Nanopore Direct RNA Sequencing Reveals the Short-Term Salt Stress Response in Maize Roots.

Transcriptome analysis, relying on the cutting-edge sequencing of cDNA libraries, has become increasingly prevalent within functional genome studies. However, the dependence on cDNA in most RNA sequencing technologies restricts their ability to detect RNA base modifications. To address this limitation, the latest Oxford Nanopore Direct RNA Sequencing (ONT DRS) technology was employed to investigate the transcriptome of maize seedling roots under salt stress. This approach aimed to unveil both the RNA transcriptional profiles and alterations in base modifications. The analysis of the differential expression revealed a total of 1398 genes and 2223 transcripts that exhibited significant variation within the maize root system following brief exposure to salt stress. Enrichment analyses, such as the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway assessments, highlighted the predominant involvement of these differentially expressed genes (DEGs) in regulating ion homeostasis, nitrogen metabolism, amino acid metabolism, and the phytohormone signaling pathways. The protein-protein interaction (PPI) analysis showed the participation of various proteins related to glycolytic metabolism, nitrogen metabolism, amino acid metabolism, abscisic acid signaling, and the jasmonate signaling pathways. It was through this intricate molecular network that these proteins collaborated to safeguard root cells against salt-induced damage. Moreover, under salt stress conditions, the occurrence of variable shear events (AS) in RNA modifications diminished, the average length of poly(A) tails underwent a slight decrease, and the number of genes at the majority of the variable polyadenylation (APA) sites decreased. Additionally, the levels of N5-methylcytosine (m5C) and N6-methyladenosine (m6A) showed a reduction. These results provide insights into the mechanisms of early salt tolerance in maize.

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.

Bisphenol A modulates proliferation, apoptosis, and wound healing process of normal prostate cells: Involvement of G2/M-phase cell cycle arrest, MAPK signaling, and transcription factor-mediated MMP regulation.

Bisphenol A (BPA) is commonly used to produce epoxy resins and polycarbonate plastics. BPA is an endocrine-disrupting chemical that is leaked from the polymer and absorbed into the body to disrupt the endocrine system. Although BPA may cause cytotoxicity in the prostate, a hormone-dependent reproductive organ, its underlying mechanism has not yet been elucidated. Here, we investigated the effects of BPA on cell proliferation, apoptosis, and the wound healing process using prostate epithelial cells (RWPE-1) and stromal cells (WPMY-1). Observations revealed that BPA induced G2/M cell cycle arrest in both cell types through the ATM-CHK1/CHK2-CDC25c-CDC2 signaling pathway, and the IC50 values were estimated to be 150 µM. Furthermore, BPA was found to induce caspase-dependent apoptosis through initiator (caspase-8 and -9) and executioner (caspase-3 and -7) caspase cascades. In addition, BPA interfered with the wound healing process through inhibition of MMP-2 and - 9 expression, accompanied by reductions in the binding activities of AP-1 as well as NF-κB motifs. Phosphorylation of MAPKs was associated with the BPA-mediated toxicity of prostate cells. These results suggest that BPA exhibits prostate toxicity by inhibiting cell proliferation, inducing apoptosis, and interfering with the wound healing process. Our study provided new insights into the precise molecular mechanisms of BPA-induced toxicity in human prostate cells.

Effect of the layered fasciocutaneous flap in the repair of complex wounds of the lower extremity.

INTRODUCTION: Repair of complex wounds of the lower extremity remains challenging for clinicians. When repairing complex wounds, it is necessary to customize the skin flap to simultaneously repair both the superficial soft tissue defect and the deep dead space wound. OBJECTIVE: This case series describes the use of a layered fasciocutaneous flap with pedicled perforator to repair complex wounds of the lower extremity. MATERIALS AND METHODS: Three cases with complex wounds of the lower extremity underwent repair using layered fasciocutaneous flap, and clinical efficacy was observed. RESULTS: In cases 1 and 3, complete survival of the layered flaps was achieved. In case 2, congestion measuring 1 cm × 1 cm occurred at the distal end of the skin paddle, followed by superficial skin ulceration, which healed 2 weeks after a dressing change. CONCLUSION: These findings indicate that a layered fasciocutaneous flap with pedicled perforator can be used to repair complex wounds of the lower extremity.

