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.

Morin inhibits PDGF-induced proliferation, migration, and invasion of vascular smooth muscle cells via modulating p27KIP1, AKT, and MMP-9 activities.

Hyper-proliferation and migration of vascular smooth muscle cells (VSMCs) are closely associated with atherosclerosis. Recently, the flavonol morin has been reported to exhibit potent anti-oxidant and anti-inflammatory activities. Therefore, we investigated molecular mechanisms of morin in VSMCs stimulated by PDGF. Morin effectively inhibited PDGF-stimulated proliferation of VSMCs through a G1 cell-cycle arrest, leading to down-regulation of CDK2, CDK4, cyclin D1, and cyclin E proteins. Interestingly, PDGF markedly down-regulated p27KIP1 protein expression; however, morin treatment restored the p27KIP1expression to the basal level. Morin did not affect phosphorylation of MAPKs (ERK, p38, and JNK); however, phosphorylation of AKT was dramatically suppressed by morin in PDGF-stimulated VSMCs. Using the PI3K inhibitor, LY294002, we revealed that AKT is a key regulator in the inhibitory mechanism of morin against PDGF-induced proliferation of VSMCs. Morin disturbed migratory and invasive potential of VSMCs via suppression of matrix metalloproteinase-9 (MMP-9) activity. Using electrophoretic mobility shift assays, we verified that NF-κB, AP-1, and Sp-1 transcription factors are implicated in the mode of action of morin, which suppresses the MMP-9 activity in PDGF-induced VSMCs. Based on the results, we believe that morin may be a potential therapeutic agent for atherosclerosis without negative side effect.

Morin Inhibits Proliferation, Migration, and Invasion of Bladder Cancer EJ Cells via Modulation of Signaling Pathways, Cell Cycle Regulators, and Transcription Factor-Mediated MMP-9 Expression.

Preclinical Research Previous studies have shown that morin exerts diverse pharmacological activities. In this study, we investigated the inhibitory activity of morin on bladder cancer EJ cells. Morin significantly inhibited EJ cell proliferation, which was related to the G1-phase cell cycle arrest together with the reduced expression of cyclin D1, cyclin E, CDK2, and CDK4 via increased expression of p21WAF1. Morin also increased ERK1/2 phosphorylation and decreased JNK and AKT phosphorylation without altering the p38MAPK phosphorylation levels. Morin treatment suppressed the migration and invasion of EJ cells in wound-healing and transwell cell invasion assays. Zymographic and electrophoretic mobility shift assays showed that morin suppressed the expression of matrix metalloproteinase-9 (MMP-9) via repression of the binding activity of AP-1, Sp-1, and NF-κB. Collectively, these results demonstrate that morin reduced cyclin D1, cyclin E, CDK2 and CDK4 expression via the induction of p21WAF1 expression, increased ERK1/2 phosphorylation and decreased JNK, and AKT phosphorylation, and prevented MMP-9 expression via the inhibition of transcription factors AP-1, Sp-1, and NF-κB, thereby resulting in the inhibition of growth, migration, and invasion of bladder cancer EJ cells. These results provide a novel insight into the use of morin in the prevention of bladder cancer. Drug Dev Res 78 : 81-90, 2017. © 2017 Wiley Periodicals, Inc.

PMA synergistically enhances apicularen A-induced cytotoxicity by disrupting microtubule networks in HeLa cells.

BACKGROUND: Combination therapy is key to improving cancer treatment efficacy. Phorbol 12-myristate 13-acetate (PMA), a well-known PKC activator, increases the cytotoxicity of several anticancer drugs. Apicularen A induces cytotoxicity in tumor cells through disrupting microtubule networks by tubulin down-regulation. In this study, we examined whether PMA increases apicularen A-induced cytotoxicity in HeLa cells. METHODS: Cell viability was examined by thiazolyl blue tetrazolium (MTT) assays. To investigate apoptotic potential of apicularen A, DNA fragmentation assays were performed followed by extracting genomic DNA, and caspase-3 activity assays were performed by fluorescence assays using fluorogenic substrate. The cell cycle distribution induced by combination with PMA and apicularen A was examined by flow cytometry after staining with propidium iodide (PI). The expression levels of target proteins were measured by Western blotting analysis using specific antibodies, and α-tubulin mRNA levels were assessed by reverse transcription polymerase chain reaction (RT-PCR). To examine the effect of combination of PMA and apicularen A on the microtubule architecture, α-tubulin protein and nuclei were visualized by immunofluorescence staining using an anti-α-tubulin antibody and PI, respectively. RESULTS: We found that apicularen A induced caspase-dependent apoptosis in HeLa cells. PMA synergistically increased cytotoxicity and apoptotic sub-G1 population induced by apicularen A. These effects were completely blocked by the PKC inhibitors Ro31-8220 and Go6983, while caspase inhibition by Z-VAD-fmk did not prevent cytotoxicity. RNA interference using siRNA against PKCα, but not PKCß and PKCγ, inhibited cytotoxicity induced by combination PMA and apicularen A. PMA increased the apicularen A-induced disruption of microtubule networks by further decreasing α- and ß-tubulin protein levels in a PKC-dependent manner. CONCLUSIONS: These results suggest that the synergy between PMA and apicularen A is involved by PKCα activation and microtubule disruption, and that may inform the development of novel approaches to treat cancer.

