Changes In Mitochondrial Membrane Potential Research Articles

The impact of HBx protein on mitochondrial dynamics and associated signaling pathways strongly depends on the hepatitis B virus genotype.

Chronic hepatitis B virus (HBV) infections are strongly associated with liver cirrhosis, inflammation, and hepatocellular carcinoma. In this context, the viral HBx protein is considered as a major factor influencing HBV-associated pathogenesis through deregulation of multiple cellular signaling pathways and is therefore a potential target for prognostic and therapeutic applications. However, HBV-associated pathogenesis differs significantly between genotypes, with the relevant factors and in particular the contribution of the genetic diversity of HBx being largely unknown. To address this question, we studied the specific genotype-dependent impact of HBx on cellular signaling pathways, focusing in particular on morphological and functional parameters of mitochondria. To exclusively investigate the impact of HBx of different genotypes on integrity and function of mitochondria in the absence of additional viral factors, we overexpressed HBx in Huh7 or HepG2 cells. Key signaling pathways were profiled by kinome analysis and correlated with expression levels of mitochondrial and pathogenic markers. Conclusively, HBx of genotypes A and G caused strong disruption of mitochondrial morphology alongside an induction of PTEN-induced putative kinase 1/Parkin-mediated mitophagy. These effects were only moderately dysregulated by genotypes B and E, whereas genotypes C and D exhibit an intermediate effect in this regard. Accordingly, changes in mitochondrial membrane potential and elevated reactive oxygen species production were associated with the HBx-mediated dysfunction among different genotypes. Also, genotype-related differences in mitophagy induction were identified and indicated that HBx-mediated changes in the mitochondria morphology and function strongly depend on the genotype. This indicates a relevant role of HBx in the process of genotype-dependent liver pathogenesis of HBV infections and reveals underlying mechanisms.IMPORTANCEThe hepatitis B virus is the main cause of chronic liver disease worldwide and differs in terms of pathogenesis and clinical outcome among the different genotypes. Furthermore, the viral HBx protein is a known factor in the progression of liver injury by inducing aberrant mitochondrial structures and functions. Consequently, the selective removal of dysfunctional mitochondria is essential to maintain overall cellular homeostasis and cell survival. Consistent with the intergenotypic difference of HBV, our data reveal significant differences regarding the impact of HBx of different genotypes on mitochondrial dynamic and function and thereby on radical oxygen stress levels within the cell. We subsequently observed that the induction of mitophagy differs significantly across the heterogenetic HBx proteins. Therefore, this study provides evidence that HBx-mediated changes in the mitochondria dynamics and functionality strongly depend on the genotype of HBx. This highlights an important contribution of HBx in the process of genotype-dependent liver pathogenesis.

From Epimedium to Neuroprotection: Exploring the Potential of Wushanicaritin

Epimedium has been used for functional foods with many beneficial functions to human health. Wushanicaritin is one of the most important chemicals int Epimedium. This study investigated the neuroprotective effects of wushanicaritin and potential underlying mechanisms. The results demonstrated that wushanicaritin possessed superior intercellular antioxidant activity compared to icaritin. Wushanicaritin, with an EC50 value of 3.87 μM, showed better neuroprotective effect than quercetin, a promising neuroprotection agent. Wushanicaritin significantly reversed lactate dehydrogenase release, reactive oxygen species generation, cell apoptosis, and mRNA expression related to cell apoptosis and oxidative defense, in glutamate-induced PC-12 cells. Wushanicaritin could also maintain the enzymatic antioxidant defense system and mitochondrial function. The suppression of caspase-3 activation and amelioration of mitochondrial membrane potential loss and nucleus morphology changes were involved in the antiapoptotic effect of wushanicaritin. These findings suggested that wushanicaritin possesses excellent intercellular antioxidant and neuroprotective activities, showing potential promise in functional foods.

Changes in Mitochondrial Membrane Potential in In Vitro Electroporation with Nano- and Microsecond Pulses

Changes in Mitochondrial Membrane Potential in <i>In Vitro</i> Electroporation with Nano- and Microsecond Pulses

GFRA4 improves the neurogenic potential of enteric neural crest stem cells via hedgehog pathway.

