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Identifying the key molecules that enhance chemo‑ and radiosensitivity in head and neck squamous cell carcinoma (HNSCC) as well as reliable biomarkers for predicting recurrence and metastasis would be desirable to improve the prognosis of HNSCC. Previously, we have reported that Regenerating gene III (REG III) expression was associated with an improved survival rate for patients with HNSCC. In addition, resveratrol (3,4',5‑trihydroxystilbene) significantly increased REG III expression in HNSCC cells, and significantly inhibited cell growth, enhanced chemo‑ and radiosensitivity, and blocked the cancer invasion of HNSCC cells in vitro. In the present study, the effect of resveratrol on cancer progression in HNSCC was investigated in vivo using a xenograft nude mouse model. The results revealed that resveratrol increased the mRNA level of REG III in vivo, which was in agreement with our previous in vitro findings. Furthermore, REG III increased the antitumor effect of radiation or cisplatin in vivo, and resveratrol sensitized HNSCC to irradiation and cisplatin in vivo. These results indicated that resveratrol could increase the efficacy of cisplatin and irradiation through the REG III expression pathway, resulting in the inhibition of HNSCC progression in vivo.

Identification of reliable markers of chemo- and radiosensitivity and the key molecules that enhance the susceptibility of head and neck squamous cell carcinoma (HNSCC) to anticancer treatments is highly desirable. Previously, we have reported that regenerating gene (REG) Ⅲ expression was such a marker associated with an improved survival rate for HNSCC patients. In the present study, we investigated the stimulators for induction of REG Ⅲ expression using REG Ⅲ promoter assay in HNSCC cells transfected with REG Ⅲ promoter vector. We tested inflammatory cytokines, growth factors, polyphenols, PPARγ activator of thiazolidinediones, and histone deacetylase inhibitors, and found that 3,4',5-trihydroxy-trans-stilbene (resveratrol) significantly increased the REG Ⅲ promoter activity and the mRNA levels of REG Ⅲ in HNSCC cells. Moreover, we demonstrated the effect of resveratrol on cancer cell progression, such as cell proliferation, chemo‑ and radiosensitivity and cancer invasion of HNSCC cells. Resveratrol significantly inhibited cell growth, enhanced chemo‑ and radiosensitivity, and blocked cancer invasion of HNSCC cells. These data suggested that resveratrol could inhibit cancer progression through the REG Ⅲ expression pathway in HNSCC cells.

Epithelial-mesenchymal transition (EMT) is a key process involved in the invasion and metastasis of cancer cells. Furthermore, EMT can induce a cancer stem cell (CSC)-like phenotype in a number of tumor types. We demonstrated that Snail is one of the master regulators that promotes EMT and mediates cancer cell migration and invasion in many types of malignancies including head and neck squamous cell carcinoma (HNSCC). In the present study, we investigated the role of Snail in inducing and maintaining CSC-like properties through EMT in HNSCC. We established HNSCC cell lines transfected with Snail. Stem cell markers were evaluated with real-time RT-PCR and western blot analysis. CSC properties were assessed using sphere formation and WST-8 assays as well as chemosensitivity and chick chorioallantoic membrane in vivo invasion assays. Introduction of Snail induced EMT properties in HNSCC cells. Moreover, Snail-induced EMT maintained the CSC-like phenotype, and enhanced sphere formation capability, chemoresistance and invasive ability. These data suggest that Snail could be one of the critical molecular targets for the development of therapeutic strategies for HNSCC.

Head and neck squamous cell carcinoma (HNSCC) is known to have a poor prognosis. The resistance to treatment and distant metastasis are important clinical problems in HNSCC. The epithelial-mesenchymal transition (EMT) is a key process in successful execution of many steps such as the invasion and metastasis for cancer cells. Snail is one of the master regulators that promote EMT in many types of malignancies including HNSCC. Recently, it has been shown that Snail-induced EMT could induce a cancer stem cell (CSC)‑like phenotype in a number of tumor types. In this study, we investigated the role of Snail in inducing EMT properties and CSC-like phenotype in HNSCC. We established HNSCC cell lines transfected with Snail. E-cadherin was analyzed using western blot analysis and immunofluorescence staining. Cell migration and invasion were assessed using wound-healing assay and modified Boyden chamber assay, respectively. CSC markers of HNSCC, CD44 and aldehyde dehydrogenase 1 (ALDH1), were also evaluated with western blot analysis, and chemosensitivity was assessed with WST-8 assay. Introduction of Snail induced EMT properties in HNSCC cells and enhanced cell migration and invasion. Moreover, Snail-induced EMT gained CSC-like phenotype and was associated with increased chemoresistance. These results suggest that Snail could be one of the attractive targets for the development of therapeutic strategies in HNSCC.

Thyroid cancer sometimes contains poorly differentiated components, which have the potential of invasion and metastasis. We evaluated the possible roles of heparin-binding EGF-like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, in cell growth and invasion of thyroid cancer cells, and demonstrated that HB-EGF is not only a potent mitogen but also a chemotactic factor in the thyroid cancer cells 8305C and SW579. The HB-EGF-mediated chemotaxis was inhibited by neutralizing antibody against the EGF receptor (EGFR/HER1/ErbB1) or tyrphostin AG1478, a specific inhibitor of the EGFR tyrosine kinase. The HB-EGF mRNA and protein expression was also analyzed using RT-PCR and immunofluorescence methods, respectively. In addition, in clinical immunohistochemical study, increased expression of HB-EGF and its receptors, HER1 and EGFR4 (HER4/ErbB4), was observed in thyroid carcinoma cells. Our findings suggest that HB-EGF acts as a potent paracrine and/or autocrine chemotactic factor as well as a mitogen that mediates HER1 and/or HER4 in the invasion and metastasis of thyroid carcinoma cells, including poorly differentiated papillary carcinomas or undifferentiated/anaplastic carcinomas. These data may aid in the development of novel therapeutic strategies for thyroid cancer.

