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.

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 and In Vivo Antitumor Efficacy of Hizikia fusiforme Celluclast Extract against Bladder Cancer.

Various physiological benefits have been linked to Hizikia fusiforme (HF), an edible brown seaweed. Here, fucose-containing sulfated polysaccharides were extracted from celluclast-processed HF (SPHF) and their antitumor efficacy against bladder cancer was evaluated in vitro and in vivo. SPHF possesses high sulfated polysaccharide and fucose contents and free radical scavenging activities compared to those of celluclast-processed HF extracts (CHF). SPHF inhibited bladder cancer EJ cell proliferation via G1-phase cell cycle arrest. This was due to the induction of p21WAF1 expression associated with the downregulation of CDKs and cyclins. Moreover, JNK phosphorylation was identified as an SPHF-mediated signaling molecule. SPHF treatment also hindered the migration and invasion of EJ cells by inhibiting MMP-9 expression, which was attributed to the repression of transcriptional binding to NF-κB, AP-1, and Sp-1 in the MMP-9 promoter region. In an animal study, SPHF treatment suppressed EJ tumor growth in xenograft mice similarly to cisplatin. Furthermore, no toxicity signs were found after weight loss assessment, biochemical tests, and organ tissue immunostaining during oral administration of 20-200 mg/kg SPHF for 20 days. Therefore, our study demonstrates the antitumor efficacy of SPHF in vitro and in vivo, thus highlighting its potential for bladder cancer treatment development.

Citrus unshiu peel suppress the metastatic potential of murine melanoma B16F10 cells in vitro and in vivo.

The peel of Citrus unshiu Marcow. fruits (CU) has long been used as a traditional medicine that has therapeutic effects against pathogenic diseases, including asthma, vomiting, dyspepsia, blood circulation disorders, and various types of cancer. In this study, we investigated the effect of CU peel on metastatic melanoma, a highly aggressive skin cancer, in B16F10 melanoma cells, and in B16F10 cells inoculated-C57BL/6 mice. Our results show that ethanol extracts of CU (EECU) inhibited cell growth and increased the apoptotic cells in B16F10 cells. EECU also stimulated the induction of mitochondria-mediated intrinsic pathway, with reduced mitochondrial membrane potential and increased generation of intracellular reactive oxygen species. Furthermore, EECU suppressed the migration, invasion, and colony formation of B16F10 cells. In addition, the oral administration of EECU reduced serum lactate dehydrogenase activity without weight loss, hepatotoxicity, nor nephrotoxicity in B16F10 cell-inoculated mice. Moreover, EECU markedly suppressed lung hypertrophy, the number and expression of metastatic tumor nodules, and the expression of inflammatory tumor necrosis factor-alpha in lung tissue. In conclusion, our findings suggest that the inhibitory effect of EECU on the metastasis of melanoma indicates that it may be regarded as a potential therapeutic herbal drug for melanoma.

Nimbolide Represses the Proliferation, Migration, and Invasion of Bladder Carcinoma Cells via Chk2-Mediated G2/M Phase Cell Cycle Arrest, Altered Signaling Pathways, and Reduced Transcription Factors-Associated MMP-9 Expression.

Nimbolide, an active chemical constituent of Azadirachta indica, reportedly has several physiological effects. Here, we assessed novel anticancer effects of nimbolide against bladder cancer EJ and 5637 cells. Nimbolide treatment inhibited the proliferation of both bladder cancer cell lines with an IC50 value of 3 µM. Treatment of cells with nimbolide induced G2/M phase cell cycle arrest via both Chk2-Cdc25C-Cdc2/cyclin B1-Wee1 pathway and Chk2-p21WAF1-Cdc2/cyclin B1-Wee1 pathway. Nimbolide increased JNK phosphorylation and decreased p38MAPK and AKT phosphorylation. Additionally, nimbolide impeded both wound healing migration and invasion abilities by suppressing matrix metalloproteinase-9 (MMP-9) activity. Finally, nimbolide repressed the binding activity of NF-κB, Sp-1, and AP-1 motifs, which are key transcription factors for MMP-9 activity regulation. Overall, our study indicates that nimbolide is a potential chemotherapeutic agent for bladder cancer.

Anti-Proliferative and Pro-Apoptotic Effects of Licochalcone A through ROS-Mediated Cell Cycle Arrest and Apoptosis in Human Bladder Cancer Cells.

