Inhibitory Mechanism of IL-6 Production by Orento in Oral Squamous Cell Carcinoma Cell Line CAL27 Stimulated by Pathogen-Associated Molecular Patterns from Periodontopathogenic Porphyromonas gingivalis

Abstract

Orento is a traditional Japanese medicinal kampo preparation that is also prescribed in oral care. In oral squamous cell carcinoma cell line CAL27, orento significantly inhibited periodontopathogenic bacterium Porphyromonas gingivalis lipopolysaccharide (LPS) and lipoproteins (PAMP)-stimulated production of interleukin (IL)-6. This suggests that orento negatively regulates PAMP-mediated toll-like receptor (TLR) signaling. Orento significantly suppressed PAMP-stimulated activation of the IL-6 promoter, indicating that orento may suppress the production of IL-6 by PAMP at the transcriptional level. Orento also suppressed TLR-mediated activation of transcription factor nuclear factor-kappa B (NF-kB) that was stimulated by PAMP. This finding indicates that orento may suppress the function and activation of factors involved in TLR signaling, thereby suppressing NF-kB-dependent expression of various genes. Orento suppressed IL-1 receptor-associated kinase (IRAK4), IRAK1, and c-Jun N-terminal kinase (JNK) phosphorylation in PAMP-stimulated CAL27 cells. This result indicates that orento is involved in the initiation of TLR signaling by PAMP and suppresses the downstream signaling pathways of myeloid differentiation primary response gene 88 (MyD88) such as mitogen-activated protein kinase (MAPK) and NF-kB cascades. These findings suggest that orento has an inhibitory effect on the production of inflammatory cytokines.

Hangeshashinto Inhibits Porphyromonas gingivalis Pathogen-Associated Molecular Patterns-Mediated IL-6 and IL-8 Production through Toll-Like Receptors in CAL27 Cells

“…Hangeshashinto was provided by Tsumura (Tokyo, Japan). P. gingivalis LPS (InvivoGen, California, USA, referred to as “PAMP” in this study) (see Imamura et al [ 10 ]). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dexamethasone (Sigma-Aldrich, Missouri, USA); resatorvid and C29 (Selleck Biotech, Kanagawa, Japan); anti-IL-6 antibody and biotinylated anti-IL-6 antibody (Thermo Fisher Scientific (eBioscience), Massachusetts, USA); anti-IL-8 antibody and biotinylated anti-IL-8 antibody (R&D Systems, Minnesota, USA).…”

“…Then, 0.02 N HCl-isopropanol was added to the medium to dissolve and solubilize the formazan product completely. Samples were measured using a Microplate Reader model 550 (Bio-Rad Labs, Inc., Hercules, California, USA) with dual wavelengths of 595/655 nm (test/reference), as previously described [ 10 ].…”

“…Samples were measured using a Microplate Reader model 550 with dual wavelengths of 450/655 nm (test/reference). The ELISA procedure was carried out as described in the CytoSet Kit User Manual (Thermo Fisher Scientific (BioSource)) [ 10 ].…”

“…One microgram of phIL-6pro-Luc (a fusion of the human IL-6 promoter region spanning from position 2636 to 5035 of GenBank Accession Number NG_011640 linked to the luciferase gene ( Figure 2(a) ) [ 10 ], along with 0.1 μ g of pRSV- β -gal (standard plasmid), was mixed with TransIT-LT1 reagent (Mirus Bio, Wisconsin, USA). Additionally, 1 μ g of pIg κ -Luc ( Figure 3(a) ) [ 10 ] and 0.1 μ g of pRSV- β -gal were also mixed with TransIT-LT1 reagent. The mixtures were then added to the cells (3 × 10 5 ), which were subsequently cultured for 24 hours.…”

“…We aim to employ an experimental system using oral cancer cells to assess the impact of hangeshashinto on intracellular signal transduction pathways in response to stimulation by Porphyromonas gingivalis ( P. gingivalis ) pathogen-associated molecular patterns (PAMP). P. gingivalis is a bacterium associated with periodontitis and serves as a reliable stimulus for IL-6 production in oral cancer cells [ 10 ]. An experimental system has been established that utilizes P. gingivalis LPS as a model for inflammation, inducing the production of inflammatory cytokines in cultured cells [ 11 ].…”