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Sirt6 is involved in multiple biological processes, including aging, metabolism, and tumor suppression. Sirt1, another member of the sirtuin family, functionally overlaps with Sirt6, but its role in tumorigenesis is controversial. In this study, we focused on cell death in association with Sirt6/Sirt1 and reactive oxygen species (ROS) in head and neck squamous cell carcinomas (HNSCCs). Sirt6 induced cell death, as widely reported, but Sirt1 contributed to cell death only when it was suppressed by Sirt6 via regulation of MDM2. Sirt6 and Sirt6-mediated suppression of Sirt1 upregulated ROS, which further led to HNSCC cell death. These results provide insight into the molecular roles of Sirt6 and Sirt1 in tumorigenesis and could therefore contribute to the development of novel strategies to treat HNSCC., Cancer: A trio of proteins to tackle tumors New understanding of the interactions between three proteins sheds light on their role in either promoting or restricting the development of tumors called squamous cell carcinomas, which account for over 90% of all cancers in the head and neck. Researchers in South Korea led by Sang Yoon Kim and Seong Who Kim at the University of Ulsan, Seoul, investigated the role of the proteins Sirt6, Sirt1 and MDM2 in controlling the death of cancer cells caused by chemicals called reactive oxygen species (ROS). The effects of Sirt6 and Sirt1 combine to regulate ROS-induced cancer cell death. Sirt6 controls the activity of MDM2, stimulating ROS production. Sirt6 also influences MDM2 to suppress Sirt1 activity, thereby also promoting cancer cell death. Drugs affecting these three proteins could offer new approaches to anti-cancer therapy.

EPHA3, a member of the EPH family, is overexpressed in various cancers. We demonstrated previously that EPHA3 is associated with radiation resistance in head and neck cancer via the PTEN/Akt/EMT pathway, the inhibition of EPHA3 significantly enhances the efficacy of radiotherapy in vitro and in vivo. In this study, we investigated the mechanisms of PTEN regulation through EPHA3-related signaling. Increased DNA methyltransferase 1 (DNMT1) and enhancer of zeste homolog 2 (EZH2) levels, along with increased histone H3 lysine 27 trimethylation (H3K27me3) levels, correlated with decreased levels of PTEN in radioresistant head and neck cancer cells. Furthermore, PTEN is regulated in two ways: DNMT1-mediated DNA methylation, and EZH2-mediated histone methylation through EPHA3/C-myc signaling. Our results suggest that EPHA3 could display a novel regulatory mechanism for the epigenetic regulation of PTEN in radioresistant head and neck cancer cells.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has attracted attention as a potential candidate for cancer therapy. However, many primary cancers are resistant to TRAIL, even when combined with standard chemotherapy. The mechanism of TRAIL resistance in cancer cells has not been fully elucidated. The TRAIL death receptor (DR) 3&prime, untranslated region (3&prime, UTR) is reported to contain AU-rich elements (AREs) that are important for regulating DR mRNA stability. However, the mechanisms by which DR mRNA stability is determined by its 3&prime, UTR are unknown. We demonstrate that tristetraprolin (TTP), an ARE-binding protein, has a critical function of regulating DR mRNA stability. DR4 mRNA contains three AREs and DR5 mRNA contains four AREs in 3&prime, UTR. TTP bound to all three AREs in DR4 and ARE3 in DR5 and enhanced decay of DR4/5 mRNA. TTP overexpression in colon cancer cells changed the TRAIL-sensitive cancer cells to TRAIL-resistant cells, and down-regulation of TTP increased TRAIL sensitivity via DR4/5 expression. Therefore, this study provides a molecular mechanism for enhanced levels of TRAIL DRs in cancer cells and a biological basis for posttranscriptional modification of TRAIL DRs. In addition, TTP status might be a biomarker for predicting TRAIL response when a TRAIL-based treatment is used for cancer.

