Liquid plasma promotes angiogenesis through upregulation of endothelial nitric oxide synthase-induced extracellular matrix metabolism: potential applications of liquid plasma for vascular injuries.

BACKGROUND: Applications of nonthermal plasma have expanded beyond the biomedical field to include antibacterial, anti-inflammatory, wound healing, and tissue regeneration. Plasma enhances epithelial cell repair; however, the potential damage to deep tissues and vascular structures remains under investigation. RESULT: This study assessed whether liquid plasma (LP) increased nitric oxide (NO) production in human umbilical vein endothelial cells by modulating endothelial NO synthase (eNOS) phosphorylation and potential signaling pathways. First, we developed a liquid plasma product and confirmed the angiogenic effect of LP using the Matrigel plug assay. We found that the NO content increased in plasma-treated water. NO in plasma-treated water promoted cell migration and angiogenesis in scratch and tube formation assays via vascular endothelial growth factor mRNA expression. In addition to endothelial cell proliferation and migration, LP influenced extracellular matrix metabolism and matrix metalloproteinase activity. These effects were abolished by treatment with NG-L-monomethyl arginine, a specific inhibitor of NO synthase. Furthermore, we investigated the signaling pathways mediating the phosphorylation and activation of eNOS in LP-treated cells and the role of LKB1-adenosine monophosphate-activated protein kinase in signaling. Downregulation of adenosine monophosphate-activated protein kinase by siRNA partially inhibited LP-induced eNOS phosphorylation, angiogenesis, and migration. CONCLUSION: The present study suggests that LP treatment may be a novel strategy for promoting angiogenesis in vascular damage. Video Abstract.

Anhydrous Alum Inhibits α-MSH-Induced Melanogenesis by Down-Regulating MITF via Dual Modulation of CREB and ERK.

Melanogenesis, the intricate process of melanin synthesis, is central to skin pigmentation and photoprotection and is regulated by various signaling pathways and transcription factors. To develop potential skin-whitening agents, we used B16F1 melanoma cells to investigate the inhibitory effects of anhydrous alum on melanogenesis and its underlying molecular mechanisms. Anhydrous alum (KAl(SO4)2) with high purity (>99%), which is generated through the heat-treatment of hydrated alum (KAl(SO4)2·12H2O) at 400 °C, potentiates a significant reduction in melanin content without cytotoxicity. Anhydrous alum downregulates the master regulator of melanogenesis, microphthalmia-associated transcription factor (MITF), which targets key genes involved in melanogenesis, thereby inhibiting α-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis. Phosphorylation of the cAMP response element-binding protein, which acts as a co-activator of MITF gene expression, is attenuated by anhydrous alum, resulting in compromised MITF transcription. Notably, anhydrous alum promoted extracellular signal-regulated kinase phosphorylation, leading to the impaired nuclear localization of MITF. Overall, these results demonstrated the generation and mode of action of anhydrous alum in B16F1 cells, which constitutes a promising option for cosmetic or therapeutic use.

Effect of the Plasma Gas Type on the Surface Characteristics of 3Y-TZP Ceramic.

Plasma surface treatment can be an attractive strategy for modifying the chemically inert nature of zirconia to improve its clinical performance. This study aimed to clarify the effect of plasma gas compositions on the physicochemical surface modifications of 3 mol% yttria-stabilized zirconia (3Y-TZP). The cold, atmospheric plasma discharges were carried out by using four different plasma gases, which are He/O2, N2/Ar, N2, and Ar from an application distance of 10 mm for 60 s. Static contact angles were measured to define the surface free energy. Changes in elemental composition, surface crystallinity, and surface topography were assessed with X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM), respectively. A significant decrease in water contact angle was observed in all plasma groups with the lowest value of 69° in the N2/Ar group. CLSM and SEM investigations exhibited no morphological changes in all plasma groups. XPS revealed that a reduction in the surface C content along with an increase in O content was pronounced in the case of N2/Ar compared to others, which was responsible for high hydrophilicity of the surface. XRD showed that the changes in crystallite size and microstrain due to oxygen atom displacements were observed in the N2/Ar group. The N2/Ar plasma treatment may contribute to enhancing the bioactivity as well as the bonding performance of 3Y-TZP by controlling the plasma-generated nitrogen functionalities.

