Sam-Pyo Hong

Expression of matrix metalloproteinase-2 and -9 in oral squamous cell carcinomas with regard to the metastatic potential

In order to evaluate the significance of matrix metalloproteinases (MMPs) in predicting the metastatic potential of oral squamous cell carcinomas (SCCs), we compared MMP-2 and -9 expression in 19 metastasizing oral SCCs with that in 25 non-metastasizing cases by immunohistochemistry and gelatin zymography. Immunohistochemistry showed that increased MMP-2 expression was not significantly related to metastasis; increased MMP-9 expression found in oral SCCs was, however, statistically significant (oral SCCs with metastasis, 73.7%; those without metastasis, 36.0%; P<0.05). Gelatin zymography revealed no significant difference in the activated form of MMP-2 between metastasizing and non-metastasizing oral SCCs. In metastasizing SCCs, on the other hand, increases in the activated form of MMP-9 were significant. These results suggest that oral SCCs express MMP-2 and -9, and that MMP-9 may play a more important role than MMP-2 in the metastasis of oral SCCs to adjacent tissue. An analysis of MMP-9 expression may be useful for predicting the metastatic potential of oral SCCs.

Inhibition of Akt activity induces the mesenchymal-to-epithelial reverting transition with restoring E-cadherin expression in KB and KOSCC-25B oral squamous cell carcinoma cells

BackgroundThe Akt/PKB family of kinases is frequently activated in human cancers, including oral squamous cell carcinoma (OSCC). Akt-induced epithelial-to-mesenchymal transition (EMT) involves downregulation of E-cadherin, which appears to result from upregulation of the transcription repressor Snail. Recently, it was proposed that carcinoma cells, especially in metastatic sites, could acquire the mesenchymal-to-epithelial reverting transition (MErT) in order to adapt the microenvironments and re-expression of E-cadherin be a critical indicator of MErT. However, the precise mechanism and biologic or clinical importance of the MErT in cancers have been little known. This study aimed to investigate whether Akt inhibition would restore the expression of E-cadherin and β-catenin, reduce that of Vimentin, and induce the MErT in OSCC cells with low or negative expression of E-cadherin. We also investigate whether inhibition of Akt activity would affect the E-cadherin repressors and signaling molecules like NF-κB, ERK, and p38.MethodsWe screened several OSCC cell lines in order to select suitable cell line models for inducing MErT, using immunoblotting and methylation specific-PCR. We examined whether Akt inhibitor phosphatidylinositol ether lipid analogues (PIA) treatment would restore the expression of E-cadherin and β-catenin, reduce that of Vimentin, and induce the MErT in KB and KOSCC-25B cells using RT-PCR, immunoblotting, immunofluorescence analysis, and in vitro migration assay. We also investigated whether inhibition of Akt activity would affect the E-cadherin repressors, including Snail, Twist, and SIP-1/ZEB-2 and signaling molecules like NF-κB, ERK, JNK, and p38 using RT-PCR, immunoblotting, and immunofluorescence analysis.ResultsOf the 7 OSCC cell lines, KB and KOSCC-25B showed constitutively activated phosphorylated Akt and low or negative expression of E-cadherin. Inhibition of Akt activity by PIA decreased NF-κB signaling, but did not affect phosphorylation of ERK, JNK, and p38 in KB and KOSCC-25B cells. Akt inhibition led to downregulation of Snail and Twist expression. In contrast, inhibition of Akt activity by PIA did not induce any changes in SIP-1/ZEB-2 expression. PIA treatment induced the expression of E-cadherin and β-catenin, reduce that of Vimentin, restored their epithelial morphology of a polygonal shape, and reduced tumor cell migration in KB and KOSCC-25B cells, which was the corresponding feature of MErT.ConclusionAll of these findings suggest that Akt inhibition could induce the MErT through decreased NF-κB signaling and downregulation of Snail and Twist in OSCC cells. A strategy involving Akt inhibition might be a useful therapeutic tool in controlling cancer dissemination and metastasis in oral cancer patients.

Ameloblastic carcinoma: an analysis of 6 cases with review of the literature.

