Mitochondrial Membrane Depolarization Research Articles

Vallaris solanacea induces mitochondrial mediated apoptosis in HL-60 human promyelocytic leukemia cells

In the present study, the apoptosis-inducing potential of a chloroform fraction from an alcoholic extract of Vallaris solanacea aerial parts (VS) was examined using human promyelocytic leukemia HL-60 cells. We discovered a concentration and time-dependent decrease in cell growth using MTT assay. Scanning electron micrographs and fluorescence microscopy were used to observe several well-documented morphological and nuclear alterations, such as reduction in cell size, chromatin condensation, fragmentation, and the creation of cell surface blebs. A considerable rise in the Sub-G0 population was revealed by cell cycle analysis. Additionally, a dose-dependent rise in cells positive for Annexin V was observed. DCFH-DA test on VS-treated HL-60 cells showed an increase in endogenous ROS generation of up to 4.3 fold. Additionally, suppression in Bcl-2 levels and increased mitochondrial membrane depolarization in treated cells were also associated with a rise in cytosolic cytochrome-c levels that was consequently followed by the activation of the caspase cascade. Further, the DNA fragmentation assay exhibited a typical ladder formation at 25 μg/ml, which became prominent in a concentration-dependent manner. Our study revealed that VS has apoptosis-inducing potential towards HL-60 cells in vitro and is an effective candidate for further anti-cancer studies.

Vitamin C alleviates hyperglycemic stress in retinal pigment epithelial cells.

Retinal pigment epithelial cells (RPECs) are a type of retinal cells that structurally and physiologically support photoreceptors. However, hyperglycemia has been shown to play a critical role in the progression of diabetic retinopathy (DR), which is one of the leading causes of vision impairment. In the diabetic eye, the high glucose environment damages RPECs via the induction of oxidative stress, leading to the release of excess reactive oxygen species (ROS) and triggering apoptosis. In this study, we aim to investigate the antioxidant mechanism of Vitamin C in reducing hyperglycemia-induced stress and whether this mechanism can preserve the function of RPECs. ARPE-19 cells were treated with high glucose in the presence or absence of Vitamin C. Cell viability was measured by MTT assay. Cleaved poly ADP-ribose polymerase (PARP) was used to identify apoptosis in the cells. ROS were detected by the DCFH-DA reaction. The accumulation of sorbitol in the aldose reductase (AR) polyol pathway was determined using the sorbitol detection assay. Primary mouse RPECs were isolated from adult mice and identified by Rpe65 expression. The mitochondrial damage was measured by mitochondrial membrane depolarization. Our results showed that high glucose conditions reduce cell viability in RPECs while Vitamin C can restore cell viability, compared to the vehicle treatment. We also demonstrated that Vitamin C reduces hyperglycemia-induced ROS production and prevents cell apoptosis in RPECs in an AR-independent pathway. These results suggest that Vitamin C is not only a nutritional necessity but also an adjuvant that can be combined with AR inhibitors for alleviating hyperglycemic stress in RPECs.

Antifungal activity of human antimicrobial peptides targeting apoptosis in Candida auris.

Introduction. Innovative antifungal therapies are of crucial importance to combat the potentially life-threatening infections linked to the multidrug-resistant fungal pathogen Candida auris. Induction of regulated cell death, apoptosis, could provide an outline for future therapeutics. Human antimicrobial peptides (AMPs), well-known antifungal compounds, have shown the ability to induce apoptosis in pathogenic fungi.Hypothesis/Gap Statement . Although it is known that AMPs possess antifungal activity against C. auris, their ability to induce apoptosis requires further investigations.Aim. This study evaluated the effects of AMPs on the induction of apoptosis in C. auris.Methods. Human neutrophil peptide-1 (HNP-1), human β-Defensins-3 (hBD-3) and human salivary histatin 5 (His 5) were assessed against two clinical C. auris isolates. Apoptosis hallmarks were examined using FITC-Annexin V/PI double labelling assay and terminal deoxynucleotidyl transferase deoxynucleotidyl transferase nick-end labelling (TUNEL) to detect phosphatidylserine externalization and DNA fragmentation, respectively. Then, several intracellular triggers were studied using JC-10 staining, spectrophotometric assay and 2',7'-dichlorofluorescin diacetate staining to measure the mitochondrial membrane potential, cytochrome-c release and reactive oxygen species (ROS) production, respectively.Results and conclusion. FITC-Annexin V/PI staining and TUNEL analysis revealed that exposure of C. auris cells to HNP-1 and hBD-3 triggered both early and late apoptosis, while His 5 caused significant necrosis. Furthermore, HNP-1 and hBD-3 induced significant mitochondrial membrane depolarization, which resulted in substantial cytochrome c release. In contrast to His 5, which showed minimal mitochondrial depolarization and no cytochrome c release. At last, all peptides significantly increased ROS production, which is related to both types of cell death. Therefore, these peptides represent promising and effective antifungal agents for treating invasive infections caused by multidrug-resistant C. auris.

