Heat shock protein 47 expression in oral squamous cell carcinomas and upregulated by arecoline in human oral epithelial cells

J Oral Pathol Med (2010) 40 : 390–396 Background: Heat shock protein 47 (HSP47) is a product of CBP2 gene located at chromosome 11q13.5, a region frequently amplified in human cancers. Areca quid chewing is a major risk factor of oral squamous cell carcinoma (OSCC). The aim of this study was to compare HSP47 expression in normal human oral epithelium and OSCC and further to explore the potential mechanisms that may lead to induce HSP47 expression. Methods: Thirty‐two OSCC specimens and ten normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The oral epithelial cell line OC2 cells were challenged with arecoline, a major areca nut alkaloid, by using Western blot analysis. Furthermore, glutathione precursor N‐acetyl‐l ‐cysteine (NAC), extracellular signal‐regulated protein kinase (ERK) inhibitor PD98059, phosphatidylinositol 3‐kinase (PI3K) inhibitor LY294002, cyclooxygenase‐2 inhibitor NS‐398, and tyrosine kinase inhibitor herbimycin A were added to find the possible regulatory mechanisms. Results: HSP47 expression was significantly higher in OSCC specimens than normal epithelium (P < 0.05). No significant difference in HSP47 expression was observed with respect to age, sex, T category, stage, and differentiation (P > 0.05). The lower HSP47 expression was associated with lymph node metastasis (P = 0.015). Arecoline was found to elevate HSP47 expression in a dose‐ and time‐dependent manner (P < 0.05). The addition of NAC, PD98059, LY294002, NS398, and herbimycin A markedly inhibited the arecoline‐induced HSP47 expression (P < 0.05). Conclusion: Our findings demonstrated that HSP47 expression is significantly upregulated in areca quid chewing‐associated OSCCs. HSP47 could be used clinically as a marker for lymph node metastasis of oral carcinogenesis. In addition, arecoline‐induced HSP47 expression was downregulated by NAC, PD98059, LY294002, NS398, and herbimycin A.     

Heat shock protein 27 expression in areca quid chewing-associated oral squamous cell carcinomas

Oral Diseases (2012) 18 , 713–719 Objectives: Heat shock protein (HSP) 27 is a low‐molecular‐weight protein that functions as a molecular chaperone and plays a cytoprotective role through its antioxidant activity during cell stress. Areca quid chewing is associated with the high incidence of oral squamous cell carcinomas (OSCCs) in Taiwan. The aim of this study was to compare heat shock protein 27 (HSP27) expression in OSCCs and the normal oral tissues. Methods: Forty‐eight OSCCs from areca quid chewers and ten normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry for HSP27. The normal human oral keratinocytes (HOKs) were challenged with arecoline, the major alkaloid of areca nut, by Western blot for HSP27. Furthermore, epigallocatechin‐3 gallate (EGCG), glutathione precursor N‐acetyl‐l ‐cysteine (NAC), cyclooxygenase‐2 inhibitor NS‐398, HSP inhibitor quercetin, extracellular signal‐regulated protein kinase (ERK) inhibitor PD98059, and p38 inhibitor SB203580 were added to find the possible regulatory mechanisms. Results: Heat shock protein 27 exhibited higher expression in OSCCs than normal specimens (P < 0.05). Arecoline was found to elevate HSP27 expression in a dose‐ and time‐dependent manner (P < 0.05). The additions of pharmacological agents were found to inhibit arecoline‐induced HSP27 expression (P < 0.05). Conclusions: Heat shock protein 27 expression is significantly elevated in areca quid chewing‐associated OSCCs. Arecoline‐induced HSP27 expression was downregulated by EGCG, NS398, NAC, quercetin, PD98059, and SB203580.     

The Upregulation of Transglutaminase‐2 by Cyclosporin A in Human Gingival Fibroblasts Is Augmented by Oxidative Stress

Background: Transglutaminase‐2 (TGM‐2) has been implicated in several fibrotic disorders and can be induced by reactive oxygen species (ROS). Hence, the authors hypothesize that cyclosporin A (CsA) may regulate TGM‐2 via ROS, and this regulation may have a role in the pathogenesis of CsA‐induced gingival overgrowth. Methods: Cytotoxicity, 2′,7′‐dichlorodihydrofluorescein diacetate assay, and Western blot were used to investigate the effects of CsA in human gingival fibroblasts (HGFs). In addition, extracellular signal‐regulated kinase (ERK) inhibitor PD98059, phosphatidylinositol 3‐kinase inhibitor LY294002, glutathione precursor N‐acetyl‐L‐cysteine (NAC), curcumin, epigallocatechin‐3 gallate (EGCG), and p38 inhibitor SB203580 were added to find the possible regulatory mechanisms. Results: Concentrations of CsA >500 ng/mL demonstrated cytotoxicity to HGFs (P < 0.05). CsA enhanced the generation of intracellular ROS at concentrations >200 ng/mL (P <0.05). TGM‐2 protein induced by CsA was found in HGFs in a dose‐ and time‐dependent manner (P <0.05). The addition of PD98059, LY294002, NAC, curcumin, EGCG, and SB203580 markedly inhibited TGM‐2 expression induced by CsA (P <0.05). Conclusions: These results demonstrate that CsA significantly upregulates intracellular ROS generation and elevates TGM‐2 expression in HGFs. In addition, TGM‐2 induced by CsA is downregulated by PD98059, LY294002, NAC, curcumin, EGCG, and SB203580.     

