Profiling of plasma-derived exosomal RNA expression in patients with periodontitis: A pilot study.

OBJECTIVE: This study aimed to profile differentially expressed (DE) exosomal RNAs in healthy subjects and periodontitis patients and compare their levels before and after treatment. MATERIALS AND METHODS: Plasma samples from healthy subjects and patients with periodontitis (pre-/post-periodontal treatment) were collected for this case-control study. After isolation of exosomes from the plasma, the RNA was extracted and small RNA sequencing was performed (3 healthy samples, 4 pre-treatment samples, and 5 post-treatment samples). Two-way analyses were conducted according to the treatment status in the periodontitis group, unpaired analysis (grouping as pre-/post-treatment) and paired analysis (matching pre- and post-treatment in the same subject). The DE exosomal RNAs were screened by sequencing and visualized using the R software. Gene Ontology analysis was performed, and target genes were identified. RESULTS: In both paired and unpaired analyses, two DE microRNAs (DEmiRs; miR-1304-3p and miR-200c-3p) and two DE small nucleolar RNAs (DEsnoRs; SNORD57 and SNODB1771) were common, and they were found to be downregulated during periodontitis and recovered to healthy levels after treatment. The top three target genes (NR3C1, GPR158, and CNN3) commonly regulated by DEmiRs were identified. CONCLUSIONS: Plasma-derived exosomal miRs (miR-1304-3p and miR-200c-3p) and snoRs (SNORD57 and SNODB1771) could be valuable biomarkers for periodontitis.

Isobutyrylshikonin has a potentially stronger cytotoxic effect in oral cancer cells than its analogue shikonin in vitro.

OBJECTIVES: The aim of the present study was to identify the anticancer effects and the mechanisms of action of shikonin and its analogue isobutyrylshikonin in oral squamous carcinoma cells. DESIGNS: The cytotoxic effects of isobutyrylshikonin and shikonin in Ca9-22 and SCC-25 cells were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry analysis of Annexin V/Propidium Iodide (PI) staining, western blot analysis and immunohistochemistry. RESULTS: Treatment with both isobutyrylshikonin and shikonin induced dose- and time-dependent apoptotic cell death in Ca9-22 cells, although the IC50 of isobutyrylshikonin was less than that of shikonin. The induction of apoptosis by both isobutyrylshikonin and shikonin was accompanied by activation of caspase-8, -9, -3, and PARP, loss of mitochondrial trans-membrane potential, and release of cytochrome c from the mitochondria. ROS mediated the apoptosis induced by isobutyrylshikonin and shikonin, indicating that ROS may play a critical role in the distinctive cytotoxic effects of isobutyrylshikonin and shikonin in Ca9-22. Isobutyrylshikonin showed a similar cytotoxic effect in SCC-25 cells at concentrations showing the effects in Ca9 cells, but not in human normal keratinocyte cells. Although there is no biological difference between isobutyrylshikonin and shikonin, isobutyrylshikonin exerts the same cytotoxic effect at a concentration 6 times lower than shikonin. CONCLUSIONS: The present study suggest that isobutyrylshikonin may be a more potent chemotherapeutic agent against oral cancer cells than shikonin. In addition, our data exhibit that both isobutyrylshikonin and shikonin induce caspase-dependent apoptosis via the mitochondrial pathway through accumulation of ROS in oral squamous carcinoma cells.

Serum Levels of Interleukin-6 and Titers of Antibodies Against Porphyromonas gingivalis Could Be Potential Biomarkers for the Diagnosis of Oral Squamous Cell Carcinoma.

It has been suggested that Porphyromonas gingivalis (P. gingivalis), a keystone pathogen in chronic periodontitis, is associated with a variety of cancers, including oral cancer. Recently, studies have shown the effects of persistent exposure to P. gingivalis on the promotion of tumorigenic properties of oral epithelial cells, suggesting that chronic P. gingivalis infection is a potential risk factor for oral cancer. On the other hand, Fusobacterium nucleatum (F. nucleatum), one of the major periodontal pathogens, has emerged as an important factor in the colon cancer progression. Here, we investigated the diagnostic potential of serum immunoglobulin G antibody against periodontal pathogens, P. gingivalis and F. nucleatum, and serum IL-6 for oral squamous cell carcinoma (OSCC). An enzyme-linked immunosorbent assay (ELISA) was used to determine and compare the serum levels of interleukin 6 (IL-6), F. nucleatum IgG, and P. gingivalis IgG in 62 OSCC patients with 46 healthy controls. The serum levels of P. gingivalis IgG and IL-6 were higher in OSCC patients than in non-OSCC controls, and the difference was statistically significant. In addition, a high serum level of IL-6 was associated with a worse prognosis in OSCC patients. Thus, P. gingivalis IgG and IL-6 could be utilized as potential serum biomarkers for the diagnosis of OSCC, and the serum level of IL-6 contributes to improved prognostic performance.

