Potentiation of Anticancer Activity of G2/M Blockers by Mild Hyperthermia.

BACKGROUND/AIM: Hyperthermia (HT), combined with chemotherapy, has been used to treat various types of cancer. This study aimed to investigate the HT-sensitivity of malignant and non-malignant cells, and then evaluate the combination effect of docetaxel (DTX) and a newly synthesized chromone derivative (compound A) with HT. MATERIALS AND METHODS: The number of viable cells was determined using the MTT method. Cell cycle distribution was analyzed using a cell sorter, and DNA fragmentation pattern was detected using agarose gel electrophoresis. RESULTS: Among 12 cultured cells, oral squamous cell carcinoma (OSCC), especially Ca9-22 cells, and myelogenous leukemia cells showed higher sensitivity to HT than lung carcinoma and glioblastoma cell lines, while normal oral cells were the most resistant. Cytotoxicity of DTX on Ca9-22 cells was maximum at 41-42°C and 45~60 min exposure to HT. DXT, compound A, and HT induced G2/M arrest of Ca-22 cells. Mild HT enhanced the DTX- and compound A-induced subG1 arrest, in a synergistic fashion. CONCLUSION: The combination G2/M blockers and mild-HT can potentially be used for the treatment of OSCC.

Resveratrol Inhibits Proliferation and Induces Autophagy by Blocking SREBP1 Expression in Oral Cancer Cells.

Resveratrol is a polyphenolic antioxidant found in grapes, red wine, and peanuts and has been reported to have anti-neoplastic effects on various cancer types. However, the exact mechanism of its anti-cancer effects in oral cancer is not fully understood and remains controversial. Resveratrol exhibits strong hypolipidemic effects; therefore, we examined its effect on lipid metabolism in oral cancer. Resveratrol significantly reduced cell viability and induced autophagic cell death in oral cancer cells but not in normal cells. This selective effect was accompanied by significantly reduced lipogenesis, which is caused by downregulation of the transcription factor sterol regulatory element-binding protein 1 (SREBP1) gene, followed by downregulation of the epidermal fatty acid-binding protein (E-FABP). It was strongly suggested that resveratrol-induced autophagy resulted from the inhibition of SREBP1-mediated cell survival signaling. Luciferase reporter assay further indicated that resveratrol has a potent and specific inhibitory effect on SREBP1-dependent transactivation. Importantly, resveratrol markedly suppressed the growth of oral cancer cells in an animal xenograft model, without exhibiting apparent cytotoxicity. In conclusion, resveratrol induces autophagy in oral cancer cells by suppressing lipid metabolism through the regulation of SREBP1 expression, which highlights a novel mechanism of the anti-cancer effect of resveratrol.

Spatial and chronological localization of septoclasts in the mouse Meckel's cartilage.

Meckel's cartilage (MC) in the first branchial arch of mammals is a transient structure that disappears before birth, except for the most anterior and posterior portions. Recent studies reported that some congenital abnormalities in craniofacial regions are linked with the persistence or dysplasia of MC. However, the mechanisms underlying the resorption of MC have not been elucidated. Cartilage resorption in endochondral ossification is performed by multinuclear osteoclasts/chondroclasts as well as mononuclear septoclasts, which were newly added to the list of cartilage phagocytes. Septoclasts located exclusively at the chondro-osseous junction of the growth plate resorb the uncalcified cartilage matrix. We hypothesized that septoclasts participate in the resorption of MC and attempted to clarify the localization and roles of septoclasts in MC of mouse using a specific immunohistochemistry marker, epidermal type-fatty acid-binding protein (E-FABP/FABP5). E-FABP-immunopositive septoclasts were detected for the first time at the beginning of MC resorption and localized along the resorption surface. Septoclasts of MC in embryonic mice possessed several processes that elongated toward the uncalcified cartilage matrix, expressed cathepsin B, and exhibited characteristic pericapillary localization. Additionally, they localized between hypertrophied cartilage and osteoclasts/chondroclasts in the resorption surface. Confocal laser-scanning microscopy revealed a decrease in the numbers of septoclasts and their processes with the progression of MC disappearance before birth. The present study showed that E-FABP-immunopositive septoclasts participated in the disappearance of MC through the resorption of the uncalcified cartilage matrix and that they have different roles from osteoclasts/chondroclasts.

