Dysregulation Of Mitochondrial Function Research Articles

Abstract 4840: Dysregulation of mitochondrial function by PLK1-mediated PDHA1 phosphorylation promotes Cr(VI)-associated lung cancer progression

Abstract Hexavalent chromium (Cr(VI)), an class I environmental carcinogen, not only induces lung epithelial cell transformation but also promotes lung cancer progression by alterations of the cell cycle and cellular energy metabolism. Using Cr(VI)-transformed (CrT) bronchial epithelial cells (BEAS-2B) and parental BEAS-2B cells, we demonstrate that the level of polo-like kinase 1 (PLK1) is highly upregulated in CrT cells, which blocks mitochondrial function, and further promotes cell proliferation both in vitro and in vivo. Cells expressing a high level of PLK1 exhibited repressed mitochondrial activity due to defective modulation of pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1), which facilitates the carbon influx to TCA cycle via catalyzing pyruvate/Acetyl-CoA conversion. Mechanistically, we show that PDHA1 can be directly phosphorylated by PLK1 at T57, which triggers collapse of E1 and PDHA1 degradation via activation of mitophagy. These defects resulted in the inhibition of oxidative phosphorylation and reduction of mitochondrial reactive oxygen species (ROS) generation, eventually inhibiting mitochondrial-mediated apoptotic response. Defining the role of PLK1 in metabolic reprogramming in Cr(VI)-associated cancer progression may give us a new perspective and a target to inhibit Cr(VI)-induced cancer development. In addition, PLK1 inhibitors may be used to increase the chemo-sensitivity of cancer cells by restoring the normal function of mitochondria, thus alleviating the drug resistance caused by dysfunction and hyperpolarization of mitochondria. Citation Format: Qiongsi Zhang, Zhiguo Li, Xiongjian Rao, Derek B. Allison, Qi Qiao, Zhuangzhuang Zhang, Yifan Kong, Ruixin Wang, Teresa W. Fan, Richard M. Higashi, Andrew N. Lane, Xiaoqi Liu. Dysregulation of mitochondrial function by PLK1-mediated PDHA1 phosphorylation promotes Cr(VI)-associated lung cancer progression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4840.

Increased Expression of the Mitochondrial Glucocorticoid Receptor Enhances Tumor Aggressiveness in a Mouse Xenograft Model

Mitochondria are important organelles for cellular physiology as they generate most of the energy requirements of the cell and orchestrate many biological functions. Dysregulation of mitochondrial function is associated with many pathological conditions, including cancer development. Mitochondrial glucocorticoid receptor (mtGR) is proposed as a crucial regulator of mitochondrial functions via its direct involvement in the regulation of mitochondrial transcription, oxidative phosphorylation (OXPHOS), enzymes biosynthesis, energy production, mitochondrial-dependent apoptosis, and regulation of oxidative stress. Moreover, recent observations revealed the interaction of mtGR with the pyruvate dehydrogenase (PDH), a key player in the metabolic switch observed in cancer, indicating direct involvement of mtGR in cancer development. In this study, by using a xenograft mouse model of mtGR-overexpressing hepatocarcinoma cells, we showed increased mtGR-associated tumor growth, which is accompanied by reduced OXPHOS biosynthesis, reduction in PDH activity, and alterations in the Krebs cycle and glucose metabolism, metabolic alterations similar to those observed in the Warburg effect. Moreover, autophagy activation is observed in mtGR-associated tumors, which further support tumor progression via increased precursors availability. Thus, we propose that increased mitochondrial localization of mtGR is associated with tumor progression possible via mtGR/PDH interaction, which could lead to suppression of PDH activity and modulation of mtGR-induced mitochondrial transcription that ends up in reduced OXPHOS biosynthesis and reduced oxidative phosphorylation versus glycolytic pathway energy production, in favor of cancer cells.

Advances in Human Mitochondria-Based Therapies

Mitochondria are the key biological generators of eukaryotic cells, controlling the energy supply while providing many important biosynthetic intermediates. Mitochondria act as a dynamic, functionally and structurally interconnected network hub closely integrated with other cellular compartments via biomembrane systems, transmitting biological information by shuttling between cells and tissues. Defects and dysregulation of mitochondrial functions are critically involved in pathological mechanisms contributing to aging, cancer, inflammation, neurodegenerative diseases, and other severe human diseases. Mediating and rejuvenating the mitochondria may therefore be of significant benefit to prevent, reverse, and even treat such pathological conditions in patients. The goal of this review is to present the most advanced strategies using mitochondria to manage such disorders and to further explore innovative approaches in the field of human mitochondria-based therapies.

