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Inhibitory effect of Porphyromonas gingivalis-derived phosphoethanolamine dihydroceramide on acid ceramidase expression in oral squamous cells.

ABSTRACT: The maintenance of diminished acid ceramidase (ASAH1) gene expression leading to the accumulation of antiproliferative intracellular ceramides in oral squamous cell carcinoma (OSCC) has emerged as a prospective oral cancer therapeutic regimen. Our published study demonstrated that the key periodontal pathogen Porphyromonas gingivalis downregulates the expression patterns of ASAH1 mRNA in normal epithelial cells in vitro. Therefore, P. gingivalis may also beneficially diminish the expression of ASAH1 in OSCC. Because a uniquely structured P. gingivalis-derived phosphoethanolamine dihydroceramide (PEDHC) inhibits the proliferation of normal human fibroblasts, this study aimed to test the effect of PEDHC on the survival of human oral squamous OECM-1 cells in vitro. We demonstrated that the P. gingivalis dihydroceramide-null (ΔPG1780) strain upregulates the expression of ASAH1 mRNA and promotes aggressive proliferation and migration of OECM-1 cells compared to the parent P. gingivalis-W83 strain. In addition, the intracellular concentration of ceramides was dramatically elevated in OECM-1 cells exposed to PEDHC in vitro. Furthermore, PEDHC inhibited expression patterns of ASAH1 mRNA as well as some genes associated with degradation of the basement membranes and extracellular matrix, for example, MMP-2, ADAM-17 and IL-6, in OECM-1 cells. Altogether, these data indicated that PEDHC produced by P. gingivalis inhibits acid ceramidase expression, promotes intracellular ceramide accumulation and suppresses the survival and migration of OSCC cells in vitro. Further studies are needed to determine molecular mechanisms of PEDHC-mediated inhibitory effect(s) on OSCC using in vivo models of oral cancer.

SUBMITTER: Yamada C

PROVIDER: S-EPMC10148054 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature

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Inhibitory effect of Porphyromonas gingivalis-derived phosphoethanolamine dihydroceramide on acid ceramidase expression in oral squamous cells.

Yamada Chiaki C Ho Anny A Nusbaum Amilia A Xu Ruijuan R Davey Mary Ellen ME Nichols Frank F Mao Cungui C Movila Alexandru A

Journal of cellular and molecular medicine 20230405 9

The maintenance of diminished acid ceramidase (ASAH1) gene expression leading to the accumulation of antiproliferative intracellular ceramides in oral squamous cell carcinoma (OSCC) has emerged as a prospective oral cancer therapeutic regimen. Our published study demonstrated that the key periodontal pathogen Porphyromonas gingivalis downregulates the expression patterns of ASAH1 mRNA in normal epithelial cells in vitro. Therefore, P. gingivalis may also beneficially diminish the expression of A ...[more]

PMID: 37016912

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Project description:Ceramidases are a group of enzymes that degrade pro-inflammatory ceramide by cleaving a fatty acid to form anti-inflammatory sphingosine lipid. Thus far, acid, neutral and alkaline ceramidase isozymes have been described. However, the expression patterns of ceramidase isoforms as well as their role in periodontal disease pathogenesis remain unknown. In this study, expression patterns of ceramidase isoforms were quantified by real-time PCR and immunohistochemistry in gingival samples of patients with periodontitis and healthy subjects, as well as in EpiGingivalTM-3D culture and OBA-9 gingival epithelial cells both of which were stimulated with or without the presence of live Porphyromonas gingivalis (ATCC 33277 strain). A significantly lower level of acid ceramidase expression was detected in gingival tissues from periodontal patients compared to those from healthy subjects. In addition, acid-ceramidase expression in EpiGingival™ 3D culture and OBA-9 cells was suppressed by stimulation with P. gingivalis in vitro. No significant fluctuation was detected for neutral or alkaline ceramidases in either gingival samples or cell cultures. Next, to elucidate the role of acid ceramidase in P. gingivalis-induced inflammation in vitro, OBA-9 cells were transduced with adenoviral vector expressing the human acid ceramidase (Ad-ASAH1) gene or control adenoviral vector (Ad-control). In response to stimulation with P. gingivalis, ASAH1-over-expressing OBA-9 cells showed significantly lower mRNA expressions of caspase-3 as well as the percentage of Annexin V-positive cells, when compared with OBA-9 cells transduced with Ad-control vector. Furthermore, in response to stimulation with P. gingivalis, ASAH1-over-expressing OBA-9 cells produced less TNF-α, IL-6, and IL1β pro-inflammatory cytokines than observed in OBA-9 cells transduced with Ad-control vector. Collectively, our data show the novel discovery of anti-inflammatory and anti-apoptotic effects of acid ceramidase in host cells exposed to periodontal bacteria, and the attenuation of the expression of host-protective acid ceramidase in periodontal lesions.