Oral Administration of Porphyromonas gingivalis, a Major Pathogen of Chronic Periodontitis, Promotes Resistance to Paclitaxel in Mouse Xenografts of Oral Squamous Cell Carcinoma.

Chemotherapy is not a first-line therapy for oral squamous cell carcinoma (OSCC), which is the most common type of oral cancer, because most OSCC shows resistance to chemotherapeutic reagents. Inflammatory signals are suggested to be associated with chemoresistance as well as carcinogenesis in many different cancers, and thus chronic periodontitis, the most common chronic inflammatory disease of the oral cavity, could modulate responsiveness to chemotherapeutic agents used against oral cancer. This study was performed to define the role of chronic periodontitis in oral cancer progression and to determine the responsiveness of oral cancer to a chemotherapeutic reagent. First, we quantified the tumor growth rate and changes in serum cytokine profiles of mice administered Porphyromonas gingivalis, a major pathogen of chronic periodontitis. Compared with uninfected mice, the mice that were chronically administered P. gingivalis showed increased resistance to paclitaxel and a decreased tumor growth rate. In addition, P. gingivalis-treated mice exhibited higher serum levels of interleukin-6 (IL-6) than uninfected mice. Furthermore, the sensitivity of tumor xenografts to paclitaxel in mice administered P. gingivalis was dramatically increased when the mice were administered ibuprofen, an anti-inflammatory drug which supports the modulatory effect of periodontal pathogen-induced inflammation in chemoresistance.

Acetylshikonin suppresses invasion of Porphyromonas gingivalis­infected YD10B oral cancer cells by modulating the interleukin-8/matrix metalloproteinase axis.

The development of pharmaceutical agents possessing anti­invasive and anti­metastatic abilities, as well as apoptotic activity, is important in decreasing the incidence and recurrence of oral cancer. Cancer cells are known to acquire invasiveness not only through epigenetic changes, but also from inflammatory stimuli within the tumor microenvironment. Accordingly, the identification of agents that can suppress the inflammation­promoted invasiveness of cancer cells may be important in treating cancer and improving the prognosis of patients with cancer. Acetylshikonin, a flavonoid with anti­inflammatory activity, inhibits proliferation and induces apoptosis of oral cancer cells. In the present study, the anti­invasive effect of acetylshikonin on YD10B oral cancer cells infected with Porphyromonas gingivalis, a major pathogen of chronic periodontitis, and the mechanisms involved were investigated. Firstly, we examined whether P. gingivalis infection increased the invasiveness of YD10B cells. Results suggested that YD10B oral cancer cells become more aggressive when they are infected with P. gingivalis. Secondly, acetylshikonin significantly inhibited the invasion of P. gingivalis­infected YD10B cells by suppressing IL­8 release and IL­8­dependent MMP release. These data suggest that acetylshikonin may be a useful preventive and therapeutic candidate for oral cancer that is chronically infected with periodontal pathogens.

Oral cancer cells sustainedly infected with Porphyromonas gingivalis exhibit resistance to Taxol and have higher metastatic potential.

Major obstacles to improving the prognosis of patients with oral squamous cell carcinoma (OSCC) are the acquisition of resistance to chemotherapeutic agents and development of metastases. Recently, inflammatory signals are suggested to be one of the most important factors in modulating chemoresistance and establishing metastatic lesions. In addition, epidemiological studies have demonstrated that periodontitis, the most common chronic inflammatory condition of the oral cavity, is closely associated with oral cancer. However, a correlation between chronic periodontitis and chemoresistance/metastasis has not been well established. Herein, we will present our study on whether sustained infection with Porphyromonas gingivalis, a major pathogen of chronic periodontitis, could modify the response of OSCC cells to chemotherapeutic agents and their metastatic capability in vivo. Tumor xenografts composed of P. gingivalis-infected OSCC cells demonstrated a higher resistance to Taxol through Notch1 activation, as compared with uninfected cells. Furthermore, P. gingivalis-infected OSCC cells formed more metastatic foci in the lung than uninfected cells.

Porphyromonas gingivalis increases the invasiveness of oral cancer cells by upregulating IL-8 and MMPs.

