Clinical implications of forkhead box M1, cyclooxygenase-2, and glucose-regulated protein 78 in breast invasive ductal carcinoma

BACKGROUND Breast infiltrating ductal carcinoma(BIDC)represents the largest heterotypic tumor group,and an in-depth understanding of the pathogenesis of BIDC is key to improving its prognosis.AIM To analyze the expression profiles and clinical implications of forkhead box M1(FOXM1),cyclooxygenase-2(COX-2),and glucose-regulated protein 78(GRP78)in BIDC.METHODS A total of 65 BIDC patients and 70 healthy controls who presented to our hospital between August 2019 and May 2021 were selected for analysis.The peripheral blood FOXM1,COX-2,and GRP78 levels in both groups were measured and the association between their expression profiles in BIDC was examined.Additionally,we investigated the diagnostic value of FOXM1,COX-2,and GRP78 in patients with BIDC and their correlations with clinicopathological features.Furthermore,BIDC patients were followed for 1 year to identify factors influencing patient prognosis.RESULTS The levels of FOXM1,COX-2,and GRP78 were significantly higher in BIDC patients compared to healthy controls(P<0.05),and a positive correlation was observed among them(P<0.05).Receiver operating characteristic analysis demonstrated that FOXM1,COX-2,and GRP78 had excellent diagnostic value in predicting the occurrence of BIDC(P<0.05).Subsequently,we found significant differences in FOXM1,COX-2,and GRP78 levels among patients with different histological grades and metastasis statuses(with vs without)(P<0.05).Cox analysis revealed that FOXM1,COX-2,GRP78,increased histological grade,and the presence of tumor metastasis were independent risk factors for prognostic death in BIDC(P<0.001).CONCLUSION FOXM1,COX-2,and GRP78 exhibit abnormally high expression in BIDC,promoting malignant tumor development and closely correlating with prognosis.These findings hold significant research implications for the future diagnosis and treatment of BIDC.

Prostaglandin E1 protects hepatocytes against endoplasmic reticulum stress-induced apoptosis via protein kinase A-dependent induction of glucose-regulated protein 78 expression

AIM To investigate the protective effect of prostaglandin E1(PGE1) against endoplasmic reticulum(ER) stressinduced hepatocyte apoptosis, and to explore its underlying mechanisms.METHODS Thapsigargin(TG) was used to induce ER stress in the human hepatic cell line L02 and hepatocarcinomaderived cell line Hep G2. To evaluate the effects of PGE1 on TG-induced apoptosis, PGE1 was used an hour prior to TG treatment. Activation of unfolded protein response signaling pathways were detected by western blotting and quantitative real-time RTPCR. Apoptotic index and cell viability of L02 cells and Hep G2 cells were determined with flow cytometry and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium] assay. RESULTS Pretreatment with 1 μmol/L PGE1 protected against TG-induced apoptosis in both L02 cells and Hep G2 cells. PGE1 enhanced the TG-induced expression of C/EBP homologous protein(CHOP), glucose-regulated protein(GRP) 78 and spliced X box-binding protein 1 at 6 h. However, it attenuated their expressions after 24 h. PGE1 alone induced protein and m RNA expressions of GRP78; PGE1 also induced protein expression of DNA damage-inducible gene 34 and inhibited the expressions of phospho-PKR-like ER kinase, phosphoeukaryotic initiation factor 2α and CHOP. Treatment with protein kinase A(PKA)-inhibitor H89 or KT5720 blocked PGE1-induced up-regulation of GRP78. Further, the cytoprotective effect of PGE1 on hepatocytes was not observed after blockade of GRP78 expression by H89 or small interfering RNA specifically targeted against human GRP78.CONCLUSION Our study demonstrates that PGE1 protects against ER stress-induced hepatocyte apoptosis via PKA pathwaydependent induction of GRP78 expression.

