[Show abstract][Hide abstract] ABSTRACT: The tumor microenvironment is composed of accessory cells and immune cells in addition to extracellular matrix (ECM) components. The stromal compartment interacts with cancer cells in a complex crosstalk to support tumor development. Growth factors and cytokines produced by stromal cells support the growth of tumor cells and promote interaction with the vasculature to enhance tumor progression and invasion. The activation of autocrine and paracrine oncogenic signaling pathways by growth factors, cytokines, and proteases derived from both tumor cells and the stromal compartment is thought to play a major role in assisting tumor cells during metastasis. Consequently, targeting tumor-stroma interactions by RNA interference (RNAi)-based approaches is a promising strategy in the search for novel treatment modalities in human cancer. Recent advances in packaging technology including the use of polymers, peptides, liposomes, and nanoparticles to deliver small interfering RNAs (siRNAs) into target cells may overcome limitations associated with potential RNAi-based therapeutics. Newly developed nonviral gene delivery approaches have shown improved anticancer efficacy suggesting that RNAi-based therapeutics provide novel opportunities to elicit significant gene silencing and induce regression of tumor growth. This chapter summarizes our current understanding of the tumor microenvironment and highlights some potential targets for therapeutic intervention with RNAi-based cancer therapeutics.
No preview · Article · Jan 2015 · Methods in Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Heart failure (HF) is a common and often fatal complication of myocardial infarction (MI). Glutathione S-transferase P1-1 (GSTP1) has anti-apoptotic, anti-inflammatory effects and is a specific serum marker in HF patients. However, its role in HF treatment is unknown.
GSTP1 effect was examined in rat MI-induced HF model. Magnetic resonance imaging examined cardiac function. GSTP1 and tumor necrosis factor-α receptor-associated factor 2 (TRAF2) mRNA and protein expression was elevated in failing myocardium; although GSTP-1 binding activity to TRAF2 was not changed versus controls. HF was associated with higher active JNK1 and p38 protein expression but reduced GSTP-1 binding activity to JNK1 and p38. Recombinant GSTP1 inhibited JNK1 and p38 and enhanced its own binding activity to TRAF2 and JNK1 in vitro. In the HF model, single dose GSTP1 treatment reduced infarct area, apoptosis and the expression of JNK1, p38, NFκB and pro-inflammatory cytokines and improved thinning ratio, cardiac index and output, stroke volume, ejection fraction, regional wall motion and survival compared to controls.
GSTP1 application early after MI results in long-term beneficial structural and functional effects that prevent progression to HF. GSTP1 could be a novel adjunct myocardial salvage approach in patients after MI.
No preview · Article · Jan 2014 · Journal of cardiac failure
[Show abstract][Hide abstract] ABSTRACT: Rho GTPases play important roles in cytoskeleton organization, cell cycle progression and are key regulators of tumor progression. Strategies to modulate increased Rho GTPase activities during cancer progression could have therapeutic potential.
We report here the characterization of a Cdc42-selective small-molecule inhibitor AZA197 for the treatment of colon cancer that was developed based on structural information known from previously developed compounds affecting Rho GTPase activation. We investigated the effects of AZA197 treatment on RhoA, Rac1 and Cdc42 activities and associated molecular mechanisms in colon cancer cells in vitro. Therapeutic effects of AZA197 were examined in vivo using xenograft mouse models of SW620 human colon cancer cells. After treatment, tumors were excised and processed for Ki-67 staining, TUNEL assays and Western blotting to evaluate proliferative and apoptotic effects induced by AZA197.
In SW620 and HT-29 human colon cancer cells, AZA197 demonstrated selectivity for Cdc42 without inhibition of Rac1 or RhoA GTPases from the same family. AZA197 suppressed colon cancer cell proliferation, cell migration and invasion and increased apoptosis associated with down-regulation of the PAK1 and ERK signaling pathways in vitro. Furthermore, systemic AZA197 treatment reduced tumor growth in vivo and significantly increased mouse survival in SW620 tumor xenografts. Ki-67 staining and tissue TUNEL assays showed that both inhibition of cell proliferation and induction of apoptosis associated with reduced PAK/ERK activation contributed to the AZA197-induced therapeutic effects in vivo.
