PPARγ ligands, 15-deoxy-Δ12,14-prostaglandin J2 and rosiglitazone regulate human cultured airway smooth muscle proliferation through different mechanisms

1. The influence of two peroxisome proliferator-activated receptor γ (PPARγ) ligands, a thiazolidinedione, rosiglitazone (RG) and the prostaglandin D2 metabolite 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) on the proliferation of human cultured airway smooth muscle (HASM) was examined.

2. The increases in HASM cell number in response to basic fibroblast growth factor (bFGF, 300 pm) or thrombin (0.3 U ml−1) were significantly inhibited by either RG (1–10 μm) or 15d-PGJ2 (1–10 μm). The effects of RG, but not 15d-PGJ2, were reversed by the selective PPARγ antagonist GW9662 (1 μm).

3. Neither RG nor 15d-PGJ2 (10 μm) decreased cell viability, or induced apoptosis, suggesting that the regulation of cell number was due to inhibition of proliferation, rather than increased cell death.

4. Flow-cytometric analysis of HASM cell cycle distribution 24 h after bFGF addition showed that RG prevented the progression of cells from G1 to S phase. In contrast, 15d-PGJ2 caused an increase in the proportion of cells in S phase, and a decrease in G2/M, compared to bFGF alone.

5. Neither RG nor 15d-PGJ2 inhibited ERK phosphorylation measured 6 h post mitogen addition. The bFGF-mediated increase in cyclin D1 protein levels after 8 h was reduced in the presence of 15d-PGJ2, but not RG.

6. Although both RG and 15d-PGJ2 can inhibit proliferation of HASM irrespective of the mitogen used, only the antiproliferative effects of RG appear to be PPARγ-dependent. The different antimitogenic mechanisms of 15d-PGJ2 and synthetic ligands for PPARγ may be exploited to optimise the potential for these compounds to inhibit airway remodelling in asthma.

Exercise increases skeletal muscle GLUT4 gene expression in patients with type 2 diabetes

The aim of the study was to determine the effect of a single bout of exercise on GLUT4 gene expression in muscle of patients with type 2 diabetes (T2D) and control subjects, matched for age and body mass index. Nine patients with T2D and nine control subjects performed 60 min of cycling exercise at ∼55% peak power (W_max). Skeletal muscle biopsies were obtained at baseline, immediately post and 3-h post exercise. GLUT4 mRNA expression increased (p < 0.05) to a similar extent immediately post exercise in control (∼60%) and T2D (∼66%) subjects, and remained elevated (p < 0.05) 3-h post exercise with no differences between groups. Similarly, p-AMP-activated protein kinase, p38 mitogen-activated kinase and proliferator-activated receptor gamma co-activator-alpha mRNA expression were increased (p < 0.05) post exercise, and were not different between the groups. In conclusion, a single bout of exercise increased skeletal muscle GLUT4 mRNA expression in patients with T2D to a similar extent as in control subjects.

Characterization of the MEK5-ERK5 module in human neutrophils and its relationship to ERK1/ERK2 in the chemotactic response

The role of the extracellular signal-regulated kinase (ERK) 1 and ERK2 in the neutrophil chemotactic response remains to be identified since a previously used specific inhibitor of MEK1 and MEK2, PD98059, that was used to provide evidence for a role of ERK1 and ERK2 in regulating chemotaxis, has recently been reported to also inhibit MEK5. This issue is made more critical by our present finding that human neutrophils express mitogen-activated protein (MAP) kinase/ERK kinase (MEK)5 and ERK5 (Big MAP kinase), and that their activities were stimulated by the bacterial tripeptide, formyl methionyl-leucyl-phenylalanine (fMLP). Dose response studies demonstrated a bell-shaped profile of fMLP-stimulated MEK5 and ERK5 activation, but this was left-shifted when compared with the profile of fMLP-stimulated chemotaxis. Kinetics studies demonstrated increases in kinase activity within 2 min, peaking at 3–5 min, and MEK5 activation was more persistent than that of ERK5. There were some similarities as well as differences in the pattern of activation between fMLP-stimulated ERK1 and ERK2, and MEK5-ERK5 activation. The up-regulation of MEK5-ERK5 activities was dependent on phosphatidylinositol 3-kinase. Studies with the recently described specific MEK inhibitor, PD184352, at concentrations that inhibited ERK1 and ERK2 but not ERK5 activity demonstrate that the ERK1 and ERK2 modules were involved in regulating fMLP-stimulated chemotaxis and chemokinesis. Our data suggest that the MEK5-ERK5 module is likely to regulate neutrophil responses at very low chemoattractant concentrations whereas at higher concentrations, a shift to the ERK1/ERK2 and p38 modules is apparent.

