Effect of CD133 overexpression on the epithelial-to-mesenchymal transition in oral cancer cell lines.

Oral squamous cell carcinoma (OSCC) is one of the most common cancers in the world. In OSCC, CD133 promotes tumor invasion and metastasis by inducing the epithelial-to-mesenchymal transition (EMT). A small subset of cancer cells known as cancer stem cells (CSCs) are thought to give rise to differentiated tumor cells and to predict tumor recurrence and metastases, i.e., CSCs may be metastatic precursors. In this study, we show that ectopic overexpression of CD133 in OSCC cell lines KB, YD9, and YD10B cells significantly promotes the EMT and acquisition of stemness properties. CSC properties were analyzed by colony-formation assay and measurement of OCT4, SOX2, and NANOG expression, and the EMT was monitored by cell migration, a cell invasion assay, and analysis of E-cadherin, N-cadherin, and vimentin expression. CD133 overexpression led to formation of irregular spheroid colonies consistent with a stem cell phenotype and increased the expression of OCT4, SOX2, NANOG, N-cadherin, and vimentin. Taken together, these findings show that elevated levels of CD133 lead to OSCC invasiveness and metastasis, associated with the upregulation of EMT and stemness markers.

Effects of light-emitting diode irradiation on RANKL-induced osteoclastogenesis.

BACKGROUND AND OBJECTIVE: Bone homeostasis is maintained by a balance between osteoblastic bone formation and osteoclastic bone resorption, where intracellular reactive oxygen species (ROS) are crucial mediators of osteoclastogenesis. Recently, low-level light therapy (LLLT), a form of laser medicine used in various clinical fields, was shown to alleviate oxidative stress by scavenging intracellular ROS. The present study aimed to investigate the impact of 635 nm irradiation from a light-emitting diode (LED) on osteoclastogenesis from receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL)-stimulated mouse bone marrow-derived macrophages (BMMs). STUDY DESIGN/MATERIALS AND METHODS: The effects of LED irradiation on osteoclastogenesis were assessed in tartrate-resistant acid phosphatase (TRAP), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cell viability, and resorption pit formation, respectively. Quantitative real-time polymerase chain reaction (qPCR) and Western blot analyses were also performed to assess mRNA expression of osteoclastogenesis-related genes and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2), p38, and c-Jun-N-terminal kinase (JNK). NF-κB activity was assayed by luciferase reporter assay and Intracellular ROS generation was investigated by the 2',7'-dichlorodihydrofluorescein diacetate (H2 DCF-DA) detection method. RESULTS: LED irradiation significantly inhibited RANKL-mediated osteoclast differentiation from BMMs and mRNA expression of TRAP, osteoclast-associated immunoglobulin-like receptor (OSCAR), and dendrocyte-expressed seven-transmembrane protein (DC-STAMP). Exposure to LED light likewise significantly decreased RANKL-facilitated NF-κB activity, p38 and ERK phosphorylation and intracellular ROS generation, and increased gene expression of nuclear factor E2-related factor 2 (Nrf2). CONCLUSIONS: Taken together, the results presented herein show that LED irradiation downregulates osteoclastogenesis by reducing ROS production. Therefore, LED irradiation/LLLT might be useful as an alternative, conservative approach to osteoporosis management.

Focal adhesion linker proteins expression of fibroblast related to adhesion in response to different transmucosal abutment surfaces.

PURPOSE: To evaluate adherence of human gingival fibroblasts (HGFs) to transmucosal abutment of dental implant with different surface conditions with time and to investigate the roles of focal adhesion linker proteins (FALPs) involved in HGFs adhesion to abutment surfaces. MATERIALS AND METHODS: Morphologies of cultured HGFs on titanium and ceramic discs with different surface were observed by scanning electron microscopy. Biocompatibility and focal adhesion were evaluated by ultrasonic wave application and cell viability assay. FALPs expression levels were assessed by RT-PCR and western blot. RESULTS: There seemed to be little difference in biocompatibility and adhesion strength of HGFs depending on the surface conditions and materials. In all experimental groups, the number of cells remaining on the disc surface after ultrasonic wave application increased more than 2 times at 3 days after seeding compared to 1-day cultured cells and this continued until 7 days of culture. FALPs expression levels, especially of vinculin and paxillin, also increased in 5-day cultured cells compared to 1-day cultured fibroblasts on the disc surface. CONCLUSION: These results might suggest that the strength of adhesion of fibroblasts to transmucosal abutment surfaces increases with time and it seemed to be related to expressions of FALPs.

