The Neem Limonoid Nimbolide Modulates Key Components of the DNA Damage Response Signalling in Cellular and Animal Models of Oral Squamous Cell Carcinoma

BACKGROUND: Deregulated DNA damage response (DDR) network is implicated in cancer progression and therapy resistance. OBJECTIVE: The present study was designed to investigate whether nimbolide, an anticancer neem limonoid, targets key components of the DDR signalling pathway in cellular and animal models of oral squamous cell carcinoma (OSCC). METHODS: OSCC cells (SCC-4 and SCC-9), 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinoma model, chemoresistant OSCC patient-derived xenograft (PDX) model established in athymic nude mice, and tissue sections from patients with oral premalignant/malignant disease were used for the study. Key molecules that orchestrate the DDR, including the MRN complex, ATM, DNA-PKcs, H2AX, and p53, were analysed by qRT-PCR, immunoblotting, immunofluorescence, and immunohistochemistry. Cell proliferation and apoptosis indices were evaluated. RESULTS: Nimbolide significantly reduced 8-oxodG levels, expression of MRN, ATMS1891, and γ-H2AX, with an increase in p-p53S15 in OSCC cells as well as in the HBP model. Nimbolide potentiated the effect of KU-55933 in ATM inhibition. In the PDX model, nimbolide suppressed tumor formation, stimulated DDR and apoptosis, inhibited cell proliferation, and enhanced sensitivity to cisplatin. Analysis of p-ATM expression revealed a significant increase during the sequential progression of hamster and human OSCC. CONCLUSIONS: This study provides compelling evidence that nimbolide functions as a DDR inhibitor in cellular and hamster OSCC models and as a DDR activator in the PDX model primarily by targeting ATM. Small molecules like nimbolide that modulate DDR are of immense benefit in cancer therapy. The study has also unveiled p-ATM as a promising biomarker of tumour progression in human OSCCs.

Anti-cancer effects of green synthesized gold nanoparticles using leaf extract of Annona muricata. L against squamous cell carcinoma cell line 15 through apoptotic pathway.

Background: Oral cancer remains one of the most dreadful diseases in developing nations. Currently, there has been a rise in the prevalence of tongue squamous cell carcinoma (SCC), with a poor prognosis. The use of standard treatment approaches against oral cancer patients brings about several side effects. In recent years, nanomedicine has provided a versatile platform for developing new targeted therapeutic modalities. However, safety remains a concern in the synthesis of nanoparticles (NPs). Therefore, the present study aims to synthesize safer phytoconstituent-mediated gold NPs (AuNPs) utilizing leaf extracts of Annona muricata, where the biochemical components of the plant leaf act as the reducing and capping agents in the synthesis of NPs, and to evaluate its anti-cancer activity against SCC. Materials and Methods: In this in vitro experimental study, AuNPs were synthesized through an effective, simple, and ecologically sound green synthesis method. After characterization of these synthesized AuNPs, in vitro assays such as 3-(4, 5-dimethylthiazole2-yl)-2, 5-biphenyl tetrazolium bromide, wound healing, and clonogenic assays were carried out to investigate the anti-cancer potential of green synthesized AuNPs in the human tongue SCC cell line (SCC-15), and the possible mechanism of action was evaluated through gene and protein expression analysis of Bax, Bcl-2, and p53 genes. The results were expressed as mean ± standard deviation using Statistical Package for Social Sciences (SPSS) 20.0 software and Student's t-test was performed for experimental data. P ≤0.05 were considered statistically significant. Results: The in vitro assays demonstrated that the synthesized AuNPs are exhibiting anti-cancer activity by apoptosis of SCC-15 cells in a dose-dependent manner. Further, it also revealed a highly significant decrease in anti-apoptotic Bcl-2 gene expression, whereas pro-apoptotic genes p53 and Bax revealed a highly significant increase, which is statistically significant compared to the control (P < 0.05). Conclusion: Our findings demonstrated that the AuNPs synthesized from A. muricata leaf extract could act as a novel anticancer agent, particularly against SCC, after further scrutiny.

In vitro Evaluation of Cytotoxic Effects of Methanolic Leaf Extracts of Annona muricata on Oral Squamous Cell Carcinoma-15 Cell Lines and its Effect On Expression of Bcl 2-associated X protein, B-cell C/Lymphoma 2 and p53 Genes.

