Activation of focal adhesion kinase induces extracellular signal-regulated kinase-mediated osteogenesis in tensile force-subjected periodontal ligament fibroblasts but not in osteoblasts.

The exact mechanism by which focal adhesion kinase (FAK) translates mechanical signals into osteogenesis differentiation in force-subjected cells has not been elucidated. The responses to different forces differ according to the origin of cells and the type of stress applied. Therefore, the recruitment of osteoclast and osteoblast progenitor cells, and the balanced activation of these cells around and within the periodontal ligament (PDL) are essential for alveolar bone remodeling. Cells within the PDL and MG63 cells were subjected to tensile forces of -100 kPa for different periods of time. At various times during the tensile force application, they were processed for the purpose of analyzing cell viability, cell cycle, and osteogenic protein. The effect of small interfering RNA transfection targeting FAK was also evaluated. Tensile force enhanced a rapid increase in the phosphorylation of FAK and up-regulated osteogenic protein expression in PDL cells, but not in MG63 cells. Transfecting PDL cells with FAK antisense oligonucleotide diminished alkaline phosphatase and osteocalcin secretion. These findings suggest that tensile force activates FAK pathways in PDL cells, which down-regulate immune cytokine and up-regulate osteogenic protein.

Association of novel gene polymorphisms RRM1 -756T>C and -269 C>A with breast cancer.

BACKGROUND: Genetic variants are reported to play an important role in the susceptibility of breast cancer. Ribonucleotide reductase 1 (RRM1) is suggested to play an essential role in the regulation of cancer development. The purpose of this study was to identify novel gene polymorphisms of RRM1 -756T>C and RRM1 -269 C>A specific to patients with breast cancer and healthy controls. METHODS: A total of 833 subjects, including 321 healthy controls and 512 patients with breast cancer, were recruited in this study. Allelic discrimination of RRM1 -756T>C (rs11030919) and RRM1 -269C>A (rs12806698) polymorphisms of the RRM1 gene was assessed with the real-time polymerase chain reaction. RESULTS: The adjusted odds ratios and 95% confidence intervals were 1.20 (0.71-2.04) and 1.10 (0.65-1.86) to have breast cancer among individuals with CC alleles of RRM1 -756T>C and individuals with AA alleles of RRM1 -269C>A gene polymorphism, respectively, compared to individuals having wild type of RRM1 gene polymorphisms. Also, there was no significant genetic interaction effect on the susceptibility of breast cancer and nonassociation between genetic polymorphisms and clinical statuses of breast cancer. CONCLUSION: Gene polymorphisms of RRM1 -756T>C and RRM1 -269C>A may be not an important factor for the susceptibility of breast cancer.

Selaginella tamariscina extract suppresses TPA-induced invasion and metastasis through inhibition of MMP-9 in human nasopharyngeal carcinoma HONE-1 cells.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is known for its high incidence of neck lymph node metastasis, which represents poor prognosis. The present study aimed to examine the anti-metastatic properties of Selaginella tamariscina extract (STE) in human nasopharyngeal carcinoma HONE-1 cells in vitro. METHODS: Cell viability was examined by MTT assay, whereas cell motility was measured by invasive, migration and would healing assays. Real-time PCR, and promoter assays confirmed the inhibitory effects of STE on matrix metalloproteinase-9 (MMP-9) mRNA level in HONE-1 cells. RESULTS: The STE inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced HONE-1 cell migration and invasion in a concentration-dependent manner. By zymographic and Western blot analyses, STE was shown to inhibit the activities and expression of MMP-9. Treatment of STE on TPA-induced HONE-1 cells inhibited MMP-9 expression and ERK1/2 phosphorylation without affecting JNK and p38 phosphorylation. CONCLUSIONS: STE inhibits MMP-9 expression and HONE-1 cell metastasis. Its inhibitory effects may involve the Src/FAK/ERK 1/2 pathway. STE may have the potential of being an anti-metastatic agent against NPC.

Bacterial alterations in salivary microbiota and their association in oral cancer.

Oral squamous cell carcinoma (OSCC) is the most common malignant neoplasm of the oral cavity and the fourth leading malignancy and cause of cancer-related death in the male population of Taiwan. Most cases are detected at advanced stages, resulting in poor prognosis. Therefore, improved detection of early oral health disorders is indispensable. The involvement of oral bacteria in inflammation and their association with OSCC progression provide a feasible target for diagnosis. Due to the nature of oral neoplasms, the diagnosis of epithelial precursor lesions is relatively easy compared with that of other types of cancer. However, the transition from an epithelial precursor lesion to cancer is slow and requires further and continuous follow-up. In this study, we investigated microbiota differences between normal individuals, epithelial precursor lesion patients, and cancer patients with different lifestyle habits, such as betel chewing and smoking, using next-generation sequencing. Overall, the oral microbiome compositions of five genera, Bacillus, Enterococcus, Parvimonas, Peptostreptococcus, and Slackia, revealed significant differences between epithelial precursor lesion and cancer patients and correlated with their classification into two clusters. These composition changes might have the potential to constitute a biomarker to help in monitoring the oral carcinogenesis transition from epithelial precursor lesion to cancer.

