Lysosomes Of Cells Research Articles

Integrin αvβ3 and LHRH Receptor Double Directed Nano-Analogue Effective Against Ovarian Cancer in Mice Model.

The high mortality rate of malignant ovarian cancer is attributed to the absence of effective early diagnosis methods. The LHRH receptor is specifically overexpressed in most ovarian cancers, and the integrin αvβ3 receptor is also overexpressed on the surface of ovarian cancer cells. In this study, we designed LHRH analogues (LHRHa)/RGD co-modified paclitaxel liposomes (LHRHa-RGD-LP-PTX) to target LHRH receptor-positive ovarian cancers more effectively and enhance the anti-ovarian cancer effects. LHRHa-RGD-LP-PTX liposomes were prepared using the thin film hydration method. The morphology, physicochemical properties, cellular uptake, and cell viability were assessed. Additionally, the cellular uptake mechanism of the modified liposomes was investigated using various endocytic inhibitors. The inhibitory effect of the formulations on tumor spheroids was observed under a microscope. The co-localization with lysosomes was visualized using confocal laser scanning microscopy (CLSM), and the in vivo tumor-targeting ability of the formulations was assessed using the IVIS fluorescent imaging system. Finally, the in vivo anti-tumor efficacy of the formulations was evaluated in the armpits of BALB/c nude mice. The results indicated that LHRHa-RGD-LP-PTX significantly enhanced cellular uptake in A2780 cells, increased cytotoxicity, and hand a more potent inhibitory effect on tumor spheroids of A2780 cells. It also showed enhanced co-localization with endosomes or lysosome in A2780 cells, improved tumor-targeting capability, and demonstrated an enhanced anti-tumor effect in LHRHR-positive ovarian cancers. The designed LHRHa-RGD-LP-PTX liposomes significantly enhanced the tumor-targeting ability and therapeutic efficacy for LHRH receptor-positive ovarian cancers.

Comparative genomics incorporating translocation renal cell carcinoma mouse model reveals molecular mechanisms of tumorigenesis.

Translocation renal cell carcinoma (tRCC) most commonly involves an ASPSCR1-TFE3 fusion, but molecular mechanisms remain elusive and animal models are lacking. Here, we show that human ASPSCR1-TFE3 driven by Pax8-Cre (a credentialed clear cell RCC driver) disrupted nephrogenesis and glomerular development, causing neonatal death, while the clear cell RCC failed driver, Sglt2-Cre, induced aggressive tRCC (as well as alveolar soft part sarcoma) with complete penetrance and short latency. However, in both contexts, ASPSCR1-TFE3 led to characteristic morphological cellular changes, loss of epithelial markers, and an epithelial-mesenchymal transition. Electron microscopy of tRCC tumors showed lysosome expansion, and functional studies revealed simultaneous activation of autophagy and mTORC1 pathways. Comparative genomic analyses encompassing an institutional human tRCC cohort (including a hitherto unreported SFPQ-TFEB fusion) and a variety of tumorgraft models (ASPSCR1-TFE3, PRCC-TFE3, SFPQ-TFE3, RBM10-TFE3, and MALAT1-TFEB) disclosed significant convergence in canonical pathways (cell cycle, lysosome, and mTORC1) and less established pathways such as Myc, E2F, and inflammation (IL-6/JAK/STAT3, interferon-γ, TLR signaling, systemic lupus, etc.). Therapeutic trials (adjusted for human drug exposures) showed antitumor activity of cabozantinib. Overall, this study provides insight into MiT/TFE-driven tumorigenesis, including the cell of origin, and characterizes diverse mouse models available for research.

