Treatment with teriparatide for advanced bisphosphonate-related osteonecrosis of the jaw around dental implants: a case report.

We report a case of a 66-year-old severely osteoporotic woman with bisphosphonate-related osteonecrosis of the jaw (BRONJ) around her dental implants, who was treated successfully with teriparatide and sequestrectomy of the mandible. After 5 months of teriparatide therapy, the sequestrum separation had progressed and a sequestrectomy was performed under general anesthesia. Five months after the operation, new bone formation was observed around the bone defect in the region of the sequestrectomy. A repeat computed tomographic image revealed improvement in the bone defect in the mandible. These results suggest that teriparatide provides beneficial effects in the treatment of advanced BRONJ around dental implants.

[Corrigendum] Functional analysis of Zyxin in cell migration and invasive potential of oral squamous cell carcinoma cells.

Following the publication of this article, an interested reader drew to our attention an anomaly associated with the presentation of Figs. 2 and 3. Essentially, there was a direct duplication of certain of the western blotting data between Fig. 2 (the E-cadherin and Actin data) and Fig. 3C (the Zyxin and Actin data). After having re-examined our original data, we realize that we inadvertently duplicated the data from Fig. 2 in Fig. 3C (the Zyxin and Actin data). A corrected version of Fig. 3C (containing the true Zyxin and Actin data), and, by natural process, also of Fig. 6, are presented below, in which the Zyxin data in Figs. 3C and 6 are now correctly shown. Since the Zyxin data was a control for the siZyxin knockout of HOC313, this error did not affect the results of the Rho family analysis in this study. We sincerely apologize for this mistake, and thank the reader of our article who drew this matter to our attention. Furthermore, we regret any inconvenience this mistake has caused. [the original article was published in the International Journal of Oncology 42: 873-880, 2013; DOI: 10.3892/ijo.2013.1761].

Osteonecrosis of the jaw in an AIDS patient: a case report.

We report a rare case of osteonecrosis of the jaw following necrotizing gingivitis in a Japanese AIDS patient. Intraoral examination showed exposed necrotic bone in the left mandible and spontaneous loss of teeth. This patient was successfully treated with highly active anti-retroviral therapy combined with minimally invasive surgical procedures to remove the osteonecrosis of the jaw.

Dental implant treatment in a young woman after marginal mandibulectomy for treatment of mandibular gingival carcinoma: a case report.

Dental implants play an important role in postoperative rehabilitation after surgical treatment of oral cancer through the provision of prosthetic tooth replacement. Two major implant prosthesis designs are available: fixed implant-supported prostheses and implant-supported overdentures. We herein report a case of a 16-year-old female patient who underwent alveolar ridge resection for treatment of mandibular gingival carcinoma. Following surgery, oral rehabilitation was attempted using an implant-supported overdenture on a gold bar retainer splinting four implants. However, the patient was not satisfied with this prosthesis because of mucosal pain and discomfort, and she gradually ceased its use. Consequently, contact with the opposing teeth caused wear of the prosthetic screws. We elected to replace the implant-supported overdenture with an implant-fixed prosthesis approximately 16 years after insertion of the overdenture to prevent further wear of the prosthetic screws. The patient was highly satisfied with the improved stability of the implant-fixed prosthesis. This case report indicates that the clinician must occasionally re-evaluate and sometimes alter the direction of treatment, even after definitive therapy has been completed.

Effect of a nitric oxide synthase inhibitor and a CXC chemokine receptor-4 antagonist on tumor growth and metastasis in a xenotransplanted mouse model of adenoid cystic carcinoma of the oral floor.

