Dissipation Of Membrane Potential Research Articles

8381 The Pro-atrophic Effect of Vitamin D Binding Protein in Skeletal Muscle and Its Involvement in Cancer-Induced Muscle Wasting

Abstract Disclosure: N. Filigheddu: None. T. Raiteri: None. S. Reano: None. A. Scircoli: None. A. Antonioli: None. F. Prodam: None. Vitamin D binding protein (VDBP), encoded by the Gc gene, is a multifunctional serum glycoprotein synthesized by hepatocytes, whose primary function is the transport of vitamin D metabolites in the bloodstream. In addition, VDBP enhances the chemotactic activity of neutrophil chemoattractants and takes part in the actin-scavenger system by acting as a monomeric G-actin-binding protein. Several studies have reported a correlation between increased levels of VDBP and various pathologies often associated with muscle wasting, including different types of tumors. Given these findings, we hypothesized that VDBP may play a role in skeletal muscle homeostasis.In vitro, treatment of C2C12 myotubes with 100 mM VDBP for 24 h induced the perturbation of intracellular actin dynamics due to VDBP's ability to bind G-actin that, in turn, led to mitochondrial dysfunction (i.e., mitochondrial membrane potential dissipation, respiratory impairment, increased ROS production, induction of the fission machinery), exacerbation of autophagy, and, eventually, atrophy, seen as the reduction of myotube diameter. Remarkably, pharmacological intervention on myotubes with jasplakinolide (250 mM, 30 min pre-treatment) to counteract VDBP effects on intracellular actin dynamics was sufficient to prevent VDBP-induced atrophy. To assess if VDBP had a causative role in muscle atrophy in vivo, we injected VDBP (1.5 mg/Kg) every 48 h for one week in the tibialis anterior muscles of Gc knock-out mice (VDBP KO). At the end of the experimental period, we observed the induction of the mitophagic gene Bnip3, impairment of muscle performances (26% reduction of grip strength at the endpoint), and a 16% reduction of muscle mass compared to the saline-injected contralateral muscles, confirming the atrophic effect of VDBP in vivo.Coherently with the upregulation of VDBP observed in cancer patients, VDBP levels also increase in murine models of cancer cachexia. To test the hypothesis that VDBP could play a role in cancer-associated muscle wasting, we induced cancer cachexia in VDBP KO mice by inoculating 106 Lewis Lung Carcinoma (LLC) cells resuspended in 100 μl of saline on the back of mice. Tumor-bearing VDBP KO mice preserved their body weight and performances, and the muscle loss was lessened by more than 50% compared to cachectic WT mice. Notably, between the two groups, there were no differences in tumor growth or food intake, ruling out the possibility that the reduction in muscle wasting could depend on smaller tumors or differential development of anorexia in the two genotypes. In conclusion, we demonstrated that VDBP acts as a hormone per se, having a direct pro-atrophic activity on skeletal muscle. Our data suggest that VDBP could represent a potential therapeutic target to treat cancer cachexia and other pathologies in which the rise of VDBP could impinge muscle mass and functionality. Presentation: 6/1/2024

9268 The Pro-atrophic Effect of Vitamin D Binding Protein in Skeletal Muscle and Its Involvement in Cancer-Induced Muscle Wasting

