Phospho-aspirin (MDC-22) inhibits breast cancer in preclinical animal models: an effect mediated by EGFR inhibition, p53 acetylation and oxidative stress

BACKGROUND: The anticancer properties of aspirin are restricted by its gastrointestinal toxicity and its limited efficacy. Therefore, we synthesized phospho-aspirin (PA-2; MDC-22), a novel derivative of aspirin, and evaluated its chemotherapeutic and chemopreventive efficacy in preclinical models of triple negative breast cancer (TNBC). METHODS: Efficacy of PA-2 was evaluated in human breast cancer cells in vitro, and in orthotopic and subcutaneous TNBC xenografts in nude mice. Mechanistic studies were also carried out to elucidate the mechanism of action of PA-2. RESULTS: PA-2 inhibited the growth of TNBC cells in vitro more potently than aspirin. Treatment of established subcutaneous TNBC xenografts (MDA-MB-231 and BT-20) with PA-2 induced a strong growth inhibitory effect, resulting in tumor stasis (79% and 90% inhibition, respectively). PA-2, but not aspirin, significantly prevented the development of orthotopic MDA-MB-231 xenografts (62% inhibition). Mechanistically, PA-2: 1) inhibited the activation of epidermal growth factor receptor (EGFR) and suppressed its downstream signaling cascades, including PI3K/AKT/mTOR and STAT3; 2) induced acetylation of p53 at multiple lysine residues and enhanced its DNA binding activity, leading to cell cycle arrest; and 3) induced oxidative stress by suppressing the thioredoxin system, consequently inhibiting the activation of the redox sensitive transcription factor NF-κB. These molecular alterations were observed in vitro and in vivo, demonstrating their relevance to the anticancer effect of PA-2. CONCLUSIONS: Our findings demonstrate that PA-2 possesses potent chemotherapeutic efficacy against TNBC, and is also effective in its chemoprevention, warranting further evaluation as an anticancer agent

A Novel Ras Inhibitor (MDC-1016) Reduces Human Pancreatic Tumor Growth in Mice

Pancreatic cancer has one of the poorest prognoses among all cancers partly because of its persistent resistance to chemotherapy. The currently limited treatment options for pancreatic cancer underscore the need for more efficient agents. Because activating Kras mutations initiate and maintain pancreatic cancer, inhibition of this pathway should have a major therapeutic impact. We synthesized phospho-farnesylthiosalicylic acid (PFTS; MDC-1016) and evaluated its efficacy, safety, and metabolism in preclinical models of pancreatic cancer. PFTS inhibited the growth of human pancreatic cancer cells in culture in a concentration- and time-dependent manner. In an MIA PaCa-2 xenograft mouse model, PFTS at a dose of 50 and 100 mg/kg significantly reduced tumor growth by 62% and 65% (P < .05 vs vehicle control). Furthermore, PFTS prevented pancreatitis-accelerated acinar-to-ductal metaplasia in mice with activated Kras. PFTS appeared to be safe, with the animals showing no signs of toxicity during treatment. Following oral administration, PFTS was rapidly absorbed, metabolized to FTS and FTS glucuronide, and distributed through the blood to body organs. Mechanistically, PFTS inhibited Ras-GTP, the active form of Ras, both in vitro and in vivo, leading to the inhibition of downstream effector pathways c-RAF/mitogen-activated protein-extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK1/2 kinase and phosphatidylinositol 3-kinase/AKT. In addition, PFTS proved to be a strong combination partner with phospho-valproic acid, a novel signal transducer and activator of transcription 3 (STAT3) inhibitor, displaying synergy in the inhibition of pancreatic cancer growth. In conclusion, PFTS, a direct Ras inhibitor, is an efficacious agent for the treatment of pancreatic cancer in preclinical models, deserving further evaluation

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

Ptf1a Binds to and Activates Area III, a Highly Conserved Region of the Pdx1 Promoter That Mediates Early Pancreas-Wide Pdx1 Expression▿

The critical pancreatic transcription factor Pdx1 is expressed throughout the pancreas early but enriched in insulin-producing β cells postnatally. Previous studies showed that the 5′ conserved promoter regions areas I and II (Pdx1PB) direct endocrine cell expression, while an adjacent region (Pdx1XB) containing conserved area III directs transient β-cell expression. In this study, we used Cre-mediated lineage tracing to track cells that activated these regions. Pdx1PBCre mediated only endocrine cell recombination, while Pdx1XBCre directed broad and early recombination in the developing pancreas. Also, a reporter transgene containing areas I, II, and III was expressed throughout the embryonic day 10.5 (E10.5) pancreas and gradually became β cell enriched, similar to endogenous Pdx1. These data suggested that sequences within area III mediate early pancreas-wide Pdx1 expression. Area III contains a binding site for PTF1, a transcription factor complex essential for pancreas development. This site contributed to area III-dependent reporter gene expression in the acinar AR42J cell line, while PTF1 specifically trans-activated area III-containing reporter expression in a nonpancreatic cell line. Importantly, Ptf1a occupied sequences spanning the endogenous PTF1 site in area III of E11.5 pancreatic buds. These data strongly suggest that PTF1 is an important early activator of Pdx1 in acinar and endocrine progenitor cells during pancreas development

P-V blocks the mitochondrial membrane potential and induces mitochondrial cell death: The role of mitochondrial STAT3.

