Cancer therapy has traditionally focused on eliminating fast-growing populations of cells. Yet, an increasing body of evidence suggests that small subpopulations of cancer cells can evade strong ...selective drug pressure by entering a 'persister' state of negligible growth. This drug-tolerant state has been hypothesized to be part of an initial strategy towards eventual acquisition of bona fide drug-resistance mechanisms. However, the diversity of drug-resistance mechanisms that can expand from a persister bottleneck is unknown. Here we compare persister-derived, erlotinib-resistant colonies that arose from a single, EGFR-addicted lung cancer cell. We find, using a combination of large-scale drug screening and whole-exome sequencing, that our erlotinib-resistant colonies acquired diverse resistance mechanisms, including the most commonly observed clinical resistance mechanisms. Thus, the drug-tolerant persister state does not limit--and may even provide a latent reservoir of cells for--the emergence of heterogeneous drug-resistance mechanisms.
Drugs that mirror the cellular effects of starvation mimics are considered promising therapeutics for common metabolic disorders, such as obesity, liver steatosis, and for ageing. Starvation, or ...caloric restriction, is known to activate the transcription factor EB (TFEB), a master regulator of lipid metabolism and lysosomal biogenesis and function. Here, we report a nanotechnology-enabled high-throughput screen to identify small-molecule agonists of TFEB and discover three novel compounds that promote autophagolysosomal activity. The three lead compounds include the clinically approved drug, digoxin; the marine-derived natural product, ikarugamycin; and the synthetic compound, alexidine dihydrochloride, which is known to act on a mitochondrial target. Mode of action studies reveal that these compounds activate TFEB via three distinct Ca
-dependent mechanisms. Formulation of these compounds in liver-tropic biodegradable, biocompatible nanoparticles confers hepatoprotection against diet-induced steatosis in murine models and extends lifespan of Caenorhabditis elegans. These results support the therapeutic potential of small-molecule TFEB activators for the treatment of metabolic and age-related disorders.
Tumor necrosis factor α (TNFα) is a proinflammatory cytokine with important roles in regulating inflammatory responses as well as cell cycle proliferation and apoptosis. Although TNFα stimulates ...apoptosis, it also activates the transcription factor NF-κB, and studies have shown that inhibition of NF-κB potentiates the cytotoxicity of TNFα. Since several chemotherapy agents act like TNFα to both promote apoptosis and activate NF-κB, understanding the role of NF-κB in suppressing apoptosis may have significant clinical applications. To understand the effects of stimulation with TNFα and the role of NF-κB in regulating this response, a 23k human cDNA microarray was used to screen TNFα-inducible genes in HeLa cells. Real-time PCR verified expression changes in 16 of these genes and revealed three distinct temporal patterns of expression after TNFα stimulation. Using RNA interference to disrupt expression of the p65 subunit of NF-κB, all but two of the genes were shown to depend on this transcription factor for their expression, which correlated well with the existence of NF-κB binding sites in most of their promoters. Inflammatory, proapoptotic, and antiapoptotic genes were all shown to be regulated by NF-κB, demonstrating the wide variety of targets activated by NF-κB signaling and the necessity of differentiating among these genes for therapeutic purposes.
Type 1 diabetes (T1D) accounts for an estimated 5% of all diabetes in the United States, afflicting over 1.25 million individuals. Maintaining long-term blood glucose control is the major goal for ...individuals with T1D. In T1D, insulin-secreting pancreatic islet β-cells are destroyed by the immune system, but glucagon-secreting islet α-cells survive. These remaining α-cells no longer respond properly to fluctuating blood glucose concentrations. Dysregulated α-cell function contributes to hyper- and hypoglycemia which can lead to macrovascular and microvascular complications. To this end, we sought to discover small molecules that suppress α-cell function for their potential as preclinical candidate compounds. Prior high-throughput screening identified a set of glucagon-suppressing compounds using a rodent α-cell line model, but these compounds were not validated in human systems.
Here, we dissociated and replated primary human islet cells and exposed them to 24 h treatment with this set of candidate glucagon-suppressing compounds. Glucagon accumulation in the medium was measured and we determined that compounds SW049164 and SW088799 exhibited significant activity. Candidate compounds were also counter-screened in our InsGLuc-MIN6 β-cell insulin secretion reporter assay. SW049164 and SW088799 had minimal impact on insulin release after a 24 h exposure. To further validate these hits, we treated intact human islets with a selection of the top candidates for 24 h. SW049164 and SW088799 significantly inhibited glucagon release into the medium without significantly altering whole islet glucagon or insulin content. In concentration-response curves SW088799 exhibited significant inhibition of glucagon release with an IC50 of 1.26 µM.