A Case of Buccal Clear Cell Carcinoma Caused by Rare Fusion Gene: EWSR1-CREM.

Clear cell carcinoma (CCC) is a rare entity in the salivary gland tumor. So far, only 10 cases of primary CCC of the buccal mucosa have been reported. Here, we first report an extremely rare case of buccal CCC with the EWSR1-CREM fusion gene. The patient, a 69-year-old woman, presented with a painless mass in the right buccal mucosa. The tumor, which had been present for about 10 years, measured approximately 15 mm in diameter and was pedunculated, elastic hard, smooth, and mobile. Histopathological examination revealed proliferating tumor cells with vacuolated and clear cytoplasm partially surrounded by hyalinized stroma. The tumor was not encapsulated, and no contact with the overlying epithelium was evident. Duct-like structures were occasionally observed in the tumor nests composed of clear cells. The tumor had invaded into surrounding muscle and adipose tissues. Immunohistochemical examination revealed that the clear cells were positive for epithelial cell markers, and myoepithelial markers were negative. Fluorescence in situ hybridization (FISH), performed to search for genetic abnormalities, demonstrated split positivity for EWSR1, and fusion with CREM was confirmed. These findings suggested a diagnosis of CCC.

Down-regulation of Glutathione Peroxidase 4 in Oral Cancer Inhibits Tumor Growth Through SREBP1 Signaling.

BACKGROUND/AIM: This study aimed to elucidate the role of glutathione peroxidase 4 (GPX4) on the sterol regulatory element binding proteins (SREBPs)-proliferation pathway in oral cancer cells, and determine its protein expression in oral cancer tissues. MATERIALS AND METHODS: Quantitative RT-PCR and immunoblot analysis were carried out. Cell viability assay, apoptosis detection assay, immunohistochemistry and GPX4 knockdown were performed. RESULTS: The levels of both GPX4 mRNA and protein were highest in SAS cells. GPX4 knockdown in SAS cells, a human oral squamous cell carcinoma cell line, using GPX4 siRNA resulted in a reduction in cell number, which appeared to be due to non-apoptotic cell death such as ferroptosis. Furthermore, SREBP was clearly down-regulated by GPX4 knockdown in SAS cells. Immunopositivity for GPX4 was revealed on the membrane of human oral squamous cell carcinoma cells, and this was correlated with p53 immunoreactivity. CONCLUSION: GPX4 appears to play an important role in oral cancer proliferation.

Domain structures of mucosa-associated lymphoid tissue lymphoma translocation 1 protein for nuclear localization in oral carcinoma cells and the proliferation inhibition.

Mucosa-associated lymphoid tissue lymphoma translocation 1 protein (MALT1) consisting of death domain, Ig-like domains and caspase-like domain is expressed in nucleus of oral carcinoma cells, and loss of the expression closely associates with disease progression and stimulates proliferation of the cells. However, nothing is known about the molecular backgrounds. In this study, eight constructs with different domain constitution of human MALT1 and six constructs were transiently and stably transfected into oral carcinoma cell lines, respectively. The immunoblot analysis showed that constructs containing caspase-like domain was expressed in nucleus and the domain-deleted constructs in cytoplasm. Immunocytochemistry of stably transfected HSC2 oral carcinoma cells confirmed the caspase-like domain-dependent nuclear localization. Involvement of domains in proliferation of stably transfected HSC2 cells was quantified by the real-time and conventional colorimetric assays. In contrast to suppression of the proliferation by full-length wild-type MALT1, any domain-deleted constructs enhanced the proliferation. Death domain construct without caspase-like domain suppressed the proliferation when it was localized in nucleus by ligating with the nuclear localization signal. These results demonstrate that nuclear localization of MALT1 in oral carcinoma cells depends on the presence of caspase-like domain and that death domain nuclear entity is responsible for MALT1 inhibition of oral carcinoma cell proliferation. Nuclear localization of death domain led by caspase-like domain may suppress oral carcinoma progression.

Palatal Polymorphous Adenocarcinoma with High-Grade Transformation: A Case Report and Literature Review.

