Osteogenic potential of apical papilla stem cells mediated by platelet-rich fibrin and low-level laser.

To evaluate the osteogenic potential of platelet-rich fibrin (PRF) and low-level laser therapy (LLLT) on human stem cells from the apical papilla (SCAP) we isolated, characterized, and then cultured in an osteogenic medium cells with PRF and/or LLLT (660 nm, 6 J/m2-irradiation). Osteogenic differentiation was assessed by bone nodule formation and expression of bone morphogenetic proteins (BMP-2 and BMP-4), whereas the molecular mechanisms were achieved by qRT-PCR and RNA-seq analysis. Statistical analysis was performed by ANOVA and Tukey's post hoc tests (p < 0.05* and p < 0.01**). Although PRF and LLLT increased bone nodule formation after 7 days and peaked at 21 days, the combination of PRF + LLLT led to the uppermost nodule formation. This was supported by increased levels of BMP-2 and -4 osteogenic proteins (p < 0.005). Furthermore, the PRF + LLLT relative expression of specific genes involved in osteogenesis, such as osteocalcin, was 2.4- (p = 0.03) and 28.3- (p = 0.001) fold higher compared to the PRF and LLLT groups, and osteopontin was 22.9- and 1.23-fold higher, respectively (p < 0.05), after 7 days of interaction. The transcriptomic profile revealed that the combination of PRF + LLLT induces MSX1, TGFB1, and SMAD1 expression, after 21 days of osteogenic differentiation conditions exposition. More studies are required to understand the complete cellular and molecular mechanisms of PRF plus LLLT on stem cells. Overall, we demonstrated for the first time that the combination of PRF and LLLT would be an excellent therapeutic tool that can be employed for dental, oral, and craniofacial repair and other tissue engineering applications.

Synergistic Effects of New Curcumin Analog (PAC) and Cisplatin on Oral Cancer Therapy.

Oral cancer has traditionally been treated with surgery, radiotherapy, chemotherapy, or a combination of these therapies. Although cisplatin, a chemotherapy drug, can effectively kill oral cancer cells by forming DNA adducts, its clinical use is limited due to adverse effects and chemo-resistance. Therefore, there is a need to develop new, targeted anticancer drugs to complement chemotherapy, allowing for reduced cisplatin doses and minimizing adverse effects. Recent studies have shown that 3,5-Bis (4-hydroxy-3-methoxybenzylidene)-N-methyl-4-piperidine (PAC), a new curcumin analog, possesses anticancer properties and could be considered a complementary or alternative therapy. In this study, we aimed to assess the potential complementary effects of PAC in combination with cisplatin for treating oral cancer. We conducted experiments using oral cancer cell lines (Ca9-22) treated with different concentrations of cisplatin (ranging from 0.1 µM to 1 µM), either alone or in conjunction with PAC (2.5 and 5 µM). Cell growth was measured using the MTT assay, while cell cytotoxicity was evaluated using an LDH assay. Propidium iodide and annexin V staining were employed to examine the impact on cell apoptosis. Flow cytometry was used to investigate the effects of the PAC/cisplatin combination on cancer cell autophagy, oxidative stress, and DNA damage. Additionally, a Western Blot analysis was performed to assess the influence of this combination on pro-carcinogenic proteins involved in various signaling pathways. The results demonstrated that PAC enhanced the efficacy of cisplatin in a dose-dependent manner, leading to a significant inhibition of oral cancer cell proliferation. Importantly, treatment with PAC (5 µM) alongside different concentrations of cisplatin reduced the IC50 of cisplatin tenfold. Combining these two agents increased apoptosis by further inducing caspase activity. In addition, the concomitant use of PAC and cisplatin enhances oral cancer cell autophagy, ROS, and MitoSOX production. However, combined PAC with cisplatin inhibits the mitochondrial membrane potential (ΔΨm), which is a marker for cell viability. Finally, this combination further enhances the inhibition of oral cancer cell migration via the inhibition of epithelial-to-mesenchymal transition genes, such as E-cadherin. We demonstrated that the combination of PAC and cisplatin markedly enhanced oral cancer cell death by inducing apoptosis, autophagy, and oxidative stress. The data presented indicate that PAC has the potential to serve as a powerful complementary agent to cisplatin in the treatment of gingival squamous cell carcinomas.