Tumor progression locus 2 (Tpl2) deficiency does not protect against obesity-induced metabolic disease

Obesity is associated with a state of chronic low grade inflammation that plays an important role in the development of insulin resistance. Tumor progression locus 2 (Tpl2) is a serine/threonine mitogen activated protein kinase kinase kinase (MAP3K) involved in regulating responses to specific inflammatory stimuli. Here we have used mice lacking Tpl2 to examine its role in obesity-associated insulin resistance. Wild type (wt) and tpl22/2 mice accumulated comparable amounts of fat and lean mass when fed either a standard chow diet or two different high fat (HF) diets containing either 42% or 59% of energy content derived from fat. No differences in glucose tolerance were observed between wt and tpl22/2 mice on any of these diets. Insulin tolerance was similar on both standard chow and 42% HF diets, but was slightly impaired in tpl22/2 mice fed the 59% HFD. While gene expression markers of macrophage recruitment and inflammation were increased in the white adipose tissue of HF fed mice compared with standard chow fed mice, no differences were observed between wt and tpl2 mice. Finally, a HF diet did not increase Tpl2 expression nor did it activate Extracellular Signal-Regulated Kinase 1/2 (ERK1/2), the MAPK downstream of Tpl2. These findings argue that Tpl2 does not play a non-redundant role in obesity associated metabolic dysfunction.

APETALA2/ETHYLENE respnse factor and basic helix-loop-helix tobacco transcription factors cooperatively mediate jasmonate-elicited nicotine biosynthesis.

Transcription factors of the plant-specific apetala2/ethylene response factor (AP2/ERF) family control plant secondary metabolism, often as part of signalling cascades induced by jasmonate (JA) or other elicitors. Here, we functionally characterized the JA-inducible tobacco (Nicotiana tabacum) AP2/ERF factor ORC1, one of the members of the NIC2-locus ERFs that control nicotine biosynthesis and a close homologue of ORCA3, a transcriptional activator of alkaloid biosynthesis in Catharanthus roseus. ORC1 positively regulated the transcription of several structural genes coding for the enzymes involved in nicotine biosynthesis. Accordingly, overexpression of ORC1 was sufficient to stimulate alkaloid biosynthesis in tobacco plants and tree tobacco (Nicotiana glauca) root cultures. In contrast to ORCA3 in C. roseus, which needs only the GCC motif in the promoters of the alkaloid synthesis genes to induce their expression, ORC1 required the presence of both GCC-motif and G-box elements in the promoters of the tobacco nicotine biosynthesis genes for maximum transactivation. Correspondingly, combined application with the JA-inducible Nicotiana basic helix–loop–helix (bHLH) factors that bind the G-box element in these promoters enhanced ORC1 action. Conversely, overaccumulation of JAZ repressor proteins that block bHLH activity reduced ORC1 functionality. Finally, the activity of both ORC1 and bHLH proteins was post-translationally upregulated by a JA-modulated phosphorylation cascade, in which a specific mitogen-activated protein kinase kinase, JA-factor stimulating MAPKK1 (JAM1), was identified. This study highlights the complexity of the molecular machinery involved in the regulation of tobacco alkaloid biosynthesis and provides mechanistic insights about its transcriptional regulators.

