Yu Chao Chang

Markedly increased Oct4 and Nanog expression correlates with cisplatin resistance in oral squamous cell carcinoma

BACKGROUND Oral squamous cell carcinoma (OSCC) is the sixth most prevalent cancer worldwide. Cancer stem cells (CSC) model theoretically contribute to tumor growth, metastasis, and chemo-radioresistance. Cisplatin is a widely used chemotherapeutic agent for OSCC treatment. The aim of this study was to compare stemness genes expression in chemo-sensitive and chemo-resistant specimens and further explore the potential markers that may lead to induce chemo-resistance in OSCC. METHODS The study method is the treatment of OC2 cells with cisplatin select cisplatin-resistant OC2 cells. Self-renewal ability was evaluated by cultivating parental and cisplatin-resistant OC2 cells within sphere-forming assay after serial passages. Differential expression profile of stemness markers between parental and cisplatin-resistant OC2 cells was elucidated. The parental and cisplatin-resistant OC2 cells were assessed for migration/invasion/clonogenicity tumorigenic properties in vitro. Expression of stemness markers in chemo-sensitive and chemo-resistant patients with OSCC was performed by immunohistochemistry staining in vivo. RESULTS Sphere-forming/self-renewal capability was increased in cisplatin-resistant OC2 cells. Cisplatin-resistant OC2 cells highly expressed the stemness markers (Nanog, Oct4, Bmi1, CD117, CD133, and ABCG2). Furthermore, cisplatin-resistant OC2 cells increased migration/invasion/clonogenicity ability. Notably, up-regulation of Oct4 and Nanog expression was significantly observed in cisplatin-resistant patients with OSCC (**P < 0.01). CONCLUSIONS These data indicate that cancer stem-like properties were expanded during the acquisition of cisplatin resistance in OSCC. Clinically, oral cancer stemness markers (Oct4 and Nanog) overexpression may promote the OSCCs recurrence to resist cisplatin.

Let-7d functions as novel regulator of epithelial-mesenchymal transition and chemoresistant property in oral cancer

Oral squamous cell carcinoma (OSCC) is a prevalent cancer worldwide. Let-7 family has been shown to function as a tumor suppressor through regulating multiple oncogenic signaling. Recent study reported that combined underexpression of miR-205 and let-7d showed negative correlation with the survival prognosis of head and neck cancer patients. However, the let-7d-involved mechanism in regulating OSCC is still unclear. In this study, we first demonstrated that let-7d expression was significantly decreased while Twist and Snail expression was increased in OSCC cancer cell lines and primary cultures as compared to normal human oral keratinocyte cells. To further investigate the role of let-7d in OSCC, we applied the SPONGE method to knock down let-7d in OECM-1 and two primary OSCC cell types. The results showed that knockdown of let-7d promote epithelial-mesenchymal transition (EMT) traits and migratory/invasive capabilities in OSCC cells. Furthermore, down-expression of let-7d significantly activated Twist and Snail expressions and chemo-resistant abilities of OSCC cells. Notably, overexpression of let-7d effectively reversed the EMT phenotype, blocked migratory/invasive abilities, and further increased the chemosensitivity in oral cancer tumor initiating ALDH1+ cells. In sum, these results show that let-7d negatively modulates EMT expression and also plays a role in regulating chemo-resistant ability in oral cancer.

Dioscorea nipponica Makino inhibits migration and invasion of human oral cancer HSC-3 cells by transcriptional inhibition of matrix metalloproteinase-2 through modulation of CREB and AP-1 activity

Oral cancer mortality has increased during the last decade due to the difficulties in treating related metastasis. Dioscorea nipponica Makino, a popular folk medicine, exerts anti-obesity and anti-inflammation properties. However, the effect of this folk medicine on metastasis of oral cancer has yet to be fully elucidated. The present study demonstrates that D. nipponica extracts (DNE), at a range of concentrations (0-50 μg/mL), concentration-dependently inhibited migration/invasion capacities of human oral cancer cells, HSC-3, without cytotoxic effects. The anti-migration effect of DNE was also observed in two other OSCC cell lines, Ca9-22 and Cal-27. Zymography, real time PCR, and Western blotting analyses revealed that DNE inhibited matrix metalloproteinase-2 (MMP-2) enzyme activity, and RNA and protein expression. The inhibitory effects of DNE on MMP-2 proceeded by up-regulating tissue inhibitor of metalloproteinase-2 (TIMP-2), as well as suppressing nuclear translocation and DNA binding activity of cAMP response element-binding (CREB) and activating protein-1 (AP-1) on the MMP-2 promoter in HSC-3 cells. In conclusion, DNE inhibited the invasion of oral cancer cells and may have potential use as a chemopreventive agent against oral cancer metastasis.

