非小细胞肺癌体外化疗药物敏感性与GRP94表达的相关性

[目的]探讨非小细胞肺癌(NSCLC)对8种化疗药物的体外敏感性与GRP94表达的相关性。[方法]用四噻唑蓝(MTT)法检测52例手术切除新鲜NSCLC组织8种化疗药物体外敏感性;免疫组织化学方法检测肺癌组织GRP94表达。采用Spearman法进行相关性分析。[结果]NSCLC52例对紫杉醇(PTX)、阿霉素(ADM)、卡铂(CBP)、拓扑替康(TPT)、长春瑞滨(NVB)、长春新碱(VCR)、顺铂(DDP)和鬼臼乙叉苷(VP-16)8种化疗药物的耐药率分别为42.31%、57.69%、63.46%、65.38%、67.31%、73.08%、78.85%和90.38%。其中,14例表现为完全耐药;且其高表达与肺癌细胞对VP-16的耐药明显相关(P<0.05)。[结论]检测GRP94表达对推测NSCLC对VP-16的耐药性具有参考意义。     

Cucurbitacin B-induced G2/M cell cycle arrest of conjunctival melanoma cells mediated by GRP78–FOXM1–KIF20A pathway

Conjunctival melanoma (CM) is a rare and fatal malignant eye tumor. In this study, we deciphered a novel anti-CM mechanism of a natural tetracyclic compound named as cucurbitacin B (CuB). We found that CuB remarkably inhibited the proliferation of CM cells including CM-AS16, CRMM1, CRMM2 and CM2005.1, without toxicity to normal cells. CuB can also induce CM cells G2/M cell cycle arrest. RNA-seq screening identified KIF20A, a key downstream effector of FOXM1 pathway, was abolished by CuB treatment. Further target identification by activity-based protein profiling chemoproteomic approach revealed that GRP78 is a potential target of CuB. Several lines of evidence demonstrated that CuB interacted with GRP78 and bound with a K_d value of 0.11 μmol/L. Furthermore, ATPase activity evaluation showed that CuB suppressed GRP78 both in human recombinant GRP78 protein and cellular lysates. Knockdown of the GRP78 gene significantly induced the downregulation of FOXM1 and related pathway proteins including KIF20A, underlying an interesting therapeutic perspective. Finally, CuB significantly inhibited tumor progression in NCG mice without causing obvious side effects in vivo. Taken together, our current work proved that GRP78–FOXM1–KIF20A as a promising pathway for CM therapy, and the traditional medicine CuB as a candidate drug to hinder this pathway.     

Endoplasmic reticulum stress is activated in light-induced retinal degeneration

Exposure to excessive levels of light induces photoreceptor apoptosis and has previously been used as a model for the study of retinal degeneration. During the light exposure, intracellular calcium levels increase, and reactive oxygen species (ROS) are generated, which have been shown to cause endoplasmic reticulum (ER) stress. In the present study, we investigated the role of ER stress in light-induced photoreceptor apoptosis. Our study demonstrated that, after light exposure, the ER stress sensors including glucose-regulated protein-78 (GRP78/BiP), caspase-12, phospho-eukaryotic initiation factor 2 alpha (eIF2 alpha), and phospho-pancreatic ER kinase (PERK) were significantly up-regulated in a time-dependent manner. The up-regulation of these proteins coincided with or preceded the photoreceptor apoptosis indicated by TUNEL. These data showed that ER stress played an important role in light-induced photoreceptor apoptosis. Therefore, ER stress modulators could be strong candidates as therapeutic agents in the treatment of retinal degenerative diseases.     

Expression of autophagy and ER stress-related proteins in primary salivary adenoid cystic carcinoma

