Targeting Wnt/β‐catenin by anthelmintic drug niclosamide overcomes paclitaxel resistance in esophageal cancer

Esophageal cancer is an aggressive malignancy, and its current treatment strategies are plagued with high rates of recurrence. In this work, we demonstrate that niclosamide, an anthelmintic drug, is a potential sensitizing candidate for overcoming chemoresistance in esophageal cancer. Using a panel of esophageal cancer cell lines and normal cells, we show that niclosamide has anti‐esophageal cancer activity and is likely to be less effective against normal esophageal epithelial and fibroblast cells. The combination of niclosamide with paclitaxel results in much greater efficacy than paclitaxel alone, suggesting that niclosamide is active against esophageal cancer cells that are resistant to paclitaxel. This is further confirmed by our results that niclosamide is effective in inhibiting proliferation and inducing apoptosis in paclitaxel‐resistant esophageal cancer cells. In line with the findings obtained from in vitro cell culture system, niclosamide augments the in vivo efficacy of paclitaxel and significantly arrests paclitaxel‐resistant esophageal cancer growth without causing toxicity in mice. Mechanistically, we show that niclosamide decreases β‐catenin level and activity, and inhibits phosphorylation of STAT3 and mTORC1 substrate 70S6K. Stabilization of β‐catenin level by Wnt activator lithium chloride (LiCl) significantly abolishes the inhibitory effects of niclosamide in inhibiting proliferation and survival but not suppressing phosphorylation of STAT3 and 70S6K in paclitaxel‐resistant esophageal cancer cells, suggesting that niclosamide sensitizes esophageal cancer cell to paclitaxel mainly through inhibiting Wnt/β‐catenin. Our work demonstrates the efficacy of niclosamide and its underlying mechanism in paclitaxel‐resistant esophageal cancer. Our work emphasizes that Wnt/β‐catenin inhibition is a sensitizing strategy in esophageal cancer.     

Resveratrol counteracts IL‐1β‐mediated impairment of extracellular matrix deposition in 3D articular chondrocyte constructs

When aiming at cell‐based therapies in osteoarthritis (OA), proinflammatory conditions mediated by cytokines such as IL‐1β need to be considered. In recent studies, the phytoalexin resveratrol (RSV) has exhibited potent anti‐inflammatory properties. However, long‐term effects on 3D cartilaginous constructs under inflammatory conditions with regard to tissue quality, especially extracellular matrix (ECM) composition, have remained unexplored. Therefore, we employed long‐term model cultures for cell‐based therapies in an in vitro OA environment and evaluated effects of RSV. Pellet constructs made from expanded porcine articular chondrocytes were cultured with either IL‐1β (1–10 ng/ml) or RSV (50 μM) alone, or a cotreatment with both agents. Treatments were applied for 14 days, either directly after pellet formation or after a preculture period of 7 days. Culture with IL‐1β (10 ng/ml) decreased pellet size and DNA amount and severely compromised glycosaminoglycan (GAG) and collagen content. Cotreatment with RSV distinctly counteracted the proinflammatory catabolism and led to partial rescue of the ECM composition in both culture systems, with especially strong effects on GAG. Marked MMP13 expression was detected in IL‐1β‐treated pellets, but none upon RSV cotreatment. Expression of collagen type I was increased upon IL‐1β treatment and still observed when adding RSV, whereas collagen type X, indicating hypertrophy, was detected exclusively in pellets treated with RSV alone. In conclusion, RSV can counteract IL‐1β‐mediated degradation and distinctly improve cartilaginous ECM deposition in 3D long‐term inflammatory cultures. Nevertheless, potential hypertrophic effects should be taken into account when considering RSV as cotreatment for articular cartilage repair techniques.     

miR‐216a promotes breast cancer cell apoptosis by targeting PKCα

Breast cancer (BC) is the most common cause of death in women throughout the world. MicroRNAs (miRNAs, miR) have been identified as key regulators in carcinogenesis of several cancers, including BC. MicroRNA‐216a (miR‐216a) is downregulated in several cancers. Here, we evaluated the effects of miRNA‐216a on breast cancer cells and the underlying mechanisms. miR‐216a level was quantified by real‐time RT‐PCR. Cell viability was analyzed by MTT assay. Wound‐healing assay was performed for detection of cell migration. Apoptosis was detected by TUNEL and caspase‐3 activity assay. Moreover, the level of protein expression was determined by Western blot. We found that miR‐216a expression was remarkably decreased in both human BC tissues and MCF‐7 cells. miR‐216a overexpression dramatically suppressed the migration and promoted the apoptosis in cultured MCF‐7 cells. We validated PKCα (protein kinase C alpha, PRKCA) as a direct target of miR‐216a. Knockdown of PKCα induced apoptosis and inhibited migration in cultured MCF‐7 cells which were reversed by miR‐216a inhibitor. Moreover, the level of miR‐216a is negatively correlated with PKCα in cell lines. Our results collectively suggest that miR‐216a suppressed migration and promoted apoptosis in breast cancer cells by targeting PKCα. These findings indicate that manipulation of miR‐216a expression may represent a novel therapeutic strategy in the treatment of breast cancer.     

