Hongya Pan

Characteristics of a cancerous cell line, HIOEC-B(a)P-96, induced by benzo(a)pyrene from human immortalized oral epithelial cell line.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in the oral and maxillofacial region. The mechanism of carcinogenesis of OSCC is still unclear. In vitro study on OSCC cell lines, especially derived from immortalized oral epithelial cells, is a very useful strategy to understand the mechanism of carcinogenesis. Based on our previous human immortalized oral epithelial cell (HIOEC) line, obtained from normal oral epithelial cells by transfection of HPV16 E6/E7 gene, a new cancerous cell line, HIOEC-B(a)P-96 (HB96), was established from the HIOEC by induction with benzo(a)pyrene. The characteristics of the HB96 cells such as cell morphology, ultrastructure, proliferation ability, invasion ability, and tumorigenesis were studied. The HB96 cells lost contact inhibition with uncontrolled cell division and obvious cell overlap, they were polygonal in shape and ununiform in size with increased ratio between nucleus and plasma. Increased proliferative ability and invasion ability were confirmed by the cell proliferation analysis and cell invasion assay, respectively. The tumorigenicity of well to moderately differentiated squamous cell carcinoma was confirmed in the nude mice experiments pathologically. Increased expression of HPV16 E6/E7 proteins and obvious correlation with decreased expression of p53 and Rb proteins was also confirmed by Western blotting. Thus, this HB96 cell line induced by benzo(a)pyrene from the HIOEC line is a useful tool to study the mechanism of carcinogenesis of OSCC in vitro for future genomic and proteomic analyses. It is also the first in vitro cancerous cell line of OSCC in China derived from immortalized oral epithelial cells.

Yes-associated protein promotes cell proliferation by activating Fos Related Activator-1 in oral squamous cell carcinoma

In our previous study, we established an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC), including a human immortalized oral epithelial cell (HIOEC) and a cancerous cell line (HB96). Microarray analysis showed that the gene encoding Yes-associated protein (YAP) was significantly increased in HB96 cells compared with HIOEC cells. But the underlying mechanism of YAP on oncogenesis, especially its downstream targets, are still not clear. YAP expression in OSCC cell lines and tissue specimens were investigated by using real-time PCR, western blotting and immunohistochemistry staining. YAP put-back plasmid with four mutation sites after YAP-siRNA interference was constructed by site-directed mutagenesis. Cell growth and colony formation were observed after YAP-siRNA interference or YAP put-back again in CAL27 cells. YAP expression was increased in the cellular carcinogenesis models and the clinical samples from primary OSCC patients. Inhibition of YAP by siRNA interference in CAL27 cells significantly inhibited cell proliferation and colony formation in soft agar, but these abilities were rescued when YAP was put-back again. At the same time, Fos Related Activator-1 (Fra-1) was down-regulated when YAP was inhibited by siRNA interference while Fra-1 was rescued when YAP was put-back again. Immunohistochemistry results also indicated that higher levels of YAP were significantly associated with Fra-1 overexpression in OSCC clinical samples. YAP could promote cell proliferation by activating transcription factor Fra-1 in oral squamous cell carcinoma.

Magnesium ion implantation on a micro/nanostructured titanium surface promotes its bioactivity and osteogenic differentiation function

As one of the important ions associated with bone osseointegration, magnesium was incorporated into a micro/nanostructured titanium surface using a magnesium plasma immersion ion-implantation method. Hierarchical hybrid micro/nanostructured titanium surfaces followed by magnesium ion implantation for 30 minutes (Mg30) and hierarchical hybrid micro/nanostructured titanium surfaces followed by magnesium ion implantation for 60 minutes (Mg60) were used as test groups. The surface morphology, chemical properties, and amount of magnesium ions released were evaluated by field-emission scanning electron microscopy, energy dispersive X-ray spectroscopy, field-emission transmission electron microscopy, and inductively coupled plasma-optical emission spectrometry. Rat bone marrow mesenchymal stem cells (rBMMSCs) were used to evaluate cell responses, including proliferation, spreading, and osteogenic differentiation on the surface of the material or in their medium extraction. Greater increases in the spreading and proliferation ability of rBMMSCs were observed on the surfaces of magnesium-implanted micro/nanostructures compared with the control plates. Furthermore, the osteocalcin (OCN), osteopontin (OPN), and alkaline phosphatase (ALP) genes were upregulated on both surfaces and in their medium extractions. The enhanced cell responses were correlated with increasing concentrations of magnesium ions, indicating that the osteoblastic differentiation of rBMMSCs was stimulated through the magnesium ion function. The magnesium ion-implanted micro/nanostructured titanium surfaces could enhance the proliferation, spreading, and osteogenic differentiation activity of rBMMSCs, suggesting they have potential application in improving bone-titanium integration.