Recent studies indicate that chronic inflammation promotes the aggressiveness of cancers. However, the direct molecular mechanisms underlying a functional link between chronic periodontitis, the most common form of oral inflammatory diseases, and the malignancy of oral cancer remain unknown. To elucidate the role of chronic periodontitis in progression of oral cancer, we examined the effect of Porphyromonas gingivalis (P. gingivalis), a major pathogen that causes chronic periodontitis, on the invasiveness of oral squamous cell carcinoma (OSCC) cells, including SCC-25, OSC-20 and SAS cells. Exposures to P. gingivalis promoted the invasive ability of OSC-20 and SAS cells via the upregulation of matrix metalloproteinases (MMPs), specifically MMP-1 and MMP-2. However, P. gingivalis-infected SCC-25 cells did not exhibit changes in their invasive properties or the low expression levels of MMPs. In an effort to delineate the molecular players that control the invasiveness, we first assessed the level of interleukin-8 (IL-8), a well-known inflammatory cytokine, in P. gingivalis-infected OSCC cells. IL-8 secretion was substantially increased in the OSC-20 and SAS cells, but not in the SCC-25 cells, following P. gingivalis infection. When IL-8 was directly applied to SCC-25 cells, their invasive ability and MMP level were significantly increased. Furthermore, the downregulation of IL-8 in P. gingivalis-infected OSC-20 and SAS cells attenuated their invasive potentials and MMP levels. Taken together, our findings strongly suggest that P. gingivalis infection plays an important role in the promotion of the invasive potential of OSCC cells via the upregulation of IL-8 and MMPs.

Prolonged and repetitive exposure to Porphyromonas gingivalis increases aggressiveness of oral cancer cells by promoting acquisition of cancer stem cell properties.

Periodontitis is the most common chronic inflammatory condition occurring in the human oral cavity, but our knowledge on its contribution to oral cancer is rather limited. To define crosstalk between chronic periodontitis and oral cancer, we investigated whether Porphyromonas gingivalis, a major pathogen of chronic periodontitis, plays a role in oral cancer progression. To mimic chronic irritation by P. gingivalis in the oral cavity, oral squamous cell carcinoma (OSCC) cells were infected with P. gingivalis twice a week for 5 weeks. Repeated infection of oral cancer cells by P. gingivalis resulted in morphological changes of host cancer cells into an elongated shape, along with the decreased expression of epithelial cell markers, suggesting acquisition of an epithelial-to-mesenchymal transition (EMT) phenotype. The prolonged exposure to P. gingivalis also promoted migratory and invasive properties of OSCC cells and provided resistance against a chemotherapeutic agent, all of which are described as cellular characteristics undergoing EMT. Importantly, long-term infection by P. gingivalis induced an increase in the expression level of CD44 and CD133, well-known cancer stem cell markers, and promoted the tumorigenic properties of infected cancer cells compared to non-infected controls. Furthermore, increased invasiveness of P. gingivalis-infected OSCC cells was correlated with enhanced production of matrix metalloproteinase (MMP)-1 and MMP-10 that was stimulated by interleukin-8 (IL-8) release. This is the first report demonstrating that P. gingivalis can increase the aggressiveness of oral cancer cells via epithelial-mesenchymal transition-like changes and the acquisition of stemness, implicating P. gingivalis as a potential bacterial risk modifier.

Curcumin-induced autophagy contributes to the decreased survival of oral cancer cells.

Curcumin, a major active component of turmeric Curcuma longa, has been shown to have inhibitory effects on cancers. In vitro studies suggest that curcumin inhibits cancer cell growth by activating apoptosis, but the mechanism underlying the anticancer effects of curcumin is unclear. Recently, it has been suggested that autophagy may play an important role in cancer therapy. However, little data are available regarding the role of autophagy in oral cancers. In this study, we have shown that curcumin has anticancer activity against oral squamous cell carcinoma (OSCC). Induction of autophagy, marked by autophagic vacuoles formation, was detected by acridine orange staining and monodansylcadaverine (MDC) dye after exposure to curcumin. Conversion of LC3-I to LC3-II, a marker of active autophagosome formation, was also detectable by Western blot following curcumin treatment. We have also observed that curcumin induced reactive oxygen species (ROS) production and autophagic vacuoles formation by curcumin was almost completely blocked in the presence of N-acetylcystein (NAC), an antioxidant. Rescue experiments using an autophagy inhibitor suppressed curcumin-induced cell death in OSCC, confirming that autophagy acts as a pro-death signal. Furthermore, curcumin shows anticancer activity against OSCC via both autophagy and apoptosis. These findings suggest that curcumin may potentially contribute to oral cancer treatment and provide useful information for the development of a new therapeutic agent.