Nitric oxide (NO) is related to angiogenesis and tumor progression and chemokine receptor-4 (CXCR4) plays a central role in cell migration in metastasis and dissemination of cancer. The present study evaluated the effectiveness of a NOS inhibitor and a CXCR4 antagonist, given as single agents or in combination, in a xenotransplanted mouse model of adenoid cystic carcinoma (ACC) of the oral floor. A metastatic tumor (ACCIM) derived from a cervical metastatic lesion of human ACC that was transplantable in nude mice was used. ACCIM showed a high frequency of spontaneous metastasis to the lung when transplanted subcutaneously in nude mice. Mice with subcutaneous transplants of ACCIM were subdivided into six groups and intraperitoneally received one of the following treatments daily for 5 weeks: a) PBS (control), b) AMD3100 (CXCR4 antagonist), c) L-NAME (NOS inhibitor), d) 1400W (iNOS inhibitor), e) both AMD3100 and L-NAME (AMD3100+L-NAME) and f) both AMD3100 and 1400W (AMD3100+1400W). Tumor growth was evaluated during treatment and metastasis was assessed at 28 weeks. Single-agent treatment with AMD3100, L-NAME or 1400W inhibited tumor growth by 20.8, 26.5 and 54.5%, respectively. Combined treatment with AMD3100+L-NAME and AMD3100+1400W inhibited tumor growth remarkably by 48.0 and 50.2%, respectively. Immunohistochemical analysis revealed lower expression of CXCR4, iNOS and eNOS in tumor cells treated with AMD3100+L-NAME or AMD3100+1400W compared to control tumor cells and increased numbers of apoptotic tumor cells were demonstrated using the TUNEL method. CXCR4 expression decreased in 1400W-treated tumors using western blot analysis. When the effect of each agent on tumor-induced angiogenesis in tumor stroma was examined histologically, microvessel density was significantly lower in the groups treated with 1400W, AMD3100+L-NAME or AMD3100+1400W compared to the control, AMD3100 and L-NAME groups. Moreover, treatment with AMD3100 or 1400W markedly inhibited lung metastasis. Our results indicated that single-agent treatment with 1400W and combined treatment with AMD3100+L-NAME or AMD3100+1400W induced apoptosis and significantly inhibited tumor-induced angiogenesis and proliferation of ACCIM in vivo. Blockade of CXCR4 and iNOS was suggested to inhibit lung metastases from ACCIM. CXCR4 and iNOS may, thus, be important prognostic factors for long-term survival in ACC.

Functional analysis of Zyxin in cell migration and invasive potential of oral squamous cell carcinoma cells.

Zyxin is an evolutionarily conserved protein that has been implicated in the regulation of actin assembly and is mainly located at focal adhesions. However, the biological roles of Zyxin in cancer cells are incompletely understood. We analyzed the functions of Zyxin in cell migration and the invasive potential of OSCC. Zyxin expression was examined using eight OSCC cell lines with two different cell morphologies (6 epithelial type and 2 fibroblastic type). To knockdown Zyxin expression, OSCC cells were transfected with Zyxin siRNA and control siRNA. The cell lines were studied by western blot analysis, immunocytochemical analysis and cell migration and invasion assay. Epithelial type OSCC cells showed a high level of E-cadherin expression and a low level of Zyxin expression. N-cadherin as well as Zyxin were strongly expressed in fibroblastic type OSCC cells. Expression levels of LPP and TRIP6, members of the human Zyxin family, did not differ between epithelial type and fibroblastic type. Knockdown of Zyxin expression by siRNA in fibroblastic type OSCC cells was associated with cell morphological changes from spindle (fibroblastic) to polygonal (epithelial) shape and significantly inhibited cell growth as well as cell migration and invasion. Expression levels of Rac1 and Cdc42 were weaker in Zyxin siRNA-treated fibroblastic type OSCC cells than in control siRNA-treated cells, but the expression of RhoA did not differ significantly. Treatment of fibroblastic type OSCC cells with Rac1 inhibitor decreased the expression of Zyxin mRNA and protein. Zyxin is suggested to promote growth, migration and invasiveness of fibroblastic type OSCC cells by upregulating Rac1 and Cdc42.

An in vitro multistep carcinogenesis model for both HPV-positive and -negative human oral squamous cell carcinomas.

