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Sudachitin, a Polymethoxyflavone Derived From Citrus Sudachi, Suppresses Lipopolysaccharide-Induced Inflammatory Bone Resorption Because of Inhibiting Osteoclast Formation in Mice
Poster Session asymptomatic, making identification more difficult. Most OPLs do not require aggressive treatment, however, preventing the transformation to malignancy is key to impacting oral cancer morbidity and mortality. Furthermore, the high mortality rate associated with oral cancer and the low transformation rate of OPLs creates a strong need for reliable assessments that more accurately identify lesions at high-risk of transformation, separating these lesions from those at lesser transformation risk. The standard of care for OPL risk assessment, dysplasia grading by histopathology, is impacted by intraand inter-observer variation as well as significant overlap between grades, affecting its usefulness as a prognostic tool. The StraticyteTM oral prognostic assessment has been developed to meet these needs. OPL biopsy samples from 150 cases with a follow-up history of up to 12 years were used. Immunohistochemistry for the biomarker S100A7 on tissue biopsy slides and tissue microarrays was performed at Mount Sinai Hospital in Toronto, Ontario, Canada. The slides were then digitally scanned on a Hamamatsu Nanozoomer-XR slide scanner and images were visualized and analyzed using Visiopharm VIS (version 5; Horsholm, Denmark). This project was approved by the Mount Sinai Hospital Research Ethics Board. All statistical analyses and model building were conducted using the R package (version 3). Stepwise Cox Regression was used to select the parameters. A multivariate Cox Regression model was fitted to selected parameters and the C-index was used to assess the model. Estimated Log Relative-Hazards from the Cox model were referred to as risk scores and used in the cut-off selection stage to classify all cases into three risk groups: low, intermediate, and high. The Nelson-Aalen-Breslow estimate, used to calculate the baseline cancer-free survival curve, is combined with the calculated risk score to produce the expected cancer-free survival probability for each case. The AalenLink-Tsiatis estimate, used to estimate the variance of expected cancer-free survival probability, provided the 95% confidence interval (CI) of the cancer-free survival curve. From the 150 cases, the 95% CI of mild, moderate, and severe dysplasia grades based on histopathological assessment, overlapped extensively throughout the first 60 months, indicating ineffective differentiation. In contrast, the 95% CIs of the Straticyte classified groups had minimal overlaps at month 60, achieving better differentiation. The performance of Straticyte was evaluated by an internal validation study using the splitsample technique. Comparing the C-index (time-toevent response) and Area Under the Curve (AUC; binary response), Straticyte risk scores were more objective and discriminatory than histopathological dysplasia grading. Furthermore, Straticyte outperformed histopathological dysplasia grading in two clinical indices. AAOMS 2016
The sensitivity between the low-risk vs. non-low-risk groups in Straticyte was 96% compared to mild vs. non-mild dysplasia gradings of 75%, with a negative predictive value of 80% and 59%, respectively. Straticyte is intended to better categorize a patient’s 5year risk for OPLs to progress to cancer and should be regarded as a complement to conventional histopathology. Straticyte can be easily incorporated into clinical practice as no additional tissue samples are needed for the assessment. References: 1. Lingen MW, et al. Critical evaluation of diagnostic aids for the detection of oral cancer. Oral Oncol, 44(1): 10-22, 2008 2. Warnakulasuriya S. et al. Oral epithelial dysplasia classification systems: predictive value, utility, weakness and scope for improvement. J Oral Pathol Med, 37(3): 127-133, 2008
POSTER 35 Sudachitin, a Polymethoxyflavone Derived From Citrus Sudachi, Suppresses LipopolysaccharideInduced Inflammatory Bone Resorption Because of Inhibiting Osteoclast Formation in Mice Y. Ohyama: Meikai University School of Dentistry, J. Ito, Y. Hakeda, J. Shimada Objective: Inflammatory bone diseases are associated not only with production of inflammatory cytokines but also with local oxidative state. Sudachtin, a polymethoxyflavone derived from Citrus sudachi, possesses antioxidant properties and regulates various functions of mammalian cells.1 However, the mechanism for osteoclastogenesis and inflammatory bone destruction remains unknown. Extensive studies have indicated that the induction of osteoclast differentiation by RANKL requires the activation of NF-kB and MAPK signaling pathways, which leads to the