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.     

MDM2 expression in areca quid chewing-associated oral squamous cell carcinomas in Taiwan

MDM2 (murine double minute gene 2) overexpression has been implicated in the pathogenesis of human tumors via inhibition of the p53 tumor suppressor protein. To investigate the potential involvement of MDM2 overexpression in the pathogenesis of oral squamous cell carcinomas (SCCs) in Taiwan, we examined the expression of MDM2 protein and its relationship to p53 protein levels in 52 oral SCCs using antibodies to MDM2 and p53. Of the 52 patients, 36 (69 %) had tumors with positive MDM2 nuclear staining and 32 (61%) had tumors with p53 nuclear staining. Co-expression of MDM2 protein and p53 was detected in 25 (48%) cases; and 9 (17%) tumors showed neither MDM2 protein nor p53 staining. A significant correlation was observed between MDM2 protein and p53 expression in 38 cases with an areca quid (AQ) chewing habit (P=0.032). No significant correlation was found between the degree of MDM2 protein staining and the patients' ages, sex, cancer location, clinical staging, primary tumor TNM status or histological differentiation of SCC at the time of initial presentation. Kaplan-Meier analysis showed that either MDM2 protein expression or co-expression of p53 and MDM2 protein did not relate significantly to patient overall survival. Nevertheless, the high prevalence of MDM2 protein overexpression found in this study suggest that MDM2 may also participate in the carcinogenesis of AQ chewing-associated oral SCCs in Taiwan.     

Hypoxia inducible factor‐1α expression in areca quid chewing‐associated oral squamous cell carcinomas

Oral Diseases (2010) 16 , 696–701 Objectives: Hypoxia inducible factor (HIF)‐1α gene expression is mainly induced by tissue hypoxia. Overexpression of HIF‐1α has been demonstrated in a variety of cancers. The aim of this study was to compare HIF‐1α expression in normal human oral epithelium and areca quid chewing‐associated oral squamous cell carcinoma (OSCC) and further to explore the potential mechanisms that may lead to induce HIF‐1α expression. Methods: Twenty‐five OSCC from areca quid chewing‐associated OSCC 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, glutathione precursor N‐acetyl‐l ‐cysteine (NAC), AP‐1 inhibitor curcumin, extracellular signal‐regulated protein kinase inhibitor PD98059, and protein kinase C inhibitor staurosporine were added to find the possible regulatory mechanisms. Results: Hypoxia inducible factor‐1α expression was significantly higher in OSCC specimens than normal specimen (P < 0.05). Arecoline was found to elevate HIF‐1α expression in a dose‐ and time‐dependent manner (P < 0.05). The addition of NAC, curcumin, PD98059, and staurosporine markedly inhibited the arecoline‐induced HIF‐1α expression (P < 0.05). Conclusions: Hypoxia inducible factor‐1α expression is significantly upregulated in areca quid chewing‐associated OSCC and HIF‐1α expression induced by arecoline is downregulated by NAC, curcumin, PD98059, and staurosporine.     

Elevated expression of cyclooxygenase (COX)-2 in oral squamous cell carcinoma – evidence for COX-2 induction by areca quid ingredients in oral keratinocytes

BACKGROUND: Elevated expression of cyclooxygenase (COX)-2 has been demonstrated in several human cancers. Whether COX-2 is up-regulated in areca quid (AQ) related oral squamous cell carcinoma (OSCC) is unknown and the potential of AQ ingredients to induce COX-2 expression has not been studied. METHODS: COX-2 expression was analyzed by immunohistochemistry and RT-PCR in oral tissues. The COX-2 mRNA and protein induction potential of AQ ingredients were analyzed by real-time RT-PCR and Western blotting in normal human oral keratinocyte (NHOK). RESULTS: COX-2 protein expression was significantly higher (P < 0.01) in OSCC (n = 27) as compared to their adjacent non-cancerous matched tissue (NCMT). COX-2 protein was nearly undetectable in control normal oral mucosa. The level of COX-2 mRNA was markedly elevated in 63% (12/19) of OSCC compared to NCMT. Hydroxychavicol induced COX-2 mRNA and protein expression in NHOK. CONCLUSIONS: COX-2 protein as well as mRNA expression were significantly enhanced in OSCC as compared to NCMT. Hydroxychavicol, a unique ingredient in AQ, induced COX-2 expression in NHOK, which highlighted early involvement of COX-2 in AQ-associated oral oncogenesis.     