Licochalcone A (LCA) is a chalcone that is predominantly found in the root of Glycyrrhiza species, which is widely used as an herbal medicine. Although previous studies have reported that LCA has a wide range of pharmacological effects, evidence for the underlying molecular mechanism of its anti-cancer efficacy is still lacking. In this study, we investigated the anti-proliferative effect of LCA on human bladder cancer cells, and found that LCA induced cell cycle arrest at G2/M phase and apoptotic cell death. Our data showed that LCA inhibited the expression of cyclin A, cyclin B1, and Wee1, but increased the expression of cyclin-dependent kinase (Cdk) inhibitor p21WAF1/CIP1, and increased p21 was bound to Cdc2 and Cdk2. LCA activated caspase-8 and -9, which are involved in the initiation of extrinsic and intrinsic apoptosis pathways, respectively, and also increased caspase-3 activity, a typical effect caspase, subsequently leading to poly (ADP-ribose) polymerase cleavage. Additionally, LCA increased the Bax/Bcl-2 ratio, and reduced the integrity of mitochondria, which contributed to the discharge of cytochrome c from the mitochondria to the cytoplasm. Moreover, LCA enhanced the intracellular levels of reactive oxygen species (ROS); however, the interruption of ROS generation using ROS scavenger led to escape from LCA-mediated G2/M arrest and apoptosis. Collectively, the present data indicate that LCA can inhibit the proliferation of human bladder cancer cells by inducing ROS-dependent G2/M phase arrest and apoptosis.

Triacanthine exerts antitumor effects on bladder cancer in vitro and in vivo.

BACKGROUND: Numerous studies have focused on solvent extracts from locust trees (Gleditsia spp.), which contain diverse bioactive components including saponins, flavonoids, and alkaloids. However, because of the undefined nature of such phytochemicals, their clinical application as chemotherapeutic agents has often been limited. PURPOSE: This study aimed to evaluate the anti-oncogenic activity of triacanthine, an alkaloid obtained from Gleditsia triacanthos L. STUDY DESIGN: The anti-oncogenicity of triacanthine in vitro was evaluated via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell-counting kit-8 assay (CCK-8 assay), flow cytometry, imunoblot, migration and invasion assays, zymography, and electrophoretic mobility shift assay in the human bladder carcinoma cell line EJ. The in vivo efficacy of triacanthine was evaluated via oral administration to EJ-xenografted BALB/c nude mice. To identify the side effects of triacanthine, cisplatin was also administered and an acute toxicity test was performed. RESULTS: Triacanthine significantly inhibited EJ cell proliferation (IC50 600 µM). Flow cytometry analysis revealed that cells were arrested in the G1 phase, and apoptotic cells accumulated in sub-G1 phase in a dose-dependent manner. Triacanthine inhibited the G1-S transition by deterring complex formation between cyclin-dependent kinases and cyclins, thereby up-regulating cell cycle inhibitors p21WAF1 and p27KIP1. In addition, triacanthine induced a caspase-dependent extrinsic pathway of apoptosis and autophagy. Early responsive kinases, extracellular signal-regulated kinase (ERK) and Janus kinase (JNK) were up-regulated by triacanthine. Triacanthine-mediated inhibition of the migratory and invasive potential of EJ cells was attributed to reduction of matrix metalloproteinase (MMP)-9 due to suppression of binding activities of the transcription factors activator protein (AP)-1, specificity protein (Sp)-1, and nuclear factor (NF)-κB. In an in vivo study, triacanthine significantly limited growth of xenografted tumors. Interestingly, while cisplatin resulted in significant weight loss after a 5-mg/kg dose, triacanthine did not cause weight loss, behavioral abnormalities, altered biochemical parameters, or tissue staining. A single oral dose acute-toxicity test (triacanthine 2,000 mg/kg) produced no adverse cytotoxic effects via blood biochemical tests and tissue-organ staining. CONCLUSION: To our knowledge, this is the first systematic evaluation of the anti-oncogenic activity of triacanthine. Therefore, we believe that our findings may guide the development of novel chemotherapeutic agents for bladder cancers.

National practice patterns and direct medical costs for prostate cancer in Korea across a 10 year period: a nationwide population-based study using a national health insurance database.

