Protaetia brevitarsis Extract Attenuates RANKL-Induced Osteoclastogenesis by Inhibiting the JNK/NF-κB/PLCγ2 Signaling Pathway.

Protaetia brevitarsis (PB)-derived bioactive substances have been used as food and medicine in many Asian countries because of their antioxidant, antidiabetic, anti-cancer, and hepatoprotective properties. However, the effect of PB extracts (PBE) on osteoclast differentiation is unclear. In this study, we investigated the effect of PBE on RANKL-induced osteoclastogenesis in mouse bone marrow-derived macrophages (BMMs). To investigate the cytotoxicity of PBE, the viability of BMMs was confirmed via MTT assay. Tartrate-resistant acid phosphatase (TRAP) staining and pit assays were performed to confirm the inhibitory effect of PBE on osteoclast differentiation and bone resorption. The expression levels of osteoclast differentiation-related genes and proteins were evaluated using quantitative real-time PCR and Western blotting. PBE attenuated osteoclastogenesis in BMMs in TRAP and pit assays without cytotoxicity. The expression levels of osteoclast marker genes and proteins induced by RANKL were decreased after PBE treatment. PBE suppressed osteoclastogenesis by inhibiting the RANKL-induced activated JNK/NF-κB/PLCγ2 signaling pathway and the expression of NFATc1 and c-Fos. Collectively, these results suggest that PBE could be a potential therapeutic strategy or functional product for osteoclast-related bone disease.

Euonymus alatus (Thunb.) Siebold leaf extract enhanced immunostimulatory effects in a cyclophosphamide-induced immunosuppressed rat model.

Background: Euonymus alatus (Thunb.) Siebold (EA) is a medicinal plant used in some Asian countries to treat various diseases, including cancer, hyperglycemia, diabetes, urticaria, dysmenorrhea, and arthritis. Owing to the wide range of pharmacological applications of EA, various roles of EA are being studied. Objective: We evaluated the immune-enhancing effect of EA treatment in a cyclophosphamide (Cy)-induced immunosuppressed rat model. Design: We analyzed the immune enhancement effect of EA on macrophages by western blotting. In addition, cell viability and natural killer (NK) cell activity were analyzed in splenocytes following EA treatment. For in vivo studies, analysis of weekly body weight, spleen weight, immune cell count, cytokine levels, and spleen histological findings was performed following EA administration in Cy-induced immunocompromised rats. Results: EA significantly increased cell viability and phospho-nuclear factor-kappa B and phospho-extracellular signal-regulated kinase protein levels in the macrophages. EA significantly increased NK cell activity in splenocytes compared with the control group. In Cy-induced immunosuppressed rats, EA administration increased spleen tissue weight and the contents of leukocytes, lymphocytes, granulocytes, intermediate cells, and plasma cytokines (tumor necrosis factor-α and interferon-γ). In addition, improvement in the damaged spleen tissue was observed. Conclusions: These findings confirm that EA exerts an immune-enhancing effect, thereby suggesting its potential as an immunostimulatory agent or functional food.

Electrochemical lipolysis of subcutaneous adipose tissue in a porcine animal model.

OBJECTIVES: There is a considerable demand for noninvasive low-cost fat reduction methods with fewer side effects and shorter recovery times. This study aims to develop a fat-reduction method through electrochemical lipolysis of subcutaneous adipocytes using needle-based electrodes, body tissue fluids, and electrical current application. METHODS: Electrochemical lipolysis was performed by inserting a 4-pin needle electrode connected to a DC power supply into the pig's abdomen. Applied electrical current (0.5 and 1 mA) and treatment time (5 or 10 minutes) were varied systematically. Ultrasound imaging was performed before and after treatment to determine changes in fat thickness. Tissue samples were collected at 0, 2, and 4 weeks posttreatment for histological evaluation to determine the mechanism of action and the procedure's efficacy. RESULTS: Electrochemical subcutaneous adipose tissue lipolysis in a porcine model was achieved through hydrolysis of physiologic fluid within the vicinity of the inserted electrode where an electric current is applied, leading to localized disruption of fat cell membranes and necrosis. Electric current configuration 1.0 mA showed more pronounced lipolysis effects applied for 10 minutes, significantly decreasing adipocyte content per treatment area. The electrochemical treatment method also stimulates collagen synthesis, which helps reduce fat. CONCLUSIONS: Electrochemical lipolysis is a potential new noninvasive localized technique to reduce fat. The treatment method induces fat cell necrosis via in situ reduction-oxidation reaction by the electrochemical activation of physiologic fluid in the surrounding tissue. Electrochemical lipolysis is a simple, low-cost, fat-reducing treatment method without harmful side effects.

