The therapeutic potential of RNA Polymerase I transcription inhibitor, CX-5461, in uterine leiomyosarcoma.

BACKGROUND: Uterine leiomyosarcoma is a rare aggressive smooth muscle cancer with poor survival rates. RNA Polymerase I (Pol I) activity is elevated in many cancers supporting tumour growth and prior studies in uterine leiomyosarcoma revealed enlarged nucleoli and upregulated Pol I activity-related genes. This study aimed to investigate the anti-tumour potential of CX-5461, a Pol I transcription inhibitor currently being evaluated in clinical trials for several cancers, against the human uterine leiomyosarcoma cell line, SK-UT-1. METHODS: SK-UT-1 was characterised using genome profiling and western blotting. The anti-tumour effects of CX-5461 were investigated using cell proliferation assays, expression analysis using qRT-PCR, and BrdU/PI based cell cycle analysis. RESULTS: Genetic analysis of SK-UT-1 revealed mutations in TP53, RB1, PTEN, APC and TSC1 & 2, all potentially associated with increased Pol I activity. Protein expression analysis showed dysregulated p53, RB1 and c-Myc. CX-5461 treatment resulted in an anti-proliferation response, G2 phase cell-cycle arrest and on-target activity demonstrated by reduced ribosomal DNA transcription. CONCLUSIONS: SK-UT-1 was confirmed as a representative model of uterine leiomyosarcoma and CX-5461 has significant potential as a novel adjuvant for this rare cancer.

IRE-1-Targeting Caged Prodrug with Endoplasmic Reticulum Stress-Inducing and XBP-1S-Inhibiting Activities for Cancer Therapy.

Activation of the IRE-1/XBP-1s pathway supports tumor progression. Here, we report a novel prodrug, TC-D-F07, in which a thiol-reactive dinitrobenzenesulfonyl (Dns) cage was installed onto the C8 hydroxyl of the covalent IRE-1 inhibitor D-F07. The electron-withdrawing Dns group in TC-D-F07 stabilizes the neighboring 1,3-dioxane acetal, allowing for stimulus-mediated control of its inhibitory activity. TC-D-F07 exhibits high sensitivity to intracellular thiols. Because tumor cells exhibit higher concentrations of glutathione and cysteine, treatment with TC-D-F07 results in more sustained levels of D-F07 in transformed versus normal cells. In addition, we show that a dinitrophenyl cysteine adduct resulting from cleavage of the Dns group induces endoplasmic reticulum (ER) stress, causing tumor cells to increase the expression of XBP-1s. The accumulated levels of D-F07 and its gradual decomposition into the active IRE-1 inhibitor eventually deprive tumor cells of XBP-1s, leading to more severe apoptosis than those treated with its uncaged analogue.

Biological effects of cyclosporin A on CD3-CD161+ and CD3+CD161+ lymphocytes.

Cyclosporin A (CSA) is a widely used drug to prevent the immune cell function. It is well known that CSA blocks transcription of cytokine genes in activated T cells. The connection between T cells and CSA has been well established. However, the effect of CSA on natural killer (NK) cells is not thoroughly understood. Therefore, in the present study, splenocytes and peripheral blood mononuclear cells (PBMCs) were treated with CSA in the presence of concanavalin A (Con A) or interleukin-2 (IL-2). CSA at higher concentrations induces apoptosis and inhibition of proliferation, while lower concentrations showed synergistically enhanced proliferation in splenocytes and PBMCs. Further, CSA favored the in vitro conversion of CD3+CD161+ cells. Splenocytes and PBMC were found to have synergistic proliferation with Con A, and PBMC exhibited significantly higher expression of NKp30, NKp44, and granzyme B along with enhanced cytotoxicity against K-562 cells in CSA-treated animals. Proliferation assay also showed that proliferation of CD161+ cells was higher in CSA-treated animals. Collectively, our results suggest that CSA differentially influences the population, function, and expression of the NK cell phenotype.

Access to Enantiopure α-Hydrazino Acids for N-Amino Peptide Synthesis.