Role of Glucose-regulated Protein 78 in Early Brain Injury after Experimental Subarachnoid Hemorrhage in Rats

Early brain injury（EBI） plays a key role in the pathogenesis of subarachnoid hemorrhage（SAH）. This study investigated the role of glucose-regulated protein 78（GRP78） in EBI after SAH. Male Sprague-Dawley rats（n=108） weighing 260±40 g were divided into control, sham-operated, and operated groups. Blood was injected into the prechiasmatic cistern of rats in the operated group. Neurological scores, ultrastructures of neurons, apoptosis, and GRP78 expression in the hippocampus were examined using Garcia scoring system, transmission electron microscopy, terminal deoxynucleotidyl transferase-mediated d UTP nick-end labelling, and Western blotting at 1, 6, 12, 24, 48, and 72 h after SAH, respectively. The results showed that neurological scores were significantly decreased in the operated group as compared with those in control and sham-operated groups at 12, 24, 48, and 72 h. Metachromatin, chromatin pyknosis at the edge, endoplasmic reticulum swelling, and invagination of nuclear membrane were observed at 24 h in the operated group, indicating the early morphological changes of apoptosis. The number of apoptotic cells was significantly increased in the operated group as compared with that in control and sham-operated groups at 6, 12, 24, 48, and 72 h. The GRP78 protein expression levels in the operated group were significantly elevated at all time points and reached the peak at 12 h. GRP78 expression was positively associated with apoptosis cells and negatively with neurological scores. In conclusion, EBI was demonstrated to occur after SAH and GRP78 was involved in the development of EBI after SAH.

心衰合剂对大鼠心肌细胞肥大模型内质网应激因子Grp78、Caspase-12表达的影响

目的观察心衰合剂对大鼠体外心肌细胞肥大模型细胞内质网应激因子Grp78及Caspase-12表达的影响。方法培养大鼠乳鼠原代心肌细胞,通过血管紧张素Ⅱ(AngⅡ)建立肥大心肌细胞模型(模型组),分别用心衰合剂(10%含药血清组)卡托普利(卡托普利组)干预细胞。Western-blot测定胞内Grp78及Caspase-12表达的蛋白表达水平。结果模型组心肌细胞Grp78、Caspase-12蛋白表达水平明显上调(P<0.01);与模型组比较,10%含药血清组及卡托普利组可以显著下调Grp78、Caspase-12蛋白表达水平(P<0.01),2组组间比较差异无统计学意义(P>0.05)。结论心衰合剂可下调内质网应激相关蛋白Grp78、Caspase-12表达水平,提示心衰合剂通过减轻内质网应激防止细胞发生凋亡,保护心肌细胞。

GRP78在肿瘤治疗及预后中的作用

肿瘤细胞为了适应低糖、低氧和低钙等慢性应激反应,可诱导葡萄糖调节蛋白78（glucose regulated protein,GRP78）的表达。GRP78作为内质网主要分子伴侣之一,在蛋白质的折叠和转运过程及内质网应激反应中发挥重要作用。目前研究表明,GRP78还可增强肿瘤细胞的增殖、侵袭能力以及对抗癌药物的耐受性,

人食管鳞状细胞癌组织中GRP78的表达及其与肿瘤生物学行为的关系

目的:探讨葡萄糖调节蛋白78(glucose-regulated proteins78,GRP78)在食管鳞状细胞癌组织及正常鳞状上皮组织中的表达情况,并分析其与临床病理特征的关系。方法:取自2007年10月至2008年11月在山东大学附属济南中心医院胸外科手术切除的新鲜食管鳞状细胞癌标本59例及距癌组织5cm以上的手术远端切缘的正常食管鳞状上皮组织20例,RT-PCR检测GRP78mRNA的表达,应用Western blotting检测GRP78蛋白的表达,并从mRNA和蛋白水平分析GRP78的表达与患者性别、肿瘤长度、浸润深度、分化程度、病理分期及淋巴转移等临床病理特征之间的关系。结果:食管鳞状细胞癌组织中GRP78的表达在mRNA和蛋白水平均明显高于食管正常鳞状上皮组织(均P<0.01)。GRP78在食管鳞状细胞癌组织中的高表达与食管鳞状细胞癌的浸润深度(P<0.05或P<0.01)、分化程度(P<0.05或P<0.01)、病理分期(pTMN,P<0.01)及淋巴转移密切相关(P<0.01),而与患者性别及肿瘤长径无关(P>0.05)。结论:GRP78参与了人类食管鳞状细胞癌的发生、发展,表达水平随着食管鳞状细胞癌组织恶性程度的增高而增高,其可作为衡量食管鳞状细胞癌恶性程度的一种有潜在价值的分子标志物。

