布雷菲德菌素A调控IRE1-XBP1信号通路增强顺铂诱导GLC-82细胞凋亡的作用

长时间剧烈的内质网应激（endoplasmic reticulum stress,ERS）可启动ERS相关通路诱导细胞凋亡;而IREl-XBP1(肌醇需求蛋白1α-X盒结合蛋白1)为高度保守的ERS基本通路。它是最有前景的肿瘤治疗靶点之一。我们研究已证明,布雷菲德菌素A（brefeldin A,BFA）能增强顺铂（cis-dichlorodiamine platinum,CDDP）的肺癌细胞杀伤作用,但其分子机制尚不清楚。本研究证明了IRE1-XBP1通路在该协同效应中的作用。AnnexinV/PI双染结合流式细胞分析显示,与BFA或CDDP单独处理比较,BFA联合CDDP处理能增强人肺癌GLC-82细胞的凋亡。RT-qPCR和Western印迹揭示,与单独处理比较,BFA联合CDDP处理能增强GLC-82细胞的procaspase3转录本（mRNA）和蛋白质的表达,以及procaspase3的裂解激活,进一步证明了BFA联合CDDP处理可增强GLC-82细胞凋亡。最重要的是,RT-qPCR和Western印迹结果证明,与单独CDDP处理比较,BFA处理的GLC-82细胞中IRE1、XBP1水平明显上调（P < 0.05）,而BFA+CDDP处理的细胞中IRE1、XBP1水平进一步上调,超过BFA组（P < 0.01）。结果提示BFA和BFA+CDDP处理细胞可激活ERS的IRE1-XBP1通路。这些结果证明,BFA可通过激活ERS中的IRE1-XBP1通路增强顺铂诱导的肿瘤细胞凋亡。本研究结果为IREl-XBP1是颇具前景的肿瘤治疗靶点提供了新的证据。     

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。     

布雷菲德菌素A通过线粒体凋亡途径增强顺铂抗肺癌细胞的作用

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(cisdichlorodiamine 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,IC_(50))分别是100 ng/mL和400 ng/mL,CDDP对GLC-82和NCI-H1299细胞的IC_(50)分别是4μg/mL和15μg/mL;而分别采用半量的BFA和CDDP联合处理GLC-82或NCIH1299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。     

单宁酸协同顺铂增强肝癌HepG2细胞内质网应激PERK-ATF4通路的激活水平

本文主要研究单宁酸(TA)联合顺铂(CDDP)对肝癌Hep G2细胞内质网应激PERK-ATF4通路的影响,以探究TA协同CDDP抗肝癌的分子机制。用180μM TA、0.9μg/m L CDDP单独用药或者联合用药处理肝癌Hep G2细胞24 h和48 h后,利用实时荧光定量PCR技术(q-RT-PCR)、蛋白免疫印迹(Western Blot)技术检测细胞PERK、p-PERK、ATF4、CHOP、Procaspase3与Cleaved caspase3分子的表达水平。q-RT-PCR及Western Blot结果显示,TA与CDDP联合用药能显著上调细胞PERK、ATF4、CHOP的表达水平和Caspase3的剪切活化水平,下调PERK的磷酸化水平(P0.01或P0.05)。TA协同CDDP增强了肝癌Hep G2细胞内质网应激PERK-ATF4通路的激活水平,其可能是通过激活细胞ERS,促进PERK-ATF4通路相关分子和ERS标志性分子CHOP的表达,抑制PERK的磷酸化,从而促进Caspase3的剪切活化来诱导细胞凋亡的发生。     

单宁酸联合顺铂增强肝癌HepG2细胞IRE1-XBP1通路的激活水平

本文主要研究单宁酸(TA)联合顺铂(CDDP)对肝癌HepG2细胞内质网应激IRE1-XBP1通路的影响。用180μM单宁酸、0.9μg/m L顺铂单独用药或者联合用药处理肝癌HepG2细胞24 h或48 h后,应用流式细胞技术测定HepG2细胞的凋亡率,实时荧光定量PCR技术(q-RT-PCR)、蛋白免疫印迹(western blot)技术检测IRE1α和XBP-1分子的表达水平。MTT结果显示,单宁酸和顺铂均能显著抑制HepG2细胞的生长,且均呈剂量性依赖;二者联合用药能够显著增加HepG2细胞的生长抑制率;流式细胞术结果显示,单宁酸与顺铂联合用药能够显著抑制HepG2细胞的增殖,并诱导细胞凋亡的发生;q-RT-PCR及Western blot结果显示,单宁酸与顺铂联合用药能显著上调细胞IRE1α和XBP-1的表达水平。结果表明单宁酸能够联合顺铂增强肝癌HepG2细胞内质网应激IRE1-XBP1通路的激活水平,提示IRE1-XBP1通路可能是单宁酸和顺铂协同抗肝癌HepG2细胞的分子机制之一。     

