Hae Ryoun Park

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.

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.

Porphyromonas gingivalis-induced autophagy suppresses cell proliferation through G1 arrest in oral cancer cells

OBJECTIVES We investigated the response of oral cancer cells to intracellular invasion of Porphyromonas gingivalis to define changes in the biological characteristics of oral cancer cells evoked by the presence of oral pathogenic bacteria within a tumour microenvironment. DESIGNS The proliferative activity, cell cycle, and autophagic response were evaluated in oral cancer cells infected with P. gingivalis 381. ROS generation was detected in these cells by DCFDA assay, and its role in the responses of oral cancer cells to P. gingivalis infection was further investigated. RESUTLS P. gingivalis inhibited proliferation of oral cancer cells by inducing G1 cell cycle arrest, but had no effect on apoptosis. Following infection with P. gingivalis, the expression of cyclin D1 and cdk4 was decreased in oral cancer cells, whereas p21, a Cdk inhibitor, was upregulated, in comparison with non-infected controls. Autophagy was prominently enhanced in these infected cells, presumably contributing to the suppressed proliferation. Further experiments revealed that such autophagic response was activated by the formation of reactive oxygen species, as evidenced by the lack of autophagic response and cell proliferation upon removal of reactive oxygen species. CONCLUSIONS These findings provide a novel insight into the mechanism by which cancer cells are influenced by tumour microenvironment including oral bacteria.

Acetylshikonin inhibits growth of oral squamous cell carcinoma by inducing apoptosis.

OBJECTIVES Recently, shikonin derivatives from Lithospermum erythrorhizon have been suggested as potential chemotherapeutic agents against numerous types of cancers in addition to their traditional uses, e.g., as anti-inflammatory agents. Acetylshikonin, one of shikonin derivatives, has also been reported to possess anticancer activity. However, few studies of the effectiveness of acetylshikonin against cancer cells have been conducted, and there are no studies of oral cancers. In this study, we investigated the usefulness of acetylshikonin as a treatment regimen for oral cancers by observing the growth inhibitory function of acetylshikonin and the involved mechanisms. DESIGNS The viability, cell cycle, and ratio of apoptotic cells of oral squamous cell carcinoma (OSCC) cells were observed after treatment with acetylshikonin using MTT assay, flow cytometric analysis, and Annexin V/PI staining, respectively. In addition, molecular changes of apoptosis-related pathways and the role of reactive oxygen species (ROS) were analyzed in acetylshikonin-treated cells. RESULTS We observed that acetylshikonin significantly suppressed the growth of OSCC cells by inducing apoptotic cell death, and acetylshikonin affected the viability of a normal keratinocyte cell line HaCaT to a lesser degree, suggesting that acetylshikonin may be a good chemotherapeutic reagent with less toxicity to normal tissues. In addition, we found that acetylshikonin-induced apoptosis of OSCC cells is mediated by ROS as well as G2 cell cycle arrest. ROS production in response to acetylshikonin treatment enhanced the phosphorylation of JNK and p38 MAPK, which are in the major pathways of apoptotic cell death mechanisms. CONCLUSIONS In summary, our data suggest that acetylshikonin is a strong candidate for use as a selective chemotherapeutic agent for the treatment of OSCC.

Agomelatine, a MT1/MT2 melatonergic receptor agonist with serotonin 5-HT2C receptor antagonistic properties, suppresses Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages

OBJECTIVE This study was performed in an attempt to examine the influence of agomelatine in mitigating the generation of proinflammatory mediators in RAW264.7 murine macrophages exposed to lipopolysaccharide (LPS) obtained from Prevotella intermedia, a gram-negative anaerobic bacterium that is related with various types of periodontal diseases, and the molecular mechanisms behind its effects. DESIGN LPS from P. intermedia strain ATCC 25611 was prepared employing the conventional phenol-water procedure. Conditioned culture media were analyzed for the levels of nitric oxide (NO), interleukin-1β (IL-1β) and IL-6. Real-time PCR analysis was carried out to determine the mRNA levels of inducible NO synthase (iNOS), IL-1β, IL-6 and SOCS1. Protein expression levels were evaluated by immunoblot test. NF-κB-dependent SEAP reporter assay was performed using a reporter cell line. DNA-binding activities of NF-κB subunits were analyzed utilizing the ELISA-based kits. RESULTS Agomelatine was found to down-regulate significantly the generation of iNOS-derived NO, IL-1β and IL-6 as well as the expression of their mRNAs in cells activated with P. intermedia LPS. Agomelatine decreased NF-κB-dependent SEAP release caused by P. intermedia LPS. Agomelatine did not inhibit NF-κB transcription induced by LPS at the level of IκB-α degradation. Instead, LPS-induced nuclear translocation and DNA binding of NF-κB p50 subunit was blocked by agomelatine. P. intermedia LPS-elicited activation of STAT1 and STAT3 was reduced notably by co-treatment with agomelatine. Agomelatine showed a tendency to enhance mRNA level of SOCS1 in LPS-activated cells as well. CONCLUSIONS Agomelatine merits further evaluation to reveal its usefulness on the host modulation of periodontal disease.

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.