BACKGROUND: A complete enumeration study was conducted to evaluate trends in national practice patterns and direct medical costs for prostate cancer (PCa) in Korea over a 10-year retrospective period using data from the Korean National Health Insurance Service. METHODS: Reimbursement records for 874,924 patients diagnosed between 2002 and 2014 with primary PCa according to the International Classification of Disease (ICD) 10th revision code C61 were accessed. To assess direct medical costs for patients newly diagnosed after 2005, data from 68,596 patients managed between January 2005 and 31 December 2014 were evaluated. RESULTS: From 2005 to 2014, the total number of PCa patients showed a 2.6-fold increase. Surgery and androgen deprivation therapy were the most common first-line treatment, alone or within the context of combined therapy. Surgery as a monotherapy was performed in 23.5% of patients in 2005, and in 39.4% of patients in 2014. From 2008, the rate of robot-assisted RP rose sharply, showing a similar rate to open RP in 2014. Average total treatment costs in the 12 months post-diagnosis were around 10 million Korean won. Average annual treatment costs thereafter were around 5 million Korean won. Out-of-pocket expenditure was highest in the first year post-diagnosis, and ranged from 12 to 17% thereafter. CONCLUSIONS: Between 2005 and 2014, a substantial change was observed in the national practice pattern for PCa in Korea. The present data provide a reliable overview of treatment patterns and medical costs for PCa in Korea.

Medical Travel among Non-Seoul Residents to Seek Prostate Cancer Treatment in Medical Facilities of Seoul.

PURPOSE: This study aims to investigate the trend in medical travel by non-Seoul residents to Seoul for treatment of prostate cancer and also to investigate the possible factors affecting the trend. MATERIALS AND METHODS: This study represents a retrospective cohort study using data from theKoreanNationalHealth Insurance System from 2002 to 2015. Annual trends were produced for proportions of patients who traveled according to the age group, economic status and types of treatment. Multiple logistic analysiswas used to determine factors affecting surgeries at medical facilities in Seoul among the non-Seoul residents. RESULTS: A total of 68,543 patients were defined as newly diagnosed prostate cancer cohorts from 2005 to 2014. The proportion of patients who traveled to Seoul for treatment, estimated from cases with prostate cancer-related claims, decreased slightly over 9 years (28.0 at 2005 and 27.0 at 2014, p=0.02). The average proportion of medical travelers seeking radical prostatectomy increased slightly but the increase was not statistically significant (43.1 at 2005 and 45.4 at 2014, p=0.26). Income level and performance ofrobot-assisted radical prostatectomy were significant positive factors for medical travel to medical facilities in Seoul. Combined comorbidity diseases and year undergoing surgery were significant negative factors for medical travel to medical facilities in Seoul. CONCLUSION: The general trend of patients travelling from outside Seoul for prostate cancer treatment decreased from 2005 to 2014. However, a large proportion of traveling remained irrespective of direct distance from Seoul.

Induction of Apoptosis by Citrus unshiu Peel in Human Breast Cancer MCF-7 Cells: Involvement of ROS-Dependent Activation of AMPK.

The fruit of Citrus unshiu MARKOVICH used for various purposes in traditional medicine has various pharmacological properties including antioxidant, anti-inflammatory, and antibacterial effects. Recently, the possibility of anti-cancer activity of the extracts or components of this fruit has been reported; however, the exact mechanism has not yet been fully understood. In this study, we evaluated the anti-proliferative effect of water extract of C. unshiu peel (WECU) on human breast cancer MCF-7 cells and investigated the underlying mechanism. Our results showed that reduction of MCF-7 cell survival by WECU was associated with the induction of apoptosis. WECU-induced apoptotic cell death was related to the activation of caspase-8 and -9, representative initiate caspases of extrinsic and intrinsic apoptosis pathways, respectively, and increase in the Bax : Bcl-2 ratio accompanied by cleavage of poly(ADP-ribose) polymerase (PARP). WECU also increased the mitochondrial dysfunction and cytosolic release of cytochrome c. In addition, AMP-activated protein kinase (AMPK) and its downstream target molecule, acetyl-CoA carboxylase, were activated in a concentration-dependent manner in WECU-treated cells. In contrast, compound C, an AMPK inhibitor, significantly inhibited WECU-induced apoptosis, while inhibiting increased expression of Bax and decreased expression of Bcl-2 by WECU and inhibition of WECU-induced PARP degradation. Furthermore, WECU provoked the production of reactive oxygen species (ROS); however, the activation of AMKP and apoptosis by WECU were prevented, when the ROS production was blocked by antioxidant N-acetyl cysteine. Therefore, our data indicate that WECU suppresses MCF-7 cell proliferation by activating the intrinsic and extrinsic apoptosis pathways through ROS-dependent AMPK pathway activation.

