Mol Cancer Ther. 2005;4:1569-1576
© 2005 American Association for Cancer Research
The anticancer activity of the fungal metabolite terrecyclic acid A is associated with modulation of multiple cellular stress response pathways
Thomas J. Turbyville1,2,
E.M. Kithsiri Wijeratne1,
Luke Whitesell2 and
A.A. Leslie Gunatilaka1
1 Southwest Center for Natural Products Research and Commercialization, Office of Arid Lands Studies, College of Agriculture and Life Sciences and 2 Section of Pediatric Hematology/Oncology, Steele Memorial Children's Research Center, The University of Arizona, Tucson, Arizona
Requests for reprints: A.A. Leslie Gunatilaka, Southwest Center for Natural Products Research, The University of Arizona, 250 East Valencia Road, Tucson, AZ 85706-6800. Phone: 520-741-1691; Fax: 520-741-1468. E-mail: leslieg{at}ag.arizona.edu
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Abstract
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Tumors are dependent on cellular stress responses, in particular the heat shock response, for survival in their hypoxic, acidotic, and nutrient-deprived microenvironments. Using cell-based reporter assays, we have identified terrecyclic acid A (TCA) from Aspergillus terreus, a fungus inhabiting the rhizosphere of Opuntia versicolor of the Sonoran desert, as a small-molecule inducer of the heat shock response that shows anticancer activity. Further characterization suggested that TCA also affects oxidative and inflammatory cellular stress response pathways. The presence of an 
-methylene ketone moiety suggested that TCA may form adducts with sulfhydryl groups of proteins. Reaction with labile intracellular cysteines was supported by our finding that the glutathione precursor N-acetyl-cysteine protected tumor cells from the cytotoxic effects of TCA whereas the glutathione-depleting agent buthionine sulfoximine enhanced its activity. Related sesquiterpenes have been shown to increase levels of reactive oxygen species (ROS) and to inhibit nuclear factor 
B (NF-B) transcriptional activity. To assess whether TCA could have similar activities, we used a ROS-sensitive dye and flow cytometry to show that TCA does indeed increase ROS levels in 3LL cells. When tested in cells carrying NF-B reporter constructs, TCA also exhibited concentration-dependent inhibition of cytokine-induced NF-B transcriptional activity. These findings suggest that TCA modulates multiple stress pathways—the oxidative, heat shock, and inflammatory responses—in tumor cells that promote their survival. Small-molecule natural products such as TCA may serve as useful probes for understanding the relationships between these pathways, potentially providing leads for the design of novel and effective anticancer drugs.
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Introduction
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Within a tumor where physiologic conditions are often poor, cancer cells have an increased requirement for heat shock protein (Hsp) function (1). Moreover, Hsps, especially Hsp90, are known to regulate a number of oncogene products within the disordered signal transduction pathways responsible for many of the malignant properties of the cancer cell (1, 2). In eukaryotic cells, stress-induced Hsp gene expression is regulated predominantly by the homotrimeric DNA-binding transcription factor HSF-1 (3, 4). Under basal conditions, HSF-1 is maintained as an inactive monomer by an Hsp90-containing multichaperone complex in the cytoplasm. Activation of the transcription factor during heat shock leads to release of monomeric HSF-1 from this complex, followed by its trimerization, translocation to the nucleus, recognition of and binding to heat shock response elements, phosphorylation, and target gene activation. A current hypothesis holds that Hsp90 plays an autoregulatory role in the heat shock response because one downstream consequence of HSF-1 activation is increased Hsp90 expression.
Two small-molecule natural products known to target Hsp90, geldanamycin (5) and radicicol (6), also activate HSF-1 by causing the chaperone to release the transcription factor allowing trimerization and subsequent transactivation of target genes including the canonical heat shock response genes (7). Using mouse fibroblasts stably transfected with a reporter construct encoding enhanced green fluorescent protein under the transcriptional control of a consensus heat shock response element, we have evaluated ethyl acetate extracts of Sonoran desert plant-associated microorganisms for their ability to induce enhanced green fluorescent protein expression (heat shock induction assay). Using this approach, we have identified several extracts active in heat shock induction assay, some of which on bioassay-guided fractionation afforded small molecules, including radicicol, known to target Hsp90.3 However, a number of extracts active in heat shock induction assay contained small molecules with no apparent Hsp90 activity. One such extract, from the fungus Aspergillus terreus, occurring in the rhizosphere of the Sonoran desert cactus Optunia versicolor (staghorn cholla), on cytotoxicity-guided fractionation afforded terrecyclic acid A (TCA; Fig. 1) and two closely related analogues with significant antitumor activity against a panel of human cancer cell lines (8) and moderate activity in preliminary experiments with 3LL Lewis Lung tumor implants in mice (data not shown).
