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. Author manuscript; available in PMC: 2015 Mar 1.
Published in final edited form as: Cancer Res. 2014 Jan 3;74(5):1506–1517. doi: 10.1158/0008-5472.CAN-13-2939

Bufalin is a potent small molecule inhibitor of the steroid receptor coactivators SRC-3 and SRC-1

Ying Wang 1,#, David M Lonard 1,*,#, Yang Yu 1, Dar-Chone Chow 2, Timothy G Palzkill 2, Jin Wang 2, Ruogu Qi 2, Alexander J Matzuk 2, Xianzhou Song 2, Franck Madoux 3, Peter Hodder 3, Peter Chase 3, Patrick R Griffin 3, Suoling Zhou 1, Lan Liao 1, Jianming Xu 1, Bert W O’Malley 1,*
PMCID: PMC3947477  NIHMSID: NIHMS553061  PMID: 24390736

Abstract

Virtually all transcription factors partner with coactivators that recruit chromatin remodeling factors and interact with the basal transcription machinery. Coactivators have been implicated in cancer cell proliferation, invasion and metastasis, including the p160 steroid receptor coactivator (SRC) family comprised of SRC-1 (NCOA1), SRC-2 (TIF2/GRIP1/NCOA2), and SRC-3 (AIB1/ACTR/NCOA3). Given their broad involvement in many cancers, they represent candidate molecular targets for new chemotherapeutics. Here we report on the results of a high throughput screening effort which identified the cardiac glycoside bufalin as a potent small molecule inhibitor for SRC-3 and SRC-1. Bufalin strongly promoted SRC-3 protein degradation and was able to block cancer cell growth at nanomolar concentrations. When incorporated into a nanoparticle delivery system, bufalin was able to reduce tumor growth in a mouse xenograft model of breast cancer. Our work identifies bufalin as a potentially broad-spectrum small molecule inhibitor for cancer.

Keywords: Natural products, screening strategies, nuclear receptors, structure and function, new targets

Introduction

The Steroid Receptor Coactivator (SRC) family comprises three members, SRC-1 (NCOA1) (1), SRC-2 (NCOA2) (2, 3) and SRC-3 (NCOA3) (4-7). Numerous studies have established broad molecular and physiological roles for the SRC family in activating nuclear receptor (NR) mediated gene expression (8). Besides NRs, they also coactivate other transcriptional factors (TFs) including NF-κB (NFKB1), E2F1 and insulin-like growth factor (IGF1) dependent TFs (9-11). The expansive roles that they play in mediating gene expression is consistent with a breadth of studies pointing to their regulation of diverse physiological and pathophysiological processes, including cancers where they are frequently overexpressed (8).

Among the SRCs, SRC-3 has been the family member most prominently linked to a wide variety of cancers. SRC-3 overexpression in the mouse mammary gland leads to spontaneous tumor formation (12). In contrast, loss of SRC-3 suppressed oncogene- and carcinogen-induced breast cancer initiation, progression and metastasis in mouse tumor models (13, 14). In humans, SRC-3 gene amplification has been found in 9.5% breast cancers and the mRNA for SRC-3 is overexpressed 64% of the time (4). Clinical and preclinical studies have shown that overexpression of SRC-3 and SRC-1 is linked to resistance to endocrine therapies in breast cancers. For instance, high expression of SRC-3, especially conjunction with high levels of EGFR and HER2 (ERBB2), is associated with poor outcome and recurrence after tamoxifen treatment (15). In ERBB2-overexpressing breast cancer cells, overexpression of SRC-3 also contributes to resistance against the ERBB2 targeting drug trastuzumab (Herceptin) (16). SRC-3 overexpression also has been observed in a wide range of other cancers including ovarian (17), endometrial (18), prostate (19, 20), liver (21), pancreatic (22), colorectal (23) and lung cancers (24).

SRC-1 also has been clearly implicated in cancer progression. In a mouse mammary tumor virus polyoma middle T (MMTV-PyMT) model system, loss of SRC-1 markedly reduces tumor cell metastasis (25). Consistent with this, elevated SRC-1 expression has been reported in approximately 20% primary human breast cancers, with higher expression positively correlating with disease recurrence and poor survival (26, 27). A positive association between high SRC-1 expression and tumor recurrence in patients with breast cancer who received tamoxifen therapy also has been reported (26, 27).