<p>(<b>A</b>) Knocking-down STAT3 increases mitochondrial O₂⁻ levels in BxPC-3 cells. Control-siRNA and STAT3-siRNA cells were preloaded with MitoSOX Red, and mitochondrial O₂⁻ was determined by flow cytometry (<i>left</i>) or confocal microscopy (<i>right</i>; x40). (<b>B</b>) STAT3 overexpression reduces the increase in mitochondrial O₂⁻ induced by P-V. BxPC-3 cells were transfected with a STAT3-expressing plasmid or a control plasmid (empty vector) for 48 h, and then treated with P-V for 4 h followed by addition of the probe MitoSOX Red. Cells were then examined by confocal microscopy (x40). (<b>C</b>) P-V collapses the mitochondrial membrane potential (ΔΨm) in a time- (<i>top</i>) and concentration-dependent (<i>bottom</i>) manner. Fluorescence histograms were quantified and results are shown as mean±SEM; *<i>p</i><<i>0.05 vs</i>. control. (<b>D</b>) Immunoblots for cytochrome c, pro-caspases and caspases 9 and 3 in cytosolic protein extracts from BxPC-3 cells treated with P-V. (<b>E</b>) BxPC-3 mitochondria-less (ρ⁰) cells are markedly more resistant to P-V-induced apoptosis. <i>Top</i>: Immunoblots for COX IV and STAT3 in parental (−) and ρ⁰ BxPC-3 cells. Loading control: β-actin.</p

P-V inhibits STAT3 mitochondrial localization and selectively induces mitochondrial ROS in PC cells.

<p>(<b>A</b>) Immunoblots for STAT3, α-tubulin or COX IV in cytosolic (CF) and mitochondrial (MF) fractions from MIA PaCa-2 cells treated with P-V for 5 h. (<b>B</b>) Immunoblots for STAT3, Hsp90, Hsp60, α-tubulin and COX IV in mitochondrial fractions from MIA PaCa-2 cells treated with P-V, VPA or phospho-aspirin (P-A) for 3 h. (<b>C</b>) Immunoblots for p-STAT3^Ser727, STAT3 and Hsp90 in mitochondrial fractions isolated from MIA PaCa-2 xenografts from control or 150 mg/kg P-V-treated mice. β-tubulin  =  cytosolic cross-contamination control; COX IV  =  mitochondrial loading control. Each lane corresponds to a different tumor sample. (<b>D</b>) P-V decreases the activity of the mitochondrial electron transfer chain complex I (NADPH dehydrogenase). The activity of the mitochondrial complex I was measured as described in Methods. Values are mean±SEM (n = 3); *<i>p</i><<i>0.05 vs</i>. control. (<b>E</b>) MitoSOX Red, DCFDA and DHE fluorescence was measured by flow cytometry in BxPC-3 cells treated with P-V for 1 h. As positive controls, we treated cells with phospho-aspirin (P-A) for 1 h, which induces both DCFDA and DHE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061532#pone.0061532-Zhao1" target="_blank">[13]</a>. <i>Right panel</i>: Mitochondrial O₂⁻ levels in a panel of six PC cells treated with P-V 1.5×IC₅₀ for 1 h. (<b>F</b>) BxPC-3 cells were treated with P-V for up to 2 h, followed by addition of the MitoSOX Red probe and cells were analyzed by confocal microscopy (x40).</p

Proposed mechanism of action of P-V.

<p>Proposed mechanism of action of P-V.</p

Mitochondrial STAT3 overexpression abrogates the anticancer effect of P-V in vivo.

<p>(<b>A</b>) MIA PaCa-2 cells with basal (STAT3<i>^normal</i>) or overexpressed STAT3 (STAT3<i>^high</i>) levels were orthotopically implanted in nude mice, which were then treated without (control) or with P-V 150 mg/kg for 18 days. <i>Top</i>: Images of representative pancreatic tumors. <i>Bottom</i>: Pancreatic tumor weight (mean ± SEM). *<i>p</i><<i>0.01 vs</i>. control; ^#<i>p</i><<i>0.01 vs</i>. STAT3<i>^high</i> P-V-treated group. (<b>B</b>) STAT3 and Mcl-1 expression in STAT3<i>^normal</i> and STAT3<i>^high</i> MIA PaCa-2 orthotopic tumor tissue sections from control and P-V-treated mice (x20). (<b>C</b>) Immunoblots for STAT3 and Bcl-x_L in orthotopic tumor samples. Each lane represents a different tumor sample. Loading control: β-actin. (<b>D</b>) MIA PaCa-2 cells with overexpressed STAT3 Y705F mutant (STAT3<i>^Y705F</i>) levels were orthotopically implanted in nude mice, which were then treated without (control) or with P-V 150 mg/kg for 18 days. Pancreatic tumor weight (mean±SEM). (<b>E</b>) AsPC-1 cells with basal (STAT3<i>^normal</i>) or overexpressed mitochondria-targeted STAT3 (MLS-STAT3) levels were orthotopically implanted in nude mice, which were then treated without (control) or with P-V 150 mg/kg for 21 days. Pancreatic tumor weight (mean±SEM). *<i>p</i><<i>0.01 vs</i>. control; ^#<i>p</i><<i>0.01 vs</i>. MLS-STAT3 P-V-treated group.</p