sirolimus and pepstatin

We compared the expression levels of 307 miRNAs in six different B16F1 melanoma cell lines of differing malignant properties and found that the miR-290-295 cluster showed a strong upregulation in the more malignant B16F1 daughter cell lines. Its overexpression in B16F1 cells had no major effects on cell proliferation, migration or anchorage-independent growth, but conferred resistance to glucose starvation. This was mediated by miR-290-295-induced downregulation of several essential autophagy genes, including Atg7 and ULK1, which resulted in inhibition of autophagic cell death induced by glucose starvation. Similar effects were observed after knockdown of Atg7 or ULK1 in B16F1 melanoma cells, and after treatment with two chemical inhibitors of autophagy. Together, these results indicate that autophagy mediates cell death of melanoma cells under chronic nutrient deprivation, and they reveal an unanticipated role of the miR-290-295 cluster in conferring a survival advantage to melanoma cells by inhibiting autophagic cell death.

Topics: 3' Untranslated Regions; Animals; Autophagy; Autophagy-Related Protein 7; Autophagy-Related Protein-1 Homolog; Cell Line, Tumor; Down-Regulation; Leucine; Melanoma; Mice; MicroRNAs; Microtubule-Associated Proteins; Multigene Family; Pepstatins; Protein Serine-Threonine Kinases; Sirolimus; Starvation

Expanded polyglutamine 72 repeat (polyQ72) aggregates induce endoplasmic reticulum (ER) stress-mediated cell death with caspase-12 activation and vesicular formation (autophagy). We examined this relationship and the molecular mechanism of autophagy formation. Rapamycin, a stimulator of autophagy, inhibited the polyQ72-induced cell death with caspase-12 activation. PolyQ72, but not polyQ11, stimulated Atg5-Atg12-Atg16 complex-dependent microtubule-associated protein 1 (MAP1) light chain 3 (LC3) conversion from LC3-I to -II, which plays a key role in autophagy. The eucaryotic translation initiation factor 2 alpha (eIF2alpha) A/A mutation, a knock-in to replace a phosphorylatable Ser51 with Ala51, and dominant-negative PERK inhibited polyQ72-induced LC3 conversion. PolyQ72 as well as ER stress stimulators upregulated Atg12 mRNA and proteins via eIF2alpha phosphorylation. Furthermore, Atg5 deficiency as well as the eIF2alpha A/A mutation increased the number of cells showing polyQ72 aggregates and polyQ72-induced caspase-12 activation. Thus, autophagy formation is a cellular defense mechanism against polyQ72-induced ER-stress-mediated cell death by degrading polyQ72 aggregates, with PERK/eIF2alpha phosphorylation being involved in polyQ72-induced LC3 conversion.

Topics: Adenine; Animals; Autophagy; Autophagy-Related Protein 5; Caspase 12; Cell Death; eIF-2 Kinase; Endoplasmic Reticulum; Enzyme Activation; Eukaryotic Initiation Factor-2; Gene Expression Regulation; Leucine; Lysosomes; Mice; Microtubule-Associated Proteins; Models, Biological; Pepstatins; Peptides; Phosphorylation; Protein Structure, Quaternary; RNA, Messenger; Sirolimus