Elucidating Gender-Specific Distribution of Imipramine, Chloroquine, and Their Metabolites in Mice Kidney Tissues through AP-MALDI-MSI.

Knowledge of gender-specific drug distributions in different organs are of great importance for personalized medicine and reducing toxicity. However, such drug distributions have not been well studied. In this study, we investigated potential differences in the distribution of imipramine and chloroquine, as well as their metabolites, between male and female kidneys. Kidneys were collected from mice treated with imipramine or chloroquine and then subjected to atmospheric pressure matrix-assisted laser desorption ionization-mass spectrometry imaging (AP-MALDI-MSI). We observed differential distributions of the drugs and their metabolites between male and female kidneys. Imipramine showed prominent distributions in the cortex and medulla in male and female kidneys, respectively. Desipramine, one of the metabolites of imipramine, showed significantly higher (*** p < 0.001) distributions in the medulla of the male kidney compared to that of the female kidney. Chloroquine and its metabolites were accumulated in the pelvis of both male and female kidneys. Interestingly, they showed a characteristic distribution in the medulla of the female kidney, while almost no distributions were observed in the same areas of the male kidney. For the first time, our study revealed that the distributions of imipramine, chloroquine, and their metabolites were different in male and female kidneys.

Identification of extracellular polymeric substances layer barrier in chloroquine phosphate-disturbed anammox consortia and mechanism dissection on cytotoxic behavior by computational chemistry.

The over-dosing use of chloroquine phosphate (CQ) poses severe threats to human beings and ecosystem due to the high persistence and biotoxicity. The discharge of CQ into wastewater would affect the biomass activity and process stability during the biological processes, e.g., anammox. However, the response mechanism of anammox consortia to CQ remain unknown. In this study, the accurate role of extracellular polymeric substances barrier in attenuating the negative effects of CQ, and the mechanism on cytotoxic behavior were dissected by molecular spectroscopy and computational chemistry. Low concentrations (≤6.0 mg/L) of CQ hardly affected the nitrogen removal performance due to the adaptive evolution of EPS barrier and anammox bacteria. Compact protein of EPS barrier can bind more CQ (0.24 mg) by hydrogen bond and van der Waals force, among which O-H and amide II region respond CQ binding preferentially. Importantly, EPS contributes to the microbiota reshape with selectively enriching Candidatus_Kuenenia for self-protection. Furthermore, the macroscopical cytotoxic behavior was dissected at a molecular level by CQ fate/distribution and computational chemistry, suggesting that the toxicity was ascribed to attack of CQ on functional proteins of anammox bacteria with atom N17 (f-=0.1209) and C2 (f+=0.1034) as the most active electrophilic and nucleophilic sites. This work would shed the light on the fate and risk of non-antibiotics in anammox process.

Impact of Suramin on Key Pathological Features of Sporadic Alzheimer's Disease-Derived Forebrain Neurons.

Background: Alzheimer's disease (AD) is characterized by disrupted proteostasis and macroautophagy (hereafter "autophagy"). The pharmacological agent suramin has known autophagy modulation properties with potential efficacy in mitigating AD neuronal pathology. Objective: In the present work, we investigate the impact of forebrain neuron exposure to suramin on the Akt/mTOR signaling pathway, a major regulator of autophagy, in comparison with rapamycin and chloroquine. We further investigate the effect of suramin on several AD-related biomarkers in sporadic AD (sAD)-derived forebrain neurons. Methods: Neurons differentiated from ReNcell neural progenitors were used to assess the impact of suramin on the Akt/mTOR signaling pathway relative to the autophagy inducer rapamycin and autophagy inhibitor chloroquine. Mature forebrain neurons were differentiated from induced pluripotent stem cells (iPSCs) sourced from a late-onset sAD patient and treated with 100µM suramin for 72 h, followed by assessments for amyloid-ß, phosphorylated tau, oxidative/nitrosative stress, and synaptic puncta density. Results: Suramin treatment of sAD-derived neurons partially ameliorated the increased p-Tau(S199)/Tau ratio, and fully remediated the increased glutathione to oxidized nitric oxide ratio, observed in untreated sAD-derived neurons relative to healthy controls. These positive results may be due in part to the distinct increases in Akt/mTOR pathway mediator p-p70S6K noted with suramin treatment of both ReNcell-derived and iPSC-derived neurons. Longer term neuronal markers, such as synaptic puncta density, were unaffected by suramin treatment. Conclusions: These findings provide initial evidence supporting the potential of suramin to reduce the degree of dysregulation in sAD-derived forebrain neurons in part via the modulation of autophagy.

