Yi-Ting Deng

Histone deacetylase 2 expression predicts poorer prognosis in oral cancer patients

Histone deacetylase 2 (HDAC2) has been implicated in the development and progression of several human tumors. We immunohistochemically examined the expression of HDAC2 protein in 20 cases of oral epithelial dysplasia (OED) and 93 cases of oral squamous cell carcinoma (OSCC). Positive HDAC2 nuclear staining was observed in 80 of the 93 (86.02%) cases of SCC and 11 of the 20 (55%) cases of ED. The labeling index (LI) for HDAC2 nuclear staining increased significantly from ED (25.8+/-26.5%) to SCCs (59.8+/-28.5%) (p<0.001). No significant correlation was found between the HDAC2 expression level and patients age, sex, oral habits in oral SCC patients. However, cancer with advanced stage, larger tumor size, or positive lymph node metastasis had higher level of HDAC2 protein expression. Kaplan-Meier curves showed oral SCC patients with high HDAC2 expression (LI>50%), advanced stage, larger tumor size, or positive lymph node metastasis had significantly shorter overall survival (p=0.0158, 0.0267, 0.0029 and 0.02514, respectively by log-rank test) than others. The results of this study show for the first time that overexpression of the HDAC protein is a frequent event in oral cancer and could be used as a prognostic factor in oral SCC.

Arecoline-stimulated connective tissue growth factor production in human buccal mucosal fibroblasts: Modulation by curcumin.

Connective tissue growth factor (CTGF) is associated with the onset and progression of fibrosis in many human tissues. Areca nut (AN) chewing is the most important etiological factor in the pathogenesis of oral submucous fibrosis (OSF). We immunohistochemically examined the expression of CTGF protein in 20 cases of OSF and found positive CTGF staining in fibroblasts and endothelial cells in all cases. Western blot analysis showed that arecoline, a main alkaloid found in AN, stimulated CTGF synthesis in a dose- and time-dependent manner in buccal mucosal fibroblasts. Constitutive overexpression of CTGF during AN chewing may enhance the fibrotic activity in OSF and play a role in the pathogenesis of OSF. Pretreatment with NF-kappaB inhibitor Bay 11-7082, JNK inhibitor SP600125, p38 MAPK inhibitor SB203580 and antioxidant N-acetyl-l-cysteine, but not ERK inhibitor PD98059, significantly reduced arecoline-induced CTGF synthesis. Furthermore, curcumin completely inhibited arecoline-induced CTGF synthesis and the inhibition is dose-dependent. These results indicated that arecoline-induced CTGF synthesis was mediated by ROS, NF-kappaB, JNK, P38 MAPK pathways and curcumin could be a useful agent in controlling OSF.

Suberoylanilide hydroxamic acid sensitizes human oral cancer cells to TRAIL-induced apoptosis through increase DR5 expression

Suberoylanilide hydroxamic acid has been shown to selectively induce tumor apoptosis in cell cultures and animal models in several types of cancers and is about as a promising new class of chemotherapeutic agents. In addition, suberoylanilide hydroxamic acid showed synergistic anticancer activity with radiation, cisplatin, and tumor necrosis factor–related apoptosis–inducing ligand (TRAIL) in some cancers. Here, we report suberoylanilide hydroxamic acid also induced apoptosis in human oral cancer cells. Western blotting showed suberoylanilide hydroxamic acid increased Fas, Fas ligand, DR4, and DR5 protein expression and activated caspase-8 and caspase-9. The apoptosis was almost completely inhibited by caspase-8 inhibitor Z-IETD-FMK and attenuated by caspase-9 inhibitor Z-LEHD-FMK. Human recombinant DR5/Fc chimera protein but not Fas/Fc or DR4/Fc significantly inhibited apoptosis induced by suberoylanilide hydroxamic acid. These results suggest that suberoylanilide hydroxamic acid induces apoptosis mainly through activation of DR5/TRAIL death pathway. Furthermore, subtoxic concentrations of suberoylanilide hydroxamic acid sensitize two TRAIL resistant human oral cancer cells, SAS and Ca9-22, to exogenous recombinant TRAIL-induced apoptosis in a p53-independent manner. Combined treatment of suberoylanilide hydroxamic acid and TRAIL may be used as a new promising therapy for oral cancer. [Mol Cancer Ther 2009;8(9):2718–25]

