他莫昔芬(tamoxifen,TAM)作为雌激素受体阳性(estrogen receptor,ER+)乳腺癌的一线化疗药物使大多数患者受益,但原发性和继发性耐药问题严重影响临床治疗效果。深入研究ER+乳腺癌TAM耐药机制,改善治疗效果是当前亟待解决的问题。抑癌因子NDRG2(N-myc downstream regulated gene 2,NDRG2)在肿瘤发生发展中发挥重要作用,但是否参与ER+乳腺癌TAM耐药尚不清楚。本研究旨在探明NDRG2在ER+乳腺癌TAM耐药中发挥的作用和机制。通过RT-PCR与免疫印迹分析对比TAM敏感型和耐药型ER+乳腺癌细胞发现,NDRG2的mRNA转录水平和蛋白质翻译水平在TAM耐药细胞中表达显著下调,且与耐药能力负相关(P<0.001);CCK-8细胞毒性实验和软琼脂克隆形成实验证实,在耐药细胞中过表达NDRG2可显著降低TAM药物半抑制浓度IC 50和软琼脂克隆形成率(P<0.001),逆转耐药表型。分子机制上,X-box结合蛋白1(X-box binding protein 1,XBP1)mRNA剪切实验与内质网相关降解(endoplasmic-reticulum associated degradation,ERAD)报告蛋白的结果显示,过表达NDRG2可增强耐药细胞中剪切型XBP1s mRNA转录与ERAD报告蛋白CD3ε-YFP表达(P<0.001),引发耐药细胞内质网强应激反应;免疫印迹检测结果显示,过表达NDRG2可显著提高耐药细胞中内质网应激感受器肌醇需要激酶1α(inositol requiring enzyme 1,IRE1α)的磷酸化水平及其下游因子,例如内质网EIP辅助因子(endoplasmic reticulum-localized DnaJ 4,ERdj4)、PKR蛋白激酶的细胞抑制剂(cellular Inhibitor of the PKR protein kinase,P58 IPK)、α甘露糖苷酶样应激蛋白(er degradation enhancingαmannosidase likeprotein,EDEM)和蛋白质二硫键异构酶家族A成员5(protein disulfide isomerase family a member 5,PDIA5)的表达水平(P<0.001)。小鼠异种移植瘤研究进一步证实,在耐药细胞中过表达NDRG2可增强TAM治疗效果,显著抑制耐药移植瘤生长(P<0.001)。以上研究结果表明,通过提高耐药细胞中NDRG2表达,
The protein,N-myc downstream-regulated gene 2(NDRG2),a tumor suppressor,is significantly decreased or absent in many types of cancer.There is a significant negative correlation between the levels of NDRG2 and the development and progression of cancer tumor recurrence and tumor invasion,in different cancers.In contrast,the in vitro and in vivo overexpression of the NDRG2 protein decreases the proliferation,growth,adhesion and migration of many types of cancer cells.The in vitro overexpression of NDRG2 increases the efficacy of certain anticancer drugs in specific types of cancer cells.We hypothesize that the delivery of the mRNA of the NDRG2 protein,encapsulated by lipid nanoparticles,could represent a potential treatment of metastatic and drug-resistant cancers.This would be accomplished using a self-amplifying mRNA that encodes the NDRG2 protein and an RNA-dependent-RNA polymerase,obtained from an in vitrotranscribed(IVT)mRNA.The IVT mRNA would be encapsulated in a lipid nanoformulation.The efficacy of the nanoformulation would be determined in cultured cancer cells and if the results are positive,nude mice transplanted with either drug-resistant or metastatic drug-resistant cancer cells,would be treated with the nanoformulation and monitored for efficacy and adverse effects.If the appropriate preclinical studies indicate this formulation is efficacious and safe,it is possible it could be evaluated in clinical trials.
Sandra E.ReznikAmit K.TiwariVivek ChavdaCharles R.Ashby Jr.
Background:Renal ischemia–reperfusion(R-I/R)injury is the most prevalent cause of acute kidney injury,with high mortality and poor prognosis.However,the underlying pathological mechanisms are not yet fully understood.Therefore,this study aimed to investigate the role of N‐myc downstream‐regulated gene 2(Ndrg2)in R-I/R injury.Methods:We examined the expression of Ndrg2 in the kidney under normal physiological conditions and after R-I/R injury by immunofluorescence staining,real-time polymerase chain reaction,and western blotting.We then detected R-I/R injury in Ndrg2-deficient(Ndrg2^(-/-))mice and wild type(Ndrg2+/+)littermates in vivo,and detected oxygen and glucose deprivation and reperfusion(OGD-R)injury in HK-2 cells.We further conducted transcriptomic sequencing to investigate the role of Ndrg2 in R-I/R injury and detected levels of oxidative stress and mitochondrial damage by dihydroethidium staining,biochemical assays,and western blot.Finally,we measured the levels of mitophagy in Ndrg2+/+and Ndrg2^(-/-)mice after R-I/R injury or HK-2 cells in OGD-R injury.Results:Ndrg2 was primarily expressed in renal proximal tubules and its expression was significantly decreased 24 h after R-I/R injury.Ndrg2^(-/-)mice exhibited significantly attenuated R-I/R injury compared to Ndrg2+/+mice.Transcriptomics profiling showed that Ndrg2 deficiency induced perturbations of multiple signaling pathways,downregulated inflammatory responses and oxidative stress,and increased autophagy following R-I/R injury.Further studies revealed that Ndrg2 deficiency reduced oxidative stress and mitochondrial damage.Notably,Ndrg2 deficiency significantly activated phosphatase and tensin homologue on chromosome ten-induced putative kinase 1(PINK1)/Parkin-mediated mitophagy.The downregulation of NDRG2 expression significantly increased cell viability after OGD-R injury,increased the expression of heme oxygenase-1,decreased the expression of nicotinamide adenine dinucleotide phosphate oxidase 4,and increased the expression of the PINK1/Park
Min LiuJianwen ChenMiao SunLixia ZhangYao YuWeidong MiYulong MaGuyan Wang
