Wedelolactone, an Anti-inflammatory Botanical, Interrupts c-Myc Oncogenic Signaling and Synergizes with Enzalutamide to Induce Apoptosis in Prostate Cancer Cells

The c-Myc oncogene undergoes frequent deregulation during neoplastic transformation and plays a causal role both in the development as well as progression phases of cancer. It is upregulated in almost all cancer types and is the subject of intense investigation because of its pleiotropic effects controlling a broad spectrum of functions, including cell proliferation, metabolism, differentiation, sensitization to apoptotic stimuli, and genetic instability, which are events intimately associated with initiation, promotion, and progression phases of cancer (1, 2). Myc is a basic helix–loop–helix leucine zipper transcription factor that dimerizes with its binding partner MAX and associates with gene promoters containing the E-box motifs (CACGTG or CACATG) to induce gene transcription (2, 3). Because of its central role in oncogenesis, Myc has emerged as a promising, stand-alone molecular target for therapy of cancers afflicted with cells addicted to the c-Myc oncogene. Alhough Myc has been identified more than 30 years ago and anti-Myc agents, such as antisense oligonucleotides, siRNA, or phosphorodiamidate morpholino oligomers, have been developed, which induce tumor cell growth arrest, differentiation, or trigger apoptosis (4–7), development of direct Myc-targeting agents has yielded very limited success for clinical use, underscoring that novel therapeutic approaches should be developed to more effectively block the overactivity of c-Myc in cancer cells. However, due to its action as a transcription factor, c-Myc enjoys the benefit of elusiveness because common approaches of active site targeting do not apply to its nonenzymatic nature. This challenge is compounded by the fact that Myc regulates a plethora of target genes, the combined action of which encompasses cellular activities starting from cell proliferation to metabolic control, including cellular senescence, thus keeping both normal as well as cancer cells in its area of activity. Myc has also been well recognized to promote androgen-independent prostate cancer cell growth, suggesting that Myc-driven signaling plays a major role in advanced stages of prostate cancer (8, 9). As targeting c-Myc to block its protein–protein or protein–DNA interaction is a massive challenge, exploration of newer avenues should be encouraged, which may yield effective Myc-targeting agents and bring Myc back into the realm of “druggable targets.”

Natural products from terrestrial and aquatic plants have been a rich source of compounds for drug discovery. However, technical barriers in high-throughput assays and challenges associated with characterization of products against molecular targets have hindered proper use of these compounds in the past two decades. However, recently growing appreciation of functional assays and phenotypic screenings has contributed to the revival of interests in studying natural products for discovery and development of new drugs (10). Earlier, we reported that wedelolactone (WDL), a plant-derived natural product, strongly inhibits the growth and survival characteristics of prostate cancer cells, but spares noncancer cells in the same experimental conditions (11). The PI3K–Akt pathway is known as a promoter of cell survival and preventer of cell death (12–16). Interestingly, it was observed that WDL kills prostate cancer cells without affecting the function of Akt but does so via downregulating the activity of PKCϵ (11). Not only these findings provided indication about the involvement of a new mechanism in the anticancer action of WDL, but also suggested about the existence of an Akt-independent, PKCϵ–dependent mechanism of survival, which appears to be fundamental to the viability of prostate cancer cells. Various formulations of the source plant of WDL, Eclipta alba, have been used as remedies of a number of human ailments (17–22). Interestingly, the pure compound, WDL, has been reported to effectively inhibit the activity of 5-lipoxygenase (5-Lox) in some cells (23, 24), and 5-Lox has been recently characterized to be a regulator of c-Myc oncogenic signaling in prostate cancer cells (25). However, details of the molecular mechanisms underlying the anticancer effects of WDL in prostate cancer cells are unknown, and possible involvement of the c-Myc oncogene as a downstream target of WDL's action has never been addressed before.

