GRP78表达下调可降低肺腺癌细胞对VP-16的耐药

背景与目的 GRP78(Glucose-regulated protein 78)是GRPs家族的成员之一,其高表达与某些肿瘤细胞化疗耐药相关.本研究旨在探讨在BAPTA-AM作用下肺腺癌SPCA-1细胞GRP78表达水平的变化及GRP78表达下调与细胞对VP-16耐药的相关性.方法 将SPCA-1细胞随机分为BAPTA-AM处理组、A23187处理组及对照组.采用RT-PCR和免疫荧光细胞化学方法 分别检测各组细胞中GRP78在核酸和蛋白水平的表达.采用流式细胞仪测定细胞在化疗药物足叶乙甙(VP-16)作用下的细胞生存率.结果 与对照组和A23187处理组相比,BAPTA-AM处理组细胞GRP78在核酸和蛋白水平的表达均明显降低,而其在VP-16作用下的细胞凋亡率则明显升高(细胞凋亡率:BAPTA-AM处理组为10.84±0.86;对照组为6.85±0.20;A23187处理组为4.95±0.19;P＜0.01).结论 BAPTA-AM能够抑制SPCA-1细胞GRP78的表达,GRP78表达下调能够提高SPCA-1细胞对VP-16的敏感性.因此,采用化学药物或反义RNA等方法抑制GRP78的表达可能成为未来治疗肺癌的新手段.     

Hsp90表达上调与肺癌细胞化疗耐药的相关性研究

背景与目的热休克蛋白(heatshockprotein,Hsp)90是Hsps家族中的重要成员,其高表达与肿瘤的发生、发展及化疗耐药密切相关。本研究旨在探讨替普瑞酮(geranylgeranylacetone,GGA)作用下人肺癌细胞SPCA-1及H446中Hsp90的表达水平变化及与肺癌细胞对顺铂化疗耐药的相关性。方法将细胞分成实验组与对照组,分别用含有不同浓度(0μM,10μM,50μM,100μM,500μM,1,000μM)的诱导剂GGA处理6h。采用免疫荧光细胞化学及West-ernblot方法检测各组细胞中Hsp90在蛋白水平的表达;应用MTT法测定细胞在化疗药物顺铂作用下生存率,并分析Hsp90的表达在两种肺癌细胞对顺铂耐药中的作用。结果 SPCA-1及H446实验组细胞中Hsp90的表达水平均明显高于相应的对照组细胞,且与GGA具有一定的浓度依赖性。MTT显示两种实验组细胞对顺铂的生存率均明显高于相应的对照组细胞,亦呈一定的GGA浓度依赖性。结论 GGA可以诱导人肺癌细胞SPCA-1及H446中的Hsp90的表达上调,且其表达水平都与GGA具有一定的浓度依赖性。Hsp90高表达的细胞对顺铂的生存率明显高于低表达的细胞,表明Hsp90的表达上调与肺癌细胞对顺铂的耐药有一定的相关性。     

P型铜转运ATP酶(ATP7B)在肺腺癌细胞株A549中的表达与顺铂耐药的关系

背景与目的 顺铂作为肺癌的基础化疗药物,顺铂耐药是导致肺癌患者化疗失败的重要原因.本实验通过检测P型铜转运ATP酶在肺腺癌细胞A549不同水平耐药株中的表达,以评估ATP7B与A549细胞顺铂耐药的关系.方法采用逐步增加顺铂剂量的方法,诱导出3株不同水平耐顺铂A549细胞株A549/DDP0.5、A549/DDP1.0、A549/DDP2.0,MTT方法检测各组别细胞对顺铂敏感性,应用RT-PCR及Western Blot方法分别检测各组别细胞的ATP7B的mRNA及蛋白表达水平,分析A549细胞顺铂敏感性与ATP7B表达水平的关系.结果相对于亲本A549细胞,3组耐药细胞的顺铂耐药指数分别达到了1.7、3.2、5.2(P＜0.001),与此相对应ATP7B的mRNA表达水平分别达到了亲本A549细胞的1.6、4.9、10.1倍(P＜0.001),同样地ATP7B的蛋白表达水平也呈现出与顺铂耐药性相平行的递增性高表达.结论肺腺癌细胞A549的顺铂耐药与细胞ATP7B高表达有关,后者的高表达有可能促成了A549细胞的获得性顺铂耐药.     

