Enhanced TLR4 Expression on Colon Cancer Cells After Chemotherapy Promotes Cell Survival and Epithelial-Mesenchymal Transition Through Phosphorylation of GSK3β

Abstract

Background: Phosphorylation of glycogen synthase kinase 3 beta (GSK3 beta) by phosphatidyl-inositide 3-kinase (PI3K)/protein kinase B (AKT) or inhibition of GSK3 beta with small-molecule inhibitor attenuates cell survival and proliferation and increases apoptosis in most cancer cell lines. In this study, we investigated the role of phosphorylated GSK3 beta activated by enhanced toll-like receptor 4 (TLR4) expression in drug-treated colon cancer cells as a model of post-chemotherapy cancer cells. Materials and Methods: The effect of TLR4 stimulation on metastasis and apoptosis in drug-exposed colon cancer cells was determined by real-time polymerase chain reaction (PCR) and immunoblotting. Results: Despite the induction of apoptosis after treatment with oxaliplatin and 5-fluorouracil, lipopolysaccharide (LPS) stimulation via increased TLR4 in drug-treated cancer cells effectively inhibited apoptosis through up-regulation of expression of anti-apoptosis-related B-cell lymphoma 2 (BCL2) family proteins [X-linked inhibitor of apoptosis protein (XIAP), BCL2, and survivin] and drug-resistance proteins [multidrug-resistance protein 1 (MDR1), multidrug resistance-associated protein (MRP) 1/2/3]. LPS-mediated signaling in drug-treated cancer cells elevated the expression of phosphorylated GSK3 beta, extracellular signal-regulated kinase (ERK), and the p65 subunit of nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-kappa B). Pharmacological inhibition of GSK3 beta (using SB216763) reduced phosphorylation of GSK3 beta, re-activated caspase-dependent apoptosis, and blocked the expression of cancer stem cell markers and invasive characteristics in LPS-stimulated drug-treated cells. In addition, the ERK-specific inhibitor, PD98059, triggered the apoptosis of TLR4-activated drug-exposed colon cancer cells, whereas there was no effect on the expression of epithelial-mesenchymal transition markers or GSK3 beta phosphorylation. Conclusion: These results suggest that TLR4-induced GSK3 beta and ERK phosphorylation independently controls cancer cell survival and regulation of GSK3 beta and ERK after chemotherapy, making TLR4 a critical target for reducing drug resistance and metastasis in patients with colon cancer.