Mcl-1 determines the imiquimod-induced apoptosis but not imiquimod-induced autophagy in skin cancer cells

Background: Imiquimod had been shown to induce apoptosis and autophagy in several skin cancer cells, especially basal cell carcinoma (BCC) cells. Objective: We evaluate the molecular mechanisms of imiquimod-induced apoptosis and autophagy in skin cancer cell lines. Methods: The Mcl-1, Bcl-2 and Bcl-xL proteins were determined by immunoblotting. The Mcl-1 mRNA level was examined by RT-PCR and real-time PCR. The mechanisms of imiquimod-induced decrease in Mcl-1 protein were evaluated by addition of cycloheximide. MG132 proteasome inhibitor or pan-caspase inhibitor. The phosphorylation of eIF4E, 4E-BP1 and eEF2 in imiquimod treated cells were examined by immunoblotting. The imiquimod-induced apoptosis and autophagy were evaluated in Mcl-1-overexpressing cells by XTT test, mitochondrial membrane potential measurement, DNA content assay, LC3 immunoblotting, EGFP-LC3 puncta formation and quantification of acidic vesicular organelle with acridine orange staining. Results: The decrease in the Mcl-1 protein level was faster and stronger than the decrease in Bcl-2 and Bcl-xL in imiquimod-treated skin cancer cells. The imiquimod-induced decrease in Mcl-1 protein was not caused by blocked transcription or the promotion of degradation but was associated with inactivation of translation factors in BCC cells. The Mcl-1-overexpressing BCC cells were more resistant to intrinsic cellular apoptosis than control BCC cells during imiquimod treatment. Mcl-1 overexpression in BCC cells resulted in the basal activation of autophagy but did not modulate imiquimod-induced autophagy or rescue imiquimod-induced autophagic cell death in BCC cells. Conclusions: Imiquimod may rapidly downregulate Mcl-1 protein levels by inhibiting translation in skin cancer cells. Mcl-1 may act to protect against apoptosis but not autophagy and autophagic cell death during imiquimod treatment in skin cancer cells. (C) 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved

Modification of Alternative Splicing of Mcl-1 Pre-mRNA Using Antisense Morpholino Oligonucleotides Induces Apoptosis in Basal Cell Carcinoma Cells

Myeloid cell leukemia-1 (Mcl-1, Mcl-1L) is an anti-apoptotic protein of the Bcl-2 family that acts as a critical molecule in apoptosis control. Mcl-1 pre-mRNA can undergo alternative splicing to yield the short isoform, Mcl-1S, which resembles BH3-only pro-apoptotic proteins and induces apoptosis. Overexpression of Mcl-1 may play a role in various human tumors, and Mcl-1 may serve as a target in cancer therapy. In this study, we found an imbalance between the expression levels of Mcl-1L and Mcl-1S in the skin basal cell carcinoma (BCC) cell line when compared with primary keratinocytes. We showed that overexpression of Mcl-1S induces apoptosis in BCC cells. Finally, we showed that Mcl-1 antisense morpholino oligonucleotides (AMOs) can specifically target Mcl-1 pre-mRNA and shift the splicing pattern from Mcl-1L to Mcl-1S mRNA and protein. This shift increases the level of pro-apoptotic Mcl-1S and reduces the level of anti-apoptotic Mcl-1L, which induces apoptosis in BCC cells and AGS cells, a human gastric adenocarcinoma epithelial cell line. Thus, this report provides a strategy for cancer therapy in which AMOs change the alternative splicing pattern of Mcl-1 pre-mRNA and thereby induce apoptosis

Operon structure and cotranslational subunit association direct protein assembly in bacteria

Assembly of protein complexes is considered a post-translational process involving random collision of subunits. We show that within the Escherichia coli cytosol, bacterial luciferase subunits LuxA and LuxB assemble into complexes close to the site of subunit synthesis. Assembly efficiency decreases markedly if subunits are synthesized on separate mRNAs from genes integrated at distant chromosomal sites. Subunit assembly initiates cotranslationally on nascent LuxB in vivo. The ribosome-associated chaperone Trigger Factor delays the onset of cotranslational interactions until the LuxB dimer interface is fully exposed. Protein assembly thus is directly coupled to the translation process, and involves spatially confined, actively chaperoned cotranslational subunit interactions. Bacterial gene organization into operons therefore reflects a fundamental mechanism for spatiotemporal regulation vital to effective cotranslational protein complex assembly

