In vitro targeting of Polo-like kinase 1 in bladder carcinoma: Comparative effects of four potent inhibitors

Despite the improvements in neoadjuvant chemotherapy, the outcome of patients with advanced bladder cancer has changed very little over the past 30 years. In the present study we tested and compared the in vitro antitumor activities of four different inhibitors of Polo-like kinase 1 (PLK1) (BI 2536, BI 6727, GW843682X, and GSK461364), against 3 bladder carcinoma cell lines RT4, 5637 and T24. The impact on radiosensitivity and drug interactions in simultaneous treatments with cisplatin, methotrexate, and doxorubicin were also investigated. Our results showed that PLK1 inhibition prevented cell proliferation and clonogenicity, causing significant inhibition of invasion of tumor cells, though modest differences were observed between drugs. Moreover, all PLK1 inhibitors induced G2/M arrest, with the subsequent induction of death in all 3 cell lines. Drug interactions studies showed auspicious results for all PLK1 inhibitors when combined with the commonly used cisplatin and methotrexate, though combinations with doxorubicin showed mostly antagonistic effects. Comparably, the four PLK1 inhibitors efficiently sensitized cells to ionizing radiation. Our findings demonstrate that irrespective of the inhibitor used, the pharmacological inhibition of PLK1 constrains bladder cancer growth and dissemination, providing new opportunities for future therapeutic intervention. However, further laboratorial and preclinical tests are still needed to corroborate the usefulness of using them in combination with other commonly used chemotherapeutic drugs. © 2013 Landes Bioscience.

Binding of BIS like and other ligands with the GSK-3 beta kinase: a combined docking and MM-PBSA study

Binding of several bisindolylmaleimide (BIS) like (BIS-3, BIS-8 and UCN1) and other ligands (H89, SB203580 and Y27632) with the glycogen synthase kinase-3 (GSK-3 beta) has been studied using combined docking, molecular dynamics and Poisson-Boltzmann surface area analysis approaches. The study generated novel binding modes of these ligands that can rationalize why some ligands inhibit GSK-3 beta while others do not. The relative binding free energies associated with these binding modes are in agreement with the corresponding measured specificities. This study further provides useful insight regarding possible existence of multiple conformations of some ligands like H89 and BIS-8. It is also found that binding modes of BIS-3, BIS-8 and UCN1 with GSK-3 beta and PDK1 kinases are similar. These new insights are expected to be useful for future rational design of novel, more potent GSK-3 beta-specific inhibitors as promising therapeutics.

Chiral gold(I) vs chiral silver complexes as catalysts for the enantioselective synthesis of the second generation GSK-hepatitis C virus inhibitor

The synthesis of a GSK 2(nd) generation inhibitor of the hepatitis C virus, by enantioselective 1,3-dipolar cycloaddition between a leucine derived iminoester and tert-butyl acrylate, was studied. The comparison between silver(I) and gold(I) catalysts in this reaction was established by working with chiral phosphoramidites or with chiral BINAP. The best reaction conditions were used for the total synthesis of the hepatitis C virus inhibitor by a four step procedure affording this product in 99% ee and in 63% overall yield. The origin of the enantioselectivity of the chiral gold(I) catalyst was justified according to DFT calculations, the stabilizing coulombic interaction between the nitrogen atom of the thiazole moiety and one of the gold atoms being crucial.

Differential activity of GSK-3 isoforms regulates NF-kappa B and TRAIL- or TNF alpha induced apoptosis in pancreatic cancer cells

While TRAIL is a promising anticancer agent due to its ability to selectively induce apoptosis in neoplastic cells, many tumors, including pancreatic ductal adenocarcinoma (PDA), display intrinsic resistance, highlighting the need for TRAIL-sensitizing agents. Here we report that TRAIL-induced apoptosis in PDA cell lines is enhanced by pharmacological inhibition of glycogen synthase kinase-3 (GSK-3) or by shRNA-mediated depletion of either GSK-3 alpha or GSK-3 beta. In contrast, depletion of GSK-3 beta, but not GSK-3 alpha, sensitized PDA cell lines to TNF alpha-induced cell death. Further experiments demonstrated that TNF alpha-stimulated I kappa B alpha phosphorylation and degradation as well as p65 nuclear translocation were normal in GSK-3 beta-deficient MEFs. Nonetheless, inhibition of GSK-3 beta function in MEFs or PDA cell lines impaired the expression of the NF-kappa B target genes Bcl-xL and cIAP2, but not I kappa B alpha. Significantly, the expression of Bcl-xL and cIAP2 could be reestablished by expression of GSK-3 beta targeted to the nucleus but not GSK-3 beta targeted to the cytoplasm, suggesting that GSK-3 beta regulates NF-kappa B function within the nucleus. Consistent with this notion, chromatin immunoprecipitation demonstrated that GSK-3 inhibition resulted in either decreased p65 binding to the promoter of BIR3, which encodes cIAP2, or increased p50 binding as well as recruitment of SIRT1 and HDAC3 to the promoter of BCL2L1, which encodes Bcl-xL. Importantly, depletion of Bcl-xL but not cIAP2, mimicked the sensitizing effect of GSK-3 inhibition on TRAIL-induced apoptosis, whereas Bcl-xL overexpression ameliorated the sensitization by GSK-3 inhibition. These results not only suggest that GSK-3 beta overexpression and nuclear localization contribute to TNF alpha and TRAIL resistance via anti-apoptotic NF-kappa B genes such as Bcl-xL, but also provide a rationale for further exploration of GSK-3 inhibitors combined with TRAIL for the treatment of PDA.

