Phospho-aspirin-2 (MDC-22) inhibits estrogen receptor positive breast cancer growth both in vitro and in vivo by a redox-dependent effect.

Phospho-aspirin (PA-2) is a novel aspirin derivative that exhibits promising anticancer properties and is considerably safer than conventional aspirin. In this study, we investigated the chemotherapeutic efficacy of PA-2 in preclinical models of estrogen receptor positive (ER+) breast cancer and elucidated its mechanism of action. PA-2 inhibited the growth of ER+ cells more potently than aspirin in vitro, and exerted a triple cytokinetic effect that includes induction of apoptosis and cell cycle arrest as well as the inhibition of cell proliferation. PA-2 is highly efficacious in vivo, as treatment of established MCF7 xenografts with PA-2 induced tumor stasis (98.2% inhibition, p<0.01). PA-2 triggered the activation of p53-dependent apoptosis via two distinct mechanisms: 1) acetylation of p53 (at K373), which disrupts its interaction with its transcription repressor MDM2, and 2) translocation of p53 to the mitochondria leading to the dissipation of mitochondrial transmembrane potential (ΔΨ(m)). Consistent with these observations, both the RNAi-mediated knockdown of p53 and forced deactylation via HDAC1 over-expression attenuated the anticancer effect of PA-2 in MCF7 cells. An upstream mediator of the signaling effects of PA-2 is RONS. PA-2 induced oxidative stress in vitro and in mice bearing MCF7 xenografts; its induction effect appears to be tumor-specific. Crucially, administration of N-acetylcysteine, a ROS scavenger, abrogated the effect of PA-2 on p53 acetylation and mitochondria translocation, thus identifying RONS as proximal molecules mediating the anticancer effect of PA-2. In summary, our findings demonstrate that PA-2 is a promising antineoplastic compound against ER+ breast cancer, warranting further evaluation as an anticancer agent.

Introduction

Breast cancer is the one of the most common cancers with more than a million cases worldwide each year and is the second leading cause of cancer deaths in females [1]. Estrogen receptor expressing (ER+) breast cancer accounts for over two-thirds of all the breast cancer cases, and they are usually sensitive to anti-estrogen agents including tamoxifen and aromatase inhibitors. However, many of the tumors eventually develop drug resistance in advanced disease, leading to poor prognosis [2]. While the mechanisms leading to drug resistance remain poorly understood, the development of alternative therapeutic agents against ER+ breast cancer is urgently needed.

Aspirin is one of the oldest and most widely used anti-inflammatory medications [3], [4]. Widely perceived as a chemopreventive agent in the prevention of colon, breast and lung cancers [5], [6], aspirin may be useful as a chemotherapeutic agent, according to recent evidence. Regular aspirin use is associated with longer survival among patients with PIK3CA-mutant colorectal cancer [7], [8], suggesting that adjuvant aspirin therapy may be beneficial in this particular subset of patients [9]. Meanwhile, PIK3CA is one of the most commonly mutated genes in estrogen receptor positive (ER+) human breast cancer with mutation frequencies of 45% in the luminal A subtype and 29% in the liminal B subtype [10]. Epidemiological [11] and experimental studies [12] both supported a role for aspirin in the treatment of ER+ breast cancer.

Despite having shown promising anticancer activity, the gastrointestinal toxicity caused by aspirin use remains a significant health concern. In an effort to reduce the gastrointestinal toxicity and to improve the efficacy of aspirin, we have developed phospho-aspirin (Fig. 1A, PA-2; MDC-22) in which the -COOH group has been covalently modified by a glycerol linker containing two diethyl-phosphate moieties. Phospho-aspirin has demonstrated a much improved gastrointestinal safety profile compared to aspirin and is more efficacious in the treatment of cancer and experimental arthritis [13], [14]. In light of these previous findings, in this study we assessed the efficacy of phospho-aspirin in the treatment of ER+ breast cancer.

Figure 1

Phospho-aspirin-2 inhibits the growth of ER+ breast cancer cells.