Areca nut extracts enhance the development of CD11b+Gr‐1+ cells with the characteristics of myeloid‐derived suppressor cells in antigen‐stimulated mice

J Oral Pathol Med (2011) 40 : 769–777 Background: Areca quid chewing is an etiological factor contributing to the development of oral cancer and pre‐cancers, whose pathophysiology has been linked to inflammation and immune deterioration. Myeloid‐derived suppressor cells (MDSC) play a key role in the regulation of immunity under certain pathological conditions, such as inflammation and cancer. As areca nut extracts (ANE) have been reported to induce a proinflammatory effect in antigen‐stimulated mice, we hypothesized that ANE might enhance the development of MDSC. Methods: Ovalbumin (OVA)‐sensitized BALB/c mice were daily administered with ANE (5–50 mg/kg), polyphenol‐enriched ANE (PANE; 25 mg/kg) or arecoline (5 mg/kg) by intraperitoneal injection for 10 doses. The mouse footpads were then subcutaneously challenged with OVA to induce local inflammatory responses. Results: ANE and PANE treatment significantly increased the spleen index and the population of CD11b⁺Gr‐1⁺ cells in the spleen and peripheral blood, whereas arecoline was inactive. In addition, ANE and PANE treatment enhanced the expression of cytokines and enzymes associated with the immunosuppressive function of MDSC, including IL‐10, arginase‐I and iNOS in splenic CD11b⁺ cells. Concordantly, ANE and PANE treatment augmented the infiltration of Gr‐1⁺IL‐10⁺ cells in the footpads challenged with OVA. Conclusions: Our results suggested that areca nut constituents, in particular, polyphenols enhanced the development of myeloid‐derived suppressor cells in vivo, which may be a critical mechanism linking inflammation and the compromised immunity reported to be associated with the pathophysiology of areca‐related oral diseases.     

Elevated transglutaminase-2 expression mediates fibrosis in areca quid chewing-associated oral submucocal fibrosis via reactive oxygen species generation

Objectives

Transglutaminase-2 (TGM-2) protein is involved in the cross-linking of matrix proteins resulting in several fibrotic disorders and can be induced by reactive oxygen species (ROS). Little is known about its role in the development of oral submucocal fibrosis (OSF). Hence, we hypothesize that TGM-2 may have a role in the pathogenesis of areca quid chewing-associated OSF and arecoline, a major areca nut alkaloid, could regulate TGM-2 via ROS generation.

Materials

Forty OSF specimens from areca quid chewing-associated OSF and ten normal buccal mucosa biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The expression of TGM-2 from fibroblasts cultured from OSF and normal buccal mucosa was evaluated by Western blot. The effect of arecoline on normal buccal mucosa fibroblasts (BMFs) was used to elucidate whether TGM-2 expression could be affected by arecoline by using 2′, 7′-dichlorofluorescein diacetate assay and Western blot. In addition, glutathione precursor N-acetyl-l-cysteine (NAC) and epigallocatechin-3 gallate (EGCG) were added to find the possible regulatory mechanisms.

Results

TGM-2 expression was significantly higher in OSF specimens than normal specimens (p < 0.05). Fibroblasts derived from OSF were found to exhibit higher TGM-2 expression than BMFs in protein levels (p < 0.05). Arecoline significantly upregulated the intracellular ROS generation in a dose-dependent manner (p < 0.05). TGM-2 protein induced by arecoline was found in BMFs in a dose-dependent manner (p < 0.05). Treatment with NAC and EGCG markedly inhibited TGM-2 expression induced by arecoline (p < 0.05).

Conclusions

Our results suggest that TGM-2 expression is significantly upregulated in OSF tissues from areca quid chewers. Arecoline-upregulated TGM-2 expression may be mediated by ROS generation.

Clinical relevance

TGM-2 protein is upregulated in areca quid chewing-associated OSF. Using this in vitro model, antioxidants could inhibit arecoline-upregulated TGM-2 expression. NAC and EGCG may serve as a useful agent in controlling OSF.

