Identification and characterization of a highly metastatic epithelial cancer cell line from rat tongue cancer

ObjectivesTongue squamous cell carcinoma (TSCC) is a clinically devastating disease. However, most established TSCC cell lines currently show undesirable malignant behaviours. The purpose of this study is to establish a highly metastatic TSCC cell line to serve as a useful tool for basic research.Materials and methodsTSCCs were induced by 4-nitroquinoline-1-oxide (4NQO) in Sprague-Dawley rats. Tumor cells were obtained from the cancer tissues by primary culture and were then purified by an in vitro invasion assay and a limiting dilution assay. The growth rate, cell cycle distribution, apoptotic rate, tumorigenicity and distant metastatic phenotypes of the rat tongue cancer cells were fully investigated and characterized.ResultsTo date, the rat tongue cancer cell line, named Rca-T, has been continuously cultured in vitro for over 210 passages and exhibit a long spindle-shaped morphology, adherent growth, and a stable epithelial phenotype. The population doubling time of Rca-T cells is 23.35 h. Approximately 39.8% of these cells are in S phase, and the apoptosis rate of Rca-T cells is 7.46%. Furthermore, in immunodeficient nude mice, both the xenograft rate and the incidence of experimental lung metastasis are 100%. The in vitro assays further reveal the highly malignant and epithelial-mesenchymal transition-like properties of Rca-T cells.ConclusionIn this study, the tumorigenic and highly distant metastatic TSCC cell line Rca-T was established. The malignant features of this cell line, especially its metastatic potential, will enable a wealth of functional studies on the molecular mechanisms of TSCC metastasis in the future.     

Identification and characterization of neural crest-derived cells in adult periodontal ligament of mice

Objective

Cells derived from the neural crest (NC) contribute to the development of several adult tissues, including tooth and periodontal tissue. Here, two transgenic lines, Wnt1-Cre/ZEG and P0-Cre/ZEG, were analysed to determine the fate and distribution of neural crest cells (NCCs) in adult mouse periodontal ligament (PDL).

Design

Paraffin-embedded and decalcified histology samples were prepared from Wnt1-Cre/ZEG and P0-Cre/ZEG mice that were 4-, 8-, or 12-weeks old. Expression of GFP (NC-derived cells), NC-markers (Slug, AP-2 alpha, HNK-1, p75NTR and Nestin), and mesenchymal stem cell markers (CD29 and STRO-1) were examined using immunohistochemistry.

Results

In four-week-old Wnt1-Cre/ZEG mice, GFP⁽⁺⁾ NC-derived cells were specifically detected in the mid-zone of PDL. The GFP⁽⁺⁾ cells constituted 1.4% of all cells in PDL, and this percentage decreased as the mice aged. The distribution and prevalence of GFP⁽⁺⁾ cells were comparable between Wnt1-Cre/ZEG and P0-Cre/ZEG mice. NC-marker⁽⁺⁾ cells were expressed only in GFP⁽⁺⁾ cells while MSC markers were detected only in GFP⁽⁻⁾ cells.

Conclusion

The prevalence and specific distribution of NC-derived cells in adult PDL of Wnt1-Cre/ZEG and P0-Cre/ZEG mouse were examined. Interestingly, various NC markers, including markers for undifferentiated NCCs, were still expressed at high levels in GFP⁽⁺⁾ cells. These observations may indicate that labelled cells in the Wnt1-Cre/ZEG and P0-Cre/ZEG mice did not constituted all NC-derived cells, but rather an interesting subset of NC-derived cells. These findings may be useful in understanding the homeostatic character of the PDL and contribute to establishing successful periodontal tissue maintenance.     

Apigenin inhibited hypoxia induced stem cell marker expression in a head and neck squamous cell carcinoma cell line

