Promotion of hematogenous metastatic potentials in human KB carcinoma cells with overexpression of cyclooxygenase-2.

To understand the role of cyclooxygenase (COX)-2 in metastatic potential of oral cancer, COX-2 overexpressing KB/COX-2 cells were inoculated orthotopically into the masseter muscle or injected into the left cardiac ventricle of nude mice. KB/COX-2 showed about 4-fold increase of COX-2 protein expression as compared to KB/Neo which was a mock transfected control. In orthotopic inoculation, metastasis to the regional lymph nodes occurred in 2 out of 15 mice, and metastasis to the lung in 3 out of 15 mice. On the other hand, in intra-cardiac injection, hematogenous metastasis to the lung and bone occurred in 8 out of 10 mice in KB/COX-2, but no metastasis occurred except for only one metastasis to the femur bone out of 10 mice in KB/Neo. Treatment of KB/COX-2 with COX-2 small interfering RNA (siRNA) inhibited the colony formation but not cell growth in vitro, and suppressed tumorigenicity and hematogenous metastasis in nude mice. When expression of adhesion molecules such as E-cadherin, alpha-catenin, beta-catenin and CD44 was examined, there was no difference in alpha- and beta-catenin between the cells. However, expression of E-cadherin was detected in KB/Neo, but not in KB/COX-2. In contrast, expression of CD44 was markedly increased in KB/COX-2 as compared to KB/Neo. Treatment with COX-2 siRNA resulted in suppression of CD44 expression and detectable expression of E-cadherin in KB/COX-2. These findings suggested that overexpression of COX-2 increased hematogenous metastasis, at least in KB cells, via down-regulating E-cadherin and up-regulating CD44 expression.

Keratotic basal cell carcinoma of the tongue: case report.

Basal cell carcinoma (BCC) is the most common skin cancer. Its occurrence in the oral mucosa is extremely rare. We report the clinical course of BCC arising in the inferior surface of the tongue. We performed a super selective intra-arterial chemotherapy combined with radiotherapy for this case. Local tumor response showed CR, and no recurrence was seen after the treatment.

Relations among expression of CXCR4, histological patterns, and metastatic potential in adenoid cystic carcinoma of the head and neck.

Adenoid cystic carcinoma (ACC) may acquire a chemokine-mediated mechanism during the process of metastasis. To investigate the involvement of chemokines in metastasis from ACC, expression of CXCR4 in surgical specimens of ACC and two tumor lines transplantable to nude mice was examined immunohistochemically. In addition, the expression levels of CXCR4 protein and mRNA were examined by Western blotting and reverse-transcription polymerase chain reaction. Our results showed that patients whose tumors expressed high levels of CXCR4 had metastases to the regional lymph nodes and the lung, resulting in poor outcomes. ACCs showing a solid or cribriform pattern with distant metastasis were strongly positive for CXCR4, while those showing a tubular or cribriform pattern without metastasis were weakly positive for CXCR4. In the in vivo model, ACCY tumor showed increasing expression levels of CXCR4 with tumor growth, and the histological pattern changed from cribriform to solid. The histological pattern of ACCI, associated with spontaneous metastasis to the neck, changed from cribriform to undifferentiated carcinoma and was highly metastatic to the lung. This tumor showed high levels of CXCR4 protein and mRNA. These results suggest that CXCR4 expression, histological patterns, and metastatic potential are closely related in ACC.

Establishment of a nude mouse transplantable model of a human malignant fibrous histiocytoma of the mandible with high metastatic potential to the lung.

Malignant fibrous histiocytoma (MFH) is one of the highest-grade sarcomas arising in bone and soft tissue. Its prognosis is poor because of chemoresistance and high metastatic potential to various organs. Few cases arising of MFH of the mandible or oral cavity have been documented. We established a tumor line in nude mice (MFH-N), which was derived from human MFH of the mandible and examined the characteristics of this tumor line. Histologically, MFH-N was identical to the original tumor and showed a storiform-pleomorphic pattern, but had low metastatic potential. Immunohistochemically, both the original and xenografted tumors expressed vimentin, S-100, alpha-SMA, and histiocytic marker CD68. Lysozyme was expressed by the original tumor, but only sporadically by the xenografted tumor. RT-PCR analysis demonstrated human beta-actin in this tumor line, indicating the human origin. In a parallel experiment, we established a new MFH cell line (MFH-NC) from MFH-N. Tumor cells inoculated into the flanks and submandibular region of nude mice developed into tumors histologically similar to MFH-N and the original tumor; multiple lung metastases were detected approximately 5 months after inoculation. The expression levels of various metastasis-related molecules differed between MFH-N and MFH-NC on Western blotting. In MFH-NC, the expressions of MMP7, MMP9, MT1-MMP, CXCR4, COX-2 and integrin alpha4 were up-regulated, while those of MMP2 and TIMP1 were down-regulated. Expression of TIMP2, integrinalphaL and sialyl lewis X were not detected in either line. Our findings suggest that the MFH-N tumor line transplantable in nude mice is a useful model for studying the biological behavior of MFH.