We ruled out the direct interaction of TCA with Hsp90 and its cochaperones using a well-characterized assay (9) based on the ATP-dependent refolding of heat denatured luciferase (data not shown). Thus, it was of interest to elucidate the molecular mechanism(s) of action responsible for the biological activities of TCA. In a limited structure-activity relationship study designed to determine the key structural features responsible for the observed activity of TCA, we prepared the derivatives, dihydro-TCA and TCA methyl ester (Me-TCA), and compared their antiproliferative activities with TCA. To characterize the biological activities of TCA, we examined three key stress responses—the heat shock, oxidative, and inflammatory responses—that seem to be integral to its activity. As a measure of TCA reactivity with cellular thiols, we evaluated the effects of the glutathione-depleting agent L-buthionine-(S,R)-sulfoximine (BSO) and the glutathione precursor N-acetyl-L-cysteine (NAC) on the cytotoxicity of TCA and Me-TCA. We also examined directly whether TCA induced changes in the redox state of the cell using flow cytometry to quantitate cellular levels of reactive oxygen species (ROS). Elevated ROS are known to transcriptionally activate the heat shock response and seem to be a major factor in TCA induction of this response.
Interestingly, two sesquiterpene natural products structurally related to TCA [i.e., parthenolide from the medicinal plant feverfew (10) and helenalin from the plant Arnica montana (11)] have been reported to exhibit activity profiles similar to TCA including inhibition of DNA synthesis, cell cycle arrest, and induction of apoptosis. Moreover, parthenolide has been shown to increase the levels of ROS by glutathione depletion in hepatocellular carcinoma cells lines (10) and, in a separate study (in HeLa cells), to inhibit nuclear factor B (NF-B) transcriptional activity by forming direct adducts with a cysteine in the kinase activation loop of IB kinase ß (11). When tested in HeLa and 293T cells carrying NF-B reporter constructs, TCA exhibited a dose-dependent inhibition of cytokine-induced NF-B transcriptional activity. Our findings show that there is overlap between the oxidative, heat shock, and inflammatory responses in tumor cells, which promotes their survival, and that TCA exerts its anticancer effects by modulating these pathways. Understanding how these responses are interconnected may be key to understanding how cancer cells survive in the harsh tumor microenvironment and could prove useful in identifying new therapeutic targets.
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Materials and Methods
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Reagents
The isolation of TCA and the preparation of dihydro-TCA and Me-TCA derivatives (Fig. 1) have been previously described (8). The oxidation-sensitive fluorescein 5,6-carboxy-2',7'-dichlorofluorescein diacetate (DCFH-DA) and the oxidation-insensitive carboxy dichlorodihydrofluorescein diacetate (carboxyDCHF-DA) were purchased form Molecular Probes (Eugene, OR). All other reagents were obtained from Sigma (St. Louis, MO).
Cell Lines and Culture
3T3-Y9-B12 and HeLa cells were grown in DMEM and 3LL (mouse Lewis Lung) cells were grown in RPMI 1640 containing 10% fetal bovine serum supplemented with 2 mmol/L glutamine and incubated in a 37°C humidified atmosphere (10% or 5% CO2 DMEM and RPMI 1640, respectively). 293T/NF-B-luciferase cells (Panomics, Redwood City, CA) were grown in DMEM as above with 100 µg/mL hygromycin.
Cell Proliferation Assays
3LL cells were seeded into 96-well plates at a density of 2,000 cells/well and allowed to reattach overnight. Cells were treated with the compounds at the indicated concentrations continuously for 48 hours followed by assay for relative viable cell number using the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and a microplate spectrophotometer as previously described (8).