Considering the ability of SRC coactivators to activate multiple growth factor signaling pathways that drive cancer cell growth and promote resistance to endocrine therapy, SRC small molecule inhibitors (SMIs) are anticipated to be effective new agents to treat cancer. In an initial “proof-of-principle” study, we recently showed that gossypol is able to inhibit SRC-3 and SRC-1 (28), demonstrating that these oncogenic coactivators are a class of accessible targets for SMI-based chemotherapy. This work established the feasibility for engaging in a high throughput compound library screen to identify more effective SRC SMIs. As a result of these high throughput screens, here we describe the characterization of the cardiac glycoside (CG) bufalin as potent SRC SMI that is able to effectively reduce SRC-3 and SRC-1 cellular protein concentration and block cancer cell growth in cell culture and animal models.

Materials and Methods

Chemicals

For high throughput screening, a MLSMR library was provided by Evotec (South San Francisco, CA) through the NIH’s Roadmap Molecular Libraries Initiative. Details regarding compound selection for this library can be found online (29). Digoxin, bufalin, ouabain and digitoxin were obtained from Sigma (St. Louis, MO) and dissolved in ethanol. Cinobufagin, cinobufotalin, cycloheximide and MG132 were obtained from Sigma and dissolved in dimethyl sulfoxide (DMSO). Strophanthidin and resibufogenin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and dissolved in ethanol and DMSO respectively. MK-2206 was purchased from SelleckChem (Houston, TX) and dissolved in DMSO. Antibodies to SRC-1, SRC-3 and GAPDH were purchased from Cell Signaling (Danvers, MA). Antibodies to CARM1 and SRC-2 were obtained from Bethyl Laboratories (Montgomery, TX).

Cell culture

Human cancer cell lines were obtained from the American Type Culture Collection (ATCC) (Manassas, VA). Cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (HeLa; MCF-7) and RPMI1640 (A549) supplemented with 10% fetal calf serum, penicillin and streptomycin (100 U/ml), unless otherwise indicated. All cells were cultured at 37°C under 5% CO2. All cells were used within six months from the time when they were obtained from ATCC, expanded and resuscitated except for MDA-MB-231-LM3.3 which were verified to be derived from MB-MBA-231 cells by short tandem repeat DNA profiling prior to use in xenograft animal model experiments.

Plasmids and transfections

The expression vectors for the GAL4-responsive luciferase reporter pG5-LUC and Gal4 DBD fusion proteins with SRC-1, SRC-2 and SRC-3 were described previously (30). Twenty-four hours before transfection, HeLa cells were plated in 24-well dishes. Cells were transfected with the indicated expression vector plasmids using Lipofectamine LTX reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol before incubation with chemicals at the indicated concentrations.

Cell extraction and assays

For luciferase assays, collected cell pellets after treatments were lysed and assayed for luciferase activity. Luciferase activities were normalized against Renilla luciferase activities according to the manufacturer’s recommendations (Promega, Madison WI). For Western blotting, cells were harvested and lysed in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% NP40) and then centrifuged for 15 min at 21,000 × g at 4°C. After total cellular protein concentration was determined by Bradford analysis (Bio-Rad, Hercules, CA), protein lysates were resolved by sodium dodecyl sulfate-7.5% polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (BioRad). Membranes were blocked and incubated with indicated antibodies as previously described (28). All experiments were repeated at least three times. Intensities of the bands of interest in the Western blotting analysis were quantitated using Image J software (31).

1,536-well plate SRC-1 and SRC-3 HTS assays

A detailed protocol for the HTS screening assay can be found on the PubChem Bioassay website (32).

Quantitative PCR analysis

MCF-7 cell total RNAs were isolated from 12-well culture dishes using the RNeasy mini Kit (Qiagen, Valencia, CA). The mRNAs for SRC-1, SRC-2, SRC-3 and GAPDH were quantitated by Taqman-based reverse transcriptase PCR (RT-PCR) using the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA). For SRC-1, the primer pair 5′-gcaaccagctctcatccact-3′ and 5′-gacgtcagcaaacacctgaa-3′ was used along with Universal Roche Probe #3 (Roche, Indianapolis, IN). For SRC-2, the primer pair 5′-aggcaacctgttcccaaac-3′ and 5′-actggcttcagcagtgtcag-3′ was used along with Universal Roche Probe #27. For SRC-3 the primer pair 5′-agctgagctgcgaggaaa-3′ and 5′-gagtccaccatccagcaagt-3′ was used with Universal Roche Probe #70. For GAPDH, the primer pair 5′-agccacatcgctcagacac-3′ and 5′-gcccaatacgaccaaatcc-3′ was used along with Universal Roche Probe #60. RT-PCR was performed using 1× TaqMan Universal PCR Master Mix (Applied BioSystems). All mRNA quantities were normalized against GAPDH RNA and experiments were repeated two times.