Biotin-stapled mixed micelles of lapatinib: Unleashing apoptosis power in triple-negative breast cancer

The treatment options currently offered for triple-negative breast cancer (TNBC) encounter numerous obstacles, including disappointing overall survival rates, unavoidable side effects, and the development of drug resistance. Additionally, there is a growing need for nutrients, like vitamins (e.g., biotin), to sustain the unregulated growth of TNBC cells. Hence, targeted therapy (LPT B-MM) for enhancing the treatment of TNBC was explored which encompassed hydrophobized [using α-TOS (α-Tocopheryl succinate] Pluronic-based mixed micelles having inherent sodium-dependent multivitamin transporter (SMVT) targeting (biotin-stapled) ability. LPT B-MM had optimal size (210.72± 25.88 nm), PDI (0.272± 0.023), and enhanced drug loading capacity (∼13.31± 2.12%). Transmission electron microscopy validated the spherical shape of these micelles. In vitro release studies demonstrated drug release in a sustained manner without the risk of hemolysis. Importantly, LPT B-MM displayed heightened cellular uptake, increased cytotoxicity, a lower IC50 value, elevated reactive oxygen species, induced mitochondrial membrane depolarization, and a greater apoptosis index in TNBC cell lines compared to free LPT. This study highlights the considerable potential of LPT B-MM to induce apoptosis, showcasing its promise in enhancing payload capabilities and enabling SMVT specific targeting within the context of TNBC. LPT B-MM demonstrated the synergistic effects of LPT and α-TOS in inducing apoptosis in TNBC cells, leading to enhanced therapeutic efficacy. This novel combination therapy offers a promising strategy for improving treatment outcomes in TNBC patients and represents a significant advancement in the field of TNBC therapy.

An In Vitro Study on the Cytotoxic, Antioxidant, and Antimicrobial Properties of Yamogenin-A Plant Steroidal Saponin and Evaluation of Its Mechanism of Action in Gastric Cancer Cells.

Yamogenin is a steroidal saponin occurring in plant species such as Asparagus officinalis, Dioscorea collettii, Trigonella foenum-graecum, and Agave sp. In this study, we evaluated in vitro cytotoxic, antioxidant, and antimicrobial properties of yamogenin. The cytotoxic activity was estimated on human colon cancer HCT116, gastric cancer AGS, squamous carcinoma UM-SCC-6 cells, and human normal fibroblasts with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The amount of apoptotic and dead AGS cells after treatment with yamogenin was estimated with flow cytometry. Also, in yamogenin-treated AGS cells we investigated the reactive oxygen species (ROS) production, mitochondrial membrane depolarization, activity level of caspase-8 and -9, and gene expression at mRNA level with flow cytometry, luminometry, and RT-PCR, respectively. The antioxidant properties of yamogenin were assessed with DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assays. The antimicrobial potential of the compound was estimated on Staphylococcus aureus, Bacillus cereus, Klebsiella pneumoniae, Escherichia coli, Salmonella enterica, Helicobacter pylori, Campylobacter coli, Campylobacter jejuni, Listeria monocytogenes, Lactobacillus paracasei, and Lactobacillus acidophilus bacteria strains. Yamogenin showed the strongest cytotoxic effect on AGS cells (IC50 18.50 ± 1.24 µg/mL) among the tested cell lines. This effect was significantly stronger in combinations of yamogenin with oxaliplatin or capecitabine than for the single compounds. Furthermore, yamogenin induced ROS production, depolarized mitochondrial membrane, and increased the activity level of caspase-8 and -9 in AGS cells. RT-PCR analysis revealed that this sapogenin strongly up-regulated TNFRSF25 expression at the mRNA level. These results indicate that yamogenin induced cell death via the extrinsic and intrinsic way of apoptosis. Antioxidant study showed that yamogenin had moderate in vitro potential (IC50 704.7 ± 5.9 µg/mL in DPPH and 631.09 ± 3.51 µg/mL in ABTS assay) as well as the inhibition of protein denaturation properties (with IC50 1421.92 ± 6.06 µg/mL). Antimicrobial test revealed a weak effect of yamogenin on bacteria strains, the strongest one being against S. aureus (with MIC value of 350 µg/mL). In conclusion, yamogenin may be a potential candidate for the treatment and prevention of gastric cancers.

