2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside-stimulated dental pulp stem cells-derived conditioned medium enhances cell activity and anti-inflammation.

BACKGROUND/PURPOSE: Dental pulp stem cells (DPSCs) contribute to the regeneration of various tissues and have superior proliferation, immune privilege, and anti-inflammation properties to other mesenchymal stem cells. 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside (THSG) not only enhances the aforementioned properties of DPSCs but also promotes self-renewal and reprogramming-like ability. However, whether THSG enhances the aforementioned properties and abilities through direct or indirect interaction mechanisms remains unclear. To address this knowledge gap, we examined the effects of THSG-stimulated DPSC-derived conditioned medium (THSG-CM) on the activity and anti-inflammation properties of cells. MATERIALS AND METHODS: DPSCs were treated with various concentrations of THSG to produce THSG-CM, which was then collected, analyzed, and lyophilized. A cytokine profiling antibody assay was used to compare protein components between THSG-treated and nontreated CM. Human skin fibroblasts (HSFs) and human gingival fibroblasts (HGFs) were used to investigate the effect of THSG-CM on cell proliferation, anti-inflammation, and wound healing abilities; for this investigation, MTS assay, quantitative real-time PCR analysis, and 2-well silicone inserts wound model were conducted. RESULTS: We observed that THSG enhanced the secretion of growth- and immune-associated proteins in THSG-CM and increased the proliferation of HSFs and HGFs. Furthermore, THSG-CM significantly attenuated lipopolysaccharide-stimulated mRNA levels of cytokines in both cells and improved wound healing abilities. CONCLUSION: We conclude that THSG-CM had more beneficial effects on cell activity and anti-inflammation in the HSFs and HGFs than DPSC-derived CM. DPSC-derived CM can be developed into a cell-free regenerative strategy in the future, and its therapeutic efficacy may be improved by THSG-CM.

2,3,5,4'-tetrahydroxystilbene-2-O-b-D-glucoside triggers the pluripotent-like possibility of dental pulp stem cells by activating the JAK2/STAT3 axis: Preliminary observations.

Abstract. BACKGROUND/PURPOSE: Although 2,3,5,4'-Tetrahydroxystilbene-2-O-beta-glucoside (THSG) reportedly has anti-inflammatory properties, its role in inducing the dedifferentiation of human dental pulp stem cells (DPSC) into pluripotent-like stem cells remains to be determined. The purpose of this study is to evaluate the effects of THSG on the pluripotent-like possibility and mechanism of DPSC. MATERIALS AND METHODS: DPSCs were treated with THSG, and cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTS) assay. Real-time polymerase chain reaction was used to analyze the mRNA expression levels of pluripotency-associated genes and oncogenes and to detect telomerase activity in the cells. Embryoid body formation assay was conducted, and pluripotency-related proteins were identified using Western blotting. Data were analyzed using one-way analysis of variance. RESULTS: Cell viability, telomerase activity, and embryoid body formation were enhanced in THSG-treated DPSCs. The mRNA expression levels of pluripotent-like genes (including Nanog homeobox [NANOG], SRY-box 2 [SOX2], and POU class 5 homeobox 1 [POU5F1/OCT4]) significantly increased after THSG treatment. The expression levels of pluripotency-related genes (Janus kinase-signal transducer 2 [JAK2] and signal transducer and activator of transcription 3 [STAT3]) increased, whereas those of oncogenes (Ras, SRC, HER2, and C-sis) decreased. Furthermore, the expression levels of the phosphorylated JAK2 and STAT3 proteins significantly increased after THSG treatment. CONCLUSION: THSG treatment may enhance the pluripotent-like possibility of DPSC through the JAK2/STAT3 axis. Hence, it may be used as an alternative cell-based therapeutic strategy in regenerative dentistry.

2,3,5,4'-Tetrahydroxystilbene-2-O-ß-d-glucoside promotes the effects of dental pulp stem cells on rebuilding periodontal tissues in experimental periodontal defects.