OTUB1 knockdown promotes apoptosis in melanoma cells by upregulating TRAIL expression.

Melanoma, the most serious type of skin cancer, exhibits a high risk of metastasis. Although chemotherapeutic treatment for metastatic melanoma improves disease outcome and patient survival, some patients exhibit resistance or toxicity to the drug treatment regime. OTUB1 is a deubiquitinating enzyme overexpressed in several cancers. In this study, we investigated the effects of inhibiting OTUB1 expression on melanoma-cell proliferation and viability and identified the underlying molecular mechanism of action of OTUB1. We did endogenous OTUB1 knockdown in melanoma cells using short interfering RNA, and assessed the resulting phenotypes via MTT assays, Western blotting, and cell-cycle analysis. We identified differentially expressed genes between OTUB1-knockdown cells and control cells using RNA sequencing and confirmed them via Western blotting and reverse transcription polymerase chain reaction. Furthermore, we investigated the involvement of apoptotic and cell survival signaling pathways upon OTUB1 depletion. OTUB1 depletion in melanoma cells decreased cell viability and caused simultaneous accumulation of cells in the sub-G1 phase, indicating an increase in the apoptotic-cell population. RNA sequencing of OTUB1-knockdown cells revealed an increase in the levels of the apoptosis-inducing protein TRAIL. Additionally, OTUB1-knockdown cells exhibited increased sensitivity to PLX4032, a BRAF inhibitor, implying that OTUB1 and BRAF act collectively in regulating apoptosis. Taken together, our findings show that OTUB1 induces apoptosis of melanoma cells in vitro, likely by upregulating TRAIL, and suggest that approaches targeting OTUB1 can be developed to provide novel therapeutic strategies for treating melanoma. [BMB Reports 2021; 54(12): 608-613].

DIM-C-pPhtBu induces lysosomal dysfunction and unfolded protein response - mediated cell death via excessive mitophagy.

Despite technological advances in cancer treatment, the survival rate of patients with head and neck cancer (HNC) has not improved significantly. Many studies have shown that endoplasmic reticulum (ER) stress-related signals are associated with mitochondrial damage and that these signals determine whether cells maintain homeostasis or activate cell death programs. The unfolded protein response (UPR) is regulated by ER membrane proteins such as double-stranded RNA-activated protein kinase R(PKR)-like ER kinase (PERK), which directly activate transcription of chaperones or genes that function in redox homeostasis, protein secretion, or cell death programs. In this study, we focused on the role of mitophagy and ER stress-mediated cell death induced by DIM-C-pPhtBu in HNC cancer. We found that DIM-C-pPhtBu, a compound that activates ER stress in many cancers, induced lysosomal dysfunction, excessive mitophagy, and cell death in HNC cells. Moreover, DIM-C-pPhtBu strongly inhibited HNC progression in a xenograft model by altering mitophagy related protein expression. Taken together, the results demonstrate that DIM-C-pPhtBu induces excessive mitophagy and eventually UPR-mediated cell death in HNC cells, suggesting that new anti-cancer drugs could be developed based on the connection between mitophagy and cancer cell death.

Protective Effects of N-Acetylcysteine against Radiation-Induced Oral Mucositis In Vitro and In Vivo.