Impaired lysosomal acidity maintenance in acid lipase-deficient cells leads to defective autophagy

The lysosome is an acid organelle that contains a variety of hydrolytic enzymes and plays a significant role in intracellular degradation to maintain cellular homeostasis. Genetic variants in lysosome-related genes can lead to severe congenital diseases, such as lysosomal storage diseases. In the present study, we investigated the impact of depleting lysosomal acid lipase A (LIPA), a lysosomal esterase that metabolizes esterified cholesterol or triglyceride, on lysosomal function. Under nutrient-rich conditions, LIPA gene knockout (LIPAKO) cells exhibited impaired autophagy, whereas, under starved conditions, they showed normal autophagy. The cause underlying the differential autophagic activity was increased sensitivity of LIPAKO cells to ammonia which was produced from L-glutamine in the medium. Further investigation revealed that ammonia did not affect upstream signals involved in autophagy induction, autophagosome-lysosome fusion, and hydrolytic enzyme activities in LIPAKO cells. On the other hand, LIPAKO cells showed defective lysosomal acidity upon ammonia loading. Microscopic analyses revealed that lysosomes of LIPAKO cells enlarged, whereas the amount of lysosomal proton pump V-ATPase did not proportionally increase. Since the enlargement of lysosomes in LIPAKO cells was not normalized under starved conditions, this is the primary change that occurred in the LIPAKO cells, and autophagy was affected by impaired lysosomal function under the specific conditions. These findings expand our comprehension of the pathogenesis of Wolman's disease, which is caused by a defect in the LIPA gene, and suggest that conditions, such as hyperlipidemia, may easily disrupt lysosomal functions.

ADCT-602, a Novel PBD Dimer-containing Antibody-Drug Conjugate for Treating CD22-positive Hematologic Malignancies.

Relapsed or refractory B-cell acute lymphoblastic leukemia (R/R B-ALL) and lymphomas have poor patient outcomes; novel therapies are needed. CD22 is an attractive target for antibody-drug conjugates (ADCs), being highly expressed in R/R B-ALL with rapid internalization kinetics. ADCT-602 is a novel CD22-targeting ADC, consisting of humanized mAb hLL2-C220, site specifically conjugated to the pyrrolobenzodiazepine dimer-based payload tesirine. In preclinical studies, ADCT-602 demonstrated potent, specific cytotoxicity in CD22-positive lymphomas and leukemias. ADCT-602 was specifically bound, internalized, and trafficked to lysosomes in CD22-positive tumor cells; after cytotoxin release, DNA interstrand crosslink formation persisted for 48 hours. In the presence of CD22-positive tumor cells, ADCT-602 caused bystander killing of CD22-negative tumor cells. A single ADCT-602 dose led to potent, dose-dependent, in vivo antitumor activity in subcutaneous and disseminated human lymphoma/leukemia models. Pharmacokinetic analyses (rat and cynomolgus monkey) showed excellent stability and tolerability of ADCT-602. Cynomolgus monkey B cells were efficiently depleted from circulation after one dose. Gene signature association analysis revealed IRAK1 as a potential marker for ADCT-602 resistance. Combining ADCT-602 + pacritinib was beneficial in ADCT-602-resistant cells. Chidamide increased CD22 expression on B-cell tumor surfaces, increasing ADCT-602 activity. These data support clinical testing of ADCT-602 in R/R B-ALL (NCT03698552) and CD22-positive hematologic cancers.

Identification of Low-Density Lipoprotein Receptor-Related Protein 1 as a CXCL14 Receptor Using Chemically Synthesized Tetrafunctional Probes.

CXCL14 is a primordial CXC-type chemokine that transports CpG oligodeoxynucleotides (ODN) into endosomes and lysosomes in dendritic cells, thereby leading to the activation of the Toll-like receptor 9 (TLR9)-mediated innate immune system. However, the underlying molecular mechanism by which the CXCL14-CpG ODN complex enters cells remains elusive. Herein, we describe the chemical synthesis of CXCL14-derived photoaffinity probes and their application to the identification of target receptors for CXCL14 using quantitative proteomics. By utilizing native chemical ligation and maleimide-thiol coupling chemistry, we synthesized site-specifically modified CXCL14-based photoaffinity probes that contain photoreactive 2-aryl-5-carboxytetrazole (ACT) and a hydrazine-labile cleavable linker. CXCL14-based probes were found to be capable of binding CpG ODN to immune cells, whose bioactivities were comparable to native CXCL14. Application of CXCL14-derived probes to quantitative proteomic experiments enabled the identification of dozens of target receptor candidates for CXCL14 in mouse macrophage-derived RAW264.7 cells, and we discovered that low-density lipoprotein receptor-related protein 1 (LRP1) is a novel receptor for CXCL14 by competitive proteome profiling. We further showed that disruption of LRP1 affected the incorporation of the CXCL14-CpG ODN complex in the cells. Overall, this report highlights the power of synthetic CXCL14-derived photoaffinity probes combined with chemical proteomics to discover previously unidentified receptors for CXCL14, which could promote an understanding of the molecular functions of CXCL14 and the elaborate machinery of innate immune systems.

