鼻咽癌组织中高表达的GSDME对TME免疫浸润和患者预后的影响

目的：探究细胞焦亡相关基因GSDME在鼻咽癌组织中的表达及其对肿瘤微环境（TME）和患者预后的影响。方法：从TCGA数据库获得548例鼻咽癌患者的基因表达数据及临床资料,运用R语言对GSDME进行差异表达分析、GO-KEGG富集分析;从人类蛋白质图谱（HPA）数据库中获取GSDME蛋白在鼻咽癌组织和相应癌旁组织中的表达数据,通过STRING数据库探究与GSDME相互作用的蛋白质网络;应用ssGSEA算法分析GSDME在鼻咽癌组织中的表达及其与24种免疫细胞浸润的相关性,运用Spearman法分析GSDME与免疫检查点分子的相关性,通过TISIDB数据库分析GSDME表达与细胞因子基因表达的相关性。单因素和多因素COX回归分析筛选预后风险因素,基于风险因素GSDME绘制预后预测列线图和校准图,根据GSDME的表达水平对鼻咽癌患者进行生存分析和风险分析。qPCR法验证中国人鼻咽癌组织中GSDME及4种趋化因子mRNA的表达。结果：数据库数据分析显示,与癌旁组织相比,鼻咽癌组织中GSDME呈高表达（P<0.01）,GO-KEGG富集分析显示GSDME参与免疫反应、细胞焦亡相关信号通路,GSDME表达与免疫细胞浸润、细胞因子及免疫检查点分子表达等相关,qPCR检查结果验证了中国人鼻咽癌组织中GSDME呈高表达。GSDME表达量、N分期和M分期是鼻咽癌患者预后的风险因素,基于风险因素建立的列线图和校正图能较好地预测鼻咽癌患者OS,鼻咽癌组织中GSDME高表达的患者预后差。结论：GSDME在鼻咽癌癌组织中呈高表达且是患者预后风险因素,基于GSDME建立的列线图预测预后效能良好;GSDME高表达与TME免疫浸润有关,其可能是免疫治疗的潜在靶点。     

新补骨脂异黄酮通过caspase-3/GSDME通路诱导肝细胞癌Huh-7细胞焦亡

目的：探讨新补骨脂异黄酮（NBIF）对肝细胞癌（HCC）Huh-7细胞焦亡的影响及其分子机制。方法：体外培养Huh-7细胞,用CCK-8法检测不同浓度的NBIF处理48 h时对细胞存活率的影响,光学显微镜下观察NBIF处理后Huh-7细胞的形态变化,乳酸脱氢酶（LDH）释放实验检测细胞的LDH释放量,WB实验检测细胞中GSDME、caspase-3的蛋白水平变化。采用si RNA干扰Huh-7细胞中caspase-3、GSDME表达后,CCK-8法检测NBIF处理对细胞存活率的影响,WB实验检测GSDME蛋白表达水平,观察NBIF处理对细胞形态的影响,并检测细胞LDH释放量。结果：60μmol/L以上的NBIF均能显著抑制Huh-7细胞的增殖（均P<0.01）,光学显微镜下观察到NBIF处理后的细胞出现肿胀、吐泡现象,且LDH释放增加（P<0.01）;WB实验结果表明,NBIF能够激活caspase-3蛋白并切割GSDME蛋白,增加GSDME-N的表达（均P<0.01）。干扰caspase-3、GSDME表达后,NBIF对细胞的抑制作用减弱（均P<0.01）,G...     

Acute cadmium exposure induces GSDME-mediated pyroptosis in triple-negative breast cancer cells through ROS generation and NLRP3 inflammasome pathway activation

Cadmium (Cd) exposure can exert an impact on carcinogenicity of breast cancer, however, the mechanism is not fully understood in triple-negative breast cancer (TNBC). We performed a TNBC MDA-MB-231 cell model and assessed the toxic effect of Cd exposure (0, 10, 20, 50, 60, 80 μM). Cd reduced cell viability in a time- and dose-dependent manner, followed by cell cycle arrest in S phase with alterations of cyclin 1A1, cyclin 1D1 and CDK2. Lactate dehydrogenase (LDH) release, apoptosis and pyroptosis were increased, which were relieved by z-VAD. Elevated ROS and NLRP3, caspase-1, IL-1β and IL-18 were detected, which was attenuated by N-acetylcysteine. Increased bax and decreased caspase-8, caspase-9 and caspase-3 were found. gasdermin E (GSDME) was activated with cleavage of GSDME-NT, which was retarded by z-VAD. Additionally, p38 MAPK signaling pathway was activated. Our data demonstrate GSDME-activated pyroptosis in Cd toxicity, implying a potential impact on TNBC.     

