Mitochondrial 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.

Ionic reverberation modulates the cellular fate of CD8+tissue resident memory T cells (TRMs) in patients with renal cell carcinoma: A novel mechanism

In metastatic renal cell carcinoma (mRCC), existing treatments including checkpoint inhibitors are failed to cure and/or prevent recurrence of the disease. Therefore, in-depth understanding of tumor tissue resident memory T cells (TRMs) dysfunction are necessitated to enrich efficacy of immunotherapies and increasing disease free survival in treated patients. In patients, we observed dysregulation of K+, Ca2+, Na2+ and Zn2+ ion channels leads to excess infiltration of their respective ions in tumor TRMs, thus ionic gradients are disturbed and cells became hyperpolarized. Moreover, overloaded intramitochondrial calcium caused mitochondrial depolarization and trigger apoptosis of tumor TRMs. Decreased prevalence of activated tumor TRMs reflected our observations. Furthermore, disruptions in ionic concentrations impaired the functional activities and/or suppressed anti-tumor action of circulating and tumor TRMs in RCC. Collectively, these findings revealed novel mechanism behind dysfunctionality of tumor TRMs. Implicating enrichment of activated TRMs within tumor would be beneficial for better management of RCC patients.

Neurotransmitter accumulation and Parkinson's disease-like phenotype caused by anion channelrhodopsin opto-controlled astrocytic mitochondrial depolarization in substantia nigra pars compacta.

Parkinson's disease (PD) is a mitochondria-related neurodegenerative disease characterized by locomotor deficits and loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc). Majority of PD research primarily focused on neuronal dysfunction, while the roles of astrocytes and their mitochondria remain largely unexplored. To bridge the gap and investigate the roles of astrocytic mitochondria in PD progression, we constructed a specialized optogenetic tool, mitochondrial-targeted anion channelrhodopsin, to manipulate mitochondrial membrane potential in astrocytes. Utilizing this tool, the depolarization of astrocytic mitochondria within the SNc in vivo led to the accumulation of γ-aminobutyric acid (GABA) and glutamate in SNc, subsequently resulting in excitatory/inhibitory imbalance and locomotor deficits. Consequently, in vivo calcium imaging and interventions of neurotransmitter antagonists demonstrated that GABA accumulation mediated movement deficits of mice. Furthermore, 1h/day intermittent astrocytic mitochondrial depolarization for 2 weeks triggered spontaneous locomotor dysfunction, α-synuclein aggregation, and the loss of DA neurons, suggesting that astrocytic mitochondrial depolarization was sufficient to induce a PD-like phenotype. In summary, our findings suggest the maintenance of proper astrocytic mitochondrial function and the reinstatement of a balanced neurotransmitter profile may provide a new angle for mitigating neuronal dysfunction during the initial phases of PD.

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.

A novel sunflower-like nanocarrier based on dual milk-derived proteins for improved bio-accessibility, stability and antioxidant activity of anthocyanin

Anthocyanin (ACN), as a hydrophilic compound, has excellent antioxidant and anti-inflammatory biological activities, but its poor stability and low bioavailability limit widespread application in the food industry. In the current study, a novel nanocarrier based “one stone for multiple birds” response strategy was designed to enhance the in vitro bio-accessibility, targeting capabilities, and antioxidant activity of ACN. The forementioned strategy entailed the utilization of triphenylphosphonium bromide (TPP)-modified dual-natural milk-derived proteins (WPCA)-fucoidan (FD) conjugates as a wall material via an amide reaction. Subsequently, ACN-loaded nanocarriers were prepared by assembly assisted with ultrasound method through hydrogen bonding, ionic complexation, and hydrophobic interaction. Transmission electron microscopy revealed that the nanocarriers exhibited a sunflower-like shape with a size of around 936 nm (7 mg/mL of ACN). Exhilaratingly, a sunflower-like nanocarrier (SNC) achieved dual pH- and redox-responsive release in the presence of 10 mM of glutathione (GSH) at pH 7.4, exhibiting intestine-responsive release behavior, high bio-accessibility, environmental stability, colon-targeting safety. Meanwhile, the nanocarrier demonstrated superior targeting of mitochondria, as evidenced by a Pearson's correlation coefficient of 0.80 at 6 h. In vitro cell experiments showed that the ACN-loaded SNC significantly effectively suppressed the generation of ROS and mitochondrial depolarization caused by oxidative stress. These findings hold significant importance in the potential ability of the anthocyanin-loaded natural bioactive nanocarriers, particularly and can potentially mitigate exogenous oxidative stress effectively.

