The effect of MC-12 on DSS- and TNBS-induced colitis in mice.

<p>Mice (7/group) received 2% DSS in drinking water or 2.5% TNBS intra-colonically to induce colitis and were treated with vehicle or MC-12 given IP, PO or IR. <b><i>A:</i></b> The body weight of mice of DSS model during treatment by IP, PO or IR, expressed as percentage of baseline (day 0). <b><i>B:</i></b> The colon length of DSS model with MC-12 treatments by IP, PO or IR. <b><i>C:</i></b> The body weight of mice of TNBS model during treatment, expressed as percentage of baseline (day 0). <b><i>D:</i></b> The colonic length of mice of TNBS model in three treatment groups. These studies were repeated at least once giving similar results. Values are mean ± SEM. *, statistically significant difference from the vehicle-treated group.</p

MC-12 reduces the serum levels of cytokines.

<p>The serum levels of TNF-α, IFN-γ, IL-1β, IL-6 and IL-10 were measured by ELISA as in Methods. Both DSS and TNBS induced their levels and treatments with MC-12, 25 mg/kg administered PO, IP or IR reduced these levels 1.6- to 5.1-fold. *p<0.01, #p<0.05 compared to vehicle-treated controls.</p

MC-12 ameliorates colitis induced by both DSS and TNBS.

<p>Paraffin sections of colonic tissues were stained with hematoxylin & eosin and their histological scores were determined as in Methods. <b><i>A:</i></b> Normal mucosa of the C57BL/6 mouse. <b><i>B:</i></b> Severe inflammation including infiltration by inflammatory cells, edema, loss of crypts and ulcerations are seen in a DSS + vehicle-treated mouse. <b><i>C, D:</i></b> MC-12, 5 or 25 mg/kg, IP, significantly decreased DSS-induced colonic inflammation. <b><i>E:</i></b> Treatment with MC-12, 25 mg/kg PO, decreased DSS-induced colonic inflammation. <b><i>F:</i></b> Treatment with MC-12, 25 mg/kg, IR, markedly reduced DSS-induced colitis. <b><i>G:</i></b> Colonic mucosa from a TNBS vehicle-treated mouse, showing severe crypt loss and inflammatory cell infiltration (arrow). <i>Inset:</i> normal mucosa of a healthy SJL/J mouse. <b><i>H:</i></b> Treatment with MC-12 25 mg/kg for 2 days decreased the inflammatory cell infiltration and crypt loss (arrow indicates remaining crypts). <b><i>I, J:</i></b> The histological score of the various study groups of both DSS- and TNBS-induced colitis. Values are mean ± SEM. H&E staining; magnification 100x.</p

The effect of MC-12 on parameters of inflammation.

<p>MPO (<b><i>A, B</i></b>), cPLA₂ (<b><i>C, D</i></b>) activity and the mRNA levels of proinflammatory and anti-inflammatory cytokines (<b><i>E, F</i></b>) were measured in colon tissue samples of both DSS- and TNBS-induced colitis mouse models. In both models MC-12 significantly inhibited MPO activity and the mRNA levels of all cytokines but not cPLA₂ activity. Values are mean ± SEM. *p<0.05 compared to vehicle-treated group, **p<0.01 compared to vehicle-treated group.</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

MC-12 inhibits COX-2 induction and decreases PGE₂ levels in DSS- and TNBS-induced colitis. <i>A:</i>

<p>Representative photomicrographs of tissue sections with COX-2 immunohistochemical staining on colon tissue from normal (no treatment), vehicle- or MC-12-treated mice from the DSS (upper panels) or TNBS (lower panel) groups. MC-12 treatment: 25 mg/kg ip. <b><i>B:</i></b> The COX-2 expression scores in the various groups of animals (n = 8/group). Differences were evaluated by Pearson’s χ² method. <b><i>C, D:</i></b> MC-12 decreased the levels of PGE₂ in colonic mucosa and of PGE₂ metabolite in serum. Values are mean ± SEM. **p<0.01 compared to vehicle-treated group, *p<0.05 compared to vehicle-treated group.</p

MC-12 inhibits NF-κB activation induced by DSS and TNBS <i>in vivo</i> and <i>in vitro</i>.

<p>Colon tissue sections were immunohistochemically stained with anti-phospho-NF-κB p65 antibody. Normal colon mucosa from C57BL/6 mice (<b><i>A</i></b>) or from SJL/J mice (<b><i>F</i></b>) shows a few cells with p-p65 nuclear positive staining. Colon mucosa with DSS (<b><i>B</i></b>) or TNBS (<b><i>G</i></b>) shows increased NF-κB nuclear-positive cells, most of which are crypt epithelial cells. Markedly less p-p65 nuclear translocation was shown in colon mucosa from DSS model treated with MC-12, 5 mg/kg ip (<b><i>C</i></b>), 25 mg/kg ip (<b><i>D</i></b>) or 25 mg/kg po (<b><i>E</i></b>) or from TNBS model treated with MC-12 25 mg/kg ip (<b><i>H</i></b>). Changes in p-p65 nuclear positive, evident in the photos are quantified (<b><i>I, J</i></b>). DSS increased NF-κB-DNA binding in NCM460 cells determined by EMSA, and MC-12 30 and 300 µM significantly blocked this effect (<b><i>K</i></b>). Values are mean ± SEM. **p<0.01 compared to vehicle-treated group. IHC staining; magnification 200x.</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

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

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