Diarylureas are small-molecule inhibitors of insulin-like growth factor I receptor signaling and breast cancer cell growth

Receptor tyrosine kinases (RTK) play a critical role in breast cancer growth and survival (1–6), and thus have become targets for antitumor therapy (7–10). Like other RTKs, the insulin-like growth factor receptor (IGF-IR) is a transmembrane protein containing an extracellular ligand binding domain and an intracellular tyrosine kinase domain. Signaling via the tyrosine kinase domain of the IGF-IR is important for normal cell growth and differentiation (11). In addition, the IGF-IR stimulates mitogenesis and suppresses apoptosis of cancer cells (11).

A critical initial step in IGF-IR signal transduction resulting from ligand binding is the conformational change of the receptor that results in trans-autophosphorylation of the β-subunits on select tyrosine residues. This autophosphorylation step is required for the subsequent activation of IGF-IR tyrosine kinase activity (11). Phosphorylation of several target substrates activates divergent signaling cascades, although many of the biological effects of IGF-I are mediated by tyrosine phosphorylation of the insulin receptor substrate (IRS) family of proteins. Phosphorylation of IRS-1 and IRS-2 allows binding of the regulatory subunit of phosphatidylinositol 3-kinase via SH2 domains. Activated phosphatidylinositol 3-kinase serine phosphorylates and activates the serine kinase Akt (12). The antiapoptotic effects of the IGF-IR are primarily mediated via the Akt/protein kinase B pathway (13), as Akt phosphorylates the protein BAD, which prevents BAD from forming a proapoptotic complex with Bcl-2 proteins (14).

Interruption of the IGF-IR signaling system, either by reducing effective IGF-I levels or targeting the receptor, can block growth and proliferation of cancer cells (15–19). Whereas overexpression of the IGF-IR can drive transformation and mitogenesis, a more important feature of the IGF-IR is the requirement for its constitutive presence in cancer cells (20). Thus, studies employing a variety of strategies to block IGF-IR signaling in a range of cancer cell types show that the IGF-IR is an effective target in cells even when tumorigenesis is driven by a variety of molecular mechanisms.

Due to this requirement for IGF-IR signaling in the establishment and maintenance of the transformed phenotype and its major role in regulating cancer cell survival, inhibitors of the IGF-IR and related RTKs are important targets for drug development (9, 21, 22). We have recently identified a new class of small-molecule compounds, diaryl ureas, which inhibit the IGF-IR. Herein we describe the biological effects of the diaryl urea compound PQ401 on the growth and survival of breast cancer cells in culture and in vivo.

Compounds that compose the chemical library employed in an initial screen against the IGF-IR, as well as additional diaryl urea compounds, were obtained from Telik Corp. (Palo Alto, CA). IGF-IR kinase domain peptide was obtained from Upstate USA (Charlottesville, VA). Antibodies against the IGF-IR (C-20), phosphospecific antibodies recognizing phosphotyrosine (PY20), and horseradish peroxidase–conjugated antiphosphotyrosine antibody (PY20HRP) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). αIR3, a monoclonal antibody against the IGF-IR, was obtained from Calbiochem (San Diego, CA) and the total protein and phosphospecific antibodies (Ser⁴⁷³) against Akt were obtained from Cell Signaling (Beverly, MA). All other reagents were from Sigma (St. Louis, MO) except where indicated.

To study the effects of diaryl ureas on IGF-IR signaling in breast cancer cells, MCF-7 cells were grown in either six-well or 96-well plates. Upon reaching 80% confluence, cells were serum starved for 18 hours. diaryl urea compounds were dissolved in DMSO and diluted with culture medium before being added to cells for 2.5 hours at 37°C. The final concentration of DMSO during the incubation was 0.3%. Cells were then stimulated with 3 nmol/L IGF-I for 10 minutes at 37°C. Reactions were terminated by rapidly aspirating medium and washing cells thrice with ice-cold PBS. Cells were harvested and solubilized in 50 mmol/L HEPES, 150 mmol/L NaCl, 1% Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride, and 2 mmol/L vanadate for 1 hour at 4°C. Protein was determined by bicinchoninic acid assay (Pierce, Rockford, IL).

