布雷菲德菌素A通过线粒体凋亡途径增强顺铂抗肺癌GLC-82细胞的作用

本研究证明了线粒体凋亡途径在布雷菲德菌素A（brefeldin A,BFA）联合顺铂（cis-dichlorodiamine platinum,CDDP）抗非小细胞肺癌（non-small cell lung cancer,NSCLC）中的作用。MTT结果显示,BFA对肺癌GLC-82和NCI-H1299细胞的半数有效抑制浓度（half maximal inhibitory concentration,IC50）分别是100 ng/mL和400 ng/mL,CDDP对GLC-82和NCI-H1299细胞的IC50分别是4 μg/mL和15 μg/mL;而分别采用半量的BFA和CDDP联合处理GLC-82或NCI-H1299细胞后,抑制作用均进一步加强。DAPI染色结果进一步证明了二者的协同作用——与单独用药组相比,细胞核染色质固缩加剧,核裂解碎片增多,乃至形成凋亡小体,表明细胞凋亡的发生。与单药组比较,联合用药导致肺癌GLC-82细胞线粒体膜电位显著下降;q-RT-PCR及Western印迹结果显示,在联合用药早期（24 h）,GLC-82细胞可能通过提高Bcl2表达以促进存活;而在联合用药晚期（48 h）,细胞已发生不可逆转的凋亡,Bcl2表达受抑制,同时二者通过促进Bax表达来诱导细胞色素C释放,使胱天蛋白酶 3发生剪切激活,最终诱导细胞凋亡发生。提示线粒体凋亡途径可能是BFA协同CDDP抗非小细胞肺癌的分子机制之一,为肺癌的临床治疗方案提供了更多的理论依据。     

Brefeldin A通过内质网应激途径协同增强顺铂的抗肺癌细胞GLC-82作用

已有研究证明,内质网应激（endoplasmic reticulum stress,ERS）启动的未折叠蛋白质反应（unfolded protein response,UPR）信号途径有助细胞存活;然而,长时间剧烈ERS可诱导细胞凋亡,因此,ERS是肿瘤治疗的新靶点。抗癌药物具有严重的毒副作用,而药物的协同作用是减少抗癌药物使用剂量、减少毒副作用的重要手段之一。本研究证明布雷菲德菌素A（brefeldin A,BFA）与顺铂（cis dichlorodiamine platinum,CDDP）的协同抗肺癌细胞的作用。MTT试验显示,单独应用BFA和顺铂时,它们抑制肺癌细胞GLC 82生长的半数有效浓度（IC50）分别是100 ng/mL和4 μg/mL;而采用半量的BFA和CDDP联合处理GLC 82细胞 24 h后,其抑制生长作用进一步加强;等效线图解法及联合作用指数分析进一步证明二者具有协同作用。两药的这种协同作用进一步被形态学检查证明——与单独用药比较,除了细胞皱缩,出现了更多的染色质固缩,细胞质及细胞核裂解碎片,乃至形成凋亡小体,提示细胞凋亡的发生。实时定量PCR及蛋白质印迹证明,与单独用药比较,联合用药导致内质网应激标志分子GRP78、CHOP表达水平明显增加,提示有内质网应激通路激活。上述结果证明,BFA与CDDP联合用药可增强对肺癌细胞GLC 82的生长抑制作用,其协同机制可能与内质网应激通路的激活有关。我们的结果支持“ERS是肿瘤治疗新靶点”学说,对肺癌的新化疗方案有一定的指导意义。     