Background: Due to their wide spectrum of phytochemical components and lack of side effects, the use of plants for the prevention and treatment of cancer has recently attracted increased attention. One among them is Annona muricata, commonly called soursop. According to recent investigations, several types of cancer have been successfully treated using this plant's extracts. However, studies on oral squamous cell carcinoma (SCC) are very limited. Aim: In the present study, we aimed to investigate the cytotoxic potential of leaf extract of A. muricata (LEAM) against oral tongue SCC-15 cell lines, using in vitro assays. Materials and Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-dipenyltetrazolium bromide assay was performed to assess cytotoxic activity, and the apoptotic effect was determined using gene expression analyses of Bcl 2-associated X protein (Bax), B-cell C/lymphoma 2 (Bcl-2), and tumor-suppressor phosphoprotein (p53). Results: Significant cytotoxicity (P ≤ 0.05) with a minimum inhibitory concentration value of 40 µg/ml was observed with the LEAM on SCC-15 cell lines. A highly significant decrease was observed in Bcl-2 gene expression (P < 0.05), whereas p53 and BAX genes revealed a highly significant increase (P < 0.05) when SCC-15 cell lines were treated with LEAM in the study group compared to the control. Conclusion: These results show that LEAM has the potential for development as a therapeutic agent for cytotoxicity, particularly on oral SCC cells, following further investigation.

Evaluation of the cytotoxic, anti-proliferative, anti-metastatic and pro-apoptotic effect of aqueous leaf extract of Annona muricata on oral tongue squamous cell carcinoma cell line (SCC-15): An in vitro study.

Background: Oral cancer still represents the leading cause of mortality in India. Due to the drawbacks of current treatment options, a safe, low-cost therapy is the need of the hour. Recently, novel plant extracts with anti-cancer properties have gained greater attention. One among them is Annona muricata and its leaf extract, which has been studied for its anti-cancer effect against various cancers. However, studies on oral cancer cells are very much limited and hence the study. Aims: To evaluate the cytotoxic, anti-proliferative, anti-metastatic and pro-apoptotic effect of aqueous leaf extract of Annona muricata (ALEAM) against SCC-15 cell lines through in vitro assays. Materials and Methods: In vitro assays such as MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], colony formation and wound healing assays were performed. Furthermore, to evaluate the underlying mechanism, gene and protein expression analysis of apoptotic/anti-apoptotic marker genes Bax, P53 and Bcl2, were done using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. Student's t-test has been performed for analysis of experimental data. Results: The results showed that ALEAM exhibited significant cytotoxic activity in a dose-dependent manner as well as inhibited colony formation and cell migration. The pro-apoptotic properties were affirmed by a highly significant drop in Bcl-2 gene expression and a highly significant rise in P53 and Bax genes in the study group compared to the control (P < 0.05). Conclusion: The current study provides evidence that ALEAM has the potential to be developed as a novel anti-cancer drug for the treatment of SCC after further clinical studies.

Protein Drug Delivery Using a Novel Maxillofacial Technique Targeting the Visual Pathway in the Brain, the Optic Nerve, and the Retina.

Protein drugs are used for treating many diseases of the eye and the brain. The formidable blood neural barriers prevent the delivery of these drugs into the eye and the brain. Hence, there is a need for a protein drug delivery system to deliver large proteins across blood-neural barriers. Low half-life, poor penetration of epithelial barriers, low stability, and immunogenicity limit the use of non-invasive systemic routes for delivering proteins. In this pre-clinical study, the efficacy of a new maxillofacial route for administering protein drugs using a novel drug delivery system is compared with systemic administration through intra-peritoneal injection and ocular administration through topical eye drops and subconjunctival and intravitreal injections. Bevacizumab and retinoschisin proteins were administered using the maxillofacial technique along with systemic and ocular routes in wild-type male C57BL/6J mice. Liquid chromatography with tandem mass spectrometry and western blot was used to detect bevacizumab in tissue samples. Furthermore, immunohistochemistry was performed to detect the presence and localization of bevacizumab and retinoschisin in the retina and brain. The maxillofacial route of delivery could target the brain including regions involved in the visual pathway and optic nerve. The maxillofacial technique and intravitreal injection were effective in delivering the drugs into the retina. A new concept based on the glymphatic pathway, cerebrospinal fluid drug distribution, and the crossover of ipsilateral optic nerve fibers at optic chiasma is proposed to explain the presence of the drug in contralateral eye following maxillofacial administration and intravitreal injection.

Cyclooxygenase-2 (COX-2) Expression in Oral Submucous Fibrosis and Oral Squamous Cell Carcinoma: An Immunohistochemical Study.