Fluorescence spectrometry

The GST fusion proteins of different portions of SRC-3 were expressed and purified as described previously (33). Fluorescence spectrometric measurements were performed using a SLM 48000S fluorescence spectrophotometer (SLM-Aminco, Rochester, NY) and an Agilent Cary Eclipse Fluorescence spectrophotometer (Agilent Technologies, Inc., Santa Clara, CA). . A total of 1.5 μM of GST SRC-3 RID, CID or bHLH was placed in a fluorescence cuvette and excited with UV light at a wavelength of 278 nm with a 2 nm bandwidth and the emission spectra were recorded from 295 nm to above 400 nm with a bandwidth of 4 nm. The aliquot size of test compound was maintained below 5% of the total sample volume in order to minimize the effects of dilution.

Cell viability assays

Cells were seeded in 96-well plates in medium supplemented with 10% fetal calf serum and allowed to reach 70% to 80% confluence while relative numbers of viable cells were measured with the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega) after compound treatment. IC50 values were determined using the Prism 4 software (Graphpad Software, Inc.).

Bufalin PLGA nanoparticles

A block copolymer of poly(lactic-co-glycolic acid) and poly(ethylene glycol) (PLGA-PEG) was chosen as a nanocarrier material. PLGA-PEG was synthesized and bufalin containing nanoparticles (NPs) were produced using a nano-precipitation method. Specifically, a mixture of bufalin and PLGA-PEG in trifluoroethanol (TFE) was dripped into water under with constant stirring. The formed bufalin NPs were verified for proper formation by dynamic light scattering (DLS) and transmission electron micrography (TEM). The amount of drug encapsulated in NPs was determined to be 1% by weight using high performance liquid chromatography (HPLC) followed by detection of absorption at a wavelength of 210 nm.

MDA-MB-231-LM3.3 xenograft tumor model

Six to seven week old SCID mice were obtained from Charles River laboratories. 750 thousand MDA-MB-231-LM3.3 cells were injected into one of the second mammary fat pads on day 1 with tumors becoming palpable in six days. At this time, animals were treated with either phosphate buffered saline (PBS), empty nanoparticles (NPs) or bufalin-loaded NPs three times per week. Tumor length and width were measured daily, with volume estimated using the formula (length × width × height)/2.

Results

High throughput screening of SRC-3 and SRC-1 inhibitors

Through a high throughput luciferase assay-based screen, a MLPCN chemical library containing 359,484 compounds (34) was screened to identify compounds capable of inhibiting the intrinsic transcriptional activities of SRC-3 (PubChem AID:588362) and SRC-1 (PubChem AID:588354) (Fig. 1). Here, compounds were evaluated by measuring luciferase expression from cells transiently transfected with a GAL4 responsive luciferase reporter (pG5-LUC) and an expression vector for either a GAL4 DNA binding (DBD) SRC-3 or GAL4 DBD SRC-1 fusion protein (30). Compounds that inhibited luciferase gene expression greater than 3σ over DMSO were counted as SMI hits. In this primary screen, the transfected HEK293 cells were treated with test compounds at a concentration of 3.6 μM in 0.36% DMSO. 36 μM gossypol was used as a positive control which was able to elicit 100% inhibition. Based upon a 3s cut-off, 620 (0.17%) compounds were able to inhibit SRC-3 and 428 (0.12%) compounds were able to inhibit SRC-1. These active compounds were then tested in the confirmatory screen in the same manner as the primary screen in quadruplicate and in a counter screen using cells transfected with an expression vector for a GAL4 DBD VP16 fusion protein instead of an expression vector for either GAL4 DBD SRC-3 or SRC-1 to exclude general inhibitors of transcription and/or luciferase activity.

