Oral Squamous Cell Carcinogenesis Research Articles

HNRNPA2B1 promotes oral squamous cell carcinogenesis via m6 A-dependent stabilization of FOXQ1 mRNA stability.

Oral squamous cell carcinoma (OSCC), as a common type of oral malignancy, has an unclear pathogenesis. N6 methyladenosine (m6 A) is a reversible and dynamic process that participates in the modulation of cancer pathogenesis and development. As an m6 A recognition protein (reader), heterogeneous nuclear ribonucleoproteins A2/B1 (HNRNPA2B1) show abnormally high expression in cancers. Forkhead box Q1 (FOXQ1), an oncogenic transcription factor, controls multiple biological processes (e.g., embryonic development, cell differentiation, and apoptosis, impacting the initiation and progression of cancers by mediating signaling pathways together with epithelial-mesenchymal transition). Through the Cancer Genome Atlas database screening along with clinical and laboratory experiments, in head and neck squamous cell carcinoma, we found a correlation between HNRNPA2B1 and FOXQ1 gene expression, with shared m6 A motifs between HNRNPA2B1 and FOXQ1 mRNA sequences. Silencing or overexpression of HNRNPA2B1 in OSCC cells affected the malignant phenotypes of OSCC cells in vitro, and depletion of HNRNPA2B1 retarded tumor growth in vivo. HNRNPA2B1 could bind to m6 A-modified FOXQ1 mRNA to enhance its mRNA stability, resulting in up-regulation of FOXQ1 protein expression. To conclude, HNRNPA2B1 was upregulated in OSCC and enhanced OSCC cell malignant phenotypes by stabilizing m6 A-modified FOXQ1 mRNA, eventually aggravating the malignancy and tumorigenicity of OSCC. This study accelerates the recognition of the potency of m6 A modification in OSCC and paves the path for OSCC's targeted diagnosis and therapy.

Molecular mechanisms underlying the epigallocatechin-3-gallate-mediated inhibition of oral squamous cell carcinogenesis

ObjectivesTo reveal the mechanisms underlying the epigallocatechin-3-gallate (EGCG)-mediated inhibition of carcinogenesis and the related regulatory signaling pathways. DesignThe effect of EGCG on the proliferation of OSCC cells was examined. SuperPred, ChEMBL, Swiss TargetPrediction, DisGeNET, GeneCards, and National Center for Biotechnology Information databases were used to predict the EGCG target genes and oral leukoplakia (OL)-related, oral submucosal fibrosis (OSF)-related, and OSCC-related genes. The binding of EGCG to the target proteins was simulated using AutoDock and PyMOL. The Cancer Genome Atlas (TCGA) dataset was subjected to consensus clustering analysis to predict the downstream molecules associated with these targets, as well as their potential functions and pathways. ResultsEGCG significantly inhibited OSCC cell proliferation (p < 0.001). By comparing EGCG target genes with genes linked to oral potentially malignant disorder (OPMD) and OSCC, a total of eleven potential EGCG target genes were identified. Furthermore, EGCG has the capacity to bind to eleven proteins. Based on consensus clustering and enrichment analysis, it is suggested that EGCG may hinder the progression of cancer by altering the cell cycle and invasive properties in precancerous lesions of the oral cavity. Some possible strategies for modifying the cell cycle and invasive properties may include EGCG-mediated suppression of specific genes and proteins, which are associated with cancer development. ConclusionsThis study investigated the molecular mechanisms and signaling pathways associated with the EGCG-induced suppression of OSCC. The identification of specific pharmacological targets of EGCG during carcinogenesis is crucial for the development of innovative combination therapies involving EGCG.