Hirschsprung disease (HSCR) is a congenital intestinal disease characterised by functional obstruction of the colon. Herein, we investigated the role and mechanism of the gene GFRA4 in HSCR. GFRA4 expression in the ganglionic and aganglionic segment tissues in patients with HSCR and healthy colon tissues were detected using qRT-PCR, western blot, and immunohistochemistry. Cell proliferation, cycle distribution, apoptosis, changes in mitochondrial membrane potential, and differentiation were assessed in mouse enteric neural crest stem cells (ENCSCs) using the CCK-8 assay, EdU staining, flow cytometry, JC-1 probe, and immunofluorescence, respectively. GSEA analysis was performed to screen the signaling pathways regulated by GFRA4. GFRA4 was downregulated in aganglionic segment tissues compared to control and ganglionic segment tissues. GFRA4 overexpression promoted proliferation and differentiation, and inhibited apoptosis in ENCSCs, while GFRA4 down-regulation had the opposite result. GFRA4 activated the hedgehog pathway. GFRA4 overexpression enhanced the expression of key factors of the hedgehog pathway, including SMO, SHH, and GLI1. However, GFRA4 down-regulation reduced their expression. An antagonist of hedgehog pathway, cyclopamine, attenuated the effect of GFRA4 overexpression on proliferation, differentiation, and apoptosis of ENCSCs. GFRA4 promotes proliferation and differentiation but inhibits apoptosis of ENCSCs via the hedgehog pathway in HSCR. This study confirms that GFRA4 improves the proliferation and differentiation of ENCSCs via modulation of the hedgehog pathway. This study for the first time revealed the role and the mechanism of the action of GFRA4 in HSCR, which indicates that GFRA4 may play a role in the pathological development of HSCR. Our findings may lay the foundation for further investigation of the mechanisms underlying HSCR development and into targets of HSCR treatment.

ROS formation, mitochondrial potential and osmotic stability of the lamprey red blood cells: effect of adrenergic stimulation and hypoosmotic stress.

Semi-anadromous animals experience salinity fluctuations during their life-span period. Alterations of environmental conditions induce stress response where catecholamines (CA) play a central role. Physiological stress and changes in external and internal osmolarity are frequently associated with increased production of reactive oxygen species (ROS). In this work, we studied the involvement of the cAMP/PKA pathway in mediating catecholamine-dependent effects on osmoregulatory responses, intracellular production of ROS, and mitochondrial membrane potential of the river lamprey (Lampetra fluviatilis, Linnaeus, 1758) red blood cells (RBCs). We also investigated the role of hypoosmotic shock in the process of ROS production and mitochondrial respiration of RBCs. For this, osmotic stability and the dynamics of the regulatory volume decrease (RVD) following hypoosmotic swelling, intracellular ROS levels, and changes in mitochondrial membrane potential were assessed in RBCs treated with epinephrine (Epi, 25 μM) and forskolin (Forsk, 20 μM). Epi and Forsk markedly reduced the osmotic stability of the lamprey RBCs whereas did not affect the dynamics of the RVD response in a hypoosmotic environment. Activation of PKA with Epi and Forsk increased ROS levels and decreased mitochondrial membrane potential of the lamprey RBCs. In contrast, upon hypoosmotic shock enhanced ROS production in RBCs was accompanied by increased mitochondrial membrane potential. Overall, a decrease in RBC osmotic stability and the enhancement of ROS formation induced by β-adrenergic stimulation raises concerns about stress-associated changes in RBC functions in agnathans. Increased ROS production in RBCs under hypoosmotic shock indicates that a decrease in blood osmolarity may be associated with oxidative damage of RBCs during lamprey migration.

Versatile Fluorescence Lifetime-Based Copper Probe to Quantify Mitochondrial Membrane Potential and Reveal Its Interaction with Protein Aggregation.

Mitochondria play a crucial role in maintaining cellular homeostasis, and the depolarization of mitochondrial membrane potential (MMP) is an important signal of apoptosis. Additionally, protein misfolding and aggregation are closely related to diseases including neurodegenerative diseases, diabetes, and cancers. However, the interaction between MMP changes and disease-related protein aggregation was rarely studied. Herein, we report a novel "turn-on" fluorescent probe MitoRhB that specifically targets to mitochondria for Cu2+ detection in situ. The fluorescence lifetime (τ) of MitoRhB exhibits a positive correlation with MMP changes, allowing us to quantitatively determine the relative MMP during SOD1 (A4 V) protein aggregation. Finally, we found that (1) the increasing concentrations of copper will accelerate the depolarization of mitochondria and reduce MMP; (2) the depolarization of mitochondria can intensify the degree of protein aggregation, suggesting a new routine of copper-induced cell death mediated through abnormal MMP depolarization and protein aggregation.

Organophosphorus Flame Retardant TPP-Induced Human Corneal Epithelial Cell Apoptosis through Caspase-Dependent Mitochondrial Pathway.