Heat Shock Induction Analysis
Heat shock induction at the transcriptional level was measured in mouse fibroblasts (3T3-Y9-B12 cells) that have been stably transfected with a plasmid encoding enhanced green fluorescent protein under the transcriptional control of a minimal consensus heat shock response element derived from the human HSP70B gene as described (7). Serial dilutions of TCA, dihydro-TCA, and Me-TCA were applied in triplicate to 3T3-Y9-B12 reporter cell monolayers established in 96-well plates and incubation was continued overnight. Control wells were treated with an equal volume of DMSO vehicle, not exceeding 0.1% v/v. After rinsing wells with PBS, fluorescence intensity was quantitated using a plate reader (Fluoroskan Ascent, Lab Systems, Milford, MA) with excitation/emission filters of 485/525 nm. To complement the quantitative data generated by heat shock induction assay, 3T3-Y9-B12 reporter cell monolayers established in a chamber slide and incubated overnight with TCA (10 µg/mL), dihydro-TCA (10 µg/mL), and Me-TCA (10 µg/mL) were also evaluated qualitatively by confocal microscopy (MRC 1024; Bio-Rad, Hercules, CA). After rinsing the chambers with PBS, confocal images were acquired using a 10x objective (Nikon TE 300) with identical gain and iris settings.
Measurement of Intracellular ROS
To measure the level of intracellular ROS, 3LL cells were seeded into 60-mm dishes (5 x 105 cells/dish) and incubated overnight to allow cells to reattach. Cells were treated in triplicate for 1 hour with 10 µg/mL of TCA or Me-TCA, 10 µmol/L H2O2 as a positive control, or with DMSO as a vehicle control. Cells were washed in RPMI 1640 containing 0.5% serum and incubated with either the oxidation-sensitive fluorescein DCFH-DA (5 µmol/L), oxidation-insensitive carboxyDCHF-DA (5 µmol/L), or vehicle control for 2 hours at 37°C. Cells were then analyzed by flow cytometry using excitation at 488 nm and emission at 525 nm (University of Arizona Cancer Center). DCFH-DA fluorescence was corrected for relative cellular dye uptake under different drug treatment conditions using carboxyDCFH-DA fluorescence (Molecular Probes).
NF-B Inhibition Assays
To evaluate NF-B inhibitory activity, we transiently transfected cells with pNF-B-luc, a reporter vector encoding firefly luciferase, under the transcriptional control of a consensus NF-B response element (Clontech, Mountain View, CA). Cotransfection with the constitutively expressed Renilla luciferase vector pRL-TKT was done to normalize for transfection efficency and cell viability. HeLa cells were seeded into 12-well plates (3.0 x 105 cells/well) and allowed to reattach overnight. Cells were cotransfected with pNF-B-luc and pRL-TK using Lipofecatamine (Invitrogen, Carlsbad, CA) according to the protocol of the manufacturer. After 18 hours, cells were pretreated for 1 hour with 1 µg/mL parthenolide as a positive control, or 10 and 5 µg/mL TCA, followed by treatment with 10 ng/mL of human tumor necrosis factor- (TNF-). After 5 hours, cell lysates were assayed using a dual luciferase assay kit (Promega). As an alternative to transient transfection assays, the stably transfected cell line 293T/NF-B-luc was used. This line carries a luciferase reporter vector regulated by six copies of a consensus NF-B response element (Panomics). These cells were seeded into 96-well plates at 3.0 x 104 cells per well and allowed to reattach overnight. Cells were then treated with various concentrations of TCA for 30 minutes, followed by addition of 10 ng/mL of TNF- for an additional 5 hours. A parallel set of cells were treated with the same concentrations of TCA but not with cytokine. After the treatment interval, lysates were assayed for protein content (bicinchoninic acid assay, Pierce Biotechnology, Rockford, IL) and luciferase activity was measured using an assay buffer consisting of 25 mmol/L Tricine-HCl (pH 7.8), 8 mmol/L MgSO4, 0.1 mmol/L EDTA, 12 mmol/L DTT, 100 µmol/L luciferin, 240 µmol/L CoA, and 0.5 mmol/L ATP. Specific luciferase activity was expressed for each treatment condition as relative light units normalized to protein content.
