Ulcerative colitis is an autoimmune-inflammatory disease characterized by increased proliferation of colonic epithelial cells, dysregulation of signal transduction pathways, elevated mucosal T cell activation, increased production of proinflammatory cytokines, and enhanced leukocyte infiltration into colonic interstitium. Several compounds that possess antiproliferative properties and/or inhibit cytokine production exhibit a therapeutic effect in murine models of colitis. Mammalian target of rapamycin (mTOR), a protein kinase regulating cell proliferation, is implicated in colon carcinogenesis. In this study, we report that a novel haloacyl aminopyridine-based molecule (P2281) is a mTOR inhibitor and is efficacious in a murine model of human colitis. In vitro studies using Western blot analysis and cell-based ELISA assays showed that P2281 inhibits mTOR activity in colon cancer cells. In vitro and in vivo assays of proinflammatory cytokine production revealed that P2281 diminishes induced IFN-γ production but not TNF-α production, indicating preferential inhibitory effects of P2281 on T cell function. In the dextran sulfate sodium (DSS) model of colitis, 1) macroscopic colon observations demonstrated that P2281 significantly inhibited DSS-induced weight loss, improved rectal bleeding index, decreased disease activity index, and reversed DSS-induced shortening of the colon; 2) histological analyses of colonic tissues revealed that P2281 distinctly attenuated DSS-induced edema, prominently diminished the leukocyte infiltration in the colonic mucosa, and resulted in protection against DSS-induced crypt damage; and 3) Western blot analysis showed that P2281 blocks DSS-induced activation of mTOR. Collectively, these results provide direct evidence that P2281, a novel mTOR inhibitor, suppresses DSS-induced colitis by inhibiting T cell function and is a potential therapeutic for colitis. Given that compounds with anticancer activity show promising anti-inflammatory efficacy, our findings reinforce the cross-therapeutic functionality of potential drugs.
- T cells
- mammalian target of rapamycin
- dextran sulfate sodium
ulcerative colitis (UC)1 is an autoimmune-inflammatory disease affecting millions of people worldwide. Neither the initiating event nor the sequence of propagating events that lead to and sustain colitis have been fully elucidated. Nevertheless, it is increasingly clear that a dysfunctional immune response, involving Toll-like receptor-4 (17) and components of normal gastrointestinal enteric bacteria (24, 49), plays a key role in the pathogenesis of colitis. Thus an early step is macrophage antigen presentation to activated mucosal T cells (53) that leads to interferon (IFN) production and release (6). Bacterial components (e.g., LPS) and IFNs trigger signal transduction cascades [e.g., NF-κB pathway; mammalian target of rapamycin (mTOR)-signal transducer and activator 1 (STAT1) pathway] (4, 13) leading to increased proliferation of colonic epithelial cells (56) and elevated expression of proinflammatory genes (e.g., cytokines such as TNF-α) (8). Proinflammatory cytokines stimulate leukocytes and endothelium leading to aberrant leukocyte recruitment and enhanced infiltration into damaged colonic interstitium (29, 38).
The above observations have led to therapeutic approaches that seek to diminish colitis (and related diseases) by attenuating the immune-inflammatory response. Indeed, in various experimental models of acute and/or chronic colitis, 1) suppression of T cell function (e.g., by cyclosporin A) (37), 2) blockade of signal transduction pathways (e.g., mTOR pathway, NF-κB pathway) (15, 34, 52), 3) inhibition of proinflammatory cytokine expression (3, 46), and 4) attenuation of leukocyte-endothelial interactions (1) separately provide a beneficial effect.
mTOR is a serine-threonine protein kinase that regulates protein synthesis, cell growth, and cell proliferation in response to growth factors and nutrients (18, 40). It is well established that mTOR plays a crucial role in tumorigenesis (40). More recently, accumulating evidence causally links increased mTOR activity to heightened inflammatory responses. Indeed, LPS stimulation of macrophages leads to the phosphorylation and activation of p70S6K1 as well as that of 4EBP1/PHAS-1 (13); both proteins are bona fide targets of mTOR. Moreover, the mTOR pathway regulates the production of nitric oxide (58) and activates STAT1-dependent transcription in macrophages in response to LPS (27). Interestingly, a recent study showed that rapamycin, a mTOR inhibitor, blunts leukocyte adhesion and extravasation in the gut mucosa, leading to suppression of experimental chronic colitis (15). In a complementary study, treatment with everolimus (another mTOR inhibitor) reduced the number of T cells in lamina propria and blocked lymphocytic IFN-γ release, thereby ameliorating established murine colitis (34). These findings suggest that mTOR inhibitors may be useful for treatment of UC.