The manner in which p53 maintains redox homeostasis and the means by which two key metabolic elements, glucose and glutamine, contribute to p53-dependent redox stability remain unclear. To elucidate the manner in which p53 deals with glucose-deprived, reactive oxygen species (ROS)-prone conditions in this regard, two isogenic cancer subclones (HN3R-A and HN3R-B) bearing distinct p53 mutations as an in vitro model of intratumoral p53 heterogeneity were identified. Following cumulative irradiation, the subclones showed a similar metabolic shift to aerobic glycolysis and increasing NADPH biogenesis for cellular defense against oxidative damage irrespective of p53 status. The radioresistant cancer cells became more sensitive to glycolysis-targeting drugs. However, in glucose-deprived and ROS-prone conditions, HN3R-B, the subclone with the original p53 increased the utilization of glutamine by GLS2, thereby maintaining redox homeostasis and ATP. Conversely, HN3R-A, the p53-deficient radioresistant subclone displayed an impairment in glutamine usage and high susceptibility to metabolic stresses as well as ROS-inducing agents despite the increased ROS scavenging system. Collectively, our findings suggest that p53 governs the alternative utilization of metabolic ingredients, such as glucose and glutamine, in ROS-prone conditions. Thus, p53 status may be an important biomarker for selecting cancer treatment strategies, including metabolic drugs and ROS-inducing agents, for recurrent cancers after radiotherapy.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive head and neck malignancy. The aim of this study was to elucidate the role of periostin (POSTN) in the epithelial-to-mesenchymal transition (EMT) process mediating the acquisition of radioresistance in HNSCC.The expression levels of EMT hallmark genes including POSTN and Erk/Akt signaling pathways were compared between radiosensitive and radioresistant HNSCC cells.POSTN mRNA expression was higher in radioresistant HNSCC cells, and silencing POSTN significantly impaired their invasiveness under the effect of EMT process represented by up-regulation of mesenchymal markers and down-regulation of an epithelial marker. Expression levels of Erk and Akt were higher in radioresistant cells.POSTN in association with the Erk and Akt signaling pathways was up-regulated during the EMT process, leading to the conversion of radiosensitive to radioresistant HNSCC cells. POSTN may be a key marker for predicting the radioresistance and therapeutic target of HNSCC.

Metronomic chemotherapy, which is defined as a low-dose and frequent administration of cytotoxic drugs without drug-free breaks, has been recently emerged as an alternative to traditional MTD therapy and has shown therapeutic benefit in breast cancer patients in numbers of clinical studies. Unlike MTD, metronomic chemotherapy acts by multiple mechanisms including antiangiogenic effect and immunomodulation, but the direct cytotoxic effect only playing a minor role due to the lowered dose. In this light, within the limits of p53-deficient breast cancer, we demonstrate the enhanced anticancer effect of metronomic chemotherapy using doxorubicin when combined with Chk1 inhibitor MK-8776 by specifically augmenting the direct cytotoxic effect on cancer cells. Since the oral drug is greatly favored in metronomic chemotherapy due to the frequent and potential long-term administration, we prepared an oral doxorubicin by producing an ionic complex with deoxycholic acid, which showed sufficient bioavailability and anticancer effect when administered orally. MK-8776 selectively enhanced the cytotoxic effect of low-concentration doxorubicin in p53-deficient breast cancer cells by abrogating the Chk1-dependent cell cycle arrest in vitro. Consistently, combining MK-8776 significantly improved the anticancer effect of the daily administered oral doxorubicin in p53-deficient breast cancer xenografts especially in a lower dose of doxorubicin without evident systemic toxicities. Combination therapy of MK-8776 and metronomic oral doxorubicin would be thus promising in the treatment of p53-deficient breast cancer benefited from the augmented direct cytotoxic effect and low risk of toxicities.