Protective effect of traditional Korean fermented soybean foods (doenjang) on a dextran sulfate sodium-induced colitis mouse model.

Objective: The cause of ulcerative colitis (UC) is unknown, and the use of anti-inflammatory and immunosuppressive drugs with certain side effects is currently replacing treatment. Therefore, it is important to find new healthy foods or ingredients that exhibit potential protective and anti-inflammatory effects on UC. This study investigated the potential protective effect of doenjang on dextran sulfate sodium (DSS)-induced colitis in a mouse model. Materials and methods: Four doenjang samples (TCD21-51-1, TCD21-55-1, TMD21-16-1, and TFD21-1-1) were used. To examine the effects of the four doenjang samples on UC caused by DSS in a mouse model, the clinical symptoms of UC, such as body weight, disease activity index (DAI), and colon macroscopic damage index (CMDI) were analyzed. Moreover, immune-related blood cell counts, serum levels and protein expression of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), and nitric oxide (NO) production were measured in DSS-induced UC in mice for analysis. Results: The four doenjang samples increased the colon length shortened by DSS, reduced DAI (diarrhea and hemoccult), CMDI (ulceration, inflammation, and hemorrhage) and the content of immune-related cells in the blood. Moreover, the levels of TNF-α, IL-6, and NO increased by DSS were decreased by doenjang, and tissue damage was significantly reduced. Conclusions: These findings confirmed that doenjang exerts protective effects against UC, suggesting its possible use in developing therapeutic strategies or functional products.

Immune-Enhancing Effects of Co-treatment With Kalopanax pictus Nakai Bark and Nelumbo nucifera Gaertner Leaf Extract in a Cyclophosphamide-Induced Immunosuppressed Rat Model.

Objective: Immune system disorders can result in various pathological conditions, such as infections and cancer. Identifying therapies that enhance the immune response might be crucial for immunocompromised individuals. Therefore, we assessed the immune-enhancing effect of co-treatment with Kalopanax pictus Nakai Bark and Nelumbo nucifera Gaertner leaf extract (KPNN) in a cyclophosphamide (Cy)-induced immunosuppressed rat model. Materials and Methods: For in vitro studies, macrophages and splenocytes were treated with various KPNN doses in the presence or absence of Cy. Macrophage viability, nitric oxide production, splenocyte viability, cytokine production and natural killer (NK) cell activity were analyzed. For in vivo studies, analysis of weekly body weight, dietary intake, tissue weight, immune-related blood cell count, cytokine levels, and spleen biopsy was performed in a Cy-induced immunocompromised animal model. Results: KPNN significantly increased phospho-NF-κB and phospho-ERK protein levels and cell viability in macrophages. KPNN significantly increased the NK cell activity in splenocytes compared to that in the control. Cy treatment decreased tumor necrosis factor (TNF)-α, interleukin (IL)-6, and interferon-γ production. In the Cy-induced immunosuppression rat model, KPNN-treated rats had significantly higher body weights and tissue weights than the Cy-treated rats. Additionally, KPNN treatment restored the immune-related factors, such as total leukocyte, lymphocyte, and intermediate cell contents, to their normal levels in the blood. The blood cytokines (TNF-α and IL-6) were increased, and spleen tissue damage was significantly alleviated. Conclusions: Collectively, KPNN exerts an immune-enhancing effect suggesting their potential as an immunostimulatory agent or functional food.

An in vitro study on anti-carcinogenic effect of remdesivir in human ovarian cancer cells via generation of reactive oxygen species.