     

Salivary arecoline levels during areca nut chewing in human volunteers

J Oral Pathol Med (2010) 39 : 465–469 Background: Arecoline stimulates cultured cells above 0.1 μg/ml and is cytotoxic above 10 μg/ml. Although this alkaloid seems important for areca nut induced oral carcinogenesis, little is known of the levels achieved during chewing. Materials and methods: Saliva was collected in 3‐ to 5‐min intervals over 50 min in 32 habitual chewers: before, for 25 min during, and for 20 min after chewing areca nut (0.5 g) without any other additives. Salivary arecoline was quantitated by HPLC‐MS. Controls comprised six subjects who denied areca nut use, and who were given rubber‐base material to chew during experiments instead. Results: Arecoline was detected before chewing in 22 subjects, exceeding the 0.1 μg/ml threshold in 20 cases. Salivary arecoline exceeded either the 0.1 or 10 μg/ml thresholds in all participants during chewing (P < 0.001). Maximum concentrations ranged from 5.66 to 97.39 μg/ml. All subjects reached 0.1 μg/ml salivary arecoline in at least 85% of time points studied (P < 0.0001), whereas 10 μg/ml was reached in 11 participants in at least 30% of the time points (P < 0.003). Arecoline concentrations varied greatly over time between individuals, and levels were much lower when peak concentrations were reached before 3 min, than in cases where arecoline peaked later (P < 0.02). No salivary arecoline was found in control saliva. Conclusions: Areca nut users have persistent background salivary arecoline levels long after chewing, whereas concentrations achieved are highly variable and consistent with a role in oral pre‐malignancy and malignancy.     

Co‐expression of Hif1α and CAIX is associated with poor prognosis in oral squamous cell carcinoma patients

J Oral Pathol Med (2010) 39 : 313–317 Background: This study investigates the prognostic impact of the expression of hypoxia‐inducible factor 1α (Hif1α) and carbonic anhydrase IX (CAIX) detected by immunohistochemistry in oral squamous cell carcinoma (OSCC). Methods: Statistical analysis of immunohistochemical results with clinical parameters including survival outcomes was performed for 80 OSCC patients. Results: Patients with a low expression of both proteins survived on average 54.8 months, whereas those with an increased expression of Hif1α in their tumors combined with a low expression of CAIX survived on average only 37.6 months (P = 0.026). In multivariate Cox’s regression hazard analysis, again patients with a low expression of Hif1α/CAIX had the best prognosis, whereas patients with increased Hif1α and low CAIX expression carried a 4.97‐fold increased risk of tumor‐related death (P = 0.042). Conclusion: A co‐detection of low Hif1α/CAIX expression is significantly correlated with a better prognosis for OSCC patients, which may have implications for therapy options for these patients.     

Synergistic effects of nicotine on arecoline-induced cytotoxicity in human buccal mucosal fibroblasts

Areca quid chewing has been linked to oral submucous fibrosis and oral cancer. Arecoline, a major areca nut alkaloid, is considered to be the most important etiologic factor in the areca nut. In order to elucidate the pathobiological effects of arecoline, cytotoxicity assays, cellular glutathione S-transferase (GST) activity and lipid peroxidation assay were employed to investigate cultured human buccal mucosal fibroblasts. To date, there is a large proportion of areca quid chewers who are also smokers. Furthermore, nicotine, the major product of cigarette smoking, was added to test how it modulated the cytotoxicity of arecoline. At a concentration higher than 50 microg/ml, arecoline was shown to be cytotoxic to human buccal fibroblasts in a dose-dependent manner by the alamar blue dye colorimetric assay (P<0.05). In addition, arecoline significantly decreased GST activity in a dose-dependent manner (P<0.05). At concentrations of 100 microg/ml and 400 microg/ml, arecoline reduced GST activity about 21% and 46%, respectively, during a 24 h incubation period. However, arecoline at any test dose did not increase lipid peroxidation in the present human buccal fibroblast test system. The addition of extracellular nicotine acted synergistically on the arecoline-induced cytotoxicity. Arecoline at a concentration of 50 microg/ml caused about 30% of cell death over the 24 h incubation period. However, 2.5 mM nicotine enhanced the cytotoxic response and caused about 50% of cell death on 50 microg/ml arecoline-induced cytotoxicity. Taken together, arecoline may render human buccal mucosal fibroblasts more vulnerable to other reactive agents in cigarettes via GST reduction. The compounds of tobacco products may act synergistically in the pathogenesis of oral mucosal lesions in areca quid chewers. The data presented here may partly explain why patients who combined the habits of areca quid chewing and cigarette smoking are at greater risk of contracting oral cancer.     