Radiotherapy is a well-established therapeutic modality used in the treatment of many cancers. However, radioresistance remains a serious obstacle to successful treatment. Radioresistance can cause local recurrence and distant metastases in some patients after radiation treatment. Thus, many studies have attempted to identify effective radiosensitizers. Eph receptor functions contribute to tumor development, modulating cell-cell adhesion, invasion, neo-angiogenesis, tumor growth and metastasis. However, the role of EphA3 in radioresistance remains unclear. In the current study, we established a stable radioresistant head and neck cancer cell line (AMC HN3R cell line) and found that EphA3 was expressed predominantly in the radioresistant head and neck cancer cell line through DNA microarray, real time PCR and Western blotting. Additionally, we found that EphA3 was overexpressed in recurrent laryngeal cancer specimens after radiation therapy. EphA3 mediated the tumor invasiveness and migration in radioresistant head and neck cancer cell lines and epithelial mesenchymal transition- related protein expression. Inhibition of EphA3 enhanced radiosensitivity in the AMC HN 3R cell line in vitro and in vivo study. In conclusion, our results suggest that EphA3 is overexpressed in radioresistant head and neck cancer and plays a crucial role in the development of radioresistance in head and neck cancers by regulating the epithelial mesenchymal transition pathway.

Background/aim Recurrent laryngeal cancer often shows an aggressive phenotype after radiotherapy and does not respond to conventional therapeutic strategies. In this study, we investigated the contribution of furin to cellular invasiveness in radio-resistant laryngeal cancer. Materials and methods Using previously established AMC-HN-3 and AMC-HN-8 cell lines from laryngeal carcinoma patients, recurrent laryngeal cancer models were generated by cumulative irradiation (AMC-HN-3-70Gy and AMC-HN-8-70Gy). Immunocytochemistry and western blotting were used to determine the epithelial-mesenchymal transition (EMT). Invasion capacity was assessed using an in vitro invasion assay. Zymography was used to assess metalloproteinase-2 (MMP-2) activity. Tumor xenografts were developed to compare growth rate and furin expression in vivo. Furin expression in 35 patients (45 samples) with salvage total laryngectomy after radiation-based treatment was assessed by laryngeal cancer tissue microarray. Results Both AMC-HN-3-70Gy and AMC-HN-8-70Gy cell lines underwent EMT following radiation. However, AMC-HN-3-70Gy cells showed increased cellular invasiveness, whereas AMC-HN-8-70Gy cells showed no difference. AMC-HN-3-70Gy cells also exhibited elevated furin expression with up-regulated expression of the active form of membrane type 1-matrix metalloproteinase (MT1-MMP)/MMP-2, whereas AMC-HN-8-70Gy cells did not show significant changes. After administration of a furin inhibitor (chloromethyl ketone (CMK)), AMC-HN-3-70Gy cells showed a significant decrease in MT1-MMP/MMP-2 expression and cellular invasiveness. Nine of 22 samples (40.9%) from salvage total laryngectomy and one of 13 pre-radiation samples (7.7%) had high furin expression. Post-radiation, furin expression increased in seven of 10 patients whose pre- and post-radiation samples were available; all-cancer mortality (three patients) was observed in this group. Conclusion Together with EMT, furin activity may serve as an indicator of an aggressive cancer phenotype, suggesting that furin is a potentially useful target for recurrent laryngeal cancer.

Objectives: PI3K/Akt/mTOR pathway is commonly activated in most cancers and is correlated with resistance to anticancer therapies such as radiotherapy. Therefore, PI3K is an attractive target for treating PI3K-associated cancers. Material and Methods: We investigated the basal expression and the expression after treatment of PI3K inhibitor or Src inhibitor of PI3K/Akt pathway-related proteins in AMC-HN3, AMC-HN3R, HN30 and HN31 cells by performing immunoblotting analysis. The sensitivity to PI3K inhibitors or Src inhibitor was analyzed by MTT assay and clonogenic assay. To determine the antitumoral activity of combination treatment with PI3K inhibitor and Src inhibitor, we used using xenograft mouse model. Results: We found that PI3K regulatory subunit p85 was predominantly phosphorylated in radioresistant head and neck cancer cell line (HN31), which showed resistance to PI3K inhibitors. Next, we investigated mechanism through which PI3K p85 phosphorylation modulated response to PI3K inhibitors. Of note, constitutive activation of Src was found in HN31 cells and upon PI3K inhibitor treatment, restoration of p-Src was occurred. Src inhibitor improved the efficacy of PI3K inhibitor treatment and suppressed the reactivation of both Src and PI3K p85 in HN31 cells. Furthermore, downregulation of PI3K p85 expression by using a specific siRNA suppressed Src phosphorylation. Conclusions: Together, our results imply the novel role of the PI3K regulatory subunit p85 in the development of resistance to PI3K inhibitors and suggest the presence of a regulatory loop between PI3K p85 and Src in radioresistant head and neck cancers with constitutively active PI3K/Akt pathway.