Figure 1.

Figure 1

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A flowchart of the high throughput screen to identify inhibitors targeting SRC-3 and SRC-1. The PubChem ID for each individual assay (AID) has been indicated and summaries results from these assays have been deposited to the PubChem database (50).

Structure-activity cluster analyses identify cardiac glycosides as a functional group of SRC-3 and SRC-1 SMIs

Active compounds retrieved from the primary screens were clustered according to structural similarities in PubChem (data not shown). The biggest cluster contains 25 compounds, sharing a common steroid nucleus, the majority of which contain a lactone moiety characteristic of CGs (35). CGs are known to inhibit the Na+/K+ ATPase in cardiac myocytes, leading to an increase in intracellular Ca2+ and stronger myocardial contraction (36). Interestingly, numerous epidemiological studies revealed that patients taking CGs have either better outcome or lower risk for various cancers, including breast cancers (37, 38), leukemia/lymphoma (39) and prostate cancer (40), although the mechanism has hitherto been unknown.

Evaluation of CG family members for their efficacy as SRC SMIs

Because a cluster of CG series was identified as active compounds in our high throughput screens, we sought to evaluate a panel of CGs to identify which were most potent as SRC SMIs. We examined SRC-3 protein concentrations in MCF-7 cells treated for 24 hours with CGs, including ouabain, digitoxin, strophanthidin, cinobufagin, cinobufotalin and resibufogenin. All tested compounds down regulated cellular SRC-3 protein levels, but at varying doses (Fig. 2). Ouabain and digitoxin appeared to be more effective than digoxin while bufalin was found to be the most potent of all. In comparison, strophanthidin also reduced SRC-3 protein, but at a higher dose than digoxin. Bufalin was the most potent bufadienolide, followed (in order) by cinobufagin, cinobufotalin and resibufogenin.

Figure 2.

Figure 2

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A panel of cardiac glycosides can reduce cellular SRC-3 protein concentrations in MCF-7 breast cancer cells. Cells were treated with plant derived CGs (ouabain, digitoxin, digoxin and strophanthidin) and Asiatic Toad derived GGs (bufalin, cinobufagin, cinobufotalin and resibufogenin) at increasing doses for 24 hours, then SRC-3 protein levels were determined by Western analysis.

Inhibition of SRC-3 and SRC-1 by digoxin and bufalin

Next, we chose to investigate the effects of CGs on the intrinsic transcriptional activities of SRC coactivators. HeLa cells were transiently transfected with a pGL5-LUC reporter and expression vectors for pBIND, pBIND-SRC-1, -SRC-2 or -SRC-3, followed by 24 hours treatment with the plant based CG digoxin or the toad CG bufalin. Digoxin reduced luciferase reporter activities in cells transfected with pBIND-SRC-1 and pBIND-SRC-3 (Fig. 3A). In contrast, reporter activities driven by the GAL4 DBD alone were only slightly affected (pBIND, inset to the right). Similarly, administration of bufalin led to a significant decrease in pBIND-SRC-1 and pBIND-SRC-3 activities in a dose dependent manner, while only minimally affecting the activity of pBIND (GAL4 DBD alone) and influencing pBIND-SRC-2 less strongly (Fig. 3B). This result suggests that both digoxin and bufalin preferentially inhibit the intrinsic transcriptional activities of SRC-3 and SRC-1, while inhibiting SRC-2 to a lesser extent.

Figure 3.

Figure 3

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Digoxin and bufalin can selectively reduce the intrinsic transcriptional activities of SRC-1 and SRC-3. Luciferase assays were performed in HeLa cells transiently transfected with the reporter vector pG5-LUC in combination with expression vectors for pBIND, pBIND SRC-1, SRC-2 or SRC-3 prior to incubation with (A) digoxin (0, 500nM and 1μM) and (B) bufalin of different doses (0, 2, 5 and 7.5 nM) for 24 hours. Difference between the vehicle treated control and compound treated samples were analyzed for significance by a Student’s t-test, with the number of p value indicated above the column. Only p values less than 0.05 were shown in the chart. Insets in the right show the outputs with the empty vector pBIND.