Chloroquine Alleviates Atherosclerosis by Modulating Regulatory T Cells Through the ATM/AMPK/mTOR Signaling Pathway in ApoE -/- Mice.

BACKGROUND: Clinical observation suggests the atheroprotective effect of chloroquine and its derivatives, while its mechanism remains unclear. This study aimed to observe the protective effect of chloroquine against atherosclerosis and explore the underlying mechanism. METHODS: Ataxia telangiectasia mutated (ATM) wild-type or haploinsufficient apolipoprotein-E-knockout (ATM+/+ApoE-/- or ATM+/-ApoE-/-) mice were treated with different dosages of chloroquine. Anti-CD25 antibody was used to deplete natural Tregs in ATM+/+ApoE-/- mice. The atherosclerotic burden in different groups of mice was comprehensively evaluated by H&E staining and Masson staining. The effect of chloroquine on the regulatory T cells (Tregs) was assessed in vivo and in vitro by flow cytometry and immunohistochemical staining. The expression of related proteins was detected by real-time polymerase chain reaction and western blotting. RESULTS: In ATM+/+ApoE-/- mice, chloroquine alleviated atherosclerotic lesions, stabilized the plaque, and increased Treg counts in the atherosclerotic lesions and spleens. However, in ATM haploinsufficient mice (ATM+/-ApoE-/-), chloroquine no longer prevented atherosclerosis or impacted Treg counts. Abolishing Treg cells using an anti-CD25 antibody in vivo abrogated the atheroprotective effect of chloroquine. In vitro, chloroquine promoted the differentiation of Tregs from naïve T cells, which was accompanied by enhanced ATM/AMP-activated protein kinase (AMPK) activity and reduced downstream mammalian target of rapamycin (mTOR) activity. DISCUSSION: These findings suggest that chloroquine ameliorates atherosclerosis and stabilizes plaque by modulating Tregs differentiation through the regulation of the ATM/AMPK/mTOR pathway.

Chloroquine inhibited Helicobacter pylori-related gastric carcinogenesis by YAP-ß-catenin-autophagy axis.

YAP participates in autophagy associated with many diseases. In this study, we demonstrate that YAP promotes autophagy by interacting with beclin 1, upregulating beclin 1 and LC3B-II protein expression, and promoting autophagosome formation after H. pylori infection in a vacuolating cytotoxin A-dependent manner. The protein levels of ß-catenin in the cytoplasm and nuclei of GES-1 cells and the mRNA levels of Axin2, Myc, Lgr5, and Ccnd1 were increased in H. pylori-infected cells or YAP-overexpressed cells, but were decreased in YAP-silenced cells. The ß-catenin inhibitor XAV939 significantly downregulated autophagy, whereas the activator LiCl showed opposite effects. An H. pylori-infected mouse model of gastric carcinoma was successfully established. The mouse model showed that H. pylori infection, when combined with NMU, promoted the tumorigenesis of gastric tissues; increased IL-1ß, IL-6, and TNF-α levels; promoted NO release; and increased the expression of beclin 1, LC3B-II more than NMU alone. Chloroquine inhibited these phenomena, but did not completely attenuate the effects of H. pylori. These results demonstrate that chloroquine can be used as a drug for the treatment of H. pylori-related gastric cancer, but the treatment should simultaneously remove H. pylori.

Characterization of LysoTracker Red uptake by in vitro model cells of the outer blood-retinal barrier: Implication of lysosomal trapping with cytoplasmic vacuolation and cytotoxicity.