Polymorphous adenocarcinoma (PAC) is the second most common intraoral malignant neoplasm of the minor salivary glands. However, it is very rare for PAC to show high-grade transformation (HGT) and to our knowledge, the English literature only seven reported cases. HGT tends to be observed when PAC recurs, and it is extremely rare to be seen at initial presentation. Here we report a 43-year-old Japanese male patient with PAC of the right palate showing HGT at initial presentation. Histopathologically, the tumor was characterized by a prominent solid and papillary-cystic growth pattern, with nuclear atypia and necrosis in area of HGT. The immunohistochemical staining pattern was consistent with PAC, as the tumor cells showed diffuse positivity for cytokeratin, vimentin and S-100, and focal positivity for bcl-2, ɑ-SMA and EMA. The tumor cells in HGT areas were markedly positive for AR and Ki-67 (about 40%/HPF), and also focally positive for cyclin D1 and p53, whereas HER2/neu, ER, PgR, p63, D2-40, GCDFP-15, and mitochondria were negative. Here we present a very rare case of palatal PAC with HGT at initial presentation.

A large-scale study of treatment methods for foreign bodies in the maxillary sinus.

Recently, reports regarding a foreign body in the maxillary sinus have considerably increased, with the majority being iatrogenic cases resulting from dental treatment. This study involves an extensive review of the Japanese literature, including 112 papers from 1978 to 2017. These papers documented total 407 cases of a foreign body in the maxillary sinus. Among the 392 cases for which treatment details were available, the Caldwell-Luc approach was used for 216, the alveolar approach for 116, extraction using nasal endoscopy for 15, and extraction using oral endoscopy for eight. Spontaneous passage occurred in 19 cases, follow-up with medication was used in 17, and "other" was noted in one. This study determined that surgical removal remains the most common method for treating both tooth roots and other foreign bodies and that the Caldwell-Luc approach is used in majority of the surgeries. No marked differences were noted among the removal methods used in relation to the foreign body type.

Initial-Stage Primary Intraosseous Squamous Cell Carcinoma Derived from Odontogenic Keratocyst with Unusual Keratoameloblastomatous Change of the Maxilla: A Case Report and Literature Discussion.

Primary intraosseous squamous cell carcinoma (PIOSCC) is a rare malignant neoplasm derived from odontogenic epithelial remnants in the central jaw bone. Most PIOSCCs originate from odontogenic cysts with a nonkeratinized epithelial lining, especially from radicular/residual and dentigerous cysts. There have been few reports of PIOSCCs derived from the odontogenic keratocyst (OKC), particularly those describing pathological features at the initial stage. The diagnosis of PIOSCC is difficult and based on exclusion of other carcinomas, including metastatic tumors from other primary sites. Here, we report an extremely rare case of initial-stage PIOSCC derived from the OKC with unusual keratoameloblastomatous change of the maxilla.

Synergism between α-mangostin and TRAIL induces apoptosis in squamous cell carcinoma of the oral cavity through the mitochondrial pathway.

Mangosteen (Garcinia mangostana) is a tree found in South-East Asia and the pericarp of its fruit has been used in folk medicine for the treatment of many human illnesses. Mangosteen fruit rinds contain a high concentration of xanthone, which is a type of polyphenol. One type of xanthone, α-mangostin, has been reported to exert chemopreventive effects against chemically-induced colon cancer through the decrease of c-Myc expression, suppressing tumor growth in a mouse model of mammary cancer. A recent study demonstrated the inhibitive effect of α-mangostin on the growth of prostate cancer. However, it remains unclear whether α-mangostin induces cell death in oral cancer. The present study examined the impact of α-mangostin on human oral squamous cell carcinoma (HOSCC). Firstly we analyzed the expression of c-Myc in five HOSCC cell lines. The highest expression level of c-Myc mRNA was observed in SAS cells and the lowest in HSC-4 cells. Therefore, SAS cells were treated with α-mangostin, which was found to exert a weak cytocidal effect. Since α-mangostin has been reported to exert synergistic effects on cancers when combined with anticancer drugs, we attempted to evaluate such synergistic effects of α-mangostin when used with a cytokine, tumor necrosis factor (TNF)-related apoptosis­inducing ligand (TRAIL). We found that the combination of α-mangostin with TRAIL induced apoptosis of SAS cells through the mitochondrial pathway via activation of caspase-9 and -3/7, following release of cytochrome c. This apoptosis was induced by S/G2/M-phase arrest. Immunopositivity for c-Myc was observed in the cytoplasm of tumor cells in 16 (40%) of the 40 cases of HOSCC. These data revealed that the combination of α-mangostin and TRAIL may have a considerable potential for the treatment of oral cancer.

