Elucidating Korean meadowsweet (Filipendula glaberrima Nakai)-derived arabinogalactan protein-induced macrophage activation and its associated mechanism of action.

This study aimed to confirm macrophage-stimulatory component from Korean meadowsweet (Filipendula glaberrima; FG) and characterize its compositional and structural properties. FG-CWH, prepared via cool-water extraction and ethanol precipitation, induced the highest secretion of NO (6.0-8.0 µM), TNF-α (8.7-9.5 ng/mL), and IL-6 (1.0-5.7 ng/mL) compared to other samples at 0.4-10 µg/mL in RAW 264.7 cells. Analytical results revealed that FG-CWH is a high-molecular-weight component with an average molecular weight of 220 kDa, constituting a polysaccharide-protein mixture. Chemical and enzymatic treatment of FG-CWH indicated its primary composition as arabinogalactan protein (AGP)-rich glycoprotein, with activity likely associated with the chemical and structural characteristics of AGP. FG-CWH treatment resulted in significant and concentration-dependent increases in iNOS (20.0-29.6 folds), TNFα (10.6-18.6 folds) and IL6 (10.9-155.6 folds) gene expression, as well as the secretion of NO (5.3-6.3 µM), TNF-α (35.4-44.3 ng/mL), and IL-6 (4.1-8.4 ng/mL) secretion, even at a reduced concentration range of 125-500 ng/mL, compared to the negative control group. Immunoblotting analysis indicated FG-CWH-induced macrophage stimulation significantly associated with the activation of MAPK (ERK, JNK, and p38) and NF-κB (p65 and IκBα). These findings can serve as valuable groundwork for developing FG-derived AGP as novel functional ingredients to enhance human immunity.

Comprehensive workflow encompassing discovery, verification, and quantification of indicator peptide in snail mucin using LC-quadrupole Orbitrap high-resolution tandem mass spectrometry.

Peptidomics analysis was conducted using high-resolution tandem mass spectrometry (MS2) to determine the peptide profile of snail-derived mucin extract (SM). The study was also aimed to identify an indicator peptide and validate a quantification method for this peptide. The peptide profiling and identification were conducted using discovery-based peptidomics analysis employing data-dependent acquisition, whereas the selected peptides were verified and quantified using parallel reaction monitoring acquisition. Among the 16 identified peptides, the selected octapeptide (TEAPLNPK) was quantified via precursor ion ionization (m/z 435.2400), followed by quantification of the corresponding quantifier ion fragment (m/z 639.3824) using MS2. The quantification method was optimized and validated in terms of specificity, linearity, accuracy, precision, and limit of detection/quantification. The validated method accurately quantified the TEAPLNPK content in the SM as 7.5 ± 0.2 µg/g. Our study not only identifies an indicator peptide from SM but also introduces a novel validation method, involving precursor ion ionization and quantification of specific fragments. Our findings may serve as a comprehensive workflow for the monitoring, selection, and quantification of indicator peptides from diverse food resources.

Bisphenol A regulates bladder cells responses via control of G2/M-phase cell cycle, apoptotic signaling, MAPK pathway, and transcription factor-associated MMP modulation.

Bisphenol A (BPA), an exogenous endocrine-disrupting chemical, is widely used to produce polycarbonate plastics. The widely used BPA has been detected in human urine samples, raising public anxiety about the detrimental effects of BPA on the bladder. In this study, we explored regulatory mechanisms for the adverse effects of BPA in human bladder BdFC and T24 cells. BPA induced extrinsic and intrinsic apoptosis and G2/M cell cycle arrest caused by the ATM-CHK1/CHK2-CDC25c-CDC2 signaling, which ultimately inhibited the growth of human bladder cells. We also found that BPA decreased the binding activity of AP-1 and NF-κB transcription factors in human bladder cells, which inhibited migration and invasion through matrix metallopeptidase-2 and -9 inactivation. Phosphorylation of MAPKs was implicated with BPA-mediated detrimental effects in human bladder cells. Collectively, our results provide a novel explanation for the underlying molecular mechanisms that BPA induces cytotoxicity in human bladder cells.

COL6A1 expression as a potential prognostic biomarker for risk stratification of T1 high grade bladder cancer: Unveiling the aggressive nature of a distinct non-muscle invasive subtype.