OBJECTIVES The purpose of this study is to report 6 cases of ameloblastic carcinoma and to analyze all published cases regarding demographic features, clinical behavior, treatment, and prognosis. STUDY DESIGN We reviewed our 6 cases of ameloblastic carcinoma and 98 cases previously reported in the English literature. Available follow-up data from 59 cases was analyzed. RESULTS In the analysis of our cases, the average age of 6 patients was 61 years and there was a predilection for the maxilla and male. Five cases arose de novo, whereas 1 case was secondary type of ameloblastic carcinoma. Microscopically, our cases showed malignant cytologic features with some histologic features of ameloblastoma. Malignant cytologic features included nuclear pleomorphism, hyperchromatism, and high mitotic activity (33.3%). Necrosis and vascular invasion were also seen in 4 cases and 1 case, respectively. Immunostaining for cytokeratins 5, 14, and 18 was all positive and labeling index of Ki-67 was 13.91%. Two patients (33.3%) had a metastatic lesion in the regional lymph nodes without distant metastasis. In the analysis of the literature, the mean age was 49.2 years with a wide age range (7-91 years). The rate of occurrence was higher in males, and the most common site of occurrence was the mandible. The male-to-female ratio was 1.97:1 and the mandible-to-maxilla ratio was 1.71:1. Most cases (70%) involved the posterior portion of the jaw. The most common symptom was swelling, followed by pain, ulceration, paresthesia, and trismus. The recurrence rate in patients treated with surgical resection was 28.3%, whereas the rate in cases of conservative therapy was 92.3%. There was a significant correlation between treatment modality and recurrence (P = 0.000). Metastatic lesions were detected in 22% of patients during follow-up, and the lung was the most common area of distant metastasis. The 5- and 10-year survival rates were 72.9% and 56.8%, respectively. There was a significant decrease in the 5-year survival rate in patients with metastasis (21.4%; P = 0.0000). CONCLUSION Metastasis from an ameloblastic carcinoma is significantly correlated with poor prognosis. Therefore, diagnosis at an early stage and close periodic screening for metastasis are necessary to improve patient prognosis.

Anti-cancer effect of genistein in oral squamous cell carcinoma with respect to angiogenesis and in vitro invasion

Oral squamous cell carcinoma (OSCC) is one of the most common head and neck cancers. OSCC generally has a poor prognosis due to its tendency towards local invasion and subsequent metastasis, which is mediated by multiple proteolytic enzymes and angiogenesis. The purpose of this study was to evaluate the anti‐cancer effects of genistein, a soybean product known to be an effective natural anti‐angiogenic agent with respect to tumor growth, angiogenesis and in vitro invasion in an OSCC model. Northern blot analysis for vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and matrix metallo‐proteinase‐2 (MMP‐2), in vitro invasion assay and gelatin zymog‐raphy were carried out for HSC‐3 cells treated with genistein (27.3 μg/ml). In the animal experiment, genistein (0.5 mg/kg) was injected into tumor (HSC‐3)‐bearing mice (male balb/c/nu). The tumor growth rate and metastasis to lymph node or lung were compared and the microvessel density (CD31) was subsequently analyzed by immunohistochemistry. The genistein‐treated group showed a down‐regulation in VEGF mRNA expression, but not in bFGF and MMP‐2 mRNA expression. Genistein reduced in vitro invasion through the artificial basement membrane and gelatin zymography also showed a reduced gelatinolytic activity in the genistein‐treated group. In the genistein‐treated mice, a significantly lower CD31 immunoreactivity was found. However, the tumor growth and metastatic behavior in the experimental group and the control group were similar and there were no significant differences. These results suggest the possible use of genistein as an anti‐cancer agent in oral squamous cell carcinoma. However, it appears from the present study that anti‐angiogenic therapy consisting of a single application of genistein may not provide a satisfactory treatment for OSCC. As a result, further research is recommended to confirm that genistein may be employed as an adjunct treatment modality for OSCC. (Cancer Sci 2003; 94: 215–220)

Evaluation of the anti-tumor and anti-angiogenic effect of paclitaxel and thalidomide on the xenotransplanted oral squamous cell carcinoma