RNF13通过IRE1/XBP-1 通路调控内质网应激介导的细胞凋亡

目的:在研究内质网应激介导的细胞凋亡过程中,我们发现Ring finger protein13(RNF13)具有促进细胞凋亡的功能。我们拟研究沉默RNF13后细胞对Tunicamycin等引起的细胞凋亡的影响,以及RNF13对活性形式的caspase3,XBP1(X-box binding protein 1)的剪切以及IRE1(Endoplasmic reticulum to nucleus signaling 1)磷酸化的影响以有助于了解RNF13促进细胞凋亡的信号通路的研究。方法:基因沉默RNF13,利用MTT方法研究RNF13沉默后对细胞增殖的影响,RNF13基因沉默后对XBP1剪切的影响,免疫印迹观察RNF13对IRE1磷酸化的影响。结果:RNF13基因沉默效率在80%以上。RNF13基因沉默后明显抑制细胞凋亡;敲低RNF13的细胞可抵抗衣霉素以及毒胡萝卜素的诱导的细胞凋亡。Caspase-3是细胞凋亡的关键蛋白。敲低RNF13后caspase-3的活性形式明显降低(降低70%,P0.001)。在加入衣霉素引起内质网应激的情况下,敲除RNF13的细胞XBP1的切割活性明显降低。敲除RNF13的细胞中IREl的磷酸化明显降低(降低90%,P0.001)。结论:RNF13通过IRE1-XBP1信号通路调节细胞凋亡。     

利用CRISPRi技术沉默MRP1基因增强A549/DDP细胞对顺铂的敏感性*

目的: 采用CRISPRi技术沉默人肺癌A549/DDP细胞MRP1基因表达,观察细胞对顺铂敏感性的变化。方法: 采用生物信息学软件分析MRP1启动子序列,设计合成3对sgRNA干扰片段,定向克隆到pSPgRNA载体中,构建靶向MRP1的干扰表达载体,分别与dCas9表达载体共转染A549/DDP细胞。实验共分为5组,分别为A549/DDP细胞组,Scrambled组,sgRNA-MRP1-1组,sgRNA-MRP1-2组和sgRNA-MRP1-3组,每组设置3个复孔,处理48 h后进行后续实验。通过qRT-PCR和Western blot检测MRP1 mRNA和蛋白表达水平,MTT法检测细胞对药物的敏感性,激光共聚焦显微镜下观察细胞的形态变化。结果: 成功构建了sgRNA-MRP1-1、sgRNA-MRP1-2和sgRNA-MRP1-3 3种干扰载体,分别与dCas9表达载体共转染A549/DDP细胞后,均能显著降低细胞MRP1基因表达(P<0.01),其中sgRNA-MRP1-2干扰效果最好,MRP1 mRNA水平降低了72%,蛋白水平降低了53%。将sgRNA-MRP1-2转染细胞后,细胞对顺铂的敏感性显著增加,IC₅₀值由74.26±3.71降低至34.29±2.51,细胞形态由梭形变为椭圆形,染色质高度凝聚、边缘化,产生凋亡小体。结论: 成功构建3种靶向MRP1的干扰表达载体,均能有效沉默A549/DDP细胞中MRP1基因表达,可增强细胞对顺铂的敏感性。     

Brefeldin A Is a Potent Inducer of Apoptosis in Human Cancer Cells Independently of p53

Brefeldin A (BFA) is a natural product that affects the structure and function of the Golgi apparatus and is in development for cancer chemotherapy. We observed that a wide range of cancer cells could undergo DNA fragmentation associated with apoptosis after BFA treatment. This DNA fragmentation was induced within 15 h in HL60 leukemia cells and after 48 h in K562 leukemia and HT-29 colon carcinoma cells with BFA concentrations as low as 0.1 μM.The DNA fragmentation had the typical internucleosomal pattern in HL60 and HT-29 cells. Apoptotic cells were also detected by microscopy. BFA-induced apoptosis is p53-independent as HL60 and K562 cells are p53 null and HT-29 are p53 mutant cells. BFA could potentiate UCN-01 and staurosporine-induced DNA fragmentation in HL60 cells. Cyclin B1/Cdc2 kinase activity decreased after BFA treatment in HL60 cells, indicating that BFA-induced DNA fragmentation was independent of a cyclin B1/Cdc2 kinase upregulation pathway. Cycloheximide could not prevent BFA-induced DNA fragmentation in HL60 cells, suggesting that protein synthesis is not needed for HL60 cells to undergo apoptosis. On the contrary, cycloheximide blocked BFA-induced DNA fragmentation in HT-29 cells, indicating that apoptosis in HT-29 cells requires macromolecular synthesis. Cell-free system experiments suggested that cytosolic proteins play an important role in triggering DNA fragmentation during apoptosis induced by BFA. Our results show that transduction signaling pathways play central roles in apoptotic regulation.     