Expression of cyclooxygenase-2 and DNA topoisomerase II alpha in precancerous and cancerous lesions of the oral mucosa.

The involvement of cyclooxygenase (COX)-2 in oral carcinogenesis and outcome of the patients is not fully understood. To determine whether COX-2 expression could serve as an indicator for them, we examined the expression of COX-2 and DNA topoisomerase (DNA-Topo) II alpha as an index of cell proliferating activity in precancerous and cancerous lesions of the oral mucosa. A 164 samples composed of 60 intraepithelial dysplasias (IEDs), 12 carcinomas in situ (CISs), 72 squamous cell carcinomas (SCCs) including 12 early invasive SCCs, 10 undifferentiated carcinomas (UCs), and 10 epithelial hyperplasias (EHPs) in the oral mucosa were examined immunohistochemically for COX-2 and DNA-Topo II alpha. Normal squamous epithelium as the control showed no COX-2 expression, whereas 41% of IEDs, 67% of CISs, 74% of SCCs, and 86% of UCs demonstrated increased COX-2 expression with elevated DNA-Topo II alpha labeling index (LI). High COX-2 expression was also observed in 61% of EHPs, but DNA-Topo II alpha LI was very low. Increased expression of COX-2 protein correlated with elevated DNA-Topo II alpha LI, indicating that COX-2 may contribute to malignant transformation and tumor growth. These two enzyme activities were increased as T, N, and M categories and stages proceeded. The patients with high expression of both COX-2 and DNA-Topo II alpha showed poor prognosis. Our results suggested that COX-2 expression become a possible indicator in oral carcinogenesis and may reflect the outcome of the patients.

Prognostic significance of cyclooxygenase-2 and DNA topoisomerase IIalpha expression in oral carcinoma.

BACKGROUND: Despite recent advances in the diagnosis and treatment of oral carcinoma, outcomes remain disappointing. The identification of new prognostic factors is necessary to improve survival. To determine the prognostic significance of cyclooxygenase (COX)-2 and DNA topoisomerase (DNA-Topo) IIalpha expression in patients with oral carcinoma, we immunohistochemically examined these enzymes and studied their relation to overall 5-year survival. METHODS: Surgical specimens were obtained from 160 patients with oral carcinoma, 80 with and 80 without regional lymph node metastasis. The specimens were immunostained for COX-2 and DNA-Topo IIalpha as an index of cell proliferative activity. COX-2 immunoreactivity and clinicopathological data were analyzed, and 5-year survival was calculated by the Kaplan-Meier method. RESULTS: COX-2 expression in primary lesions was higher in cases with lymph node metastasis than in those without lymph node metastasis. An increase in tumor size was associated with increased COX-2 expression. In most cases with lymph node metastasis, COX-2 expression was higher in metastatic lesions than in primary lesions. As COX-2 expression increased, the DNA-Topo IIalpha labeling index significantly increased and the overall 5-year survival rate decreased. CONCLUSION: Expression of COX-2 and DNA-Topo IIalpha were related to lymph node metastasis, cell proliferative activity, and overall 5-year survival rate in oral carcinoma. These enzymes may therefore be valuable diagnostic and prognostic indices in oral carcinoma.

Establishment and characterization of a human adenoid cystic carcinoma cell line forming colonies cultured in collagen gel and transplantable in nude mice.

We established a new cell line (ACCNS) from human adenoid cystic carcinoma (AdCC) of the maxilla, using tissue culture techniques and it has been successfully subcultured for more than 100 passages during 2 years. The population doubling time was approximately 39.2 h on a plastic dish. In type I collagen gel culture, the cells formed spherical colonies by day 7 after seeding. The colonies showed tubular and solid structures, and eosinophilic material stained with mucicarmine was revealed in the inner space. Immunohistochemically, ACCNS cells demonstrated expressions of keratin, alpha-smooth muscle actin, vimentin and S-100 protein, similar to those of original AdCC. These findings indicate that ACCNS cells possess the characteristics of AdCC. In addition, inoculation of 4.0x10(6) cells into nude mice developed tumors that were histologically confirmed as undifferentiated carcinoma. Therefore, ACCNS is the first AdCC cell line with tumorigenicity in nude mice. Based on these results, ACCNS provides a useful culture model of AdCC to analyze the biological characteristics and behavior of this tumor.