Given the set of tested candidates were all top hits from the primary screen in rodent α-cells, this suggests some conservation of mechanism of action between human and rodents, at least for SW088799. Future structure-activity relationship studies of SW088799 may aid in elucidating its protein target(s) or enable its use as a tool compound to suppress α-cell activity
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Autosis is a distinct form of cell death that requires both autophagy genes and the Na+,K+-ATPase pump. However, the relationship between the autophagy machinery and Na+,K+-ATPase is unknown. We ...explored the hypothesis that Na+,K+-ATPase interacts with the autophagy protein Beclin 1 during stress and autosis-inducing conditions. Starvation increased the Beclin 1/Na+,K+-ATPase interaction in cultured cells, and this was blocked by cardiac glycosides, inhibitors of Na+,K+-ATPase. Increases in Beclin 1/Na+,K+-ATPase interaction were also observed in tissues from starved mice, livers of patients with anorexia nervosa, brains of neonatal rats subjected to cerebral hypoxia-ischemia (HI), and kidneys of mice subjected to renal ischemia/reperfusion injury (IRI). Cardiac glycosides blocked the increased Beclin 1/Na+,K+-ATPase interaction during cerebral HI injury and renal IRI. In the mouse renal IRI model, cardiac glycosides reduced numbers of autotic cells in the kidney and improved clinical outcome. Moreover, blockade of endogenous cardiac glycosides increased Beclin 1/Na+,K+-ATPase interaction and autotic cell death in mouse hearts during exercise. Thus, Beclin 1/Na+,K+-ATPase interaction is increased in stress conditions, and cardiac glycosides decrease this interaction and autosis in both pathophysiological and physiological settings. This crosstalk between cellular machinery that generates and consumes energy during stress may represent a fundamental homeostatic mechanism.
The originally published version of this Article contained an error in the spelling of the author Nathaniel W. Oswald, which was incorrectly given as Nathaniel W. Olswald. This has now been corrected ...in both the PDF and HTML versions of the Article.
Autophagy, a lysosomal degradation pathway, plays a crucial role in cellular homeostasis, development, immunity, tumor suppression, metabolism, prevention of neurodegeneration, and lifespan ...extension. Thus, pharmacological stimulation of autophagy may be an effective approach for preventing or treating certain human diseases and/or aging. We sought to establish a method for developing new chemical compounds that specifically induce autophagy. To do this, we developed two assays to identify compounds that target a key regulatory node of autophagy inductionspecifically, the binding of Bcl-2 (a negative regulator of autophagy) to Beclin 1 (an allosteric modulator of the Beclin 1/VPS34 lipid kinase complex that functions in autophagy initiation). These assays use either a split-luciferase assay to measure Beclin 1/Bcl-2 binding in cells or an AlphaLISA assay to directly measure direct Beclin 1/Bcl-2 binding in vitro. We screened two different chemical compound libraries, comprising ∼300 K compounds, to identify small molecules that disrupt Beclin 1/Bcl-2 binding and induce autophagy. Three novel compounds were identified that directly inhibit Beclin 1/Bcl-2 interaction with an IC50 in the micromolar range and increase autophagic flux. These compounds do not demonstrate significant cytotoxicity, and they exert selectivity for disruption of Bcl-2 binding to the BH3 domain of Beclin 1 compared with the BH3 domain of the pro-apoptotic Bcl-2 family members, Bax and Bim. Thus, we have identified candidate molecules that serve as lead templates for developing potent and selective Beclin 1/Bcl-2 inhibitors that may be clinically useful as autophagy-inducing agents.
Correction to: Oncogene (2003) 22, 2054–2064. doi: 10.1038/sj.onc.1206262 Since the publication of the above manuscript, the authors have identified an error in the sequence of the p65 siRNA ...oligonucleotide as listed in the study. The error is on page 2062, under Materials and methods, SiRNAi transfection and RNA preparation.
The automobile recall has been implemented in China for more than 10 years, the recall process has entered a mature stage and the recall rate also tends to be stable. However, the automobile recall, ...quite common in abroad, has a serious impact on the consumer’s purchase decision in the country. In this paper, firstly, analyze the data of automobile recall in the past 10 years; then using the Bayesian formula, to study the possibility change of consumer’s purchase decision under the condition of the automobile recall or not, and to study the impact of the recall on the final purchase decision making under consideration of performance and price.
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