Angiogenesis is an essential process for the growth and invasion of cancer. However, it is uncertain that anti-angiogenic effects can be a major treatment strategy of oral cancer. The aim of this study was to investigate whether thalidomide and paclitaxel, which are known to be potent inhibitors of angiogenesis, have inhibitory effects on the growth of oral squamous cell carcinoma (OSCC) xenotransplanted into nude mice and whether anti-angiogenesis can be included as a major treatment strategy of oral cancer. After human OSCC cell line, KB, was subcutaneously inoculated into 32 nude mice, the volume of tumor was measured every 3 days. When the tumor mass reached 300-500 mm3, thalidomide (200 mg/kg) and paclitaxel (13 mg/kg) were administered into the animals and tumor volume change was checked. The excised tumor masses on the 30th day after administration were frozen and processed for immunohistochemistry using vascular endothelial growth factor (VEGF) and CD31, and for real-time reverse transcription-polymerase chain reaction (RT-PCR). We evaluated VEGF expression and the expression of its mRNA and CD31 for vessel density. Paclitaxel showed an inhibitory effect on the growth of transplanted human OSCC and reduced the immunohistochemical expression of VEGF and CD31 and VEGF mRNA (P<0.01). Thalidomide also lowered remarkably VEGF expression (P<0.01) and CD31 (P<0.01) as well as VEGF mRNA (P<0.05), but it did not show statistically significant inhibitory effect on the tumor growth. These results suggest that the growth of human OSCC is not simply dependent on VEGF-induced angiogenesis and that anti-angiogenic therapy alone is not likely to be effective for the treatment of OSCC, but might be regarded as adjuvant chemotherapeutic strategy.

Comparative immunohistochemical study of ameloblastoma and ameloblastic carcinoma

OBJECTIVE Ameloblastic carcinoma combines the histologic features of ameloblastoma with cytologic atypia, regardless of whether it has metastasized. Because of its rarity, there are few immunoprofile studies of ameloblastic carcinoma and few comparative studies of ameloblastic carcinoma and ameloblastoma. In this study, we compared the expression levels of cytokeratins (CKs), matrix metalloproteinases (MMPs), and Ki-67 between ameloblastoma and ameloblastic carcinoma, and assessed the usefulness of these markers for differentiating the tumors. STUDY DESIGN We assessed CK7, CK14, CK18, CK19, MMP-2, MMP-9, and Ki-67 expression by immunohistochemistry in 10 cases of ameloblastoma and 7 cases of ameloblastic carcinoma and then compared expression patterns between the 2 groups. RESULTS Immunostaining for CK14 and CK19 was diffuse and strongly positive in both tumor types, but staining for CK7 was focally positive in only 1 case of ameloblastoma and absent in all cases of ameloblastic carcinoma. However, there was a significant difference in CK18 expression between the 2 tumors (P = .000). Whereas 80% of ameloblastomas showed negative reactivity for CK18, most cases of ameloblastic carcinomas showed a moderate to strong intensity of immunostaining for CK18. Regarding the expression of MMPs, there were significant differences in parenchymal MMP-2 and stromal MMP-9 expression between the 2 tumors. Compared to ameloblastoma, ameloblastic carcinoma showed significantly strong expression of MMP-2 in parenchymal cells (P = .001) and MMP-9 in stromal cells (P = .013). However, there were no differences in MMP-2 expression of stromal cells and MMP-9 expression of parenchymal cells between ameloblastoma and ameloblastic carcinoma. The mean Ki-67 labeling index (LI) of ameloblastic carcinomas was 17.21%, which was significantly higher than that of ameloblastomas (3.57%; P = .002). CONCLUSIONS The significant expression of CK18, parenchymal MMP-2, stromal MMP-9, and Ki-67 could provide useful markers for differentiating ameloblastic carcinoma from ameloblastoma.

Inactivation patterns of p16/INK4A in oral squamous cell carcinomas

The p16/INK4A is one of the major target genes in carcinogenesis and its inactivation has frequently been reported in other types of tumors. The purpose of this study is to evaluate inactivation patterns of p16/INK4A in oral squamous cell carcinoma. Six different oral cancer cell lines, SCC-4, SCC-9, SCC-15, SCC-25, KB, and SNUDH- 379 were examined for inactivation of p16/INK4A genes. In the analysis of p16/INK4A gene inactivation, PCR amplification, direct sequencing, and methylation-specific PCR methods were adopted for evaluation of homozygous deletion, point mutation, and promoter hypermethylation, respectively. Homozygous deletion was detected in SCC-25 and SCC-9. SCC-15 showed hypermethylated promoter region within p16/INK4A gene. It is suggestive in the present study that inactivation patterns of p16/INK4A were mainly homozygous deletion, promoter methylation rather than point mutation in oral squamous cancer cell lines, so treatment modalities of oral squamous cell carcinoma should be focused on these types of inactivation.