非小细胞肺癌体外化疗药物敏感性与GRP78表达的相关性

背景和目的 糖调节蛋白78（GRP78）在乳腺癌细胞中表达增高并与其对化疗药物的耐药性有关，而非小细胞肺癌（NSCLC）中GRP78的表达与其对化疗药物的耐药性是否相关，研究较少。本研究拟探讨NSCLC对8种化疗药物的体外敏感性与GRP78表达的相关性。方法 采用四噻唑蓝（MTT）法对52例手术切除的新鲜NSCLC组织进行8种化疗药物体外敏感性检测；采用免疫组织化学方法检测其GRP78的表达水平。采用Spearman相关分析对NSCLC的耐药性与GRP78表达的相关性进行分析。结果 52例NSCLC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％、90．38％，其中14例表现为完全耐药。GRP78的表达水平随着肺癌恶性程度的提高而增加，且其高表达与肺癌细胞对VP-16、ADM、VCR和TPT的耐药具有相关性（P〈0．05）。结论 MTT法体外化疗药物敏感性实验对NSCLC的治疗具有一定的指导作用，GRP78对判定NSCLC的恶性程度及其耐药性具有一定的参考意义。     

GRP78在不同亚型宫颈癌细胞株中表达情况及对顺铂敏感性影响

目的：研究不同亚型宫颈癌细胞株中葡萄糖调节蛋白78（glucose regulated protein 78,GRP78）表达情况，以及GRP78表达水平对宫颈癌细胞顺铂敏感性的影响。方法：采用免疫组织化学法检测人乳头瘤病毒（human papillomavirus，HPV）16（+）SiHa、HPV18（+）HeLa以及HPV（-）C33a宫颈癌细胞中的GRP78表达，MMT、Western blot法检测以上3种细胞在不同浓度顺铂、衣霉素+顺铂处理后的细胞抑制率及GRP78表达水平，采用单因素方差分析，分析相同浓度下单用顺铂、衣霉素+顺铂联合处理后3种细胞的细胞抑制率及GRP78蛋白表达的差异。结果：SiHa、HeLa细胞GRP78阳性表达水平明显高于C33a细胞，SiHa细胞GRP78阳性表达水平最高（P＜0.01）。随着顺铂浓度增加，3种宫颈癌细胞抑制率增加呈浓度依赖性，且HeLa细胞抑制率最高（P＜0.05）。与单独顺铂组相比，衣霉素联合顺铂药物处理组中HeLa及SiHa细胞抑制率显著升高（P＜0.05），并且GRP78表达水平也明显升高（P＜0.01），且各细胞株中GRP78表达水平均随顺铂浓度增加而增加。结论：GRP78表达水平与宫颈癌细胞亚型有关，SiHa、HeLa细胞对顺铂敏感性高于C33a细胞。衣霉素导致GRP78明显高表达，增加了SiHa、HeLa细胞对顺铂的敏感性。     

SOX4对非小细胞肺癌细胞A549的顺铂耐药作用的影响

背景与目的 肺癌是最严重的恶性肿瘤之一,其中非小细胞肺癌发病率在肺癌中居首位.部分患者对顺铂耐受是导致化疗失败的主要原因.SOX4是转录调节因子,在多种肿瘤的发生发展过程中发挥着重要的作用,通过调控β-catenin的表达对Wnt信号通路进行调控.本研究旨在探讨SOX4对非小细胞肺癌细胞A549的顺铂耐药作用的影响.方法 体外诱导法建立顺铂耐药的肺癌细胞株A549/DDP,CCK8法检测A549及A549/DDP细胞对顺铂的耐药性.绘制A549与A549/DDP细胞生长曲线.Western blot检测耐药细胞株中SOX4的蛋白表达水平.CCK8法检测敲减SOX4后耐药细胞株A549/DDP对顺铂耐药性.实时定量PCR和免疫印迹法检测敲减SOX4后A549/DPP细胞中β-catenin和Survivin蛋白的表达.结果 成功构建非小细胞肺癌顺铂耐药细胞株A549/DDP,其耐药性是亲本细胞的13.7倍,差异有统计学意义(P<0.001).生长曲线显示耐药细胞株与亲本细胞增殖速度没有统计学差异(P<0.05);与A549细胞相比,耐药细胞A549/DDP细胞中SOX4的表达显著增高(P<0.001).敲减SOX4后,A549/DDP细胞的耐药性显著降低;敲减SOX4后,A549/DDP细胞中β-catenin和Survivin的mRNA和蛋白表达水平显著降低.结论 SOX4的表达水平影响非小细胞肺癌细胞A549对顺铂的耐药性.     