These data indicate the therapeutic potential of the small-molecule inhibitor AZA197 based on targeting Cdc42 GTPase activity to modulate colorectal cancer growth.
Full-text · Article · Nov 2013 · Journal of Translational Medicine
[Show abstract][Hide abstract] ABSTRACT: Deregulated Rho GTPases Rac1 and Cdc42 have been discovered in various tumors, including prostate and Rac protein expression significantly increases in prostate cancer. The Rac and Cdc42 pathways promote the uncontrolled proliferation, invasion and metastatic properties of human cancer cells. We synthesized the novel compound AZA1 based on structural information of the known Rac1 inhibitor NSC23766. In the current study we investigated the effects of inhibition of these pathways by AZA1 on prostate tumorigenicity by performing preclinical studies using a xenograft mouse model of prostate cancer. In androgen-independent prostate cancer cells, AZA1 inhibited both Rac1 and Cdc42 but not RhoA GTPase activity in a dose-dependent manner and blocked cellular migration and proliferation. Cyclin D1 expression significantly decreased following Rac1/Cdc42 inhibition in prostate cancer cells. AZA1 treatment also down-regulated PAK and AKT activity in prostate cancer cells, associated with induction of the pro-apoptotic function of BAD by suppression of serine-112 phosphorylation. Daily systemic administration of AZA1 for 2 weeks reduced growth of human 22Rv1 prostate tumor xenografts in mice and improved the survival of tumor-bearing animals significantly. These data suggest a role of AZA1 in blocking Rac1/Cdc42-dependent cell cycle progression, cancer cell migration and increase of cancer cell apoptosis involving down-regulation of the AKT and PAK signaling pathway in prostate cancer cells. We therefore propose that a small-molecule inhibitor therapy targeting Rac1/Cdc42 Rho GTPase signaling pathways may be used as a novel treatment for patients with advanced prostate cancer.
[Show abstract][Hide abstract] ABSTRACT: Colony-stimulating factor-1 (CSF1) is a key regulator of mammary gland development, and a modulator of tissue macrophages. Expression of the CSF-1 receptor gene c-fms is strongly associated with poor outcome in breast cancer and results in tumor cell invasiveness and pro-metastatic behavior in vitro. However, CSF-1's role as a predictive factor in breast cancer remains unclear. We have prospectively measured circulating CSF-1 with ELISA in 572 women with early breast cancer and in 688 women with benign breast lesions, and correlated these concentrations with overall survival, nodal status and other clinical and histological parameters. Serum CSF-1 concentrations were significantly elevated in patients with early breast cancer when compared to those with benign tumors (p<0.0001). Within breast cancer patients, CSF-1 was higher in women with involved axillary lymph nodes (p=0.03). Serum CSF-1 correlated with tumor size (p=0.002), age (p<0.001), and Ki67 expression (p=0.006). Log CSF-1 serum concentrations were predictive of poor survival in both univariate (HR: 3.77, CI: 1.65-8.65, p=0.002) and multivariate analyses (HR: 3.1, CI: 1.03-9.33, p=0.04). Post- but not premenopausal women with CSF-1 serum concentrations >873 pg/ml experienced a significantly poorer outcome (p=0.004 log rank test). Serum CSF1 concentrations are elevated in women with malignant breast tumors. In early breast cancer, elevated serum CSF-1 is associated with nodal involvement, and in postmenopausal women also with poor overall survival.