It is interesting to note that prostate cancer cells show several fold increase in the expression of c-Myc gene and a multifold increase in c-Myc mRNA compared with normal cells, such as lymphocytes (26–29). Ectopic expression of c-Myc in human androgen-dependent prostate cancer cells leads them to grow without androgen stimulation and to keep their tumorigenic activity in androgen-depleted conditions (8, 29, 30), suggesting that Myc is a bona fide target for management of advanced castration-resistant prostate cancer. It has been documented that downregulation of c-Myc slows the growth of prostate cancer cells, suggesting a physiologic role for c-Myc in prostate cancer and a possible use of c-Myc as a stand-alone target for therapeutic intervention (7–9). However, c-Myc being a transcription factor, poses great difficulty to be targeted by agents to block its protein–protein or protein–DNA interactions. To get an insight into the molecular effects of WDL in prostate cancer cells, we have found that WDL dramatically decreases the mRNA and protein levels of c-Myc in prostate cancer cells in a clear dose- and time-dependent manner. Moreover, the transcriptional activity of c-Myc as well as the invasive and soft agar colony–forming abilities of prostate cancer cells are also downregulated upon treatment with WDL. We reported earlier that WDL inhibits PKCϵ while inducing apoptosis in prostate cancer cells (11) and that PKCϵ has emerged as a newly characterized signaling intermediate in the 5-oxoETE/OXER1–driven prostate cancer cell survival pathway (31, 32). PKCϵ has been found to phosphorylate and activate Stat3 (33, 34), and inhibition of Stat3 downregulates expression of c-Myc gene in prostate cancer cells (25), suggesting that the inhibitory effect of WDL on c-Myc function in prostate cancer cells may be mediated via inhibition of PKCϵ and Stat3-mediated transcription of the c-Myc gene. Altogether, these findings revealed a new mechanism of action of WDL in prostate cancer cells, and as c-Myc is intimately associated with aggressive features in prostate cancer cells, our recent findings suggest that WDL may turn out to be a novel agent to downregulate c-Myc oncogenic signaling for development of an effective strategy for prostate cancer therapy.

LNCaP and PC3 human prostate cancer cells were purchased from ATCC and certified by STR analysis (2012). Cells were grown in RPMI1640 medium (Invitrogen). All the media were supplemented with 10% FBS and antibiotics. Antibodies against c-Myc and survivin were purchased from R&D Systems, and antibodies against TMPRSS2, cyclin D1, CDK4, ATF3, TNFα, GADD45-α, and nucleoporin were from Santa Cruz Biotechnology. Phospho-c-Myc antibodies were obtained from Abcam. Polyclonal antibody against Aurora kinase was purchased from Cell Signaling Technology. Monoclonal anti-β-actin antibody, WDL, 10058-F4, MG132, calpain inhibitor, and ibuprofen were purchased from Sigma Chemical CO. Enzalutamide was purchased from Selleck Chemicals.

Cell viability was measured by MTS/PES One Solution Cell Titer Assay (Promega Corp) as described previously (11, 25).

Cells (∼3 × 10⁵) were plated overnight in RPMI1640 medium supplemented with 10% FBS onto 60-mm diameter tissue culture plates (Falcon) and allowed to grow for 48 hours. On the day of experiment, the spent culture medium was replaced with 2 mL fresh RPMI medium and the cells were treated with inhibitors. Control cells were treated with solvent (DMSO). Photographs were taken with a Nikon digital camera attached to a LEICA microscope at ×400. Image acquisition and data processing were done with a Dell computer attached to the microscope using Q-Capture 7 software.

LNCaP cells were plated and treated with 30 μmol/L WDL at 37°C. Then, the cells were harvested, washed, and total RNA was isolated from exponentially growing cells using RNeasy Mini Kit from Qiagen. For the real-time qPCR, one microgram of total RNA was used from treated and untreated samples for the reverse transcriptase reaction using High-Capacity cDNA-RT Kit from Applied Biosystems/Life Technologies. Then, the qPCR reactions were performed in triplicates using TaqMan Gene Expression Assay Kits from Applied Biosystems/Life Technologies using ABI-7500 Fast Real-Time qPCR machine.

LNCaP or PC3 cells (∼3 × 10⁵) were plated in 60-mm diameter plates and allowed to grow for 48 hours. The old medium was then replaced with 2 mL fresh RPMI medium and the cells were treated with inhibitors. After treatment, cells were harvested, washed, and lysed in lysis buffer (50 mmol/L HEPES buffer, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L orthovanadate, 10 mmol/L sodium pyrophosphate, 10 mmol/L sodium fluoride, 1% NP-40, and a cocktail of protease inhibitors). Proteins were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat milk and then blotted with appropriate primary antibody, followed by peroxidase-labeled secondary antibody. Bands were visualized by Enhanced Chemiluminescence Detection Kit from Pierce Biotechnology and analyzed with a densitometer using Kodak imaging software. Unless otherwise mentioned, protein blots were analyzed in three independent experiments.