PURPOSE: Radiation-induced oral mucositis limits delivery of high-dose radiation to targeted cancers. Therefore, it is necessary to develop a treatment strategy to alleviate radiation-induced oral mucositis during radiation therapy. We previously reported that inhibiting reactive oxygen species (ROS) generation suppresses autophagy. Irradiation induces autophagy, suggesting that antioxidant treatment may be used to inhibit radiation-induced oral mucositis. MATERIALS AND METHODS: We determined whether treatment with N-acetyl cysteine (NAC) could attenuate radiation-induced buccal mucosa damage in vitro and in vivo. The protective effects of NAC against oral mucositis were confirmed by transmission electron microscopy and immunocytochemistry. mRNA and protein levels of DNA damage and autophagy-related genes were measured by quantitative real-time polymerase chain reaction and western blot analysis, respectively. RESULTS: Rats manifesting radiation-induced oral mucositis showed decreased oral intake, loss of body weight, and low survival rate. NAC intake slightly increased oral intake, body weight, and the survival rate without statistical significance. However, histopathologic characteristics were markedly restored in NAC-treated irradiated rats. LC3B staining of rat buccal mucosa revealed that NAC treatment significantly decreased the number of radiation-induced autophagic cells. Further, NAC inhibited radiation-induced ROS generation and autophagy signaling. In vitro, NAC treatment significantly reduced the expression of NRF2, LC3B, p62, and Beclin-1 in keratinocytes compared with that after radiation treatment. CONCLUSION: NAC treatment significantly inhibited radiation-induced autophagy in keratinocytes and rat buccal mucosa and may be a potentially safe and effective option for the prevention of radiation-induced buccal mucosa damage.

GnRH impairs diabetic wound healing through enhanced NETosis.

It has been reported that neutrophil extracellular traps (NETs) impair wound healing in diabetes and that inhibiting NET generation (NETosis) improves wound healing in diabetic mice. Gonadotropin-releasing hormone (GnRH) agonists are associated with a greater risk of diabetes. However, the role of GnRH in diabetic wound healing is unclear. We determined whether GnRH-promoted NETosis and induced more severe and delayed diabetic wound healing. A mouse model of diabetes was established using five injections with streptozotocin. Mice with blood glucose levels >250 mg/dL were then used in the experiments. GnRH agonist treatment induced delayed wound healing and increased NETosis at the skin wounds of diabetic mice. In contrast, GnRH antagonist treatment inhibited GnRH agonist-induced delayed wound healing. The expression of NETosis markers PAD4 and citrullinated histone H3 were increased in the GnRH-treated diabetic skin wounds in diabetic mice and patients. In vitro experiments also showed that neutrophils expressed a GnRH receptor and that GnRH agonist treatment increased NETosis markers and promoted phorbol myristate acetate (PMA)-induced NETosis in mouse and human neutrophils. Furthermore, GnRH antagonist treatment suppressed the expression of NETosis markers and PMA-induced NETosis, which were increased by GnRH treatment. These results indicated that GnRH-promoted NETosis and that increased NETosis induced delayed wound healing in diabetic skin wounds. Thus, inhibition of GnRH might be a novel treatment of diabetic foot ulcers.

Non-thermal plasma inhibits mast cell activation and ameliorates allergic skin inflammatory diseases in NC/Nga mice.

Non-thermal plasma (NTP) has many functional activities such as, sterilization, wound healing and anti-cancer activity. Despite of its wide spread biomedical application, the effect of NTP on immune cells and allergic response has not been well studied. In this study, we determined whether NTP suppresses mast cell activation, which is important for allergic response, and ameliorates an atopic dermatitis (AD)-like skin inflammatory disease in mice. Exposure to NTP-treated medium during mast cell activation inhibited the expression and production of IL-6, TNF-α and suppressed NF-κB activation. We also investigated whether NTP treatment ameliorates house dust mite (HDM)-induced AD-like skin inflammation in mice. NTP treatment inhibited increases in epidermal thickness and recruitment of mast cells and eosinophils, which are important cell types in AD pathogenesis. In addition, Th2 cell differentiation was induced by application of HDM and the differentiation was also inhibited in the draining lymph node of NTP-treated mice. Finally, the expression of AD-related cytokines and chemokines was also decreased in NTP-treated mice. Taken together, these results suggest that NTP might be useful in the treatment of allergic skin diseases, such as AD.

Nonthermal atmospheric plasma enhances myoblast differentiation by eliciting STAT3 phosphorylation.