Molecular and cellular cues of diet‐associated oral carcinogenesis—with an emphasis on areca‐nut‐induced oral cancer development

In modern times, potent dietary carcinogens are key contributors for neoplastic development. For oral squamous cell carcinoma (OSCC), one of the leading cancer types in developing countries, main oncogenic inducers/enhancers, including areca nut chewing, tobacco smoking, and alcohol consumption, were shown to promote cancer initiation/progression. Over decades, studies from different laboratories have identified underlying cellular and molecular mechanisms for carcinogen‐induced OSCC. In this review, we will give an overview of where we are in understanding potential oral carcinogenic factors stimulated OSCC tumorigenesis, especially those associated with areca nut chewing in Asians, aiming to provide future scope of possible interception.     

Increased plasminogen activator inhibitor-1/tissue type plasminogen activator ratio in oral submucous fibrosis

OBJECTIVE: Plasminogen activators and their inhibitors are thought to be key participants in the balance of proteolytic and antiproteolytic activities that regulate extracellular matrix (ECM) turnover. However, little is known about the expression of plasminogen/plasmin system at the site of oral submucous fibrosis (OSF). METHODS: We compared the activities of tissue type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) between fibroblasts derived from normal buccal mucosa and OSF by using an enzyme-linked immunosorbent assay. Furthermore, arecoline, a major areca nut alkaloid, was challenged with normal buccal mucosal fibroblasts (BMFs) to elucidate whether the activities of t-PA and PAI-1 could be affected by arecoline. RESULTS: Both t-PA and PAI-1 were found to be increased in OSF than in BMFs (P < 0.01). In addition, there was a statistically significant difference in PAI-1/t-PA ratio between OSF and BMF (P < 0.01). The addition of arecoline upregulated not only PAI-1, but also t-PA in BMFs (P < 0.05). In addition, the ratio between PAI-1 and t-PA was found to be significantly increased by a linear regression assay (P < 0.01). CONCLUSION: These results suggest that OSF caused by areca quid chewing may be the result of an imbalance in the plasminogen/plasmin system, the net result of which is increased deposition of ECM.     

Impact of interleukin-8 gene polymorphisms and environmental factors on oral cancer susceptibility in Taiwan

Oral Diseases (2012) 18 , 307–314 Objectives: Interleukin‐8 (IL‐8), which is an angiogenic chemokine with a high expression level in tumor tissues, plays important roles in developing many human malignancies including oral squamous cell carcinoma (OSCC). This study was designed to examine the association of IL‐8 gene polymorphisms with the susceptibility and clinicopathological characteristics of OSCC. Methods: A total of 270 patients with OSCC and 350 healthy control subjects were recruited. Four single nucleotide polymorphisms (SNPs) of IL‐8 genes were analyzed using polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) genotyping analysis. Results: Results showed that four IL‐8 SNPs (?251 T/A, +781 C/T, +1633 C/T, and +2767 A/T) were not associated with oral cancer susceptibility as well as clinicopathological parameters. But among 345 smokers, IL‐8 polymorphisms carriers with betel quid chewing were found to have a 17.41‐ to 23.14‐fold risk to have oral cancer compared to IL‐8 wild‐type carriers without betel quid chewing. Among 262 betel quid chewers, IL‐8 polymorphisms carriers with smoking have a 10.54‐ to 20.44‐fold risk to have oral cancer compared to those who carried wild type without smoking. Conclusions: Our results suggest that the combination of IL‐8 gene polymorphisms and environmental carcinogens might be highly related to the risk of oral cancer.     

Fos‐related activator‐1 is overexpressed in oral squamous cell carcinoma and associated with tumor lymph node metastasis

J Oral Pathol Med (2010) 39 : 470–476 Background: Previously, we established an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC), including the human immortalized oral epithelia cells (HIOECs) and its derived cancerous HB cells. Then, expression microarray analysis showed that the gene encoding fos‐related activator‐1 (Fra‐1) was significantly upregulated in the cancerous HB cells compared with HIOECs. Methods: To confirm the expression of Fra‐1 at mRNA and protein levels by real‐time PCR and western blotting analysis in the carcinogenesis model of OSCC and CAL27 cells. To investigate Fra‐1 expression in clinical samples from 30 primary OSCC patients by immunohistochemistry. Results: Fra‐1 expression was increased both at mRNA and protein levels in this carcinogenesis model of OSCC and CAL27 cells. Nuclear and cytoplasmic Fra‐1 protein expressions both increased in the cancerous tissues compared with those in the paired adjacent non‐malignant epithelia (nuclear: P < 0.001, cytoplasmic: P = 0.003). A higher level of nuclear Fra‐1 expression was seen in the tumor samples of patients with lymph node metastasis than those without lymph node metastasis (5.07 ± 1.33 vs 3.81 ± 1.33, P = 0.023). Higher level of Fra‐1 expression was also found in the tumor invasive margin than tumor center. Conclusions: Fra‐1 is a positive gene of OSCC development and progression, Fra‐1 can be used as a potential therapeutic target gene and an additional marker for evaluation of lymph node metastasis.