PURPOSE: T1 high grade (T1HG) bladder cancer (BC) is a type of non-muscle invasive BC (NMIBC) that is recognized as an aggressive subtype with a heightened propensity for progression. Current risk stratification methods for NMIBC rely on clinicopathological indicators; however, these approaches do not adequately capture the aggressive nature of T1HG BC. Thus, new, more accurate biomarkers for T1HG risk stratification are needed. Here, we enrolled three different patient cohorts and investigated expression of collagen type VI alpha 1 (COL6A1), a key component of the extracellular matrix, at different stages and grades of BC, with a specific focus on T1HG BC. MATERIALS AND METHODS: Samples from 298 BC patients were subjected to RNA sequencing and real-time polymerase chain reaction. RESULTS: We found that T1HG BC and muscle invasive BC (MIBC) exhibited comparable expression of COL6A1, which was significantly higher than that by other NMIBC subtypes. In particular, T1HG patients who later progressed to MIBC had considerably higher expression of COL6A1 than Ta, T1 low grade patients, and patients that did not progress, highlighting the aggressive nature and higher risk of progression associated with T1HG BC. Moreover, Cox and Kaplan-Meier survival analyses revealed a significant association between elevated expression of COL6A1 and poor progression-free survival of T1HG BC patients (multivariate Cox hazard ratio, 16.812; 95% confidence interval, 3.283-86.095; p=0.001 and p=0.0002 [log-rank test]). CONCLUSIONS: These findings suggest that COL6A1 may be a promising biomarker for risk stratification of T1HG BC, offering valuable insight into disease prognosis and guidance of personalized treatment decisions.

Urban aerosol particulate matter promotes mitochondrial oxidative stress-induced cellular senescence in human retinal pigment epithelial ARPE-19 cells.

Environmental exposure to urban particulate matter (UPM) is a serious health concern worldwide. Although several studies have linked UPM to ocular diseases, no study has reported effects of UPM exposure on senescence in retinal cells. Therefore, this study aimed to investigate the effects of UPM on senescence and regulatory signaling in human retinal pigment epithelial ARPE-19 cells. Our study demonstrated that UPM significantly promoted senescence, with increased senescence-associated ß-galactosidase activity. Moreover, both mRNA and protein levels of senescence markers (p16 and p21) and the senescence-associated secretory phenotype, including IL-1ß, matrix metalloproteinase-1, and -3 were upregulated. Notably, UPM increased mitochondrial reactive oxygen species-dependent nuclear factor-kappa B (NF-κB) activation during senescence. In contrast, use of NF-κB inhibitor Bay 11-7082 reduced the level of senescence markers. Taken together, our results provide the first in vitro preliminary evidence that UPM induces senescence by promoting mitochondrial oxidative stress-mediated NF-κB activation in ARPE-19 cells.

N-methylsansalvamide elicits antitumor effects in colon cancer cells in vitro and in vivo by regulating proliferation, apoptosis, and metastatic capacity.

N-methylsansalvamide (MSSV), a cyclic pentadepsipeptide, was obtained from a strain of Fusarium solani f. radicicola. The current study investigated the anti-colorectal cancer effect of MSSV. MSSV exhibited the inhibition of the proliferation in HCT116 cells via induction of G0/G1 cell cycle arrest by downregulating CDK 2, CDK6, cyclin D, and cyclin E, and upregulating p21WAF1 and p27KIP1. Decreased phosphorylation of AKT was observed in MSSV-treated cells. Moreover, MSSV treatment induced caspase-mediated apoptosis through elevating the level of cleaved caspase 3, cleaved PARP, cleaved caspase 9, and pro-apoptotic Bax. MSSV revealed the declined MMP-9 level mediated by reduction in the binding activity of AP-1, Sp-1, and NF-κB motifs, which led to the migration and invasion of HCT116 cells. In vitro metabolism with rat liver S9 fractions was performed to examine the effect of MSSV metabolites. The metabolic process enhanced the inhibitory effect of MSSV on the HCT116 cell proliferation via decline of cyclin D1 expression and AKT phosphorylation. Finally, oral administration of MSSV inhibited the tumor growth of HCT116 xenograft mice. These results suggest that MSSV is a potential anti-tumor agent in colorectal cancer treatment.