Identification of a reliable biomarker for predicting prognosis in head and neck cancers is highly desirable and has long been sought. There have been several reports that members of the regenerating gene (REG) family are highly expressed in chronic inflammation and in tumors of the digestive organs. In addition, it has been described in several reports that REG expression is associated with the progression of digestive cancers. In the present study, we evaluated the effect of REG expression on the prognosis of hypopharyngeal cancer. We investigated 37 cases with hypopharyngeal squamous cell carcinoma, determined REG mRNA expression, which is easily detected in formalin-fixed paraffin-embedded tissues using the real-time reverse transcriptase-polymerase chain reaction method, and evaluated the survival rate using the Kaplan-Meier method. According to these results, REG III mRNA expression was significantly associated with prolonged survival. Therefore, we constructed hypopharyngeal cancer cell lines transfected with REG III and assessed the cell proliferation and chemosensitivity and/or radiosensitivity in vitro. Cells transfected with REG III exhibited significantly lower cell proliferation and higher chemosensitivity and/or radiosensitivity compared with the control cells. These data suggest that REG III may be a reliable biomarker of prognosis in hypopharyngeal cancer. This is the first report concerning the association of REG III expression and the prognosis of head and neck squamous cell carcinoma including hypopharyngeal cancer.

Activation to a large extent of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and mutations in the p53 gene are involved in lung cancer therapeutic resistance. The mammalian target of rapamycin (mTOR) acts as a downstream effector for Akt. Activation of the Akt/mTOR signal is a contributing factor to decreased radiation sensitivity. The purpose of this study was to examine whether the effect of rapamycin on radiation sensitivity is affected by cellular p53 gene status. Cellular radiation sensitivity was evaluated by using two human non-small cell lung cancer (NSCLC) cell lines with the same genetic background except for their p53 gene status (H1299/wtp53 and H1299/mp53). The cells were treated with rapamycin and/or radiation. Cell viability, cell proliferation, apoptosis, cell cycle and Akt/mTOR signaling activity were explored. Rapamycin synergistically enhanced the cytotoxicity of radiation, promoting the induction of apoptosis. Moreover, the combined treatment augmented the cytostatic effects of radiation regardless of cellular p53 gene status. Rapamycin in combination with radiation increased G1 arrest and suppressed progression to S phase in both cell lines. Furthermore, the combined treatment conduced to a prominent p53-independent down-regulation of the mTOR signal and pro-survival molecule, cyclin D1. Rapamycin can enhance the effect of radiation through the repression of pro-survival signals and the reduction in the apoptotic threshold. Taken together, inhibition of the mTOR signal may be a promising strategy for radiosensitization with no relevance to p53 gene status from the aspects of cell lethality and cell growth depression.

To increase the chemo-sensitivity of anaplastic thyroid carcinoma, we examined the effects of glycerol on the tumor growth after CDDP treatment. The cultured cells of an anaplastic thyroid carcinoma cell line (8305c) carrying a mutated p53 gene (mp53) were transplanted into the thighs of nude mice. Tumor growth was evaluated until 24 days after intraperitoneal injection of CDDP and/or pre-injection of glycerol to the tumor. We treated the mice with half the tumor volume of glycerol (1.2 M) and/or CDDP at 6 mg/kg (BW) either of which hardly inhibited tumor growth by itself. When we treated the mice with the combination of glycerol and CDDP at these concentrations, however, a clear delay of the tumor growth was observed. We also immunohistochemically analyzed the effects of glycerol on the induction of caspase-3 activity and apoptosis. Cells positive for cleavage to active caspase-3 and 85 kDa PARP, and apoptosis were hardly observed in the tumors when they were treated with glycerol or CDDP alone. In contrast, when they were treated with CDDP combined with glycerol, such positive cells were significantly increased. It has been shown that glycerol synergistically enhanced the effects of CDDP as a tumor suppressive agent through the induction of caspase-3-mediated apoptosis in 8305c tumors. Therefore, glycerol might be useful for chemotherapy in patients with mp53 cancer cells.

We have investigated the CDDP sensitivities of two tongue cancer cell lines with differing p53 genetic status, one with wild-type p53 (SAS) and the other with mutant-type p53 (HSC-4). SAS was about 2 times more sensitive at the D10 dose and demonstrated increased p53 and Bax protein levels at 10 h after CDDP treatment on Western blot analysis. On the other hand, overexpression of p53 in HSC-4 was observed without CDDP treatment and no elevation of Bax could be detected. Apoptosis was observed after CDDP treatment in SAS but not in HSC-4 by Hoechst 33342-staining and electrophoresis methods. These findings indicate that p53 plays an important role in apoptosis as a positive regulator of Bax expression. It is suggested that p53 status may have predictive potential with regard to response to CDDP therapy.