Enhanced light output power in InGaN light-emitting diodes by fabricating inclined undercut structure

The InGaN-based light-emitting diode (LED) with an inclined undercut structure is fabricated through the photoelectrochemical two-step process to increase light extraction efficiency. In the first step the sidewall-undercut structure at the p-type and n-type GaN interface is created by selective wet oxidation on an n-type GaN surface in pure H2O solution. In the second step an inclined undercut structure through a crystallographic wet-etching process is formed by immersion in hot KOH solution. This crystallographic wet-etching process can remove the Ga2O3 layer and form a {1011} p-type GaN stable plane, {1010} n-type GaN stable plane on the mesa sidewall. This inclined p-type GaN plane of LED structure can provide the higher overlap of incident light beam core and extraction core overlap on the mesa sidewall, and the total light output power of the treated LED is 2.10 times higher than the standard LED. Consequently, this inclined undercut LED structure is suitable for high-efficiency nitride-based LED application. (c) 2006 The Electrochemical Society

Imiquimod simultaneously induces autophagy and apoptosis in human basal cell carcinoma cells

P>Background Imiquimod shows antitumour activity through the stimulation of cell-mediated immunity in vivo. Recent studies have shown that imiquimod promotes apoptosis in melanoma cells and induces autophagy in macrophage cell lines. Objectives To evaluate the imiquimod-induced apoptosis, autophagy and their relationship in a basal cell carcinoma (BCC) cell line. Methods Cell viability was determined by XTT test. Apoptosis was evaluated by DNA content assay, annexin V/propidium iodide staining assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling assay. Autophagy was determined by LC3 immunoblotting, EGFP-LC3 puncta formation and quantification of acidic vesicular organelles with acridine orange staining. The temporal and spatial differences of imiquimod-induced apoptosis and autophagy were examined by immunoblotting and simultaneously monitored by staining the EGFP-LC3 transfected cells with caspase 3 fluorogenic substrate. We inhibited the apoptosis and autophagy by pancaspase inhibitor and siRNA for Beclin 1 or Atg5, respectively, to evaluate the interplay between imiquimod-induced apoptosis and autophagy. Results We found that imiquimod induces autophagy and apoptosis in BCC cells in a time- and dose-dependent manner. Imiquimod not only induced EGFP-LC3 puncta formation for autophagy, but also simultaneously activated an apoptotic caspase cascade in the same cells. Both apoptosis and autophagy induced by imiquimod cooperate to cause BCC cell death. However, inhibition of imiquimod-induced apoptosis increased the strength of autophagy, and inhibition of imiquimod-induced autophagy further promoted cell apoptosis. Conclusions This study not only demonstrates that imiquimod can directly induce autophagy and apoptosis in BCC cells, but also shows the cooperation and coordination between these two processes to induce cell death

Mechanical properties of highly porous PDLLA/Bioglass (R) composite foams as scaffolds for bone tissue engineering

This study developed highly porous degradable composites as potential scaffolds for bone tissue engineering. These scaffolds consisted of poly-d,l-lactic acid filled with 2 and 15 vol.% of 45S5 Bioglass® particles and were produced via thermally induced solid–liquid phase separation and subsequent solvent sublimation. The scaffolds had a bimodal and anisotropic pore structure, with tubular macro-pores of 100 μm in diameter, and with interconnected micro-pores of 10–50 μm in diameter. Quasi-static and thermal dynamic mechanical analysis carried out in compression along with thermogravimetric analysis was used to investigate the effect of Bioglass® on the properties of the foams. Quasi-static compression testing demonstrated mechanical anisotropy concomitant with the direction of the macro-pores. An analytical modelling approach was applied, which demonstrated that the presence of Bioglass® did not significantly alter the porous architecture of these foams and reflected the mechanical anisotropy which was congruent with the scanning electron microscopy investigation. This study found that the Ishai–Cohen and Gibson–Ashby models can be combined to predict the compressive modulus of the composite foams. The modulus and density of these complex foams are related by a power-law function with an exponent between 2 and 3