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

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 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

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 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 (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

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 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

Targeting Mitochondrial STAT3 with the Novel Phospho-Valproic Acid (MDC-1112) Inhibits Pancreatic Cancer Growth in Mice

<div><p>New agents are needed to treat pancreatic cancer, one of the most lethal human malignancies. We synthesized phospho-valproic acid, a novel valproic acid derivative, (P-V; MDC-1112) and evaluated its efficacy in the control of pancreatic cancer. P-V inhibited the growth of human pancreatic cancer xenografts in mice by 60%–97%, and 100% when combined with cimetidine. The dominant molecular target of P-V was STAT3. P-V inhibited the phosphorylation of JAK2 and Src, and the Hsp90-STAT3 association, suppressing the activating phosphorylation of STAT3, which in turn reduced the expression of STAT3-dependent proteins Bcl-x_L, Mcl-1 and survivin. P-V also reduced STAT3 levels in the mitochondria by preventing its translocation from the cytosol, and enhanced the mitochondrial levels of reactive oxygen species, which triggered apoptosis. Inhibition of mitochondrial STAT3 by P-V was required for its anticancer effect; mitochondrial STAT3 overexpression rescued animals from the tumor growth inhibition by P-V. Our results indicate that P-V is a promising candidate drug against pancreatic cancer and establish mitochondrial STAT3 as its key molecular target.</p></div