Summary Tumor hypoxia is associated with poorer outcome in patients with head and neck carcinomas, but little is known about hypoxia biomarkers in oral tongue cancer. We evaluated whether hypoxia biomarkers and clinicopathologic variables were prognostic predictors in patients with T2-staged squamous cell carcinoma (SCC) of the oral tongue. Tissue microarrays were constructed from formalin-fixed tumor blocks of 43 patients with T2-staged tongue SCCs treated by surgical resection and neck dissection. Tissue samples were stained with monoclonal antibodies to hypoxia-inducible factor (HIF)-1α, HIF-2α, carbonic anhydrase (CA)-9, glucose transporter (GLUT)-1, and erythropoietin receptor (EPOR). Locoregional control and survival rates were calculated by the Kaplan-Meier method, and prognostic factors were calculated from uni- and multivariate analyses. Tumor thickness was correlated with expression of CA-9 and GLUT-1 and nodal classification was correlated with GLUT-1 expression. The nodal metastasis rate was 51%, and the 5-year locoregional control and disease-specific survival (DSS) rates were 59% and 69%, respectively. Univariate analysis showed that HIF-1α and EPOR expression were significantly related to DSS. Multivariate analysis showed that EPOR expression was an independent predictor of DSS ( P = 0.030). EPOR expression may be an independent predictor for DSS in patients with T2-staged SCC of the oral tongue.

We found four proteins in the AMC-HN-9 cells that might perform important functions in radio-resistance. These results also suggest that this cell line may provide us with important information about cancer cells regarding the roles of reactive oxygen species (ROS and antioxidants) in irradiation.Radiation susceptibility can be determined by the expression of some proteins. To identify such proteins involved in radiation susceptibility, we analyzed two cell lines with different radiation sensitivity.The AMC-HN-3 and -9 cell lines established from head and neck cancer patients were employed. They were irradiated with 4 Gy and two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was carried out. Then matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) was performed to identify 20 proteins. Western blotting was used to confirm the significantly expressed proteins.After 2-D PAGE, five spots were differentially expressed in the AMC-HN-9. Heat shock protein 27 kDa (HSP27), peroxiredoxin (Prx) II, glutathione S-transferase pi (GSTP), and an unknown protein were detected through MALDI-TOF MS. By using Western blotting, remarkable increments of the band intensity of HSP27, GSTP, Prx II, and Prx IV were confirmed in the AMC-HN-9 cell line.

It has been proposed that Wnt signaling pathway may be a key radioprotective mechanism in irradiated cancer cells; however, the specific radioresistance mechanisms remain not to be fully clarified. Here we elucidate a novel signaling pathway of radioresistance in head and neck cancer (HNC) cell lines involving interactions among the Wnt signaling pathway, cyclooxygenase-2 (COX-2) and Ku expression. Activation of the Wnt signaling pathway by (2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO) resulted in beta-catenin cytoplasmic accumulation and translocation to the nucleus, upregulated Ku expression and increased radioresistance in the COX-2-expressing HNC cell line. In contrast, Wnt singaling activation by BIO had no effects on Ku expression and radiosensitivity in a HNC cell line negative for COX-2. Interactions between Wnt singaling and Ku were indirectly regulated by COX-2. Blockage of COX-2 signaling led to the suppression of beta-catenin-induced Ku expression, and to consequent recovery of the radiosensitivity in HNC cells. Our results conclusively suggest that beta-catenin plays a pivotal role in the regulation of Ku expression via the proposed COX-2 intracellular pathway, thus supporting a novel radioresistance mechanism of HNC.

