FBXL19 Targeted STK11 Degradation Enhances Cigarette Smoke-Induced Airway Epithelial Cell Cytotoxicity.

Objective: To investigate the effects of cigarette smoke (CS) on Serine/Threonine Kinase 11 (STK11) and to determine STK11's role in CS-induced airway epithelial cell cytotoxicity.Methods: STK11 expression levels in the lung tissues of smokers with or without COPD and mice exposed to CS or room air (RA) were determined by immunoblotting and RT-PCR. BEAS-2Bs-human bronchial airway epithelial cells were exposed to CS extract (CSE), and the changes in STK11 expression levels were determined by immunoblotting and RT-PCR. BEAS-2B cells were transfected with STK11-specific siRNA or STK11 expression plasmid, and the effects of CSE on airway epithelial cell cytotoxicity were measured. To determine the specific STK11 degradation-proteolytic pathway, BEAS-2Bs were treated with cycloheximide alone or combined with MG132 or leupeptin. Finally, to identify the F-box protein mediating the STK11 degradation, a screening assay was performed using transfection with a panel of FBXL E3 ligase subunits.Results: STK11 protein levels were significantly decreased in the lung tissues of smokers with COPD relative to smokers without COPD. STK11 protein levels were also significantly decreased in mouse lung tissues exposed to CS compared to RA. Exposure to CSE shortened the STK11 mRNA and protein half-life to 4 h in BEAS-2B cells. STK11 protein overexpression attenuated the CSE-induced cytotoxicity; in contrast, its knockdown augmented CSE-induced cytotoxicity. FBXL19 mediates CSE-induced STK11 protein degradation via the ubiquitin-proteasome pathway in cultured BEAS-2B cells. FBXL19 overexpression led to accelerated STK11 ubiquitination and degradation in a dose-dependent manner.Conclusions: Our results suggest that CSE enhances the degradation of STK11 protein in airway epithelial cells via the FBXL19-mediated ubiquitin-proteasomal pathway, leading to augmented cell death.HIGHLIGHTSLung tissues of COPD-smokers exhibited a decreased STK11 RNA and protein expression.STK11 overexpression attenuates CS-induced airway epithelial cell cytotoxicity.STK11 depletion augments CS-induced airway epithelial cell cytotoxicity.CS diminishes STK11 via FBXL19-mediated ubiquitin-proteasome degradation.

Loss of the yeast transporter Agp2 upregulates the pleiotropic drug-resistant pump Pdr5 and confers resistance to the protein synthesis inhibitor cycloheximide.

The transmembrane protein Agp2, initially shown as a transporter of L-carnitine, mediates the high-affinity transport of polyamines and the anticancer drug bleomycin-A5. Cells lacking Agp2 are hyper-resistant to polyamine and bleomycin-A5. In these earlier studies, we showed that the protein synthesis inhibitor cycloheximide blocked the uptake of bleomycin-A5 into the cells suggesting that the drug uptake system may require de novo synthesis. However, our recent findings demonstrated that cycloheximide, instead, induced rapid degradation of Agp2, and in the absence of Agp2 cells are resistant to cycloheximide. These observations raised the possibility that the degradation of Agp2 may allow the cell to alter its drug resistance network to combat the toxic effects of cycloheximide. In this study, we show that membrane extracts from agp2Δ mutants accentuated several proteins that were differentially expressed in comparison to the parent. Mass spectrometry analysis of the membrane extracts uncovered the pleiotropic drug efflux pump, Pdr5, involved in the efflux of cycloheximide, as a key protein upregulated in the agp2Δ mutant. Moreover, a global gene expression analysis revealed that 322 genes were differentially affected in the agp2Δ mutant versus the parent, including the prominent PDR5 gene and genes required for mitochondrial function. We further show that Agp2 is associated with the upstream region of the PDR5 gene, leading to the hypothesis that cycloheximide resistance displayed by the agp2Δ mutant is due to the derepression of the PDR5 gene.

Changes in the conflicting nongenomic effects of progesterone in rat myometrium during pregnancy.