GRP78 inhibits macrophage adhesion via SR-A

Class A scavenger receptor （SR-A） plays an important role in macrophage adhesion. However, the underlying mechanism remains unclear. We previously found that 78 kDa glucose-regulated protein （GRP78） inhibited SR- A-mediated ligand internalization into macrophage by binding to SR-A. The aim of the study was to investigate whether GRP78 could regulate SR-A-mediated cell adhesion. We demonstrated that GRP78 bound directly to SR-A by fluorescence resonance energy transfer （FRET） assay. Overexpression of GRP78 inhibited macrophage adhesion via SR-A. These results suggest that GRP78 may act as an inhibitor of macrophage adhesion via SR-A.

A new polymorphism in the GRP78 is not associated with HBV invasion

AIM:To examine the association between -86 bp(T>A) in the glucose-regulated protein 78 gene(GRP78) and hepatitis B virus(HBV) invasion.METHODS:DNA was genotyped for the single-nucleotide polymorphism by polymerase chain reaction followed by sequencing in a sample of 382 unrelated HBV carriers and a total of 350 sex-and age-matched healthy controls.Serological markers for HBV infection were determined by enzyme-linked immunosorbent as-say kits or clinical chemistry testing.RESULTS:The distributions of allelotype and genotype in cases were not significantly different from those in controls.In addition,our fi ndings suggested that neither alanine aminotransferase/hepatitis B e antigen nor HBV-DNA were associated with the allele/genotype variation in HBV infected individuals.CONCLUSION:-86 bp T>A polymorphism in GRP78 gene is not related to the clinical risk and acute exacerbation of HBV invasion.

Rational design of a turn-on fluorescent probe for visualization of GRP78 protein in tumor models

Fluorescence image for accurate tumor label still faces challenges in cancer detection and diagnostics.Emerging evidence is indicating that glucose-regulated protein 78(GRP78), a stress-inducible protein chaperone, is a great potential biomarker and therapeutic target for cancer. However, currently available probe for image tumor based on GRP78 has not been reported, owning to no obvious strategy in probe design towards this protein. In this paper, a hairpin-shaped peptidyl probe(pep FAM) conjugated with a 5-FAM fluorophore and a dabcyl quencher at both ends was developed, respectively. The probe was designed by performing a traditional fluorescence resonance energy transfer mechanism and employing a GRP78 specifically-binding peptide. Furthermore, the probe was used to specifically image cancer cells,and accurately image xenograft tumors in mice models. The novel fluorescent probe is expected to be a useful tool for the diagnostics of cancer.

不同组织学类型食管癌GRP78的表达及其临床病理特征的相关性研究

食管癌（Esophageal cancinoma,EC）是由食管粘膜上皮和腺体发生的恶性肿瘤,也是最常见的消化管恶性肿瘤之一。为了早发现、早诊断、早治疗、已达到延长生存期和改善预后的目的,探索一种经济实用、特异性高、敏感性强、适用于人群普查的早期诊断食管癌的方法,成为医务工作者关注的焦点。近期研究发现,葡萄糖调节蛋白78（glucose regulated protein 78kD,GRP78）与肿瘤的发生、发展、耐药、