Agr function is upregulated by photodynamic therapy for Staphylococcus aureus and is related to resistance to photodynamic therapy.

Photodynamic therapy (PDT) has been considered a feasible alternative for antimicrobial therapy of multidrug-resistant pathogens. However, bacterial response mechanisms against PDT-generated photo-oxidative stress remain largely unknown. Herein, it is shown that the accessory gene regulator Agr is involved in Staphylococcus aureus response to photo-oxidative stress generated by laser-induced PDT with the photosensitizer chlorin e6 . Transcriptional profiling revealed that sublethal PDT induces a general stress response and also activates Agr-dependent gene regulation. Moreover, mutant S. aureus lacking Agr function showed hypersusceptibility to two independent PDT conditions with higher energy densities, demonstrating Agr-dependent S. aureus resistance against PDT.

In vitro and in vivo antimicrobial effect of photodynamic therapy using a highly pure chlorin e6 against Staphylococcus aureus Xen29.

Photodynamic therapy (PDT) has been recommended as an alternative therapy for various diseases including microbial infection. Recently, we developed a new method for the preparation of highly pure chlorin e(6) (Ce(6)), which has been widely used as a second-generation photosensitizer. PDT using Ce(6) was very effective for inhibition of in vitro growth of several bacterial strains. To clarify a possibility for its clinical application, in this study, we examined in vitro and in vivo antimicrobial effects of Ce(6)-mediated PDT in mice model of skin infection of Staphylococcus aureus Xen29. Inhibition zone analysis and colony forming unit (CFU) count revealed that Ce(6)-mediated PDT inhibited effectively in vitro bacterial growth. In addition, biofilm formation ability of S. aureus Xen29 was decreased by Ce(6)-mediated PDT. In vivo experiment, mice receiving Ce(6)-mediated PDT exhibited less intensity of bioluminescent signal, showing significant inhibition of bacterial growth. Furthermore, in histopathological examination, marked neutrophilic infiltration and massive bacterial colonies were seen in control mice and mice receiving laser or Ce(6) alone, but not in mice treated with PDT. These results suggest that PDT using Ce(6) extracted by our new method can be clinically useful against bacterial infectious diseases.

Relaxin is up-regulated in the rat ovary by orthodontic tooth movement.

Relaxin (Rln) is an ovarian hormone that stimulates osteoclastic and osteoblastic activities and connective tissue turnover. To investigate the expression of Rln during orthodontic tooth movement, rats were implanted with orthodontic appliances that connected a spring from the upper incisors to the first molar with a 70 cN force. Rats in each group were killed 6, 48, and 144 h after activating the appliance, and the levels of Rln1 and Rln3 expression in the ovary were determined by real-time RT-PCR, northern blots, western blots, and immunofluorescence analyses. The amount of tooth movement induced by the orthodontic force increased in a time-dependent manner. The levels of Rln1 mRNA increased by 12-, 41-, and 263-fold at 6, 48, and 144 h, respectively, after orthodontic tooth movement. The time-dependent increase in the concentration of Rln 1 protein in the ovary was also confirmed by western blotting. Rln 1 was localized in the granulosa cells of the ovarian follicles, and the immunoreactivity against Rln 1 was increased by the movement. In contrast, the concentration of Rln 3 was below the level of detection. The results of this study suggest that local changes in periodontal tissues induced by orthodontic tooth movement may affect Rln1 expression in the ovary. However, further studies are needed to decipher the mechanisms involved and the possible contribution of the increased level of expression of Rln 1 to the tooth movement.

Photodynamic therapy with hexenyl ester of 5-aminolevulinic acid induces necrotic cell death in salivary gland adenocarcinoma cells.