Chikungunya virus infection in human microglial C20 cells induces mitochondria-mediated apoptosis.

Chikungunya virus (CHIKV) infection is associated with acute clinical manifestations and chronic joint inflammation. CHIKV has emerged as a significant causative agent of central nervous system (CNS) complications, including encephalitis and related sequelae. Microglial cells, crucial for immune responses and tissue repair in the CNS, play a vital role in the host response to viral infections, with their activation potentially leading to either protection or pathology. In this study, the infection biology of CHIKV in the C20 human microglial cell line was investigated. The permissiveness of C20 cells to CHIKV infection was assessed, and viral replication kinetics were compared to Vero E6 cells. Cytopathic effects of CHIKV infection on C20 cells were examined, along with ultrastructural changes using transmission electron microscopy. Additionally, apoptosis induction, mitochondrial membrane potential, and alterations in cell surface marker expression were evaluated by flow cytometry. CHIKV infection demonstrated permissiveness in C20 cells, similar to Vero cells, resulting in robust viral replication and cytopathic effects. Ultrastructural analysis revealed viral replication, mature virion formation, and distinctive cytoplasmic and nuclear changes in infected C20 cells. CHIKV infection induced significant apoptosis in C20 cells, accompanied by mitochondrial membrane depolarization and altered expression of cell surface markers such as CD11c, CD14, and HLA-DR. Notably, decreased CD14 expression was observed in CHIKV-infected C20 cells. The study findings suggest that CHIKV infection induces apoptosis in C20 microglial cells via the mitochondrial pathway, with significant alterations in cell surface marker expression, particularly CD14 that is linked with apoptosis induction. These observations provide valuable insights into the role of human microglial cells in the host response to CHIKV infection and contribute to the knowledge on the neuropathogenesis of this virus.

TSPO deficiency exacerbates acute lung injury via NLRP3 inflammasome-mediated pyroptosis.

Acute respiratory distress syndrome (ARDS) is a common cause of respiratory failure in many critically disease patients. Although inflammasome activation plays an important role in the induction of acute lung injury (ALI) and ARDS, the regulatory mechanism of this process is still unclear. When cells are stimulated by inflammation, the integrity and physiological function of mitochondria play a crucial part in pyroptosis. However, the underlying mechanisms and function of mitochondrial proteins in the process of pyroptosis are largely not yet known. Here, we identified the 18-kDa translocator protein (TSPO), a mitochondrial outer membrane protein, as an important mediator regulating nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation in macrophages during ALI. TSPO gene knockout (KO) and lipopolysaccharide (LPS)-induced ALI/ARDS mouse models were employed to investigate the biological role of TSPO in the pathogenesis of ARDS. Murine macrophages were used to further characterize the effect of TSPO on the NLRP3 inflammasome pathway. Activation of NLRP3 inflammasome through LPS+adenosine triphosphate (ATP) co-stimulation, followed by detection of mitochondrial membrane potential, reactive oxygen species (ROS) production, and cell death was preformed to evaluate the potential biological function of TSPO. Comparisons between two groups were performed with a two-sided unpaired t-test. TSPO-KO mice exhibited more severe pulmonary inflammation in response to LPS-induced ALI. TSPO deficiency resulted in enhanced activation of the NLRP3 inflammasome pathway, promoting more proinflammatory cytokine production of macrophages in LPS-injured lung tissue, including interleukin (IL)-1β, IL-18, and macrophage inflammatory protein (MIP)-2. Mitochondria in TSPO-KO macrophages tended to depolarize in response to cellular stress. The increased production of mitochondrial damage-associated molecular pattern (mtDAMP) led to enhanced mitochondrial membrane depolarization and pyroptosis in TSPO-KO cells. TSPO may be the key regulatory of cellular pyroptosis, it plays a vital protective role in ARDS occurrence and development.

