Effect of RAB25 knockdown on ferroptosis of colorectal cancer cells

doi:10.16150/j.1671-2870.2022.06.08

Abstract

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

Key words: Colorectal cancer, Ras-associated binding protein 25, Ferroptosis

CLC Number:

R735.3

LI Jiaxi, WANG Jinjiang, YU Liping, YUAN Ying, QIAO Guanglei, MA Lijun. Effect of RAB25 knockdown on ferroptosis of colorectal cancer cells[J]. Journal of Diagnostics Concepts & Practice, 2022, 21(06): 710-718.

Figures/Tables 9

References 37

[1]	Biller L H, Schrag D. Diagnosis and Treatment of metastatic colorectal cancer: a review[J]. JAMA, 2021, 325(7):69-685. doi: 10.1001/jama.2020.18936 URL
[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. doi: 10.3322/caac.v71.3 URL
[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. doi: 10.1016/j.cell.2012.03.042 pmid: 22632970
[4]	Dixon S J, Stockwell B R. The Hallmarks of Ferroptosis[J]. Annual Review of Cancer Biology, 2019, 3(1):35-54. doi: 10.1146/cancerbio.2019.3.issue-1 URL
[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. doi: 10.1016/j.cell.2013.12.010 pmid: 24439385
[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. doi: 10.1016/j.biomaterials.2021.121315 URL
[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. doi: 10.1186/s12943-022-01530-y pmid: 35151318
[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. doi: 10.1111/cpr.2019.52.issue-3 URL
[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. doi: 10.1007/s10495-020-01635-z
[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. doi: 10.3322/caac.v68.6 URL
[12]	Li J, Cao F, Yin H L, et al. Ferroptosis: past, present and future[J]. Cell Death Dis, 2020, 11(2):88. doi: 10.1038/s41419-020-2298-2 pmid: 32015325
[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. pmid: 25368241
[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. doi: 10.1038/s41418-019-0352-3
[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. doi: 10.1007/s10549-013-2473-6 URL
[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. doi: 10.1126/science.1140321 pmid: 17525332
[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. doi: 10.1038/onc.2015.60 pmid: 25798841
[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. doi: 10.1021/acschembio.9b00939 pmid: 31899616
[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. doi: 10.3892/ol.2019.9888 pmid: 30854078
[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. doi: 10.1158/2159-8290.CD-19-0338 pmid: 31554642
[21]	Papke B, Der C J. Drugging RAS: Know the enemy[J]. Science, 2017, 355(6330):1158-1163. doi: 10.1126/science.aam7622 pmid: 28302824
[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. doi: 10.1093/carcin/bgt187 pmid: 23722651
[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. pmid: 25187796
[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. doi: 10.1038/emm.2017.248 URL
[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. doi: 10.18632/oncotarget.1711 pmid: 24658031
[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. doi: 10.1007/s13277-015-4606-5 URL
[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. doi: 10.1186/s12935-017-0411-0 pmid: 28400705
[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.
	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. doi: 10.3727/096504016X14736286083065 pmid: 28281975
[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. doi: 10.1158/1078-0432.CCR-12-2858 pmid: 23340300
[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. doi: 10.1126/science.aal5240 pmid: 29170280
[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. doi: 10.1016/j.biopha.2020.110710 pmid: 33568263
[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. doi: 10.1074/jbc.M004410200 pmid: 10869360
[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. doi: 10.1186/s13045-019-0720-y
[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. doi: 10.1016/j.freeradbiomed.2022.01.013 URL
[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. doi: 10.1038/s41419-021-04367-3 pmid: 34775496