Mutations of the adenomatous polyposis coli gene in areca quid and tobacco-associated oral squamous cell carcinomas in Taiwan.

BACKGROUND: Adenomatous polyposis coli (APC) gene mutations have been demonstrated not only in colorectal tumors but also in a variety of human cancers. METHODS: To elucidate the possible roles of APC gene mutations in oral squamous cell carcinomas (OSCCs), we examined 40 untreated human primary OSCCs using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing assays. RESULTS: By screening nearly one-half of the coding region (codons 279-1673, including the MCR) of the APC gene, five missense mutations and a 1-base pair deletion were detected in five (12.5%) tumors, resulting in five amino-acid substitutions or a truncation of the APC protein. All patients with APC mutations were both areca quid chewers and tobacco smokers (P = 0.049). CONCLUSIONS: These results suggest that APC mutations may also contribute to the carcinogenesis of at least some OSCCs in Taiwan, especially for the users of areca quid and tobacco.     

Expression of p53 protein correlates with decreased survival in patients with areca quid chewing and smoking-associated oral squamous cell carcinomas in Taiwan

Expression of p53 protein was examined in oral squamous cell carcinoma (SCC) from patients who were areca quid (AQ) chewers and/or tobacco smokers, using anti-p53 antibodies with an immunoperoxidase technique. Positive p53 stain was observed in 47 of 81 (58%) cases of oral SCC. p53 overexpression was found to higher in patients without AQ chewing and smoking habits than in patients with these two habits (80% vs 52%, P=0.076). No significant correlation was found between p53 expression and the patients' age, sex, cancer location, clinical staging, primary tumor TNM status, or histological differentiation of SCC. The Kaplan-Meier analysis showed that the prognosis for patients with p53-negative tumors was significantly better than that for patients with p53-positive tumors (P<0.05). A significant correlation was also observed between positive lymph node status and poor prognosis (P<0.05). These results suggest that p53 may serve as an adjuvant marker of poor survival in patients with oral SCCs in Taiwan.     

Prognostic role of p21WAF1 expression in areca quid chewing and smoking-associated oral squamous cell carcinoma in Taiwan.

BACKGROUND: Alterations in p21WAF1 protein expression have been observed in a wide variety of human cancers by immunohistochemistry, and both decreased and increased levels of p21WAF1 protein expression have been shown to correlate with poor prognosis. METHOD: To examine the relation between p21WAF1 protein expression and prognosis in oral squamous cell carcinomas (SCCs), we performed an immunohistochemical study with antip21WAF1 antibody on 43 oral SCCs. Immunostaining results were then correlated with p53 protein levels, clinicopathological parameters of the tumors and overall patient survival. RESULTS: Of the 43 patients, 31 (72%) had tumors with positive p21WAF1 nuclear staining and 27 (63%) had tumors with p53 nuclear staining. There was no significant correlation between p21WAF1 and p53 protein expressions and both mutant p53-containing oral SCCs overexpressed p21WAF1 protein. In addition, no significant correlation was found between the p21WAF1 expression and the patients' age, sex, oral habit, cancer location, or primary tumor TNM status at the time of initial presentation. The Kaplan-Meier analysis showed a significant correlation between p21WAF1 protein overexpression and poor patient overall survival (P = 0.049). When p53 and p21WAF1 were evaluated together, the 5-year overall survival was lowest in p53(+)-p21WAF1(+) patients and highest in p53(-)-p21WAF1(-) patients (P = 0.057). CONCLUSION: Combined evaluation of p21WAF1 and p53 expressions may be useful in estimating the prognosis of patients with oral SCCs in Taiwan.     