C-terminal region of Rv1039c (PPE15) protein of Mycobacterium tuberculosis targets host mitochondria to induce macrophage apoptosis.

Mycobacterium tuberculosis (Mtb) genome possesses a unique family called Proline-Glutamate/Proline-Proline-Glutamate (PE/PPE) gene family, exclusive to pathogenic mycobacterium. Some of these proteins are known to play role in virulence and immune response modulation, but many are still uncharacterized. This study investigated the role of C-terminal region of Rv1039c (PPE15) in inducing mitochondrial perturbations and macrophage apoptosis. Our in-silico studies revealed the disordered, coiled, and hydrophobic C-terminal region in Rv1039c has similarity with C-terminal of mitochondria-targeting pro-apoptotic host proteins. Wild type Rv1039c and C-terminal deleted Rv1039c (Rv1039c-/-Cterm) recombinant proteins were purified and their M. smegmatis knock-in strains were constructed which were used for in-vitro experiments. Confocal microscopy showed localization of Rv1039c to mitochondria of PMA-differentiated THP1 macrophages; and reduced mitochondrial membrane depolarization and production of mitochondrial superoxides were observed in response to Rv1039c-/-Cterm in comparison to full-length Rv1039c. The C-terminal region of Rv1039c was found to activate caspases 3, 7 and 9 along with upregulated expression of pro-apoptotic genes like Bax and Bim. Rv1039c-/-Cterm also reduced the Cytochrome-C release from the mitochondria and the expression of AnnexinV/PI positive and TUNEL positive cells as compared to Rv1039c. Additionally, Rv1039c was observed to upregulate the TLR4-NF-κB-TNF-α signalling whereas the same was downregulated in response to Rv1039c-/-Cterm. These findings suggested that the C-terminal region of Rv1039c is a molecular mimic of pro-apoptotic host proteins which induce mitochondria-dependent macrophage apoptosis and evoke host immune response. These observations enhance our understanding about the role of PE/PPE proteins at host-pathogen interface.

Advancing apoptosis induction in triple negative breast cancer: Empowering treatment with tyrosine-stapled mixed micelles of lapatinib

The available treatment modalities for triple-negative breast cancer (TNBC) face several challenges, including discouraging overall survival outcomes, unavoidable toxicities, and the emergence of drug resistance. Further, there is an amplified requirement for various amino acids to support the uncontrolled proliferation of TNBC. Hence, targeted therapy for enhancing the treatment of TNBC was explored which encompassed hydrophobized (using α-linolenic acid) Pluronic-based mixed micelles having inherent L-type/Large neutral amino acid transporter 1 targeting (tyrosine-stapled) ability (LPT T-MM). LPT T-MM had optimal size (142.34 ± 30.23 nm), polydispersity index (0.224 ± 0.01), and enhanced drug loading capacity (∼10 %). Transmission electron microscopy confirmed their spherical shape. In vitro release studies showed sustained drug release without hemolysis risk. LPT T-MM had higher cellular uptake and cytotoxicity than free LPT. IC50 values for LPT T-MM were significantly reduced in MDA-MB-231 (∼1.64-fold) and 4 T1 (∼1.88-fold) cell lines; apoptosis index was superior in MDA-MB-231 (∼2.04-fold) and 4 T1 cell line (∼2.09-fold) compared to free LPT. LPT T-MM generated more reactive oxygen species and caused mitochondrial membrane depolarization, indicating increased cell death by apoptosis. Thus, LPT T-MM had the ability to promote apoptosis, showed promise in enhancing the payload capabilities and targeting in TNBC.

