Whole cigarette smoke promotes human gingival epithelial cell apoptosis and inhibits cell repair processes

Semlali A, Chakir J, Goulet J‐P, Chmielewski W, Rouabhia M. Whole cigarette smoke promotes human gingival epithelial cell apoptosis and inhibits cell repair processes. J Periodont Res 2011; 46: 533–541.
©2011 John Wiley & Sons A/S Background and Objective: Smoking cigarettes increases the risk of developing various types of human diseases, including cancers and periodontitis. As gingival epithelial cells are known to play an active role in innate immunity via the secretion of a wide variety of mediators, and as these cells are the first ones exposed to environmental stimuli such as cigarette smoke, we sought to investigate the effects of whole cigarette smoke on normal human gingival epithelial cells and tissue. Material and Methods: Human gingival epithelial cells were extracted from healthy nonsmokers and used either as a monolayer or as an engineered human oral mucosa to investigate the effect of whole cigarette smoke on cell growth, apoptosis and wound repair/migration. Results: Our findings show that when gingival epithelial cells were exposed once to whole cigarette smoke, this resulted in a significant inhibition of cell growth through an apoptotic pathway, as confirmed by an increase of Bax and a decrease of Bcl‐xL and caspase‐3 activity. Cigarette smoke also inhibited epithelial cell migration. These effects may explain the disorganization of the engineered human oral mucosa tissue when exposed to whole cigarette smoke. Conclusion: Exposure to whole cigarette smoke markedly inhibits epithelial cell growth through an apoptosis/necrosis pathway that involves Bax and Bcl‐xL proteins and caspase‐3 activity. Cigarette smoke also disrupts epithelial cell migration, which may negatively affect periodontal wound healing.     

Cyclosporine A Inhibits Apoptosis of Rat Gingival Epithelium

Background: The use of cyclosporine A (CsA) induces hyperplasia of the gingival epithelium in a site‐specific response manner, but the molecular mechanism via which the lesion occurs is unclear. The present research aims to investigate the site‐specific effect of CsA on the apoptosis of gingival epithelium associated with gingival hyperplasia. Methods: Forty Wistar rats were divided into CsA‐treated and non‐treated groups. Paraffin‐embedded sections of mandibular first molars were selected for hematoxylin and eosin staining, immunohistochemistry analyses of bcl‐2 and caspase‐3, and the staining of terminal deoxynucleotidyl transfer‐mediated dUTP nick‐end labeling (TUNEL). The area of the whole gingival epithelium and the length of rete pegs were measured, and the number of bcl‐2‐ and caspase‐3‐positive cells in the longest rete peg were counted. The analysis of variance for factorial designs and Fisher least significant difference test for post hoc analysis were used to determine the significance levels. Results: In CsA‐treated rats, bcl‐2 expression was significantly upregulated, whereas caspase‐3 expression was downregulated, along with a reduced number of TUNEL‐positive cells. The site‐specific distribution of bcl‐2 was consistent with the site‐specific hyperplasia of the gingival epithelium in CsA‐treated rats. Conclusions: CsA inhibited gingival epithelial apoptosis via the mitochondrial pathway and common pathway. The antiapoptotic protein bcl‐2 might play a critical role in the pathogenesis of the site‐specific hyperplasia of gingival epithelium induced by CsA. There were mechanistic differences in the regulation of apoptosis for cells in the attached gingival epithelium, free gingival epithelium, and junctional epithelium.     

Variants of Porphyromonas gingivalis lipopolysaccharide alter lipidation of autophagic protein,microtubule‐associated protein 1 light chain 3, LC3