Establishment of nude mouse transplantable model of a human adenoid cystic carcinoma of the oral floor showing metastasis to the lymph node and lung.

Adenoid cystic carcinoma (ACC) is a generally slow-growing but highly malignant salivary gland neoplasm with remarkable capacities for local invasion and lung metastasis. The precise characteristics of ACC are not fully understood because there was no suitable animal model. We have successfully established a new human tumor line (ACCI) derived from ACC of the oral floor, which showed a cribriform pattern histologically and serially transplantable into nude mice. This tumor developed spontaneous metastasis to the neck at the second passage level, and the histological feature changed from ACC to undifferentiated carcinoma (ACCIM). ACCIM caused spontaneous metastasis to the lung at high incidence when transplanted subcutaneously in nude mice. In this study, we examined the characteristics of this interesting human ACC metastatic line. Tumor fragments were subcutaneously transplanted into nude mice and tumor growth was measured at 1-week intervals. Histological and immunohistochemical examinations were performed. As a result, the tumor growth rate of ACCIM increased as compared to that of ACCI, and the PCNA labeling index was elevated. Furthermore, ACCIM produced multiple metastases to lymph nodes and lungs 5 months after transplantation, and all mice died within 6 months. These multiple metastases were also confirmed in orthotopic transplantation to the tongue. RT-PCR analysis revealed that ACCIM expressed human beta-actin, indicating its human origin. From these findings, ACCIM transplanted into nude mice would provide a useful model for investigating the biological behaviour of ACC.

Elevated cell migration, invasion and tumorigenicity in human KB carcinoma cells transfected with COX-2 cDNA.

In order to investigate the involvement of cyclooxygenase (COX)-2 in cell growth and invasion of oral cancer, a human epidermoid carcinoma cell line KB minimally expressing COX-2 protein was transfected with COX-2 cDNA and these activities were compared with mock-transfected KB in vitro and in vivo. KB/COX-2 clones showed a similar growth rate in vitro compared to KB/neo clones, but demonstrated significantly increased PGE2 production, cell migration and invasion. These KB/COX-2 clones markedly expressed MMP-9, pro-MMP-2 and activated-MMP-2 as compared to KB/neo clones in gelatin zymography. Western blot analysis showed that expression of MT1-MMP, Rho and Rac 1 in KB/COX-2 clones were stronger than that in KB/neo clones, but expression of TIMP-1 and TIMP-2 were weaker in KB/COX-2 clones than in KB/neo clones. When these cells were inoculated subcutaneously into nude mice, tumorigenicity and tumor growth were significantly elevated in KB/COX-2 tumors than in KB/neo tumors, and the gelatinase activity was much stronger in KB/COX-2 tumor tissues than in KB/neo tumor tissues in film in situ zymography. The orthotopic inoculation of cells to the oral floor showed that local invasion was pronounced in KB/COX-2 tumors. These results indicated that overexpression of COX-2 elevated tumorigenicity, tumor growth and invasion of human KB carcinoma cells via up-regulated MMP and Rho family small GTPases and down-regulated TIMP activities.

Mucinous adenocarcinoma of the temporal region initially diagnosed as temporomandibular disorders: a case report.

Adenocarcinoma occurring in the temporal region has not previously been reported. We present a case of mucinous adenocarcinoma of the temporal region. A 62-year-old female patient was diagnosed as having temporomandibular disorders because of severe trismus and joint pain. Although trismus progressively worsened, there were no abnormal findings on diagnostic imaging studies including magnetic resonance imaging (MRI) and bone scintigraphy. As swelling of the temporal region was observed, biopsy was performed. Histologic examination showed chronic inflammation of the striated muscle. Approximately 6 months later, follow-up MRI demonstrated an ill-defined mass lesion in the infratemporal region extending to the intracranium. Histologic diagnosis of the biopsy showed that this mass lesion was moderately differentiated mucinous adenocarcinoma.

Changes of histological and biological features by serial passages in a human adenoid cystic carcinoma line transplantable in nude mice.