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Results
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TCA Inhibits Cancer Cell Growth and Induces a Heat Shock Response
Having identified the secondary metabolite responsible for anticancer and heat shock induction assay activity in A. terreus extract, we did dose-response analyses of TCA comparing its cytotoxicity and its ability to induce a heat shock response at the transcriptional level. As measured by MTT assay using 3LL cells, TCA showed antiproliferative activity in a concentration-dependent manner (Fig. 2). To compare its antiproliferative activity to its heat shock induction activity, 3T3-Y9-B12 reporter cells were treated with serial dilutions of TCA. The resulting increased levels of enhanced green fluorescent protein as measured by mean fluorescence (up to 2-fold over the vehicle-treated cells) indicate induction of the heat shock transcriptional response within the range of the relevant antiproliferative dose (Fig. 2). Conventional cytotoxic chemotherapeutics such as doxorubicin and cisplatin do not induce a heat shock response, indicating that the induction of the heat shock response by TCA is not simply a result of cytotoxicity (7).
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Figure 2. TCA activates a transcriptional heat shock response and inhibits tumor cell growth. Reporter cells stably transfected with a plasmid encoding enhanced green fluorescent protein under the control of a minimal consensus heat shock response element were treated overnight with the indicated concentrations of TCA. Fluorescence was then measured by microplate reader. Alternatively, 3LL Lewis Lung cells were treated with the same drug concentrations and after 48 h of continuous incubation in the presence of the drugs, MTT was added and absorbance values were collected as a measure of relative viable cell number. Points, mean of triplicate determinations, expressed as a percentage of the solvent vehicle control; bars, SD. Representative of three independent experiments.
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Structure-Activity Relationship Studies
The Reduced Form of TCA Has No Anticancer Activity. Based on comparison with other small molecules with similar functional group organization, we suspected that TCA could form adducts with sulfhydryl groups in proteins via its exocyclic methylene. To examine the role of this -methylene ketone moiety in the biological activity of TCA, we prepared dihydro-TCA by catalytic hydrogenation of TCA (Fig. 1). Dihydro-TCA was found to be inactive in the MTT assay using 3LL cells (Fig. 3A), suggesting that the exocyclic methylene serves as a site for Michael addition of a nucleophilic molecule—possibly a thiol group of a protein or polypeptide.
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Figure 3. Structure-activity relationship studies. A, concentration-dependent inhibition of tumor cell proliferation and survival. After 48 h of continuous incubation in the presence of the indicated drugs, MTT was added and absorbance values were collected. Points, mean of triplicate determinations, expressed as percent of vehicle control–treated wells; bars, SD. Representative of three independent experiments. B, Me-TCA activates the heat shock response more effectively than TCA whereas dihydro-TCA has no activity. Reporter cells were treated with TCA, dihydro-TCA, and Me-TCA. After overnight treatment, fluorescence was measured by fluorescence plate reader. Points, mean of triplicate determinations, expressed as a percentage of the negative control; bars, SD. Representative of two independent experiments. C, representative confocal images from TCA-, dihydro-TCA–, and Me-TCA–treated 3T3-Y9-B12 cells obtained using identical magnification, gain, and iris settings.
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The Methyl Ester of TCA Is More Active than TCA. We prepared the methyl ester derivative of TCA (Me-TCA) to examine whether the carboxylic acid group of the molecule contributes to its activity (Fig. 1). As seen in Fig. 3A, Me-TCA showed markedly enhanced antiproliferative activity compared with TCA. This finding suggests that the carboxylic acid group is not responsible for its activity. It also suggests that in cell culture, the carboxylic acid moiety of TCA may participate in an intramolecular reaction with the exocyclic methylene, leading to an inactive form of the molecule, or that the methyl ester enhances the cellular uptake of the small molecule.