Ibuprofen treatment blunts early translational signaling responses in human skeletal muscle following resistance exercise.

Cyclooxygenase-1 and -2 pathway-derived prostaglandins (PGs) have been implicated in adaptive muscle responses to exercise, but the role of PGs in contraction-induced muscle signaling has not been determined. We investigated the effect of inhibition of cyclooxygenase-1 and -2 activities with the nonsteroidal anti-inflammatory drug ibuprofen on human muscle signaling responses to resistance exercise. Subjects orally ingested 1,200 mg ibuprofen (or placebo control) in three 400-mg doses administered ∼30 min before and ∼6 h and ∼12 h following a bout of unaccustomed resistance exercise (80% one repetition maximum). Muscle biopsies were obtained at rest (preexercise), immediately postexercise (0 h), 3 h postexercise, and at 24 h of recovery. In the placebo (PLA) group, phosphorylation of ERK1/2 (Thr202/Tyr204), ribosomal protein S6 kinase (RSK, Ser380), mitogen-activated kinase 1 (Mnk1, Thr197/202), and p70S6 kinase (p70S6K, Thr421/Ser424) increased at both 0 and 3 h postexercise, with delayed elevation of phospho (p)-p70S6K (Thr389) and p-rpS6 (Ser235/S36 and Ser240/244) at 3 h postexercise. Only p-ERK1/2 (Thr202/Tyr204) remained significantly elevated in the 24-h postexercise biopsy. Ibuprofen treatment prevented sustained elevation of MEK-ERK signaling at 3 h (p-ERK1/2, p-RSK, p-Mnk1, p-p70S6K Thr421/Ser424) and 24 h (p-ERK1/2) postexercise, and this was associated with suppressed phosphorylation of ribosomal protein S6 (Ser235/236 and Ser240/244). Early contraction-induced p-Akt (Ser473) and p-p70S6K (Thr389) were not influenced by ibuprofen, but p-p70S6K (Thr389) remained elevated 24 h postexercise only in those receiving ibuprofen treatment. Early muscle signaling responses to resistance exercise are, in part, ibuprofen sensitive, suggesting that PGs are important signaling molecules during early postexercise recovery.

The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells

Danusertib (Danu) is a pan-inhibitor of Aurora kinases and a third-generation breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (Bcr-Abl) tyrosine kinase inhibitor, but its antitumor effect and underlying mechanisms in the treatment of human breast cancer remain elusive. This study aimed to investigate the effects of Danu on the growth, apoptosis, autophagy, and epithelial-to-mesenchymal transition (EMT) and the molecular mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. The results demonstrated that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both breast cancer cell lines. Danu arrested MCF7 and MDA-MB-231 cells in G2/M phase, accompanied by the downregulation of cyclin-dependent kinase 1 and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53. Danu significantly decreased the expression of B-cell lymphoma-extra-large (Bcl-xl) and B-cell lymphoma 2 (Bcl-2), but increased the expression of Bcl-2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and promoted the cleavage of caspases 3 and 9. Furthermore, Danu significantly increased the expression levels of the membrane-bound microtubule-associated protein 1A/1B-light chain 3 (LC3-II) and beclin 1 in breast cancer cells, two markers for autophagy. Danu induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1 and 2 (Erk1/2) and inhibited the activation of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways in breast cancer cells. Treatment with wortmannin (a phosphatidylinositol 3-kinase inhibitor) markedly inhibited Danu-induced activation of p38 MAPK and conversion of cytosolic LC3-I to membrane-bound LC3-II. Pharmacological inhibition and small interfering RNA-mediated knockdown of p38 MAPK suppressed Akt activation, resulting in LC3-II accumulation and enhanced autophagy. Pharmacological inhibition and small interfering RNA-mediated knockdown of Erk1/2 also remarkably increased the level of LC3-II in MCF7 cells. Moreover, Danu inhibited EMT in both MCF7 and MDA-MB-231 cells with upregulated E-cadherin and zona occludens protein 1 (ZO-1) but downregulated N-cadherin, zinc finger E-box-binding homeobox 1 (TCF8/ZEB1), snail, slug, vimentin, and β-catenin. Notably, Danu showed lower cytotoxicity toward normal breast epithelial MCF10A cells. These findings indicate that Danu promotes cellular apoptosis and autophagy but inhibits EMT in human breast cancer cells via modulation of p38 MAPK/Erk1/2/Akt/mTOR signaling pathways. Danu may represent a promising anticancer agent for breast cancer treatment. More studies are warranted to fully delineate the underlying mechanisms, efficacy, and safety of Danu in breast cancer therapy.