内质网应激及其在糖尿病肾病中的作用机制

糖尿病肾病（diabetic nephropathy,DN）是糖尿病最常见的微血管并发症,是导致终末期肾脏疾病（end-stage renal disease,ESRD）的继发性肾脏疾病的主要病因之一。多种因素如缺氧、氧化应激、病毒感染、遗传突变等,可导致内质网内稳态失衡,大量未折叠蛋白和错误折叠引起蛋白堆积,即形成内质网应激(endoplasmic reticulum stress, ERS),从而激活未折叠蛋白反应(unfolded protein response, UPR)介导的三条经典的细胞适应性应答通路以恢复内质网稳态和细胞活性。但如果刺激过强或持续存在,便会启动细胞凋亡信号通路。大量研究表明ERS与DN的发生发展相关,并参与不同类型肾细胞损伤的过程,因此ERS作为治疗DN的有效靶点具有很重要的研究前景,调控ERS可为DN的治疗提供新的理论支持。从ERS相关信号通路及其在DN中的作用和新进展领域作一综述,以期为DN的治疗研究提供参考。     

内质网应激与胰岛β细胞凋亡的研究进展

胰岛β细胞具有高度发达的内质网,对内质网应激(endoplasmic reticulum stress,ERS)非常敏感。多种因素可以打破内质网稳态,引起蛋白质折叠障碍或错误折叠以及Ca2+代谢紊乱,进一步触发内质网应激。适度的内质网应激有利于细胞内环境的恢复,过度的内质网应激可会导致内质网功能受损,诱导胰岛β细胞凋亡,从而介导糖尿病的发生、发展。本文就内质网应激与胰岛β细胞凋亡的研究进展做一综述。     

内质网应激介导的细胞凋亡

内质网是细胞内重要的细胞器,内质网功能的损伤引起ER应激(ERS).内质网通过激活未折叠蛋白质反应(UPR)以保护由内质网应激所引起的细胞损伤,恢复细胞功能,包括暂停早期蛋白质合成、内质网分子伴侣和折叠酶的转录激活、内质网相关性降解(ERAD)的诱导.长期过强的内质网应激诱导内质网相关性细胞凋亡,清除受损细胞,包括内质网应激诱导CHOP/GADD153表达、JNK的激活以及caspase-12蛋白水解酶的活化等一系列生物学效应.     

内质网应激诱导的自噬在肝脏疾病中的研究进展

错误/未折叠蛋白的积累可导致内质网(endoplasmic reticulum, ER)结构和功能紊乱,从而诱发内质网应激(endoplasmic reticulum stress, ERS),激活未折叠蛋白反应(unfolded protein response, UPR)。UPR作为适应性机制可恢复早期的ERS,重建ER稳态;当UPR不足以缓解ERS时,会通过UPR介导的3个跨膜蛋白(IRE1α、PERK、ATF6)诱发细胞凋亡或自噬。自噬作为ERS的另一种保护性反应,可通过降解错误折叠蛋白和清除受损细胞器来减轻ERS。另外,自噬是ERS重要的下游事件并处于凋亡的上游,ERS介导的自噬可通过调节细胞凋亡发挥促凋亡或抗凋亡双重作用。ERS与自噬间的相互作用在酒精/非酒精性脂肪肝、肝纤维化、肝癌等多种肝脏疾病中扮演着重要角色,但二者在肝病发生发展过程中的具体机制尚不明确。因此,探讨ERS与自噬通过复杂的网络通路调控肝脏疾病的机制有助于改善相关肝脏疾病,这可能成为治疗肝病的有效靶点。     

藻蓝蛋白联合全反式维甲酸对HeLa细胞生长抑制作用的研究(英文)

