Indraneel Bhattacharyya

Overexpression of Dicer as a Result of Reduced let-7 microRNA Levels Contributes to Increased Cell Proliferation of Oral Cancer Cells

Recent reports have demonstrated that Dicer, an RNase III endonuclease required for microRNA (miRNA) maturation, is aberrantly expressed in different types of cancer. Furthermore, Dicer has been reported to be regulated by the let‐7 family of miRNA genes. We hypothesize that Dicer is aberrantly expressed in oral cancer cells due to altered expressions of let‐7 and that Dicer contributes to the development and progression of the disease. Western blot examination of Dicer protein levels in four head and neck squamous cell carcinoma (HNSCC) cell lines, including two oral cancer cell lines, demonstrated that Dicer had between 4‐ and 24‐fold higher expression levels when compared to normal human primary gingival epithelial cells. Furthermore, five of six oral cancer tissues analyzed by indirect immunofluorescence had increased Dicer protein expression, compared to normal gingival epithelial tissue. The Dicer mRNA levels were not found to correlate well with protein expression in the HNSCC cell lines, suggesting that Dicer protein expression was post‐transcriptionally regulated. Analysis of let‐7a and let‐7b levels in HNSCC cell lines by real‐time PCR demonstrated that let‐7b, but not let‐7a, was significantly reduced in the HNSCC cell lines compared to control cells. Lastly, transfection of oral cancer cells with chemically synthesized let‐7b and small interfering RNAs targeting Dicer significantly inhibited cell proliferation up to 83% and >100%, respectively, as early as 3 days post‐transfection. Together, these data demonstrate that elevated expression levels of Dicer in oral cancer cells correlate with downregulation of let‐7b and increased cell proliferation.

Identification of genes and molecular pathways involved in the progression of premalignant oral epithelia

An early interventional effort in oral premalignancy requires novel molecular targets and diagnostic biomarkers to delay or reverse incidences of malignant progression. Microarray-based transcriptional profiling in disease states provides global insight into the causal biomolecular processes and novel pathways involved. In this study, we investigated transcript profiles in precancerous oral lesions to identify nearly 1,700 genes as significantly overexpressed or underexpressed and a primarily affected metabolic pathway that may be responsible for irreversible transition to progressive stages of oral cancer. For the first time, we show a convergence of several genes and pathways known for their oncogenic capabilities, in progression of premalignant oral epithelial tissues. This study consequently provides a molecular basis for persistent proinflammatory conditions in oral premalignant tissues. We found that lipocalin-type prostaglandin D2 synthase (PTGDS), a key enzyme in the arachidonic acid metabolism pathway, as repressed in premalignant stages. We show the protective role of these enzyme-derived metabolites in inhibiting cell proliferation using an in vitro oral cancer progression model. We have also confirmed the overexpression of two invasion-related biomarkers, psoriasin (PSOR1) and versican (CSPG2), in oral premalignant and malignant archival tissues. Our results clearly indicate that pharmacologic intervention with anti-inflammatory prostaglandin D2–like analogues may help prevent or delay oral epithelial carcinogenesis because of metabolic restoration of a negative feedback regulatory loop through its several cognate receptors or target molecules. Further studies directed toward a multitude of possible protective mechanisms of this lipocalin-type enzyme or its products in oral cancer progression are warranted.

Active Invasion of Oral and Aortic Tissues by Porphyromonas gingivalis in Mice Causally Links Periodontitis and Atherosclerosis

Atherosclerotic vascular disease is a leading cause of myocardial infarction and cerebrovascular accident, and independent associations with periodontal disease (PD) are reported. PD is caused by polymicrobial infections and aggressive immune responses. Genomic DNA of Porphyromonas gingivalis, the best-studied bacterial pathogen associated with severe PD, is detected within atherosclerotic plaque. We examined causal relationships between chronic P. gingivalis oral infection, PD, and atherosclerosis in hyperlipidemic ApoEnull mice. ApoEnull mice (n = 24) were orally infected with P. gingivalis for 12 and 24 weeks. PD was assessed by standard clinical measurements while the aorta was examined for atherosclerotic lesions and inflammatory markers by array. Systemic inflammatory markers serum amyloid A, nitric oxide, and oxidized low-density lipoprotein were analyzed. P. gingivalis infection elicited specific antibodies and alveolar bone loss. Fluorescent in situ hybridization detected viable P. gingivalis within oral epithelium and aorta, and genomic DNA was detected within systemic organs. Aortic plaque area was significantly increased in P. gingivalis-infected mice at 24 weeks (P<0.01). Aortic RNA and protein arrays indicated a strong Th2 response. Chronic oral infection with P. gingivalis results in a specific immune response, significant increases in oral bone resorption, aortic inflammation, viable bacteria in oral epithelium and aorta, and plaque development.