Biocompatibility and proteomic profiling of DMSA-coated iron nanocubes in a human glioblastoma cell line.

Background: Superparamagnetic iron core iron oxide shell nanocubes have previously shown superior performance in magnetic resonance imaging T2 contrast enhancement compared with spherical nanoparticles. Methods: Iron core iron oxide shell nanocubes were synthesized, stabilized with dimercaptosuccinic acid (DMSA-NC) and physicochemically characterized. MRI contrast enhancement and biocompatibility were assessed in vitro. Results: DMSA-NC showed a transverse relaxivity of 122.59mM-1·s-1 Fe. Treatment with DMSA-NC did not induce cytotoxicity or oxidative stress in U-251 cells, and electron microscopy demonstrated DMSA-NC localization within endosomes and lysosomes in cells following internalization. Global proteomics revealed dysregulation of iron storage, transport, transcription and mRNA processing proteins. Conclusion: DMSA-NC is a promising T2 MRI contrast agent which, in this preliminary investigation, demonstrates favorable biocompatibility with an astrocyte cell model.

Fluorogenic Rhodamine Probes with Pyrrole Substitution Enables STED and Lifetime Imaging of Lysosomes in Live Cells.

Fluorogenic dyes with high brightness, large turn-on ratios, excellent photostability, favorable specificity, low cytotoxicity, and high membrane permeability are essential for high-resolution fluorescence imaging in live cells. In this study, we endowed these desirable properties to a rhodamine derivative by simply replacing the N, N-diethyl group with a pyrrole substituent. The resulting dye, Rh-NH, exhibited doubled Stokes shifts (54 nm) and a red-shift of more than 50 nm in fluorescence spectra compared to Rhodamine B. Rh-NH preferentially exists in a non-emissive but highly permeable spirolactone form. Upon binding to lysosomes, the collective effects of low pH, low polarity, and high viscosity endow Rh-NH with significant fluorescence turn-on, making it a suitable candidate for wash-free, high-contrast lysosome tracking. Consequently, Rh-NH enabled us to successfully explore stimulated emission depletion (STED) super-resolution imaging of lysosome dynamics, as well as fluorescence lifetime imaging of lysosomes in live cells.

Mobilization of cholesterol induces the transition from quiescence to growth in Caenorhabditis elegans through steroid hormone and mTOR signaling

Recovery from the quiescent developmental stage called dauer is an essential process in C. elegans and provides an excellent model to understand how metabolic transitions contribute to developmental plasticity. Here we show that cholesterol bound to the small secreted proteins SCL-12 or SCL-13 is sequestered in the gut lumen during the dauer state. Upon recovery from dauer, bound cholesterol undergoes endocytosis into lysosomes of intestinal cells, where SCL-12 and SCL-13 are degraded and cholesterol is released. Free cholesterol activates mTORC1 and is used for the production of dafachronic acids. This leads to promotion of protein synthesis and growth, and a metabolic switch at the transcriptional level. Thus, mobilization of sequestered cholesterol stores is the key event for transition from quiescence to growth, and cholesterol is the major signaling molecule in this process.

Tumor-Targeting Multiple Metabolic Regulations for Bursting Antitumor Efficacy of Chemodynamic Therapy.