Increase in placental glutathione S-transferase in human oral epithelial dysplastic lesions and squamous cell carcinomas

This study investigated the immunohistochemical expression of human placental glutathione S-transferase (GST-φ) in the epithelium of oral premalignant and malignant lesions. Epithelial lining of normal oral mucosa, hyperplastic lesions and oral epithelium exhibiting mild dysplasia showed weak to moderate GST-φ staining. Moderate epithelial dysplasia revealed an increased antibody content while severe dysplasia. carcinoma- in-situ (CIS) and squamous cell carcinoma (SCC) demonstrated markedly increased antibody binding. The GST-φ staining was evident mainly in the cytoplasm. Severe dysplasia. CIS and SCC were also characterized by areas of cells with intensive nuclear GST-φ staining. These findings support the hypothesis that GST-φ plays a role in human oral carcinogenesis and may be used as a tumor marker for human oral premalignant and malignant lesions.     

Expression of cyclin D1 is correlated with poor prognosis in patients with areaca quid chewing-related oral squamous cell carcinomas in Tiwan

Abnormal expression of cell cycle regulatory proteins, particularly cyclin D1, has been implicated in the pathogenesis of several types of cancer. We have examined the expression of cyclin D1 in histological sections of oral squamous cell carcinomas (SCCs) using anti-cyclin D1 antibodies with an immunoperoxidase technique. Cyclin D1 nuclear staining was observed in 73 of 88 (83%) cases of oral SCC. In 54 of these 73 (74%) cases, positive cyclin D1 staining was also found in the normal appearing epithelium immediately adjacent to the cyclin D1-positive SCCs. No significant correlation was found between the expression of cyclin D1 and the patients' age, sex, oral habits, cancer location and STNM status. The Kaplan-Meier analysis showed that patients with tumors containing more than 10% cyclin D1-positive cells had significantly shorter overall survival than those with tumors containing less than 10% cyclin D1-positive cells or with cyclin D1-negative tumors (P<0.05). Patients with positive lymph node status also had significantly shorter overall survival (P<0.01). These results indicate that cyclin D1 may play an important role in the genesis of oral SCC and may serve as an adjuvant marker of worse prognosis in patients with oral SCCs in Taiwan.     

Heat shock protein expression in oral lichen planus

To assess the potential role of heat shock protein (HSP) in the pathogenesis of oral lichen planus (OLP), sections of OLP, normal oral mucosa, non-specific oral ulceration (NSOU) and dysplastic OLP were assessed for HSP expression using avidin-biotin complex immunohistochemistry with an anti-HSP 70 polyclonal antibody. There were statistically significant differences in both the vertical and horizontal staining distribution when other groups were compared with the OLP group (p<0.01). Using microdensitometry, the mean staining intensity in OLP, dysplastic OLP and NSOU was elevated in comparison with normal oral mucosa (p<0.001). In a standard tritiated thymidine uptake assay, lymphocytes extracted from nine OLP lesions demonstrated significant proliferation when stimulated with purified protein derivative (PPD), of which HSP is a major constituent, with stimulation indices ranging from 2 to 132. These results are consistent with the hypothesis that, in OLP patients, diverse exogenous agenst may cause upregulated expression of HSP by oral mucosal keratinocytes. A reaction of cytotoxic T lymphocytes to these activated keratinocytes may then result in the tissue destruction which is characteristic of OLP lesions.     