Me-TCA and TCA Induce a Heat Shock Response in 3T3-Y9-B12 Cells. To evaluate the ability of Me-TCA, dihydro-TCA, and TCA to induce a heat shock response at the transcriptional level, we incubated 3T3-Y9-B12 cells with serial dilutions of the three compounds overnight. The increased levels of enhanced green fluorescent protein indicate induction of the heat shock transcriptional response by TCA and especially by Me-TCA (Fig. 3B and C), which showed a 10-fold induction of enhanced green fluorescent protein expression compared with the vehicle control. Moreover, dihydro-TCA was inactive (Fig. 3B and C), indicating that the same structural features responsible for cytotoxicity are also involved in induction of the heat shock response.
Redox Activity of TCA
NAC Inhibits TCA Activity whereas BSO Enhances Me-TCA Activity. Oxidative stress is one of several mechanisms through which the heat shock response can be activated (4). Studies undertaken with HSF-1 in vivo and in vitro indicate that it is a redox-sensitive protein. In particular, two labile cysteine residues (disulfide bridge forming) near the DNA-binding domain are required for the trimerization of HSF-1 (12). Moreover, the generation of ROS in response to environmental conditions, such as heat, physical, or chemical insults, or to disturbances in cellular metabolism, such as ATP depletion, can lead to the transcriptional activation of HSF-1. The highly electrophilic nature of TCA suggests that it may react with nucleophilic sulfhydryl groups in the proteins that it targets. One such group is the highly reactive thiol of the peptide glutathione. To examine whether TCA forms adducts with cellular thiols, we examined the effect of the antioxidant and glutathione precursor NAC on inhibition of the cytotoxicity of TCA (Fig. 4A). 3LL cells treated continuously for 48 hours with TCA and NAC in a fixed ratio across a broad concentration range showed improved survival compared with the cells treated with TCA alone. NAC, by itself, had no effect on cell survival at any concentration. In a separate experiment, NAC, as expected, inhibited the activity of Me-TCA (Fig. 4B). On the other hand, 3LL cells treated with the glutathione-depleting agent BSO in combination with TCA were more sensitive to the cytotoxic effects of the drug than to the drug alone (Fig. 4A) whereas the cells treated with increasing concentrations of BSO were unaffected (data not shown). BSO also enhanced the activity of Me-TCA (Fig. 4B). Taken together, these data indicate that TCA and Me-TCA form adducts with cellular thiols and could be depleting cellular antioxidant defenses through this mechanism.
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Figure 4. Effect of NAC and BSO on the activity of TCA and Me-TCA. A, the glutathione precursor NAC inhibits the activity of TCA and the glutathione-depleting agent BSO enhances the activity of TCA against 3LL cells. Serial dilutions of TCA alone, TCA and NAC together (concentrations of NAC were 0.2, 0.4, 0.8, 1.6, and 3.2 mmol/L), and TCA and BSO together (concentrations of BSO were 0.125, 0.25, 0.5, 1, and 2 mmol/L) were added to the cells in triplicate. B, the glutathione precursor NAC inhibits the activity of Me-TCA and the glutathione-depleting agent BSO enhances the activity of Me-TCA against 3LL cells. Serial dilutions of Me-TCA alone, Me-TCA and NAC together, and Me-TCA and BSO together were added to the cells in triplicate as in A. Relative viable cell number was measured for both A and B after 48 h of continuous incubation in the presence of the drugs by MTT assay. BSO and NAC alone had no effect on cell survival at concentrations selected for these experiments (data not shown). Points, mean of triplicate determinations, expressed as percent of vehicle control–treated wells; bars, SD. Representative of three independent experiments.
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TCA Induces Increased Levels of ROS. To directly examine whether TCA induces the cellular accumulation of ROS, intracellular levels were examined in 3LL cells treated for 1.5 hours with TCA, Me-TCA, DMSO vehicle, or H2O2 (positive control) using the ROS-sensitive fluorescent dye DCFH-DA and a second fluorescent dye (carboxy-DCFH-DA) to control for cellular uptake. Flow cytometric analysis of treated cells indicated that 10 µg/mL of TCA led to a >2-fold increase in levels of ROS whereas an equivalent concentration of Me-TCA showed approximately thrice greater induction of ROS than TCA (Fig. 5). In a separate experiment, dihydro-TCA had no effect on cellular levels of ROS (data not shown). These findings suggest that the cytotoxicity of TCA and Me-TCA is at least in part mediated through the induction of increased levels of oxidative stress either through depletion of antioxidant thiols or through direct effects on ROS production within the cell.