Presence of Porphyromonas gingivalis in gingival squamous cell carcinoma

Periodontal disease has been recently linked to a variety of systemic conditions such as diabetes, cardiovascular disease, preterm delivery, and oral cancer. The most common bacteria associated with periodontal disease, Porphyromonas gingivalis (P. gingivalis) has not yet been studied in the malignant gingival tissues. The objective of this study was to investigate the presence of P. gingivalis in specimens from squamous cell carcinoma patients. We have performed immunohistochemical staining to investigate the presence of P. gingivalis and Streptococcus gordonii (S. gordonii), a non invasive oral bacteria, in paraffin embedded samples of gingival squamous cell carcinoma (n=10) and normal gingiva (n=5). Staining for P. gingivalis revealed the presence of the bacteria in normal gingival tissues and gingival carcinoma, with higher levels (more than 33%, P<0.05) detected in the carcinoma samples. The staining intensity was also significantly enhanced in the malignant tissue by 2 folds (P<0.023) compared to specimens stained for the non‐invasive S. gordonii. P. gingivalis is abundantly present in malignant oral epithelium suggesting a potential association of the bacteria with gingival squamous cell carcinoma.

Polymicrobial Infection with Major Periodontal Pathogens Induced Periodontal Disease and Aortic Atherosclerosis in Hyperlipidemic ApoEnull Mice

Periodontal disease (PD) and atherosclerosis are both polymicrobial and multifactorial and although observational studies supported the association, the causative relationship between these two diseases is not yet established. Polymicrobial infection-induced periodontal disease is postulated to accelerate atherosclerotic plaque growth by enhancing atherosclerotic risk factors of orally infected Apolipoprotein E deficient (ApoEnull) mice. At 16 weeks of infection, samples of blood, mandible, maxilla, aorta, heart, spleen, and liver were collected, analyzed for bacterial genomic DNA, immune response, inflammation, alveolar bone loss, serum inflammatory marker, atherosclerosis risk factors, and aortic atherosclerosis. PCR analysis of polymicrobial-infected (Porphyromonas gingivalis [P. gingivalis], Treponema denticola [T. denticola], and Tannerella forsythia [T. forsythia]) mice resulted in detection of bacterial genomic DNA in oral plaque samples indicating colonization of the oral cavity by all three species. Fluorescent in situ hybridization detected P. gingivalis and T. denticola within gingival tissues of infected mice and morphometric analysis showed an increase in palatal alveolar bone loss (p<0.0001) and intrabony defects suggesting development of periodontal disease in this model. Polymicrobial-infected mice also showed an increase in aortic plaque area (p<0.05) with macrophage accumulation, enhanced serum amyloid A, and increased serum cholesterol and triglycerides. A systemic infection was indicated by the detection of bacterial genomic DNA in the aorta and liver of infected mice and elevated levels of bacterial specific IgG antibodies (p<0.0001). This study was a unique effort to understand the effects of a polymicrobial infection with P. gingivalis, T. denticola and T. forsythia on periodontal disease and associated atherosclerosis in ApoEnull mice.

Role of Porphyromonas gingivalis phosphoserine phosphatase enzyme SerB in inflammation, immune response, and induction of alveolar bone resorption in rats.

ABSTRACT Porphyromonas gingivalis secretes a serine phosphatase enzyme, SerB, upon contact with gingival epithelial cells in vitro. The SerB protein plays a critical role in internalization and survival of the organism in epithelial cells. SerB is also responsible for the inhibition of interleukin-8 (IL-8) secretion from gingival epithelial cells infected with P. gingivalis. This study examined the ability of a P. gingivalis SerB mutant to colonize the oral cavity and induce gingival inflammation, immune responses, and alveolar bone resorption in a rat model of periodontal disease. Both P. gingivalis ATCC 33277 and an isogenic ΔSerB mutant colonized the oral cavities of rats during the 12-week experimental period. Both of the strains induced significant (P < 0.05) systemic levels of immunoglobulin G (IgG) and isotypes IgG1, IgG2a, and IgG2b, indicating the involvement of both T helper type 1 (Th1) and Th2 responses to infection. Both strains induced significantly (P < 0.05) higher levels of alveolar bone resorption in infected rats than in sham-infected control rats. However, horizontal and interproximal alveolar bone resorption induced by the SerB mutant was significantly (P < 0.05) lower than that induced by the parental strain. Rats infected with the ΔSerB mutant exhibited significantly higher levels of apical migration of the junctional epithelium (P < 0.01) and polymorphonuclear neutrophil (PMN) recruitment (P < 0.001) into the gingival tissues than rats infected with the wild type. In conclusion, in a rat model of periodontal disease, the SerB phosphatase of P. gingivalis is required for maximal alveolar bone resorption, and in the absence of SerB, more PMNs are recruited into the gingival tissues.

