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.


| INTRODUC TI ON
Oral squamous cell carcinoma (OSCC) is one of the most common malignant cancers of the head and neck and is among the top 10 common cancers worldwide. 1 Notably, OSCC can alter sphingolipid metabolism towards increasing proliferative species such as sphingosine-1-phosphate (S1P) while decreasing antiproliferative species such as ceramide. Furthermore, the ceramide/S1P ratio is regulated by acid ceramidase (ASAH1). 2,3 Therefore, the maintenance of diminished ASAH1 gene expression leading to the accumulation of intracellular ceramide in OSCC has emerged as a potential objective for oral cancer therapy.

We previously reported that infection with oral bacteria
Porphyromonas gingivalis downregulates the expression of ASAH1 mRNA in normal epithelial cells, thus promoting inflammation and cell apoptosis associated with periodontal disease. 4 Emerging evidence demonstrated that a uniquely structured phosphoethanolamine dihydroceramide (PEDHC), produced by microbes from Bacteroides spp., including P. gingivalis, contributes to intracellular ceramide accumulation in normal epithelial tissue. 5,6 While the association of P. gingivalis with oral and neck cancer pathology was intensively studied, 7,8 the effects of P. gingivalis-derived PEDHC on the OSCC remains unclear.
In this study, we investigated the impact of P. gingivalis-W83, W83-dihydroceramide null mutant strain (ΔPG1780) and PEDHC on the proliferation and migration of OSCC in vitro. Here, we also addressed whether PEDHC affects intracellular ceramide metabolism in OSCC.

| MATERIAL S AND ME THODS
Detailed materials and methods are provided in the Supplementary Material and Methods.

| RE SULTS AND D ISCUSS I ON
Among the known clinically relevant P. gingivalis strains, the W83 strain contributes to severe periodontitis in various experimental mouse models. 9,10 Therefore, P. gingivalis-W83 and ΔPG1780 were used to infect healthy human oral gingival OBA-9 and squamous cell carcinoma OECM-1 cells in vitro. To our knowledge, exposure of OBA-9 cells to either P. gingivalis-W83 or ΔPG1780 significantly inhibited their proliferation. In contrast, no or little effect of P. gingivalis-W83 was observed on the proliferation of OECM-1 cells, whereas exposure to ΔPG1780 increased the proliferation of OECM-1 cells ( Figure 1A). We also observed that exposure to live P. gingivalis-W83 or ΔPG1780 significantly elevated migration of OECM-1 cells compared to a non-infected controls, with the mutant strain being more effective than the wild-type strain ( Figure 1B The critically low concentration of intracellular ceramide sphingolipids in oral squamous carcinoma could be caused by increased ceramidases activity which occupies a powerful position in the catabolism of pro-apoptotic ceramide and generation of pro-survival S1P bioactive lipid. 14-16 Among the known ceramidases, the expression pattern of acid ceramidase (ASAH1) mRNA was dramatically elevated in OECM-1 cells compared to OBA-9 cells ( Figure S2). Since we reported that only ASAH1 was inhibited in normal OBA-9 cells by P. gingivalis-ATCC3327 strain, 4 we validated the effect(s) of the acid ceramidase chemical inhibitor LCL-521 on the proliferation and migration of oral OECM-1 squamous cells. As expected, LCL-521 significantly reduced the expression of ASAH1 mRNA in OECM1 cells in a concentration-dependent manner ( Figure S3A). Furthermore, expression of NF-kB, MMP2, ADAM17 and IL6 genes were diminished in OECM-1 cells exposed to LCL-521 ( Figure S3B). We also detected that LCL-521 downregulated expression of S1PR1, and S1PR3 mRNAs ( Figure S3C). These S1PR1 and S1PR3 congenic receptors are significant in the S1P-mediated survival and chemotaxis of cancer cells. 14,17 Finally, we also observed that the viability and migration of OECM-1 were significantly inhibited by LCL-521 compared to the sham control ( Figure S3B-D).
Because our data indicated that acid ceramidase plays an essential role in the regulation of cancer cell viability, we postulated next that PEDHC affects OECM-1 survival via downregulation of ASAH1 mRNA. Exposure of OECM-1 cells to the ΔPG1780 strain significantly elevated the expression of ASAH1 mRNA when compared to P. gingivalis-W83. Furthermore, we observed no significant effect of P. gingivalis-W83 on the ASAH1 expression in OECM-1 cells ( Figure S4A). In addition, a concentration-dependent downregulation of ASAH1 was observed in OECM-1 cells exposed to the PEDHC ( Figure S4B). These data agree with earlier published observations indicating that acid ceramidase is critical in the survival of cancer stem cells in melanoma, glioblastoma and colon cancer. 2,18 F I G U R E 1 Effect of live Porphyromonas gingivalis (P.g) dihydroceramide sphingolipids null ΔPG1780 (P.g-Sph Null ), and P. gingivalis-W83 wild-type (P.g-WT) control strains, and isolated PEDHC on the proliferation and migration of oral squamous OECM-1. (A) Proliferation of oral squamous cancer OECM-1 cells in the presence of P.g-WT and P.g-Sph Null strains. (B) Representative images of scratched and recovered wounded areas (marked by black lines) on confluence monolayers of OECM-1 cancer cells at different time points exposed to P.g-WT and P.g-Sph Null strains. Furthermore, it was also demonstrated that genetic and pharmacological inhibition of acid ceramidase prevents asymmetric cell division, which is frequently observed in cancer patients. 19 Therefore, the maintenance of diminished acid ceramidase expression or activity leading to the accumulation of intracellular ceramide in OSCC and other types of cancer has emerged as a potential objective for cancer therapy. 20,21 Finally, we tested the impact of PEDHC on the S1P-mediated migration of OECM1 and the expression of S1PR1 and S1PR3 receptors.
To our knowledge, migration of OECM-1 cells significantly elevated in response to S1P (Figure 2A, B). In contrast, the S1P-mediated migration of OECM-1 was diminished in the presence of PEDHC in vitro. In addition, PEDHC downregulated the expression patterns of NF-kB, and IL6 mRNAs as well as S1PR1 and S1PR3 mRNAs in OECM-1 cells elicited by S1P ( Figure 2C, D; Figure S4B). Besides the positive pleiotropic impact of S1P on physiological chemoattraction, several studies also reported that the ligation of S1P to its S1PR1 and S1PR3 congenic receptors promotes chemotactic migration of F I G U R E 2 Porphyromonas gingivalis-derived PEDHC inhibits S1P-mediated migration of OECM-1 cells. Representative images (A) and migration quantification (B) of oral squamous OECM-1 cells at different time points exposed to S1P (1 μM) alone or in combination with PEDHC (1 μg/mL). Expression patterns of S1PR receptors, S1PR1 and S1PR3, (C) and genes associated with degradation of the basement membrane and extracellular matrix, including NF-kB, MMP2, ADAM17, and IL-6 (D) in OECM-1 cells. Data are shown from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. cancer cells. 17 More specifically, S1P/S1PR1 axis accelerates tumour progression by upregulating IL-6. 22 In addition, S1PR1 can also activate ERK to enhance cell survival and promote cell migration in fibrosarcoma and Hodgkin lymphoma. 23 Furthermore, human breast cancers predominantly express S1PR3. 24 Our data also agree with earlier published observations indicating that the bacterial-derived lipids induce host immune responses and contribute to the S1Pmediated signalling. 5,25 Collectively, these data indicated that PEDHC downregulates

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors have no conflicts of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data available on request from the authors.