Introduction: Growing evidence has shown that cyclooxygenase-2 (COX-2), an enzyme capable of catalyzing prostaglandin production, plays a key role in carcinogenesis. Selective COX-2 inhibitors have been shown to reduce the establishment of tumors such as oral squamous cell carcinoma (OSCC) and premalignant conditions such oral submucous fibrosis (OSMF) in experimental models. The aim of this study was to investigate the immunohistochemical expression of COX-2 in OSCC and OSMF with the normal oral mucosa as control. Material and Methods: Forty-five formalin-fixed paraffin-embedded samples comprising 20 OSCC, 20 OSMF, and 5 normal oral mucosa specimens were withdrawn from the archives of the Department of Oral and Maxillofacial Pathology for immunohistochemical examination for COX-2 expression. Negative and less than 5% COX-2 positivity was considered negative expressions, while greater than or equal to 5% COX-2 positivity was considered positive expression. The data obtained were statistically analyzed. Results: The difference in percentages of expression in normal mucosa, OSCC, and OSMF was highly significant (P < 0.01). In comparison to normal mucosa, OSCC and OSMF had an increased level of COX-2 expression. However, there was an insignificant difference between the various histological gradings of OSCC and OSMF. Conclusion: The results of the present study confirm the role of COX-2 in carcinogenesis and in the progression of premalignant conditions to malignancy.

Quantitative assessment of platelet rich fibrin for the repair of extraction socket in a rat model.

The present study evaluated the quantitative effects of platelet-rich fibrin (PRF) for the repair of extraction socket in Sprague Dawley (SD) rat model by assessing several key clinical parameters. Seventy two male SD rats were subjected to surgical extraction of the maxillary right incisor. Rats were randomly divided into four groups with eighteen rats in each group based on the treatment received: extraction socket without treatment of PRF was taken as control (group I). Extraction socket implanted with 0.1, 0.2, and 0.4 mL of PRF was taken as study groups (groups II, III, and IV). The obtained results demonstrated that, low dose of PRF efficiently enhanced the natural healing cascade. Whereas, high dose interfered with native tissue contribution and altered the natural healing process. The beneficial effects of quantity-based application of PRF may raise the possibility of a new approach as complementary therapy besides conventional treatment.

Activation of PI3K/Akt/NF-kB Signaling Mediates Swedish Snus Induced Proliferation and Apoptosis Evasion in the Rat Forestomach: Modulation by Blueberry.

BACKGROUND AND OBJECTIVES: The present study was undertaken to ascertain whether the modulatory effects of blueberries on cell proliferation induced by Swedish snus in the rat forestomach epithelium is mediated via abrogation of the PI3K/Akt/NFκB signaling axis that regulates cell fate decision. METHODS: The transcript and protein expression of genes involved in cell cycle progression and apoptosis, as well as canonical PI3K/Akt/NF-κB signaling pathways, were analyzed by qRT-PCR, immunoblotting and ELISA. Expression profiling of noncoding RNAs (ncRNAs) that influence PI3K/Akt/NF-κB signaling was undertaken. TUNEL assay was performed using flow cytometry. RESULTS: Administration of snus induced basal cell hyperplasia in the rat forestomach with increased cell proliferation and inhibition of apoptosis. This was associated with the activation of PI3K/Akt/NFκB signaling. Coadministration of blueberries significantly suppressed snus-induced hyperplasia. Analysis of the molecular mechanisms revealed that blueberries suppress the phosphorylation of Akt, NF-κB and IKKß, prevent nuclear translocation of NF-κB and modulate the expression of microRNAs that influence PI3K/Akt/NF-κB signaling. CONCLUSION: Taken together, the results of the current study provide compelling evidence that blueberries exert significant protective effects against snus-induced soft tissue changes in the rat forestomach epithelium mediated by inhibiting key molecular players in the PI3K/Akt/NF-κB signaling axis. Long-term studies on the impact of snus exposure on various cellular processes, signaling pathways, and the interplay between genetic and epigenetic mechanisms are however warranted. The results of this investigation may contribute to the development of protection against soft tissue changes induced by smokeless tobacco in the human oral cavity.

Immunohistochemical analysis of Nuclear Factor-kappa B (NF-κB) between follicular and plexiform ameloblastomas: A pilot study.