A: Chemical structure of phospho-aspirin-2 (PA-2, MDC-22). B: 24 h-IC50 values of PA-2 and aspirin in ER+ breast cancer cell lines. C: Chemotherapeutic effect of PA-2 on subcutaneous MCF7 xenografts in nude mice. Nude mice bearing established MCF7 xenografts were treated with vehicle or PA-2 (500 mg/kg in corn oil) for five times a week. *, p<0.01, compared to control; n = 16 tumors/group.

Modified NSAIDs, in particular phospho-NSAIDs, appear to exert their antineoplastic effect via mechanisms distinct from those of conventional NSAIDs [15]. Our recent studies have attempted to decipher the molecular targets of modified NSAIDs [16]–[18]; and we have identified induction of oxidative stress as a key mechanism mediating the therapeutic effect of this class of novel anticancer drugs. Indeed, an increased level of reactive oxygen and nitrogen species (RONS) preceded the signaling changes in response to phospho-NSAIDs. On the other hand, intracellular RONS levels are intimately linked to the activation of the p53 tumor suppressor [19], which, in turn, suppresses tumor growth via modulating cell cycle progression and apoptosis. Hence, we evaluated the effect of PA-2 on oxidative stress in ER+ breast cancer, and its relationship to the re-activation of p53 and tumor growth inhibition.

Herein, we demonstrate that PA-2 is efficacious against breast cancer growth in vitro and in vivo, an effect that is superior to that of aspirin. We also show that PA-2 induces a state of oxidative stress in breast cancer cells, leading to the acetylation and re-activation of p53 tumor suppressor which is accompanied by reduced growth of ER+ breast cancer cells in vitro and in vivo.

Materials and Methods

Reagents

PA-2 was kindly provided by Medicon Pharmaceuticals, Inc., Setauket, NY. Aspirin was purchased from Sigma-Aldrich (St Louis, MO). We prepared 500 mM stock solutions of both in DMSO for cell culture studies. Antibodies used in this study were from Cell Signaling Technology (Danvers, MA). All other reagents, unless otherwise stated, were from Sigma-Aldrich.

Cell culture

Human ER+ breast cancer cell lines (MCF-7 and T-47D) (American Type Culture Collection (ATCC), Manassas, VA) were grown as monolayers in the specific medium and conditions suggested by ATCC. All the cell lines were passaged in our laboratory less than 6 months after receipt.

Cell viability assay

We determined cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay according to the manufacturer's protocol (Promega, Madison, WI).

Cytokinetic analysis

For apoptosis, cells were treated with vehicle or PA-2 for 24 h, stained with Annexin V-FITC (Invitrogen, Carlsbad, CA) and PI (0.5 µg/ml) and analyzed by flow cytometry on a FACScaliber (BD Biosciences, San Jose, CA). To determine cell proliferation, we measured incorporation of 5-bromo-2′-deoxyuridine (BrdU) into newly synthesized cellular DNA following the manufacturer's protocol (BD Biosciences) and the cells were analyzed by flow cytometry. Cell cycle distribution was analyzed by flow cytometry as described [15].

Plasmid and siRNA transfection

P53 pooled siRNA was purchased from Santa Cruz Biotechnology (Dallas, TX) and HDAC1 plasmid was obtained from Addgene (Cambridge, MA). Transient transfection was performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer's instructions.

Mitochondrial DNA depleted cells

MCF-7 cells depleted of mitochondrial DNA (ρ0) were generated by incubating cells with 200 ng/ml ethidium bromide, 100 µg/ml sodium pyruvate and 50 µg/ml uridine for 8 weeks as previously described [20].

Determination of reactive oxygen and nitrogen species (RONS)

After treatment with vehicle or PA-2, cells were trypsinized, re-suspended in 10 µM of MitoSOX Red (Invitrogen) or DAF-FM Diacetate (DAF-2, Invitrogen). After incubation at 37°C for 30 min in the dark, their fluorescence intensity was determined by flow cytometry.