AIMS: Although the functions of progesterone in the myometrium are well-established, the nongenomic effects of progesterone in pregnant myometrial contractions are still unclear. Therefore, this study aimed to investigate changes in the nongenomic effects of progesterone during pregnancy. MAIN METHODS: Myometrial strips were obtained from non-pregnant, pregnant, and postpartum rats, and the nongenomic effects of progesterone in the myometrium during pregnancy were examined. Additionally, the influence of actinomycin D and cycloheximide and the effects of Org OD-02-0 (a specific membrane progesterone receptor (mPR) agonist) in the myometrium were investigated. Moreover, DNA microarray and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to identify genes involved in progesterone-induced effects in the myometrium. KEY FINDINGS: Progesterone did not cause rhythmic contractions in non-pregnant myometrium but induced rhythmic contractions in pregnant myometrium, with the effects peaking at 20 d + 8 h of pregnancy. However, myometrial contractions decreased after delivery and were restored to non-pregnant levels at 7 d postpartum. Additionally, progesterone stably inhibited high KCl-induced myometrial contractions during pregnancy. Moreover, the nongenomic effects of progesterone were unaffected by actinomycin D or cycloheximide, and Org OD-02-0 effectively mimicked these effects. DNA microarray analysis and qRT-PCR revealed a significant increase in mPRß gene expression during pregnancy. However, mPRα, mPRγ, mPRδ, and mPRε expression levels remained unchanged. SIGNIFICANCE: The stimulatory nongenomic effect of progesterone, which was inducible and mPRß-dependent during pregnancy, may be involved in parturition. The inhibitory effect, which was constitutive and depended on other mPRs, may be involved in pregnancy maintenance.

Pharmacokinetics of cycloheximide in rats and evaluation of its effect as a blocker of intestinal lymph formation.

Cycloheximide (CHX) has been used to reduce the flow of intestinal lymph and as a non-surgical tool to study drug absorption via the intestinal lymphatics. Pharmacokinetic information on the agent, and its relationship to effect and toxicity, have not been examined. The goal of this study was to provide pharmacokinetic data and link it to lymph-blocking and toxicological effects. Jugular-vein cannulated (JVC) adult Sprague-Dawley male rats were administered 0.5 mg/kg CHX by oral, intraperitoneal (ip), and intravenous routes followed by blood draws, and CHX was assayed using LC-MS/MS. Another four JVC rats were given peanut oil (2 mL/kg) without and then with CHX to measure effects on lipid absorption as a surrogate indicator of lymph flow. One-week later plasma biochemistry measures were obtained. The results indicated that CHX had a high clearance and volume of distribution, and oral absolute bioavailability of 0.47 with 0.5 mg/kg. CHX was associated with dose- and route-dependent pharmacokinetics. The relative bioavailability after ip doses was over 3. CHX had low plasma protein binding and minor urinary excretion. Metabolism appeared to be occur by oxidation and glucuronidation. Reductions in plasma lipids (24-40 %) were seen after 2.5 mg/kg orally with signs of inflammation and increased liver enzymes persisting for a week after the dose. CHX was associated with a reduction in lipid absorption after oral doses of 2.5 mg/kg, which seems to justify its use as a non-surgical tool to evaluate the lymphatic pathway of absorption of drugs. However, it also possesses hepatotoxicity, which should be taken into consideration in its use.

Influence of reconsolidation in maintenance of cocaine-associated contextual memories formed during adolescence or adulthood.

Adolescents are at increased risk to develop substance use disorders and suffer from relapse throughout life. Targeted weakening of drug-associated memories has been shown to reduce relapse-like behavior in adult rats, however this process has been understudied in adolescents. We aimed to examine whether adolescent-formed, cocaine-associated memories could be manipulated via reconsolidation mechanisms. To accomplish this objective, we used an abbreviated operant cocaine self-administration paradigm (ABRV Coc-SA). Adult and adolescent rats received jugular catheterization surgery followed by ABRV Coc-SA in a distinct context for 2 h, 2×/day over 5 days. Extinction training (EXT) occurred in a second context for 2 h, 2×/day over 4 days. To retrieve cocaine-context memories, rats were exposed to the cocaine-paired context for 15 min, followed by subcutaneous injection of vehicle or the protein synthesis inhibitor cycloheximide (2.5 mg/kg). Two additional EXT sessions were conducted before a 2 h reinstatement test in the cocaine-paired context to assess cocaine-seeking behavior. We find that both adult and adolescent cocaine-exposed rats show similar levels of cocaine-seeking behavior regardless of post-reactivation treatment. Our results suggest that systemic treatment with the protein synthesis inhibitor cycloheximide does not impair reconsolidation of cocaine-context memories and subsequent relapse during adulthood or adolescence.

Redefining pleiotropic drug resistance in a pathogenic yeast: Pdr1 functions as a sensor of cellular stresses in Candida glabrata.