Ganglion is a common benign lesion that usually arises adjacent to the joints or tendons of the hand. However, an intratendinous ganglion is a rare condition. We report two cases of intratendinous ganglion of the extensor tendon of the hand which were treated with excision.

We attempted to elucidate the mechanism of cell death after radiation by studying how β-catenin silencing controls the radiation sensitivity of radioresistant head and neck cancer cells.The most radioresistant cancer cell line (AMC-HN-9) was selected for study. Targeted silencing of β-catenin was used on siRNAs. Sensitivity to radiation was examined using clonogenic and methylthiazol tetrazolium (MTT) assays.A combination of irradiation plus β-catenin silencing led to a significant reduction in the inherent radioresistance of AMC-HN-9 cells. Although expression of Ku70/80 was upregulated in AMC-HN-9 cells after irradiation, Ku70/80 was dramatically decreased in a combination of irradiation and β-catenin silencing. Interestingly, irradiation-induced Ku70/80 was completely prevented by β-catenin silencing-induced LKB1/AMP-activated protein kinase (LKB1/AMPK) signal.The LKB1/AMPK pathway might relay the signal between the Wnt/β-catenin pathway and the Ku70/Ku80 DNA repair machinery, and play a decisive role in fine-tuning the responses of cancer cells to irradiation. © 2015 Wiley Periodicals, Inc. Head Neck 38: E1909-E1917, 2016.

The Ku protein is essential for the repair of a majority of DNA double-strand breaks in mammalian cells. The purpose of this study was to investigate the relationship between the expression of Ku70/80 and sensitivity to radiation in cancer cell lines of the head and neck. The sensitivity to radiation in various head and neck cancer cell lines (AMC-HN-1 to -9) was analyzed by colony forming assay. Of the nine cell lines examined, the most radiosensitive cell line (AMC-HN-3) and the most radioresistant cell line (AMC-HN-9) were selected for this experiments. The expression of Ku70/80 was examined after irradiation using real time PCR, Western blotting and immunofluorescence in two different cell lines. Cell cycle distribution after irradiation were analysed. A differential radioresponse was demonstrated by expression of Ku70/80 in AMC-HN-3 and AMC-HN-9 cells. While the expression of Ku70 was slightly increased in the radioresistant AMC-HN-9 cell line, the expression of Ku80 was remarkably increased, suggesting a correlation between Ku80 expression and radiation resistance. Overexpression of Ku80 plays an important role in the repair of DNA damage induced by radiation. Ku80 expression may provide an effective predictive assay of radiosensitivity in head and neck cancers.

Although intramedullary nailing is an ideal treatment for subtrochanteric femoral fractures, it is technically challenging in fractures extending into the nail entry area and/or involving the lesser trochanter. Although the application of circumferential wire may facilitate reduction in these situations, its use remains controversial due to possible blood supply disturbances to underlying bone. In the present study, we evaluated complex subtrochanteric fractures treated by percutaneous cerclage wiring followed by intramedullary (IM) nailing for anatomical fracture reduction and union. Twelve patients (mean age 48.3 years) with an unstable subtrochanteric fracture were prospectively treated. Indications of percutaneous cerclage wiring followed by IM nailing were a fracture extending proximally into the nail entry area deemed difficult to treat by anatomical reconstruction by IM nailing or a fracture with long oblique or spiral component. One or two cerclage wires were percutaneously applied for the temporary reduction of main fragments, and then, a cephalo-medullary or a reconstruction nail was fixed. We assessed radiologic results (union time, alignment), functional results, and complications. All 12 cases healed, without a bone graft, at an average of 19.1 weeks after surgery (range 16–24). In 11 cases, acceptable alignment was achieved (mean, valgus 0.3° extension 0.6°) with minimal leg-length discrepancy; the other exhibited 1 cm of shortening. All patients were able to return to pre-injury activity levels, and median Merle d’Aubigne score was 16.9 (15–18). No infection or implant-related complication was encountered to latest follow-up (minimum 12 months postoperatively). Temporary reduction by percutaneous wiring offers a means of satisfactory nailing in difficult subtrochanteric femoral fractures, and affords anatomical reconstruction and favorable bony union.

