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Updated: Jun 8, 2026
Discovery of Driver Genes in Colorectal HT29-derived Cancer Stem-Like Tumorspheres
Published on: July 22, 2020
[Study on the molecular expression and regulation of toll pathway in HT-29 cells]
1Department of Gastroenterology, West China Hospital, Sichuan University, Chengdu 610041, China.
Objective:
To investigate the changes of TLR4, MD2 and HBD2 in transcription and protein expression levels in HT-29 colon cancer cell line after inducing by cytokines TNF-alpha, IL-1beta, IFN-gamma and LPS, and its relationship with NF-kappaB activation.
Methods:
HT-29 cells were divided into 8 groups, by adding RPMI 1640, TNF-alpha (20 ng/mL), IL-1beta (20 ng/mL), IFN-gamma (20 ng/mL), LPS (50 ng/mL), TNF-alpha (20 ng/mL) + LPS (50 ng/ mL), IL-1beta (20 ng/mL) + LPS (50 ng/mL), IFN-gamma (20 ng/mL) + LPS(50 ng/mL) respectively for intervention. ELISA was applied to detect the IL-8 in the supernatants of each group. The level of TLR4, MD2 and HBD2 mRNA were assayed by RT-PCR. The expressions of TLR4 and NF-kappaB protein of each group were determined by western blot.
Results:
IL-8 expressions in supernatant of cytokines and cytokine plus LPS group were higher than that of control (P < 0.01). Pre-incubation with cytokines, following by LPS stimulation, HT-29 cells further augmented IL-8 secretion (P < 0.01). Increased levels of TLR4 and MD2 mRNA in HT-29 cells with Cytokines and cytokine plus LPS stimulating were observed (P < 0.01). The level of HBD2 mRNA were elevated in IL-1beta and LPS plus (TNF-alpha, IL-1beta, IFN-gamma) groups (P < 0.05). Elevated expressions of TLR4 and NF-kappaB protein in HT-29 cells with cytokine and cytokine plus LPS were also observed (P < 0.01). Pre-incubation with cytokines, following by LPS stimulation, HT-29 cells further up-regulated NF-kappaB protein expression (P < 0.05).
Conclusions:
Cytokines (TNF-alpha, IL-1beta, IFN-gamma) can enhance TLR4 and MD-2 expressions and promote the reaction with LPS in intestinal epithelial cells (IEC), which leads to over activity of IEC with commensal bacteria and initiation or aggravation of intestinal inflammation. Inflammatory stimuli (IL-1beta, LPS + TNF-alpha, LPS + IFN-gamma) may induce the transcription and expression of HBD2 gene in IEC, which leads to enhancing intestinal adaptive immune responses and affects the progress of intestinal inflammation.
Insights
Cytokines and LPS increase TLR4, MD-2, and NF-kappaB activation in colon cancer cells. This suggests a role in intestinal inflammation by enhancing immune responses and bacterial interactions.
Area of Science:
- Immunology
- Cell Biology
- Gastroenterology
Context:
- Intestinal epithelial cells (IECs) play a crucial role in maintaining gut homeostasis and immune surveillance.
- Toll-like receptor 4 (TLR4) and its co-receptor MD-2 are key sensors of bacterial components like lipopolysaccharide (LPS).
- Cytokines such as TNF-alpha, IL-1beta, and IFN-gamma are critical mediators of inflammation in the gut.
Purpose:
- To investigate the impact of specific cytokines (TNF-alpha, IL-1beta, IFN-gamma) and LPS on the expression of TLR4, MD-2, and human beta-defensin 2 (HBD2) in HT-29 colon cancer cells.
- To examine the relationship between these molecular changes and the activation of the NF-kappaB signaling pathway.
- To elucidate the role of these interactions in the context of intestinal inflammation.
Summary:
- Treatment of HT-29 cells with cytokines and/or LPS significantly increased IL-8 secretion.
- Elevated mRNA levels of TLR4 and MD-2 were observed following cytokine and LPS stimulation.
- HBD2 mRNA levels were upregulated by IL-1beta and combined cytokine-LPS treatments, while TLR4 and NF-kappaB protein expressions were increased across multiple treatment groups.
Impact:
- Cytokines enhance TLR4/MD-2 expression and LPS responsiveness in IECs, potentially leading to overactivation and inflammation.
- Inflammatory stimuli can induce HBD2 expression, modulating adaptive immune responses and influencing intestinal inflammation progression.
- These findings highlight molecular mechanisms underlying cytokine and bacterial component interactions in intestinal inflammation, relevant for colon cancer research.
