Ursolic acid impairs cellular lipid homeostasis and lysosomal membrane integrity in breast carcinoma cells

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cells11244079/s1, Figure S1: UA kills MCF7 cells partly through apoptosis; Figure S2: UA causes LC3 puncta formation in MCF7 and HCT15 cells; Figure S3: UA causes LMP prior to MOMP and alters lysosomal localization in HeLa cells; Figure S4: Additional lipidomics data; Table S1: List of resources; Table S2: Internal lipid and drug standards; Table S3: Precursor ion, fragment ion, and neutral loss for lipid identification; Table S4: All experiments.Cancer is one of the leading causes of death worldwide, thus the search for new cancer therapies is of utmost importance. Ursolic acid is a naturally occurring pentacyclic triterpene with a wide range of pharmacological activities including anti-inflammatory and anti-neoplastic effects. The latter has been assigned to its ability to promote apoptosis and inhibit cancer cell proliferation by poorly defined mechanisms. In this report, we identify lysosomes as the essential targets of the anti-cancer activity of ursolic acid. The treatment of MCF7 breast cancer cells with ursolic acid elevates lysosomal pH, alters the cellular lipid profile, and causes lysosomal membrane permeabilization and leakage of lysosomal enzymes into the cytosol. Lysosomal membrane permeabilization precedes the essential hallmarks of apoptosis placing it as an initial event in the cascade of effects induced by ursolic acid. The disruption of the lysosomal function impairs the autophagic pathway and likely partakes in the mechanism by which ursolic acid kills cancer cells. Furthermore, we find that combining treatment with ursolic acid and cationic amphiphilic drugs can significantly enhance the degree of lysosomal membrane permeabilization and cell death in breast cancer cells.This work was supported by grants from the Danish National Research Foundation (DNRF125), European Research Council (AdG340751), Danish Cancer Society (R167-A11061) and, Novo Nordisk Foundation (NNF19OC0054296), to M.J., K.M. was supported by the Independent Research Fund Denmark (6108–00542B) and Novo Nordisk Foundation (NNF17OC0029432).info:eu-repo/semantics/publishedVersio

Neurodegenerative Diseases and Autophagy

Most neurodegenerative diseases are characterized by the accumulation of aggregated proteins within neurons. These aggregate-prone proteins cause toxicity, a phenomenon that is further exacerbated when there is defective protein clearance. Autophagy is an intracellular clearance pathway that can clear these protein aggregates and has been shown to be beneficial in the treatment of neurodegenerative diseases in a variety of model systems. Here, we introduce the key components of the autophagy machinery and signaling pathways that control this process and discuss the evidence that autophagic flux may be impaired and therefore a contributing factor in neurodegenerative disease pathogenesis. Finally, we review the use of autophagy upregulation as a therapeutic strategy to treat neurodegenerative disorders

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

DNA-dependent protein kinase regulates lysosomal AMP-dependent protein kinase activation and autophagy

Macroautophagy/autophagy is a central component of the cytoprotective cellular stress response. To enlighten stress-induced autophagy signaling, we screened a human kinome siRNA library for regulators of autophagic flux in MCF7 human breast carcinoma cells and identified the catalytic subunit of DNA-dependent protein kinase PRKDC/DNA-PKcs as a positive regulator of basal and DNA damage-induced autophagy. Analysis of autophagy-regulating signaling cascades placed PRKDC upstream of the AMP-dependent protein kinase (AMPK) complex and ULK1 kinase. In normal culture conditions, PRKDC interacted with the AMPK complex and phosphorylated its nucleotide-sensing γ1 subunit PRKAG1/AMPKγ1 at Ser192 and Thr284, both events being significantly reduced upon the activation of the AMPK complex. Alanine substitutions of PRKDC phosphorylation sites in PRKAG1 reduced AMPK complex activation without affecting its nucleotide sensing capacity. Instead, the disturbance of PRKDC-mediated phosphorylation of PRKAG1 inhibited the lysosomal localization of the AMPK complex and its starvation-induced association with STK11 (serine/threonine kinase 11). Taken together, our data suggest that PRKDC-mediated phosphorylation of PRKAG1 primes AMPK complex to the lysosomal activation by STK11 in cancer cells thereby linking DNA damage response to autophagy and cellular metabolism