Aryl-isoquinoline as a Potential Scaffold for Novel Antitumor Agents against Glioblastoma Cells

Background: Glioblastoma is one of the most aggressive types of tumors, which occurs in the central nervous system, and has a high fatality rate. Among the cellular changes observed in glioblastoma is the overexpression of certain anti-apoptotic proteins, such as Bcl-xL. Recently, the alkaloid sanguinarine (SAN) was identified as a potent inhibitor of this class of proteins. Objective: In this work, the antitumor activity of ten aryl-isoquinolines that were synthesized based on molecular simplification of SAN was investigated. Methods: The SAN derivatives were prepared by Suzuki reaction and bimolecular nucleophilic substitution. The compounds were tested against glioblastoma (U87MG) and melanoma (A375) tumor lines in the MTT and SRB assay. The cell death mechanism was evaluated by flow cytometry. The molecular modeling study was used to evaluate the interactions between the prepared compounds and the Bcl-xL protein. Results: Analogues presented IC50 values against glioblastoma lower than temozolomide. Evaluation against astrocytes and fibroblasts indicated that the analogues were significantly superior to SAN regarding selectivity. The most active compound, 2e, induced phosphatidylserine externalization and mitochondrial membrane depolarization, indicating apoptotic death by the intrinsic pathway. In addition, 2e provides cell cycle arrest at the G2/M phase. Molecular dynamics suggested that 2e interacts with Bcl-xL mainly by hydrophobic interactions. Conclusion: In our study, aryl-isoquinoline represents a relevant scaffold to be explored by medicinal chemists to develop potential anti-glioblastoma agents.

Thioredoxin is a metabolic rheostat controlling regulatory B cells

Metabolic programming is important for B cell fate, but the bioenergetic requirement for regulatory B (Breg) cell differentiation and function is unknown. Here we show that Breg cell differentiation, unlike non-Breg cells, relies on mitochondrial electron transport and homeostatic levels of reactive oxygen species (ROS). Single-cell RNA sequencing analysis revealed that TXN, encoding the metabolic redox protein thioredoxin (Trx), is highly expressed by Breg cells, unlike Trx inhibitor TXNIP which was downregulated. Pharmacological inhibition or gene silencing of TXN resulted in mitochondrial membrane depolarization and increased ROS levels, selectively suppressing Breg cell differentiation and function while favoring pro-inflammatory B cell differentiation. Patients with systemic lupus erythematosus (SLE), characterized by Breg cell deficiencies, present with B cell mitochondrial membrane depolarization, elevated ROS and fewer Trx+ B cells. Exogenous Trx stimulation restored Breg cells and mitochondrial membrane polarization in SLE B cells to healthy B cell levels, indicating Trx insufficiency underlies Breg cell impairment in patients with SLE.

Role of KCNK3 Dysfunction in Dasatinib-associated PAH and Endothelial Cell Dysfunction.

Pulmonary arterial hypertension (PAH) is severe cardiopulmonary disease that may be triggered by exposure to drugs such as dasatinib or facilitated by genetic predispositions. The incidence of dasatinib-associated PAH is estimated at 0.45%, suggesting individual predispositions. The mechanisms of dasatinib-associated PAH are still incomplete. We discovered a KCNK3 gene (coding for outward K+ channel) variant in a patient with dasatinib-associated PAH, and we investigated the impact of this variant on KCNK3 function. Additionally, we assessed the effects of dasatinib exposure on KCNK3 expression. In control-human in pulmonary arterial smooth muscle cells (hPASMCs) and pulmonary endothelial cells (hPECs), we evaluated the consequence of KCNK3 knockdown on cell migration, mitochondrial membrane potential, ATP production, and in vitro tube formation. Using mass spectrometry, we determined the KCNK3 interactome. Patch-clamp revealed that the KCNK3 variant represents a loss-of-function variant. Dasatinib contributed to pulmonary artery constriction by decreasing KCNK3 function and expression. In control-hPASMCs, KCNK3 knockdown promotes mitochondrial membrane depolarization and glycolytic shift. Dasatinib exposure or KCNK3 knockdown reduced the number of caveolae in hPECs. Moreover, KCNK3 knockdown in control-hPECs reduced migration, proliferation, and in vitro tubulogenesis. Using proximity labeling and mass spectrometry, we identified the KCNK3 interactome, revealing that KCNK3 interacts with various proteins across different cellular compartments. We identified a novel pathogenic variant in the KCNK3 and showed that dasatinib downregulates KCNK3, emphasizing the relationship between dasatinib-associated PAH and KCNK3 dysfunction. We demonstrated that loss of KCNK3-dependent signaling contributes to endothelial dysfunction in PAH and glycolytic switch of hPASMCs.

