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Background/purpose: Cancer stem cells (CSCs) have been known to be implicated in tumorigenesis, metastasis, and drug resistance in oral squamous cell carcinomas (OSCC). In this study, we aimed to investigate whether magnolol, a polyphenolic component derived from Magnolia officinalis, exhibited the anti-CSCs properties., Methods: The cytotoxicity of magnolol was tested using normal gingival epithelioid SG cells and sphere-forming OSCC-CSCs isolated from SAS, OECM1, and GNM cells. Secondary sphere-forming ability, the proportion of ALDH1 positive cells, Transwell migration, and invasion capacities were examined as well. The chemosensitive effects of magnolol were investigated using MTT, secondary sphere-forming, and invasion assays., Results: Magnolol exerted a higher cytotoxicity of OSCC-CSCs and cancer stemness features, including self-renewal ability, the expression CSC marker, migration, and invasion capacities were all downregulated in magnolol-treated OSCC-CSCs. Moreover, administration of magnolol potentiated the effect of cisplatin, including a decrease in cell viability, self-renewal, and invasion activities. In addition, we observed that the secretion of IL-6 and phosphorylation of Stat3 were decreased in OSCC-CSCs treated with magnolol., Conclusion: Our data suggest that magnolol is able to target CSCs and suppress the cancer stemness properties, at least in part, via IL-6/Stat3 signaling. Besides, a dietary supplement of magnolol may function as an adjunct to cisplatin treatment., Competing Interests: Declaration of competing interest The authors have no conflicts of interest relevant to this article., (Copyright © 2021. Published by Elsevier B.V.)

Background/purpose: Oral submucous fibrosis (OSF) a well-recognized oral premalignant disorder. Several studies have demonstrated that periostin, a matricellular protein, is involved in the development and pathogenesis of fibrosis diseases. Nevertheless, the contribution of periostin in OSF remains to be uncovered. The purpose of the study was to illustrate the functional role of periostin involved in OSF pathogenesis., Methods: RNA-sequencing was employed to screen for differentially expressed genes in normal and OSF tissues. Validation of the upregulation of periostin in OSF specimens and fibrotic buccal mucosal fibroblasts (fBMFs) was conducted by qRT-PCR. The correlation of the gene expression of periostin and various fibrosis markers was analyzed. In addition, the functional role of periostin in myofibroblast features was tested using collagen gel contraction and transwell migration assays., Results: We observed overexpression of periostin in OSF specimens using RNA-sequencing and confirmed its upregulation in OSF tissues and patient-derived fBMFs. Besides, there was a positive relationship between the expression of periostin and several fibrosis-associated markers, including ACTA2 (α-smooth muscle actin; α-SMA), COL1A1 (type 1 collagen α1 chain), TGFB1 (TGF-β1), and FN1 (fibronectin). Furthermore, we examined the effect of silencing periostin on the maintenance of myofibroblast characteristics and showed that knockdown of periostin suppressed the expression of α-SMA. Also, inhibition of periostin markedly downregulated the myofibroblast activities (collagen gel contraction and migration capacities)., Conclusion: Our results indicate the aberrant expression of periostin in OSF tissues and myofibroblasts. Moreover, the expression of periostin is positively associated with fibrosis markers, and repression of periostin may be a promising direction to alleviate the progression of OSF., Competing Interests: Declaration of competing interest The authors have no conflicts of interest relevant to this article., (Copyright © 2021. Published by Elsevier B.V.)

Background/purpose: Oral cancer stem cells (CSCs) have been considered as the key cells that are implicated in tumor recurrence and metastasis. In recent years, great attention has been paid to the significance of various non-coding RNAs due to their regulatory roles in oral CSCs. Although the function of long non-coding RNA MEG3 in various cancers has been investigated, its effects on the features of oral CSCs remained to be determined., Methods: The expression levels of MEG3 in tongue squamous cell carcinomas and prognostic effect have been evaluated. We assessed the expression of MEG3 in sphere cells (oral CSCs) using qRT-PCR. Secondary sphere formation and invasion assays were conducted to evaluate the self-renewal and metastatic abilities, respectively. Bioinformatics software and luciferase reporter assay were used to predict and verify the relationship between MEG3 and miR-421., Results: MEG3 was downregulated in the tissues of oral cancer and associated with a poor prognosis. In oral CSCs, the expression of MEG3 was repressed and overexpression of MEG3 resulted in suppression of self-renewal and invasion abilities. Luciferase reporter assay showed that miR-421 directly interacted with MEG3, and our subsequent experiment demonstrated that elevation of miR-421 reversed the MEG3-inhibited characteristics of oral CSCs., Conclusion: Our findings suggest that MEG3 can serve as a tumor suppressor in oral CSCs by impeding the action of miR-421. Moreover, targeting MEG3-miR-421 axis has the potential to mitigate the tumor recurrence and metastasis., Competing Interests: Declaration of competing interest The authors have no conflicts of interest relevant to this article., (Copyright © 2020. Published by Elsevier B.V.)