ObjectiveCancer stem cells contribute to tumor recurrence, and a hypoxic environment is critical for maintaining cancer stem cells. Apigenin is a natural product with anticancer activity. However, the effect of apigenin on cancer stem cells remains unclear. Our aim was to investigate the effect of apigenin on cancer stem cell marker expression in head and neck squamous cell carcinoma cells under hypoxia.DesignWe used three head and neck squamous cell carcinoma cell lines; HN-8, HN-30, and HSC-3. The mRNA expression of cancer stem cell markers was determined by semiquantitative RT-PCR and Real-time PCR. The cytotoxic effect of apigenin was determined by MTT colorimetric assay. Flow cytometry was used to reveal the number of cells expressing cancer stem cell surface markers.ResultsHN-30 cells, a cancer cell line from the pharynx, showed the greatest response to hypoxia by increasing their expression of CD44, CD105, NANOG, OCT-4, REX-1, and VEGF. Apigenin significantly decreased HN-30 cell viability in dose- and time-dependent manners. In addition, 40 μM apigenin significantly down-regulated the mRNA expression of CD44, NANOG, and CD105. Consistent with these results, the hypoxia-induced increase in CD44⁺ cells, CD105⁺ cells, and STRO-1⁺ cells was significantly abolished by apigenin.ConclusionApigenin suppresses cancer stem cell marker expression and the number of cells expressing cell surface markers under hypoxia.     

Effects of low-level laser therapy on the proliferation and apoptosis of gingival fibroblasts treated with zoledronic acid

Low-level laser therapy (LLLT) has been indicated as an adjuvant therapy for bisphosphonate-induced osteonecrosis. However, the effects of LLLT on bisphosphonate-treated cells are not yet clear. This study evaluated the effects of LLLT on the proliferation and apoptosis of gingival fibroblasts treated with zoledronic acid (ZA). Cells were exposed to ZA at 5 μM for 48 h. Irradiation was performed using a laser diode prototype (LaserTABLE, InGaAsP; 780 nm ± 3 nm, 25 mW) at 0.5 or 3 J/cm², three times every 24 h. Cell proliferation and apoptosis were evaluated by fluorescence microscopy. Data were analyzed by Mann–Whitney test at the 5% level of significance. ZA decreased cell proliferation to 47.62% (interquartile range (IQR) 23.80–57.14%; P = 0.007) and increased apoptosis of gingival fibroblasts to 27.7% (IQR 20.9–33.4%; P = 0.0001). LLLT increased cell proliferation compared with non-irradiated cells, at 0.5 J/cm² (57.14%, IQR 57.14–71.43%; P = 0.003) and at 3 J/cm² (76.19%, IQR 61.90–76.19%; P = 0.0001), but did not increase cell proliferation in ZA-treated cells. Irradiated fibroblasts presented lower apoptosis rates than the ZA-treated cells, but apoptosis was no different in ZA-treated cells compared to those that were ZA-treated and also irradiated.     

Caffeine increases the expression of cystatin SN in human submandibular acinar-like HSG cells

ObjectiveThe study aimed at evaluating in vitro the effect of caffeine on expression of cystatin SN, a potential marker of sensitivity to bitterness in humans.MethodsDifferentiation of human submandibular gland (HSG) cells was induced by culturing cells on Matrigel. Caffeine cytotoxicity was assessed over 3 days by the Resazurin test. Finally, effects of 5, 50 and 100 μM caffeine exposure on cystatin SN expression were explored over 3 days by ELISA.ResultsAt concentrations relevant to human adult plasma levels (5, 50 and 100 μM), caffeine did not affect cell viability whether cells were differentiated or not. Cystatin SN levels were overall higher in differentiated cells and increased with time in both conditions. There was a significant (p < 0.001) effect of caffeine on cystatin SN expression specifically in differentiated cells.ConclusionsThe HSG cell line proved to be a relevant tool to study in vitro the effect of caffeine at concentrations consistent with dietary intake in human subjects. The results suggest that salivary cystatin SN abundance may depend on caffeine intake, with possible consequences on taste sensitivity.     

Establishment of a highly metastatic buccal squamous cell carcinoma cell line from a Sprague-Dawley Rat

ObjectivesThe incidence of buccal squamous cell carcinoma (buccal SCC) is considered to be the second highest out of all oral cancers, but the unsatisfactory in vivo tumorigenicity and metastatic potential of the widely used cell lines have greatly delayed studies on the mechanisms of tumor progression. This study aimed to establish a highly metastatic buccal SCC cell line, which may serve a useful tool in buccal SCC research.Materials and methodsBuccal SCC was induced by 4-nitroquinoline-1-oxide (4NQO) in Sprague-Dawley rats. The cancer samples were collected, and the tumor cells were purified in vitro. A highly aggressive cell line termed “Rca-B” was established by an invasion assay. Its proliferative ability, cell cycle distribution, baseline level of apoptosis, carcinogenicity and metastatic behavior in nude mice were investigated.ResultsTo date, Rca-B cells have been stably cultured in vitro for more than 180 passages. These cells were polygonal or spindle-shaped, grew adhesively, and exhibited a stable epithelial phenotype as characterized by positive expression of cytokeratin. The population doubling time was 25.09 h. Cells in S-phase of the cell cycle accounted for 31.17% of the total number of cells, and the baseline level of apoptosis was 8.52%. The in vitro migration and invasion assays revealed highly aggressive features of Rca-B cells. In addition, the rate of xenograft formation was 100%, and the incidence of experimental lung metastasis was 81.8% in immunodeficient nude mice.ConclusionThe Rca-B cell line was established as a highly metastatic rat buccal SCC cell line, and its in-depth characterization, which includes malignant behaviors, allows for a wealth of functional studies on the molecular mechanisms of buccal SCC progression and targeted therapy.     