Autophagy is frequently activated in radioresistant cancer cells. In the present study, we evaluated the role of autophagy and transforming growth factor-activated kinase 1 (TAK1) in radioresistance.TAK1 phosphorylation in MDA-MB231 breast cancer cells was evaluated by western blotting. The regulatory effects of the TAK1 inhibitor and autophagy inhibitor were assessed by cell morphology, cell survival and induction of apoptosis.Radiation induced the phosphorylation of TAK1, whereas the inhibition of TAK1 activity enhanced the cytotoxicity of radiation in MDA-MB231 cells. Autophagy inhibitors significantly enhanced radiation-induced apoptosis of MDA-MB231 cells. This augmentation in radiosensitivity seemed to result from the suppression of TAK1 activation.Inhibition of autophagy enhanced radiosensitivity through suppression of radiation-induced TAK1 activation, suggesting that the modulation of TAK1-induced autophagy may be a good therapeutic strategy to treat radioresistant breast cancer.

There are limited therapeutic options for patients with recurrent head and neck cancer after radiation therapy failure. To assess the use of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a salvage chemotherapeutic agent for recurrent cancer after radiation failure, we investigated the effect of clinically relevant cumulative irradiation on TRAIL-induced apoptosis.Using a previously established HN3 cell line from a laryngeal carcinoma patient, we generated a chronically irradiated HN3R isogenic cell line. Viability and apoptosis in HN3 and HN3R cells treated with TRAIL were analyzed with MTS and PI/annexin V-FITC assays. Western blotting and flow cytometry were used to determine the underlying mechanism of TRAIL resistance. DR4 expression was semiquantitatively scored in a tissue microarray with 107 laryngeal cancer specimens. Methylation-specific polymerase chain reaction and bisulfite sequencing for DR4 were performed for genomic DNA isolated from each cell line.HN3R cells were more resistant than HN3 cells to TRAIL-induced apoptosis because of significantly reduced levels of the DR4 receptor. The DR4 staining score in 37 salvage surgical specimens after radiation failure was lower in 70 surgical specimens without radiation treatment (3.03 ± 2.75 vs 5.46 ± 3.30, respectively; P.001). HN3R cells had a methylated DR4 CpG island that was partially demethylated by the DNA demethylating agent 5-aza-2'-deoxycytidine.Epigenetic silencing of the TRAIL receptor by hypermethylation of a DR4 CpG island might be an underlying mechanism for TRAIL resistance in recurrent laryngeal carcinoma treated with radiation.

Autophagy is frequently activated in radioresistant cancer cells where it provides a cell survival strategy. The mTOR inhibitor rapamycin activates autophagy but paradoxically it also enhances radiosensitivity. In this study, we investigated the mechanisms of these opposing actions in radiation-resistant glioma or parotid carcinoma cells. Radiation treatment transiently enhanced autophagic flux for a period of 72 hours in these cells and treatment with rapamycin or the mTOR inhibitor PP242 potentiated this effect. However, these treatments also increased heterochromatin formation, irreversible growth arrest, and premature senescence, as defined by expression of senescence-associated β-galactosidase activity. This augmentation in radiosensitivity seemed to result from a restoration in the activity of the tumor suppressor RB and a suppression of RB-mediated E2F target genes. In tumor xenografts, we showed that administering rapamycin delayed tumor regrowth after irradiation and increased senescence-associated β-galactosidase staining in the tumor. Our findings suggest that a potent and persistent activation of autophagy by mTOR inhibitors, even in cancer cells where autophagy is occurring, can trigger premature senescence as a method to restore radiosensitivity. Cancer Res; 73(14); 4267–77. ©2013 AACR.

