Possible mechanism of betel-quid-extract-induced expression of matrix metalloproteinase-2.

BACKGROUND/PURPOSE: Betel quid extract (BQE) has been demonstrated to induce matrix metalloproteinase (MMP)-2 expression. This study aimed to establish the possible mechanism involved in this event. METHODS: Western blotting, reverse-transcription polymerase chain reaction, and gelatin zymography were used to study the expression level of MMP-2. LY294002, PD98059, U0126, N-acetyl-L-cysteine, SB203580, SP600125, and Bay 11-7082 were used to pretreat OECM-1 cells before BQE treatment and MMP-2 detection. RESULTS: OECM-1 cells were subjected to short-term (10 minutes) or long-term (24 hours) BQE treatment (designated as SBT and LBT, respectively), and we found that both treatments increased MMP-2 protein and extracellular signal-regulated kinase (ERK) phosphorylation levels in a concentration- and time-dependent manner. LBT also increased MMP-2 mRNA level. LBT-induced MMP-2 secretion was not inhibited by PD98059 (up to 50 µM) when ERK was effectively blocked, but was attenuated by LY294002 (0-10 µM) in a concentration-dependent manner. This LBT effect was inhibited strongly by SB203580 (10 µM), SP600125 (10 µM), and Bay 11-7082 (10 µM) and mildly by N-acetyl-L-cysteine (5 mM), but not by U0126 (10 µM). CONCLUSION: Both SBT and LBT upregulate MMP-2 expression, and LBT-induced MMP-2 expression might be mediated by phosphoinositide 3-kinase, p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and nuclear factor-κB, and to a lesser extent, by reactive oxygen species, rather than by ERK.

Arecoline and the 30-100 kDa fraction of areca nut extract differentially regulate mTOR and respectively induce apoptosis and autophagy: a pilot study.

Areca nut (AN) is recognized as a human carcinogen; however, few studies of the cytotoxic effects of AN ingredients on cells have been reported. In Taiwan, AN, lime and inflorescence of Piper betle are the common components of betel quid (BQ). We recently noticed that extract of AN (ANE), but not those of lime and inflorescence of Piper betle, induces rounding cell morphology and nuclear shrinkage in different types of carcinoma cells. In this study, the rounding cell activity was first traced to the partially purified >or=10 kDa fraction (ANE >or= 10 K) and subsequently to the 30-100 kDa fraction (ANE 30-100 K). ANE and ANE >or=10 K stimulated nuclear shrinkage (P < 0.001 in both cases) and the clearance of the cytoplasm. ANE, ANE >or= 10 K, and ANE 30-100 K induced the cleavage of LC3-I (P < 0.05, 0.01, and 0.05, respectively) and the emergence of autophagic vacuoles (AVs) and acidic vesicles. On the other hand, arecoline (Are, the major alkaloid of AN) triggered caspase-3 activation, peri-nuclear chromatin condensation, and micronucleation. Meanwhile, ANE 30-100 K, but not Are, inhibited the phosphorylation of the mammalian target of rapamycin (mTOR)-Ser(2448). In conclusion, this study demonstrates that different AN ingredients exerting differential impact on mTOR-Ser(2448) phosphorylation are capable of triggering apoptosis and autophagy.

Upregulation of matrix metalloproteinase-1 (MMP-1) expression in oral carcinomas of betel quid (BQ) users: roles of BQ ingredients in the acceleration of tumour cell motility through MMP-1.