雷公藤甲素对GSDME依赖性肝癌细胞焦亡的影响

目的:探究雷公藤甲素对肝癌细胞Bel-7402焦亡的影响及其分子机制。方法:体外培养肝癌细胞Bel-7402,CCK-8检测不同浓度的雷公藤甲素(2.5、5.0、10.0μg/mL)对肝癌细胞活力影响;PI/DAPI染色观察细胞焦亡形态变化;ELISA试剂盒检测IL-1β和IL-18释放量;Western blotting检测Caspase-3、GSDME、IL-1β和IL-18表达;转染GSDME siRNA 24 h后PI/DAPI染色检测细胞焦亡形态变化。结果:CCK-8检测发现,随着雷公藤甲素浓度增加,Bel-7402细胞活力明显降低(P<0.01);给予雷公藤甲素(10.0μg/mL)能够明显促进细胞焦亡,升高细胞上清液中IL-1β和IL-18含量,促进Caspase-3、GSDME-N、IL-1β和IL-18表达(P<0.05或P<0.01);siGSDME能够明显抑制细胞焦亡形态变化,降低细胞焦亡数目。结论:雷公藤甲素能够促进GSDME依赖性细胞焦亡,抑制肝癌细胞增殖。     

GSDME引起的细胞焦亡在肿瘤治疗中的研究进展

细胞焦亡是由Gasdermin家族蛋白介导的程序性死亡方式,并伴随炎症及免疫反应。Gasdermin含有一个功能性的N端结构域、C端结构域,以及 一个被激活的炎性半胱天冬酶（caspases）识别和裂解的连接子,此结构可在膜上穿孔,导致膜肿胀和破裂。Gasdermin家族蛋白均可诱导细胞发生焦亡,但只 有Gasdermin E(GSDME)启动子甲基化存在于多数肿瘤中,使其有可能成为新的预测肿瘤和化疗药物选择的因子,并且其诱导的焦亡常与自然杀伤细胞有密切联 系。除此以外,GSDME引起的细胞焦亡也与其他类型疾病有密切联系,如嵌合抗原受体(CAR)T细胞治疗癌症的疗效和糖尿病性肾病（DKD）等。本文将对GSDME蛋 白在各种疾病中的研究现状进行综述,以期对临床肿瘤及其他类型疾病的治疗有所帮助。     

基于细胞焦亡机制的肿瘤免疫治疗新策略

细胞焦亡是近年来发现的一种全新的调节性细胞死亡形式,在肿瘤免疫中发挥重要作用。肿瘤发生发展进程中的细 胞焦亡包含免疫细胞焦亡（ICP）和肿瘤细胞焦亡（CCP）两种类型,并可能发挥促癌或抑癌作用。Caspase-1介导的经典途径和 caspase-4/5/11介导的非经典途径均可以介导ICP的发生,ICP参与的肿瘤免疫中,炎症小体和细胞因子IL-18、IL-1β发挥了重要 作用。Caspase-3和颗粒酶B切割并激活GSDME途径介导了CCP的发生,在肿瘤发生过程中自发且持久的CCP促进肿瘤生长, 而在化疗等过程中GSDME激活介导的CCP则具有显著的抑癌作用。细胞焦亡的发生可以刺激肿瘤微环境中的炎症反应,提高 肿瘤细胞的免疫原性,促进抗肿瘤免疫的发生。因此,细胞焦亡及其引发的炎症反应在肿瘤免疫中发挥重要作用,对该领域的研 究可能为抗肿瘤免疫治疗提供新思路。     

奥沙利铂促进过表达GSDME的结直肠癌Lovo细胞发生细胞焦亡

目的：观察过表达Gasdermin蛋白E（gasdermin E, GSDME）基因的结直肠癌Lovo细胞经奥沙利铂处理后发生细胞焦 亡的现象。方法：通过qPCR检测结直肠癌细胞株Lovo、正常结直肠上皮细胞HCOEPIC中GSDME基因的表达水平，构建过表 达野生型和突变型GSDME的GSDME-WT和GSDME-D270A重组质粒，包装为相应慢病毒后感染Lovo细胞构建GSDME稳定 高表达细胞株，Western blotting检测WT、 D270A和空载体组中GSDME表达水平。用不同浓度奥沙利铂（0、 4、 8、16、32、64 μg/ ml）作用于WT、 D270A组Lovo 细胞和 HCOEPIC 细胞，显微镜下观察细胞的形态变化。结果：GSDME在HCOEPIC 细胞中表 达显著高于Lovo细胞（P<0.01）。成功构建GSDME-WT和GSDME-D270A高表达重组质粒和相应的Lovo细胞株，与空载体组 相比，WT和D270A组Lovo细胞中GSDME表达水平明显升高（均P<0.05）。镜下观察发现， 64 μg/ml奥沙利铂处理各组细胞9和 12 h时，WT组的Lovo细胞和HCOEPIC细胞体积逐渐增大并向一侧“吹泡”，表现出明显的细胞焦亡现象，细胞焦亡率显著高于 无奥沙利铂处理的对照组[Lovo 细胞：（7.405±1.010） % vs（3.441±0.401）% ， P<0.05；HCOEPIC 细胞： (7.203±1.020)% vs (4.201±0.302)%， P<0.05]。结论：奥沙利铂促进过表达GSDME基因的结直肠癌Lovo细胞发生细胞焦亡。     

RETRACTED: Alpinumisoflavone triggers GSDME-dependent pyroptosis in esophageal squamous cell carcinomas