IL-28A/IL-10Rß axis promotes angiogenesis via eNOS/AKT signaling and AP-1/NF-κB/MMP-2 network by regulating HSP70-1 expression.

INTRODUCTION: Angiogenesis plays a significant role in the development of tumor progression and inflammatory diseases. The role of IL-28A in angiogenesis and its precise regulatory mechanisms remain rarely elucidated. OBJECTIVES: We report the novel regulatory role of IL-28A in physiological angiogenesis. The study aimed to elucidate the regulatory mechanisms involved in IL-28A-mediated angiogenesis and identify key genes associated with IL-28A-induced angiogenic responses. METHODS: To know the effect of IL-28A on angiogenesis, HUVECs were applied to perform proliferation, migration, invasion, tube formation, immunoblot, and EMSA. Gene expression changes in HUVECs following IL-28A treatment were analyzed by NGS. The functional role of HSP70-1 and IL-10Rß in IL-28A-induced angiogenic responses was evaluated using PCR and siRNA knockdown. Animal studies were conducted by aortic ring ex vivo assays, Matrigel plug in vivo assays, and immunochemistry using HSP70-1 knockout and transgenic mice models. The efficacy of IL-28A in angiogenesis was confirmed in a hind-limb ischemia model. RESULTS: Autocrine/paracrine actions in HUVECs regulated IL-28A protein expression. Exogenous IL-28A increased the proliferation of HUVECs via eNOS/AKT and ERK1/2 signaling. IL-28A treatment promoted migration, invasion, and capillary tube formation of HUVECs through induction of the AP-1/NF-κB/MMP-2 network, which was associated with eNOS/AKT and ERK1/2 signaling. The efficacy of IL-28A-induced angiogenic potential was confirmed by aortic ring and Matrigel plug assay. HSP70-1 was identified as an IL-28A-mediated angiogenic effector gene using bioinformatics. Knockdown of HSP70-1 abolished angiogenic responses and eNOS/AKT signaling in IL-28A-treated HUVECs. IL-28A-induced microvessel sprouting formation was testified in HSP70-1-deficient and HSP70-1 transgenic mice. Flow recovery in hind-limb ischemia mice was accelerated by IL-28A injection. Finally, ablation of the IL-10Rß gene impeded the angiogenic responses and eNOS/AKT signaling stimulated by IL-28A in HUVECs. CONCLUSION: HSP70-1 drives the progression of angiogenesis by the IL-28A/IL-10Rß axis via eNOS/AKT signaling and the AP-1/NF-κB/MMP-2 network.

Ethanol Extracts of Cornus alba Improve Benign Prostatic Hyperplasia by Inhibiting Prostate Cell Proliferation through Modulating 5 Alpha-Reductase/Androgen Receptor Axis-Mediated Signaling.

PURPOSE: The aim of this study was to investigate the efficacy of ethanol extracts of Cornus alba (ECA) against benign prostatic hyperplasia (BPH) in vitro and in vivo. MATERIALS AND METHODS: The prostate stromal cells (WPMY-1) and epithelial cells (RWPE-1) were used to examine the action mechanism of ECA in BPH in vitro. ECA efficacy was evaluated in vivo using a testosterone propionate (TP)-induced BPH rat model. RESULTS: Treatment with ECA inhibited the proliferation of prostate cells by inducing G1-phase cell cycle arrest through the regulation of positive and negative proteins. Treatment of prostate cells with ECA resulted in alterations in the mitogen-activated protein kinases and protein kinase B signaling pathways. The transcriptional binding activity of the NF-κB motif was suppressed in both ECA-treated prostate cells. In addition, treatment with ECA altered the level of BPH-associated axis markers (5α-reductase, fibroblast growth factor-2, androgen receptor, epidermal growth factor, Bcl-2, and Bax) in both cell lines. Finally, the administration of ECA attenuated the enlargement of prostatic tissues in the TP-induced BPH rat model, accompanied by histology, immunoblot, and serum dihydrotestosterone levels. CONCLUSIONS: These results demonstrated that ECA exerted beneficial effects on BPH both in vitro and in vivo and might provide valuable information in the development of preventive or therapeutic agents for improving BPH.

