Agents that interfere with tumoral immune tolerance may be useful to prevent or treat cancer. Brassinin is a phytoalexin, a class of natural products derived from plants that includes the widely known compound resveratrol. Brassinin has been demonstrated to have chemopreventive activity in preclinical models but the mechanisms underlying its anticancer properties are unknown. Here, we show that brassinin and a synthetic derivative 5-bromo-brassinin (5-Br-brassinin) are bioavailable inhibitors of indoleamine 2,3-dioxygenase (IDO), a pro-toleragenic enzyme that drives immune escape in cancer. Like other known IDO inhibitors, both of these compounds combined with chemotherapy to elicit regression of autochthonous mammary gland tumors in MMTV-Neu mice. Furthermore, growth of highly aggressive melanoma isograft tumors was suppressed by single agent treatment with 5-Br-brassinin. This response to treatment was lost in athymic mice, indicating a requirement for active host T-cell immunity, and in IDO-null knockout mice, providing direct genetic evidence that IDO inhibition is essential to the antitumor mechanism of action of 5-Br-brassinin. The natural product brassinin thus provides the structural basis for a new class of compounds with in vivo anticancer activity that is mediated through the inhibition of IDO.
Indoleamine 2,3-dioxygenase (IDO) is a monomeric, heme-containing enzyme that catabolizes the essential amino acid tryptophan (Hayaishi et al., 1984). Whereas a second liver-specific enzyme, tryptophan dioxygenase (TDO2), is responsible for maintaining tryptophan homeostasis, IDO has an immunomodulatory role that is mediated through effects of tryptophan catabolism on T cells (Mellor and Munn, 2004). An interferon-inducible enzyme, IDO is elevated at sites of inflammation and immune privilege. IDO was first established as an important pro-toleragenic enzyme in a seminal in vivo study that utilized the bioavailable IDO inhibitor 1-methyl-tryptophan (1MT) to elicit immune rejection of allogenic concepti during pregnancy (Munn et al., 1998). Significantly, IDO plays a similar pro-toleragenic role in the pathophysiological context of tumors. The IDO enzyme was first identified, in part, through findings of elevated tryptophan catabolism in cancer patients that could not be ascribed to TDO2 (Hayaishi et al., 1984) and recent reports have associated IDO elevation with less favorable outcomes in certain cancers (Okamoto et al., 2005; Brandacher et al., 2006; Ino et al., 2006). Pharmacological intervention in tumoral immune escape is a novel concept for which IDO is a leading target (Muller and Scherle, 2006) based on preclinical evidence that small molecule inhibitors of IDO can effectively cooperate with chemotherapy to elicit regression of established tumors in mice (Muller et al., 2005).
Plants produce a vast array of chemically complex compounds that have been a valuable source for the discovery of novel chemotherapeutic agents and currently there is particular interest in the development of botanicals for chemoprevention (Chemoprevention Working Group, 1999; Park and Pezzuto, 2002). Among the potentially active components identified, some of the most promising are phytoalexins. Resveratrol is perhaps the best known of this class of anti-microbial compounds, which are synthesized by plants in response to various stresses (Muller, 1958). Brassinin ([3-(S-methyldithiocarbamoyl)aminomethyl indole]), first isolated from Chinese cabbage inoculated with Pseudomonas chichorii (Takasugi et al., 1986, 1988), belongs to a group of sulfur-containing, tryptophan-derived phytoalexins that are unique to crucifers (Mezencev et al., 2003). Brassinin has been shown to inhibit the formation of carcinogen-induced preneoplastic lesions in mouse mammary gland organ culture and to suppress papilloma formation in the classical two-stage DMBA/TPA skin carcinogenesis model (Mehta et al., 1995). Recently, we have shown that brassinin is a micromolar inhibitor of the IDO enzyme and have evaluated the IDO inhibitory activity of a large set of derivatized variations of the brassinin core structure in order to investigate structure–activity relationships (Gaspari et al., 2006). In this study, we provide direct in vivo evidence that brassinin-based compounds can act as anticancer agents through their ability to inhibit IDO.