Candida glabrata is a prominent opportunistic fungal pathogen of humans. The increasing incidence of C. glabrata infections is attributed to both innate and acquired resistance to antifungals. Previous studies suggest the transcription factor Pdr1 and several target genes encoding ABC transporters are critical elements of pleiotropic defense against azoles and other antifungals. This study utilizes Hermes transposon insertion profiling to investigate Pdr1-independent and Pdr1-dependent mechanisms that alter susceptibility to the frontline antifungal fluconazole. Several new genes were found to alter fluconazole susceptibility independent of Pdr1 (CYB5, SSK1, SSK2, HOG1, TRP1). A bZIP transcription repressor of mitochondrial function (CIN5) positively regulated Pdr1 while hundreds of genes encoding mitochondrial proteins were confirmed as negative regulators of Pdr1. The antibiotic oligomycin activated Pdr1 and antagonized fluconazole efficacy likely by interfering with mitochondrial processes in C. glabrata. Unexpectedly, disruption of many 60S ribosomal proteins also activated Pdr1, thus mimicking the effects of the mRNA translation inhibitors. Cycloheximide failed to fully activate Pdr1 in a cycloheximide-resistant Rpl28-Q38E mutant. Similarly, fluconazole failed to fully activate Pdr1 in a strain expressing a low-affinity variant of Erg11. Fluconazole activated Pdr1 with very slow kinetics that correlated with the delayed onset of cellular stress. These findings are inconsistent with the idea that Pdr1 directly senses xenobiotics and support an alternative hypothesis where Pdr1 senses cellular stresses that arise only after engagement of xenobiotics with their targets. IMPORTANCE Candida glabrata is an opportunistic pathogenic yeast that causes discomfort and death. Its incidence has been increasing because of natural defenses to our common antifungal medications. This study explores the entire genome for impacts on resistance to fluconazole. We find several new and unexpected genes can impact susceptibility to fluconazole. Several antibiotics can also alter the efficacy of fluconazole. Most importantly, we find that Pdr1-a key determinant of fluconazole resistance-is not regulated directly through binding of fluconazole and instead is regulated indirectly by sensing the cellular stresses caused by fluconazole blockage of sterol biosynthesis. This new understanding of drug resistance mechanisms could improve the outcomes of current antifungals and accelerate the development of novel therapeutics.

Degradation of MYC by the mutant p53 reactivator drug, COTI-2 in breast cancer cells.

TP53 (p53) and MYC are amongst the most frequently altered genes in cancer. Both are thus attractive targets for new anticancer therapies. Historically, however, both genes have proved challenging to target and currently there is no approved therapy against either. The aim of this study was to investigate the effect of the mutant p53 reactivating drug, COTI-2 on MYC. Total MYC, pSer62 MYC and pThr58 MYC were detected using Western blotting. Proteasome-mediated degradation was determined using the proteasome, inhibitor MG-132, while MYC half-life was measured using pulse chase experiments in the presence of cycloheximide. Cell proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Treatment of 5 mutant p53 breast cancer cell lines with COTI-2 resulted in dose-dependent MYC degradation. Addition of the proteasome inhibitor, MG132, rescued the degradation, suggesting that this proteolytic system was at least partly responsible for the inactivation of MYC. Using cycloheximide in pulse chase experiments, COTI-2 was found to reduce the half-life of MYC in 2 different mutant p53 breast cancer cell lines, i.e., from 34.8 to 18.6 min in MDA-MB-232 cells and from 29.6 to 20.3 min in MDA-MB-468 cells. Co-treatment with COTI-2 and the MYC inhibitor, MYCi975 resulted in synergistic growth inhibition in all 4 mutant p53 cell lines investigated. The dual ability of COTI-2 to reactivate mutant p53 and degrade MYC should enable this compound to have broad application as an anticancer drug.

Self-Nanoemulsifying Drug Delivery System for Enhanced Bioavailability of Madecassic Acid: In vitro and in vivo Evaluation.