Determination of mitochondrial transmembrane potential (ΔΨm) by JC-1 staining

After treatment with PA-2 for 3 h, cells were trypsinized and stained with 5 µM JC-1 for 30 min at 37°C and their fluorescence intensity was analyzed with a FACScalibur.

Urinary 15-F2t-isoprostane assay

Urine was collected after treatment for 2 days and 5 days, respectively. Levels of 15-F2t-isoprostane and creatinine in the urine were determined by ELISA (Oxford Biomedical Research, MA). 15-F2t-isoprostane values were normalized to creatinine levels.

Immunoblotting

After treatment with vehicle or PA-2 (as indicated), cells were lysed in RIPA lysis buffer and the protein concentration was determined using the Bradford method (Bio-Rad, Hercules, CA). Electrophoresis of cell lysates was performed on 10% SDS-polyacrylamide gel electrophoresis and protein was transferred onto nitrocellulose membranes as described. Antibodies, except β-actin (Sigma-Aldrich), were obtained from Cell Signaling Technology (Beverly, MA).

Breast cancer xenograft model

All animal experiments were approved by the Institutional Animal Care and Use Committee of Stony Brook University. Female Balb/c nude mice were obtained from Charles River Laboratories (Wilmington, MA). Mice used in this study were euthanized using CO2. Prior to the implantation of MCF-7 cells, each mouse was inoculated subcutaneously with a 0.72 mg β-estradiol pellet (Innovative Research of America, Sarasota, FL). After 3 days, the mice were inoculated subcutaneously into each of their flanks with 2.5×106 MCF-7 breast cancer cells suspended in Matrigel (BD Biosciences, Franklin Lakes, NJ). When the tumor reached approximately 280 mm3, animals were randomized into the control and treatment groups (n = 10/group). The animals were treated with vehicle or PA-2 500 mg/kg i.p. in corn oil 5 times/wk. Tumors were measured twice a week with a caliper, and tumor volumes were calculated using the following formula: tumor volume  =  [length × width × (length + width/2) ×0.56]. At the end of the treatment period, the animals were sacrificed, and their tumors were removed. To calculate tumor growth inhibition, we subtracted the baseline tumor volume from the final one.

Immunohistochemical analysis

Immunohistochemical staining for Ki-67, Ac-p53 (K373) and phospho-NF-κB (p-p65, activated form of NF-κB) was performed on human breast xenograft tissue samples as previously described [18]. Apoptosis was determined by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay.

Statistical analysis

Results are expressed as mean ± SEM. Differences between groups were determined by one-factor analysis of variance followed by Tukey's test for multiple comparisons. p<0.05 was considered statistically significant.

Results

PA-2 inhibits the growth of human ER+ breast cancer via a strong cytokinetic effect

We first compared the growth inhibitory effect of PA-2 and aspirin in ER+ breast cancer cell lines by MTT assay. PA-2 suppressed the growth of MCF-7 and T-47D cells with IC50 values of 248 and 208 µM, respectively, representing a 8- to 9-fold enhancement compared to aspirin (>2 mM). We next assessed the therapeutic efficacy of PA-2 in vivo using a subcutaneous MCF-7 xenograft model in nude mice. As shown in Fig. 1C, PA-2 significantly inhibited the growth of MCF-7 xenografts starting on day 12 of treatment until the end of the study (p<0.05). On day 22, the tumor volume was 564±56 mm3 for the vehicle group while the tumor volume was 285±36 mm3 for the PA-2-treated group (98.2% inhibition, p<0.01), indicating that PA-2 is highly effective in suppressing the growth of ER+ breast cancer in vivo.