Backbone N-methylation of α-peptides has been widely employed to enhance the bioavailability and bioactivity of parent sequences. Heteroatomic peptide amide substituents have received less attention due, in part, to the lack of practical synthetic strategies. Here, we report the synthesis of α-hydrazino acids derived from 19 out of the 20 canonical proteinogenic amino acids and demonstrate their use in the solid-phase synthesis of N-amino peptide derivatives.

Aronia melanocarpa (Black Chokeberry) Reduces Ethanol-Induced Gastric Damage via Regulation of HSP-70, NF-κB, and MCP-1 Signaling.

Aronia melanocarpa (Michx.) Ell. belongs to the Rosaceae family. The purpose of this study is to explore the gastroprotective effect of the Aronia melanocarpa hydro-alcoholic extract (AMHAE) against ethanol-induced gastric ulcer in a rat model. Different concentrations (50, 100, and 200 mg/kg) of AMHAE, or 30 mg/kg of omeprazole, significantly inhibited the gastric injury formation. The ethanol-induced ulcer group showed significant increases of malondialdehyde (MDA), myeloperoxidase (MPO), tumor necrosis factor (TNF)-α, nuclear factor-kappaB p65 (NF-κB p65), and monocyte chemoattractant protein (MCP)-1, and decreased activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-px), and interleukin (IL)-4. However, AMHAE (200 mg/kg) pretreatment significantly reversed the altered pathophysiological levels of these biomolecules to near normal stages. The gastroprotective activity of AMHAE was abolished by pretreatment with l-NAME, naloxone, capsazepine, and indomethacin, demonstrating the participation of nitric oxide (NO), opioids, TRPV (vanilloid receptor-related transient receptor potential), and prostaglandins in AMHAE-assisted gastroprotection against ethanol-induced gastric injuries. This gastroprotective effect of AMHAE might be due to the downregulation of TNF-α-based NF-κB, MCP-1 signaling and strong antioxidant properties.

Peptide N-Amination Supports ß-Sheet Conformations.

The conformational heterogeneity of backbone N-substituted peptides limits their ability to adopt stable secondary structures. Herein, we describe a practical synthesis of backbone aminated peptides that readily adopt ß-sheet folds. Data derived from model N-amino peptides suggest that extended conformations are stabilized through cooperative steric, electrostatic, and hydrogen-bonding interactions.

Apoptotic Cell Death Induced by ofLBP6A, Lipopolysaccharide Binding Protein Model Peptide, Derived from Paralichthy olivaceus on MKN-28 Cells.

The aim of this study was to evaluate the anti-cancer effects of lipopolysaccharide binding protein (LBP) analogs derived from the marine resource Paralichthy olivaceus on MKN-28 gastric cancer cells. Five LBP analogs were used: ofLBP1N, ofLBP2A, ofLBP4N, ofLBP5A, and ofLBP6A. ofLBP6A induced cell death of MKN-28 cells at a concentration of 40 µM. While the anti-proliferation effects ofLBP6A showed on MKN-28 cells at concentration of 40 µM, it did not affect non-cancerous HEK-293 cells at the same concentration. The mechanism study showed that ofLBP6A lead to the inhibition of cell proliferation by apoptosis along with morphological changes. The phosphorylation of Fas associated death domain (FADD) as well as the expressions of cleaved caspase-8, -7, and -3 were increased by ofLBP6A treatment. Increased the expression level of cleaved caspase-3 was confirmed by immunofluorescence staining. The expressions of Bid, Bax, and cytochrome C were also increased by the treatment. However, the expressions of cellular FLICE (FADD-like IL-1ß-converting enzyme)-inhibitory protein (FLIP), Bcl-XL, and Bcl-2 were decreased by ofLBP6A treatment. The results of this study were the first to demonstrate the apoptotic anti-cancer effects of ofLBP6A, derived from P. olivavaceus on gastric cancer cells.

Growth Performance, Relative Meat and Organ Weights, Cecal Microflora, and Blood Characteristics in Broiler Chickens Fed Diets Containing Different Nutrient Density with or without Essential Oils.