Purpose: Madecassic acid (MCA) is a natural triterpenoid isolated from centellae herba that has diverse biological effects, such as anti-inflammatory, antioxidant, and anticancer activities. However, the efficacy of MCA is limited by low oral bioavailability caused by its extremely poor aqueous solubility. This study aimed to develop a self-nanoemulsifying drug delivery system (SNEDDS) for MCA to improve its oral absorption. Methods: The utilized oil phases, surfactants, and co-surfactants for SNEDDS were selected based on the solubility of MCA and emulsification efficiency. The optimized formulation was characterized for pharmaceutical properties and its pharmacokinetic behavior was examined in rats. Besides, the intestinal absorption property of MCA was investigated using in situ single-pass intestinal perfusion and intestinal lymphatic transport. Results: The optimized nanoemulsion formula consists of Capryol 90:Labrasol:Kolliphor ELP:Transcutol HP in a weight ratio of 1:2.7:2.7:3.6 (w/w/w/w). MCA-loaded SNEDDS presented a small droplet size (21.52 ± 0.23 nm), with a zeta potential value of -3.05 ± 0.3 mV. Compared with pure MCA, SNEDDS had a higher effective permeability coefficient and showed 8.47-fold and 4.01-fold of maximum plasma concentration (Cmax) and area under the plasma concentration-time curve (AUC), respectively. Cycloheximide was pretreated before the experiment to evaluate the degree of lymphatic uptake. The results showed that cycloheximide greatly influenced the absorption of SNEDDS, resulting in 82.26% and 76.98% reduction in Cmax and AUC, respectively. Conclusion: This study reports the MCA-loaded SNEDDS with distinctly enhanced in vitro and in vivo performance compared with pure MCA and concludes that the SNEDDS formulation could be a viable and effective strategy for improving the dissolution rate and bioavailability of poor aqueous-soluble ingredients.

The Yeast Permease Agp2 Senses Cycloheximide and Undergoes Degradation That Requires the Small Protein Brp1-Cellular Fate of Agp2 in Response to Cycloheximide.

The Saccharomyces cerevisiae Agp2 is a plasma membrane protein initially reported to be an uptake transporter for L-carnitine. Agp2 was later rediscovered, together with three additional proteins, Sky1, Ptk2, and Brp1, to be involved in the uptake of the polyamine analogue bleomycin-A5, an anticancer drug. Mutants lacking either Agp2, Sky1, Ptk2, or Brp1 are extremely resistant to polyamines and bleomycin-A5, suggesting that these four proteins act in the same transport pathway. We previously demonstrated that pretreating cells with the protein synthesis inhibitor cycloheximide (CHX) blocked the uptake of fluorescently labelled bleomycin (F-BLM), raising the possibility that CHX could either compete for F-BLM uptake or alter the transport function of Agp2. Herein, we showed that the agp2Δ mutant displayed striking resistance to CHX as compared to the parent, suggesting that Agp2 is required to mediate the physiological effect of CHX. We examined the fate of Agp2 as a GFP tag protein in response to CHX and observed that the drug triggered the disappearance of Agp2 in a concentration- and time-dependent manner. Immunoprecipitation analysis revealed that Agp2-GFP exists in higher molecular weight forms that were ubiquitinylated, which rapidly disappeared within 10 min of treatment with CHX. CHX did not trigger any significant loss of Agp2-GFP in the absence of the Brp1 protein; however, the role of Brp1 in this process remains elusive. We propose that Agp2 is degraded upon sensing CHX to downregulate further uptake of the drug and discuss the potential function of Brp1 in the degradation process.

Thoracic perivascular adipose tissue inhibits VSMC apoptosis and aortic aneurysm formation in mice via the secretome of browning adipocytes.

Abdominal aortic aneurysm (AAA) is a dangerous vascular disease without any effective drug therapies so far. Emerging evidence suggests the phenotypic differences in perivascular adipose tissue (PVAT) between regions of the aorta are implicated in the development of atherosclerosis evidenced by the abdominal aorta more vulnerable to atherosclerosis than the thoracic aorta in large animals and humans. The prevalence of thoracic aortic aneurysms (TAA) is much less than that of abdominal aortic aneurysms (AAA). In this study we investigated the effect of thoracic PVAT (T-PVAT) transplantation on aortic aneurysm formation and the impact of T-PVAT on vascular smooth muscle cells. Calcium phosphate-induced mouse AAA model was established. T-PVAT (20 mg) was implanted around the abdominal aorta of recipient mice after removal of endogenous abdominal PVAT (A-PVAT) and calcium phosphate treatment. Mice were sacrificed two weeks after the surgery and the maximum external diameter of infrarenal aorta was measured. We found that T-PVAT displayed a more BAT-like phenotype than A-PVAT; transplantation of T-PVAT significantly attenuated calcium phosphate-induced abdominal aortic dilation and elastic degradation as compared to sham control or A-PVAT transplantation. In addition, T-PVAT transplantation largely preserved smooth muscle cell content in the abdominal aortic wall. Co-culture of T-PVAT with vascular smooth muscle cells (VSMCs) significantly inhibited H2O2- or TNFα plus cycloheximide-induced VSMC apoptosis. RNA sequencing analysis showed that T-PVAT was enriched by browning adipocytes and anti-apoptotic secretory proteins. We further verified that the secretome of mature adipocytes isolated from T-PVAT significantly inhibited H2O2- or TNFα plus cycloheximide-induced VSMC apoptosis. Using proteomic and bioinformatic analyses we identified cartilage oligomeric matrix protein (COMP) as a secreted protein significantly increased in T-PVAT. Recombinant COMP protein significantly inhibited VSMC apoptosis. We conclude that T-PVAT exerts anti-apoptosis effect on VSMCs and attenuates AAA formation, which is possibly attributed to the secretome of browning adipocytes.