The use of nonthermal atmospheric plasma (NTP) in the biomedical field has recently expanded into cell death induction in cancer, infection prevention, inflammation treatment, and wound-healing enhancement. NTP has been demonstrated to enhance skin and muscle regeneration, but its effects on tissue regeneration, following deep tissue or muscle damage, remains underinvestigated. In this study, we determined the effects of NTP on muscle differentiation and the mechanisms of NTP's contribution to differentiation and regeneration. NTP treatment enhanced cell differentiation in primary normal human skeletal muscle myoblast cells and increased the relative expression of mRNA levels of MyoD which is one of the earliest markers of myogenic commitment, and myogenin, which are important transcription factors required for myogenic differentiation. Furthermore, NTP treatment induced increases in the levels of myosin heavy chain, a differentiated muscle-specific protein, and in myotube formation of myoblasts. We observed that signal transducer and activator of transcription 3 (STAT3) activation induced by NTP treatment affects the myogenic differentiation. In addition, STAT3 phosphorylation was also enhanced by NTP treatment in injured animal muscle. These findings indicate that NTP could enhance musculoskeletal differentiation by acting as an external stimulus for myoblast differentiation, suggesting its treatment potential in promoting regeneration of damaged muscle.-Park, J. K., Kim, Y. S., Kang, S. U., Lee, Y. S., Won, H.-R., Kim, C.-H. Nonthermal atmospheric plasma enhances myoblast differentiation by eliciting STAT3 phosphorylation.

Non-thermal plasma treated solution with potential as a novel therapeutic agent for nasal mucosa regeneration.

Adequate and rapid mucosal regeneration is one of the most important factors in the healing process of nasal mucosa after surgery or trauma. In particular, delayed mucosal regeneration after surgery is an important cause of surgical failure. However, no effective treatment is available yet. Non-thermal plasma (NTP) has several medical effects, but the existing probe type is limited to local direct treatment. Therefore, we investigated the various effects using liquid type plasma to overcome this limitation. In addition, the therapeutic effects of non-thermal plasma treated solution (NTS) on nasal mucosa have yet to be determined. Experiments were carried out using BEAS-2B, a human bronchial epithelial cell line similar to nasal mucosa epithelium. NTS had no cytotoxicity to the BEAS-2B cells and enhanced cell proliferation. NTS also promoted migration of BEAS-2B cells. NTS increased cell proliferation and migration via epidermal growth factor receptor (EGFR) activities and epithelial-to-mesenchymal transition (EMT) signaling. Furthermore, NTS enhanced wound healing of nasal mucosa in an animal model. Accordingly, NTS promotes nasal mucosa wound healing by increasing cell proliferation and migration. These findings suggest the therapeutic potential of NTS in nasal mucosa wound healing.

Comparative Effects of Non-Thermal Atmospheric Pressure Plasma on Migration and Invasion in Oral Squamous Cell Cancer, by Gas Type.

PURPOSE: The fourth state of matter, plasma is known as an ionized gas with electrons, radicals and ions. The use of non-thermal plasma (NTP) in cancer research became possible because of the progresses in plasma medicine. Previous studies on the potential NTP-mediated cancer therapy have mainly concentrated on cancer cell apoptosis. In the present study, we compared the inhibitory effect of NTP on cell migration and invasion in the oral squamous cancer cell lines. MATERIALS AND METHODS: We used oral squamous cancer cell lines (SCC1483, MSKQLL1) and different gases (N2, He, and Ar). To investigate the mechanism of plasma treatment, using different gases (N2, He, and Ar) which induces anti-migration and anti-invasion properties, we performed wound healing assay, invasion assay and gelatin zymography. RESULTS: The results showed that NTP inhibits cancer cell migration and invasion of oral squamous cancer cell. In addition, focal adhesion kinase expression and matrix metalloproteinase-2/9 activity were also inhibited. CONCLUSION: The suppression of cancer cell invasion by NTP varied depending on the type of gas. Comparison of the three gases revealed that N2 NTP inhibited cell migration and invasion most potently via decreased expression of focal adhesion kinase and matrix metalloproteinase activity.