The present study was conducted to investigate whether dietary essential oils could affect growth performance, relative organ weights, cecal microflora, immune responses and blood profiles of broiler chickens fed on diets containing different nutrient densities. A total of eight hundred-forty 1-d-old male broiler chicks were randomly allotted into twenty-eight pens (7 pens per treatment, 30 chicks per pen). There were four experimental diets containing two different nutrient densities and supplemented with or without essential oils. Experimental period lasted for 35 days. No clear interaction between nutrient density and essential oils on any of growth performance-related parameters was observed. Live body weights were affected (p<0.05) by nutrient density at 21 days and by dietary essential oils at 35 days. Essential oils significantly (p<0.05) increased daily body weight gain and feed conversion ratio during the periods of 22 to 35 and 1 to 35 days, but failed to affect feed intake during the entire experimental period. Daily weight gain at 1 to 21 days and feed intake at 1 to 21 and 1 to 35 days were significantly impaired (p<0.05) by nutrient density. There were significant treatment interactions (p<0.05) on relative weights of bursa of Fabricius and abdominal fat contents. Finally, either essential oil or nutrient density did not influence the relative percentages of breast and leg meats, the population of cecal microflora, blood parameters and antibody titers against Newcastle disease and infectious bronchitis in broiler chickens. It was concluded that dietary essential oils, independent to nutrient density, failed to stimulate feed intake, but increased growth performance in broiler chickens.

Antimetastatic effects of gambogic acid are mediated via the actin cytoskeleton and NF-κB pathways in SK-HEP1 cells.

Hepatocellular carcinoma (HCC) is one of the most malignant and frequent cancers with a high metastatic potential. The prevention of HCC metastasis is a critical target for effective therapies in HCC. Gambogic acid (GA), a natural compound obtained from Garcinia hanburyi has reported anticancer activity in cell lines. However, the antimetastatic mechanisms of GA are unclear, particularly with respect to HCC. In this study, the influence of GA on migration and invasion of SK-HEP1 cells was evaluated. At concentrations above 0.6 µM, GA reduced cell proliferation in SK-HEP1 cells without affecting proliferation of noncancerous HEK-293 cells. GA also suppressed migration and invasion of SK-HEP1 cells. GA downregulated the expression of the integrin ß1/rho family GTPase signaling pathway, suppressed the actin rearrangement related to cell cytoskeleton and migration and decreased matrix metalloproteinases MMP-2, MMP-9, and NF-κB expression involved in cancer invasion. These results suggest that GA may be a potential lead in developing an antimetastatic therapeutic for the treatment of HCC.

Overexpression of TCL1 activates the endoplasmic reticulum stress response: a novel mechanism of leukemic progression in mice.

Chronic lymphocytic leukemia (CLL) represents 30% of adult leukemia. TCL1 is expressed in ~ 90% of human CLL. Transgenic expression of TCL1 in murine B cells (Eµ-TCL1) results in mouse CLL. Here we show for the first time that the previously unexplored endoplasmic reticulum (ER) stress response is aberrantly activated in Eµ-TCL1 mouse and human CLL. This includes activation of the IRE-1/XBP-1 pathway and the transcriptionally up-regulated expression of Derlin-1, Derlin-2, BiP, GRP94, and PDI. TCL1 associates with the XBP-1 transcription factor, and causes the dysregulated expression of the transcription factors, Pax5, IRF4, and Blimp-1, and of the activation-induced cytidine deaminase. In addition, TCL1-overexpressing CLL cells manufacture a distinctly different BCR, as we detected increased expression of membrane-bound IgM and altered N-linked glycosylation of Igα and Igß, which account for the hyperactive BCR in malignant CLL. To demonstrate that the ER stress-response pathway is a novel molecular target for the treatment of CLL, we blocked the IRE-1/XBP-1 pathway using a novel inhibitor, and observed apoptosis and significantly stalled growth of CLL cells in vitro and in mice. These studies reveal an important role of TCL1 in activating the ER stress response in support for malignant progression of CLL.

Imidazo[1,2-a]pyridine-based peptidomimetics as inhibitors of Akt.