Background/purpose: MicroRNA-200c (miR-200c) recently emerged as an important regulator of tumorigenesis and cancer metastasis, however, its role in regulating oral submucous fibrosis (OSF) remains unknown. In this study, we investigated the functional role of miR-200c in myofibroblastic differentiation activity and identified its potential target., Methods: qRT-PCR was applied to assess the expression of miR-200c in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs). Arecoline, a major areca nut alkaloid, was utilized to explore whether the expression of miR-200c would alter following stimulation. Collagen gel contraction, migration and invasion capabilities were examined in arecoline-stimulated BMFs as wells as in fBMFs. Luciferase reporter assay was conducted to show the relationship between miR-200c and ZEB1., Results: Our results showed that the expression of miR-200c was downregulated in OSF specimen and fBMFs. Arecoline treatment dose-dependently reduced the relative expression of miR-200c in normal BMFs. Overexpression of miR-200c impeded the arecoline-induced collagen gel contraction, migration, invasion and wound healing capacities. Moreover, ectopic expression of miR-200c in fBMFs successfully reduced the increased collagen gel contractility and invasion abilities. Our results demonstrated that ZEB1 was a direct target of miR-200c, and overexpression of miR-200c inhibited the expression of ZEB1 and α-SMA., Conclusion: These findings suggest that downregulation of miR-200c in OSF may be involved in the pathogenesis of areca nut-associated OSF through regulation of ZEB1., (Copyright © 2018. Published by Elsevier B.V.)

Background/purpose: Emerging research findings suggest that long non-coding RNAs (lncRNAs) are key regulators to fibrosis formation. Nevertheless, the role of lncRNA GAS5-AS1 in the progression of precancerous oral submucous fibrosis (OSF) remains to be elucidated., Methods: Quantitative real-time PCR were used to examine the expression of GAS5-AS1 in OSF tissues. The activities of myofibroblasts, including collagen contractility and cell migration, as well as the marker α-smooth muscle actin (SMA) were assessed following overexpression of GAS5-AS1. Also, we analyzed the expression of Smad activity in order to gain insight into the downstream regulator., Results: The level of GAS5-AS1 was found significantly downregulated in the OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs). Ectopic expression of GAS5-AS1 significantly reduced the abilities of collagen gel contraction and migration in fBMFs or arecoline-treated BMFs. Moreover, we have shown that overexpression of GAS5-AS1 inhibited the expression of p-Smad and the marker of myofibroblasts., Conclusion: We showed the reduced expression of GAS5-AS1 in OSF tissues and demonstrated its effect on the myofibroblast activities and the level of p-Smad and α-SMA, indicating its potential contribution in OSF pathogenesis., (Copyright © 2017. Published by Elsevier B.V.)

Background/purpose: Cumulative evidence suggest that microRNAs (miRNAs) function as biosignatures of oral squamous cell carcinomas (OSCC). However, the functional roles of miR-1 as well as its downstream targets in the regulation of tumorigenicity in OSCC remain unclear., Methods: miRNAs RT-PCR analysis was performed to identify miR-1 as a putative candidate on mediating invasiveness of OSCC cells. Consequently, we elucidated the tumorigenicity of OSCC cells with miR-1 downregulation or overexpression, respectively. Finally, miR-1 on OSCC tumor tissues was examined., Results: miR-1 levels were significantly downregulated in the malignant OSCC cells. Overexpression of miR-1 significantly reduced migration/invasiveness of OSCC cells. In addition, overexpression of miR-1 decreased cancer stem cells properties. Conversely, downregulation of miR-1 promotes migration and invasiveness in OSCC cells. We have shown that miR-1 is able to target Slug, suppressing their expression. Clinically, lower miR-1 expression was found in patients with advanced nodal metastasis OSCC., Conclusion: miR-1 as novel biosignatures in OSCC lymph node metastatic patients, supporting the development of novel strategies for OSCC treatment., (Copyright © 2017. Published by Elsevier B.V.)

