从白屈菜乙醇提取物中分离出的白屈菜碱在诱导 HeLa细胞凋亡中的作用及参与其作用的主要信号转导通路。方法:细胞先以不同浓度白屈菜碱处理48h,用噻唑蓝法分析确定半数致死量(median lethal dose,LD50)。用4′,6-二脒基-2-苯基吲哚染色,追踪分析核浓染以及 DNA 损伤和碎片的形态学变化,并用流式细胞术分析检测活性氧(reactive oxygen species,ROS)的产生以及细胞周期阻滞和线粒体膜电位的变化。用圆二色光谱分析寻找白屈菜碱和小牛胸腺 DNA可能的相互作用。用逆转录聚合酶链反应和蛋白免疫印迹法测定p38、p53、蛋白激酶B(protein kinase B,AKT)、磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinases,PI3K)、Janus激酶3(Janus kinase 3,JAK3)、信号转导及转录激活因子3(signal transducer and activator oftranscription 3,STAT3)等的mRNA和蛋白表达,以及E6、E7癌基因和促凋亡基因、抗凋亡基因的mRNA和蛋白表达。结果:根据白屈菜碱的LD50(30μg/mL),选定3种实验剂量,即22.5、30和37.5μg/mL。结果显示,白屈菜碱抑制了 HeLa细胞增殖,诱发其细胞凋亡,表现为 ROS的产生,细胞亚 G1和 G0/G1周期阻滞,线粒体膜电位变化和 DNA 碎片产生。圆二色光谱分析结果显示白屈菜碱和小牛胸腺 DNA 间存在有效的相互作用。信号通路的研究显示白屈菜碱通过上调p38、p53和其他促凋亡基因的表达,以及下调 AKT、PI3K、JAK3、STAT3、E6、E7和其他抗凋亡基因的表达,有效诱发细胞凋亡。结论:从白屈菜中分离出的白屈菜碱能通过改变p38-p53及 AKT/PI3激酶信号转导通路有效地诱发 HeLa细胞凋亡。

Homeopathic mother tincture of Phytolacca decandra induces apoptosis in skin melanoma cells by activating caspase-mediated signaling via reactive oxygen species elevation

OBJECTIVE： Preventive measures against skin melanoma like chemotherapy are useful but suffer from chronic side effects and drug resistance. Ethanolic extract of Phytolacca decandra （PD）, used in homeopathy for the treatment of various ailments like chronic rheumatism, regular conjunctivitis, psoriasis, and in some skin diseases was tested for its possible anticancer potential. METHODS： Cytotoxicity of the drug was tested by conducting 3-（4,5-dimethylthiazol-2-yl）-2,5- diphenyltetrazolium bromide assay on both normal （peripheral blood mononuclear cells） and A375 cells. Fluorescence microscopic study of 4＇,6-diamidino-2-phenylindole dihydrochloride-stained cells was conducted for DNA fragmentation assay, and changes in cellular morphology, if any, were also recorded. Lactate dehydrogenase activity assay was done to evaluate the percentages of apoptosis and necrosis. Reactive oxygen species （ROS） accumulation, if any, and expression study of apoptotic genes also were evaluated to pin-point the actual events of apoptosis. RESULTS： Results showed that PD administration caused a remarkable reduction in proliferation of A375 cells, without showing much cytotoxicity on peripheral blood mononuclear cells. Generation of ROS and DNA damage, which made the cancer cells prone to apoptosis, were found to be enhanced in PD-treated cells. These results were duly supported by the analytical data on expression of different cellular and nuclear proteins, as for example, by down- regulation of Akt and Bcl-2, up-regulation of p53, Bax and caspase 3, and an increase in number of cell deaths by apoptosis in A375 cells. CONCLUSION： Overall results demonstrate anticancer potentials of PD on A375 cells through activation of caspase-mediated signaling and ROS generation.

Diarylheptanoid-myricanone isolated from ethanolic extract of Myrica cerifera shows anticancer effects on HeLa and PC3 cell lines:signalling pathway and drug-DNA interaction

OBJECTIVE： To test if myricanone （02H2405）, a cyclic diarylheptanoid, has anticancer effects on two different cancer cell lines HeLa and PC3. The present study was conducted with a note on the drug-DNA interaction and apoptotic signalling pathway. METHODS： Several studies like cytotoxicity, nuclear damage, annexin-V-fluorescein isothiocyanate （FITC）/propidium iodide （PI）-Iabelled apoptotic assay and cell cycle arrest, immunoblot and reverse transcriptase-polymerase chain reaction （RT-PCR） were used following standard protocols. Circular dichroism （CD） spectroscopy was also done to evaluate whether myricanone effectively interacted with DNA to bring about conformational changes that could strongly inhibit the cancer cell proliferation. RESULTS： Myricanone showed a greater cytotoxic effect on PC3 cells than on HeLa cells. Myricanone promoted G0/G arrest in HeLa cells and S phase arrest in PC3 cells. Nuclear condensation and annexin V-FITC/PI studies revealed that myricanone promoted apoptotic cell death. CD spectroscopic data indicated that myricanone had an interaction with calf thymus DNA that changed DNA structural conformation. RT-PCR and immunoblot studies revealed that myricanone activated the apoptotic signalling cascades through down-regulation of transcription factors like nuclear factor-KB （NF-KB） （p65）, and signal transducers and activators of transcription 3 （STAT3）; cell cycle regulators like cyclin D1, and survivin and other signal proteins like Bcl-2 and up-regulation of Bax, caspase-9 and caspase-3. CONCLUSION： Myricanone induced apoptosis in both types of cancer cells by triggering caspase activation, and suppression of cell proliferation by down-regulation of NF-KB and STAT3 signalling cascades, which makes it a suitable candidate for possible use in the formulation of therapeutic alent for combatin cancer.