IGF-IR autophosphorylation was then determined by ELISA as previously described for the IR (23). Briefly, 20 μg of lysate protein were added to duplicate wells in a 96-well plate coated with monoclonal antibody to the IGF-IR (αIR3; 2 μg/mL) and incubated for 18 hours at 4°C. Plates were washed five times, and then horseradish peroxidase–conjugated antiphosphotyrosine antibody (0.3 μg/mL), diluted in Solution B [50 mmol/L HEPES (pH 7.6), 150 mmol/L NaCl, 0.05% Tween 20, 1 mmol/L phenylmethylsulfonyl fluoride, 2 mmol/L vanadate and 1 mg/mL bacitracin], was added for 2 hours at 22°C. Plates were washed five times before color development with 3,3′,5,5′-tetramethly benzidine peroxidase substrate, which was terminated with 1.0 mol/L H₃PO₄. Values for receptor autophosphorylation were determined by measuring absorbance at 450 nm.

One microgram of constitutively active IGF-IR kinase domain peptide was incubated +/− varying concentrations of PQ401 in 2% DMSO in 40 mmol/L Tris (pH 7.4), 80 μmol/L EGTA, 0.25% 2-mercaptoethanol, 80 μmol/L Na₃VO₄, 10 mmol/L MgCl₂, and 2 mmol/L MnCl₂ for 20 minutes. ATP was then added at a final concentration of 20 μmol/L. Autophosphorylation of the kinase domain peptide was allowed to occur for 20 minutes at 22°C. The reaction was stopped by the addition of SDS-reducing buffer and the samples were run on SDS-PAGE. Following transfer to nitrocellulose membrane, peptide autophosphorylation was determined by Western blotting employing an antibody against phosphotyrosine (PY20).

Proliferation assays were conducted with MCF-7 or MCNeuA breast cancer cells. The MCNeuA cell line is a mammary carcinoma cell line we have recently established from a spontaneously arising tumor in a neu transgenic female mouse (24) and is thus driven by HER2/Neu overexpression but which also expresses IGF-IR. The inhibitory effects of diaryl urea on breast cancer cell growth were determined using a CyQuant cell proliferation assay kit (Molecular Probes, Eugene, OR). MCF-7 or MCNeuA cells were plated in 96-well plates (5 × 10³ per well) in phenol red–free DMEM supplemented with 10% FCS. One plate was prepared for each harvest day. Cells were allowed to adhere overnight and were then treated with various concentrations of diaryl urea or DMSO as a vehicle control. Microplate cultures were harvested on days 0, 1, 2, and 3 by inverting the microplate onto paper towels with gentle blotting to remove growth medium without disrupting adherent cells. Each plate was kept at −80°C until the end of the experiment (day 3) when all of the plates were thawed and assayed together. After thawing, 200 μL of CyQuant GR solution were added to each well and the plates were incubated in the dark for 2 to 5 minutes. Fluorescence was measured with a SpectraMax Gemini XS fluorescence microplate reader (Molecular Devices, Sunnyvale, CA) with 480-nm excitation and 520-nm emission. Proliferation index was calculated as the percent of nucleotide content versus control cells at day 0.

MCF-7 cells were harvested by washing thrice with PBS and dissociating with 1 mL 0.05% trypsin. Cells were resuspended in 5 mL defined medium (1:1 Ham's F12/DMEM 4.5 g/L glucose; 1 mg/mL bovine serum albumin; 10 μg/mL transferrin; 15 mmol/L HEPES pH 7.2; 2 mmol/L l-glutamine; 100 units/mL penicillin G; 100 μg/mL streptomycin SO₄; 2.5 μg/mL fungizone) containing 200 μg soybean trypsin inhibitor. Cells were plated in 96-well collagen-coated plates (Sigma) at a density of 5,000 per well in 100-μL medium. Twenty hours later, defined medium with or without IGF-I (10 nmol/L) was added. Four hours later, PQ401 diluted in defined medium was added. Plates were harvested on day 3, as described above, for determination of cell number by CyQuant assay.

Inhibition of IGF-I-stimulated activation of the serine kinase Akt was determined from the lysates prepared from MCF-7 cells grown in six-well plates and employed in the IGF-IR phosphotyrosine ELISA described above. Twelve micrograms of sample were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and phosphorylated Akt was quantified by blotting with a phosphospecific antibody to Akt(Ser⁴⁷³). Total Akt content was determined in these samples by blotting with an antibody against the Akt protein.