Abstract Disclosure: N. Filigheddu: None. T. Raiteri: None. S. Reano: None. A. Scircoli: None. A. Antonioli: None. F. Prodam: None. Vitamin D binding protein (VDBP), encoded by the Gc gene, is a multifunctional serum glycoprotein synthesized by hepatocytes, whose primary function is the transport of vitamin D metabolites in the bloodstream. In addition, VDBP enhances the chemotactic activity of neutrophil chemoattractants and takes part in the actin-scavenger system by acting as a monomeric G-actin-binding protein. Several studies have reported a correlation between increased levels of VDBP and various pathologies often associated with muscle wasting, including different types of tumors. Given these findings, we hypothesized that VDBP may play a role in skeletal muscle homeostasis.In vitro, treatment of C2C12 myotubes with 100 mM VDBP for 24 h induced the perturbation of intracellular actin dynamics due to VDBP's ability to bind G-actin that, in turn, led to mitochondrial dysfunction (i.e., mitochondrial membrane potential dissipation, respiratory impairment, increased ROS production, induction of the fission machinery), exacerbation of autophagy, and, eventually, atrophy, seen as the reduction of myotube diameter. Remarkably, pharmacological intervention on myotubes with jasplakinolide (250 mM, 30 min pre-treatment) to counteract VDBP effects on intracellular actin dynamics was sufficient to prevent VDBP-induced atrophy. To assess if VDBP had a causative role in muscle atrophy in vivo, we injected VDBP (1.5 mg/Kg) every 48 h for one week in the tibialis anterior muscles of Gc knock-out mice (VDBP KO). At the end of the experimental period, we observed the induction of the mitophagic gene Bnip3, impairment of muscle performances (26% reduction of grip strength at the endpoint), and a 16% reduction of muscle mass compared to the saline-injected contralateral muscles, confirming the atrophic effect of VDBP in vivo.Coherently with the upregulation of VDBP observed in cancer patients, VDBP levels also increase in murine models of cancer cachexia. To test the hypothesis that VDBP could play a role in cancer-associated muscle wasting, we induced cancer cachexia in VDBP KO mice by inoculating 106 Lewis Lung Carcinoma (LLC) cells resuspended in 100 μl of saline on the back of mice. Tumor-bearing VDBP KO mice preserved their body weight and performances, and the muscle loss was lessened by more than 50% compared to cachectic WT mice. Notably, between the two groups, there were no differences in tumor growth or food intake, ruling out the possibility that the reduction in muscle wasting could depend on smaller tumors or differential development of anorexia in the two genotypes. In conclusion, we demonstrated that VDBP acts as a hormone per se, having a direct pro-atrophic activity on skeletal muscle. Our data suggest that VDBP could represent a potential therapeutic target to treat cancer cachexia and other pathologies in which the rise of VDBP could impinge muscle mass and functionality. Presentation: 6/1/2024

New Mitochondria-Targeted Fisetin Derivative Compromises Mitophagy and Limits Survival of Drug-Induced Senescent Breast Cancer Cells.

Mitochondria are considered as promising targets for cancer treatment. In the present study, triphenyl phosphonium cationic group-conjugated fisetin (mito-fisetin) was synthesized, and its anticancer activity was investigated in several cellular models of estrogen receptor (ER)-positive breast cancer in vitro and in vivo in proliferating and tamoxifen-promoted senescent states. Mito-fisetin, when used at low micromolar concentrations, stimulated the dissipation of mitochondrial membrane potential and oxidative stress, and affected mitochondrial function, resulting in apoptosis induction in senescent breast cancer cells. Mito-fisetin-mediated cytotoxicity was due to increased levels of phosphorylated AMPK, decreased levels of AKT and HSP90, and impaired mitophagic response, as judged by the analysis of the markers of mitophagosome formation. Senescent breast cancer cells were found to be more sensitive to mito-fisetin treatment than proliferating ones. We postulate that mitochondrial targeting in the case of fisetin may be considered as a promising anticancer and senotherapeutic strategy to eliminate drug-resistant senescent breast cancer cells.

Protective Effects of Anethole in Foeniculum vulgare Mill. Seed Ethanol Extract on Hypoxia/Reoxygenation Injury in H9C2 Heart Myoblast Cells.

Active compounds from plants and herbs are increasingly incorporated into modern medical systems to address cardiovascular diseases (CVDs). Foeniculum vulgare Mill., commonly known as fennel, is an aromatic medicinal plant and culinary herb that is popular worldwide. Protective effects against cellular damage were assessed in the H9C2 cardiomyocyte hypoxia/reoxygenation (H/R) experimental model. The identities of phytochemicals in FVSE were determined by GC-MS analysis. The phytochemical's potential for nutrients and pharmacokinetic properties was assessed by ADMET analysis. GC-MS analysis of the ethanol extracts of F. vulgare identified 41 bioactive compounds, with four prominent ones: anethole, 1-(4-methoxyphenyl)-2-propanone, ethoxydimethylphenylsilane, and para-anisaldehyde diethyl acetal. Among these, anethole stands out due to its potential for nutrients and pharmacokinetic properties assessed by ADMET analysis, such as bioavailability, lipophilicity, flexibility, and compliance with Lipinski's Rule of Five. In the H/R injury model of H9C2 heart myoblast cells, FVSE and anethole suppressed H/R-induced reactive oxygen species (ROS) generation, DNA double-strand break damage, nuclear condensation, and the dissipation of mitochondrial membrane potential (ΔΨm). These findings highlight the therapeutic potential of FVSE and its prominent component, anethole, in the treatment of CVDs, particularly those associated with hypoxia-induced damage.