Autophagy is the endogenous cellular pathway that facilitates cellular survival by maintaining energy homeostasis and macromolecular synthesis during cellular stress and nutrient deprivation. Endoplasmic reticulum (ER) stress is the process in which disruption of these physiological functions leads to an accumulation of unfolded proteins and induces the unfolded protein response (UPR). ER stress and autophagy are involved in human cancer. We investigated the expression of autophagic proteins (LC3 and beclin 1) and ER stress-related protein (GRP78) in head and neck adenoid cystic carcinoma tissue. Tissue samples from 79 cases of head and neck adenoid cystic carcinoma tissue were utilized for immunohistochemistry. LC3 expression was significantly correlated with lymph node involvement (P = .016) and TNM (P = .021). Beclin 1 expression was significantly correlated with the histological growth pattern (P = .002), the histological grade (P = .000), and longer survival (P = .000). GRP78 expression was significantly correlated with the histological growth pattern (P = .019), the histological grade (P = .019), and longer survival (P = .001). LC3 expression was positively correlated with beclin 1 expression (P = .000); LC3 and beclin 1 expressions were positively correlated with GRP78 expression respectively (P = .035) (P = .008). Our study describes the expression of LC3, beclin 1, and GRP78 in adenoid cystic carcinoma and its relationship with clinicopathologic factors and overall survival. These results suggest that LC3, beclin 1, and GRP78 may play an important role in the tumorigenesis of adenoid cystic carcinoma, and that beclin 1 and GRP78 may serve as new prognostic indicators for the outcome of patients with adenoid cystic carcinoma.     

人HUT78细胞褪黑素受体的基因与蛋白表达的研究

为了检测人HUT78细胞是否存在褪黑素受体（melatonin receptor,MR)及其亚细胞分布特点，采用异硫氢酸胍－苯酚－氯仿一步法抽提总RNA，进一步通过RT－PCR方法检测MR亚型mt1和MT2的mRNA，并将RT－PCR的阳性产物通过自动测序仪测序；同时应用免疫组化染色方法检测mt1和MT2受体亚型蛋白在HUT78细胞内的分布特点，RT－PCR方法则得到了368bp mt1 cDNA片段，无321bp MP2 cDNA片段，同时将mt1阳性条带通过胶回收，测序，结果显示扩增产物与人mt1受体亚型的基因序列相吻合，免疫组化结果显示，人HUT78细胞存在mt1受体蛋白，主要分布于细胞膜和细胞浆，呈棕褐色阳性颗粒，而细胞上未见阳性颗粒，MT2受体蛋白则未检出，提示褪黑素（melatonin,Mel)对T淋巴细胞具有直接调节作用，为研究生理及病理情况下Mel调节免疫系统的作用机制奠定了基础。     

Detection of opiate-enhanced increases in DNA damage,HPRT mutants,and the mutation frequency in human HUT-78 cells

In previous studies we have shown highly significant increases in chromosome damage and sister chromatid exchanges in heroin addicts, particularly when caffeine and metabolic inhibitors are added to the medium. Using human HUT-78 T-cell cultures, we now find direct in vitro evidence of opiate-induced or opiate-promoted mutagenesis via several assay systems. First, with microgel electrophoresis (MGE), we observed graded, dose-dependent, significant increases (P < .0001) in the frequency of comet tails of fragmented DNA when cells were treated with morphine alone (5 × 10^?9M up to 10^?7M) or when co-treated with the more potent mutagen, ethylmethanesulfonate (EMS). There were also dose-dependent increases in the lengths and densities of the comet tails observed. These findings were confirmed by a series of MGE experiments in which several days of morphine exposure preceded a 2-hr pulse of EMS. Second, mutant frequency (MF) assays also indicated significant opiate effects. These studies required separate assessment of cloning efficiencies and the frequencies of TG-resistant, HPRT-deficient mutant clones under four test conditions: no treatment, morphine alone for 4 days, morphine plus EMS, and EMS alone. Prior to the treatment phase, aminopterin was used to eliminate background HPRT mutations. The medium was changed after the treatment phase, the cells were allowed to express mutant pheno-types, and then TG was added and resistant mutant clones counted after 16 days. The background MF level for controls and for cells treated with EMS alone were negligible at 5.12 × 10^?8 and 7.25 × 10^?8, respectively. In the cells treated with morphine alone or morphine plus EMS, MF levels increased very significantly (P < .001) by >100-fold to 5.1 × 10^?6 and 7.0 × 10^?6, respectively. Cloning efficiency also decreased significantly with both morphine-exposed conditions. Preliminary analysis with the single strand conformational polymorphism (SSCP) procedure following 6-thioguanine (TG) selection, also confirmed the occurrence of Exon 3 mutants of the HPRT gene in cells exposed to morphine plus EMS. It appears that brief EMS exposure can be repaired, whereas, if morphine exposure persists through one or more cell cycles, direct or indirect mutagenesis is initiated. © 1994 Wiley-Liss, Inc.     