BACKGROUND: Ameloblastoma among benign tumors holds a unique position by its locally destructive and invasive nature. Tumors that originate from the odontogenic apparatus or its remnants in the jaws show diverse clinical presentations, behavior and histologic patterns. The differed biological behavior behind follicular and plexiform ameloblastomas has never attained completeness because of the lack of rhythmic correlation regarding the exact mechanism. Nuclear factor-kappa B (NF-κB) pathways play a crucial role in survival, death and differentiation during physiologic and pathologic conditions. With this background, the study has been aimed to investigate the expression of NF-κB in follicular and plexiform ameloblastomas. OBJECTIVE: The objective of this study was to analyze the immunohistochemical expression pattern of NF-κB in ameloblastoma and to compare the immunohistochemical expression pattern of NF-κB among the histological types of ameloblastoma, follicular and plexiform patterns. METHODOLOGY: Total 20 ameloblastomas (10 follicular, 10 plexiform) were immunostained with antihuman NF-κB p65 mouse IgG monoclonal antibody, and the pattern of staining is statistically analyzed using Chi-square test with the level of significance (P < 0.05). RESULTS: Twelve (3 follicular, 9 plexiform) out of 20 ameloblastomas showed immunoreactivity to NF-κB p65. In ameloblastoma, only the peripheral preameloblast-like tall columnar cells showed reactivity, whereas the stellate reticulum-like cells are immunonegative. The staining pattern was membranous in the immunoreactive cells. The results were studied with the associated and inducing pathways from the literature, and a possible mechanism has been proposed. CONCLUSION: The expression pattern of NF-κB was found to be higher in plexiform ameloblastoma than follicular ameloblastoma.

Astaxanthin inhibits hallmarks of cancer by targeting the PI3K/NF-κΒ/STAT3 signalling axis in oral squamous cell carcinoma models.

Aberrant activation of the PI3K/Akt signalling pathway, a major driving force of diverse cellular processes has been implicated in tumour development and progression. Here, we report that astaxanthin (AXT), a potent antioxidant ketocarotenoid prevents cancer hallmarks by inhibiting PI3K/Akt and the associated downstream NF-κB and STAT-3 signalling pathways in SCC131 and SCC4 oral cancer cells as well as in the hamster buccal pouch carcinogenesis model. Using small molecule inhibitors of NF-κB, STAT-3 and PI3K and by overexpression of PI3K, we provide evidence to show that AXT inhibits NF-κB and STAT-3 signalling and cancer hallmarks by restraining the kinase activity of PI3K/Akt. Additionally, AXT downregulated the noncoding RNAs (ncRNAs), miR-21 and HOTAIR that influence PI3K/Akt signalling emphasising its modulatory effects on epigenetic regulation. Ethyl cellulose-based AXT nanoparticles showed greater chemotherapeutic efficacy in the hamster oral carcinogenesis model compared to native AXT. We suggest that AXT prevents cell proliferation, apoptosis evasion, invasion and angiogenesis by intercepting the crosstalk between the PI3K/Akt, NF-κB and STAT-3 signalling circuits both in vitro and in vivo. Astaxanthin that abrogates the PI3K/Akt signalling axis, a central hub that orchestrates acquisition of cancer hallmarks is a promising candidate for anticancer drug development.

Expression of Cluster of Differentiation 1a-Positive Langerhans Cells in Oral Lichen Planus.

BACKGROUND/PURPOSE: Lichen planus is a T-cell-mediated mucocutaneous disorder characterized histopathologically by a band of chronic inflammatory cells in the subepithelial zone and degeneration of basal layer. The present study was aimed to evaluate the distribution and quantitative assessment of cluster of differentiation 1a (CD1a)-positive Langerhans cells (LCs) in oral lichen planus (OLP), thus to determine the role of LCs pertaining to the changes occurring in OLP. MATERIALS AND METHODS: Five cases of normal oral mucosa and 20 cases of OLP were immunostained with CD1a antibody; the positive cells were counted manually in the photomicrographs and statistically analyzed using t-test, Mann-Whitney test, and Wilcoxon signed-rank test. RESULTS: The average percentage of CD1a-positive LCs in normal subjects was 0.9%, and in the OLP cases higher percentage was observed (3.93%). The statistical comparison of these two parameters was significant (P=0.018). The degree of basal cell degeneration and density of subepithelial infiltrate on statistical comparison with the concentration of CD1a-positive LCs showed significant results. CONCLUSION: LCs play a pivotal role in the recruitment of CD4+ and CD8+ cells to the subepithelial region and basal keratinocytes apoptosis. A small number of study subjects, assessment of only CD1a molecule and LCs in the epidermis only were a few of the drawbacks of the study.