The basic histologic patterns of adenoid cystic carcinoma (ACC) are classified into three types (tubular, cribriform and solid), but clinical significance of the histological type is unclear. We have successfully established a human tumor line derived from ACC that is serially transplantable in nude mice. This tumor showed an increased growth rate as the passage levels proceeded, and the histological type was changed from a cribriform pattern in the initial stage to a solid one. In this study, we investigated the relationship between histological type and biological characteristics by analyzing the serially transplantable ACC tumor model. As a result, the tumor growth rate at the 15th passage level was increased approximately 5-fold compared with that at the initial passage level. In the histological type, approximately 30% of the cribriform pattern in the initial level was changed to a solid one at the 15th passage level, and the PCNA labeling index was elevated 4-fold. Concomitant with this, expression of Ki-67, p53 and bcl-2 proteins was increased, and apoptotic cells were decreased as demonstrated by the TUNEL method. From these findings, it was suggested that cell proliferation and histological change of this ACC tumor are related to the inhibition of apoptosis. This tumor line would provide a useful model for investigating the biological behavior of ACC.

Promotion of cell differentiation, and suppression of cell growth and cyclooxygenase-2 expression by differentiation-inducing agents in human oral squamous carcinoma SCC25 cells.

We investigated the relationship between cell growth and differentiation and COX-2 expression in oral squamous cell carcinoma (SCC) in vitro and in vivo. Treatment of SCC25 oral squamous carcinoma cells with sodium butyrate (SB) at 0.5-5 mM or all-trans retinoic acid (ATRA) at 3-300 microM inhibited cell growth and induced apoptosis in a dose-dependent manner with concomittant increases in expression of keratin 13, p21WAF1/Cip1 and p27Kip1 and decreases in expression of COX-2. These effects were more pronounced with SB than with ATRA. Injection of SB or ATRA near SCC25-derived tumors in nude mice resulted in inhibition of growth and elevation of differentiation of the tumor accompanied by marked keratinization and increased expression of keratin 13 and decreased expression of COX-2. These results show that the differentiation-inducing agents, particularly SB, suppress growth of oral squamous carcinoma cells through apoptosis and induce cell differentiation possibly through mechanisms involving COX-2, p27Kip1 and/or p21WAF1/Cip1 in vitro and in vivo.

Increased expression of cyclooxygenase (COX)-2 in DMBA-induced hamster cheek pouch carcinogenesis and chemopreventive effect of a selective COX-2 inhibitor celecoxib.

BACKGROUND: In recent years, overexpression of cyclooxygenase (COX)-2 protein and mRNA has been reported in various cancer tissues. Therefore, it has been suggested that COX-2 is related to carcinogenesis. METHODS: Hamsters were treated by painting a buccal pouch with a 0.5% DMBA solution dissolved in acetone. Basal diet or diets containing 150, 500 and 1500 ppm of celecoxib, a selective COX-2 inhibitor, were given ad libitum to hamsters, and tumor development was observed. RESULTS: Immunohistochemical and Western blot analyses revealed that COX-2 expression was increased toward the carcinogenesis. Although all hamsters developed squamous cell carcinoma, the onset of tumor formation was delayed in a dose-dependent manner. Also, tumor growth was retarded and survived animals were increased in the group of celecoxib treatment. Histologically, administration of celecoxib increased the apoptotic cells in the tumor parenchyma and significantly inhibited the angiogenesis in the stroma. CONCLUSIONS: The COX-2 expression was increased during hamster cheek pouch chemical carcinogenesis. Administration of celecoxib demonstrated the chemopreventive potential against the carcinogenesis.

Establishment of a new cell line with neuronal differentiation derived from small cell neuroendocrine carcinoma of the maxillary sinus.

OBJECTIVE: It is well known that small cell neuroendocrine carcinoma (SNEC) arising at extrapulmonary sites has a poor prognosis and an interesting biological characterization. To understand biological characterization and elucidation of the origin of the histogenesis of SNEC, we report the establishment of a new SNEC cell line and characteristics of neuroendocrine properties including neuronal differentiation by treatment with dibutyryl cyclic AMP (db-cAMP). METHODS: We established a new cell line (SNEC-MI) derived from SNEC of the maxillary sinus by a modified spill-out method, and verified neuroendocrine properties including neuronal differentiation by immunocytochemical and immunoblotting methods. RESULTS: The established cell line showed spherical or spindle shape in monolayer culture and was positive for neuron-specific enolase (NSE), neuronal cell adhesion protein (N-CAM, CD56) and gastrin-releasing peptide. NSE was also demonstrated in the cultured medium and dense-core neuroendocrine granules were detected ultrastructurally in the cytoplasm. Treatment of cells with db-cAMP markedly induced the development and elongation of neuronal processes, which formed a netlike arrangement. Characterization of these elongated neuronal processes revealed them immunoreacting intensely with high molecular-weight neurofilament, and a time-dependent increase of microtubule-associated protein-2 in cell lysates. CONCLUSIONS: These findings indicated that this cell line possesses the capability to differentiate into neuronal cells, and supported the hypothesis that extrapulmonary SNEC might be derived from a pluripotent stem cell.

