Purpose: We investigated the mechanism and clinical significance of the epithelial-mesenchymal transition (EMT)-induced chemoresistance in head and neck squamous cell carcinoma (HNSCC).Experimental Design: The correlation between the expression of different EMT regulators and chemoresistance genes, such as excision repair cross complementation group 1 (ERCC1), was evaluated in cancer cell lines from the NCI-60 database and four human HNSCC cell lines. Ectopic expression of Snail or short-interference RNA-mediated repression of Snail or ERCC1 was done in HNSCC cell lines. Cell viability was examined for cells after cisplatin treatment. A luciferase reporter assay and chromatin immunoprecipitation were used to identify the transcriptional regulation of ERCC1 by Snail. Immunohistochemical analysis of Snail, Twist1, ERCC1, hypoxia inducible factor-1 α (HIF-1α), and NBS1 were done in samples from 72 HNSCC patients receiving cisplatin-based chemotherapy.Results: The correlation between the expression of Snail and ERCC1 was confirmed in different cell lines, including HNSCC cells. In HNSCC cell lines, overexpression of Snail in the low endogenous Snail/ ERCC1 cell lines FaDu or CAL-27 increased ERCC1 expression, and hypoxia or overexpression of NBS1 also upregulated ERCC1. Knockdown of Snail in the high endogenous Snail/ERCC1 cell line OECM-1 downregulated ERCC1 expression and attenuated cisplatin resistance. Furthermore, suppression of ERCC1 in Snail-or NBS1-overexpressing HNSCC cells enhanced sensitivity to cisplatin. Snail directly regulated ERCC1 transcription. In patients with HNSCC, coexpression of Snail and ERCC1 correlated with cisplatin resistance and a poor prognosis.Conclusions: Activation of ERCC1 by Snail is critical in the generation of cisplatin resistance of HNSCC cells. Clin Cancer Res; 16(18); 4561-71. ©2010 AACR.The epithelial-mesenchymal transition (EMT), a major mechanism of cancer metastasis, is initiated by repression of the epithelial adhesion molecule E-cadherin by several transcription factors, including Snail (also known as Snail1), Slug (also known as Snail2), Twist1, Zeb1, SIP1, and E47 (1). In most human cancers, metastatic tumors are resistant to chemotherapy; therefore, patients with such tumors typically have poor outcomes. Emerging evidence suggests a correlation between EMT and the resistance to chemotherapy of cancer cells. For example, colorectal cancer cells that are resistant to oxaliplatin undergo phenotypic changes indicative of an EMT (2). Direct regulation of Akt2 by Twist contributes to paclitaxel resistance in breast cancer cells (3). Induction of EMT in breast cancer cells leads to an enrichment of cells with stem-like properties and chemoresistance (4). A recent report found that Zeb1 and other EMT regulators allow pancreatic cancer cells to maintain drug resistance (5). Taken together, these studies suggest that diverse types of cancer cells acquire drug-resistant phenotypes during EMT. Although there is an evident association among EMT, metastasis, and chemoresis...

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The hypoxic tumor environment has been shown to be critical to cancer metastasis through the promotion of angiogenesis, induction of epithelial-mesenchymal transition (EMT), and acquisition of invasive potential. However, the impact of hypoxia on the expression profile of the proteolytic enzymes involved in invasiveness is relatively unknown. Membrane-type 4 matrix metalloproteinase (MT4-MMP) is a glycosyl-phosphatidyl inositol-anchored protease that has been shown to be overexpressed in human cancers. However, detailed mechanisms regarding the regulation and function of MT4-MMP expression in tumor cells remain unknown. Here, we demonstrate that hypoxia or overexpression of hypoxia-inducible factor-1alpha (HIF-1alpha) induced MT4-MMP expression in human cancer cells. Activation of SLUG, a transcriptional factor regulating the EMT process of human cancers, by HIF-1alpha was critical for the induction of MT4-MMP under hypoxia. SLUG regulated the transcription of MT4-MMP through direct binding to the E-box located in its proximal promoter. Short-interference RNA-mediated knockdown of MT4-MMP attenuated in vitro invasiveness and in vivo pulmonary colonization of tumor cells without affecting cell migratory ability. MT4-MMP promoted invasiveness and pulmonary colonization through modulation of the expression profile of MMPs and angiogenic factors. Finally, coexpression of HIF-1alpha and MT4-MMP in human head and neck cancer was predictive of a worse clinical outcome. These findings establish a novel signaling pathway for hypoxia-mediated metastasis and elucidate the underlying regulatory mechanism and functional significance of MT4-MMP in cancer metastasis.