LNCaP cells were treated with WDL, and nuclei were isolated using a kit from Sigma Chemical CO. Localization of c-Myc was detected by Western blot analysis using nucleoporin-A as a control. DNA-binding activity of c-Myc was measured using an ELISA kit from Active Motif using 4 μg of nuclear extracts per assay. Free wild-type and mutated E-box DNA sequences were used in parallel as positive and negative controls for assay validation.

LNCaP cells were transfected with lentiviral E-box luciferase constructs (>90% cells transfected), expanded, and replated in 96-well culture plates in triplicates. Cells were then treated with WDL, and the luciferase activity was measured by a Luciferase Assay Kit from Promega Corporation. Ibuprofen and F4 (10058-F4, a Myc/Max binding inhibitor) were used as negative and positive controls in parallel assays.

Invasion assay was done in Matrigel-coated Boyden Transwell chambers from BD Biosciences. Transwell chambers were soaked in 50 μL serum-free RPMI medium for 30 minutes at room temperature and then approximately 4 × 10⁴ cells (in RPMI medium containing 0.1% BSA) were placed into the upper chambers with or without drugs. These chambers were then placed in a 24-well plate (one per well) on top of 500 μL RPMI medium containing 3% FBS as chemoattractant. Inhibitors were added directly to the medium and mixed. Then, the cells were incubated at 37°C in the CO₂ incubator for 16 hours. Noninvaded cells along with Matrigel in the upper chambers were scraped with a cotton tip applicator and then the membranes were fixed in methanol, stained with 0.25% crystal violet, and observed under a Leica microscope at ×200.

Colony formation assays were performed in 6-well plates by placing approximately 15,000 LNCaP cells in 0.5 mL of 0.3% soft agar on top of a 2 mL base layer of 0.6% agar. Plates were allowed to settle, and then the wells were covered with 2 mL fresh RPMI medium containing 10% FBS with or without inhibitors. Plates were incubated at 37°C in the CO₂ incubator for a maximum period of 3 weeks. Cell growth medium and inhibitors were exchanged every fourth day. At the end of incubation, cells were stained with 0.25% crystal violet. Then, pictures were taken and colonies were counted under a Leica microscope at ×150.

Balb/c nude (Nu/Nu) mice were purchased from Charles River Laboratories and injected subcutaneously with 2 × 10⁶ LNCaP cells. They were randomized into two groups when tumors grew to approximately 100 mm³ and then treated either with the solvent or with WDL at 200 mg/kg per day 5 days a week for 4 weeks. Tumors were measured once a week with slide calipers, and volumes were calculated using the formula a(b)²/2. Formalin-fixed paraffin-embedded sections from treated and untreated LNCaP prostate tumors were deparaffinized and soaked in graded concentrations of ethanol. Then, the sections were treated with antigen retrieval buffer in the microwave (power setting: high) for 1 minute. After washing, sections were blocked in 10% horse serum for 1 hour at room temperature and then treated either with control rabbit IgG or with primary rabbit polyclonal anti-c-Myc or anti-survivin or anti-cyclin D1 antibody (1:100) overnight at 4°C, followed by secondary anti-rabbit IgG labeled with HRP. After washing, slides were stained for 30 seconds using DAB as substrate and then counterstained with hematoxylin (Harris) for 30 seconds. Blocking, antibody treatment, and color development were done using Vectastain Elite Impression kit (cat. # MP-7401; Vector Laboratories). Photographs were taken with a Nikon digital camera attached to a Leica microscope at ×600.

By real-time PCR analysis, we found that treatment with WDL decreases the levels of c-Myc mRNA in prostate cancer cells, which is clearly detectable as early as 4 hours posttreatment (Fig. 1A). We also found that WDL dramatically decreases the protein level of c-Myc in a clear dose- and time-dependent manner (Fig. 1B–E). Similar effects of WDL were found in the androgen-independent PC3 human prostate cancer cells (Fig. 1F–J). To understand the biochemical nature of the rapid protein loss, we found that WDL treatment-induced decrease in c-Myc protein is inhibited by MG132 (a proteasome inhibitor), but not by calpain inhibitor, suggesting that a proteasome-mediated degradation of c-Myc protein is triggered upon WDL treatment (Fig. 1K and L). We also found that WDL treatment induces a rapid increase in the phosphorylation of c-Myc at Thr-58 and a decrease in the phosphorylation of c-Myc at Ser-62 (Fig. 1M and N), events which are known to occur before ubiquitination and degradation of the c-Myc protein (2, 35, 36).

