论著

RAB25沉默抑制结直肠癌细胞铁死亡的作用研究

展开
  • 上海交通大学医学院附属同仁医院肿瘤科,上海 200336

收稿日期: 2022-03-17

  网络出版日期: 2023-04-23

基金资助

国家自然科学基金(81672335);上海市卫生和计划生育委员会科研课题(20174Y0231)

Effect of RAB25 knockdown on ferroptosis of colorectal cancer cells

Expand
  • Department of Oncology, Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200336, China

Received date: 2022-03-17

  Online published: 2023-04-23

摘要

目的:探讨沉默RAS相关结合蛋白25(ras-associated binding protein 25, RAB25)在结直肠癌(colorectal cancer,CRC)细胞铁死亡中的作用。方法:利用GEPIA(Gene Expression Profiling Interactive Analysis)数据库分析RAB25的表达水平及与铁死亡关键基因表达之间的关联。在CRC细胞系HCT116上构建慢病毒介导的RAB25沉默细胞株(shRAB25),应用定量实时聚合酶链反应(quantitative real-time polymerase chain reaction, qRT-PCR)和蛋白印迹法检测RAB25的表达情况。应用细胞增殖与毒性检测法(CCK8)检测不同浓度的(0~20 μmol/L)铁死亡诱导剂erastin对沉默RAB25后细胞活力的影响;利用荧光显微镜和透射电镜分别观察erastin对沉默RAB25后的细胞形态和线粒体结构的影响;使用C11-BODIPY染色和流式细胞仪检测erastin对沉默RAB25后的细胞脂膜过氧化水平的影响。检测erastin与西妥昔单抗对沉默RAB25后的细胞活力的联合作用。结果:RAB25在CRC中表达升高(P<0.01);RAB25表达与铁死亡关键基因表达明显相关。当erastin≥10 μmol/L时,与阴性对照组(空载慢病毒感染阴性组)相比,RAB25沉默组抑制了铁死亡导致的细胞杀伤(P<0.000 1),细胞形态和线粒体结构更清晰完整;流式细胞术检测结果提示,细胞脂膜过氧化水平明显下降(P<0.000 1)。RAB25表达使erastin和西妥昔单抗对HCT116细胞的联合杀伤作用明显增强。结果:沉默RAB25可抑制erastin诱导的CRC细胞铁死亡;RAB25可协同铁死亡诱导剂erastin诱导铁死亡,增强西妥昔单抗的疗效。

本文引用格式

李佳曦, 汪锦江, 俞立萍, 袁英, 乔光磊, 马俐君 . RAB25沉默抑制结直肠癌细胞铁死亡的作用研究[J]. 诊断学理论与实践, 2022 , 21(06) : 710 -718 . DOI: 10.16150/j.1671-2870.2022.06.08

Abstract

Objective: To investigate the effect of RAB25 on the sensitivity of erastin-induced ferroptosis in colorectal cancer(CRC). Methods: The GEPIA database was used to analyze the mRNA expression levels of RAB25 in CRC tissues and the co-expression between RAB25 and ferroptosis related genes. The lentivirus-mediated RAB25 knockdown cell line was constructed. The expression of RAB25 was detected by quantitative real-time polymerase chain (qRT-PCR) and western blotting. After adding erastin at different concentrations(0-20 μmol/L), the cell proliferation rate was measured by CCK8 assay. The cell morphology and the mitochondrial structure were observed by inverted fluorescence microscope and transmission electron microscope, respectively. C11-BODIPY staining and flow cytometry probe were used to determine the level of lipid peroxides in cellular membranes. The combined effect of erastin and cetuximab on cellular viability in RAB25 knockdown cells were investigated. Results: The mRNA expression of RAB25 was unregulated in colorectal cancer tissues (P<0.01). The mRNA expression of RAB25 was correlated with some vital ferroptosis-associated genes. When erastin ≥10 μmol/L, the shRAB25 group inhibited the killing effect of ferroptosis on cell viability (P<0.000 1) compared to that of the control group, and cell morphology and mitochondrial structure were clearer and complete in the shRAB25 group. Flow cytometry revealed a significant decrease in lipid peroxidation levels in the shRAB25 group(P<0.000 1) compared to the control group. The expression of shRAB25 enhanced the toxic effects by erastin and cetuximab combination. Conclusions: RAB25 is highly expressed in colorectal cancer tissues and played an significant role in ferroptosis. After silencing RAB25, the erastin-induced lethal effect of colorectal cancer cells was inhibited. RAB25 and erastin may induce ferroptosis synergistically, enhancing the efficacy of cetuximab