A widely used organophosphate flame retardant (OPFR), triphenyl phosphate (TPP), is frequently detected in various environmental media and humans. However, there is little known on the human corneal epithelium of health risk when exposed to TPP. In this study, human normal corneal epithelial cells (HCECs) were used to investigate the cell viability, morphology, apoptosis, and mitochondrial membrane potential after they were exposed to TPP, as well as their underlying molecular mechanisms. We found that TPP decreased cell viability in a concentration-dependent manner, with a half maximal inhibitory concentration (IC50) of 220 μM. Furthermore, TPP significantly induced HCEC apoptosis, decreased mitochondrial membrane potential in a dose-dependent manner, and changed the mRNA levels of the apoptosis biomarker genes (Cyt c, Caspase-9, Caspase-3, Bcl-2, and Bax). The results showed that TPP induced cytotoxicity in HCECs, eventually leading to apoptosis and changes in mitochondrial membrane potential. In addition, the caspase-dependent mitochondrial pathways may be involved in TPP-induced HCEC apoptosis. This study provides a reference for the human corneal toxicity of TPP, indicating that the risks of OPFR to human health cannot be ignored.

Abstract LB267: Disrupting cytoplasmic proton motive force induces ferroptotic cell death in colorectal cancer: A novel anti-cancer mechanism

Abstract Introduction: Chemotherapeutic resistance and tumor recurrence remain the leading causes of poor prognosis in colorectal cancer (CRC). Herein, we evaluated the anti-cancer effect of a novel repurposed drug HLN* in CRC. Methods: The changes in membrane potential were studied using DiSC(3) fluorescence by confocal microscopy. Cytotoxic activity of compound was studied using Sulphorhodamine-B (SRB) assay. Changes in mitochondrial membrane potential were studied using JC-1 dye. ROS accumulation was analyzed using DCFDA fluorescence using FACS analysis. Ferrorange assay was used to check the ferrous ion influx within the cell. Western blot was used to identify mode of cell death enacted. Results: Our results show that HLN treatment reduced the survival of human and murine CRC cells (CT26, HCT116, Colo 205 and MC38) with IC50 ranging 1-4 µM. HLN further inhibited proton motive force (PMF) by sequentially depolarizing and hyperpolarizing the cell membrane, as examined by cellular membrane potential using DiSC(3) dye by confocal microscopy. Disruption of the PMF leads to changes in various cellular signaling cascades including ROS production and mitochondrial membrane potential (MMP) changes. The MMP was significantly reduced by HLN treatment in CRC cells. Furthermore, we also observed an increase in ROS production by HLN. Pretreatment with N-acetyl cysteine (NAC), an antioxidant, blocked the effects of HLN. Interestingly, ferroptotic cell death markers GPX4, GPX1, SLC7A11/xCT, NRF2 and KEAP1 were significantly inhibited by HLN treatment in CRC cells as observed by western blotting. Ferroptotic induction was then confirmed using ferrorange assay wherein we observed a concentration dependent increase in ferrorange intensity indicating an increase in Fe+2. Overall, HLN dissipates ΔpH by modulating iron influx leading to ferroptotic cell death through ROS. Conclusion: Taken together, our results demonstrate the anti-cancer activity of a novel repurposed drug HLN and its unique mechanism of action. Our results provide a promise for developing this drug as a potential treatment option for CRC, through a novel anti-cancer mechanism. * Due to Intellectual property issued, the name of the drug is not provided Citation Format: Shreyas R. Gaikwad, Mohamed A. Eltokhy, Morgan Moore, Keerthi Kailath, Sanjay K. Srivastava. Disrupting cytoplasmic proton motive force induces ferroptotic cell death in colorectal cancer: A novel anti-cancer mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB267.

Yiqigubiao pill treatment regulates Sirtuin 5 expression and mitochondrial function in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous group of pathophysiological bases of airway inflammation and its anti-inflammatory response. Aberrant mitochondrial signaling and mitochondrial dysfunction underlie the pathomechanisms leading to COPD. This study aims to investigate the effects of the Yiqigubiao (YQGB) pill, a traditional Chinese medicine (TCM), on Sirtuin 5 (SIRT5) and mitochondrial function in patients with COPD. Thirty-four patients with COPD were randomized into oral YQGB or placebo groups concurrent with a 24-week routine treatment. The pulmonary function was assessed by examining the levels of forced expiratory volume in one second (FEV1)/forced vital capacity (FVC), FEV1, and FVC. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect SIRT5 expression in mitochondria isolated from peripheral blood. Flow cytometry was used to detect changes in mitochondrial membrane potential and reactive oxygen species (ROS) in peripheral blood lymphocytes. Human bronchial epithelial (HBE) cells stimulated by cigarette smoke extract (CSE) were treated with YQGB. After SIRT5 was knocked down in cells, the changes in mitochondrial membrane potential, levels of adenosine triphosphate (ATP), and ROS were detected. YQGB treatment significantly improved lung function in patients with COPD. The expression of SIRT5 and the mitochondrial membrane potential significantly increased and ROS decreased in patients with COPD after YQGB treatment. The CSE decreased cell proliferation and SIRT5 expression, which was alleviated after YQGB treatment. Furthermore, SIRT5 was knocked down in CSE-stimulated HBE cells, and its expression was elevated upon YQGB treatment. The knockdown of SIRT5 significantly altered the CSE-stimulation-induced dysregulation of mitochondrial membrane potential, ATP levels, and ROS. This was also restored after YQGB treatment. YQGB treatment can elevate SIRT5 expression, restore mitochondrial function in COPD, and exert protective effects.