MCF-7 cells were plated in 96-well plates (5 × 10³ per well) in a volume of 200 μL. After 24 hours, cells were washed thrice with PBS and medium containing diaryl urea (final concentrations, 7.5, 15, or 30 μmol/L) or doxorubicin (final concentration, 10 μmol/L) diluted in DMSO, or DMSO alone (final concentration, 0.5%), was added. After 24 hours of incubation with the compounds, 10 μL of 10% NP40 were added per well and the plate was placed on a rotary shaker as lysis occurred for 5 minutes. The cell extract and supernatant were transferred to Eppendorf tubes and debris was removed by spinning in a microcentrifuge for 2 minutes at 5,000 rpm at 4°C. General caspase activity was ascertained by employing the M30 Apoptosense ELISA kit (Alexis Biochemicals, Lausen, Switzerland) which measures the level of a neo-epitope in the COOH-terminal domain of cytokeratin 18 (amino acids 387–396) that is revealed after apoptotic cleavage. Briefly, 25 μL of culture extract plus supernatant were added to duplicate wells in a 96-well plate coated with mouse monoclonal antibody to CK18. Then, horseradish peroxidase–conjugated M30 antibody, diluted in phosphate buffer with protein stabilizers (included in kit), was added immediately afterwards, and plate was incubated with agitation for 4 hours at 22°C. Plates were washed five times and 3,3′,5,5′-tetramethly benzidine peroxidase substrate was added, and plates were then incubated in darkness for 20 minutes at 22°C. The reaction was stopped with 1.0 mol/L H₂SO₄. Values for caspase activity were determined by measuring absorbance at 450 nm.

Our syngeneic model studies used the FVB/N-TgN(MMTVneu)202 mouse strain developed by Guy et al. (25). This strain, denoted hereafter as neuTg, expresses the wild-type rat neu proto-oncogene (a homologue of human HER2) under the control of the mouse mammary tumor virus (MMTV) long terminal repeat on an FVB mouse background. The MCNeuA mammary carcinoma cell line employed in these studies was derived from a spontaneously arising tumor in a neuTg female mouse (23). These cells are tumorigenic when transplanted back into neuTg mice. Female mice were injected s.c. with 10⁵ MCNeuA tumor cells on day 0. Treatment with PQ401 began on day 3 postinoculation. For injection, PQ401 was dissolved into 50% polysorbate 80/50% ethanol at a concentration of 25 mg/mL. This mixture was then diluted with PBS to a final concentration of 4 mg/mL PQ401 (8% polysorbate 80 and ethanol). Treated mice received 50 or 100 mg/kg PQ401 prepared in 8% polysorbate 80/ethanol/PBS, administered i.p. thrice a week. Control mice received i.p. injections of the polysorbate 80/ethanol vehicle only. Tumor growth was measured at the time of drug delivery on each treatment day with calipers and tumor volume was calculated using the equation (length × width²) × (π/6).

Statistics were calculated using MedCalc statistical software (MedCalc Software, Mariakerke, Belgium). Growth of breast cancer cells in culture and in vivo was analyzed by two-way ANOVA for treatment, time, and interaction effects with post hoc analysis by Student's t test. Dose effects of diaryl urea on receptor phosphorylation were analyzed by one-way ANOVA with post hoc analysis by Student's t test. Significance was set at P < 0.05.

To identify novel inhibitors of the IGF-IR, a chemical library was generated via the target-related affinity profiling lead identification technology (26, 27). These compounds were employed in initial screening assays for inhibitory activity against the IGF-IR in MCF-7 breast cancer cells. In an initial screening of 100 compounds at 5 to 25 μg/mL each, a compound with a general diaryl urea structure produced a dose-dependent inhibition of IGF-I-stimulated IGF-IR autophosphorylation (data not shown). To identify an effective diaryl urea compound for early biological studies, we initially constructed a small diaryl urea library consisting of four basic families, each with a distinct structure or linkage of the second aromatic ring. Based on results from a subsequent screen of these related diaryl ureas, we chose one compound from the phenyl-4-quinoyl urea class, PQ401 (Fig. 1A), to study the biological effects of this class of small molecules based on its significant potency against the IGF-IR in cell-based assays. In a series of studies in MCF-7 cells, PQ401 inhibited IGF-I-stimulated IGF-IR autophosphorylation with an IC₅₀ of 12.0 ± 0.9 μmol/L (Fig. 1B). IGF-IR inhibition was minimal or absent in eight compounds with a 3-quinoyl group replacing the methyl-substituted 4-quinoyl group seen in PQ401, suggesting the more linear conformation of the 3-linkage of these compounds reduced effectiveness against the target (data not shown). From this family of inactive compounds, PQ20 (Fig. 2), which showed no IGF-IR inhibiting capacity (Fig. 2), was subsequently employed as an IGF-IR kinase-inactive control due to its first aromatic ring being identical to PQ401. PQ20 was thus employed to test the specificity of the IGF-IR inhibitory effects of PQ401 on cell growth.