BACKGROUND: Remdesivir is an anti-viral drug that inhibits RNA polymerase. In 2020, remdesivir was recognized as the most promising therapeutic agents against coronavirus disease 2019 (COVID-19). However, the effects of remdesivir on cancers have hardly been studied. PURPOSE: Here, we reported that the anti-carcinogenic effect of remdesivir on SKOV3 cells, one of human ovarian cancer cell lines. RESEARCH DESIGN: We anlalyzed the anti-carcarcinogenic effect of remdesivir in SKOV3 cells by performing in vitro cell assay and western blotting. RESULTS: WST-1 showed that remdesivir decreased cell viability in SKOV3 cells. Experiments conducted by Muse Cell Analyzer showed that remdesivir-induced apoptosis in SKOV3 cells. We found that the expression level of FOXO3, Bax, and Bim increased, whereas Bcl-2, caspase-3, and caspase-7 decreased by remdesivir in SKOV3 cells. Furthermore, we observed that intracellular reactive oxygen species (ROS) level increased after treatment of remdesivir in SKOV3 cells. Interestingly, cytotoxicity of remdesivir decreased after treatment of N-Acetylcysteine. CONCLUSION: Taken together, our results demonstrated that remdesivir has an anti-carcinogenic effect on SKOV3 cells vis up-regulation of reactive oxygen species, which suggests that remdesivir could be a promising reagent for treatment of ovarian cancer.

Increased Anti-Allergic Effects of Secretome of Low-Level Light Treated Tonsil-Derived Mesenchymal Stem Cells in Allergic Rhinitis Mouse Model.

BACKGROUND: Low-level light therapy (LLLT) is widely used for the photobiomodulation of cell behavior. Recent studies have shown that LLLT affects the proliferation and migration of various types of mesenchymal stem cells (MSCs). However, there is a lack of studies investigating the effect of LLT on enhancing the immunomodulatory properties of tonsil-derived MSCs (T-MSCs). OBJECTIVE: The aim of this study was to investigate the immunomodulatory effects of conditioned media from T-MSCs (T-MSCs-CM) treated with LLLT in allergic inflammation. METHODS: We isolated T-MSCs from human palatine tonsils and evaluated the ingredients of T-MSCs-CM. The effect of T-MSCs-CM treated with LLLT was evaluated in a mouse model of allergic rhinitis (AR). We randomly divided the mice into four groups (negative control, positive control, T-MSCs-CM alone, and T-MSCs-CM treated with LLLT). To elucidate the therapeutic effect, we assessed rhinitis symptoms, serum immunoglobulin (Ig), the number of inflammatory cells, and cytokine expression. RESULTS: We identified increased expression of immunomodulatory factors, such as HGF, TGF-ß, and PGE, in T-MSCs-CM treated with LLLT, compared to T-MSCs-CM without LLLT. Our animal study demonstrated reduced allergic symptoms and lower expression of total IgE and OVA-specific IgE in the LLLT-treated T-MSCs-CM group compared to the AR group and T-MSCs-CM alone. Moreover, we found that T-MSCs-CM treated with LLLT showed significantly decreased infiltration of eosinophils, neutrophils, and IL-17 cells in the nasal mucosa and reduced IL-4, IL-17, and IFN-γ expression in OVA-incubated splenocytes compared to the AR group. CONCLUSIONS: The present study suggests that T-MSCs-CM treated with LLLT may provide an improved therapeutic effect against nasal allergic inflammation than T-MSCs-CM alone.

The application of SFDI and LSI system to evaluate the blood perfusion in skin flap mouse model.

The aim of this study is to evaluate whether the blood perfusion to tissues for detecting ischemic necrosis can be quantitatively monitored by spatial frequency domain imaging (SFDI) and laser speckle imaging (LSI) in a skin flap mouse model. Skin flaps were made on Institute of Cancer Research (ICR) mice. Using SFDI and LSI, the following parameters were estimated: oxyhemoglobin (HbO2), deoxyhemoglobin (Hb), total hemoglobin (THb), tissue oxygen saturation (StO2), and speckle flow index (SFI). Histologically, epithelium thickness, collagen deposition, and blood vessel count of skin flap tissues were analyzed. Then, the correlation of SFDI and histological results was assessed by application of Spearman rank correlation method. As the result, the number of blood vessels and the percentage of collagen areas showed significant difference between the necrotic tissue group and the non-necrotic one. Especially, the necrotic tissue had a complete epithelial loss and loses its normal structure. We identified that SFDI/LSI parameters were significantly different between non-necrotic and necrotic tissue groups. Especially, all SFDI and LSI parameters measured on the 1st day after surgery showed significant difference between necrotic tissue and non-necrotic tissue. In addition, the number of blood vessel and percentage of collagen area were positively correlated with HbO2 and StO2 among SFDI/LSI parameters. Meanwhile, the number of blood vessel and percentage of collagen area showed the negative correlation with Hb. By applying SFDI and LSI simultaneously to the skin flap, we could quantitatively monitor the blood perfusion and the tissue condition which can help us to detect ischemic necrosis objectively in early stage.