Pharmacological effects of biologically synthesized ginsenoside CK-rich preparation (AceCK40) on the colitis symptoms in DSS-induced Caco-2 cells and C57BL mice.

BACKGROUND: Despite the notable pharmacological potential of natural ginsenosides, their industrial application is hindered by low oral bioavailability. Recent research centers on the production of less-glycosylated minor ginsenosides. PURPOSE: This study aimed to explore the effect of a biologically synthesized ginsenoside CK-rich minor ginsenoside complex (AceCK40), on ameliorating colitis using DSS-induced colitis models in vitro and in vivo. METHODS: The ginsenoside composition of AceCK40 was determined by HPLC-ELSD and UHPLC-MS/MS analyses. In vitro colitis model was established using dextran sodium sulfate (DSS)-induced Caco-2 intestinal epithelial model. For in vivo experiments, DSS-induced severe colitis mouse model was established. RESULTS: In DSS-stimulated Caco-2 cells, AceCK40 downregulated mitogen-activated protein kinase (MAPK) activation (p < 0.05), inhibited monocyte chemoattractant protein-1 (MCP-1) production (p < 0.05), and enhanced MUC2 expression (p < 0.05), mediated via signaling pathway regulation. Daily AceCK40 administration at doses of 10 and 30 mg/kg/day was well tolerated by DSS-induced severe colitis mice. These doses led to significant alleviation of disease activity index score (> 36.0% decrease, p < 0.05), increased luminal immunoglobulin (Ig)G (> 37.6% increase, p < 0.001) and IgA (> 33.8% increase, p < 0.001), lowered interleukin (IL)-6 (> 65.7% decrease, p < 0.01) and MCP-1 (> 116.2% decrease, p < 0.05), as well as elevated serum IgA (> 51.4% increase, p < 0.001) and lowered serum IL-6 (112.3% decrease at 30 mg/kg, p < 0.001). Hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining revealed that DSS-mediated thickening of the muscular externa, extensive submucosal edema, crypt distortion, and decreased mucin droplets were significantly alleviated by AceCK40 administration. Additionally, daily administration of AceCK40 led to significant recovery of colonic tight junctions damaged by DSS through the elevation in the expression of adhesion molecules, including occludin, E-cadherin, and N-cadherin. CONCLUSION: This study presents the initial evidence elucidating the anti-colitis effects of AceCK40 and its underlying mechanism of action through sequential in vitro and in vivo systems employing DSS stimulation. Our findings provide valuable fundamental data for the utilization of AceCK40 in the development of novel anti-colitis candidates.

Apicularen A acetate induces cell death via AIF translocation and disrupts the microtubule network by down-regulating tubulin in HM7 human colon cancer cells.

Apicularen A is a novel antitumor agent and strongly induces death in tumor cells. In this study, we synthesized apicularen A acetate, an acetyl derivative of apicularen A, and investigated its antitumor effect and mechanism in HM7 colon cancer cells. Apicularen A acetate induced apoptotic cell death and caspase-3 activation; however, the pan-caspase inhibitor Z-VAD-fmk could not prevent this cell death. Apicularen A acetate induced the loss of mitochondrial membrane potential and the translocation of apoptosis-inducing factor (AIF) from mitochondria. In addition, apicularen A acetate significantly decreased tubulin mRNA and protein levels and induced disruption of microtubule networks. Taken together, these results indicate that the mechanism of apicularen A acetate involves caspase-independent apoptotic cell death and disruption of microtubule architecture.

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.

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.

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.

Protein-bound polysaccharide from Phellinus linteus inhibits tumor growth, invasion, and angiogenesis and alters Wnt/ß-catenin in SW480 human colon cancer cells.