To determine if PQ401 was acting directly against the kinase domain of the IGF-IR, we incubated a constitutively active synthetic peptide consisting of amino acids 959-end of the IGF-IR with increasing concentrations of PQ401 before the addition of 20 μmol/L ATP. PQ401 inhibited autophosphorylation of the IGF-IR kinase domain at concentrations <100 nmol/L, with an IC₅₀ <1 μmol/L (Fig. 3).

To test our hypothesis that inhibition of the IGF-IR by PQ401 would inhibit growth of breast cancer cells, we studied the effects of exposure to both PQ401 and the kinase inactive PQ20 on proliferation of MCF-7 breast cancer cells. Employing a CyQuant assay, which estimates cell number by quantifying nucleic acid content, we examined MCF-7 proliferation across 3 days of incubation with various concentrations of either diaryl urea. The compounds were added to normal medium on day 0 and the medium was not changed over the course of the study. With this protocol, PQ401, at concentrations in the range of 1 μmol/L, significantly reduced proliferation (IC₅₀, 8 μmol/L), and at concentrations >10 μmol/L, produced a dramatic reduction in cell number from pretreatment levels (Fig. 4A). In contrast, PQ20 had no effect on proliferation of MCF-7 cells at any concentration tested (Fig. 4B). PQ401 was also able to inhibit growth of MCNeuA cells with somewhat less potency (IC₅₀, 15 μmol/L; data not shown), suggesting that inhibition of IGF-IR by diaryl ureas can reduce growth in breast tumors driven by overexpression of the HER2/neu receptor.

To determine further whether the inhibitory effects of PQ401 on MCF-7 cells were specific for the effects on the IGF-IR, we examined the ability of PQ401 to specifically inhibit growth of these cells mediated by IGF-I alone. Supplementing serum-free medium with 10 nmol/L IGF-I produced a 3-fold increase in cell number over the 3 days of growth, showing the responsiveness of these cells to IGF-I (Fig. 4C). In MCF-7 cells grown in serum-free medium supplemented only with 10 nmol/L IGF-I, PQ401 inhibited proliferation with an IC₅₀ of ∼6 μmol/L (Fig. 4C). The addition of PQ401 to cells incubated in nonsupplemented, serum-free medium produced a less dramatic inhibition of growth. At concentrations of PQ401 ≥15 μmol/L, the growth stimulatory effects of IGF-I over the serum-free condition were fully abolished.

To explore the mechanisms linking the IGF-IR inhibitory actions of diaryl ureas and their cytotoxic effects in cells, we studied the effects of PQ401 on the cell cycle and apoptotic pathways that are regulated by the IGF-IR. Exposure of MCF-7 cells to varying concentrations of PQ401 for 2 hours produced a significant reduction in the IGF-I-stimulated phosphorylation of the serine kinase Akt/protein kinase B (Fig. 5). Next, we employed an ELISA for the apoptosis-associated M30 neo-epitope in total cellular extracts plus supernatant medium to assess activation of the caspase apoptotic pathway. Twenty-four hours of incubation with PQ401 induced a dose-dependent increase in caspase activation (Fig. 6) in MCF-7 cells.