Alteration of integrin expression in oral squamous cell carcinomas

OBJECTIVE: This study examines the intensity of expression of beta 1, alpha 2, alpha 3, alpha 5, alpha 6 integrin subunits in oral squamous cell carcinoma (SCC) as opposed to normal oral epithelium, and the intensity of expression and distribution pattern of the above subunits in relation to tumour differentiation grade. MATERIALS AND METHODS: Cryostat sections of 25 cases of oral SCC and 15 cases of normal oral epithelium were studied by immunohistochemistry (APAAP method). RESULTS: The intensity of expression of beta 1, alpha 2 (Pearson chi 2 P < 0.001) and alpha 6 (Test for Trend P < 0.05) integrin subunits was reduced significantly in SCC compared to normal oral epithelium. All integrin subunits were mainly expressed in the peripheral cell layer of tumour islands. No correlation was found between the intensity of integrin expression and the degree of differentiation in SCC. The same applied to the distribution pattern of the integrin subunits. By means of cross examination of all integrins, the loss of intensity of alpha 2 beta 1 integrin expression was found to have the strongest correlation with oral SCC (Ordered Logistic Regression). CONCLUSIONS: Reduced intensity of expression of all subunits was found in oral SCC compared to normal epithelium. Further investigation is needed to determine whether alpha 2 beta 1 integrin expression can be used as a prognostic evaluator for the behaviour of the disease.     

Eotaxin expression in oral squamous cell carcinomas with and without tumour associated tissue eosinophilia

AIM: Eotaxin is a powerful and selective eosinophil chemoattractant. The purpose of this study was to compare the expression of eotaxin in oral squamous cell carcinomas with and without tumour associated tissue eosinophilia (TATE). The mechanisms that control the recruitment of eosinophils to these tumours are not clearly established. METHODS: A total of 60 patients with oral squamous cell carcinomas (OSCC) with TNM stages II and III, located in the tongue, oral floor, retromolar area and inferior gingiva were divided in two groups: 1--OSCC with intense eosinophilic inflammatory infiltrate and 2--OSCC with absent/low eosinophilic inflammatory infiltrate. The eotaxin expression was analyzed by immunohistochemistry using standard streptavidin-biotin-peroxidase complex technique with monoclonal (mouse anti-human eotaxin) and polyclonal (rabbit anti-human eotaxin) antibodies. RESULTS: The eotaxin expression was identified in normal oral mucosa as well as in both OSCC groups including malignant epithelial cells, eosinophils, neutrophils, plasma cells and fibroblasts. The eosinophils showed intense immunopositivity for eotaxin. CONCLUSION: These results suggest that the eotaxin expressed in oral squamous cell carcinomas, mainly derived from eosinophils, is probably involved in the mechanisms of eosinophils chemotaxis to the tumour and in the maintenance of TATE in these malignant tumours.     

肿瘤抑制蛋白Maspin在口腔鳞状细胞癌中的表达及意义

目的探讨Maspin蛋白在口腔鳞状细胞癌（OSCC）发生发展过程中的作用,为临床应用提供理论依据。方法采用鼠抗人Maspin单克隆抗体及SP免疫组化法检测45例OSCC、33例癌旁组织及15例正常口腔组织中Maspin蛋白的表达水平,用半定量积分法判断结果。结果在正常口腔组织、癌旁组织和OSCC组织中Maspin蛋白表达阳性率分别为86.67％（13/15）、72.73%（24/33）和37.78%（17/45）。Maspin蛋白在OSCC组织、癌旁组织和正常口腔组织中表达逐渐增高,其中OSCC组织和正常口腔组织、OSCC组织和癌旁组织、癌旁组织和正常口腔组织之间表达差异有统计学意义（P<0.05）。Maspin蛋白的表达与OSCC淋巴结转移和组织学分级有关（P＜0.05）,与TNM分期无关（P＞0.05）。结论Maspin蛋白在OSCC的发生发展过程中可能起着重要作用,检测OSCC组织中Maspin蛋白的表达水平可能有助于对淋巴结转移潜能的预测。     

Infrequent p53 mutations in patients with areca quid chewing-associated oral squamous cell carcinomas in Taiwan