BACKGROUND: Polysaccharides extracted from the Phellinus linteus (PL) mushroom are known to possess anti-tumor effects. However, the molecular mechanisms responsible for the anti-tumor properties of PL remain to be explored. Experiments were carried out to unravel the anticancer effects of PL. METHODS: The anti-cancer effects of PL were examined in SW480 colon cancer cells by evaluating cell proliferation, invasion and matrix metallo-proteinase (MMP) activity. The anti-angiogenic effects of PL were examined by assessing human umbilical vein endothelial cell (HUVEC) proliferation and capillary tube formation. The in vivo effect of PL was evaluated in an athymic nude mouse SW480 tumor engraft model. RESULTS: PL (125-1000 µg/mL) significantly inhibited cell proliferation and decreased ß-catenin expression in SW480 cells. Expression of cyclin D1, one of the downstream-regulated genes of ß-catenin, and T-cell factor/lymphocyte enhancer binding factor (TCF/LEF) transcription activity were also significantly reduced by PL treatment. PL inhibited in vitro invasion and motility as well as the activity of MMP-9. In addition, PL treatment inhibited HUVEC proliferation and capillary tube formation. Tumor growth of SW480 cells implanted into nude mice was significantly decreased as a consequence of PL treatment, and tumor tissues from treated animals showed an increase in the apoptotic index and a decrease in ß-catenin expression. Moreover, the proliferation index and microvessel density were significantly decreased. CONCLUSIONS: These data suggest that PL suppresses tumor growth, invasion, and angiogenesis through the inhibition of Wnt/ß-catenin signaling in certain colon cancer cells.

Omega-3-polyunsaturated fatty acids suppress pancreatic cancer cell growth in vitro and in vivo via downregulation of Wnt/Beta-catenin signaling.

BACKGROUND/AIMS: ω3-polyunsaturated fatty acids (ω3- PUFAs) are known to possess anticancer properties. However, the relationship between ω3-PUFAs and ß-catenin, one of the key components of the Wnt signaling pathway, in human pancreatic cancer remains poorly characterized. METHODS: Human pancreatic cancer cells (SW1990 and PANC-1) were exposed to two ω3-PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), to investigate the relationship between ω3-PUFAs and the Wnt/ß-catenin signaling pathway in vitro. Mouse pancreatic cancer (PANC02) cells were implanted into fat-1 transgenic mice, which express ω3 desaturases and result in elevated levels of ω3-PUFAs endogenously. The tumor size, levels of Wnt/ß-catenin signaling molecules and apoptosis levels were analyzed to examine the influence of ω3-PUFAs in vivo. RESULTS: DHA and EPA significantly inhibited cell growth and increased cell death in pancreatic cancer cells. DHA also reduced ß-catenin expression, T cell factor/lymphoid-enhancing factor reporter activity and induced ß-catenin/Axin/GSK-3ß complex formation, a known precursor to ß-catenin degradation. Furthermore, Wnt3a, a natural canonical Wnt pathway ligand, reversed DHA-induced growth inhibition in PANC-1 cells. Immunohistochemical analysis showed aberrant upregulation and increased nuclear staining of ß-catenin in tumor tissues from pancreatic cancer patients. However, ß-catenin levels in tumor tissues from fat-1 transgenic mice were reduced with a significant increase in apoptosis compared with those from control mice. CONCLUSION: ω3-PUFAs may be an effective therapy for the chemoprevention and treatment of human pancreatic cancer. and IAP.

A Novel Cyclic Pentadepsipeptide, N-Methylsansalvamide, Suppresses Angiogenic Responses and Exhibits Antitumor Efficacy against Bladder Cancer.

Here, we explored the anti-tumor efficacy of a cyclic pentadepsipeptide, N-methylsansalvamide (MSSV), in bladder cancer. MSSV inhibited the proliferation of both bladder cancer 5637 and T24 cells, which was attributed to the G1-phase cell cycle arrest, apoptosis induction, and alteration of mitogen-activated protein kinases (MAPKs) and protein kinase b (AKT) signaling pathways. Additionally, the treatment of bladder cancer cells with MSSV suppressed migratory and invasive potential via the transcription factor-mediated expression of matrix metalloproteinase 9 (MMP-9). MSSV abrogated vascular endothelial growth factor (VEGF)-induced angiogenic responses in vitro and in vivo. Furthermore, our result showed the potent anti-tumor efficacy of MSSV in a xenograft mouse model implanted with bladder cancer 5637 cells. Finally, acute toxicity test data obtained from blood biochemical test and liver staining indicated that the oral administration of MSSV at 2000 mg/kg caused no adverse cytotoxic effects. Our preclinical data described the potent anti-angiogenic and anti-tumor efficacy of MSSV and showed no signs of acute toxicity, thereby suggesting the putative potential of oral MSSV as a novel anti-tumor agent in bladder cancer treatment.

