Search

Gambogic acid (GA) is known for its anti-cancer activity in a phase II clinical trial. However, the detailed molecular mechanisms of its anti-multidrug resistance remain unclear. The present study was designed to study the relationship between GA and multidrug-resistant protein ATP-binding cassette transporter B1 (ABCB1). GA dose dependently inhibited ABCB1 activity levels in the in vitro Pgp-Glo assay system and increased the cellular accumulation of ABCB1 substrate adriamycin. Although GA had no significant influence on ABCB1 mRNA in the real-time PCR assay, Western blot detection indicated the compound reduced ABCB1 protein levels. Further study showed the proteasome inhibitor MG-132 reversed the GA-decreased ABCB1 level and prolonged half-life of ABCB1. It was also found that GA coordinated with other anti-cancer drugs (such as adriamycin, docetaxel, verapamil and protopanaxadiol) to enhance cellular cytotoxicity on human epithelial cancer cell lines with higher ABCB1 expression levels. These data suggest that GA functions as a non-competitive inhibitor of ABCB1 by directly inhibiting and reducing its expression levels by promoting protein degradation through post-translational proteasome pathway. The results of this study will aid in the understanding of the synergistic effects of combining GA with other drugs as a new anti-multidrug-resistant agent.

Background Toll-like receptor 4 (TLR4) is expressed on immune cells as a sensor that recognizes lipopolysaccharide (LPS), a microbial conserved component. It has recently been determined that the expression of TLR4 is also found in various types of tumor cells. Cisplatin is a widely used chemotherapeutic agent for oral squamous cell carcinoma (OSCC) treatment. However, the mechanisms responsible for cisplatin resistance are not well understood. Results The present study was designed to elucidate the role of TLR4 expression in human OSCC regarding immune escape and apoptotic resistance to cisplatin. TLR4 and the myeloid differentiation primary response gene 88 (MyD88) were highly expressed in OSCC cell lines. Upon LPS stimulation both NF-κB and p38 MAPK pathways were activated in OSCC cell lines, followed by the production of large quantities of IL-6, IL-8 and VEGF compared with human immortalized oral epithelia cells (HIOECs). OSCC cell lines were found to be resistant to cisplatin-mediated apoptosis after pretreatment with LPS. Conclusions Our results suggested that TLR4 was functionally expressed in human OSCC cells and development of resistance to cisplatin in human OSCC might occur through the mechanism involving TLR4 and its signaling pathway. Suppression of TLR4 and its signaling pathway might thus elevate sensitivity to cisplatin and potentially help improve the prognosis of patients with OSCC.

To investigate the expression of cytokeratin 17 (CK17) in oral squamous cell carcinoma (OSCC) as well as its clinical significance.Detection of the mRNA level and protein expression of CK17 in the in vitro cellular carcinogenesis model of OSCC, some OSCC cell lines and tissue specimens from 30 primary OSCC patients were performed using real-time polymerase chain reaction (PCR), Western blot and immunohistochemistry, respectively.Increased CK17 mRNA level was observed in the HB56 and OSC cell lines compared with the HIOEC using real-time PCR technique. Western blot showed increased CK17 protein expression in all the cell lines compared with the HIOEC. Increased CK17 mRNA and immunoreaction levels were also observed in the cancerous tissue specimens from OSCC patients compared with normal adjacent tissues (P0.01).The significantly increased CK17 gene may be associated with the tumorigenesis and development of OSCC.

Calcium phosphate cements (CPCs), which are widely used in bone regeneration, possess good biocompatibility and osteoconductivity and have been demonstrated to be candidate carriers for bone growth factors. However, limited release of growth factors from CPCs and slow degradation of the materials are not desirable for certain clinical applications. Previous studies have shown that calcium-deficient hydroxyapatite (CDHA) from CPCs presents more rapid degradation rate than CPCs. In this study, a hybrid growth factor delivery system was prepared by using bone morphogenetic protein 2 (BMP-2) loaded CDHA porous scaffold with sulfated chitosan (SCS) coating for improved release profile. We tested the BMP-2 release characteristic of CDHA/BMP-2/SCS composite in vitro and its ability to repair rat calvarial bone defects. A higher percentage of BMP-2 was released when sulfated chitosan coating was present compared with CDHA/BMP-2 group. Eight weeks postoperation, the repaired crania were evaluated by microcomputed tomography, sequential fluorescent labeling, histological analysis, and immunohistochemistry. CDHA/BMP-2/SCS group promoted the most extensive new bone formation than CDHA/BMP-2 and CDHA groups. Our observations suggest that sulfated chitosan coating could enhance the release profile of CDHA/BMP-2 composite in vitro and promote new bone formation in vivo. The hybrid CDHA/BMP-2/SCS system is a promising growth factor delivery strategy for bone regeneration.