Interfering with intratumoral metabolic processes is proven to effectively sensitize different antitumor treatments. Here, a tumor-targeting catalytic nanoplatform (CQ@MIL-GOX@PB) loading with autophagy inhibitor (chloroquine, CQ) and glucose oxidase (GOX) is fabricated to interfere with the metabolisms of tumor cells and tumor-associated macrophages (TAMs), then realizing effective antitumor chemodynamic therapy (CDT). Once accumulating in the tumor site with the navigation of external biotin, CQ@MIL-GOX@PB will release Fe ions and CQ in the acid lysosomes of tumor cells, the latter can sensitize Fe ions-involved antitumor CDT by blocking the autophagy-dependent cell repair. Meanwhile, the GOX component will consume glucose, which not only generates many H2O2 for CDT but also once again decelerates the tumor repair process by reducing energy metabolism. What is more, the release of CQ can also drive the NO anabolism of TAMs to further sensitize CDT. This strategy of multiple metabolic regulations is evidenced to significantly improve the antitumor effect of traditional CDT nanoagents and might provide a new sight to overcome the bottlenecks of different antitumor treatments.

Singapore grouper iridovirus VP20 interacts with grouper TBK1 and IRF3 to attenuate the interferon immune response

Singapore grouper iridovirus (SGIV), belonging to genus Ranavirus, family Iridoviridae, is a highly pathogenic agent and causes heavy economic losses in the global grouper aquaculture. Recent studies demonstrated that SGIV infection attenuated antiviral immune and inflammatory response induced by poly (I:C) in vitro. However, little was known about the potential functions of the immune regulatory proteins encoded by SGIV. Here, we identified the detailed roles of VP20 and clarified the potential mechanism underlying its immune regulatory function during SGIV infection. Our results showed that VP20 was an IE gene, and partially co-localized with Golgi apparatus and lysosomes in grouper cells. Overexpression of VP20 enhanced SGIV replication, demonstrated by the increase in the transcription levels of viral core genes and the protein synthesis of MCP. Reporter gene assays showed that SGIV VP20 overexpression significantly reduced the IFN promoter activity induced by poly (I:C), grouper stimulator of interferon genes (EcSTING) and TANK-binding kinase 1 (EcTBK1). Consistently, the transcription levels of IFN related genes were significantly decreased in VP20 overexpressing cells compared to those in control cells. Co-IP assay and confocal microscopy observations indicated that VP20 co-localized and interacted with EcTBK1 and EcIRF3, but not EcSTING. In addition, VP20 was able to degrade EcIRF3 and attenuate the antiviral action of EcIRF3, while had no effect on EcTBK1. Together, SGIV VP20 was speculated to promote viral replication through attenuating the IFN response mediated by TBK1-IRF3 in vitro. Our findings provided new insights into the immune regulatory function of SGIV encoded unknown proteins.

The ubiquitin ligases Cbl and Cbl-b regulate macrophage growth by controlling CSF-1R import into macropinosomes.

The ubiquitination of transmembrane receptors regulates endocytosis, intracellular traffic, and signal transduction. Bone marrow-derived macrophages from myeloid Cbl-/- and Cbl-b-/- double knockout (DKO) mice display sustained proliferation mirroring the myeloproliferative disease that these mice succumb to. Here, we found that the ubiquitin ligases Cbl and Cbl-b have overlapping functions for controlling the endocytosis and intracellular traffic of the CSF-1R. DKO macrophages displayed complete loss of ubiquitination of the CSF-1R whereas partial ubiquitination was observed for either single Cbl-/- or Cbl-b-/- macrophages. Unlike wild type, DKO macrophages were immortal and displayed slower CSF-1R internalization, elevated AKT signaling, and a failure to transport the CSF-1R into the lumen of nascent macropinosomes, leaving its cytoplasmic region available for signaling. CSF-1R degradation depended upon lysosomal vATPase activity in both WT and DKO macrophages, with this degradation confined to macropinosomes in WT but occurring in distributed/tubular lysosomes in DKO cells. RNA-sequencing comparison of Cbl-/-, Cbl-b-/- and DKO macrophages indicated that while the overall macrophage transcriptional program remained intact, DKO macrophages had alterations in gene expression associated with growth factor signaling, cell cycle, inflammation and senescence. Cbl-b-/- had minimal effect on the transcriptional program whereas Cbl-/- led to more alternations but only DKO macrophages demonstrated substantial changes in the transcriptome, suggesting overlapping but unique functions for the two Cbl-family members. Thus, Cbl/Cbl-b-mediated ubiquitination of CSF-1R regulates its endocytic fate, constrains inflammatory gene expression, and regulates signaling for macrophage proliferation.