Stem cell differentiation-induced calcium silicate cement with bacteriostatic activity.

Calcium-based bone cements are widely used in dental and orthopaedic surgery. Those based on calcium phosphate (CPCs) or calcium silicate (CSCs) have a number of favourable properties that encourage their clinical use in bone defect repair. The purpose of the present study was to compare the in vitro osteogenesis and bacteriostatic activity of BoneSource CPCs with home-made CSCs, particularly in regard to their facility for cell differentiation. Cement in vitro osteogenic activity was evaluated by incubating the cement specimens with human mesenchymal stem cells (hMSCs). The bacteriostatic activity of the two cements against Gram-positive (S. aureus) and Gram-negative (P. aeruginosa) bacterial strains was assessed using a bacteriostasis ratio assay and by inhibition zone examination. Compared with CPC, CSC was shown to promote greater proliferation and osteogenic differentiation (alkaline phosphatase and osteocalcin), and the formation of mineralization nodules of hMSCs. It is worth noting that CSC could effectively induce hMSC differentiation, even when the culture medium did not contain osteogenetic differentiation agents. Compared with CPC, CSC also showed significantly greater bacteriostatic activity, as revealed by inhibition zones and the bacteriostasis ratio. Our findings suggest that CSC is a useful bioactive material for bone repair in terms of inducing cell differentiation, and may be considered an alternative to CPCs.

Role of the P38 pathway in calcium silicate cement-induced cell viability and angiogenesis-related proteins of human dental pulp cell in vitro.

INTRODUCTION: This study investigated that calcium silicate (CS) cement may influence the behavior of human dental pulp cells (hDPCs) via mitogen-activated protein kinase pathway, in particular p38. We have addressed that Si ion released from CS cement can influence osmolarity in the medium, which may stimulate hDPC viability and induce angiogenesis-related proteins through stimulation of the nitric oxide synthase and nitric oxide secretion. METHODS: The hDPCs was cultured with CS cement to angiogenesis. Then, cell viability, ion concentration, osmolality, nitric oxide secretion, the von Willebrand factor, and angiopoietin-1 protein expression were examined. RESULTS: CS cement elicited a significant (P < .05) increase of 15%, 20%, and 19% in viability compared with control on days 1, 3, and 5 of cell seeding, respectively. The CS cement consumed calcium and phosphate ions and released more Si ions in medium. The CS significantly (P < .05) increased the osmolality to 303.52 ± 3.07, 315.03 ± 5.80, and 319.95 ± 4.68 mOsm/kg for 1, 3, and 5 days, respectively. P38 was activated through phosphorylation; the phosphorylation kinase was investigated in our cell system after culture with CS cement. Moreover, expression levels for angiopoietin-1 and von Willebrand factor in hDPCs on CS cement were higher than those of the CS + p38 inhibitor (SB203580) group (P < .05) at all of the analyzed time points. CONCLUSIONS: This study showed that CS cement was able to activate the p38 pathway in hDPCs cultured in vitro. Moreover, Si was shown to increase osmolality required to facilitate the angiogenic differentiation of hDPCs via the p38 signaling pathway. When the p38 pathway was blocked by SB203580, the angiogenic-dependent protein secretion was decreased. These findings verified that the p38 pathway plays a key role in regulating the angiogenic behavior of hDPCs cultured on CS cement.

The role of integrin αv in proliferation and differentiation of human dental pulp cell response to calcium silicate cement.

INTRODUCTION: It has been proved that integrin αv activity is related to cell proliferation, differentiation, migration, and organ development. However, the biological functions of integrin αv in human dental pulp cells (hDPCs) cultured on silicate-based materials have not been explored. The aim of this study was to investigate the role of integrin αv in the proliferation and odontogenic differentiation of hDPCs cultured with the effect of calcium silicate (CS) cement and ß-tricalcium phosphate (TCP) cement. METHODS: In this study, hDPCs were cultured on CS and TCP materials, and we evaluated fibronectin (FN) secretion and integrin αv expression during the cell attachment stage. After small interfering RNA transfection targeting integrin αv, the proliferation and odontogenesis differentiation behavior of hDPCs were analyzed. RESULTS: The results indicate that CS releases Si ion-increased FN secretion and adsorption, which promote cell attachment more effectively than TCP. The CS cement facilitates FN and αv subintegrin expression. However, the FN adsorption and integrin expression of TCP are similar to that observed in the control dish. Integrin αv small interfering RNA inhibited odontogenic differentiation of hDPCs with the decreased formation of mineralized nodules on CS. It also down-regulated the protein expression of multiple markers of odontogenesis and the expression of dentin sialophosphoprotein protein. CONCLUSIONS: These results establish composition-dependent differences in integrin binding and its effectiveness as a mechanism regulating cellular responses to biomaterial surface.