MiRNA Expression Profiles in Isolated Ventricular Cardiomyocytes: Insights into Doxorubicin-Induced Cardiotoxicity.

Doxorubicin (DOX), widely used as a chemotherapeutic agent for various cancers, is limited in its clinical utility by its cardiotoxic effects. Despite its widespread use, the precise mechanisms underlying DOX-induced cardiotoxicity at the cellular and molecular levels remain unclear, hindering the development of preventive and early detection strategies. To characterize the cytotoxic effects of DOX on isolated ventricular cardiomyocytes, focusing on the expression of specific microRNAs (miRNAs) and their molecular targets associated with endogenous cardioprotective mechanisms such as the ATP-sensitive potassium channel (KATP), Sirtuin 1 (SIRT1), FOXO1, and GSK3β. We isolated Guinea pig ventricular cardiomyocytes by retrograde perfusion and enzymatic dissociation. We assessed cell morphology, Reactive Oxygen Species (ROS) levels, intracellular calcium, and mitochondrial membrane potential using light microscopy and specific probes. We determined the miRNA expression profile using small RNAseq and validated it using stem-loop qRT-PCR. We quantified mRNA levels of some predicted and validated molecular targets using qRT-PCR and analyzed protein expression using Western blot. Exposure to 10 µM DOX resulted in cardiomyocyte shortening, increased ROS and intracellular calcium levels, mitochondrial membrane potential depolarization, and changes in specific miRNA expression. Additionally, we observed the differential expression of KATP subunits (ABCC9, KCNJ8, and KCNJ11), FOXO1, SIRT1, and GSK3β molecules associated with endogenous cardioprotective mechanisms. Supported by miRNA gene regulatory networks and functional enrichment analysis, these findings suggest that DOX-induced cardiotoxicity disrupts biological processes associated with cardioprotective mechanisms. Further research must clarify their specific molecular changes in DOX-induced cardiac dysfunction and investigate their diagnostic biomarkers and therapeutic potential.

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.

A RAB7A phosphoswitch coordinates Rubicon Homology protein regulation of Parkin-dependent mitophagy.

Activation of PINK1 and Parkin in response to mitochondrial damage initiates a response that includes phosphorylation of RAB7A at Ser72. Rubicon is a RAB7A binding negative regulator of autophagy. The structure of the Rubicon:RAB7A complex suggests that phosphorylation of RAB7A at Ser72 would block Rubicon binding. Indeed, in vitro phosphorylation of RAB7A by TBK1 abrogates Rubicon:RAB7A binding. Pacer, a positive regulator of autophagy, has an RH domain with a basic triad predicted to bind an introduced phosphate. Consistent with this, Pacer-RH binds to phosho-RAB7A but not to unphosphorylated RAB7A. In cells, mitochondrial depolarization reduces Rubicon:RAB7A colocalization whilst recruiting Pacer to phospho-RAB7A-positive puncta. Pacer knockout reduces Parkin mitophagy with little effect on bulk autophagy or Parkin-independent mitophagy. Rescue of Parkin-dependent mitophagy requires the intact pRAB7A phosphate-binding basic triad of Pacer. Together these structural and functional data support a model in which the TBK1-dependent phosphorylation of RAB7A serves as a switch, promoting mitophagy by relieving Rubicon inhibition and favoring Pacer activation.