We report the design, synthesis, and biological evaluation of imidazopyridine-based peptidomimetics based on the substrate consensus sequence of Akt, an AGC family serine/threonine kinase hyperactivated in over 50% of human tumors. Our ligand-based approach led to the identification of novel substrate mimetic inhibitors of Akt1 featuring an unnatural extended dipeptide surrogate. Compound 11 inhibits Akt isoforms in the sub-micromolar range and exhibits improved proteolytic stability relative to a parent pentapeptide.

PLASMA DYNAMICS OF NEUTROPHIL EXTRACELLULAR TRAPS AND CELL-FREE DNA IN SEPTIC AND NONSEPTIC VASOPLEGIC SHOCK: A PROSPECTIVE COMPARATIVE OBSERVATIONAL COHORT STUDY.

ABSTRACT: Background: The association between neutrophil extracellular traps (NETs) and the requirement for vasopressor and inotropic support in vasoplegic shock is unclear. This study aimed to investigate the dynamics of plasma levels of NETs and cell-free DNA (cfDNA) up to 48 h after the admission to the intensive care unit (ICU) for management of vasoplegic shock of infectious (SEPSIS) or noninfectious (following cardiac surgery, CARDIAC) origin. Methods: This is a prospective, observational study of NETs and cfDNA plasma levels at 0H (admission) and then at 12H, 24H, and 48H in SEPSIS and CARDIAC patients. The vasopressor inotropic score (VIS), the Sequential Organ Failure Assessment (SOFA) score, and time spent with invasive ventilation, in ICU and in hospital, were recorded. Associations between NETs/cfDNA and VIS and SOFA were analyzed by Spearman's correlation (rho), and between NETs/cfDNA and ventilation/ICU/hospitalization times by generalized linear regression. Results: Both NETs and cfDNA remained elevated over 48 h in SEPSIS (n = 46) and CARDIAC (n = 30) patients, with time-weighted average concentrations greatest in SEPSIS (NETs median difference 0.06 [0.02-0.11], P = 0.005; cfDNA median difference 0.48 [0.20-1.02], P < 0.001). The VIS correlated to NETs (rho = 0.3-0.60 in SEPSIS, P < 0.01, rho = 0.36-0.57 in CARDIAC, P ≤ 0.01) and cfDNA (rho = 0.40-0.56 in SEPSIS, P < 0.01, rho = 0.38-0.47 in CARDIAC, P < 0.05). NETs correlated with SOFA. Neither NETs nor cfDNA were independently associated with ventilator/ICU/hospitalization times. Conclusion: Plasma levels of NETs and cfDNA correlated with the dose of vasopressors and inotropes administered over 48 h in patients with vasoplegic shock from sepsis or following cardiac surgery. NETs levels also correlated with organ dysfunction. These findings suggest that similar mechanisms involving release of NETs are involved in the pathophysiology of vasoplegic shock irrespective of an infectious or noninfectious etiology.

Korean Red Ginseng Induced Cardioprotection against Myocardial Ischemia in Guinea Pig.

This study was designed to evaluate the protective effect of Korean red ginseng (KRG) against ischemia/reperfusion (I/R) injury in isolated guinea pig heart. KRG has been shown to possess various ginsenosides, which are the major components of Panax ginseng. These components are known naturally occurring compounds with beneficial effects and free radical scavenging activity. The heart was induced to ischemia for 60 min, followed by 120 min reperfusion. The hearts were randomly allocated into five groups (n=8 for each group): normal control (N/C), KRG control, I/R control, 250 mg/kg KRG group and 500 mg/kg KRG group. KRG significantly increased hemodynamics parameters such as aortic flow, coronary flow and cardiac output. Moreover, KRG significantly increased left ventricular systolic pressure (LVSP), the maximal rate of contraction (+dP/dtmax) and maximal rate of relaxation (-dP/dtmax). Also, treatment of KRG ameliorated electrocardiographic index such as the QRS, QT and RR intervals. Moreover, KRG significantly suppressed the lactate dehydrogenase, creatine kinase-MB fraction and cardiac troponin I and ameliorated the oxidative stress markers such as malondialdehyde and glutathione. KRG was standardized through ultra performance liquid chromatograph analysis for its major ginsenosides. Taken together, KRG has been shown to prevent cardiac injury by normalizing the biochemical and oxidative stress.