The pyridine scaffold is a very common structural motif that can be found in many natural products and in several pharmacologically interesting compounds (20, 22, 30). Therefore, the synthesis of pyridine derivatives, with the objective of developing new drugs, is an active area of research. Indeed, 1) it has been claimed that 2-cyanopyridylurea derivative can treat hyperproliferative and angiogenesis disorders (50), 2) 3-cyano-2,6-dihydropyridine inhibits dihydrouracil dehydrogenase and its coadministration with 1-ethoxymethyl-5-fluorouracil enhances the antitumor effect (54), and 3) pyridothienopyrimidines exhibit cytotoxic activity (41). In our general search for novel anticancer agents, we found that 2-chloro-N-(6-cyanopyridin-3-yl)propanamide (P2281; Fig. 1) markedly inhibits mTOR activity in colon cancer cells. Given these observations, and the fact that mTOR inhibitors may be useful for treatment of UC, we probed the use of P2281 as a therapeutic for colitis. For this, we used the dextran sulfate sodium (DSS)-induced murine model of acute colitis, a model that is well recognized and known to mimic the pathological features of human colitis (28, 57). This model is characterized by dysregulated inflammatory response indicated by presence of edema, infiltration of inflammatory cells, and extensive mucosal damage (28, 57). We established this model in our center and used it to assess the efficacy of P2281 on the gross pathology of colitis.
MATERIALS AND METHODS
Synthesis of P2281.
To a stirring suspension of 5-amino-2-cyano pyridine (2.0 g, 16.8 mmol) in chloroform (50 ml), triethylamine (2.54 g, 25.2 mmol), and 2-chloropropanoyl chloride (2.35 g, 18.48 mmol) were added dropwise at 0°C. After complete addition of chloroacetyl chloride, the reaction mixture was allowed to come to room temperature and stirred overnight. The reaction mixture was diluted with chloroform (100 ml) and washed with water (2 × 50 ml). Subsequently, the organic layer was dried over sulfate sodium and concentrated in vacuo, and the resulting product was crystallized from chloroform-petroleum ether (1:2) to obtain the desired compound (Fig. 1) in 95% yield (3.3 g). 1H-NMR (DMSO-d6 300 MHz), δ: 10.99 (singlet, 1H), 8.86–8.87 (doublet, 1H, J = 2.7 Hz), 8.25–8.29 (doublet of doublets, 1H, J = 2.7 and 8.7), 7.98–8.01 (doublet, 1H, J = 8.7 Hz), 4.66–4.73 (multiplet, 1H), 1.60–1.62 (doublet, 3H), mass spectrometry mass-to-charge ratio 210 (M+1) calculated for C9H8N3ClO 209.05. High-resolution mass spectroscopy HPLC 99.42% (acetonitrile-ammonium acetate-triethylamine pH 5.0). The discovery and structure-activity-relationship leading to P2281 has also been investigated (S. Kumar, unpublished observations).
H460 human non-small cell lung cancer cells and HCT-116 human colon carcinoma cells were purchased from ATCC (Manassas, VA). Both the cell lines were cultured in RPMI 1640 (GIBCO-BRL; Paisley, UK) supplemented with 10% heat-inactivated fetal calf serum (FCS; JRH Australia), 100 units/ml penicillin (Sigma Aldrich; St. Louis, MO), and 100 μg/ml streptomycin (Sigma Aldrich).
Assays for analyzing mTOR activity.