Project description:BACKGROUND Oral squamous cell carcinoma (OSCC) is the most common head and neck malignancy, characterized by high recurrence rate resulting in poor prognosis. Porphyromonas gingivalis, most closely correlated with chronic periodontitis, is increasingly thought to play a significant role in OSCC development via influencing tumor-associated macrophages. However, its specific function remains unclear. In this study, we attempted to explore the mechanism of action of P. gingivalis in the recurrence of OSCC by bioinformatics analysis, to lay a foundation for subsequent basic experiments. MATERIAL AND METHODS The P. gingivalis-infected macrophage microarray dataset (GSE24897) and the OSCC advanced relapse patient microarray dataset (GSE87593) were retrieved from the Gene Expression Omnibus (GEO) database. Differentially-expressed genes (DEGs) were screened using R system, and the intersected DEGs were analyzed for functional enrichment, and protein-protein interaction (PPI) networks were constructed. The expression of significant DEG in GSE24897 microarray was assessed to determine its effect on macrophage immune infiltration in pan-cancer by applying the TIMER 2.0 repository. To detect the expression of DOK3 in OSCC specimens, immunohistochemical (ICH) assay was used. RESULTS A total of 106 co-expressed DEGs were upregulated and 131 were downregulation. The biological processes were mainly enriched in DNA-templated transcription terms, the cellular component enrichment was mainly enriched in the nucleus terms, and the molecular function enrichment was mainly enriched in protein-binding function terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that the DEGs were mainly enriched in the MAPK signaling pathway. Overall analysis of the PPI network showed a significant aggregation, with the top 10 hub co-expressed genes (CASP3, FYN, HNRNPA2B1, NR3C1, RELA, REL, POLR2F, RAN, RHOA, and STAT5B). DOK3 is significantly upregulated in P. gingivalis-infected macrophages, which is associated with macrophage infiltration and differentiation. There was more positive DOK3 staining in the group with P. gingivalis infection. CONCLUSIONS P. gingivalis can affect the recurrence of OSCC by increasing the expression of DOK3 in TAMs, which may be involved in activation of signaling pathways such as TNF and MAPK.