Endovascular papillary angioendothelioma (Dabska tumor) of the tongue: report of a case.

Endovascular papillary angioendothelioma (EPA), known as Dabska tumor, is a very rare vascular neoplasm that usually occurs in the skin or subcutis of infants and young children. There is no previous report of EPA appearing in the oral cavity. Here, we present an exceptionally rare case of EPA of the tongue in a 67-year-old man. A well-defined, reddish tumor measuring 11 mm x 8 mm x 7 mm existed at the submucosal area of the left tongue margin. Microscopic examination of an excisional biopsy specimen revealed the endothelioid tumor cells showing a papillary growth pattern, such as blood vessel-like structures. Immunohistochemical studies showed positive reactivities for CD31, CD34, alpha-smooth muscle actin, and factor VIII-related antigen in most of the tumor cells and CD68 in some tumor cells. Based on these observations, the tumor was diagnosed as an EPA.

Apoptosis induction and enhancement of cytotoxicity of anticancer drugs by celecoxib, a selective cyclooxygenase-2 inhibitor, in human head and neck carcinoma cell lines.

Colorectal carcinomas are well known to highly express COX-2 and their growth is markedly inhibited by COX-2 inhibitors, but little is known about head and neck carcinomas. In this study, we investigated the effect of a selective COX-2 inhibitor, celecoxib, on growth and apoptosis induction of four human head and neck carcinoma cell lines, SCC25, KB, HSG and HSY, in comparison with frequently used COX inhibitor sulindac. Also, we examined whether celecoxib augments the sensitivity of these cell lines to anticancer drugs such as doxorubicin (DOX), vincristine (VCR), cisplatin (CDDP), bleomycin (BLM) and 5-fluorouracil (5-FU). The growth of all cultured cell lines particularly SCC25 and HSG was inhibited by celecoxib and sulindac in a dose-dependent manner. The IC50 of celecoxib was ten times lower than that of sulindac. SCC25 produced ample PGE2 whereas KB, HSG and HSY produced a small amount of PGE2. The PGE2 production and COX-2 expression were inhibited more efficiently by celecoxib than by sulindac. Exogenous addition of PGE2 resulted in an increased cell growth of SCC25 even under the celecoxib-treated condition, but not of HSG. These results suggested that PGE2 is involved in the growth of SCC25 but not of HSG. The ability of celecoxib to induce apoptosis is greater than that of sulindac. Treatment of SCC25 and HSG with non-cytotoxic 1 micro M or less cytotoxic 5 micro M of celecoxib enhanced the sensitivity of both cell lines to anticancer drugs, particularly in DOX, VCR and BLM two to ten times as demonstrated by lowering of IC50s. The enhanced rate was almost parallel to the degree of apoptosis induction. These findings indicated that a selective COX-2 inhibitor celecoxib inhibits cell proliferation, induces apoptosis and augments sensitivity to anticancer drugs in human head and neck carcinoma cells. Therefore, celecoxib would be useful as biological modulator in treatment of head and neck cancer.

Small cell neuroendocrine carcinoma of the maxillary sinus--a case report and nude mouse transplantable model.

BACKGROUND: A rare case of small cell neuroendocrine carcinoma (SNEC) arising in the maxillary sinus is presented, and a SNEC tumor line serially transplantable in nude mice was established. Tumor marker for SNEC is also discussed. METHODS: The tumor tissues obtained from operated material were heterotransplanted subcutaneously into nude mice. Histopathologic studies and immunoradiometric assays for NSE and pro-GRP in serum were performed. RESULTS: The primary lesion was composed of tumor nests of small cells with hyperchromatic nuclei and was positive for NSE and chromogranin A immunohistochemically. Serum levels of NSE and pro-GRP changed dynamically, reflecting the clinical status. Nude mouse tumor showed similar histologic features to those of original tumor and expressed NSE. Neuroendocrine granules were detected in tumor cells in electron microscopy. Serum NSE level in nude mice was elevated in proportion to the relative tumor weight. CONCLUSIONS: Serum NSE and pro-GRP were useful tumor markers for extrapulmonary SNEC. A SNEC tumor transplantable in nude mice would provide a valuable model for characterization of this lesion.