Uncoupling as initiating event in mitochondrial dysfunction after diuron exposure

Diuron, a herbicide derived from urea, has been shown to induce urinary bladder urothelial tumors in rodents, leading the U.S. Environmental Protection Agency (USEPA) to designate it as a ‘known/likely’ human carcinogen. In our laboratory, a series of studies investigating the carcinogenic mode of action (MoA) of Diuron have consistently revealed its cytotoxic effects on the urinary bladder urothelium. Prolonged exposure to relatively high doses of Diuron results in urothelial necrosis, regenerative hyperplasia, and eventually, the development of tumors. The hypothesis posited is that Diuron and its metabolites exert toxicity by causing damage to mitochondria, a phenomenon referred to as mitotoxicity. Our research focuses on evaluating how Diuron and its metabolites affect mitochondria isolated from both the urothelium and the liver, the primary organ for Diuron biotransformation. In this context, we present and discuss data pertaining to mitochondria isolated from the liver of Wistar rats exposed to Diuron or its metabolites 3-(3,4-diclorofenil)-1-metilureia (DCPMU) or 3,4-dichloroaniline (DCA) at concentrations ranging from 0.5 to 500 µM in vitro. The findings indicate that, at concentrations of 100 and 500 µM, the tested chemicals induce uncoupling of oxidative phosphorylation, as evidenced by the dissipation of mitochondrial membrane potential and basal oxygen consumption. Notably, at 500 µM, DCA causes mitochondrial swelling, a morphofunctional indicator of severe organelle damage. These outcomes underscore the classification of Diuron and its metabolites, DCA and DCPMU, as mitotoxic to liver cells, given the pronounced mitochondrial dysfunction they induce.

Evaluation of the Antibacterial Potential of Two Short Linear Peptides YI12 and FK13 against Multidrug-Resistant Bacteria.

The accelerating spread of antibiotic resistance has significantly weakened the clinical efficacy of existing antibiotics, posing a severe threat to public health. There is an urgent need to develop novel antimicrobial alternatives that can bypass the mechanisms of antibiotic resistance and effectively kill multidrug-resistant (MDR) pathogens. Antimicrobial peptides (AMPs) are one of the most promising candidates to treat MDR pathogenic infections since they display broad-spectrum antimicrobial activities and are less prone to achieve drug resistance. In this study, we investigated the antibacterial capability and mechanisms of two machine learning-driven linear peptide compounds termed YI12 and FK13. We reveal that YI12 and FK13 exhibit broad-spectrum antibacterial properties against clinically significant bacterial pathogens, inducing no or minimal hemolysis in mammalian red blood cells. We further ascertain that YI12 and FK13 are resilient to heat and acid-base conditions, and exhibit susceptibility to hydrolytic enzymes and divalent cations under physiological conditions. Initial mechanistic investigations reveal that YI12 and FK13 compromise bacterial membrane integrity, leading to membrane potential dissipation and excessive reactive oxygen species (ROS) generation. Collectively, our findings highlight the prospective utility of these two cationic amphiphilic peptides as broad-spectrum antibacterial agents.

Colistin Induces Oxidative Stress and Apoptotic Cell Death through the Activation of the AhR/CYP1A1 Pathway in PC12 Cells.

Colistin is commonly regarded as the "last-resort" antibiotic for combating life-threatening infections caused by multidrug-resistant (MDR) gram-negative bacteria. Neurotoxicity is a potential adverse event associated with colistin application in clinical settings, yet the exact molecular mechanisms remain unclear. This study examined the detrimental impact of colistin exposure on PC12 cells and the associated molecular mechanisms. Colistin treatment at concentrations of 0-400 μM decreased cell viability and induced apoptotic cell death in both time- and concentration-dependent manners. Exposure to colistin triggered the production of reactive oxygen species (ROS) and caused oxidative stress damage in PC12 cells. N-acetylcysteine (NAC) supplementation partially mitigated the cytotoxic and apoptotic outcomes of colistin. Evidence of mitochondrial dysfunction was observed through the dissipation of membrane potential. Additionally, colistin treatment upregulated the expression of AhR and CYP1A1 mRNAs in PC12 cells. Pharmacological inhibition of AhR (e.g., using α-naphthoflavone) or intervention with the CYP1A1 gene significantly decreased the production of ROS induced by colistin, subsequently lowering caspase activation and cell apoptosis. In conclusion, our findings demonstrate, for the first time, that the activation of the AhR/CYP1A1 pathway contributes partially to colistin-induced oxidative stress and apoptosis, offering insights into the cytotoxic effects of colistin.