Sigma-1 receptor is involved in modification of ER-mitochondria proximity and Ca2+ homeostasis in cardiomyocytes

The Sigma-1 receptor (Sigmar1) is downregulated in heart failure model mice with mitochondrial dysfunction. However, the mechanism in detail has not been investigated. In this study, we investigated the role of Sigmar1 in ER-mitochondria proximity using Sigmar1-knockdown or -overexpressed neonatal rat ventricular myocytes (NRVMs). The endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy was aggravated with the dysregulation of mitochondrial function and ER-mitochondrial junctional formation in Sigmar1-knockdown NRVMs, whereas improved in Sigmar1 overexpressed NRVMs. Our data suggests that the reduction of the cardiac Sigmar1 results in decrease mitochondrial Ca2+ influx and promotes mitochondrial fission, followed by reduced ER-mitochondria proximity, exacerbating ET-1-induced cardiomyocyte injury.

Nanofibrillar peptide hydrogels for self-delivery of lonidamine and synergistic photodynamic therapy

The use of lonidamine (LND) in photodynamic therapy (PDT) provides a viable approach to develop low-dose PDT modules with high efficacy, for LND potentiates cytotoxicity of photosensitizers through dysregulation of mitochondrial function. Yet, the efficacy of LND is restricted by its low accumulation in cancer cells, especially in the mitochondrial compartments. To address the problem, we design an LND-derived self-assembling peptide molecule (LND-K) that dually targets integrin receptors and mitochondria of cancer cells. The targeted cellular delivery of LND-K gives higher efficacy in ablation of mitochondrial function in melanoma cells A375, as compared to free LND or the control molecule that lacks mitochondria-targeting moieties. To integrate LND-K in a typical PDT module, we develop a nanofibrillar hydrogel system through co-assembly of LND-K and TPPS4, an anionic photosensitizer that forms tight electrostatic interactions with cationic residues of LND-K. Notably, hydrogel formulation of LND-K/TPPS4 facilitates slow release of TPPS4 over 14 days in vitro, and displays a longer retention time than aqueous solution of TPPS4in vivo. By integrating a mitochondria-targeted molecule (LND-K) in a typical PDT module, we achieve synergistic killing of A375 cells with dual drugs, where LND-K not only serves as a chemotherapeutic drug, but also potentiates the cytotoxicities of TPPS4 toward A375 cells in vitro and in vivo. The peptide-based drug self-delivery system promises the development of efficacious combination treatments against cancer, that integrate cell sensitization with existing anticancer modules (e.g., chemotherapy and PDT) for enhanced therapeutic efficacy. Statement of significanceThis study reports the design and synthesis of a lonidamine (LND)-derived self-assembling peptide (LND-K) that dually targets integrin receptors and mitochondria of cancer cells. Under the precision guidance of a mitochondria-targeting sequence, LND-K-containing nanofibers target mitochondria and ablate mitochondrial functions. On one hand, the targeted delivery of LND-K reduces cell viabilities through a chemotherapy route; on the other hand, LND-K sensitizes cancer cells for subsequent PDT treatment with enhanced efficacy, which is mediated by induction of ROS, loss of mitochondrial membrane potential, and decrease of cellular ATP level. We believe that the design of mitochondria-targeted drug delivery systems with a self-assembling molecule provides a new approach to potentiate cytotoxicity of photosensitizers in a low-dose PDT module.

The significance of oxidative stress in the pathophysiology of Long COVID and Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS)

Long COVID is now well accepted as an ongoing post-viral syndrome resulting from infection of a single virus, the pandemic SARS-CoV-2. It mirrors the post-viral fatigue syndrome, Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome, a global debilitating illness arising mainly from sporadic geographically-specific viral outbreaks, and from community endemic infections, but also from other stressors. Core symptoms of both syndromes are post-exertional malaise (a worsening of symptoms following mental or physical activity), pervasive fatigue, cognitive dysfunction (brain fog), and sleep disturbance. Long COVID patients frequently also suffer from shortness of breath, relating to the lung involvement of the SARS-CoV-2 virus. There is no universally accepted pathophysiology, or recognized biomarkers yet for Long COVID or indeed for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Clinical case definitions with very similar characteristics for each have been defined. Chronic inflammation, immune dysfunction, and disrupted energy production in the peripheral system has been confirmed in Long COVID and has been well documented in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Neuroinflammation occurs in the brain in Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome as shown from a small number of positron emission tomography and magnetic resonance spectroscopy studies, and has now been demonstrated for Long COVID. Oxidative stress, an increase in reactive oxygen and reactive nitrogen species, and free radicals, has long been suggested as a potential cause for many of the symptoms seen in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome, resulting from both activation of the brain’s immune system and dysregulation of mitochondrial function throughout the body. The brain as a high producer of energy may be particularly susceptible to oxidative stress. It has been shown in peripheral immune cells that the balanced production of proteins involved in regulation of the reactive oxygen species in mitochondria is disturbed in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Fluctuations in the chronic low level neuroinflammation during the ongoing course of Long COVID as well as Myalgic Encephalomyelitis/Chronic Fatigue Syndrome have been proposed to cause the characteristic severe relapses in patients. This review explores oxidative stress as a likely significant contributor to the pathophysiology of Long COVID and Myalgic Encephalomyelitis/Chronic Fatigue Syndrome, and the mechanisms by which oxidative stress could cause the symptoms seen in both syndromes. Treatments that could mitigate oxidative stress and thereby lessen the debilitating symptoms to improve the life of patients are discussed.