expression of NFATc1, a critical transcription factor for osteoclast differentiation. NFATc1, in turn, induces osteoclast formation and bone-resorbing activity. Here, we examined the effect of sudachitin on osteoclast differentiation and lipopolysaccharide (LPS)induced inflammatory bone resorption, and found that sudachitin suppressed osteoclast formation and function both in vitro and in vivo. Materials and Methods: In vitro osteoclast formation has been previously described.2 Bone marrow cells obtained from 48-week-old male C57BL/6 mice were cultured for 3 days in the presence of M-CSF to isolate osteoclast precursors. The osteoclast precursors were then treated with M-CSF and soluble RANKL to form mature osteoclasts. After culturing for the desired time, e-71
Poster Session TRAP-positive multinucleated cells formed in the culture, which were considered to be osteoclastic cells, were counted. Western blotting analysis and quantitative RTPCR was performed according to the ordinary protocols. To evaluate the effect of sudachitin on in vivo LPSinduced calvarial bone destruction, 8-week-old mice were injected with LPS subperiosteally in the calvarial bone daily for 5 days. After 6 days, mCT scanning of the calvariae was performed. The mean values of the groups were compared by unpaired Student’s t test or by oneway ANOVAs. P < 0.05 was considered significant. Results: When osteoclast precursors were treated with sudachitin in the presence of sRANKL and M-CSF, the number of TRAP-positive multinucleated osteoclasts formed in the culture was decreased compared with sudachitin-untreated culture in a dose-dependent manner [stachitin (10 mM) group vs. untreated group: 6.8 0.9 cells/well vs. 625.3 18.0 cells/well, p<0.05]. Since sudachitin had no effect on the proliferation and viability of osteoclast lineage cells, the inhibitory effect of sudachitin was attributed to the direct action in osteoclastogenesis. Consistent with the inhibition, sudachitin suppressed the activation of Erk signal in the osteoclast precursors. Furthermore, sudachitin decreased expression of c-fos and NFATc1 in mRNA and protein levels. Followed by the down-regulation of those transcription factors, the expressions of osteoclast differentiationrelated molecules such as TRAP, cathepsin K, DC-STAMP and Atp6v0d2 were also decreased. In addition, sudachitin suppressed the production of intracellular reactive oxygen species. Consistent with the in vitro experiments, when inflammatory bone destruction of calvariae was induced by LPS injection, simultaneous administration of sudachitin (50 mM) with LPS reduced the elevated bone resorption as well as the increased TRAP and cathepsin K expression in vivo [stachitin (10 mM) group vs. untreated group: relative amount of TRAP mRNA; 4.69 0.93 vs. 2.66 0.30 , p<0.05; relative amount of cathepsin K mRNA: 6.0 0.27 vs 2.52 0.37 , p<0.05]. Conclusion: Sudachitin, a polymethoxyflavone, inhibits osteoclast formation and suppresses LPS-induced inflammatory bone resorption in mice. Therefore, sudachitin could be an effective component for the prevention of inflammatory bone disorders. References: 1. Yuasa K, Tada K, Harita G, Fujimoto T, Tsukayama M, Tsuji A. Sudachitin, a polymethoxyflavone from Citrus sudachi, suppresses lipopolysaccharide-induced inflammatory responses in mouse macrophage-like RAW264 cells. Biosci Biotechnol Biochem, 76:598-600, 2012 2. Nakayachi M, Ito J, Hayashida C, Ohyama Y, Kakino A, Okayasu M, Sato T, Ogasawara T, Kaneda T, Suda N, Sawamura T, Hakeda Y. Lectin-like oxidized low-density lipoprotein receptor-1 abrogation causes resistance to inflammatory bone destruction in mice, despite promoting osteoclastogenesis in the steady state. Bone, 75:170-182, 2015
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POSTER 36 Oral Melanoacanthoma: A Report of Two Cases and a Review of the Literature P. G. Tolomeo: New York University Langone Medical Center/Bellevue Hospital Center, J. S. Lee, N. Zawada, A. R. Kerr, J. A. Phelan Oral melanoacanthoma (MA) is a rare, benign pigmented lesion that presents as a painless, rapidly growing, brown-black macular lesion that commonly affects the buccal mucosa in areas that are subject to chronic trauma/irritation.1,2 MA is commonly seen in the third and fourth decades of life and primarily affects blacks with a strong female predilection.3,4 Histopathologically, the lesions exhibit proliferation of keratinocytes and dendritic melanocytes.5 This report includes two cases of oral melanoacanthoma and a review of the literature. Case 1: A 43-year-old black female presented with a slowly enlarging pigmented lesion on the right buccal mucosa. The patient did not recall any known trauma to the area or previous infection and reported that the lesion was painless but had a gradually