Matrix metalloproteinases (MMPs) are commonly expressed in carcinomas including oral squamous cell carcinomas (OSCCs). On the other hand, some evidences suggested that ingredients of betel quid (BQ) inhibit the activity and/or expression of some MMPs thought to be the pathogenesis of oral submucous fibrosis. This study was to analyse whether MMP-1 expression is inhibited in OSCC specimens from BQ users and in cell lines survived from the challenge of BQ ingredients. We found that MMP-1 mRNA was expressed in all the tested 27 OSCC. Levels of MMP-1 mRNA and protein were significantly elevated in the tested five OSCC specimens than in their adjacent tissues (P<0.001 and 0.05, respectively). Esophageal carcinoma (CE81T/VGH) and OSCC (OECM-1) cell lines survived from the cytotoxic BQ extract (BQE) and arecoline selection process were found to express higher MMP-1 mRNA and protein levels, or to exhibit a significant acceleration of two-dimensional (2D) motility than their non-selected parental cells. The enhanced motility was further demonstrated to be specifically and significantly inhibited by the MMP-1 neutralizing antibody and/or by the transfection of an MMP-1 specific antisense oligodeoxynucleotide. These results suggest that in some carcinomas of the upper aerodigestive tract, BQ usage may upregulate MMP-1 expression in the survived tumour cells, and increase their mobility in an MMP-1-dependent manner.

Clinicopathologic significance of tumor cell-lined vessel and microenvironment in oral squamous cell carcinoma.

Oral squamous cell carcinoma (SCC) can be vascularized through a process called "tumor cell-lined vessels". Currently, the tumor microenvironment, which is recognized as hypoxic and orchestrated largely by inflammatory cells and defective blood vessels, is considered an important participant in the neoplastic process. We sought to determine their clinicopathologic significance and prognostic implication in oral SCC. Vascular structure was investigated by multistaining with pan-cytokeratin, CD34, and alpha-smooth actin/type IV collagen. Immunohistochemical staining of the hypoxia inducible factor-1 alpha (HIF-1 alpha) and CD68 was used to reflect hypoxia and tumor-associated macrophages (TAM). Our results showed that in a high percentage of vessels in cancer tissue. There is absence of pericyte coverage and loss of basement membrane lining. Significant association between the integrity of vascular structure and lymph node involvement and presence of tumor cell-lined vessel was found. HIF-1 alpha overexpression was frequently observed in cancer cells (78/112) and correlated with tumor progress index. In cancer tissues, the TAM ranged from 28 to 296 cells/mm2 with a mean of 144.6+/-64.3 cells/mm2. There was a significant correlation between TAM and lymph node involvement (P=0.004) and tumor size (P=0.004). Also, a close association was found between TAM count and integrity of vascular structure. In addition, survival analysis revealed that tumor cell-lined vessels (P=0.001), HIF-1 alpha expression (P=0.004), and TAM (P=0.001) correlated significantly with poor survival. We conclude that in the cancer microenvironment, HIF-1 alpha expression and the TAM are induced and contributed to malignant behavior of tumor cells. Furthermore, the presence of tumor cell-lined vessel, HIF-1 alpha overexpression, and high TAM could be the potential markers of prognosis for patients with oral SCC.

Outcome of treatment with total main tumor resection and supraomohyoid neck dissection in oral squamous cell carcinoma.

BACKGROUND/PURPOSE: Supraomohyoid neck dissection (SOHND) is commonly used to treat oral squamous cell carcinoma (OSCC) patients with clinical N0 or selected N1 status. The purpose of this study was to evaluate the clinical outcome of OSCC patients treated with SOHND. METHODS: This retrospective study reviewed the clinical outcome of 257 patients (247 men, 10 women) with N0, N1 and N2a OSCC treated with wide excision of the main tumor and SOHND between 1992 and 1999. All patients were followed up for at least 5 years. Survival distributions were analyzed using Kaplan-Meier curves. N status was compared using chi2 and log rank tests. RESULTS: The neck failure rate was 20% for clinically false negative cases, 6.1% for clinically true negative cases, 21.8% for clinically false positive cases, and 40% for clinically true positive cases. The 3- and 5-year overall neck disease-free survival rates were 79.8% and 77.6%, respectively. The 3- and 5-year neck disease-free survival rates were 86.7% and 84.2% for pathologic N0 cases, 56.9% and 56.9% for pathologic N1 cases, and 27.5% and 27.5% for pathologic N2 cases, respectively. Log rank test showed that the p value for difference in survival at 3-5 years was 0.064 for pathologic N0 vs. N1 cases, < 0.0001 for pathologic N0 vs. N2 cases, and 0.008 for pathologic N1 vs. N2 cases. CONCLUSION: This study showed that SOHND is effective for pathologic N0 OSCC, relatively effective for pathologic N1, and less effective for pathologic N2a. These findings also support that when SOHND is used to treat N2a OSCC, postoperative radiotherapy or radical neck dissection may be needed to improve the neck disease-free survival rate.