A Glucose Oxidase-Curcumin Composite Nanoreactor for Multimodal Synergistic Cancer Therapy.

Glucose oxidase (GOx) selectively oxidizes β-d-glucose into gluconic acid and hydrogen peroxide; thus, it has emerged as a promising anticancer agent by tumor starvation and oxidative therapy. Here, we developed a nanoscale platform or "nanoreactor" that incorporates GOx and the bioactive natural product curcumin (CUR) to achieve a multimodal anticancer nanocomposite. The composite nanoreactor was formed by loading CUR in biodegradable polymeric nanoparticles (NPs) of poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-PCL). Prime-coating of the NPs with an iron(III)-tannic acid complex enabled facile immobilization of GOx on the NP surface. The NPs were monodisperse with a hydrodynamic diameter of 122 nm and a partially negative surface charge. The NPs were also associated with an excellent CUR loading efficiency and sustained release up to 96 h, which was accelerated by surface-immobilized GOx and followed supercase II transport. Viability assays were conducted on two model cancer cell lines, MCF-7 and MDA-MB-231 cells, as well as human dermal fibroblasts as a representative normal cell line. The assays revealed significantly improved potency of CUR in the composite nanoreactor, with up to 6000- and 1280-fold increase in MCF-7 and MDA-MB-231 cells, respectively, and lower toxicity toward normal cells. The NPs were also able to promote intracellular reactive oxygen species (ROS) generation and dissipation of the mitochondrial membrane potential, providing important clues on the mechanism of action of the nanoreactor. Further investigation of caspase-3 activity revealed that the nanoreactor had no effect or inhibited caspase-3 levels, signifying a caspase-independent mechanism of inducing apoptosis. Our findings present a promising nanocarrier platform that combines therapeutic agents with distinct mechanisms of action acting in synergy for more effective cancer therapy.

Synthesis of Bisindole Alkaloids and Their Mode of Action against Methicillin-Resistant Staphylococcus Aureus.

About 100,000 deaths are attributed annually to infections with methicillin-resistant Staphylococcus aureus (MRSA) despite concerted efforts toward vaccine development and clinical trials involving several preclinically efficacious drug candidates. This necessitates the development of alternative therapeutic options against this drug-resistant bacterial pathogen. Using the Masuda borylation-Suzuki coupling (MBSC) sequence, we previously synthesized and modified naturally occurring bisindole alkaloids, alocasin A, hyrtinadine A and scalaradine A, resulting in derivatives showing potent in vitro and in vivo antibacterial efficacy. Here, we report on a modified one-pot MBSC protocol for the synthesis of previously reported and several undescribed N-tosyl-protected bisindoles with anti-MRSA activities and moderate cytotoxicity against human monocytic and kidney cell lines. In continuation of the mode of action investigation of the previously synthesized membrane-permeabilizing hit compounds, mechanistic studies reveal that bisindoles impact the cytoplasmic membrane of Gram-positive bacteria by promiscuously interacting with lipid II and membrane phospholipids while rapidly dissipating membrane potential. The bactericidal and lipid II-interacting lead compounds 5c and 5f might be interesting starting points for drug development in the fight against MRSA.

Mitochondrial injury induced by triclopyr in the rat liver

The herbicide triclopyr (3,5,6-trichloro-2-pyridinyloxyacetic acid) is already considered an environmental problem due to damage caused by incorrect disposal, leaching, and aerial dispersion, which may pose risks to the environment and human health. Studies have evaluated metabolism, absorption, excretion, and active transport but there is no clear information about its mode of action (MoA) and its cytotoxic action potential remains unknown. In this context, mitochondria have been used to assess the toxicity of xenobiotics, for this reason, to identify the toxic mechanism of triclopyr, hepatic mitochondria from Wistar rats were exposed in vitro to different concentrations of triclopyr (0.5–500 µM). There was neither formation/accumulation of reactive oxygen and nitrogen species, nor lipid peroxidation or changes in the mitochondrial antioxidant system, in addition to proper functioning of oxidative phosphorylation and ATP production. Changes were found in NAD(P)H oxidation, membrane potential dissipation and mitochondrial calcium gradient. These results demonstrate that mitochondria suffer damage related to their bioenergetics and redox status but not to their structure when exposed to concentrations of triclopyr considered higher than those described as found in the environment so far. Highlights Triclopyr has a low mitochondrial uncoupling potential. The damage caused to the bioenergetics and redox state of the mitochondria is related to concentrations considered higher than those found in the environment. Even at high concentrations, triclopyr was not able to change the structure of the organelle after exposure. Oxidative phosphorylation and ATP production were not impaired after exposure. NAD(P)H oxidation resulted in potential membrane dissipation and mitochondrial calcium gradient dissipation. Triclopyr does not have RONS-forming properties, as well as it does not peroxide membrane lipids, it preserves membrane sulfhydryl groups and maintains the normality of the GSH/GSSG ratio.