Cell-based ELISA (11) detecting the phosphorylation of p70S6 kinase at Thr 389, which is a bona fide target for mTOR kinase (18), was used to characterize the mTOR activity. Briefly, H460 and/or HCT-116 cells were plated on 96-well tissue culture plates and allowed to adhere for 24 h. Subsequently, the cells were serum starved for 18–24 h. The cells were then pretreated with P2281 or 0.5% DMSO (carrier control for P2281) for 1 h, following which the cells were stimulated with 20% FCS to induce the signaling cascade via mTOR. After 30-min incubation at 37°C, the cells were washed, fixed in 3.7% formaldehyde at ambient temperature for 15 min, washed with PBS plus 0.1% Triton-X (PBS-Triton), and incubated in PBS-Triton containing 10% FCS. Unless otherwise noted, from this point on all antibody dilutions and washes were carried out in PBS-Triton. Rabbit polyclonal antibody to phospho-p70S6K1-Thr389 (Cell Signaling; Danvers, MA) was added (diluted 1:500), and the cells were incubated at ambient temperature for 1 h. After the incubation, the wells were washed and a peroxidase-conjugated polyclonal (secondary) antibody to rabbit IgG (Santa Cruz Biotechnology; Santa Cruz, CA) was added (diluted 1:500). Following 1 h incubation at room temperature, the wells were washed and treated with o-phenylene diamine dihydrochloride (Sigma Aldrich). After a 5-min incubation at room temperature, the reaction was stopped by use of H2SO4. The absorbance of the fluid in each well was determined at 490 nm by use of a microwell plate spectrophotometer (Molecular Devices, Sunnyvale, CA). In each experiment, rapamycin (0.2 μM; Sigma Aldrich) was used as a positive control for mTOR inhibition. In every experiment, each condition was run in triplicate wells.
Western blot analysis (45) was used to confirm the mTOR activity. H460 and/or HCT-116 cells were serum starved for 24 h. Subsequently, the cells were pretreated with P2281 or DMSO for 1 h, following which the cells were stimulated with 20% FCS. After 30-min incubation at 37°C, the cells were washed extensively and lysed with mammalian cell lytic reagent (Sigma Aldrich) supplemented with protease inhibitor cocktail (Sigma Aldrich). In experimental colitis (described below), colon samples from various groups of mice were homogenized and lysed with mammalian cell lytic reagent supplemented with protease inhibitor cocktail. Lysates were used immediately or stored at −20°C for later use. The protein levels in lysates were quantified by using Bradford reagent (Bio-Rad Laboratories; Hercules, CA). For SDS-PAGE and Western blotting, equal amounts of lysates (40 μg) were diluted in reducing sample buffer, boiled, and then separated on 10% Tris·HCl SDS-PAGE gels. Subsequently, the resolved proteins were transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore) and membrane blocked with 5% nonfat milk. Membranes were washed and incubated with primary antibodies (anti-phospho-p70S6K1 and anti-phospho-4E-BP1; Cell Signaling) overnight at 4°C. After extensive washing with PBS plus 0.1% Tween 20, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology). Following 1-h incubation, membranes were washed and proteins of interest detected by using the chemiluminescent substrate (SuperSignal West Femto Substrate; Pierce Biotechnology, Rockford, IL). The images were captured by a Kodak FX documentation system. In each in vitro experiment, rapamycin (1 μM) was used as a positive control for mTOR inhibition. To quantify the relative differences in phosphorylation, densitometry analysis was performed using the Quantity One software (Bio-Rad Laboratories) with high-resolution “tiff” images. The optical density of each band was determined with the area and the pixel number kept constant. Following background subtraction and normalization to the β-actin band, the percent phosphorylation of the relevant protein (p4E-BP1 or p70S6K1) was calculated.
Proinflammatory cytokine production assay using hPBMCs.