Granulocyte colony‐stimulating factor produces a decrease in IFNγ and increase in IL‐4 when administrated to healthy donors

Hematopoietic stem cells transplantation (HSCT) is the leading curative therapy for a variety of hematological and hereditary diseases; however, graft versus host disease (GVHD), an immunologic phenomenon that is favored by Th1 cytokines and cytotoxic cells from donors, is present frequently and is one of the most important causes of transplant related mortality. Peripheral blood HSCT is the preferred source of stem cells in almost 100% of the cases of autologous HSCT and in 70% of allogeneic transplants. The best mobilizing agent to get the stem cells out from the bone marrow is the Granulocyte‐Colony Stimulating Factor (G‐CSF). In this work, our main objective was to study a possible correlation between the graft cell dose and the patient's clinical outcome. We evaluated the immunologic changes produced by G‐CSF in the lymphocyte and cytokine profiles in allogeneic HSC donors. HSC from twelve donors were mobilized with G‐CSF at 16 μg/kg/day, for 5 days. Basal Peripheral Blood (BPB), Mobilized Peripheral Blood (MPB), and aphaeresis mononuclear cells (G‐MNC) samples were taken from all donors. Using flow cytometry, we quantified CD19⁺, CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺, NK, NKT, DC1, and DC2 cells. Cytokines were determined by ELISA in culture supernatants. CD19⁺ (p = 0.001), DC1 (p < 0.002) and DC2 (p < 0.001) cells were increased in MPB with respect to BPB. An increase in Th2 cytokines such as (IL‐4) and a decrease in Th1 cytokines (IFNγ, IL‐2) were also found in MPB samples. In conclusion, Th1 and Th2 cytokines are relevant in predicting the clinical outcome after allogeneic peripheral blood HSCT. J. Clin. Apheresis 25:181–187, 2010. © 2010 Wiley‐Liss, Inc.     

Electromagnetic fields counteract IL‐1β activity during chondrogenesis of bovine mesenchymal stem cells

Osteoarthritis (OA) is a common joint disease associated with articular cartilage degeneration. To improve the therapeutic options of OA, tissue engineering based on the use of mesenchymal stem cells (MSCs) has emerged. However, the presence of inflammatory cytokines, such as interleukin‐1β (IL‐1β), during chondrogenesis reduces the efficacy of cartilage engineering repair procedures by preventing chondrogenic differentiation. Previous studies have shown that electromagnetic fields (EMFs) stimulate anabolic processes in OA cartilage and limit IL‐1β catabolic effects. We investigated the role of EMFs during chondrogenic differentiation of MSCs, isolated from bovine synovial fluid, in the absence and presence of IL‐1β. Pellets of MSCs were differentiated for 3 and 5 weeks with transforming growth factor‐β3 (TGFβ3), in the absence and presence of IL‐1β and exposed or unexposed to EMFs. Biochemical, quantitative real‐time RT–PCR and histological results showed that EMFs alone or in the presence of TGFβ3 play a limited role in promoting chondrogenic differentiation. Notably, in the presence of IL‐1β and TGFβ3 a recovery of proteoglycan (PG) synthesis, PG content and aggrecan and type II collagen mRNA expression in the EMF‐exposed compared to unexposed pellets was observed. Also, histological and immunohistochemical results showed an increase in staining for alcian blue, type II collagen and aggrecan in EMF‐exposed pellets. In conclusion, this study shows a significant role of EMFs in counteracting the IL‐1β‐induced inhibition of chondrogenesis, suggesting EMFs as a therapeutic strategy for improving the clinical outcome of cartilage engineering repair procedures, based on the use of MSCs. Copyright © 2012 John Wiley & Sons, Ltd.     

