Artesunate activates mitochondrial apoptosis in breast cancer cells via iron-catalyzed lysosomal reactive oxygen species production.
ABSTRACT: The antimalarial agent artesunate (ART) activates programmed cell death (PCD) in cancer cells in a manner dependent on the presence of iron and the generation of reactive oxygen species. In malaria parasites, ART cytotoxicity originates from interactions with heme-derived iron within the food vacuole. The analogous digestive compartment of mammalian cells, the lysosome, similarly contains high levels of redox-active iron and in response to specific stimuli can initiate mitochondrial apoptosis. We thus investigated the role of lysosomes in ART-induced PCD and determined that in MCF-7 breast cancer cells ART activates lysosome-dependent mitochondrial outer membrane permeabilization. ART impacted endolysosomal and autophagosomal compartments, inhibiting autophagosome turnover and causing perinuclear clustering of autophagosomes, early and late endosomes, and lysosomes. Lysosomal iron chelation blocked all measured parameters of ART-induced PCD, whereas lysosomal iron loading enhanced death, thus identifying lysosomal iron as the lethal source of reactive oxygen species upstream of mitochondrial outer membrane permeabilization. Moreover, lysosomal inhibitors chloroquine and bafilomycin A1 reduced ART-activated PCD, evidencing a requirement for lysosomal function during PCD signaling. ART killing did not involve activation of the BH3-only protein, Bid, yet ART enhanced TNF-mediated Bid cleavage. We additionally demonstrated the lysosomal PCD pathway in T47D and MDA-MB-231 breast cancer cells. Importantly, non-tumorigenic MCF-10A cells resisted ART-induced PCD. Together, our data suggest that ART triggers PCD via engagement of distinct, interconnected PCD pathways, with hierarchical signaling from lysosomes to mitochondria, suggesting a potential clinical use of ART for targeting lysosomes in cancer treatment.
Project description:Upon apoptotic stimuli, lysosomal proteases, including cathepsins and chymotrypsin, are released into cytosol due to lysosomal membrane permeabilization (LMP), where they trigger apoptosis via the lysosomal-mitochondrial pathway of apoptosis. Herein, the mechanism of LMP was investigated. We found that caspase 8-cleaved Bid (tBid) could result in LMP directly. Although Bax or Bak might modestly enhance tBid-triggered LMP, they are not necessary for LMP. To study this further, large unilamellar vesicles (LUVs), model membranes mimicking the lipid constitution of lysosomes, were used to reconstitute the membrane permeabilization process in vitro. We found that phosphatidic acid (PA), one of the major acidic phospholipids found in lysosome membrane, is essential for tBid-induced LMP. PA facilitates the insertion of tBid deeply into lipid bilayers, where it undergoes homo-oligomerization and triggers the formation of highly curved nonbilayer lipid phases. These events induce LMP via pore formation mechanisms because encapsulated fluorescein-conjugated dextran (FD)-20 was released more significantly than FD-70 or FD-250 from LUVs due to its smaller molecular size. On the basis of these data, we proposed tBid-PA interactions in the lysosomal membranes form lipidic pores and result in LMP. We further noted that chymotrypsin-cleaved Bid is more potent than tBid at binding to PA, inserting into the lipid bilayer, and promoting LMP. This amplification mechanism likely contributes to the culmination of apoptotic signaling.
Project description:Hypoxia-inducible pro-death protein BNIP3 (BCL-2/adenovirus E1B 19-kDa interacting protein 3), provokes mitochondrial permeabilization causing cardiomyocyte death in ischemia-reperfusion injury. Inhibition of autophagy accelerates BNIP3-induced cell death, by preventing removal of damaged mitochondria. We tested the hypothesis that stimulating autophagy will attenuate BNIP3-induced cardiomyocyte death. Neonatal rat cardiac myocytes (NRCMs) were adenovirally transduced with BNIP3 (or LacZ as control; at multiplicity of infection = 100); and autophagy was stimulated with rapamycin (100 nM). Cell death was assessed at 48 h. BNIP3 expression increased autophagosome abundance 8-fold and caused a 3.6-fold increase in cardiomyocyte death as compared with control. Rapamycin treatment of BNIP3-expressing cells led to further increase in autophagosome number without affecting cell death. BNIP3 expression led to accumulation of autophagosome-bound LC3-II and p62, and an increase in autophagosomes, but not autolysosomes (assessed with dual fluorescent mCherry-GFP-LC3 expression). BNIP3, but not the transmembrane deletion variant, interacted with LC3 and colocalized with mitochondria and lysosomes. However, BNIP3 did not target to lysosomes by subcellular fractionation, provoke lysosome permeabilization or alter lysosome pH. Rather, BNIP3-induced autophagy caused a decline in lysosome numbers with decreased expression of the lysosomal protein LAMP-1, indicating lysosome consumption and consequent autophagosome accumulation. Forced expression of transcription factor EB (TFEB) in BNIP3-expressing cells increased lysosome numbers, decreased autophagosomes and increased autolysosomes, prevented p62 accumulation, removed depolarized mitochondria and attenuated BNIP3-induced death. We conclude that BNIP3 expression induced autophagosome accumulation with lysosome consumption in cardiomyocytes. Forced expression of TFEB, a lysosomal biogenesis factor, restored autophagosome processing and attenuated BNIP3-induced cell death.