Caffeic acid phenethyl ester restores mitochondrial homeostasis against peritoneal fibrosis induced by peritoneal dialysis through the AMPK/SIRT1 pathway

Increasing evidence suggests that peritoneal fibrosis induced by peritoneal dialysis (PD) is linked to oxidative stress. However, there are currently no effective interventions for peritoneal fibrosis. In the present study, we explored whether adding caffeic acid phenethyl ester (CAPE) to peritoneal dialysis fluid (PDF) improved peritoneal fibrosis caused by PD and explored the molecular mechanism. We established a peritoneal fibrosis model in Sprague–Dawley rats through intraperitoneal injection of PDF and lipopolysaccharide (LPS). Rats in the PD group showed increased peritoneal thickness, submesothelial collagen deposition, and the expression of TGFβ1 and α-SMA. Adding CAPE to PDF significantly inhibited PD-induced submesothelial thickening, reduced TGFβ1 and α-SMA expression, alleviated peritoneal fibrosis, and improved the peritoneal ultrafiltration function. In vitro, peritoneal mesothelial cells (PMCs) treated with PDF showed inhibition of the AMPK/SIRT1 pathway, mitochondrial membrane potential depolarization, overproduction of mitochondrial reactive oxygen species (ROS), decreased ATP synthesis, and induction of mesothelial-mesenchymal transition (MMT). CAPE activated the AMPK/SIRT1 pathway, thereby inhibiting mitochondrial membrane potential depolarization, reducing mitochondrial ROS generation, and maintaining ATP synthesis. However, the beneficial effects of CAPE were counteracted by an AMPK inhibitor and siSIRT1. Our results suggest that CAPE maintains mitochondrial homeostasis by upregulating the AMPK/SIRT1 pathway, which alleviates oxidative stress and MMT, thereby mitigating the damage to the peritoneal structure and function caused by PD. These findings suggest that adding CAPE to PDF may prevent and treat peritoneal fibrosis.

Folic acid-tripeptide-conjugated synthetic biodegradable nanoparticle-loaded with Ormeloxifene potentially inhibited breast cancer xenograft tumor

ObjectivesOrmeloxifene (OLF) is an estrogen receptor modulator used in breast cancer treatment with highly favorable pharmacodynamics and pharmacokinetic properties. Folic acid (FA) receptor overexpresses in breast cancer cells. Hence, the study was intended to evaluate the therapeutic potential of OLF-encapsulated FA-peptide-conjugated nanoparticles (FA-Pep-OLF-NP) for breast cancer treatment. MethodsWe prepared folic acid tripeptide conjugated OLF-encapsulated PLGA nanoparticles. The surface conjugation of folic acid-tripeptide was assessed by FTIR and XPS analysis. Selectivity, cytotoxicity, apoptosis, mitochondrial depolarization, reactive oxygen species, and cell cycle analysis of the experimental nanoparticles were tested on human breast cancer cells, MDA-MB-231 and MCF-7. The therapeutic efficacy of FA-Pep-OLF-NP and unconjugated nanoparticle OLF-NP was compared in the MDA-MB-231-xenograft tumor animal model. ResultsOLF-NP and FA-Pep-OLF-NP exhibited 4.8 % and 4.5 % w/w of drug loading, respectively. The particles were spherical with a smooth surface and uniform size distribution. FA-Pep-OLF-NP revealed higher cytotoxicity, mitochondrial depolarization, and apoptosis potential in MDA-MB-231 cells compared to MCF-7 cells. Tumor uptake and tumor volume reduction by FA-Pep-OLF-NP and OLF-NP were established in MDA-MB-231 tumor-bearing nude mice. ConclusionFA-Pep-OLF-NP showed preferential delivery of OLF to the breast cancer cells and in breast tumors compared to OLF-NP and showed more drug accumulation in breast cancer xenograft models. FA-Pep-OLF-NP delayed the progress of folate receptor overexpressed breast cancer.