Lysosomal trapping, a physicochemical process in which lipophilic cationic compounds are sequestered in lysosomes, can affect drug disposition and cytotoxicity. To better understand lysosomal trapping at the outer blood-retinal barrier (BRB), we investigated the distribution of LysoTracker Red (LTR), a probe compound for lysosomal trapping, in conditionally immortalized rat retinal pigment epithelial (RPE-J) cells. LTR uptake by RPE-J cells was dependent on temperature and attenuated by ammonium chloride and protonophore, which decreased the pH gradient between the lysosome and cytoplasm, suggesting lysosomal trapping of LTR in RPE-J cells. The involvement of lysosomal trapping in response to cationic drugs, including neuroprotectants such as desipramine and memantine, was also suggested by an inhibition study of LTR uptake. Chloroquine, which is known to show ocular toxicity, induced cytoplasmic vacuolization in RPE-J cells with a half-maximal effective concentration of 1.35 µM. This value was 59 times lower than the median lethal concentration (= 79.1 µM) of chloroquine, suggesting that vacuolization was not a direct trigger of cell death. These results are helpful for understanding the lysosomal trapping of cationic drugs, which is associated with drug disposition and cytotoxicity in the outer BRB.

Chloroquine resistance evolution in Plasmodium falciparum is mediated by the putative amino acid transporter AAT1.

Malaria parasites break down host haemoglobin into peptides and amino acids in the digestive vacuole for export to the parasite cytoplasm for growth: interrupting this process is central to the mode of action of several antimalarial drugs. Mutations in the chloroquine (CQ) resistance transporter, pfcrt, located in the digestive vacuole membrane, confer CQ resistance in Plasmodium falciparum, and typically also affect parasite fitness. However, the role of other parasite loci in the evolution of CQ resistance is unclear. Here we use a combination of population genomics, genetic crosses and gene editing to demonstrate that a second vacuolar transporter plays a key role in both resistance and compensatory evolution. Longitudinal genomic analyses of the Gambian parasites revealed temporal signatures of selection on a putative amino acid transporter (pfaat1) variant S258L, which increased from 0% to 97% in frequency between 1984 and 2014 in parallel with the pfcrt1 K76T variant. Parasite genetic crosses then identified a chromosome 6 quantitative trait locus containing pfaat1 that is selected by CQ treatment. Gene editing demonstrated that pfaat1 S258L potentiates CQ resistance but at a cost of reduced fitness, while pfaat1 F313S, a common southeast Asian polymorphism, reduces CQ resistance while restoring fitness. Our analyses reveal hidden complexity in CQ resistance evolution, suggesting that pfaat1 may underlie regional differences in the dynamics of resistance evolution, and modulate parasite resistance or fitness by manipulating the balance between both amino acid and drug transport.

Salivary gland LAMP3 mRNA expression is a possible predictive marker in the response to hydroxychloroquine in Sjögren's disease.

Hydroxychloroquine (HCQ) is a lysosomotropic agent that is commonly used for treating Sjögren's disease (SjD). However, its efficacy is controversial because of the divergent response to the drug among patients. In a subgroup of SjD patients, lysosome-associated membrane protein 3 (LAMP3) is elevated in expression in the salivary glands and promotes lysosomal dysregulation and lysosome-dependent apoptotic cell death. In this study, chloroquine (CQ) and its derivative HCQ were tested for their ability to prevent LAMP3-induced apoptosis, in vitro and on a mouse model of SjD. In addition, efficacy of HCQ treatment was retrospectively compared between high LAMP3 mRNA expression in minor salivary glands and those with LAMP3 mRNA levels comparable with healthy controls. Study results show that CQ treatment stabilized the lysosomal membrane in LAMP3-overexpressing cells via deactivation of cathepsin B, resulting in decreased apoptotic cell death. In mice with established SjD-like phenotype, HCQ treatment also significantly decreased apoptotic cell death and ameliorated salivary gland hypofunction. Retrospective analysis of SjD patients found that HCQ tended to be more effective in improving disease activity index, symptom severity and hypergammaglobulinemia in patients with high LAMP3 expression compared those with normal LAMP3 expression. Taken together, these findings suggested that by determining salivary gland LAMP3 mRNA level, a patient's response to HCQ treatment could be predicted. This finding may provide a novel strategy for guiding the development of more personalized medicine for SjD.

Chloroquine reduces neutrophil extracellular trap (NET) formation through inhibition of peptidyl arginine deiminase 4 (PAD4).