Blood was collected from normal healthy volunteers after informed consent. Human peripheral blood mononuclear cells (hPBMCs) were harvested by Ficoll-Hypaque density gradient centrifugation (1.077 g/ml; Sigma Aldrich) (10). hPBMCs were resuspended in RPMI 1640 culture medium (GIBCO-BRL) containing 10% FCS, 100 U/ml penicillin (Sigma Chemical, St. Louis, MO), and 100 μg/ml streptomycin (Sigma Chemical) at 1 × 106 cells/ml. 1 × 105 hPBMCs/well were pretreated with P2281 or 0.5% DMSO (carrier control for P2281) for 30 min at 37°C. Subsequently, these cells were stimulated with 1 μg/ml LPS (Escherichia coli 0111:B4, Sigma Chemical) or concanavalin A (Sigma Chemical). Following 5-h (for LPS experiments) or 18-h (for concanavalin A experiments) incubation at 37°C, supernatants were collected and stored at −70°C until assayed for human TNF-α, IL-1, IL-6, IL-8, or IFN-γ by ELISA as described by the manufacturer (OptiEIA ELISA sets, BD BioSciences). In every experiment, positive controls were used for inhibiting induced proinflammatory cytokine production: rolipram (300 μM; Sigma Aldrich) for TNF-α and cyclosporine (1 μM; Sigma Aldrich) for IFN-γ. In every experiment, each condition was run in triplicate wells.
In vivo proinflammatory cytokine production assay.
BALB/c mice (6 wk of age, weighing 18–22 g) were obtained from Jackson Laboratories (Bar Harbor, ME) and housed in individually ventilated cages. All animal experiments were carried out in accordance with the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals and “Guide for Care and Use of Laboratory Animals” (National Institutes of Health Publication No. 85-23, revised 1985). All animal experiments were approved by Institutional Animal Ethics Committee of Piramal Life Sciences Limited. In preliminary experiments, we ascertained the pharmacokinetic profile of P2281 administered intraperitoneally (ip) to mice. These studies revealed that a dose of 100 mg/kg of P2281 results in a maximal concentration (Cmax) of 180 μM in the plasma of mice (data not shown). A rough extrapolation of these findings suggests that a dose of 15 mg/kg ip P2281 would result in a Cmax of ∼30 μM (concentration at which mTOR inhibition is observed in HCT116 colon cancer cells; see results). Accordingly, the effect of P2281 on experimental colitis was studied at a dose of 15 mg/kg (ip) (described below). Furthermore, given that P2281 did not inhibit induced TNF-α production in vitro (essentially negative data), we investigated the effect of P2281 on induced TNF-α production in vivo at 30 mg/kg (i.e., 2× the dose used in efficacy studies) to rule out any dose-related issues. P2281, 30 mg/kg suspended in 0.5% carboxymethylcellulose (CMC; Sigma Aldrich containing Tween 80), was administered ip to mice. One hour later, LPS (E. coli serotype 0127:B8; 1 mg/kg; Sigma Aldrich) dissolved in sterile pyrogen-free normal saline was administered ip. The negative control group received normal saline as an ip injection, whereas all other groups received LPS. Rolipram (30 mg/kg po) was used as a positive control for inhibiting induced TNF-α production. After 1.5 h, blood was collected from the abdominal artery by using a 1-ml syringe flushed with heparinized saline (50 IU/ml). Plasma was separated by centrifugation at 2,000 g at room temperature, aliquoted, and stored at −70°C until assayed for mouse TNF-α levels by ELISA per manufacturer's instructions (OptiEIA ELISA sets, BD BioSciences). In each experiment, every group consisted of six mice.
Induction of colitis and P2281 treatment.
C57BL/6J mice (6 wk of age, weighing 18–22 g) were obtained from Jackson Laboratories and housed in individually ventilated cages. Colitis was induced in mice by giving 3% (wt/vol) DSS (molecular mass 30–40 kDa; ICN Biomedicals, Aurora, OH) in drinking water ad libitum as reported by others (26, 35, 48). Importantly, 30- to 40-kDa DSS was utilized in this study because it is known to induce more severe colitis than 5-kDa DSS and 500-kDa DSS (25). For each mouse, weight and rectal bleeding were determined every day following the introduction of DSS. DSS-induced colitis was assessed by macroscopic and histological analyses of the colon (described below). To probe the efficacy of P2281, a group of six mice were given daily ip injections of 15 mg/kg P2281 suspension in CMC. On the day of euthanasia, various parameters indicative of clinical disease were graded as mentioned in Table 1. All these parameters were lumped to obtain an overall clinical disease activity index.
Macroscopic colon assessment.