The presence of bacteria in the synovial fluid of the temporomandibular joint and clinical significance: preliminary study

PURPOSE The objective of this study was to find any relation between the presence of specific bacterial species in the synovial fluid of the temporomandibular joint (TMJ) and clinical parameters. PATIENTS AND METHODS We studied 43 patients (male-to-female ratio, 1:1.69; average age, 34.37 +/- 14.55 years). Thirty-three patients had a displaced disc in the TMJ (DD group), and 10 patients did not have a displaced disc of the TMJ or any symptom related to TMJ disorders (NDD group). Clinical examinations were made to determine maximum mouth opening, joint sounds, previous trauma history, systemic disease, and TMJ pain. Six bacterial species that were reported in other studies were chosen to evaluate the presence of bacteria in the TMJ for this study. RESULTS Mycoplasma genitalium was most frequently detected in synovial fluid (86.0%). Staphylococcus aureus, Mycoplasma fermentans/orale, Actinobacillus actinomycetemcomitans, and Streptococcus mitis were detected in 51.2%, 37.2%, 25.6%, and 7.0% of samples, respectively. beta-Hemolytic Streptococcus was not detected. The prevalence of S aureus was significantly higher in the DD group than in the NDD group (P <.05). The patients who had M. fermentans/orale were 5.40 times more likely to be younger than 30 years than were those without M. fermentans/orale (P <.05). Those with M. genitalium were 5.81 times more likely to be female than were those without M. genitalium (P <.05). CONCLUSION The presence of S. aureus in TMJ synovial fluid was related to TMJ disorder symptoms and clinical parameters seemed to be influenced by bacterial presence in TMJ synovial fluid.

Prognostic significance of CXCR-4 expression in oral squamous cell carcinoma

OBJECTIVE We investigated the prognostic significance of CXC chemokine receptor CXCR-4 expression in patients with oral squamous cell carcinoma (OSCC) and its relationship with matrix metalloproteinase 2 (MMP-2), MMP-9, and Ki-67 expression. STUDY DESIGN The CXCR-4, MMP-2, MMP-9, and Ki-67 expression was assessed immunohistochemically in 74 OSCC patients. The results were analyzed in connection with clinicopathologic factors. RESULTS The CXCR-4 expression was positive in 45 cases and significantly correlated with lymph node metastasis (P = .037), MMP-9 expression (P = .025), and Ki-67 expression (P = .001). Univariate analysis showed that CXCR-4 expression, MMP-9 expression, Ki-67 expression, tumor size, lymph node metastasis, clinical stage, and recurrence positively correlated with prognosis. Multivariate analysis indicated that CXCR-4 expression was an independent prognostic factor for poor survival in patients with OSCC. CONCLUSION Expression of CXCR-4 is a significant prognostic indicator for poor survival in patients with OSCC and correlates with expression of MMP-9 and Ki-67. The inhibition of CXCR-4 represents a possible molecular approach to the treatment of OSCC.

Evaluation of survivin as a prognostic marker in oral squamous cell carcinoma

BACKGROUND Poor prognosis of oral squamous cell carcinoma (OSCC) is partly attributed to the lack of significant tumor marker for accurate staging and prognostication. We have evaluated survivin, which is a member of the inhibitor of apoptosis family as a cancer marker associated with proliferation, angiogenesis, oral carcinogenesis, and OSCC patient survival, as we reported a prognostic significance of survivin expression in lymph node previously. METHODS To evaluate survivin expression in six OSCC cell lines, Western blotting was performed. Hamster oral carcinogenesis model was used to observe changes of survivin expression in oral carcinogenesis. Finally, we assessed the diagnostic and prognostic significance of survivin in a series of 38 primary OSCC through immunohistochemistry (CD31, PCNA) and Kaplan-Meiers test. RESULTS Survivin expression was detected in all OSCC cell lines at a varying level but not observed in normal gingival keratinocyte cells. In hamster model, survivin expression was observed from 8 weeks through 16 weeks and the intensity of expression became strong until 16 weeks. Clinicopathological analysis revealed a significant correlation between survivin expression and lymph node metastasis (P = 0.006) and proliferation (P < 0.001). However, there was no significant relationship with differentiation, micro vessel density, and cancer stage based on TNM. Survivin overexpression had a significant negative effect on survival of patients. CONCLUSIONS These results demonstrate the significant relationship between survivin expression and oral carcinogenesis and aggressiveness of OSCC including survival rate of patient. Survivin therefore may be used as a significant cancer marker to gain prognostic information of OSCC.