No preview · Article · Sep 2013 · Endocrine Related Cancer
[Show abstract][Hide abstract] ABSTRACT: The growth and vascularization of prostate cancer is dependent on interactions between cancer cells and supporting stromal cells. The primary stromal cell type found in prostate tumors is the carcinoma-associated fibroblast, which produces placental growth factor (PlGF). PlGF is a member of the vascular endothelial growth factor (VEGF) family of angiogenic molecules and PlGF mRNA levels increase after androgen deprivation therapy in prostate cancer. In this study, we show that PlGF has a direct dose-dependent proliferative effect on human PC-3 prostate cancer cells in vitro and fibroblast-derived PlGF increases PC-3 proliferation in co-culture. In xenograft tumor models, intratumoral administration of murine PlGF siRNA reduced stromal-derived PlGF expression, reduced tumor burden and decreased the number of Ki-67 positive proliferating cells associated with reduced vascular density. These data show that targeting stromal PlGF expression may represent a therapeutic target for the treatment of prostate cancer.
Full-text · Article · Sep 2013 · International Journal of Molecular Sciences
[Show abstract][Hide abstract] ABSTRACT: Purpose: Shock waves were shown to induce angiogenesis in ischemic myocardium in rodent models. In the present experiments we aimed to address safety and efficacy of direct epicardial shock wave therapy in a preclinical large animal model and to further evaluate mechanism of action of this novel therapy.
Methods: Four weeks after left anterior descending (LAD) artery ligation in pigs, animals underwent re-thoracotomy with (SW group, n=6) or without (CTR, n=5) direct epicardial shock waves (300 impulses at 0.38mJ/mm2) applied to the infarcted anterior wall. Efficacy endpoints were improvement of LVEF and induction of angiogenesis 6 weeks after shock wave therapy. Safety endpoints were hemodynamic stability during treatment and myocardial damage.
A receptor tyrosine kinase profiler was performed in human coronary artery endothelial cells to proof receptor activation.
Results: Four weeks after LAD ligation, LVEF decreased in both shock wave (43±3%, p<0.001) and control (41±4%, p=0.012) group. LVEF markedly improved in shock wave animals 6 weeks after treatment (62±9%, p=0.006), no improvement was observed in controls (41±4%, p=0.36). Quantitative histology revealed significant angiogenesis six weeks after treatment as shown by number of arterioles (CTR 2±0.4 arterioles/high power field vs. SW 9±3, p=0.004), number of capillaries (CTR 9±4 vs. SWT 53±12, p<0.001) and number of vital cells (CTR 212±32 vs. SWT 310±48, p=0.003). No acute or chronic adverse effects were observed. In-vitro experiments revealed a two- and four-fold increase in VEGF receptor 1 and 2 phosphorylation in human coronary artery endothelial cells.
Conclusions: Direct epicardial shock wave treatment in a large animal model of ischemic heart failure exerted positive effect on LVEF improvement and did not show any adverse effects. Angiogenesis was induced by stimulation of VEGF receptors. It may serve in future as an adjunct to CABG surgery.
Preview · Article · Aug 2013 · European Heart Journal
[Show abstract][Hide abstract] ABSTRACT: Background:
Various techniques of orthotopic single lung transplantation in rats have been reported; however, their widespread use has been limited owing to the complexity of the procedure. We report a novel microsurgical lung transplantation model in rats with a high survival rate that can be performed by one surgeon alone.
A total of 90 left lung allografts were transplanted from Fischer to Wistar Kyoto rats. We developed a triple axis precision system to place and stabilize the vascular clips intrathoracically to clamp the bronchovascular structures, thereby avoiding interference with the heart and contralateral lung movement. A single-suture bronchial anastomosis technique and proximal cuffing approach for vascular anastomosis was used, rendering surgical assistance unnecessary.
In our recent series, both short-term (12 h) and long-term (21 d) survival was 100%. The lungs showed excellent perfusion and ventilation immediately on transplantation. Blood gas samples drawn from the left pulmonary vein and the histologic sections revealed excellent graft function. The donor operation lasted 20 ± 2 min, donor left lung dissection required 20 ± 2 min, and implantation required 90 ± 5 min.
The present innovative method of left orthotopic single lung transplantation can be performed by one experienced surgeon alone, with excellent results and a high degree of reproducibility.
