Epithelial-Mesenchymal Transition in Oral Squamous Cell Carcinoma

Oral cancer is one of the drastic human cancers due to its aggressiveness and high mortality rate. Of all oral cancers, squamous cell carcinoma is the most common accounting for more than 90%. Epithelial-mesenchymal transition (EMT) is suggested to play an important role during cancer invasion and metastasis. Recently, emerging knowledge on EMT in carcinogenesis is explosive, tempting us to analyze previous studies on EMT in oral squamous cell carcinoma (OSCC). In this paper, we have first addressed the general molecular mechanisms of EMT, evidenced by alterations of cell morphology during EMT, the presence of cadherin switching, turning on and turning off of many specific genes, the activation of various signaling pathways, and so on. The remaining part of this paper will focus on recent findings of the investigations of EMT on OSCC. These include the evidence of EMT taking place in OSCC and the signaling pathways employed by OSCC cells during their invasion and metastasis. Collectively, with the large body of new knowledge on EMT in OSCC elaborated here, we are hopeful that targeting treatment for OSCC will be developed

Host-microbial interactions in health and disease

published_or_final_versio

An in vitro anti-inflammatory effect of Thai propolis in human dental pulp cells

Objective: To explore the potential for development of Thai propolis extract as a pulp capping agent to suppress pulpal inflammation from dental pulp infections. This study aimed to examine the anti-inflammatory effect of the propolis extract on the arachidonic acid pathway, activated by interleukin (IL)-1β, in cultured human dental pulp cells.  Methodology: Dental pulp cells, isolated from three freshly extracted third molars, were first characterized for their mesenchymal origin and treated with 10 ng/ml of IL-1β in the presence or absence of non-toxic concentrations of the extract from 0.08 to 1.25 mg/ml, as determined by the PrestoBlue cytotoxic assay. Total RNA was harvested and analyzed for mRNA expressions of 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2). Western blot hybridization was performed to investigate COX-2 protein expression. Culture supernatants were assayed for released prostaglandin E2 levels. Immunofluorescence was conducted to determine involvement of nuclear factor-kappaB (NF-kB) in the inhibitory effect of the extract. Results: Stimulation of the pulp cells with IL-1β resulted in the activation of arachidonic acid metabolism via COX-2, but not 5-LOX. Incubation with various non-toxic concentrations of the propolis extract significantly inhibited upregulated COX-2 mRNA and protein expressions upon treatment with IL-1β (p<0.05), resulting in a significant decrease in elevated PGE2 levels (p<0.05). Nuclear translocation of the p50 and the p65 subunits of NF-kB upon treatment with IL-1β was also blocked by incubation with the extract. Conclusions: Upregulated COX-2 expression and enhanced PGE2 synthesis upon treatment with IL-1β in human dental pulp cells were suppressed by incubation with non-toxic doses of Thai propolis extract via involvement of the NF-kB activation. This extract could be therapeutically used as a pulp capping material due to its anti-inflammatory properties

The Antimicrobial Peptide, LL-37, Inhibits in vitro Osteoclastogenesis

Uncoupled bone resorption leads to net alveolar bone loss in periodontitis. The deficiency of LL-37, the only human antimicrobial peptide in the cathelicidin family, in patients with aggressive periodontitis suggests that LL-37 may play a pivotal role in the inhibition of alveolar bone destruction in periodontitis. We aimed to investigate a novel function of LL-37 in osteoimmunity by blocking osteoclastogenesis in vitro. Human osteoclast progenitor cells were isolated from a buffy coat of blood samples. The cells were cultured in the presence of various concentrations of LL-37 during an in vitro induction of osteoclastogenesis. Non-toxic doses of LL-37 could block multinuclear formation of the progenitor cells and significantly diminish the number of tartrate-resistant acid-phosphatase-positive cells and the formation of resorption pits (p < 0.05), whereas these concentrations induced cellular proliferation, as demonstrated by increased expression of proliferating cell nuclear antigen. Expression of several osteoclast genes was down-regulated by LL-37 treatment. It was demonstrated that nuclear translocation of nuclear- factor-activated T-cells 2 (NFAT2) was blocked by LL-37 treatment, consistent with a significant reduction in the calcineurin activity (p < 0.005). Collectively, our findings demonstrate that LL-37 inhibits the in vitro osteoclastogenesis by inhibiting the calcineurin activity, thus preventing nuclear translocation of NFAT2. Abbreviations: CALCR, calcitonin receptor; ClC-7, chloride-proton exchanger; CTSK, cathepsin K; DAPI, 4′,6-diamidino-2-phenylindole; EGTA, ethylene glycol tetraacetic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M-CSF/CSF1, macrophage-colony- stimulating factor; MMP-9, matrix metalloproteinase-9; MTT, [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]; NFAT2, nuclear factor of activated T-cells 2; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; RANK, receptor activator of nuclear factor kappa-B; RANKL, receptor activator of nuclear factor kappa-B ligand; RT-PCR, reverse-transcription polymerase chain- reaction; TBS, Tris-buffered saline; TCIRG1, T-cell, immune regulator 1, ATPase, H+ transporting, lysosomal V0 subunit A3; TRAcP, tartrate-resistant acid phosphatase