Effects of digoxin and bufalin on cellular protein concentrations of SRCs and other coactivators

Because the steady-state levels of coactivator proteins have been shown to correlate with their transcriptional activities and with cancer progression (33), we sought to examine the effects of digoxin and bufalin on SRC protein levels in MCF-7 breast and A549 lung cancer cells after 24 hours of incubation. As shown in Figure 4 A-C, SRC-3 and SRC-1 protein levels were significantly reduced both by digoxin and bufalin in a dose-dependent manner, suggesting that the loss in SRC-3 and SRC-1 intrinsic activities seen above is due to the reduced cellular concentration of either coactivator. In agreement with the finding that SRC-2 activities are less strongly affected, both digoxin and bufalin have more modest effects on cellular level of the SRC-2 protein. We then investigated the steady-state level of CARM1 that has been shown to exist in a multi-protein coactivator complex along with SRC-3 (41). In contrast to the noticeable decrease in SRC-3 protein level, CARM1 protein level is not altered after treatment in A549 cells but was reduced moderately in MCF-7 cells.

Figure 4.

Figure 4

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Digoxin and bufalin selectively reduce cellular protein levels of SRC-3 and SRC-1, but not that of SRC-2. (A) MCF-7 cells were treated with digoxin (0, 0.5, 1 and 2μM) for 24 hours. Cell extracts were then blotted and probed using antibodies against SRC-1, SRC-2, SRC-3, CARM1, actin and GAPDH. (B) A549 cells were treated with bufalin (0, 1, 2 and 5 nM) and (C) MCF-7 cells treated with bufalin (0, 1, 2, 3, 4 and 5 nM), and analyzed as described in (A). Relative intensities of bands were normalized by either GAPDH or by Actin as shown.

Digoxin and bufalin inhibit cancer cell proliferation

Next, we assessed the effects of digoxin and bufalin on cancer cell proliferation. First, MTS assays were performed on MCF-7 cells treated with digoxin at different doses for 24 hours. This revealed that digoxin can block MCF-7 cell growth with IC50 of about 500 nM (Fig. 5A left panel), in line with the dose of digoxin required to reduce SRC-3 protein levels in the cell (Fig. 5A right panel). In contrast, the IC50 of bufalin in MCF-7 and A549 cells were below 5 nM in either cell lines (Fig. 5B and C, left panels). Again, the dose of bufalin required to block cancer cell proliferation corresponds with the concentration required to cause downregulation of the SRC-3 protein (Fig. 5B and C, right panels). Differential dependence of distinct cell types toward each of the three SRCs could account for the differences in bufalin effect on cell viability and SRC-3 cellular protein concentration. Importantly, the concentrations of bufalin required to kill cancer cells tested here is less than the reported human maximum tolerated dose (MTD) of 8.75 nM (42), pointing to its potential clinical use as an anticancer agent. For this reason, we chose to focus on bufalin for further characterization as a SRC SMI.

Figure 5.

Figure 5

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Digoxin and bufalin block breast and lung cancer cell proliferation. (A) Digoxin reduced MCF-7 cell viabilities in line with SRC-3 protein inhibition in a dose-dependent manner. (Left panel) Cells were treated with different doses (0, 1, 2, 5, 10, 20, 50, 100, 200, 500 nM, 1 and 2 μM) of digoxin for 24 hours, then cell viability was determined by MTS assays. Collected data were normalized to the vehicle treated control. Each data point represented the average of relative values derived from three wells of digoxin treated cells at the indicated dose. Nonlinear regression was used to determine the point of 50% inhibition for cell viability (IC50). (Right panel) Relative SRC-3 protein levels after digoxin (0.5, 1 and 2 μM) treatment compared with the vehicle control (derived from Western data in Figure 4) are shown as the blue dashed line. (B-C) Bufalin reduced (B) A549 and (C) MCF-7 cell viabilities in accordance with SRC-3 protein degradation with increasing doses. (Left panels) The dose responses for cell viabilities upon bufalin treatment (0, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500 and 1000 nM) were analyzed as described above. (Right panels) Relative SRC-3 protein content after bufalin treatment (0, 1, 2 and 5 nM) in A549 cells (derived from blotting shown in Figure 4) and treatment (0, 1, 2, 5 and 10 nM) in MCF-7 cells are shown as the blue dashed line. (D) Knockout of the SRC-3 protein attenuates the inhibitory effects of bufalin on cell viability. HeLa parental and SRC-3 knockout cells (SRC-3KO) were treated with bufalin at different doses (0, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500 and 1000 nM) for 24 hours and cell viability was determined by MTS assay. HeLa parental cells are shown as the red solid line and SRC-3KO are shown as the blue dashed line.