Korean Red Ginseng modulates immune function by upregulating CD4+CD8+ T cells and NK cell activities on porcine.

In the present study, we investigated whether treatment with KRG improve the parameters of immune activity such as the cytotoxicity, populations of CD4+ CD8+T cell, CD3-CD172-CD8+ NK cell and CD172+ monocyte as well as natural cytotoxicity receptors such as Nkp46, Nkp44, Nkp30. In results, KRG significantly increased these immune activities. These results indicate that KRG has distinct immune-enhancing effects by increasing the roles of T cells and NK cell in porcine.

Bone marrow stem cells incubated with ellipticine regenerate articular cartilage by attenuating inflammation and cartilage degradation in rabbit model.

BACKGROUND: Ellipticine (Ellip.) was recently reported to have beneficial effects on the differentiation of adipose-derived stem cells into mature chondrocyte-like cells. On the other hand, no practical results have been derived from the transplantation of bone marrow stem cells (BMSCs) in a rabbit osteoarthritis (OA) model. OBJECTIVES: This study examined whether autologous BMSCs incubated with ellipticine (Ellip.+BMSCs) could regenerate articular cartilage in rabbit OA, a model similar to degenerative arthritis in human beings. METHODS: A portion of rabbit articular cartilage was surgically removed, and Ellip.+BMSCs were transplanted into the lesion area. After two and four weeks of treatment, the serum levels of proinflammatory cytokines, i.e., tumor necrosis factor α (TNF-α) and prostaglandin E2 (PGE2), were analyzed, while macroscopic and micro-computed tomography (CT) evaluations were conducted to determine the intensity of cartilage degeneration. Furthermore, immuno-blotting was performed to evaluate the mitogen-activated protein kinases, PI3K/Akt, and nuclear factor-κB (NF-κB) signaling in rabbit OA models. Histological staining was used to confirm the change in the pattern of collagen and proteoglycan in the articular cartilage matrix. RESULTS: The transplantation of Ellip.+BMSCs elicited a chondroprotective effect by reducing the inflammatory factors (TNF-α, PGE2) in a time-dependent manner. Macroscopic observations, micro-CT, and histological staining revealed articular cartilage regeneration with the downregulation of matrix-metallo proteinases (MMPs), preventing articular cartilage degradation. Furthermore, histological observations confirmed a significant boost in the production of chondrocytes, collagen, and proteoglycan compared to the control group. Western blotting data revealed the downregulation of the p38, PI3K-Akt, and NF-κB inflammatory pathways to attenuate inflammation. CONCLUSIONS: The transplantation of Ellip.+BMSCs normalized the OA condition by boosting the recovery of degenerated articular cartilage and inhibiting the catabolic signaling pathway.

Targeting RNA Polymerase I Transcription Activity in Osteosarcoma: Pre-Clinical Molecular and Animal Treatment Studies.

The survival rate of patients with osteosarcoma (OS) has not improved over the last 30 years. Mutations in the genes TP53, RB1 and c-Myc frequently occur in OS and enhance RNA Polymerase I (Pol I) activity, thus supporting uncontrolled cancer cell proliferation. We therefore hypothesised that Pol I inhibition may be an effective therapeutic strategy for this aggressive cancer. The Pol I inhibitor CX-5461 has demonstrated therapeutic efficacy in different cancers in pre-clinical and phase I clinical trials; thus, the effects were determined on ten human OS cell lines. Following characterisation using genome profiling and Western blotting, RNA Pol I activity, cell proliferation and cell cycle progression were evaluated in vitro, and the growth of TP53 wild-type and mutant tumours was measured in a murine allograft model and in two human xenograft OS models. CX-5461 treatment resulted in reduced ribosomal DNA (rDNA) transcription and Growth 2 (G2)-phase cell cycle arrest in all OS cell lines. Additionally, tumour growth in all allograft and xenograft OS models was effectively suppressed without apparent toxicity. Our study demonstrates the efficacy of Pol I inhibition against OS with varying genetic alterations. This study provides pre-clinical evidence to support this novel therapeutic approach in OS.

