Electrolytically generated acid functional water inhibits NF-κB activity by attenuating nuclear-cytoplasmic shuttling of p65 and p50 subunits

Background: Human β-defensin 2 (hBD2) gene expression is dependent on nuclear factor kappa B (NF-κB) activity. We have previously demonstrated that electrolytically generated acid functional water (FW) induces the expression of hBD2 in the human oral squamous cell carcinoma (OSCC) cell line Ca9-22. However, the induction was not dependent on NF-κB activity; in fact, FW inhibited NF-κB activity. Therefore, we hypothesized that FW might reduce spontaneous interleukin 8 (IL-8) secretion by Ca9-22 cells, which is heavily dependent on NF-κB activity. This study aimed at demonstrating the inhibitory effect of FW on NF-κB activity. Methods: Ca9-22 cells were incubated with FW, and spontaneous IL-8 secretion was observed by enzyme-linked immunosorbent assay. Luciferase assay was performed using the 5′-untranslated region of the IL-8 gene. The steps of NF-κB activation blocked by FW were evaluated by localization of the NF-κB subunits p65 and p50 by immunofluorescence staining. Western blotting was further performed to confirm the changes in NF-κB subunit localization. Results: The Ca9-22 cells spontaneously secreted IL-8, which was rapidly and drastically inhibited by FW treatment. The luciferase assay demonstrated the inhibitory action of FW, which was diminished by deletion of the NF-κB binding site from this construct. FW treatment altered the distribution of both the p65 and p50 subunits. P65, which was localized in the nucleus during the resting state, moved to the cytoplasm after FW treatment, whereas, p50, localized in the cytoplasm during the resting state, moved to the nucleus subsequent to FW treatment. Conclusions: The results from this study indicate that FW might inhibit spontaneous IL-8 secretion by redistribution of the NF-κB subunits within the cells.

Acid-electrolyzed functional water induces extracellular matrix metalloproteinase inducer, a possible novel alarmin, secretion from oral squamous cell carcinoma cell lines

Extracellular matrix metalloproteinase inducer (EMMPRIN) secretion was induced in the oral squamous cell carcinoma cell line HSC3 cell by acid-electrolyzed functional water (FW) stimulation. Augmented EMMPRIN secretion was not under transcriptional control; rather, it was derived from the intracellular storages. EMMPRIN secretion was also induced under oxidative stress and accompanied by the release of lactate dehydrogenase (LDH). The molecules released from cells undergoing necrosis are called as alarmins, and the secretion of IL-1α, a typical alarmin, was induced by FW stimulation and oxidative stress. Intracellular localization was examined by cell fractionation. A significant amount of EMMPRIN was localized in the triton X-100 and DNase sensitive fractions; the levels were drastically reduced following FW treatment. The function of the released EMMPRIN was examined using the monocytic cell line THP1. Culture supernatant derived from FW-treated HSC3 cells induced the expression of matrix metalloproteinases (MMPs) 1, 2, 8, 9, 13, and 14, platelet-derived growth factor, and interleukin-8. In contrast, vascular endothelial growth factor expression was reduced. Induction of these factors was abolished following eliminating of EMMPRIN by immunoprecipitation. These results indicate that EMMPRIN might be considered as a type of alarmin that transduces danger signals to the surrounding cells.

Acid-electrolyzed functional water-induces Interleukin-1α release from Intracellular Storage Sites in Oral Squamous Cell Carcinoma

The aim of this study was to examine the acid-electrolyzed functional water (FW)-mediated cytokine release in an oral squamous cell carcinoma-derived cell line (OSCC) following treatment with FW. FW is generated by the electrolysis of a sodium chloride solution and accelerate the burn wound healing. To elucidate the underlying mechanisms, the cytokine/chemokine secretion profile of HSC3 cells was examined using a cytokine array. FW treatment significantly induced interleukin (IL)-1α secretion, which was confirmed by enzyme-linked immunosorbent assay. Subsequently, the HSC3 cells were pre-treated with cycloheximide (CHX) for 1 h prior to FW stimulation to determine whether the augmented IL-1α secretion was due to enhanced protein synthesis. CHX pre-treatment did not affect IL-1α secretion suggesting that the secreted IL-1α might have been derived from intracellular storage sites. The amount of IL-1α in the cell lysate of the FW-treated HSC3 cells was significantly lower than that of the non-treated cells. Immunofluorescence staining using a polyclonal antibody against full-length IL-1α revealed a drastic reduction in IL-1α inside the FW- treated cells. IL-1α is synthesized in its precursor form (pIL-1α) and cleaved to produce pro-piece and mature IL-1α (ppIL-1α and mIL-1α) inside the cells. In the present study, only pIL-1α was detected within the HSC3 cells in its resting state. However, FW stimulation resulted in the release of the 33 kDa and two other smaller forms (about 19 kDa) of the protein. These results indicates that FW treatment induces IL-1α secretion, a typical alarmin, from the intracellular storage in OSCC cells.