参考文献

[1] Biller L H, Schrag D. Diagnosis and Treatment of metastatic colorectal cancer: a review[J]. JAMA, 2021, 325(7):69-685.
[2] Sung H, Ferlay J, Siegel R L, et al. Global cancer statistics 2020: GLOBOCAN estimates of Incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2021, 71(3):209-249.
[3] Dixon S J, Lembrg K M, Lamprecht M R, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death[J]. Cell, 2012, 149(5):1060-1072.
[4] Dixon S J, Stockwell B R. The Hallmarks of Ferroptosis[J]. Annual Review of Cancer Biology, 2019, 3(1):35-54.
[5] Yang W S, Sriramaratnam R, Welsch M E, et al. Regulation of ferroptotic cancer cell death by GPX4[J]. Cell, 2014, 156(1-2):317-331.
[6] Han W, Duan X, Ni K, et al. Co-delivery of dihydroartemisinin and pyropheophorbide-iron elicits ferroptosis to potentiate cancer immunotherapy[J]. Biomaterials, 2022, 280:121315.
[7] Li Y, Chen W, Qi Y, et al. H2 S-Scavenged and Activated Iron Oxide-Hydroxide Nanospindles for MRI-Guided Photothermal Therapy and Ferroptosis in Colon Cancer[J]. Small, 2020, 16(37):e2001356.
[8] Zhang C, Liu X, Jin S, et al. Ferroptosis in cancer therapy: a novel approach to reversing drug resistance[J]. Mol Cancer, 2022, 21(1):47.
[9] Wang J, Zhou P, Wang X, et al. Rab25 promotes erlotinib resistance by activating the β1 integrin/AKT/β-catenin pathway in NSCLC[J]. Cell Prolif, 2019, 52(3):e12592.
[10] Temel S G, Giray A, Karakas B, et al. RAB25 confers resistance to chemotherapy by altering mitochondrial apoptosis signaling in ovarian cancer cells[J]. Apoptosis, 2020, 25(11-12):799-816.
[11] Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2018, 68(6):394-424.
[12] Li J, Cao F, Yin H L, et al. Ferroptosis: past, present and future[J]. Cell Death Dis, 2020, 11(2):88.
[13] Lachaier E, Louandre C, Godin C, et al. Sorafenib induces ferroptosis in human cancer cell lines originating from different solid tumors[J]. Anticancer Res, 2014, 34(11):6417-6422.
[14] Chen G Q, Benthani F A, Wu J, et al. Artemisinin compounds sensitize cancer cells to ferroptosis by regulating iron homeostasis[J]. Cell Death Differ, 2020, 27(1):242-254.
[15] Bjarnadottir O, Romero Q, Bendahl P O, et al. Targeting HMG-CoA reductase with statins in a window-of-opportunity breast cancer trial[J]. Breast Cancer Res Treat, 2013, 138(2):499-508.
[16] Matsuoka S, Ballif B A, Smogorzewska A, et al. ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage[J]. Science, 2007, 316(5828):1160-1166.
[17] Sleire L, Skeie B S, Netland I A, et al. Drug repurposing: sulfasalazine sensitizes gliomas to gamma knife radiosurgery by blocking cystine uptake through system Xc-, leading to glutathione depletion[J]. Oncogene, 2015, 34(49):5951-5959.
[18] Ye L F, Chaudhary K R, Zandkarimi F, et al. Radiation-Induced Lipid Peroxidation Triggers Ferroptosis and Synergizes with Ferroptosis Inducers[J]. ACS Chem Biol, 2020, 15(2):469-484.
[19] Pan X, Lin Z, Jiang D, et al. Erastin decreases radioresistance of NSCLC cells partially by inducing GPX4-mediated ferroptosis[J]. Oncol Lett, 2019, 17(3):3001-3008.