The effect of electroacupuncture on insulin-like growth factor-I and oxidative stress in an animal model of renal failure-induced hypertension.

BACKGROUND/AIMS: The present study was performed to demonstrate the effect of electroacupuncture (EA) and acupuncture on renal failure (RF)-induced hypertension. METHODS: We stimulated the Zusanli (ST36) and Taixi (KI3) acupoints in a rat model of RF-induced hypertension. RESULTS: EA significantly reduced RF-induced hypertension (p < 0.05). In a histopathological study, increments in RF-induced glomerulosclerosis and tubulointerstitial fibrosis were attenuated by EA treatment (p < 0.05). The increase in albuminuria in the RF group was also reduced by EA treatment (p < 0.05). In blood analysis, the increment in RF-induced serum BUN and creatinine concentrations were decreased by EA (p < 0.05), and the decreases in RF-induced insulin-like growth factor-I (IGF-I) mRNA and protein levels were increased by EA in both the kidney and the serum (p < 0.05). The increases in RF-induced oxidative stress, e.g. inducible nitric oxide synthase, heme oxygenase and thiobarbituric acid-reactive substance expression, were significantly decreased in the RF-EA group (p < 0.01). CONCLUSION: These findings suggest that the anti-hypertensive mechanism of EA could be related to the effects of oxidative stress on IGF-I in RF-induced hypertension.

Ginsenoside-Rb1 prevents bone cartilage destruction through down-regulation of p-Akt, p-P38, and p-P65 signaling in rabbit.

BACKGROUND: Osteoarthritis (OA) is the most common joint complaint resulting in pain, disability, and loss of quality of life. On the other hand, ginsenoside-Rb1 is a plant product derived from ginseng that possesses immune-regulation and anti-inflammatory activities. However, it has been reported that different rout of administration but hydrogel-based Ginsenoside-Rb1 in an OA rabbit model has not been investigated. PURPOSE: The aim of this study was to investigate the potential effects of ginsenoside-Rb1 such as anti-arthritic activity in a rabbit knee OA model via NF- κB, PI3K/Akt, and P38/(MAPK) pathways. STUDY DESIGN: In the current study, rabbit osteoarthritis was induced by hollow trephine on the femur trochlea and the hydrogel-based Ginsenoside-Rb1 sheets were inserted on the rabbit knee to assess the anti-arthritis activity of ginsenoside-Rb1 which is sustained release. METHODS: After the hydrogel-based Rb1 sheet insert on the rabbit knee, macroscopic and micro CT was performed for investigation of chondroprotective effect. Matrix metalloproteinases (MMPs) and apoptotic expression were assessed through Immunohistochemistry and RT-PCR assay. In addition, the flow cytometry technique was used for the investigation of intracellular reactive oxygen species (ROS) production and histological changes were examined by HE, safranin O, and Masson trichrome staining method. Furthermore, the NF- κB, PI3K/Akt, and P38/(MAPK) pathways were investigated using Western blot analysis. RESULTS: Macroscopic and micro CT investigation of hydrogel-Rb1 treatment showed a dose-dependent chondroprotective effect. Immunohistochemistry and RT-PCR revealed that expression of matrix metalloproteinases (MMPs) and apoptotic markers TNF-α, caspase-3, and bax are down-regulated in a dose-dependent fashion following implantation of hydrogel-Rb. Higher levels of intracellular reactive oxygen species (ROS) were observed in the OA group. In histopathological investigation of hydrogel-Rb1 exhibited larger amounts of chondro cells, glycosaminoglycan's, and collagen compared to the defect group. Furthermore, the NF- κB, PI3K/Akt, and P38/(MAPK) pathways were downregulated by hydrogel-Rb1 while the disease model showed upstream. In the meantime, MMP expression level was considerably down-regulated. CONCLUSIONS: Our results indicate the protective effect of ginsenoside-Rb1 against OA pathogenesis through prevention of apoptosis with suppression of ROS production and activation of NF-κB signaling through downregulation of the MAPK and PI3K/Akt signaling pathways.