The ethanol extract of Cyperus exaltatus var. iwasakii exhibits cell cycle dysregulation, ERK1/2/p38 MAPK/AKT phosphorylation, and reduced MMP-9-mediated metastatic capacity in prostate cancer models in vitro and in vivo.

BACKGROUND: Prostate cancer is the second most common cause of cancer death worldwide in men. The development of novel and highly efficient therapeutic strategies is strongly recommended to treat prostate cancer. Cyperaceae are an ecologically and economically important family of plants with several pharmacological effects. However, the biological efficacy of Cyperus exaltatus var. iwasakii (CE) is unknown. PURPOSE: This study aimed to investigate the antitumor effect of the ethanol extract of CE against prostate cancer. METHODS: In vitro antitumor efficacy of CE was explored by the MTT assay, cell counting assay, FACS analysis, immunoblot, wound-healing migration, invasion assay, zymographic assay, and EMSA in prostate cancer cells, DU145 and LNCaP. For in vivo experiments, xenograft mice were injected with LNCaP cells. Histology (H&E and Ki-67) and biochemical enzyme assay were then performed. The toxicity test was evaluated by an acute toxicity assay. The phytochemical constituents of CE were identified by spectrometric and chromatographic analyses. RESULTS: CE exerted a significant antiproliferative effect against prostate cancer cells. CE-induced antiproliferative cells were associated with cell cycle arrest at G0/G1 (cyclin D1/CDK4, cyclin E/CDK2, p21Waf1) in DU145 cells, but G2/M (ATR, CHK1, Cdc2, Cdc25c, p21Waf1, and p53) in LNCaP cells. CE stimulated the phosphorylation of ERK1/2, p38 MAPK, and AKT in DU145 cells, but only p38 MAPK phosphorylation was increased in LNCaP cells. CE treatment suppressed migration and invasion in the two types of prostate cancer cells by inhibiting MMP-9 activity through the regulation of transcription factors, such as AP-1 and NF-κB. In vivo experiments showed a reduction in tumor weight and size following oral CE administration. Histochemistry confirmed that CE inhibited tumor growth in the mouse LNCaP xenograft model. The administration of CE had no adverse effects on body weight, behavioral patterns, blood biochemistry, and histopathology findings of vital organs in mice. Finally, a total of 13 phytochemical constituents were identified and quantified in CE. The most abundant secondary metabolites in CE were astragalin, tricin, and p-coumaric acid. CONCLUSION: Our results demonstrated the antitumor efficacy of CE against prostate cancer. These findings suggest that CE might be a potential candidate for prostate cancer prevention or treatment.

Phloroglucinol Inhibits Oxidative-Stress-Induced Cytotoxicity in C2C12 Murine Myoblasts through Nrf-2-Mediated Activation of HO-1.

Phloroglucinol is a class of polyphenolic compounds containing aromatic phenyl rings and is known to have various pharmacological activities. Recently, we reported that this compound isolated from Ecklonia cava, a brown alga belonging to the family Laminariaceae, has potent antioxidant activity in human dermal keratinocytes. In this study, we evaluated whether phloroglucinol could protect against hydrogen peroxide (H2O2)-induced oxidative damage in murine-derived C2C12 myoblasts. Our results revealed that phloroglucinol suppressed H2O2-induced cytotoxicity and DNA damage while blocking the production of reactive oxygen species. We also found that phloroglucinol protected cells from the induction of apoptosis associated with mitochondrial impairment caused by H2O2 treatment. Furthermore, phloroglucinol enhanced the phosphorylation of nuclear factor-erythroid-2 related factor 2 (Nrf2) as well as the expression and activity of heme oxygenase-1 (HO-1). However, such anti-apoptotic and cytoprotective effects of phloroglucinol were greatly abolished by the HO-1 inhibitor, suggesting that phloroglucinol could increase the Nrf2-mediated activity of HO-1 to protect C2C12 myoblasts from oxidative stress. Taken together, our results indicate that phloroglucinol has a strong antioxidant activity as an Nrf2 activator and may have therapeutic benefits for oxidative-stress-mediated muscle disease.

Bisphenol A exposure inhibits vascular smooth muscle cell responses: Involvement of proliferation, migration, and invasion.