Cryopreserved bone-derived osteoblasts (CBOs) have been considered as a promising cell source for bone regeneration. Previous studies have demonstrated that CBOs had good proliferation and osteogenicity. However, the long-term outcome of CBOs in vivo still remains unknown. In this experiment, we applied CBOs combined with calcium phosphate cement (CPC) to augment maxillary sinus in canine, computer tomography, polychrome labeling, biomechanical tests, fluorescent immunohistochemistry staining and histological analysis were used to analyze the property and mineralization process of the tissue-engineered bone preclinical application. Our results showed that CBOs combined with CPC could promote bone regeneration, dramatically maintain the height, volume and biomechanical property of augmented maxillary sinus. Furthermore, the tissue-engineered bone was more mature than scaffold alone or autogenous bone, and bone formation and remodeling were still apparent 20 months postoperatively. Additionally, 4 months after surgery might be the suitable time point for implants placement in the regenerated bone. These results also indicate that cryopreserved bone may be a potential source of osteoblasts for maxillary sinus augmentation.

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.

The emerging fields of tissue engineering and regenerative medicine require large numbers of cells for therapy. Although the properties of cells obtained from a variety of fresh tissues have been delineated, the knowledge regarding cryopreserved grafts-derived cells remains elusive. Previous studies have shown that living cells could be isolated from cryopreserved bone grafts. However, whether cryopreserved bone-derived cells can be applied in regenerative medicine is largely unknown. The present study was to evaluate the potential application of cryopreserved grafts-derived cells for tissue regeneration. We showed that cells derived from cryopreserved bone grafts could maintain good proliferation activity and osteogenic phenotype. The biological phenotype of these cells could be well preserved. The transplantation of cryopreserved bone-derived cells on scaffold could promote new bone formation in nude mice and enhance the osteointegration for dental implants in canine, which confirmed their osteogenic capacity, and showed that cells derived from cryopreserved bone were comparable to that of fresh bone in terms of the ability to promote osteogenesis in vivo. This work demonstrates that cryopreserved bone grafts may represent a novel, accessible source of cells for tissue regeneration therapy, and the results of our study may also stimulate the development of other cryopreservation techniques in basic and clinical studies.

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. Based on the previous cell line, HIOEC-B(a)P-96 (HB96), which we obtained by HPV16 E6/E7-immortalized human oral epithelial cells (HIOEC) and benzo(a)pyrene inducement, we prepared a new HB-second generation cancer cell line (HB-2) by continuous passage. Its characteristics such as morphology, proliferation activity, karyotype, and tumorigenesis were studied. The HB-2 cells displayed uncontrolled cell division and lost contact inhibition leading to cell overlap. Cells were polygonal and unevenly shaped, with an increased nucleus versus plasma ratio. Increased proliferative activity was confirmed by MTT assays. The tumorigenicity was confirmed by tumor growth experiments in nude mice. Therefore, the HB-2 and HB96 cell lines are useful tools to study the mechanism of carcinogenesis of OSCC in vitro for future genomic and proteomic analyses.

MHC class I peptide loading complex defects are frequently observed in tumor cells which facilitate tumor cells escaping from immune surveillance. Tapasin plays an important role in the assembly of MHC class I molecules with peptides in the endoplasmic reticulum. The aim of this study was to investigate the expression of tapasin in primary oral squamous cell carcinoma (OSCC) and its potential clinical implication. Formalin-fixed, paraffin-embedded tumor biopsies from 67 patients with primary OSCC were analyzed for tapasin expression using immunohistochemistry. Tapasin promoter methylation status in OSCC cell lines was determined using ethylation-specific polymerase chain reaction, bisulphate genomic sequencing, and expression reactivation assay. Lack of tapasin expression was observed in 30 of 67 (43%) tumors and was significantly associated with poor pathologic differentiation grade of OSCC (P = 0.028). The cumulative 5-year survival rate was also significantly correlated with pathologic differentiation grade (P = 0.001) and tapasin expression level (P = 0.015). Decreased tapasin expression was an indicator of poor survival (P = 0.048). Tapasin promoter methylation was observed in all three OSCC cell lines examined, and the mRNA and protein levels of tapasin increased markedly after treatment with 5-aza-2′-deoxycytidine. Downregulation of tapasin is associated with a poor clinical outcome for OSCC patients and may serve as a prognostic biomarker. The promoter methylation may contribute to the tapasin downregulation in OSCC.