RNA干扰GRP75表达改善人肺腺癌细胞对顺铂耐药性

背景与目的 GRP75(glucose regulated protein75)属于热休克蛋白(hot shock protein,HSP)家族,是一种主要位于线粒体的分子伴侣,在某些耐药的肿瘤细胞中高表达。本研究通过慢病毒介导的RNA干扰技术沉默GRP75基因的表达,观察下调GRP75的表达对肿瘤细胞耐药性的影响并探讨GRP75在肿瘤细胞耐药机制中的作用。方法以顺铂为诱导药物,人肺腺癌细胞系A549为诱导对象,采用逐步增加剂量与大剂量冲击相结合的方法,诱导建立耐顺铂细胞株A549/CDDP。分别将包裹GRP75-shRNA和不含干扰序列的慢病毒转染A549和A549/CDDP细胞,在荧光显微镜下观察各组细胞转染率,应用M比色法检测干扰前后各组细胞对顺铂的敏感性,应用Western blot检测干扰前后各组细胞GRP75、p53、bcl-2的表达。结果各组细胞感染效率均在90%以上,转染后A549/CDDP和A549细胞中GRP75的表达均明显下调(P<0.05)。转染前后A549/CDDP细胞对顺铂的耐药指数分别为21.52和4.14。转染后A549/CDDP细胞内p53的表达上调(P<0.05),bcl-2表达下调(P<0.05)。结论 GRP75是A549细胞对顺铂耐药机制的相关蛋白之一,其在耐药机制中的作用与其对p53和bcl-2的调控有关。     

基于TCGA数据库筛选肺腺癌顺铂耐药的分子标志物及功能验证

目的 探究在肺腺癌顺铂耐药过程中起关键调控作用的相关基因。方法 利用生物信息学方法在TCGA数据库和GDSC数据库下载肺腺癌患者顺铂敏感组与耐药组之间的差异表达基因,对差异基因进行GO功能分析和KEGG通路富集分析,构建蛋白质互作网络,并进行层次聚类分析,筛选得到关键基因并利用实时荧光定量PCR和ELISA法在细胞水平进行验证。通过siRNA沉默A549/DDP细胞中CXCL10基因的表达并检测其对顺铂的敏感度。结果 筛选得到178个差异表达基因,经过聚类分析最终获得肺腺癌顺铂耐药的关键基因CXCL9、CXCL10、NKX2-1以及SFTPA1。暂时选择CXCL10进行后续验证及功能实验。实时荧光定量PCR结果显示CXCL10在A549/DDP细胞中的mRNA表达量显著高于A549细胞（P<0.001）,A549/DDP细胞上清液中CXCL10蛋白的表达量高于A549细胞,均与生物信息学预测一致。MTT结果显示沉默CXCL10表达后,A549/DDP细胞对DDP的敏感度增加。结论 CXCL10是调控肺腺癌顺铂耐药的关键基因,下调CXCL10表达可成为逆转肺腺癌顺铂耐药的潜在靶点。     