Anti-cancer effect of (-)-epigallocatechin-3-gallate (EGCG) in head and neck cancer through repression of transactivation and enhanced degradation of ß-catenin.

BACKGROUND AND PURPOSE: Aberrant expression of ß-catenin is highly associated with progression of various cancers including head and neck cancer (HNC). Green tea is most commonly used beverage in the world and one of the more bioactive compounds is the antioxidant epigallocatechin gallate (EGCG). This study was performed to investigate the mechanism by which EGCG inhibits the growth of HNC, focusing on the modulation of the expression and activity of ß-catenin. METHODS: In vitro effects of EGCG on the transcription, translation, or degradation of ß-catenin were investigated. Antitumor effects of EGCG in vivo were evaluated in a syngeneic mouse model and ß-catenin expression was checked in HNC patients' samples. RESULTS: ß-catenin expression was elevated in tumor samples of HNC patients. EGCG induced apoptosis in KB and FaDu cells through the suppression of ß-catenin signaling. Knockdown of ß-catenin using siRNA enhanced the proapoptotic activities of EGCG. EGCG decreased mRNA and transcriptional activity of ß-catenin in p53 wild-type KB cells. EGCG also enhanced the ubiquitination and proteasomal degradation of ß-catenin. The suppression of ß-catenin and consequent apoptosis were observed in response to EGCG treatment in a syngeneic mouse model. In conclusion, we report that EGCG inhibits ß-catenin expression through multiple mechanisms including decreased transcription and increased ubiquitin-mediated 26S proteasomal degradation. CONCLUSION: This study proposes a novel molecular rationale for antitumor activities of green tea in HNCs.

Anti-cancer Effect of Luminacin, a Marine Microbial Extract, in Head and Neck Squamous Cell Carcinoma Progression via Autophagic Cell Death.

PURPOSE: The purpose of this study is to determine whether luminacin, a marine microbial extract from the Streptomyces species, has anti-tumor effects on head and neck squamous cell carcinoma (HNSCC) cell lines via autophagic cell death. MATERIALS AND METHODS: Inhibition of cell survival and increased cell death was measured using cell viability, colony forming, and apoptosis assays. Migration and invasion abilities of head and cancer cells were evaluated using wound healing, scattering, and invasion assays. Changes in the signal pathway related to autophagic cell death were investigated. Drug toxicity of luminacin was examined in in vitro HaCaT cells and an in vivo zebrafish model. RESULTS: Luminacin showed potent cytotoxicity in HNSCC cells in cell viability, colony forming, and fluorescence-activated cell sorting analysis. In vitro migration and invasion of HNSCC cells were attenuated by luminacin treatment. Combined with Beclin-1 and LC3B, Luminacin induced autophagic cell death in head and neck cancer cells. In addition, in a zebrafish model and human keratinocyte cell line used for toxicity testing, luminacin treatment with a cytotoxic concentration to HNSCC cells did not cause toxicity. CONCLUSION: Taken together, these results demonstrate that luminacin induces the inhibition of growth and cancer progression via autophagic cell death in HNSCC cell lines, indicating a possible alternative chemotherapeutic approach for treatment of HNSCC.

Combination of NTP with cetuximab inhibited invasion/migration of cetuximab-resistant OSCC cells: Involvement of NF-κB signaling.

Although the epidermal growth factor receptor (EGFR) is an established target in head-and-neck cancer (HNC), resistance to EGFR-targeted therapy mediated by various mechanisms has been reported. Therefore, a combination strategy to overcome resistance to EGFR mono-targeted therapy is clinically required. We have previously demonstrated that non-thermal atmospheric pressure plasma (NTP) induces death of various cancer cells, including oral squamous cancer (OSCC) cells. In this study, we report for the first time that combining NTP treatment with cetuximab led to inhibition of migration and invasion in cetuximab-resistant OSCC cells, which could be a promising strategy to overcome resistance to anti-EGFR therapy. NTP induced deactivation of NF-κB in SCCQLL1 cells, but not in MSKQLL1 cells. In addition, NTP increased the expression level of E-cadherin, and decreased those of vimentin, Slug, Snail, matrix metalloproteinase (MMP)-2, -9, and activities of MMPs. Moreover, NF-κB upregulation using cDNA diminished the combination effect of NTP on invasion, migration and related signals. Taken together, these results indicate that the combination of NTP with cetuximab can decrease invasiveness in cetuximab-resistant OSCCs through a novel mechanism involving the NF-κB pathway. These findings show the therapeutic potential of treatment that combines NTP and cetuximab in OSCC.