KZL204, a Trifluoromethylated Quinazoline Derivative, Exhibits High Potent Anti-Tumor Effects on Glioblastoma Multiforme U251MG Cells.

OBJECTIVE: Recently, there has been much interest in quinazoline derivatives due to their unique anti-tumor effects. In this study, we aimed to investigate the effects of KZL204, an active trifluoromethylated quinazoline derivative, on a human glioblastoma multiforme (GBM) cell line U251MG. Additionally, we tried to identify the potential target of KZL204 for treating GBM. METHODS: Cell counting kit-8 (CCK-8) assay for cytotoxicity, 5-ethynyl-2-deoxyuridine (EdU) staining for cell proliferation, flow cytometry for cell apoptosis and cell cycle, wound scratch test for cell migration, and transwell assay for cell invasion were carried out on U251MG cells after exposing them to different concentrations of KZL204. In addition, western blot analysis, network pharmacology-based analysis, molecular docking assay, cellular thermal shift assay (CETSA), and cycloheximide chase assay were performed. RESULTS: Our results showed that KZL204 concentration-dependently inhibited U251MG cell proliferation, induced apoptosis, arrested cell cycle in the G2/M phase, and inhibited cell invasion and migration capacity. Further network pharmacology-based analysis revealed that epidermal growth factor receptor (EGFR), FYN, YES1, LYN, ephrin type-A receptor 2 (EPHA2), and EPHA4 are the top 6 core targets for inhibiting cell growth, apoptosis, cell cycle, and metastasis of the GBM cells. Molecular docking and CETSA showed that KZL204 had a strong targeting binding affinity with EPHA2. Cycloheximide chase assay and western blot results demonstrated that KZL204 could down-regulate the protein level of EPHA2. CONCLUSIONS: KZL204 exhibits potent inhibitory activity for glioblastoma multiforme cells, which may be related to its role in promoting the degradation of EPHA2.

Sodium arsenite but not aluminum chloride stimulates ABC transporter activity in renal proximal tubules of killifish (Fundulus heteroclitus).

ABC export proteins including Multidrug resistance-related protein 2 (Mrp2) serve as detoxification mechanism in renal proximal tubules due to active transport of xenobiotics and metabolic waste products into primary urine. The environmental pollutants aluminum and arsenic interfere with a multitude of regulatory mechanisms in the body and here their impact on ABC transporter function was studied. NaAsO2 but not AlCl3 rapidly stimulated Mrp2-mediated Texas Red (TR) transport in isolated renal proximal tubules from killifish, a well-established laboratory model for the determination of efflux transporter activity by utilizing fluorescent substrates for the ABC transporters of interest and confocal microscopy followed by image analysis. This observed stimulation remained unaffected by the translation inhibitor cycloheximide (CHX), but it was abrogated by antagonists and inhibitors of the endothelin receptor type B (ETB)/nitric oxide synthase (NOS)/protein kinase C (PKC) signaling pathway. NaAsO2-triggered effects were abolished as a consequence of PKCα inhibition through Gö6976 and PKCα inhibitor peptide C2-4. Phosphatidylinositol 3-kinase (PI3K) inhibitor LY 294,002 as well as the mammalian target of rapamycin (mTOR) inhibitor rapamycin suppressed NaAsO2-triggered stimulation of luminal TR transport. In addition, the stimulatory effect of NaAsO2 was abolished by GSK650394, an inhibitor of serum- and glucocorticoid-inducible kinase 1 (SGK1), which is an important downstream target. Environmentally relevant concentrations of NaAsO2 further stimulated transport function of P-glycoprotein (P-gp), Multidrug resistance-related protein 4 (Mrp4) and Breast cancer resistance protein (Bcrp) while AlCl3 was ineffective. To our knowledge, this is the first report engaging in the impact of NaAsO2 on efflux transporter signaling and it may contribute to the understanding of defense mechanisms versus this worrying pollutant.

Resveratrol Promotes LRSAM1 E3 Ubiquitin Ligase-Dependent Degradation of Misfolded Proteins Linked with Neurodegeneration.