At the end of DSS treatment period, mice were anesthetized with urethane (1.5 g/kg ip). Maximum possible quantity of blood was collected through the abdominal aorta in a heparinized tube. Subsequently, the whole colon was excised. The colon was macroscopically assessed by determining 1) the presence or absence of blood and 2) the length. The whole colon was utilized for histological analyses and/or Western blot analysis.
Histological analysis of colon.
Colon biopsies from proximal, medial, and distal colon were collected and fixed in neutral buffered formalin. Paraffin-embedded sections (5-μm thickness) of the colon specimens were stained with Mayer's hematoxylin (Sigma Aldrich) and eosin (Loba Chemie, Mumbai, India) and graded by an investigator blinded to the treatment groups. Histopathological scoring was performed based on presence of inflammatory cells, extent of crypt damage, erosions, and overall architectural damage, each scored on a scale of 0 to 3 as described elsewhere (12). Sections were scored for each feature separately and the scores were added to arrive at the final histopathological scoring for individual colon specimen. Histological assessment of disease activity was carried out by analyzing the average histopathological score of a group.
For analyzing differences between two groups, Student's t-test was used. For analyzing differences among multiple (more than two) groups, a single-factor ANOVA followed by Bonferroni's multiple pairwise comparison tests was used. P values < 0.05 were considered statistically significant. Unless stated otherwise, all error bars represent standard error of mean.
P2281, a haloacyl aminopyridine-based molecule, inhibits mTOR activity in cancer cells.
Prior studies have shown that pyridine-based molecules are known to possess antiproliferative properties (20, 22, 30). mTOR is a protein kinase that regulates cell proliferation (18, 40). Thus in our attempt to identify novel anticancer agents we ascertained the effect of library compounds on mTOR activity. As part of this general screening process, we investigated the effect of P2281, a haloacyl aminopyridine-based molecule (Fig. 1), on mTOR activity. Initially, we performed cell-based ELISA and used the phosphorylation of p70S6 kinase at Thr389 as a measure of mTOR activity. Consistent with results of prior studies (36), naive H460 cells exhibited little if any mTOR activity (Fig. 2A). Upon stimulation with 20% FCS the phosphorylation of p70S6K1 at Thr389 (18) was markedly increased, indicating elevated mTOR activation (Fig. 2A). Pretreatment of cancer cells with P2281 or with positive control rapamycin each led to a significant decrease in the phosphorylation of p70S6K1 at Thr389 (Fig. 2A). To further characterize the mTOR inhibitory activity of P2281, we examined its effect on phosphorylation of 4E-BP1 protein. Western blot analysis of lysates from FCS-stimulated H460 cells showed prominent upregulation in phosphorylation status of 4E-BP1 compared with lysates from naive cells. In line with observations from cell-based ELISA, pretreatment with P2281 or with positive controls LY294002 and rapamycin each led to a distinct decrease of 4E-BP1 phosphorylation (Fig. 2B). Of note, P2281 inhibited the FCS-induced phosphorylation of p70S6K1 (Thr389) in HCT-116 colon cancer cells (Fig. 2C). The relative differences in phosphorylation were quantified by densitometry. For this, the phosphorylation of the protein in the serum-stimulated, DMSO-treated samples (Fig. 2, B and C, lane 2) was considered to be 100%. The phosphoprotein levels in the remaining samples of the respective experiment were calculated with reference to the serum-stimulated, DMSO-treated samples. Thus, following treatment of H460 cells with P2281 (Fig. 2B, lanes 3 and 4), the residual phosphorylation of 4E-BP1 was 47% whereas the residual phosphorylation of 4E-BP1 following treatment with LY294002 (Fig. 2B, lane 5) and rapamycin (Fig. 2B, lane 6) was 33 and 48%, respectively. Similarly, the residual phosphorylation of p70S6K1 in HCT-116 cells following P2281 treatment (Fig. 2C, lane 3) was 55% compared with 17% following LY294002 treatment (Fig. 2C, lane 4). It is well established that, in addition to being phosphorylated by mTOR, p70S6K1 can also be phosphorylated via the phosphatidylinositol 3′-kinase (PI3K) pathway by PDK1 at Thr389 (47, 55). Given these findings of earlier studies, it is not entirely surprising that treatment of HCT-116 cells with LY294002 (which inhibits both PI3K and mTOR) results in more potent abrogation of p70S6K1 phosphorylation. Interestingly, P2281 did not significantly inhibit the PI3K activity, as evidenced by lack of effect on phosphorylation of Akt (18% inhibition; data not shown), suggesting that is a selective preferential mTOR inhibitor.