BAG-1蛋白在衣霉素诱导人肺癌A549细胞内质网应激及提高顺铂敏感性中的作用

目的:体外观察低剂量衣霉素对人肺腺癌A549细胞内质网应激(endoplasmic reticulum stress,ERS)的诱导作用,以及此过程中Bcl-2结合抗凋亡基因1(Bcl-2 associated athanogene 1,BAG-1)表达的变化;同时,观察衣霉素作用后A549细胞对顺铂敏感性的变化以及此过程中BAG-1蛋白表达的变化.方法:采用蛋白质印迹法检测低剂量衣霉素作用A549细胞后ERS标志性蛋白GRP78及抗凋亡蛋白BAG-1的表达变化;MTT法测定衣霉素作用后顺铂对A549细胞的半数抑制浓度(half inhibitory concentration,IC50)变化;FCM法检测低剂量衣霉素作用后A549细胞对顺铂的敏感性变化;蛋白质印迹法检测衣霉素作用前后顺铂对A549细胞中BAG-1和procaspase-12蛋白表达的影响.结果:1.25 μg/mL衣霉素作用A549细胞8h后,能诱导细胞发生ERS,即ERS标志性蛋白GRP78表达上调,同时BAG-1蛋白表达也明显上调(P均＜0.05).衣霉素诱导A549细胞ERS能增加细胞对顺铂的敏感性(P＜0.05).细胞发生ERS前,1.25、2.5和5 μg/mL 3个质量浓度的顺铂均上调BAG-1蛋白的表达(P均＜ 0.05),但2.5和5μg/mL顺铂诱导BAG -1蛋白上调量均小于1.25 μg/mL组(P＜0.05);细胞发生ERS后,与单纯ERS未给予顺铂干预的细胞组比,1.25、2.5和5μg/mL 3个质量浓度的顺铂均下调BAG -1蛋白的表达(P均＜0.05),且下调量与顺铂浓度呈正比.2.5和5μg/mL顺铂能通过ERS途径诱导细胞发生凋亡,在此过程中BAG-1和procaspase-12蛋白表达均下调(P均＜0.05).结论:BAG-1蛋白可能是ERS和顺铂诱导肺癌细胞凋亡的一个重要调控因子之一,且ERS凋亡途径可能是顺铂诱导肺癌细胞凋亡的重要途径之一.     

人肺腺癌细胞耐顺铂株A549/CDDP的比较蛋白质组学研究

背景与目的 化疗是肺癌综合治疗的重要部分,但是肿瘤细胞耐药常导致化疗失败.本研究采用蛋白组学方法研究人肺腺癌细胞A549对顺铂产生耐药性前后的蛋白表达差异,筛选有价值的靶位.方法 以顺铂为诱导药物,人肺腺癌细胞系A549为诱导对象,采用逐步增加剂量与大剂量冲击相结合的方法,诱导建立耐顺铂细胞株A549/CDDP.对A549与A549/CDDP进行蛋白组学研究,即利用双向凝胶电泳(2-DE)分离两组总蛋白后,通过图像分析寻找表达差异的蛋白,对其进行MALDI-TOF质谱分析.结果 比较两者2-DE图谱,得到差异蛋白点82个;对其中6个差异蛋白点进行肽质量指纹图分析,鉴定出葡萄糖调节蛋白75、核糖体蛋白S4、线粒体F1-ATP合酶β亚单位、免疫球蛋白重链可变区;以上差异蛋白均在耐药株中过表达,而在对照组细胞中表达下调或不表达.结论 所鉴定的差异蛋白将为阐明肺癌细胞对顺铂耐药性产生的机制提供线索,也为筛选具体潜在价值的肺癌化疗靶位提供理论依据.     

Retrovirus-mediated transduction of a short hairpin RNA gene for GRP78 fails to downregulate GRP78 expression but leads to cisplatin sensitization in HeLa cells

Glucose-regulated protein 78 (GRP78) is expressed abundantly in various types of cancer cells and is believed to contribute to chemotherapeutic resistance. In this study, we investigated the effect of a continuous approach for the expression of a short hairpin RNA (shRNA) targeted to GRP78 with retrovirus transduction on the sensitivity to the anticancer drugs VP-16 and cisplatin. The reduction of GRP78 expression failed, and the expression of GRP94 and P5 chaperon mRNA increased; this increase was associated with a mild activation of the unfolded protein response in HeLa cells, which were stably transduced with GRP78 shRNA gene. The transduced cells exhibited similar sensitivity to VP-16-induced cell death when compared to control GFP shRNA gene-transduced cells. However, sensitivity to cisplatin-induced cell death was higher in GRP78 shRNA gene-transduced cells compared to control cells. These results demonstrate that the continuous or prolonged approach targeting GRP78 confers sensitization of HeLa cells to cisplatin independently of the down-regulation of GRP78 expression. The role of the unfolded protein response in sensitization to cisplatin is discussed.     