As the protein product of c-Myc is a transcription factor, we examined whether inhibition of 5-Lox affects the nuclear accumulation and DNA-binding activity of c-Myc. Results are depicted in Fig. 2A and B, showing significant decrease in the nuclear accumulation of c-Myc upon treatment with WDL in a dose-dependent manner. We also observed that the DNA-binding activity of c-Myc in nuclear extracts is decreased accordingly in WDL-treated cells (Fig. 2C). Moreover, we transfected prostate cancer cells with lentiviral constructs of the consensus c-Myc–binding sequence (E-box luciferase) to measure the transcriptional activity of c-Myc, which revealed that the transcriptional activity of c-Myc is reduced by WDL in a dose and time-dependent manner (Fig. 2D and E). Ibuprofen (a cyclooxygenase inhibitor) and 10058-F4 (a Myc/Max-binding inhibitor) were used as negative and positive controls, respectively, to verify measurement of Myc-specific effects.

c-Myc is well known for its strong oncogenic activity, regulating transcription of a set of genes that in turn regulate important cell functions, such as cell survival, proliferation, invasion, and metastasis. Thus, we analyzed expression of some well-characterized c-Myc target genes after WDL treatment. As the oncogenic function of c-Myc involves promotion of cell division via activation of cyclins and CDKs, and prevention of cell death via activation of antiapoptotic proteins, we examined the function of c-Myc by analyzing the regulation of well-known c-Myc targets. We observed that WDL dramatically decreases mRNA expression (ΔC_t higher) of survivin, cyclin D1, Aurora kinase, TMPRSS2, ADAMTS1, PCNA, and Gemin4, whereas it increases expression of the proapoptotic gene ATF3 (ΔC_t lower) in a time-dependent manner (Fig. 3A). We also observed that treatment with WDL decreases protein levels c-Myc targets (survivin, cyclin D1, aurora kinase, CDK4) and increases the level of ATF3 (Fig. 3B and C).

The c-Myc oncoprotein plays a pivotal role in promoting invasion and metastasis in various types of cancer, including prostate cancer (37–42). Thus, we analyzed the effect of WDL treatment on metastasis-related cell functions, which are mediated by c-Myc activity. We found that WDL decreases the Matrigel-invasive ability of prostate cancer cells in a dose-dependent manner (Fig. 4A and B). We also observed that WDL severely affects the soft agar colony–forming abilities of prostate cancer cells, suggesting that the metastatic abilities of prostate cancer cells could be significantly inhibited by WDL treatment (Fig. 4C and D). Ibuprofen, a cyclooxygenase inhibitor, was used as a negative control, which was found to be completely ineffective to inhibit invasion or colony formation under the same experimental conditions.

In a pilot experiment, we tested the in vivo effects of WDL on tumor growth, and on protein levels of c-Myc and targets using LNCaP prostate tumor xenografts treating Balb/c nude (Nu/Nu) mice with WDL at a dose of 200 mg/kg per day 5 days a week for 4 weeks via oral gavage. By immunohistochemical analysis, we found that WDL treatment significantly reduced tumor growth and decreased the protein levels of c-Myc and its targets (survivin, cyclin D1) in prostate tumor xenografts (Fig. 5A–D). As c-Myc and its targets are well known to promote all stages of cancer, including tumor formation, tumor growth as well as metastasis, our findings suggest that WDL may turn out to be an effective agent in reducing prostate tumor burden in vivo via downregulation of c-Myc oncogenic signaling. The dark yellow color in cells of treated tumors is presumably due to excessive accumulation of WDL in them.

The c-Myc oncogene is well characterized to promote androgen-independent growth of prostate cancer cells, and overactivation of c-Myc is frequently observed to be associated with castration-resistant prostate tumors, suggesting that activated c-Myc supports the development of castration-resistant prostate cancer (8, 26–30). Thus, we wanted to test our hypothesis whether WDL (by virtue of its inhibition of c-Myc) may enhance the effects of enzalutamide, an FDA-approved inhibitor of androgen receptor function, which is commonly used in the clinic to treat disseminated prostate cancers that are not manageable by surgery or radiotherapy. Interestingly, we observed that WDL cooperates with enzalutamide to inhibit prostate cancer cell growth and viability (Fig. 6A). Moreover, we observed that WDL and enzalutamide synergistically induce apoptosis to kill prostate cancer cells (Fig. 6B–D). These findings indicate that coadministration of WDL may enhance the effects of enzalutamide to reduce prostate cancer cell viability and suggest that a combination of WDL and enzalutamide may yield better clinical outcome to inhibit prostate tumor growth by killing prostate cancer cells via induction of apoptosis.

