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LPS receptor subunits have antagonistic roles in epithelial apoptosis and colonic carcinogenesis.

ABSTRACT: Colorectal carcinoma (CRC) is characterized by unlimited proliferation and suppression of apoptosis, selective advantages for tumor survival, and chemoresistance. Lipopolysaccharide (LPS) signaling is involved in both epithelial homeostasis and tumorigenesis, but the relative roles had by LPS receptor subunits CD14 and Toll-like receptor 4 (TLR4) are poorly understood. Our study showed that normal human colonocytes were CD14(+)TLR4(-), whereas cancerous tissues were CD14(+)TLR4(+), by immunofluorescent staining. Using a chemical-induced CRC model, increased epithelial apoptosis and decreased tumor multiplicity and sizes were observed in TLR4-mutant mice compared with wild-type (WT) mice with CD14(+)TLR4(+) colonocytes. WT mice intracolonically administered a TLR4 antagonist displayed tumor reduction associated with enhanced apoptosis in cancerous tissues. Mucosa-associated LPS content was elevated in response to CRC induction. Epithelial apoptosis induced by LPS hypersensitivity in TLR4-mutant mice was prevented by intracolonic administration of neutralizing anti-CD14. Moreover, LPS-induced apoptosis was observed in primary colonic organoid cultures derived from TLR4 mutant but not WT murine crypts. Gene silencing of TLR4 increased cell apoptosis in WT organoids, whereas knockdown of CD14 ablated cell death in TLR4-mutant organoids. In vitro studies showed that LPS challenge caused apoptosis in Caco-2 cells (CD14(+)TLR4(-)) in a CD14-, phosphatidylcholine-specific phospholipase C-, sphingomyelinase-, and protein kinase C-?-dependent manner. Conversely, expression of functional but not mutant TLR4 (Asp299Gly, Thr399Ile, and Pro714His) rescued cells from LPS/CD14-induced apoptosis. In summary, CD14-mediated lipid signaling induced epithelial apoptosis, whereas TLR4 antagonistically promoted cell survival and cancer development. Our findings indicate that dysfunction in the CD14/TLR4 antagonism may contribute to normal epithelial transition to carcinogenesis, and provide novel strategies for intervention against colorectal cancer.

SUBMITTER: Kuo WT

PROVIDER: S-EPMC4563778 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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LPS receptor subunits have antagonistic roles in epithelial apoptosis and colonic carcinogenesis.

Kuo W-T WT Lee T-C TC Yang H-Y HY Chen C-Y CY Au Y-C YC Lu Y-Z YZ Wu L-L LL Wei S-C SC Ni Y-H YH Lin B-R BR Chen Y Y Tsai Y-H YH Kung J T JT Sheu F F Lin L-W LW Yu L C-H LC

Cell death and differentiation 20150130 10

Colorectal carcinoma (CRC) is characterized by unlimited proliferation and suppression of apoptosis, selective advantages for tumor survival, and chemoresistance. Lipopolysaccharide (LPS) signaling is involved in both epithelial homeostasis and tumorigenesis, but the relative roles had by LPS receptor subunits CD14 and Toll-like receptor 4 (TLR4) are poorly understood. Our study showed that normal human colonocytes were CD14(+)TLR4(-), whereas cancerous tissues were CD14(+)TLR4(+), by immunofluo ...[more]

PMID: 25633197

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Project description:A microarray analysis of an animal model with experimental sepsis induced by caecal ligation and puncture revealed that the level of microRNA?195 (miR?195) was upregulated. However, to the best of our knowledge, the role of miR?195 in sepsis remains unknown. The present study investigated the effect of miR?195 on apoptosis in sepsis and investigated the underlying mechanism. The level of miR?195 was measured in human intestinal epithelial cells following exposure to lipopolysaccharide (LPS). Cell viability and apoptosis were detected using Cell Counting kit?8 and flow cytometry assays. The expression levels of apoptosis?associated proteins were determined using western blot analysis. In addition, a dual?luciferase reporter assay was employed to verify the association between miR?195 and sirtuin 1 (SIRT1). Furthermore, the SIRT1 inhibitor EX527 was applied to further confirm the regulatory network of miR?195/SIRT1 in LPS?induced apoptosis. It was demonstrated that LPS significantly inhibited cell viability and promoted cell apoptosis in NCM460 cells in a dose?dependent manner. In addition, miR?195 was significantly upregulated following LPS treatment. The present results revealed that silencing miR?195 prevented apoptosis and alleviated cell injury in LPS?induced NCM460 cells. Further investigation demonstrated that miR?195 bound directly to and negatively regulated SIRT1. Inhibition of SIRT1 reversed the protective effects of miR?195?silencing on the apoptosis and viability of NCM460 cells. Furthermore, silencing miR?195 prevented endoplasmic reticulum (ER) stress?induced apoptosis via a downregulation of SIRT1 and its downstream effectors, including activating transcription factor 4, C/EBP homologous protein, glucose?regulated protein 78 and growth arrest and DNA?damage protein 34, as well as the phosphorylation of eukaryotic translation initiation factor 2A. In conclusion, the present study revealed a novel mechanism by which miR?195 regulates SIRT1?mediated downstream effectors in ER stress?induced apoptosis in sepsis.

Project description:Toll-like receptors (TLRs) belong to the group of pathogen recognition receptors known to play a crucial role in the innate immune system. In cancer, TLR expression is still debated controversially due to contradictory results reporting that both induction of apoptosis as well as tumor progression could depend on TLR signaling, whereby recent data rather indicate a pro-tumorigenic effect. The biological phenomenon of cell fusion has been associated with cancer progression due to findings revealing that fusion-derived hybrid cells could exhibit properties like an increased metastatogenic capacity and an increased drug resistance. Thus, M13MDA435 hybrid cell lines, which derived from spontaneous fusion events between human M13SV1-EGFP-Neo breast epithelial cells and human MDA-MB-435-Hyg breast cancer cells, were investigated. Cultivation of cells in the presence of the TLR4 ligand LPS potently induced apoptosis in all hybrid clones, but not in parental cells, which was most likely attributed to differential kinetics of the TLR4 signal transduction cascade. Activation of this pathway concomitant with NF-κB nuclear translocation and TNF-α expression was solely observed in hybrid cells. However, induction of LPS mediated apoptosis was not TNF-α dependent since TNF-α neutralization was not correlated to a decreased amount of dead cells. In addition to TNF-α, LPS also caused IFN-β expression in hybrid clones 1 and 3. Interestingly, hybrid clones differ in the mode of LPS induced apoptosis. While neutralization of IFN-β was sufficient to impair the LPS induced apoptosis in M13MDA435-1 and -3 hybrids, the amount of apoptotic M13MDA435-2 and -4 hybrid cells remained unchanged in the presence of neutralizing IFN-β antibodies. In summary, the fusion of non-LPS susceptible parental human breast epithelial cells and human breast cancer cells gave rise to LPS susceptible hybrid cells, which is in view with the cell fusion hypothesis that hybrid cells could exhibit novel properties.

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LPS receptor subunits have antagonistic roles in epithelial apoptosis and colonic carcinogenesis.

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LPS receptor subunits have antagonistic roles in epithelial apoptosis and colonic carcinogenesis.

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