AKT inhibition mitigates GRP78 (glucose‐regulated protein) expression and contribution to chemoresistance in endometrial cancers

Overexpression of the unfolded protein response master regulator GRP78 is associated with poor prognosis and therapeutic resistance in numerous human cancers, yet its role in endometrial cancers (EC) is undefined. To better understand the contribution of GRP78 to EC, we examined its expression levels in EC patient samples and EC cell lines. We demonstrate that GRP78 overexpression occurs more frequently in EC tissues compared with that found in normal endometrium, and that GRP78 expression occurs in most EC cell lines examined. Functional analysis demonstrated that GRP78 is inducible by cisplatin in EC cells, and siRNA knockdown of GRP78 augments chemotherapy‐mediated cell death. Examination of AKT and GRP78 expression demonstrated that inhibition of AKT activity by MK2206 blocks GRP78 expression in EC cells. SiRNA studies also revealed that knockdown of GRP78 reduces but does not abrogate AKT activity, demonstrating that GRP78 is required for optimal AKT activity. In the presence of MK2206, siRNA knockdown of GRP78 does not augment AKT mediated survival in response to cisplatin treatment, suggesting that GRP78′s antiapoptosis functions are part of the AKT survival pathway. Targeted therapies that reduce GRP78 expression or activity in cancers may serve to increase the effectiveness of current therapies for EC patients.     

GRP78 is overexpressed in glioblastomas and regulates glioma cell growth and apoptosis

We characterized the expression and function of the endoplasmic reticulum protein GRP78 in glial tumors. GRP78 is highly expressed in glioblastomas but not in oligodendrogliomas, and its expression is inversely correlated with median patient survival. Overexpression of GRP78 in glioma cells decreases caspase 7 activation and renders the cells resistant to etoposide- and cisplatin-induced apoptosis, whereas silencing of GRP78 decreases cell growth and sensitizes glioma cells to etoposide, cisplatin, and gamma-radiation. Thus, GRP78 contributes to the increased apoptosis resistance and growth of glioma cells and may provide a target for enhancing the therapeutic responsiveness of these tumors.     

Akt is the downstream target of GRP78 in mediating cisplatin resistance in ER stress-tolerant human lung cancer cells

Cisplatin [cis-diaminodichloroplatinum (II) (CDDP)] is the cornerstone of lung cancer chemotherapy. However, its efficacy is limited due to the development of drug resistance in cancer cells. This study was designed to uncover the mechanisms under CDDP resistance in lung cancer cells involving endoplasmic reticulum (ER) stress tolerance-induced and GRP78-dependant Akt activation. In this study we established ER stress-tolerant (ERST) human lung cancer lines H460et and A549et. We found that the ERST Lung cancer cells are resistant to CDDP treatment. We further showed that, compared to the parental cell lines, H460et and A549et show significantly increased GRP78 and phospho(p)-Akt levels. And phosphorylation of Akt, which can be regulated by GRP78, is essential to the ERST-associated CDDP resistance. Our findings identify a new mechanism of regulating Akt activity and a new mechanism through which CDDP resistance is formed in lung cancer cells.     