Triton X-100 counteracts antibiotic resistance of Enterococcus faecalis: An in vitro study

ObjectivesThe high prevalence of antibiotic-resistant bacteria poses a threat to the global public health. The appropriate use of adjuvants to restore the antimicrobial activity of antibiotics against resistant bacteria could be an effective strategy for combating antibiotic resistance. In this study, we investigated the counteraction of Triton X-100 (TX-100) and the mechanisms underlying the antibiotic resistance of Enterococcus faecalis (E. faecalis). MethodsStandard, wild-type (WT), and induced antibiotic-resistant E. faecalis strains were used in this study. In vitro antibacterial experiments were conducted to evaluate the antimicrobial activities of gentamicin sulfate and ciprofloxacin hydrochloride in the presence and absence of 0.02 % TX-100 against both planktonic and biofilm bacteria. Transcriptomic and untargeted metabolomic analyses were performed to explore the molecular mechanisms of TX-100 as an antibiotic adjuvant. Additionally, membrane permeability, membrane potential, glycolysis-related enzyme activity, intracellular adenosine triphosphate (ATP), and expression levels of virulence genes were assessed. The biocompatibility of different drug combinations was also evaluated. ResultsA substantially low TX-100 concentration improved the antimicrobial effects of gentamicin sulfate or ciprofloxacin hydrochloride against antibiotic-resistant E. faecalis. Mechanistic studies demonstrated that TX-100 increased cell membrane permeability and dissipated membrane potential. Moreover, antibiotic resistance and pathogenicity of E. faecalis were attenuated by TX-100 via downregulation of the ABC transporter, phosphotransferase system (PTS), and ATP supply. ConclusionsTX-100 enhanced the antimicrobial activity of gentamicin sulfate and ciprofloxacin hydrochloride at a low concentration by improving antibiotic susceptibility and attenuating antibiotic resistance and pathogenicity of E. faecalis. Clinical significanceThese findings provide a theoretical basis for developing new root canal disinfectants that can reduce antibiotic resistance.

Antibacterial Activity and Mechanism of Candesartan Cilexetil against Enterococcus faecalis.

Enterococcus faecalis infections pose a significant clinical challenge due to their multidrug resistance and propensity for biofilm formation. Exploring alternative treatment options, such as repurposing existing drugs, is crucial in addressing this issue. This study investigates the antibacterial activity of candesartan cilexetil against E. faecalis and elucidates its mechanism of action. Candesartan cilexetil exhibited notable antibacterial activity against both E. faecalis and Enterococcus faecium, with minimum inhibitory concentration (MIC) of ≤25 μM. Time-kill curves demonstrated concentration-dependent bactericidal effects. Candesartan cilexetil could significantly inhibited biofilm formation at the concentration of 1/4× MIC and induced alterations in biofilm structure. Permeability assays revealed compromised bacterial membranes, accompanied by the dissipation of membrane potential in E. faecalis cells after treatment with candesartan cilexetil. Checkerboard analysis showed that bacterial membrane phospholipids phosphatidylglycerol and cardiolipin could neutralize the antibacterial activity of candesartan cilexetil in a dose-dependent manner. Biolayer interferometry (BLI) assay indicated specific interactions between candesartan cilexetil and phosphatidylglycerol or cardiolipin. This study demonstrates the promising antibacterial and antibiofilm activities of candesartan cilexetil against multidrug-resistant E. faecalis. The mechanism of action involves disruption of bacterial membranes, possibly by interacting with membrane phospholipids. These findings underscore the potential utility of candesartan cilexetil as an effective therapeutic agent for combating E. faecalis infections, offering a valuable strategy in the battle against antibiotic-resistant pathogens.