To determine the Galectin-1 protein expression in oral squamous cell carcinoma (OSCC).Comparative proteomic analysis of an in vitro cellular carcinogenesis model of OSCC we previously established was performed to identify differentially expressed proteins. Galectin-1 was further validated in vitro (human immortalized oral epithelia cell line and OSCC lines) and in vivo (tissue samples from OSCC patients) by Western blotting and immunohistochemistry, respectively.Increased Galectin-1 protein expression was identified in the cancerous cell line compared with the immortalized oral epithelial cell line in the in vitro cellular carcinogenesis model, and then validated in the OSCC lines and cancerous tissues. Galectin-1 protein expression was negatively correlated with the tumor pathologic differentiation grades, a higher Galectin-1 protein expression indicating a poorer pathologic differentiation grade.Galectin-1 protein expression level increases in OSCC, it may serve as a candidate marker for pathologic differentiation grade of OSCC.

To assess the influence of high extracellular glucose levels on the osteogenic differentiation of bone marrow stromal cells (BMSCs) and to determine if Sonic hedgehog (Shh) protein can alleviate those effects. BMSCs were incubated with NG (normal glucose), NG+Shh (200 ng/ml Shh in normal glucose), NG+Shh+Gan (200 ng/ml Shh and 5 micromol/L GANT61 in normal glucose), HG (high glucose), HG+Shh (200 ng/ml Shh in high glucose), and HG+Shh+Gan (200 ng/ml Shh and 5 micromol/L GANT61 in high glucose). The expression levels of Shh signaling pathway genes Patched 1 (PTCH1) and osteogenesis-related genes were tested, which included bone morphogenetic protein 4 (BMP4), runt-related transcription factor 2 (Runx2), and osteopontin (OPN). Alkaline phosphatase (ALPase) activity and mineralized matrix formation were also investigated. Immunofluorescent staining of Gli1 was tested for Shh signaling activation. We found that recombinant Shh in normal-glucose medium could promote osteogenic differentiation of BMSCs, while inhibiting Shh signaling by GANT61 could antagonize this differentiation. Besides that high glucose impaired the Shh signaling as well as osteogenic differentiation of BMSCs, reactivation of Shh signal pathway by addition of Shh protein could mitigate the inhibition while further deactivation by Shh inhibitor GANT61 could retain their osteogenic inhibitions. The above data suggest that Shh pathway activity is involved in the HG condition mediated osteoblastic differentiation deficiency for BMSCs and that recombinant Shh could alleviate this inhibitory effect.

Despite advances in the development of human papillomavirus (HPV) prophylactic vaccines, little progress has been made in the field of therapeutic vaccines in recent years. In the present study, we found a significant accumulation of immature myeloid cells (ImC) in large TC-1 tumors and demonstrated that a HPV therapeutic vaccine restored antitumor immune responses with the correction of aberrant myeloid cell differentiation by all-trans retinoic acid (ATRA). Our study demonstrated that combining ATRA with vaccination not only decreased the number of Gr-1+ CD11b+ ImC, but for the first time also suppressed the function of Gr-1+ CD11b+ ImC with decreased expression of CD80. Furthermore, large numbers of CD11c+ CD80+, CD11c+ CD86+, and CD11c+ MHCII+ mature dendritic cells were recruited. The combination therapy generated significantly increased numbers of functional E7-specific T cells with elevated interferon-γ secretion and enhanced cytotoxic T-cell activity. These findings suggest potential clinical benefits for the combined use of ATRA and HPV therapeutic vaccines. (Cancer Sci 2009; 100: 334–340)

A tissue-engineered bone has become a viable alternative to autologous bone for bone augmentation in atrophy alveolar ridge. The aim of the present study was to evaluate porous beta-tricalcium phosphate (beta-TCP) combined with autologous osteoblasts to augment edentulous alveolar ridge in a canine model. Autologous osteoblasts were expanded and combined with beta-TCP scaffold to fabricate a tissue-engineered bone. 12 bilateral alveolar ridge augmentation surgeries were carried out in 6 beagle dogs with the following 3 groups: beta-TCP/osteoblasts, beta-TCP alone and autogenous iliac bone control (n=4 per group). Sequential fluorescent labeling and radiographs were used to compare new bone formation and mineralization in each group. 24 weeks later, animals were sacrificed and non-decalcified and decalcified sections were evaluated histologically and histomorphometrically. Results indicated that the tissue-engineered bone dramatically enhanced new bone formation and mineralization, increase the new bone area, and maintain the height and thickness of the augmented alveolar ridge when compared with beta-TCP alone group. More importantly, the tissue-engineered bone achieved an elevated bone height and thickness comparable to that of autogenous iliac bone graft. This study demonstrated the potential of porous beta-TCP as a substrate for autogenous osteoblasts in bone tissue engineering for alveolar ridge augmentation.