Porphyromonas gingivalis often subverts host cell autophagic processes for its own survival. Our previous studies document the association of the cargo sorting protein, melanoregulin (MREG), with its binding partner, the autophagic protein, microtubule‐associated protein 1 light chain 3 (LC3) in macrophages incubated with P. gingivalis (strain 33277). Differences in the lipid A moiety of lipopolysaccharide (LPS) affect the virulence of P. gingivalis; penta‐acylated LPS₁₆₉₀ is a weak Toll‐like receptor 4 agonist compared with Escherichia coli LPS, whereas tetra‐acylated LPS_1435/1449 acts as an LPS₁₆₉₀ antagonist. To determine how P. gingivalis LPS₁₆₉₀ affects autophagy we assessed LC3‐dependent and MREG‐dependent processes in green fluorescent protein (GFP)‐LC3‐expressing Saos‐2 cells. LPS₁₆₉₀ stimulated the formation of very large LC3‐positive vacuoles and MREG puncta. This LPS₁₆₉₀‐mediated LC3 lipidation decreased in the presence of LPS_1435/1449. When Saos‐2 cells were incubated with P. gingivalis the bacteria internalized but did not traffic to GFP‐LC3‐positive structures. Nevertheless, increases in LC3 lipidation and MREG puncta were observed. Collectively, these results suggest that P. gingivalis internalization is not necessary for LC3 lipidation. Primary human gingival epithelial cells isolated from patients with periodontitis showed both LC3II and MREG puncta whereas cells from disease‐free individuals exhibited little co‐localization of these two proteins. These results suggest that the prevalence of a particular LPS moiety may modulate the degradative capacity of host cells, so influencing bacterial survival.     

Butyrate induces reactive oxygen species production and affects cell cycle progression in human gingival fibroblasts

Background and Objective: Short‐chain fatty acids, such as butyric acid and propionic acid, are metabolic by‐products generated by periodontal microflora such as Porphyromonas gingivalis, and contribute to the pathogenesis of periodontitis. However, the effects of butyrate on the biological activities of gingival fibroblasts (GFs) are not well elucidated. Material and Methods: Human GFs were exposed to various concentrations of butyrate (0.5–16 mm ) for 24 h. Viable cells that excluded trypan blue were counted. Cell cycle distribution of GFs was analyzed by propidium iodide‐staining flow cytometry. Cellular reactive oxygen species (ROS) production was measured by flow cytometry using 2’,7’‐dichlorofluorescein (DCF). Total RNA and protein lysates were isolated and subjected to RT‐PCR using specific primers or to western blotting using specific antibodies, respectively. Results: Butyrate inhibited the growth of GFs, as indicated by a decrease in the number of viable cells. This event was associated with an induction of G0/G1 and G2/M cell cycle arrest by butyrate (4–16 mm ) in GFs. However, no marked apoptosis of GFs was noted in this experimental condition. Butyrate (> 2 mm ) inhibited the expression of cdc2, cdc25C and cyclinB1 mRNAs and reduced the levels of Cdc2, Cdc25C and cyclinB1 proteins in GFs, as determined using RT‐PCR and western blotting, respectively. This toxic effect of butyrate was associated with the production of ROS. Conclusion: These results suggest that butyrate generated by periodontal pathogens may be involved in the pathogenesis of periodontal diseases via the induction of ROS production and the impairment of cell growth, cell cycle progression and expression of cell cycle‐related genes in GFs. These events are important in the initiation and prolongation of inflammatory processes in periodontal diseases.     

Porphyromonas gingivalis infection sequesters pro‐apoptotic Bad through Akt in primary gingival epithelial cells

Porphyromonas gingivalis, a self‐limiting oral pathogen, can colonize and replicate in gingival epithelial cells (GECs). P. gingivalis‐infected GECs are protected from mitochondrion‐dependent apoptosis, partially through activation of phosphatidyl inositol 3‐kinase/Akt signaling. Biochemical events associated with P. gingivalis‐induced inhibition of apoptosis include the blocking of mitochondrial membrane permeability and cytochrome‐c release. We studied functional importance of Akt and the status of associated key mitochondrial molecules, pro‐apoptotic Bad and caspase‐9, during infection of GECs. We found that P. gingivalis infection caused significant phosphorylation of Bad progressively, while messenger RNA levels for Bad slowly decreased. Fluorescence microscopy showed translocation of the mitochondrial Bad to the cytosol post‐infection. Conversely, P. gingivalis lost the ability to promote phosphorylation and translocation of Bad in Akt‐deficient GECs. Caspase‐9 activation induced by a chemical inducer of apoptosis was significantly inhibited by infection over time. However, Akt depletion by small interfering RNA did not reverse inhibition of caspase‐9 activation by infection. Hence, P. gingivalis inactivates pro‐apoptotic Bad through Akt. The inhibition of caspase‐9 activation appears to be independent of Akt. Overall, our findings suggest that Akt is a key component of anti‐apoptotic pathways stimulated by P. gingivalis. The P. gingivalis uses other mitochondrial pathways to protect host cells from cell‐death and to ensure its survival in gingival epithelium.     