Neutrophil extracellular traps (NETs) occur when chromatin is decondensed and extruded from the cell, generating a web-like structure. NETs have been implicated in the pathogenesis of several sterile disease states and thus are a potential therapeutic target. Various pathways have been shown to induce NETs, including autophagy, with several key enzymes being activated like peptidyl arginine deiminase 4 (PAD4), an enzyme responsible for citrullination of histones, allowing for DNA unwinding and subsequent release from the cell. Pre-clinical studies have already demonstrated that chloroquine (CQ) and hydroxychloroquine (HCQ) are able to reduce NETs and slow disease progression. The exact mechanism as to how these drugs reduce NETs has yet to be elucidated. CQ and HCQ decrease NET formation from various NET activators, independent of their autophagy inhibitory function. CQ and HCQ were found to inhibit PAD4 exclusively, in a dose-dependent manner, confirmed with reduced CitH3+ NETs after CQ or HCQ treatment. Circulating CitH3 levels were reduced in pancreatic cancer patients after HCQ treatment. In silico screening of PAD4 protein structure identified a likely binding site interaction at Arg639 for CQ and Trp347, Ser468, and Glu580 for HCQ. SPR analysis confirmed the binding of HCQ and CQ with PAD4 with KD values of 54.1 µM (CQ) and 88.1 µM (HCQ). This data provide evidence of direct PAD4 inhibition as a mechanism for CQ/HCQ inhibition of NETs. We propose that these drugs likely reduce NET formation through multiple mechanisms; the previously established TLR9 and autophagy inhibitory mechanism and the novel PAD4 inhibitory mechanism.

Chloroquine is a safe autophagy inhibitor for sustaining the expression of antioxidant enzymes in trophoblasts.

Inhibition of autophagy contributes to the pathophysiology of preeclampsia. Although chloroquine (CHQ) is an autophagy inhibitor, it can reduce the occurrence of preeclampsia in women with systemic lupus erythematosus. To clarify this important clinical question, this study aimed to address the safety of CHQ in trophoblast cells from the viewpoint of homeostasis, in which the anti-oxidative stress (OS) response and autophagy are involved. We used Western blotting to evaluate the protein levels in the trophoblast cells. The expression levels of heme oxygenase-1 (HO-1), an anti-OS enzyme, mediate resistance to OS induced by hydrogen peroxide (H2O2) in trophoblast cell lines. Among the autophagy modulators, bafilomycin A1 (BAF), an autophagy inhibitor, but not autophagy activators, suppressed HO-1 expression in BeWo cells; CHQ did not suppress HO-1 expression in BeWo cells. To clarify the role of autophagy in HO-1 induction, we observed no difference in HO-1 induction by H2O2 between autophagy-normal and autophagy-deficient cells. As for the mechanism of HO-1 induction by OS, BAF suppressed HO-1 induction by downregulating the expression of neighbor of BRCA1 gene 1 (NBR1) in the selective p62-NBR1-nuclear factor erythroid 2-related factor 2 (Nrf2) autophagy pathway. CHQ did not inhibit HO-1 expression by sustaining NBR1 expression in human villous tissues compared to BAF treatment. In conclusion, CHQ is a safer medicine than BAF for sustaining NBR1, which resist against OS in trophoblasts by connecting selective autophagy and the anti-OS response.

Altered Lysosomal Function Manipulates Cellular Biosynthetic Capacity By Remodeling Intracellular Cholesterol Distribution.

Lysosomes are known to influence cholesterol trafficking into endoplasmic reticulum (ER) membranes. Though intracellular cholesterol levels are known to influence the lipid biosynthetic responses in ER, the specific effects of lysosomal modulation on these outcomes is not known. To demonstrate this, C2C12 cells were treated with chloroquine, a lysosomotropic agent, and its effects on cellular biosynthetic capacity, structural and functional status of ER was determined. In addition to its known effects on autophagy reduction, chloroquine treatment induced accumulation of total cellular lipid and ER-specific cholesterol content. It was also observed that chloroquine caused an increase in smooth-ER content with defects in overall protein turnover. Further, since ER and mitochondria function in close association through ER membrane contact sites, it is likely that lysosomal modulation also brings about associated changes in mitochondria. In this regard, we found that chloroquine reduces mitochondrial membrane potential and mitochondrial dynamics. Collectively, the differential biosynthetic response of rise in lipid content, but not protein content, cannot be accounted by merely considering that chloroquine induced suppression of autophagy causes defects in organelle function. In this defective autophagy scenario, both biosynthetic responses such as lipid and protein synthesis are expected to be reduced rather than only the latter, as observed with chloroquine. These findings suggest that cholesterol trafficking/distribution within cellular organelles could act as an intracellular mediator of differential biosynthetic remodelling in interconnected organelles.