increased in size. Oral examination revealed a 2.0 x 2.0 cm. brown macule on the right buccal mucosa. A punch biopsy was taken of the pigmented area. The tissue was placed in 10% formalin and submitted for microscopic examination. The tissue was stained with hematoxylin and eosin and exhibited acanthotic, stratified squamous epithelium with dendritic melanocytes dispersed throughout the epithelium consistent with a diagnosis of melanoacanthoma. Case 2: A-35 year-old black female presented with a rapidly growing pigmented lesion on the left buccal mucosa. Two years prior to presentation the patient had noted a brown lesion on the buccal mucosa adjacent to a fractured tooth. The lesion remained unchanged and asymptomatic for approximately two years. One week prior to presentation, the patient noted that the lesion was enlarging, but remained painless. Oral examination revealed a 1.5 x 1.5 cm. brown macule surrounded by erythema on the left buccal mucosa adjacent to a fractured tooth. A punch biopsy was taken that included both the pigmented and erythematous areas. The tissue was placed in 10% formalin and submitted for microscopic examination. The tissue was stained with hematoxylin and eosin and exhibited similar histopathologic features to the previous case. Immunohistochemical staining with S-100 and Melan-A dramatically demonstrated the dendritic melanocytes. Review of the literature revealed a total of 50 cases of oral melanoacanthoma. These lesions were reported in black females on the buccal mucosa with subsequent resolution. The cases here demonstrate similar clinical AAOMS 2016
The sensitivity between the low-risk vs. non-low-risk groups in Straticyte was 96% compared to mild vs. non-mild dysplasia gradings of 75%, with a negative predictive value of 80% and 59%, respectively. Straticyte is intended to better categorize a patient’s 5year risk for OPLs to progress to cancer and should be regarded as a complement to conventional histopathology. Straticyte can be easily incorporated into clinical practice as no additional tissue samples are needed for the assessment. References: 1. Lingen MW, et al. Critical evaluation of diagnostic aids for the detection of oral cancer. Oral Oncol, 44(1): 10-22, 2008 2. Warnakulasuriya S. et al. Oral epithelial dysplasia classification systems: predictive value, utility, weakness and scope for improvement. J Oral Pathol Med, 37(3): 127-133, 2008
POSTER 35 Sudachitin, a Polymethoxyflavone Derived From Citrus Sudachi, Suppresses LipopolysaccharideInduced Inflammatory Bone Resorption Because of Inhibiting Osteoclast Formation in Mice Y. Ohyama: Meikai University School of Dentistry, J. Ito, Y. Hakeda, J. Shimada Objective: Inflammatory bone diseases are associated not only with production of inflammatory cytokines but also with local oxidative state. Sudachtin, a polymethoxyflavone derived from Citrus sudachi, possesses antioxidant properties and regulates various functions of mammalian cells.1 However, the mechanism for osteoclastogenesis and inflammatory bone destruction remains unknown. Extensive studies have indicated that the induction of osteoclast differentiation by RANKL requires the activation of NF-kB and MAPK signaling pathways, which leads to the expression of NFATc1, a critical transcription factor for osteoclast differentiation. NFATc1, in turn, induces osteoclast formation and bone-resorbing activity. Here, we examined the effect of sudachitin on osteoclast differentiation and lipopolysaccharide (LPS)induced inflammatory bone resorption, and found that sudachitin suppressed osteoclast formation and function both in vitro and in vivo. Materials and Methods: In vitro osteoclast formation has been previously described.2 Bone marrow cells obtained from 48-week-old male C57BL/6 mice were cultured for 3 days in the presence of M-CSF to isolate osteoclast precursors. The osteoclast precursors were then treated with M-CSF and soluble RANKL to form mature osteoclasts. After culturing for the desired time, e-71
Poster Session TRAP-positive multinucleated cells formed in the culture, which were considered to be osteoclastic cells, were counted. Western blotting analysis and quantitative RTPCR was performed according to the ordinary protocols. To evaluate the effect of sudachitin on in vivo LPSinduced calvarial bone destruction, 8-week-old mice were injected with LPS subperiosteally in the calvarial bone daily for 5 days. After 6 days, mCT scanning of the calvariae was performed. The mean values of the groups were compared by unpaired Student’s t test or by oneway ANOVAs. P < 0.05 was considered significant. Results: When osteoclast precursors were treated with sudachitin in the presence of sRANKL and M-CSF, the number of TRAP-positive multinucleated osteoclasts formed in the culture was