Effect of cannabis smoke condensate on human nasal epithelial cell adhesion, growth, and migration.

OBJECTIVE: When inhaled, cannabis smoke interacts with airway tissues, including the nasal mucosa, which may lead to nasal pathologies. We examined the effect of cannabis smoke condensate (CSC) on nasal epithelial cell and tissue behaviors. METHODS: Human nasal epithelial cells were exposed or not to CSC at different concentrations (1, 5, 10, and 20 %) and for different durations. Cell adhesion and viability were assessed, as well as post-wound cell migration and lactate dehydrogenase (LDH) release. RESULTS: The nasal epithelial cells showed a larger cell size and a faint nucleus following exposure to CSC, compared to that observed in that control. This was supported by fewer adherent cells present after exposure for either 1 or 24 h to 5, 15, and 20 % CSC. CSC also had a significant toxic effect by reducing cell viability after both 1 and 24 h of exposure. This toxic effect was significant even at a low concentration (1 %) of CSC. The effects on nasal epithelial cell viability were confirmed by the decrease in cell migration. After the scratch and subsequent exposure to CSC for either 6 or 24 h, a complete inhibition of nasal epithelial cell migration was observed, compared to that found in the controls. CSC was toxic to the nasal epithelial cells, as the level of LDH significantly increased following cell exposure all CSC concentrations. CONCLUSION: Cannabis smoke condensate had a negative effect on several nasal epithelial cell behaviors. These findings indicate that cannabis smoke could be a threat to nasal tissues and ultimately lead to nasal and sinus disorders.

Electronic cigarette vapor increases Streptococcus mutans growth, adhesion, biofilm formation, and expression of the biofilm-associated genes.

OBJECTIVE: It still not known whether electronic cigarettes (e-cigarettes) contribute to dental caries. This study aimed to evaluate the effect of e-cigarettes on the growth of Streptococcus mutans, the formation of biofilm, and the expression of certain virulence genes. MATERIALS AND METHODS: Streptococcus mutans cells were exposed or not to e-cigarettes with and without nicotine or to cigarette smoke twice a day for 15 min each exposure period. The bacterial growth and the expression of glucosyltranferase, competence, and glucan-binding genes were evaluated after 24 hr. Biofilm formation was assessed after 1, 2, and 3 days. S. mutans adhesion and growth to e-cigarette exposed human teeth were assessed. RESULTS: We observed an increase in S. mutans growth with e-cigarettes, mainly at the early culture period. This was confirmed by an increase of biofilm mass ranging from 8 ± 0.5 mg with the control to 47 ± 5 mg after six exposures to nicotine-rich e-cigarettes. S. mutans cells adhered better to e-cigarette exposed teeth. E-cigarettes increased the expression of glucosyltranferase, competence, and glucan-binding genes. CONCLUSIONS: E-cigarettes increased the growth of S. mutans and the expression of virulent genes. E-cigarettes promoted the adhesion to, and formation of biofilms on teeth surfaces.

Effect of e-cigarettes on nasal epithelial cell growth, Ki67 expression, and pro-inflammatory cytokine secretion.

OBJECTIVE: Upon use, e-cigarette aerosol comes in contact with various mucosal tissues, including the nasal epithelium, which may lead to nasal pathologies. We therefore assessed the effect of e-cigarettes on nasal epithelial cell and tissue behaviours. METHODS: Human primary nasal epithelial cells and engineered 3D nasal mucosa tissues were exposed or not to either e-cigarette aerosol or standard cigarette smoke. We then evaluated cell viability and lactate dehydrogenase (LDH) activity. With the tissues analysed tissue structure, the expression of Ki67 proliferating marker, and the secretion of pro-inflammatory cytokines by the engineered nasal mucosa. RESULTS: The nasal epithelial cells exposed to e-cigarettes displayed a larger cell size and a faint nucleus following exposure to e-cigarettes. This is supported by the increased levels of LDH activity following exposure to e-cigarettes, compared to that observed in the control. Tissues exposed to e-cigarette aerosol displayed a structural deregulation, with more large-sized cells, fewer Ki67-positive cells, and a reduced proliferation rate, compared to that observed in the non-exposed tissues. Cytokine measurements showed high levels of IL-6, IL-8, TNFα, and MCP-1, demonstrating that e-cigarettes activated pro-inflammatory cytokine responses. CONCLUSION: E-cigarette aerosol showed adverse effects on nasal epithelial cells and nasal engineered mucosa tissue. These findings indicate that e-cigarettes could be a threat to nasal tissues and may impair the innate immune function of nasal epithelial cells.