Imeglimin attenuates NLRP3 inflammasome activation by restoring mitochondrial functions in macrophages

Imeglimin is a novel oral antidiabetic drug for treating type 2 diabetes. However, the effect of imeglimin on NLRP3 inflammasome activation has not been investigated yet. Here, we aimed to investigate whether imeglimin reduces LPS-induced NLRP3 inflammasome activation in THP-1 macrophages and examine the associated underlying mechanisms. We analyzed the mRNA and protein expression levels of NLRP3 inflammasome components and IL-1β secretion. Additionally, reactive oxygen species (ROS) generation, mitochondrial membrane potential, and mitochondrial permeability transition pore (mPTP) opening were measured by flow cytometry. Imeglimin inhibited NLRP3 inflammasome-mediated IL-1β production in LPS-stimulated THP-1-derived macrophages. In addition, imeglimin reduced LPS-induced mitochondrial ROS production and mitogen-activated protein kinase phosphorylation. Furthermore, imeglimin restored the mitochondrial function by modulating mitochondrial membrane depolarization and mPTP opening. We demonstrated for the first time that imeglimin reduces LPS-induced NLRP3 inflammasome activation by inhibiting mPTP opening in THP-1 macrophages. These results suggest that imeglimin could be a promising new anti-inflammatory agent for treating diabetic complications.

OMA1 protease eliminates arrested protein import intermediates upon mitochondrial depolarization.

Most mitochondrial proteins originate from the cytosol and require transport into the organelle. Such precursor proteins must be unfolded to pass through translocation channels in mitochondrial membranes. Misfolding of transported proteins can result in their arrest and translocation failure. Arrested proteins block further import, disturbing mitochondrial functions and cellular proteostasis. Cellular responses to translocation failure have been defined in yeast. We developed the cell line-based translocase clogging model to discover molecular mechanisms that resolve failed import events in humans. The mechanism we uncover differs significantly from these described in fungi, where ATPase-driven extraction of blocked protein is directly coupled with proteasomal processing. We found human cells to rely primarily on mitochondrial factors to clear translocation channel blockage. The mitochondrial membrane depolarization triggered proteolytic cleavage of the stalled protein, which involved mitochondrial protease OMA1. The cleavage allowed releasing the protein fragment that blocked the translocase. The released fragment was further cleared in the cytosol by VCP/p97 and the proteasome.

Novel hybrid compounds of sclareol and doxorubicin as potential anticancer nanotherapy for glioblastoma

Two novel hybrid compounds, CON1 and CON2, have been developed by combining sclareol (SC) and doxorubicin (DOX) into a single molecular entity. These hybrid compounds have a 1:1 molar ratio of covalently linked SC and DOX. They have demonstrated promising anticancer properties, especially in glioblastoma cells, and have also shown potential in treating multidrug-resistant (MDR) cancer cells that express the P-glycoprotein (P-gp) membrane transporter. CON1 and CON2 form nanoparticles, as confirmed by Zetasizer, transmission electron microscopy (TEM), and chemical modeling. TEM also showed that CON1 and CON2 can be found in glioblastoma cells, specifically in the cytoplasm, different organelles, nucleus, and nucleolus. To examine the anticancer properties, the U87 glioblastoma cell line, and its corresponding multidrug-resistant U87-TxR cell line, as well as patient-derived astrocytoma grade 3 cells (ASC), were used, while normal human lung fibroblasts were used to determine the selectivity. CON1 and CON2 exhibited better resistance and selectivity profiles than DOX, showing less cytotoxicity, as evidenced by real-time cell analysis, DNA damage determination, cell death induction, mitochondrial respiration, and mitochondrial membrane depolarization studies. Cell cycle analysis and the β-galactosidase activity assay suggested that glioblastoma cells die by senescence following CON1 treatment. Overall, CON1 and CON2 showed great potential as they have better anticancer features than DOX. They are promising candidates for additional preclinical and clinical studies on glioblastoma.