Solanum trilobatum leaf extract-derived silver nanoparticles downregulate the PI3K/AKT/mTOR signaling pathway and attenuate oral squamous cell carcinoma cell proliferation

Introduction: Oral cancer’s aggressive nature poses a significant health risk, demanding timely diagnosis and effective treatment. Despite progress in conventional therapies like chemotherapy and surgery, their limitations drive the exploration of alternative strategies. This study assessed Solanum trilobatum-derived silver nanoparticles (AgNPs) in impacting cancer cell cycles, inducing apoptosis, and modulating key pathways, including the phosphoinositide 3-kinase (PI3K)/Protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway, leveraging phytotherapy and nanotechnology—a promising frontier in cancer treatment. Methods: AgNPs were synthesized through the reduction of ions and stabilized using aqueous leaf extracts of S. trilobatum. After the characterization of AgNPs, the mRNA Gene Expression and mitochondrial membrane potentials (MMPs) were assessed. DNA fragmentation was done and DNA pattern by gel documentation system was observed. The study also assessed the modulation of the PI3K/Akt/mTOR cascade, impacting tumor growth and proliferation. Results: Biosynthesized AgNPs were characterized using UV-visible spectrophotometry (UVvis), energy-dispersive X-ray spectroscopy (EDX), scanning electron microscopy (SEM), and Fourier-transform infrared (FTIR) spectroscopy. DNA fragmentation exhibited a typical ladder pattern. Dose-dependent changes in MMP were observed in the treated oral cancer cells. The effect of S. trilobatum-derived AgNPs in targeting the cell signaling pathway correlated significantly with their anticancer potency (P&lt;0.001). Conclusion: This study reveals S. trilobatum leaf extract-based AgNPs as a natural cytostatic agent against oral squamous carcinoma. Utilizing nature’s resources and nanoscale science, they hold promise for enhancing oral cancer treatment outcomes and survival rates.

Caffeic acid phenethyl ester inhibits neuro-inflammation and oxidative stress following spinal cord injury by mitigating mitochondrial dysfunction via the SIRT1/PGC1α/DRP1 signaling pathway

BackgroundThe treatment of spinal cord injury (SCI) has always been a significant research focus of clinical neuroscience, with inhibition of microglia-mediated neuro-inflammation as well as oxidative stress key to successful SCI patient treatment. Caffeic acid phenethyl ester (CAPE), a compound extracted from propolis, has both anti-inflammatory and anti-oxidative effects, but its SCI therapeutic effects have rarely been reported.MethodsWe constructed a mouse spinal cord contusion model and administered CAPE intraperitoneally for 7 consecutive days after injury, and methylprednisolone (MP) was used as a positive control. Hematoxylin–eosin, Nissl, and Luxol Fast Blue staining were used to assess the effect of CAPE on the structures of nervous tissue after SCI. Basso Mouse Scale scores and footprint analysis were used to explore the effect of CAPE on the recovery of motor function by SCI mice. Western blot analysis and immunofluorescence staining assessed levels of inflammatory mediators and oxidative stress-related proteins both in vivo and in vitro after CAPE treatment. Further, reactive oxygen species (ROS) within the cytoplasm were detected using an ROS kit. Changes in mitochondrial membrane potential after CAPE treatment were detected with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide. Mechanistically, western blot analysis and immunofluorescence staining were used to examine the effect of CAPE on the SIRT1/PGC1α/DRP1 signaling pathway.ResultsCAPE-treated SCI mice showed less neuronal tissue loss, more neuronal survival, and reduced demyelination. Interestingly, SCI mice treated with CAPE showed better recovery of motor function. CAPE treatment reduced the expression of inflammatory and oxidative mediators, including iNOS, COX-2, TNF-α, IL-1β, 1L-6, NOX-2, and NOX-4, as well as the positive control MP both in vitro and in vivo. In addition, molecular docking experiments showed that CAPE had a high affinity for SIRT1, and that CAPE treatment significantly activated SIRT1 and PGC1α, with down-regulation of DRP1. Further, CAPE treatment significantly reduced the level of ROS in cellular cytoplasm and increased the mitochondrial membrane potential, which improved normal mitochondrial function. After administering the SIRT1 inhibitor nicotinamide, the effect of CAPE on neuro-inflammation and oxidative stress was reversed.On the contrary, SIRT1 agonist SRT2183 further enhanced the anti-inflammatory and antioxidant effects of CAPE, indicating that the anti-inflammatory and anti-oxidative stress effects of CAPE after SCI were dependent on SIRT1.ConclusionCAPE inhibits microglia-mediated neuro-inflammation and oxidative stress and supports mitochondrial function by regulating the SIRT1/PGC1α/DRP1 signaling pathway after SCI. These effects demonstrate that CAPE reduces nerve tissue damage. Therefore, CAPE is a potential drug for the treatment of SCI through production of anti-inflammatory and anti-oxidative stress effects.Graphical

