ATR‐101 inhibits cholesterol efflux and cortisol secretion by ATP‐binding cassette transporters, causing cytotoxic cholesterol accumulation in adrenocortical carcinoma cells

Background and PurposeTo further the development of new agents for the treatment of adrenocortical carcinoma (ACC), we characterized the molecular and cellular mechanisms of cytotoxicity by the adrenalytic compound ATR‐101 (PD132301‐02).Experimental ApproachWe compared the effects of ATR‐101, PD129337, and ABC transporter inhibitors on cholesterol accumulation and efflux, on cortisol secretion, on ATP levels, and on caspase activation in ACC‐derived cell lines. We examined the effects of these compounds in combination with methyl‐β‐cyclodextrin or exogenous cholesterol to determine the roles of altered cholesterol levels in the effects of these compounds.Key ResultsATR‐101 caused cholesterol accumulation, ATP depletion, and caspase activation within 30 minutes after addition to ACC‐derived cells, whereas PD129337 did not. Suppression of cholesterol accumulation by methyl‐β‐cyclodextrin or exogenous cholesterol, prevented ATP depletion and caspase activation by ATR‐101. ATR‐101 blocked cholesterol efflux and cortisol secretion, suggesting that it inhibited ABCA1, ABCG1, and MDR1 transporters. Combinations of ABCA1, ABCG1, and MDR1 inhibitors were also cytotoxic. Combinations of ATR‐101 with inhibitors of ABCG1, MDR1, or mitochondrial functions had increased cytotoxicity. Inhibitors of steroidogenesis reduced ATP depletion by ATR‐101, whereas U18666A enhanced cholesterol accumulation and ATP depletion together with ATR‐101. ATR‐101 repressed ABCA1, ABCG1, and IDOL transcription by mechanisms that were distinct from the mechanisms that caused cholesterol accumulation.Conclusions and ImplicationsInhibition of multiple ABC transporters and the consequent accumulation of cholesterol mediated the cytotoxicity of ATR‐101. Compounds that replicate these effects in tumours are likely to be useful in the treatment of ACC.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138270/1/bph13951-sup-0001-supplementary_material.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138270/2/bph13951_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138270/3/bph13951.pd

Apoptosis: A Four-Week Laboratory Investigation for Advanced Molecular and Cellular Biology Students

Over the past decade, apoptosis has emerged as an important field of study central to ongoing research in many diverse fields, from developmental biology to cancer research. Apoptosis proceeds by a highly coordinated series of events that includes enzyme activation, DNA fragmentation, and alterations in plasma membrane permeability. The detection of each of these phenotypic changes is accessible to advanced undergraduate cell and molecular biology students. We describe a 4-week laboratory sequence that integrates cell culture, fluorescence microscopy, DNA isolation and analysis, and western blotting (immunoblotting) to follow apoptosis in cultured human cells. Students working in teams chemically induce apoptosis, and harvest, process, and analyze cells, using their data to determine the order of events during apoptosis. We, as instructors, expose the students to an environment closely simulating what they would encounter in an active cell or molecular biology research laboratory by having students coordinate and perform multiple tasks simultaneously and by having them experience experimental design using current literature, data interpretation, and analysis to answer a single question. Students are assessed by examination of laboratory notebooks for completeness of experimental protocols and analysis of results and for completion of an assignment that includes questions pertaining to data interpretation and apoptosis

An assessment of acute insecticide toxicity loading (AITL) of chemical pesticides used on agricultural land in the United States.

We present a method for calculating the Acute Insecticide Toxicity Loading (AITL) on US agricultural lands and surrounding areas and an assessment of the changes in AITL from 1992 through 2014. The AITL method accounts for the total mass of insecticides used in the US, acute toxicity to insects using honey bee contact and oral LD50 as reference values for arthropod toxicity, and the environmental persistence of the pesticides. This screening analysis shows that the types of synthetic insecticides applied to agricultural lands have fundamentally shifted over the last two decades from predominantly organophosphorus and N-methyl carbamate pesticides to a mix dominated by neonicotinoids and pyrethroids. The neonicotinoids are generally applied to US agricultural land at lower application rates per acre; however, they are considerably more toxic to insects and generally persist longer in the environment. We found a 48- and 4-fold increase in AITL from 1992 to 2014 for oral and contact toxicity, respectively. Neonicotinoids are primarily responsible for this increase, representing between 61 to nearly 99 percent of the total toxicity loading in 2014. The crops most responsible for the increase in AITL are corn and soybeans, with particularly large increases in relative soybean contributions to AITL between 2010 and 2014. Oral exposures are of potentially greater concern because of the relatively higher toxicity (low LD50s) and greater likelihood of exposure from residues in pollen, nectar, guttation water, and other environmental media. Using AITL to assess oral toxicity by class of pesticide, the neonicotinoids accounted for nearly 92 percent of total AITL from 1992 to 2014. Chlorpyrifos, the fifth most widely used insecticide during this time contributed just 1.4 percent of total AITL based on oral LD50s. Although we use some simplifying assumptions, our screening analysis demonstrates an increase in pesticide toxicity loading over the past 26 years, which potentially threatens the health of honey bees and other pollinators and may contribute to declines in beneficial insect populations as well as insectivorous birds and other insect consumers