Impact of Electronic Cigarettes on Oral Health: a Review.

Electronic cigarettes (e­cigarettes) are widely available, and their use is increasing worldwide. They are promoted as a safer alternative to combustible cigarette smoking and as an effective smoking cessation aid. E­cigarettes are designed to provide smokers with the desired nicotine dose without burning tobacco. They contain flavoured humectants that include nicotine in concentrations of 0-36 mg/mL. Evidence suggests that e­cigarettes are a better nicotine delivery method than combustible cigarettes and have reduced adverse general and oral health effects, compared with combustible cigarettes. However, although e­cigarettes might be an acceptable harm-reduction strategy, the differential effects of e­cigarettes and combustible cigarettes have been based on self-reported perceptions. In addition, a growing number of young people, who have never engaged in combustible cigarette smoking, are smoking e­cigarettes, which may not be harmless. We analyzed peer-reviewed publications available through PubMed to summarize the effects of e­cigarettes on oral health.

Antimicrobial peptides Pep19-2.5 and Pep19-4LF inhibit Streptococcus mutans growth and biofilm formation.

With this study, we investigated the effect of synthetic antimicrobial peptides Pep19-2.5 and Pep194LF alone or in combination with antibiotics on S. mutans growth and biofilm formation/disruption. We also examined the cytotoxic effect of each peptide on monocytes. S. mutans was cultured in the presence of different concentrations of each peptide. We showed that Pep19-2.5 and Pep19-4LF were able to significantly (p ≤ 0.01) inhibit the growth of S. mutans. The synthetic peptides also decreased biofilm formation by S. mutans. Furthermore, both peptides reduced the viability of S. mutans in already formed biofilms. The combination of each peptide with antibiotics (penicillin/streptomycin, P/S) produced additive interactions which inhibited S. mutans growth and biofilm formation. Pep19-2.5 and Pep19-4LF were nontoxic, as they did not decrease monocyte viability and did not increase the lactate dehydrogenase activity of the exposed cells. In conclusion, synthetic peptides Pep19-2.5 and Pep19-4LF did inhibit S. mutans growth and its capacity to form biofilm. Both peptides were found to be nontoxic to monocytes. These data provide new insight into the efficacy of synthetic peptides Pep19-2.5 and Pep19-4LF against S. mutans. These peptides may thus be useful in controlling the adverse effects of this cariogenic bacterium in human.

Cigarette Smoke and E-Cigarette Vapor Dysregulate Osteoblast Interaction With Titanium Dental Implant Surface.

The purpose of this study was to determine the possible deleterious effects of e-cigarette vapor on osteoblast interaction with dental implant material. Osteoblasts were cultured onto Ti6Al4V titanium implant disks and were then exposed or not to whole cigarette smoke (CS), as well as to nicotine-rich (NR) or nicotine-free (NF) e-vapor for 15 or 30 minutes once a day for 1, 2, or 3 days, after which time various analyses were performed. Osteoblast growth on the titanium implant disks was found to be significantly ( P < .001) reduced following exposure to CS and to the NR and NF e-vapors. Osteoblast attachment to the dental implant material was also dysregulated by CS and the NR and NF e-vapors through a decreased production of adhesion proteins such as F-actin. The effects of CS and e-cigarette vapor on osteoblast growth and attachment were confirmed by reduced alkaline phosphatase (ALP) activity and tissue mineralization. The adverse effects of CS and the NR and NF e-vapors on osteoblast interaction with dental implant material also involved the caspase-3 pathway, as the caspase-3 protein level increased following exposure of the osteoblasts to CS or e-vapor. It should be noted that the adverse effects of CS on osteoblast growth, attachment, ALP, and mineralized degradation were greater than those of the NR and NF e-vapors, although the latter did downregulate osteoblast interaction with the dental implant material. Overall results suggest the need to consider e-cigarettes as a possible contributor to dental implant failure and/or complications.

