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A Mixture of Atropisomers Enhances Neutral Lipid Degradation in Mammalian Cells with Autophagy Induction.

ABSTRACT: Atropisomers with a biaryl dihydronaphthopyranone structure, dinapinones A1 (DPA1) (M position) and A2 (DPA2) (P position), were isolated from the fungus culture broth of Talaromyces pinophilus FKI-3864 as inhibitors of [¹⁴C]neutral lipid ([¹⁴C]triacylglycerol (TG) and [¹⁴C]cholesteryl ester (CE)) synthesis from [¹⁴C]oleic acid in Chinese hamster ovary-K1 (CHO-K1) cells. DPA2 inhibited [¹⁴C]TG and [¹⁴C]CE synthesis (IC₅₀s_, 0.65 and 5.6??M, respectively), but DPA1 had no inhibitory activity on [¹⁴C]TG and [¹⁴C]CE synthesis even at 12??M. However, a 1:1 mixture of DPA1 and DPA2 (DPA_mix) had the most potent inhibitory activity on [¹⁴C]TG and [¹⁴C]CE synthesis (IC₅₀s, 0.054 and 0.18??M, respectively). The mechanism of action of DPA_mix was investigated. DPA_mix had no effects on the enzymes involved in neutral lipid synthesis, while DPA_mix enhanced the degradation of [¹⁴C]neutral lipids with concomitant decrease in cytosolic lipid droplets accumulated in CHO-K1 cells. From analysis of autophagy marker proteins, DPA_mix caused dose-dependent induction of microtubule-associated protein light chain 3-II (LC3-II) and degradation of p62. In the autophagic flux assay using bafilomycin A₁, DPA_mix upregulated autophagosome turnover. These results reveal that DPA_mix enhances neutral lipid degradation together with induction of autophagy.

SUBMITTER: Kobayashi K

PROVIDER: S-EPMC6092391 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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A Mixture of Atropisomers Enhances Neutral Lipid Degradation in Mammalian Cells with Autophagy Induction.

Kobayashi Keisuke K Ohte Satoshi S Ohshiro Taichi T Ugaki Narihiro N Tomoda Hiroshi H

Scientific reports 20180814 1

Atropisomers with a biaryl dihydronaphthopyranone structure, dinapinones A1 (DPA1) (M position) and A2 (DPA2) (P position), were isolated from the fungus culture broth of Talaromyces pinophilus FKI-3864 as inhibitors of [14C]neutral lipid ([14C]triacylglycerol (TG) and [14C]cholesteryl ester (CE)) synthesis from [14C]oleic acid in Chinese hamster ovary-K1 (CHO-K1) cells. DPA2 inhibited [14C]TG and [14C]CE synthesis (IC50</sub ...[more]

PMID: 30108268

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Project description:Two pathways are responsible for the majority of regulated protein catabolism in eukaryotic cells: the ubiquitin-proteasome system (UPS) and lysosomal self-degradation through autophagy. Both processes are necessary for cellular homeostasis by ensuring continuous turnover and quality control of most intracellular proteins. Recent studies established that both UPS and autophagy are capable of selectively eliminating ubiquitinated proteins and that autophagy may partially compensate for the lack of proteasomal degradation, but the molecular links between these pathways are poorly characterized.Here we show that autophagy is enhanced by the silencing of genes encoding various proteasome subunits (?, ? or regulatory) in larval fat body cells. Proteasome inactivation induces canonical autophagy, as it depends on core autophagy genes Atg1, Vps34, Atg9, Atg4 and Atg12. Large-scale accumulation of aggregates containing p62 and ubiquitinated proteins is observed in proteasome RNAi cells. Importantly, overexpressed Atg8a reporters are captured into the cytoplasmic aggregates, but these do not represent autophagosomes. Loss of p62 does not block autophagy upregulation upon proteasome impairment, suggesting that compensatory autophagy is not simply due to the buildup of excess cargo. One of the best characterized substrates of UPS is the ? subunit of hypoxia-inducible transcription factor 1 (HIF-1?), which is continuously degraded by the proteasome during normoxic conditions. Hypoxia is a known trigger of autophagy in mammalian cells, and we show that genetic activation of hypoxia signaling also induces autophagy in Drosophila. Moreover, we find that proteasome inactivation-induced autophagy requires sima, the Drosophila ortholog of HIF-1?.We have characterized proteasome inactivation- and hypoxia signaling-induced autophagy in the commonly used larval Drosophila fat body model. Activation of both autophagy and hypoxia signaling was implicated in various cancers, and mutations affecting genes encoding UPS enzymes have recently been suggested to cause renal cancer. Our studies identify a novel genetic link that may play an important role in that context, as HIF-1?/sima may contribute to upregulation of autophagy by impaired proteasomal activity.

Project description:BACKGROUND:Altered lipid metabolism is an emerging hallmark of aggressive breast cancers. The N-myc downstream regulated gene (NDRG1) gene plays a critical role in peripheral nervous system myelination, as inactivating mutations cause severe demyelinating neuropathy. In breast cancer, elevated NDRG1 expression has been linked to clinical outcomes, but its functional role in breast cancer physiology has remained unclear. METHODS:A meta-analysis of NDRG1 expression in multiple large publicly available genomic databases was conducted. Genome-wide expression correlation and Cox proportional hazards and Kaplan-Meier modeling of clinical outcomes associated with elevated expression were assessed. To study NDRG1 function, gene silencing and overexpression phenotypic studies were carried out in a panel of cell lines representing all major breast cancer molecular subtypes. Changes in cell proliferation, morphology, and neutral lipid accumulation due to altered NDRG1 expression were assessed by high throughput, quantitative microscopy. Comprehensive lipidomics mass spectrometry was applied to characterize global changes in lipid species due to NDRG1 silencing. Labeled fatty acids were used to monitor cellular fatty acid uptake and subcellular distribution under nutrient replete and starvation culture conditions. RESULTS:NDRG1 overexpression correlated with glycolytic and hypoxia-associated gene expression, and was associated with elevated rates of metastasis and patient mortality. Silencing NDRG1 reduced cell proliferation rates, causing lipid metabolism dysfunction including increased fatty acid incorporation into neutral lipids and lipid droplets. Conversely, NDRG1 expression minimized lipid droplet formation under nutrient replete and starvation conditions. CONCLUSIONS:Here we report that NDRG1 contributes to breast cancer aggressiveness by regulating the fate of lipids in cells that exhibit an altered lipid metabolic phenotype. In line with its role in promoting myelination and its association with altered metabolism in cancer, our findings show that NDRG1 is a critical regulator of lipid fate in breast cancer cells. The association between NDRG1 and poor prognosis in breast cancer suggests it should play a more prominent role in patient risk assessment. The function of NDRG1 in breast cancer lipid metabolism may represent a promising therapeutic approach in the future.

Dataset Information

A Mixture of Atropisomers Enhances Neutral Lipid Degradation in Mammalian Cells with Autophagy Induction.

Publications

A Mixture of Atropisomers Enhances Neutral Lipid Degradation in Mammalian Cells with Autophagy Induction.

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