IDO has wide substrate specificity for compounds containing an indole structure (Malachowski et al., 2005). Evaluating commercially available indole-containing compounds, we have identified several molecules with IDO inhibitory potencies of less than 100 μM (Figure 1), including a methylthiohydantoin derivative of tryptophan that has been described previously (Muller et al., 2005). Of particular interest among these molecules were two natural products with chemopreventive properties—3,3′-diindolylmethane, the primary metabolic product of indole-3-carbinol and brassinin (Figure 1). Based on its superior potency, specificity and bioavailability, we focused work in this study on brassinin and a synthetic derivative, 5-bromo-brassinin (5-Br-brassinin; Figure 1).
Brassinin and 5-Br-brassinin both behaved as competitive inhibitors of the tryptophan catabolic activity of recombinant human IDO enzyme in a cell-free enzyme assay, with Ki values (Table 1) below the 35 μM value obtained for the widely used IDO inhibitor D,L-1MT (Hou et al., 2007). The potency of 1MT is substantially attenuated in cell-based assays, with EC50 values in the 100 μM range (Hou et al., 2007), but this was not a significant issue for the two brassinin compounds. Tryptophan catabolism by both human and mouse IDO expressed ectopically in the COS-1 cell line was inhibited by both compounds with EC50 values in the 25–35 μM range (Table 1). In this same cell-based assay, activity of recombinant human tryptophan 2,3-dioxygenase (TDO2), the hepatic enzyme which catabolizes tryptophan in the same manner as IDO, was not significantly affected at concentrations of either compound up to 100 μM (data not shown). Cell viability profiles for both COS-1 cells, used to perform the cell-based enzyme assay, and B16-F10 mouse melanoma-derived cells, used in tumor experiments described below, indicated no evidence of cytotoxicity or growth suppression associated with exposure up to 100 μM of brassinin or 5-Br-brassinin (Figure 2).
Serum analysis from mice indicated that both brassinin and 5-Br-brassinin are orally bioavailable. When formulated for oral gavage in 50% hydroxypropyl β-cyclodextran (HPBCD), an excipient that can improve drug delivery (Davis and Brewster, 2004), 5-Br-brassinin was found to have a superior pharmacologic profile (Figure 3), exhibiting sustained levels in serum for up to 8 h while brassinin was essentially cleared by 3 h.
In mouse mammary tumor virus (MMTV)-Neu transgenic mice, HER2/Neu expression controlled by the MMTV promoter drives the development of focal mammary gland carcinomas (Guy et al., 1992) which histopathologically resemble human ductal carcinoma in situ (Cardiff and Wellings, 1999). We have previously reported that continuous administration of IDO inhibitory compounds, delivered by subcutaneously implanted time-release pellets, can cooperate with paclitaxel as well as other cytotoxic chemotherapeutic agents to elicit regression of established mammary gland tumors in this very stringent autochthonous tumor model (Muller et al., 2005). More recently, we have demonstrated that bolus oral delivery of the IDO inhibitor 1MT on a twice a day (b.i.d.) schedule can also cooperate with paclitaxel to regress these tumors (Hou et al., 2007). Based on their oral bioavailability in HPBCD, we evaluated brassinin and 5-Br-brassinin administered by oral bolus dosing at 400 mg kg−1 twice a day, a treatment regimen that likely approached the maximum tolerated dose as the mice exhibited clear evidence of compound-related toxicity which manifested outwardly as the development of a scruffy appearance. Delivered in this manner, neither compound alone demonstrated significant single agent activity. Brassinin in combination with paclitaxel did produce tumor regressions; however, the effect was not of sufficient magnitude to infer a statistically significant benefit relative to paclitaxel treatment alone with the number of tumors evaluated (Figure 4b). Rapid clearance of this compound following bolus dose delivery may account for this rather limited response relative to our previous experience with other IDO inhibitory compounds (Muller et al., 2005). In contrast, the more pharmacologically stable compound 5-Br-brassinin in combination with paclitaxel did produce an effect that was significantly better than paclitaxel treatment alone (Figure 4b). Collectively, these data are consistent with the conclusion that these brassinin-based compounds are able to target IDO effectively in vivo and add to the number of structurally distinct compounds with IDO inhibitory activity that can cooperate with the cytotoxic chemotherapeutic drug paclitaxel to regress autochthonous mammary carcinomas in this mouse breast cancer model.
