Inhibition of Ion Channels and Heart Beat in Drosophila by Selective COX-2 Inhibitor SC-791

Recent findings suggest that modulation of ion channels might be implicated in some of the clinical effects of coxibs, selective inhibitors of cyclooxygenase-2 (COX-2). Celecoxib and its inactive analog 2,5-dimethyl-celecoxib, but not rofecoxib, can suppress or augment ionic currents and alter functioning of neurons and myocytes. To better understand these unexpected effects, we have recently investigated the mechanism of inhibition of human Kv2.1 channels by a highly selective COX-2 inhibitor SC-791. In this study we have further explored the SC-791 action on ion channels and heartbeat in Drosophila, which lacks cyclooxygenases and thus can serve as a convenient model to study COX-2-independent mechanisms of coxibs. Using intracellular recordings in combination with a pharmacological approach and utilizing available Drosophila mutants, we found that SC-791 inhibited voltage-activated K+ and L-type Ca2+ channels in larval body-wall muscles and reduced heart rate in a concentration-dependent manner. Unlike celecoxib and several other K+ channel blockers, SC-791 did not induce arrhythmia. Instead, application of SC-791 resulted in a dramatic slowing of contractions and, at higher concentrations, in progressively weaker contractions with gradual cessation of heartbeat. Isradipine, a selective blocker of L-type Ca2+ channels, showed a similar pattern of heart arrest, though no prolongation of contractions was observed. Ryanodine was the only channel modulating compound of those tested additionally that was capable of slowing contractions. Like SC-791, ryanodine reduced heart rate without arrhythmia. However, it could not stop heartbeat completely even at 500 µM, the highest concentration used. The magnitude of heart rate reduction, when SC-791 and ryanodine were applied together, was smaller than expected for independent mechanisms, raising the possibility that SC-791 might be interfering with excitation-contraction coupling in Drosophila heart

Single Exposure of Human Oral Mucosa Fibroblasts to Ultraviolet B Radiation Reduces Proliferation and Induces COX-2 Expression and Activation

The lip vermillion constitutes a transition tissue, between oral mucosa and skin, where oral mucosal cells from epithelial and connective tissue compartments are exposed to ultraviolet (UV) sunlight. Fibroblasts are abundant resident cells of the connective tissue which are key regulators of extracellular matrix composition, as well as, epithelial and endothelial cell function. UVB light, an inherent component of sunlight, causes several alterations in skin fibroblasts, including premature senescence and increased cyclooxygenase (COX)-2 expression. To assess if UVB irradiation had similar effects on fibroblasts derived from human oral mucosa (HOM), primary cultures of HOM fibroblasts were irradiated with a single dose of 30 or 60 mJ/cm²of UVB light or sham-irradiated. Fibroblast proliferation was assessed from 3 to 48 hrs after UVB-irradiation utilizing [³H]-thymidine incorporation and MTT assays. In addition, COX-2 mRNA expression was detected by RT-PCR, and PGE2 production was assessed using enzyme immunoassay from 0.5 to 24 hrs after UVB-irradiation. The results showed a significant decrease in proliferation of UVB-irradiated HOM fibroblasts as compared to controls as measured by both [³H]-thymidine incorporation and MTT assays (p<0.001). HOM fibroblasts had increased COX-2 mRNA expression at 0.5 and 12 hrs after irradiation, and PGE2 production was elevated at 12 and 24 hrs post-irradiation as compared to controls (p<0.05). The results showed an inhibitory effect of a single dose of UVB irradiation on HOM fibroblast proliferation with an increase in COX-2 expression and activation. Therefore, photodamaged fibroblasts may play and important role in the pathogenesis of UV-induced lesions of the lip

Tetrahydrohyperforin Inhibits the Proteolytic Processing of Amyloid Precursor Protein and Enhances Its Degradation by Atg5-Dependent Autophagy.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) peptide. We have previously shown that the compound tetrahydrohyperforin (IDN5706) prevents accumulation of Aβ species in an in vivo model of AD, however the mechanism that explains this reduction is not well understood. We show herein that IDN5706 decreases the levels of ER degradation enhancer, mannosidase alpha-like 1 (EDEM1), a key chaperone related to endoplasmic-reticulum-associated degradation (ERAD). Moreover, we observed that low levels of EDEM1 correlated with a strong activation of autophagy, suggesting a crosstalk between these two pathways. We observed that IDN5706 perturbs the glycosylation and proteolytic processing of the amyloid precursor protein (APP), resulting in the accumulation of immature APP (iAPP) in the endoplasmic reticulum. To investigate the contribution of autophagy, we tested the effect of IDN5706 in Atg5-depleted cells. We found that depletion of Atg5 enhanced the accumulation of iAPP in response to IDN5706 by slowing down its degradation. Our findings reveal that IDN5706 promotes degradation of iAPP via the activation of Atg5-dependent autophagy, shedding light on the mechanism that may contribute to the reduction of Aβ production in vivo

IDN5706 disrupts glycosylation of APP.

<p>(A) H4 cells stably expressing an amyloidogenic version of APP tagged to GFP (APP-GFP) were treated with 250 μM IDN5706 for the indicated periods of time. Cell extracts were subjected to Western blot analysis with an antibody to GFP. Western blotting with antibody to β-actin was used as loading control. (B) H4 cells stably expressing APP-GFP, were left untreated (lanes 1–3) or treated for 16 h with 250 μM IDN5706 (lanes 4–8). Cell extracts were subjected to immunoprecipitation with a an antibody to GFP, followed by denaturation and digestion with the indicated glycosidases for 1 h at 37°C. Immunoprecipitated proteins were subjected to Western blot analysis with anti-GFP-HRP. (A-B) The position of molecular mass markers is indicated on the left. mAPP, mature APP; iAPP, immature APP.</p

IDN5706 reduces the levels of EDEM1 in a time-dependent manner.