Accumulating evidence has shown that targeting SRC-3 expression impairs cancer cell growth in multiple cancer types (8). Given the fact that the decreased cell viability induced by bufalin is accompanied by reduced SRC-3 protein levels, we sought to investigate the specific role of the SRC-3 protein in blocking cancer cell proliferation. To do this, we used a zinc finger nuclease (ZFN) to knockout both SRC-3 alleles in the HeLa cell line (SRC-3KO cells). It was difficult to obtain viable SRC-3KO cells initially, however we noted that after two months, they proliferated at a rate comparable to wild type cells, likely after adapting genetically to the loss of SRC-3. As shown in Figure 5D, SRC-3 protein is abolished in these SRC-3KO knockout cells. Compared with parental SRC-3+/+ cells, the response of SRC-3KO cells to bufalin administration is blunted. This finding supports the idea that SRC-3 protein is involved in mediating the cell response to bufalin treatment. However, the remaining response of SRC-3KO cells to bufalin, including the lack of an increase in the effective concentration required for these cells to respond to treatment, is likely due to the SRC-1 and SRC-2 that continues to be expressed in these cells and that also responds to bufalin at a similar dose as SRC-3, in addition to any unknown off-target actions of the compound.

Bufalin promotes proteasome-mediated degradation of SRC-3 and binds directly to the coactivator

To gain insights into the mechanism of bufalin-mediated SRC-3 protein downregulation, we assessed whether 24 hours of bufalin treatment affected the production of mRNAs for each SRC family member in MCF-7 cells. Quantitative PCR revealed that mRNAs levels for SRC-1 and SRC-2 were not significantly altered, whereas the mRNAs for SRC-3 actually was increased upon bufalin incubation (Fig. 6A). This result suggested that bufalin reduces SRC-3 and SRC-1 protein levels posttranscriptionally.

Figure 6.

Figure 6

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Bufalin promotes proteasome-mediated degradation of the SRC-3 protein. (A) Effects of bufalin on the mRNA levels of SRC-1, SRC-2 and SRC-3. MCF-7 cells were incubated with bufalin (0, 1, 2 and 5 nM) for 24 hours and total RNA was extracted and analyzed by quantitative-PCR. Differences between the control and the treated samples were analyzed for significance using a Student’s t-test. Only p values less than 0.05 are shown in the chart above its corresponding column. (B) Effects of bufalin on SRC-3 protein turnover rates. MCF-7 cells were treated with cycloheximide (CHX) (200 μg/ml) and bufalin (50 nM), individually or simultaneously, and then harvested at 0, 0.5, 1, 1.5, 2, 2.5, 3 and 3.5 hours thereafter and visualized by Western blotting analysis. (C) The proteasome inhibitor MG132 prevents SRC-3 protein downregulation induced by bufalin. MCF-7 were changed to medium supplemented with 0.5% stripped FCS and penicillin and streptomycin (100 U/ml) overnight, then incubated with bufalin (0, 1, 3 and 5 nM) in the absence or presence of 5 μM MG132 for 24 hours. Cell extracts were collected and analyzed by Western blotting using antibodies against SRC-3 and GAPDH. (D). Bufalin directly binds to the receptor interacting domain (RID) of SRC-3. Intrinsic tryptophan fluorescence emission spectra of SRC-3 RID (λex=278 nm) was quenched and red-shifted with increasing concentrations of bufalin.

To further investigate the underlying mechanism responsible for bufalin-mediated SRC-3 protein downregulation, we compared the turnover rates of the SRC-3 protein in MCF-7 cells between vehicle and bufalin treatment in the presence of the protein synthesis inhibitor cycloheximide (CHX). Cells were treated with CHX and bufalin, individually or simultaneously, and then harvested at the indicated time points for Western blotting analysis. In line with a previous report (43), SRC-3 decays with a four hour half-life (Fig. 6B). Addition of bufalin accelerated the rate of SRC-3 turnover, indicating that bufalin promotes degradation of the SRC-3 protein.