Pulsed electrical stimulation benefits wound healing by activating skin fibroblasts through the TGFß1/ERK/NF-κB axis.

BACKGROUND: Dermal fibroblasts activated by conductive polymer-mediated electrical stimulation (ES) have shown myofibroblast characteristics that favor wound healing. However, the signaling pathway related to this phenotype switch remains unclear, and the in vivo survival of the electrically activated cells has never been studied. METHODS: Primary human skin fibroblasts were exposed to pulsed-ES mediated through polypyrrole (PPy) coated fabrics. The expression of α-smooth muscle actin (α-SMA) and the signaling pathways were investigated by ELISA, Western blot and specific inhibition test, and immunocytochemistry staining as well as qRT-PCR analysis. In vivo implantation was performed in a mouse model to clarify the cell fate or contractile phenotype maintenance following ES stimulation. RESULTS: We demonstrated the upregulation of TGFß1 and phosph-ERK, and the NF-κB nuclear enrichment in the ES-activated cells. The ES-activated fibroblasts retained high level of α-smooth muscle actin expression even after prolonged subculture. Subcutaneous implantation for 15 days revealed more human myofibroblasts in the experimental groups. CONCLUSIONS: These findings demonstrate for the first time the involvement of the TGFß1/ERK/NF-κB signaling pathway in ES-activated fibroblasts. The ES induced phenotype switch proves stable in subculture and in animal, pointing potential application in wound healing. GENERAL SIGNIFICANCE: Reveal of how ES activates cells and the implication of ES activated cells in wound healing.

E-Cigarette Vapor Induces an Apoptotic Response in Human Gingival Epithelial Cells Through the Caspase-3 Pathway.

Electronic cigarettes represent an increasingly significant proportion of today's consumable tobacco products. E-cigarettes contain several chemicals which may promote oral diseases. The aim of this study was to investigate the effect of e-cigarette vapor on human gingival epithelial cells. Results show that e-cigarette vapor altered the morphology of cells from small cuboidal form to large undefined shapes. Both single and multiple exposures to e-cigarette vapor led to a bulky morphology with large faint nuclei and an enlarged cytoplasm. E-cigarette vapor also increased L-lactate dehydrogenase (LDH) activity in the targeted cells. This activity was greater with repeated exposures. Furthermore, e-cigarette vapor increased apoptotic/necrotic epithelial cell percentages compared to that observed in the control. Epithelial cell apoptosis was confirmed by TUNEL assay showing that exposure to e-cigarette vapor increased apoptotic cell numbers, particularly after two and three exposures. This negative effect involved the caspase-3 pathway, the activity of which was greater with repeated exposure and which decreased following the use of caspase-3 inhibitor. The adverse effects of e-cigarette vapor on gingival epithelial cells may lead to dysregulated gingival cell function and result in oral disease. J. Cell. Physiol. 232: 1539-1547, 2017. © 2016 Wiley Periodicals, Inc.

Ex Vivo Assay of Electrical Stimulation to Rat Sciatic Nerves: Cell Behaviors and Growth Factor Expression.

Neurite outgrowth and axon regeneration are known to benefit from electrical stimulation. However, how neuritis and their surroundings react to electrical field is difficult to replicate by monolayer cell culture. In this work freshly harvested rat sciatic nerves were cultured and exposed to two types of electrical field, after which time the nerve tissues were immunohistologically stained and the expression of neurotrophic factors and cytokines were evaluated. ELISA assay was used to confirm the production of specific proteins. All cell populations survived the 48 h culture with little necrosis. Electrical stimulation was found to accelerate Wallerian degeneration and help Schwann cells to switch into migratory phenotype. Inductive electrical stimulation was shown to upregulate the secretion of multiple neurotrophic factors. Cellular distribution in nerve tissue was altered upon the application of an electrical field. This work thus presents an ex vivo model to study denervated axon in well controlled electrical field, bridging monolayer cell culture and animal experiment. It also demonstrated the critical role of electrical field distribution in regulating cellular activities.