Josamycin suppresses Prevotella intermedia lipopolysaccharide-induced production of nitric oxide and interleukin-1β in murine macrophages

AIMS Josamycin has immunomodulatory properties independent of its antibacterial actions. This study was designed to explore the influences and associated mechanisms of josamycin upon the generation of proinflammatory mediators in murine macrophages activated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogenic bacterium associated with periodontal disease. MAIN METHODS LPS was purified by employing phenol-water extraction protocol. Culture supernatants were analyzed for nitric oxide (NO) and interleukin (IL)-1β. Real-time PCR and immunoblotting were conducted to quantify mRNA and protein expression, respectively. The expression levels of IL-1β were analyzed by confocal laser scanning microscopy. NF-κB-dependent SEAP levels were estimated by reporter assay. KEY FINDINGS Josamycin significantly attenuated NO production elicited by P. intermedia LPS as well as induction of iNOS protein and mRNA in RAW264.7 cells. While the release of IL-1β from cells stimulated by LPS was suppressed in the presence of josamycin, josamycin failed to reduce the degree of IL-1β mRNA expression. Josamycin did not reduce the stability of IL-1β mRNA induced by P. intermedia LPS, at the same time josamycin also failed to suppress the LPS-induced intracellular IL-1β expression. Josamycin augmented HO-1 induction in cells exposed to P. intermedia LPS, and SnPP, an inhibitor of HO-1 activity, reversed the suppressive impact of josamycin upon NO generation induced by LPS. Josamycin diminished NF-κB transcriptional activity induced by P. intermedia LPS. Further, josamycin enhanced SOCS1 mRNA level in cells activated with LPS. SIGNIFICANCE Josamycin suppressed P. intermedia LPS-induced generation of NO and IL-1β in RAW264.7 macrophages via the inhibition of NF-κB activation as well as HO-1 and SOCS1 induction. Josamycin may have benefits as a host modulatory agent in treating periodontal disease.

Acetylshikonin suppresses invasion of Porphyromonas gingivalis‑infected YD10B oral cancer cells by modulating the interleukin-8/matrix metalloproteinase axis

The development of pharmaceutical agents possessing anti-invasive and anti-metastatic 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 inflammation-promoted invasiveness of cancer cells may be important in treating cancer and improving the prognosis of patients with cancer. Acetylshikonin, a flavonoid with anti-inflammatory activity, inhibits proliferation and induces apoptosis of oral cancer cells. In the present study, the anti-invasive 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. gingivalis-infected YD10B cells by suppressing IL-8 release and IL-8-dependent 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.

NCX 4040, a nitric oxide-donating aspirin derivative, inhibits Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages.

In this study, the effects and underlying mechanisms of NCX 4040, a nitric oxide (NO)-donating aspirin derivative, on the production of proinflammatory mediators were examined using murine macrophages exposed to lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in the etiology of periodontal disease. NCX 4040 significantly reduced P. intermedia LPS-induced production of inducible NO synthase (iNOS)-derived NO, IL-1β and IL-6 as well as their mRNA expression in RAW264.7 cells. Notably, NCX 4040 was much more effective than the parental compound aspirin in reducing LPS-induced production of inflammatory mediators. NCX 4040 induced the expression of heme oxygenase-1 (HO-1) in cells treated with P. intermedia LPS, and the suppressive effect of NCX 4040 on LPS-induced NO production was significantly reversed by SnPP, a competitive HO-1 inhibitor. NCX 4040 did not influence LPS-induced phosphorylation of JNK and p38. IκB-α degradation as well as nuclear translocation and DNA-binding activities of NF-κB p65 and p50 subunits induced by P. intermedia LPS were significantly reduced by NCX 4040. Besides, LPS-induced phosphorylation of STAT1 and STAT3 was significantly down-regulated by NCX 4040. Further, NCX 4040 elevated the SOCS1 mRNA in cells stimulated with LPS. This study indicates that NCX 4040 inhibits P. intermedia LPS-induced production of NO, IL-1β and IL-6 in murine macrophages through anti-inflammatory HO-1 induction and suppression of NF-κB, STAT1 and STAT3 activation, which is associated with the activation of SOCS1 signaling. NCX 4040 could potentially be a promising tool in the treatment of periodontal disease, although further studies are required to verify this.

Abstract B13: Porphyromonas gingivalis-induced autophagy suppresses cell proliferation through G1 arrest in oral cancer cells

Epidemiological studies have shown that chronic inflammation predisposes individuals to different types of cancer and that microbial infections can trigger chronic inflammation. Porphyromonas gingivalis is a major microbial pathogen in periodontitis, which is a chronic inflammatory condition of the oral cavity and one of the most common infectious diseases. In this study, we examined the effect of P. gingivalis on oral cancer cells to define the role of chronic periodontitis on the development and/or progression of oral cancers. P. gingivalis-infected cells showed reduced proliferation rates and a concomitant G1 arrest compared with non-infected cells. One mechanism through which P. gingivalis blocked cell cycle progression in oral cancer cells was through ROS production, which induced autophagy. We conclude that the suppression of cell proliferation and cell cycle progression through autophagy may serve as an alternative to apoptosis to eliminate transformed cells. Autophagy represents a survival strategy against environmentally stressful conditions, such as the pathogenic invasion of P. gingivalis, and the prolonged survival of P. gingivalis-infected cells may contribute to the malignant progression of oral cancer cells. Citation Format: Tae Jin Cho, Vok Hee Woo, Jeom Il Choi, Seung Jo Kim, Hong In Shin, Bong Soo Park, Dae Geun Park, Hae Ryoun Park. Porphyromonas gingivalis -induced autophagy suppresses cell proliferation through G1 arrest in oral cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B13.