Periostitis Ossificans Arising in the Mandibular Bone of a Young Patient: Report of an Unusual Case and Review of the Literature.

Periostitis ossificans, also known as Garré osteomyelitis, is a specific type of chronic osteomyelitis that forms new bone under the periosteum resulting from a periosteal reaction to chronic inflammation or infections. It commonly affects the mandible secondary to odontogenic infection. The therapeutic approach involves eliminating the infectious cause and antibiotic administration. This report describes an unusual case of periostitis ossificans arising from the mandible of an 11-year-old boy. The cause of infection was correlated with a lower right unerupted third molar, which had no obvious connection with the oral cavity. The histologic diagnosis was chronic osteomyelitis with proliferative periostitis. The patient has been followed for 1 year, without any evidence of recurrence. Periostitis ossificans can be diagnostically problematic, and various conditions must be considered in the differential diagnosis.

Detection of Epstein-Barr virus genome and latent infection gene expression in normal epithelia, epithelial dysplasia, and squamous cell carcinoma of the oral cavity.

A relationship between Epstein-Barr virus (EBV) infection and cancer of lymphoid and epithelial tissues such as Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma (NPC), gastric carcinoma, and oral cancer has been reported. EBV is transmitted orally and infects B cells and epithelial cells. However, it has remained uncertain whether EBV plays a role in carcinogenesis of oral mucosal tissue. In the present study, we detected the EBV genome and latent EBV gene expression in normal mucosal epithelia, epithelial dysplasia, and oral squamous cell carcinoma (OSCC) to clarify whether EBV is involved in carcinogenesis of the oral cavity. We examined 333 formalin-fixed, paraffin-embedded tissue samples (morphologically normal oral mucosa 30 samples, gingivitis 32, tonsillitis 17, oral epithelial dysplasia 83, OSCC 150, and NPC 21). EBV latent infection genes (EBNA-2, LMP-1) were detected not only in OSCC (50.2 %, 10.7 %) but also in severe epithelial dysplasia (66.7 %, 44.4 %), mild to moderate epithelial dysplasia (43.1 %, 18.5 %), gingivitis (78.1 %, 21.9 %), and normal mucosa (83.3 %, 23.3 %). Furthermore, the intensity of EBV latent infection gene expression (EBER, LMP-1) was significantly higher in severe epithelial dysplasia (94.4 %, 72.2 %) than in OSCC (34.7 %, 38.7 %). These results suggest that EBV latent infection genes and their increased expression in severe epithelial dysplasia might play an important role in the dysplasia-carcinoma sequence in the oral cavity.

Comparative study of cytokeratin and langerin expression in keratinized cystic lesions of the oral and maxillofacial regions.

Dermoid cysts (DMCs) and epidermoid cysts (EDMCs) usually arise in soft tissues, whereas orthokeratinized odontogenic cysts (OOCs) and keratocystic odontogenic tumors (KCOTs) develop in the jaw. In this study, we performed immunohistochemical analysis of cytokeratins (CKs) to examine differences in the lining epithelium of DMCs, EDMCs, OOCs, and KCOTs. In addition, we carried out immunohistochemical examination of langerin to clarify the biological characteristics of the orthokeratinized lining epithelium of DMCs, EDMCs, and OOCs. Seven DMCs, 30 EDMCs, 11 OOCs, and 28 KCOTs were examined immunohistochemically using antibodies against CK10, 13, 14, 16, 17, 19, and langerin. Immunoreactivities for CKs and langerin in oral DMCs and EDMCs were similar to those of lesions affecting the skin. Positive reactivity for CK13 and 17 was evident in OOCs, but not in DMCs/EDMCs. CK10 was significantly positive in all layers except for the basal layer in OOCs, but was negative in KCOTs. CK17 was positive in all layers in KCOTs, and in all layers except for the basal layer in both OOCs and dentigerous cysts. CK19 was negative in OOCs. Langerhans cells were found mainly in OOCs, but were hardly evident in KCOTs. These results suggest that DMCs/EDMCs, OOCs and KCOTs are independent diseases.