Abstract 4558: A triterpenoid derivative has strong antitumor activities in gemcitabine-resistant advanced bladder cancer models and promotes immune cell infiltration in a high grade murine syngeneic model

Abstract Bladder cancer (BC) presents a formidable challenge in treatment and patient survival, requiring particular attention to advanced stages, such as muscle-invasive stage (MIBC), due to its potential for rapid progression and substantial impact on patient morbidity and mortality. The five-year survival rate for MIBC is a meager 30%, which drops further down to 8% once tumors metastasize. Advanced bladder cancers (High grade T1) are treated with intravesical therapy with Gemcitabine and Cisplatin (G+C) combination and Stage T2 are treated neoadjuvant with G+C and cystectomy, thereby decreasing the quality of life for patients with BC. Furthermore, tumors progress beyond responding to other chemotherapy regimens such as dose-dense treatment with Methotrexate +Vinblastine + Adriamycin and Cis-platin (MVAC), all of which will further decrease the quality of life with persistent adverse toxicity. Our laboratory has developed a novel compound derived from the pentacyclic triterpenoid, Ursolic acid, termed N-methyl piperazine conjugated-Ursolic acid, or UA4, and have demonstrated its antitumor activities against both Gem-Sensitive (Gem-S) and Gem-Resistant (Gem-R) human advanced BC cell lines, T24 and 5637. We tested the combination of Gem, UA4 and Gem + UA4 in a murine non-MIBC model (MB49) to investigate the role of immune cell infiltration during tumor development and treatment response. UA4 treatment was equally effective in chemo-sensitive and chemo-resistant cell lines at dosages between 4-5µM. Previous in vitro mechanistic studies on UA4 and UA4+Gem combination treatments revealed cell death occurred through apoptosis and autophagy, along with a highly selective form of mitochondrial-mediated autophagy (mitophagy). These findings were confirmed with transmission electron microscopy ultrastructural analysis, cell cycle phase analyses, excess, changes in mitochondrial membrane potential and reactive oxygen species measurements. Gem-S and Gem-R xenografts in athymic mice further supported UA4’s efficacy in vivo, demonstrating a significant reduction in both tumor weight and volume following oral gavage (100mg/kg), with mice exhibiting no signs of systemic toxicity. Combination treatment with UA4 and Gem synergistically increased antiproliferative activity. MB49 syngeneic mice model further supported UA4 as an effective therapy, and immunofluorescence assays with immune-related markers, suggested an anti-inflammatory effect in vivo. Overall, we demonstrate UA4 has a potential to be used as an efficacious, non-toxic compound for further adjuvant use in clinical settings. Citation Format: Juliette Rose Seremak, Kunj Bihari Gupta, Siva S. Panda, Bal L. Lokeshwar. A triterpenoid derivative has strong antitumor activities in gemcitabine-resistant advanced bladder cancer models and promotes immune cell infiltration in a high grade murine syngeneic model [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 4558.