Previous studies have associated bisphenol A (BPA) with malignant tumor formation, infertility, and atherosclerosis in vitro and in vivo. However, the precise mechanisms through which BPA affects the cardiovascular system under normal conditions remain unclear. Therefore, this study investigated the biological mechanisms through which BPA affects the responses of aortic vascular smooth muscle cells (VSMCs). BPA treatment inhibited the proliferative activity of VSMCs and induced G2/M-phase cell cycle arrest via stimulation of the ATM-CHK2-Cdc25C-p21WAF1-Cdc2 cascade in VSMCs. Furthermore, BPA treatment upregulated the phosphorylation of mitogen-activated protein kinase (MAPK) pathways such as ERK, JNK, and p38 MAPK in VSMCs. However, the phosphorylation level of AKT was down-regulated by BPA treatment. Additionally, the phosphorylation of ERK, JNK, and p38 MAPK was suppressed when the cells were treated with their respective inhibitors (U0126, SP600125, and SB203580). BPA suppressed MMP-9 activity by reducing the binding activity of AP-1, Sp-1, and NF-κB, thus inhibiting the invasive and migratory ability of VSMCs. These data demonstrate that BPA interferes with the proliferation, migration, and invasion capacities of VSMCs. Therefore, our findings suggest that overexposure to BPA can lead to cardiovascular damage due to dysregulated VSMC responses.

Utility of a Molecular Signature for Predicting Recurrence and Progression in Non-Muscle-Invasive Bladder Cancer Patients: Comparison with the EORTC, CUETO and 2021 EAU Risk Groups.

To evaluate the utility of different risk assessments in non-muscle-invasive bladder cancer (NMIBC) patients, a total of 178 NMIBC patients from Chungbuk National University Hospital (CBNUH) were enrolled, and the predictive value of the molecular signature-based subtype predictor (MSP888) and risk calculators based on clinicopathological factors (EORTC, CUETO and 2021 EAU risk scores) was compared. Of the 178 patients, 49 were newly analyzed by the RNA-sequencing, and their MSP888 subtype was evaluated. The ability of the EORTC, MSP888 and two molecular subtyping systems of bladder cancer (Lund and UROMOL subtypes) to predict progression of 460 NMIBC patients from the UROMOL project was assessed. Cox regression analyses showed that the MSP888 was an independent predictor of NMIBC progression in the CBNUH cohort (p = 0.043). Particularly in patients without an intravesical BCG immunotherapy, MSP888 significantly linked with risk of disease recurrence and progression (both p < 0.05). However, the EORTC, CUETO and 2021 EAU risk scores showed disappointing results with respect to estimating the NMIBC prognosis. In the UROMOL cohort, the MSP888, Lund and UROMOL subtypes demonstrated a similar capacity to predict NMIBC progression (all p < 0.05). Conclusively, the MSP888 is favorable for stratifying patients to facilitate optimal treatment.

Study on the use of Nanostring nCounter to analyze RNA extracted from formalin-fixed-paraffin-embedded and fresh frozen bladder cancer tissues.

Formalin-fixed paraffin-embedded (FFPE) tissue is the most common source of archived material for genomic medicine. However, FFPE tissue is suboptimal for high-throughput analyses, such as RNA sequencing, because the quality of nucleic acids in FFPE tissues is low. We compared RNA-seq with the nCounter system to evaluate use of FFPE tissue for genomic medicine. Twelve fresh frozen bladder cancer samples were analyzed by both RNA sequencing and nCounter, and matched FFPE samples, by nCounter. Gene-expression values obtained by these two platforms were compared by calculating Pearson correlation coefficients for each sample (across the set of matched genes) and for each matched gene (across the set of samples). For each sample, gene-expression levels measured by RNA sequencing highly correlated with those measured by nCounter (all Pearson's R > 0.8, P < 0.0001), as seen by hierarchical clustering. RNA sequencing results for fresh frozen tissues positively correlated with nCounter results for FFPE tissues (R ranged from 0.675 to 0.873, all P < 0.0001). Correlation and hierarchical-clustering analyses of nCounter data from the two specimens demonstrated a strong positive correlation between each group (R ranged from 0.779 to 0.977, all P < 0.0001). Our findings suggest that the nCounter system is useful for assaying archived-FFPE samples and that the gene-expression signatures obtained from FFPE samples represent those from fresh frozen tissues.

Structural properties and anti-dermatitis effects of flavonoids-loaded gold nanoparticles prepared by Eupatorium japonicum.