In vitro cellular model is an important tool to be used to investigate the cellular events related to pathophysiological conditions in humans. We have developed an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC). In this study, we performed comparative proteomic analysis using 2-DE and LC-tandem mass chromatography to separate and identify differentially expressed proteins. Forty-five proteins were identified, including 24 proteins with decreased expression and 19 proteins with increased expression during carcinogenesis from immortalized oral epithelial cells to squamous cancerous cells. The identified known proteins were classified into three ontologies of cellular component, molecular function, and biological process. Further validation of five identified proteins (ANXA1, ANXA2, CTSB, KRT17, and S100A6) in the cellular carcinogenesis model and cancerous tissues from OSCC patients confirmed the comparative proteomic results. Moreover, Annexin A1 and A2 expression levels correlated with the pathological differentiation grade of cancerous tissues. Thus, this work provides a dynamic protein file of differentially expressed proteins in oral squamous carcinoma cells, which could provide clues to study the mechanisms of OSCC carcinogenesis and possibly be developed as potential biomarkers for clinical diagnosis or prognostic monitoring.

BackgroundThe study was designed to investigate immunocompetence in relation to cancer progression in rat and to assess the effect of the traditional Chinese anti-cancer medicine, "Shenyang" prescription, on immunity.Methods4-Nitroquinoline-1-oxide (4NQO) was administered to 80 Sprague-Dawley (SD) rats via the drinking water for up to 36 weeks. Tongue squamous cell carcinoma (SCC) was confirmed by pathological examination in 61 rats. "Shenyang" prescription was administered to subgroups of these rats, and blood samples were taken before and after treatment. Lymphocyte subsets were determined by flow cytometry. Serum Th1 and Th2-type cytokines were assessed by an enzyme-linked immunosorbent assay.ResultsAs the cancer progressed at the tongue root, the percentage of CD3+CD4+ T lymphocytes and NK cells and the levels of IFN-γ and IL-2 decreased gradually, while the percentage of CD3+CD8+ T lymphocytes and the levels of IL-4 and IL-10 increased. The CD4+/CD8+ ratios were lower in the cancer groups than in the control group. However, after administering "Shenyang" prescription, the levels of CD3+CD4+ T lymphocytes, NK cells, IFN-γ and IL-2 increased, while the CD3+CD8+ T lymphocyte counts and the levels of IL-4 and IL-10 decreased.Conclusion4NQO-induced lesions were good models for exploring oral cavity carcinogenesis. The rats with 4NQO-induced SCC demonstrated abnormalities in lymphocyte subsets and a shift from Th1-type to Th2-type, which were good models for assessing the effect of anticancer agent on immunity. Oral cancer progression was associated with an aggressive disturbance of immune function. "Shenyang" prescription has the ability to improve the disturbance of immune function.

Calcium phosphate (Ca-P) scaffolds have been widely employed as a supportive matrix and delivery system for bone tissue engineering. Previous studies using osteoinductive growth factors loaded Ca-P scaffolds via passive adsorption often experience issues associated with easy inactivation and uncontrolled release. In present study, a new delivery system was fabricated using bone morphogenetic protein-2 (BMP-2) loaded calcium-deficient hydroxyapatite (CDHA) scaffold by lyophilization with addition of trehalose. The in vitro osteogenesis effects of this formulation were compared with lyophilized BMP-2/CDHA construct without trehalose and absorbed BMP-2/CDHA constructs with or without trehalose. The release characteristics and alkaline phosphatase (ALP) activity analyses showed that addition of trehalose could sufficiently protect BMP-2 bioactivity during lyophilization and achieve sustained BMP-2 release from lyophilized CDHA construct in vitro and in vivo. However, absorbed BMP-2/CDHA constructs with or without trehalose showed similar BMP-2 bioactivity and presented a burst release. Quantitative real-time PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that lyophilized BMP-2/CDHA construct with trehalose (lyo-tre-BMP-2) promoted osteogenic differentiation of bone marrow stromal cells (bMSCs) significantly and this formulation could preserve over 70% protein bioactivity after 5 weeks storage at 25°C. Micro-computed tomography, histological and fluorescent labeling analyses further demonstrated that lyo-tre-BMP-2 formulation combined with bMSCs led to the most percentage of new bone volume (38.79% ± 5.32%) and area (40.71% ± 7.14%) as well as the most percentage of fluorochrome stained bone area (alizarin red S: 2.64% ± 0.44%, calcein: 6.08% ± 1.37%) and mineral apposition rate (4.13 ± 0.62 µm/day) in critical-sized rat cranial defects healing. Biomechanical tests also indicated the maximum stiffness (118.17 ± 15.02 Mpa) and load of fracture (144.67 ± 16.13 N). These results lay a potential framework for future study by using trehalose to preserve growth factor bioactivity and optimize release profile of Ca-P based delivery system for enhanced bone regeneration.