Abstract 5909: Sulindac exhibits anti-proliferative effects in pre-clinical models of ovarian cancer

Abstract Objectives: Given the intricate relationship between inflammation and ovarian cancer (OC) development and progression, anti-inflammatory drugs may have potential in the prevention and treatment of OC. Sulindac exhibits potent anti-inflammatory effects through inhibition of COX1/COX2 pathways and promising anti-tumor activity in pre-clinical models of colorectal and endometrial cancer. Thus, we investigated the efficacy of sulindac as an anti-tumorigenic agent in OC pre-clinical cell and mouse models. Methods: The human OC cell lines, MES and OVCAR5, and the K18-gT121+/-;p53fl/fl;Brca1fl/fl (KpB) mouse model of high-grade serous OC were used. Cellular proliferation was assessed by MTT and colony count assays. Apoptosis was assessed using cleaved caspase 3, 8, and 9 assays. To measure cellular stress, production of reactive oxygen species was measured by DCFH-DA assay, and the JC1 assay was used to assess changes in mitochondrial membrane potential. Cellular adhesion was evaluated by laminin assay, and cellular migration was determined by wound healing assay. Western immunoblotting was used to assess the effects of sulindac on downstream targets related to cellular stress, apoptosis, cellular invasion potential, cell cycle control, and inflammation. The KpB mice were treated with sulindac (7.5 mg/kg, oral gavage, daily) or placebo for four weeks. Results: Sulindac inhibited cellular proliferation in a dose-dependent manner in the MES and OVCAR5 cells, with IC50s of 75.3 and 75.69 μM, respectively. Sulindac (100 μM) reduced colony formation by 81% in the MES cells and by 49% in the OVCAR5 cells (p&amp;lt;0.01). Treatment of MES and OVCAR5 cells with sulindac increased ROS production and decreased mitochondrial membrane potential, in a dose-dependent manner (p&amp;lt;0.01). Sulindac induced activity of cleaved caspase 3, 8, and 9, in MES and OVCAR5 cells (p&amp;lt;0.01). In addition, sulindac (100 μM) effectively decreased cellular adhesion by 31% in MES cells and by 23% in OVCAR5 cells (p&amp;lt;0.01). These results were confirmed by Western immunoblotting demonstrating that sulindac downregulated the anti-apoptotic proteins Bcl-xl and Mcl-1 and upregulated the pro-apoptotic proteins Bax and cellular stress proteins Bip, ATF-4, and PDI, in both cell lines. Sulindac downregulated expression of several epithelial-mesenchymal transition proteins in both cell lines, including beta-catenin, snail, and slug. Sulindac also downregulated expression of cell cycle proteins CDK4, CDK5, and Cyclin D1 and inflammatory protein COX-2 in both cell lines. After 4 wks of treatment, sulindac significantly inhibited tumor growth by 71% (1.89 g vs 0.54 g, p&amp;lt;0.01) in KpB mice. Conclusions: Our results find that sulindac exhibited anti-tumorigenic effects in OC cell lines and the KpB OC mouse model. Given these promising pre-clinical results, further studies on the repurposing of sulindac for the prevention and treatment of OC is warranted. Citation Format: Nikita Sinha, Shuning Chen, Jennifer Haag, Xiaochang Shen, Boer Deng, Ziyi Zhao, Chunxiao Zhou, Victoria Bae-Jump. Sulindac exhibits anti-proliferative effects in pre-clinical models of ovarian 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 5909.

Multi-omics characterization of partial chemical reprogramming reveals evidence of cell rejuvenation

Partial reprogramming by cyclic short-term expression of Yamanaka factors holds promise for shifting cells to younger states and consequently delaying the onset of many diseases of aging. However, the delivery of transgenes and potential risk of teratoma formation present challenges for in vivo applications. Recent advances include the use of cocktails of compounds to reprogram somatic cells, but the characteristics and mechanisms of partial cellular reprogramming by chemicals remain unclear. Here, we report a multi-omics characterization of partial chemical reprogramming in fibroblasts from young and aged mice. We measured the effects of partial chemical reprogramming on the epigenome, transcriptome, proteome, phosphoproteome, and metabolome. At the transcriptome, proteome, and phosphoproteome levels, we saw widescale changes induced by this treatment, with the most notable signature being an upregulation of mitochondrial oxidative phosphorylation. Furthermore, at the metabolome level, we observed a reduction in the accumulation of aging-related metabolites. Using both transcriptomic and epigenetic clock-based analyses, we show that partial chemical reprogramming reduces the biological age of mouse fibroblasts. We demonstrate that these changes have functional impacts, as evidenced by changes in cellular respiration and mitochondrial membrane potential. Taken together, these results illuminate the potential for chemical reprogramming reagents to rejuvenate aged biological systems and warrant further investigation into adapting these approaches for in vivo age reversal.

Multi-omics characterization of partial chemical reprogramming reveals evidence of cell rejuvenation.