目的:探讨全反式维甲酸和藻蓝蛋白单独及联合用药对HeLa细胞生长的影响,并揭示两者联合用药对细胞周期和细胞凋亡影响的分子机制。方法:MTT法检测全反式维甲酸和藻蓝蛋白单独及联合用药对HeLa细胞生长的影响,原位杂交法检测用药前后细胞内CDK-4基因mRNA的表达情况,免疫组化法检测用药前后bcl-2基因的表达情况,TUNEL法检测用药前后细胞凋亡情况。结果:全反式维甲酸和藻蓝蛋白均具有抑制HeLa细胞生长的作用,当达到相同的抑制率时,联合藻蓝蛋白使用可以显著降低全反式维甲酸的使用剂量从而达到降低毒副作用的目的。两者联合用药可以显著降低CDK-4的表达量从而对HeLa细胞的细胞周期产生影响。两者联合用药可以显著下调bcl-2的表达水平从而引发细胞凋亡。结论:通过联合藻蓝蛋白,可以显著降低全反式维甲酸的使用剂量从而降低毒副作用。全反式维甲酸和藻蓝蛋白联合用药抑制HeLa细胞生长的分子机制可能是通过抑制CDK-4和bcl-2的表达来影响细胞周期并最终导致细胞凋亡。     

Antiproliferation effect of Rosemary (Rosmarinus officinalis) on human ovarian cancer cells in vitro

Rosemary (Rosmarinus officinalis L.) is a popular culinary/medicinal herb. Recent studies have shown it has pharmacologic activities for cancer chemoprevention and therapy. This study evaluated the antiproliferation activity of rosemary extract (RE) against human ovarian cancer cells, and whether the extract and its three main active ingredients carnosol (CS), carnosic acid (CA) and rosmarinic acid (RA) can enhance the antiproliferation activity of cisplatin (CDDP). Our study showed that RE has significant antiproliferation activity on human ovarian cancer A2780 and its CDDP resistant daughter cell line A2780CP70, with IC(50) (50% inhibitory concentration) estimated at 1/1000 and 1/400 dilutions respectively. RE enhanced the antiproliferation effect with CDDP on both A2780 and A2780CP70 cells. A2780 cells were consistently more sensitive to CS, CA, and RA than A2780CP70 cells between 2.5 and 20μg/ml. CS and RA also showed synergistic antiproliferation effect with CDDP on A2780 cells at some concentrations. RE treated by ultrafiltration, dialysis, and removal of phenolics lost the antiproliferation activity suggested that the activity resides in phenolics with MW<1000Da. Apoptosis array study of A2780 cells treated with RE showed that the expression of a number of genes regulating apoptosis were modulated by the treatment. This study showed that RE inhibited the proliferation of ovarian cancer cell lines by affecting the cell cycle at multiple phases. It induced apoptosis by modifying the expression of multiple genes regulating apoptosis, and holds potential as an adjunct to cancer chemotherapy.     

叶酸靶向载顺铂羧甲基-β-环糊精纳米复合物的制备及对鼻咽癌的体外抑制效应

为达到鼻咽癌（nasopharyngeal carcinoma,NeC）的靶向化疗,该研究通过酰胺化反应和配位偶联技术制备叶酸（folicacid,FA）分子靶向载川页铂（cisplatin,CDDP）羧甲基-β-环糊精（carboxymethyl-β-cyclodextrin,CM—β—CD）纳米复合物（FA-CM—β—CD—CDDP）,采用邻苯二胺（o-phenylenediamine,OPDA）比色法检测复合物中CDDP含量,紫外分光光谱检测FA含量,透射电镜观察复合物形态,激光粒度仪测定复合物粒径大小。荧光显微镜观察NPC叶酸受体（folatereceptor,FR）阳性HNE-1细胞及FR阴性CNE-2细胞对偶联FITC的复合物的吞噬及OPDA比色法检测细胞内CDDP的浓度。通过MTT法、集落形成实验和流式细胞术检测复合物对HNE-1细胞增殖能力和凋亡的影响。研究结果显示,复合物中偶联的FA和CDDP浓度分别为340gg／mL和2mg／mL,CDDP包封率达20．00％,复合物粒径均匀且大小为157．8nm。HNE-1细胞内见较多FITC,细胞内CDDP浓度为6．24ng／mL,而CNE-2细胞内FITC较少,细胞内CDDP浓度仅约2．01ng／mL。HNE-1生长抑制率在24h明显高于对照组（CM—β—CD—CDDP）,其IC50（4．80μg／mL）明显低于对照组（6．97μg／mL）,但当所载的CDDP终浓度达到16．00μg／mL时,两组抑制率均达到80％以上;作用48h两组抑制率无明显差异。在24h,当复合物的CDDP终浓度为1．00μg／mL时,HNE—1的集落形成率为33．21％,明显低于对照组（52．27％）。当复合物的CDDP终浓度为0．25μg／mL和1．00μg／mL时,HNE-1的凋亡率分别达12．65％和22．35％,明显高于对照组（6．91％和14.21％）。研究结果表明,成功构建的FA—CM—β—CD．cDDP纳米复合物能够靶向抑制FR阳性的NPC细胞增殖并促进其凋亡。     