Exposure to long-term evolution radiofrequency electromagnetic fields decreases neuroblastoma cell proliferation via Akt/mTOR-mediated cellular senescence.

The aim of this study was to examine the potential effects of long-term evolution (LTE) radiofrequency electromagnetic fields (RF-EMF) on cell proliferation using SH-SY5Y neuronal cells. The growth rate and proliferation of SH-SY5Y cells were significantly decreased upon exposure to 1760 MHz RF-EMF at 4 W/kg specific absorption rate (SAR) for 4 hr/day for 4 days. Cell cycle analysis indicated that the cell cycle was delayed in the G0/G1 phase after RF-EMF exposure. However, DNA damage or apoptosis was not involved in the reduced cellular proliferation following RF-EMF exposure because the expression levels of histone H2A.X at Ser139 (γH2AX) were not markedly altered and the apoptotic pathway was not activated. However, SH-SY5Y cells exposed to RF-EMF exhibited a significant elevation in Akt and mTOR phosphorylation levels. In addition, the total amount of p53 and phosphorylated-p53 was significantly increased. Data suggested that Akt/mTOR-mediated cellular senescence led to p53 activation via stimulation of the mTOR pathway in SH-SY5Y cells. The transcriptional activation of p53 led to a rise in expression of cyclin-dependent kinase (CDK) inhibitors p21 and p27. Further, subsequent inhibition of CDK2 and CDK4 produced a fall in phosphorylated retinoblastoma (pRb at Ser807/811), which decreased cell proliferation. Taken together, these data suggest that exposure to RF-EMF might induce Akt/mTOR-mediated cellular senescence, which may delay the cell cycle without triggering DNA damage in SH-SY5Y neuroblastoma cells.

Activation of matrix metalloproteinases and FoxO3a in HaCaT keratinocytes by radiofrequency electromagnetic field exposure.

As the skin is the largest body organ and critically serves as a barrier, it is frequently exposed and could be physiologically affected by radiofrequency electromagnetic field (RF-EMF) exposure. In this study, we found that 1760 MHz RF-EMF (4.0 W/kg specific absorption rate for 2 h/day during 4 days) exposure could induce intracellular reactive oxygen species (ROS) production in HaCaT human keratinocytes using 2',7'-dichlorofluorescin diacetate fluorescent probe analysis. However, cell growth and viability were unaffected by RF-EMF exposure. Since oxidative stress in the skin greatly influences the skin-aging process, we analyzed the skin senescence-related factors activated by ROS generation. Matrix metalloproteinases 1, 3, and 7 (MMP1, MMP3, and MMP7), the main skin wrinkle-related proteins, were significantly increased in HaCaT cells after RF-EMF exposure. Additionally, the gelatinolytic activities of secreted MMP2 and MMP9 were also increased by RF-EMF exposure. FoxO3a (Ser318/321) and ERK1/2 (Thr 202/Tyr 204) phosphorylation levels were significantly increased by RF-EMF exposure. However, Bcl2 and Bax expression levels were not significantly changed, indicating that the apoptotic pathway was not activated in keratinocytes following RF-EMF exposure. In summary, our findings show that exposure to 1760 MHz RF-EMF induces ROS generation, leading to MMP activation and FoxO3a and ERK1/2 phosphorylation. These data suggest that RF-EMF exposure induces cellular senescence of skin cells through ROS induction in HaCaT human keratinocytes.

Effects of Wnt signaling on epithelial to mesenchymal transition in chronic rhinosinusitis with nasal polyp.