Partial reprogramming by cyclic short-term expression of Yamanaka factors holds promise for shifting cells to younger states and consequently delaying the onset of many diseases of aging. However, the delivery of transgenes and potential risk of teratoma formation present challenges for in vivo applications. Recent advances include the use of cocktails of compounds to reprogram somatic cells, but the characteristics and mechanisms of partial cellular reprogramming by chemicals remain unclear. Here, we report a multi-omics characterization of partial chemical reprogramming in fibroblasts from young and aged mice. We measured the effects of partial chemical reprogramming on the epigenome, transcriptome, proteome, phosphoproteome, and metabolome. At the transcriptome, proteome, and phosphoproteome levels, we saw widescale changes induced by this treatment, with the most notable signature being an upregulation of mitochondrial oxidative phosphorylation. Furthermore, at the metabolome level, we observed a reduction in the accumulation of aging-related metabolites. Using both transcriptomic and epigenetic clock-based analyses, we show that partial chemical reprogramming reduces the biological age of mouse fibroblasts. We demonstrate that these changes have functional impacts, as evidenced by changes in cellular respiration and mitochondrial membrane potential. Taken together, these results illuminate the potential for chemical reprogramming reagents to rejuvenate aged biological systems and warrant further investigation into adapting these approaches for in vivo age reversal.

Abstract 5579: MGAloss-of-function regulates mitochondrial oxidative metabolism-induced cancer stem cell phenotype during lymphomagenesis with therapeutic potential

Abstract As an antagonist of MYC oncogene, Max gene associated (MGA) gene are implicated in multiple cancers, proposing the potential role of tumor suppressor. Using multi-omics data of The Cancer Genome Atlas (TCGA, n=10,295) and lymphoma datasets from our group (n=702), we defined genetic features associated with MGA and its related MYC/MAX/MXD network members across 36 cancers. Pan-cancer, MGA mutation, predominant as nonsense or frameshift, was observed in 4.39% of all samples. MGA expression was significantly downregulated in mutated samples than that of wild type samples. Moreover, MGA mutation was positively associated with inferior clinical outcomes in diffuse large B-cell lymphoma (P=0.001 and P&amp;lt;0.001 in 2 independent datasets), peripheral T-cell lymphoma (P=0.044) and natural killer/T-cell lymphoma (P=0.012), encouraging in-depth study on the important role of MGA in lymphomagenesis. Conditional knockout of MGA in hematopoietic system using Vav-iCre (Mgafl/fl, Vav-iCre mice) led to reduction in the numbers of peripheral blood lymphocyte due to blockade differentiation and enhanced stemness as evidenced by flowcytometry and RNA-sequencing (RNA-seq). To investigate the molecular mechanism, we performed pulldown assay in NK-92 cells molecular silencing of MGA or scramble control, and subsequently subjected for mass spectrum analysis. A total of 97 proteins were identified to interaction with MGA and mainly enriched to nucleosome (26), cytosolic ribosome (16), immune response (13), and glycolysis (6). Molecular silencing of MGA, localizing in the nucleolus, interfered with ribosomal RNA processing, enhanced “onco-ribosome” biogenesis, and contributed to aberrant translational program characterized by mitochondrial oxidative metabolism. Changes in mitochondrial respiration and membrane potential are key to maintaining stemness in cancer cells. Considering that stemness is important for tumorigenesis, recurrence, metastasis, and drug resistance, we exploited high-throughput drug screening platform and found that targeted inhibition of Exportin 1 (XPO1) significantly suppressed the viability of tumor cells molecular silencing MGA, which were resistant to chemotherapeutic agents such as gemcitabine, etoposide, etc. Mechanically, molecular silencing MGA induced abnormalities of nucleocytoplasmic localization in 739 proteins involving in mitochondrial respiratory chain complex I assembly, oxidative phosphorylation, and mitochondrial translation, 50% (332/739) of which were rescued by treatment with XPO1 inhibitor Selinexor. In summary, MGA initiated lymphoma cell stemness via onco-ribosome mediated activation of mitochondrial oxidative metabolism. Selinexor targeted the aberrant nucleocytoplasmic localization and served as a promising therapeutic agent. Citation Format: Jie Xiong, Wen-Fang Wang, Wei-Li Zhao. MGAloss-of-function regulates mitochondrial oxidative metabolism-induced cancer stem cell phenotype during lymphomagenesis with therapeutic potential [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 5579.