Our lab has previously shown that all SRC coactivators are targets of the proteasome, as evidenced by the observation that treatment with the proteasome inhibitor MG132 increases their protein levels (30). When we examined the effects of concomitant treatment with MG132 and bufalin ranging from 1 nM to 5 nM on SRC-3 protein levels in MCF-7 cells (Fig. 6C), MG132 treatment alone was able to strongly elevate the SRC-3 protein levels, consistent with that previously reported. The reduction in SRC-3 protein level induced by bufalin treatment was blocked by MG132, leading to protein levels up to a point comparable to cells treated with MG132 alone.

Next, we sought to determine if bufalin promoted SRC-3 protein degradation through physical interaction with the SRC-3 protein. Through fluorescence shift analysis of SRC-3 protein fragments, bufalin was found to quench the intrinsic fluorescence and shift the emission maximum of the receptor interacting domain (RID) of SRC-3 (Fig. 6D), indicating that bufalin binds directly to the RID of SRC-3. In contrast, there are no changes in the intrinsic fluorescence observed for the CBP interaction domain or the basic helix-loop-helix domain of SRC-3 upon the addition of bufalin (data not shown), which mitigates the possibility that the fluorescence changes seen for the RID fragment are due to non-specific interactions with bufalin. Due to the high affinity of bufalin binding to the SRC-3 RID, it is difficult to precisely determine its affinity, however binding was detected even with 5 nM of bufalin, consistent with its potency in cell culture experiments.

Bufalin sensitizes cancer cells to other targeted therapeutics

Accumulating evidence shows that SRC coactivators sit at a nexus linking diverse growth signaling cascades (see Introduction). This role for SRCs supports the notion that a SRC SMI such as bufalin should break crosstalk between different growth factor pathways, attenuating their ability to signal to downstream pathway components responsible for enacting cellular transcriptional programs that drive cell proliferation, invasion and metastasis. SRC-3 has been shown to activate PI3K/AKT/mTOR signaling (44) and we wanted to ask whether the inhibitory effects of an AKT inhibitor on cell viability could be enhanced by co-treatment with bufalin. To test this, we treated A549 cells with a low dose of bufalin in combination with the AKT inhibitor MK-2206. As shown in Figure 7A, combined treatment with bufalin and MK-2206 exhibited a markedly higher ability to block A549 cell proliferation than either agent alone.

Figure 7.

Figure 7

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(A) Bufalin sensitizes cancer cells to other targeted chemotherapeutic agents and blocks tumor growth in vivo. When combined with 1 nM or 2 nM bufalin, co-treatment with the Akt inhibitor MK-2206 significantly reduced lung cancer A549 cell viability (1, 2 and 5 μM). Cell viability was determined by MTS assays, with data normalized to the vehicle treatment control, set to 100%. Each data point represented the average from three data points. (B) Non-transformed TM4 Sertoli cells were treated with bufalin at the indicated concentrations. 72 hours after treatment, cell viability was determined via MTS assay. (C) Bufalin NP inhibition of tumor growth in vivo. SCID mice were injected with 750 thousand LM3.3 cells into the 2nd mammary gland (cleared) with two sites per mice. Six days after tumor cell injection, mice were treated with PBS vehicle, bufalin NP (1.5 mg/kg) or with blank NP that were not loaded with bufalin three times per week (p<0.05 *, p<0.01 ** for comparisons between blank NP and bufalin-loaded NP).

A bufalin nanoparticle formation can block tumor cell growth in a xenograft model

Bufalin is an excellent candidate for incorporation into a nanoparticle (NP) based delivery system because it can specifically kill cancer cells (IC50 3-5 nM) without observable toxicity in non-transformed TM4 Sertoli cells at doses up to 10 μM (Fig. 7B) or primary hepatocytes even at doses as high as 300 nM (data not shown), but possesses significant cardiotoxicity – a fact we substantiated in our own animal studies with free bufalin (data not shown), and it has a short in vivo half-life. With “free bufalin”, we observed a response in metastatic tumor lesions using an in vivo model described below, but we were unable to achieve a satisfactory response in primary tumors, possibly due to dose limiting toxicities or to inadequate drug penetration into larger tumors (data not shown). Already, a wheat germ agglutinin-grafted lipid bufalin nanoparticle has been developed to allow for its oral delivery and improve its stability (45). Another formulation consists of a methoxy polyethylene glycol (PEG), polylactic-co-glycolic acid (PLGA), poly-L-lysine and cyclic arginine-glycine-aspartic acid carrier loaded with bufalin that has been shown to possess a favorable biodistribution profile and has anti-tumor activity in vivo (45). Here, we chose to test a similar and established PEG-PLGA based NP delivery system (see Material and Methods) (46) that has already been successfully used produce doxorubicin PEG-PLGA NPs that has been designed to avoid distribution to cardiac tissue.