EGCG blocks TGFβ1-induced CCN2 by suppressing JNK and p38 in buccal fibroblasts

ObjectivesTransforming growth factor β (TGFβ) has been suggested as the main trigger for the increased collagen production and decreased matrix degradation pathways in oral submucous fibrosis (OSF). Connective tissue growth factor (CTGF/CCN2) and cyclooxygenase-2 (COX-2) were found to overexpress in OSF. The aim of this study was to investigate the molecular mechanism underlying the TGFβ-induced CCN2 expressions in human buccal mucosal fibroblasts (BMFs) to identify the potential targets for drug intervention or chemoprevention of OSF.Materials and methodsTGFβ-induced CCN2 expression and its signaling pathways were assessed by Western blot analyses in BMFs.ResultsTGFβ1 stimulated CCN2 synthesis in BMFs. Pretreatment with c-Jun NH2-terminal kinase (JNK) inhibitor SP600125, p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, and activin receptor-like kinase 5 (ALK5) inhibitor SB431542 significantly reduced TGFβ1-induced CCN2 synthesis. Epigallocatechin-3-gallate (EGCG) completely blocked TGFβ1-induced CCN2 synthesis by inhibiting the phosphorylation of JNK and p38 MAPK. Prostaglandin E2 (PGE2) inhibited the TGFβ1-induced CCN2 synthesis in human fetal lung fibroblasts IMR90 but not in BMFs.ConclusionsThe TGFβ1-induced CCN2 synthesis in BMFs could be mediated by the ALK5, JNK, and p38 MAPK pathways. EGCG blocks TGFβ1-induced CCN2 by suppressing JNK and p38 in BMFs.Clinical relevanceThe exceptional signal transduction pathways of TGFβ1-induced CCN2 production in BMFs contribute to the resistance of PGE2 downregulation of CCN2 expression; therefore, the CTGF/CCN2 levels are maintained in the OSF tissues in the presence of COX-2. EGCG may serve as a useful agent in controlling OSF.

Thrombin‐stimulated connective tissue growth factor (CTGF/CCN2) production in human buccal mucosal fibroblasts: Inhibition by epigallocatechin‐3‐gallate

Connective tissue growth factor (CTGF/CCN2) is associated with many human fibrotic disorders and was found to overexpress in oral submucous fibrosis (OSF). OSF is the result of persistent chemical irritation and microtrauma to oral mucosa from areca nut. Microtrauma could lead to the release of thrombin.

Curcumin Inhibits TGFβ1-induced CCN2 via Src, JNK, and Smad3 in Gingiva

Transforming growth factor β (TGFβ) is a key regulator associated with the pathogenesis of gingival overgrowth (GO). Connective tissue growth factor (CTGF/CCN2) is overexpressed in GO tissues. CCN2 promotes and sustains fibrosis initiated by TGFβ. Previous studies have shown that JNK and Smad3 activation is required for TGFβ-induced CCN2 expressions in human gingival fibroblasts (HGFs). In this study, we have found that Src is a major signaling mediator for TGFβ-induced CCN2 expressions in HGFs. Pre-treatment with 2 Src kinase inhibitors (PP2, Src inhibitor-1) significantly reduced TGFβ1-induced CCN2 synthesis and JNK and Smad3 activation in HGFs. These results suggest that Src is an upstream signaling transducer of JNK and Smad3 with respect to TGFβ1-stimulated CCN2 expression in HGFs. We further found that curcumin significantly abrogated the TGFβ1-induced CCN2 in HGFs by inhibiting the phosphorylations of Src, JNK, and Smad3. Furthermore, curcumin inhibited TGFβ1-induced HGF migration and α-SMA expression. Curcumin potentially qualifies as a useful agent for the control of GO.

NADPH Oxidase 4 Mediates TGFβ1-induced CCN2 in Gingival Fibroblasts

Transforming growth factor β (TGFβ) plays a central role in the pathogenesis of gingival overgrowth (GO). Connective tissue growth factor (CTGF; or CCN2) is induced by TGFβ in human gingival fibroblasts (HGFs) and is overexpressed in GO tissues. CCN2 creates an environment favorable for fibrogenesis and is required for the maximal profibrotic effects of TGFβ. We previously showed that Src, JNK, and Smad3 mediate TGFβ1-induced CCN2 protein expression in HGFs. Moreover, Src is an upstream signaling transducer of JNK and Smad3. Recent studies suggested that NADPH oxidase (NOX)–dependent redox mechanisms are involved in mediating the profibrotic effects of TGFβ. In this study, we demonstrated that TGFβ1 upregulated NOX4 protein expression and increased reactive oxygen species (ROS) production in HGFs. Genetic or pharmacologic targeting of NOX4 abrogated TGFβ1-induced ROS production; Src, JNK, and Smad3 activation; and CCN2 and type I collagen protein expression in HGFs. Our results indicated that NOX4-derived ROS play pivotal roles in activating Src kinase activity leading to the activation of canonical (Smad3) and noncanonical (JNK) cascades that cooperate to attain maximum CCN2 expression. Furthermore, we demonstrated that curcumin significantly inhibited the TGFβ1-induced NOX4 protein expression in HGFs. Curcumin potentially qualifies as an agent to control GO by suppressing TGFβ1-induced NOX4 expression in HGFs.