过表达NOLC1诱导人非小细胞肺癌细胞凋亡

人核仁磷酸化蛋白1 (Nucleolar and coiledbody phosphoprotein 1,NOLC1)在癌症的发生发展过程中起着至关重要的调控作用,为探讨NOLC1对肺癌细胞的作用,本研究通过Gateway系统构建重组NOLC1腺病毒载体,成功包装NOLC1腺病毒后,分别感染正常人类胚胎肺细胞(HEL)和非小细胞肺癌细胞(A549细胞),过表达NOLC1。通过MTT实验、AnnexinV-APC/PI双染法和线粒体膜电位实验,证明与HEL细胞相比,NOLC1的过表达对A549细胞的活性降低、凋亡增加、线粒体膜电位下降影响较为显著;通过Real-time PCR检测Caspase家族、TNF与受体家族和BCL2家族基因的表达,发现过表达NOLC1明显上调了A549细胞中促凋亡基因的表达,下调了抗凋亡基因的表达,其中两种重要的促凋亡蛋白CASP8和BAX均显著上调,但是在HEL细胞中这种影响不明显。研究结果表明过表达NOLC1蛋白通过对线粒体通路和死亡受体通路的共同作用,对非小细胞癌具有显著的抗肿瘤活性。     

Pinecone of Pinus koraiensis Inducing Apoptosis in Human Lung Cancer Cells by Activating Caspase‐3 and its Chemical Constituents

Pinecones from Pinus koraiensisSiebold & Zucc . (Pinaceae), which have historically been treated as an undesired waste by‐product in the processing of seeds, have recently been shown to contain ingredients with potent biological activities, such as polyphenols exhibiting antitumor activity. With this study, we seek to broaden our understanding of antitumor compounds contained in these pinecones beyond just polyphenols. We found that the water extract of P. koraiensis pinecones exhibits significant cytotoxic activity, with IC₅₀ values ranging from 0.62 to 1.73 mg/ml in four human lung cancer cell lines, A549, H1264, H1299, and Calu‐6, irrespective of their p53 status. We also demonstrate that pinecone water extract induces apoptosis associated with caspase‐3 activation in the same cancer cell lines. Chemical investigation of the pinecone water extract revealed eight main components ( 1  –  8 ), and their structures were identified as dehydroabietic acid ( 1 ), 15‐hydroxy‐7‐oxodehydroabietic acid ( 2 ), 7β,15‐dihydroxydehydroabietic acid ( 3 ), β‐d ‐glucopyranosyl labda‐8(17,13)‐diene‐(15,16)‐lactone‐19‐oate ( 4 ), 7α,15‐dihydroxydehydroabietic acid ( 5 ), (+)‐(1S,2S,4R)‐limonene‐1,2‐diol ( 6 ), sobrerol ( 7 ), and 4‐hydroxybenzoic acid ( 8 ). These findings suggest a novel biological application of P. koraiensis pinecones in combatting human lung cancer, and further identify the major compounds that could contribute to this anticancer activity.     

E2F1 介导8-氯-腺苷引起的人肺癌细胞H1299的凋亡

8-氯-腺苷（8-Cl-adenosine,8-Cl-Ado）可诱导人非小细胞性肺癌细胞H1299发生凋亡,但其分子机制还没有阐明．首先用四唑盐（MTT）比色法检测了8-Cl-Ado 对H1299 细胞的生长抑制作用．进一步采用蛋白质免疫印迹法(Western blotting) 检测了8-Cl-Ado 处理H1299细胞后,procaspase-3 的激活情况以及E2F1的蛋白水平．通过用pcDNA-HA-E2F1表达载体和pSUPER-E2F1 RNA 干扰载体分别转染H1299 细胞,研究在E2F1 过表达和RNA 干扰（RNA interference, RNAi）两种情况下对凋亡的影响．实验结果表明,8-Cl-Ado可抑制H1299 细胞的生长,激活凋亡关键执行蛋白procaspase-3,升高E2F1 蛋白水平．当E2F1 过表达后,同时伴有procaspase-3 的激活,而E2F1 表达受到抑制后,与对照相比,8-Cl-Ado 引起的procaspase-3 的激活被明显抑制,说明E2F1 介导8-Cl-Ado 引起的人肺癌细胞H1299 的凋亡．     