Alisertib, an Aurora kinase A inhibitor, induces apoptosis and autophagy but inhibits epithelial to mesenchymal transition in human epithelial ovarian cancer cells.

Ovarian cancer is a leading killer of women, and no cure for advanced ovarian cancer is available. Alisertib (ALS), a selective Aurora kinase A (AURKA) inhibitor, has shown potent anticancer effects, and is under clinical investigation for the treatment of advanced solid tumor and hematologic malignancies. However, the role of ALS in the treatment of ovarian cancer remains unclear. This study investigated the effects of ALS on cell growth, apoptosis, autophagy, and epithelial to mesenchymal transition (EMT), and the underlying mechanisms in human epithelial ovarian cancer SKOV3 and OVCAR4 cells. Our docking study showed that ALS, MLN8054, and VX-680 preferentially bound to AURKA over AURKB via hydrogen bond formation, charge interaction, and π-π stacking. ALS had potent growth-inhibitory, proapoptotic, proautophagic, and EMT-inhibitory effects on SKOV3 and OVCAR4 cells. ALS arrested SKOV3 and OVCAR4 cells in G2/M phase and induced mitochondria-mediated apoptosis and autophagy in both SKOV3 and OVCAR4 cell lines in a concentration-dependent manner. ALS suppressed phosphatidylinositol 3-kinase/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase pathways but activated 5'-AMP-dependent kinase, as indicated by their altered phosphorylation, contributing to the proautophagic activity of ALS. Modulation of autophagy altered basal and ALS-induced apoptosis in SKOV3 and OVCAR4 cells. Further, ALS suppressed the EMT-like phenotype in both cell lines by restoring the balance between E-cadherin and N-cadherin. ALS downregulated sirtuin 1 and pre-B cell colony enhancing factor (PBEF/visfatin) expression levels and inhibited phosphorylation of AURKA in both cell lines. These findings indicate that ALS blocks the cell cycle by G2/M phase arrest and promotes cellular apoptosis and autophagy, but inhibits EMT via phosphatidylinositol 3-kinase/Akt/mTOR-mediated and sirtuin 1-mediated pathways in human epithelial ovarian cancer cells. Further studies are warranted to validate the efficacy and safety of ALS in the treatment of ovarian cancer.

LNA aptamer based multi-modal, Fe3O4-saturated lactoferrin (Fe3O4-bLf) nanocarriers for triple positive (EpCAM, CD133, CD44) colon tumor targeting and NIR, MRI and CT imaging