Resveratrol Attenuates the Proliferation of Prostatic Stromal Cells in Benign Prostatic Hyperplasia by Regulating Cell Cycle Progression, Apoptosis, Signaling Pathways, BPH Markers, and NF- κB Activity.

Resveratrol can inhibit cell proliferation and metastasis and induce apoptosis. However, the mechanisms of action through which resveratrol inhibits the abnormal proliferation of prostate stromal cells, causing prostatic hyperplasia, have not been fully elucidated. Here, we evaluated the inhibitory effects of resveratrol on cell° proliferation associated with prostatic hyperplasia using WPMY-1 cells. Our results showed that resveratrol inhibited the proliferation of WPMY-1 cells via the induction of G0/G1-phase cell cycle arrest, which was caused by downregulated expression of cyclins and cyclin-dependent kinases regulated by increased p21WAF1 and p27KIP1 expression level. In addition, resveratrol treatment suppressed the phosphorylation of phosphatidylinositol 3-kinase/AKT and extracellular signal-regulated kinase 1/2. The expression levels of molecular markers affecting prostate development were also reduced by treatment with resveratrol. Finally, resveratrol attenuated the binding activity of the transcription factor nuclear factor-κB in WPMY-1 cells, and accelerated apoptotic cell death via intrinsic cascade pathway. These results indicate that resveratrol may be useful for the prevention or treatment of prostatic hyperplasia.

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.

HIF-1α Enhances Vascular Endothelial Cell Permeability Through Degradation and Translocation of Vascular Endothelial Cadherin and Claudin-5 in Rats With Burn Injury.

The mechanism underlying burn injury-induced enhanced vascular endothelial permeability and consequent body fluid extravasation is unclear. Here, the rat aortic endothelial cells (RAECs) were treated with the serum derived from rats with burn injury to elucidate the mechanism. Sprague-Dawley (SD) rats were grouped as follows (10 rats/group): control, 2, 4, 8, 12, and 24 hours postburn groups. The heart, liver, kidney, lung, jejunum, and ileum of rats injected with 2% Evans blue (EB) through the tail vein were excised to detect the EB level in each organ. The serum levels of hypoxia-inducible factor-1α (HIF-1α) and endothelin-1 (ET-1) were examined using enzyme-linked immunosorbent assay (ELISA). The effect of serum from 12-hour postburn group on the membrane permeability of RAEC monolayer, as well as on the mRNA and protein levels of ET-1, endothelin receptor A (ETA), ETB, and zonula occludens (ZO-1), was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. The membrane permeability of GV230/HIF-1α-transfected or shRNA-HIF-1α-transfected RAECs, as well as the expression levels of HIF-1α, ET-1, ETA, ETB, vascular endothelial (VE)-cadherin, and claudin-5, was analyzed using qRT-PCR and western blotting, whereas the localization of VE-cadherin and claudin-5 was examined using immunofluorescence. The serum HIF-1α and ET-1 levels in the burn groups, which peaked at 12 hours postburn, were significantly upregulated (P < .01) when compared with those in the control group. Additionally, the serum HIF-1α levels were positively correlated with vascular permeability. Compared with the shRNA-negative control-transfected RAECs, the shRNA-II/HIF-1α-transfected RAECs exhibited downregulated expression of HIF-1α, ET-1, ETA, and ETB (P < .01), and upregulated expression of ZO-1, claudin-5, and VE-cadherin (P < .05). Compared with the GV230-transfected RAECs, the GV230/HIF-1α-transfected RAECs exhibited upregulated expression of HIF-1α, ET-1, ETA, and ETB (P < .01), and downregulated expression of ZO-1, claudin-5, and VE-cadherin (P < .05). The GV230/HIF-1α-transfected RAECs exhibited degradation and translocation of VE-cadherin and claudin-5. In addition to degradation of VE-cadherin and claudin-5, HIF-1α mediated enhanced endothelial cell permeability through upregulation of ET-1, ETA, and ETB, and downregulation of ZO-1 and VE-cadherin in rats with burn injury.