Full-text · Article · May 2013 · Journal of Surgical Research
[Show abstract][Hide abstract] ABSTRACT: The early growth response transcription factor Egr-1 controls cell specific responses to proliferation, differentiation and apoptosis. Expression of Egr-1 and downstream transcription is closely controlled and cell specific upregulation induced by processes such as hypoxia and ischemia has been previously linked to multiple aspects of cardiovascular injury. In this study, we showed constitutive expression of Egr-1 in cultured human ventricular cardiac fibroblasts, used adenoviral mediated gene transfer to study the effects of continuous Egr-1 overexpression and studied downstream transcription by Western blotting, immunohistochemistry and siRNA transfection. Apoptosis was assessed by fluorescence microscopy and flow cytometry in the presence of caspase inhibitors. Overexpression of Egr-1 directly induced apoptosis associated with caspase activation in human cardiac fibroblast cultures in vitro assessed by fluorescence microscopy and flow cytometry. Apoptotic induction was associated with a caspase activation associated loss of mitochondrial membrane potential and transient downstream transcriptional up-regulation of the pro-apoptotic gene product Siva-1. Suppression of Siva-1 induction by siRNA partially reversed Egr-1 mediated loss of cell viability. These findings suggest a previously unknown role for Egr-1 and transcriptional regulation of Siva-1 in the control of cardiac accessory cell death.
Full-text · Article · Jan 2013 · International Journal of Molecular Sciences
[Show abstract][Hide abstract] ABSTRACT: Glutathione S-transferase P1 1 (GSTP1) belongs to the multigene isozyme family involved in cellular response to oxidative stress and apoptosis. Our initial retrospective proteomic analysis suggested that GSTP1 is associated with heart failure (HF). Although pro-B-type natriuretic peptide (proBNP) serves currently as a surrogate diagnostic and prognostic parameter in HF patients, its specificity remains uncertain. We hypothesized that GSTP1 might be a useful serum marker in the monitoring of HF patients.
Serum GSTP1 and proBNP were prospectively measured in 193 patients subdivided based on their ejection fraction (EF) either in equal-sized quintiles or predefined EF groups >52%, 43%-52%, 33%-42%, 23%-32% and ≤22%. At a cutoff of ≥231 ng/mL, GSTP1 identified HF patients with EF ≤22% with 81% sensitivity and 83% specificity, and at a cutoff of ≥655 pg/mL, proBNP identified the same patient group with 84% sensitivity and 22% specificity. GSTP1 at a ≥126 ng/mL cutoff identified EF ≤42% with 90% sensitivity and 95% specificity, or proBNP at a ≥396 pg/mL cutoff had 97% sensitivity and 20% specificity. In regression analyses, GSTP1, but not proBNP, discriminated between EF ≤42% and EF >42% in HF patients.
These results suggest that GSTP1 is strongly associated with HF and could serve as a sensitive and specific marker to predict the ventricular function in HF patients.
Full-text · Article · Mar 2012 · Journal of cardiac failure
[Show abstract][Hide abstract] ABSTRACT: Proteomic analysis of myocardial tissue from patient population is suited to yield insights into cellular and molecular mechanisms taking place in cardiovascular diseases. However, it has been limited by small sized biopsies and complicated by high variances between patients. Therefore, there is a high demand for suitable model systems with the capability to simulate ischemic and cardiotoxic effects in vitro, under defined conditions. In this context, we established an in vitro ischemia/reperfusion cardiac disease model based on the contractile HL-1 cell line. To identify pathways involved in the cellular alterations induced by ischemia and thereby defining disease-specific biomarkers and potential target structures for new drug candidates we used fluorescence 2D-difference gel electrophoresis. By comparing spot density changes in ischemic and reperfusion samples we detected several protein spots that were differentially abundant. Using MALDI-TOF/TOF-MS and ESI-MS the proteins were identified and subsequently grouped by functionality. Most prominent were changes in apoptosis signalling, cell structure and energy-metabolism. Alterations were confirmed by analysis of human biopsies from patients with ischemic cardiomyopathy.
