Dataset Information

Clostridioides difficile Toxin A-Induced Wnt/?-Catenin Pathway Inhibition Is Mediated by Rac1 Glucosylation.

ABSTRACT: Clostridioides difficile toxin A (TcdA) has been shown to inhibit cellular Wnt signaling, the major driving force behind the proliferation of epithelial cells in colonic crypts, likely through the inhibition of ?-catenin nuclear translocation. Herein, we aimed to advance the understanding of this mechanism by replicating the findings in vivo and by investigating the specific role of Rac1, a member of the Rho GTPase family, on the inhibition of the Wnt-induced ?-catenin nuclear translocation triggered by TcdA. To investigate the effects of TcdA on the Wnt/?-catenin pathway in vivo, we injected the ileal loops of C57BL/6 mice with TcdA [phosphate-buffered saline (PBS) as the control] to induce C. difficile disease-like ileitis. After 4 h post-injection, we obtained ileum tissue samples to assess Wnt signaling activation and cell proliferation through Western blotting, immunohistochemistry, and qPCR. To assess the role of Rac1 on Wnt signaling inhibition by TcdA, we transfected rat intestinal epithelial cells (IEC-6) with either a constitutively active Rac1 plasmid (pcDNA3-EGFP-Rac1-Q61L) or an empty vector, which served as the control. We incubated these cells with Wnt3a-conditioned medium (Wnt3a-CM) to induce Wnt/?-catenin pathway activation, and then challenged the cells with TcdA. We assessed Wnt signaling activation in vitro with TOP/FOPflash luciferase assays, determined nuclear ?-catenin translocation by immunofluorescence, measured cyclin D1 protein expression by Western blotting, and quantified cell proliferation by Ki67 immunostaining. In vivo, TcdA decreased ?-catenin, cyclin D1, and cMYC expression and inhibited the translocation of ?-catenin into the nucleus in the ileum epithelial cells. In addition, TcdA suppressed cell proliferation and increased Wnt3a expression, but did not alter Rac1 gene expression in the ileum tissue. In vitro, constitutively active Rac1 prevented Wnt signaling inhibition by enabling the ?-catenin nuclear translocation that had been blocked by TcdA. Our results show that TcdA inhibits Wnt/?-catenin pathway in vivo and demonstrate that this inhibition is likely caused by a Rac1-mediated mechanism.

SUBMITTER: Martins CS

PROVIDER: S-EPMC7483921 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Clostridioides difficile Toxin A-Induced Wnt/β-Catenin Pathway Inhibition Is Mediated by Rac1 Glucosylation.

Martins Conceição S CS Costa Deiziane V S DVS Lima Bruno B BB Leitäo Renata F C RFC Freire Gildênio E GE Silva Guilherme F M GFM Pacífico Dvison M DM Abreu José G JG Brito Gerly A C GAC

Frontiers in microbiology 20200828

Clostridioides difficile toxin A (TcdA) has been shown to inhibit cellular Wnt signaling, the major driving force behind the proliferation of epithelial cells in colonic crypts, likely through the inhibition of β-catenin nuclear translocation. Herein, we aimed to advance the understanding of this mechanism by replicating the findings in vivo and by investigating the specific role of Rac1, a member of the Rho GTPase family, on the inhibition of the Wnt-induced β-catenin nuclear tran ...[more]

PMID: 32983019

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Project description:BackgroundAlthough there are many treatments for breast cancer, such as surgery, radiotherapy, chemotherapy, estrogen receptor antagonists, immune checkpoint inhibitors and so on. However, safer and more effective therapeutic drugs for breast cancer are needed. Sinensetin, a safer therapeutic drugs, come from citrus species and medicinal plants used in traditional medicine, while its role and underlying mechanism in breast cancer remain unclear. Our study aimed to investigate the role and mechanism of sinensetin in breast cancer.MethodsCell Counting Kit-8 (CCK-8) was used to determine the safe concentration of sinensetin in MCF-10A, MCF7 and MDA-MB-231 cells; 120 μM sinensetin was used in subsequent experiments. Real time polymerase chain reaction (RT-PCR), Western blotting, Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling (TUNEL) apoptosis assay, Transwell invasion assay and Clone formation assay were used in this study to determine cell viability, mRNA expression, protein levels, apoptosis, proliferation, invasion and so on.ResultsHerein, our results showed that 120 μM sinensetin suppressed the cell viability and promoted apoptosis of MCF7 and MDA-MB-231 cells. Treatment with 120 µM sinensetin for 24 h showed no significant toxicity to normal mammary cells; 120 μM sinensetin decreased cell proliferation, invasion, and epithelial-mesenchymal transition (EMT), and downregulated β-catenin, lymphatic enhancing factor 1 (LEF1), T-cell factor (TCF) 1/TCF7, and TCF3/TCF7L1 expression in MCF7 and MDA-MB-231 cells. The Wnt agonist SKL2001 reversed the inhibitory effect of sinensetin on cell survival, metastasis, and EMT. Sinensetin-induced downregulation of β-catenin, LEF1, and TCF1/TCF7 expression were upregulated by SKL2001 in MCF7 and MDA-MB-231 cells.ConclusionsIn summary, sinensetin suppressed the metastasis of breast cancer cell via inhibition of Wnt/β-catenin pathway and there were no adverse effects on normal breast cells. Our study confirmed the role of sinensetin in breast cancer cells and provided a better understanding of the underlying mechanism.

Dataset Information

Clostridioides difficile Toxin A-Induced Wnt/?-Catenin Pathway Inhibition Is Mediated by Rac1 Glucosylation.

Publications

<i>Clostridioides difficile</i> Toxin A-Induced Wnt/β-Catenin Pathway Inhibition Is Mediated by Rac1 Glucosylation.

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