Sprouty 4 expression in human oral squamous cell carcinogenesis

Background/purposeReviewing literature, sprouty 4 (SPRY4) has not been studied in human oral squamous cell carcinomas (OSCCs). The study aimed to examine SPRY4 expression in human oral squamous cell carcinogenesis. Materials and methodsA total of 95 OSCCs, 10 OPMDs with malignant transformation (MT), 17 OPMDs without MT, and six normal oral mucosa (NOM) samples were recruited for immunohistochemical staining; three OSCC tissues with normal tissue counterpart NOM were employed for Western blotting. Three human oral cancer cell lines (OCCLs), an oral precancer cell line (dysplastic oral keratinocyte, DOK), and a primary culture of normal oral keratinocytes (HOK) were used for Western blotting; OCCLs and HOK were employed for real-time quantitative reverse transcription-polymerase chain reaction. OCCLs were evaluated in terms of proliferation, migration, and invasion assays. ResultsSPRY4 protein expression was significantly increased in OSCCs compared with NOM. Protein and mRNA SPRY4 expression in OCCLs were significantly elevated compared with HOK. Significant increases in the degrees of proliferation, migration, and invasion were noted in OCCLs with SPRY4 siRNA transfection compared with those without transfection. SPRY4 protein level was increased in OPMD with MT compared to OPMD without MT. SPRY4 protein was significant increase in DOK in comparison with HOK. SPRY4 protein expression was significantly increased from NOM and OPMD without MT to OSCC. SPRY4 protein expression in OCCLs was significantly enhanced compared with DOK and HOK respectively. ConclusionOur results indicate that SPRY4 expression is possibly involved in human oral squamous cell carcinogenesis.

Overexpression of transient receptor potential melastatin 6 during human oral squamous cell carcinogenesis

Background/purposeTransient receptor potential melastatin (TRPM) channel is involved in cell proliferation and cell survival. Eight members (TRPM1–8) are within the TRPM subfamily. The current study is aimed to investigate TRPM6 expression in human oral carcinogenesis. Materials and methodsSixty-six oral squamous cell carcinomas (OSCCs), 47 oral potentially malignant disorders (OPMD) with moderate-severe epithelial dysplasia (ED), 28 OPMD with mild ED, and 33 normal oral mucosa (NOM) samples were subjected to immunohistochemical staining. Two human oral cancer cell lines (OCCLs), an oral premalignant cell line (DOK), and a normal oral keratinocyte culture (HOK) were used for Western blot analysis. OCCLs were evaluated for proliferation, migration, invasion assays, and intracellular calcium concentration. ResultsTRPM6 protein expression in OSCC was significantly increased as compared with normal samples. Protein expression of TRPM6 in OCCLs was significantly higher as compared with HOK. Significant decreases in degrees of proliferation, migration, invasion, and intracellular calcium concentration were noted in OCCLs with TRPM6 siRNA transfection as compared with those without transfection. Significantly increased TRPM6 protein level was noted in OPMD with moderate-severe ED as compared with those with mild ED. ConclusionOur results implicate that TRPM6 overexpression is potentially related to human oral carcinogenesis.

Expression of Orai1 and STIM1 in human oral squamous cell carcinogenesis

Background/purposeReturn of Ca2+ to endoplasmic reticulum is mediated by Orai/STIM-mediated store-operated Ca2+ entry (SOCE) channel. We aimed to investigate Orai1 and STIM1 expressions in human oral carcinogenesis. Materials and methodsSixty-six oral squamous cell carcinomas (OSCCs), 14 oral potentially malignant disorders (OPMD) with moderate-severe oral epithelial dysplasia (OED), 19 OPMD with mild OED, and 14 normal oral mucosa (NOM) samples were subjected to immunohistochemical staining. Two human oral cancer cell lines (OCCLs), an oral premalignant cell line (DOK), and a normal oral keratinocyte culture (HOK) were used for Western blot and real-time quantitative reverse transcription-polymerase chain reaction. OCCLs were evaluated for proliferation, migration, and invasion assays. ResultsOrai1 and STIM1 protein and mRNA expressions in OSCC were significantly enhanced as compared with normal samples. Protein expressions of Orai1 and STIM1 in OCCLs were significantly enhanced as compared with HOK. Significant decreases in degrees of proliferation, migration and invasion were noted in OCCLs with Orai1 and STIM1 siRNA transfection as compared with those without transfection. Significantly increased Orai1 and STIM1 protein levels were noted in OPMD with moderate-severe OED as compared with those with mild OED. ConclusionOur results indicate that Orai1 and STIM1 overexpression is associated with human oral carcinogenesis.