Dengue virus entry into liver (HepG2) cells is independent of hsp90 and hsp70

Recently, several stress-related proteins including GRP78, hsp70, and hsp90 have been implicated as dengue virus receptors in various cell types, with hsp90/70 being implicated as a receptor complex in monocytes and macrophages, while GRP78 has been implicated as a liver cell expressed dengue virus receptor. To assess whether the hsp90/70 complex plays a role in the internalization of the dengue viruses into liver cells, we undertook infection inhibition studies with lipopolysaccharide and antibodies directed against both hsp70 and hsp90, individually and in combination. No inhibition of any dengue serotype was seen in the presence of lipopolysaccharide or antibodies directed against either hsp70 or hsp90 either singly or in combination. A moderate inhibition of dengue virus serotype 2 entry into liver cells was observed in the presence of antibodies directed against GRP78. These results confirm a proposed role for GRP78 as a dengue virus serotype 2 receptor protein and suggest that the recently identified hsp90/70 complex does not play a role in dengue virus internalization into liver cells.     

sGRP78在恶性肿瘤中的研究进展

随着对葡萄糖调节蛋白78（GRP78）的不断研究,发现GRP78不仅存在于内质网,还存在于多种肿瘤细胞的表面,而细胞表面GRP78(sGRP78)作为一种多功能蛋白质,能够与组织型纤溶酶原激活剂（t-PA）、纤溶蛋白酶原（PG）、α2-巨球蛋白、PI3K的调节亚基p85等多种配体结合,参与多种恶性肿瘤细胞的生长、侵袭、转移及耐药等多种生物学特性的调节。越来越多的研究表明,sGRP78可作为恶性肿瘤的生物标志物和治疗靶点。全文就sGRP78在恶性肿瘤中的研究进展作一综述。     

Activity of artemether and OZ78 against triclabendazole-resistant Fasciola hepatica

Triclabendazole is the drug of choice against Fasciola hepatica infections in humans and animals. However, parasite resistance against triclabendazole is spreading in the veterinary field, and there are no drugs of comparable activity currently available for the treatment and control of fascioliasis. We investigated the efficacy of single oral doses of artemether and OZ78 against adult triclabendazole-resistant F. hepatica harboured in rats, and compared the results with triclabendazole administered at two different doses. Single oral doses of 100 mg/kg OZ78 and 200 mg/kg artemether resulted in worm burden reductions of 100%. Whereas a single 10 mg/kg dose of triclabendazole achieved a worm burden reduction of only 4.0%, a five-fold higher dose yielded a significant worm burden reduction of 60.9%. However, the lower dose of triclabendazole administered to rats harbouring a triclabendazole-sensitive F. hepatica isolate resulted in a worm burden reduction of 95.3%. Our findings confirm that artemether and OZ78 possess good fasciocidal properties, even against a triclabendazole-resistant F. hepatica isolate, and hence these drugs might become useful in areas where triclabendazole resistance is common.     

AT1受体自身抗体对糖尿病肾病大鼠肾脏内质网应激通路相关分子葡萄糖调节蛋白78和CCAAT/增强子结合蛋白同源蛋白表达的影响

目的 探讨AT1受体自身抗体(AT1-AA)对DN大鼠肾脏内质网应激(ERS)通路相关分子葡萄糖调节蛋白78(GRP78)和CCAAT/增强子结合蛋白同源蛋白(CHOP)表达的影响. 方法 制备DN大鼠模型,ELISA检测血清AT1-AA,根据ELISA结果随机选择AT1-AA阳性和阴性DN大鼠纳入DN组(n=12),同时设立正常对照组(NC,n=6).电镜观察肾脏超微结构变化;TUNEL法检测肾脏细胞凋亡;RT-PCR测定大鼠肾组织GRP78和CHOP mRNA水平;Western blot分析肾组织中GRP78和CHOP蛋白的表达量. 结果 DN组肾脏细胞凋亡率较NC组升高,其中,AT1-AA阳性大鼠凋亡率高于AT1-AA阴性大鼠[(20.05±1.71)％vs(13.24±4.93)％](P＜0.01).与NC组相比,DN组肾组织GRP78、CHOP蛋白和mRNA水平均上调;进一步比较发现,AT1-AA阳性DN大鼠GRP78,CHOP蛋白及mRNA水平升高较AT1-AA阴性DN大鼠更明显. 结论 AT1-AA可能通过诱导DN大鼠肾脏ERS反应,并经ERS相关的CHOP凋亡信号通路而促进肾脏细胞凋亡,加重肾脏损害.