We show here that H89 can also suppress IL-1b-mediated release of the NF-kB -dependent gene IL-6 from peritoneal macrophages ex vivo (Fig. S2A and Methods S1), suggesting a role for H89 on the inflammatory macrophage phenotype. OVA-treatment increased the number of mast cells in both asthma models. Interestingly, H89 treatment had no effect on lung mast cell numbers in the acute model where AHR and lung inflammation can develop in the absence of mast cells as shown from studies in mast cell-deficient animals [25]. By contrast, H89 significantly reduced lung mast cell numbers in the moderate asthma model which is highly dependent on the presence and activation of mast cells for airway inflammation and remodeling [24,25]. We show that treatment of bone marrow-derived cultured mast cells (BMCMCs) with H89 does not inhibit antigen- and IgEinduced mast cell degranulation and IL-6 production in vitro (Fig. S2B & S2C and Methods S1). Thus it is likely that the decrease in mast cell numbers observed in H89-treated mice in the moderate model reflects the lower levels of IgE in these animals and the subsequent reduced IgE-dependent mast cell activation rather than direct effects of H89 on mast cells. We finally show that although H89 is a potent anti-inflammatory drug when administered before each challenge, a single treatment with H89 before the last challenge has no effect on AHR or numbers of inflammatory cells in BAL fluids in the acute asthma model (Figure S3 and Methods S1). As a positive control, we included a group receiving a single administration of the clinically efficient glucocorticoid dexamethasone (DEX). Although single treatment with DEX was less efficient than treatment with DEX before each challenge, as we reported previously [27], it reduced AHR (although the difference did not reach significance) and slightly but significantly reduced numbers of eosinophils in BAL fluid (Figure S3). In conclusion, we here demonstrate that the AGC kinase inhibitor H89 inhibits airway inflammation and hyperresponsiveness in two murine models of asthma when administered before each challenge. Although particular care must be taken when attempting to extrapolate findings from animal models of a disease to their human counterparts, our results suggest that H89 or other AGC kinase inhibitors might be candidates for alternate treatment in glucocorticoid-resistant asthma patients.
Abstract
Rapamycin is an allosteric inhibitor of mammalian target of rapamycin, and inhibits tumor growth and angiogenesis. Recent studies suggested a possibility that rapamycin renormalizes aberrant tumor vasculature and improves tumor oxygenation. The longitudinal effects of rapamycin on angiogenesis and tumor oxygenation were evaluated in murine squamous cell carcinoma (SCCVII) by electron paramagnetic resonance imaging (EPRI) and magnetic resonance imaging (MRI) to identify an optimal time after rapamycin treatment for enhanced tumor radioresponse. Rapamycin treatment was initiated on SCCVII solid tumors 8 days after implantation (500?50 mm3) and measurements of tumor pO2 and blood volume were conducted from day 8 to 14 by EPRI/MRI. Microvessel density was evaluated over the same time period by immunohistochemical analysis. Tumor blood volume as measured by MRI significantly decreased 2 days after rapamycin treatment. Tumor pO2 levels modestly but significantly increased 2 days after rapamycin treatment; whereas, it decreased in non-treated control tumors. Furthermore, the fraction of hypoxic area (pixels with pO2,10 mm Hg) in the tumor region decreased 2 days after rapamycin treatments. Immunohistochemical analysis of tumor microvessel density and pericyte coverage revealed that microvessel density decreased 2 days after rapamycin treatment, but pericyte coverage did not change, similar to what was seen with anti-angiogenic agents such as sunitinib which cause vascular renormalization. Collectively, EPRI/MRI co-imaging can provide non-invasive evidence of rapamycin-induced vascular renormalization and resultant transient increase in tumor oxygenation. Improved oxygenation by rapamycin treatment provides a temporal window for anti-cancer therapies to realize enhanced response to radiotherapy.
Citation: Saito K, Matsumoto S, Yasui H, Devasahayam N, Subramanian S, et al. (2012) Longitudinal Imaging Studies of Tumor Microenvironment in Mice Treated with the mTOR Inhibitor Rapamycin. Editor: Kwan Man, The University of Hong Kong, Hong Kong Received June 19, 2012; Accepted October 9, 2012; Published November 20, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was supported by the Intramural Research Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
Introduction
Multiple genetic and epigenetic events are known to result in the dysregulation of several signaling pathways that have an impact on neoplastic disease progression, such as squamous cell carcinomas (SCC) [1,2]. One such pathway, the phosphatidylinositol 3-kinase (PI3K)-Akt pathway is frequently activated in many cancers, and controls cellular metabolism, growth, and proliferation [3?]. The mammalian target of rapamycin (mTOR) is an atypical serine/ threonine kinase, which acts downstream of PI3K/Akt and, therefore has become an attractive therapeutic target [7?0]. It follows that inhibitors of mTOR, such as rapamycin and its derivatives are currently being evaluated for molecular targeted therapy of neoplastic diseases [9]. The inhibition of mTOR with its specific allosteric inhibitor, rapamycin, provokes a rapid death of squamous xenografts, resulting in tumor regression [11]. The molecular basis of this is currently an active area of research [12]. For example, a recent study using a reverse-pharmacology approach, which involved the expression of a rapamycin-insensitive form of mTOR in squamous cancer cells, showed that cancer cells are the primary targets of rapamycin in vivo, and that mTOR controls the expression of hypoxia-inducible factor-1a (HIF-1a), a key transcription factor that orchestrates the cellular response to hypoxic stress, including the regulation of the expression of angiogenic factors, thus providing a likely mechanism by which rapamycin exerts its tumor suppressive and antiangiogenic effects [13]. Blocking mTOR pathway in SCC tumors was also shown to prevent accumulation of HIF-1a resulting in inhibition of processes involved in glucose metabolism as well as decrease in proangiogenic factors such as vascular endothelial growth factor (VEGF) [13]. Recent studies using magnetic resonance imaging (MRI) showed that treatment with mTOR inhibitors results in strong antiangiogenic and anti-vascular effects in solid tumors [12].