BACKGROUND: This study aimed to investigate the regenerative effects of 2,3,5,4'-tetrahydroxystilbene-2-O-ß-d-glucoside (THSG)-treated human dental pulp stem cells (DPSC) on the healing of experimental periodontal defects in rats. METHODS: The maxillary first molars of 30 male Sprague-Dawley rats were extracted, and after healing, bilateral periodontal defects were surgically created mesially in second molars. The defects were treated with Matrigel (as control), DPSC, or DPSC + THSG. After 2 weeks, the healed defects were evaluated using microcomputed tomography and through histological and immunohistochemical analyses. RESULTS: In the microcomputed tomography analysis, more new bone formation in the DPSC and DPSC + THSG groups was observed compared with the control group. The periodontal bone supporting ratio in site with DPSC + THSG was significantly higher than that in DPSC. Histologically, an enhanced new bone formation and more significant periodontal attachment were observed in the DPSC + THSG group. The expression levels of proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), and osteopontin (OPN) in the DPSC + THSG group were significantly greater than those in other groups. CONCLUSIONS: THSG-revolutionized DPSCs significantly shortened the regenerative period of periodontal defects by enhancing the cell recruitment and possibly the angiogenesis in rat models, which illustrate the critical implications for a clinical application and provide a novel tactic for periodontitis treatment.

Combined Treatment of Heteronemin and Tetrac Induces Antiproliferation in Oral Cancer Cells.

BACKGROUND: Heteronemin, a marine sesterterpenoid-type natural product, possesses an antiproliferative effect in cancer cells. In addition, heteronemin has been shown to inhibit p53 expression. Our laboratory has demonstrated that the thyroid hormone deaminated analogue, tetrac, activates p53 and induces antiproliferation in colorectal cancer. However, such drug mechanisms are still to be studied in oral cancer cells. METHODS: We investigated the antiproliferative effects by Cell Counting Kit-8 and flow cytometry. The signal transduction pathway was measured by Western blotting analyses. Quantitative PCR was used to evaluate gene expression regulated by heteronemin, 3,3',5,5'-tetraiodothyroacetic acid (tetrac), or their combined treatment in oral cancer cells. RESULTS: Heteronemin inhibited not only expression of proliferative genes and Homo Sapiens Thrombospondin 1 (THBS-1) but also cell proliferation in both OEC-M1 and SCC-25 cells. Remarkably, heteronemin increased TGF-ß1 expression in SCC-25 cells. Tetrac suppressed expression of THBS-1 but not p53 expression in both cancer cell lines. Furthermore, the synergistic effect of tetrac and heteronemin inhibited ERK1/2 activation and heteronemin also blocked STAT3 signaling. Combined treatment increased p53 protein and p53 activation accumulation although heteronemin inhibited p53 expression in both cancer cell lines. The combined treatment induced antiproliferation synergistically more than a single agent. CONCLUSIONS: Both heteronemin and tetrac inhibited ERK1/2 activation and increased p53 phosphorylation. They also inhibited THBS-1 expression. Moreover, tetrac suppressed TGF-ß expression combined with heteronemin to further enhance antiproliferation and anti-metastasis in oral cancer cells.

Resveratrol antagonizes thyroid hormone-induced expression of checkpoint and proliferative genes in oral cancer cells.

BACKGROUND/PURPOSE: Dysregulation of cell cycle checkpoint control may lead to the independence of growth regulating signals. Checkpoint protein such as the PD-1/PD-L1 immune checkpoint involving tumor cells and host immune defense lymphocytes is a well-studied therapeutic target in oncology. Acting at a cell surface receptor on plasma membrane integrin αvß3, thyroxine stimulates intracellular accumulation of PD-L1 in cancer cells. Although resveratrol also binds to integrin αvß3, it reduces PD-L1 expression. MATERIALS AND METHODS: In current studies, we investigated the roles of resveratrol and thyroxine in regulating expression of proliferation-related genes and checkpoint genes, PD-L1, BTLA in two oral cancer cell lines. RESULTS: Thyroxine suppressed the expression of pro-apoptotic BAD but induced proliferative CCND1 expression in SSC-25 cells and OEC-M1 cells. It activated expression of PD-L1 and BTLA in both cell lines. On the other hand, resveratrol suppressed the expression of all. Alternatively, it activated BAD expression. Thus thyroxine induces checkpoint gene expression which may promote proliferation in cancer cells. Alternatively, resveratrol reverses the stimulatory effects of thyroid hormone to induce anti-proliferation. CONCLUSION: These findings provide new insights into the antagonizing effect of resveratrol on the thyroxine-induced expression of checkpoint genes and proliferative genes in oral cancers.