Intracellular cholesterol transport inhibition Impairs autophagy flux by decreasing autophagosome-lysosome fusion.

BACKGROUND: Autophagy is an intracellular degradation process crucial for homeostasis. During autophagy, a double-membrane autophagosome fuses with lysosome through SNARE machinery STX17 to form autolysosome for degradation of damaged organelle. Whereas defective autophagy enhances cholesterol accumulation in the lysosome and impaired autophagic flux that results Niemann-Pick type C1 (NPC1) disease. However, exact interconnection between NPC1 and autophagic flux remain obscure due to the existence of controversial reports. RESULTS: This study aimed at a comparison of the effects of three autophagic inhibitor drugs, including chloroquine, U18666A, and bafilomycin A1, on the intracellular cholesterol transport and autophagy flux. Chloroquine, an autophagic flux inhibitor; U1866A, a NPC1 inhibitor, and bafilomycin A, a lysosomotropic agent are well known to inhibit autophagy by different mechanism. Here we showed that treatment with U1866A and bafilomycin A induces lysosomal cholesterol accumulation that prevented autophagic flux by decreasing autophagosome-lysosome fusion. We also demonstrated that accumulation of cholesterol within the lysosome did not affect lysosomal pH. Although the clearance of accumulated cholesterol by cyclodextrin restored the defective autophagosome-lysosome fusion, the autophagy flux restoration was possible only when lysosomal acidification was not altered. In addition, a failure of STX17 trafficking to autophagosomes plays a key role in prevention of autophagy flux caused by intracellular cholesterol transport inhibitors. CONCLUSIONS: Our data provide a new insight that the impaired autophagy flux does not necessarily result in lysosomal cholesterol accumulation even though it prevents autophagosome-lysosome fusion. Video abstract.

Piperaquine-resistant PfCRT mutations differentially impact drug transport, hemoglobin catabolism and parasite physiology in Plasmodium falciparum asexual blood stages.

The emergence of Plasmodium falciparum parasite resistance to dihydroartemisinin + piperaquine (PPQ) in Southeast Asia threatens plans to increase the global use of this first-line antimalarial combination. High-level PPQ resistance appears to be mediated primarily by novel mutations in the P. falciparum chloroquine resistance transporter (PfCRT), which enhance parasite survival at high PPQ concentrations in vitro and increase the risk of dihydroartemisinin + PPQ treatment failure in patients. Using isogenic Dd2 parasites expressing contemporary pfcrt alleles with differential in vitro PPQ susceptibilities, we herein characterize the molecular and physiological adaptations that define PPQ resistance in vitro. Using drug uptake and cellular heme fractionation assays we report that the F145I, M343L, and G353V PfCRT mutations differentially impact PPQ and chloroquine efflux. These mutations also modulate proteolytic degradation of host hemoglobin and the chemical inactivation of reactive heme species. Peptidomic analyses reveal significantly higher accumulation of putative hemoglobin-derived peptides in the PPQ-resistant mutant PfCRT isoforms compared to parental PPQ-sensitive Dd2. Joint transcriptomic and metabolomic profiling of late trophozoites from PPQ-resistant or -sensitive isogenic lines reveals differential expression of genes involved in protein translation and cellular metabolism. PPQ-resistant parasites also show increased susceptibility to an inhibitor of the P. falciparum M17 aminopeptidase that operates on short globin-derived peptides. These results reveal unique physiological changes caused by the gain of PPQ resistance and highlight the potential therapeutic value of targeting peptide metabolism in P. falciparum.

Chloroquine alleviates the heat-induced to injure via autophagy and apoptosis mechanisms in skin cell and mouse models.