Kirenol regulates the cell proliferative and inflammatory markers in DMBA-induced oral squamous cell carcinogenesis in hamster.

This present findings hypothesized the modulatory effects of kirenol on expression pattern of cell proliferative and inflammatory markers during DMBA induced HBP carcinogenesis. The machinery pathways for chemomodulatory effect of kirenol was investigated by analyzing the levels of antioxidants histological changes, lipid peroxidation and molecular expression pathway of PCNA, NF-κB in the DMBA only painted HBPC. Oral cancer was developed in the HBP model by DMBA (0.5%) three times a week for 14th weeks. We analyzed body weight with deregulated molecular expressions pattern of PCNA and NF-κB was noticed in the DMBA induced hamsters compared to control hamsters. Oral administration of kirenol 30 mg/kg bw, to DMBA induced hamster models reverted the activity of the biochemical markers in Group 4. Besides, tumor tissues of hamsters receive antioxidant capability from kirenol exclaimed significant modifications in DMBA induced causes: inhibits cell proliferation (inhibits PCNA expression) and suppresses inflammation (decreased NF-κB expression) of markers. Taken together, the protective effect of that kirenol an augmenting inflammation of the started cells and exhibited antiproliferative, anti-inflammatory, antilipid peroxidative and restores the xenobiotic enzymes levels (phase I and II) system and enhances antioxidant properties in oral carcinoma hamsters, in which turn, is reflected diminished tumor burden, volume, and multiplicity.

PLAC8 inhibits oral squamous cell carcinogenesis and epithelial-mesenchymal transition via the Wnt/β-catenin and PI3K/Akt/GSK3β signaling pathways.

Placenta-specific 8 (PLAC8) is closely associated with the proliferation, apoptosis and autophagy of several tumor cells. However, the expression and function of PLAC8 in oral squamous cell carcinoma (OSCC) remain unknown. Therefore, the present study investigated the function and mechanism of PLAC8 in OSCC. Reverse transcription-quantitative PCR and western blot analyses were performed to quantify the expression of PLAC8 in OSCC cell lines. The function of PLAC8 in OSCC was investigated via transfection, the Transwell and Cell Counting Kit-8 assays, immunofluorescence staining and western blotting. The results demonstrated that PLAC8 exspression was downregulated in OSCC cell lines. PLAC8 inhibited the cell proliferation in OSCC. In addition, PLAC8 restrained invasion and epithelial-mesenchymal transition of OSCC cells. Furthermore, β-catenin helped to repress PLAC8 expression by regulating the Wnt/β-catenin and PI3K/Akt/GSK3β signaling pathways in OSCC cells. Collectively, the results of the present study suggest that PLAC8 acts as a tumor suppressor in OSCC by downregulating β-catenin.

Overexpression of sprouty 1 protein in human oral squamous cell carcinogenesis

Background/purposeSprouty (SPRY) has four isoforms, SPRY1–4, and its deficiency produces haphazard ‘sprouting’ of tracheal tubules. This study investigated SPRY1 protein expression in human oral potentially malignant disorders (OPMDs) and oral squamous cell carcinomas (OSCCs).Materials and methods90 OSCCs, 10 OPMDs with malignant transformation (MT), 17 OPMDs without MT, and six normal oral mucosa (NOM) tissue samples were subjected to immunohistochemical staining. Three human oral cancer cell lines (OCCLs), an oral precancer cell line (DOK), and a primary culture of normal oral keratinocytes (HOK) were used for western blotting.ResultsSignificantly increased expression of SPRY1 protein from NOM and OPMD without MT to OSCC was observed. The protein expressions of SPRY1 in OCCLs were significantly enhanced as compared with DOK and HOK. Increased phosphor/total-ERK expression was observed in OCCLs as compared with HOK. A significantly increased SPRY1 protein level was noted in OPMDs with MT as compared with those without MT, in addition to a significant increase in DOK in comparison with HOK.ConclusionOur results indicated that overexpression of SPRY1 protein is potentially associated with human oral squamous cell carcinogenesis.