Effect of verapamil, a calcium channel blocker, on the odontogenic activity of human dental pulp cells cultured with silicate-based materials.

INTRODUCTION: This study examines how calcium silicate cement extracts influence the behavior of human dental pulp cells (hDPCs) through calcium channels and active mitogen-activated protein kinase pathways, in particular extracellular signal-related kinase (ERK). METHODS: HDPCs are treated with various silicon concentrations both with and without verapamil, after which the cells' viability and odontogenic differentiation markers are determined by using PrestoBlue assay and Western blot, respectively. RESULTS: The silicon promoted cell proliferation and inhibited calcium channel blockers. It was also found that silicon increased ERK and p38 activity in a dose-dependent manner. Furthermore, it raised the expression and secretion of alkaline phosphatase, osteocalcin, dentin sialophosphoprotein, and dentin matrix protein-1. In addition, statistically significant differences (P < .05) have been found in the secretion of osteocalcin in ERK inhibitor + verapamil between the silicon concentrations; these varations are dose-dependent and indicate that ERK signaling is involved in the silicon-induced odontogenic differentiation of hDPCs. CONCLUSIONS: The current study shows that silicon ions released from calcium silicate substrates play a key role in odontoblastic differentiation of hDPCs through calcium channels and modulate ERK activation.

Aspirin-induced inhibition of adipogenesis was p53-dependent and associated with inactivation of pentose phosphate pathway.

Obesity has become a major public health problem of global significance. Today, aspirin remains the most commonly used medication for the treatment of pyrexia, pain, inflammation and antiplatelet. The present study aims at evaluating the possible existence of a putative p53-dependent pathway underlying the aspirin-induced inhibition of adipogenesis. Cell migration assay was identified by the ability to migrate through Transwell insert. Oil Red O staining was employed to quantify adipose accumulation. The concentration of glucose and triglyceride were measured by using assay kits. The expression levels of several master regulatory molecules controlling various signal pathways were monitored using the immunoblotting techniques. Aspirin significantly inhibited preadipocyte migration and adipose accumulation. The p53-p21 signaling and the expression of differentiation marker glycerol-3-phosphate dehydrogenase were increased in a dose-dependent manner. It indicated that aspirin induced adipocyte differentiation through p53-p21 pathway. The oncogenic ERK 1/2 MAPK signaling was induced, whereas, the expression of adipogenic markers peroxisome proliferator-activated receptor γ (PPARγ), adipocyte fatty acid-binding protein (A-FABP) and inflammatory factors cyclooxygenase-2 (Cox-2), tumor necrosis factor α (TNFα) and inducible nitric oxide synthase (iNOS) were inhibited. Aspirin negatively regulated the pentose phosphate pathway (PPP) by inhibiting the expression of rate-limiting enzyme glucose-6-phosphate dehydrogenase. Knockdown the expression of oncogenic ERK 1/2 MAPK by using 10 µM PD98059 significantly increased triglyceride synthesis, adipose accumulation and activated PPP, however, decreased glucose uptake. Diverted the glucose flux to PPP, rather than increased glucose uptake, was associated with adipogenesis. Down-regulated the expression of tumor suppressor p53 by 10 µM pifithrin-α (PFTα) alone had no effect on adipose accumulation. However, administration of aspirin accompanied with PFTα abolished aspirin-induced inhibition of adipogenesis. We demonstrated that aspirin-induced inhibition of adipogenesis was p53-dependent and associated with inactivation of PPP. Blockade PPP may be a novel strategy for obesity prevention and therapy. Moreover, when use aspirin in therapeutic strategy, the p53 status should be considered.

Comparative Osteogenesis of Radiopaque Dicalcium Silicate Cement and White-Colored Mineral Trioxide Aggregate in a Rabbit Femur Model.

The radiopaque dicalcium silicate cement (RDSC) displayed a shortened setting time and good biocompatibility. This study aimed to compare the regenerative potential of RDSC and white-colored mineral trioxide aggregate (WMTA) using a rabbit femur model. The animals were sacrificed at one, three and six months to accomplish histological and biochemical analyses. The results indicated that after one month of implantation, WMTA was associated with a greyish color alteration within its mass, while RDSC presented color stability even at six months. Histological assay with Masson's Trichrome and Von Kossa stains showed the presence of newly formed bone surrounding the implanted sites in the rabbit femur. The histochemical data revealed that the RDSC group had significantly more bone regeneration than did the WMTA groups at three and six months. The conclusion drawn is that the encouraging results support the potential applications of RDSC as an improved alternative to WMTA for endodontic uses.