The Wnt/β-catenin pathway regulates the viability and radiosensitivity of head and neck squamous cancer cells (HNSCC). Increased β-catenin predisposes HNSCC patients to poor prognosis and survival. This study was conducted to determine the mechanism by which β-catenin regulates the viability of HNSCC. AMC-HN-3, -HN-8, UM-SCC-38, and -SCC-47 cells, which were established from human head and neck cancer specimens, and underwent cell death following β-catenin silencing. β-Catenin silencing significantly induced G1 arrest and increased the expression of Bax and active caspase-3, which demonstrates the sequential activation of apoptotic cascades following treatment of HNSCC with targeted siRNA. Intriguingly, β-catenin silencing also induced autophagy. Here, we confirm that the number of autophagic vacuoles and the expression of type II light chain 3 were increased in cells that were treated with β-catenin siRNA. These cell death modes are most likely due to the activation of LKB1-dependent AMPK following β-catenin silencing. The activated LKB1/AMPK pathway in AMC-HN-3 cells caused G1 arrest by phosphorylating p53 and suppressing mTOR signaling. In addition, treating AMC-HN-3 cells with LKB1 siRNA preserved cell viability against β-catenin silencing-induced cytotoxicity. Taken together, these results imply that following β-catenin silencing, HNSCC undergo both apoptotic and autophagic cell death that are under the control of LKB1/AMPK. To the best of our knowledge, these results suggest for the first time that novel crosstalk between β-catenin and the LKB1/AMPK pathway regulates the viability of HNSCC. This study thus presents new insights into our understanding of the cellular and molecular mechanisms involved in β-catenin silencing-induced cell death.

The issue of whether aberrant expression of β1-integrin is associated with cancer progression and development of resistance to cytotoxic therapy is of considerable interest. Studies to date have shown that the anchorage-independent survival of cancer is attributed, in part, to epithelial-to-mesenchymal transition (EMT). Here, we have reported a novel alternative mechanism of anchorage-independent survival of cancer cells. Cell lines derived from head and neck cancer patients (AMC-HN-3 and AMC-HN-9) and the well-known EMT cancer cell line, MDA-MB231, were examined. The EMT features of AMC-HN-9 cells were comparable to those of MDA-MB231, whereas AMC-HN-3 cells showed no EMT characteristics. Although the pattern and degree of β1-integrin expression were similar in all three cell lines, sensitivities of the cells to β1-integrin knockdown with small interfering RNA (siRNA) were different. Cancer cells with no EMT features underwent cell death to a more significant extent following β1-integrin silencing than those with EMT. Intriguingly, we observed reactive activation of the p53–p21 pathway after β1-integrin silencing in AMC-HN-9 cells lacking an apparent cell death response. Simultaneous knockdown of wild-type p53 and β1-integrin in this cell line promoted cell death. Our data collectively indicate that β1-integrin-related cell death is closely associated with EMT phenotypes and activation of the p53–p21 pathway is partly involved in the acquisition of resistance to apoptosis induced by β1-integrin silencing. Further clarification of the mechanisms underlying p53 integration with β1-integrin signaling may facilitate the development of novel anti-cancer strategies.

Targeting hypoxia-inducible factor-1 (HIF-1) active cells in tumors may be an excellent strategy to improve the outcome of radiation therapy. On the basis of the reported role of YC-1 as a HIF-1 inhibitor with anti-cancer activity, we tested the therapeutic efficacy of YC-1 against radioresistance in vitro. The AMC-HN3 cancer cell line, developed from squamous cell carcinoma of the larynx, was cultured under hypoxic conditions or in the presence of cobalt chloride. Both treatments induced nuclear accumulation of HIF-1alpha protein. Cells cultured under normoxic or hypoxic conditions with and without YC-1 treatment were irradiated and analyzed using flow cytometry and clonogenic assays. In the absence of YC-1 treatment, irradiation induced a greater cytotoxic effect in normoxic cells than in cobalt-treated cells. Treatment of cobalt-treated cells with YC-1 effectively inhibited HIF-1alpha expression, and enhanced the sensitivity of cells to radiation, decreasing the surviving fraction to that of normoxic cells. Flow cytometry confirmed these results, showing that the sub-G1 fraction was increased in YC-1-treated hypoxic cells after irradiation. Our results suggest that YC-1 treatment may be an effective therapeutic strategy for overcoming the radioresistance of HIF-1alpha-expressing, hypoxic cancer cells.