Valproate reduces retinal ganglion cell apoptosis in rats after optic nerve crush

The retinal ganglion cells of the optic nerve have a limited capacity for self-repair after injury.Valproate is a histone deacetylase inhibitor and multitarget drug,which has been demonstrated to protect retinal neurons.In this study,we established rat models of optic nerve-crush injury and injected valproate into the vitreous cavity immediately after modeling.We evaluated changes in the ultrastructure morphology of the endoplasmic reticulum of retinal ganglion cells over time via transmission electron microscope.Immunohistochemistry and western blot assay revealed that valproate upregulated the expression of the endoplasmic reticulum stress marker glucose-regulated protein 78 and downregulated the expression of transcription factor C/EBP homologous protein,phosphorylated eukaryotic translation initiation factor 2α,and caspase-12 in the endoplasmic reticulum of retinal ganglion cells.These findings suggest that valproate reduces apoptosis of retinal ganglion cells in the rat after optic nerve-crush injury by attenuating phosphorylated eukaryotic translation initiation factor 2α-C/EBP homologous protein signaling and caspase-12 activation during endoplasmic reticulum stress.These findings represent a newly discovered mechanism that regulates how valproate protects neurons.

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 Kdvalue of0.11 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.

针刺对戊四唑诱发惊厥大鼠海马葡萄糖调节蛋白78和C/EBP同源蛋白表达的影响

目的:观察针刺对惊厥大鼠海马神经元内葡萄糖调节蛋白78(Grp 78)和C/EBP同源蛋白(CHOP)蛋白表达的影响,探讨针刺抗惊厥的可能机制。方法:SD大鼠随机分为正常对照组(n=6)、模型组(n=18)、针刺组(n=18)。腹腔注射戊四唑(50mg/kg)复制惊厥大鼠模型。针刺组立即针刺"百会""大椎"30min。各组分别于造模后2、12、48h采用免疫组化法观察各组大鼠海马CA 1区Grp 78和CHOP蛋白表达情况。结果:模型组大鼠在惊厥发作2、12h海马CA 1区Grp 78蛋白表达与正常组比较明显增强(P<0.01);针刺组大鼠在惊厥发作12h和48h与模型组比较Grp 78蛋白表达显著增加(P<0.01,P<0.05)。在惊厥发作各个时段与正常组比较,模型组大鼠海马CA 1区CHOP蛋白表达明显上调(P<0.01);针刺组海马CA 1区CHOP蛋白阳性表达在惊厥发作各个时段与模型组比较明显减少(P<0.05,P<0.01)。结论:针刺能明显调节惊厥大鼠海马神经元Grp 78蛋白和CHOP蛋白的表达,从而起到保护惊厥性脑损伤作用。

二甲双胍对人口腔癌KB细胞增殖及凋亡的影响

目的研究二甲双胍(Metformin)对人口腔癌KB细胞增殖及凋亡的影响,以期为口腔癌的治疗开辟新的路径。方法 MTT法检测不同浓度(1.25、2.5、5、10、20 mmol/L)二甲双胍处理口腔癌KB细胞24、48、72 h后对细胞增殖的影响;分别用0.25、0.5、1 mmol/L的二甲双胍处理KB细胞,8 d后观察其对集落克隆形成的影响;5 mmol/L二甲双胍处理细胞24 h,线粒体膜电位检测试剂盒(JC-1)检测线粒体膜电位的变化;PI单染检测二甲双胍对KB细胞凋亡的影响;Western blot检测二甲双胍(5 mmol/L)处理KB细胞不同时间(0、6、16、24 h)GRP78以及caspase-3的表达。结果二甲双胍处理细胞后,对KB细胞的增殖有明显的抑制作用,并且呈药物浓度和时间依赖性。5 mmol/L的二甲双胍处理KB细胞24、48、72 h的存活率为68.0%、36.9%、14.5%,但24 h细胞凋亡率仅为11.99%,将浓度加大后凋亡率为24.11%,高于对照组;二甲双胍抑制KB细胞集落克隆的形成;JC-1荧光检测,红绿荧光的相对比例降低,提示膜电位下降;二甲双胍刺激口腔癌KB细胞GRP78的表达先上调后有减弱趋势,并且能诱导Caspase-3的激活。结论二甲双胍能显著抑制KB细胞的增殖并且诱导细胞凋亡,其机制与线粒体凋亡途径的激活以及过度的内质网应激有关。

