The expression and significance of MRP1, LRP,TOPOIIβ, and BCL2 in tongue squamous cell carcinoma

J Oral Pathol Med (2012) 41 : 141–148 Background: Multiple drug resistance protein 1 (MRP1), lung resistance protein (LRP), topoisomerase IIβ (TOPOIIβ) and B‐cell lymphoma 2 (BCL2) are well known in the development of drug resistance in cancer cells. The aim of this study was to evaluate the relationship between them and the clinicopathological features, their expression differences between tumor tissue and experimental drug‐resistant model in tongue carcinoma. Materials and methods: Multiple drug resistance protein 1, LRP, TOPOIIβ, and BCL2 expression was examined by immunohistochemistry in specimens from radical surgeries of 65 patients with tongue carcinoma. A cisplatin‐resistance cell line, SCC‐15/cisplatin, was established from a cisplatin‐sensitive cell line, SCC‐15. A MTT‐based method was used to analyze drug potencies. Immunofluorescence was used to detect protein expression in both cell lines. Western blot was used to compare the protein expressions in specimens and SCC‐15/cisplatin cells. Results: We found higher expression of MRP1, LRP, and BCL2 and lower expression of TOPOIIβ in tongue carcinoma compared with adjacent non‐neoplastic tongue tissues (P < 0.05). In addition, MRP1 and TopoIIβ expression were significantly associated with clinical stage, lymph node metastasis and histologic grade, and LRP was significantly associated with histologic grade in the samples (P < 0.05). Finally, Western blot showed that higher expressions of MRP1, LRP, and BCL2 and lower expression of TopoIIβ were observed in SCC‐15/cisplatin cells than in clinical samples. Conclusion: Our results suggest that the high expressions of MRP1, LRP, and BCL2 and low expression of TOPOIIβ in patients with tongue carcinoma indicates that intrinsic drug resistance may exist in tongue carcinoma, and is associated with tumor differentiation and cisplatin resistance in tongue carcinoma.     

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.     

Phospholipase C‐γ1 expression correlated with cancer progression of potentially malignant oral lesions

Background: Phospholipase C‐γ1 (PLCγ1) is required for cellular migration during tumor progression and invasion of oral squamous cell carcinoma (OSCC) cells. The objective of the current study was to determine immunoexpression pattern of PLCγ1 in oral potentially malignant lesions (OPLs) and evaluate PLCγ1 usefulness as a biomarker for predicting clinical behavior in the carcinogenesis of OPL. Methods: In a retrospective follow‐up study, the expression pattern of PLCγ1 protein was determined using immunohistochemistry in samples from 68 patients, including untransformed cases (n = 38) and malignant‐transformed cases (n = 30). The corresponding post‐malignant lesions (OSCCs) were also performed. Results: We observed that elevated expression of PLCγ1 in 40 of 68 (59%) general OPLs and 23 of 30 (77%) OSCCs compared with that in normal oral mucosa. Kaplan–Meier analysis revealed that patients with PLCγ1 positivity had a significantly higher incidence of OSCC than those with PLCγ1 negativity. Cox regression analysis revealed that PLCγ1 expression patterns were significantly associated with increased risk of malignant progression. In addition, the correlation between PLCγ1 expression in pre‐malignant OPL and that in post‐malignant OSCC was significant (P = 0.004). Conclusion: These data indicate that PLCγ1 expression in OPL correlated with oral cancer progression, and PLCγ1 may serve as a useful marker for the identification of high‐risk OPL into OSCC.     

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.     

The role of α9β1 integrin in modulating epithelial cell behaviour

J Oral Pathol Med (2011) 40 : 755–761 Background: Integrins initiate signalling in response to the extracellular matrix (ECM), which is important in wound healing and cancer. Previous studies have shown that over‐expression of the αvβ6 integrin in oral squamous cell carcinoma (OSCC) cells results in enhanced motility and expression of matrix‐degrading proteases, and the aim of this study was to investigate whether this is also the case for the α9β1 integrin. Methods: H357 OSCCcells were transfected with the α9 integrin subunit and proliferation, adhesion and migration assays were performed on these along with null vector control and wild‐type cells. The effect of ligand engagement on matrix metalloproteinase expression and the plasminogen activator system was measured using ELISA and chromogenic assays. Expression of α9 integrin was examined in oral squamous cell carcinoma tissue by immunohistochemistry. Results: Functionally active α9 integrin mediated specific upregulation of adhesion and migration towards the TNfn3RAA fragment of tenascin‐C but reduced proliferation. Migration towards collagen I was also enhanced in transfected cells. Matrix metalloproteinase‐2 and metalloproteinase‐9 expression was increased upon TNfn3RAA ligand engagement. Cell surface plasmin generation was also enhanced in α9‐expressing cells and was the result of enhanced expression of urokinase receptor. In normal oral mucosa, α9 integrin expression was restricted to the suprabasal and prickle cell layers, and expression was heterogeneous in tumours but present in islands infiltrating connective tissue particularly in moderately and well‐differentiated lesions. Conclusions: The α9β1 integrin may play a key role in modulation of tumour behaviour including enhanced cell migration and expression of matrix‐degrading proteases.     