Background Gambogic acid (GA) is a major active ingredient of gamboge, a widely used traditional Chinese medicine that has been reported to be a potent cytotoxic agent against some malignant tumors. Many studies have shown that the NF-kappa B signaling pathway plays an important role in anti-apoptosis and the drug resistance of tumor cells during chemotherapy. In this study, the effects and mechanisms of GA and the NF-kappa B inhibitor celastrol on oral cancer cells were investigated. Methods Three human oral squamous cell carcinoma cell lines, Tca8113, TSCC and NT, were treated with GA alone, celastrol alone or GA plus celastrol. Cytotoxicity was assessed by MTT assay. The rate of apoptosis was examined with annexin V/PI staining as well as transmission electronic microscopy in Tca8113 cells. The level of constitutive NF-kappa B activity in oral squamous cell carcinoma cell lines was determined by immunofluorescence assays and nuclear extracts and electrophoretic mobility shift assays (EMSAs) in vitro. To further investigate the role of NF-kappa B activity in GA and celastrol treatment in oral squamous cell carcinoma, we used the dominant negative mutant SR-IκBα to inhibit NF-kappa B activity and to observe its influence on the effect of GA. Results The results showed that GA could inhibit the proliferation and induce the apoptosis of the oral squamous cell carcinoma cell lines and that the NF-kappa B pathway was simultaneously activated by GA treatment. The minimal cytotoxic dose of celastrol was able to effectively suppress the GA-induced NF-kappa B pathway activation. Following the combined treatment with GA and the minimal cytotoxic dose of celastrol or the dominant negative mutant SR-IκBα, proliferation was significantly inhibited, and the apoptotic rate of Tca8113 cells was significantly increased. Conclusion The combination of GA and celastrol has a synergistic antitumor effect. The effect can be primarily attributed to apoptosis induced by a decrease in NF-kappa B pathway activation. The NF-kappa B signaling pathway plays an important role in this process. Therefore, combining GA and celastrol may be a promising modality for treating oral squamous cell carcinoma.

Background G protein–coupled receptor family C group 5 member A (GPRC5A), a retinoic acid-inducible gene, is a lung tumor suppressor. Previously, we showed that repression of GPRC5A expression was associated with pathologic differentiation grade of oral squamous cell carcinomas (OSCC) and overexpression of GPRC5A gene inhibited the malignant phenotype in OSCC cells, suggesting that GPRC5A also functions as a tumor suppressor in oral cancer. However, the molecular mechanisms underlying GPRC5A deficiency in head and neck squamous cell carcinoma (HNSCC) are still unclear. Methods In this study, we used Western blot analysis and immunohistochemical (IHC) staining to investigate the expression of GPRC5A in both HNSCC cell lines and clinical samples. GPRC5A stable transfectants and their parental HNSCC cells were characterized for their biological activities in anchorage-independent growth. Results IHC analysis showed that, GPRC5A expression was high in normal tissue, but gradually decreased in oral leukoplakia, a precancerous stage, and greatly suppressed in primary cancer. Repression of GPRC5A was correlated with activated STAT3, which associates with aggressive clinicopathological features in HNSCC patients. Moreover, overexpression of GPRC5A suppressed IL-6-induced-STAT3 activation and inhibited anchorage-independent growth in HNSCC cells. Conclusions Repressed GPRC5A associates with increased tumor grade and activated STAT3, which may be used as a prognostic marker for tumor progression of HNSCC.