Electrically Activated Primary Human Fibroblasts Improve In Vitro and In Vivo Skin Regeneration.

Electrical stimulation (ES) changes cellular behaviors and thus constitutes a potential strategy to promote wound healing. However, well-controlled in vitro findings have yet to be translated to in vivo trials. This study was to demonstrate the feasibility and advantages of transplanting electrically activated cells (E-Cells) to help wound healing. Primary human skin fibroblasts were activated through well defined ES and cultured with keratinocytes to generate engineered human skin (EHS), which were transplanted to nu/nu mice. The electrically activated EHS grafts were analyzed at 20 and 30 days post-grafting, showing faster wound closure, thick epidermis, vasculature, and functional basement membrane containing laminin and type IV collagen that were totally produced by the implanted human cells. Because a variety of cells can be electrically activated, E-Cells may become a new cell source and the transplantation of E-Cells may represent a new strategy in wound healing and tissue engineering. J. Cell. Physiol. 231: 1814-1821, 2016. © 2015 Wiley Periodicals, Inc.

Cigarette smoke condensate increases C. albicans adhesion, growth, biofilm formation, and EAP1, HWP1 and SAP2 gene expression.

BACKGROUND: Smokers are more prone to oral infections than are non-smokers. Cigarette smoke reaches the host cells but also microorganisms present in the oral cavity. The contact between cigarette smoke and oral bacteria promotes such oral diseases as periodontitis. Cigarette smoke can also modulate C. albicans activities that promote oral candidiasis. The goal of this study was to investigate the effect of cigarette smoke condensate on C. albicans adhesion, growth, and biofilm formation as well as the activation of EAP1, HWP1 and secreted aspartic protease 2. RESULTS: Cigarette smoke condensate (CSC) increased C. albicans adhesion and growth, as well as biofilm formation. These features may be supported by the activation of certain important genes. Using quantitative RT-PCR, we demonstrated that CSC-exposed C. albicans expressed high levels of EAP1, HWP1 and SAP2 mRNA and that this gene expression increased with increasing concentrations of CSC. CONCLUSION: CSC induction of C. albicans adhesion, growth, and biofilm formation may explain the increased persistence of this pathogen in smokers. These findings may also be relevant to other biofilm-induced oral diseases.

Cellular mechanisms mediating the anti-cancer effects of carnosol on gingiva carcinoma.

Carnosol, a rosemary polyphenol, displays anticancer properties and is suggested as a safer alternative to conventional surgery, radiotherapy, and chemotherapy. Given that its effects on gingiva carcinoma have not yet been investigated, the aim of this study was to explore its anti-tumor selectivity and to unravel its underlying mechanisms of action. Hence, oral tongue and gingiva carcinoma cell lines exposed to carnosol were analyzed to estimate cytotoxicity, cell viability, cell proliferation, and colony formation potential as compared with those of normal cells. Key cell cycle and apoptotic markers were also measured. Finally, cell migration, oxidative stress, and crucial cell signaling pathways were assessed. Selective anti-gingiva carcinoma activity was disclosed. Overall, carnosol mediated colony formation and proliferation suppression in addition to cytotoxicity induction. Cell cycle arrest was highlighted by the disruption of the c-myc oncogene/p53 tumor suppressor balance. Carnosol also increased apoptosis, oxidative stress, and antioxidant activity. On a larger scale, the alteration of cell cycle and apoptotic profiles was also demonstrated by QPCR array. This was most likely achieved by controlling the STAT5, ERK1/2, p38, and NF-ĸB signaling pathways. Lastly, carnosol reduced inflammation and invasion ability by modulating IL-6 and MMP9/TIMP-1 axes. This study establishes a robust foundation, urging extensive inquiry both in vivo and in clinical settings, to substantiate the efficacy of carnosol in managing gingiva carcinoma.

Eugenol as a potential adjuvant therapy for gingival squamous cell carcinoma.