P2281 inhibits induced IFN-γ production but not TNF-α production.
It is increasingly being recognized that anticancer therapeutics can possess anti-inflammatory properties (23, 31, 39). More importantly, a growing body of evidence associates increased mTOR activation to inflammatory complications (27, 58). Accordingly, we sought to investigate the effects of P2281 in inflammation assays. LPS stimulation of macrophages leads to not only activation of mTOR but also secretion of proinflammatory cytokines including TNF-α. This fact, combined with the documented role of bacterial endotoxin and TNF-α in the pathogenesis of inflammatory disorders including UC (5, 9), led us to initially probe the effect of P2281 on the LPS-induced expression of TNF-α. Freshly isolated hPBMCs were pretreated with P2281 or 0.5% DMSO (carrier control) and subsequently challenged with LPS for 5 h. ELISA of supernatants revealed that LPS stimulation induced TNF-α production from hPBMCs (Fig. 3A). Rolipram, the positive control, significantly inhibited LPS-induced production of TNF-α (Fig. 3A). However, P2281 had little, if any, effect on LPS-induced production of TNF-α (Fig. 3A). Similar results were obtained with rapamycin (data not shown). These in vitro findings were corroborated by in vivo studies wherein P2281 did not inhibit LPS-induced TNF-α production (Fig. 3B).
Given that everolimus, a mTOR inhibitor, inhibits IFN-γ production (34), we next investigated the effect of P2281 on the induced production of IFN-γ. Freshly isolated hPBMCs were pretreated with P2281 or 0.5% DMSO and subsequently stimulated with concanavalin A for 18 h. ELISA of supernatants revealed that concanavalin stimulation induced IFN-γ production from hPBMCs (Fig. 4A). Cyclosporine A, the positive control, significantly inhibited concanavalin-induced production of IFN-γ (Fig. 4A). More importantly, P2281 also significantly inhibited concanavalin-induced production of IFN-γ (Fig. 4A). Accordingly, dose-response studies were carried out. P2281 inhibited induced IFN-γ production in a dose-dependent manner with significant inhibition being observed at P2281 concentration ≥30 μM (Fig. 4B). Rapamycin also inhibited concanavalin-induced IFN-γ production in a dose-dependent manner (data not shown). Collectively, these results clearly demonstrate that P2281 inhibits induced IFN-γ production but not induced TNF-α production.
P2281 suppresses DSS-induced colitis.
The observations that rapamycin and everolimus (both mTOR inhibitors) are efficacious in animal models of colitis (15, 34), combined with the findings that blocking IFN-γ production elicits a therapeutic effect in experimental colitis (16), led us to hypothesize that P2281 (which inhibits mTOR activation as well as IFN-γ production) would be efficacious in a murine model of colitis. Accordingly, we investigated the effect of P2281 in an experimental model of colitis. A group of six mice was given DSS solution from days 1 to 10. As a control for DSS treatment, a group of six mice was given regular drinking water from days 1 to 10 (normal mice; naive). Separate groups of mice (6 mice per group) were given DSS solution from days 1 to 10 and received daily injections of P2281 (15 mg/kg ip) or 0.5% CMC (vehicle control for P2281). The P2281 and CMC administration were started on the same day as DSS was added to the water. All mice were euthanized after day 10, and macroscopic and histological analysis of the colon was performed.