Dental Pulp Stem Cell Transplantation with 2,3,5,4'-Tetrahydroxystilbene-2-O-ß-D-glucoside Accelerates Alveolar Bone Regeneration in Rats.

INTRODUCTION: Although the therapeutic potential of human dental pulp stem cells (hDPSCs) has been studied for bone regeneration, the therapeutic efficiency needs further consideration and examinations for clinical applications. Thus, the aims of this study were to evaluate the effect of 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside (THSG) on the osteogenic differentiation of hDPSCs and to examine the therapeutic efficiency of the THSG-enhanced osseous potential of hDPSCs in alveolar bony defects of rats. METHODS: Expressions of osteogenic messenger RNAs (including ALP, RUNX2, BGLAP, and AMBN) were examined by quantitative real-time polymerase chain reaction. Alizarin red S staining was conducted to analyze THSG-induced mineralization of hDPSCs. To investigate the regenerative effects of THSG-treated hDPSCs on dental alveolar bone, bony defects were created in male Sprague-Dawley rats. Defects were treated with Matrigel (Corning Inc, Corning, NY), hDPSCs, or hDPSCs + THSG. After 2 weeks, defect healing was evaluated by micro-computed tomographic and histologic analyses. RESULTS: In the cell model, THSG induced osteogenesis-associated genes (ALP, RUNX2, and BGLAP) and an enamel-related gene (AMBN), resulting in mineralization as detected by alizarin red S staining after 2 weeks of treatment. In the animal model, THSG increased all parameters of bone formation (the relative bone volume, trabecular thickness, trabecular number, and trabecular separation) in alveolar bony defects of rats. THSG not only improved the quality of newly formed bone but also the quantity of new bone. CONCLUSIONS: These results showed important findings in revealing the THSG-enhanced osteogenic differentiation of hDPSCs and THSG-facilitated bone regeneration, which may provide an alternative option for cell-based regenerative therapy.

Implication for second primary cancer from visible oral and oropharyngeal premalignant lesions in betel-nut chewing related oral cancer.

BACKGROUND: Visible oral and oropharyngeal premalignant lesions may be used to monitor for a second primary oral cancer. To control for bias, we focused on the visible oral and oropharyngeal premalignant lesions of patients with oral cancer with a positive betel-nut chewing habit. Visible oral and oropharyngeal premalignant lesions that can predict second primary oral cancers were studied. METHODS: Nine hundred ninety-seven patients with positive betel-nut chewing habits and oral cancer were enrolled in this retrospective cohort study. We analyzed the relevance of their visible oral and oropharyngeal premalignant lesion incidence and relative clinicopathological variables to the development of a second primary oral cancer. RESULTS: Second primary oral cancer risk was significantly higher in patients with positive visible oral and oropharyngeal premalignant lesions (P < .0001), especially in younger patients (P = .0023; ≤40 years: adjusted odds ratio [OR] 2.66; 40-60 years: adjusted OR 2.61). The heterogeneous leukoplakia was (adjusted OR 2.17) higher than homogeneous leukoplakia. CONCLUSION: The predictive value and practicality of visible oral and oropharyngeal premalignant lesions make it a potentially valuable marker in follow-ups of patients with a positive betel-nut chewing habit with oral cancer, especially young patients with heterogeneous leukoplakia.

Cell-based semiquantitative assay for sulfated glycosaminoglycans facilitating the identification of chondrogenesis.

Glycosaminoglycans (GAGs), in particular chondroitin sulfate, are an accepted marker of chondrogenic cells. In this study, a cell-based sulfated GAG assay for identifying the chondrogenesis of mesenchymal stem cells was developed. Based on fluorescent staining using safranin O and 4',6-diamidino-2-phenylindole (DAPI), this method was highly sensitive. The results were both qualitative and quantitative. The method is suitable for identifying the chondrogenic process and also for screening compounds. The method may be helpful for discovering novel bioactive compounds for cartilage regeneration.