PA-2 suppressed the growth of ER+ breast cancer cells via a strong cytokinetic effect. In vitro, PA-2 a) inhibited cell proliferation at 1xIC50 by 59.8% in MCF-7 cells (Fig 2A) and by 58% in T-47D cells (Figure S1A), b) induced apoptosis at 1.5xIC50 by 2.4-fold in MCF-7 cells (Fig 2B) and by 2.5-fold in T-47D cells (Figure S1B), and c) suppressed the G1 to S cell cycle phase transition, leading to an increased accumulation (15.8% in MCF-7 cells and 29% in T-47D cells) of cells in the G1 phase (Fig. 2C and Figure S1C). We also evaluated the cytokinetic effect of PA-2 in vivo. We assessed cell proliferation and apoptosis in the MCF7 xenografts using Ki-67 staining (Fig 2A) and TUNEL assay, respectively (Fig. 2B). Compared to the vehicle, PA-2 reduced cell proliferation from 16% to 5.6% (p<0.007); and increased apoptosis from 3.6% to 6.1% (p<0.0002). These data indicate that PA-2 exerted a strong cytokinetic effect on ER+ breast cancer xenografts in vivo. PA-2 triggered apoptosis via the caspase cascade (Fig. 3), which involved the activation of caspase-2 (2.4-fold increase over control in MCF-7 cells and 1.5-fold in T-47D cells, Fig. 3A), caspase-8 (Fig. 3B), caspase-9 (1.3-fold increase over control in MCF-7 cells, Fig. 3C), and eventually the effector caspase-3 (2.8-fold increase over control in MCF-7 cells, Fig. 3D).

Figure 2

Phospho-aspirin-2 exerts a triple cytokinetic effect on MCF7 cells.

A: Left: MCF7 cells were treated PA-2 for 24 h and the percentage of proliferating cells was determined by BrdU incorporation. Right: The percentage of proliferating cells in vehicle or PA-2 treated MCF7 xenografts were determined by Ki-67 staining. Representative images (top) and the quantification (bottom) of Ki-67 expression in tumor sections, * p<0.007. B: Left: MCF7 cells treated with PA-2 for 24 h were stained with Annexin V/PI, and the percentage of apoptotic cells was determined by flow cytometry. Right: The percentage of apoptotic cells in MCF7 xenografts were determined by TUNEL assay. Representative images (top) and the quantification (bottom) of TUNEL positive cells in tumor sections, * p<0.002. All values are mean ± SEM. C: PA-2 blocks the G1/S cell cycle phase transition after 24 h treatment in MCF7 cells, determined by flow cytometry following PI staining.

Figure 3

Phospho-aspirin-2 activates caspase cascades.

PA-2 treatment in MCF7 or T-47 D cells resulted in decreased expression of pro-caspase-2, -8, -9 and -3 and increased expression of their cleaved forms, as determined by western blot.

PA-2 induces acetylation of p53

In ER+ breast cancer, the activity p53 is frequently silenced either by mutational inactivation and/or by the amplification of MDM2 that binds to p53 and represses its stability and transcriptional activity [21]. The p53-MDM2 interaction is finely controlled by post-translational modification of p53. In particular, acetylation of p53 is an indispensable post-translational modification that mediates dissociation of p53 from MDM2, leading to p53 activation. In MCF7 cells we observed that p53 is deacetylated at K373. On the other hand, treatment of MCF7 cells with 1.5xIC50 PA-2 induced acetylation of p53 at K373 in a time-dependent manner, reaching maximal acetylation after 16 h of treatment (Fig. 4A). Similar results were obtained with T-47D cells (Fig. 4A). In MCF7 xenografts, PA-2 also increased the p53 acetylation (K373) by over 2.2-fold, as determined by immunohistochemical staining (Fig. 4A), suggesting that PA-2 is an inducer of p53 acetylation in vitro and in vivo.

Figure 4

Phospho-aspirin-2 acetylates p53.

A: Left: PA-2 induced the acetylation of p53 in MCF7 or T-47D cells (K373) in a time-dependent manner. Right: PA-2 induced p53 acetylation in MCF7 xenograft (K373). Representative images (right) and the quantification (left) of acetylated p53 (K373) positive cells in tumor sections, * p<0.001. B: Left: The effect of PA-2 on p53-MDM2 interaction as determined by immuno-precipitation (top) and the expression of p21 by western blot (bottom). Right: siRNA-mediated knockdown of p53 attenuated the pro-apoptotic effect of PA-2 in MCF7 cells (results are the average of three independent experiments, *p<0.05). Knockdown of p53 was determined by western blot. C: HDAC1 overexpression suppressed the anticancer activity of PA-2. Left: HDAC1 overexpression abrogated PA-2-induced the acetylation of p53 at K373 (top) and mildly suppressed its ability to induce apoptosis in MCF7 cells (bottom, results are the average of three independent experiments, *p<0.05). Right: HDAC1 overexpression suppressed the effect of PA-2 on cell proliferation in MCF7 cells (results are the average of three independent experiments, *p<0.05).