Non-thermal plasma induces AKT degradation through turn-on the MUL1 E3 ligase in head and neck cancer.

Recent research on non-thermal plasma (NTP, an ionized gas) has identified it as a novel cancer therapeutic tool. However, the molecular mechanism remains unclear. In this study, we demonstrated NTP induced cell death of head and neck cancer (HNC) through the AKT ubiquitin-proteasome system. NTP increased the gene expression of mitochondrial E3 ubiquitin protein ligase 1 (MUL1), an E3 ligase for AKT, and NTP-induced HNC cell death was prevented by MUL1 siRNA. We also showed that MUL1 inhibited the level of AKT and p-AKT and MUL1 expression was increased by NTP-induced ROS. Furthermore, we optimized and manufactured a new type of NTP, a liquid type of NTP (LTP). In syngeneic and xenograft in vivo tumor models, LTP inhibited tumor progression by increasing the MUL1 level and reducing p-AKT levels, indicating that LTP also has an anti-cancer effect through the same mechanism as that of NTP. Taken together, our results suggest that NTP and LTP have great potential for HNC therapy.

Nonthermal plasma induces apoptosis in ATC cells: involvement of JNK and p38 MAPK-dependent ROS.

PURPOSE: To determine the effects of nonthermal plasma (NTP) induced by helium (He) alone or He plus oxygen (O2) on the generation of reactive oxygen species (ROS) and cell death in anaplastic thyroid cancer cells. MATERIALS AND METHODS: NTP was generated in He alone or He plus O2 blowing through a nozzle by applying a high alternating current voltage to the discharge electrodes. Optical emission spectroscopy was used to identify various excited plasma species. The apoptotic effect of NTP on the anaplastic thyroid cancer cell lines, such as HTH83, U-HTH 7, and SW1763, was verified with annexin V/propidium staining and TUNEL assay. ROS formation after NTP treatment was identified with fluorescence-activated cell sorting with DCFDA staining. The mitogen-activated protein kinase pathways and caspase cascade were investigated to evaluate the molecular mechanism involved and cellular targets of plasma. RESULTS: NTP induced significant apoptosis in all three cancer cell lines. The plasma using He and O2 generated more O2-related species, and increased apoptosis and intracellular ROS formation compared with the plasma using He alone. NTP treatment of SW1763 increased the expression of phosphor-JNK, phosphor-p38, and caspase-3, but not phosphor-ERK. Apoptosis of SW1763 as well as expressions of elevated phosphor-JNK, phosphor-p38, and caspase-3 induced by NTP were effectively inhibited by intracellular ROS scavengers. CONCLUSION: NTP using He plus O2 induced significant apoptosis in anaplastic cancer cell lines through intracellular ROS formation. This may represent a new promising treatment modality for this highly lethal disease.

Non-thermal atmospheric pressure plasma inhibits thyroid papillary cancer cell invasion via cytoskeletal modulation, altered MMP-2/-9/uPA activity.