A polar viscosity-sensitive fluorescent probe with large Stokes shifts for simultaneous imaging of lipid droplets and lysosomes in tobacco leaf vein cells and biological systems.

Lipid droplets (LDs) and lysosomes were dynamic organelles present in most eukaryotic cells that were interconnected and worked closely together to ensure the smooth physiological activities of organisms. The interaction between lipid droplets and lysosomes was thought to play a role in the development of certain diseases. In this paper we designed and synthesised a lipid droplet lysosomal probe. The Nap-Lyso-Ph-OH probe was constructed according to the ICT mechanism and exhibited sensitivity to both polarity and viscosity. The probe exhibited low cytotoxicity, a large Stokes shift, excellent selectivity and photostability. The probe was successfully used for labelling and imaging of lipid droplets and lysosomes in cells and zebrafish. Interestingly, we used tobacco seedling cells to explore the ability of Nap-Lyso-Ph-OH for imaging lipid droplet labelling in plant cells.

Water-soluble red fluorescent protein dimers for hypoxic two-photon photodynamic therapy.

In this study, two water-soluble red fluorescent protein (RFP) dimers, FP2R' and FP2R'', were synthesized by linking two phenothiazine-based RFP chromophore analogues through alkyl chains or alkoxy chains for hypoxic two-photon photodynamic therapy. RFP dimers are heavy-atom-free two-photon photosensitizers in which the intersystem crossing process is boosted by S and N heteroatoms. In terms of the aqueous solubility, the saturation concentration of FP2R'' was 3.5 mM, the emission wavelength was 677 nm, the singlet oxygen yield was 18%, and the two-photon absorption coefficient (β) was 2.1 × 10-11 cm W-1. Further, the RFP dimer FP2R'' showed excellent biocompatibility, negligible dark toxicity, and could produce 1O2 and O2˙- simultaneously. Under 460 nm illumination, the photosensitizer FP2R'' showed high phototoxicity with an IC50 value of 4.08 μM in an hypoxia environment, indicating that the photosensitizer FP2R'' has an excellent anti-hypoxia ability. In addition, the photosensitizer FP2R'' demonstrated a precise localization ability to lysosomes and its Pearson's colocalization coefficient was 0.94, which could guide the aggregation of photosensitizers in the lysosomes of tumor cells to effectively improve its photodynamic therapy (PDT) effect. In particular, when exposed to 800 nm two-photon excitation, FP2R'' effectively produced 1O2 and O2˙- in zebrafish and exhibited a bright two-photon fluorescence imaging capability. At the same time, the efficacy of two-photon photodynamic therapy mediated by the photosensitizer FP2R'' was verified in the tumor zebrafish model, and the growth of tumor cells in zebrafish was significantly inhibited under a two-photon laser irradiation. The water-soluble two-photon photosensitizer FP2R'' that was reasonably constructed in this study can be used as a high-efficiency hypoxic two-photon photosensitizer to inhibit deep tumor tissues.

Real-Time Visualization of Cholesterol Trafficking in Human Granulosa Cells Using Confocal Live Cell Microscopy as a Tool to Study the Novel Role of Autophagy in Sex Steroid Synthesis.

Autophagy is an evolutionarily conserved process that aims to maintain the energy homeostasis of the cell by recycling long-lived proteins and organelles. We have very recently demonstrated that lipophagy, a special form of autophagy, mediates the association of the lipid droplets (LDs) with lysosomes to deliver the lipid cargo within the LDs to lysosomes for degradation in order to release free cholesterol required for steroid synthesis in human ovary and testis. In this chapter, we describe live cell confocal microscopy technique that allows us to monitor real-time cholesterol trafficking and the association of cholesterol-laden LDs with lysosome (lipophagy) in human granulosa cells.