Acetylation of p53 by PA-2 in MCF7 cells disrupted the association between p53 and MDM-2, which in turn, may lead to p53 activation (Fig. 4B). We also observed induction of p21, a downstream target of p53 after treatment with 1.5xIC50 PA-2 in both cell lines (3.8-fold increase over control in MCF-7 cells at 2 h, and 2.4-fold in T-47D cells at 4 h; Fig. 4B). Consistent with a role of p53 activation in the anticancer effect of PA-2, siRNA-mediated knockdown of p53 attenuated PA-2 (1.5xIC50 and 2xIC50) induced apoptosis in MCF7 cells by 52% (Fig. 4B). To further assess the functional role of p53 acetylation in the cell death induction by PA-2, we over-expressed histone deacetylase 1 (HDAC1) in MCF7 cells (Fig. 4C). HDAC1 has been shown to be recruited to the MDM2-p53 complex, where it mediates the deacetylation and the subsequent degradation of p53 [22]. The forced expression of HDAC1 profoundly suppressed p53 acetylation by PA-2 treatment. As a consequence, HDAC1 overexpression attenuated the antineoplastic effect of this drug, as manifested in reduced inhibition of cell growth by PA-2 (19% inhibition of apoptosis at 2× IC50, and 6-fold decrease of proliferation at 1× IC50 in MCF-7 cells; Fig. 4C). PA-2 therefore mediates its anticancer effect, at least in part, via the induction of p53 acetylation, and subsequent activation of p53 and its downstream signaling pathways.

PA-2 induces the translocation of p53 to mitochondria

Recent evidence has indicated that p53 translocates to the mitochondria upon various stress stimuli to trigger apoptosis via opening of the mitochondrial permeability transition pore [23]. Hence, we analyzed the subcellular localization (cytosolic, mitochondria and nuclear) of p53 after treatment with 1.5xIC50 PA-2. As shown in Fig. 5A, PA-2 treatment resulted in significant accumulation of p53 in the mitochondria and cytochrome c release into cytosol in both cell lines (the mitochondrial p53 level increased 3.2-fold in MCF-7 cells and 2.4-fold in T-47D cells; the cytosolic cytochrome c level increased 1.8-fold in MCF-7 and 1.7-fold in T-47D cells), whereas the levels of cytosol and nuclear p53 remained unchanged. This observation indicates that PA-2 induced shuttling of p53 to the mitochondria. To evaluate whether PA-2 also regulates opening of the mitochondrial permeability transition pore (PTP), we measured mitochondrial membrane potential (Δψm) using the JC-1 fluorescence probe. Indeed, treatment with PA-2 in MCF7 cells significantly depolarized the mitochondrial membrane by 43% (Fig. 5B).

Figure 5

Phospho-aspirin-2 induces mitochondrial translocation of p53 and mitochondria-dependent apoptosis.

A: The mitochondrial, cytosolic and nuclear levels of p53 and cytochrome c were determined after treatment with PA-2 by western blot. B: PA-2 caused the collapse of mitochondrial membrane potential (Δψm), as indicated by the increased JC-1 fluorescence relative to the control. C: Mitochondria-depleted MCF7 cells (ρ0) showed resistance to PA-2. Left: Immunoblotting of control and ρ0 MCF7 cell lysates for the specific marker mitochondrial protein COXIV (top) and the effect of PA-2 on the viability of parental and ρ0 MCF7 cells was determined by the MTT assay (bottom). Right: ρ0 MCF7 cells were resistant to PA-2-induced apoptotic cell death, as measured by Annexin V/PI staining and flow cytometry (results are the average of three independent experiments, *p<0.05).