The Anticancer Effects of Garlic Extracts on Bladder Cancer Compared to Cisplatin: A Common Mechanism of Action via Centromere Protein M.

Although garlic induces apoptosis in cancer cells, it is unclear whether the effects are similar to those of cisplatin against bladder cancer (BC). Therefore, this study investigated whether garlic extracts and cisplatin show similar activity when used to treat BC. The effect of garlic on T24 BC cell line was examined in a BALB/C-nude mouse xenograft model and compared with that of cisplatin. Tissue microarray analysis and gene network analysis were performed to identify differences in gene expression by control tumors and tumors exposed to garlic extract or cisplatin. Investigation of gene expression based on tissues from 165 BC patients and normal controls was then performed to identify common targets of garlic and cisplatin. Tumor volume and tumor weight in cisplatin (0.05[Formula: see text]mg/kg)- and garlic-treated mice were significantly smaller than those in negative control mice. However, cisplatin-treated mice also showed a significant reduction in body weight. Microarray analysis of tumor tissue identified 515 common anticancer genes in the garlic and cisplatin groups ([Formula: see text]). Gene network analysis of 252 of these genes using the Cytoscape and ClueGo software packages mapped 17 genes and 9 gene ontologies to gene networks. BC (NMIBC and MIBC) patients with low expression of centromere protein M (CENPM) showed significantly better progression-free survival than those with high expression. Garlic extract shows anticancer activity in vivo similar to that of cisplatin, with no evident of side effects. Both appear to act by targeting protein-DNA complex assembly; in particular, expression of CENPM.

Induction of apoptosis by ethanol extract of Citrus unshiu Markovich peel in human bladder cancer T24 cells through ROS-mediated inactivation of the PI3K/Akt pathway.

Citrus unshiu peel has been used to prevent and treat various diseases in traditional East-Asian medicine including in Korea. Extracts of C. unshiu peel are known to have various pharmacological effects including antioxidant, anti-inflammatory, and antibacterial properties. Although the possibility of their anti-cancer activity has recently been reported, the exact mechanisms in human cancer cells have not been sufficiently studied. In this study, the inhibitory effect of ethanol extract of C. unshiu peel (EECU) on the growth of human bladder cancer T24 cells was evaluated and the underlying mechanism was investigated. The present study demonstrated that the suppression of T24 cell viability by EECU is associated with apoptosis induction. EECU-induced apoptosis was found to correlate with an activation of caspase-8, -9, and -3 in concomitance with a decrease in the expression of the inhibitor of apoptosis family of proteins and an increase in the Bax:Bcl-2 ratio accompanied by the proteolytic degradation of poly(ADP-ribose) polymerase. EECU also increased the generation of reactive oxygen species (ROS), collapse of mitochondrial membrane potential, and cytochrome c release to the cytosol, along with a truncation of Bid. In addition, EECU inactivated phosphatidylinositol 3-kinase (PI3K) as well as Akt, a downstream molecular target of PI3K, and LY294002, a specific PI3K inhibitor significantly enhanced EECU-induced apoptosis and cell viability reduction. However, N-acetyl cysteine, a general ROS scavenger, completely reversed the EECU-induced dephosphorylation of PI3K and Akt, as well as cell apoptosis. Taken together, these findings suggest that EECU inhibits T24 cell proliferation by activating intrinsic and extrinsic apoptosis pathways through a ROS-mediated inactivation of the PI3K/Akt pathway.

Induction of apoptosis by Moutan Cortex Radicis in human gastric cancer cells through the activation of caspases and the AMPK signaling pathway