5-aryl-pyrazolo[3,4-b]pyridazines: Potent inhibitors of glycogen synthase kinase-3 (GSK-3)

Introduction of a nitrogen atom into the 6-position of a series of pyrazolo[3,4-b]pyridines led to a dramatic improvement in the potency of GSK-3 inhibition. Rationalisation of the binding mode suggested participation of a putative structural water molecule, which was subsequently confirmed by X-ray crystallography. (C) 2003 Elsevier Science Ltd. All rights reserved.

3-anilino-4-arylmaleimides: Potent and selective inhibitors of glycogen synthase kinase-3 (GSK-3)

Potent 3-anilino-4-arylmaleimide glycogen synthase kinase-3 (GSK-3) inhibitors have been prepared using automated array methodology. A number of these are highly selective, having little inhibitory potency against more than 20 other protein kinases. (C) 2001 Elsevier Science Ltd. All rights reserved.

Chronic resistance training decreases MuRF-1 and Atrogin-1 gene expression but does not modify Akt, GSK-3 beta and p70S6K levels in rats

Long-term adaptation to resistance training is probably due to the cumulative molecular effects of each exercise session. Therefore, we studied in female Wistar rats the molecular effects of a chronic resistance training regimen (3 months) leading to skeletal muscle hypertrophy in the plantaris muscle. Our results demonstrated that muscle proteolytic genes MuRF-1 and Atrogin-1 were significantly decreased in the exercised group measured 24 h after the last resistance exercise session (41.64 and 61.19%, respectively; P < 0.05). Nonetheless, when measured at the same time point, 4EBP-1, GSK-3 beta and eIF2B epsilon mRNA levels and Akt, GSK-3 beta and p70S6K protein levels (regulators of translation initiation) were not modified. Such data suggests that if gene transcription constitutes a control point in the protein synthesis pathway this regulation probably occurs in early adaptation periods or during extreme situations leading to skeletal muscle remodeling. However, proteolytic gene expression is modified even after a prolonged resistance training regimen leading to moderate skeletal muscle hypertrophy.

Angiotensin type 1 receptor mediates thyroid hormone-induced cardiomyocyte hypertrophy through the Akt/GSK-3 beta/mTOR signaling pathway

Several studies have implicated the renin angiotensin system in the cardiac hypertrophy induced by thyroid hormone. However, whether Angiotensin type 1 receptor (AT(1)R) is critically required to the development of T(3)-induced cardiomyocyte hypertrophy as well as whether the intracellular mechanisms that are triggered by AT(1)R are able to contribute to this hypertrophy model is unknown. To address these questions, we employed a selective small interfering RNA (siRNA, 50 nM) or an AT(1)R blocker (Losartan, 1 mu M) to evaluate the specific role of this receptor in primary cultures of neonatal cardiomyocytes submitted to T(3) (10 nM) treatment. The cardiomyocytes transfected with the AT(1)R siRNA presented reduced mRNA (90%, P < 0.001) and protein (70%, P < 0.001) expression of AT(1)R. The AT(1)R silencing and the AT(1)R blockade totally prevented the T(3)-induced cardiomyocyte hypertrophy, as evidenced by lower mRNA expression of atrial natriuretic factor (66%, P < 0.01) and skeletal alpha-actin (170%, P < 0.01) as well as by reduction in protein synthesis (85%, P < 0.001). The cardiomyocytes treated with T(3) demonstrated a rapid activation of Akt/GSK-3 beta/mTOR signaling pathway, which was completely inhibited by the use of PI3K inhibitors (LY294002, 10 mu M and Wortmannin, 200 nM). In addition, we demonstrated that the AT(1)R mediated the T(3)-induced activation of Akt/GSK-3 beta/mTOR signaling pathway, since the AT(1)R silencing and the AT(1)R blockade attenuated or totally prevented the activation of this signaling pathway. We also reported that local Angiotensin I/II (Ang I/II) levels (120%, P < 0.05) and the AT(1)R expression (180%, P < 0.05) were rapidly increased by T(3) treatment. These data demonstrate for the first time that the AT(1)R is a critical mediator to the T(3)-induced cardiomyocyte hypertrophy as well as to the activation of Akt/GSK-3 beta/mTOR signaling pathway. These results represent a new insight into the mechanism of T(3)-induced cardiomyocyte hypertrophy, indicating that the Ang I/II-AT(1)R-Akt/GSK-3 beta/mTOR pathway corresponds to a potential mediator of the trophic effect exerted by T(3) in cardiomyocytes.