The profibrotic cytokine transforming growth factor‐β1 increases endothelial progenitor cell angiogenic properties

Summary. Background: Transforming growth factor‐β1 (TGF‐β1) is a profibrotic cytokine that plays a major role in vascular biology, and is known to regulate the phenotype and activity of various vascular cell populations. Because most fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), are associated with vascular remodeling, and as endothelial progenitor cells (EPCs) may be involved in this process, we investigated the impact of TGF‐β1 modulation of EPC angiogenic properties. Methods: TGF‐β1 plasma levels were determined in 64 patients with IPF and compared with those in controls. The effect of TGF‐β1 on angiogenesis was studied in vivo in a Matrigel plug model and in vitro on endothelial colony‐forming cells (ECFCs). We studied the effects of inhibiting the expression of the three main receptors of TGF‐β1 in ECFCs by using short interfering RNA. Results: Total TGF‐β1 plasma levels were significantly increased in patients with IPF as compared with controls (P < 0.0001). TGF‐β1 had proangiogenic effects in vivo by increasing hemoglobin content and blood vessel formation in Matrigel plugs implanted in C57/Bl6 mice, and in vitro by enhancing ECFC viability and migration. The effects were abolished by silencing the three main TGF‐β1 receptors. Conclusions: TGF‐β1 is proangiogenic in vivo and induces ECFC angiogenic properties in vitro, suggesting that TGF‐β1 may play a role during vascular remodeling in fibrotic disease states via EPCs.     

Interleukin (IL)‐1β, IL‐1 receptor antagonist,IL‐6, IL‐8, IL‐10, and tumor necrosis factor α gene polymorphisms in patients with febrile seizures

Inflammation and genetics may play a role in the pathogenesis of febrile seizures (FSs). We aimed to test whether interleukin‐1β (IL‐1β), IL‐1 receptor antagonist (IL‐1 Ra), IL‐6 promoter, IL‐8, IL‐10, or tumor necrosis factor (TNF) gene polymorphisms could be used as markers of susceptibility to FSs. An association study was performed among a cohort of 104 patients with FSs and 143 normal control subjects. There was no significant difference between patients and controls in the distribution of allele frequencies of the IL‐1β promoter, IL‐1β exon 5, IL‐6 promoter, IL‐8, IL‐10, or TNF‐α gene polymorphisms. In contrast, the IL‐1 Ra‐I homozygote was more frequent in patients with FSs than in healthy controls (93.2% vs. 83.92%, χ²=4.51, P=0.034). In addition, individuals homozygous for the IL‐1 Ra‐I genotype were more than twice as likely to develop FSs than individuals heterozygous for the IL‐1 Ra‐I/II genotype (OR, 2.63, 95% CI: 1.08–6.39; χ²=4.55, P=0.033). We conclude that the IL‐1 Ra gene might be one of the useful markers for predicting susceptibility to FSs. J. Clin. Lab. Anal. 24:154–159, 2010. © 2010 Wiley‐Liss, Inc.     

Enhancement of fibrinolysis by inhibiting enzymatic cleavage of precursor α2‐antiplasmin

Summary. Background and objective: Resistance of thrombi to plasmin digestion depends primarily on the amount of α₂‐antiplasmin (α₂AP) incorporated within fibrin. Circulating prolyl‐specific serine proteinase, antiplasmin‐cleaving enzyme (APCE), a homologue of fibroblast activation protein (FAP), cleaves precursor Met‐α₂AP between ‐Pro12‐Asn13‐ to yield Asn‐α₂AP, which is crosslinked to fibrin approximately 13× more rapidly than Met‐α₂AP and confers resistance to plasmin. We reasoned that an APCE inhibitor might decrease conversion of Met‐α₂AP to Asn‐α₂AP and thereby enhance endogenous fibrinolysis. Methods and results: We designed and synthesized several APCE inhibitors and assessed each vs. plasma dipeptidyl peptidase IV (DPPIV) and prolyl oligopeptidase (POP), which have amino acid sequence similarity with APCE. Acetyl‐Arg‐(8‐amino‐3,6‐dioxaoctanoic acid)‐d ‐Ala‐l ‐boroPro selectively inhibited APCE vs. DPPIV, with an apparent K_i of 5.7 nm vs. 6.1 μm , indicating that an approximately 1000‐fold greater inhibitor concentration is required for DPPIV than for APCE. An apparent K_i of 7.4 nm was found for POP inhibition, which is similar to 5.7 nm for APCE; however, the potential problem of overlapping FAP/APCE and POP inhibition was negated by our finding that normal human plasma lacks POP activity. The inhibitor construct caused a dose‐dependent decrease of APCE‐mediated Met‐α₂AP cleavage, which ultimately shortened plasminogen activator‐induced plasma clot lysis times. Incubation of the inhibitor with human plasma for 22 h did not lessen its APCE inhibitory activity, with its IC₅₀ value in plasma remaining comparable to that in phosphate buffer. Conclusion: These data establish that inhibition of APCE might represent a therapeutic approach for enhancing thrombolytic activity.