荞麦中光毒物质的转化及荞麦光敏素对人结肠癌细胞HCT-116的抑制活性

荞麦光敏素（fagopyrins）具有光毒性和酪氨酸激酶抑制活性,可作为光动力治疗中的光敏剂,用于癌症的治疗。本研究采用电喷雾质谱法和荧光光度法,探究了荞麦中光毒物存在的型式,并分析了光敏素前体(protofagopyrin)转化为荞麦光敏素的条件。通过MTT细胞实验,研究荞麦光敏素提取物对人结直肠癌细胞和人结肠上皮细胞生长增殖的影响。结果表明,荞麦中光毒物以光敏素前体为主,并且有微量的荞麦光敏素,前者质谱的离子强度是后者的400倍。光敏素前体的转化发生在光照提取过程中,而且提取液在光照放置过程中,光敏素前体继续转化,尤其是前0.5 h对于转化的影响显著超过了提取过程。荞麦光敏素提取物与细胞作用,避光培养时,对HCT-116癌细胞有轻度的抑制作用;而对FHC正常细胞不仅未见抑制还有促进作用。光照结合避光培养时,荞麦光敏素对癌细胞的抑制随着提取物浓度的增加而增强,当提取物浓度达到200 μg/mL时,抑制率达到50%;而对正常细胞提取物,浓度低于200 μg/mL时未见抑制,500 ～ 1 000 μg/mL时抑制率达到50%。光敏素提取物与金丝桃素抑制细胞的能力相比,光照结合避光培养时,金丝桃素对正常细胞的抑制作用强于对癌细胞,而荞麦光敏素的提取物正好相反。提示荞麦光敏素比金丝桃素对癌细胞的抑制更具特异性。上述结果为从荞麦叶中开发出有利于肿瘤治疗的药物提供了实验依据。     

Involvement of the Golgi region in the intracellular trafficking of cholera toxin

The intracellular pathway following receptor-mediated endocytosis of cholera toxin was studied using brefeldin A (BFA), which inhibited protein secretion and induced dramatic morphological changes in the Golgi region. In both mouse Y1 adrenal cells and CHO cells, BFA at 1 μg/ml caused a 80–90% inhibition of the cholera toxin (CT)-elevation of intracellular cAMP. The inhibition of the cytotoxicity of CT by BFA was also observed in a rounding assay of Y1 adrenal cells. The inhibition of CT cytotoxicity by BFA was dose dependent, with the ID₅₀ value similar to the LD₅₀ of BFA in Y1 adrenal cells. Binding and internalization of [¹²⁵I]-cholera toxin in Y1 adrenal cells was not affected by BFA. Unlike the BFA-sensitive cell lines such as Y1 adrenal and CHO cells, BFA at 1 μg/ml did not inhibit the cytotoxicity of CT in PtK₁ cells, of which the Golgi structure was BFA-resistant. These results strongly suggest that a BFA-sensitive Golgi is required for the protection of CT cytotoxicity by BFA. In contrast, elevation of the intracellular cAMP by forskolin, which acts directly on the plasma membrane adenylate cyclase, was not affected by BFA. These observations indicate that the intoxication of target cells by CT requires an intact Golgi region for its intracellular trafficking and/or processing. In this respect, CT shares a common intracellular pathway with ricin, Pseudomonas toxin, and modeccin, even though their structures and modes of action are very different. © 1993 Wiley-Liss, Inc.     