The toxic effects induced by benzethonium chloride on Daphnia carinata over two generations: Resistance and higher sublethal toxicity

With the widespread utilization of the sanitizing product benzethonium chloride (BEC) throughout the coronavirus pandemic, concerns have emerged regarding its potential hazards. Nevertheless, the long-term and multigenerational toxic effects of BEC on aquatic organisms remains unexplored. This study investigates acute and chronic toxicity, oxidative stress, mitochondrial membrane potential, ATP concentrations, and gene expression using Daphnia carinata as the model organism. Meanwhile, hierarchical clustering analysis was utilized to investigate phenotypic effects among different treatment groups. The integrated biomarker response index version 2 (IBRv2) was employed to estimate the deviation in toxic effects over two generations. These results indicated that D. carinata in the second generation exhibited higher survival rate and lower levels of oxidative stress than the first generation. However, the higher sublethal effects were found in the second generation as follows, the weakened growth performance, mitochondrial membrane potential depolarization, reduced ATP concentrations, and down-regulated gene expression. The mitochondrial toxicity induced by BEC may account for the distinct toxic effects exhibited in two generations. The findings here can assist with the evaluation of potential risk for BEC on aquatic organisms, and provide new insight into the cross-generational toxicity mechanisms of pollutants in aquatic ecosystems.

Essential oil of oregano (Origanum vulgare L.) reduces infection and proliferation of Toxoplasma gondii in BeWo cells with induction of autophagy and death of tachyzoites through a mechanism similar to necrosis.

Toxoplasmosis poses a global health threat, ranging from asymptomatic cases to severe, potentially fatal manifestations, especially in immunocompromised individuals and congenital transmission. Prior research suggests that oregano essential oil (OEO) exhibits diverse biological effects, including antiparasitic activity against Toxoplasma gondii. Given concerns about current treatments, exploring new compounds is important. This study was to assess the toxicity of OEO on BeWo cells and T. gondii tachyzoites, as well as to evaluate its effectiveness in in vitro infection models and determine its direct action on free tachyzoites. OEO toxicity on BeWo cells and T. gondii tachyzoites was assessed by MTT and trypan blue methods, determining cytotoxic concentration (CC50), inhibitory concentration (IC50), and selectivity index (SI). Infection and proliferation indices were analyzed. Direct assessments of the parasite included reactive oxygen species (ROS) levels, mitochondrial membrane potential, necrosis, and apoptosis, as well as electron microscopy. Oregano oil exhibited low cytotoxicity on BeWo cells (CC50: 114.8µg/mL ± 0.01) and reduced parasite viability (IC50 12.5 ± 0.06µg/mL), demonstrating 9.18 times greater selectivity for parasites than BeWo cells. OEO treatment significantly decreased intracellular proliferation in infected cells by 84% after 24h with 50μg/mL. Mechanistic investigations revealed increased ROS levels, mitochondrial depolarization, and lipid droplet formation, linked to autophagy induction and plasma membrane permeabilization. These alterations, observed through electron microscopy, suggested a necrotic process confirmed by propidium iodide labeling. OEO treatment demonstrated anti-T. gondii action through cellular and metabolic change while maintaining low toxicity to trophoblastic cells.