Specific Deletion of p16INK4a with Retention of p19ARF Enhances the Development of Invasive Oral Squamous Cell Carcinoma

The cyclin-dependent kinase inhibitor 2A (CDKN2A)/alternate reading frame (ARF) locus consists of two overlapping tumor suppressor genes, p16INK4a and p14ARF (p19ARF in mice), encoding two unrelated proteins in alternative reading frames. Previous reports suggest that p16INK4a and p14ARF alterations independently exhibit differential roles, and p16INK4a is more closely associated with a poor prognosis in oral cancer. However, the role of p16INK4a-specific loss in oral squamous cell carcinogenesis remains unclear. The authors assessed chemical carcinogen 4-nitroquinoline 1-oxide (4NQO)-induced multistep oral squamous cell carcinogenesis in mice carrying p16INK4a-specific loss with retention of the p19ARF gene (p16INK4a-/-). 4NQO-treated p16-/- mice exhibited a higher incidence and multiplicity of oral squamous cell carcinoma (OSCC) development relative to 4NQO-treated wild-type mice. 4NQO-treated p16INK4a-/- OSCC cells exhibited higher proliferation and up-regulation of Arf, transcription factor E2f1, tumor protein p63 (tp63), and oncogenic ΔNp63, an isoform p63, compared with observations in 4NQO-treated wild-type OSCC cells. Furthermore, the overexpression of oncogenic ΔNp63 was associated with human OSCC. In conclusion, these results in mice indicate the biological significance of p16INK4a-specific loss with retention of p19ARF in oral squamous cell carcinogenesis, and ΔNp63 may be a potential target for OSCC.

Role of atypical chemokine receptor ACKR2 in experimental oral squamous cell carcinogenesis

BackgroundChemokines and chemokine receptors are critical in oral tumourigenesis. The atypical chemokine receptor ACKR2 is a scavenger of CC chemokines controlling the availability of these molecules at tumour sites, but the role of ACKR2 in the context of oral carcinogenesis is unexplored. MethodsIn this study, wild-type (WT) and ACKR2 deficient mice (ACKR2−/−) were treated with chemical carcinogen 4-nitroquinoline-1-oxide (4NQO) for induction of oral carcinogenesis. Tongues were collected for macro and microscopic analysis and to evaluate the expression of ACKRs, CC chemokines and its receptors, inflammatory cytokines, angiogenic factors, adhesion molecules and extracellular matrix components. ResultsAn increased expression of ACKR2 in squamous cell carcinoma (SCC) lesions of 4NQO-treated WT mice was observed. No significant differences were seen in the ACKR1, ACKR3 and ACKR4 mRNA expression comparing SCC lesions from WT and ACKR2−/− treated mice. Significantly higher expression of CCL2, IL-6 and IL-17 was detected in ACKR2−/− treated mice. In contrast, the expression of other CC-chemokines, and receptors, angiogenic factors, adhesion molecules and extracellular matrix components were similarly increased in SCC lesions of both groups. Clinical and histopathological analysis revealed no differences in inflammatory cell recruitment and in the SCC incidence comparing WT and ACKR2−/− treated mice. ConclusionThe results suggest that ACKR2 expression regulates inflammation in tumour-microenvironment but the absence of ACKR2 does not impact chemically-induced oral carcinogenesis.

Promotion of cell proliferation by the proto-oncogene DEK enhances oral squamous cell carcinogenesis through field cancerization.