Tetracycline-Loaded Electrospun Poly(L-lactide-co-ε-caprolactone) Membranes for One-Step Periodontal Treatment

In this research, a one-step periodontal membrane, with the required function and properties, has been designed as an alternative method of tissue regenerative treatments. Designed nanoporous prototypes from poly(l-lactide-co-ε-caprolactone) (PLCL, 70:30 mol %) were fabricated by electrospinning, denoted as S-PLCL. They were subsequently loaded with tetracycline (TC) in order to enhance periodontal regeneration and deliver an anti-inflammatory and antibiotic drug. It was found that TC loading did not have any significant effect on the fiber diameter but did increase hydrophilicity. With the increase in TC loading, the water vapor permeability (WVP) of the S-PLCL membrane decreased within the range of 31–56% when compared with neat S-PLCL membranes, while in the solvent-cast film (F-PLCL), no significant change in WVP was observed. Moreover, S-PLCL demonstrated a controllable slow release rate of TC. S-PLCL loaded with 1500 μg/mL of TC showed a release concentration of 30 ppm over a certain time period to promote greater levels of human oral fibroblast and human oral keratinocyte cell proliferation and plaque inhibition. In conclusion, a TC-loaded S-PLCL fibrous membrane has been designed and fabricated to provide the ideal conditions for cell proliferation and antibiotic activity during treatment, outperforming nonfibrous F-PLCL loaded with TC at the same concentration

Understanding the roles of gingival beta-defensins

Gingival epithelium produces β-defensins, small cationic peptides, as part of its contribution to the innate host defense against the bacterial challenge that is constantly present in the oral cavity. Besides their functions in healthy gingival tissues, β-defensins are involved in the initiation and progression, as well as restriction of periodontal tissue destruction, by acting as antimicrobial, chemotactic, and anti-inflammatory agents. In this article, we review the common knowledge about β-defensins, coming from in vivo and in vitro monolayer studies, and present new aspects, based on the experience on three-dimensional organotypic culture models, to the important role of gingival β-defensins in homeostasis of the periodontium

Expression of human beta-defensins 1 and 2 in kidneys with chronic bacterial infection

BACKGROUND: Constitutive expression and localization of antimicrobial human β-defensin-1 (HBD-1) in human kidneys as a potential mechanism of antimicrobial defense has been previously reported. Inducible expression of human β-defensin-2 (HBD-2) has been described in various epithelial organs but not for the urogenital tract. METHODS: We investigated the gene- and protein expression of HBD-1 and HBD-2 by reverse transcriptase-polymerase chain reaction, and immunohistochemistry in 15 normal human kidney samples and 15 renal tissues with chronic bacterial infection. Additionally, cell culture experiments were performed to study HBD gene expression by real-time RT-PCR in response to inflammatory cytokines TNFα and IL-1β as well as lipopolysaccharide from Gram-negative bacteria. RESULTS: Constitutive HBD-1 gene- and protein expression was detected in normal renal tissue and kidneys with chronic infection. As a novel finding, inducible HBD-2 gene- and protein expression was demonstrated in tubulus epithelia with chronic infection but not in normal renal tissue. In pyelonephritic kidneys HBD-1 and HBD-2 expression showed a similar pattern of localizaton in distal tubules, loops of Henle and in collecting ducts of the kidney. Furthermore, real-time RT-PCR of kidney derived cell lines stimulated with inflammatory agents TNF-α, IL-1β and LPS revealed a strong increase in relative HBD-2 transcription level and also a slight increase in relative HBD-1 transcription level. CONCLUSIONS: Upregulated HBD-2 expression in renal tubulus epithelium indicates a role of a wider range of human defensins for antimicrobial host defense in the urogenital tract than previously recognized