To verify the role of the mitochondria in PA-2-induced cell death, we generated mitochondria-null (ρ0) MCF7 cells, confirmed by the absence of mitochondrial protein cytochrome c oxidase subunit IV (COXIV; Fig. 5C). Compared to the parental MCF7 cells, ρ0 cells were significantly more resistant to PA-2-induced apoptosis (Fig. 5C). While PA-2 induced apoptosis in parental cells by 3- and 6-fold at 1.5xIC50 and 2xIC50, respectively, at equimolar concentration PA-2 only induced apoptosis by 2- and 3-fold in the ρ0 cells. Accordingly, the ρ0 cells were significantly more resistant to the cytotoxicity of PA-2, indicated by a 1.5-fold increase in its 24-hour IC50 (336 vs. 222 µM for parental cells). Our results substantiate the role of mitochondria in the cancer cell killing effect of PA-2.

PA-2 induces oxidative stress in vitro and in vivo

RONS play a significant role in the mechanism of action of phospho-NSAIDs [24]. We determined the effect of PA-2 on RONS stress using MitoSOX Red (mitochondrial O2 •-) and DAF2 (NO•). Compared to control, PA-2 1.5xIC50 increased mitochondrial O2 •- by 58% in both MCF-7 and T-47D cells, and NO• by 42% in MCF7 and 75% in T-47D cells (Fig. 6A and figure S2A). To assess the effect of PA-2 on oxidative stress in vivo, we measured urinary 15-F2t-Isoprostane [25], [26]. In mice bearing MCF7 xenografts, PA-2 (500 mg/kg, i.p.) induced a significant increase in urinary F2-isoprostane as early as 3 days after initiation of the treatment. 15-F2t-isoprostane levels on day 3 were 33.1±6.8 ng/mg creatinine in controls and 13.5±3.7 ng/mg creatinine in the PA-2 group, representing a nearly 1.5-fold increase (p<0.05). A similar phenomenon was also observed on day 10 of treatment (p<0.05) (Fig. 6B). We also measured another RONS biomarker, 8-hydroxydeoxyguanosine (8-OHdG), in MCF-7 xenografted tumors and found the level of 8-OHdG significantly elevated (2.7-fold) in the treated group compared to its vehicle group (figure S2B). In contrast, PA-2 treatment failed to promote oxidative stress in the equivalent mice without MCF7 xenografts (Fig. 6B), suggesting that the induction of RONS by PA-2 is specific to the MCF7 xenografts while sparing the normal tissues. Furthermore, we observed a significant inverse correlation between the individual tumor sizes and the urinary 15-F2t-isoprostane levels (p<0.05), which implies that a stronger RONS response is associated with greater efficacy for PA-2.

Figure 6

Phospho-aspirin-2 induces oxidative stress.

A: Left: Mitochondria superoxide was measured by MitoSOX Red dye after treatment of MCF7 cells with PA-2. Right: Nitric oxide levels was determined by DAF2 staining after treatment with PA-2. B: The levels of 15-F2t-isoprostane in 24-h urine of nude mice were determined using an enzyme-linked immunosorbent assay kit. Left: results from nude mice bearing MCF7 xenografts on day 3 (top) and day 10 (bottom). Right: results from nude mice bearing no xenografts on day 3 (top). The association between tumor volume and urinary 15-F2t-isoprostane levels in the nude mice from the xenograft study is shown (bottom).

PA-2 activates p53 and inhibits NF-κB signaling through enhanced oxidative stress

To further decipher the role of RONS in mediating the anticancer effect of PA-2, we examined the impact of RONS blockade on p53 acetylation and mitochondrial translocation. N-acetylcysteine (NAC, 10 mM), a RONS scavenger, blocked RONS induction by PA-2 in MCF7 cells (data not shown). Importantly, the co-incubation of PA-2 with NAC abrogated the effect of the former on p53 acetylation and mitochondrial translocation (Fig. 7A), which indicates that RONS induction is an upstream event in the action of PA-2. Correspondingly, co-incubation with NAC (10 mM) also suppressed the pro-apoptotic effect of PA-2 in MCF7 cells by 33% (Fig. 7B). Another crucial downstream effect of RONS induction by PA-2 is inactivation of NF-κB, a redox-sensitive dimer that modulates cell growth and inflammation in cancer [27]. Treatment of MCF7 xenografts with PA-2 suppressed NF-κB activation, as evidenced by a decreased expression of phosphorylated p65 (P-p65) by 32% compared to control (Fig. 7C). These results suggest that RONS are the proximal molecules mediating the anticancer effect of PA-2 in breast cancer.