Glucose-regulated protein 78 mediates hormone-independent prostate cancer progression and metastasis through maspin and COX-2 expression

Glucose-regulated protein 78 (GRP78) plays an essential role in embryonic development and in the progression and therapeutic resistance of many cancers. However, little is known about the function of GRP78 in hormone-independent prostate cancer. Here, we found that the expression levels of GRP78 were higher in PC-3 cells than in DU-145 cells. When the expression of GRP78 was silenced using a GRP78-specific small interfering RNA in PC-3 cells, the growth rate and adhesive ability were reduced. Cell migration was dramatically decreased in GRP78-depleted cells. Dissection of the involved signal pathways revealed that maspin expression was upregulated after silencing GRP78 expression. The upregulation of maspin and downregulation of COX-2 may cause the decrease in cell proliferation and migration observed after silencing GRP78 expression. Silencing GRP78 expression may suppress the proliferation, adhesion, and migration of prostate cancer cells via maspin and COX-2 regulation.     

Overexpression of endoplasmic reticulum molecular chaperone GRP94 and GRP78 in human lung cancer tissues and its significance

BACKGROUND: To investigate the relationship between the expression of glucose-regulated protein94 (GRP94) and GRP78 at the level of mRNA and protein in vivo and in human lung cancer. METHODS: RT-PCR, real-time PCR, immunohistochemistry and/or Western blot were used in 54 cases of lung cancer and corresponding normal lung tissue. RESULTS: The expression pattern of GRP94 and GRP78 was similar. There was a significant overexpression of GRP94 and GRP78 at both mRNA and protein levels in cancer tissues as compared to normal tissues. The relative levels of GRP94 and GRP78 mRNA evaluated by RT-PCR in cancer and normal lung tissue were: GRP94: 3.48+/-2.06 versus 2.01+/-1.83; GRP78: 3.64+/-1.87 versus 2.21+/-1.54; by real-time PCR were: GRP94: 2.89+/-0.64 versus 1.12+/-0.54; GRP78: 2.56+/-0.82 versus 0.96+/-0.42. The relative level of GRP94 and GRP78 protein by Western blot in cancer and normal lung tissue were: GRP94: 3.46+/-1.72 versus 1.81+/-0.92; GRP78: 4.84+/-2.55 versus 1.91+/-1.15, indicating an approximate 2-fold and a 3-fold increase in GRP94 and GRP78 protein in cancer tissue as compared with normal tissue. Immunohistochemistry result for GRP94 and GRP78 in cancer and normal tissue was similar, that is: a stronger stain was observed in cancer tissue (main intensity of staining ++ to +++) compared to normal tissue (main intensity of staining + to ++). All the difference for GRP94 and GRP78 between the two tissues were significant (p<0.05). Furthermore, the overexpression of GRP94 and GRP78 in the cancer tissue correlated with grade of differentiation and stage of tumors. There was stronger expression in poorly differentiated tumors than in well-moderately differentiated tumors (p<0.05). There was also stronger expression in stage III than in stages I and II tumors (p<0.05). No statistically significant differences were found among various pathologic types of tumors. Correlation analysis showed that there is a positive correlation between GRP94 and GRP78. CONCLUSION: The expression pattern of GRP94 and GRP78 was similar in human lung cancer. They both were related with the differentiation and progression of the cancer. The expression at mRNA and protein level may be valuable in evaluating the grade of differentiation and clinical stage of human lung cancer.     

