BACKGROUND-AIM. Increased reactive oxygen species (ROS) levels play critical roles in chronic inflammation, and predispose to colon carcinogenesis. Wnt signaling is essential for gut morphogenesis, tissue homeostasis and self-renewal, but its aberrant activation may drive the colorectal cancer (CRC). The ROS production seems to induce the Wnt/β-Catenin pathways, 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 (MSI) and SW480 (MSS) cells were treated with H2O2 [2 mM and 10 mM] for 15’and 30’. We assayed cell viability by MTS and cell cycle by FACS. Gene expression was evaluated by SYBR Green qRT-PCR, and protein expression was analyzed by IHC. Statistical analysis was performed by T-test (p value<0.05). RESULTS. MTS revealed different inhibition rates of cell growth at H2O2 concentrations. Acute stress induced by H2O2 [2mM] up-regulated gene expression of canonical LRP6 and LEF1, and non canonical ROR2 and JUN/AP1 molecules in SW480, while reduced ROR2 and LRP6 expression in HCT116. Both pathways showed a dose dependent increase in SW480, at H2O2 [10mM]. In HCT116 down-regulated gene expression of APC, LRP6, LEF1, and p65-NFkB was dependent on treatment time, in opposition to non-canonical ROR2. MUTYH, OGG1, NRF2, COX2 and JUN/AP1 expression significantly increased. H2O2 treatment induced FZD6 protein expression in HCT116 cytoplasm and E-cadherin protein expression in SW480 cytoplasm, while beta-catenin increased in both cell lines. Intriguingly we relieved a de novo APC expression in both cell lines cytoplasm. FACS analysis of cell cycle showed time dependent changes: upon 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. CONCLUSIONS. In MSI and MSS CRC cells, oxidative stress differently affects the WNT pathways at gene and protein expression levels. Our results could unravel a new scenario for innovative CRC therapeutic approaches.
Oxidative stress induces Wnt canonical/non-canonical pathways modulation in colon cancer cell models
Ferlazzo Guido;
2019-01-01
Abstract
BACKGROUND-AIM. Increased reactive oxygen species (ROS) levels play critical roles in chronic inflammation, and predispose to colon carcinogenesis. Wnt signaling is essential for gut morphogenesis, tissue homeostasis and self-renewal, but its aberrant activation may drive the colorectal cancer (CRC). The ROS production seems to induce the Wnt/β-Catenin pathways, 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 (MSI) and SW480 (MSS) cells were treated with H2O2 [2 mM and 10 mM] for 15’and 30’. We assayed cell viability by MTS and cell cycle by FACS. Gene expression was evaluated by SYBR Green qRT-PCR, and protein expression was analyzed by IHC. Statistical analysis was performed by T-test (p value<0.05). RESULTS. MTS revealed different inhibition rates of cell growth at H2O2 concentrations. Acute stress induced by H2O2 [2mM] up-regulated gene expression of canonical LRP6 and LEF1, and non canonical ROR2 and JUN/AP1 molecules in SW480, while reduced ROR2 and LRP6 expression in HCT116. Both pathways showed a dose dependent increase in SW480, at H2O2 [10mM]. In HCT116 down-regulated gene expression of APC, LRP6, LEF1, and p65-NFkB was dependent on treatment time, in opposition to non-canonical ROR2. MUTYH, OGG1, NRF2, COX2 and JUN/AP1 expression significantly increased. H2O2 treatment induced FZD6 protein expression in HCT116 cytoplasm and E-cadherin protein expression in SW480 cytoplasm, while beta-catenin increased in both cell lines. Intriguingly we relieved a de novo APC expression in both cell lines cytoplasm. FACS analysis of cell cycle showed time dependent changes: upon 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. CONCLUSIONS. In MSI and MSS CRC cells, oxidative stress differently affects the WNT pathways at gene and protein expression levels. Our results could unravel a new scenario for innovative CRC therapeutic approaches.
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