Project description:Lysosome-activated apoptosis represents an alternative method of overcoming tumor resistance compared to traditional forms of treatment. Pulsed magnetic fields open a new avenue for controlled and targeted initiation of lysosomal permeabilization in cancer cells via mechanical actuation of magnetic nanomaterials. In this study we used a noninvasive tool; namely, a benchtop pulsed magnetic system, which enabled remote activation of apoptosis in liver cancer cells. The magnetic system we designed represents a platform that can be used in a wide range of biomedical applications. We show that liver cancer cells can be loaded with superparamagnetic iron oxide nanoparticles (SPIONs). SPIONs retained in lysosomal compartments can be effectively actuated with a high intensity (up to 8 T), short pulse width (~15 µs), pulsed magnetic field (PMF), resulting in lysosomal membrane permeabilization (LMP) in cancer cells. We revealed that SPION-loaded lysosomes undergo LMP by assessing an increase in the cytosolic activity of the lysosomal cathepsin B. The extent of cell death induced by LMP correlated with the accumulation of reactive oxygen species in cells. LMP was achieved for estimated forces of 700 pN and higher. Furthermore, we validated our approach on a three-dimensional cellular culture model to be able to mimic in vivo conditions. Overall, our results show that PMF treatment of SPION-loaded lysosomes can be utilized as a noninvasive tool to remotely induce apoptosis.
Project description:The lysosome is an acidic multi-functional organelle with roles in macromolecular digestion, nutrient sensing, and signaling. However, why cells require acidic lysosomes to proliferate and which nutrients become limiting under lysosomal dysfunction are unclear. To address this, we performed CRISPR-Cas9-based genetic screens and identified cholesterol biosynthesis and iron uptake as essential metabolic pathways when lysosomal pH is altered. While cholesterol synthesis is only necessary, iron is both necessary and sufficient for cell proliferation under lysosomal dysfunction. Remarkably, iron supplementation restores cell proliferation under both pharmacologic and genetic-mediated lysosomal dysfunction. The rescue was independent of metabolic or signaling changes classically associated with increased lysosomal pH, uncoupling lysosomal function from cell proliferation. Finally, our experiments revealed that lysosomal dysfunction dramatically alters mitochondrial metabolism and hypoxia inducible factor (HIF) signaling due to iron depletion. Altogether, these findings identify iron homeostasis as the key function of lysosomal acidity for cell proliferation.
Project description:OBJECTIVES:The genotoxicity of cisplatin towards nuclear DNA is not sufficient to explain the cisplatin resistance of hepatocellular carcinoma (HCC) cells; cisplatin interacts with many organelles, which can influence the sensitivity. Here, we explored the role of mitochondrial-lysosomal crosstalk in the cisplatin resistance of HCC cells. MATERIALS AND METHODS:Huh7 and HepG2 cells were subjected to different treatments. Flow cytometry was conducted to detect mitochondrial reactive oxygen species, mitochondrial mass, lysosomal function, mitochondrial membrane potential and apoptosis. Western blotting was performed to evaluate protein levels. The oxygen consumption rate was measured to evaluate mitochondrial function. RESULTS:Cisplatin activated mitophagy and lysosomal biogenesis, resulting in crosstalk between mitochondria and lysosomes and cisplatin resistance in HCC cells. Furthermore, a combination of cisplatin with the phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) inhibitor PKI-402 induced lysosomal membrane permeabilization. This effect changed the role of the lysosome from a protective one to that of a cell death promoter, completely destroying the mitochondrial-lysosomal crosstalk and significantly enhancing the sensitivity of HCC cells to cisplatin. CONCLUSIONS:This is the first evidence of the importance of mitochondrial-lysosomal crosstalk in the cisplatin resistance of HCC cells and of the destruction of this crosstalk by a PI3K/mTOR inhibitor to increase the sensitivity of HCC cells to cisplatin. This mechanism could be developed as a novel target for treatment of HCC in the future.
Project description:Both mitochondria and lysosomes are essential for maintaining cellular homeostasis, and dysfunction of both organelles has been observed in multiple diseases. Mitochondria are highly dynamic and undergo fission and fusion to maintain a functional mitochondrial network, which drives cellular metabolism. Lysosomes similarly undergo constant dynamic regulation by the RAB7 GTPase, which cycles from an active GTP-bound state into an inactive GDP-bound state upon GTP hydrolysis. Here we have identified the formation and regulation of mitochondria-lysosome membrane contact sites using electron microscopy, structured illumination microscopy and high spatial and temporal resolution confocal live cell imaging. Mitochondria-lysosome contacts formed dynamically in healthy untreated cells and were distinct from damaged mitochondria that were targeted into lysosomes for degradation. Contact formation was promoted by active GTP-bound lysosomal RAB7, and contact untethering was mediated by recruitment of the RAB7 GTPase-activating protein TBC1D15 to mitochondria by FIS1 to drive RAB7 GTP hydrolysis and thereby release contacts. Functionally, lysosomal contacts mark sites of mitochondrial fission, allowing regulation of mitochondrial networks by lysosomes, whereas conversely, mitochondrial contacts regulate lysosomal RAB7 hydrolysis via TBC1D15. Mitochondria-lysosome contacts thus allow bidirectional regulation of mitochondrial and lysosomal dynamics, and may explain the dysfunction observed in both organelles in various human diseases.