Matrix metalloproteinase 7 and perlecan in oral epithelial dysplasia and carcinoma in situ: an aid for histopathologic recognition of their cell proliferation centers

Background:  As one of the valuable tools for differential diagnoses of oral epithelial dysplasia, carcinoma in situ (CIS) and squamous cell carcinoma (SCC), we have proposed the immunohistochemistry for perlecan, a heparan sulfate proteoglycan (HSPG). As HSPGs have been shown to be extracellular docking molecules for matrix metalloproteinase (MMP) 7, our aim was to determine the expression mode of MMP-7 in these lesions for its possible diagnostic aid for oral borderline malignancies.
Methods:  Twenty cases each of moderate dysplasia, CIS, SCC, and normal/hyperplastic/mild dysplastic epithelia of the tongue and buccal mucosa were immunohistochemically examined for MMP-1, -2 and -7 in reference to their perlecan immunolocalization.
Results:  The expression of all three MMPs in the normal mucosal epithelium was restricted mainly to the parabasal layers. The most striking finding was strong expression of MMP-7 in epithelial dysplasia with a two-phase appearance: a clear demarcation of MMP-7-immunopositive (+) lower dysplastic/basaloid cells from non-positive upper keratinized cells. MMP-7+ cells were spread over the whole epithelial layer of CIS. In SCC, MMP-7 positivity was reduced from carcinoma cells but instead appeared in stromal cells. These expression profiles of MMP-7 resembled those of perlecan. MMP-1 and MMP-2 exhibited a similar but much weaker staining than MMP-7.
Conclusion:  These results suggest that the enhanced metabolism of perlecan associated with MMP-7 plays an important role in the cell proliferation of oral epithelia in their malignant transformation process, and that MMP-7 immunohistochemistry may be a valuable aid for identification of the cell proliferation center in oral CIS and dysplasia.     

Porphyromonas gingivalis inhibits M2 activation of macrophages by suppressing α‐ketoglutarate production in mice

Reprograming of metabolic pathways is critical in governing the polarization of macrophages into classical proinflammatory M1 or alternative anti‐inflammatory M2 phenotypes in metabolic diseases, such as diabetes. Porphyromonas gingivalis, a keystone pathogen of periodontitis, causes an imbalance in M1/M2 activation, resulting in a hyperinflammatory environment that promotes the pathogenesis of periodontitis. However, whether P. gingivalis infection modulates metabolic pathways to alter macrophage polarization remains unclear. Bone‐marrow‐derived macrophages (BMDMs) were collected from 6‐week‐old female C57BL/6 mice and stimulated with P. gingivalis, P. gingivalis‐derived LPS or IL‐4. Relative gene expression and protein production were measured by quantitative real‐time PCR, RNA sequencing and western blotting. Colorimetric assays were also performed to assess the amounts of α‐ketoglutarate (α‐KG) and succinate. P. gingivalis or P. gingivalis‐derived LPS‐induced inflammatory responses enhanced M1 macrophages and suppressed M2 macrophages, even in the presence of IL‐4. P. gingivalis inhibited Idh1/2 and Gpt1/2 mRNA expression, and increased Akgdh mRNA expression, thus decreasing the ratio of α‐KG/succinate. Supplementation of cell‐permeable dimethyl‐α‐KG dramatically restored M2 activation during P. gingivalis infection. Our study suggests that P. gingivalis maintains a hyperinflammatory state by suppressing the production of α‐KG by M2 macrophages.     