To directly test the antitumor mechanism of action of the brassinin compounds in vivo, we have conducted studies using B16-F10 melanoma isografts which do not express IDO directly in the tumor but rather accumulate IDO-expressing, toleragenic plasmacytoid DCs in the tumor draining lymph node (Munn et al., 2004). The IDO inhibitor 1MT, although lacking significant single agent activity, cooperatively suppressed growth of B16-F10 tumors in combination with chemotherapeutic agents or tumor irradiation (Hou et al., 2007), consistent with IDO-expression in host stromal cells being the relevant target in this tumor model. We confirmed that IDO expression was undetectable in the B16-F10 cell line (Figure 5a). Neither the D nor L isomer of 1MT produced significant growth inhibition when administered as single agents and a similar outcome was obtained with brassinin (data not shown). On the other hand, 5-Br-brassinin treatment produced significant suppression of B16-F10 tumor outgrowth (Figure 5b). This effect of 5-Br-brassinin treatment on tumor outgrowth was not evident in athymic nude mice (Figure 5c), indicating that its mechanism of action requires T cell-based immunity and is not mediated through a direct cytotoxic effect on the tumor. Because B16-F10 tumors do not express any detectable IDO, such that the stromal compartment is the only source of IDO activity (Munn et al., 2004), it was possible also to directly test the relevance of IDO as a target by performing the experiment in genetically modified, syngeneic mice in which both alleles of the Indo gene were functionally disrupted (IDO-null). In the context of the IDO-null host, 5-Br-brassinin had no impact on tumor growth (Figure 5d). The results of this experiment, therefore, genetically define IDO as a therapeutically essential molecular target of 5-Br-brassinin in this in vivo tumor model.
In this study, we have shown that, as with other IDO inhibitory compounds, brassinins can be delivered in vivo to leverage the effectiveness of chemotherapy against established tumors in an autochthonous mouse model of breast cancer. Moreover, we have directly demonstrated that the brassinin-based compound 5-Br-brassinin can suppress tumor outgrowth through a T cell-dependent mechanism that obligately involves IDO. Cruciferous vegetables have garnered a great deal of attention due to their anticancer properties (Murillo and Mehta, 2001), and brassinin is a constituent of crucifers with demonstrated anticancer activity in mouse tumor models. While a variety of possible molecular mechanisms have been proposed to explain this activity, none has been directly validated in vivo. In particular, brassinin is cited as an inducer of quinone reductase (QR) based on data from a mouse mammary gland organ culture model in which incubation with 220 μM brassinin for 3 days resulted in a fourfold increase in QR activity (Mehta et al., 1995). QR is a phase II enzyme that detoxifies mutagenic carcinogens, and as an inducer of this enzyme, brassinin would be predicted to act as an ‘anti-initiator’. However, the same study found that, in the two stage DMBA/TPA skin carcinogenesis model, brassinin acted instead as an ‘anti-promoter’ with no apparent impact on the initiation stage of carcinogenesis (Mehta et al., 1995). Unlike QR, IDO is likely to affect the promotional stage of carcinogenesis and, indeed, TPA is a powerful proinflammatory contact-sensitizer that would be expected to substantially elevate levels of IFNγ, the principle cytokine inducer of IDO, in the draining lymph nodes (Thomson et al., 1993).