Oct4 mediates tumor initiating properties in oral squamous cell carcinomas through the regulation of epithelial-mesenchymal transition.

Background Overexpression of Oct4, an important transcription factor of embryonic stem cells (ESC), has been reported in several cancers. The aim of this study was to determine the emerging role of Oct4 in oral squamous cell carcinoma (OSCC) both in vitro and in vivo. Methodology/Principal Finding Tumourigenic activity and molecular mechanisms of Oct4 overexpression or knockdown by lentiviral infection in OSCC was investigated in vitro and in vivo. Initially, we demonstrated that Oct4 expression was increased in OSCC cell lines as compared to a normal oral epithelial cell line SG. Overexpression of Oct4 was demonstrated to enhance cell proliferation, invasiveness, anchorage-independent growth and xenotransplantation tumourigenicity. These findings were coupled with epithelial-mesenchymal transition (EMT) transformation in OSCCs. In contrast, the silence of Oct4 significantly blocked the xenograft tumorigenesis of OSCC-derived cancer stem cells (OSCC-CSCs) and significantly improved the recipient survival. Clinically, the level of Oct4 expression was higher in recurrent and metastatic OSCC specimens but lower in primary OSCC specimens. Conclusion/Significance Our results suggest that Oct4-mediated tumorigenecity is associated with the regulation of EMT. Oct4 might be a therapeutic target for OSCC.

Quercetin in elimination of tumor initiating stem-like and mesenchymal transformation property in head and neck cancer.

Previously, we enriched a subpopulation of head and neck cancer–derived tumor initiating cells (HNC‐TICs) presented high tumorigenic, chemo‐radioresistant, and coupled with epithelial–mesenchymal transition (EMT) properties. The purpose of this study was to investigate the therapeutic effect and molecular mechanisms of quercetin on HNC‐TICs.

β-catenin expression in areca quid chewing-associated oral squamous cell carcinomas and upregulated by arecoline in human oral epithelial cells

BACKGROUND/PURPOSE Nuclear localization of β-catenin is known to associate with malignant transformation of many squamous cell carcinomas. The aim of this study was to compare β-catenin expression in normal human oral epithelium and areca quid chewing associated oral squamous cell carcinomas (OSCCs) and further to explore the potential mechanisms that may lead to induce β-catenin expression. METHODS A total of 40 areca quid chewing-associated OSCCs and 10 normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The oral epithelial cell line GNM cells were challenged with arecoline, a major areca nut alkaloid, by using Western blot analysis. Furthermore, extracellular signal-regulated protein kinase inhibitor PD98059, glutathione precursor N-acetyl-l-cysteine (NAC), tyrosine kinase inhibitor herbimycin-A, p38 inhibitor SB203580, and phosphatidylinositaol 3-kinase inhibitor LY294002 were added to find the possible regulatory mechanisms. RESULTS β-catenin expression was significantly higher in OSCC specimens than that in normal oral epithelial specimens (p < 0.05). It was demonstrated that normal oral epithelium showed only membranous staining for β-catenin, and membranous staining was lost or reduced with an increase in cytoplasmic/nuclear staining in OSCCs. Arecoline was found to elevate β-catenin expression in a dose-dependent manner (p < 0.05). The addition of PD98059, NAC, herbimycin-A, SB203580, and LY294002 markedly inhibited the arecoline-induced β-catenin expression (p < 0.05). CONCLUSION β-catenin expression is significantly upregulated in areca quid chewing-associated OSCC. The localization of β-catenin expression is correlated with the tumor size and clinical stage. In addition, β-catenin expression induced by arecoline is downregulated by PD98059, NAC, herbimycin-A, SB203580, and LY294002.

Concomitant upregulation of matrix metalloproteinase-2 in lesions and circulating plasma of oral lichen planus

Background/purpose Oral lichen planus (OLP) is a chronic inflammatory disorder characterized by a T cell-mediated immune response against epithelial cells. Matrix metalloproteinases (MMPs) are an important group of zinc enzymes and are thought to play an important role in the degradation of components of the extracellular matrix. The aim of this study was to assess the expression of MMP-2 and MMP-9 in both tissue specimens and circulating plasma. Materials and methods Twelve cases of OLP were collected. In addition, six cases with chronic inflammation and six normal subjects were also recruited. Oral tissue specimens were collected for measurement of MMPs by immunohistochemistry, and sera prepared from peripheral blood were used for gelatin zymography to determine MMP levels in plasma. Results The level of MMP-2 in patients with OLP was significantly higher than that in the other two groups, both in tissues and sera (P Conclusion MMP-2 overexpression in OLP is consistent with its upregulation in peripheral serum. This result also indicates that MMP-2 might play a role in the pathogenesis of OLP.