We employed MCNeuA breast cancer cells in a syngeneic mouse model of breast cancer. This cell line is a mammary carcinoma cell line that we have recently established from a spontaneously arising tumor in a neuTg female mouse (23). These cells display the characteristics of an estrogen-insensitive, p53-null, HER2-positive breast tumor. In culture, these cells displayed similar sensitivity as MCF-7 cells to growth inhibition by PQ401 as well as inhibition of IGF-IR autophosphorylation (data not shown). Thus, we employed these cells for in vivo studies to examine PQ401 effects against an aggressive tumor phenotype observed in ∼30% of human breast cancers that is recalcitrant to antiestrogen therapy. In addition, when these cells are implanted s.c. into the strain from which they were derived, all injected animals develop tumors. Thus, we could initiate treatment 3 days following implantation, before the development of palpable tumors. Injection of either 50 or 100 mg/kg PQ401 thrice per week in a polysorbate 80/ethanol vehicle (28) resulted in a significant dose-dependent reduction in tumor growth over the course of the study (Fig. 7). Tumor growth in the animals treated with 100 mg/kg PQ401 thrice a week was 20% of that in the vehicle-treated controls. This dosing protocol was well tolerated by the animals.

In the present study, we observed that diaryl ureas are direct inhibitors of the IGF-IR in isolated receptor preparations and cultured breast cancer cells. Diaryl urea treatment reduces breast cancer cell growth in cell culture in a manner specific for IGF-IR inhibition and reduces tumor growth in vivo. These studies suggest that diaryl ureas are potential anticancer agents acting via inhibition of the IGF-IR.

The IGF-IR is an essential component of tumorigenesis and an active target in the development of anticancer therapies (5). Expression of the IGF-IR is a requirement for transformation in cultured cells (29). IGF-IR overexpression is a feature of many types of transformed cells and tumors (20, 30). The IGF-IR is overexpressed and hyperphosphorylated in breast tumors in vivo (31), and hyperactivation of the IGF-IR is associated with the early stages of breast cancer (3, 32, 33).

A number of approaches have been employed to target the IGF-IR in cultured breast cancer cells. Antisense RNA for the IGF-IR (16), monoclonal antibodies (34, 35), transfection with a dominant negative IGF-IR (17), and small-molecule catechol mimics (21, 36) have all shown effectiveness at inhibiting proliferation of breast cancer cells in vitro. Expressing inactive IGF-IR in breast cancer cells inhibits tumor growth in vivo (17). The effectiveness of these anti-IGF-I therapies has also been shown in a variety of other tumor cell types including human prostate cancers (37), human lung cancers (38), human papillomavirus–positive and –negative endometrial cancers (39), ovarian cancer (40), glioblastomas (41), melanomas (42), and rhabdomyosarcomas (43). Nakamura et al. (39) showed that initial overexpression of IGF-IR was not required for growth inhibition by antisense RNA-mediated down-regulation of the IGF-IR. In their studies, C33a cervical cancer cells, which have a p53 mutation, very low IGF-IR expression, and less IGF-I-stimulated growth than other cervical cancer cell lines, showed significant reductions in monolayer growth and soft agar colony formation after IGF-IR down-regulation (39). These results highlight the requirement of IGF-IR signaling for survival of cells of which the growth may be driven primarily by a variety of other kinases or oncogenes (3).

In the present study, PQ401 was able to inhibit growth of both MCF-7 and MCNeuA breast cancer cells. Whereas transformation and growth of MCNeuA cells is driven by overexpression of the HER2/Neu receptor, the effectiveness of IGF-IR inhibition is not surprising considering the generalized requirement of IGF-I signaling for cancer cell growth and survival. It is not clear, however, the extent to which inhibition of the IGF-IR by PQ401 bypasses the stimulatory effects of HER2 in these cells, whether cross talk between the RTKs might modulate the HER2 signal, or whether effects beyond those observed against the IGF-IR might block growth of the MCNeuA cells in vivo.

Various other RTKs have been employed as targets in recent and ongoing attempts to create anticancer pharmaceutical agents (reviewed in ref. 9). Many of these compounds have been generated against the platelet-derived growth factor receptor, vascular endothelial growth factor receptor, and epidermal growth factor receptor family of kinases, either as antiproliferative or antiangiogenic agents (9). Recently, the anticancer effects of a small-molecule inhibitor of the IGF-IR have been reported (44, 45). These compounds are reported to show selectivity for the IGF-IR that is greater against isolated receptors than in cell-based assays (44, 45). Both compounds showed effectiveness at inhibiting proliferation of multiple cancer cell lines, and each compound inhibited the growth of xenograft models of multiple myeloma or fibroid sarcoma. One compound, NVP-ADW-742, showed cell growth–inhibitory activity similar to that observed with a monoclonal antibody against the IGF-IR, suggesting IGF-IR-specific effects (44).