Oxidized low‐density lipoprotein increases interleukin‐8 production in human gingival epithelial cell line Ca9‐22

Suzuki K, Sakiyama Y, Usui M, Obama T, Kato R, Itabe H, Yamamoto M. Oxidized low‐density lipoprotein increases interleukin‐8 production in human gingival epithelial cell line Ca9‐22. J Periodont Res 2010; 45: 488–495. © 2010 John Wiley & Sons A/S Background and Objective: Recent epidemiological studies have shown a correlation between periodontitis and hyperlipidemia. We have found high levels of oxidized low‐density lipoprotein (OxLDL) in the gingival crevicular fluid of dental patients. In the present study, we tried to examine the possible role of OxLDL in periodontal inflammation in vitro. Material and Methods: Cells of the human gingival epithelial cell line Ca9‐22 were cultured in media containing OxLDL, and the amounts of interleukin‐8 (IL‐8) and prostaglandin E₂ (PGE₂) produced were measured using ELISAs. Results: Production of IL‐8 by Ca9‐22 cells was significantly increased when the cells were treated with OxLDL, but not with native LDL or acetylated LDL. Production of PGE₂ by Ca9‐22 cells was enhanced by co‐incubation with OxLDL and interleukin‐1β (IL‐1β). Scavenger receptor inhibitors, fucoidan and dextran sulfate, inhibited the OxLDL‐induced IL‐8 and PGE₂ production in the presence of IL‐1β. The p³⁸ MAPK inhibitors SB203580 and SB202190 and the ERK inhibitor PD98059 inhibited the OxLDL‐induced IL‐8 production. Among oxidized lipids and chemically modified LDL, 7‐ketocholesterol enhanced IL‐8 production. Conclusion: This is the first report to show that OxLDL enhances IL‐8 production in epithelial cells.     

Inhibition of autophagy augments apoptosis in human oral squamous cell carcinoma under nutrient depletion

There has been little research conducted regarding autophagy in oral squamous cell carcinoma (OSCC). Given the prevalence of oral cancers which are OSCC and the severe side effects of current treatments, there is a pressing need to develop effective alternative therapies. In this study, we have endeavored to explore the biological characteristics of oral squamous cell carcinoma cell line KB cells, in particular with regard to the role played by autophagy in their survival. Autophagy was activated by nutrient depletion via culturing cells in Earle's balanced salts (EBSS) and was measured via indices relating to Beclin 1, microtubule‐associated protein light chain 3 (MAPLC3, LC3), p62, and Green fluorescent protein–light chain 3 plasmid transfection (GFP‐LC3). Cell death and apoptosis induced by nutrient depletion was measured using both MTT assay and flow cytometry (FCM). Compared to initial levels at 0 h, Beclin 1 density in EBSS‐treated cells was found to have increased at 6, 12, and 18 h in a time‐dependent manner and was found to have subsequently declined at 24 and 48 h. p62 levels, LC3‐II/LC3‐I ratio, and GFP‐LC3 levels increased at 6, 12, 18, 24, and 48 h in a time‐dependent manner. 3‐methyladenine (3‐MA) was found to inhibit autophagy and the expression of Beclin 1 and significantly enhanced nutrient depletion‐induced apoptosis and death. We concluded that nutrient depletion enhances OSCC cell autophagy in time‐course patterns and that the inhibition of autophagy augments apoptosis in OSCC cells. We also deduced that Beclin 1 takes part in the development and progression of autophagy, potentially playing an important role in the crosstalk between apoptosis and autophagy in OSCC cells. These findings suggest that nutrient depletion may be an effective way to explore autophagy and that autophagy inhibitors should be investigated as a potential novel agent for the adjuvant treatment of human OSCC.     