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.

Efficacy of Treating Chronic Tibial Osteomyelitis With Bone Defect Using a Pedicled Perforator-Layered Flap and Fasciocutaneous Flap of the Posterior Tibial Artery: A Case Report.

INTRODUCTION: Tibial osteomyelitis is a common complication of bone tissue trauma. Obtaining good soft tissue coverage and effective infection management is key to the treatment of chronic osteomyelitis of the tibia accompanied with bone defect and bone exposure. The pedicled posterior tibial artery perforator layered fasciocutaneous flap can be used to repair soft tissue defects and can be used as a long-term, localized anti-infective. CASE REPORT: A 54-year-old male presented with an ulcer, purulent discharge at the left anterior tibia, and a fever 28 years after complete healing of the scar site. The patient received debridement and negative pressure wound therapy (NPWT) in a hospital setting. After presenting to the authors' department, there was difficulty in closing the exposed bone marrow cavity. On the basis of systemic use of intravenous antibiotics, multiple debridements and NPWT were used to effectively remove necrotic tissue and control infection. Afterward, the pedicled posterior tibial artery perforator layered fasciocutaneous flap was designed to fill the bone marrow cavity as well as cover and seal the wound of bone exposure and soft tissue defect simultaneously. The layered fasciocutaneous flap was well established after operation, and no recurrence of osteomyelitis was found. CONCLUSION: Debridement with negative pressure wound therapy can be an effective treatment for the wound bed preparation in advance of surgery, and the pedicled posterior tibial artery perforator layered fasciocutaneous flap can be used for the treatment of several soft tissue defects.

Carnosine exerts antitumor activity against bladder cancers in vitro and in vivo via suppression of angiogenesis.

Carnosine, a naturally occurring dipeptide, was recently reported to exhibit anticancer activity; however, the molecular mechanisms and regulators underlying its activity against tumor-associated angiogenesis remain unidentified. In this study, we evaluated the in vitro and in vivo antitumor effects of carnosine in EJ bladder cancer cells and EJ-xenografted BALB/c nude mice, respectively. In addition, in vitro capillary tube formation of HUVECs, ex vivo aortic ring and in vivo Matrigel plug assays were employed to examine the antiangiogenic potential of carnosine. Carnosine significantly inhibited EJ cell proliferation. Flow cytometric and immunoblot analyses indicated that carnosine modulated regulators of the G1 cell cycle phase, including cyclin D1, CDK4 and p21WAF1. The mitogen-activated protein kinases, ERK and p38, but not JNK or AKT, responded to carnosine. Carnosine inhibited the migratory and invasive potential of EJ cells by inhibiting MMP-9 activity, which was associated with suppression of binding activity of NF-κB, SP-1 and AP-1. In xenograft tumors, carnosine exhibited antitumor activity equivalent to cisplatin, but no weight loss occurred in carnosine-treated mice. In HUVECs, carnosine inhibited VEGF-mediated proliferation, colony tube formation, migration and invasion. The antiangiogenic activity of carnosine was partially due to the suppression of VEGFR-2-mediated ERK/AKT/eNOS signaling and MMP-2. Furthermore, using aortic ring and Matrigel plug assays, we confirmed the antiangiogenic activity of carnosine. Given that targeting tumor-associated angiogenesis is a proven effective therapeutic strategy, our results may provide valuable information for the development of preventive or therapeutic agents for bladder cancer patients.

Effects of hypoxia inducible factor-1α on expression levels of MLCK, p-MLC and ZO-1 of rat endothelial cells.