Two Keggin-type Germanotungstates: Synthesis, characterization, and antitumor mechanism of mitochondrial apoptosis

Germanotungstates have been studied variously and widely in antitumor activity aganist various cancer cells; however, their mode of action remains unclear. In this study, we integrated transition metals and organic ligands into the Keggin polyoxometalates via covalence and metal coordination and crystallized two Keggin-type germanotungstates compounds 1 [Mn(2,2ʹ-bpy)3]2[GeW12O40]·0·5H2O and 2 [Fe(2,2ʹ-bpy)2Cl2]2(Hbpy)2[GeW12O40]. Compounds 1 and 2 exhibited high anti-proliferative activities against gastric cancer AGS and MGC803, colon cancer SW480, and liver cancer HepG2 cells, with IC50 values below 50 μmol/L. Further investigation of the molecular mechanism in AGS cells revealed that compounds 1 and 2 repressed the STAT3 pathway and its downstream Bcl-2 family proteins. Downregulation of anti-apoptotic Bcl-2 family proteins leads to the dissipation of mitochondrial membrane potential, eventually triggering caspase-dependent apoptosis. These results demonstrate that compounds 1 and 2 exert antitumor activity via mitochondria-mediated apoptosis. Overall, our findings provide evidence for the development of antitumor germanotungstate-type drugs.

Nr1h4 and Thrb ameliorate ER stress and provide protection in the MPTP mouse model of Parkinson's.

Elevated ER stress has been linked to the pathogenesis of several disease conditions including neurodegeneration. In this study, we have holistically determined the differential expression of all the nuclear receptors (NRs) in the presence of classical ER stress inducers. Activation of Nr1h4 and Thrb by their cognate ligands (GW4064 and T3) ameliorates the tunicamycin (TM)-induced expression of ER stress genes. A combination of both ligands is effective in mitigating cell death induced by TM. Further exploration of their protective effects in the Parkinson's disease (PD) model shows that they reduce MPP+-induced dissipation of mitochondrial membrane potential and ROS generation in an in vitro PD model in neuronal cells. Furthermore, the generation of an experimental murine PD model reveals that simultaneous treatment of GW4064 and T3 protects mice from ER stress, dopaminergic cell death, and functional deficits in the MPTP mouse model of PD. Thus, activation of Nr1h4 and Thrb by their respective ligands plays an indispensable role in ER stress amelioration and mounts protective effects in the MPTP mouse model of PD.

Hibiscus manihot L. flower extract induces anticancer activity through modulation of apoptosis and autophagy in A549 cells

Lung cancer is a major public health issue and heavy burden in China and worldwide due to its high incidence and mortality without effective treatment. It’s imperative to develop new treatments to overcome drug resistance. Natural products from food source, given their wide-ranging and long-term benefits, have been increasingly used in tumor prevention and treatment. This study revealed that Hibiscus manihot L. flower extract (HML) suppressed the proliferation and migration of A549 cells in a dose and time dependent manner and disrupting cell cycle progression. HML markedly enhanced the accumulation of ROS, stimulated the dissipation of mitochondrial membrane potential (MMP) and that facilitated mitophagy through the loss of mitochondrial function. In addition, HML induced apoptosis by activation of the PTEN-P53 pathway and inhibition of ATG5/7-dependent autophagy induced by PINK1-mediated mitophagy in A549 cells. Moreover, HML exert anticancer effects together with 5-FU through synergistic effect. Taken together, HML may serve as a potential tumor prevention and adjuvant treatment for its functional attributes.

Mechanism of Cell-Killing Activity of Plantaricin LD1 Against Escherichia coli ATCC 25922.

Plantaricin LD1 was purified from a potential probiotic strain, Lactobacillus plantarum LD1 previously isolated from indigenous food, Dosa. In this study, we have performed a detailed mechanism of action of plantaricin LD1 against Escherichia coli ATCC 25922 considering Micrococcus luteus MTCC 106 as control. The plantaricin LD1 showed a minimum inhibitory concentration (MIC) of 34.57µg/mL and a minimum bactericidal concentration (MBC) of 138.3µg/mL against M. luteus MTCC 106, whereas MIC 69.15µg/mL and MBC 276.6µg/mL were found against E. coli ATCC 25922. The efflux of potassium ions, dissipation of membrane potential (∆ψ), and transmembrane pH gradient (∆pH) of plantaricin LD1-treated cells suggested the membrane-acting nature of plantaricin LD1. Plantaricin LD1 also caused degradation of the genomic DNA of the target strains tested. The cell killing was confirmed by staining with propidium iodide and visualized under light and electron microscopes. The bacteriocin-treated cells were found to be ruptured, swollen, and elongated. Thus, the findings indicate plantaricin LD1 kills E. coli ATCC 25922 by interacting with the cell membrane resulting in the efflux of intracellular contents and also causing degradation of nucleic acids leading to cell death.

