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Chronic kidney disease (CKD) induces several systemic effects including the accumulation and production of uremic toxins responsible for the activation of various damaging processes. The abundant discharge of urea and other waste substances in the gut favors the selection of an altered gut microbiota in patients with CKD. The prevalence of aerobic bacteria leads to the release and accumulation in the gut and blood of various substances derived from tryptophan and tyrosine metabolism, such as Indoxyl Sulphate (IS) and P-cresyl sulphate (P-cs). P-cs and IS play a key role in the activation of various pro-tumourigenic processes such as chronic systemic inflammation, increased free radical production and immune dysfunction. An up to two-fold increase in colon cancer (CRC) incidence with worse prognosis in CKD has been reported in the literature, although the pathogenetic mechanisms underlying this association have not yet been described. Considering the pro-oncogenic activities that P-cs and IS carry out at the systemic and intestinal levels, we evaluated the action of these toxins on CRC cells lines proliferation, invasion, and migration. Moreover, the cellular expression of Vimentin and E-cadherin (proteins involved in epithelial to mesenchymal transition), was assessed to discover possible molecular mechanisms underlying the increased cancer aggressiveness.
CRC cell lines (HT29, Hct116 and LoVo) were cultured at different IS and p-CS concentrations, similar to those detected in CKD patients with gut dysbiosis. Cells proliferation was assessed by Clonogenic Assay and colorimetric assay (WST-8), while invasion and migration were tested by Wound Healing assay and Invasion Assay respectively. Cellular expression of Vimentin and E-cadherin was assessed by Western blot.
All tested IS and P-cs concentrations induced a significant increase in CRC cells proliferation, migration and invasion compared to controls. However, IS 0.0175mg/ml and P-cs 0.0206mg/ml were associated with the highest increase of these cancer aggressiveness parameters. Linearly, vimentin and cadherin cellular expression were respectively increased and decreased at the same IS and P-cs concentrations for peak cells growth, migration, and invasion activities. This expected modulation of Vimentin and E-cadherin is expression of uremic toxins-related epithelial to mesenchymal transition of cultured cancer cells.
Our study demonstrates the ability of IS and P-cs in inducing an increased CRC aggressiveness and the transformation to a less differentiated cancer phenotype. In conclusion, gut dysbiosis-linked uremic toxins appear to have a pivotal role in CRC progression in CKD patients.