OBJECTIVE: To examine the aberrant expression of endothelial permeability associated proteins including MLCK, p-MLC and ZO-1 in presence of different levels of hypoxia-inducible factor 1 alpha (HIF-1α). METHODS: We established monolayer vascular endothelial cell model with the primary rat endothelial cells. Over-expressed or under-expressed HIF-1α cell lines were made by endothelial cells transfected with plasmid vector constructed with HIF-1α gene or HIF-1α-specific short hairpin RNA (shRNA). Levels of mRNA and protein of MLCK, p-MLC and ZO-1 were determined using Real-Time PCR and Western blot. All data were analyzed using by One-Way ANOVA method and LSD. RESULTS: We successfully cultured the rat endothelial primary cells for four days. The mRNA and protein levels of MLCK and p-MLC were significantly increased in the HIF-1α over-expression group than that in the blank control group and the empty plasmid GV230 group (P＜0.05). ZO-1 was significantly lower in the HIF-1α over-expression group than that in the blank control group and the GV230 group. On the contrary, the mRNA and protein levels of MLCK and p-MLC were significantly lower in the HIF-1α under-expression group than that in the blank control group and the shRNA-NC group (P＜0.05). ZO-1 was significantly higher in the HIF-1α low-expression group than that in the blank control group and the shRNA-NC group. CONCLUSION: HIF-1α positively regulates the expression of MLCK and p-MLC and negatively regulates the expression of ZO-1 in rat monolayer endothelial cells.

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.

[Study on the mechanism of hypertonic salt solution alleviates lung injury of rats at the early stage of severe scald].

OBJECTIVE: To explore the mechanism of hypertonic salt solution (HS) alleviates lung injury of rats at the early stage of severe scald. METHODS: Thirty-two female Sprague-Dawley (SD) rats were randomly assigned to sham group, lactated Ringer solution (LR) group, HS200 group (200 mmol/L HS group, 1 L 200 mmol/L HS contained 955 mL LR and 45 mL 10% NaCl) and HS400 group (400 mmol/L HS group, 1 L 400 mmol/L HS contained 846 mL LR and 154 mL 10% NaCl), with 8 rats in each group. A 30% total body surface area (TBSA) III degree scalded model was reproduced by scalded on the back with 98 centigrade boiling water for 12 seconds, whereas those in the sham group were exposed to 37 centigrade water without liquid resuscitation. Rats in the three drug intervention groups were resuscitated with LR, 200 mmol/L HS and 400 mmol/L HS by caudal vein according to the Parkland formula, respectively. All rats were sacrificed at 8 hours after scald injury to harvest abdominal aorta blood and lung tissues. Interleukins (IL-6, IL-10 and IL-17) in serum were determined by enzyme-linked immunosorbent assay (ELISA). Samples from the lung tissue were used to measure malondialdehyde (MDA) and superoxide dismutase (SOD) levels by ultraviolet spectrophotometer. Expressions of p38 mitogen-activated protein kinase (p38MAPK) and extracellular regulated protein kinase 1/2 (ERK1/2) in the lung were determined by Western Blot. The lung tissue was stained with hematoxylin and eosin (HE), and the pathological changes were observed with a light microscope. RESULTS: Compared with the sham group, the lung tissues in the LR group were damage obviously, which accompanied with more inflammatory cell infiltration, cell edema and pulmonary septum thickening, and the levels of IL-6, IL-10, IL-17 in serum and MDA content, the phosphorylation of p38MAPK and ERK1/2 in lung tissues were increased whereas the activity of SOD was decreased. Compared with the LR group, the lung injury was significantly alleviated, the levels of IL-6, IL-17 in serum and MDA content and the phosphorylation of p38MAPK and ERK1/2 were decreased, and the levels of IL-10 and SOD were increased in both HS groups with a dose-dependent manner. There were significant difference in above parameters between HS400 group and LR group [serum IL-6 (ng/L): 3.76±0.12 vs. 6.72±0.90, serum IL-10 (ng/L): 33.76±3.71 vs. 16.77±3.19, serum IL-17 (ng/L): 103.52±2.78 vs. 124.96±4.96, lung MDA (nmol/mg): 5.59±0.24 vs. 7.09±0.39, lung SOD (U/mg): 226.7±3.9 vs. 172.7±3.4, lung phosphorylation of p38MAPK (p-p38MAPK)/p38MAPK: 0.15±0.09 vs. 0.35±0.19, lung phosphorylation of ERK1/2 (p-ERK1/2)/ERK1/2: 0.27±0.01 vs. 0.70±0.01, all P < 0.01]. CONCLUSIONS: HS protected against lung injury induced by severe burns in rats with a dose-dependent manner, and it was better than LR, and its possible mechanism was related with reducing the expression of p38MAPK and ERK1/2 pathway in lung tissue, increasing the level of anti-inflammatory cytokines and decreasing the release of pro-inflammatory cytokines, thus inhibiting excessive inflammation and oxidative stress injury in lung.