Oral squamous cell carcinoma (OSCC) develops through a multistep carcinogenic process involving field cancerization. The DEK gene is a proto‐oncogene with functions in genetic and epigenetic modifications, and has oncogenic functions, including cellular proliferation, differentiation, and senescence. DEK overexpression is associated with malignancies; however, the functional roles of DEK overexpression are unclear. We demonstrated that DEK‐expressing cells were significantly increased in human dysplasia/carcinoma in situ and OSCC. Furthermore, we generated ubiquitous and squamous cell‐specific doxycycline (DOX)‐inducible Dek mice (iDek and iDek‐e mice respectively). Both DOX+ iDek and iDek‐e mice did not show differences in the oral mucosa compared with DOX‐ mice. In the environment exposed to carcinogen, DOX‐treated (DOX+) iDek mice showed field cancerization and OSCC development. Microarray analysis revealed that DEK overexpression was mediated by the upregulation of DNA replication‐ and cell cycle‐related genes, particularly those related to the G 1/S transition. Tongue tumors overexpressing DEK showed increased proliferating cell nuclear antigen and elongator complex protein 3 expression. Our data suggest that DEK overexpression enhanced carcinogenesis, including field cancerization, in OSCC by stimulating the G 1/S phase transition and promoting DNA replication, providing important insights into the potential applications of DEK as a target in the treatment and prevention of OSCC.

Blueberry inhibits invasion and angiogenesis in 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral squamous cell carcinogenesis in hamsters via suppression of TGF-β and NF-κB signaling pathways

Aberrant activation of oncogenic signaling pathways plays a pivotal role in tumor initiation and progression. The purpose of the present study was to investigate the chemopreventive and therapeutic efficacy of blueberry in the hamster buccal pouch (HBP) carcinogenesis model based on its ability to target TGF-β, PI3K/Akt, MAPK and NF-κB signaling and its impact on invasion and angiogenesis. Squamous cell carcinomas were induced in the HBP by 7,12-dimethylbenz[a]anthracene (DMBA). The effect of blueberry on the oncogenic signaling pathways and downstream events was analyzed by quantitative real-time PCR and immunoblotting. Experiments with the ECV304 cell line were performed to explore the mechanism by which blueberry regulates angiogenesis. Blueberry supplementation inhibited the development and progression of HBP carcinomas by abrogating TGF-β and PI3K/Akt pathways. Although blueberry failed to influence MAPK, it suppressed NF-κB activation by preventing nuclear translocation of NF-κB p65. Blueberry also modulated the expression of the oncomiR miR-21 and the tumor suppressor let-7. Collectively, these changes induced a shift to an anti-invasive and anti-angiogenic phenotype as evidenced by downregulating matrix metalloproteinases and vascular endothelial growth factor. Blueberry also inhibited angiogenesis in ECV304 cells by suppressing migration and tube formation. The results of the present study suggest that targeting oncogenic signaling pathways that influence acquisition of cancer hallmarks is an effective strategy for chemointervention. Identification of modulatory effects on phosphorylation, intracellular localization of oncogenic transcription factors and microRNAs unraveled by the present study as key mechanisms of action of blueberry is critical from a therapeutic perspective.

Lyophilized strawberries prevent 7,12-dimethylbenz[α]anthracene (DMBA)-induced oral squamous cell carcinogenesis in hamsters

This study evaluated the ability of lyophilized strawberries to prevent 7,12-dimethylbenz[α]anthracene (DMBA)-induced tumor development in hamster cheek pouch. The experimental diet containing 5% strawberries was given to hamsters during or after DMBA treatment, or throughout the entire bioassay. Our data indicate that tumor incidence, multiplicity, volume and histologic grade of oral precancerous lesions were reduced in animals fed the experimental diet compared to those fed the control diet. We chemically identified and quantified the main phytochemicals by high performance liquid chromatography/tandem mass spectrometry. Fourteen quantified compounds were classified to three categories: anthocyanins (48.8%, w/w of the phenolics), ellagitannin/ellagic acid/ellagic acid derivates (42.9%) and flavonols (8.3%). We further elucidated the molecular mechanisms underlying preventive activities of strawberries. Our mechanistic studies show that strawberries suppress cell proliferation, angiogenesis and oncogenic signaling. Moreover, feeding 5% strawberries also significantly decreased arachidonic acid metabolism and formation of 8-hydroxydeoxyguanosine. These observations suggest that strawberries inhibit DMBA-induced oral cancer in hamsters through suppression of multiple oncogenic events, and thus may serve as alterative agents for oral cancer prevention in humans.