Orally absorbable anticancer medications have great advantages for conventional cancer therapies to patients. Here we evaluated the potent anticancer effect of orally absorbable LHD, a chemical conjugate of low-molecular-weight heparin and deoxycholic acid, on tumor graft growth models.We characterized the angiogenic factors, such as VEGF, heparanase, and MMPs, of murine squamous cell carcinoma (SCC7), melanoma (B16F10) or lung carcinoma (LLC1). Two weeks after oral administration of LHD into these cancer-cell-bearing mice, we evaluated the antiangiogenic activity of LHD.Although all cancer cells expressed the angiogenic factors, SCC7 cells had much higher angiogenic potential and grew rapidly after implantation into mice. When orally administered, LHD delayed tumor graft growth regardless of cancer types. Particularly, LHD powerfully diminished the SCC7-derived tumor growth. Also, the expression of angiogenic factors in all kinds of tumor tissues was decreased, thereby attenuating the neovascularization in tumor tissue.Our study shows that LHD has potent anticancer and antiangiogenic effect on at least three kinds of tumor cells. LHD can be specifically used for preventing neovascularization in tumor tissue because it has therapeutical potential as an antiangiogenic drug and can be orally absorbed.

Purpose: Human peroxiredoxins (Prxs) are known as a family of thiol-specific antioxidant enzymes, among which Prx-I and -II play an important role in protecting cells from irradiation-induced cell death. It is not known whether Prx-IV also protects cells from ionizing radiation (IR). Methods and Materials: To evaluate the protective role of Prx-IV in IR, we transfected full-length Prx-IV cDNA into AMC-HN3 cells, which weakly express endogenous Prx-IV, and knocked down the expression of Prx-IV with siRNA methods using AMC-HN7 cells, which express high levels of endogenous Prx-IV. Radiosensitivity profiles in these cells were evaluated using clonogenic assay, FACS analysis, cell viability, and TUNEL assay. Results: Three Prx-IV expressing clones were isolated. Prx-IV regulated intracellular reactive oxygen species (ROS) levels and made cells more resistant to IR-induced apoptosis. Furthermore, the knockdown of Prx-IV with siRNA made cells more sensitive to IR-induced apoptosis. Conclusion: The results of these studies suggest that Prx-IV may play an important role in protecting cells from IR-induced apoptosis in head-and-neck squamous cell carcinoma.

The inhibitory efficacies of new bile acid acylated-heparin derivative (heparin-DOCA) were evaluated on experimental lung metastasis. We evaluated the effect of heparin-DOCA on intercellular interactions including those between B16F10 and thrombin-activated platelets and TNF-alpha-activated HUVECs, and between B16F10 and immobilized mouse P-selectin. In addition, the inhibitory effects of heparin-DOCA on adhesion and invasion of B16F10 to Matrigel were studied. In an animal mouse study, the blood clot formation and the retention of red fluorescence protein (RFP)-B16F10 in lungs were assessed after heparin-DOCA and RFP-B16F10 intravenous administration. Furthermore, we investigated the anti-metastatic effect of heparin-DOCA against lung metastasis induced by B16F10 and SCC7. Heparin-DOCA inhibited intercellular interactions between B16F10 and activated platelets or activated HUVECs by blocking P- and E-selectin-mediated interactions. Moreover, it reduced adhesion and invasion of B16F10 to ECM, thereby affecting the reduction of early retention of B16F10 in the lung. Heparin-DOCA attenuated lung colony formation on the surfaces and in interior of the lung, and attenuated metastasis by B16F10 and SCC7. These results suggest that heparin-DOCA may have potentials as therapeutic agent that prevents tumor metastasis and progression.