Abstract 2122: Enhanced anticancer efficacy via ROS-dependent ferroptosis: Synergy between surufatinib and cisplatin in small cell lung cancer

Abstract Objective: Small cell lung cancer (SCLC), a highly malignant subset of neuroendocrine carcinoma, accounts for 13% of lung cancer cases and is characterized by its high aggressiveness and poor prognosis. The longstanding first-line treatment, a platinum-etoposide chemotherapy regimen, is increasingly compromised by resistance issues, highlighting the critical need for new therapeutic approaches. Surufatinib, an inhibitor selectively targeting vascular endothelial growth factor receptors (VEGFR) 1, 2, and 3, fibroblast growth factor receptor type 1 (FGFR1), and colony-stimulating factor-1 receptor (CSF-1R), has demonstrated efficacy in a spectrum of solid tumors, including neuroendocrine neoplasm, thyroid cancers and biliary cancers. Recently, ferroptosis, an iron-dependent cell death pathway, offers therapeutic promise, especially by inhibiting the xCT system, effective in pancreatic and lung adenocarcinomas. Yet, surufatinib's potential for inducing ferroptosis in SCLC remains to be investigated. Methods: Cytotoxicity assays were performed on SCLC cell lines to assess the combinatorial effect of surufatinib and cisplatin, revealing a synergistic interaction. Ferroptosis inhibitors were applied to assess the reversal of cytotoxic effects. Mitochondrial morphology, reactive oxygen species (ROS) accumulation, membrane potential dissipation (JC-1 assay), lipid peroxidation (MDA levels), and intracellular iron accumulation (Fe2+ quantification) assays confirmed ferroptosis induction. Western blot analyses were conducted to explore the mechanistic pathways, supplemented by in vivo antitumor efficacy evaluations. Results: In vitro assays revealed that surufatinib, when combined with cisplatin, exerts a pronounced synergistic effect on small cell lung cancer (SCLC) cell lines by inducing ferroptosis. The combination therapy strategically modulated ferroptosis-related pathways: cisplatin effectively decreased the expression of glutathione peroxidase 4 (GPX4), critical for cellular antioxidant defenses, while surufatinib increased the expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), which is essential for the accumulation of lipid peroxides. This dual pathway engagement significantly amplified the ferroptotic response, culminating in heightened antitumor activity. Corroborative in vivo experiments substantiated these findings, validating the combined treatment's efficacy in a living model and underscoring its potential as a robust therapeutic strategy for SCLC. Conclusion: Our findings reveal that surufatinib triggers ferroptosis in small cell lung cancer—a novel discovery. Additionally, it also unveils that surufatinib and cisplatin together enhance anti-cancer efficacy via the ROS-mediated ferroptosis pathway, marking a significant advance in SCLC therapy. Citation Format: Xiaolin Li, Ziting Zhang, Qiyun Tang, Huae Xu. Enhanced anticancer efficacy via ROS-dependent ferroptosis: Synergy between surufatinib and cisplatin in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2122.

Evaluation of Efficacy of Curcumin and Caffeic Acid Phenethyl Ester in Breast Cancer by Preclinical Studies.

CAPE and curcumin are a class of phenolics. While curcumin is obtained from turmeric, CAPE is found in Baccharis sarothroides and Populus deltoides. Both agents are reported to produce activities in some cancer types. The combined and comparative effects of the two agents in breast cancer have not yet reported. We evaluated the potential of CAPE and curcumin in both in vitro and in vivo breast cancer models. Human breast cancer cell lines, MDA-MB-231 and MCF-7, were exposed to CAPE and curcumin, followed by functional assays such as cell cytotoxicity, cell proliferation and colony formation, cell cycle, mitochondrial membrane potential, apoptosis, and monodansylcadaverine (MDC) staining for autophagy. Computational analyses and mouse models were also used. Employing computational analyses, both agents were found to exhibit drug-like properties. Both molecules interacted with the key molecules of the NF-κB pathway. CAPE and curcumin inhibited cell proliferation, colony formation, and invasion, triggering apoptosis in breast cancer cells. CAPE was found to be more effective than curcumin. Two agents working together were more effective than each agent working alone. Both agents suppressed the expression of survivin, Bcl-xL and GLUT-1. The level of cleaved PARP was increased by both agents. Both phenolics observed an induction in ROS generation. Further, both molecules triggered a dissipation in mitochondrial membrane potential. In mice models implanted with Ehrlich-Lettre ascites carcinoma (EAC) cells, both drugs inhibited the growth of the tumour. The phenolics also modulated the metabolic parameters in tumour-bearing mice. The observations suggest that the combination of curcumin plus CAPE may be better in comparison to individual molecules. Other: The study opens a window for analysing the efficacy of the combination of CAPE and curcumin in animal studies. This will provide a basis for examining the combined efficacy of two agents in a clinical trial.

