Tyrosine kinase inhibitors (TKI) such as sunitinib and pazopanib display their efficacy in a variety of solid tumours. However, their use in therapy is limited by the lack of evidence about the ...ability to induce cell death in cancer cells. Our aim was to evaluate cytotoxic effects induced by sunitinib and pazopanib in 5637 and J82 bladder cancer cell lines.
Cell viability was tested by MTT assay. Autophagy was evaluated by western blot using anti-LC3 and anti-p62 antibodies, acridine orange staining and FACS analysis. Oxygen radical generation and necrosis were determined by FACS analysis using DCFDA and PI staining. Cathepsin B activation was evaluated by western blot and fluorogenic Z-Arg-Arg-AMC peptide. Finally, gene expression was performed using RT-PCR Profiler array.
We found that sunitinib treatment for 24 h triggers incomplete autophagy, impairs cathepsin B activation and stimulates a lysosomal-dependent necrosis. By contrast, treatment for 48 h with pazopanib induces cathepsin B activation and autophagic cell death, markedly reversed by CA074-Me and 3-MA, cathepsin B and autophagic inhibitors, respectively. Finally, pazopanib upregulates the α-glucosidase and downregulates the TP73 mRNA expression.
Our results showing distinct cell death mechanisms activated by different TKIs, provide the biological basis for novel molecularly targeted approaches.
Driving pressure (ΔP) represents tidal volume normalised to respiratory system compliance (CRS) and is a novel parameter to target ventilator settings. We conducted a study to determine whether CRS ...and ΔP reflect aerated lung volume and dynamic strain during general anaesthesia.
Twenty non-obese patients undergoing open abdominal surgery received three PEEP levels (2, 7, or 12 cm H2O) in random order with constant tidal volume ventilation. Respiratory mechanics, lung volumes, and alveolar recruitment were measured to assess end-expiratory aerated volume, which was compared with the patient's individual predicted functional residual capacity in supine position (FRCp).
CRS was linearly related to aerated volume and ΔP to dynamic strain at PEEP of 2 cm H2O (intraoperative FRC) (r=0.72 and r=0.73, both P<0.001). These relationships were maintained with higher PEEP only when aerated volume did not overcome FRCp (r=0.73, P<0.001; r=0.54, P=0.004), with 100 ml lung volume increases accompanied by 1.8 ml cm H2O−1 (95% confidence interval 1.1–2.5) increases in CRS. When aerated volume was greater or equal to FRCp (35% of patients at PEEP 2 cm H2O, 55% at PEEP 7 cm H2O, and 75% at PEEP 12 cm H2O), CRS and ΔP were independent from aerated volume and dynamic strain, with CRS weakly but significantly inversely related to alveolar dead space fraction (r=–0.47, P=0.001). PEEP-induced alveolar recruitment yielded higher CRS and reduced ΔP only at aerated volumes below FRCp (P=0.015 and 0.008, respectively).
During general anaesthesia, respiratory system compliance and driving pressure reflect aerated lung volume and dynamic strain, respectively, only if aerated volume does not exceed functional residual capacity in supine position, which is a frequent event when PEEP is used in this setting.
A ruthenium(
ii
)
p
-cymene derivative of quercetin was synthesized and functionally tested for cholesterol-lowering ability
via
direct 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) inhibition. ...Ruthenium complexation dramatically increased the inhibition potency of the parent quercetin toward HMGR, with a consequent enhancement of the cholesterol-lowering effect in hepatic cells.
A ruthenium(
ii
)
p
-cymene derivative of quercetin was synthesized and functionally tested for cholesterol-lowering ability
via
direct 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) inhibition.
Mangiferin is a natural xanthone glycoside with therapeutic potential. Herein, its cytotoxic properties were explored in a human cell model of breast adenocarcinoma. The results supported the ...multi-target nature of mangiferin action, as the inhibition of three enzymatic systems, namely HMG-CoA reductase, the proteasome and plasmin, respectively in charge of regulating cholesterol homeostasis, protein turnover and cell adhesion, was documented for the first time. Globally, mangiferin was able to selectively block breast cancer cell growth by inducing apoptosis and by arresting cell proliferation through a combined action on cholesterol and proteasome pathways, as well as to inhibit plasmin-mediated mechanisms of cell migration.
The effect of a group of natural flavonoids on human thrombin amidolytic activity was investigated using a spectrophotometric inhibition assay while information on the kinetics and thermodynamics was ...obtained using optical biosensor techniques. All the flavonoids tested acted as reversible inhibitors, and the quercetin–thrombin complex was found to be most stable at pH=7.5. Docking analysis indicated that quercetin's inhibitory behavior could be related to its planar structure and low steric hindrance, and to its ability to form a critical H-bond with thrombin His57.
Targeted protein degradation refers to the use of small molecules that recruit a ubiquitin ligase to a target protein for ubiquitination and subsequent proteasome-dependent degradation. While ...degraders have been developed for many targets, key questions regarding degrader development and the consequences of acute pharmacological degradation remain, specifically for targets that exist in obligate multi-protein complexes. Here, we synthesize a pan-histone deacetylase (HDAC) degrader library for the chemo-proteomic exploration of acute degradation of a key class of chromatin-modifying enzymes. Using chemo-proteomics, we not only map the degradability of the zinc-dependent HDAC family identifying leads for targeting HDACs 1–8 and 10 but also explore important aspects of degrading epigenetic enzymes. We discover cell line-driven target specificity and that HDAC degradation often results in collateral loss of HDAC-containing repressive complexes. These findings potentially offer a new mechanism toward controlling chromatin structure, and our resource will facilitate accelerated degrader design and development for HDACs.
