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

<p>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.</p

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

<p>A: <i>Left</i>: MCF7 cells were treated PA-2 for 24 h and the percentage of proliferating cells was determined by BrdU incorporation. <i>Right</i>: 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: <i>Left</i>: 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. <i>Right</i>: 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.</p

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

<p><b>A:</b> The mitochondrial, cytosolic and nuclear levels of p53 and cytochrome c were determined after treatment with PA-2 by western blot. <b>B:</b> PA-2 caused the collapse of mitochondrial membrane potential (Δψ_m), as indicated by the increased JC-1 fluorescence relative to the control. <b>C:</b> Mitochondria-depleted MCF7 cells (ρ⁰) showed resistance to PA-2. <i>Left</i>: 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). <i>Right</i>: 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).</p

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

<p><b>A:</b><i>Left</i>: Levels of total and acetylated p53 in MCF7 whole cell lysate after PA-2 treatment with or without 10 mM N-acetylcysteine. <i>Right</i>: Levels of total and acetylated p53 in MCF7 mitochondria after PA-2 treatment with or without 10 mM N-acetylcysteine. <b>B:</b> Co-incubation of PA-2 with N-acetylcysteine attenuated its apoptotic effect in MCF7 cells. <b>C:</b> 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.</p

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

<p>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. *, <i>p</i><0.01, compared to control; <i>n</i> = 16 tumors/group.</p

Phospho-aspirin-2 induces oxidative stress.

<p><b>A:</b><i>Left</i>: Mitochondria superoxide was measured by MitoSOX Red dye after treatment of MCF7 cells with PA-2. <i>Right</i>: Nitric oxide levels was determined by DAF2 staining after treatment with PA-2. <b>B:</b> The levels of 15-F_2t-isoprostane in 24-h urine of nude mice were determined using an enzyme-linked immunosorbent assay kit. <i>Left</i>: results from nude mice bearing MCF7 xenografts on day 3 (top) and day 10 (bottom). <i>Right</i>: results from nude mice bearing no xenografts on day 3 (top). The association between tumor volume and urinary 15-F_2t-isoprostane levels in the nude mice from the xenograft study is shown (bottom).</p

Phospho-aspirin-2 activates caspase cascades.

<p>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.</p

Phospho-Aspirin-2 (MDC-22) Inhibits Estrogen Receptor Positive Breast Cancer Growth Both <i>In Vitro</i> and <i>In Vivo</i> by a Redox-Dependent Effect

<div><p>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 <i>in vitro</i>, 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 <i>in vivo</i>, as treatment of established MCF7 xenografts with PA-2 induced tumor stasis (98.2% inhibition, <i>p</i><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 <i>in vitro</i> 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.</p></div

Phospho-aspirin-2 acetylates p53.

<p><b>A:</b><i>Left</i>: PA-2 induced the acetylation of p53 in MCF7 or T-47D cells (K373) in a time-dependent manner. <i>Right</i>: 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>B:</b><i>Left</i>: The effect of PA-2 on p53-MDM2 interaction as determined by immuno-precipitation (top) and the expression of p21 by western blot (bottom). <i>Right</i>: 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. <b>C:</b> HDAC1 overexpression suppressed the anticancer activity of PA-2. <i>Left</i>: 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). <i>Right</i>: 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).</p

MC-12, an Annexin A1-Based Peptide, Is Effective in the Treatment of Experimental Colitis

<div><p>Annexin A1 (ANXA1) inhibits NF-κB, a key regulator of inflammation, the common pathophysiological mechanism of inflammatory bowel diseases (IBD). MC-12, an ANXA1-based tripeptide, suppresses NF-κB activation. Here, we determined the efficacy of MC-12 in the control of IBD. Mice with colitis induced by dextran sodium sulfate (DSS) or 2,4,6-trinitro benzene sulfonic acid (TNBS) were treated with various doses of MC-12 administered intraperitoneally, orally or intrarectally. We determined colon length and the histological score of colitis, and assayed: in colon tissue the levels of TNF-α, IFN-γ, IL-1β, IL-6 and IL-10 by RT-PCR; prostaglandin E₂ (PGE₂), cytoplasmic phospholipase A₂ (cPLA₂) and myeloperoxidase by immunoassay; and COX-2 and NF- κB by immunohistochemistry; and in serum the levels of various cytokines by immunoassay. In both models MC-12: reversed dose-dependently colonic inflammation; inhibited by up to 47% myeloperoxidase activity; had a minimal effect on cytoplasmic phospholipase A₂; reduced significantly the induced levels of TNF-α, IFN-γ, IL-1β, IL-6 and IL-10, returning them to baseline. DSS and TNBS markedly activated NF-κB in colonic epithelial cells and MC-12 decreased this effect by 85.8% and 72.5%, respectively. MC-12 had a similar effect in cultured NCM460 normal colon epithelial cells. Finally, MC-12 suppressed the induction of COX-2 expression, the level of PGE₂ in the colon and PGE₂ metabolite in serum. In conclusion, MC-12, representing a novel class of short peptide inhibitors of NF-κB, has a strong effect against colitis in two preclinical models recapitulating features of human IBD. Its mechanism of action is complex and includes pronounced inhibition of NF-κB. MC-12 merits further development as an agent for the control of IBD.</p> </div