Development and regression of the thyroglossal duct in mice.

The thyroid anlage develops in the foramen caecum area of the tongue, and migrates through the anterior neck towards its final position in front of the laryngeal cartilages. During migration, the thyroglossal duct, a temporary structure connecting the thyroid anlage and the foramen caecum, is recognized. In the present study, chronological changes and apoptosis in the thyroglossal duct of mice were investigated histochemically using an antibody against Nkx2-1, initially identified as a thyroid transcription factor 1 (TTF1), and the TUNEL reaction in consecutive serial sagittal sections. At embryonic day 10.00 (E10.00), the thyroid anlage was Nkx2-1-immunoreactive and located just below the foramen caecum. As the thyroid anlage descended, the thyroglossal duct was formed at E10.25, being less than 10µm in diameter. By E10.75, the Nkx2-1-positive thyroglossal duct had progressively elongated up to 100µm. At E11.00 the thyroglossal duct began to disappear, beginning in its mid-portion, and finally became invisible at E11.50. At E11.00-12.00, apoptotic cells were found in an area where the thyroglossal duct was partially discontinuous. After E12.00, cartilaginous tissue of the hyoid bone anlage developed in the mid-portion of the area where the thyroglossal duct had regressed. Immunoreactivity for thyroglobulin, a marker of differentiated thyroid endocrine cells, was detected at E13.00. These results strongly suggest that the mouse thyroglossal duct disappears as a result of apoptosis before differentiation of the endocrine thyroid.

Podoplanin expressing cancer-associated fibroblasts in oral cancer.

Podoplanin is a mucin-type glycoprotein widely used as a lymphatic endothelial marker. It is well known that podoplanin is expressed in various neoplasms including oral squamous cell carcinoma (OSCC). Apart from podoplanin expression in cancer cells, recent studies have suggested that podoplanin expression in stromal cancer-associated fibroblasts (CAFs) may be an indicator of poor prognosis in various cancers. In the present study, we performed immunohistochemical analyses of podoplanin and alpha-smooth muscle actin (α-SMA) in OSCC in order to clarify the significance of podoplanin-positive CAFs. Paraffin-embedded tissue specimens of 69 primary and 29 corresponding metastatic lesions in lymph nodes were examined immunohistochemically using antibodies against podoplanin and α-SMA. Podoplanin-positive stromal fibroblasts were detected in 51 (73.9%) of the 69 primary OSCCs and 24 (82.8%) of the 29 lymph nodes metastases. α-SMA immunoreactivity was observed in 39 (56.5%) of the primaries and 24 (82.8%) of the metastases. Further examination showed that 38 (74.5%) of the primary lesions and 23 (95.8%) of the metastases with podoplanin positivity were also positive for α-SMA. In addition, the intensity of α-SMA immunoreactivity increased as that of podoplanin became stronger. Podoplanin-positive CAFs are considered to be myofibroblasts that may contribute to progression of oral cancer.

Differential expression of fatty acid-binding proteins and pathological implications in the progression of tongue carcinoma.

Tongue carcinomas are common malignancies of the oral cavity. Understanding the molecular mechanisms behind the disease progression is a prerequisite for improving patient prognosis. Fatty acid-binding proteins (FABPs) are cytoplasmic lipid chaperones that affect cellular organization and energy production. Although their aberrant expression is involved in carcinoma progression, its role in the pathology of tongue carcinomas remains unclear. In the present study, the immunohistochemical expression of FABP4 and FABP5 in tongue carcinomas (n=58) and its involvement in the clinicopathological parameters were examined. Normal tongue epithelial cells expressed FABP5, an epidermal-type FABP, but not FABP4, an adipocyte-type FABP. The cytoplasmic staining of FABP5 was increased in carcinomas with advanced T-stage (P<0.05) and clinical stage (P<0.05). Ectopic expression of FABP4 was detected in almost all carcinomas, although its role in disease progression remains undetermined. Upregulation of FABP5 in the wounded skin of genetically normal mice indicated that microenvironmental tissue factors induce FABP5 expression. The results of the present study demonstrated the aberrant expression of FABP4 and FABP5 in tongue carcinomas and suggested the involvement of FABP5 in disease progression.