Impact of stainless-steel welding fumes on proteins and non-coding RNAs regulating DNA damage response in the respiratory tract of Sprague-Dawley rats.

Substantial evidence has established the negative impact of inhalation exposure to welding fumes on respiratory functions. The aim of the present study was to investigate the effect of welding fume inhalation on expression of molecules that function as sensors, transducers and effectors of DNA damage response (DDR) in the respiratory tract of male Sprague-Dawley rats. Animals were exposed to 50 mg/m3 stainless steel welding fumes for 1 h/d for 4, 8, and 12 weeks, respectively. Histological examination demonstrated preneoplastic changes in trachea and bronchi with focal atelectasis and accumulation of chromium (Cr) in the lungs. This was associated with elevated levels of DNA damage markers (8-oxodG, γH2AX), ATM phosphorylation, cell cycle arrest, apoptosis induction, activation of homologous recombination (HR), non-homologous end joining (NHEJ), and Nrf2 signaling, as well as altered expression of noncoding RNAs (ncRNAs). However, after 12 weeks of exposure, DDR was compromised as reflected by resumption of the cell cycle, repair inhibition, and failure of apoptosis. Data demonstrate that exposure to welding fumes influences two crucial layers of DDR regulation, phosphorylation of key proteins in NHEJ and HR, as well as the ncRNAs that epigenetically modulate DDR. Evidence indicates that marked DNA damage coupled with non-productive DNA repair and apoptosis avoidance may be involved in neoplastic transformation.

Expression of ß1integrin in normal epithelium, oral submucous fibrosis and oral squamous cell carcinoma.

INTRODUCTION AND AIMS: The possible reason suggested for epithelial atrophy in oral submucous fibrosis (OSMF) is ischemia. Dysregulation in the epithelial proliferation and maturation is also thought to be a cause. The ß1 integrin identifies the oral epithelial stem cells. The changes induced by the arecanut on these cells may result in epithelial alterations. The aim of this study is to evaluate the stem cells distribution and percentage by assessing the ß1 integrin expression. MATERIALS AND METHODS: The study included normal oral mucosa (15 cases) and disease group (97 cases). The disease group was further subdivided into early (29 cases), moderate (34 cases), advanced OSMF (18 cases) and oral squamous cell carcinoma(OSCC) associated with OSMF (16 cases). The tissues were stained for ß1 integrin antibodies. The positive cells and staining intensities were analysed to determine the staining index, and statistically evaluated using KW test statistics. RESULTS: ß1 integrin was observed in retepegs region and the percentage of positive cells was 14%- 30% in the control. In OSMF, the ß1 integrin positivity was observed in basal and suprabasal layers, and the percentage was ranged from 2%-71%. ß1 integrin expression in OSCC was observed both in central and peripheral cells and ranged from 17%-85%. On comparison, the difference in staining index among normal, OSMF and carcinomas was significant at p<0.01. The stem cells percentage was increased both in OSMF and carcinomas. The non-dysplastic epithelium of OSMF with severe atrophy showed lowest percentage. It is inferred that absence of stem cells and proliferation may attribute for the atrophy.

Caspase-3 expression in normal oral epithelium, oral submucous fibrosis and oral squamous cell carcinoma.

CONTEXT: The epithelium atrophy, as the oral submucous fibrosis (OSMF) progresses, is believed to be an after effect of stromal fibrosis, hyalinization, decrease in vascularity and cellularity and is considered as "ischemic atrophy." Due to hypoxia, caspase-3 get activation and subsequent decrease in viable cell count can occur. AIMS AND OBJECTIVES: To determine caspase-3 expression in various grades of OSMF and oral squamous cell carcinoma (OSCC) to find out whether upregulation of apoptosis is responsible for the epithelial changes in OSMF. SUBJECTS AND METHODS: The control tissue (15 samples from normal oral mucosa) and study group comprising 97 cases of OSMF of different grades and OSCC associated with OSMF were stained with caspase-3 antibody, and the percentage of positive cells was calculated using ImageJ software. STATISTICAL ANALYSIS: The results obtained were statistically analyzed using ANOVA and Tukey's honest significance difference test and Mann-Whitney U-test. RESULTS: There was a nuclear expression of caspase-3 in basal and parabasal layers of normal epithelium. There was cytoplasmic expression of caspase-3 in OSMF without dysplasia, total absence of caspase-3 expression in dysplastic epithelium and in majority cases of OSCC. The caspase-3 percentage was increased in OSMF (0%-53%) when compared with OSCC (0%-8%). The statistical comparison of caspase-3 among normal, OSMF and OSCC patients revealed significant correlation (P < 0.00010). The comparison within different grades of OSMF and between dysplastic and nondysplastic epithelium OSMF also showed significance (P < 0.019). CONCLUSIONS: The decreased expression of caspase-3 in disease progression reflects its role in the malignant transformation.