Recently, green synthesis-based nanoformulations using plants or microorganisms have attracted great interest because of their several advantages. Nanotechnology-based biological macromolecules are emerging materials with potential applications in cosmetics and medications for ameliorating and treating inflammatory skin diseases (ISDs). Eupatorium japonicum (EJ), a native Korean medicinal plant belonging to the family Asteraceae, has been traditionally used to prepare prescriptions for the treatment of various inflammatory diseases. EJ-based gold nanoparticles (EJ-AuNPs) were biosynthesized under optimal conditions and characterized their physicochemical properties using various microscopic and spectrometric techniques. Additionally, the effects of EJ-AuNPs on ISDs as well as their underlying mechanisms were investigated in the tumor necrosis factor-α/interferon-γ (T+I)-induced skin HaCaT keratinocytes. The MTT and live/dead cell staining assays showed that EJ-AuNP treatment was considerably safer than EJ treatment alone in HaCaT cells. Moreover, EJ-AuNP treatment effectively suppressed the production of T+I-stimulated inflammatory cytokines (RANTES, TARC, CTACK, IL-6, and IL-8) and intracellular reactive oxygen species, and such EJ-driven anti-inflammatory effects were shown to be associated with the downregulation of intracellular mitogen-activated protein kinase and nuclear factor-κB signaling pathways. The present study provides preliminary results and a valuable strategy for developing novel anti-skin dermatitis drug candidates using plant extract-based gold nanoparticles.

Biologically Synthesized Rosa rugosa-Based Gold Nanoparticles Suppress Skin Inflammatory Responses via MAPK and NF-κB Signaling Pathway in TNF-α/IFN-γ-Induced HaCaT Keratinocytes.

Nanotechnology-applied materials and related therapeutics have gained attention for treating inflammatory skin diseases. The beach rose (Rosa rugosa), belonging to the family Rosaceae, is a perennial, deciduous woody shrub endemic to northeastern Asia. In this study, R. rugosa-based gold nanoparticles (RR-AuNPs) were biologically synthesized under optimal conditions to explore their potential as anti-inflammatory agents for treating skin inflammation. The synthesized RR-AuNPs were analyzed using field emission-transmission electron microscopy, energy-dispersive X-ray spectrometry, selected-area electron diffraction, and X-ray diffraction. The uniformly well-structured AuNPs showed near-spherical and polygonal shapes. Cell viability evaluation and optical observation results showed that the RR-AuNPs were absorbed by human keratinocytes without causing cytotoxic effects. The effects of RR-AuNPs on the skin inflammatory response were investigated in human keratinocytes treated with tumor necrosis factor-α/interferon-γ (T + I). The results showed that T + I-stimulated increases in inflammatory mediators, including chemokines, interleukins, and reactive oxygen species, were significantly suppressed by RR-AuNP treatment in a concentration-dependent manner. The western blotting results indicated that the RR-AuNP-mediated anti-inflammatory effects were highly associated with the suppression of inflammatory signaling, mitogen-activated protein kinase, and nuclear factor-κB. These results demonstrate that plant extract-based AuNPs are novel anti-inflammatory candidates for topical application to treat skin inflammation.

Immunostimulatory Effect of Postbiotics Prepared from Phellinus linteus Mycelial Submerged Culture via Activation of Spleen and Peyer's Patch in C3H/HeN Mice.

Medicinal mushrooms are an important natural resource promoting health benefits. Herein, Phellinus linteus mycelia were prepared under submerged cultivation, the mycelium-containing culture broth was extracted as a whole to obtain the postbiotic materials (PLME), and its effect on the immune system was evaluated in normal C3H/HeN mice. Oral administration of PLME for 4 weeks was well tolerated and safe. In the PLME-administered groups, in addition to the production of immunostimulatory cytokines, such as interferon gamma (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6), the mitogenic activity was significantly increased. PLME administration also significantly increased the levels of serum immunoglobulin G (IgG) and IgA in the small intestinal fluid and Peyer's patches and enhanced Peyer's patch-mediated bone marrow cell proliferation activity and cytokine production (IL-2, IL-6, and IFN-γ). Histomorphometric analyses showed an increase in immune cells in the spleen and small intestinal tissues of mice administered PLME, supporting the rationale for its immune system activation. PLME mainly contained neutral sugar (969.1 mg/g), comprising primarily of glucose as a monosaccharide unit. The ß-glucan content was 88.5 mg/g. Data suggest that PLME effectively promote immune function by stimulating the systemic immune system through the spleen and intestinal immune tissues. PLME can thus be developed as a functional ingredient to enhance immune functions.