Oral malodorous compound triggers mitochondrial-dependent apoptosis and causes genomic DNA damage in human gingival epithelial cells

Calenic B, Yaegaki K, Murata T, Imai T, Aoyama I, Sato T, Ii H. Oral malodorous compound triggers mitochondrial‐dependent apoptosis and causes genomic DNA damage in human gingival epithelial cells. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2008.01199.x. © 2009 John Wiley & Sons A/S Background and Objective: Volatile sulfur compounds are the main compounds causing halitosis. One of these compounds, hydrogen sulfide (H₂S), which is responsible for physiological halitosis, is reported also to have periodontal pathogenic activities. Hydrogen sulfide has been shown to activate the apoptotic process in different tissues. Apoptosis plays an important role in the development of periodontitis. The aim of this study was to determine whether H₂S causes apoptosis in human gingival epithelial cells and to examine the cellular signaling pathway initiating the process. Material and Methods: Human gingival epithelial cells were incubated with 50 ng/mL H₂S in air contining 5% CO₂ for 24, 48 or 72 h. To detect apoptosis, the cells were stained with annexin V and 7‐amino actinomycin D, and analyzed using flow cytometry. Reactive oxygen species, mitochondrial membrane depolarization and release of cytochrome C into the cytosol were assessed using flow cytometry and enzyme‐linked immunosorbent assay. Activity levels for the key apoptotic enzymes caspase‐9, ‐8 and ‐3 were also determined. Genomic DNA damage was detected using single‐cell gel electrophoresis. Results: Apoptosis was significantly increased to 24.5 ± 5.7 at 24 h and 41.5 ± 8.9% at 48 h (p < 0.01). Reactive oxygen species were enhanced and mitochondrial membrane depolarization was collapsed. Cytochrome C release was dramatically increased (0.12 ± 0.02 vs. 0.02 ± 0.01 at 24 h and 0.21 ± 0.02 vs. 0.02 ± 0.01 ng/mL at 48 h; p < 0.05). Caspase‐9 and ‐3 were strongly activated, while caspase‐8 activity remained low. The percentage of DNA strand breaks increased, especially at 48 h. Conclusion: Hydrogen sulfide induces apoptosis in human gingival epithelial cells by activating the mitochondrial pathway.     

β-Phenethyl isothiocyanate induces death receptor 5 to induce apoptosis in human oral cancer cells via p38

Oral Diseases (2012) 18 , 513–519 Objectives: β‐Phenylethyl isothiocyanate (PEITC) has been demonstrated to fight many types of cancers through various molecular pathways. In this study, we focused on its effect on the induction of apoptosis to inhibit cell growth and molecular mechanism in oral cancer. Materials and methods: 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(2,4‐disulfophenyl)‐2‐(4 sulfophenyl)‐2H‐tetrazolium (MTS) assay was used to examine cell viability. The apoptotic effect was investigated using 4′‐6‐Diamidino‐2‐phenylindole (DAPI) staining or Western blotting. Inhibitors were used to determine the molecular target and mechanism of PEITC‐mediated apoptosis. Results: β‐Phenylethyl isothiocyanate inhibited the growth of HN22 human oral cancer cells and induced caspase‐dependent apoptosis in HN22 cells as evidenced by nuclear fragmentation and the activation of caspase 3. It increased cleaved caspase 8, truncated BID, and death receptor 5 (DR5) through the activation of p38 MAPK. This result was confirmed by blockage of PEITC‐induced cleavages of Poly(ADP‐ribose) Polymerase, caspase‐3, caspase‐8, and DR5 by p38 MAPK inhibitor, SB203580. We also found that PEITC activated p38 and augmented DR5 to induce apoptosis in other human oral cancer cells. Conclusions: These results suggest that DR5 is a potential molecular target for PEITC‐induced apoptosis in oral cancer via p38 MAPK.     