Akt signalling through GSK-3beta, mTOR and Foxo1 is involved in human skeletal muscle hypertrophy and atrophy

Skeletal muscle size is tightly regulated by the synergy between anabolic and catabolic signalling pathways which, in humans, have not been well characterized. Akt has been suggested to play a pivotal role in the regulation of skeletal muscle hypertrophy and atrophy in rodents and cells. Here we measured the amount of phospho-Akt and several of its downstream anabolic targets (glycogen synthase kinase-3β (GSK-3β), mTOR, p70s6k and 4E-BP1) and catabolic targets (Foxo1, Foxo3, atrogin-1 and MuRF1). All measurements were performed in human quadriceps muscle biopsies taken after 8 weeks of both hypertrophy-stimulating resistance training and atrophy-stimulating de-training. Following resistance training a muscle hypertrophy (∼10%) and an increase in phospho-Akt, phospho-GSK-3β and phospho-mTOR protein content were observed. This was paralleled by a decrease in Foxo1 nuclear protein content. Following the de-training period a muscle atrophy (5%), relative to the post-training muscle size, a decrease in phospho-Akt and GSK-3β and an increase in Foxo1 were observed. Atrogin-1 and MuRF1 increased after the hypertrophy and decreased after the atrophy phases. We demonstrate, for the first time in human skeletal muscle, that the regulation of Akt and its downstream signalling pathways GSK-3β, mTOR and Foxo1 are associated with both the skeletal muscle hypertrophy and atrophy processes.

New drug targets in depression : inflammatory, cell-mediated immune, oxidative and nitrosative stress, mitochondrial, antioxidant, and neuroprogressive pathways. And new drug candidates—Nrf2 activators and GSK-3 inhibitors

This paper reviews new drug targets in the treatment of depression and new drug candidates to treat depression. Depression is characterized by aberrations in six intertwined pathways: (1) inflammatory pathways as indicated by increased levels of proinflammatory cytokines, e.g. interleukin-1 (IL-1), IL-6, and tumour necrosis factor α. (2) Activation of cell-mediated immune pathways as indicated by an increased production of interferon γ and neopterin. (3) Increased reactive oxygen and nitrogen species and damage by oxidative and nitrosative stress (O&NS), including lipid peroxidation, damage to DNA, proteins and mitochondria. (4) Lowered levels of key antioxidants, such as coenzyme Q10, zinc, vitamin E, glutathione, and glutathione peroxidase. (5) Damage to mitochondria and mitochondrial DNA and reduced activity of respiratory chain enzymes and adenosine triphosphate production. (6) Neuroprogression, which is the progressive process of neurodegeneration, apoptosis, and reduced neurogenesis and neuronal plasticity, phenomena that are probably caused by inflammation and O&NS. Antidepressants tend to normalize the above six pathways. Targeting these pathways has the potential to yield antidepressant effects, e.g. using cytokine antagonists, minocycline, Cox-2 inhibitors, statins, acetylsalicylic acid, ketamine, ω3 poly-unsaturated fatty acids, antioxidants, and neurotrophic factors. These six pathways offer new, pathophysiologically guided drug targets suggesting that novel therapies could be developed that target these six pathways simultaneously. Both nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activators and glycogen synthase kinase-3 (GSK-3) inhibitors target the six above-mentioned pathways. GSK-3 inhibitors have antidepressant effects in animal models of depression. Nrf2 activators and GSK-3 inhibitors have the potential to be advanced to phase-2 clinical trials to examine whether they augment the efficacy of antidepressants or are useful as monotherapy.

REGULATION OF FOXC2 EXPRESSION AND FUNCTION DURING EPITHELIAL-MESENCHYMAL TRANSITION BY GSK-3β

Metastasis is the ultimate cause for the majority of cancer-related deaths. The forkhead box transcription factor FOXC2 is known to be involved in regulating metastasis as well as a variety of developmental processes, including the formation of lymphatic and cardiovascular systems. Previous studies have shown that FOXC2 protein is localized either in the nucleus and/or in the cytoplasm of human breast tumor cells. This pattern of localization is similar to that of another forkhead family member, FOXO3a. Additionally, localization of FOXO3a is known to be differentially regulated by upstream kinase AKT. Therefore, I investigated whether FOXC2 localization could also be regulated by upstream kinases. Analysis of FOXC2 protein sequence revealed two potential phosphorylation sites for GSK-3β. Furthermore, inhibition of GSK-3βsignificantly reduces FOXC2 protein. In addition, exposure of HMLE Twist cells expressing endogenous FOXC2 to the GSK-3β inhibitor, TWS119, results in accumulation of FOXC2 protein in the cytoplasm with concomitant decrease in the nucleus in a time-dependent manner. Furthermore, continued treatment with TWS119 eventually induces epithelial morphology and decreased stem cell properties including sphere formation in these cells. Further characterization of FOXC2- GSK-3β interaction and the associated signaling cascade are necessary to determine the effect of FOXC2 phosphorylation by GSK-3β on EMT and metastasis.