Involvement of insulin-like growth factor-I secretion and all-trans-retinoic acid-induced decrement in viability in MCF-7 cells.

BACKGROUND/AIM: Insulin-like growth factor-I (IGF-I) is associated with survival, apoptosis and proliferation in MCF-7 cells. All-trans-retinoic acid (RA) is used as a cancer therapeutic, but the use of RA in cancer therapy is limited by the unpredictable resistance of cancer cells to drug action. Furthermore, the relationship of IGF-I with RA-induced decrement in cell viability has yet to be elucidated. MATERIALS AND METHODS: MCF-7 cells were grown as confluent monolayers. To demonstrate the signaling pathways and roles of IGF-I, we suppressed endogenous target IGF-I with specific small interfering RNA treatment. Protein kinase C-δ and insulin receptor substrate 1 (IRS-1) protein levels were analyzed by Western blot. Radioimmunoassays were used to assess the concentration of endogenous IGF-I, and RT-PCR was used to assess IGF-I mRNA expression. Cell viability was analyzed with the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. RESULTS: We showed that RA treatment resulted in a dose- and time-dependent decrease in the secretion and synthesis of IGF-I. Subsequently, we found that suppression of IGF-I and IRS-1 protein decreased cell viability. In contrast, suppression of protein kinase C-δ and stimulation of IGF-I protected cells from RA-induced decrement in cell viability. The combination of RA treatment with IGF-I or IRS-1 small interfering RNA resulted in an additive decrease in cell viability. CONCLUSION: These results indicate that IGF-I plays an important role in the effect of RA and that suppression of IGF-I is implicated in the RA-induced inhibition of cell viability in MCF-7 cells.

IGF-1 Facilitates Cartilage Reconstruction by Regulating PI3K/AKT, MAPK, and NF-kB Signaling in Rabbit Osteoarthritis.

PURPOSE: The pathogenesis of osteoarthritis (OA) is characterized by joint degeneration. The pro-inflammatory cytokine interleukin (IL)-1ß plays a vital role in the pathogenesis of OA by stimulation of specific signaling pathways like NF-κB, PI3K/Akt, and MAPKs pathways. The catabolic role of growth factors in the OA may be inhibited cytokine-activated pathogen. The purpose of this study was to investigate the potential effects of insulin-like growth factor-1 (IGF-1) on IL-1ß-induced apoptosis in rabbit chondrocytes in vitro and in an in vivo rabbit knee OA model. METHODS: In the present study, the OA developed in chondrocyte with the treatment of IL-1ß and articular cartilage ruptures by removal of cartilage from the rabbit knee femoral condyle. After IGF-1 treatment, immunohistochemistry and qRT-PCR were identified OA expression with changes in MMPs (matrix metalloproteinases). The production of ROS (intracellular reactive oxygen species) in the OA was detected by flow cytometry. Further, the disease progression was microscopically investigated and pathophysiological changes were analyzed using histology. The NF-κB, PI3K/Akt and P38 (MAPK) specific pathways that are associated with disease progression were also checked using the Western blot technique. RESULTS: The expression of MMPs and various apoptotic markers are down-regulated following administration of IGF-1 in a dose-dependent fashion while significantly up-regulation of TIMP-1. The results showed that higher levels of ROS were observed upon treatment of chondrocytes and chondral OA with IL-1ß. Collectively, our results indicated that IGF-1 protected NF-κB pathway by suppression of PI3K/Akt and MAPKs specific pathways. Furthermore, the macroscopic and pathological investigation showed that it has a chondroprotective effect by the formation of hyaline cartilage. CONCLUSION: Our results indicate a protective effect of IGF-1 against OA pathogenesis by inhibition of NF-κB signaling via regulation of the MAPK and PI3K/Akt signaling pathways and prevention of apoptosis by suppression of ROS production.