As reported by others (48), DSS-induction of colitis was manifested with significant increase in clinical disease activity index associated with significant weight loss, presence of rectal bleeding, diarrhea, and distinct occurrence of occult blood in feces (Fig. 5). Consistent with these observations, DSS treatment significantly reduced the colon length (Fig. 5F). CMC (vehicle control for P2281), given coincident with DSS treatment, had no effect on DSS-induced disease (data not shown). Importantly, P2281, given coincident with DSS treatment, significantly inhibited DSS-induced weight loss (Fig. 5A) and significantly inhibited DSS-induced disease activity index (Fig. 5E). In line with these observations, P2281 treatment significantly inhibited the DSS-induced shortening of the colon (Fig. 5F). P2281 treatment diminished (albeit at not statistically significant levels) DSS-induced rectal bleeding (Fig. 5B) and attenuated (albeit at not statistically significant levels) DSS-induced decreases in hemoglobin levels (Fig. 5D).
Histological analysis confirmed the DSS induction of colitis. As reported by others (25), colonic tissue sections from DSS mice, but not from normal mice, revealed severe inflammation, characterized by presence of edema, distinct inflammatory cellular infiltrate, and extensive damage to mucosa and epithelium along with crypt destruction (Fig. 6A). Remarkably, tissue sections from P2281-treated DSS mice, but not CMC-treated DSS mice, revealed attenuation in inflammation, characterized by suppression of edema, reduction in inflammatory cellular infiltrate, and protection against epithelium and crypt damage (Fig. 6A and data not shown). Accordingly, the histopathological score of P2281-treated DSS mice was significantly lower than that of CMC-treated DSS mice (Fig. 6B).
Western blot analysis revealed that DSS induced phosphorylation of 4E-BP1 in the colon (Fig. 7). Consistent with in vitro observations, P2281, at concentration efficacious in suppressing colitis, prominently suppressed DSS-induced phosphorylation of 4E-BP1 in colon (Fig. 7). The relative differences in phosphorylation were quantified by densitometry. For this, the phosphorylation of the protein in the lysates from colon of CMC-treated DSS mice (Fig. 7, lane 2) was considered to be 100%. The phosphoprotein levels in the remaining samples were calculated with reference to the CMC-treated DSS mice. In P2281-treated DSS mice (Fig. 7, lane 3), the residual phosphorylation of 4E-BP1 was found to be 47% compared with 35% in naive mice (Fig. 7, lane 1). Thus P2281 treatment causes a marked reduction in DSS-induced mTOR activity.
Taken together, the above results clearly demonstrate that P2281 significantly inhibits DSS-induced macroscopic and histological abnormalities in the colon by inhibiting mTOR activation.
A critical component of UC is increased proliferation of colonic epithelial cells (56), dysregulation of signal transduction pathways (4), elevated mucosal T cell activation (53), increased production of proinflammatory cytokines (6, 8), and enhanced leukocyte infiltration into colonic interstitium (29, 38). A growing body of evidence supports the notion that kinase inhibitors, which possess antiproliferative activities, are potential therapeutics for UC (15, 34, 39). In our search for anticancer therapeutics, we have found that P2281 inhibits mTOR activity in colon cancer cells and suppresses DSS-induced colitis. Given that prior studies have demonstrated that compounds with anticancer activity show promising anti-inflammatory efficacy (39, 43, 51, 59), the findings from this study, thus, reinforce the cross-therapeutic functionality of potential drugs.
Our results clearly demonstrate that P2281 markedly suppressed DSS-induced colitis. Specifically, P2281 significantly inhibited DSS-induced weight loss and significantly reversed DSS-induced shortening of the colon (Fig. 5, A and F). P2281 also diminished the rectal bleeding index and attenuated the DSS-induced reduction in hemoglobin levels (Figs. 5, B and D); however, the effect of P2281 on these DSS-induced disease parameters did not reach statistical significance. The reason for preferential efficacy of P2281 on certain parameters of DSS-induced disease is currently unknown. However, microscopic histological analyses corroborate the macroscopic observations of P2281 efficacy in protecting mice against DSS-induced colitis. In particular, P2281 dramatically suppresses edema, reduces leukocyte infiltration, and maintains mucosal integrity in DSS-treated mice (Fig. 6A). The results of our study are in line with observations from other studies (15, 34) and suggest that marked mTOR inhibitory activity (Fig. 7) can elicit a meaningful physiological effect. Given that the macroscopic and microscopic manifestations of colitis observed in our system have been reported in other animal models of colitis and in humans (28), our data are likely broadly applicable to other experimental colitis models, and importantly to human colitis. Further experiments are warranted to confirm this hypothesis.