Osteopontin expression in reactive lesions of gingiva.

Reactive proliferations of the gingiva comprise lesions such as pyogenic granuloma (PG), inflammatory fibroepithelial hyperplasia (IFH), peripheral ossifying fibroma (POF), and peripheral giant cell lesion. Osteopontin (OPN) has a dual role, it promotes mineralization when it is bound to solid substrate, and on the other hand, it inhibits mineralization when it is seen in association with solution. Objectives The study aimed to evaluate the expression of osteopontin in normal gingival tissue and different types of focal reactive proliferations of gingival tissue, and its role in the development of calcification within it. Material and Methods The presence and distribution of osteopontin was assessed using immunohistochemistry in five cases of normal gingival tissue and 30 cases of focal reactive proliferations of gingiva. Results There was no expression of osteopontin in normal subjects. Few cases of pyogenic granuloma, inflammatory fibroepithelial hyperplasia, and all the cases of peripheral ossifying fibroma showed positivity for osteopontin in the inflammatory cells, stromal cells, extracellular matrix, and in the calcifications. Conclusion The expression of osteopontin in all the cases of peripheral ossifying fibroma speculates that the majority of the cases of peripheral ossifying fibroma originate from the periodontal ligament cells. The treatment modalities for peripheral ossifying fibroma should differ from other focal reactive proliferations of gingiva.

Osteopontin expression in reactive lesions of gingiva

Reactive proliferations of the gingiva comprise lesions such as pyogenic granuloma (PG), inflammatory fibroepithelial hyperplasia (IFH), peripheral ossifying fibroma (POF), and peripheral giant cell lesion. Osteopontin (OPN) has a dual role, it promotes mineralization when it is bound to solid substrate, and on the other hand, it inhibits mineralization when it is seen in association with solution. Objectives The study aimed to evaluate the expression of osteopontin in normal gingival tissue and different types of focal reactive proliferations of gingival tissue, and its role in the development of calcification within it. Material and Methods The presence and distribution of osteopontin was assessed using immunohistochemistry in five cases of normal gingival tissue and 30 cases of focal reactive proliferations of gingiva. Results There was no expression of osteopontin in normal subjects. Few cases of pyogenic granuloma, inflammatory fibroepithelial hyperplasia, and all the cases of peripheral ossifying fibroma showed positivity for osteopontin in the inflammatory cells, stromal cells, extracellular matrix, and in the calcifications. Conclusion The expression of osteopontin in all the cases of peripheral ossifying fibroma speculates that the majority of the cases of peripheral ossifying fibroma originate from the periodontal ligament cells. The treatment modalities for peripheral ossifying fibroma should differ from other focal reactive proliferations of gingiva. .

Chlorophyllin abrogates canonical Wnt/ß-catenin signaling and angiogenesis to inhibit the development of DMBA-induced hamster cheek pouch carcinomas.

BACKGROUND: Chlorophyllin, a water soluble semi-synthetic food-grade derivative is reported to exhibit a wide range of beneficial health effects. We investigated the effect of chlorophyllin supplementation on Wnt/ß-catenin and vascular endothelial growth factor (VEGF) signaling in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model. METHODS AND RESULTS: Hamsters were divided into 4 groups. The right buccal pouches of group 1 and 2 hamsters were painted with 0.5 % DMBA for 14 weeks. Group 2 animals received in addition chlorophyllin (4 mg/kg bw) in the diet. Group 3 animals received chlorophyllin alone and group 4 animals served as control. mRNA and protein expression of components of Wnt, VEGF, and PI3K/Akt signaling pathways were analyzed by RT-PCR and Western blot analysis. Dietary chlorophyllin administration suppressed the development of HBP carcinomas by altering the expression of several components of the Wnt/ß-catenin signaling pathway. This was associated with inhibition of angiogenesis as evidenced by decreased expression of the proangiogenic factors HIF-1α, VEGF, and VEGFR2. Chlorophyllin administration also downregulated the expression of histone deacetylases involved in epigenetic regulation of tumor angiogenesis. CONCLUSION: Dietary chlorophyllin that abrogates Wnt/ß-catenin and VEGF signaling by targeting a multitude of key signaling molecules is an attractive candidate for preventing tumor progression.