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Figure 5. ROS induction activity. A, TCA and Me-TCA induce ROS in 3LL cells after 1.5 h of treatment. 3LL Lewis Lung cells were incubated with the drugs and 10 µmol/L H2O2 as a positive control for 1.5 h, and then stained with ROS-sensitive fluorescent dye DCFH-DA or with carboxyDCFH-DA to correct for the cellular uptake of the dye in the different treatments. Cells were analyzed by flow cytometry and triplicate determinations were used to calculate the mean corrected fluorescence ± SE (a commutation of error calculation was used for SE). Representative of three independent experiments.
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TCA Inhibits NF-B Activity. Mechanistic studies on sesquiterpene lactones such as parthenolide have indicated that the ROS activity of these molecules is associated with inhibition of the NF-B inflammatory response pathway (10, 11). For example, parthenolide is known to form a stable adduct with Cys179 in IB kinase ß via its exocyclic methylene moiety, thereby inactivating IB kinase ß, a key regulator of NF-B (11). Furthermore, NF-B is normally activated by increased levels of ROS and inhibited by antioxidants (13). The dual effect of inhibition of cytokine-mediated NF-B activation and the induction of ROS may elicit a major disruption of cellular homeostatic mechanisms and may be responsible for the anticancer activity of these molecules. To determine whether TCA also has anti-NF-B activity, HeLa cells were transiently cotransfected with a NF-B-luc reporter construct and a constitutively expressed Renilla construct for normalization (pRL-TK) in a dual luciferase assay. TCA showed inhibition of TNF--induced NF-B reporter activity to near basal levels (Fig. 6A). A second set of experiments was done to better asses the concentration dependence of NF-B inhibition by TCA and to show that its activity was not just cell type specific. We used a cell line derived from 293T embryonic kidney cells immortalized with the large T antigen and stably transfected with a NF-B luciferase reporter. In these cells, TCA showed pronounced and concentration-dependent inhibition of TNF--stimulated NF-B reporter activity whereas it had no effect on basal reporter activity (Fig. 6B).
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Discussion
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Cellular stress response pathways, including the heat shock response, afford the cell multiple and overlapping systems for responding to changes in the chemical, physical, and physiologic environment. In particular, heat shock gene transcription is activated in response to conditions that denature proteins (4). Basal levels of Hsp are necessary for numerous housekeeping functions including folding of newly synthesized polypeptides and maturation of numerous receptors, kinases, and transcription factors (14). Denaturing conditions such as oxidative stress induce rapid and adaptive increases in the synthesis of Hsp to help refold improperly folded proteins or shunt irreversibly damaged proteins into the proteosomal degradation pathway. As mentioned above, these same chaperones regulate the major heat shock transcription factor HSF-1 providing a positive feedback mechanism. Thus, the activity of Hsp90 and Hsp70 in refolding denaturing proteins under the conditions of stress leads to up-regulation of the heat shock response.
Oxidative stress and thermal stress are both sensed directly by HSF-1. Two cysteine residues (Cys35 and Cys105) within the DNA-binding domain of the transcription factor remain reduced under normal cellular conditions but form disulfide bonds in response to oxidative and heat stress (12). Formation of this disulfide bond leads to trimerization of HSF-1—an early step in the transactivation of heat shock response genes. Perturbations of the redox state can have major consequences on cell regulation and oxidative stress may be a universal stress response signal. Moreover, ROS are thought to act as modulators and costimulators of cytokine-mediated inflammatory responses including the stimulation of NF-B transcriptional activation (15). The transcription factor NF-B is the central and immediate early regulator of the inflammatory response; although NF-B activation is known to suppress apoptotic signaling, it is the relative amplitudes of NF-B and ROS signaling that determine whether the cell activates the cell death machinery (15). Therefore, the perturbation of the balance between these signaling pathways would be predicted to negatively affect cell survival. Importantly, here, we show that TCA inhibits NF-B while simultaneously increasing ROS resulting in anticancer activity.