The B16-F10 tumor isograft data reported here clearly indicate that inhibition of host IDO activity is essential for the single agent suppression of tumor outgrowth by 5-Br-brassinin. This degree of efficacy is rather remarkable when compared with other agents that target immune-based pathways. For example, the costimulatory molecule CTLA4 is a powerful antagonist of T-cell activation that may act, at least in part, by elevating IDO (Grohmann et al., 2002). However, in the B16-F10 tumor model, CTLA4 monoclonal antibody blockade failed to elicit a single agent response, (although it produced significant autoimmunity), effecting tumor growth suppression only when administered in conjunction with a granulocyte-macrophage colony-stimulating factor transduced tumor cell vaccine (GVAX) (Quezada et al., 2006).
Administration of brassinin, unlike 5-Br-brassinin, did not significantly impact B16-F10 tumor growth, perhaps simply reflecting inferior pharmacokinetics. However, since the IDO inhibitor 1MT also does not show single agent activity in the B16-F10 tumor model either, it is also possible that IDO inhibition may be necessary but not sufficient to account for the ability of 5-Br-brassinin to suppress B16-F10 tumor growth. Previously, 1MT has been shown to cooperate with various cytotoxic agents (cyclophosphamide, gemcitabine, IR) to suppress B16-F10 tumor growth (Hou et al., 2007). Analogous to these agents, 5-Br-brassinin may have an intrinsic cytotoxic effect that enhances its antitumor activity. Although we observed no discernable impact of brassinin or 5-Br-brassinin at concentrations up to 100 μM on the viability of either B16-F10 or COS-1 cells, other groups have reported that at similar concentrations brassinin can reduce the number of viable cells in a 72 h assay by 25–50% relative to controls among different cancer cell lines tested including B16-F10 (Sabol et al., 2000; Pilatova et al., 2005; Csomos et al., 2006). While our finding that 5-Br-brassinin treatment had no significant impact on B16-F10 tumor growth in athymic nude mice clearly indicates that any direct cytotoxic effect that this compound may have is not sufficient to account for its antitumor activity in vivo, these data do not rule out that mild cytotoxicity might contribute to therapeutic efficacy. It will be important to further explore such mechanistic questions to fully understand how to best develop the antitumor mechanism of action associated with brassinin-based compounds.
A key finding of this study is that 5-Br-brassinin treatment substantially suppressed B16-F10 tumor growth in wild-type mice but not IDO-null mice. However, these data also apparently indicate that a complete absence of IDO in the host is irrelevant to tumor outgrowth since comparable growth rates were observed in IDO-null and wild-type mice in the absence of compound treatment. These results recapitulate observations made in pregnancy studies in which acute exposure to the IDO inhibitor 1MT resulted in immune rejection of allogeneic concepti while the viability of allogeneic concepti was normal in genetic knockout mice but was no longer affected by 1MT treatment (Munn et al., 1998; Baban et al., 2004). One possible explanation given for these apparently dichotomous results is that compensatory mechanisms may come into play in the knockout mice (Baban et al., 2004). In the context of cancer, these data suggest that, while IDO may not be the only possible immune escape mechanism, when IDO is available to tumors they may preferentially become dependent on it for continued growth in what might be termed ‘tolerance addiction’.
Because targeting tumoral immune tolerance is a unique approach to cancer treatment, the use of IDO inhibitors in combination with other types of agents may represent the best opportunity to simultaneously attack tumors on multiple fronts. Evidence from mouse tumor models already supports the possible use of IDO inhibitors with certain chemotherapeutic drugs (Muller et al., 2005; Hou et al., 2007) and it seems likely that IDO inhibitors will enhance cancer vaccines and other approaches that aim at stimulating immune effector function as well. IDO inhibitors might also be used to intervene earlier in the process of immune editing when the nascent tumor is not as plastic. For this sort of chemopreventive strategy, administration of IDO inhibitors through dietary uptake would be a particularly attractive means of delivery, and further study of the pharmacodynamic impact of cruciferous vegetable consumption on IDO activity should be pursued.
Materials and methods
Brassinin was synthesized as described (Gaspari et al., 2006). 5-Br-brassinin was synthesized by the Advanced Synthesis Group (Newark, DE, USA). HPBCD was purchased from Cargill Inc. (Cedar Rapids, IA, USA).
