Overexpression of T-type calcium channel Cav3.1 in oral squamous cell carcinoma: association with proliferation and anti-apoptotic activity.

Cav3.1, a subfamily of T-type calcium channel, is overexpressed in various human cancers and exerts important functions in tumor progression. This study is to identify the expression pattern and clinical significance of Cav3.1 in oral squamous cell carcinoma (OSCC). Firstly, the expression levels of Cav3.1 in oral mucosa (OM), dysplasia and oral squamous cell carcinoma (OSCC) were determined and compared by real-time quantitative PCR and Western blot analysis. After that, human tissue microarrays, containing 29 OM, 23 dysplasia and 122 primary OSCC samples, were applied to investigate the expression levels of Cav3.1, proliferation markers [Ki-67, proliferating cell nuclear antigen (PCNA)] and cellular anti-apoptosis markers [B cell lymphoma 2 (Bcl-2)] by immunohistochemistry and digital pathology analysis. In addition, we determined the function of Cav3.1 using knockdown assays of Cav3.1 in vitro. The results demonstrated that the mRNA and protein expression of Cav3.1 were significantly higher in OSCC specimens, and Cav3.1 expression in primary OSCCs was correlated with tumor size and pathological grade. Statistical analysis of immunohistochemical staining showed that Cav3.1 was closely correlated with Ki-67, PCNA and Bcl-2. Functional studies showed that the knockdown of Cav3.1 in OSCC cell lines using RNA interference influenced cell proliferation and apoptosis in vitro. Taken together, these findings suggested that Cav3.1 is overexpressed in OSCC tissues, also associated with proliferative and anti-apoptotic activity in oral squamous cell carcinoma.

Lymphocyte­derived microparticles stimulate osteoclastogenesis by inducing RANKL in fibroblasts of odontogenic keratocysts.

Leukocyte­derived microparticles (LMPs) include neutrophil­, lymphocyte­ and monocyte­derived MPs. LMPs act as proinflammatory mediators in autoimmune diseases, infectious diseases and vascular diseases. The present study examined the hypothesis that the percentage of LMPs was increased in patients with inflamed odontogenic keratocysts (OKCs), and investigated the biological effects of Jurkat cell­derived MPs on the fibroblasts of OKCs in vitro. Cyst fluid MPs, obtained by centrifugation of samples from 20 patients with inflamed OKCs, 3 patients with uninflamed OKCs, 15 patients with radicular cysts (RCs) and 12 patients with inflamed dentigerous cysts (DCs), were analyzed by transmission electron microscopy, dynamic light scattering and immunofluorescence staining. The percentages and concentrations of cyst fluid LMPs were further determined by flow cytometry. The cytokine levels of apoptotic Jurkat cell­derived MPs and Jurkat cell supernatants were compared by cytokine antibody arrays. Fibroblasts were isolated from 3 patients with OKC and co­cultured with apoptotic Jurkat cell­derived MPs with or without interleukin (IL)­15Rα to detect the levels of matrix metallopeptidase 9 (MMP­9) and receptor activator of nuclear factor­κB ligand (RANKL) by reverse transcription­quantitative polymerase chain reaction and enzyme­linked immunosorbent assay. The supernatant from Jurkat MPs­treated fibroblasts was collected to make conditioned medium in which the osteoclastogenesis of Raw264.7 cells was determined. Antibodies against human soluble (s)RANKL were added to the conditioned medium to investigate the inhibitory effects. Mean percentages of lymphocyte­ and neutrophil­derived MPs were significantly higher in inflamed OKCs than in DCs. Significant elevations in IL­15 were detected in apoptotic Jurkat cell­derived MPs compared with that in Jurkat cell supernatant. Furthermore, higher levels of MMP­9 and RANKL were detected in Jurkat cell MP­treated OKC fibroblasts, and this was partially blocked by IL­15Rα. Increased osteoclast­like cell formation was observed in the Jurkat MPs­treated fibroblast supernatant and Raw264.7 co­culture groups. The anti­human sRANKL antibody in the Jurkat MPs­treated fibroblast supernatant group decreased the osteoclastogenesis of the Raw264.7 cells. These results indicate that LMPs serve as novel communication tools that contribute toward the bone resorption of inflamed OKCs by inducing RANKL of OKC fibroblasts via IL­15.

Overexpression of Fra-1, c-Jun and c-Fos in odontogenic keratocysts: potential correlation with proliferative and anti-apoptotic activity.

AIMS: The purpose of this study was to explore the potential involvement of Fra-1, c-Jun and c-Fos, three vital members of the AP-1 complex, in the pathogenesis of odontogenic keratocysts (OKCs). METHODS AND RESULTS: Tissue samples, containing 10 normal oral mucosa (OM), 10 dentigerous cysts (DC) and 32 OKC specimens, were applied to investigate the expression levels of Fra-1, c-Jun and c-Fos by immunohistochemistry and real-time-quantitative polymerase chain reaction (RT-qPCR). The association between Fra-1, c-Jun and c-Fos expression levels and markers of proliferation [Ki-67, proliferating cell nuclear antigen (PCNA)], anti-apoptosis (Bcl-2) was then investigated in the OKC serial tissue sections. The results showed that Fra-1, c-Jun and c-Fos expression levels were increased significantly in OKCs compared to these in OM and DC tissue samples. Meanwhile, the expression levels of Fra-1, c-Jun and c-Fos were associated positively with the expression levels of Ki-67, PCNA and Bcl-2, as confirmed further by double-labelling immunofluorescence analysis and hierarchical analysis. CONCLUSIONS: This study revealed for the first time that Fra-1, c-Jun and c-Fos were overexpressed in OKCs and had a close correlation with proliferation and anti-apoptosis potential of OKCs.

Increased level of cell-derived microparticles in the cyst fluids of odontogenic keratocysts.

The aim of this study was to examine the level and basic characteristics of cell­derived microparticles (MPs) in the cyst fluids of odontogenic keratocysts (OKCs). For this purpose, MPs from the cyst fluids (CFMPs) of OKCs were purified by a classic differential centrifugation method and characterized by a transmission electron microscope and fluorescence microscope. Flow cytometric analysis was used to determine the size, concentration and cellular origins of the CFMPs. Moreover, the expression level of receptor activator for nuclear factor­κB ligand in the OKCs was evaluated by immunohistochemical staining and then analyzed for its correlation with the concentration of CFMPs by Spearman's rank correlation test. In addition, reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and tartaric­resistant acid phosphatase (TRAP) staining were performed to examine the osteoclastogenesis of mouse bone marrow­derived macrophages (BMMs) in response to CFMPs. The results revealed that the levels of total CFMPs were significantly elevated in OKCs compared with dentigerous cysts (DCs) and radicular cysts (RCs). In addition, in vitro experiments further revealed that CFMPs derived from the OKCs of patients could be taken up by BMMs, leading to a significant increase in the mRNA expression levels of nuclear factor of activated T­cells 1 (NFATc1) and TRAP. Moreover, TRAP­positive multinucleated osteoclasts were successfully cultured in the presence of macrophage colony­stimulating factor (M­CSF) and CFMPs with BMMs. On the whole, our findings indicate that patients with OKCs have higher levels of CFMPs compared with patients with DCs and RCs, which may be associated with the bone resorption of OKCs.