Arecoline stimulated Cyr61 production in human gingival epithelial cells: inhibition by lovastatin.

Cyr61 is associated with growth and progression of many types of tumors and is an independent poor prognostic indicator for oral cancer patients. Areca nut (AN) chewing is the most important etiological factor in the pathogenesis of oral cancer in India and many Southeast Asian countries. Yet, the molecular mechanisms involved in the AN-induced oral cancer remain largely unknown. In this study, we show that arecoline, a main alkaloid found in AN, stimulated Cyr61 synthesis in human gingival epithelial S-G cells. Constitutive overexpression of Cyr61 protein in oral epithelial cells during AN chewing may play a role in the pathogenesis of oral cancer. ERK inhibitor PD98059, N-acetyl-L-cysteine, Rho-associated protein kinase (ROCK) selective inhibitor Y-27632 and a geranylgeranyltransferase inhibitor reduced the arecoline-stimulated levels of Cyr61 protein by ∼31%, 47%, 65% and 100%, respectively. Lovastatin also completely inhibited arecoline-induced Cyr61 synthesis and the inhibition is dose-dependent. Decreased of geranylgeranylated proteins could be the mechanism that lovastatin regulates Cyr61 synthesis and lovastatin could serve as a useful agent in controlling AN-induced oral cancer.

Thrombin Activates Latent TGFβ1 via Integrin αvβ1 in Gingival Fibroblasts

Transforming growth factor β (TGFβ) regulates cell proliferation, differentiation, migration, apoptosis, and extracellular matrix production. It also plays a pivotal role in the pathogenesis of gingival overgrowth. Thrombin is a key player in tissue repair, remodeling, and fibrosis after an injury, and it exerts profibrotic effects by activating protease-activated receptors. Connective tissue growth factor (CTGF or CCN2) modulates cell adhesion, migration, proliferation, matrix production, and wound healing. It is overexpressed in many fibrotic disorders, including gingival overgrowth, and it is positively associated with the degree of fibrosis in gingival overgrowth. In human gingival fibroblasts, we previously found that TGFβ1 induced CCN2 protein synthesis through c-jun N-terminal kinase and Smad3 activation. Thrombin stimulates CCN2 synthesis through protease-activated receptor 1 and c-jun N-terminal kinase signaling. Curcumin inhibited TGFβ1- and thrombin-induced CCN2 synthesis. In this study, we demonstrated that thrombin and protease-activated receptor 1 agonist SFLLRN induced latent TGFβ1 activation and Smad3 phosphorylation in human gingival fibroblasts. Pretreatment with a TGFβ-neutralizing antibody, TGFβ type I receptor inhibitor SB431542, and Smad3 inhibitor SIS3 inhibited approximately 86%, 94%, and 100% of thrombin-induced CCN2 synthesis, respectively. Furthermore, blocking integrin subunits αv and β1 with antibodies effectively inhibited SFLLRN-induced Smad3 phosphorylation and CCN2 synthesis and increased activated TGFβ1 levels; however, similar effects were not observed for integrins αvβ3 and αvβ5. These results suggest that protease-activated receptor 1–induced CCN2 synthesis in human gingival fibroblasts is mediated through integrin αvβ1–induced latent TGFβ1 activation and subsequent TGFβ1 signaling. Moreover, curcumin dose dependently decreased thrombin-induced activated TGFβ1 levels. Curcumin-inhibited thrombin-induced CCN2 synthesis in human gingival fibroblasts is caused by the suppression of latent TGFβ1 activation.

Arecoline stimulated early growth response-1 production in human buccal fibroblasts: suppression by epigallocatechin-3-gallate.

Early growth response‐1 (Egr‐1) protein plays an important role in many human fibrotic diseases. Areca nut chewing is the most important risk factor of oral submucous fibrosis (OSF).