Porphyromonas gingivalis induces autophagy in THP‐1‐derived macrophages

Autophagy provides a mechanism for the turnover of cellular organelles and proteins through a lysosome‐dependent degradation pathway and is a possible mechanism in inflammatory disease. Periodontitis is an inflammatory disease caused by periodontal pathogens. Porphyromonas gingivalis, an important periodontal pathogen, activates cellular autophagy to provide a replicative niche while suppressing apoptosis in endothelial cells. However, the molecular basis for a causal relationship between P. gingivalis and autophagy is unclear. This research examines the involvement of P. gingivalis in autophagy through light chain 3 (LC3) and autophagic proteins, and the role of P. gingivalis‐induced autophagy in the clearance of P. gingivalis and inflammation. To investigate the molecular mechanism of autophagy induced by P. gingivalis, PMA‐differentiated THP‐1‐derived macrophages were infected with live P. gingivalis. The P. gingivalis increased the formation of autophagosomes in a multiplicity of infection‐dependent manner, as well as autophagolysosomes. Porphyromonas gingivalis activated LC3‐I/LC3‐II conversion and increased the conjugation of autophagy‐related 5 (ATG5) –ATG12 and the expression of Beclin1. The expressions of Beclin1, ATG5–ATG12 conjugate, and LC3‐II were significantly inhibited by the presence of 3‐methyladenine, an autophagy inhibitor. Interestingly, 3‐methyladenine increased the survival of P. gingivalis and proinflammatory cytokine interleukin‐1β production. The data indicate that P. gingivalis induces autophagy in PMA‐differentiated THP‐1‐derived macrophages and in turn, macrophages eliminate P. gingivalis through an autophagic response, which can lead to the restriction of an excessive inflammatory response by downregulating interleukin‐1β production. The induction of autophagy by P. gingivalis may play an important role in the periodontal inflammatory process and serve as a target for the development of new therapies.     

Toll-like receptor 7 agonist, imiquimod, inhibits oral squamous carcinoma cells through apoptosis and necrosis

J Oral Pathol Med (2012) 41 : 540–546 Background: Toll‐like receptor (TLR) agonists have anticancer effect by inducing apoptosis or activating immune cells. In this study, we investigated whether imiquimod, TLR7 agonist, inhibits the proliferation of oral cancer cells. Methods: Toll‐like receptor 7 expression and IL‐6/8 production by imiquimod were examined using RT‐PCR and Enzyme‐linked immunosorbent assay, respectively. Cell viability was examined by MTT assay. To examine apoptotic cell death, Annexin V/PI staining for flow cytometry and Western blot analysis were performed. Necrotic cell death was determined by leakage of lactate dehydrogenase (LDH), HMGB1, and PI staining in imiquimod‐treated oral squamous cell carcinoma (OSCC) cells. Results: Toll‐like receptor7 mRNA was expressed in OSCC cells. Imiquimod induced IL‐6 and IL‐8 production in OSCC cells, suggesting the functional expression of TLR7. Imiquimod inhibited cells proliferation in a dose‐dependent manner. The ratio of annexin V‐positive cells and cleaved caspase‐3/7 was increased by imiquimod treatment in OSCC cells, suggesting that imiquimod‐induced cell death in OSCC cells may be owing to apoptosis. In addition, LDH secretion and PI staining were detected in OSCC cells treated with imiquimod, showing that imiquimod also induced necrotic cell death in the OSCC cells. Conclusions: Imiquimod inhibited effectively the growth of OSCC cells by inducing apoptosis and necrosis.     

Protease‐activated receptor 2 mediates interleukin‐8 and intercellular adhesion molecule‐1 expression in response to Aggregatibacter actinomycetemcomitans

Introduction:  We investigated the mechanisms by which extracts of Aggregatibacter actinomycetemcomitans affect the inflammatory response in gingival epithelial cells. Methods:  Human gingival cells (Ca9‐22) were cultured in bacterial extracts prepared from A. actinomycetemcomitans ATCC 29522. The cells were pretreated with protease inhibitors or transfected with small interfering RNA (siRNA) specific for protease‐activated receptor 2 (PAR‐2). Results:  The pretreatment of cells with serine protease inhibitors significantly inhibited A. actinomycetemcomitans extract‐induced expression of interleukin‐8 (IL‐8) and intercellular adhesion molecule‐1 (ICAM‐1) at both the messenger RNA and protein levels. In addition, A. actinomycetemcomitans extract‐induced IL‐8 and ICAM‐1 expression was significantly decreased in PAR‐2/siRNA‐transfected cells. Conclusions:  A. actinomycetemcomitans extract‐induced IL‐8 and ICAM‐1 expression in gingival epithelial cells is mediated by PAR‐2.     