GPER agonist G1 suppresses neuronal apoptosis mediated by endoplasmic reticulum stress after cerebral ischemia/reperfusion injury

Studies have confirmed a strong association between activation of the endoplasmic reticulum stress pathway and cerebral ischemia/reperfusion(I/R) injury.In this study,three key proteins in the endoplasmic reticulum stress pathway(glucose-regulated protein 78,caspase-12,and C/EBP homologous protein) were selected to examine the potential mechanism of endoplasmic reticulum stress in the neuroprotective effect of G protein-coupled estrogen receptor.Female Sprague-Dawley rats received ovariectomy(OVX),and then cerebral I/R rat models(OVX+ I/R) were established by middle cerebral artery occlusion.Immediately after I/R,rat models were injected with 100 μg/kg E2(OVX + I/R +E2),or 100 μg/kg G protein-coupled estrogen receptor agonist G1(OVX + I/R + G1) in the lateral ventricle.Longa scoring was used to detect neurobehavioral changes in each group.Infarct volumes were measured by 2,3,5-triphenyltetrazolium chloride staining.Morphological changes in neurons were observed by Nissl staining.Terminal dexynucleotidyl transferase-mediated nick end-labeling staining revealed that compared with the OVX + I/R group,neurological function was remarkably improved,infarct volume was reduced,number of normal Nissl bodies was dramatically increased,and number of apoptotic neurons in the hippocampus was decreased after E2 and G1 intervention.To detect the expression and distribution of endoplasmic reticulum stress-related proteins in the endoplasmic reticulum,caspase-12 distribution and expression were detected by immunofluorescence,and mRNA and protein levels of glucose-regulated protein 78,caspase-12,and C/EBP homologous protein were determined by polymerase chain reaction and western blot assay.The results showed that compared with the OVX+ I/R group,E2 and G1 treatment obviously decreased mRNA and protein expression levels of glucose-regulated protein 78,C/EBP homologous protein,and caspase-12.However,the G protein-coupled estrogen receptor antagonist G15(OVX + I/R + E2 + G15) could eliminate the effect of E2 on cerebral I/R injury.These results confirm that E2 and G protein-coupled estrogen receptor can inhibit the expression of endoplasmic reticulum stress-related proteins and neuronal apoptosis in the hippocampus,thereby improving dysfunction caused by cerebral I/R injury.Every experimental protocol was approved by the Institutional Ethics Review Board at the First Affiliated Hospital of Shihezi University School of Medicine,China(approval No.SHZ A2017-171) on February 27,2017.

ER stress and ER stress-induced apoptosis are activated in gastric SMCs in diabetic rats

AIM: To investigate the gastric muscle injury caused by endoplasmic reticulum (ER) stress in rats with diabetic gastroparesis.

Mitomycin C induces apoptosis in human epidural scar fibroblasts after surgical decompression for spinal cord injury

