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Cisplatin (CDDP) is widely used to treat oral squamous cell carcinoma (OSCC), however, many patients exhibit acquired drug resistance. Yes-associated protein (YAP) is a transcriptional co-activator of the Hippo pathway that regulates organ size and promotes cell proliferation. YAP overexpression correlates with epithelial-mesenchymal transition and nodal metastasis, resulting in anti-tubulin drug resistance. Whether YAP overexpression is the cause of CDDP resistance in cancer cells is unclear, therefore, we investigated the correlation between YAP expression and CDDP sensitivity. We established three CDDP-resistant cell lines (OSC-19-R, SCCKN-R and HSC-3-R) from the OSCC parental cell lines. We also examined the expression levels of ATP7B, GST-π and ERCC1, which are strongly associated with CDDP resistance, and Hippo pathway-related proteins by western blotting. Using immunocytochemistry, we examined the cellular localization of YAP. Additionally, following knockdown of YAP using short interfering RNAs (siRNAs), we analyzed changes in sensitivity to CDDP. Compared with parental OSC-19 cells, OSC-19-R cells were obviously larger. Expression levels of YAP were not significantly different between OSC-19 and OSC-19-R. However, expression levels of phosphorylated YAP in OSC-19-R were decreased. We observed translocation of YAP from the cytoplasm to the nucleus in OSC-19-R cells. Knockdown of YAP using siRNAs revealed that sensitivity to CDDP was significantly increased. Translocation of YAP correlated with the acquisition of CDDP resistance. YAP could be a new therapeutic target for the treatment of patients with cancer that are resistant to CDDP.

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.

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.

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.

In order to investigate the involvement of cyclooxygenase (COX)-2 in oral carcinogenesis and chemoprevention for it, we examined the COX-2 expression during dimethylbenzanthracene (DMBA)-induced hamster cheek pouch carcinogenesis and the inhibitory effect of sulindac, a non-steroidal anti-inflammatory drug (NSAID), on the carcinogenesis and its derived squamous carcinoma cell line HCPC-1. From the beginning of DMBA application, basal diet or diets containing sulindac 200 or 400 ppm were given to hamsters, and observation of tumor development and measurement of body weight were performed. Immunohistochemical analysis revealed that COX-2 expression was increased toward carcinogenesis from epithelial dysplasia to squamous cell carcinoma (SCC). All hamsters developed SCC, but the onset of carcinoma formation was significantly delayed up to 14.8 and 11.8 weeks in the 200 ppm, and 400 ppm sulindac group, respectively, as compared to 8.7 weeks in the control group. In addition, tumor growth was retarded in the group of sulindac treatment, and mean survival time was 23.7 weeks in the control group and 36.3 and 33.8 weeks in the 200 and 400 ppm sulindac group, respectively. Body weight loss was not observed during the experimental period. Histologically, administration of sulindac inhibited angiogenesis in the tumor stroma. Treatment with sulindac sulfide, an active metabolite of sulindac, caused inhibition of cell growth, PGE2 production and VEGF production in HCPC-1 cells in vitro. Expression of COX-2 protein in HCPC-1 cells was also decreased 2-fold by treatment with sulindac sulfide. It was thus indicated that inhibitory effects were partly due to inhibition of tumor angiogenesis by sulindac. These findings suggested the involvement of COX-2 in DMBA-induced hamster cheek pouch carcinogenesis and the chemopreventive potential of sulindac.

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.

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.

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.

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.

Hypercalcemia of malignancy is a common problem for cancer clinicians. Treatment of hypercalcemia of malignancy with pamidronate in a patient with advanced recurrent oral cancer is reported herein. The patient was a 36-year-old man who had neck lymph node metastases due to recurrence of squamous cell carcinoma of the buccal mucosa and complained of cloudiness of consciousness due to hypercalcemia. The patient was administered pamidronate. A fine reduction in serum calcium was observed and cloudiness of consciousness was also alleviated. Although treatment of hypercalcemia with pamidronate is palliative, it is effective against the deterioration of quality of life.