Treatment of bisphosphonate-related osteonecrosis of the jaw with platelet-rich fibrin.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a well-known potential complication of bisphosphonate treatment. The clinical findings of BRONJ may vary in patients, including exposed necrotic bone, which can be accompanied by pain, swelling, paresthesia, pus discharge, and soft tissue ulceration. Currently, the management of BRONJ remains controversial, and there is no definitive treatment other than the standard protocol. Choukroun’s platelet-rich fibrin (PRF) represents a relatively new biotechnology for the stimulation and acceleration of tissue healing and bone regeneration. In this study, we report a BRONJ patient who was treated with surgical debridement, sequestrectomy, and simultaneous reconstruction using PRF as the sole grafting material. A 79-year-old woman was referred in July 2011 by a local dental clinic for a painful jaw bone lesion. Her medical history included osteoporosis since 1996, which was treated weekly with alendronate (Fosamax, 70 mg, per os; Merck, Whitehouse Station, NJ, USA) for 10 more years, and intravenous zoledronic acid (Aclasta; Norvatis Pharmaceuticals, Frimley, Camberley, UK) infusions annually, at a dose of 5 mg, from 2010 to 2011. The intraoral examination revealed a diffuse, tender, erythematous swelling over the edentulous area at the right posterior mandible with a purulent discharge

Increased expression of carbonic anhydrase IX in oral submucous fibrosis and oral squamous cell carcinoma

Abstract Background: Cumulative evidence has demonstrated that carbonic anhydrase IX (CAIX) is upregulated in many types of human cancers. We attempted to evaluate plasma levels of CAIX in patients with oral cancer and investigated whether plasma CAIX is correlated with the progression of this disease. Method: In total, 191 patients with oral cancer, 30 patients with oral submucous fibrosis and 100 controls were recruited in this study. The plasma samples were collected and the levels of soluble CAIX in plasma were determined by the enzyme-linked immunosorbent assay (ELISA). Furthermore, the normal buccal mucosa fibroblast was challenged by arecoline, the major areca nut alkaloid, to assess the relationship between the levels of CAIX and areca nut chewing in oral cancer patients. Results: Results showed that patients with oral cancer exhibited significantly higher levels of soluble CAIX compared to controls (p<0.001). Plasma levels of CAIX in oral cancer patients were associated with clinical stages after adjusting for age and areca nut chewing (p<0.05). In addition, patients with areca nuts chewing had higher CAIX levels than those who have not chewed areca nuts. Total carbonic anhydrase activity and CAIX mRNA levels were significantly higher in oral submucous fibrosis fibroblasts than in normal buccal mucosa fibroblasts. Moreover, arecoline elevated CAIX expression in a dose-dependent manner in normal buccal mucosa fibroblasts. Conclusions: Our results suggest that determining plasma levels of CAIX may be used as a non-invasive method for monitoring oral cancer progression and the involvement of areca quid chewing in oral carcinogenesis may be related to a higher expression of CAIX.

Antimetastatic potentials of salvianolic acid A on oral squamous cell carcinoma by targeting MMP-2 and the c-Raf/MEK/ERK pathway

The metastasis of oral squamous cell carcinoma (OSCC) is one of the most important causes of cancer‐related deaths. Thus, various therapeutic strategies have been developed to prevent the metastasis of OSCC. Salvianolic acid A (SAA), a traditional Chinese medicine, has antithrombosis, antiplatelet, anti‐inflammation, and antitumor activities. Here, we provide molecular evidence indicating that SAA exerts its antimetastatic effects by markedly inhibiting the invasion and migration of oral squamous SCC‐9 and SCC‐25 cells. SCC‐9 and SCC‐25 cells were treated with various concentrations of SAA to further investigate the precise involvement of SAA in cancer metastasis. The results of zymography, and Western blotting indicated that SAA treatment may decrease matrix metallopoteinase‐2 (MMP‐2) expression. SAA also inhibited p‐c‐Raf, p‐MEK1/2, and p‐ERK1/2 protein expression. In addition, treating SCC‐9 cells with U0126, a MEK‐specific inhibitor, decreased MMP‐2 expression and concomitantly inhibited cell migration. Our findings suggested that SAA inhibits the invasion and migration of OSCC by inhibiting the c‐Raf/MEK/ERK pathways that control MMP‐2 expression. Our findings provide new insights into the molecular mechanisms that underlie the antimetastatic effect of SAA and are thus valuable for the development of treatment strategies for metastatic OSCC.