Project description:Porphyromonas gingivalis has been associated with progression of periodontitis, characterized by inflammation and destruction of periodontal tissues. Here, we report that matcha, a product of Camellia sinensis, hampers the adherence and survival of P. gingivalis through multiple tactics. Matcha extract (ME) inhibited the growth not only of P. gingivalis but also of Prevotella nigrescens and Fusobacterium nucleatum, while it did not inhibit growth of nine species of oral streptococci and Aggregatibacter actinomycetemcomitans. ME-mediated P. gingivalis growth inhibition was characterized by both morphological and physiological changes at the bacterial envelope, which were accompanied by nano-particle formation and decreased membrane fluidity/permeability without loss of membrane integrity. ME also triggered autoaggregation of P. gingivalis in a major fimbriae (FimA)-dependent manner. In addition, adherence of P. gingivalis was dramatically inhibited by ME, irrespective of fimbriae. Furthermore, a structure-activity relationship study tested a series of catechins isolated from ME and identified the pyrogallol-type B-ring of catechins as essential for P. gingivalis growth inhibition. In a clinical study to assess the microbiological and therapeutic effects of matcha mouthwash in patients with periodontitis, the P. gingivalis number in saliva was significantly reduced by matcha mouthwash compared to the pre-intervention level. A tendency toward improvement in probing pocket depth was observed in the matcha group, although the difference was not statistically significant. Taken together, we present a proof of concept, based on the multimodal inhibitory effect of matcha against P. gingivalis, and that matcha may have clinical applicability for prevention and treatment of periodontitis.ImportancePeriodontitis, a multifactorial inflammatory disease of the oral cavity, results in alveolar bone destruction, and is a major cause of tooth loss of humans. In addition, emerging evidence has demonstrated associations between periodontitis and a wide range of other chronic inflammation-driven disorders, including diabetes mellitus, preterm birth, cardiovascular disease, aspiration pneumonia, rheumatoid arthritis, cognitive disorder, and cancer. In the present study, we report that matcha, a product of Camellia sinensis, hampers Porphyromonas gingivalis, a major periodontal pathobiont, in not only a series of in vitro experiments but also a pilot intervention clinical trial of patients with periodontitis, in which matcha mouthwash statistically significantly reduced the P. gingivalis number in saliva, as compared to the pre-intervention level. Taken together, we suggest that matcha may have clinical applicability for prevention and treatment of periodontitis.

Project description:BackgroundAmong different types of sphingolipids produced by human cells, the possible engagement of ceramide species in the pathogenesis of Alzheimer's disease (AD) has attracted recent attention. While ceramides are primarily generated by de novo synthesis in mammalian cells, only a limited number of bacterial species, produce ceramides, including phosphoglycerol dihydroceramide (PGDHC) that is produced by the key periodontal pathogen Porphyromonas gingivalis. Emerging evidence indicates that virulence factors produced by P. gingivalis, such as lipopolysaccharide and gingipain, may be engaged in the initiation and/or progression of AD. However, the potential role of PGDHC in the pathogenesis of AD remains unknown. Therefore, the aim of this study was to evaluate the influence of PGDHC on hallmark findings in AD.Material and methodsCHO-7WD10 and SH-SY-5Y cells were exposed to PGDHC and lipopolysaccharide (LPS) isolated from P. gingivalis. Soluble Aβ42 peptide, amyloid precursor protein (APP), phosphorylated tau and senescence-associated secretory phenotype (SASP) factors were quantified using ELISA and Western blot assays.ResultsOur results indicate that P. gingivalis (Pg)-derived PGDHC, but not Pg-LPS, upregulated secretion of soluble Aβ42 peptide and expression of APP in CHO-7WD10 cells. Furthermore, hyperphosphorylation of tau protein was observed in SH-SY-5Y cells in response to PGDHC lipid. In contrast, Pg-LPS had little, or no significant effect on the tau phosphorylation induced in SH-SY-5Y cells. However, both PGDHC and Pg-LPS contributed to the senescence of SH-SY5Y cells as indicated by the production of senescence-associated secretory phenotype (SASP) markers, including beta-galactosidase, cathepsin B (CtsB), and pro-inflammatory cytokines TNF-α, and IL-6. Additionally, PGDHC diminished expression of the senescence-protection marker sirtuin-1 in SH-SY-5Y cells.ConclusionsAltogether, our results indicate that P. gingivalis-derived PGDHC ceramide promotes amyloidogenesis and hyperphosphorylation, as well as the production of SASP factors. Thus, PGDHC may represent a novel class of bacterial-derived virulence factors for AD associated with periodontitis.

Dataset Information

Inhibitory effect of Porphyromonas gingivalis-derived phosphoethanolamine dihydroceramide on acid ceramidase expression in oral squamous cells.

Publications

Inhibitory effect of Porphyromonas gingivalis-derived phosphoethanolamine dihydroceramide on acid ceramidase expression in oral squamous cells.

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