CIP2A expression and localization in oral carcinoma and dysplasia

Aims: Oral squamous cell carcinoma (OSCC) is the most prevalent malignancy of the oral cavity resulting in severe morbidity and mortality. To date only few proteins have been suggested as potential biomarkers or targets for this type of cancer. Cancerous inhibitor of PP2A (CIP2A) is a protein expressed in epithelial tissues that stabilizes the oncogene c-Myc and causes cell transformation. This study was designed to investigate the expression of CIP2A in OSCC cell lines and tissues representing human normal, dysplasia and OSCC. Methods: Using quantitative real time PCR, mRNA quantification for CIP2A was performed in a primary gingival cell line and OSCCs CAL 27 and SCC-25. Paraffin embedded human specimen classified as normal, dysplastic or OSCC were immunohistochemically stained for CIP2A expression. EGFR and CIP2A were also stained by immunofluorescence for co-localization. Samples of human normal oral tissue and OSCC were studied by PCR for mRNA expression of CIP2A. Results: CIP2A was significantly increased in the human carcinoma cell lines compared to the primary gingival cell line. CIP2A was overexpressed in the human oral dysplasia and OSCC tissues compared to normal oral tissues. CIP2A was also preferentially localized in the dysplastic and OSCC epithelial areas compared to EGFR that was expressed mainly in areas of relatively normal epithelium and in dysplastic tissues above the basal layers. Conclusions: CIP2A may play a significant role in oral malignant transformation and therefore, it may be a potential target for chemotherapy of OSCC.

Myopericytoma: report of two cases associated with trauma

Myopericytoma is a rare, recently described tumor demonstrating a hemangiopericytoma‐like vascular pattern. We present two cases of myopericytoma associated with trauma: a 64‐year‐old man who developed several nodules on his nose four months after sustaining multiple abrasions to his forehead and nose, and a 72‐year‐old woman with a solitary growth in the alveolar ridge of unknown duration. Biopsy specimens of the lesions in both cases demonstrated a striking concentric perivascular proliferation of bland spindle‐shaped pericytic cells characteristic of myopericytoma. Despite sharing morphologic features with angioleiomyoma, myofibroma and glomus tumor, myopericytoma is thought to represent a distinct perivascular myoid neoplasm of skin and soft tissues. The tumor is characterized by a radial and perivascular arrangement of ovoid, spindled to round neoplastic cells that are immunoreactive to alpha‐smooth muscle actin, often for h‐caldesmon as well as smooth muscle myosin‐heavy chain, and usually negative for desmin antibodies. Most cases of myopericytoma are benign, however, local recurrence and malignancy have recently been reported, Myopericytoma can be multifocal involving a single or multiple anatomic regions, and tends to occur in dermal and superficial soft tissues of adults primarily on the extremities. Our cases are unusual examples of myopericytoma manifesting as multiple nodules on the nose, and a solitary growth on the buccal mucosa after trauma.

Virulence of major periodontal pathogens and lack of humoral immune protection in a rat model of periodontal disease

OBJECTIVE This study was designed to test the hypothesis that periodontal pathogens Tannerella forsythia and Porphyromonas gingivalis are synergistic in terms of virulence potential using a model of mixed-microbial infection in rats. MATERIALS AND METHODS Three groups of rats were infected orally with either T. forsythia or P. gingivalis in mono-bacterial infections or as mixed-microbial infections for 12 weeks and a sham-infected group were used as a control. This study examined bacterial infection, inflammation, immunity, and alveolar bone loss changes with disease progression. RESULTS Tannerella forsythia and P. gingivalis genomic DNA was detected in microbial samples from infected rats by PCR indicating their colonization in the rat oral cavity. Primary infection induced significantly high IgG, IgG2b, IgG1, and IgG2a antibody levels indicating activation of mixed Th1 and Th2 immune responses. Rats infected with the mixed-microbial consortium exhibited significantly increased palatal horizontal and interproximal alveolar bone loss. Histological examinations indicated significant hyperplasia of the gingival epithelium with moderate inflammatory infiltration and apical migration of junctional epithelium. The results observed differ compared to uninfected controls. CONCLUSION Our results indicated that T. forsythia and P. gingivalis exhibit virulence, but not virulence synergy, resulting in the immuno-inflammatory responses and lack of humoral immune protection during periodontitis in rats.

Porphyromonas gingivalis and Treponema denticola Mixed Microbial Infection in a Rat Model of Periodontal Disease

Porphyromonas gingivalis and Treponema denticola are periodontal pathogens that express virulence factors associated with the pathogenesis of periodontitis. In this paper we tested the hypothesis that P. gingivalis and T. denticola are synergistic in terms of virulence; using a model of mixed microbial infection in rats. Groups of rats were orally infected with either P. gingivalis or T. denticola or mixed microbial infections for 7 and 12 weeks. P. gingivalis genomic DNA was detected more frequently by PCR than T. denticola. Both bacteria induced significantly high IgG, IgG2b, IgG1, IgG2a antibody levels indicating a stimulation of Th1 and Th2 immune response. Radiographic and morphometric measurements demonstrated that rats infected with the mixed infection exhibited significantly more alveolar bone loss than shaminfected control rats. Histology revealed apical migration of junctional epithelium, rete ridge elongation, and crestal alveolar bone resorption; resembling periodontal disease lesion. These results showed that P. gingivalis and T. denticola exhibit no synergistic virulence in a rat model of periodontal disease.