Rational design of a red-emission fluorescent probe for imaging mitochondrial viscosity and fatty liver tissues

Abnormal mitochondrial viscosity is closely associated with various diseases. Thus, it is imperative to design and synthesized fluorescent probes for monitoring changes in mitochondrial viscosity. In this work, two series of fluorescence probes with triphenylamine unit and 2-hydroxyphenylbenzothiazole unit respectively were constructed. The impact of varying chain lengths on the photophysical properties of these probes was investigated. The results indicated that probe TPAC12 with dodecane group exhibited the largest fluorescence enhancement. The results of cell imaging experiments demonstrated that all these probes exhibited specific targeting towards mitochondria and were not influenced by changes in mitochondrial membrane potential. Additionally, probe TPAC12 facilitated the monitoring of viscosity alterations within mitochondria. Importantly, probe TPAC12 has been effectively utilized for the identification of both fatty liver and normal liver tissues. These results suggested that probe TPAC12 holds great potential as a valuable tool for detecting mitochondrial viscosity in biological systems.

Adaptation of the technique of determination of mitochondrial membrane potential change in C2C12 cells using the ready set “Mitochondrial Membrane Potential Kit” (Sigma-Aldrich)

   Background. We used the “Mitochondrial Membrane Potential Kit” (Sigma-Aldrich) to detected MMP but encountered difficulties by applied this kit because in manual there not JC-10 concentration and it not allowing for account cell type, size, density, differences in incubation time for different cell cultures.   Objective. Adaptation of the method for determining MMP (mitochondrial membrane potential) in C2C12 cells using in microplate reader and electron microscope.   Design and methods. MMP in C2C12 cells was measured by two way, using fluorescence microscopy (Zeiss, Zen program) and using a plate fluorimeter (CLARIOstar (BMG LABTECH). JC-10 and TMRE dyes (Sigma-Aldrich) were used as fluorescent probes.   Results. Optimal conditions for detection changes in mitochondrial membrane potential in C2C12 cells were selected. 100- fold dilution of the dye JC-10 (Dye Loading Solution) and replacement of the manufactured buffer to PBS led to repeatability and reproducibility results.   Conclusion. When using ready-made kits for measuring MMP, the method proposed by the manufacturer may not be suitable for the selected cell line. In our study to mouse myoblasts of the C2C12 line, a dilution of the dye for loading was required 100 times compared to that recommended by the manufacturer.

Antimicrobial peptide A9K as a gene delivery vector in cancer cells

Designed peptides are promising biomaterials for biomedical applications. The amphiphilic cationic antimicrobial peptide (AMP), A9K, can self-assemble into nano-rod structures and has shown cancer cell selectivity and could therefore be a promising candidate for therapeutic delivery into cancer cells. In this paper, we investigate the selectivity of A9K for cancer cell models, examining its effect on two human cancer cell lines, A431 and HCT-116. Little or no activity was observed on the control, human dermal fibroblasts (HDFs). In the cancer cell lines the peptide inhibited cellular growth through changes in mitochondrial morphology and membrane potential while remaining harmless towards HDFs. In addition, the peptide can bind to and protect nucleic acids while transporting them into both 2D cultures and 3D spheroids of cancer cells. A9K showed high efficiency in delivering siRNA molecules into the centre of the spheroids. A9K was also explored in vivo, using a zebrafish (Danio rerio) development toxicity assay, showing that the peptide is safe at low doses. Finally, a high-content imaging screen, using RNA interference (RNAi) targeted towards cellular uptake, in HCT-116 cells was carried out. Our findings suggest that active cellular uptake is involved in peptide internalisation, mediated through clathrin-mediated endocytosis. These new discoveries make A9K attractive for future developments in clinical and biotechnological applications.

Osteoblastic differentiation and changes in the redox state in pulp stem cells by laser treatment

The aim of this study was to determine the effect of low-level laser therapy (LLLT) on cell proliferation, mitochondrial membrane potential changes (∆Ψm), reactive oxygen species (ROS), and osteoblast differentiation of human dental pulp stem cells (hDPSCs). These cells were irradiated with 660- and 940-nm lasers for 5 s, 50 s, and 180 s. Cell proliferation was assessed using the resazurin assay, cell differentiation by RUNX2 and BMP2 expression, and the presence of calcification nodules using alizarin-red S staining. ROS was determined by the dichlorofluorescein-diacetate technique and changes in ∆Ψm by the tetramethylrhodamine-ester assay. Data were analyzed by a Student’s t-test and Mann–Whitney U test. The 940-nm wavelength for 5 and 50 s increased proliferation at 4 days postirradiation. After 8 days, a significant decrease in proliferation was observed in all groups. Calcification nodules were evident in all groups, with a greater staining intensity in cells treated with a 940-nm laser for 50 s, an effect that correlated with increased RUNX2 and BMP2 expression. ROS production and Δψm increased independently of irradiation time. In conclusion, photobiomodulation (PBM) with LLLT induced morphological changes and reduced cell proliferation rate, which was associated with osteoblastic differentiation and increased ROS and Δψm, independent of wavelength and time.