The mechanism whereby diaryl urea inhibits RTK activity has not yet been elucidated. Whereas most small-molecule RTK inhibitors are competitive inhibitors of ATP binding, diaryl urea does not share general structural homology with ATP analogues. Others have reported that heterocyclic ureas inhibit the p38 mitogen-activated protein serine threonine kinase by interacting with a conserved hydrophobic pocket that is spatially distinct from the ATP binding site (46, 47). In their model, binding of heterocyclic ureas within the pocket causes a side chain of Phe to block access to the ATP binding site. Thus, the compounds act as indirect inhibitors of ATP binding.

Our data are consistent with an IGF-I-specific effect of diaryl urea molecules on tumor growth. We employed a negative control diaryl urea compound, PQ20, which has an identical structure of both the aryl ring and the urea linker but features an alternate substitution position on the quinoyl ring. This compound displayed no activity against IGF-I-stimulated receptor autophosphorylation in MCF-7 cells and, correspondingly, had no negative effects on growth of MCF-7 cells in culture. Thus, it is unlikely that any reduction in cell proliferation by PQ401 was due to general toxicity of the compound. Further, inhibition of IGF-IR activation by PQ401 was accompanied by a coordinate decrease in the phosphorylation state of Akt and an activation of the caspase apoptotic pathway. Together, these data show a specific blockade of the IGF-I-mediated pathway regulating apoptosis and suggest that the reduction in cell number induced by incubating cells with PQ401 reflect a specific effect on IGF-IR-dependent cell survival.

Because serum contains numerous factors that could also be driving growth of MCF-7 cells in culture, we further tested for specificity of the diaryl urea effects for the IGF-IR by studying the ability of PQ401 to inhibit IGF-I-mediated cell growth. Growing MCF-7 cells in medium supplemented only with 10 nmol/L IGF-I resulted in an increase in the potency of PQ401 to inhibit proliferation. In these studies, PQ401 also inhibited the proliferation of cells grown in serum-free medium in the absence of added IGF-I, although to a lesser extent. MCF-7 cells are reported to not express IGF-I mRNA (48) but they do secrete IGF-II, which can stimulate cell growth via an autocrine effect (49, 50). Therefore, it is likely that much of the effect of PQ401 on MCF-7 cells incubated in serum-free medium is due to the effect on the IGF-IR as well. However, it cannot be discounted that diaryl ureas exert additional actions distinct from those on the IGF-IR.

Our studies in the syngeneic model of breast cancer indicated that PQ401 could also inhibit the growth of breast cancer cells in vivo. By employing the neuTg-derived MCNeuA cells implanted back into this strain, we were able to treat animals that were not immunocompromised, and we could initiate treatment before the development of palpable tumors, facilitating longer treatment periods. We employed a very conservative treatment protocol with PQ401, administering i.p. doses only thrice per week. Using this schedule, the animals tolerated 100 mg/kg doses without incident. Although the tumor volume in animals treated with 100 mg/kg dose of PQ401 was only 20% of that in vehicle-treated controls, it is possible that a more aggressive dosing protocol could produce even greater reduction in tumor growth rates.

The pharmacology of the diaryl urea compounds employed in these studies has not yet been studied. We cannot rule out the possibility that a metabolite or degradation product, rather than the PQ401 compound, is responsible for the effects on cells and in vivo. However, the ability of PQ401 to directly inhibit autophosphorylation of the isolated kinase domain suggests a direct effect of this compound on the IGF-IR. Overall, the metabolism of diaryl ureas has not been studied extensively. Pyridines (51) and quinolines (52) are known to be metabolically labile and tend to produce 1- and 2-oxides, as well as other metabolites. The possibility that the 4-aminoquinaldine heterocycle present in PQ401 is processed or activated in a similar fashion has not been shown but cannot be ruled out in the current study.

In summary, we have identified a novel class of IGF-IR-inhibiting compounds that are potential anticancer agents. The lead diaryl urea compound, PQ401, inhibits growth of MCF-7 breast cancer cells both in culture and in a syngeneic mouse model of breast cancer. PQ401 acts, at least in part, by inducing caspase-mediated apoptosis via inhibition of the Akt/protein kinase B pathway.