Adoption of plant-derived compounds for the management of oral cancer is encouraged by the scientific community due to emerging chemoresistance and conventional treatments adverse effects. Considering that very few studies investigated eugenol clinical relevance for gingival carcinoma, we ought to explore its selectivity and performance according to aggressiveness level. For this purpose, non-oncogenic human oral epithelial cells (GMSM-K) were used together with the Tongue (SCC-9) and Gingival (Ca9-22) squamous cell carcinoma lines to assess key tumorigenesis processes. Overall, eugenol inhibited cell proliferation and colony formation while inducing cytotoxicity in cancer cells as compared to normal counterparts. The recorded effect was greater in gingival carcinoma and appears to be mediated through apoptosis induction and promotion of p21/p27/cyclin D1 modulation and subsequent Ca9-22 cell cycle arrest at the G0/G1 phase, in a p53-independent manner. At these levels, distinct genetic profiles were uncovered for both cell lines by QPCR array. Moreover, it seems that our active component limited Ca9-22 and SCC-9 cell migration respectively through MMP1/3 downregulation and stimulation of inactive MMPs complex formation. Finally, Ca9-22 behaviour appears to be mainly modulated by the P38/STAT5/NFkB pathways. In summary, we can disclose that eugenol is cancer selective and that its mediated anti-cancer mechanisms vary according to the cell line with gingival squamous cell carcinoma being more sensitive to this phytotherapy agent.

C. albicans growth, transition, biofilm formation, and gene expression modulation by antimicrobial decapeptide KSL-W.

BACKGROUND: Antimicrobial peptides have been the focus of much research over the last decade because of their effectiveness and broad-spectrum activity against microbial pathogens. These peptides also participate in inflammation and the innate host defense system by modulating the immune function that promotes immune cell adhesion and migration as well as the respiratory burst, which makes them even more attractive as therapeutic agents. This has led to the synthesis of various antimicrobial peptides, including KSL-W (KKVVFWVKFK-NH2), for potential clinical use. Because this peptide displays antimicrobial activity against bacteria, we sought to determine its antifungal effect on C. albicans. Growth, hyphal form, biofilm formation, and degradation were thus examined along with EFG1, NRG1, EAP1, HWP1, and SAP 2-4-5-6 gene expression by quantitative RT-PCR. RESULTS: This study demonstrates that KSL-W markedly reduced C. albicans growth at both early and late incubation times. The significant effect of KSL-W on C. albicans growth was observed beginning at 10 µg/ml after 5 h of contact by reducing C. albicans transition and at 25 µg/ml by completely inhibiting C. albicans transition. Cultured C. albicans under biofilm-inducing conditions revealed that both KSL-W and amphotericin B significantly decreased biofilm formation at 2, 4, and 6 days of culture. KSL-W also disrupted mature C. albicans biofilms. The effect of KSL-W on C. albicans growth, transition, and biofilm formation/disruption may thus occur through gene modulation, as the expression of various genes involved in C. albicans growth, transition and biofilm formation were all downregulated when C. albicans was treated with KSL-W. The effect was greater when C. albicans was cultured under hyphae-inducing conditions. CONCLUSIONS: These data provide new insight into the efficacy of KSL-W against C. albicans and its potential use as an antifungal therapy.

Electrical stimulation modulates osteoblast proliferation and bone protein production through heparin-bioactivated conductive scaffolds.

Electrical fields are known to interact with human cells. This principle has been explored to regulate cellular activities for bone tissue regeneration. In this work, Saos-2 cells were cultured on conductive scaffolds made of biodegradable poly(L-lactide) and the heparin-containing, electrically conducting polypyrrole (PPy/HE) to study their reaction to electrical stimulation (ES) mediated through such scaffolds. Both the duration and intensity of ES enhanced cell proliferation, generating a unique electrical intensity and temporal "window" within which osteoblast proliferation was upmodulated in contrast to the downmodulation or ineffectiveness in other ES regions. The favourable ES intensity (200 mV/mm) was further investigated in terms of the gene activation and protein production of two important osteoblast markers characterised by extracellular matrix maturation and mineralisation, that is alkaline phosphatase (ALP) and osteocalcin (OC). Both genes were found activated and the relevant protein production increased significantly following ES. In contrast, ES in the down-modulation region (400 mV/mm) suppressed the production of both ALP and OC. This work demonstrated that important osteoblast markers can be modulated with specific ES parameters mediated through conductive polymer substrates, providing a unique strategy for bone tissue engineering.

