Targeting ETHE1 inhibits tumorigenesis in vitro and in vivo by preventing aerobic glycolysis in gastric adenocarcinoma cells

Abstract

Gastric adenocarcinoma (GAC) is a prevalent form of cancer that frequently displays abnormal metabolism characterized by increased aerobic glycolysis. Therefore, inhibition of glycolysis may exhibit therapeutic potential for the management of advanced or recurrent gastric cancer. Analysis of ethylmalonic encephalopathy protein 1 (ETHE1) expression levels in 30 pairs of cancerous and paracancerous tissues, and 50 tumor tissue sections collected from patients with GAC revealed that ETHE1 expression was upregulated in cancerous tissues compared with in paracancerous tissues. Advanced tumor stage, lymph node metastasis and Tumor-Node-Metastasis stage were associated with high ETHE1 expression. Knockdown of ETHE1 expression in GAC cells resulted in a significant inhibition of cell proliferation and in cell cycle arrest, accompanied by downregulated levels of cyclin D1 and cyclin-dependent kinase 4. ETHE1 knockdown also resulted in increased apoptosis of GAC cells, and increased caspase-3 and caspase-9 activity. Additionally, the expression levels of proteins associated with aerobic glycolysis were downregulated following ETHE1 knockdown, which may reduce glucose consumption, lactic acid production and ATP levels. In the in vivo experiments, suppressed tumor growth and increased tumor cell apoptosis were observed in the xenograft tumor model in animals injected with ETHE1-knockdown GAC cells. In summary, knockdown of ETHE1 inhibited aerobic glycolysis, promoted apoptosis and inhibited tumor cell proliferation in GAC cells. These results highlight ETHE1 as a promising molecular target for the treatment of GAC potentially using an adjuvant to target it, offering a novel approach in the exploration of targeted therapeutic drugs for GAC.

Keywords: aerobic glycolysis; apoptosis; ethylmalonic encephalopathy protein 1; gastric adenocarcinoma; tumor growth.

Figure 1.

Analysis of ETHE1 expression in clinical samples. (A) ETHE1 expression in various types of cancer was analyzed based on TCGA and Genotype-Tissue Expression databases. (B) Immunohistochemical images of ETHE1 expression in normal gastric tissue and gastric cancer tissue were obtained from the Human Protein Atlas database. ETHEI expression in GAC and paracancerous tissues was detected using (C) reverse transcription-quantitative PCR and (D) western blotting. (E) Survival analysis of ETHE1 expression in STAD was conducted based on TCGA data. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. ETHEI, ethylmalonic encephalopathy protein 1; GAC, gastric adenocarcinoma; STAD, stomach adenocarcinoma; TCGA, The Cancer Genome Atlas; ns, not significant.

Figure 2.

Expression of ETHE1 in tissues from patients with GAC and GAC cell lines. (A) Representative images of ETHE1 immunohistochemical staining in GAC tissue. Scale bar, 100 µm (magnification, ×200); 50 µm (magnification, ×400). (B) Reverse transcription-quantitative PCR and (C) western blotting of ETHEI expression in GAC cells. **P<0.01, ***P<0.001. ETHEI, ethylmalonic encephalopathy protein 1; GAC, gastric adenocarcinoma.

Figure 3.

ETHE1 knockdown inhibits CAG cell proliferation. NCI-N87 and MKN-45 cells were infected with a lentivirus carrying ETHE1-specific shRNAs or a shRNA without any target sequence. (A) Reverse transcription-quantitative PCR and western blotting of ETHEI expression in gastric adenocarcinoma cells. (B) Cell proliferation during 48 h was detected using the Cell Counting Kit-8 assay. (C) Representative EdU staining images. Scale bar, 50 µm. (D) Flow cytometric analysis of cell cycle progression. (E) Western blot analysis of CDK4 and cyclin D1. *P<0.05, **P<0.01, **P<0.01 and ***P<0.001 vs. Ctrl or as indicated. CDK4, cyclin-dependent kinase 4; Ctrl, control; ETHEI, ethylmalonic encephalopathy protein 1; sh, short hairpin.

Figure 4.

ETHE1 knockdown promotes CAG cell apoptosis. (A) Flow cytometric analysis of apoptosis. (B) Apoptosis of gastric adenocarcinoma cells was detected by TUNEL staining. Scale bar, 50 µm. (C) Caspase-3 and (D) caspase-9 activity was detected using kits. **P<0.01, ***P<0.001. Ctrl, control; sh, short hairpin.

Figure 5.

ETHE1 knockdown inhibits aerobic glycolysis in CAG cells. (A) Glucose consumption, (B) lactic acid production and (C) ATP levels were detected using kits. (D) Western blot analysis of GLUT1, LDHA, HK2 and PKM2. **P<0.01, ***P<0.001. Ctrl, control; GLUT1, glucose transporter type 1; LDHA, lactate dehydrogenase A; HK2, hexokinase 2; PKM2, pyruvate kinase isozyme type M2; sh, short hairpin.

Figure 6.

BALB/c nude mice received subcutaneous injections of stabilized infected NCI-N87 cells to induce tumor formation in vivo. (A) Tumor tissue images, tumor growth curve and tumor weight. (B) Representative immunohistochemical staining images of Ki67 and ETHE1. (C) Apoptosis in gastric adenocarcinoma tissue was detected by TUNEL staining. Scale bar, 100 µm. *P<0.05, **P<0.01 and ***P<0.001 vs. Ctrl. Ctrl, control; ETHEI, ethylmalonic encephalopathy protein 1; sh, short hairpin.

Figure 7.

Analysis of ETHE1 expression in immune regulation of STAD. Correlation analysis of ETHE1 with (A) immunosuppressive factors, (B) immune-promoting factors and (C) chemokines in STAD using the TISIDB database. (D) Expression of ETHE1 in M2 macrophages and Tregs in STAD tissues from using Gene Expression Profiling Interactive Analysis database. *P<0.05 vs. Normal. CXCL, C-X-C motif chemokine ligand; ETHEI, ethylmalonic encephalopathy protein 1; IDO1, indoleamine 2,3-dioxygenase 1; IL6R, IL-6 receptor; IL10RB, IL-10 receptor subunit β; LGALS9, galectin 9; PVRL2, nectin cell adhesion molecule 2; STAD, stomach adenocarcinoma; Tregs, regulatory T cells; ULBP1, UL16 binding protein 1.