Synthesis and activities of new indolopyrrolobenzodiazepine derivatives toward acute myeloid leukemia cells.

The synthesis of new indolopyrrolobenzodiazepine derivatives is described. Six compounds were selected for evaluation of cytotoxicity towards acute myeloid leukemia (AML) cells and normal fibroblasts. One compound (29) showed selective AML cell death induction. Its action was only partly overcome by knock-down of p53 or Bcl-2 overexpression, suggesting a strong activation of intrinsic apoptotic pathways.

A liposomal formulation of simvastatin and doxorubicin for improved cardioprotective and anti-cancer effect.

Anthracyclines such as doxorubicin (Dox) are the preferred chemotherapeutics for several cancers. However, Dox-induced cardiotoxicity limits its therapeutic potential. Liposomal encapsulation of Dox has been used for patients with risk to develop Dox induced cardiotoxicity but does not surpass the efficacy of the unencapsulated drug. Statins are widely used as cholesterol lowering drugs and have also demonstrated cardioprotective activity in cancer patients undergoing Dox therapy. We developed a liposome loaded with Dox and simvastatin (Sim) and investigated their effect on cardiomyocytes and zebrafish larvae. Furthermore, we investigated if the doses required for cardioprotection compromised the cytotoxicity of Dox in mammary and prostate cancer cells. Combination of Sim and Dox reduced ROS generation in cardiomyocytes, both given as free drugs, or co-encapsulated in liposomes. In contrast, Sim potentiated ROS-generation and cytotoxic activity of Dox towards cancer cells also when co-encapsulated in liposomes. In zebrafish larvae, Sim treatment reduced Dox-induced cardiac affection, and the liposomes did not induce any sign of Dox-induced cardiotoxicity. Our results show that liposomal co-encapsulation of Sim and Dox can be an efficient way of further reducing the risk of cardiotoxic events of liposomal Dox, while retaining, or even potentiating the anti-cancer effect of Dox.

Epac1 null mice have nephrogenic diabetes insipidus with deficient corticopapillary osmotic gradient and weaker collecting duct tight junctions.

AIM: The cAMP-mediator Epac1 (RapGef3) has high renal expression. Preliminary observations revealed increased diuresis in Epac1-/- mice. We hypothesized that Epac1 could restrict diuresis by promoting transcellular collecting duct (CD) water and urea transport or by stabilizing CD paracellular junctions to reduce osmolyte loss from the renal papillary interstitium. METHODS: In Epac1-/- and Wt C57BL/6J mice, renal papillae, dissected from snap-frozen kidneys, were assayed for the content of key osmolytes. Cell junctions were analysed by transmission electron microscopy. Urea transport integrity was evaluated by urea loading with 40% protein diet, endogenous vasopressin production was manipulated by intragastric water loading and moderate dehydration and vasopressin type 2 receptors were stimulated selectively by i.p.-injected desmopressin (dDAVP). Glomerular filtration rate (GFR) was estimated as [14 C]inulin clearance. The glomerular filtration barrier was evaluated by urinary albumin excretion and microvascular leakage by the renal content of time-spaced intravenously injected 125 I- and 131 I-labelled albumin. RESULTS: Epac1-/- mice had increased diuresis and increased free water clearance under antidiuretic conditions. They had shorter and less dense CD tight junction (TJs) and attenuated corticomedullary osmotic gradient. Epac1-/- mice had no increased protein diet-induced urea-dependent osmotic diuresis, and expressed Wt levels of aquaporin-2 (AQP-2) and urea transporter A1/3 (UT-A1/3). Epac1-/- mice had no urinary albumin leakage and unaltered renal microvascular albumin extravasation. Their GFR was moderately increased, unless when treated with furosemide. CONCLUSION: Our results conform to the hypothesis that Epac1-dependent mechanisms protect against diabetes insipidus by maintaining renal papillary osmolarity and the integrity of CD TJs.

A Kinase Inhibitor with Anti-Pim Kinase Activity is a Potent and Selective Cytotoxic Agent Toward Acute Myeloid Leukemia.

More than 40 years ago, the present standard induction therapy for acute myeloid leukemia (AML) was developed. This consists of the metabolic inhibitor cytarabine (AraC) and the cytostatic topoisomerase 2 inhibitor daunorubucin (DNR). In light of the high chance for relapse, as well as the large heterogeneity, novel therapies are needed to improve patient outcome. We have tested the anti-AML activity of 15 novel compounds based on the scaffolds pyrrolo[2,3-a]carbazole-3-carbaldehyde, pyrazolo[3,4-c]carbazole, pyrazolo[4,3-a]phenanthridine, or pyrrolo[2,3-g]indazole. The compounds were inhibitors of Pim kinases, but could also have inhibitory activity against other protein kinases. Ser/Thr kinases like the Pim kinases have been identified as potential drug targets for AML therapy. The compound VS-II-173 induced AML cell death with EC50 below 5 µmol/L, and was 10 times less potent against nonmalignant cells. It perturbed Pim-kinase-mediated AML cell signaling, such as attenuation of Stat5 or MDM2 phosphorylation, and synergized with DNR to induce AML cell death. VS-II-173 induced cell death also in patients with AML blasts, including blast carrying high-risk FLT3-ITD mutations. Mutation of nucleophosmin-1 was associated with good response to VS-II-173. In conclusion new scaffolds for potential AML drugs have been explored. The selective activity toward patient AML blasts and AML cell lines of the pyrazolo-analogue VS-II-173 make it a promising drug candidate to be further tested in preclinical animal models for AML.