Project description:Osteosarcoma cellular iron concentration is higher than that in normal bone cells and other cell types. High levels of cellular iron help catalyze the Fenton reaction to produce reactive oxygen species (ROS), which promotes cancer cell proliferation. Dihydroartemisinin (DHA), a classic anti-malarial drug, kills plasmodium through iron-dependent ROS generation. In this research, we observed the anti-osteosarcoma effects and mechanisms of DHA. We found that DHA induced ROS production, caused mitochondrial damage, and activated autophagy via stimulation of the ROS/Erk1/2 pathway. As the storage site for a pool of ferrous iron, lysosomes are often the key organelles affected by drugs targeting iron. In this study, we observed that DHA induced lysosomal superoxide production, leading lysosomal membrane permeabilization (LMP), and autophagic flux blockage. By reducing or increasing cellular iron using deferoxamine (DFO) or ferric ammonium citrate (FAC), respectively, we found that DHA inhibited osteosarcoma in an iron-dependent manner. Therefore, iron may be a potential adjuvant for DHA in osteosarcoma treatment.
Project description:The ability to control the movement of nanoparticles remotely and with high precision would have far-reaching implications in many areas of nanotechnology. We have designed a unique dynamic magnetic field (DMF) generator that can induce rotational movements of superparamagnetic iron oxide nanoparticles (SPIONs). We examined whether the rotational nanoparticle movement could be used for remote induction of cell death by injuring lysosomal membrane structures. We further hypothesized that the shear forces created by the generation of oscillatory torques (incomplete rotation) of SPIONs bound to lysosomal membranes would cause membrane permeabilization, lead to extravasation of lysosomal contents into the cytoplasm, and induce apoptosis. To this end, we covalently conjugated SPIONs with antibodies targeting the lysosomal protein marker LAMP1 (LAMP1-SPION). Remote activation of slow rotation of LAMP1-SPIONs significantly improved the efficacy of cellular internalization of the nanoparticles. LAMP1-SPIONs then preferentially accumulated along the membrane in lysosomes in both rat insulinoma tumor cells and human pancreatic beta cells due to binding of LAMP1-SPIONs to endogenous LAMP1. Further activation of torques by the LAMP1-SPIONs bound to lysosomes resulted in rapid decrease in size and number of lysosomes, attributable to tearing of the lysosomal membrane by the shear force of the rotationally activated LAMP1-SPIONs. This remote activation resulted in an increased expression of early and late apoptotic markers and impaired cell growth. Our findings suggest that DMF treatment of lysosome-targeted nanoparticles offers a noninvasive tool to induce apoptosis remotely and could serve as an important platform technology for a wide range of biomedical applications.
Project description:Cellular stresses trigger autophagy to remove damaged macromolecules and organelles. Lysosomes 'host' multiple stress-sensing mechanisms that trigger the coordinated biogenesis of autophagosomes and lysosomes. For example, transcription factor (TF)EB, which regulates autophagy and lysosome biogenesis, is activated following the inhibition of mTOR, a lysosome-localized nutrient sensor. Here we show that reactive oxygen species (ROS) activate TFEB via a lysosomal Ca(2+)-dependent mechanism independent of mTOR. Exogenous oxidants or increasing mitochondrial ROS levels directly and specifically activate lysosomal TRPML1 channels, inducing lysosomal Ca(2+) release. This activation triggers calcineurin-dependent TFEB-nuclear translocation, autophagy induction and lysosome biogenesis. When TRPML1 is genetically inactivated or pharmacologically inhibited, clearance of damaged mitochondria and removal of excess ROS are blocked. Furthermore, TRPML1's ROS sensitivity is specifically required for lysosome adaptation to mitochondrial damage. Hence, TRPML1 is a ROS sensor localized on the lysosomal membrane that orchestrates an autophagy-dependent negative-feedback programme to mitigate oxidative stress in the cell.
Project description:It is widely accepted that lysosomes are essential for cell homeostasis, and autophagy plays an important role in tumor development. Here, we found FV-429, a synthetic flavonoid compound, inhibited autophagy flux, promoted autophagosomes accumulation, and inhibited lysosomal degradation in T-cell malignancies. These effects were likely to be achieved by lysosomal dysregulation. The destructive effects of FV-429 on lysosomes resulted in blockage of lysosome-associated membrane fusion, lysosomal membrane permeabilization (LMP), and cathepsin-mediated caspase-independent cell death (CICD). Moreover, we initially investigated the effects of autophagy inhibition by FV-429 on the therapeutic efficacy of chemotherapy and found that FV-429 sensitized cancer cells to chemotherapy agents. Our findings suggest that FV-429 could be a potential novel autophagy inhibitor with notable antitumor efficacy as a single agent.