北美香柏叶的乙醇提取物阻断A549细胞增殖并引起细胞凋亡的体外研究(英文)

目的:研究北美香柏叶的乙醇提取物对非小细胞肺癌A549细胞的抗肿瘤及抗增殖作用。方法:噻唑蓝法检验不同剂量北美香柏叶的乙醇提取物对细胞活性的影响。确定半数最大抑制浓度为282μg/mL,另外选择两个浓度188μg/mL和376μg/mL进行剂量依赖性检测。进行溴脱氧尿苷结合实验和细胞迁移实验检测药物的抗肿瘤细胞增殖活性。膜联蛋白V-异硫氰酸荧光黄-碘化丙啶双染色后采用荧光激活细胞分类分析仪对细胞早期凋亡进行检测。Hoechst 33258及吖啶橙-溴化乙啶荧光染色法进行DNA片段分析。间接酶联免疫吸附法分析Bax-Bcl2的调节和表达情况。逆转录聚合酶链反应检测caspase3基因表达情况,其活性和蛋白水平的表达则使用间接酶联免疫吸附法和蛋白印迹法进行检测。结果:A549的细胞活性在暴露于北美香柏叶的乙醇提取物24h后呈剂量依赖性下降。脱氧尿苷结合实验和细胞迁移实验表明细胞的增殖活性与暴露于药物的时间有时间依赖性关系。11.72%的细胞在双染色后呈阳性反应,表明药物引起了细胞的早期凋亡。药物作用24h后DNA片段彗星尾的出现以及Hoechst 33258荧光染色的增加提示显著的DNA缺口出现以及染色质凝聚。Bax的上调及Bcl2的下调表明了细胞凋亡的出现。逆转录聚合酶链反应、间接酶联免疫吸附法以及蛋白印迹法的检测结果表明caspase3的活性随着抗聚(腺苷二磷酸-核糖)聚合酶的表达的增加而增加。结论:北美香柏叶的乙醇提取物能够促进A549细胞凋亡并抑制其增殖活性。

Oleanolic acid isolated from ethanolic extract of Phytolacca decandra induces apoptosis in A375 skin melanoma cells: drug-DNA interaction and signaling cascade

OBJECTIVE： Oleanolic acid （OA） has been reported to have anticancer effects, but the extent of its cytotoxicity, its ability to interact with nuclear DNA, its action against skin melanoma, as well as the molecular mechanism of its action against cell proliferation and in support of cell death are still unexplored. This led us to examine the efficacy of OA, a bioactive compound isolated from Phytolacca decandra, on these issues in the present investigation. METHODS： Studies related to analyses of cell viability, drug-DNA interaction, cell proliferation, cell cycle and epidermal growth factor receptor （EGFR） activity were performed. To investigate whether cells undergo apoptosis, studies like fluorescence microscopy, poly （ADP-ribose） polymerase （PARP） degradation, annexin V-fluorescein isothiocyanate/propidium iodide assay, alteration in mitochondrial membrane potential and activity of some relevant signaling proteins were performed. RESULTS： OA displayed a minimal and negligible cytotoxic effect on normal HaCaT cells （skin keratinocytes） and peripheral blood mononuclear cells but by contrast it reduced A375 cell viability significantly. OA interacted with nuclear DNA quickly after exposure. It acted as an anti- proliferative agent. It suppressed EGFR activity. OA administration led the cells to mitochondria- dependent caspase 3-mediated apoptosis. CONCLUSION： OA interacts with cellular DNA, inhibits proliferation possibly through modulating EGFR activity and induces mitochondria-dependent caspase 3-mediated apoptosis in A375 cells which would qualify it as a potent anticancer agent.

Low doses of ethanolic extract of Boldo(Peumus boldus) can ameliorate toxicity generated by cisplatin in normal liver cells of mice in vivo and in WRL-68 cells in vitro, but not in cancer cells in vivo or in vitro

OBJECTIVE： Use of cisplatin, a conventional anticancer drug, is restricted because it generates strong hepatotoxicity by accumulating in liver. Therefore its anticancer potential can only be fully exploited if its own toxicity is considerably reduced. Towards this goal, ethanolic extract of the plant, Boldo （Peumus boldus）, known for its antihepatotoxic effects, was used simultaneously with cisplatin, to test its ability to reduce cisplatin＇s cytotoxicity without affecting its anticancer potential. METHODS： The cytotoxicity of Boldo extract （BE） and cisplatin, administered alone and in combination, was determined in three cancer cell lines （A549, HeLa, and HepG2） and in normal liver cells （WRL-68）. Drug-DNA interaction, DNA damage, cell cycle, apoptosis, reactive oxygen species （ROS） and mitochondrial membrane potential （MMP, Aψ） were also studied. Hepatotoxicity and antioxidant activity levels were determined by alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and glutathione assays in mice. The cytotoxicity of related proteins was tested by Western blotting. RESULTS： Co-administration of BE and cisplatin increased viability of normal cells, but had no effect on the viability of cancer cells. Boldo protected liver from damage and normalized different antioxidant enzyme levels in vivo and also reduced ROS and re-polarized MMP in vitro. Bax and cytochrome c translocation was reduced with caspase 3 down-regulation. Further, a drug- DNA interaction study revealed that BE reduced cisplatin＇s DNA-binding capacity, resulting in a reduction in DNA damage. CONCLUSION： Results indicated that a low dose of BE could be used beneficially in combination with cisplatin to reduce its toxicity without hampering cisplatin＇s anticancer effect. These findings siqnify a potential future use of BE in cancer therapy.