BACKGROUND: Epithelial to mesenchymal transition (EMT) is associated with the pathophysiology of chronic rhinosinusitis with nasal polyp (CRSwNP). Wnt signaling is causative for EMT, whereas the mechanism in CRSwNP is not fully understood. OBJECTIVE: We sought to evaluate the role of Wnt signaling in EMT of CRSwNP using a murine nasal polyp (NP) model and human tissues. METHODS: Inflammatory markers and EMT-related molecules were evaluated in NP models using adenomatosis polyposis coli (Apc)Min/+ mice with activated Wnt signaling and NP models treated with Wnt signaling inhibitor, indocyanine green-001 (ICG-001). EMT markers and Wnt signaling-associated mediators were analysed using human sinonasal tissues from control subjects and CRSwNP patients. RESULTS: ApcMin/+ mice-induced NPs exhibited more frequent polypoid lesions and upregulation of Wnt-related molecules, including nuclear ß-catenin, WNT3A and cyclin D1. Markers of EMT were significantly overexpressed in the ApcMin/+ NP mice (p<0.001 for E-cadherin and α-smooth muscle actin), and interleukin (IL)-17A+ cells and neutrophilic infiltration were increased in ApcMin/+ NP mice (p<0.001). Inhibition of Wnt signaling via ICG-001 resulted in significantly decreased nasal polypoid lesions (p<0.001), EMT-related markers (p=0.019 for E-cadherin and p=0.002 for vimentin) and the mRNA levels of IL-4 (p<0.001) and IL-17A (p=0.004) compared with the positive control group. Finally, nuclear ß-catenin (p=0.042) was significantly increased compared with the control, and the expression levels of Wnt ligands and receptors were upregulated in human NP tissues (p=0.045 for WNT3A and p=0.042 for FZD2), suggesting increased Wnt signaling and EMT in CRSwNP. CONCLUSION: Wnt signaling may contribute to the pathogenesis of NPs through EMT. Therefore, inhibition of Wnt signaling may be a potential therapeutic strategy for patients with CRSwNP.

The Supernatant of Tonsil-Derived Mesenchymal Stem Cell Has Antiallergic Effects in Allergic Rhinitis Mouse Model.

METHODS: We isolated T-MSCs from human palatine tonsil and evaluated the ingredients of T-MSCs-CM. The effect of T-MSCs-CM was evaluated in the AR mouse model that was randomly divided into five groups (negative control, positive control, and T-MSCs-CM treated (0.1 mg, 1 mg, and 10 mg)). To investigate the therapeutic effect, we analyzed rhinitis symptoms, serum immunoglobulin (Ig), inflammatory cells, and cytokine expression. We also assessed T cell receptor signal, including MAP kinase (ERK/JNK), p65, and NFAT1. RESULTS: We identified the increment of TGF-ß1, PGE2, and HGF in the T-MSCs-CM. In an animal study, the T-MSCs-CM-treated group showed significantly reduced allergic symptoms and infiltration of eosinophils and neutrophils in the nasal mucosa, whereas there was no significant difference in total IgE and the OVA-specific IgE level. Additionally, we found that the 10 mg T-MSCs-CM-treated group showed a significantly decreased IL-4 mRNA expression, compared to the (+) Con group. In the analysis of T cell receptor signal, the phosphorylation of MAP kinases, translocation of p65, and activation of NFAT1 were inhibited after T-MSCs-CM. CONCLUSIONS: Our findings suggest that T-MSCs-CM showed a partial immunomodulatory effect on the AR mouse model by the inhibition of T cell activation via MAP kinase, p65, and NFAT1.

Role of IL-17A in Chronic Rhinosinusitis With Nasal Polyp.

PURPOSE: Th17-associated inflammation is increased in chronic rhinosinusitis with nasal polyp (CRSwNP), and is associated with disease severity and steroid resistance. Overexpressed interleukin (IL)-17A affects CRSwNP by tissue remodeling, eosinophilic accumulation, and neutrophilic infiltration. We aimed to identify the role of IL-17A in CRSwNP and to evaluate the effects of anti-IL-17A blocking antibody on nasal polyp (NP) formation using a murine NP model. Moreover, we sought to investigate whether the inhibition of mechanistic target of the rapamycin (mTOR) signal pathway could suppress IL-17A expression and NP formation. METHODS: Human sinonasal tissues from control subjects and patients with chronic rhinosinusitis (CRS) were analyzed using immunohistochemistry (IHC) and immunofluorescence staining. The effects of IL-17A neutralizing antibody and rapamycin were evaluated in a murine NP model. Mouse samples were analyzed using IHC, quantitative real-time polymerase chain reaction, and enzyme-linked immunosorbent assay. RESULTS: IL-17A⁺ inflammatory cells were significantly increased in number in NP from patients with CRSwNP compared to that in uncinate process tissues from control subjects and patients with CRS without NP or CRSwNP. CD68⁺ M1 macrophages dominantly expressed IL-17A, followed by neutrophils and T helper cells, in NP tissues. Neutralization of IL-17A effectively reduced the number of NPs, inflammatory cytokines, and IL-17A-producing cells, including M1 macrophages. Inhibition of IL-17A via the mTOR pathway using rapamycin also attenuated NP formation and inflammation in the murine NP model. CONCLUSIONS: IL-17A possibly plays a role in the pathogenesis of CRSwNP, the major cellular source being M1 macrophage in NP tissues. Targeting IL-17A directly or indirectly may be an effective therapeutic strategy for CRSwNP.

