LINE-1 ORF1 Protein Is Up-regulated by Reactive Oxygen Species and Associated with Bladder Urothelial Carcinoma Progression

Abstract

Background/aim: Reactivation of long interspersed nuclear element-1 (LINE-1) and oxidative stress are suggested to have oncogenic potential to drive tumorigenesis and cancer progression. We previously demonstrated that reactive oxygen species (ROS) caused hypomethylation of LINE-1 elements in bladder cancer cells. In this study, we investigated the expression of LINE-1-encoded protein (ORF1p) and oxidative stress marker 4-hydroxynonenal (4-HNE) in human bladder cancer tissues, as well as induction of ORF1p expression by ROS in bladder cancer cell lines.

Materials and methods: Thirty-six cancerous and 15 non-cancerous adjacent tissues were immunohistochemically stained for ORF1p and 4-HNE. ORF1p expression and cell migration were determined in bladder cancer cells exposed to H₂O₂ Results: ORF1p and 4-HNE expression was higher in cancerous than non-cancerous tissues. Elevated ORF1p expression was associated with increased 4-HNE expression and with advanced tumors. H₂O₂ provoked oxidative stress and up-regulated ORF1p expression in VM-CUB-1 compared to the untreated control, and to a lesser degree in TCCSUP. H₂O₂ exposure enhanced cell migration in UM-UC-3, TCCSUP and VM-CUB-1.

Conclusion: Elevated ORF1p expression is associated with tumor progression. ROS experimentally induce ORF1p expression and promote migration in bladder cancer cells.

Keywords: 4-HNE; LINE-1; ORF1p; bladder cancer; cancer progression; immunohistochemistry; oxidative stress.

Figure 1. Representative micrographs of ORF1p and 4-HNE expression in bladder cancer tissues. ORF1p was negative or expressed at very low levels in non-cancerous bladder tissues (A and C). In contrast, expression of ORF1p in bladder cancer tissues was remarkably increased (B and D). Similar to ORF1p, 4-HNE expression was elevated in bladder cancer tissues (F), but it was obviously low in non-cancerous bladder tissues (E). Magnifications: ×100 (A and B), ×400 (C-F).

Figure 2. Levels of ORF1p and 4-HNE expression compared between cancerous and non-cancerous bladder tissues, and correlation of ORF1p and 4-HNE expression in bladder cancer tissues. Both ORF1p expression (A) and 4-HNE (B) levels were significantly higher in bladder cancer tissues than in non-cancerous bladder tissues. In paired cancerous and non-cancerous tissue samples, significantly higher levels of ORF1p (C) and 4-HNE (D) expression were observed in cancerous areas compared to adjacent non-cancerous regions. ORF1p expression was positively correlated with 4-HNE expression in bladder cancer tissues (E).

Figure 3. Association of ORF1p expression with bladder tumor progression. ORF1p expression in muscle-invasive bladder tumors was significantly higher than that in papillary/superficial (non muscle-invasive) tumors (A). Likewise, expression of ORF1p in high-grade tumors was significantly higher than low-grade tumors (B). Left: representative immunostainings; right: quantitative evaluation.

Figure 4. Induction of ORF1p expression by ROS in bladder cancer cell lines. In VM-CUB-1 cells (A), exposure to H₂O₂ (30 μM for 72 h) caused a significant increase in ORF1p expression compared to untreated controls, and this increment was effectively normalized by co-treatment with antioxidants (for 72 h), TA (300 μM) and SAM (100 μM). In TCCSUP cells (B), ORF1p expression was slightly increased after H2O2 exposure without reaching statistical significance. Co-treatment with SAM significantly caused reduction of ORF1p expression in H₂O₂-treated cells.

Figure 5. Scratch assay showing ROS-induced cell migration in bladder cancer cell lines, UM-UC-3, TCCSUP and VM-CUB-1. UM-UC-3 and TCCSUP were treated with H₂O₂ for 72 h, but VM-CUB-1 for 6 h. All H₂O₂-treated cells migrated faster to fill the gaps than the untreated control cells. Co-treatment with TA reduced the migration capability of the H₂O₂-treated cells.