Abstract Moutan Cortex Radicis, the root bark of Paeonia × suffruticosa Andrews, Paeoniaceae, has been widely used in traditional medicine therapy. Although it has been shown to possess many pharmacological activities, the molecular mechanisms of its anti-cancer activity have not been clearly elucidated. In the present study, we investigated the pro-apoptotic effects of the ethanol extract of Moutan Cortex Radicis in human gastric cancer AGS cells. Moutan Cortex Radicis treatment inhibited the cell viability of AGS cells in a concentration-dependent manner, which was associated with apoptotic cell death. Moutan Cortex Radicis's induction of apoptosis was connected with the upregulation of death receptor 4, death receptor 5, tumor necrosis factor-related apoptosis-inducing ligand, Fas ligand, and Bax, and the downregulation of Bcl-2 and Bid. Moutan Cortex Radicis treatment also induced the loss of mitochondrial membrane potential (Δψm), the proteolytic activation of caspases (-3, -8, and -9), and the degradation of poly(ADP-ribose) polymerase, an activated caspase-3 substrate protein. However, the pre-treatment of a caspase-3 inhibitor significantly attenuated Moutan Cortex Radicis-induced apoptosis and cell viability reduction. In addition, Moutan Cortex Radicis treatment effectively activated the adenosine monophosphate-activated protein kinase signaling pathway; however, a specific inhibitor of AMPK significantly reduced Moutan Cortex Radicis-induced apoptosis. Overall, the results suggest that the apoptotic activity of Moutan Cortex Radicis may be associated with a caspase-dependent cascade through the activation of both extrinsic and intrinsic signaling pathways connected with adenosine monophosphate-activated protein kinase activation, and Moutan Cortex Radicis as an activator of adenosine monophosphate-activated protein kinase could be a prospective application to treat human cancers.

Garlic extract in bladder cancer prevention: Evidence from T24 bladder cancer cell xenograft model, tissue microarray, and gene network analysis.

There is a growing interest in the use of naturally occurring agents in cancer prevention. This study investigated the garlic extract affects in bladder cancer (BC) prevention. The effect of garlic extract in cancer prevention was evaluated using the T24 BC BALB/C-nude mouse xenograft model. Microarray analysis of tissues was performed to identify differences in gene expression between garlic extract intake and control diet, and gene network analysis was performed to assess candidate mechanisms of action. Furthermore, we investigated the expression value of selected genes in the data of 165 BC patients. Compared to the control group, significant differences in tumor volume and tumor weight were observed in the groups fed 20 mg/kg (p<0.05), 200 mg/kg, and 1000 mg/kg of garlic extract (p<0.01). Genes (645) were identified as cancer prevention-related genes (fold change >2 and p<0.05) by tissue microarray analysis. A gene network analysis of 279 of these genes (p<0.01) was performed using Cytoscape/ClueGo software: 36 genes and 37 gene ontologies were mapped to gene networks. Protein kinase A (PKA) signaling pathway including AKAP12, RDX, and RAB13 genes were identified as potential mechanisms for the activity of garlic extract in cancer prevention. In BC patients, AKAP12 and RDX were decreased but, RAB13 was increased. Oral garlic extract has strong cancer prevention activity in vivo and an acceptable safety profile. PKA signaling process, especially increasing AKAP12 and RDX and decreasing RAB13, are candidate pathways that may mediate this prevention effect.

HSPA6 augments garlic extract-induced inhibition of proliferation, migration, and invasion of bladder cancer EJ cells; Implication for cell cycle dysregulation, signaling pathway alteration, and transcription factor-associated MMP-9 regulation.

Although recent studies have demonstrated the anti-tumor effects of garlic extract (GE), the exact molecular mechanism is still unclear. In this study, we investigated the molecular mechanism associated with the inhibitory action of GE against bladder cancer EJ cell responses. Treatment with GE significantly inhibited proliferation of EJ cells dose-dependently through G2/M-phase cell cycle arrest. This G2/M-phase cell cycle arrest by GE was due to the activation of ATM and CHK2, which appears to inhibit phosphorylation of Cdc25C (Ser216) and Cdc2 (Thr14/Tyr15), this in turn was accompanied by down-regulation of cyclin B1 and up-regulation of p21WAF1. Furthermore, GE treatment was also found to induce phosphorylation of MAPK (ERK1/2, p38MAPK, and JNK) and AKT. In addition, GE impeded the migration and invasion of EJ cells via inhibition of MMP-9 expression followed by decreased binding activities of AP-1, Sp-1, and NF-κB motifs. Based on microarray datasets, we selected Heat shock protein A6 (HSPA6) as the most up-regulated gene responsible for the inhibitory effects of GE. Interestingly, overexpression of HSPA6 gene resulted in an augmentation effect with GE inhibiting proliferation, migration, and invasion of EJ cells. The augmentation effect of HSPA6 was verified by enhancing the induction of G2/M-phase-mediated ATM-CHK2-Cdc25C-p21WAF1-Cdc2 cascade, phosphorylation of MAPK and AKT signaling, and suppression of transcription factor-associated MMP-9 regulation in response to GE in EJ cells. Overall, our novel results indicate that HSPA6 reinforces the GE-mediated inhibitory effects of proliferation, migration, and invasion of EJ cells and may provide a new approach for therapeutic treatment of malignancies.