Effects of Low-Level Laser Irradiation in a Mouse Model of Allergic Rhinitis.

BACKGROUND AND OBJECTIVES: To evaluate the antiallergic effect of low-level laser irradiation (LLLI) at 650 nm in a mouse model of allergic rhinitis (AR), and to examine the underlying mechanisms. STUDY DESIGN/MATERIALS AND METHODS: BALB/c mice were sensitized with ovalbumin (OVA) and alum and challenged intranasally with OVA. Straight- and diffusion-type LLLI were applied directly into the intranasal cavity of the mice once daily for 10 days (650 nm, 5 mW, 15 min/day) and multiple allergic parameters were evaluated. RESULTS: LLLI reduced allergic symptoms, such as rubbing and sneezing, and suppressed the serum total immunoglobulin E (IgE), OVA-specific IgE, and OVA-specific IgG1 levels. Diffusion-type LLLI significantly reduced eosinophil infiltration of nasal mucosa and lymph nodes (LNs). LLLI reduced the expression of interleukin-4 (IL-4) and IL-17 in cervical LN and splenocyte culture supernatant, as well as their messenger RNA levels in nasal mucosa. However, the expression of interferonγ (IFN-γ) and IL-6 was unaffected by LLLI. The levels of reactive oxygen species (ROS) and nitric oxide (NO) in LN cells and the nasal mucosa, which were increased in the AR group, were reduced by LLLI, suggesting involvement of ROS and NO within their mechanism. CONCLUSIONS: LLLI exerted an antiallergic effect by decreasing local and systemic IL-4, IL-17, and IgE levels, as well as eosinophilic infiltration into the nasal mucosa, in a mouse model of AR by modulating ROS and NO levels. Diffusion-type LLLI exhibited greater efficacy against AR than straight-type LLLI. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

FAM83H is involved in stabilization of ß-catenin and progression of osteosarcomas.

BACKGROUND: FAM83H was initially identified as a protein essential for dental enamel formation. Recent reports have shown that FAM83H is also involved in the progression of human cancers in conjunction with tumor-associated molecules, such as MYC and ß-catenin. However, the role of FAM83H in sarcoma has not yet been investigated. METHODS: The expression and roles of FAM83H and ß-catenin were evaluated in human osteosarcomas from 34 patients and osteosarcoma cells. RESULTS: The expression of nuclear FAM83H, cytoplasmic FAM83H, and ß-catenin were significantly associated with each other and significantly associated with shorter survival of osteosarcoma patients by univariate analysis. In multivariate analysis, cytoplasmic expression of FAM83H was an independent indicator of shorter survival of osteosarcoma patients (overall survival; P <  0.001, relapse-free survival; P <  0.001). In U2OS, MG63, and KHOS/NP osteosarcoma cells, the knock-down of FAM83H decreased proliferation and invasion activity and overexpression of FAM83H increased proliferation and invasion activity. In KHOS/NP cells, knock-down of FAM83H significantly inhibited, and overexpression of FAM83H significantly increased in vivo growth of cells. In addition, the knock-down of FAM83H decreased protein expression of ß-catenin, active ß-catenin, cyclin D1, vimentin, and snail. Overexpression of FAM83H increased protein expression of ß-catenin, active ß-catenin, cyclin D1, vimentin, and snail. However, the expression of ß-catenin mRNA was not significantly altered with knock-down or overexpression of FAM83H. In addition, FAM83H and ß-catenin shown to directly interact via immunoprecipitation and nuclear and cytoplasmic localization of ß-catenin was decreased with knock-down of FAM83H. Moreover, the ubiquitination and proteasomal degradation of ß-catenin was increased with knock-down of FAM83H. CONCLUSIONS: This study suggests that FAM83H is involved in the progression of osteosarcomas via a mechanism involving the stabilization of ß-catenin and the promotion of proliferation and invasiveness of osteosarcomas.