Increased expression of matrix metalloproteinase-2 in oral cells after short-term stimulation and long-term usage of areca quid.

BACKGROUND AND PURPOSE: Arecoline, an areca quid (AQ) component, has been shown to inhibit the secretion and activity of matrix metalloproteinase-2 (MMP-2) in fibroblast cultures. This study assessed whether MMP-2 expression was inhibited in the saliva samples and tumor specimens of oral tumor patients with a long-term history of AQ consumption. The net effect of crude AQ extract (AQE) on MMP-2 expression by oral cells was also investigated. METHODS: Western blot analysis, zymography, and reverse transcriptase-polymerase chain reaction were used to detect MMP-2 protein and mRNA in saliva and tumor samples, as well as in the conditioned media (CM) of oral cell cultures. RESULTS: The level of MMP-2 protein was significantly higher in the saliva samples of 12 oral tumor patients who had a minimum 10-year AQ-consuming history than in those of 12 non-AQ-using healthy controls (p < 0.05). MMP-2 mRNA was expressed in 26 of 28 oral squamous cell carcinoma (OSCC) specimens. MMP-2 protein was also detectable in the tested OSCC homogenates. Short-term stimulation with 10% AQE increased the secretion of MMP-2 protein in the CM of oral epidermoid carcinoma cell Meng-1 (an OSCC cell line) and oral fibroblasts. CONCLUSIONS: MMP-2 expression is elevated rather than inhibited in most oral tumor patients with long-term AQ usage. Short-term AQE stimulation also increases the secretion of MMP-2 by oral epithelial cells and fibroblasts. Our results suggest that AQ consumption may promote oral tumor progression through the induction of MMP-2 secretion.

Areca quid chewing enhances the expression of salivary matrix metalloproteinase-9.

BACKGROUND AND PURPOSE: The effects of areca quid (AQ) consumption on salivary matrix metalloproteinases (MMPs) which may participate in tumor invasion and metastasis remains unclear. This study assessed the change in salivary MMP-9 protein levels 2 hours after 5-minute AQ chewing stimulation (AQCS) in non-AQ users and the expression profile of this proteinase in saliva and tumor specimens of oral squamous cell carcinoma (OSCC) patients with a history of AQ use. METHODS: MMP-9 transcript was analyzed by reverse transcription-polymerase chain reaction. MMP-9 protein level was measured by both Western blot and gelatin zymography. RESULTS: The protein level of salivary MMP-9 was 3.1- to 8.9-fold enhanced 2 h after AQCS in 3 healthy volunteers as revealed by Western blot and zymography. As a control, gum chewing did not significantly change salivary MMP-9 protein level. Expression of MMP-9 transcript was found in 25 of 28 OSCC specimens and significantly correlated with cervical lymph node metastasis (p = 0.037). All of the 8 tested OSCC tissue homogenate samples available and all 12 saliva samples from 12 oral tumor outpatients were positive for MMP-9 protein. CONCLUSIONS: Elevation of MMP-9 may be one of the net effects of AQCS in vivo, which may play a role in the pathogenesis of oral mucosal lesions. Furthermore, the association of MMP-9 expression with neck-lymph-node metastasis may imply a significant role of MMP-9 in the progression of OSCC among patients with a history of AQ use in Taiwan.