Ras gene mutation is not related to tumour invasion during rat tongue carcinogenesis induced by 4‐nitroquinoline 1‐oxide

J Oral Pathol Med (2011) 40 : 325–333 Background: The aim of this study was to investigate whether mutations in the genes H‐ras and K‐ras were related to the mechanism of invasion as a result of the immunoexpression of H‐Ras, Ki‐67, alpha‐smooth muscle actin (SMA) and vascular endothelial growth factor (VEGF) during 4‐nitroquinoline 1‐oxide (4NQO)‐induced rat tongue carcinogenesis. Methods: Male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution through their drinking water for 4, 12 and 20 weeks. Ten animals were used as negative control. Results: Although no histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure, Ki‐67 was overexpresssed in the ‘normal’ oral epithelium. In pre‐neoplastic lesions at 12 weeks following carcinogen exposure, the levels of Ki‐67 were increased (P < 0.05) when compared to negative control. Ki‐67, alpha‐SMA and VEGF were also overexpressed in squamous cell carcinomas induced after 20 weeks of treatment with 4NQO. No significant statistical differences (P > 0.05) were found in H‐ras protein expression for all experimental periods evaluated that corresponded to normal oral mucosa, hyperplasia, dysplasia and squamous cell carcinomas. In the same way, no mutations in H‐ras or K‐ras genes were found. Conclusions: Our results support the idea that expression of Ki‐67 plays a crucial role during malignant transformation being closely related to neoplastic conversion of the oral mucosa cells. However, it seems that mutations in the ras genes are not involved to experimental tongue carcinogenesis induced by 4NQO.     

Molecular mimicry of Aggregatibacter actinomycetemcomitans with β2 glycoprotein I

Introduction: β2‐Glycoprotein I (β2GPI) is important in the suppression of coagulation, and antibodies against TLRVYK peptides on the β2GPI molecule are related to thrombosis. According to the Swiss‐Prot database, Aggregatibacter actinomycetemcomitans leukotoxin c has sequences (SIRVYK) that are homologous to the TLRVYK peptides. The aim of this study was to investigate the effects of A. actinomycetemcomitans infection on the antibody response against SIRVYK peptides in patients with periodontitis. Methods: Serum immunoglobulin G (IgG) antibody and IgG subclass antibody titers against SIRVYK or TLRVYK peptides were measured by enzyme‐linked immunosorbent assay in 46 patients with aggressive periodontitis (eight with localized disease, 38 with generalized disease), 28 patients with chronic periodontitis, and 20 periodontally healthy subjects. The presence of A. actinomycetemcomitans in plaque and saliva samples was determined using polymerase chain reaction. Results: The level of anti‐SIRVYK antibodies was significantly higher in patients who were A. actinomycetemcomitans‐positive than in A. actinomycetemcomitans‐negative patients (P < 0.05) in the chronic periodontitis group. A similar trend was found in the antibody response to TLRVYK peptide; however, no statistically significant difference was seen between A. actinomycetemcomitans‐positive and ‐negative patients. The A. actinomycetemcomitans‐positive patients displayed significantly higher levels of anti‐SIRVYK IgG2 and IgG3 antibodies than A. actinomycetemcomitans‐negative patients (P < 0.05 and P < 0.05, respectively). The level of IgG2 was highest among the four IgG subclasses and it predominantly increased in patients who were A. actinomycetemcomitans‐positive. Anti‐TLRVYK antibody levels were significantly correlated with anti‐SIRVYK IgG antibody levels. Conclusion: The results suggest that A. actinomycetemcomitans infection may elicit anti‐SIRVYK IgG antibodies and modify the anti‐TLRVYK antibody response in patients with periodontitis by molecular mimicry with β2GPI.     

Effects of interleukin‐1β and tumor necrosis factor‐α on macrophage inflammatory protein‐3α production in synovial fibroblast‐like cells from human temporomandibular joints

Background

Interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) are key mediators of the intracapsular pathological conditions of the temporomandibular joint (TMJ). Therefore, the gene expression profiles in synovial fibroblast‐like cells (SFCs) from patients with internal derangement of the TMJ were examined after they were stimulated with IL‐1β or TNF‐α to determine which genes were altered.

Methods

Ribonucleic acid was isolated from SFCs after IL‐1β or TNF‐α treatment. Gene expression profiling was performed using oligonucleotide microarray analysis. On the basis of the results of this assay, we investigated the kinetics of macrophage inflammatory protein‐3α (MIP‐3α) gene expression using PCR, and protein production in TMJ SFCs stimulated by IL‐1β or TNF‐α using an ELISA. Inhibition experiments were performed with MAPK and NFκB inhibitors. SFCs were stimulated with IL‐1β or TNF‐α after treatment with inhibitors. The MIP‐3α levels were measured using an ELISA.