Plasma, the fourth state of matter, is defined as a partially or completely ionized gas that includes a mixture of electrons and ions. Advances in plasma physics have made it possible to use non-thermal atmospheric pressure plasma (NTP) in cancer research. However, previous studies have focused mainly on apoptotic cancer cell death mediated by NTP as a potential cancer therapy. In this study, we investigated the effect of NTP on invasion or metastasis, as well as the mechanism by which plasma induces anti-migration and anti-invasion properties in human thyroid papillary cancer cell lines (BHP10-3 and TPC1). Wound healing, pull-down, and Transwell assays demonstrated that NTP reduced cell migration and invasion. In addition, NTP induced morphological changes and cytoskeletal rearrangements, as detected by scanning electron microscopy and immunocytochemistry. We also examined matrix metalloproteinase (MMP)-2/-9 and urokinase-type plasminogen activator (uPA) activity using gelatin zymography, uPA assays and RT-PCR. FAK, Src, and paxillin expression was detected using Western blot analyses and immunocytochemistry. NTP decreased FAK, Src, and paxillin expression as well as MMP/uPA activity. In conclusion, NTP inhibited the invasion and metastasis of BHP10-3 and TPC1 cells by decreasing MMP-2/-9 and uPA activities and rearranging the cytoskeleton, which is regulated by the FAK/Src complex. These findings suggest novel actions for NTP and may aid in the development of new therapeutic strategies for locally invasive and metastatic cancers.

Non-thermal atmospheric pressure plasma induces apoptosis in oral cavity squamous cell carcinoma: Involvement of DNA-damage-triggering sub-G(1) arrest via the ATM/p53 pathway.

Recent advances in physics have made possible the use of non-thermal atmospheric pressure plasma (NTP) in cancer research. Although increasing evidence suggests that NTP induces death of various cancer cell types, thus offering a promising alternative treatment, the mechanism of its therapeutic effect is little understood. In this study, we report for the first time that NTP led to apoptotic cell death in oral cavity squamous cell carcinoma (OSCC). Interestingly, NTP induced a sub-G(1) arrest in p53 wild-type OSCCs, but not in p53-mutated OSCCs. In addition, NTP increased the expression levels of ATM, p53 (Ser 15, 20 and 46), p21, and cyclin D1. A comet assay, Western blotting and immunocytochemistry of γH2AX suggested that NTP-induced apoptosis and sub-G(1) arrest were associated with DNA damage and the ATM/p53 signaling pathway in SCC25 cells. Moreover, ATM knockdown using siRNA attenuated the effect of NTP on cell death, sub-G(1) arrest and related signals. Taken together, these results indicate that NTP induced apoptotic cell death in p53 wild-type OSCCs through a novel mechanism involving DNA damage and triggering of sub-G(1) arrest via the ATM/p53 pathway. These findings show the therapeutic potential of NTP in OSCC.

Tolfenamic acid induces apoptosis and growth inhibition in anaplastic thyroid cancer: Involvement of nonsteroidal anti-inflammatory drug-activated gene-1 expression and intracellular reactive oxygen species generation.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are usually used for the treatment of inflammatory diseases. However, certain NSAIDs also have antitumor activities in various cancers, including head and neck cancer, through cyclooxygenase-dependent or independent pathways. Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1), a TGF-ß superfamily protein, is induced by NSAIDs and has been shown to be induced by several antitumorigenic compounds and to exhibit proapoptotic and antitumorigenic activities. In this report, we demonstrate for the first time that tolfenamic acid (TA) transcriptionally induced the expression of NAG-1 during TA-induced apoptosis of anaplastic thyroid cancer (ATC) cells. TA reduced the viability of ATC cells in a dose-dependent manner and induced apoptosis, findings that were coincident with NAG-1 expression. Overexpression of the NAG-1 gene using cDNA enhanced the apoptotic effect of TA, whereas suppression of NAG-1 expression by small interfering RNA attenuated TA-induced apoptosis. Subsequently, we found that intracellular ROS generation plays an important role in activating the proapoptotic protein NAG-1. Then, we confirmed antitumorigenic effects of TA in a nude mouse orthotopic ATC model, and this result accompanied the augmentation of NAG-1 expression and ROS generation in tumor tissue. Taken together, these results demonstrate that TA induces apoptosis via NAG-1 expression and ROS generation in in vitro and in vivo ATC models, providing a novel mechanistic explanation and indicating a potential chemotherapeutic approach for treatment of ATC.