二甲双胍逆转人卵巢癌细胞株对顺铂耐药的实验研究

目的 探讨二甲双胍对人卵巢癌耐顺铂细胞株SKOV3／DDP的耐药逆转作用及机制。方法 将人卵巢癌细胞株SKOV3细胞设为亲本组,耐顺铂细胞株SKOV3／DDP细胞分为空白对照组、顺铂组、二甲双胍组和联合用药组（顺铂+二甲双胍组）;采用CCK-8法检测不同浓度二甲双胍对SKOV3／DDP细胞的作用,RT-PCR和Western blotting检测各组细胞中内质网应激相关蛋白的表达。结果 不同浓度二甲双胍作用24h后,对SKOV3细胞和SKOV3／DDP细胞均有抑制作用,且呈浓度依赖性（P＜0.05）;当二甲双胍＞5 mmol／L时,其对SKOV3／DDP细胞的增殖抑制率高于SKOV3细胞（P＜0.05）。顺铂对SKOV3细胞和SKOV3／DDP细胞的半数抑制浓度（IC50）分别为15.25 μg／ml和118.68 μg／ml,SKOV3／DDP的耐药倍数为7.78。联合用药组SKOV3／DDP细胞IC50为 73.45 μg／ml,耐药倍数为4.81。二甲双胍对SKOV3／DDP细胞对顺铂耐药性的逆转倍数为1.62。亲本组中GRP78 mRNA和蛋白的相对表达水平分别为0.38±0.02和0.24±0.04;顺铂组、二甲双胍组和联合用药组的GRP78 mRNA的相对表达水平分别为0.93±0.02、0.68±0.02、0.49±0.07,与空白对照组的0.83±0.04比较,联合用药组明显降低,其次为二甲双胍组,顺铂组升高,差异均有统计学意义（P＜0.05）;GRP78蛋白在各组中表达与其mRNA表达趋势一致,差异均有统计学意义（P＜0.05）。顺铂组、二甲双胍组、联合用药组CHOP蛋白的表达量分别为0.42±0.03、0.69±0.03、0.84±0.01,均高于空白对照组的0.39±0.05,差异均有统计学意义（P＜0.05）。结论 二甲双胍对人卵巢癌顺铂耐药细胞的耐药性有调节作用,其可能作用机制为通过降低GRP78表达,提高CHOP蛋白表达的内质网应激途径促进细胞凋亡来降低耐药细胞对顺铂的耐药性。     

Involvement of GRP78 in the Resistance of Ovarian Carcinoma Cells to Paclitaxel

Background: Glucose regulated protein 78 (GRP78) is a type of molecular chaperone. It is a possible candidateprotein that contributes to development of drug resistance. We first examined the involvement of GRP78 inchemotherapy-resistance in human ovarian cancer cell. Materials and Methods: The expression of GRP78mRNA and protein were examined by RT-PCR and western blotting, respectively, in human ovarian cancer cellsline (HO-8910). Sensitivity of HO-8910 to paclitaxel was determined with methyl thiazolyl tetrazolium (MTT).Suppression of GRP78 expression was performed using specific small-interfering RNA (siRNA) in HO-8910cells, and cell apoptosis was assessed by flow cytometry. Statistical analysis was performed using the SPSS 15.0statistical package. Results: HO-8910 cells, with high basal levels of GRP78, exhibited low sensitivity to paclitaxel.The mRNA and protein levels of GRP78 were dramatically decreased at 24h, 48h and 72h after transfection andthe sensitivity to paclitaxel was increased when the GRP78 gene was disturbed by specific siRNA transfection.Conclusions: The results suggested that high GRP78 expression might be one of the molecular mechanismscausing resistance to paclitaxel, and therefore siRNA of GRP78 may be useful in tumor-specific gene therapyfor ovarian cancer.     

Glucose-regulated protein 78 as a novel effector of BRCA1 for inhibiting stress-induced apoptosis

The tumor suppressor BRCA1 is mutated in a high percentage of familial breast and ovarian cancer, but our understanding of its mechanisms of action remains incomplete. We report here that glucose-regulated protein (GRP)-78, a critical regulator of the unfolded protein response (UPR), is a novel downstream target of BRCA1. We showed that overexpression of wild-type BRCA1 suppressed the expression of GRP78, whereas expression of mutant BRCA1 gene or targeted inhibition of endogenous BRCA1 using small-interfering RNA (siRNA) enhanced GRP78 expression. Knockdown of BRCA1 also led to induction of other components of UPR, such as GRP94 and CHOP. Consistent with a role of BRCA1 knockdown in mediating cell survival, forced expression of GRP78 stimulated cell proliferation and prevented apoptosis, including that induced by endoplasmic reticulum stress and chemotherapy, in ovarian OVCAR-3 and breast MCF-7 cancer cells. Overexpression of wild-type BRCA1 could increase the apoptosis of GRP78-overexpressing cells. Conversely, knockdown GRP78 by siRNA sensitized ovarian and breast cancer cells to apoptosis. This effect was reduced when the expression of BRCA1 was simultaneously knockdown by siRNA, indicating that BRCA1 also negatively regulates GRP78-mediated cell survival and resistance to apoptosis.