Results

Macrophage inflammatory protein‐3α was the gene most upregulated by IL‐1β‐ or TNF‐α stimulation. The mRNA and protein levels of MIP‐3α increased in response to IL‐1β in a time‐dependent manner. In contrast, during TNF‐α stimulation, the MIP‐3α mRNA levels peaked at 4 h, and the protein levels peaked at 8 h. In addition, the IL‐1β‐ and TNF‐α‐stimulated MIP‐3α production was potently reduced by the MAPK and NFκB signaling pathway inhibitors.

Conclusion

Interleukin‐1β and TNF‐α increased the MIP‐3α production in SFCs via the MAPK and NFκB pathways. These results suggest that the production of MIP‐3α from stimulation with IL‐1β or TNF‐α is one factor associated with the inflammatory progression of the internal derangement of the TMJ.     

Expression of integrin α9 subunit and tenascin in oral leukoplakia, lichen planus, and squamous cell carcinoma

OBJECTIVES: Integrin alpha9 subunit is a member of beta1 integrin family and binds tenascin (TN). It is expressed by stratified squamous epithelium and may be associated with cell differentiation and growth. We studied if the expression of alpha9 integrin and TN is altered in leukoplakia, lichen planus, and squamous cell carcinoma (SCC).METHODS: Frozen sections of tissue samples obtained from normal human keratinized (16 subjects) and non-keratinized (three subjects) oral mucosa, oral leukopakias with dysplasia (19 subjects), reticular type lichen planus (nine subjects), or oral mucosal SCC (23 subjects) were stained immunohistochemically with antibodies against alpha9 integrin and TN.RESULTS: In contrast to its most prominent localization at the cell membranes of the basal epithelial cells in the normal mucosa, alpha9 integrin was localized in a more diffuse pattern with focal loss of expression at the epithelial cell membranes in leukoplakic dysplasia, lichen planus, and SCC. In some areas of SCC, alpha9 integrin localized throughout all cell layers of the tumor epithelium. In most areas, alpha9 integrin colocalized with TN but in heavily inflamed areas there was focal loss of TN and alpha9 integrin at the basement membrane zone. CONCLUSIONS: The findings show that alpha9 integrin expression is altered in leukoplakic dysplasia, lichen planus, and SCC.     

Loss of human β‐defensin 1, 2, and 3 expression in oral squamous cell carcinoma

Introduction: Human β‐defensins (HBDs) are cationic, antimicrobial peptides produced by epithelial cells and involved in various aspects of the innate and acquired immune responses. They are expressed by oral tissues as constitutive and inducible genes. Recently, single nucleotide polymorphisms (SNPs) of β‐defensins have been correlated with increased susceptibility to certain diseases. Studies have reported altered expression of β‐defensins in cancers suggesting their involvement in carcinogenesis. The purpose of this study was to evaluate the regulation of HBD‐1 (also published as DEFB1), HBD‐2 (DEFB4) and HBD‐3 (DEFB103A) ( http://www.genenames.org/index.html ) and HBD‐1 SNPs in oral squamous cell carcinoma cell lines (OSCC) and healthy gingival keratinocytes. Methods: β‐defensin expression was quantitatively assessed using real‐time polymerase chain reactions in OSCC and control cell lines after exposure to interleukin‐1β, tumor necrosis factor‐α, and interferon‐γ. Control data were obtained in a previous study. DNA from 19 OSCC cell lines and 44 control subjects were extracted and the HBD‐1 region spanning the 5′ untranslated region to the first intron was sequenced and analysed for SNP identification and distribution. Results: HBD‐1 and HBD‐2 basal messenger RNA expression were significantly lower in OSCC. In addition, the ability to be induced was significantly reduced in OSCC for all three β‐defensins. Four HBD‐1 SNPs were differentially distributed between cancer and control populations. Genotype distribution at the HBD‐1 locus also suggested loss of heterozygosity in OSCC. Conclusions: The genetic variation observed in OSCC compared with that in control cell lines may account for differences in β‐defensin expression. These results suggest a putative role for β‐defensins in carcinogenesis and indicate that β‐defensins may be useful markers of OSCC.     