BACKGROUND/AIMS: Cells require regular maintenance of proteostasis. Synthesis of new polypeptides and elimination of damaged or old proteins is an uninterrupted mechanism essential for a healthy cellular environment. Impairment in the removal of misfolded proteins can disturb proteostasis; such toxic aggregation of misfolded proteins can act as a primary risk factor for neurodegenerative diseases and imperfect ageing. The critical challenge is to design effective protein quality control (PQC) based molecular tactics that could potentially eliminate aggregation-prone protein load from the cell. Still, targeting specific components of the PQC pathway for the suppression of proteotoxic insults retains several challenges. Earlier, we had observed that LRSAM1 promotes the degradation of aberrant proteins. Here, we examined the effect of resveratrol, a stilbenoid phytoalexin compound, treatment on LRSAM1 E3 ubiquitin ligase, involved in the spongiform neurodegeneration. METHODS: In this study, we reported induction of mRNA and protein levels of LRSAM1 in response to resveratrol treatment via RT-PCR, immunoblotting, and immunofluorescence analysis. The LRSAM1-mediated proteasomal-based clearance of misfolded proteins was also investigated via proteasome activity assays, immunoblotting and immunofluorescence analysis. The increased stability of LRSAM1 by resveratrol was demonstrated by cycloheximide chase analysis. RESULTS: Here, we show that resveratrol treatment induces LRSAM1 E3 ubiquitin ligase expression levels. Further, our findings suggest that overexpression of LRSAM1 significantly elevates proteasome activities and improves the degradation of bona fide heat-denatured luciferase protein. Exposure of resveratrol not only slows down the turnover of LRSAM1 but also effectively degrades abnormal proteinaceous inclusions, which eventually promotes cell viability. CONCLUSION: Our findings suggest that resveratrol facilitates LRSAM1 endogenous establishment, which consequently promotes the proteasome machinery for effective removal of intracellular accumulated misfolded or proteasomal-designated substrates. Altogether, our study proposes a promising molecular approach to specifically trigger PQC signaling for efficacious rejuvenation of defective proteostasis via activation of overburdened proteolytic machinery.

Activation of the ß­TrCP/IκBα/inflammation axis limits the sensitivity of liver cancer cells to neddylation inhibition.

Upregulation of protein neddylation occurs in numerous types of human cancer, including liver cancer. MLN4924, a potent neddylation­inhibiting pharmacological agent, demonstrates anticancer ability in numerous cancers. However, the sensitivity of MLN4924 in liver cancer remains unsatisfactory due to factors causing resistance. RT­qPCR and western blotting were utilized to assess the mRNA and protein levels of genes, respectively. Cell Counting Kit­8 assay and colony formation assays were employed to assess cell viability and proliferation. The pathway of protein degradation and stability were determined by western blotting after treatment with MG132 and cycloheximide. An immunoprecipitation assay was utilized to detect the ubiquitination of protein. An in vitro ubiquitination assay was used to determine the ubiquitin linkage. To the best of our knowledge, the present study was the first to demonstrate that NF­κB inhibitor α (IκBα) downregulation and subsequent inflammation in response to MLN4924 limited the antitumor potential of MLN4924. Ectopic expression of IκBα enhanced the antitumor potential of MLN4924 in liver cancer cells. Moreover, the results of the present study demonstrated that MLN4924 decreased IκBα via promoting the K48 linkage of ubiquitin to IκBα. Mechanistic studies demonstrated that MLN4924 enhanced the protein stability of ß­transducin repeat­containing protein (ß­TrCP), promoting the ubiquitination of IκBα, which led to the ubiquitin­mediated degradation of IκBα. In addition, the results of the present study also demonstrated that ß­TrCP knockdown markedly inhibited MLN4924 from suppressing the growth of liver cancer cells, via attenuating MLN4924­mediated IκBα downregulation and inflammation. Collectively, these results indicated that the ß­TrCP/IκBα/inflammation pathway may act as a novel resistance factor of MLN4924, and targeting ß­TrCP may be beneficial for the treatment of liver cancer.

Novel Synergistic Anti-Enteroviral Drug Combinations.

Background: Enterovirus infections affect people around the world, causing a range of illnesses, from mild fevers to severe, potentially fatal conditions. There are no approved treatments for enterovirus infections. Methods: We have tested our library of broad-spectrum antiviral agents (BSAs) against echovirus 1 (EV1) in human adenocarcinoma alveolar basal epithelial A549 cells. We also tested combinations of the most active compounds against EV1 in A549 and human immortalized retinal pigment epithelium RPE cells. Results: We confirmed anti-enteroviral activities of pleconaril, rupintrivir, cycloheximide, vemurafenib, remdesivir, emetine, and anisomycin and identified novel synergistic rupintrivir-vemurafenib, vemurafenib-pleconaril and rupintrivir-pleconaril combinations against EV1 infection. Conclusions: Because rupintrivir, vemurafenib, and pleconaril require lower concentrations to inhibit enterovirus replication in vitro when combined, their cocktails may have fewer side effects in vivo and, therefore, should be further explored in preclinical and clinical trials against EV1 and other enterovirus infections.