In vivo effects of zoledronic acid on oral mucosal epithelial cells

Oral Diseases (2011) 17 , 291–297 Objective: Osteonecrosis of the jaw is a serious complication of bisphosphonate treatment for which the pathophysiology is unknown. The purpose of this study was to investigate whether in vivo zoledronic acid (ZA) induces alterations in cell proliferation, apoptosis, and matrix metalloproteinases (MMPs) expression in oral mucosal epithelial cells. Methods: One‐year‐old dogs were either untreated (control group) or given high doses of intravenous ZA (ZA group) for 3 months. The doses of ZA were equivalent to those given to cancer patients, yet were administered two times more frequently (every 2 weeks). Mucosal tissues were assessed immunohistochemically for cell proliferation (proliferating cell nuclear antigen, PCNA), matrix metalloproteinase (MMP) expression, and apoptosis (caspase 3 and TUNEL). Results: There were no significant differences between the groups with respect to PCNA, MMP‐2, MMP‐14, and TUNEL positive cells. However, the expression of MMP‐9 was significantly higher in the control group than in the ZA group (P < 0.05), whereas the expression of caspase 3 was significantly lower in the control group than in the ZA group (P < 0.05). Conclusion: These results suggest that high doses of ZA resulted in higher levels of apoptosis and lower levels of MMP‐9 in the oral epithelial cells supporting the idea of bisphosphonate treatment affects the oral mucosa.     

5‐aminolevulinic acid induce apoptosis via NF‐κB/JNK pathway in human oral cancer Ca9–22 cells

J Oral Pathol Med (2010) 40 : 483–489 Background: 5‐aminolevulinic acid‐based photodynamic therapy (5‐ALA‐PDT) is being used to treat oral pre‐cancerous and cancerous lesions with some encouraging clinical outcomes. However, the exact mechanisms behind the photodynamic treatment are still not fully elucidated. Method: Flow cytometry, TdT‐mediated dUTP nick end labeling assay and Western blot analysis were used to investigate the effects of 5‐ALA‐PDT on human oral cancer Ca9–22 cells. Results: We found that 5‐ALA‐PDT induces apoptosis in Ca9–22 cells. Western blotting showed that 5‐ALA‐PDT activates both the caspase‐8 and caspase‐9 pathways, which differed from previous studies conducted in other cell types. Activation of JNK was evident as early as 30 min. The caspases activation was inhibited by JNK inhibitor SP600125. Treatment with NF‐κB inhibitor Bay 11‐7082 (Bay) completely abrogated ALA‐PDT‐induced JNK activation. In addition, Bay and SP600125 almost completely abolished ALA‐PDT‐induced apoptosis. Conclusion: These results demonstrate significant involvement of caspase‐8 and ‐9 and their upstream NF‐κB‐JNK pathways in ALA‐PDT‐induced apoptosis. Future studies on how NF‐κB and JNK activity regulate ALA‐PDT response should provide a better strategy for the treatment of oral cancer.     

Inhibition of autophagy augments chemotherapy in human salivary adenoid cystic carcinoma

Although cisplatin (DDP)‐based adjuvant chemotherapy is widely used in the treatment of salivary adenoid cystic carcinoma (SACC), SACCs have developed resistance to cisplatin, resulting in chemotherapy failure. Autophagy serves as a critical adaptive response, which was increased in tumor cells in chemotherapy. However, the function of autophagy is not clear in SACC. In this study, apoptosis induced by DDP in SACC high metastatic cell line (ACC‐M) was revealed using MTT assay, flow cytometry, and caspase‐3 immunoblotting. The autophagy activation induced by DDP treatment was measured by transmission electron microscopy, green fluorescent protein–light chain 3 plasmid transfection LC3 immunoblotting and p62 immunoblotting. 3‐methyladenine (3‐MA) or small interference RNA targeting beclin 1 (beclin 1 siRNA) inhibited autophagy and significantly enhanced DDP‐induced apoptosis. ACC‐M xenografts in nude mice further verified the synergistic effect of DDP and 3‐MA. In conclusion, autophagy activation was caused to protect cancer cells from DDP‐induced apoptosis and autophagy inhibition could be a promising strategy for adjuvant chemotherapy in SACC.     