Deletion of exchange proteins directly activated by cAMP (Epac) causes defects in hippocampal signaling in female mice.

Previous studies demonstrate essential roles for the exchange proteins directly activated by cAMP 1 and 2 (Epac1 and Epac2; here collectively referred to as Epac) in the brain. In the hippocampus, Epac contributes to the control of neuronal growth and differentiation and has been implicated in memory and learning as well as in anxiety and depression. In the present study we address the hypothesis that Epac affects hippocampal cellular responses to acute restraint stress. Stress causes activation of the hypothalamus-pituitary-adrenal (HPA)-axis, and glucocorticoid receptor (GR) signaling is essential for proper feedback regulation of the stress response, both in the brain and along the HPA axis. In the hippocampus, GR expression is regulated by cAMP and the brain enriched micro RNA miR-124. Epac has been associated with miR-124 expression in hippocampal neurons, but not in regulation of GR. We report that hippocampal expression of Epac1 and Epac2 increased in response to acute stress in female wild type mice. In female mice genetically deleted for Epac, nuclear translocation of GR in response to restraint stress was significantly delayed, and moreover, miR-124 expression was decreased in these mice. Male mice lacking Epac also showed abnormalities in miR-124 expression, but the phenotype was less profound than in females. Serum corticosterone levels were slightly altered immediately after stress in both male and female mice deleted for Epac. The presented data indicate that Epac1 and Epac2 are involved in controlling cellular responses to acute stress in the mouse hippocampus and provide novel insights into the underlying transcriptional and signaling networks. Interestingly, we observe sex specific differences when Epac is deleted. As the incidence and prevalence of stress-related diseases are higher in women than in men, the Epac knockout models might serve as genetic tools to further elucidate the cellular mechanisms underlying differences between male and female with regard to regulation of stress.

Epac1-deficient mice have bleeding phenotype and thrombocytes with decreased GPIbß expression.

Epac1 (Exchange protein directly activated by cAMP 1) limits fluid loss from the circulation by tightening the endothelial barrier. We show here that Epac1-/- mice, but not Epac2-/- mice, have prolonged bleeding time, suggesting that Epac1 may limit fluid loss also by restraining bleeding. The Epac1-/- mice had deficient in vitro secondary hemostasis. Quantitative comprehensive proteomics analysis revealed that Epac1-/- mouse platelets (thrombocytes) had unbalanced expression of key components of the glycoprotein Ib-IX-V (GPIb-IX-V) complex, with decrease of GP1bß and no change of GP1bα. This complex is critical for platelet adhesion under arterial shear conditions. Furthermore, Epac1-/- mice have reduced levels of plasma coagulation factors and fibrinogen, increased size of circulating platelets, increased megakaryocytes (the GP1bß level was decreased also in Epac1-/- bone marrow) and higher abundance of reticulated platelets. Viscoelastic measurement of clotting function revealed Epac1-/- mice with a dysfunction in the clotting process, which corresponds to reduced plasma levels of coagulation factors like factor XIII and fibrinogen. We propose that the observed platelet phenotype is due to deficient Epac1 activity during megakaryopoiesis and thrombopoiesis, and that the defects in blood clotting for Epac1-/- is connected to secondary hemostasis.

Cyanobacteria as a Source for Novel Anti-Leukemic Compounds.

Cyanobacteria are an inspiring source of bioactive secondary metabolites. These bioactive agents are a diverse group of compounds which are varying in their bioactive targets, the mechanisms of action, and chemical structures. Cyanobacteria from various environments, especially marine benthic cyanobacteria, are found to be rich sources for the search for novel bioactive compounds. Several compounds with anticancer activities have been discovered from cyanobacteria and some of these have succeeded to enter the clinical trials. Varying anticancer agents are needed to overcome increasing challenges in cancer treatments. Different search methods are used to reveal anticancer compounds from natural products, but cell based methods are the most common. Cyanobacterial bioactive compounds as agents against acute myeloid leukemia are not well studied. Here we examined our new results combined with previous studies of anti-leukemic compounds from cyanobacteria with emphasis to reveal common features in strains producing such activity. We report that cyanobacteria harbor specific anti-leukemic compounds since several studied strains induced apoptosis against AML cells but were inactive against non-malignant cells like hepatocytes. We noted that particularly benthic strains from the Baltic Sea, such as Anabaena sp., were especially potential AML apoptosis inducers. Taken together, this review and re-analysis of data demonstrates the power of maintaining large culture collections for the search for novel bioactivities, and also how anti-AML activity in cyanobacteria can be revealed by relatively simple and low-cost assays.