Numerous studies have shown that topical application of mitomycin C after surgical decompression effectively reduces scar adhesion. However, the underlying mechanisms remain unclear. In this study, we investigated the effect of mitomycin C on the proliferation and apoptosis of human epidural scar fibroblasts. Human epidural scar fibroblasts were treated with various concentrations of mitomycin C （1, 5, 10, 20, 40 μg/mL） for 12, 24 and 48 hours. Mitomycin C suppressed the growth of these cells in a dose- and time-dependent manner. Mitomycin C upregulated the expression levels of Fas, DR4, DR5, cleaved caspase-8/9, Bax, Bim and cleaved caspase-3 proteins, and it downregulated Bcl-2 and Bcl-xL expression. In addition, inhibitors of caspase-8 and caspase-9 （Z-IETD-FMK and Z-LEHD-FMK, respectively） did not fully inhibit mitomycin C-induced apoptosis. Furthermore, mitomycin C induced endoplasmic reticulum stress by increasing the expression of glucose-regulated protein 78, CAAT/enhancer-binding protein homologous protein （CHOP） and caspase 4 in a dose-dependent manner. Salubrinal significantly inhibited the mitomycin C-induced cell viability loss and apoptosis, and these effects were accompanied by a reduction in CHOP expression. Our results support the hypothesis that mitomycin C induces human epidural scar fibroblast apoptosis, at least in part, via the endoplasmic reticulum stress pathway.

Resveratrol reduces brain injury after subarachnoid hemorrhage by inhibiting oxidative stress and endoplasmic reticulum stress

Previous studies have shown that resveratrol,a bioactive substance found in many plants,can reduce early brain injury after subarachnoid hemorrhage,but how it acts is still unclear.This study explored the mechanism using the experimental subarachnoid hemorrhage rat model established by injecting autologous blood into the cerebellomedullary cistern.Rat models were treated with an intraperitoneal injection of 60 mg/kg resveratrol 2,6,24 and 46 hours after injury.At 48 hours after injury,their neurological function was assessed using a modified Garcia score.Brain edema was measured by the wet-dry method.Neuronal apoptosis in the prefrontal cortex was detected by terminal deoxyribonucleotidyl transferase-mediated biotin-16-dUTP nick-end labeling assay.Levels of reactive oxygen species and malondialdehyde in the prefrontal cortex were determined by colorimetry.CHOP,glucose-regulated protein 78,nuclear factor-erythroid2-related factor 2 and heme oxygenase-1 mRNA expression levels in the prefrontal cortex were measured by reverse transcription polymerase chain reaction.Tumor necrosis factor-alpha content in the prefrontal cortex was detected by enzyme linked immunosorbent assay.Immunohistochemical staining was used to detect the number of positive cells of nuclear factor-erythroid 2-related factor 2,heme oxygenase 1,glucose-regulated protein 78,CHOP and glial fibrillary acidic protein.Western blot assay was utilized to analyze the expression levels of nuclear factor-erythroid 2-related factor 2,heme oxygenase 1,glucose-regulated protein 78 and CHOP protein expression levels in the prefrontal cortex.The results showed that resveratrol treatment markedly alleviated neurological deficits and brain edema in experimental subarachnoid hemorrhage rats,and reduced neuronal apoptosis in the prefrontal cortex.Resveratrol reduced the levels of reactive oxygen species and malondialdehyde,and increased the expression of nuclear factor-erythroid 2-related factor 2,heme oxygenase-1 mRNA and protein in the prefrontal cortex.Resveratrol decreased glucose-regulated protein 78,CHOP mRNA and protein expression and tumor necrosis factor-alpha level.It also activated astrocytes.The results suggest that resveratrol exerted neuroprotective effect on subarachnoid hemorrhage by reducing oxidative damage,endoplasmic reticulum stress and neuroinflammation.The study was approved by the Animals Ethics Committee of Shandong University,China on February 22,2016(approval No.LL-201602022).

Artificial intelligence and machine learning could support drug development for hepatitis A virus internal ribosomal entry sites

Hepatitis A virus(HAV)infection is still an important health issue worldwide.Although several effective HAV vaccines are available,it is difficult to perform universal vaccination in certain countries.Therefore,it may be better to develop antivirals against HAV for the prevention of severe hepatitis A.We found that several drugs potentially inhibit HAV internal ribosomal entry site-dependent translation and HAV replication.Artificial intelligence and machine learning could also support screening of anti-HAV drugs,using drug repositioning and drug rescue approaches.