Fox Chase SCID Beige mice were injected with 750 thousand MDA-MB-231 derived LM3.3 cells into the 2nd mammary gland (cleared) with two sites per mice. Six days after tumor cell injection, mice were separated into three groups and given the following treatments: 1) PBS vehicle control-treated (n=5) 2) bufalin NP (1.5 mg/kg)-treated (n=10) and 3) blank NP-treated (n=5). Mice were treated three times per week via intravenous injection. After this, primary tumor volume was measured to assess the ability of the bufalin NP to inhibit tumor growth (Fig. 7C). Aggressive tumor growth was observed in the PBS and blank NP-treated control mice, while the bufalin NP-treated mice showed a significant inhibition in the rate of tumor growth four days after initial treatment that was sustained throughout the experiment.

Discussion

The SRC family has been widely implicated in carcinogenesis providing a strong impetus to develop SRC SMIs as novel and effective therapeutic agents. Using a luminescence-based assay to assess coactivator intrinsic transcriptional activities, we conducted a high throughput screen of a MLPCN compound library (34) to identify SRC-3 and SRC-1 SMI hits. Do to the large size of this library, we were able to gain insight into the comparative activities of many structurally related compounds, revealing CGs as the largest class of compounds with SRC SMI activities. Our data demonstrate that both digoxin and bufalin selectively reduced intrinsic activities of SRC-3 and SRC-1, consistent with that observed in the primary screen. Marked differences in the potencies of CGs were observed, with most inhibiting SRCs at concentrations higher than their maximum tolerated dose (MTD). However, bufalin was found to have better potency and inhibited SRCs at concentrations below its MTD of 8.75 nM, leading to our focus on it as a potentially clinically useful SRC SMI.

For centuries, CGs have been used to treat patients with edematous states, irregular heartbeats or chronic heart failure and epidemiological evidence has shown that patients who take CGs are at lower risk for various cancers, pointing to their potential as cancer therapeutic agents (47). Interestingly, cardiac glycosides also have been shown to inhibit the androgen receptor (AR) but were found not to bind to the receptor itself (48).

In spite of their anti-cancer properties, CG effectiveness is limited by their narrow therapeutic window. For example, the non-toxic plasma concentration of digoxin for cardiac disease patients is 2.6 nM or less (49). In our study, the concentration of digoxin required to inhibit SRC-3/SRC-1 is greater than 200 nM. In contrast, we show here that bufalin is effective at a low nanomolar range (~3-5 nM) which is within the concentration range observed in patient plasma where no cardiac toxicity was observed (~9 nM) (42). Given the enhanced effectiveness when combined with the AKT inhibitor MK-2206, bufalin’s ability to block tumor growth might be achievable at even lower doses. Another approach we pursued to avoid the dose limiting toxicity associated with bufalin toward the heart was to deliver bufalin in a PEG-PLGA delivery particle, where we were able to effectively block breast tumor growth in vivo. Bufalin has already been tested in several small clinical trials (42) and the data presented here that characterize it as a SRC SMI provide new insight into its mechanism of action. Results presented here demonstrating that SRC-3 and SRC-1 are targets of bufalin promise to more rationally guide its future use as a novel therapeutic agent.

Acknowledgments

This work was performed using funding from the Susan G. Komen Foundation (PG12221410), the Prostate Cancer Foundation, the Clayton Foundation and the Dunn Foundation to BWO, Cancer Prevention and Research Institute of Texas (RP100348 and RP101251 to BWO and CPRIT R1104 to JW) and from the National Institutes of Health (HD076596 to DML) and (DK059820 to BWO). High throughput screening was supported through the National Institutes of Health Molecular Libraries Program (U54 MH084512 to PH and FM).

Footnotes

Conflict of interest disclosure statement: The authors have no conflict of interest to declare.

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