Rosa hybrida Petal Extract Exhibits Antitumor Effects by Abrogating Tumor Progression and Angiogenesis in Bladder Cancer Both In Vivo and In Vitro.

The edible Rosa hybrida (RH) petal is utilized in functional foods and cosmetics. Although the biological function of RH petal extract is known, mechanism of action studies involving tumor-associated angiogenesis have not yet been reported. Herein, we investigated the regulatory effect of the ethanol extract of RH petal (EERH) on tumor growth and tumor angiogenesis against bladder cancer. EERH treatment inhibited the bladder carcinoma T24 cell and 5637 cell proliferation because of G1-phase cell cycle arrest by inducing p21WAF1 expression and reducing cyclins/CDKs level. EERH regulated signaling pathways differently in both cells. EERH-stimulated suppression of T24 and 5637 cell migration and invasion was associated with the decline in transcription factor-mediated MMP-9 expression. EERH oral administration to xenograft mice reduced tumor growth. Furthermore, no obvious toxicity was observed in acute toxicity test. Decreased CD31 levels in EERH-treated tumor tissues led to examine the angiogenic response. EERH alleviated VEGF-stimulated tube formation and proliferation by downregulating the VEGFR2/eNOS/AKT/ERK1/2 cascade in HUVECs. EERH impeded migration and invasion of VEGF-induced HUVECs, which is attributed to the repressed MMP-2 expression. Suppression of neo-microvessel sprouting, induced by VEGF, was verified by treatment with EERH using the ex vivo aortic ring assay. Finally, kaempferol was identified as the main active compound of EERH. The present study demonstrated that EERH may aid the development of antitumor agents against bladder cancer.

In vitro assessment of the anti-inflammatory and skin-moisturizing effects of Filipendula palmata (Pall.) Maxim. On human keratinocytes and identification of its bioactive phytochemicals.

ETHNOPHARMACOLOGICAL RELEVANCE: The meadowsweet family (genus Filipendula) includes about 30 species, which have been traditionally used in folk medicine to treat various inflammatory diseases. Particularily, F. palmata (Pall.) Maxim. (Siberian meadowsweet) were traditionally and widely used as an ethnic herb in the Oroqen application. AIM OF THE STUDY: Limited studies have been documented on most species, except for two main species, F. ulmaria (L.) Maxim. and F. vulgaris Moench. Thus, this study aimed to investigate the anti-inflammatory and skin-moisturizing effects of 70% ethanolic extract (FPE) of F. palmata on human epidermal keratinocytes. MATERIALS AND METHODS: HaCaT keratinocytes were treated with FPE under different conditions. Quantitative real time-PCR, enzyme-linked immunosorbent assay, western blotting methods were used to evaluate the effect and molecular mechanism of the cells treated with FPE. The bioactive compounds in FPE, which are responsible for biological activities, was explored using mass spectrometric analysis. RESULTS: FPE did not show a cytotoxic effect on the cells at concentrations below 200 µg/mL. FPE significantly suppressed the intracellular reactive oxygen species and mitochondrial superoxide of inflamed HaCaT cells induced by tumor necrosis factor-α and interferon-γ (T + I) and inflammatory chemokine genes and proteins, such as CC chemokine ligands (CCL5, CCL17, and CCL27) and CXC chemokine ligand (CXCL8). These anti-inflammatory activities of FPE were mediated by the downregulation of mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways. In normal HaCaT cells, FPE significantly promoted the production of hyaluronic acid (HA) via the downregulation of hyaluronidase (HYAL1 and HYAL2) and upregulation of hyaluronic acid synthase (HAS1, HAS2, and HAS3) genes, and these effects seemed to be associated with the PI3K/Akt/NF-κB signaling. Ultraperformance liquid chromatography-tandem mass spectrometry indicated that FPE contains four flavonoids, including (+)-catechin, miquelianin, scutellarin, and quercitrin, as its major phytochemicals. Finally, we demonstrated that miquelianin and quercitrin contribute partially to the anti-inflammatory and HA-producing activity of FPE without cytotoxic effects on HaCaT cells. CONCLUSIONS: Our findings suggest that topical applications of FPE can be utilized as an alternative therapy for treating skin inflammation. Additionally, our findings serve as a reference in applying FPE as a functional ingredient to treat inflammatory skin diseases and promote skin health.