Induction of apoptosis in oral epithelial cells by Candida albicans

During infection, interactions between Candida albicans and oral epithelial cells result in oral epithelial cell death. This is clinically manifested by the development of oral mucosal ulcerations generally associated with discomfort. In vitro studies have shown that C. albicans induces early apoptotic alterations in oral epithelial cells; however, these studies have also shown that treatment of infected cells with caspase inhibitors does not prevent their death. The reasons for these contradictory results are unknown and it is still not clear if C. albicans stimulates oral epithelial signaling pathways that promote apoptotic cell death. Activation of specific death pathways in response to microbial organisms plays an essential role in modulating the pathogenesis of a variety of infectious diseases. The aim of this study was to (i) characterize C. albicans‐induced apoptotic morphological alterations in oral epithelial cells, and (ii) investigate the activation of apoptotic signaling pathways and expression of apoptotic genes during infection. Candida albicans induced early apoptotic changes in over 50% of oral epithelial cells. However, only 15% of those showed mid‐late apoptotic alterations. At the molecular level, C. albicans caused a loss of the mitochondrial transmembrane potential and translocation of mitochondrial cytochrome c. Caspase‐3/9 activities increased only during the first hours of infection. Moreover, poly[ADP ribose] polymerase 1 was cleaved into apoptotic and necrotic‐like fragments. Finally, five anti‐apoptotic genes were significantly upregulated and two pro‐apoptotic genes were downregulated during infection. Altogether, these findings indicate that epithelial apoptotic pathways are activated in response to C. albicans, but fail to progress and promote apoptotic cell death.     

Sex Differences in Inflammatory and Apoptotic Signaling Molecules In Normal and Diseased Human Gingiva

Background: The purpose of this study is to determine whether sex dimorphism exists in the expression of inflammatory and apoptotic mediators in gingiva obtained from normal and diseased sites of periodontal disease. Methods: Gingival papillae were obtained from individuals (56 males and 62 females) who required extraction of adjacent teeth. Gingival samples were grouped by adjacent sulcus depth: 1 to 3 mm (normal), 3 mm with bleeding on probing (slight disease), 3 to 6 mm (moderate disease), and >6 mm (severe disease). The tissue concentrations of cysteine‐requiring aspartate‐directed protease 3 (caspase‐3), interleukin‐2, tumor necrosis factor–related apoptosis‐inducing ligand, Fas ligand, p38α mitogen‐activated protein kinase, extracellular signal‐related kinase 1/2, and survivin were determined by enzyme‐linked immunosorbent assay. These mediator concentrations, age of donor, sex of donor, and gingival sulcular depth were the outcome variables. Data were compared by factorial analysis of variance, post hoc Tukey, and Pearson correlation test. P <0.05 was used to indicate significant differences among the outcome variables. Results: The mean gingival sulcular depth was significantly greater in male than in female groups (P <0.05). The majority of the tested mediators were significantly correlated with both sex and sulcular depth and with caspase‐3 (P <0.05). The concentration of caspase‐3 in female gingiva at all diseased sites was significantly greater than in gingiva derived from male sites (P <0.05). Conclusions: These data suggest sex dimorphism in the presence of gingival apoptosis at sites of periodontal disease, with females having the highest incidence of apoptosis. Because apoptosis clears inflammatory cells and promotes healing, this phenomenon could provide a mechanism for sex dimorphism for the incidence of periodontal disease.     

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.     

Delivery of cytolethal distending toxin B induces cell cycle arrest and apoptosis in gingival squamous cell carcinoma in vitro

The cytolethal distending toxin (Cdt) from Actinobacillus actinomycetemcomitans consists of three proteins, CdtA, CdtB, and CdtC, which are responsible for cell cycle arrest and apoptosis. In the present study, local delivery systems of recombinant CdtB and CdtB-expressing plasmid were established using Ca9-22, human gingival squamous cell carcinoma cell line. When CdtB was delivered to Ca9-22 cells using a BioPORTER, a 32-kDa protein was detected by Western blotting, and G2 cell cycle arrest and apoptosis occurred. In addition, the CdtB delivered upregulated the expression of phosphorylated p53 and the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) in Ca9-22 cells, suggesting that these intracellular molecules might contribute to the induction of G2 cell cycle arrest and apoptosis. When the CdtB-expressing plasmid was transfected into Ca9-22 cells by lipofection or electroporation, CdtB (32 kDa) was clearly detected. Further, TdT-mediated dUTP nick end labeling positive cells were observed after transfection of the CdtB-expressing plasmid. These findings indicated that delivery of the CdtB protein and transfection of the cdtB gene induced cell cycle arrest and apoptosis in Ca9-22 cells in vitro, and we conclude that it may be possible to induce apoptosis in human gingival squamous cell carcinoma by electroporation of the cdtB gene.