The role of SPP1 as a prognostic biomarker and therapeutic target in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignancies and has a low 5-year survival rate. Mounting evidence suggests that oral potentially malignant disorders, such as oral leukoplakia (OLK), may progress to HNSCC. Given that OLK and HNSCC are often insidious and asymptomatic, the identification of markers of OLK malignant transformation and therapeutic targets in HNSCC is critical. Using various online tools and publicly available gene expression datasets, the secreted phosphoprotein 1 gene (SPP1) was identified as a significant differentially expressed gene among OLK, HNSCC, and non-cancerous tissues. SPP1 mRNA levels were elevated in HNSCC tissues and were associated with cancer stage, tumor grade, and human papillomavirus infection status. High SPP1 mRNA levels were correlated with poor overall survival of HNSCC patients. In contrast, SPP1 mutations were not significantly associated with overall survival, although their frequency in HNSCC was very low (0.6%). Furthermore, SPP1 expression levels in HNSCC were positively correlated with the infiltration of CD4⁺ cells, macrophages, neutrophils, and dendritic cells. The study results suggest that SPP1 may represent a diagnostic and prognostic biomarker, as well as a potential therapeutic target in HNSCC.     

Immunohistochemical analysis of lymphatic vessel density and mast cells in oral tongue squamous cell carcinoma

The aim of this study was to analyze lymphangiogenesis and the presence of mast cells in oral tongue squamous cell carcinoma (OTSCC), correlating the findings with clinicopathological parameters (clinical stage, tumor size, nodal metastasis, histological grade of malignancy, local recurrence, and clinical outcome). Fifty-six cases of primary OTSCC were selected. Lymphatic vessels and mast cells were identified by immunostaining with anti-podoplanin (D2-40) and anti-tryptase antibody, respectively. Lymphatic vessel density (LVD) and mast cell density (MCD) were determined in the intratumoral and peritumoral areas. Intratumoral LVD was higher in advanced clinical stages (III/IV) when compared to early-stage (p = 0.017) and in metastatic cases compared to non-metastatic tumors (p = 0.013). Peritumoral LVD and intratumoral or peritumoral MCD did not differ significantly according to the clinicopathological parameters of OTSCCs (p > 0.05). No significant correlations between LVD and MCD were observed at the intratumoral (r = ?0.014; p = 0.918) or peritumoral level (r = 0.156; p = 0.251). Our findings suggest that intratumoral lymphatic vessels, compared to peritumoral lymphatic vessels, appear to be more related to the progression of OTSCC. MCD alone does not seem to be determinant for lymphangiogenesis or for the biological behavior of OTSCC, indicating multiple pro- and antitumor effects of these inflammatory cells.     

Dynamics of Bromodeoxyuridine Label–retaining Dental Pulp Cells during Pulpal Healing after Cavity Preparation in Mice

Introduction

This study aimed at clarifying the dynamics of bromodeoxyuridine (BrdU) label-retaining cells (LRCs) and their relationship to cell proliferation and apoptosis during pulpal healing after cavity preparation in mice.

Methods

A groove-shaped cavity was prepared on the mesial cervical surface of the upper first molars, and the samples were collected at intervals of 12 hours–14 days. The demineralized paraffin sections were processed for immunohistochemistry for BrdU, nestin, and Ki-67 and apoptosis assay using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) and in situ hybridization for dentin sialophosphoprotein (Dspp).

