Identification, characterization and microRNA expression profiling of side population cells in human oral squamous cell carcinoma Tca8113 cell lines.

The present study aimed to evaluate the stem cell markers, characteristics and biological functions of cancer stem­like side population (SP) cells in human oral cancer. SP cells were isolated from the human oral squamous cell carcinoma Tca8113 cell line by Hoechst 33342 fluorescence dye and flow cytometry. The colony forming and proliferative capability of SP and non­SP cells were detected using a live­cell analysis system in vitro. The number of cells expressing stem cell markers was compared between SP cells and non­SP cells by flow cytometry. Reverse transcription­quantitative polymerase chain reaction and western blotting were used to detect the mRNA and protein expression levels of stem cell genes, respectively. Differential expression of microRNAs (miRNAs) in SP and non­SP cells was determined by microarray hybridization and an miRNA regulation network was produced. With regard to the proliferation capability, SP cells reached 60.0% confluence after 40 h of growth compared with 35.1% confluence for non­SP cells (P<0.05). The number of colonies in SP cells was 43.1±9.2 compared with 33.0±8.2 of non­SP cells (P<0.05). The aldehyde dehydrogenase­1 (ALDH1)­positive cell number in the SP cells was increased by 10 times compared with the non­SP cells (P<0.01). The mRNA and protein expression levels of ALDH1, SRY­box 2, POU class 5 homeobox 1 and Nanog homeobox in SP cells were significantly higher compared with non­SP cells (P<0.05). Microarray hybridization demonstrated that 21 miRNAs were upregulated and 13 miRNAs were downregulated in SP cells compared with non­SP cells. SP cells in Tca8113 demonstrated greater capability of proliferation and colony formation compared with non­SP cells in vitro. Stem cell markers were overexpressed in SP cells compared with non­SP cells.

[Changes of cementum endotoxin levels in different teeth with periodontitis treated with root conditioning].

PURPOSE: To observe the changes of endotoxin levels after different teeth with periodontitis were treated with different methods. METHODS： Six healthy premolars extracted for orthodontic reasons and 36 posterior teeth extracted due to severe periodontitis were selected. Each tooth was processed from two 4 mm×4 mm×1 mm cementum pieces 2 mm under the cementum-enamel junction, each tooth with periodontitis was numbered. Healthy teeth served as negative control group, one of the two tablets from each tooth with periodontitis was selected in the periodontitis group, which was not treated with root surface treatment. The remaining 36 teeth with periodontitis were randomly divided into 6 groups: SRP group, SRP + antimicrobial peptide A group , SRP + antimicrobial peptide B group, SRP + EDTA group, SRP + Er:YAG laser group and SRP + Nd:YAG laser group. Endotoxin concentration in each tooth was determined by chromogenic substrate limulus reagent. The endotoxin concentration in each tooth was recorded according to the serial number, and the changes of endotoxin concentration were calculated before and after treatment. SPSS17.0 software package was used to analyze the data. RESULTS: Compared with the teeth with periodontitis, endotoxin concentration decreased to varying degrees, there were significant differences in each treatment group(P<0.01). Compared with SRP group, endotoxin levels in SRP + antimicrobial peptide A group, SRP + antimicrobial peptide B group, SRP + Er:YAG laser group were significantly decreased(P<0.01). No significant difference decreased was from between SRP + EDTA group and SRP + Nd:YAG laser group(P>0.05). CONCLUSIONS: Treatment of teeth with periodontitis using different methods can decrease the level of endotoxin, and the treatment of periodontitis root surface with antimicrobial peptide A + SRP may be more effective.

Subcellular location and photodynamic therapeutic effect of chlorin e6 in the human tongue squamous cell cancer Tca8113 cell line.

The present study aimed to investigate the distribution and photodynamic therapeutic effect of chlorin e6 (Ce6) in the human tongue squamous cell carcinoma Tca8113 cell line in vitro. The distribution of Ce6 in the Tca8113 cells was observed in situ combined with mitochondrial and lysosomal fluorescent probes. Next, 630-nm semiconductor laser irradiation was performed. The MTS colorimetric method was used to determine cell survival. Annexin V fluorescein isothiocyanate/propidium iodide (PI) double staining was used to detect early apoptosis following photodynamic therapy (PDT). The flow cytometer was used to analyze the DNA content subsequent to PI-staining. It was observed that Ce6 could combine with the cellular membrane following 30 min of incubation with the Tca8113 cells. As the length of incubation increased, Ce6 gradually entered the cells in a particular distribution and reached saturation by 3 h. Co-localization analysis demonstrated that Ce6 was more likely to be present in the mitochondria than in the lysosomes. The cells incubated with 5 µg/ml Ce6 for 24 h exhibited a low toxicity of 5%, however, following light irradiation, Ce6-PDT was able to kill the Tca8113 cells in vitro. The cell toxicity was positively correlated with Ce6 concentration and light dose, therefore, the effect of Ce6 was concentration/dose-dependent (P<0.01). The lower Ce6 concentrations and light doses could significantly induce apoptosis in the Tca8113 cells, while higher doses increased necrosis/percentage of dead cells. In summary, Ce6 saturated the Tca8113 cells following 3 h of incubation. Furthermore, Ce6-PDT effectively killed the cultured Tca8113 cells in vitro at a safe concentration. At a low concentration and light dose, Ce6 is more likely to induce cell apoptosis via the mitochondria than the lysosomes.