In the previous studies, (-)-epigallocatechin-3-gallate (EGCG) has been shown to have anticarcinogenic effects via modulation in protein expression of p53. Using p53 positive Hep G2 and p53 negative Hep 3B cells, we found that treatment of EGCG resulted in dose-dependent inhibition of cellular proliferation, which suggests that the interaction of EGCG with p53 may not fully explain its inhibitory effect on proliferation. Caloric restriction (CR) reduces the incidence and progression of spontaneous and induced tumors in laboratory rodents. EGCG has multiple beneficial activities similar to those associated with CR. One key enzyme thought to be activated during CR is AMP-activated kinase (AMPK), a sensor of cellular energy levels. Here, we showed that EGCG activated AMPK in both p53 positive and negative human hepatoma cells. The activation of AMPK suppressed downstream substrates, such as mammalian target of rapamycin (mTOR) and eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) and a general decrease in mRNA translation. Moreover, EGCG activated AMPK decreases the activity and/or expression of lipogenic enzymes, such as fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC). Interestingly, the decision between apoptosis and growth arrest following AMPK activation is greatly influenced by p53 status. In p53 positive Hep G2 cells, EGCG blocked the progression of cell cycle at G1 phase by inducing p53 expression and further up-regulating p21 expression. However, EGCG inducted apoptosis in p53 negative Hep 3B cells. Based on these results, we have demonstrated that EGCG has a potential to be a chemoprevention and anti-lipogenesis agent for human hepatoma cells.

Prostate carcinoma is the most frequently diagnosed malignancy and the second leading cause of cancer-related death of men in the United States. Epidermal growth factor (EGF) generated from bone tissue contributes to prostate cancer metastasis through stimulating matrix metalloproteinase (MMP) secretions from prostate cancer cells. In this study, in vitro invasion assay was performed by incubating penta-O-galloyl-β-D-glucose (5GG) at various concentrations with 2 × 10 4 PC-3 cells for 48 h. The anti-invasive and cytotoxic effects of 5GG were found and evaluated on the human androgen-independent prostate cancer PC-3 cell line by MTT assays and Western blot analyses. 5GG inhibited the EGF-induced cell invasiveness and MMP-9 expression in a dose-and timedependent manner by reducing the MMP-9 transcriptional activity. To explore the mechanisms for the 5GG-mediated regulation of MMP-9, we further examined the effects of 5GG on transcription factors, including NF-κB, AP-1, and mitogen-activated protein kinase (MAPK) activities. The results showed that 5GG suppressed the EGF-induced NF-κB nuclear translocation and also abrogated the EGF-induced activation of c-jun N-terminal kinase (JNK), an upstream modulator of NF-κB. Moreover, we showed that 5GG reduced EGFR expression through the proteasome pathway. These results suggest that 5GG may exert at least part of its anti-invasive effect in androgen-independent prostate cancer by controlling MMP-9 expression through the suppression of the EGFR/JNK pathway. Finally, 5GG suppresses invasion and tumorigenesis in nude mice treatment with intratibia injection of PC-3 cells. These in vitro and in vivo results suggest that 5GG may be a therapeutic candidate for the treatment of advanced prostate cancer.

Transforming growth factor-β (TGF-β)-mediated epithelial mesenchymal transition (EMT) of human lung cancer cells may contribute to lung cancer metastasis. It has been reported that EGCG can inhibit tumorigenesis and cancer cell growth in lung cancer; however, the effect of EGCG on EMT in nonsmall cell lung cancer (NSCLC) cells has not been investigated. In this study, we found that NSCLC cells A549 and H1299 were converted to the fibroblastic phenotype in response to TGF-β. Epithelial marker E-cadherin was down-regulated, and mesenchymal marker vimentin was up-regulated simultaneously. Our results illustrated that TGF-β was able to induce EMT in NSCLC cells, and EGCG would reverse TGF-β-induced morphological changes, up-regulate the expression of E-cadherin, and down-regulate the expression of vimentin. Immunofluorescent staining also demonstrated that E-cadherin was up-regulated and that vimentin was down-regulated by EGCG pretreatment. Moreover, wound-healing and the in vitro invasion assay showed that EGCG could inhibit TGF-β-induced migration and invasion of NSCLC cells. By using the dual-luciferase reporter assay, we demonstrated that EGCG inhibited TGF-β-induced EMT at the transcriptional level. EGCG decreased the phosphorylation of Smad2 and Erk1/2, inhibited the nuclear translocation of Smad2, and repressed the expression of transcription factors ZEB1, Snail, Slug, and Twist, and up-regulated the expression of E-cadherin. In summary, our results suggest that EGCG can inhibit TGF-β-induced EMT via down-regulation of phosphorylated Smad2 and Erk1/2 in NSCLC cells.

In this study, a chlorine dioxide solution (UC-1) composed of chlorine dioxide was produced using an electrolytic method and subsequently purified using a membrane. UC-1 was determined to contain 2000 ppm of gaseous chlorine dioxide in water. The efficacy and safety of UC-1 were evaluated. The antimicrobial activity was more than 98.2% reduction when UC-1 concentrations were 5 and 20 ppm for bacteria and fungi, respectively. The half maximal inhibitory concentrations (IC50) of H1N1, influenza virus B/TW/71718/04, and EV71 were 84.65 ± 0.64, 95.91 ± 11.61, and 46.39 ± 1.97 ppm, respectively. A 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test revealed that the cell viability of mouse lung fibroblast L929 cells was 93.7% at a 200 ppm UC-1 concentration that is over that anticipated in routine use. Moreover, 50 ppm UC-1 showed no significant symptoms in a rabbit ocular irritation test. In an inhalation toxicity test, treatment with 20 ppm UC-1 for 24 h showed no abnormality and no mortality in clinical symptoms and normal functioning of the lung and other organs. A ClO2 concentration of up to 40 ppm in drinking water did not show any toxicity in a subchronic oral toxicity test. Herein, UC-1 showed favorable disinfection activity and a higher safety profile tendency than in previous reports.

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