Legionella bononiensis sp. nov., isolated from a hotel water distribution system in northern Italy.

Legionella-like isolates, strains 27fs60, 30fs61 and 30cs62T, were isolated from a hotel water distribution system in the Emilia-Romagna region, Italy. Isolates were Gram- and Ziehl Neelsen-stain-negative, rod-shaped, with transitory flagella presence and able to grow at 32-37 °C (with an optimum at 32 °C) on buffered charcoal-yeast extract agar with l-cysteine, glycine-vancomycin-polymyxin B-cycloheximide agar and Wadowsky-Yee medium agar. The strains showed positive reactions for oxidase, hippurate and gelatinase and a weakly positive reaction for catalase. Based on the EUCAST cut-off, strain 30cs62T was resistant to ciprofloxacin (5 mg l-1). The mip and rpoB gene sequences of the three strains showed close matches to those of Legionella quateirensis ATCC 49507T with similarity values of 98.2 and 94.5 %, respectively. Whole genome sequencing of the three strains was performed, resulting in G+C contents of 39.0, 39.1 and 39.0 mol%, respectively. The identity percentage measured by average nucleotide identity between the three strains and their respective closest strains were: 91.32 % L. quateirensis NCTC 12376T, 91.45 % L. quateirensis ATCC 49507T and 91.45 % L. quateirensis ATCC 49507T, respectively. The digital DNA-DNA hybridization analysis demonstrated how the isolates were separated from the most related phylogenetic Legionella species (L. quateirensis ATCC 49507T, ≤40.10 % DNA-DNA relatedness). The concatenated phylogenetic tree based on 16S rRNA, mip, rpoB and rnpB genes, shows a close relationship with L. quateirensis ATCC 49507T. The results obtained confirm the status of an independent species. The name proposed for this species is Legionella bononiensis sp. nov. with 30cs62T (=ATCC TSD-262T=DSM 112526T) as the type strain.

Colletofragarone A2 Inhibits Cancer Cell Growth In Vivo and Leads to the Degradation and Aggregation of Mutant p53.

Mutant p53 not only loses its original tumor suppressor function but also acquires new abilities regarding oncogenic progression. Therefore, the strategy of targeting mutant p53 has attracted attention for cancer therapy. We isolated colletofragarone A2 (CF) from the fungus Colletotrichum sp. (13S020), which decreases mutant p53 levels in cells, and herein examine its effect on mutant p53. CF showed more potent cytotoxic activities on cells with p53R175H structural mutants than those with different p53 statuses such as a DNA-contact mutant, wild-type, and null cells. CF markedly decreased tumor cell growth in vivo using a mouse xenograft model with HuCCT1 (p53R175H) cells. Cotreatment of SK-BR-3 (p53R175H) cells with CF and cycloheximide decreased mutant p53 levels by promoting p53 degradation. In the presence of MG-132, CF induced the accumulation of the aggregated mutant p53. These results suggest that CF inhibits the function of molecular chaperones such as HSP90.

Epimedokoreanin B inhibits the growth of lung cancer cells through endoplasmic reticulum stress-mediated paraptosis accompanied by autophagosome accumulation.

Epimedokoreanin B (EKB) is a prenylated flavonoid isolated from Epimedium koreanum. In this article, we described the anti-cancerous effects of EKB and its underlying mechanism in human non-small cell lung cancer (NSCLC) A549 and NCI-H292 cells. EKB treatment inhibited cell proliferation and migration accompanied by cytoplasmic vacuolation in both cell lines. The cell death induced by EKB lacked the features of apoptosis like chromatin condensation, phosphatidyl serine exposure and caspase cleavage. The vacuoles stimulated by EKB predominantly derived from endoplasmic reticulum (ER) and mitochondria dilation, which are the characteristics of paraptosis. Down-regulation of Alix and up-regulation of ER stress-related proteins after EKB treatment further supported the occurrence of paraptosis. ER stress inhibitor 4-phenylbutyric acid (4-PBA) and protein synthesis inhibitor cycloheximide (CHX) treatment antagonized the vacuoles formation as well as cell death induced by EKB, indicating that ER stress was involved in EKB induced paraptosis. In addition, autophagosome accumulation accompanied with autophagy flux blocking was observed in EKB treated cells, this was consistent with the occurrence of ER stress. Collectively, EKB was demonstrated as a paraptosis-like cell death inducer in A549 and NCI-H292 cells. The inhibitory effect of EKB on lung cancer cell proliferation was further demonstrated in a zebrafish xenograft model. These findings raise the possibility that paraptosis inducers may be considered as alternative choices for lung cancer therapy.