A new model of ischemia/reperfusion injury using precision-cut liver slices

Background: Primary cells isolated from the liver are widely used to investigate hepatic pathophysiology. However, these cells do not represent cell-to-cell interactions, three dimensional architecture, and zonal structure in the liver. Furthermore, characterization of cellular and molecular events during ischemia/reperfusion (I/R) is challenging in liver biopsies. Thus, a new model of I/R injury resembling the native liver is needed. Methods: We generated precision-cut liver slices (PCLS) from mouse and human livers using a Krumdieck automatic tissue slicer (Alabama R & D, Munford, AL). Thickness of individual PLCL was 200 microns. To simulate anoxia, nutrient depletion, and tissue acidosis during ischemia, PCLS were incubated in Krebs-Ringer-N-(2-Hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) (HEPES) solution at pH 6.2 in the anaerobic chamber (Coy Laboratory, Grass Lake, MI) for 30 min. To simulate normoxia, nutrient repletion, and restoration to normal pH during reperfusion, ischemic PCLS were aerobically incubated in Dulbecco's modified eagle medium (DMEM) at pH 7.4 for up to 4 h. Changes in tissue viability were assessed with the release of lactate dehydrogenase (LDH) into the extracellular medium. Some PCLS were labeled with green fluorescent rhodamine-123 (Rd-123, mitochondrial membrane potential indicator) and red fluorescent propidium iodide (PI, necrosis marker). Confocal images of PCLS were collected after I/R. Results: LDH release assay revealed that while PCLS from chow diet (CD)-fed lean mice tolerated I/R injury well, those from high fat diet (HFD)-fed steatotic mice were susceptible to I/R injury. Confocal microscopy showed substantial mitochondrial depolarization (loss of Rd-123) and necrosis (nuclear labeling of PI) in the HFD group, which was, however, minimally observed in the CD group. Of importance, mitochondrial dysfunction and cell death after reperfusion occurred predominantly in hepatocytes. Heightened I/R injury in steatotic livers was also confirmed in primary hepatocytes isolated from either CD- or HFD-fed mice. To compare the similarities and differences between mouse and human livers, PCLS were generated from discarded human livers deemed unsuitable for transplant and subjected to 30 min of in vitro ischemia. The loss of mitochondrial membrane potential and hepatocyte death progressively increased after I/R in discarded human livers. At 1 h after reperfusion, diffusive fluorescence of Rd-123 and nuclear labeling of PI became evident, indicative of mitochondrial dysfunction and tissue injury after I/R. SUMMARY: LDH assay and confocal analysis of PCLS demonstrated heightened susceptibility of steatotic livers to I/R injury. In contrast to PCLS from lean livers, those from steatotic livers rapidly developed mitochondrial depolarization and onset of necrosis, cardinal features of I/R injury, even after short ischemia. The early phase of reperfusion injury in steatotic livers occurred predominantly in parenchymal cells. CONCLUSION: In vitro I/R with PCLS is a model suitable for hepatic I/R injury. This work (J-S K) was funded by the National Institutes of Health (DK079878), Mid-America Transplant Foundation (07201903), Foundation for Barnes-Jewish Hospital (4776, 5153), and Washington University Department of Surgery Pilot Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Female Sex Protects Cerebral Arteries from Mitochondrial Membrane Potential Depolarization and Cell Death Induced by Reactive Oxygen Species

Stroke and traumatic brain injury augment the production of reactive oxygen species (ROS) leading to apoptosis in cerebral arteries, and females are more resilient to vascular damage than males. Depolarization of mitochondrial membrane potential (ΔΨm) is a key event in apoptosis, however, when ΔΨm is suffciently depolarized, ATP synthase can reverse directions and act as a proton pump to limit ΔΨm depolarization. We hypothesized that ATP synthase attenuates depolarization of ΔΨm in cerebral arteries during acute oxidative stress thus limiting cell death. Posterior cerebral arteries (PCA; ~80 μm diameter) from male and female mice (age, 4-6 mo) were isolated, cannulated, and pressurized to 90 cm H2O at 36°C. Cell death was quantified with Hoechst 33342 (1 μM, labels all nuclei) and propidium iodide (2 μM, labels dead nuclei), and ΔΨm was evaluated with tetramethylrhodamine methyl ester (TMRM, 10 nM). Vessels were exposed to H2O2 (200 μM) in the presence/absence of ATP synthase inhibitor oligomycin (2 μM). H2O2 exposure (50 min) led to significantly (p<0.05) greater smooth muscle cell death in males compared to females (30±7.4% vs. 7±3%; n=8); there was a similar trend for endothelial cell death. Oligomycin greatly augmented apoptosis in PCAs from both males and females to ~80% and eliminated differences between sexes. Consistently, H2O2 evoked a more robust depolarization of ΔΨm in males vs. females and oligomycin enhanced ΔΨm depolarization to H2O2 leading to a similar response in both sexes. To directly depolarize ΔΨm, PCAs were treated with the protonophore FCCP (10 μM). Surprisingly, depolarization of ΔΨm to FCCP was greater in males vs. females, and oligomycin augmented depolarization and eliminated differences between sexes. FCCP initiated minimal (<5%) cell death at 50-min or 3-h exposure; oligomycin did not alter cell death to FCCP. We conclude that: 1) cerebral vessels from female mice possess greater resilience to H2O2 -induced apoptosis than males by limiting depolarization of ΔΨm; and 2) oxidative stress triggers cell death through pathways in addition to ΔΨm depolarization. Support: NIH R01NS134690. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