Naturopathic Care for Anxiety: A Randomized Controlled Trial ISRCTN78958974

BACKGROUND: Anxiety is a serious personal health condition and represents a substantial burden to overall quality of life. Additionally anxiety disorders represent a significant cost to the health care system as well as employers through benefits coverage and days missed due to incapacity. This study sought to explore the effectiveness of naturopathic care on anxiety symptoms using a randomized trial. METHODS: Employees with moderate to severe anxiety of longer than 6 weeks duration were randomized based on age and gender to receive naturopathic care (NC) (n = 41) or standardized psychotherapy intervention (PT) (n = 40) over a period of 12 weeks. Blinding of investigators and participants during randomization and allocation was maintained. Participants in the NC group received dietary counseling, deep breathing relaxation techniques, a standard multi-vitamin, and the herbal medicine, ashwagandha (Withania somnifera) (300 mg b.i.d. standardized to 1.5% with anolides, prepared from root). The PT intervention received psychotherapy, and matched deep breathing relaxation techniques, and placebo. The primary outcome measure was the Beck Anxiety Inventory (BAI) and secondary outcome measures included the Short Form 36 (SF-36), Fatigue Symptom Inventory (FSI), and Measure Yourself Medical Outcomes Profile (MY-MOP) to measure anxiety, mental health, and quality of life respectively. Participants were blinded to the placebo-controlled intervention. RESULTS: Seventy-five participants (93%) were followed for 8 or more weeks on the trial. Final BAI scores decreased by 56.5% (p<0.0001) in the NC group and 30.5% (p<0.0001) in the PT group. BAI group scores were significantly decreased in the NC group compared to PT group (p = 0.003). Significant differences between groups were also observed in mental health, concentration, fatigue, social functioning, vitality, and overall quality of life with the NC group exhibiting greater clinical benefit. No serious adverse reactions were observed in either group. RELEVANCE: Many patients seek alternatives and/or complementary care to conventional anxiety treatments. To date, no study has evaluated the potential of a naturopathic treatment protocol to effectively treat anxiety. Knowledge of the efficacy, safety or risk of natural health products, and naturopathic treatments is important for physicians and the public in order to make informed decisions. INTERPRETATION: Both NC and PT led to significant improvements in patients' anxiety. Group comparison demonstrated a significant decrease in anxiety levels in the NC group over the PT group. Significant improvements in secondary quality of life measures were also observed in the NC group as compared to PT. The whole system of naturopathic care for anxiety needs to be investigated further including a closer examination of the individual components within the context of their additive effect. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN78958974

Sphingosine Kinase-1 Is Required for Toll Mediated β-Defensin 2 Induction in Human Oral Keratinocytes

Host defense against invading pathogens is triggered by various receptors including toll-like receptors (TLRs). Activation of TLRs is a pivotal step in the initiation of innate, inflammatory, and antimicrobial defense mechanisms. Human beta-defensin 2 (HBD-2) is a cationic antimicrobial peptide secreted upon gram-negative bacterial perturbation in many cells. Stimulation of various TLRs has been shown to induce HBD-2 in oral keratinocytes, yet the underlying cellular mechanisms of this induction are poorly understood.Here we demonstrate that HBD-2 induction is mediated by the Sphingosine kinase-1 (Sphk-1) and augmented by the inhibition of Glycogen Synthase Kinase-3beta (GSK-3beta) via the Phosphoinositide 3-kinase (PI3K) dependent pathway. HBD-2 secretion was dose dependently inhibited by a pharmacological inhibitor of Sphk-1. Interestingly, inhibition of GSK-3beta by SB 216763 or by RNA interference, augmented HBD-2 induction. Overexpression of Sphk-1 with concomitant inhibition of GSK-3beta enhanced the induction of beta-defensin-2 in oral keratinocytes. Ectopic expression of constitutively active GSK-3beta (S9A) abrogated HBD-2 whereas kinase inactive GSK-3beta (R85A) induced higher amounts of HBD-2.These data implicate Sphk-1 in HBD-2 regulation in oral keratinocytes which also involves the activation of PI3K, AKT, GSK-3beta and ERK 1/2. Thus we reveal the intricate relationship and pathways of toll-signaling molecules regulating HBD-2 which may have therapeutic potential