We observed that WDL, a medicinal plant–derived coumestan compound, dramatically decreases the expression and function of c-Myc in prostate cancer cells. This is especially significant because the c-Myc oncoprotein (with many of its target genes, encoding proteins that help maintain the transformed state) can initiate or promote almost all human cancers. Expression of c-Myc is frequently deregulated in a wide range of human cancers, including prostate cancer, and is often associated with aggressive, poorly differentiated tumors (1–5, 26–30). The importance of Myc as a cancer promoter stems from the fact that the Myc oncoprotein is a pleiotropic basic helix–loop–helix leucine zipper transcription factor, which coordinates expression of diverse cellular programs that together regulate a variety of cellular processes, including cell growth and proliferation, transcription, differentiation, cell motility, and apoptosis (29, 37–42). Myc coordinates a vast functionally diverse repertoire of genes that are required for orderly proliferation of cells and is functionally nonredundant. Interestingly, Myc is required by both normal and cancer cells for efficient proliferation. In normal cells, Myc is turned on by growth factor signaling, which instructs cell division cycle; however, its function in cancer cells is almost always compromised either by gene amplification or by mutation that promotes uncontrollable cell proliferation and tumor formation. Thus, Myc has repeatedly been recognized as an elusive molecular target for cancer therapy, which triggered interest both in searching for its upstream and downstream regulators and also in discovering agents that counteract the role it plays in transformation and therapy resistance.

Previously, we reported that WDL reduces the viability of both androgen-sensitive (LNCaP) as well as androgen-independent (PC3, DU145) human prostate cancer cells, without affecting normal, noncancer prostate epithelial cells in the same experimental conditions (11). As many prostate cancer cells (such as LNCaP, PC3) bear Myc overactivation and are driven by Myc function for maintaining their cancer phenotype, we took advantage of using these cell culture models to address the question of the modulation of c-Myc by WDL. Our observation of the decrease in c-Myc protein level as well as its DNA-binding and E-box luciferase activities suggest that c-Myc in prostate cancer cells can be effectively downregulated by WDL (Figs. 1 and 2). Moreover, the inhibition of c-Myc by WDL in prostate cancer cells is correlated with downregulation of the expression of its target genes, such as survivin, aurora kinase, and cyclin D1 (Fig. 3). These findings opened up the possibility to control the expression and function of c-Myc in prostate (and possibly other types of) cancer cells by small-molecule chemical inhibitors of 5-Lox, such as WDL. Ibuprofen, a cyclooxygenase inhibitor, was used as a negative control, which was found to be ineffective. c-Myc has been well characterized to play a pivotal role in promoting metastasis in various types of cancer cells through defined mechanisms (37–42). Thus, we analyzed the effect of WDL treatment on the invasive and soft agar colony–forming abilities of prostate cancer cells. We observed that WDL effectively blocked both invasion and anchorage-independent colony formation by prostate cancer cells on soft agar (Fig. 4). Moreover, we observed that oral administration of WDL exerts significant in vivo effect in inhibiting growth of tumors and decreasing expression of c-Myc and its targets in nude mice xenografts (Fig. 5), which suggests that WDL possesses all the desired characteristics of a standard chemotherapeutic agent, such as good solubility, bioavailability, and in vivo efficacy.

Transition of prostate cancer from an androgen-dependent to an androgen-independent phenotype is a complex process that presumably involves both the selection of preexisting clones of androgen-independent cells as well as selection for genetic events that help the cancer cells survive and grow in an environment devoid of androgenic signaling. One molecular mechanism of developing androgen independence by which prostate cancer cells grow and proliferate independently of AR and androgen has been suggested to be based on Myc activation. c-Myc has been strongly implicated in the development and progression of castration-resistant prostate cancer. Several studies have detected a common amplicon during the conversion to androgen-independent prostate cancer in a short region spanning chromosome 8q and containing the c-Myc gene (26, 27). In clinical samples, amplifications of c-Myc gene has been found in more than 70% of androgen-independent prostate tumors by FISH, and a significant increase in c-Myc amplification was observed after anti-androgen treatment (26–30). Bernard and colleagues examined the effect of c-Myc in androgen-independent prostate cancer progression by using LNCaP cells treated with the antiandrogen Casodex, which showed that overexpression of c-Myc was sufficient to induce androgen-independent growth in Casodex-treated cells (8). Interestingly, we observed that low-dose WDL synergizes with enzalutamide to reduce viability and to induce apoptosis in prostate cancer cells (Fig. 6), suggesting that WDL may yield better results in prostate cancer therapy when used in combination with enzalutamide.