COS-1 monkey cells and B16-F10 mouse melanoma cells (ATCC, Manassas, VA, USA) were cultured with Dulbecco's modified Eagle's medium (DMEM) (Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) and 1% penicillin-streptomycin (Invitrogen) at 37 °C in 5% CO2.
C57BL/6 and FVB-strain MMTV-Neu transgenic mice were obtained from the Jackson Laboratory. Athymic NCr-nu/nu (nude mice) were obtained from NCI-Frederick. IDO knockout mice have previously been described (Baban et al., 2004). Studies involving mice were approved by the institutional animal use committee of the Lankenau Institute for Medical Research.
IDO enzyme assays
The cell-free IDO enzyme assay was performed in a 96-well microtiter plate with active recombinant human his6-IDO enzyme purified by sequential chromatography over phosphocellulose and Ni-NTA agarose columns from E.coli strain BL21DE3pLysS transformed with pet5Ahis6huIDO as described (Gaspari et al., 2006). The reaction mixture for carrying out the enzyme assay contained 50 mM potassium phosphate buffer (pH 6.5), 40 mM ascorbic acid, 400 μg ml−1 catalase, 20 μM methylene blue. Enzyme activity was assessed for each IDO preparation and the amount of enzyme used in the assay was based on this determination. The substrate L-tryptophan (100 mM stock in 0.1 N HCl) was serially diluted from 200 to 25 μM. Inhibitors were dissolved in DMSO to make 100 mM stock solutions and assessed at final concentrations of 100 and 50 μM in a total reaction volume of 200 μl. Reactions were carried out at 37 °C for 60 min, stopped by adding 30% (w/v) trichloroacetic acid, and then heated at 65 °C for 15 min to convert kynurenine to N-formyl-kynurenine. Plates were then spun at 6000 g for 5 min, 100 μl supernatant from each well was transferred to a new 96-well plate and mixed with 2% (w/v) p-dimethyl benzaldehyde (Sigma-Aldrich, St Louis, MO, USA) in acetic acid. The yellow color generated from the reaction with N-formyl-kynurenine was quantitated at 490 nm using a Synergy HT microtiter plate reader (Bio-Tek, Winnooski, VT, USA). The data were analysed by using Prism 4 software (Graph Pad software, Inc., San Diego, CA, USA).
Cellular activity of selected compounds was assessed against both the human and mouse IDO enzymes transiently expressed in COS-1 monkey cells in a 96-well assay as described (Muller et al., 2005). COS-1 cells at 2.5 × 104 cells per well were transected overnight with pcDNA3.1-based expression plasmids in Opti-MEM I media (Invitrogen) using polyethyleneimine (Sigma-Aldrich), replaced the next day with standard growth medium (DMEM supplemented with 10% FBS and antibiotics). The following day, compounds solubilized in DMSO were serially diluted into plate wells (final DMSO concentration was no more than 1:1000). Plates were sealed in plastic wrap and incubated 16 h at 37 °C in a humified CO2 incubator. Reactions were terminated by withdrawing 140 μl media per well and mixing thoroughly into 10 μl 26% trichloroacetic acid (TCA) in wells of a new plate. Stopped reactions were heated at 65 °C for 15 min to convert kynurenine to N-formyl-kynurenine, which was processed and quantitated as above. The data were analysed by using Prism 4 software.
In vitro cytotoxicity assay
Compound cytotoxicity was assessed using the sulforhodamine B (SRB) viable cell assay (Skehan et al., 1990). Cells were seeded into 96-well tissue culture plates at densities (2000 cells per well) which allowed untreated cells to reach a nearly confluent state after 4 days. Cells were treated with serial dilutions of brassinin and its 5-Br analogue 24 h after seeding. The SRB cytotoxicity assay was performed following 72 h of compound exposure. Cells were fixed with 50% trichloroacetic acid and stained with 0.4% (w/v) SRB (Sigma-Aldrich) dissolved in 1% acetic acid. Unbound dye was removed by four washes with 1% acetic acid, and protein bound dye was extracted with 10 mM unbuffered Tris base (pH 10.5) for 5 min. Optical density was read at 570 nm using a Synergy HT microtiter plate reader.