Esophageal squamous cell carcinoma (ESCC) presents a common human malignancy in the digestive system. We aimed to explore the critical effects of alpinumisoflavone (AIF) on ESCC in vitro and in vivo. The cell counting kit-8 assay was used to determine cell viability. Colony formation assay was employed to examine the effect of AIF on the long-term growth of ESCC cells. Cell apoptosis was determined by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Cell morphologies were observed by light microscopy. The enzyme-linked immunosorbent assay was performed to examine the lactate dehydrogenase release from AIF-treated cells. Immunofluorescent labeling was utilized to examine AIF-induced GSDME expression. Western blot was employed to determine the expression levels of the associated proteins. Immunohistochemistry was performed to determine the localization and expression of the associated proteins in mice tumor tissues. AIF inhibited ESCC cell viability and suppressed cell growth in a dose- and time-dependent fashion. Results showed that AIF promoted apoptosis in ESCC cells. Meanwhile, our results also showed that AIF triggered pyroptotic cell death in ESCC, which was mediated by gasdermin E (GSDME) cleavage. In addition, our experiments provided experimental evidence that AIF-induced GSDME cleavage was dependent on caspase-3 activation. Moreover, the inhibition of GSDSE by knockdown was able to switch the form of cell death from pyroptosis to apoptosis. Furthermore, the results from the xenograft animal model also supported our findings in vitro that AIF was able to promote GSDME-mediated pyroptotic cell death in ESCC. AIF inhibited ESCC growth in vitro and in vivo by triggering GSDME-mediated pyroptotic cell death, which is dependent on caspase-3 activation.     

白藜芦醇改善高糖引起肾小球系膜细胞损伤的作用研究

目的:探讨白藜芦醇（RSV）对体外高糖刺激引起大鼠肾小球系膜细胞（RGMCs）损伤的影响及其潜在分子保护机制。方法:取处于对数生长期的RGMCs,分为甘露醇高渗透压对照组（OC）、正常对照组（NG）、高糖组（HG）以及高糖RSV处理组,分别处理24、48 h。试剂盒检测不同时间各组细胞上清液中LDH的水平;Western 印迹法检测不同时间各组凋亡相关B淋巴细胞瘤-2基因（Bcl-2）、Bcl-2相关X蛋白（Bax）、半胱氨酸天冬氨酸蛋白酶（Caspase）-1、Caspase-1活性片段（p20）、Gasdermin E（GSDME）、GSDME活性片段（GSDME-N）、Caspase-3、Caspase-3活性片段（p17）、炎性相关细胞因子白细胞介素-1β（IL-1β）及其活化形式（mIL-1β）的表达;ELISA检测不同时间各组细胞培养上清IL-1β的水平。结果:LDH结果显示,RSV可降低高糖刺激48 h后细胞的死亡率（F=31.48,P<0.05）。Western 印迹法结果显示,仅在RSV处理24 h后,Bax表达较HG组显著降低,同时伴随Bcl-2表达升高（F=143.1,P<0.05）;RSV处理24 h后HG组p20、mIL-1β表达降低（F=26.74、25.76,均P<0.05）;此外,RSV处理48 h后p17、GSDME-N的表达较HG组明显下降（F=25.84、18.49,均P<0.05）。ELISA结果显示,与HG组相比,RSV处理48 h后上清中IL-1β的水平显著降低（F=80.22,P<0.01）。上述指标在OC组及NG组间的变化均无统计学意义（均P＞0.05）。结论:RSV可通过减少炎症因子的产生及抑制细胞早期凋亡、晚期焦亡过程来改善高糖引起的RGMCs损伤。     

Miltirone induces cell death in hepatocellular carcinoma cell through GSDME-dependent pyroptosis

Pyroptosis is a form of programmed cell death, and recently described as a new molecular mechanism of chemotherapy drugs in the treatment of tumors. Miltirone, a derivative of phenanthrene-quinone isolated from the root of Salvia miltiorrhiza Bunge, has been shown to possess anti-cancer activities. Here, we found that miltirone inhibited the cell viability of either HepG2 or Hepa1-6 cells, and induced the proteolytic cleavage of gasdermin E (GSDME) in each hepatocellular carcinoma (HCC) cell line, with concomitant cleavage of caspase 3. Knocking out GSDME switched miltirone-induced cell death from pyroptosis to apoptosis. Additionally, the induction effects of miltirone on GSDME-dependent pyroptosis were attenuated by siRNA-mediated caspase three silencing and the specific caspase three inhibitor Z-DEVD-FMK, respectively. Miltirone effectively elicited intracellular accumulation of reactive oxygen species (ROS), and suppressed phosphorylation of mitogen-activated and extracellular signal-regulated kinase (MEK) and extracellular regulated protein kinases 1/2 (ERK1/2) for pyroptosis induction. Moreover, miltirone significantly inhibited tumor growth and induced pyroptosis in the Hepa1-6 mouse HCC syngeneic model. These results provide a new insight that miltirone is a potential therapeutic agent for the treatment of HCC via GSDME-dependent pyroptosis.