Silver nanoparticles stimulate 5-Fluorouracil-induced colorectal cancer cells to kill through the upregulation TRPV1-mediated calcium signaling pathways.

The involvement of the TRP vanilloid 1 (TRPV1)cation channel on the 5-Fluorouracil (5-FU)-caused Ca2+ signals through the activation of the apoptotic signaling pathway and stimulating the mitochondrial Ca2+ and Zn2+ accumulation-induced reactive oxygen species (ROS) productions in several cancer cells, except the colorectal cancer (HT-29) cell line, was recently reported. I aimed to investigate the action of silver nanoparticles (SiNPs) and 5-FU incubations through the activation of TRPV1 on ROS, apoptosis, and cell death in the HT-29 cell line. The cells were divided into four groups: control, SiNP (100 µM for 48 h), 5-FU (25 μM for 24 h), and 5-FU + SiNP. SiNP treatment through TRPV1 activation (via capsaicin) stimulated the oxidant and apoptotic actions of 5-FU in the cells, whereas they were diminished in the cells by the TRPV1 antagonist (capsazepine) treatment. The apoptotic and cell death actions of 5-FU were determined by increasing the propidium iodide/Hoechst rate, caspase-3, -8, and -9 activations, mitochondrial membrane depolarization, lipid peroxidation, and ROS, but decreasing the glutathione and glutathione peroxidase. The increase of cytosolic free Ca2+ and Zn2+ into mitochondria via the stimulation of TRPV1 current density increased oxidant and apoptotic properties of 5-FU in the cells. For the therapy of HT-29 tumor cells, I found that the combination of SiNPs and 5-FU was synergistic via TRPV1 activation.

Mitochondrial perturbation in immune cells enhances cell-mediated innate immunity in Drosophila

BackgroundMitochondria participate in various cellular processes including energy metabolism, apoptosis, autophagy, production of reactive oxygen species, stress responses, inflammation and immunity. However, the role of mitochondrial metabolism in immune cells and tissues shaping the innate immune responses are not yet fully understood. We investigated the effects of tissue-specific mitochondrial perturbation on the immune responses at the organismal level. Genes for oxidative phosphorylation (OXPHOS) complexes cI-cV were knocked down in the fruit fly Drosophila melanogaster, targeting the two main immune tissues, the fat body and the immune cells (hemocytes).ResultsWhile OXPHOS perturbation in the fat body was detrimental, hemocyte-specific perturbation led to an enhanced immunocompetence. This was accompanied by the formation of melanized hemocyte aggregates (melanotic nodules), a sign of activation of cell-mediated innate immunity. Furthermore, the hemocyte-specific OXPHOS perturbation induced immune activation of hemocytes, resulting in an infection-like hemocyte profile and an enhanced immune response against parasitoid wasp infection. In addition, OXPHOS perturbation in hemocytes resulted in mitochondrial membrane depolarization and upregulation of genes associated with the mitochondrial unfolded protein response.ConclusionsOverall, we show that while the effects of mitochondrial perturbation on immune responses are highly tissue-specific, mild mitochondrial dysfunction can be beneficial in immune-challenged individuals and contributes to variation in infection outcomes among individuals.

STING dependent BAX-IRF3 signaling results in apoptosis during late-stage Coxiella burnetii infection

STING (STimulator of Interferon Genes) is a cytosolic sensor for cyclic dinucleotides (CDNs) and initiates an innate immune response upon binding to CDNs. Coxiella burnetii is a Gram-negative obligate intracellular bacterium and the causative agent of the zoonotic disease Q fever. The ability of C. burnetii to inhibit host cell death is a critical factor in disease development. Previous studies have shown that C. burnetii inhibits host cell apoptosis at early stages of infection. However, during the late-stages of infection, there is host cell lysis resulting in the release of bacteria to infect bystander cells. Thus, we investigated the role of STING during late-stages of C. burnetii infection and examined STING’s impact on host cell death. We show that the loss of STING results in higher bacterial loads and abrogates IFNβ and IL6 induction at 12 days post-infection. The absence of STING during C. burnetii infection significantly reduces apoptosis through decreased caspase-8 and -3 activation. During infection, STING activates IRF3 which interacts with BAX. BAX then translocates to the mitochondria, which is followed by mitochondrial membrane depolarization. This results in increased cytosolic mtDNA in a STING-dependent manner. The presence of increased cytosolic mtDNA results in greater cytosolic 2′-3′ cGAMP, creating a positive feedback loop and leading to further increases in STING activation and its downstream signaling. Taken together, we show that STING signaling is critical for BAX-IRF3-mediated mitochondria-induced apoptosis during late-stage C. burnetii infection.