A zinc metal complex as an NIR emissive probe for real-time dynamics and in vivo embryogenic evolution of lysosomes using super-resolution microscopy.

Zinc (Zn) based fluorescent metal complexes have gained increasing attention due to their non-toxicity and high brightness with marked fluorescence quantum yield (QY). However, they have rarely been employed in super-resolution microscopy (SRM) to study live cells and in vivo dynamics of lysosomes. Here, we present an NIR emissive highly photostable Zn-complex as a multifaceted fluorescent probe for the long-term dynamical distribution of lysosomes in various cancerous and non-cancerous cells in live condition and in vivo embryogenic evolution in Caenorhabditis elegans (C. elegans). Apart from the normal fission, fusion, and kiss & run, the motility and the exact location of lysosomes at each point were mapped precisely. A notable difference in the lysosomal motility in the peripheral region between cancerous and non-cancerous cells was distinctly observed. This is attributed to the difference in viscosity of the cytoplasmic environment. On the other hand, along with the super-resolved structure of the smallest size lysosome (∼77 nm) in live C. elegans, the complete in vivo embryogenic evolution of lysosomes and lysosome-related organelles (LROs) was captured. We were able to capture the images of lysosomes and LROs at different stages of C. elegans, starting from a single cell and extending to a fully matured adult animal.

Modular development of organelle-targeting fluorescent probes for imaging formaldehyde in live cells.

Formaldehyde (FA) is endogenously generated via fundamental biological processes in living systems. Aberrant FA homeostasis in subcellular microenvironments is implicated in numerous pathological conditions. Fluorescent probes for detecting FA in specific organelles are thus of great research interest. Herein, we present a modular strategy to construct diverse organelle-targeting FA probes by incorporating selective organelle-targeting moieties into the scaffold of a 1,8-naphthalimide-derived FA fluorescent probe. These probes react with FA through the 2-aza-Cope arrangement and exhibit highly selective fluorescence increases for detecting FA in aqueous solutions. Moreover, these organelle-targeting probes, i.e., FFP551-Nuc, FFP551-ER, FFP551-Mito, and FFP551-Lyso, allow selective localization and imaging of FA in the nucleus, endoplasmic reticulum, mitochondria, and lysosomes of live mammalian cells, respectively. Furthermore, FFP551-Nuc has been successfully employed to monitor changes of endogenous FA levels in the nucleus of live mammalian cells. Overall, these probes should represent new imaging tools for studying the biology and pathology associated with FA in different intracellular compartments.

Fluorescent protein chromophores modified with aromatic heterocycles for photodynamic therapy and two-photon fluorescence imaging.

In this paper, three fluorescent protein chromophore analogs PFPAr (PFPP, PFPC, and PFPT) were synthesized and proved to be useful for photodynamic therapy and two-photon fluorescence imaging. By adding five- or six-membered aromatic heterocycles to the photosensitizer PFP, we obtained three fluorescent protein photosensitizers PFPAr with better performances. As a demonstration, compared with the reported photosensitizer PFP, photosensitizer PFPP exhibits larger emission wavelengths (701 nm) and achieves a slight enhancement in the efficiency of singlet oxygen (ΦΔ = 23%). Notably, PFPP can perform good two-photon fluorescence imaging with an 800 nm femtosecond laser in zebrafish. In in vitro cytotoxicity assays, PFPP shows good phototoxicity (IC50 = 4.12 μM) and acceptable dark toxicity (cell viability assay >90%). The reactive oxygen imaging experiments and AO/EB double staining assay indicate that PFPP can generate singlet oxygen to eliminate A-549 tumor cells effectively with photoexcitation of 460 nm blue light (20 mW cm-2). Furthermore, PFPP can label the lysosomes of tumor cells with high specificity for lysosomes (Pearson's correlation coefficient of 0.91). Thus, our study demonstrated that the rational introduction of aromatic heterocycles into fluorescent protein photosensitizers can effectively enhance the key parameters of photosensitivity and pave the way for further two-photon photodynamic therapy.

A novel pH-activated AIEgen probe for dynamic lysosome tracking and high-efficiency photodynamic therapy.