PLEK2 promotes cancer stemness and tumorigenesis of head and neck squamous cell carcinoma via the c-Myc-mediated positive feedback loop.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is one of the most frequent malignancies worldwide and is characterized by unfavorable prognosis, high lymph node metastasis and early recurrence. However, the molecular events regulating HNSCC tumorigenesis remain poorly understood. Therefore, uncovering the underlying mechanisms is urgently needed to identify novel and promising therapeutic targets for HNSCC. In this study, we aimed to explore the role of pleckstrin-2 (PLEK2) in regulating HNSCC tumorigenesis. METHODS: The expression pattern of PLEK2 and its clinical significance in HNSCC were determined by analyzing publicly assessable datasets and our own independent HNSCC cohort. In vitro and in vivo experiments, including cell proliferation, colony formation, Matrigel invasion, tumor sphere formation, ALDEFLUOR, Western blotting assays and xenograft mouse models, were used to investigate the role of PLEK2 in regulating the malignant behaviors of HNSCC cells. The underlying molecular mechanisms for the tumor-promoting role of PLEK2 were elucidated using co-immunoprecipitation, cycloheximide chase analysis, ubiquitination assays, chromatin immunoprecipitation-quantitative polymerase chain reaction, luciferase reporter assays and rescue experiments. RESULTS: The expression levels of PLEK2 mRNA and protein were significantly increased in HNSCC tissues, and PLEK2 overexpression was strongly associated with poor overall survival and therapeutic resistance. Additionally, PLEK2 was important for maintaining the proliferation, invasion, epithelial-mesenchymal transition, cancer stemness and tumorigenesis of HNSCC cells and could alter the cellular metabolism of the cancer cells. Mechanistically, PLEK2 interacted with c-Myc and reduced the association of F-box and WD repeat domain containing 7 (FBXW7) with c-Myc, thereby avoiding ubiquitination and subsequent proteasome-mediated degradation of c-Myc. Moreover, the c-Myc signaling activated by PLEK2 was important for sustaining the aggressive malignant phenotypes and tumorigenesis of HNSCC cells. c-Myc also directly bounded to the PLEK2 promoter and activated its transcription, forming a positive feedback loop. CONCLUSIONS: Collectively, these findings uncover a previously unknown molecular basis of PLEK2-enhanced c-Myc signaling in HNSCC, suggesting that PLEK2 may represent a promising therapeutic target for treating HNSCC.

The Multivalent Polyampholyte Domain of Nst1, a P-Body-Associated Saccharomyces cerevisiae Protein, Provides a Platform for Interacting with P-Body Components.

The condensation of nuclear promyelocytic leukemia bodies, cytoplasmic P-granules, P-bodies (PBs), and stress granules is reversible and dynamic via liquid-liquid phase separation. Although each condensate comprises hundreds of proteins with promiscuous interactions, a few key scaffold proteins are required. Essential scaffold domain sequence elements, such as poly-Q, low-complexity regions, oligomerizing domains, and RNA-binding domains, have been evaluated to understand their roles in biomolecular condensation processes. However, the underlying mechanisms remain unclear. We analyzed Nst1, a PB-associated protein that can intrinsically induce PB component condensations when overexpressed. Various Nst1 domain deletion mutants with unique sequence distributions, including intrinsically disordered regions (IDRs) and aggregation-prone regions, were constructed based on structural predictions. The overexpression of Nst1 deletion mutants lacking the aggregation-prone domain (APD) significantly inhibited self-condensation, implicating APD as an oligomerizing domain promoting self-condensation. Remarkably, cells overexpressing the Nst1 deletion mutant of the polyampholyte domain (PD) in the IDR region (Nst1∆PD) rarely accumulate endogenous enhanced green fluorescent protein (EGFP)-tagged Dcp2. However, Nst1∆PD formed self-condensates, suggesting that Nst1 requires PD to interact with Dcp2, regardless of its self-condensation. In Nst1∆PD-overexpressing cells treated with cycloheximide (CHX), Dcp2, Xrn1, Dhh1, and Edc3 had significantly diminished condensation compared to those in CHX-treated Nst1-overexpressing cells. These observations suggest that the PD of the IDR in Nst1 functions as a hub domain interacting with other PB components.