Identification of Cytoskeleton-Associated Proteins Essential for Lysosomal Stability and Survival of Human Cancer Cells

<div><p>Microtubule-disturbing drugs inhibit lysosomal trafficking and induce lysosomal membrane permeabilization followed by cathepsin-dependent cell death. To identify specific trafficking-related proteins that control cell survival and lysosomal stability, we screened a molecular motor siRNA library in human MCF7 breast cancer cells. SiRNAs targeting four kinesins (KIF11/Eg5, KIF20A, KIF21A, KIF25), myosin 1G (MYO1G), myosin heavy chain 1 (MYH1) and tropomyosin 2 (TPM2) were identified as effective inducers of non-apoptotic cell death. The cell death induced by KIF11, KIF21A, KIF25, MYH1 or TPM2 siRNAs was preceded by lysosomal membrane permeabilization, and all identified siRNAs induced several changes in the endo-lysosomal compartment, <em>i.e.</em> increased lysosomal volume (KIF11, KIF20A, KIF25, MYO1G, MYH1), increased cysteine cathepsin activity (KIF20A, KIF25), altered lysosomal localization (KIF25, MYH1, TPM2), increased dextran accumulation (KIF20A), or reduced autophagic flux (MYO1G, MYH1). Importantly, all seven siRNAs also killed human cervix cancer (HeLa) and osteosarcoma (U-2-OS) cells and sensitized cancer cells to other lysosome-destabilizing treatments, <em>i.e.</em> photo-oxidation, siramesine, etoposide or cisplatin. Similarly to KIF11 siRNA, the KIF11 inhibitor monastrol induced lysosomal membrane permeabilization and sensitized several cancer cell lines to siramesine. While KIF11 inhibitors are under clinical development as mitotic blockers, our data reveal a new function for KIF11 in controlling lysosomal stability and introduce six other molecular motors as putative cancer drug targets.</p> </div

Effect of the identified siRNAs on autophagy and dextran uptake.

<p>(A–D) tfLC3-MCF7 cells (A, B) or MCF7 cells (C, D) were left untreated, treated with Oligofectamine (Oligo) or transfected with control siRNA (CT) or indicated siRNA pools (3×6.67 nM). (A, B) After 48 h, tfLC3-MCF7 cells were analyzed by confocal microscopy. Representative images (A; <i>Bars</i>, 10 µm) and quantification of puncta (B) are shown. Raptor siRNA (RPTOR) served as a control for increased autophagic flux. Closed arrows indicate AVd, open arrows indicate AVi. (C) After 60 h, the level of p62/SQSTM1 (p62), which is degraded by autophagy, was examined by Western blot. Rapamycin (20 nM, 4 h) was used to induce autophagy. Numbers represent p62 levels as percentage of the level in untreated control siRNA-transfected cells. (D) <i>Top</i>, After 60 h, MCF7 cells were treated with 100 µg/ml Alexa Fluor 488-dextran (dextran-488) for 1 h and analyzed by flow cytometry (FL1-H). The threshold for high intensity staining was defined so that 88% of control siRNA-transfected cells were below. <i>Bottom</i>, Example histograms showing dextran-488 content of cells transfected with control or KIF20A siRNA. M1 = gate for high intensity staining. Values represent means + SEM of 20 cells in one representative experiment (B, n = 3) or means + SD of three independent experiments (D). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, vs. control siRNA-transfected cells.</p

Sensitization to lysosome-disrupting drugs by the identified siRNAs and monastrol.

<p>(A, B) MCF7 cells were left untreated, treated with Oligofectamine (Oligo) or transfected with control siRNA or indicated siRNA pools (3×6.67 nM). After 48 h, cells were left untreated or treated with 2 µM siramesine (<i>top</i>), 50 µM etoposide (<i>middle</i>) or 10 µM cisplatin (<i>bottom</i>) for additional 48 h before light microscopy pictures were taken (representative images in B) and cell death was quantified by the LDH release assay (A). (C) MCF7 cells were left untreated or treated with 2 µM siramesine together with indicated concentrations of monastrol for 72 h and cell death was quantified by the LDH release assay. Values represent means + SD of a minimum of three independent experiments. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, vs. control siRNA-transfected cells (A) or cells treated with 2 µM siramesine alone (C).</p