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•Pan-HDAC degrader library enabled chemo-proteomics map of HDAC degradability•HDAC degradation results in degradation of HDAC-containing repressive complexes•Cell line-dependent HDAC isoform specificity
Xiong et al. report the design and synthesis of a diverse multi-targeted library of HDAC degraders and assess their activity in cells. The findings provide a roadmap for future design of HDAC-targeted degraders and reveal important insights regarding the biological role of this epigenetic family of enzymes.
Nitric oxide and prostaglandins are among the numerous substances released by activated glial cells. The aim of this study was to evaluate the effect of high-level aspirin on iNOS expression in ...cultured rat glial cells treated with lipopolysaccharide (LPS) as pathological stimulator. Using Western Blotting, we verified that aspirin enhanced LPS-induced iNOS expression and the presence of 15-deoxy-Δ
12,14-prostaglandin (15d-PGJ
2) suppressed this aspirin effect. However, the exposure of LPS-treated glial cells to aspirin resulted in a decrease of NO production. These results suggest that aspirin interferes with the cross-talk of prostaglandins and NO, blocking the endogenous negative control exerted by COX products on iNOS expression. On the other side, aspirin seems to act directly on iNOS reducing its activity, even if it does not completely block NO release by LPS-stimulated glial cells. Then aspirin could maintain homeostatic functions of NO, while it prevents toxic effects, corresponding to high NO concentrations.
Electromagnetic fields are an assessed cause of prolonging free radicals lifespan. This study was carried out to investigate the influence of extremely low frequency electromagnetic fields on protein ...oxidation and on the 20S proteasome functionality, the complex responsible for the degradation of oxidized proteins. Caco 2 cells were exposed, for 24–72 hours, to 1 mT, 50 Hz electromagnetic fields. The treatment induced a time-dependent increase both in cell growth and in protein oxidation, more evident in the presence of TPA, while no changes in cell viability were detected. Exposing the cells to 50 Hz electromagnetic fields caused a global activation of the 20S proteasome catalytic components, particularly evident at 72 hours exposure and in the presence of TPA. The finding that EGCG, a natural antioxidant compound, counteracted the field-related pro-oxidant effects demonstrates that the increased proteasome activity was due to an enhancement in intracellular free radicals.
Wheat sprouts contain a very high level of organic phosphates and a powerful cocktail of different molecules such as enzymes, reducing glycosides and polyphenols.
The antioxidant properties of wheat ...sprouts have been widely documented and it has been shown that they are able to protect DNA against free-radicals mediated oxidative damage. Furthermore, we have recently reported on the effects of several polyphenols on 20S proteasomes, underlying the dual role of epigallocatechin-3-gallate as an antioxidant and a proteasome effector in cancer cells.
The aim of this study was to investigate the effects of wheat sprout extracts on 20S proteasome functionality. Wheat sprout extracts have been analysed and characterized for their polyphenolic content using the Folin-Ciocalteau reagent and RP-HPLC technique. Comparing our data with a polyphenol standard mixture we identified five different polyphenols: gallic acid, epigallocatechin-3-gallate, epigallocatechin, epicatechin and catechin.
The treatment of isolated 20S proteasomes with the extract induced a gradual inhibition of all the tested components, ChT-L, T-L, PGPH and BrAAP, in both the complexes. At low extract concentration a slight activation of the enzyme was evident only for the BrAAP component of the constitutive enzyme and the ChT-L activity of the immunoproteasome. β-casein degradation rate decreased, particularly with the immunoproteasome. Human Colon adenocarcinoma (Caco) cells, stimulated with 12-
O-tetradecanoylphorbol-13-acetate, showed activation of the 20S proteasome activities at short incubation times and an increase in intracellular oxidative proteins. Cells treatment with wheat sprout extract led to proteasome inhibition in unstimulated cells and attenuated the effects mediated by TPA. Finally, exposure to the extract affected the expression levels of pro-apoptotic proteins.
Targeted protein degradation (TPD) refers to the use of small molecules to induce ubiquitin-dependent degradation of proteins. TPD is of interest in drug development, as it can address previously ...inaccessible targets. However, degrader discovery and optimization remains an inefficient process due to a lack of understanding of the relative importance of the key molecular events required to induce target degradation. Here, we use chemo-proteomics to annotate the degradable kinome. Our expansive dataset provides chemical leads for ∼200 kinases and demonstrates that the current practice of starting from the highest potency binder is an ineffective method for discovering active compounds. We develop multitargeted degraders to answer fundamental questions about the ubiquitin proteasome system, uncovering that kinase degradation is p97 dependent. This work will not only fuel kinase degrader discovery, but also provides a blueprint for evaluating targeted degradation across entire gene families to accelerate understanding of TPD beyond the kinome.
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•A global map of kinase degradability provides chemical leads for >200 kinases•Open-access chemical proteomics resource (https://proteomics.fischerlab.org)•Large-scale chemical exploration of key variables for targeted protein degradation•Multi-targeted degraders uncover fundamentals of ubiquitin-mediated protein turnover
A synthetic chemistry and chemo-proteomics platform used to annotate the “degradable kinome” provides chemical leads for developing degraders of approximately 200 distinct kinase targets and offers new general design principles for developing future kinase degraders.
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