Rosa hybrida extract suppresses vascular smooth muscle cell responses by the targeting of signaling pathways, cell cycle regulation and matrix metalloproteinase-9 expression.

The pharmacological effects of Rosa hybrida are well known in the cosmetics industry. However, the role of Rosa hybrida in cardiovascular biology had not previously been investigated, to the best of our knowledge. The aim of the present study was to elucidate the effect of water extract of Rosa hybrida (WERH) on platelet­derived growth factor (PDGF)-stimulated vascular smooth muscle cells (VSMCs). VSMC proliferation, which was stimulated by PDGF, was inhibited in a non-toxic manner by WERH treatment, which also diminished the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT. Treatment with WERH also induced G1-phase cell cycle arrest, which was due to the decreased expression of cyclins and cyclin-dependent kinases (CDKs), and induced p21WAF1 expression in PDGF-stimulated VSMCs. Moreover, WERH treatment suppressed the migration and invasion of VSMCs stimulated with PDGF. Treatment with WERH abolished the expression of matrix metalloproteinase-9 (MMP-9) and decreased the binding activity of nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and specificity protein 1 (Sp1) motifs in PDGF-stimulated VSMCs. WERH treatment inhibited the proliferation of PDGF­stimulated VSMCs through p21WAF1­mediated G1-phase cell cycle arrest, by decreasing the kinase activity of cyclin/CDK complexes. Furthermore, WERH suppressed the PDGF-induced phosphorylation of ERK1/2 and AKT in VSMCs. Finally, treatment with WERH impeded the migration and invasion of VSMCs stimulated by PDGF by downregulating MMP-9 expression and a reduction in NF-κB, AP-1 and Sp1 activity. These results provide new insights into the effects of WERH on PDGF-stimulated VSMCs, and we suggest that WERH has the potential to act as a novel agent for the prevention and/or treatment of vascular diseases.

Esculetin Inhibits VEGF-Induced Angiogenesis Both In Vitro and In Vivo.

Esculetin is known to inhibit tumor growth, but its effect in angiogenesis has not been studied. Here, we report the efficacy of esculetin on VEGF-induced angiogenesis. Esculetin treatment inhibited VEGF-induced proliferation and DNA synthesis of HUVECs with no cell toxicity. G1-phase cell-cycle arrest was associated with a decreased expression of cyclins and CDKs via the binding of p27KIP1. Esculetin down-regulated the MMP-2 expression in VEGF-stimulated HUVECs, which suppressed colony tube formation and migration. Esculetin reduced the phosphorylation of VEGFR-2 and the downstream signaling of VEGFR-2, including ERK1/2 and eNOS/Akt pathways. Esculetin suppressed microvessel outgrowth from an aortic ring ex vivo model treated with VEGF, and blocked the VEGF-induced formation of new blood vessels and hemoglobin content in an in vivo Matrigel plug model. Collectively, VEGF-stimulated responses in angiogenesis were inhibited in vitro and in vivo, providing a theoretical basis for effective use against anti-angiogenic therapies.

An exploration of the antioxidant effects of garlic saponins in mouse-derived C2C12 myoblasts.