[20] Lang X, Green M D, Wang W, et al. Radiotherapy and Immunotherapy Promote Tumoral Lipid Oxidation and Ferroptosis via Synergistic Repression of SLC7A11[J]. Cancer Discov, 2019, 9(12):1673-1685.
[21] Papke B, Der C J. Drugging RAS: Know the enemy[J]. Science, 2017, 355(6330):1158-1163.
[22] Zhang J, Wei J, Lu J, et al. Overexpression of Rab25 contributes to metastasis of bladder cancer through induction of epithelial-mesenchymal transition and activation of Akt/GSK-3β/Snail signaling[J]. Carcinogenesis, 2013, 34(10):2401-2408.
[23] Liu L, Ding G. Rab25 expression predicts poor prognosis in clear cell renal cell carcinoma[J]. Exp Ther Med, 2014, 8(4):1055-1058.
[24] Jeong B Y, Cho K H, Jeong K J, et al. Rab25 augments cancer cell invasiveness through a β1 integrin/EGFR/VEGF-A/Snail signaling axis and expression of fascin[J]. Exp Mol Med, 2018, 50(1):e435.
[25] Jo U, Park K H, Whang Y M, et al. EGFR endocytosis is a novel therapeutic target in lung cancer with wild-type EGFR[J]. Oncotarget, 2014, 5(5):1265-1278.
[26] Geng D, Zhao W, Feng Y, et al. Overexpression of Rab25 promotes hepatocellular carcinoma cell proliferation and invasion[J]. Tumour Biol, 2016, 37(6):7713-7718.
[27] Hu C, Chen B, Zhou Y, et al. High expression of Rab25 contributes to malignant phenotypes and biochemical recurrence in patients with prostate cancer after radical prostatectomy[J]. Cancer Cell Int, 2017, 17:45.
[28] 关明珺, 任笑云, 王大路, 等. Rab25蛋白在结肠癌中的表达及临床意义[J]. 现代肿瘤医学, 2017, 25(12):1935-1937.
[28] Guan M J, Ren X Y, Wang D L, et al. The expressions and clinical significance of Rab25 in colon cancer[J]. J Modern Oncol, 2017, 25(12):1935-1937.
[29] Ding B, Cui B, Gao M, et al. Knockdown of Ras-related protein 25 (Rab25) inhibits the in vitro cytotoxicity and in vivo antitumor activity of human glioblastoma multiforme cells[J]. Oncol Res, 2017, 25(3):331-340.
[30] Amornphimoltham P, Rechache K, Thompson J, et al. Rab25 regulates invasion and metastasis in head and neck cancer[J]. Clin Cancer Res, 2013, 19(6):1375-1388.
[31] Cheng J M, Volk L, Janaki D K, et al. Tumor suppressor function of Rab25 in triple-negative breast cancer[J]. Int J Cancer, 2010, 126(12):2799-2812.
[32] Bullman S, Pedamallu C S, Sicinska E, et al. Analysis of Fusobacterium persistence and antibiotic response in colorectal cancer[J]. Science, 2017, 358(6369):1443-1448.
[33] Li B, Yang L, Peng X, et al. Emerging mechanisms and applications of ferroptosis in the treatment of resistant cancers[J]. Biomed Pharmacother, 2020, 130:110710.
[34] Wang X, Kumar R, Navarre J, et al. Regulation of vesicle trafficking in madin-darby canine kidney cells by Rab11a and Rab25[J]. J Biol Chem, 2000, 275(37):29138-29146.
[35] Mou Y, Wang J, Wu J, et al. Ferroptosis, a new form of cell death: opportunities and challenges in cancer[J]. J Hematol Oncol, 2019, 12(1):34.
[36] Tang Q, Chen H, Mai Z, et al. Bim- and Bax-mediated mitochondrial pathway dominates abivertinib-induced apoptosis and ferroptosis[J]. Free Radic Biol Med, 2022, 180:198-209.
[37] Yang J, Mo J, Dai J, et al. Cetuximab promotes RSL3-induced ferroptosis by suppressing the Nrf2/HO-1 signalling pathway in KRAS mutant colorectal cancer[J]. Cell Death Dis, 2021, 12(11):1079.
文章导航

/