decreased compared with sudachitin-untreated culture in a dose-dependent manner [stachitin (10 mM) group vs. untreated group: 6.8 0.9 cells/well vs. 625.3 18.0 cells/well, p<0.05]. Since sudachitin had no effect on the proliferation and viability of osteoclast lineage cells, the inhibitory effect of sudachitin was attributed to the direct action in osteoclastogenesis. Consistent with the inhibition, sudachitin suppressed the activation of Erk signal in the osteoclast precursors. Furthermore, sudachitin decreased expression of c-fos and NFATc1 in mRNA and protein levels. Followed by the down-regulation of those transcription factors, the expressions of osteoclast differentiationrelated molecules such as TRAP, cathepsin K, DC-STAMP and Atp6v0d2 were also decreased. In addition, sudachitin suppressed the production of intracellular reactive oxygen species. Consistent with the in vitro experiments, when inflammatory bone destruction of calvariae was induced by LPS injection, simultaneous administration of sudachitin (50 mM) with LPS reduced the elevated bone resorption as well as the increased TRAP and cathepsin K expression in vivo [stachitin (10 mM) group vs. untreated group: relative amount of TRAP mRNA; 4.69 0.93 vs. 2.66 0.30 , p<0.05; relative amount of cathepsin K mRNA: 6.0 0.27 vs 2.52 0.37 , p<0.05]. Conclusion: Sudachitin, a polymethoxyflavone, inhibits osteoclast formation and suppresses LPS-induced inflammatory bone resorption in mice. Therefore, sudachitin could be an effective component for the prevention of inflammatory bone disorders. References: 1. Yuasa K, Tada K, Harita G, Fujimoto T, Tsukayama M, Tsuji A. Sudachitin, a polymethoxyflavone from Citrus sudachi, suppresses lipopolysaccharide-induced inflammatory responses in mouse macrophage-like RAW264 cells. Biosci Biotechnol Biochem, 76:598-600, 2012 2. Nakayachi M, Ito J, Hayashida C, Ohyama Y, Kakino A, Okayasu M, Sato T, Ogasawara T, Kaneda T, Suda N, Sawamura T, Hakeda Y. Lectin-like oxidized low-density lipoprotein receptor-1 abrogation causes resistance to inflammatory bone destruction in mice, despite promoting osteoclastogenesis in the steady state. Bone, 75:170-182, 2015
e-72
POSTER 36 Oral Melanoacanthoma: A Report of Two Cases and a Review of the Literature P. G. Tolomeo: New York University Langone Medical Center/Bellevue Hospital Center, J. S. Lee, N. Zawada, A. R. Kerr, J. A. Phelan Oral melanoacanthoma (MA) is a rare, benign pigmented lesion that presents as a painless, rapidly growing, brown-black macular lesion that commonly affects the buccal mucosa in areas that are subject to chronic trauma/irritation.1,2 MA is commonly seen in the third and fourth decades of life and primarily affects blacks with a strong female predilection.3,4 Histopathologically, the lesions exhibit proliferation of keratinocytes and dendritic melanocytes.5 This report includes two cases of oral melanoacanthoma and a review of the literature. Case 1: A 43-year-old black female presented with a slowly enlarging pigmented lesion on the right buccal mucosa. The patient did not recall any known trauma to the area or previous infection and reported that the lesion was painless but had a gradually increased in size. Oral examination revealed a 2.0 x 2.0 cm. brown macule on the right buccal mucosa. A punch biopsy was taken of the pigmented area. The tissue was placed in 10% formalin and submitted for microscopic examination. The tissue was stained with hematoxylin and eosin and exhibited acanthotic, stratified squamous epithelium with dendritic melanocytes dispersed throughout the epithelium consistent with a diagnosis of melanoacanthoma. Case 2: A-35 year-old black female presented with a rapidly growing pigmented lesion on the left buccal mucosa. Two years prior to presentation the patient had noted a brown lesion on the buccal mucosa adjacent to a fractured tooth. The lesion remained unchanged and asymptomatic for approximately two years. One week prior to presentation, the patient noted that the lesion was enlarging, but remained painless. Oral examination revealed a 1.5 x 1.5 cm. brown macule surrounded by erythema on the left buccal mucosa adjacent to a fractured tooth. A punch biopsy was taken that included both the pigmented and erythematous areas. The tissue was placed in 10% formalin and submitted for microscopic examination. The tissue was stained with hematoxylin and eosin and exhibited similar histopathologic features to the previous case. Immunohistochemical staining with S-100 and Melan-A dramatically demonstrated the dendritic melanocytes. Review of the literature revealed a total of 50 cases of oral melanoacanthoma. These lesions were reported in black females on the buccal mucosa with subsequent resolution. The cases here demonstrate similar clinical AAOMS 2016