Collagen type VI­α1 and 2 repress the proliferation, migration and invasion of bladder cancer cells.

The bladder cancer (BCa) microenvironment comprises heterogeneous tumor cell populations, the surrounding stroma and the extracellular matrix (ECM). Collagen, the scaffold of the tumor microenvironment, regulates ECM remodeling to promote tumor infiltration, angiogenesis, invasion and migration. The present study examined how collagen type VI­α (COL6A) 1 and 2 function during BCa pathogenesis and progression, with the aim of facilitating the development of precision therapeutics, risk stratification and molecular diagnosis. COL6A1 and COL6A2 mRNA expression in non­muscle invasive BCa (NMIBC) and MIBC tissue samples was measured using reverse transcription­quantitative PCR. In addition, the tumor­suppressive effects of COL6A1 and COL6A2 in human BCa EJ cells (MGH­U1) were assessed. Compared with normal controls, COL6A1 and COL6A2 mRNA expression was downregulated in both NMIBC and MIBC tissue samples (P<0.05, respectively). COL6A1 and COL6A2 effectively inhibited the proliferation of human BCa EJ cells (MGH­U1) and induced cell cycle arrest at the G1 phase. Additionally, COL6A1 and COL6A2 served roles in MAPK and AKT signaling by increasing p38 MAPK phosphorylation and decreasing AKT phosphorylation. Finally, COL6A1 and COL6A2 inhibited wound healing and invasion by suppressing the activity of matrix metalloproteinase (MMP)­2 and MMP­9. In conclusion, COL6A1 and COL6A2 may act as classical collagens by forming a physical barrier to inhibit BCa tumor growth and invasion.

Expression regulation and function of Pref-1 during adipogenesis of human mesenchymal stem cells (MSCs).

Preadipocyte Factor 1 (Pref-1), also known as Delta-like Protein 1 (DLK-1) is an epidermal growth factor-like domain-containing trans-membrane protein that is involved in adipogenesis and cell fate decision. Its function in adipogenesis is reported inconsistently based on different cellular model systems. Here, by using human mesenchymal stem cells (MSCs), we show that Pref-1 is modulated by both dexamethasone and 3-isobutyl-1methylxanthine (IBMX), two components of the adipogenic induction mixture during the adipogenesis in vitro. IBMX induces the expression of Pref-1 in a time- and dose-dependent manner through cyclic AMP and cyclic GMP independent pathway and attenuates adipocyte differentiation by down-regulating PPARgamma (peroxisome proliferator activated receptor gamma) expression. Dexamethasone, on the other hand, is capable of subduing the inhibitory effect of IBMX-induced Pref-1 and initiating the adipogenesis by up-regulating PPARgamma expression. Moreover, the treatment of IBMX or dexamethasone alone fails to develop MSCs into mature adipocytes, however, treating cells with both IBMX and dexamethasone leads to a complete adipocyte differentiation as evaluated by lipid-droplet formation. Taken together, our study demonstrates that IBMX accelerates accumulation of lipid in MSCs only under the circumstance that the negative effect of Pref-1 induced by IBMX on the adipogenesis is overcome by dexamethasone.

Downregulation of APE1/Ref-1 is involved in the senescence of mesenchymal stem cells.