Results

During hour 12–day 1, odontoblasts and subodontoblastic cells beneath the affected dentin showed degenerative features and TUNEL-positive reactions, and the expressions of nestin and Dspp were lost in the damaged odontoblasts. TUNEL-positive reactions were observed even in the center of the pulp tissue, whereas dense and granular LRCs remained there. On days 2–3, Ki-67–positive cells were significantly increased in number in the center of mesial dental pulp. During days 3–5, granular and few dense LRCs were committed into some (not all) nestin-positive newly differentiated odontoblast-like cells, and these differentiated cells began to express nestin and Dspp. Until day 14, tertiary dentin formation occurred next to the preexisting dentin at the mesial pulp floor in addition to the mesial coronal pulp.

Conclusions

These results suggest that odontoblasts and subodontoblastic cells degenerate after tooth drilling, and, subsequently, dental pulp stem/progenitor cells actively proliferate and differentiate into new odontoblast-like cells.     

The use of green tea extract as a storage medium for the avulsed tooth

Introduction

Green tea extract (GTE) has been reported to have remarkable anti-inflammatory, antioxidant, and anticarcinogenic effects and to prolong allograft survivals. The purpose of the present study is to investigate in vitro the efficacy of GTE as a storage medium for avulsed teeth. We estimated the possibility for storage medium by maintaining the viability of human periodontal ligament (PDL) cells.

Methods

Human PDL cells were cultured and stored in the following media: (1) Hank’s balanced salt solution (HBSS), (2) tap water, (3) milk, (4) GTE, and (5) commercial green tea. After 1, 3, 6, 12, and 24 hours, cells in different media were examined under the optical microscope, and their viabilities were analyzed by using a nucleocounter and 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium assay. The data were statistically analyzed by analysis of variance tests with post analysis using the Duncan method (P < .05).

Results

The result indicates that there was no difference in cell viability between GTE and HBSS media, whereas GTE showed higher cell viability than other media (P < .05).

Conclusions

Our study shows that the efficacy of GTE in maintaining the viability of human PDL cells is similar to that of HBSS and higher than that of milk. Therefore, we conclude that GTE could be a suitable, alternative storage medium for avulsed teeth.     

CCR7-CCL21 axis promotes the cervical lymph node metastasis of tongue squamous cell carcinoma by up-regulating MUC1

This study aims at investigating the potential role of MUC1 in CCR7-CCL21 axis-induced metastasis of tongue squamous cell carcinoma (TSCC).TSCC patients were selected for epidemiologic trends. The expression of CCR7 and MUC1 was detected via immunohistochemistry. SCC15 and CAL27 cells were induced by CCL21 and specific antibody to CCR7. Gene and protein expression was detected using qRT-PCR and western blotting. Migration and invasion capacities of TSCC cells were determined using wound healing and Transwell invasion assays.The male:female ratio of 78 patients was 1.6:1. Metastasis rate of cervical lymph nodes (CLNs) was 42.3%. CLN metastasis significantly correlated with T staging (P = 0.026), clinical staging (P = 0.024), and depth of invasion (DOI, P = 0.001). DOI significantly influenced CLN metastasis (P = 0.033, OR = 10.919) of TSCC, as did CCR7 (P = 0.041) and MUC1 (P = 0.026). The consistency of CCR7 and MUC1 expression was fairly good (Kappa = 0.683, P < 0.001). Reduced survival was significantly associated with higher expression of CCR7 (P = 0.039) and MUC1 (P = 0.030). CCL21 up-regulated MUC1 in SCC15 cells, which was inhibited when CCR7 was blocked. MUC1 positively correlated with TSCC cell migration and invasion.CCR7-CCL21 axis might promote CLN metastasis of TSCC by up-regulating MUC1. CCR7 and MUC1 show promise as potential biomarkers for TSCC treatment.     

Inhibition of microbial adhesion to plastic surface and human buccal epithelial cells by Rhodomyrtus tomentosa leaf extract

Objective

The adherence of oral pathogenic microorganisms to host tissues is the initial step for successful process of oral diseases. This study aimed to determine the effect of the Rhodomyrtus tomentosa leaf extract and rhodomyrtone, an antibacterial compound from R. tomentosa leaf, on adhesion of some oral pathogens to polystyrene plastic surface and human buccal epithelial cells.

Methods

The minimum inhibitory concentration (MIC) was evaluated using broth microdilution method. The microbial adhesion to the plastic surface and buccal cells was determined using microtiter plate method and microscopy technique.