A novel AIEgen molecular probe (N-3QL) with typical AIE effects, good biocompatibility, lysosome targeting, pH activation, excellent photostability, and high brightness was synthesized using two simple synthetic steps. Spectroscopic and cytotoxicity experiments indicate that N-3QL can not only be used for the dynamic monitoring of cancer cell lysosomes, but also for photodynamic therapy (PDT) ablation of cancer cells.

Biodegradable persistent ROS-generating nanosonosensitizers for enhanced synergistic cancer therapy by inducing cascaded oxidative stress.

Sonodynamic therapy (SDT) is gaining popularity in cancer treatment due to its superior controllability and high tissue permeability. Nonetheless, the efficacy of SDT is severely diminished by the transient generation of limited reactive oxygen species (ROS). Herein, we introduce an acid-activated nanosonosensitizer, CaO2@PCN, by the controllable coating of porphyrinic metal-organic frameworks (PCN-224) on CaO2 to induce cascaded oxidative stress in tumors. The PCN-224 doping can generate ROS during SDT to induce intracellular oxidative stress and abnormal calcium channels. Meanwhile, the ultrasound also promotes extracellular calcium influx. In addition, CaO2@PCN sequentially degrades in the tumor cell lysosomes, releasing Ca2+ and H2O2 to induce further abnormal calcium channels and elevate the levels of Ca2+. Insufficient catalase (CAT) in tumor cells promotes intracellular calcium overload, which can induce persistent ROS generation and mitochondrial dysfunction through ion interference therapy (IIT). More importantly, PCN-224 also protects CaO2 against significant degradation under neutral conditions. Hence, the well-designed CaO2@PCN produces synergistic SDT/IIT effects and persistent ROS against cancer. More notably, the acidity-responsive biodegradability endows CaO2@PCN with excellent biosafety and promising clinical potential.

Cleavage of Hsp70.1 causes lysosomal cell death under stress conditions.

Autophagy mediates the degradation of intracellular macromolecules and organelles within lysosomes. There are three types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy. Heat shock protein 70.1 (Hsp70.1) exhibits dual functions as a chaperone protein and a lysosomal membrane stabilizer. Since chaperone-mediated autophagy participates in the recycling of ∼30% cytosolic proteins, its disorder causes cell susceptibility to stress conditions. Cargo proteins destined for degradation such as amyloid precursor protein and tau protein are trafficked by Hsp70.1 from the cytosol into lysosomes. Hsp70.1 is composed of an N-terminal nucleotide-binding domain (NBD) and a C-terminal domain that binds to cargo proteins, termed the substrate-binding domain (SBD). The NBD and SBD are connected by the interdomain linker LL1, which modulates the allosteric structure of Hsp70.1 in response to ADP/ATP binding. After the passage of the Hsp70.1-cargo complex through the lysosomal limiting membrane, high-affinity binding of the positive-charged SBD with negative-charged bis(monoacylglycero)phosphate (BMP) at the internal vesicular membranes activates acid sphingomyelinase to generate ceramide for stabilizing lysosomal membranes. As the integrity of the lysosomal limiting membrane is critical to ensure cargo protein degradation within the acidic lumen, the disintegration of the lysosomal limiting membrane is lethal to cells. After the intake of high-fat diets, however, β-oxidation of fatty acids in the mitochondria generates reactive oxygen species, which enhance the oxidation of membrane linoleic acids to produce 4-hydroxy-2-nonenal (4-HNE). In addition, 4-HNE is produced during the heating of linoleic acid-rich vegetable oils and incorporated into the body via deep-fried foods. This endogenous and exogenous 4-HNE synergically causes an increase in its serum and organ levels to induce carbonylation of Hsp70.1 at Arg469, which facilitates its conformational change and access of activated μ-calpain to LL1. Therefore, the cleavage of Hsp70.1 occurs prior to its influx into the lysosomal lumen, which leads to lysosomal membrane permeabilization/rupture. The resultant leakage of cathepsins is responsible for lysosomal cell death, which would be one of the causative factors of lifestyle-related diseases.