The Expression Patterns of FAM83H and PANX2 Are Associated With Shorter Survival of Clear Cell Renal Cell Carcinoma Patients.

FAM83H is primarily known for its role in amelogenesis; however, recent reports suggest FAM83H might be involved in tumorigenesis. Although the studies of FAM83H in kidney cancer are limited, a search of the public database shows a significant association between FAM83H and pannexin-2 (PANX2) in clear cell renal cell carcinomas (CCRCCs). Therefore, we evaluated the clinicopathological significance of the immunohistochemical expression of FAM83H and PANX2 in 199 CCRCC patients. The expression of FAM83H and PANX2 were significantly associated with each other. In univariate analysis, individual, and co-expression pattern of FAM83H and PANX2 was significantly associated with shorter overall survival (OS) and relapse-free survival (RFS) of CCRCC patients: nuclear expression of FAM83H (OS; P < 0.001, RFS; P < 0.001), cytoplasmic expression of FAM83H (OS; P < 0.001, RFS; P < 0.001), nuclear expression of PANX2 (OS; P < 0.001, RFS; P < 0.001), cytoplasmic expression of PANX2 (OS; P < 0.001, RFS; P < 0.001), co-expression pattern of nuclear FAM83H and nuclear PANX2 (OS; P < 0.001, RFS; P < 0.001). In multivariate analysis, nuclear expression of FAM83H (OS; P < 0.001, RFS; P = 0.003) and the co-expression pattern of nuclear FAM83H and PANX2 (OS; P < 0.001, RFS; P < 0.001) were independent indicators of shorter survival of CCRCC patients. Cytoplasmic expression of FAM83H was associated with shorter RFS (P = 0.030) in multivariate analysis. In Caki-1 and Caki-2 CCRCC cells, knock-down of FAM83H decreased PANX2 expression and cell proliferation, and overexpression of FAM83H increased PANX2 expression and cell proliferation. These results suggest that FAM83H and PANX2 might be involved in the progression of CCRCC in a co-operative manner, and their expression might be used as novel prognostic indicators for CCRCC patients.

SIRT6 Is Involved in the Progression of Ovarian Carcinomas via ß-Catenin-Mediated Epithelial to Mesenchymal Transition.

SIRT6 is involved in various cellular signaling pathways including those involved in tumorigenesis in association with ß-catenin. However, the role of SIRT6 in tumorigenesis has been controversially reported and the studies on the role of SIRT6 in ovarian cancers is limited. In this study, we evaluated the expression and roles of SIRT6 in conjunction with the expression of active ß-catenin in 104 human ovarian carcinomas and ovarian cancer cells. In human ovarian carcinomas, the expressions of SIRT6 and active ß-catenin were associated with higher tumor stage, higher histologic grade, and platinum-resistance. Moreover, nuclear expression of SIRT6 (104 ovarian carcinomas; P = 0.010, 63 high-grade serous carcinomas; P = 0.040), and activated ß-catenin (104 ovarian carcinomas; P = 0.013, 63 high-grade serous carcinomas; P = 0.005) were independent indicators of shorter overall survival of ovarian carcinoma patients in multivariate analysis. In OVCAR3 and OVCAR5 ovarian cancer cells, knock-down of SIRT6 significantly inhibited the migration and invasion of cells, but did not inhibit the proliferation of cells. SIRT6-mediated invasiveness of ovarian cancer cells was associated with the expression of epithelial-to-mesenchymal transition-related signaling molecules such as snail, vimentin, N-cadherin, E-cadherin, and activated ß-catenin. Especially, SIRT6-mediated increase of invasiveness and activation of epithelial-to-mesenchymal transition signaling was attenuated by knock-down of ß-catenin. In conclusion, this study suggests that SIRT6-ß-catenin signaling is involved in the epithelial-to-mesenchymal transition of ovarian cancer cells, and the expression of SIRT6 and active ß-catenin might be used as indicators of poor prognosis of ovarian carcinoma patients. In addition, our results suggest that SIRT6-ß-catenin signaling might be a new therapeutic target of ovarian carcinomas.