Emetine induces oxidative stress, cell differentiation and NF-κB inhibition, suppressing AML stem/progenitor cells

Acute myeloid leukemia (AML) is a fatal malignancy of the blood and bone marrow. Leukemic stem cells (LSCs) are a rare subset of leukemic cells that promote the development and progression of AML, and eradication of LSCs is critical for effective control of this disease. Emetine is an FDA-approved antiparasitic drug with antitumor properties; however, little is known about its potential against LSCs. Herein, we explored the antileukemic potential of emetine, focusing on its effects on AML stem/progenitor cells. Emetine exhibited potent cytotoxic activity both in hematologic and solid cancer cells and induced AML cell differentiation. Emetine also inhibited AML stem/progenitor cells, as evidenced by decreased expression of CD34, CD97, CD99, and CD123 in KG-1a cells, indicating anti-AML stem/progenitor cell activities. The administration of emetine at a dosage of 10 mg/kg for two weeks showed no significant toxicity and significantly reduced xenograft leukemic growth in vivo. NF-κB activation was reduced in emetine-treated KG-1a cells, as shown by reduced phospho-NF-κB p65 (S529) and nuclear NF-κB p65. DNA fragmentation, YO-PRO-1 staining, mitochondrial depolarization and increased levels of active caspase-3 and cleaved PARP (Asp214) were detected in emetine-treated KG-1a cells. Moreover, treatment with the pancaspase inhibitor Z-VAD(OMe)-FMK partially prevented the apoptotic cell death induced by emetine. Emetine treatment also increased cellular and mitochondrial reactive oxygen species, and emetine-induced apoptosis in KG-1a cells was partially prevented by the antioxidant N-acetylcysteine, indicating that emetine induces apoptosis, at least in part, by inducing oxidative stress. Overall, these studies indicate that emetine is a novel potential anti-AML agent with promising activity against stem/progenitor cells, encouraging the development of further studies aimed at its clinical application.

ONC212 enhances YM155 cytotoxicity by triggering SLC35F2 expression and NOXA-dependent MCL1 degradation in acute myeloid leukemia cells