With the establishment of our in vitro disease model for ischemia injury target identification via proteomic research becomes independent from rare human material and will create new possibilities in cardiac research.
[Show abstract][Hide abstract] ABSTRACT: Cardiac allograft vasculopathy (CAV) has an incidence of 43% at 8 years after heart transplantation with extremely limited treatment options and unclear pathogenesis. CAV constitutes a significant complication that limits the long-term survival of heart recipients. Insulin-like growth factor-1 (IGF-1) is associated with different cardiovascular diseases; however, its role in CAV pathogenesis remains unknown.
Serum samples of 10 matched recipients with CAV and 10 with no-CAV were initially screened with a protein array. Subsequently, IGF-1- and IGF-binding protein-3 (IGFBP-3) were analyzed using enzyme-linked immunosorbent assay in 44 randomly selected CAV and 50 no-CAV patients at two time points.
The initial screening showed that IGF-1 and IGFBP-3 are differentially expressed in CAV compared with no-CAV patients (P=0.037 and P<0.0001, respectively). Subsequent enzyme-linked immunosorbent assay analyses indicated that serum IGF-1 protein concentrations were significantly lower in CAV patients (159.7±114 ng/mL) as compared with no-CAV patients (234.1±136 ng/mL; P=0.02). Serum IGFBP-3 protein concentrations were significantly lower in CAV (0.46±0.37 mg/L) as compared with no-CAV patients (1.03±0.73 mg/L; P=0.04). Multivariate logistic regression analyses showed that IGF-1 (odds ratio, 0.89; P=0.04) and IGFBP-3 (odds ratio, 0.09; P=0.03) are independent risk factors for CAV.
Low IGF-1 and IGFPB-3 serum concentrations are associated with CAV. The assessment of serum IGF-1 and IGFPB-3 might be beneficial in identifying cardiac allograft recipients who are prone to develop CAV. Moreover, IGF-1 might be a useful therapy that could protect cardiac allografts against CAV.
[Show abstract][Hide abstract] ABSTRACT: The pathogenesis of cardiac allograft vasculopathy after heart transplant remains controversial. Histologically, cardiac allograft vasculopathy is characterized by intimal hyperplasia of the coronary arteries induced by infiltrating cells. The origin of these infiltrating cells in cardiac allograft vasculopathy is unclear. Endothelial progenitor cells are reportedly involved in cardiac allograft vasculopathy; however, the role of CD14(+) monocyte-derived progenitor cells in cardiac allograft vasculopathy pathogenesis remains unknown.
Monocyte-derived progenitor cells were isolated from blood mononuclear cell fractions obtained from 25 patients with cardiac allograft vasculopathy and 25 patients without cardiac allograft vasculopathy.
Both patients with cardiac allograft vasculopathy and those without cardiac allograft vasculopathy had CD45(+), CD34(+), CD14(+), CD141(-), CD31(-) monocyte-derived progenitor cells that differentiated into mesenchymal lineages. Monocyte-derived progenitor cells formed significantly higher numbers of colonies in patients with cardiac allograft vasculopathy than in those without cardiac allograft vasculopathy; this correlated with posttransplant follow-up time. Importantly, monocyte-derived progenitor cells from patients with cardiac allograft vasculopathy expressed significantly more α smooth muscle actin and proliferated at a higher rate than did monocyte-derived progenitor cells of patients without cardiac allograft vasculopathy. In vitro experiments suggested a paracrine control mechanism in proliferation of monocyte-derived progenitor cells in cardiac allograft vasculopathy.
These results indicate that monocyte-derived progenitor cells are associated with cardiac allograft vasculopathy, have the ability to transdifferentiate into smooth muscle cells, and thus may contribute to intimal hyperplasia of coronary arteries in cardiac allograft vasculopathy. Targeting monocyte-derived progenitor cell recruitment could be beneficial in cardiac allograft vasculopathy treatment.
Full-text · Article · Nov 2011 · The Journal of thoracic and cardiovascular surgery