5-脂氧合酶激活蛋白小干扰RNA诱导人乳腺癌细胞凋亡的初步研究

目的：研究5-脂氧合酶激活蛋白（FLAP）的表达抑制对乳腺癌细胞凋亡的诱导作用。方法：通过小干扰RNA（siRNA）抑制乳腺癌细胞MDA-MB-231中FLAP的表达,用流式细胞仪检测膜联蛋白（annexin）-V标记的早期凋亡细胞,用Western印迹检测细胞凋亡相关蛋白的水平。结果：转染了FLAP siRNA的乳腺癌细胞,24h后FLAP的表达被抑制,17％的细胞出现早期凋亡;48h时早期凋亡细胞增加到32．1％;72h时早期凋亡细胞下降到13．8％,而死亡或凋亡晚期细胞占到61．3％。在细胞凋亡过程中,Bcl-2水平下降,而细胞色素c、胱冬蛋白酶（caspase）-3的水平逐渐增高。结论：FLAP的表达抑制可以诱导乳腺癌细胞通过Bcl-2和胱冬蛋白酶-3途径发生凋亡。     

Depletion of HDAC6 Enhances Cisplatin-Induced DNA Damage and Apoptosis in Non-Small Cell Lung Cancer Cells

Histone deacetylase inhibitors (HDACi) are promising therapeutic agents which are currently used in combination with chemotherapeutic agents in clinical trials for cancer treatment including non-small cell lung cancer (NSCLC). However, the mechanisms underlying their anti-tumor activities remain elusive. Previous studies showed that inhibition of HDAC6 induces DNA damage and sensitizes transformed cells to anti-tumor agents such as etoposide and doxorubicin. Here, we showed that depletion of HDAC6 in two NSCLC cell lines, H292 and A549, sensitized cells to cisplatin, one of the first-line chemotherapeutic agents used to treat NSCLC. We suggested that depletion of HDAC6 increased cisplatin-induced cytotoxicity was due to the enhancement of apoptosis via activating ATR/Chk1 pathway. Furthermore, we showed that HDAC6 protein levels were positively correlated with cisplatin IC(50) in 15 NSCLC cell lines. Lastly, depletion of HDAC6 in H292 xenografts rendered decreased tumor weight and volume and exhibited increased basal apoptosis compared with the controls in a xenograft mouse model. In summary, our findings suggest that HDAC6 is positively associated with cisplatin resistance in NSCLC and reveal HDAC6 as a potential novel therapeutic target for platinum refractory NSCLC.     

Knockdown of TRPM8 Suppresses Cancer Malignancy and Enhances Epirubicin-induced Apoptosis in Human Osteosarcoma Cells

As the function of transient receptor potential melastatin member 8 (TRPM8) in osteosarcoma is still unknown, we aim to investigate the possible effects and potential mechanisms of TRPM8 on cell proliferation, metastasis and chemosensitivity in osteosarcoma cells. We find that TRPM8 is aberrantly over-expressed in human osteosarcoma tissues and cell lines. Knockdown of TRPM8 by siRNA in osteosarcoma cells leads to the impaired regulation of intracellular Ca²⁺ concentration and then the Akt-GSK-3β pathway and the phosphorylation of p44/p42 and FAK are suppressed. Knockdown of TRPM8 not only negatively influences the cell proliferation and metastasis but also enhances epirubicin-induced cell apoptosis. Such results reveal that TRPM8 is worthy further investigation for its potential as a clinical biomarker and therapeutic target in osteosarcoma.