Photodynamic therapy has been developed as an alternative therapy of cancer. The aim of this study was to examine whether PDT with hexenyl ester of 5-aminolaevulinic acid (ALA-hx) inhibits the proliferation of the salivary gland adenocarcinoma SGT cells. Cell proliferation was examined by MTT assay. The gene expression of Coproporphyrinogen oxidase (CPO) and ROS production was also examined. Flow cytometry and in vivo Chorioallantoic membrane (CAM) assay was performed. ALA-hx PDT inhibited effectively the proliferation of SGT cells. Treatment of ALA-hx induced CPO mRNA expression and ROS was produced by ALA-hx PDT in SGT cells. Flow cytometry and LDH assay showed that ALA-hx PDT induced necrotic cell death rather than apoptosis in SGT cells. In vivo CAM assay showed that ALA-hx PDT induced tumor destruction by inducing necrosis. These results indicate that ALA-hx PDT effectively inhibits the proliferation of SGT cells by inducing necrosis.

Antimicrobial effect of photodynamic therapy using a highly pure chlorin e6.

The aim of the present study is to evaluate the antimicrobial effect of photodynamic therapy (PDT) using a highly pure chlorin e(6) (Ce(6)), against various pathogenic bacteria. To examine the antimicrobial effect of Ce(6)-mediated PDT against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Salmonella enterica serovar Typhimurium, inhibition zone formation, CFU quantification, and bacterial viability were evaluated. Inhibition zone analysis showed that Ce(6)-mediated PDT is very effective to inhibit the growth of S. aureus and P. aeruginosa, but has only minor effect to E. coli and S. Typhimurium, which was dependent on the energy density of laser and dose of Ce(6). Ce(6)-mediated PDT also nearly inhibited the colony formation of S. aureus and P. aeruginosa, and partially inhibited that of E. coli and S. Typhimurium. In addition, the number of viable bacteria decreased greatly after PDT application with LS-chlorin e6 of 10 microM and laser and energy density of 20 J/cm(2). These results show that Ce(6)-mediated PDT can be an effective alternative for antimicrobial treatment.

Anticancer effect of photodynamic therapy with hexenyl ester of 5-aminolevulinic acid in oral squamous cell carcinoma.

BACKGROUND: Five-aminolaevulinic acid (ALA) and its derivatives act as precursors of the photosensitizer protoporphyrin IX (PpIX). In this study, the effect of photodynamic therapy (PDT) with hexenyl ester of ALA (ALA-hx) was examined in a human oral squamous cell carcinoma, YD10B cells. METHODS: PpIX accumulation and mRNA expression of coproporphyrinogen oxidase (CPO) by ALA and ALA-hx was examined. Cell viability was examined by MTT assay and the molecular mechanism was investigated. RESULTS: The PpIX synthesis and mRNA expression of CPO was much higher in the cells treated with ALA-hx than ALA. At the concentration that PDT with ALA did not affect cell growth, ALA-hx PDT effectively produced reactive oxygen species (ROS) and suppressed cell growth. Growth inhibition by ALA-hx PDT was due to mitochondrial-dependent apoptosis. CONCLUSION: Our results suggest that ALA-hx PDT effectively induced apoptosis of YD-10B cells and can be considered as a therapeutic alternative for oral cancer.

Efficient preparation of highly pure chlorin e6 and its photodynamic anti-cancer activity in a rat tumor model.

Photodynamic therapy (PDT) is currently being used as an alternative therapeutic modality for a variety of malignant tumors. This study was performed to show an efficient preparation of second generation of photosensitizer chlorin e6 (Ce6) with high yield and purity, and to test antitumor activity of Ce6-induced PDT (Ce6-PDT) both in vitro and in vivo using a rat tumor model. Three-week-old male Sprague-Dawley (SD) rats were inoculated s.c. on the right flank with 5x10(6) RK3E-ras cells. The animals were administered i.v. with Ce6 (10 mg/kg) and 24 h later, PDT was performed using a laser diode at a light dose of 100 J/cm2. Ce6-PDT generated reactive oxygen species and led to significant growth inhibition in RK3E-ras cell. In addition, Ce6-PDT induced apoptosis through the activation of caspase-3 and its downstream target, PARP cleavage. The protein level of anti-apoptotic bcl-2 was also reduced by Ce6-PDT in RK3E-ras cells. In in vivo experiments, application of Ce6-PDT led to a significant reduction of tumor size. PCNA immunostaining and TUNEL assay revealed that Ce6-PDT inhibited tumor cell proliferation and increased apoptosis. These findings suggest that the newly purified Ce6-PDT can effectively arrest tumor growth by inhibiting cell proliferation and inducing apoptosis.