The current therapies for inflammatory bowel disease include immunomodulating agents such as mesalazine, corticosteroids, and cyclosporine A (5). The latter is believed to act primarily through effects on T cell function. P2281 inhibits mTOR activity and also suppresses induced production of IFN-γ (cytokine released by T cells) (Figs. 2 and 4). Interestingly, we found that P2281 failed to block in vitro and in vivo LPS-induced production of TNF-α (cytokine released by macrophages). The inability of P2281 to inhibit in vivo LPS-induced TNF-α production was not because of pharmacokinetic issues because sufficient levels (Cmax: 38 μg/ml, i.e., 180 μM) of P2281 were seen in the plasma of mice after administration of 100 mg/kg P2281 (data not shown). These observations, combined with the fact that LPS stimulates mTOR activity (13, 27), indicate that mTOR activation plays little if any role in induced TNF-α production. Furthermore, P2281 had little if any effect on LPS-induced IL-6, IL-8, and IL-1 production from hPBMCs (data not shown). Taken together, these results suggest that P2281 may have preferential effects on T cells compared with macrophages. Our findings are not entirely surprising particularly since mTOR inhibitors (such as rapamycin) are known to be potent inhibitors of T cell function (7). In view of our observations, it will, thus, be of interest to determine the efficacy of P2281 in other T cell-mediated disorders [e.g., psoriasis (33)].
The findings from this study have important implications for oncology too. It is well established that mTOR is a critical integrator of various signals emanating from growth factors, cytokines, hormones, and nutrients (18). Activation of mTOR leads to phosphorylation of two major effector molecules, p70S6 kinase and 4E-BP1, which in turn enhances protein translation and cell growth. Several studies have implicated mTOR in tumorigenesis (40), and the fact that mTOR inhibitors such as rapamycin, temserolimus, and CCI-779 are showing promising results in clinical trials (18) is in itself a fair indication of the importance of mTOR in oncology. Moreover, because of their antiproliferative effects on T and B cells, rapamycin (7) and its analogs have also been successfully used as immunosuppressants, indicating that mTOR also plays a crucial role in functioning of the immune system. The activation of mTOR is governed by Akt (protein kinase B), which in turn is activated by the PI3K pathway (19, 32). Thus triggering of the PI3K-Akt-mTOR arm of the cellular signaling machinery in many cases is necessary and sufficient for tumorigenesis. Of note, the PI3K-Akt pathway has been implicated in colon carcinogenesis (44). Interestingly, P2281 did not significantly inhibit the phosphorylation of Akt (18% inhibition; data not shown), indicating that it is a preferential mTOR inhibitor. Separately, overexpression of the protein synthesis initiation factor eIF4E has been documented in colon cancers (42). Given that mTOR positively regulates the function of eIF4E, it follows that mTOR is a critical target for colon cancer. A connection between Wnt and mTOR has also been reported. The Wnt pathway can inactivate TSC1/2 proteins, which are the negative regulators of mTOR, thus relieving the inhibition on mTOR (21). The enhanced activation of mTOR would then drive colon carcinogenesis. mTOR activation was also shown to lead to chromosomal instability and the formation of colonic polyposis (2). Taken together, these findings demonstrate the importance of targeting mTOR in colorectal cancer. Moreover, inflammatory disorders such as Crohn's disease and UC may also lead to colon cancer in the long run (14). Our findings thus suggest that targeting mTOR may not only prevent colitis but also exert a further inhibitory effect on colon cancer development in inflammatory bowel disease patients.
In conclusion, we have demonstrated that P2281 is a novel mTOR inhibitor and that systemic ip application of P2281 significantly suppresses chemically induced murine colitis. Thus P2281 may be a potential therapeutic for UC with important implications for colon cancer too.
We thank Rai Ajit Srivastava for critical review of the manuscript, Kumar Nemmani for useful discussions, and Lyle Fonseca, Divya Kamath, and Merlene Ann Babu for expert technical assistance.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2008 the American Physiological Society