Role of nitric oxide in human pulmonary microvascular endothelial cell adhesion

Cyclopentenylcytosine (CPEC) is cytotoxic to HT-29 cells in vitro and in vivo. Treatment with CPEC resulted in sensitizing HT-29 cells to cisplatin (CDDP), as evidenced by synergistic cytotoxicity. CPEC exhibits potent cytotoxicity to HT-29 cells in vitro, 2 and 24 h exposure providing an LC50 of 2.4 and 0.46 microM, respectively. Exposure of HT-29 cells to CDDP for 2 h resulted in an LC50 of 26 microM. Treatment of HT-29 cells with 1.0 or 1.25 microM CPEC and then incubating with CDDP showed synergistic cytotoxicity. Lesser synergy at very high concentrations of CPEC was demonstrated when HT-29 cells were first exposed to CDDP and then incubated with CPEC. Combination index calculations showed synergistic cytotoxicity in HT-29 cells when CPEC was combined with CDDP. Synergistic antitumor activity was demonstrable in vivo in mice transplanted with HT-29 tumor when treated with a combination of CPEC and CDDP without undue toxicity, since no excessive loss in mouse body weight or overt pathology was observed. CPEC had no influence on the total DNA adduct formation and CDDP did not affect the intracellular levels of CPEC or its metabolites, suggesting that enhanced CDDP cytotoxicity resulted from a step subsequent to excision of platinum-cross-linked DNA. These studies support a new approach for augmenting cytotoxic effect of CPEC with CDDP in treating human colon carcinoma.     

The effect of bee bread (Perga) with chemotherapy on MDA-MB-231 cells

Bee bread (BB) is a bee product like propolis and honey. It is the main food for larvae and bees producing royal jelly in the hive. It also known as Perga. As with other bee products, it is increasingly popular due to its antioxidant properties. The aim of this study was to examine the effects of BB on MDA-MB-231 breast cancer cells and the effects on these cells when administered together with Doxorubicin (DOX) and Cisplatin (CDDP), used in cancer treatment. The proliferation of the cells was determined by applying 5 mg/mL BB together with different concentrations of DOX and CDDP. In addition to these studies, the effect of DOX+BB and CDDP+BB combinations on the migration of MDA-MB-231 cells was determined by the wound healing method. The expression levels of Bid and Bcl-2 were determined by RtqPCR. According to these studies, as expected, BB did not show a significant toxic effect on MDA-MB-231 cells at different concentrations. BB significantly suppressed the effect of DOX and CDDP on the proliferation of MDA-MB-231 cells. BB with DOX and CDDP suppressed the proapoptotic Bid gene while overexpressing the anti-apoptotic Bcl-2 gene, separately. Interestingly, BB blocked the migration of MDA-MB-231 cells by 50% even after 72 h. As a result, BB significantly reduced the toxicity of DOX and CDDP on MDA-MB-231 cells. The most interesting result of the study is that BB prevented the migration of cancer cells.

     

反相HPLC离子对色谱法测定植物组织中的甜菜碱

A reverse phase ion-pair HPLC assay has been developed for screening glycinebetaine content in plant tissues in studies of its relation to salinity tolerance. Separation was performed on a 250 mm×4.6 mm stainless steel column (packed with 10 μm irregular-H) eluted with 50　mmol/L KH 2PO4 (pH 4.45) containing ion-pair agent 0.1% PIC B-8 (1-octane sulfonic acid, Waters). Detection was performed by UV absorbance at 192 nm. The recovery rate of glycinebetaine in tissue extracts ranged from 85% to 96%. Glycinebetaine concentrations in leaf and root of Populus euphratica and P.`popularis 35-44' varied between 0.06 and 0.75 μg/g fresh weight (FW) with higher values in leaf tissue. Glycinebetaine level in suspension cells of P. euphratica increased from 0.45 to 0.77 μg/g FW following NaCl stress. The main assay procedure described here offers a number of advantages over other methods of estimating betaines: a) It increases precision and accuracy as compared with the periodide. b) Using a reverse HPLC column thus it decreases the expense for purchasing special equipment (such as strong cation-exchange columns). c) It avoids the need to generate derivatives thus allows rapid assay for betaines. This technique is not, however, suitable for use on crude, unpurified extracts and an ion-exchange clean up procedure is required. In the author's experiment, the extracts were passed through a 5 mL column of Dowex 1×8 (100-200 mesh, OH- form, Sigma) in series with a second column of a 5 mL Dowex 50W×2 (50-100 mesh, H+ form, Serva). Bound betaine was recovered from Dowex 50W×2 with 10mL of 2mol/L NH4OH and the eluate was evaporated to dryness at 65℃ in vacuum. 1 mL solution for the HPLC elution was used for dissolving betaine samples before separation on column. 