Mutations in the conserved regions (exons 5-9) of the p53 gene were investigated in 37 untreated human primary oral squamous cell carcinomas (SCCs) using polymerase chain reaction-single strand conformation polymorphism and DNA sequencing analyses. P53 mutations were detected in 2 of 37 (5.4%) oral SCC cases. One tumor sample (case 23) showed a mis-sense point mutation at codon 177, changing CCC to CTC, which resulted in a substitution of proline to leucine in the p53 protein. The other tumor (case 33) had a point mutation at codon 266, changing GGA to AGA and causing a substitution of glycine to arginine in the p53 protein. These two patients with p53 mutations did not have an areca quid chewing habit. These results suggest that mutations in the p53 gene may not play a role in the pathogenesis of human oral SCCs in Taiwan. Recently, we have shown that positive p53 staining was observed in 47 of 81 (58%) cases of oral SCC. The discrepancies between positive p53 protein staining and the low prevalence of p53 mutation in oral SCCs indicate that other mechanism(s) are involved in p53 overexpression.     

Apoptosis-associated markers and clinical outcome in human oral squamous cell carcinomas

Background:  Apoptosis is a genetically regulated cell death involved in the deletion of cells in normal or malignant tissues. Proteins of the Bcl-2 family play a key role in the control of apoptosis and carry out both pro-apoptotic and anti-apoptotic functions. The present study evaluated the prognostic value of Bcl-2 and Bax expression at the invasive front of oral squamous cell carcinoma (OSCC), taking clinicopathological findings into account.
Methods:  Fifty-six specimens of OSCC were randomly selected, and Bcl-2 and Bax expression was evaluated by immunohistochemistry in formalin-fixed, paraffin-embedded pre-treated specimens at the invasive front of OSCC. Clinicopathological data were gathered and patient survival was analysed.
Results:  No significant relationship was found between Bcl-2 or Bax expression and clinical variables. Patients with Bcl-2 expression had a worse prognosis than those without Bcl-2 expression, but the difference did not reach statistical significance. Patients with Bax expression had a significantly better prognosis than those without Bax expression ( P  <   0.05). In univariate analyses, T category, mode of cancer invasion and Bax expression showed significant correlations. Multivariate analysis revealed that the mode of cancer invasion and Bax expression were significant and independent variables. Bax expression was found to be the strongest independent prognostic parameter. Patients with negative Bcl-2 expression and positive Bax expression had a significantly better prognosis ( P  <   0.005).
Conclusion:  We suggest that Bax expression at the invasive front of OSCC is a significant predictor of prognosis and that it is therefore important to investigate the expression of Bcl-2 and Bax in this disease.     

舌鳞癌细胞热处理后HSP27的表达变化

目的 观察热疗后舌鳞癌Tscca细胞中热休克蛋白27 (heat shock protein 27,HSP27)的变化及其对凋亡的作用.方法 常规培养的Tscca细胞分6组,对照组不加热,其余5组43℃水浴法热处理40 min后分别常规培养2、4、8、12、24h,应用蛋白组学方法检测HSP27变化.运用空载质粒(pcDNA3)、pcDNA3-HSP27质粒转染Tscca细胞24h,免疫印迹分析靶细胞中的HSP27表达.43℃水浴处理未转染及转染组细胞0 min、40 min,再培养24 h后流式细胞仪检测细胞凋亡率.结果 加热后12 h内细胞内HSP27持续升高.HSP27质粒转染细胞中HSP27蛋白表达增强.0 min热处理,未转染组、pcDNA3转染组及HSP27质粒转染组细胞凋亡率无差异;40min热处理,未转染组与pcDNA3转染组凋亡率无差异,HSP27质粒转染组分别与未转染组及空载质粒转染组比较,均有差异(P＜0.05).结论 热处理后舌鳞癌Tscca细胞中HSP27含量升高.HSP27的变化与Tscca细胞凋亡的变化有关.