The senescence of human mesenchymal stem cells (hMSCs) causes disruption of tissue and organ maintenance, and is thus an obstacle to stem cell-based therapies for disease. Although some researchers have studied changes in the characteristics of hMSCs (decreases in differentiation ability and self-renewal), comparing young and old ages, the mechanisms of stem cell senescence have not yet been defined. In this study, we developed a growth curve for human bone marrow derived MSCs (hBMSCs) which changes into a hyperbolic state after passage number 7. Senescence associated beta-galactosidase (SA beta-gal) staining of hBMSCs showed 10% in passage 9 and 45% in passage 11. We detected an increase in endogenous superoxide levels during senescence that correlated with senescence markers (SA beta-gal, hyperbolic growth curve). Interestingly, even though endogenous superoxide increased in a replicative senescence model, the expression of APE1/Ref-1, which is sensitive to intracellular redox state, decreased. These effects were confirmed in a stress-induced senescence model by exogenous treatment with H(2)O(2). This change is related to the p53 activity that negatively regulates APE1/Ref-1. p21 expression levels, which represent p53 activity, were transiently increased in passage 9, meaning that they correlated with the expression of APE1/Ref-1. Overexpression of APE1/Ref-1 suppressed superoxide production and decreased SA beta-gal in hBMSCs. In conclusion, intracellular superoxide accumulation appears to be the main cause of the senescence of hBMSCs, and overexpression of APE1/Ref-1 can rescue cells from the senescence phenotype. Maintaining characteristics of hBMSCs by regulating intracellular reactive oxygen species production can contribute to tissue regeneration and to improved cell therapy.

Carnosine Impedes PDGF-Stimulated Proliferation and Migration of Vascular Smooth Muscle Cells In Vitro and Sprout Outgrowth Ex Vivo.

Carnosine, a naturally producing dipeptide, exhibits various beneficial effects. However, the possible role of carnosine in vascular disorders associated with pathological conditions, including proliferation and migration of vascular smooth muscle cells (VSMCs), largely remains unrevealed. Here, we investigated the regulatory role and mechanism of carnosine in platelet-derived growth factor (PDGF)-induced VSMCs. Carnosine inhibited the proliferation of PDGF-induced VSMCs without any cytotoxic effects. Carnosine treatment also induced G1-phase cell cycle arrest by causing a p21WAF1-mediated reduction in the expression of both cyclin-dependent kinases (CDKs) and cyclins in PDGF-treated VSMCs. Carnosine treatment suppressed c-Jun N-terminal kinase (JNK) phosphorylation in PDGF-stimulated signaling. Additionally, carnosine significantly prevented the migration of VSMCs exposed to PDGF. Carnosine abolished matrix metalloproteinase (MMP)-9 activity via reduced transcriptional binding activity of NF-κB, Sp-1, and AP-1 motifs in PDGF-treated VSMCs. Moreover, using aortic assay ex vivo, it was observed that carnosine addition attenuated PDGF-stimulated sprout outgrowth of VSMCs. Taken together, these results demonstrated that carnosine impeded the proliferation and migration of PDGF-stimulated VSMCs by regulating cell cycle machinery, JNK signaling, and transcription factor-mediated MMP-9 activity as well as prevented ex vivo sprout outgrowth of blood vessels. Thus, carnosine may be a potential candidate for preventing vascular proliferative disease.

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.

Peanut Sprout Extracts Cultivated with Fermented Sawdust Medium Inhibits Benign Prostatic Hyperplasia In Vitro and In Vivo.

PURPOSE: In this study, we tested whether the resveratrol-enriched peanut sprout extracts cultivated with fermented sawdust medium (PSEFS) could suppress benign prostatic hyperplasia (BPH) in vitro and in vivo. MATERIALS AND METHODS: The mode of action of PSEFS was estimated by employing high-performance liquid chromatography analysis, MTT assay, cell counting, cell cycle analysis, immunoblots, and immunoprecipitation and electrophoretic mobility shift assay. In vivo efficacy of PSEFS was analyzed in BPH animal model via immunostaining and enzyme-linked immunosorbent assay. RESULTS: We selected the Yesan peanut sprout variety, which contains the highest level of resveratrol. The resveratrol levels in PSEFS were higher than those obtained with hydroponic technology. PSEFS treatment induced cell cycle arrest at the G1-phase by downregulating CDK4 and cyclin D1 via p21WAF1 induction in the RWPE-1 and WPMY prostate cells, thereby decreasing their proliferation. Treatment with PSEFS decreased ERK1/2 phosphorylation and increased JNK phosphorylation. The levels of DNA-bound transcription factors associated with proliferation (nuclear factor-κB, Sp-1, and AP-1) decreased upon PSEFS treatment in both prostate cells. Additionally, the levels of the molecular markers of BPH development (5α-reductase, androgen receptor, fibroblast growth factor, Bcl-2, and Bax) also changed by the addition of PSEFS. Finally, in a testosterone propionate-induced BPH model in rats, PSEFS administration attenuated the size, weight, and thickness of prostate tissues with no signs of death. CONCLUSIONS: These results showed that PSEFS inhibited BPH both in vitro and in vivo and might be useful in the development of a potential BPH therapy.