Figure 7

Induction of oxidative stress mediates the effect of phospho-aspirin-2 on p53.

A: Left: Levels of total and acetylated p53 in MCF7 whole cell lysate after PA-2 treatment with or without 10 mM N-acetylcysteine. Right: Levels of total and acetylated p53 in MCF7 mitochondria after PA-2 treatment with or without 10 mM N-acetylcysteine. B: Co-incubation of PA-2 with N-acetylcysteine attenuated its apoptotic effect in MCF7 cells. C: Activated NF-kB (p-p65) levels from MCF7 tumors, determined by immunohistochemistry using anti-p-p65 antibody, were suppressed in PA-2-treated group compared with its vehicle group. The percentage of p-p65 positive cells in 10 fields was determined and averaged for each tumor. * P<0.05, compared to vehicle. Representative image are shown; magnification 200 x. All values are Mean ± SEM.

Discussion

Our findings indicate that the novel aspirin derivative PA-2 is an efficacious agent against ER+ breast cancer in preclinical models. PA-2 induced a potent cytokinetic effect on ER+ breast cancer cells in vitro and in vivo, mediated via a cascade of events involving a) profound induction of oxidative stress; b) p53 acetylation; and c) translocation of p53 to the mitochondria, culminating in p53-dependent apoptosis and cell cycle arrest, the net result of which is the potent inhibition of tumor growth (Fig. 8).

Figure 8

Proposed mechanism for the anticancer effect of phospho-aspirin-2 in ER+ breast cancer.

PA-2 inhibits ER+ breast cancer through (i) induction of oxidative stress; (ii) acetylation of p53, which disrupts p53-MDM2 interaction and increases the expression of p21; and (iii) translocation of p53 to the mitochondria; which ultimately results in mitochondrial-dependent apoptosis and cell growth arrest.

Aspirin use is associated with an improved outcome in breast, colon and prostate cancers [8], [28], [29]; however, its application as an adjuvant therapy and a chemopreventive is limited by its significant side effects involving the gastrointestinal tract. PA-2 is a novel compound that we have designed to improve the therapeutic index of aspirin. The phospho-modification of the carboxylic acid moiety of aspirin results in a remarkable safety profile [13], as well as improved efficacy [15], [30]–[33]. In ER+ breast cancer cell lines, PA-2 exhibits 6- to 8-fold improvement in potency as compared to aspirin. In vivo, administration of PA-2 has a dramatic effect on the growth of ER+ MCF7 xenografts, achieving a complete inhibition of tumor growth, triggered by a cytokinetic effect involving apoptosis induction, inhibition of cell proliferation and induction of cell cycle arrest.

Our work has identified an oxidative stress-initiated and p53-dependent signaling cascade that plays a fundamental role in the anticancer effect of PA-2. Modulation of oxidative stress is increasingly recognized to be a potential anticancer strategy [34]. ER+ breast cancer cell lines such as MCF7 displayed an increased susceptibility to cell death in response to the induction of RONS [35]. Phospho-NSAIDs, as a class of novel anticancer drugs, elevate oxidative stress in cancer cells as a common proximal event that causes the induction of cancer cell death [18]. Indeed, PA-2 induces oxidative stress in ER+ breast cancer cells in vitro and in MCF7 xenografts in vivo. The major RONS species involved were mitochondrial superoxide anion and nitric oxide. Remarkably, induction of oxidative stress by PA-2 appears to be exclusive to tumor tissues, as there was no significant elevation of oxidative stress in tumor-free animals. Elevation of oxidative stress at the beginning of PA-2 treatment was inversely correlated with tumor volume at the end point, suggesting that oxidative stress appears to be a dominant mediator of the anticancer effect of PA-2. It should be noted that the detection of mitochondrial superoxide anion and nitric oxide as reported here does have limitations, being in many instances qualitative indicators due to possible additional oxidation products with overlapping fluorescence spectra (review in [36]). Nevertheless, given its strong predictive value, oxidative stress induction, as determined here, may serve as a potential biomarker in future studies for identifying patient populations sensitive or resistant to the anticancer effect of PA-2.