Proteomic identification of keratin alterations with enhanced proliferation of oral carcinoma cells by loss of mucosa-associated lymphoid tissue 1 expression.

Progression of oral carcinomas associates with aberrant activation and inactivation of molecules that work in established or unknown pathways. Although mucosa­associated lymphoid tissue 1 (MALT1) expressed in normal oral epithelium is inactivated in the aggressive subset of carcinomas with worse prognosis, phenotypic changes of carcinoma cells upon the loss of expression is unknown. We performed a proteomic analysis to identify MALT1­regulated proteins in oral carcinoma cells. Four different keratins were included in the ten most abundantly changed proteins. K8/18 were upregulated in MALT1 stably­expressing carcinoma cells and K5/14 in MALT1­marginal control cells. K8/18 upregulation and K5/14 downregulation were MALT1 dose­dependent and observed in a series of oral carcinoma cells. MALT1 suppressed cell proliferation (0.52-fold, P<0.01) and its dominant-negative form stimulated it (1.33-fold, P<0.01). The decreased proliferation associated with reduction of cyclin D1, which was recovered by the short interfering RNA against MALT1. Taken together, loss of MALT1 expression alters keratin expression and enhances proliferation of carcinoma cells, and may progress oral carcinomas into the advanced state.

Podoplanin promotes cell migration via the EGF-Src-Cas pathway in oral squamous cell carcinoma cell lines.

Human podoplanin is a type-1 transmembrane sialomucin-like glycoprotein that is involved in cell migration, tumor cell invasion and metastasis. Our recent study of oral squamous cell carcinoma (OSCC) demonstrated that the degree of immunohistochemical expression of podoplanin was correlated with the severity of epithelial dysplasia and significantly associated with a poor pathologic grade of differentiation. Furthermore, it has been reported that Src directly associates with the epidermal growth factor receptor (EGFR) in OSCC cells upon stimulation with EGF and phosphorylates Crk-associated substrate (Cas), podoplanin acting downstream of Src and Cas to promote cell migration. However, the molecular function of podoplanin remains unclear. In this study we performed real-time RT-PCR, Western blotting and scratch assay using OSCC cell lines in order to clarify the molecular biological function of podoplanin expression associated with various growth factors including EGF and with the Src-Cas signaling pathway. Podoplanin was found to have a marked influence on cancer cell migration and the expression of matrix metalloprotease-9 (MMP-9) in the oral cavity upon stimulation with EGF. Podoplanin promotes oral cancer cell migration, and the EGF-Src-Cas pathway is one of the possible mechanisms responsible for progression of cancer in the oral cavity.

Immunohistochemical expression of podoplanin in so-called hard α-keratin-expressing tumors, including calcifying cystic odontogenic tumor, craniopharyngioma, and pilomatrixoma.

Podoplanin, a transmembrane sialomucin-like glycoprotein, is a specific marker of lymphatic vessels, and its expression is also considered to be associated with tumor invasion and tooth development. In this study, we examined the expression of podoplanin in calcifying cystic odontogenic tumor (CCOT) in comparison with that in other so-called hard α-keratin-expressing tumors such as craniopharyngioma (CP) and pilomatrixoma (PM). Immunohistochemical staining for podoplanin was carried out using surgical specimens of 15 CCOTs of the jaw, 19 CPs of the pituitary gland, and 15 PMs of the skin. Positivity for hard α-keratin was evident in ghost, shadow and transitional cells in all of these tumors (100%). The podoplanin expression in CCOTs was evident in the periphery of ameloblastoma-like epithelium (86.6%) and the epithelial cells adjacent to ghost cells (60%). On the other hand, in adamantinomatous-type CPs, podoplanin expression was observed in epithelial components corresponding to the stratum intermedium (100%), but not in the periphery of ameloblastoma-like epithelium (0%). In squamous-type CPs podoplanin was expressed in basal cells (100%), but all of the PMs were podoplanin-negative (0%). In the periphery of the ameloblastoma-like epithelium or basophilic cell layer, podoplanin was expressed more strongly in CCOTs than in CPs or PMs. These findings suggest that the expression of podoplanin in CCOTs may reflect rapid turnover of cytoskeletal filaments and local invasiveness.