Burns can cause cell death and irreversible tissue damage. We examined the pathway of human dermis fibroblasts cell death caused by skin burns and the roles of chloroquine in human skin keratinocytes HaCaT wound healing. Western blot assays were performed to assess expression of proteins associated with autophagy, apoptosis, and endoplasmic reticulum stress in skin cells following burns. Changes in apoptosis-related proteins were assessed using flow cytometry, and wound cell migration was examined using wound healing assays. The burn animal model was used to test whether chloroquine would promote wound healing. In human burned fibroblasts, expression of LC3B-II and Cleave-caspase-7 was increased, whereas expression of Beclin-1, p62, and Grp78 was decreased. Severe burn induced ER stress and ERK phosphorylation, but PD98059 or necrostatin-1 treatment cells did not affect expression of autophagy LC3B-II protein and can induce apoptosis. Even though added with TGF-ß and FGF did not repair autophagy caused by burns. Suggesting that autophagy and apoptosis were involved in heat-injured mechanism. Recombinant Wnt3a protein can help restore expression of ß-catenin which reduced following burns in keratinocytes. Wnt3a protein can promote migration of keratinocytes after burns. Interesting, chloroquine increased expression of LC3B-II protein and restored cell migration activity after 24 h of burns. Consistently, surgical dressing containing chloroquine promoted wound healing in a burn animal mode. Autophagy and Wnt/ß-catenin is two signalling pathways that participate in cell repair and wound healing in human fibroblasts, keratinocytes. Surgical dressing containing chloroquine can recover wound healing in burned rats.

Palmitic acid hinders extracellular traps of neutrophil from postpartum dairy cow in vitro.

Peripartum dairy cows experience negative energy balance, characterized by high concentrations of blood free fatty acids (FFA) and immune dysfunction. Palmitic acid (PA), the most abundant saturated fatty acid in cow blood, is not only an energy precursor, but causes cellular dysfunction when in excess. Neutrophil extracellular traps (NET) are one of the arsenals of weapons neutrophils use to fight invading pathogens. However, given the marked increase in circulating PA during the peripartum period, it remains to be determined what effect (if any) PA has on NET release. Thus, the objective of this study was to evaluate the effect of PA on NET release and the underlying mechanism in vitro. Phorbol-12-myristate-13-acetate (PMA; 100 ng/mL, 3 h) was used to induce the release of NET in vitro. We isolated neutrophils from the peripheral blood of 5 healthy postpartum dairy cows with similar parity (median = 3, range = 2-4), milk yield (median = 27.84 kg/d per cow, range = 25.79-31.43 kg/d per cow), days in milk (median = 7 d, range = 4-10 d), and serum FFA <0.25 mM, ß-hydroxybutyric acid <0.6 mM, and glucose >3.5 mM. Inhibition of double-stranded DNA (dsDNA) level, a marker of NET release, in response to PA was used to determine an optimal incubation time and concentration for in vitro experiments. Cells were maintained in RPMI-1640 basic medium without phenol red, treated with 600 µM PA for different times (4, 5, 6, and 7 h) in the presence or absence of PMA. There was a decrease for dsDNA level in the supernatant due to increased duration of PA treatment, with a peak response at 6 h. Thus, 6 h was selected as the challenge time. Then, cells were treated with different concentrations of PA (100, 200, 400, and 600 µM) for 6 h in the presence or absence of PMA. There was a decrease for dsDNA level in the supernatant due to increased dose of PA, with a peak response at 400 µM. Finally, 400 µM PA for 6 h was selected as the treatment for subsequent experiments. Protein abundance of citrullinated histone in the presence or absence of PMA was markedly lower in response to incubation with PA. Morphological observations by laser confocal microscopy and scanning electron microscopy showed that the ratio of NET-releasing cells decreased in response to incubation with PA. Autophagy is a potential key intermediate process in the regulation of NET by PA. To investigate the effect of PA on autophagy, we used chloroquine to block lysosomal degradation. Exogenous PA led to accumulation of sequestosome-1 and microtubule-associated protein 1 light chain 3-II, and no further accumulation in the presence of chloroquine, all of which suggested an impairment of autophagic flux. To verify the role of autophagy in NET, we used rapamycin to promote autophagic flux; 100 nM rapamycin attenuated the suppressive effect of PA on NET release indicated by greater dsDNA levels, accumulation of citrullinated histone, and ratio of NET-releasing neutrophils. Overall, these data demonstrate PA inhibits NET release by suppressing autophagic flux, which provides information for understanding the immune dysfunction in postpartum cows.