Expression of growth differentiation factor 15 is positively correlated with histopathological malignant grade and in vitro cell proliferation in oral squamous cell carcinoma

Previously, we established an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC) and expression microarray analysis showed that the gene encoding growth differentiation factor 15 (GDF15) was significantly upregulated in this model. In this study, we confirmed that expression of GDF15 was increased both at mRNA and protein levels in a panel of OSCC lines and clinical samples from primary OSCC patients. We also observed that expression of GDF15 was positively correlated with the malignancy of the disease: a higher level of GDF15 expression indicates a higher malignant grade of OSCC. Treatment of OSCC cell line (Tca3118) with siRNA against GDF15 significantly inhibited cellular proliferation and colony formation. Based on these observations, we conclude that GDF15 is a positive gene of OSCC development and progression and GDF15 can be used as an additional marker for histopathologic evaluation of OSCC differentiation.

Antibacterial property, angiogenic and osteogenic activity of Cu-incorporated TiO2 coating

Numerous efforts have been made to modify the surface topography and chemical composition of biomedical implants in order to enhance the antibacterial ability and the osteointegration between implants and surrounding bone tissue. In the present work, copper-incorporated TiO2 coatings were fabricated by combining micro-arc oxidation and hydrothermal treatment together to functionalize the surface of Ti implants. The as-prepared surfaces exhibited a hierarchical structure comprising nanoneedles nearly perpendicular to the microrough surface of the TiO2 coating. The Cu-loaded TiO2 coating possessed strong antimicrobial ability against Gram-negative Escherichia coli. In vitro cytocompatibility evaluation suggests that no significant cytotoxicity appeared on the Cu-incorporated TiO2 coating. Furthermore, the addition of the copper element could stimulate the expression of angiogenic genes, including the hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in rat bone marrow stem cells (BMSCs). Moreover, they tended to undergo osteogenic differentiation, indicated by the up-regulation expression of osteogenic markers and the higher level of alkaline phosphatase activity. This study provides insight for the surface modification of biomedical Ti-based implants. To the best of our best knowledge, this is a successful attempt for the first time to combine micro-arc oxidation and hydrothermal treatment to introduce copper nutrient element to functionalize Ti-based implant surfaces with enhanced angiogenesis potential, osteostimulation and antimicrobial properties that can better meet clinical needs.

Correlation of increased twist with lymph node metastasis in patients with oral squamous cell carcinoma.

PURPOSE To investigate the protein expression of Twist, Snail, and Slug in oral squamous cell carcinoma (OSCC) samples and evaluate the potential correlation between the expression status and clinicopathologic features in patients with OSCC. PATIENTS AND METHODS Twist, Snail, and Slug protein expression was assessed by immunohistochemistry in a total of 60 OSCC samples and 10 normal oral mucosal samples. The associations between the protein expression and clinicopathologic parameters were mainly detected using the χ(2) test. The survival analysis was performed using the Kaplan-Meier method, and the prognostic analysis was performed using Cox regression models. RESULTS Immunohistochemistry stain analysis showed that positive Twist, Snail, and Slug protein expression was observed in 70%, 63.3%, and 58.3% of the cases, respectively. Twist protein expression was positively associated with lymph node metastasis, pathologic grade, and tumor stage (P = .012, P = .008, and P = .004, respectively, χ(2) test). All patients were followed up for 6 to 59 months (mean 37). A correlation between Twist protein expression and tumor recurrence was detected (log-rank test, P = .025). Nevertheless, no correlation was found between the Snail and Slug protein expression and the clinicopathologic parameters. CONCLUSIONS Twist might serve as a useful molecular marker for lymph node metastasis and a poor prognosis in OSCC.

Increased expression of Annexin A2 in oral squamous cell carcinoma.

Previously, in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC) was established with a line of human immortalized oral epithelia cells (HIOECs), a line of cancerous HB96 cells, and another kind of cells (HB56 cells) at the early stage of carcinogenesis. In this study, comparative proteomic analysis identified a panel of differentially expressed proteins among these cells, and Annexin A2 shown as one of the significantly up-regulated proteins accompanying cellular transformation. Annexin A2 was further validated for its expression in the three kinds of cells and in the clinical samples of tumour tissues and their adjacent normal epithelia from primary OSCC patients. Western blot analysis and real-time PCR detected increased Annexin A2 protein and mRNA levels in cancerous HB56 and HB96 cells over HIOECs. Immunohistochemistry showed elevated Annexin A2 protein expression in tumour tissues over the adjacent non-malignant epithelia from OSCC patients; however, the mRNA levels between tumour and normal tissues did not change significantly. Interestingly, levels of Annexin A2 protein expression negatively correlated with the tumour differentiation grades. The results presented here suggest that Annexin A2 protein may play important roles in carcinogenesis of OSCC, and it may also serve as a candidate biomarker for pathologic differentiation grade of OSCC.