<p>(A) H4 cells were left untreated or treated for different periods of time with 250 μM IDN5706, and subsequently analyzed by Western blotting with an EDEM1 antibody. The antibody specificity was validated on protein extracts from cells stably expressing either luciferase shRNA, used as positive control (shLuc), or EDEM1 shRNA (shEDEM1). Specific antibodies against ER proteins including Calnexin, Gp78 and Bip were tested. (B) Densitometric quantification of the levels of EDEM1 in cells left untreated or treated with IDN5706 for 16 h, as shown in A. Bars represent the mean ± SD of three independent experiments (***, <i>P</i> < 0.001). (C) H4 cells were either left untreated (lane 1) or transfected with a plasmid encoding HA-epitope-tagged EDEM1 (lanes 2 and 3). After 16 h cells were left untreated or treated with 250 μM IDN5706 for 8 h, and cellular extracts were subjected to SDS-PAGE followed by immunoblot with mouse antibody anti-HA-epitope. In A and C, Western blotting with antibody to β-actin was used as loading control. The position of molecular mass markers is indicated on the left.</p

IDN5706 reduces phosphorylation in S2448 of mTOR.

<p>(A) N2a cells were left untreated (lane 1) or treated 4 h with 250 μM IDN5706 (lane 2), and cellular extracts were subjected to SDS-PAGE followed by immunoblot with rabbit antibody to phosphorylated mTOR at serine 2448 (p-mTOR (S2448)), or rabbit antibody to total mTOR. Western blotting with antibody to β-actin was used as loading control. The position of a molecular mass marker is indicated on the left. (B) Densitometric analysis of the levels of p-mTOR (S2448) shown in (A). Values were normalized to the levels of total mTOR, and presented as the mean ± SD of three independent experiments. ***, <i>P</i> < 0.001.</p

Effects of IDN5706 on APP turnover at the ER.

<p>(A) Similar to unfolded luminal glycoproteins that are recruited by EDEM1 from the Calnexin/Calreticulum (CNX/CLR) folding cycle (<b>1</b>), a fraction of newly-synthesized, immature APP (iAPP) is substrate of the endoplasmic reticulum-associated protein degradation (ERAD) (<b>2</b>). (B) The levels of EDEM1 are reduced upon IDN5706 treatment by a process independent on the levels of Atg5. This is a mechanism that needs further elucidation (<b>?</b>). Accumulation of iAPP in the ER elicited by IDN5706 activates an alternative early degradation of iAPP by Atg5-dependent autophagy (<b>3</b>).</p

IDN5706 inhibits proteolytic processing of APP to CTFs, and production of Aβ species.

<p>(A) Schematic representation of APP and carboxy-terminal fragments (CTFs) indicating their topological domains and the position of the proteolytic cleavage sites by α, β and γ secretases. The cytosolic region, recognized by the anti-tail antibody, and the γ-secretase inhibitor, DAPT, are indicated. (B) H4 cells were left untreated (lane 1) or treated for 16 h either with 250 μM IDN5706 (lane 2), 1 μM DAPT (lane 3), or with a combination of 250 μM IDN5706 and 1 μM DAPT (lane 4). Cell extracts were subjected to Western blot analysis using the anti-tail antibody to the cytosolic C-terminal region of APP. Western blotting with antibody to β-actin was used as loading control. mAPP, mature APP; iAPP, immature APP. The position of molecular mass markers is indicated on the left. (C-D) CHO 7AP2 cells were cultured in DMEM containing low glucose and without fetal bovine serum, in the absence or presence of 250 μM IDN5706 for 16 h. The amount of Aβ40 and Aβ42 peptides in the culture medium was analyzed by ELISA. (E) Ratio of the amount of Aβ42 and Aβ40 peptides as an indicator of toxicity. (C-E) Values are presented as the mean ± SD of three independent experiments. *, <i>P</i> < 0,05 and **, <i>P</i> < 0,005.</p

IDN5706 activates autophagy.

<p>H4 cells stably expressing mRFP-EGFP-LC3 were left untreated (A) or treated with either EBSS (B), 0.1 mM Chloroquine (C) or 250 μM IDN5706 (D) for the indicated time, and analyzed by fluorescence microscopy. Merging of the images in the green and red channels generated the third image in each row; in merged images red indicates presence of LC3 in acidic compartments (mRFP⁺EGFP^-), and yellow indicates presence of LC3 in non-acidic compartments (mRFP⁺EGFP⁺). Bar, 10 μm.</p

Depletion of Atg5 increases the accumulation of APP in response to IDN5706.

<p>H4 cells stably expressing an amyloidogenic version of APP tagged to GFP, and stably expressing either luciferase shRNA (control; shLuc) (A and B) or Atg5 shRNA (shAtg5) (C and D), were left untreated (Control; A and C) or treated with 250 μM IDN5706 for 8 h (IDN5706; B and D). Cells were fixed, and labeled with a mouse monoclonal antibody to Calnexin, followed by Alexa-594-conjugated donkey anti-mouse IgG (red channel; A-D). Stained cells were analyzed by fluorescence microscopy. Merging of the images in the green and red channels generated the third image in each row; yellow indicates overlapping localization of the green and red channels. Arrows indicate puncta of colocalization. Bar, 10 μm.</p