Epithelial Splicing Regulatory Proteins 1 (ESRP1) and 2 (ESRP2) Suppress Cancer Cell Motility via Different Mechanisms

ESRP1 (epithelial splicing regulatory protein 1) and ESRP2 regulate alternative splicing events associated with epithelial phenotypes of cells, and both are down-regulated during the epithelial-mesenchymal transition. However, little is known about their expression and functions during carcinogenesis. In this study, we found that expression of both ESRP1 and ESRP2 is plastic: during oral squamous cell carcinogenesis, these proteins are up-regulated relative to their levels in normal epithelium but down-regulated in invasive fronts. Importantly, ESRP1 and ESRP2 are re-expressed in the lymph nodes, where carcinoma cells metastasize and colonize. In head and neck carcinoma cell lines, ESRP1 and ESRP2 suppress cancer cell motility through distinct mechanisms: knockdown of ESRP1 affects the dynamics of the actin cytoskeleton through induction of Rac1b, whereas knockdown of ESRP2 attenuates cell-cell adhesion through increased expression of epithelial-mesenchymal transition-associated transcription factors. Down-regulation of ESRP1 and ESRP2 is thus closely associated with a motile phenotype of cancer cells.

Abstract 477: The dynamic changes of KLF4 in the oral squamous cell carcinogenesis

Abstract KLF4 (formerly GKLF) is a zinc-finger transcription factor expressed in the epithelia of the skin, lungs, gastrointestinal tract and several other organs. In vitro studies have suggested that KLF4 plays an important role in cell proliferation and/or differentiation. Several studies implied that KLF4 is associated with both tumour suppression and oncogenesis, but the function of KLF4 in the oral carcinogenesis has not been investigated. To address this issue we have generated an animal model of experimental tongue cancerous lesions in C57BL/6J mouse by using carcinogen 4-nitroquinoline-1-oxide (4NQO) in drinking water .And we detected the KLF4 expression in mouse tongue precancerous and cancer lesions by immunohistochemistry. Normally KLF4 was strongly expressed in the nuclei of mice tongue epithelial cells. The staining of KLF4 was gradually decreased from normal epithelial to hyperplasia to dysplasia, and markedly decreased in the squamous cell carcinomas. The results indicated that KLF4 could served as a tumour-suppressor functions in the oral carcinogenesis. Citation Format: Man Liu, Ying Su, Xinyan Zhang. The dynamic changes of KLF4 in the oral squamous cell carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 477. doi:10.1158/1538-7445.AM2014-477

Altered epigenetic regulation of homeobox genes in human oral squamous cell carcinoma cells

To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNAseq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes.

Proteomic profile in serum of hamster during DMBA-induced oral squamous cell carcinogenesis

Proteomic profile in serum of hamster during DMBA-induced oral squamous cell carcinogenesis

Epithelial to mesenchymal transition (EMT) biomarkers – E-cadherin, beta-catenin, APC and Vimentin – in oral squamous cell carcinogenesis and transformation