Induction of apoptotic cell death in human bladder cancer cells by ethanol extract of Zanthoxylum schinifolium leaf, through ROS-dependent inactivation of the PI3K/Akt signaling pathway.

BACKGROUND/OBJECTIVES: Zanthoxylum schinifolium is traditionally used as a spice for cooking in East Asian countries. This study was undertaken to evaluate the anti-proliferative potential of ethanol extracts of Z. schinifolium leaves (EEZS) against human bladder cancer T24 cells. MATERIALS/METHODS: Subsequent to measuring the cytotoxicity of EEZS, the anti-cancer activity was measured by assessing apoptosis induction, reactive oxygen species (ROS) generation, and mitochondrial membrane potential (MMP). In addition, we determined the underlying mechanism of EEZS-induced apoptosis through various assays, including Western blot analysis. RESULTS: EEZS treatment concentration-dependently inhibited T24 cell survival, which is associated with apoptosis induction. Exposure to EEZS induced the expression of Fas and Fas-ligand, activated caspases, and subsequently resulted to cleavage of poly (ADP-ribose) polymerase. EEZS also enhanced the expression of cytochrome c in the cytoplasm by suppressing MMP, following increase in the ratio of Bax:Bcl-2 expression and truncation of Bid. However, EEZS-mediated growth inhibition and apoptosis were significantly diminished by a pan-caspase inhibitor. Moreover, EEZS inhibited activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway, and the apoptosis-inducing potential of EEZS was promoted in the presence of PI3K/Akt inhibitor. In addition, EEZS enhanced the production of ROS, whereas N-acetyl cysteine (NAC), a ROS scavenger, markedly suppressed growth inhibition and inactivation of the PI3K/Akt signaling pathway induced by EEZS. Furthermore, NAC significantly attenuated the EEZS-induced apoptosis and reduction of cell viability. CONCLUSIONS: Taken together, our results indicate that exposure to EEZS exhibits anti-cancer activity in T24 bladder cancer cells through ROS-dependent induction of apoptosis and inactivation of the PI3K/Akt signaling pathway.

In vitro and in vivo anti-tumor efficacy of krill oil against bladder cancer: Involvement of tumor-associated angiogenic vasculature.

Krill oil (KO) obtained from Euphausia superba is nutrient-rich and has a positive effect on human health. Here, we explored the efficacy of KO in inhibiting tumor progression and tumor vascularization. KO (100-300 µg/mL) repressed the proliferation of bladder tumor cell lines MBT-2 and T24. Treatment of cells with KO raised cell-cycle arrest at G0/G1-phase via modulation of positive regulators and negative regulators in bladder cancer cells. KO treatment regulated phosphorylation of proteins involved in PI3K/AKT and MAPK signaling pathways, including ERK, JNK, and p38 MAPK. Additionally, KO hampered the invasion and migration of both cell lines via reduction of MMP-9 expression levels by disrupting transcriptional binding of Sp-1, AP-1, and NF-κB motifs. Moreover, in animal studies, KO (150-300 mg/kg) significantly decreased tumor growth in xenograft mice bearing T24 tumor cells. No significant toxic effects were observed in acute toxicity tests, including biological analysis and H&E staining. The reduced level of CD31 expression in KO-treated tumor tissues prompted us to investigate the effect of KO on tumor angiogenesis. KO (5-40 µg/mL) treatment impeded VEGF-induced capillary tube formation and proliferation by inhibiting VEGFR2-modulated eNOS/AKT/ERK1/2 signaling axis in HUVECs. Treatment of HUVECs with KO inhibited VEGF-stimulated migration and invasion by reducing MMP-2 expression level. VEGF-driven sprouting capacity of neo-microvessels was suppressed in the presence of KO (20-40 µg/mL), as determined via an ex vivo aortic ring assay. Our results indicated that KO can regulate both tumor growth and tumor-associated angiogenesis via a novel mechanism. Thus, KO may be a promising antitumor candidate, potentially useful to prevent or treat bladder cancer.