Quercetin Promotes TFEB Nuclear Translocation and Activates Lysosomal Degradation of Ferritin to Induce Ferroptosis in Breast Cancer Cells.

Objective. To investigate the antiproliferative efficacy of quercetin on breast cell lines and its mechanism of ferroptosis regulation. Cells (MCF-7 and MDA-231) were treated with quercetin at 0.1, 1, and 10 µM, respectively. The cell counting kit-8 (CCK-8) assay was applied to assess cell viability, and the intracellular iron level, malondialdehyde (MDA), and carbonylated protein were measured. After treating the cells with quercetin, western blot was applied to determine the level of transcription factor EB (TFEB) and lysosomal-associated membrane protein 1 (LAMP-1) in cells. Meanwhile, western blot was performed to assess the nuclear translocation of TFEB protein in cells. TFEB siRNA and autophagy lysosomal inhibitor, chloroquine, were used to block ferroptosis induced by quercetin. Quercetin induced breast cancer cell death and upregulated the level of iron, MDA, and carbonyl protein in a concentration-dependent manner. Meanwhile, TFEB was highly expressed in the nucleus and lowly expressed in the cytoplasm. The high expression of TFEB promoted the expression of lysosome-related gene LAMP-1, which in turn promoted the degradation of ferritin and the release of ferric ions. The above pharmacodynamic effects of quercetin can be blocked by TFEB siRNA or chloroquine. Quercetin promotes TFEB expression and nuclear transcription, induces the onset of iron death, and thus exerts a pharmacological effect on killing breast cancer cells.

Rhein induces changes in the lysosomal compartment of HeLa cells.

Rhein is an anthraquinone found in Rheum palmatum, used in Chinese medicine. Due to potential anticancer properties, the study assessed its effect on the lysosomal compartment, which indirectly influences cell death. The experiment was performed on HeLa cells by treating them with rhein at concentrations of 100-300 µM. LC3-II protein and caspase 3/7 activity, level of apoptosis, the concentration of reactive oxide species (ROS), and mitochondrial potential (Δψm) were evaluated by the cytometric method. To evaluate the permeability of the lysosomal membrane (LMP), staining with acridine orange and the assessment of activity of cathepsin D and L in the lysosomal and extralysosomal fractions were used. Cell viability was assessed by -(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (MTT) and neutral red (NR) assays. Changes in cells were also demonstrated at the level of electron, optical, confocal, and fluorescence microscopy. Inhibition of autophagy was done using chloroquine. Rhein-induced degradation processes were confirmed by an increase in the number of primary lysosomes, autophagosomes, and autolysosomes. At high concentrations, rhein caused the generation of ROS, which induced LMP expressed by quenching of acridine orange fluorescence. These results correlated with a reduction of lysosomes, as visualized in graphical modeling, with the decreased uptake of NR by lysosomes, and increased activity of cathepsin D and L in the extralysosomal fraction. The studies also showed an increase in the activity of caspase 3/7 and a decrease in the expression of Bcl-2 protein, indicative of rhein-stimulated apoptosis. At the same time, we demonstrated that preincubation of cells with chloroquine inhibited rhein-induced autophagy and contributed to increased cytotoxicity to HeLa cells. Rhein also induced DNA damage and led to cycle arrest in the S phase. Our results indicate that rhein, by inducing changes in the lysosomal compartment, indirectly affects apoptosis of HeLa cells and in combination with autophagy inhibitors may be an effective form of anticancer therapy.

The Knock-Down of the Chloroquine Resistance Transporter PfCRT Is Linked to Oligopeptide Handling in Plasmodium falciparum.