Constitutive activation of the NF-B pathway is a common molecular feature of cancer cells and has been identified in patient-derived tumor samples of both hematopoietic and solid tumor origins (16). The consequences of this constitutive activation include increased survival signaling, proliferation, angiogenesis, and invasion, which are key features of the malignant phenotype (17). Moreover, tumors with constitutive NF-B activity have inherent resistance to many anticancer therapies; antineoplastic agents such as paclitaxel, vinblastine, and doxorubicin activate NF-B through a protein kinase C–dependent mechanism (18). Molecules such as TCA could serve to improve the efficacy of these conventional agents by blocking the activation of NF-B (19).
The findings presented here show that TCA represents a class of molecules that destabilize the mechanisms that enable the cell to resist cellular stress and to respond to pathogenic stimuli or infection. Cancer cells may be particularly dependent on such pathways for their survival, thus providing the basis for a potentially useful therapeutic index for compounds like TCA. We present evidence that TCA and Me-TCA induce a transcriptional heat shock response in mammalian cells (Figs. 2 and 3B). This activity is associated with increased levels of ROS (Fig. 5) and can be rescued to some extent by the antioxidant glutathione (Fig. 4). We also show that TCA leads to inhibition of cytokine-mediated activation of NF-B (Fig. 6). Further studies are warranted to learn whether TCA forms adducts with NF-B or HSF-1 directly or alters their activity indirectly by binding some other key cellular protein via its reactive methylene group. On examination of the chemical structure of TCA, however, it may be that its unique sesquiterpene carbon backbone serves to target the compound to a specific protein(s) containing a complementary binding site within its three-dimensional configuration, rather than acting as a general and highly reactive electrophile. For instance, we did not observe TCA forming adducts with DNA which contains nucleophilic groups that readily form adducts with available electrophiles (data not shown). The exocyclic methylene of the compound, in conjugation with the carbonyl group, probably then functions as an effector by forming a stable adduct with the sulfhydryl groups of cysteines of the targeted protein(s).
Evidence is rapidly accumulating that inflammatory signaling within tissues is responsible in part for the progression of transformed cells to the malignant phenotype (20) and that this process is mediated by TNF signaling and NF-B (16). In transgenic mice, in which the ability to activate NF-B has been turned off either through deletion of the IB kinase ß activator enzyme (21) or through selective activation of the NF-B inhibitor IB (22), the incidence of tumor formation is markedly reduced compared with the animals in which the pathway is active. The ability of TCA to inhibit cytokine-mediated activation of NF-B may provide a means of limiting the malignant transformation of cells or inhibiting their progression through selective inactivation of this pathway (23). Further, as ROS are known to induce cell death signaling, the inhibition of NF-B signaling in combination with the increased levels of cellular ROS in TCA-treated cells may shift tumor cells from an equilibrium that favors cell survival and proliferation to one that favors cell death and apoptosis. The findings presented here provide evidence that TCA modulates multiple cellular stress response pathways in mammalian cells and that modulation of these pathways can lead to cytotoxicity and antitumor activity. Therefore, TCA and its more potent derivative Me-TCA seem to provide attractive leads for the development of new anticancer drugs with novel mechanism(s) of action.
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Acknowledgments
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We thank Marilyn Marron and Manping X. Liu for technical assistance and for conducting heat shock induction assays, R. Dorr and M.A. Raymond for preliminary in vivo TCA activity data, and M. Tome for technical assistance with ROS assays.
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Footnotes
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Grant support: NIH/National Cancer Institute grant 1RO1 CA09025-01 A1, Arizona Disease Control Research Commission contract 30004 (A.A.L. Gunatilaka), and graduate fellowship (T.J. Turbyville) and postdoctoral support (E.M.K. Wijeratne) from the BIO5 Institute and College of Agriculture and Life Sciences, respectively, of The University of Arizona.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
3 Turbyville TJ, Wijeratne EMK, Liu MX, et al. Search for Hsp90 inhibitors with potential anticancer activity: isolation and structure-activity relationship studies of radicicol and monocillin 1 from two plant-associated fungi of the Sonoran desert. J Nat Prod, submitted for publication. 
Received 2/17/05;
revised 7/28/05;
accepted 8/11/05.
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Abstract
Introduction