Expression of oxidized protein tyrosine phosphatase and γH2AX predicts poor survival of gastric carcinoma patients.

BACKGROUND: Oxidative stress induces various intracellular damage, which might be correlated with tumorigenesis. Accumulated oxidative stresses might inactivate protein tyrosine phosphatase (PTP) by oxidizing it, and inducing the phosphorylation of H2AX (γH2AX) in response to DNA damage. METHODS: We evaluated the clinical significance of the expression of oxidized-PTP and γH2AX in 169 gastric carcinomas. RESULTS: Immunohistochemical expression of nuclear oxidized-PTP, cytoplasmic oxidized-PTP, and γH2AX expression were significantly associated with each other, and their expressions predicted shorter survival of gastric carcinoma patients. In multivariate analysis, nuclear oxidized-PTP (overall survival; p <  0.001, relapse-free survival; P <  0.001) was an independent indicator of poor prognosis of gastric carcinoma patients. In addition, co-expression patterns of nuclear oxidized-PTP and γH2AX were independent indicators of poor prognosis of gastric carcinoma patients (overall survival; P <  0.001, relapse-free survival; P <  0.001). CONCLUSIONS: This study suggests that oxidative stress-mediated oxidation of PTP might be involved in the progression of gastric carcinomas. In addition, this study suggests that individual and co-expression pattern of nuclear oxidized-PTP and γH2AX might be used as a prognostic marker of gastric carcinomas.

The PARP inhibitor olaparib potentiates the effect of the DNA damaging agent doxorubicin in osteosarcoma.

BACKGROUND: PARP1 facilitates the recovery of DNA-damaged cells by recruiting DNA damage response molecules such as γH2AX and BRCA1/2, and plays a role in resistance to antitumor therapies. Therefore, PARP inhibition being evaluated as an anti-cancer therapy. However, there are limited studies regrading PARP inhibition in osteosarcoma. METHODS: We evaluated the expression of DNA damage response molecules in 35 human osteosarcomas and investigated the effects of co-treatment of the PARP inhibitor, olaparib, and doxorubicin in osteosarcoma cells. RESULTS: The expression patterns of PARP1, γH2AX, BRCA1, and BRCA2 were significantly associated with shorter survival of osteosarcoma patients. In osteosarcoma cells, knock-down of PARP1 and treatment of olaparib significantly inhibited proliferation of cells and induced apoptosis. Moreover, the anti-tumor effect was more significant with co-treatment of olaparib and doxorubicin in vitro and in vivo. CONCLUSIONS: This study suggests that combined use of a PARP inhibitor with doxorubicin, a DNA damaging agent, might be effective in the treatment of osteosarcoma patients, especially in the poor-prognostic subgroups of osteosarcoma expressing PARP1, γH2AX, or BRCA1/2.

Expression of ANO1/DOG1 is associated with shorter survival and progression of breast carcinomas.

The expression of ANO1 is considered to have diagnostic specificity for gastrointestinal stromal tumors. However, its function as a calcium-activated chloride channel suggests that the expression of ANO1 is not restricted to gastrointestinal stromal tumors. Recently, it has been reported that ANO1 has roles in the progression of human malignant tumors. However, the role of ANO1 in breast carcinoma has been controversial. Therefore, we investigated the expression of ANO1 in 139 breast carcinoma patients and the role of ANO1 in vitro. The immunohistochemical expression of ANO1 was significantly associated with the expression of ß-catenin, cyclin D1, MMP9, snail, and E-cadherin. Especially, ANO1 expression was an independent indicator of poor prognosis of shorter overall survival and relapse-free survival of breast carcinoma patients by multivariate analysis. In MCF7 and MDA-MB-231 breast carcinoma cells, inhibition of ANO1 with T16Ainh-A01 or siRNA for ANO1 significantly suppressed the proliferation of cells. Knock-down of ANO1 with siRNA induced G0/G1 cell cycle arrest and significantly inhibited the invasiveness of breast carcinoma cells. Knock-down of ANO1 decreased the expression of ß-catenin, cyclin D1, MMP9, snail, and N-cadherin, and increased the expression of E-cadherin. In conclusion, this study demonstrates that ANO1 expression is an indicator of poor prognosis of breast carcinoma patients and suggests that ANO1 might be a therapeutic target for breast carcinoma patients with ANO1-positive tumors and poor prognosis.