Oral squamous cell carcinomas (OSCCs) are considered to arise from human oral keratinocytes. DNAs of human papillomaviruses (HPVs), predominantly types 16 and 18, etiological agents of cervical cancer, have been detected in approximately 25% of OSCCs. In accordance with the established role of E6 and E7 in inactivating p53 and pRB, respectively, mutations of p53 and inactivation of p16(INK4a) are frequently observed in HPV-negative OSCCs. In addition, other alterations such as overexpression of epidermal growth factor receptor (EGFR) are often observed in both HPV-positive and -negative OSCCs. However, causal-relationships between accumulation of these abnormalities and multi-step carcinogenesis are not fully understood. To elucidate underlying processes, we transduced either HPV16 E6/E7 or mutant CDK4 (CDK4(R24C)), cyclin D1 and human telomerase reverse transcriptase (TERT) into primary human tongue keratinocytes (HTK), and obtained immortal cell populations, HTK-16E6E7 and HTK-K4DT. Additional transduction of oncogenic HRAS or EGFR together with MYC into the HTK-16E6E7 and dominant-negative p53 expressing HTK-K4DT resulted in anchorage-independent growth and subcutaneous tumor formation in nude mice. These results indicate that either HRAS mutation or activation of EGFR in cooperation with MYC overexpression play critical roles in transformation of HTKs on a background of inactivation of the pRB and p53 pathways and telomerase activation. This in vitro model system recapitulating the development of OSCCs should facilitate further studies of mechanisms of carcinogenesis in the oral cavity.

Up-regulation of neutrophil gelatinase-associated lipocalin in oral squamous cell carcinoma: relation to cell differentiation.

Neutrophil gelatinase-associated lipocalin (NGAL, also known as lipocalin2, LCN2) is a secreted glycoprotein with increased expression in solid tumors. The expression and functions of NGAL in oral cancer, however, remain unclear. We investigated the expression of NGAL in oral cancer tissues and oral cancer cell lines. By immunohistochemical examinations, NGAL expression was strongly up-regulated in well-differentiated OSCC tissues and moderately to weakly up-regulated in moderately to poorly differentiated OSCC tissues. In contrast, NGAL expression was weak or very weak in normal mucosa and leukoplakia. By western blot analysis, NGAL expression levels positively correlated with cell morphology patterns and loss of E-cadherin. In addition, the enzymatic activity of the NGAL/MMP-9 complex significantly correlated with the results obtained by zymographic analysis. In conclusion, NGAL expression is high in well-differentiated cancer, suggesting that NGAL may be a useful diagnostic marker of tumor-cell differentiation.

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.

In vitro susceptibility to anticancer agents of the human KB carcinoma cell line transfected with COX-2 cDNA.

In order to investigate the malignant phenotype of cyclooxygenase (COX)-2 overexpressing cancer cells, a human epidermoid KB carcinoma cell line minimally expressing COX-2 protein was transfected with human COX-2 cDNA. In this study, we used a COX-2 transfected clone KB/COX-2 and a neomycin-transfected clone KB/neo as the control. When we examined the susceptibility to anticancer agents, there was no difference between these two clones in vincristine, bleomycin and 5-fluorouracil, although KB/COX-2 showed a 2.5-fold resistance to cisplatin (CDDP) as compared with KB/neo. The IC50 for CDDP was 4.3 microM in KB/COX-2 and 1.7 microM in KB/neo. Treatment with small interfering RNA (siRNA) mediated the inhibition of COX-2 significantly increasing the level of susceptibility to CDDP in COX-2 siRNA as compared to that of the control siRNA. The expression of MRP1 and MRP2 was stronger in KB/COX-2 than in KB/neo by Western blot analysis. In addition, apoptosis induction by CDDP was at a lower level in KB/COX-2 (31%) than in KB/neo (38%). These results suggested that the overexpression of COX-2 increases the intracellular production of MRP1 and MRP2 and causes drug resistance to CDDP.

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.

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.