Epithelial-mesenchymal transition transcription factor ZEB1/ZEB2 co-expression predicts poor prognosis and maintains tumor-initiating properties in head and neck cancer.

OBJECTIVES: Both epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties may be involved in metastasis, which contributes to the high mortality rate of patients with head and neck cancers (HNCs). However, the mechanisms through which the EMT transcription factors ZEB1 and ZEB2 regulate HNC are still unclear. METHODS: Tumor initiating capability of HNC-CH133(+) cells with ZEB1/2 knockdown or co-overexpression was presented in vitro and in vivo. RESULTS: In the present study, we demonstrated that ZEB1/ZEB2 expression was significantly increased in HNC-CD133(+) CSC-like cells compared with HNC-CD133(-) cells. The small interfering RNA (siRNA)-mediated co-knockdown of ZEB1 and ZEB2 (siZEB1/2) in HNC-CH133(+) cells suppressed their CSC-like properties, including self-renewal ability, the expression of stemness markers, and drug resistance. In contrast, the co-overexpression of ZEB1/ZEB2 in HNC-CD133(-) cells enhanced their sphere-forming ability and increased the percentage of CD44-positive cells and side population cells. In vivo studies showed that the delivery of siZEB1/2 to xenograft tumors in nude mice reduced tumor growth and the rate of distant metastasis. In clinical samples, the levels of ZEB1/ZEB2 expression were low in local lesions but high in metastatic lymph nodes in HNC tissues. Patients with tumors that co-expressed ZEB1(high) and ZEB2(high) had especially poor survival rates. CONCLUSION: Therapies targeting ZEB1/ZEB2 in HNC-CD133(+) cells may provide a new approach for HNC therapy in the future.

BPDE-like DNA adduct level in oral tissue may act as a risk biomarker of oral cancer.

OBJECTIVE: Most reports have shown that PAH-related DNA adducts are positively correlated with the smoking status of oral cancer patients. However, these reports did not focus on a specific carcinogen in cigarette smoke. The purpose of this study was to elucidate the role of the BPDE (7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene)-DNA adduct in the development of oral cancer in Taiwanese patients. DESIGN: We enrolled 158 oral cancer patients and 64 non-cancer controls to investigate whether there were differences in susceptibility to cigarette smoke exposure in the formation of DNA adducts between cancer patients and controls. Immunohistochemistry and ELISA (enzyme-linked immunosorbent assay) were used to evaluate BPDE-DNA adduct levels in this study. RESULTS: Our data showed that the BPDE-DNA adduct levels were positively correlated with gender, smoking status, betel nut chewing and alcohol consumption. The difference in DNA adduct levels could be explained by genetic polymorphisms of glutathione S-transferase M1 (GSTM1), but not by cytochrome P-4501A1 (CYP1A1). Patients with high DNA adduct levels (≧34.03 adducts/10(8) nucleotides) had an approximately 9.936-fold risk of oral cancer compared with those with low DNA adduct levels (<34.03 adducts/10(8) nucleotides) (p<0.001). CONCLUSIONS: We suggest that genetic background and carcinogen exposure may increase the risk of developing oral cancer.

Differential impact of IL-10 expression on survival and relapse between HPV16-positive and -negative oral squamous cell carcinomas.

Human papillomavirus (HPV) is a risk factor in a subset of oropharyngeal cancer; however, the contribution of HPV in the malignancy of oral squamous cell carcinomas (OSCC) is not fully understood in Taiwanese. Herein, 61 patients with no risk factors and 117 patients with one or more risk factors were enrolled in this study. HPV16/18 infection rate in non-smokers, non-drinkers and non-betel quid chewers was higher than their counterparts. The development of HPV-infected cancer has been shown to be associated with interleukin-10 (IL-10) expression. To this end, IL-10 mRNA expression in OSCC tumors was evaluated by real-time RT-PCR. Data showed that HPV-positive patients had higher IL-10 mRNA levels than in HPV-negative patients. Kaplan-Meier and Cox-regression analysis indicated that the prognostic significance of IL-10 mRNA on overall survival and relapse free survival was only observed in HPV-positive OSCC, but not in HPV-negative OSCC. Mechanistically, the elevation of IL-10 by E6 was responsible for increased colony formation and migration capability in OSCC cells. Therefore, we suggest that IL-10 induced by E6 promotes cell growth and migration capability and consequent poor survival and relapse in HPV-positive OSCC.