The chloroquine resistance transporter, PfCRT, is an essential factor during intraerythrocytic development of the human malaria parasite Plasmodium falciparum. PfCRT resides at the digestive vacuole of the parasite, where hemoglobin taken up by the parasite from its host cell is degraded. PfCRT can acquire several mutations that render PfCRT a drug transporting system expelling compounds targeting hemoglobin degradation from the digestive vacuole. The non-drug related function of PfCRT is less clear, although a recent study has suggested a role in oligopeptide transport based on studies conducted in a heterologous expression system. The uncertainty about the natural function of PfCRT is partly due to a lack of a null mutant and a dearth of functional assays in the parasite. Here, we report on the generation of a conditional PfCRT knock-down mutant in P. falciparum. The mutant accumulated oligopeptides 2 to at least 8 residues in length under knock-down conditions, as shown by comparative global metabolomics. The accumulated oligopeptides were structurally diverse, had an isoelectric point between 4.0 and 5.4 and were electrically neutral or carried a single charge at the digestive vacuolar pH of 5.2. Fluorescently labeled dipeptides and live cell imaging identified the digestive vacuole as the compartment where oligopeptides accumulated. Our findings suggest a function of PfCRT in oligopeptide transport across the digestive vacuolar membrane in P. falciparum and associated with it a role in nutrient acquisition and the maintenance of the colloid osmotic balance. IMPORTANCE The chloroquine resistance transporter, PfCRT, is important for the survival of the human malaria parasite Plasmodium falciparum. It increases the tolerance to many antimalarial drugs, and it is essential for the development of the parasite within red blood cells. While we understand the role of PfCRT in drug resistance in ever increasing detail, the non-drug resistance functions are still debated. Identifying the natural substrate of PfCRT has been hampered by a paucity of functional assays to test putative substrates in the parasite system and the absence of a parasite mutant deficient for the PfCRT encoding gene. By generating a conditional PfCRT knock-down mutant, together with comparative metabolomics and uptake studies using fluorescently labeled oligopeptides, we could show that PfCRT is an oligopeptide transporter. The oligopeptides were structurally diverse and were electrically neutral or carried a single charge. Our data support a function of PfCRT in oligopeptide transport.

Lipopolysaccharide reduces melanin synthesis in vitiligo melanocytes by regulating autophagy.

Vitiligo is an autoimmune-related disease with a complex aetiology that involves innate immunity. Toll-like receptors (TLRs) are important parts of innate immunity and are related to a variety of autoimmune diseases, including vitiligo, through an unknown mechanism. In this study, we found that the TLR4 gene expression was increased in blood samples of patients with advanced stage vitiligo, and then, we evaluated the effect of TLR4 ligand lipopolysaccharide (LPS) on melanin synthesis in a vitiligo melanocyte cell line PIG3V and along with its mechanism. LPS suppressed melanin synthesis, downregulated the expression of melanin synthesis-related proteins and activated autophagy in vitiligo melanocytes. Inhibiting autophagy with 3-methyladenine or chloroquine blocked these effects. This suggests that LPS inhibits skin pigmentation by modulating autophagy, thus providing novel insights into the pathogenesis of vitiligo.

SIRT2 Affects Cell Proliferation and Apoptosis by Suppressing the Level of Autophagy in Renal Podocytes.

Purpose: Despite the discovery of many important molecules in diabetic nephropathy, there has been very limited progress in the management of diabetic kidney diseases and the design of new drugs. To fill this gap, the present study explored the expression of SIRT2 in high-glucose murine kidney foot cells and its impact on cell biological functions. Methods: Expression levels of SIRT2 in the MPC-5 of murine kidney foot cells after high and normal glucose treatment or in cells targeted with siRNA were detected using qRT-PCR. Cellular proliferation and programmed cell death were analyzed via the CCK8 assay and flow cell technique, separately. Levels of autophagy markers were measured by western blotting, and chloroquine treatment was applied to the cells to observe the effect of SIRT2 on cell proliferation and apoptosis after treatment. Results: The expression level of SIRT2 was remarkably upregulated in the high-GLU group in contrast to the low-GLU group. The cell proliferation and autophagy levels were significantly reduced, and apoptosis was remarkably reinforced in the high-GLU group in contrast to the normal GLU group. However, knocking down the expression level of SIRT2 caused an increase in cell proliferation and cell autophagy levels and significantly weakened apoptosis. Chloroquine influenced cell proliferation and apoptosis in cells targeted with SIRT2 siRNA. Conclusion: SIRT2 expression was upregulated in hyperglycaemic murine kidney foot cells, and knocking down the expression level of SIRT2 affected the biological function of the cells. We found that SIRT2 may modulate cell proliferation and apoptosis by regulating cell autophagy.