Results

The ethanol extract of leaf demonstrated antibacterial activity against oral microorganisms including Staphylococcus aureus ATCC 25923, Streptococcus mutans (clinical isolate), and Candida albicans ATCC 90028 with the MIC values of 31.25, 15.62, and 1000 μg/ml, respectively. Rhodomyrtone displayed activity with the MIC values of 0.78 and 0.39 μg/ml against S. aureus ATCC 25923 and S. mutans, respectively. The MIC value of the compound against C. albicans ATCC 90028 was more than 100 μg/ml which was the highest test concentration. All pathogenic microorganisms treated with the extract and rhodomyrtone at their subinhibitory concentrations resulted in a decrease in their adherence ability to both plastic surface and buccal cells.

Conclusion

It is suggested that R. tomentosa extract and rhodomyrtone may be useful in therapy or as prophylaxis in infections involving oral pathogens.     

Differences of isolated dental stem cells dependent on donor age and consequences for autologous tooth replacement

Objective

Autologous therapy via stem cell-based tissue regeneration is an aim to rebuild natural teeth. One option is the use of adult stem cells from the dental pulp (DPSCs), which have been shown to differentiate into several types of tissue in vitro and in vivo, especially into tooth-like structures. DPSCs are mainly isolated from the dental pulp of third molars routinely extracted for orthodontic reasons. Due to the extraction of third molars at various phases of life, DPSCs are isolated at different developmental stages of the tooth.

Design

The present study addressed the question whether DPSCs from patients of different ages were similar in their growth characteristics with respect to the stage of tooth development. Therefore DPSCs from third molars of 12–30 year-old patients were extracted, and growth characteristics, e.g. doubling time and maximal cell division potential were analysed. In addition, pulp and hard dental material weight were recorded.

Results

Irrespective of the age of patients almost all isolated cells reached 40–60 generations with no correlation between maximal cell division potential and patient age. Cells from patients <22 years showed a significantly faster doubling time than the cells from patients ≥22 years.

Conclusion

The age of patients at the time of stem cell isolation is not a crucial factor concerning maximal cell division potential, but does have an impact on the doubling time. However, differences in individuals regarding growth characteristics were more pronounced than age-dependent differences.     

Evaluation of Cd8+ and natural killer cells defense in oral and oropharyngeal squamous cell carcinoma

Purpose

The aim of this study was to evaluate the population of CD8+ and natural killer (NK) cells in samples of oral (OSCC) and oropharyngeal (OPSCC) squamous cell carcinoma.

Characterization of a new spinel Li–Cr–Mn–O for secondary lithium batteries

A new chromium-substituted Li₂O–yMnO₂ (y=4) spinel phase has been prepared at low temperature using a sol–gel process. The electrochemical behavior of the obtained material is investigated in liquid-electrolyte lithium cells. This material presents a plateau at around 3 V versus Li ∣ Li⁺ with a large discharge capacity of 140 mAh g^?1 and fairly good cycle ability.     

Differentiation of dental pulp stem cells into a neural lineage

We previously investigated whether dental pulp-derived cells possess similar pluripotency to bone marrow cells, and reported their capacity to differentiate into osteoblasts, as well as the characteristics of the stem cells present in dental pulp. In the present study, we hypothesized that neural stem cells would also exist in rat dental pulp, similar to bone marrow and the brain, and attempted to induce their differentiation into a neural lineage by applying an in vitro study design previously reported to induce differentiation of human bone marrow cells. Before inducing differentiation, we detected cells expressing nestin (Nes), which is known to be a marker for neural stem cells, within primary cultures of rat dental pulp-derived cells, suggesting the existence of neural stem cells in dental pulp. Quantitative analyses of the mRNA and protein expression levels revealed downregulation of both the Nes mRNA and protein levels to about 68.1% and 12.4%, respectively, after the induction of differentiation compared to the corresponding levels before induction. Conversely, the glial fibrillary acidic protein (Gfap) mRNA level was elevated by 1.3-fold after the induction of differentiation compared with the level before induction. The reduced number of Nes-positive cells and decreased Nes mRNA and protein levels after the induction of differentiation may be attributed to differentiation of neural stem cells into a neural lineage. Moreover, the increased number of Gfap-positive cells and increased Gfap mRNA level after the induction of differentiation most likely support their progressive differentiation into a glial cell lineage, since Gfap is a marker that is upregulated in glial cells. Our present data demonstrate the existence of neural stem cells in tissues other than the central nervous system, and may represent a significant step toward providing more diverse and multiple sources of stem cells for future regenerative medicine.