In this study, we aimed to confirm the protective effects of garlic saponins against oxidative stress-induced cellular damage and to further elucidate the underlying mechanisms in mouse-derived C2C12 myoblasts. Relative cell viability was determined by 3-(4.5-dimethylthiazol-2-yl)-2.5 diphenyltetrazolium bromide assay. Comet assay was used to measure DNA damage and oxidative stress was determined using 2',7'-dichlorofluorescein diacetate to measure intracellular reactive oxygen species (ROS) generation. Western blot analysis and small interfering RNA (siRNA)-based knockdown were used in order to investigate the possible molecular mechanisms. Our results revealed that garlic saponins prevented hydrogen peroxide (H2O2)-induced growth inhibition and exhibited scavenging activity against intracellular ROS. We also observed that garlic saponins prevented H2O2-induced comet tail formation and decreased the phosphorylation levels of γH2AX expression, suggesting that they can prevent H2O2-induced DNA damage. In addition, garlic saponins increased the levels of heme oxygenase-1 (HO-1), a potent antioxidant enzyme associated with the induction and phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2) and the translocation of Nrf2 from the cytosol into the nucleus. However, the protective effects of garlic saponins on H2O2-induced ROS generation and growth inhibition were significantly reduced by zinc protoporphyrin Ⅸ, an HO-1 competitive inhibitor. In addition, the potential of garlic saponins to mediate HO-1 induction and protect against H2O2­mediated growth inhibition was adversely affected by transient transfection with Nrf2-specific siRNA. Garlic saponins activated extracellular signal­regulated kinase (ERK) signaling, whereas a specific ERK inhibitor was able to inhibit HO-1 upregulation, as well as Nrf2 induction and phosphorylation. Taken together, the findings of our study suggest that garlic saponins activate the Nrf2/HO-1 pathway by enabling ERK to contribute to the induction of phase Ⅱ antioxidant and detoxifying enzymes, including HO-1 in C2C12 cells.

Anti-inflammatory potential of saponins derived from cultured wild ginseng roots in lipopolysaccharide-stimulated RAW 264.7 macrophages.

Ginseng, namely the root of Panax ginseng Meyer, is a well-known traditional medicine that has been used in Asian countries for thousands of years. Ginseng saponins have been shown to exert a variety of prominent pharmacological effects in a number of diseases. The aim of the present study was to identify the anti-inflammatory effects of total saponins extracted from cultured wild ginseng roots (TSWG) on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. An elevated production of nitric oxide (NO) was detected in the RAW 264.7 cells in response to stimulation with LPS, as shown by NO detection assay using Griess reagent. However, pre-treatment with TSWG inhibited the production of NO through the suppression of inducible NO synthase gene expression. Furthermore, the LPS-induced gene expression and production of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were significantly reduced by treatment with TSWG, as shown by ELISA, and western blot analysis and RT-PCR, respectively. In the LPS-stimulated RAW 264.7 cells, nuclear factor-κB (NF-κB) was translocated from the cytosol to the nucleus, while pre-treatment with TSWG induced the sequestration of NF-κB in the cytosol through the inhibition of the inhibitor of κB degradation, as shown by immunofluorescence staining. TSWG also contributed to the downregulation of mitogen-activated protein kinases and Akt in the LPS-stimulated RAW 264.7 cells. Additionally, in the TSWG-treated RAW 264.7 cells, we observed the activation of nuclear factor (erythroid-derived 2)-like 2 and an increase in heme oxygenase-1 expression; these effects were associated with the inhibition of the generation of reactive oxygen species. The results from the present study indicate that TSWG exerts anti-inflammatory and antioxidant effects, suggesting that TSWG may be an effective therapeutic agent for inflammatory diseases and prevent cellular damage induced by oxidative stress.

Anti-Inflammatory Effects of 3-(4'-Hydroxyl-3',5'-Dimethoxyphenyl)Propionic Acid, an Active Component of Korean Cabbage Kimchi, in Lipopolysaccharide-Stimulated BV2 Microglia.

We investigated the protective ability of 3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid (HDMPPA), an active principle in Korean cabbage kimchi, against the production of proinflammatory mediators and cytokines, and the mechanisms involved in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. HDMPPA significantly suppressed the production of nitric oxide (NO) and prostaglandin E2, along with the expression of inducible NO synthase and cyclooxygenase-2 in LPS-stimulated BV2 cells, at concentrations with no cytotoxicity. HDMPPA also attenuated the LPS-induced expression and secretion of proinflammatory cytokines, such as tumor necrosis factor-α and interleukin-1ß. Furthermore, HDMPPA inhibited LPS-induced nuclear factor-κB (NF-κB) activation, which was associated with the abrogation of IκB-α degradation and phosphorylation, and subsequent decreases in NF-κB p65 levels. Moreover, the phosphorylation of mitogen-activated protein kinases (MAPKs) and Akt, a downstream molecule of phosphatidylinositol-3-kinase (PI3K), in LPS-stimulated BV2 cells was suppressed markedly by HDMPPA. This effect was associated with a significant reduction in the formation of intracellular reactive oxygen species. The findings in this study suggest that HDMPPA may exert anti-inflammatory responses by suppressing LPS-induced expression of proinflammatory mediators and cytokines through blockage of NF-κB, MAPKs, and PI3K/Akt signaling pathways and oxidative stress in microglia.