How WDL downregulates a strong oncogene like c-Myc in prostate cancer cells is an intriguing but open question. Previous findings documented that WDL is a potent inhibitor of 5-Lox (IC₅₀ = 2.5 μmol/L), which inhibits the enzymatic activity of 5-Lox by an oxygen radical scavenger mechanism (23, 24), although WDL also inhibits other molecules at higher concentrations (22, 43). The enzyme 5-Lox is well known to process arachidonic acid to generate leukotrienes, which play a major role in asthma. Recent studies have demonstrated an important role of 5-Lox in the regulation of c-Myc oncogenic signaling in prostate cancer cells (25, 44). Thus, 5-Lox has emerged as a potential molecular target for therapeutic development against Myc-driven prostate cancer. However, lingering problems with solubility and bioavailability of several available 5-Lox inhibitors have limited their use for prostate cancer therapy. On the basis of published reports about the 5-Lox–inhibitory effect of WDL, we expected that WDL would inhibit c-Myc and induce apoptosis in prostate cancer cells as we recently observed with other 5-Lox inhibitors (31, 32). Here, we observed that WDL induces dramatic loss of c-Myc in prostate cancer cells, and this phenomenon is correlated with our previous observation of the inhibition of PKCϵ by WDL (11). PKCϵ activates Stat3 via phosphorylation at Ser-727, which has been found to be intimately linked with prostate cancer (33, 34). As Stat3 regulates c-Myc in prostate cancer cells (25), WDL may downregulate c-Myc via inhibition of Stat3-mediated transcription.

Prostate cancer is the most common form of malignancy and second leading cause of cancer-related deaths in men in the United States (45). Although prostate cancer initially responds to antiandrogenic therapy, androgen-resistant disease almost always develops (46, 47). Development of androgen-resistant metastatic prostate cancer always ends up with a fatal outcome because currently there is no effective therapy available for this type of prostate cancer. Thus, novel agents and strategies are urgently needed to improve treatment options for androgen-resistant prostate cancer. The universal deregulation of c-Myc gene expression in tumor cells makes inhibition of c-Myc an attractive pharmacologic approach for treating diverse types of cancer, including prostate cancer, although direct targeting of c-Myc yielded no significant clinical benefit (48, 49). Plus the enthusiasm of direct targeting of c-Myc has been muted by lack of evidence that c-Myc inhibition would be therapeutically efficacious, raising concerns that it would induce serious side effects by inhibiting normal, noncancer cells where c-Myc is also functional. Under the circumstances, our findings revealed a novel strategy to silence c-Myc by WDL in prostate cancer cells, which may open up new directions to monitor the oncogenic action of c-Myc and, thus, may help overcome practical difficulties in designing direct inhibitors of Myc. On the basis of the potency, solubility, and selectivity profile of WDL against prostate cancer cells in vitro and in vivo, it appears that WDL is a promising candidate drug and should be tested further for development of a novel prostate cancer treatment strategy. As Myc plays a critical role both in the development and maintenance of transformed phenotype, our observations of the regulation of expression and function of c-Myc not only reveal an unknown mechanism of action of WDL but also open up a new avenue to utilize natural agents in prevention and treatment of prostate cancer, especially Myc-driven advanced prostate cancer.

No potential conflicts of interest were disclosed.

Conception and design: S. Sarveswaran, J. Ghosh

Development of methodology: S. Sarveswaran, J. Ghosh

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R. Ghosh, R. Parikh, J. Ghosh

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Sarveswaran, J. Ghosh

Writing, review, and/or revision of the manuscript: S. Sarveswaran, J. Ghosh

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Ghosh

Study supervision: J. Ghosh

The authors thank Dr. Zhou for his help with the qPCR experiments.

This study was supported by the NCI of the NIH under award number RO1 CA 152334, the Department of Defense Prostate Cancer Research ProgramW81-XWH-05-1-0022, and the Henry Ford Health System internal grant A10203 (to J. Ghosh).

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.