Background-aim. It has long been recognised that the aberrant regulation of WNT/β-Catenin signaling is involved in the pathogenesis of colorectal cancer. Chronic inflammation predisposes to colon carcinogenesis by increased reactive oxygen species (ROS) levels and can impair the Wingless/It (WNT)/β-Catenin. This pathway is essential for gut morphogenesis, tissue homeostasis and self-renewal and its aberrant activation may drive the colorectal cancer (CRC), but the molecular mechanisms involved in CRC progression are still undefined. To evaluate the molecular relationship among oxidative stress and canonical/non-canonical Wnt pathways, we analyzed the response to ROS exposure in CRC cell lines with different Wnt signaling behaviour. Methods. HCT116 (β-Catenin) and SW480 (APC) cells were exposed to Hydrogen Peroxide (H2O2) as oxidizing agent for different times and concentrations (from 50 µM to 10 mM). We assayed cell viability/mitochondria activity by MTS and cell cycle by FACS. Gene expression was evaluated by SYBR Green qRT-PCR in cells under acute stress conditions [2 mM and 10 mM] for 15’and 30’. Protein expression was analysed by IHC. Statistical analysis was performed by T-test (p value<0.05). Results. MTS revealed rates of cell inhibition and growth at different H2O2 concentrations. FACS analysis of cell cycle showed time dependent changes: after H2O2 [2mM] treatment at 15’, SW480 increased in G1 and G2 and decreased in S, whereas HCT116 increased in G1 and slightly reduced in G2; after 30’, SW480 enhanced in G1 and S, and reduced in G2 while HCT116 diminished in G1 and increased in S/G2. In SW480 cells, acute stress induced by lower H2O2 concentration [2mM] up-regulated gene expression of canonical LRP6 and LEF1, and non-canonical ROR2 and JUN/AP1 molecules. While in HCT116 the same H2O2 concentration [2mM] reduced ROR2 and LRP6 expression of WNT co-receptor and differently affected the WNT transcription factors, upregulating LEF1 (canonical) and downregulating JUN/AP1 (non-canonical). As regard APC, it showed a behaviour dependent on time and concentration of H2O2 treatment. IHC protein expression analysis showed that H2O2 treatment induced FZD6 in HCT116 cytoplasm and E-cadherin in SW480 cytoplasm, while beta-catenin increased in both cell lines. Intriguingly oxidative stress induced a de novo APC expression in cytoplasm of both cell lines. Conclusions. In CRC cells with mutations in APC or B-catenin, oxidative stress differently affects the WNT pathways at gene and protein expression levels. Our results could unravel an APC central role in driving and maintaining tumorigenesis and a novel scenario for innovative CRC therapeutic approaches.

Oxidative Stress Induces WNT Canonical/Non-Canonical Pathway Modulation in Colon Cancer Cells with APC or beta-Catenin Mutation

Ferlazzo G;
2020-01-01

Abstract

Background-aim. It has long been recognised that the aberrant regulation of WNT/β-Catenin signaling is involved in the pathogenesis of colorectal cancer. Chronic inflammation predisposes to colon carcinogenesis by increased reactive oxygen species (ROS) levels and can impair the Wingless/It (WNT)/β-Catenin. This pathway is essential for gut morphogenesis, tissue homeostasis and self-renewal and its aberrant activation may drive the colorectal cancer (CRC), but the molecular mechanisms involved in CRC progression are still undefined. To evaluate the molecular relationship among oxidative stress and canonical/non-canonical Wnt pathways, we analyzed the response to ROS exposure in CRC cell lines with different Wnt signaling behaviour. Methods. HCT116 (β-Catenin) and SW480 (APC) cells were exposed to Hydrogen Peroxide (H2O2) as oxidizing agent for different times and concentrations (from 50 µM to 10 mM). We assayed cell viability/mitochondria activity by MTS and cell cycle by FACS. Gene expression was evaluated by SYBR Green qRT-PCR in cells under acute stress conditions [2 mM and 10 mM] for 15’and 30’. Protein expression was analysed by IHC. Statistical analysis was performed by T-test (p value<0.05). Results. MTS revealed rates of cell inhibition and growth at different H2O2 concentrations. FACS analysis of cell cycle showed time dependent changes: after H2O2 [2mM] treatment at 15’, SW480 increased in G1 and G2 and decreased in S, whereas HCT116 increased in G1 and slightly reduced in G2; after 30’, SW480 enhanced in G1 and S, and reduced in G2 while HCT116 diminished in G1 and increased in S/G2. In SW480 cells, acute stress induced by lower H2O2 concentration [2mM] up-regulated gene expression of canonical LRP6 and LEF1, and non-canonical ROR2 and JUN/AP1 molecules. While in HCT116 the same H2O2 concentration [2mM] reduced ROR2 and LRP6 expression of WNT co-receptor and differently affected the WNT transcription factors, upregulating LEF1 (canonical) and downregulating JUN/AP1 (non-canonical). As regard APC, it showed a behaviour dependent on time and concentration of H2O2 treatment. IHC protein expression analysis showed that H2O2 treatment induced FZD6 in HCT116 cytoplasm and E-cadherin in SW480 cytoplasm, while beta-catenin increased in both cell lines. Intriguingly oxidative stress induced a de novo APC expression in cytoplasm of both cell lines. Conclusions. In CRC cells with mutations in APC or B-catenin, oxidative stress differently affects the WNT pathways at gene and protein expression levels. Our results could unravel an APC central role in driving and maintaining tumorigenesis and a novel scenario for innovative CRC therapeutic approaches.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11567/1117980
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