Although the anticancer activity of ONC212 has been reported, the precise mechanism underlying its apoptotic effects remains unclear. In this study, we investigated the apoptotic mechanism of ONC212 in acute myeloid leukemia (AML) cells. ONC212 induces apoptosis, MCL1 downregulation, and mitochondrial depolarization in AML U937 cells. Ectopic MCL1 expression alleviates mitochondria-mediated apoptosis in ONC212-treated U937 cells. ONC212 triggers AKT phosphorylation, inducing NOX4-dependent ROS production and promoting HuR transcription. HuR-mediated ATF4 mRNA stabilization stimulates NOXA and SLC35F2 expression; ONC212-induced upregulation of NOXA leads to MCL1 degradation. The synergistic effect of ONC212 on YM155 cytotoxicity was dependent on increased SLC35F2 expression. In addition, YM155 feedback facilitated the activation of the ONC212-induced signaling pathway. A similar mechanism explains ONC212- and ONC212/YM155-induced AML HL-60 cell death. The continuous treatment of U937 cells with the benzene metabolite hydroquinone (HQ) generated U937/HQ cells, exhibiting enhanced responsiveness to the cytotoxic effects of ONC212. In U937/HQ cells, ONC212 triggered apoptosis through NOXA-mediated MCL1 downregulation, enhancing YM155 cytotoxicity. Collectively, our data suggested that ONC212 upregulated SLC35F2 expression and triggered NOXA-mediated MCL1 degradation in U937, U937/HQ, and HL-60 cells by activating the AKT/NOX4/HuR/ATF4 pathway. The ONC212-induced signaling pathway showed anti-AML activity and enhanced YM155 cytotoxicity.

Mitochondrial responses to constant and cyclic hypoxia depend on the oxidized fuel in a hypoxia-tolerant marine bivalve Crassostrea gigas

Sessile benthic organisms like oysters inhabit the intertidal zone, subject to alternating hypoxia and reoxygenation (H/R) episodes during tidal movements, impacting respiratory chain activities and metabolome compositions. We investigated the effects of constant severe hypoxia (90 min at ~ 0% O2 ) followed by 10 min reoxygenation, and cyclic hypoxia (5 cycles of 15 min at ~ 0% O2 and 10 min reoxygenation) on isolated mitochondria from the gill and the digestive gland of Crassostrea gigas respiring on pyruvate, palmitate, or succinate. Constant hypoxia suppressed oxidative phosphorylation (OXPHOS), particularly during Complex I-linked substrates oxidation. It had no effect on mitochondrial reactive oxygen species (ROS) efflux but increased fractional electron leak (FEL). In mitochondria oxidizing Complex I substrates, exposure to cyclic hypoxia prompted a significant drop after the first H/R cycle. In contrast, succinate-driven respiration only showed significant decline after the third to fifth H/R cycle. ROS efflux saw little change during cyclic hypoxia regardless of the oxidized substrate, but Complex I-driven FEL tended to increase with each subsequent H/R cycle. These observations suggest that succinate may serve as a beneficial stress fuel under H/R conditions, aiding in the post-hypoxic recovery of oysters by reducing oxidative stress and facilitating rapid ATP re-synthesis. The impacts of constant and cyclic hypoxia of similar duration on mitochondrial respiration and oxidative lesions in the proteins were comparable indicating that the mitochondrial damage is mostly determined by the lack of oxygen and mitochondrial depolarization. The ROS efflux in the mitochondria of oysters was minimally affected by oxygen fluctuations indicating that tight regulation of ROS production may contribute to robust mitochondrial phenotype of oysters and protect against H/R induced stress.

Hydroxytyrosol acetate from olive leaves (Olea Europaea L.) induces apoptosis via mitochondrial pathway in BEL7402 cell line

Hydroxytyrosol acetate is one of the polyphenolic compounds in olive leaves. Hydroxytyrosol acetate has a variety of biological activities, such as antibacterial, antioxidant, anti-inflammatory, cognitive improvement and neuroprotective effects. However, there is no report on the antitumor activity and the antitumor mechanism of hydroxytyrosol acetate. In our study, we studied the antitumor activity of hydroxytyrosol acetate by MTT assay and determined the antitumor mechanism by DNA ladder assay, mitochondrial membrane potential assay and western blot assay. We found that hydroxytyrosol acetate could inhibit cell proliferation, and the inhibition rate was 78.08%. The further researches showed that hydroxytyrosol acetate could downregulate Bcl-2 protein while upregulate Bax protein. It also could induce mitochondrial depolarisation and release of cytochrome C. These results indicated that hydroxytyrosol acetate might induce BEL7402 cells apoptosis via mitochondrial pathway.

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.