Vacuum extraction enhances rhPDGF-BB immobilization on nanotubes to improve implant osseointegration in ovariectomized rats

UNLABELLED Nanotube morphology has been previously applied to improve osseointegration in osteoporosis, but the osteogenic capability of the technique requires further improvements. This study aimed to investigate the effects of vacuum extraction on the loading of rhPDGF-BB on nanotube arrays as well as its effects on the osseointegration of ovariectomized (OVX) rats. More rhPDGF-BB protein particles aggregated on the nanotube surface and into the nanotube after vacuum extraction for 10 min. The immobilized protein could be slowly released for at least 14 days and still kept its biological activity. In vitro, the immobilized rhPDGF-BB enhanced cell adhesion, proliferation and osteogenic differentiation. In vivo, more rhPDGF-BB immobilized on the nanotube surface also promoted the osseointegration. These results suggest that the enhanced immobilization of rhPDGF-BB on nanotube arrays can potentially be used in the future as an implant surface modification strategy in dental and orthopedic applications in osteoporotic patients. FROM THE CLINICAL EDITOR This study presents convincing evidence that enhanced immobilization of recombinant human PDGF-BB protein particles on nanotubes lead to improved osteogenic differentiation in an experimental system. When used as a surface modification strategy for dental or orthopedic implants, this method was able to promote osseointegration even in an osteoporotic animal model, raising the likelihood for potential future clinical applications.

Strontium delivery on topographical titanium to enhance bioactivity and osseointegration in osteoporotic rats

Osseointegration remains a major clinical challenge in osteoporotic patients. Strontium (Sr) has been shown to be a significant therapy to favor bone growth by both increasing new bone formation and reducing bone resorption. In this study, we attempt to chemically functionalize Ti implants by micro-arc oxidation, alkali treatment and ion exchange. This functionalized Ti surface possessed a hierarchical topography with Sr incorporation, which can release Sr ions at a slow rate. To our knowledge, this work is the first to use this type of Sr-doped Ti surface to address osteoporotic bone mesenchymal stem cells (BMSCs) in the dual directions of bone regeneration, bone formation and bone resorption. The modified surface was demonstrated to remarkably enhance the adhesion, spreading, and osteogenic differentiation of BMSCs in vitro. The effect of the wash-out solution from various groups on osteoporotic BMSCs was also investigated. The Sr-doped group can improve the ALP activity and osteogenic gene expression. Moreover, the Sr-doped group and the wash-out solution show the most inhibition in osteoclast formation and maturation. Furthermore, the increased bioactivity of the hierarchical structure was also confirmed with the ovariectomized rat femur model in vivo. The outcome of fluorescence labeling, histology and histomorphometric analysis demonstrated a significant promotion of osseointegration in ovariectomized rats. Altogether, the experimental data indicate that the fabrication of a Sr-doped hierarchical Ti surface is a meaningful attempt to incorporate the Sr nutrient element into Ti-based implants, and it is expected to be exploited in developing better osseointegration for osteoporotic patients.

Fos-related activator-1 is overexpressed in oral squamous cell carcinoma and associated with tumor lymph node metastasis

BACKGROUND Previously, we established an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC), including the human immortalized oral epithelia cells (HIOECs) and its derived cancerous HB cells. Then, expression microarray analysis showed that the gene encoding fos-related activator-1 (Fra-1) was significantly upregulated in the cancerous HB cells compared with HIOECs. METHODS To confirm the expression of Fra-1 at mRNA and protein levels by real-time PCR and western blotting analysis in the carcinogenesis model of OSCC and CAL27 cells. To investigate Fra-1 expression in clinical samples from 30 primary OSCC patients by immunohistochemistry. RESULTS Fra-1 expression was increased both at mRNA and protein levels in this carcinogenesis model of OSCC and CAL27 cells. Nuclear and cytoplasmic Fra-1 protein expressions both increased in the cancerous tissues compared with those in the paired adjacent non-malignant epithelia (nuclear: P < 0.001, cytoplasmic: P = 0.003). A higher level of nuclear Fra-1 expression was seen in the tumor samples of patients with lymph node metastasis than those without lymph node metastasis (5.07 +/- 1.33 vs 3.81 +/- 1.33, P = 0.023). Higher level of Fra-1 expression was also found in the tumor invasive margin than tumor center. CONCLUSIONS Fra-1 is a positive gene of OSCC development and progression, Fra-1 can be used as a potential therapeutic target gene and an additional marker for evaluation of lymph node metastasis.