This is the first ever attempt to combine anti-cancer therapeutic effects of emerging anticancer biodrug bovine lactoferrin (bLf), and multimodal imaging efficacy of Fe3O4 nanoparticles (NPs) together, as a saturated Fe3O4-bLf. For cancer stem cell specific uptake of nanocapsules/nanocarriers (NCs), Fe3O4-bLf was encapsulated in alginate enclosed chitosan coated calcium phosphate (AEC-CP) NCs targeted (Tar) with locked nucleic acid (LNA) modified aptamers against epithelial cell adhesion molecule (EpCAM) and nucleolin markers. The nanoformulation was fed orally to mice injected with triple positive (EpCAM, CD133, CD44) sorted colon cancer stem cells in the xenograft cancer stem cell mice model. The complete regression of tumor was observed in 70% of mice fed on non-targeted (NT) NCs, with 30% mice showing tumor recurrence after 30 days, while only 10% mice fed with Tar NCs showed tumor recurrence indicating a significantly higher survival rate. From tumor tissue analyses of 35 apoptotic markers, 55 angiogenesis markers, 40 cytokines, 15 stem cell markers and gene expression studies of important signaling molecules, it was revealed that the anti-cancer mechanism of Fe3O4-bLf was intervened through TRAIL, Fas, Fas-associated protein with death domain (FADD) mediated phosphorylation of p53, to induce activation of second mitochondria-derived activator of caspases (SMAC)/DIABLO (inhibiting survivin) and mitochondrial depolarization leading to release of cytochrome C. Induction of apoptosis was observed by inhibition of the Akt pathway and activation of cytokines released from monocytes/macrophages and dendritic cells (interleukin (IL) 27, keratinocyte chemoattractant (KC)). On the other hand, the recurrence of tumor in AEC-CP-Fe3O4-bLf NCs fed mice mainly occurred due to activation of alternative pathways such as mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinases (ERK) and Wnt signaling leading to an increase in expression of survivin, survivin splice variant (survivin 2B) and other anti-apoptotic proteins Bad, Bcl-2 and XIAP. Apart from the promising anti-cancer efficacy and the exceptional tumor targeting ability observed by multimodal imaging using near-infrared (NIR) imaging, magnetic resonance imaging (MRI) and computerized tomographic (CT) techniques, these NCs also maintained the immunomodulatory benefits of bLf as they were able to increase the RBC, hemoglobin, iron calcium and zinc levels in mice.

Controllable drug uptake and nongenomic response through estrogen-anchored cyclodextrin drug complex

Breast cancer is a leading killer of women worldwide. Cyclodextrin-based estrogen receptor-targeting drug-delivery systems represent a promising direction in cancer therapy but have rarely been investigated. To seek new targeting therapies for membrane estrogen receptor-positive breast cancer, an estrogen-anchored cyclodextrin encapsulating a doxorubicin derivative Ada-DOX (CDE1-Ada-DOX) has been synthesized and evaluated in human breast cancer MCF-7 cells. First, we synthesized estrone-conjugated cyclodextrin (CDE1), which formed the complex CDE1-Ada-DOX via molecular recognition with the derivative adamantane-doxorubicin (Ada-DOX) (Kd =1,617 M(-1)). The structure of the targeting vector CDE1 was fully characterized using (1)H- and (13)C-nuclear magnetic resonance, mass spectrometry, and electron microscopy. CDE1-Ada-DOX showed two-phase drug-release kinetics with much slower release than Ada-DOX. The fluorescence polarization analysis reveals that CDE1-Ada-DOX binds to recombinant human estrogen receptor α fragments with a Kd of 0.027 µM. Competition assay of the drug complex with estrogen ligands demonstrated that estrone and tamoxifen competed with CDE1-Ada-DOX for membrane estrogen receptor binding in MCF-7 cells. Intermolecular self-assembly of CDE1 molecules were observed, showing tail-in-bucket and wire-like structures confirmed by transmission electronic microscopy. CDE1-Ada-DOX had an unexpected lower drug uptake (when the host-guest ratio was >1) than non-targeting drugs in MCF-7 cells due to ensconced ligands in cyclodextrins cavities resulting from the intermolecular self-assembly. The uptake of CDE1-Ada-DOX was significantly increased when the host-guest ratio was adjusted to be less than half at the concentration of CDE1 over 5 µM due to the release of the estrone residues. CDE1 elicited rapid activation of mitogen-activated protein kinases (p44/42 MAPK, Erk1/2) in minutes through phosphorylation of Thr202/Tyr204 in MCF-7 cells. These results demonstrate a targeted therapeutics delivery of CDE1-Ada-DOX to breast cancer cells in a controlled manner and that the drug vector CDE1 can potentially be employed as a molecular tool to differentiate nongenomic from genomic mechanism.