Synergistic Effect of Anethole and Platinum Drug Cisplatin against Oral Cancer Cell Growth and Migration by Inhibiting MAPKase, Beta-Catenin, and NF-κB Pathways.

Cisplatin is a common drug used to treat patients with oral squamous cell carcinoma. However, cisplatin-induced chemoresistance poses a major challenge to its clinical application. Our recent study has shown that anethole possesses an anti-oral cancer effect. In this study, we examined the combined effect of anethole and cisplatin on oral cancer therapy. Gingival cancer cells Ca9-22 were cultured in the presence of various concentrations of cisplatin with or without anethole. The cell viability/proliferation and cytotoxicity were evaluated, respectively, by MTT, Hoechst staining, and LDH assay, while colony formation was measured by crystal violet. Oral cancer cell migration was evaluated by the scratch method. Apoptosis, caspase activity, oxidative stress, MitoSOX, and mitochondrial membrane potential (ΔΨm) levels were evaluated by flow cytometry, and the inhibition of signaling pathways was investigated by Western blot. Our results show that anethole (3 µM) potentiates cisplatin-induced inhibition of cell proliferation and decreases the ΔΨm on Ca9-22 cells. Furthermore, drug combination was found to inhibit cell migration and enhanced cisplatin cytotoxicity. The combination of anethole and cisplatin potentiates cisplatin-induced oral cancer cell apoptosis through the activation of caspase, while we also found anethole and cisplatin to enhance the cisplatin-induced generation of reactive oxygen species (ROS) and mitochondrial stress. In addition, major cancer signaling pathways were inhibited by the combination of anethole and cisplatin such as MAPKase, beta-catenin, and NF-κB pathways. This study reports that the combination of anethole and cisplatin might provide a beneficial effect in enhancing the cisplatin cancer cell-killing effect, thus lowering the associated side effects.

Antagonistic effect of Candida albicans and IFNγ on E-cadherin expression and production by human primary gingival epithelial cells.

Caused mainly by Candida albicans, oropharyngeal candidiasis is the most common oral complication associated with HIV disease worldwide. Host defenses against C. albicans essentially fall into two categories: specific immune mechanisms and local oral mucosal epithelial cell defenses. Since oral mucosa is the first line of defense in the form of a physical barrier against C. albicans invasion, and since epithelial cells are involved in anti-Candida innate immunity through different cytokines, we wanted to determine whether C. albicans alters E-cadherin expression and production, and whether interferon-γ (INFγ), a TH1 cytokine, is involved in the anti-Candida defense. Using primary human gingival epithelial cells, we demonstrated that C. albicans significantly decreased E-cadherin mRNA expression and protein production. This effect was basically obtained at later infective periods (24 and 48h). Interestingly, when IFNγ was added to C. albicans infected epithelial cell cultures, it prevented the side effect of C. albicans on E-cadherin mRNA expression and protein production and deposition. All together, these results suggested concomitant interactions between oral epithelial cells and IFNγ against C. albicans infection.

Phenotypic stress response of Pseudomonas aeruginosa following culture in water microcosms.

The purpose of the present study was to explore the potential behavioural changes of Pseudomonas aeruginosa following growth in different aquatic environmental conditions. To achieve this, P. aeruginosa was cultured in various water microcosms for 12 months under fixed (pH, nutrients and temperature) factors. P. aeruginosa responses to these conditions were investigated using colony morphotype, biochemical and enzymatic characterisation, pyocin typing, serotyping, sensitivity to different classes of antibiotics and molecular identification. Results show that starvation in water microcosms lead to unusual phenotypes. Of interest is that the pyocin changed from 24/n in the wild type to 83/a following culture in the water microcosms, and the serotype changed from O6 in the wild type to O1 in microcosm-cultured P. aeruginosa. Furthermore, the starvation period in various aquatic microcosms enhanced the resistance of P. aeruginosa against beta-lactam antibiotics. Compared to the other aquatic environments, the seawater microcosm produced the greatest amount of variations in P. aeruginosa. Overall, data demonstrated a high adaptability of P. aeruginosa to environmental changes. This may explain the unusual antibiotic-resistant phenotypes belonging to P. aeruginosa species, and their capacity for spreading that leads to human infections.