Induction of oxidative stress by PA-2 in ER+ breast cancer is highly consequential, as it activates redox-sensitive downstream signaling that ultimately contributes to its growth inhibitory effect (Fig. 8). A pivotal downstream target is the tumor suppressor, p53. Intracellular levels of p53 are tightly regulated through its ubiquitylation by MDM2 and subsequent degradation by the 26S proteasome [37], and p53 expression is frequently silenced in ER+ breast cancer [38]. PA-2 strongly induced p53 acetylation, a post-translational modification that destabilizes the p53-MDM2 interaction, leading to the accumulation of p53 and its transcriptional activation. Furthermore, PA-2 prompted the mitochondrial translocation of p53, where it can regulate the opening of the PTP to directly induce mitochondrial-dependent cell death [39]. siRNA-mediated knockdown of p53, or its forced deacetylation by the over-expression of HDAC1 diminished the anticancer effect of PA-2, thereby confirming the important role of p53 in PA-2 induced apoptosis and growth arrest. The effect of PA-2 on apoptosis and p53 can be attenuated by co-incubation with NAC, an antioxidant, suggesting that it occurs downstream of oxidative stress induction. Induction of oxidative stress also has repercussions on the activity of NF-κB, which is strongly inhibited by PA-2 administration.

The signaling cascades induced by PA-2 culminate in apoptotic cell death in ER+ breast cancer. PA-2 treatment triggered the rapid dissipation of mitochondria transmembrane potential, consistent with mitochondria-dependent apoptotic cell death. Indeed, PA-2-induced apoptosis in MCF7 cells showed a marked dependence on the mitochondria, evidenced by the resistance of mitochondria depleted cells (p0) to PA-2 induced apoptotic cell death. The centrality of mitochondria in the induction of apoptosis by PA-2 is also evident from the enhanced mitochondrial ROS levels, and activation of caspase-2, -8,-9 and -3.

Conclusions

Our work indicates that PA-2 is effective and safe against ER+ breast cancer in preclinical models and establishes the induction of oxidative stress as a critical mediator of its anticancer effect. Oxidative stress is accompanied by the downstream acetylation and mitochondrial translocation of p53, leading to a strong cytokinetic effect. The promising efficacy of PA-2 in vivo suggests that its anticancer properties in ER+ breast cancer warrants further evaluation.

Contains the following files: Figure S1. Phospho-aspirin-2 exerts a triple cytokinetic effect onT-47D cells. A: T-47D cells were treated PA-2 for 24 h and the percentage of proliferating cells was determined by BrdU incorporation B: T-47D cells treated with PA-2 for 24 h were stained with Annexin V/PI, and the percentage of apoptotic cells was determined by flow cytometry. C: PA-2 blocks the G1/S cell cycle phase transition after 24 h treatment in T-47D cells, determined by flow cytometry following PI staining. Figure S2. Phospho-aspirin-2 induced RONS production in T-47D cells and increased 8-OHdG level in MCF-7 xenografts. A: Left: Mitochondria superoxide was measuredby MitoSOX Red dye after treatment of T-47D cells with PA-2. Right: Nitric oxide levels was determined byDAF2 staining after treatment with PA-2. B: 8-OHdG level from MCF-7 xenografts was determined by IHC. The percentage of p-p65 positive cells in 10 fields was determined and averaged for each tumor. * P<0.009, compared to vehicle. Representative image are shown; magnification 200 x. All values are Mean ± SEM.

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