Unlocking apoptosis in triple negative breast cancer: Harnessing “glutamine trap” to amplify the efficacy of lapatinib-loaded mixed micelles

Certain aggressive cancers, such as triple-negative breast cancer (TNBC), heavily bank on glutamine for their proliferation and survival. In this context, TNBC functions as a “glutamine trap,” extracting circulating glutamine at a rate surpassing that of any other organ. Moreover, the overexpression of Alanine, Serine, Cysteine Transporter 2 (ASCT2), a key player in glutamine uptake, further underscores the significance of targeted therapy to enhance TNBC treatment. This led to the exploration of a novel approach involving hydrophobized Pluronic-based mixed micelles achieved through the use of docosahexaenoic acid and stapled with glutamine for displaying inherent ASCT2 targeting ability—a formulation termed LPT G-MM. LPT G-MM exhibited optimal characteristics, including a size of 163.66 ± 10.34 nm, a polydispersity index of 0.237 ± 0.083, and an enhanced drug loading capacity of approximately 15 %. Transmission electron microscopy validated the spherical shape of these micelles. In vitro release studies demonstrated drug release in a sustained manner without the risk of hemolysis. Importantly, LPT G-MM displayed heightened cellular uptake, increased cytotoxicity, a lower IC50 value, elevated reactive oxygen species, induced mitochondrial membrane depolarization, and a greater apoptosis index in TNBC cell lines compared to free LPT. The pharmacokinetic profile of LPT G-MM revealed a substantial rise in half-life (t1/2) by approximately 1.48-fold and an elevation in the area under the curve [AUC(0→∞)] by approximately 1.19-fold. Moreover, there was a significant reduction in the percentage of tumor volume by approximately 7.26-fold, along with decreased serum toxicity markers compared to free LPT. In summary, LPT G-MM demonstrated promising potential in boosting payload capacities and targeting specificity in the context of TNBC treatment.

Anti-Tumor Efficacy of Oleuropein-Loaded ZnO/Au Mesoporous Silica Nanoparticle in 5-FU-Resistant Colorectal Cancer Cells.

5-fluorouracil (5-FU) is a first-line chemotherapeutic agent used to treat colorectal cancer (CRC). However, 5-FU induces drug resistance and activation of cancer stem cells (CSCs). In the present study, we designed a novel biocompatible nanomedicine system with high efficacy and biocompatibility by synthesizing mesoporous silica nanoparticle (MSN)-structured ZnO and gold ions. Oleuropein (OLP) is a phenolic compound derived from olive leaves that exerts anti-cancer effects. Therefore, we synthesized OLP-loaded ZnO/Au MSNs (ZnO/Au/OLP MSNs) and examined their anti-cancer effects on 5-FU-resistant CRC cells. ZnO/Au MSNs were synthesized and functionalized, and their physical and chemical compositions were characterized using UV-visible spectroscopy, dynamic light scattering, and transmission electron microscopy (TEM). Their effects were assessed in terms of cellular proliferation capacity, migration and invasion ability, colony-forming ability, spheroid-forming ability, reactive oxygen species (ROS) production, and mitochondrial membrane depolarization. ZnO/Au MSNs were mostly composed of various ions containing ZnO and gold ions, had a spheroid phenotype, and exhibited no cytotoxicity. ZnO/Au/OLP MSNs reduced cell viability and CSC formation and induced apoptosis of 5-FU-resistant CRC cells via necrosis via ROS accumulation and DNA fragmentation. ZnO/Au/OLP MSNs exhibited anti-cancer activity by upregulating necrosis. These results revealed that ZnO/Au/OLP MSNs are a novel drug delivery system for 5-FU CRC therapy.