To investigate immunohistochemical (IHC) analysis of E-cadherin, β-catenin, APC and Vimentin for prediction of oral malignant transformation. Immunoreactivity for E-cadherin, β-catenin, APC and Vimentin were determined for 100 oral biopsies classified as normal, mild dysplasia, moderate-severe dysplasia or OSCC, using the IHC scoring or label index scoring systems. Co-expression of biomarkers and correlation with histopathological grading was analysed. Vimentin and E-cadherin results were confirmed by RT-PCR and further investigated in vitro using a novel organotypic cell invasion model based on human dermis. A trend for decreased E-cadherin expression but increased Vimentin expression that correlated with increased disease severity was observed. Epithelial β-catenin localisation shifted from being membranous to cytoplasmic/nuclear with increased histopathological grade severity. Relative to normal, APC expression was decreased for mild dysplasia but increased for OSCC. Co-expression of β-catenin, APC and Vimentin (Spearman rank correlation) suggests interdependence of these molecules and involvement of the Wnt pathway in oral malignant transformation. Relative mRNA expression of E-cadherin for dysplasia and OSCC were less than 1% of normal tissue values, and mRNA expression of Vimentin was 3.7 times higher for OSCC than normal. After 63 days of organotypic culture neoplastic oral keratinocytes (PE/CA-PJ15) lost expression of E-cadherin and gained expression of Vimentin relative to their non-invasive counterparts in the epithelium. Trends in the expression of EMT markers - E-cadherin, β-catenin, APC and Vimentin - suggest their involvement in oral carcinogenesis via Wnt pathway dysregulation. Aberrant expression of β-catenin, APC and Vimentin are potential markers of malignant transformation.

Andrographolide Inhibits Oral Squamous Cell Carcinogenesis through NF-κB Inactivation

The NF-κB family of transcription factors is essential for promoting cell proliferation and preventing cell apoptosis. We have previously shown that Andrographolide (Andro) isolated from an herbal plant, Andrographis paniculata, covalently modifies reduced cysteine62 in the oligonucleotide binding pocket of p50 for inhibition of NF-κB activation. Here we report that Andro, but not its inactive structural analog 4H-Andro, potently suppressed squamous cell carcinogenesis induced by 7,12-dimethyl-1,2-benzanthracene (DMBA) in the hamster model of cheek buccal pouch. Compared with 4H-Andro, Andro reduced phosphorylation of p65 (Ser536) and IκBα (Ser32/36) for inhibiting aberrant NF-κB activation, suppressed c-Myc and cyclin D1 expression and attenuated neoplastic cell proliferation, promoted cancerous cell apoptosis, and mitigated tumor-induced angiogenesis. Consistently, Andro retarded growth, decreased proliferation, and promoted apoptosis of Tb cells, a human tongue squamous cell carcinoma cell line, in time- and dose-dependent manners, with concomitant reduction of the expression of NF-κB targeting molecules in vitro. Our results thus demonstrate that NF-κB activation plays important roles in the pathogenesis of chemically induced squamous cell carcinoma. By inhibition of aberrant NF-κB activation, Andro treats chemically induced oral squamous cell carcinogenesis.

Expression of inhibitor of apoptosis family proteins in human oral squamous cell carcinogenesis

The purpose of this study was to determine inhibitor of apoptosis (IAP) expression, its relationship with p53, and epigenetic change in oral carcinogenesis that remain to be elucidated. We measured IAP and p53 expression in 44 oral potentially malignant disorders and their corresponding malignant-transformed oral squamous cell carcinomas (OSCCs), and in 44 other non-transformed oral potentially malignant disorders. IAP and p53 expression in 10 fresh OSCCs, together with epigenetic change of their mutation, were also determined. Normal mucosa did not express IAP/mutated p53. Oral potentially malignant disorders that underwent transformation exhibited high IAPs (>90%) and less-consistent mutated-p53 (34%) expression, whereas transformed OSCCs exhibited high IAP and mutated-p53 expression. Fresh OSCCs exhibited 80% to 100% IAP mRNA expression and 50% protein, mRNA, and p53 mutation expression. Normal tissues revealed DNA methylation of IAP, whereas cancerous tissues overexpressing IAP exhibited hypomethylation. This study showed that IAP expression is an early event in oral carcinogenesis and that epigenetic and genetic pathways are associated with IAP expression in OSCC.