MMTV-Neu mice were orally gavaged with 0.1 ml of a sonicated suspension of the desired compound (400 mg kg−1) in 50% HPBCD. Blood was collected at different intervals and serum prepared using the Stat Sampler Kit (Statspin, Norwood, MA, USA) following the vendor's instructions. Serum samples were stored at −80 °C. Samples were processed by extracting twice with 300 μl of tert-butyl methyl ether (Sigma-Aldrich) per 100 μl of serum. Organic and aqueous phases were separated by centrifugation (2800 g for 10 min), transferred to a fresh microfuge tube, and evaporated to dryness in the presence of 15 μl DMSO. Extracted samples in DMSO were diluted to 110 μl with 1:4 mixture of acetonitrile:water and then analysed by high-pressure liquid chromatography on a 250 × 4.5 mm Luna 5u C18 column (Phenomenex, Torrance, CA, USA). The mobile phase consisting of acetonitrile-water and solutes was eluted at a flow rate of 1.0 ml per minute in a 0–90% acetonitrile gradient for the first 7 min and in 90% acetonitrile for an additional 8 min. Columns were re-equilibrated with water for 20 min between samples. Serially diluted solutions of brassinin and 5-Br brassinin in 1:4 acetonitrile:water served as standards. The analyte was detected by UV detector at 278 nm and the peak area was quantified using Windaq software (DataQ Instrument, Akron, OH, USA).
Tumor formation and drug response
For autochthonous mammary gland tumor treatment studies, parous, FVB-strain MMTV-Neu mice expressing the wild-type form of the rat HER2/Neu proto-oncogene were used as described (Muller et al., 2005). When subjected to two rounds of pregnancy and lactation, the incidence of palpable tumors in this model is ∼80% by 7 months of age and increases to nearly 95% by 8 months. Tumor-bearing animals were enrolled randomly in control and experimental groups when tumors reached 0.5–1.0 cm in diameter for 2-week treatment response studies. Brassinin compounds were delivered for the first five consecutive days in 50% HPBCD excipient by oral gavage b.i.d. at 400 mg kg−1 while control animals received vehicle only. For those animals receiving paclitaxel, treatment was initiated concurrent with the administration of brassinin compounds and delivered by bolus i.v. injection into the tail vein on a schedule of 3 × per week. At the end of the 2-week treatment period, tumors were excised from euthanized animals and volumes were determined. Graphing and statistical analysis of the data was performed by using Prism 4 software.
B16-F10 melanoma-derived cell line isograft tumor challenge experiments were carried out as described (Hou et al., 2007). 1 × 105 cells were injected subcutaneously into mice at day 0 of the experiment, and treatment was initiated at day 7 following initial tumor cell engraftment. Tumor growth was monitored by performing caliper measurements of orthogonal diameters and the estimated tumor volume was calculated based on the formula for determining a prolapsed elliptoid (d2 × l/0.52) where d is the shorter of the two orthogonal measurements. Graphing and statistical analysis of the data was performed by using Prism 4 software.
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AJM is the recipient of grants from the DoD Breast Cancer Research Program (BC044350), the State of Pennsylvania Department of Health (CURE/Tobacco Settlement Award), the Lance Armstrong Foundation and the Concern Foundation. GCP is the recipient of NIH R01 grants CA82222, CA100123 and CA109542. Additional support for this project was provided by grants to GCP from the Charlotte Geyer Foundation and the Lankenau Hospital Foundation.
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Banerjee, T., DuHadaway, J., Gaspari, P. et al. A key in vivo antitumor mechanism of action of natural product-based brassinins is inhibition of indoleamine 2,3-dioxygenase. Oncogene 27, 2851–2857 (2008). https://doi.org/10.1038/sj.onc.1210939
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