Background/purpose: Cancer stem cells (CSCs) are deemed as the driving force of tumorigenesis in oral squamous cell carcinomas (OSCCs). In this study, we investigated the chemotherapeutic effect of sulforaphane, a dietary component from broccoli sprouts, on targeting OSCC-CSCs., Methods: The effect of sulforaphane on normal oral epithelial cells (SG) and sphere-forming OSCC-CSCs isolated from SAS and GNM cells was examined. ALDH1 activity and CD44 positivity of OSCC-CSCs with sulforaphane treatment was assessed by flow cytometry analysis. In vitro and in vivo tumorigenicity assays of OSCC-CSCs with sulforaphane treatment were presented., Results: We observed that the sulforaphane dose-dependently eliminated the proliferation rate of OSCC-CSCs, whereas the inhibition on SG cells proliferation was limited. Cancer stemness properties including self-renewal, CD44 positivity, and ALDH1 activity were also decreased in OSCC-CSCs with different doses of sulforaphane treatment. Moreover, sulforaphane treatment of OSCC-CSCs decreased the migration, invasion, clonogenicity, and in vivo tumorigenicity of xenograghts. Sulforaphane treatment resulted in a dose-dependent increase in the levels of tumor suppressive miR200c., Conclusion: These lines of evidence suggest that sulforaphane can suppress the cancer stemness and tumor-initiating properties in OSCC-CSCs both in vitro and in vivo., (Copyright © 2016. Published by Elsevier B.V.)

Background/purpose: Previously, we successfully identified oral cancer stem cells (OCSC) displaying enhanced stemness and tumorigenic potentials. In the study, we investigated the chemotherapeutic effect of the flavonoid luteolin, commonly found in fruits and vegetables, on targeting OCSC., Methods: Oralspheres was applied to isolate OCSC. aldehyde dehydrogenase 1 activity and CD44 positivity of OCSC with luteolin treatment were assessed by flow cytometry analysis. Radio-sensitivity of OCSC treated with luteolin was examined. Invasion and colony-forming assays were performed to assess oncogenicity in OCSC. The expression of interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) was examined by enzyme-linked immunosorbent assay and western blot analysis., Results: We showed that luteolin effectively inhibited the proliferation rate, self-renewal, aldehyde dehydrogenase 1 activity, and CD44 positivity of OCSC but did not cause significant cytotoxicity of normal epithelial cells. Moreover, luteolin restored radio-sensitivity in OCSC. Combined treatment with luteolin and radiation displayed synergistic effect on invasiveness and clonogenicity of OCSC. Mechanistically, treatment of luteolin resulted in inactivation of IL-6/STAT3 signaling., Conclusion: These results suggest that combined treatment of luteolin and radiation therapy can attenuate tumorigenicity of OCSC through IL-6/STAT3 signaling inactivation., (Copyright © 2016. Published by Elsevier B.V.)

Background/purpose: Oral submucous fibrosis (OSF), a chronic progressive scarring disease, has been considered as a precancerous condition of oral mucosa. In this study, we investigated the functional role of Twist, an epithelial-mesenchymal transition (EMT) transcriptional factor, in myofibroblastic differentiation activity of OSF., Methods: Arecoline, a major areca nut alkaloid, was used to explore whether expression of Twist could be changed dose-dependently in human primary buccal mucosal fibroblasts (BMFs). Collagen gel contraction and migration capability in arecoline-stimulated BMFs and primary oral submucous fibrosis-derived fibroblasts (OSFs) with Twist knockdown was presented., Results: We observed that the treatment of arecoline dose-dependently increased Twist expression transcript and protein levels in BMFs. The myofibroblast activity including collagen gel contraction and migration capability also induced by arecoline, while knockdown of Twist reversed these phenomena. Importantly, inhibition of Twist led to the suppression collagen contraction and wound healing capability of primary cultivated OSFs. Clinically, Twist transcript and protein expression was higher in areca quid chewing-associated OSF tissues than in normal oral mucosa tissues., Conclusion: This evidence suggests that upregulation of Twist might be involved in the pathogenesis of areca quid-associated OSF through dysregulation of myofibroblast activity., (Copyright © 2015. Published by Elsevier B.V.)

Intraosseous verrucous carcinoma (IOVC) arising from an odontogenic cyst is extremely rare. We report a case of intraosseous verrucous carcinoma in a 74-year-old male who presented with a left mandibular swelling with recurrent pus discharge from gingiva of tooth #35. Panoramic radiography revealed an impacted tooth #34 and a large well-defined, radiolucent lesion surrounding the crown of tooth #34. The clinical diagnosis was an infected dentigerous cyst. Surgical excision of the cyst together with extraction of tooth #34 was performed. Histopathological examination showed proliferation of hyperparakeratotic stratified squamous cyst lining epithelium and down-growth of broad and bulbous epithelial ridges with pushing border invasion into the fibrous cystic wall. A verrucous carcinoma arising from an infected dentigerous cyst was diagnosed. There was no recurrence of the tumor 5 months after surgery., (Copyright © 2012. Published by Elsevier B.V.)