Angiopoietin-like protein 4 potentiates DATS-induced inhibition of proliferation, migration, and invasion of bladder cancer EJ cells; involvement of G2/M-phase cell cycle arrest, signaling pathways, and transcription factors-mediated MMP-9 expression.

Background: Diallyl trisulfide (DATS), a bioactive sulfur compound in garlic, has been highlighted due to its strong anti-carcinogenic activity. Objective: The current study investigated the molecular mechanism of garlic-derived DATS in cancer cells. Additionally, we explored possible molecular markers to monitoring clinical responses to DATS-based chemotherapy. Design: EJ bladder carcinoma cells were treated with different concentration of DATS. Molecular changes including differentially expressed genes in EJ cells were examined using immunoblot, FACS cell cycle analysis, migration and invasion assays, electrophoresis mobility shift assay (EMSA), microarray, and bioinformatics analysis. Results: DATS inhibited EJ cell growth via G2/M-phase cell cycle arrest. ATM-CHK2-Cdc25c-p21WAF1-Cdc2 signaling cascade, MAPKs, and AKT were associated with the DATS-mediated growth inhibition of EJ cells. DATS-induced inhibition of migration and invasion was correlated with down-regulated MMP-9 via reduced activation of AP-1, Sp-1, and NF-κB. Through microarray gene expression analysis, ANGPTL4, PLCXD1, and MMP3 were identified as candidates of molecular targets of DATS. Introduction of each gene to EJ cells revealed that ANGPTL4 was associated with the DATS-induced inhibition of cell growth, migration, and invasion. Conclusions: ANGPTL4 regulates DATS-mediated inhibition of proliferation, migration, and invasion of EJ cells, and thus, has potential as a prognostic marker for bladder cancer patients.

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.

Inhibitory effects of fermented extract of Ophiopogon japonicas on thrombin-induced vascular smooth muscle cells.

Ophiopogon japonicus is known to have various pharmacological effects. The present study investigated the effects of an extract of fermented Ophiopogon japonicas (FEOJ) on thrombin­treated vascular smooth muscle cells (VSMCs). FEOJ treatment inhibited the proliferation of VSMCs treated with thrombin as indicated by an MTT assay. These inhibitory effects were associated with decreased phosphorylation of AKT, reduced expression of cyclin D1 and increased expression of p27KIP1 in thrombin­induced VSMCs. In addition, FEOJ treatment suppressed the thrombin­stimulated migration of VSMCs as demonstrated by a wound­healing migration assay. Furthermore, zymographic analyses demonstrated that treatment of FEOJ with VSMCs suppressed the thrombin­induced expression of matrix metalloproteinase (MMP)­2, which was attributed to the reduction of nuclear factor (NF)­κB binding activity. Collectively, these results demonstrated that FEOJ induced p27KIP1 expression, reduced cyclin D1 expression and AKT phosphorylation, and inhibited MMP­2 expression mediated by downregulation of NF­κB binding activity in thrombin­treated VSMCs, which led to growth inhibition and repression of migration. These results supported the use of FEOJ for the prevention of vascular diseases and provided novel insight into the underlying mechanism of action.