Cytisine–N-methylene-(5,7,4’-trihydroxy)- isoflavone ameliorates ischemic stroke-induced brain injury in mouse by regulating the oxidative stress and BDNF-Trkb/Akt pathway

BackgroundA novel compound Cytisine–N-methylene-(5,7,4′-trihydroxy)- isoflavone (LY01) found in the Sophora alopecuroides L is a neuroprotective agent. However, the effect and potential mechanism of LY01 treatment for ischemic stroke (IS) have not been fully elucidated. Aim of the studyThe aim of this study is to demonstrate whether LY01 can rescue ischemic stroke-induced brain injury and oxygen–glucose deprivation/reperfusion (OGD/R). ResultsOur results show that intragastric administration of LY01 improves ischemic stroke behaviors in mice, as demonstrated by neurological score, infarct volume, cerebral water content, rotarod test for activity. Compared with the model group, the ginkgo biloba extract (EGb) and LY01 reversed the neurological score, infarct volume, cerebral water content, rotarod test in model mice. Further analysis showed that the LY01 rescued oxidative stress in the model mice, which was reflected in the increased levels of catalase, superoxide dismutase, total antioxidant capacity and decreased levels of malondialdehyde in the serum of the model mice. Moreover, the expression of the brain-derived neurotrophic factor brain-derived neurotrophic factor (BDNF), phosphorylated protein kinase B (p-Akt), Bax, Bcl-2, (p)-tropomysin related kinase B (p-Trkb) was restored and the expression of Bax, glial fibrillary acidic protein (GFAP) in the brains of the model mice was inhibited through LY01 treatment. In the polymerase chain reaction (PCR) data, after giving LY01, the expression in the brains of model mice was that, IL-10 increased and IL-1β, Bax, Bcl-2 decreased. Furthermore, the results indicated that LY01 improved cell viability, reactive oxygen species content, and mitochondrial membrane potential dissipation induced by OGD/R in primary culture of rat cortical neurons. Bax and caspase-3 activity was upregulated compared to the before after treatment with LY01. ConclusionsOur study suggests that LY01 reversed ischemic stroke by reducing oxidative stress and activating the BDNF-TrkB/Akt pathway and exerted a neuroprotective action against OGD/R injury via attenuation, a novel approach was suggested to treat ischemic stroke. Our observations justify the traditional use of LY01 for a treatment of IS in nervous system.

A Comparative Analysis of the Antibacterial Spectrum of Ultrasmall Manganese Ferrite Nanozymes with Varied Surface Modifications.

Bacterial infectious diseases pose a significant global challenge. However, conventional antibacterial agents exhibit limited therapeutic effectiveness due to the emergence of drug resistance, necessitating the exploration of novel antibacterial strategies. Nanozymes have emerged as a highly promising alternative to antibiotics, owing to their particular catalytic activities against pathogens. Herein, we synthesized ultrasmall-sized MnFe2O4 nanozymes with different charges (MnFe2O4-COOH, MnFe2O4-PEG, MnFe2O4-NH2) and assessed their antibacterial capabilities. It was found that MnFe2O4 nanozymes exhibited both antibacterial and antibiofilm properties attributed to their excellent peroxidase-like activities and small sizes, enabling them to penetrate biofilms and interact with bacteria. Moreover, MnFe2O4 nanozymes effectively expedite wound healing within 12 days and facilitate tissue repair and regeneration while concurrently reducing inflammation. MnFe2O4-COOH displayed favorable antibacterial activity against Gram-positive bacteria, with 80% bacterial removal efficiency against MRSA by interacting with phosphatidylglycerol (PG) and cardiolipin (CL) of the membrane. By interacting with negatively charged bacteria surfaces, MnFe2O4-NH2 demonstrated the most significant and broad-spectrum antibacterial activity, with 95 and 85% removal efficiency against methicillin-resistant Staphylococcus aureus (MRSA) and P. aeruginosa, respectively. MnFe2O4-PEG dissipated membrane potential and reduced ATP levels in MRSA and P. aeruginosa, showing relatively broad-spectrum antibacterial activity. To conclude, MnFe2O4 nanozymes offer a promising therapeutic approach for treating wound infections.