Experience with anti‐TNF‐α therapy for orofacial granulomatosis

J Oral Pathol Med (2011) 40 : 14–19 Background: Orofacial granulomatosis (OFG) can be challenging to treat and experience with anti‐TNF‐α therapy is limited. We report our experience with infliximab (IFX) and adalimumab (ADA) for OFG in 14 patients, the largest reported series to date. Methods: A review of patients receiving induction and maintenance IFX for OFG +/? Crohn’s disease (CD) for active oral disease failing other therapies was performed. Clinical response defined by global physician assessment, aided by oral disease activity scores, was assessed at 2 months, 1 and 2 years. ADA was considered for patients failing IFX. Adverse events were recorded. Predictors of need for anti‐TNF‐α therapy were determined by comparison with OFG patients not requiring anti‐TNF‐α from our overall OFG database (n = 207). Results: Fourteen patients (9 men) were treated with IFX [OFG only (n = 7), OFG with CD (n = 7)]. Nine patients received concomitant immunosuppression. Median duration of treatment was 18 months. Short‐term response was achieved in 10/14 (71%) patients. Eight of 14 (57%) and 4/12 (33%) patients remained responsive at 1 and 2 years, respectively. Two patients who failed IFX responded to ADA. Factors predicting need for anti‐TNF‐α therapy were oral sulcal involvement, intestinal CD and a raised C‐reactive protein (CRP). Oral sulcal involvement predicted response at 1 and 2 years. Intestinal CD did not predict response. The only significant adverse event was an IFX infusion reaction. Conclusion: IFX provided good short‐term response for most OFG patients; however, a significant proportion lost response long term. Adverse events were uncommon. Patients failing IFX may respond to ADA.     

Clinicopathological and immunohistochemical features of oral spindle cell carcinoma

J Oral Pathol Med (2010) 39 : 335–341 Background: Oral spindle cell carcinoma (SpCC) is a rare variant of oral squamous cell carcinoma (SCC). The aims of this study were to compare the clinicopathologic and immunohistochemical features of oral SpCC with conventional oral SCC. Methods: Five cases of oral SpCC and 10 cases of oral SCC (five well‐differentiated and five poorly differentiated) were evaluated through conventional hematoxylin and eosin staining and immunohistochemical reactions to cytokeratins (CK), vimentin, desmin, smooth muscle actin, muscle‐specific actin, S‐100 protein, epithelial membrane antigen (EMA), p53, and ki‐67. Results: Oral SpCC showed predilection for males on their sixth decade of life, presenting clinically as painful infiltrative ulcers or ulcerated exophytic polypoid masses, preferably located on the alveolar mucosa. Mesenchymal markers were expressed in the spindle cell but not in the carcinomatous component of SpCC, and it was negative in all SCC. CKs AE1/AE3, 6, 14, and EMA were positive on both carcinomatous and spindle cell components of most SpCCs. These tumors also presented higher p53 and ki‐67 expression and no CK 1 expression in contrast to well‐differentiated SCC. Conclusion: Oral SpCC presented a different clinical profile than conventional SCC and histopathologic features and p53 and ki‐67 expression closer to poorly differentiated SCC. Besides mesenchymal markers, CK AE1/AE3, 6, 14, and EMA expression on spindle cells may be useful as an adjunct on microscopical differential diagnosis of SpCC.     

Short telomeres in an oral precancerous lesion: Q-FISH analysis of leukoplakia

J Oral Pathol Med (2012) 41 : 372–378 Objectives: A precancerous condition is a lesion that, if left untreated, leads to cancer or can be induced to become malignant. In the oral region, leukoplakia is a lesion that has been regarded as precancerous. In cases of oral carcinoma, we have frequently noticed that a type of leukoplakia histologically demonstrating hyper‐orthokeratosis and mild atypia (ortho‐keratotic dysplasia; OKD) is often associated with carcinoma, either synchronously or metachronously. Therefore, we consider OKD‐type leukoplakia to be a true precancerous lesion. Materials and Methods: In an attempt to clarify the relationship between OKD as a precancerous condition in the oral mucosa and telomere length, we estimated telomere lengths in this type of leukoplakia using quantitative fluorescence in situ hybridization, and also quantified the frequency of anaphase–telophase bridges (ATBs) in comparison with squamous cell carcinoma in situ (CIS) and the background tissues of CIS and OKD. Results: Ortho‐keratotic dysplasia was frequently associated with squamous cell carcinoma (45.0%) and showed significantly shorter telomeres than normal control epithelium, CIS, or the background of CIS or OKD. The frequency of ATBs was much higher in OKD than in control epithelium or CIS. Conclusion: Ortho‐keratotic dysplasia appears to be frequently associated with carcinoma, chromosomal instability, and excessively shortened telomeres, not only in the lesion itself but also in the surrounding background. Therefore, when this type of leukoplakia is recognized in the oral region, strict follow‐up for oral squamous cell carcinoma is necessary, focusing not only on the areas of leukoplakia, but also the surrounding background.