Placental mitochondrial function as a driver of angiogenesis and placental dysfunction.

The placenta is a highly vascularized and complex foetal organ that performs various tasks, crucial to a healthy pregnancy. Its dysfunction leads to complications such as stillbirth, preeclampsia, and intrauterine growth restriction. The specific cause of placental dysfunction remains unknown. Recently, the role of mitochondrial function and mitochondrial adaptations in the context of angiogenesis and placental dysfunction is getting more attention. The required energy for placental remodelling, nutrient transport, hormone synthesis, and the reactive oxygen species leads to oxidative stress, stemming from mitochondria. Mitochondria adapt to environmental changes and have been shown to adjust their oxygen and nutrient use to best support placental angiogenesis and foetal development. Angiogenesis is the process by which blood vessels form and is essential for the delivery of nutrients to the body. This process is regulated by different factors, pro-angiogenic factors and anti-angiogenic factors, such as sFlt-1. Increased circulating sFlt-1 levels have been linked to different preeclamptic phenotypes. One of many effects of increased sFlt-1 levels, is the dysregulation of mitochondrial function. This review covers mitochondrial adaptations during placentation, the importance of the anti-angiogenic factor sFlt-1in placental dysfunction and its role in the dysregulation of mitochondrial function.

Latent HIV‐1 Exosomes Induce Mitochondrial Hyperfusion due to Loss of Phosphorylated Dynamin‐related Protein 1 in Brain Endothelium

Background Damage to the cerebral vascular endothelium is a critical initiating event in the development of HIV-1-associated neurocognitive disorders (HAND). To study the role of mitochondria in cerebral endothelial dysfunction, we investigated how exosomes, isolated from both cell lines with integrated provirus and HIV-1 infected primary cells (HIV-exosomes), accelerate the dysfunction of either primary human brain microvascular endothelial cells (HBMVECs) in vitro or mice brain microvessels (MVs), ex vivo. Methods Exosomes were isolated by either ultracentrifugation from uninfected and latent HIV-1 infected T-cells (Jurkat Exo and J-Lat(9.2) Exo, respectively) and monocytes (U937 Exo and U1 Exo, respectively) or by using QIAGEN exoEasy Maxi kit from HIV-1 infected human peripheral blood mononuclear cells (HIV+PBMCs Exo). Exosomes were characterized by the qNano-IZON system. Initially, the assessment of mitochondrial characteristics was performed in primary HBMVECs, and this study was further extended in extracted cerebral MVs from mice brain expressing mito-Dendra2+ fluorescence protein (Green) in endothelial mitochondria. Results Quantitative analysis of the extracellular vesicles (EVs) indicates that the isolated EVs were predominantly exosomes. This was further supported by the detection of exosomal markers, e.g. CD63, CD81, Alix, TSG101 and the absence of large EV-related protein, GRP94. These exosomes were readily taken up by both primary HBMVECs and mice cerebral MVs. HIV-exosomes induced cellular and mitochondrial superoxide production but reduced mitochondrial membrane potential in HBMVECs. HIV-exosomes increased mitochondrial hyperfusion, possibly due to the loss of phosphorylated dynamin-related protein 1 (p-DRP1). We further confirmed that HIV-exosomes, containing the HIV-Tat protein, and viral Tat protein accelerate brain endothelial dysfunction due to reduction in the expressions of (a) p-DRP1, (b) phosphorylated endothelial nitric oxide synthase (p-eNOS), and (c) blood brain barrier related tight junction proteins. Finally, HIV-1 infected primary human PBMCs produced more exosomes than uninfected controls, and exposure to HIV+PBMCs Exo also reduced both p-DRP1 and p-eNOS expressions in primary HBMVECs. Conclusions Our novel findings reveal the significant role of HIV-exosomes on dysregulation of mitochondrial function, which induces adverse changes in the function of the brain microvascular endothelium. These mechanistic studies using HIV-exosomes may elucidate the etiology of HAND and provide novel therapeutic strategies.

Latent HIV-Exosomes Induce Mitochondrial Hyperfusion Due to Loss of Phosphorylated Dynamin-Related Protein 1 in Brain Endothelium

Damage to the cerebral vascular endothelium is a critical initiating event in the development of HIV-1-associated neurocognitive disorders. To study the role of mitochondria in cerebral endothelial dysfunction, we investigated how exosomes, isolated from both cell lines with integrated provirus and HIV-1 infected primary cells (HIV-exosomes), accelerate the dysfunction of primary human brain microvascular endothelial cells (HBMVECs) by inducing mitochondrial hyperfusion, and reducing the expression of phosphorylated endothelial nitric oxide synthase (p-eNOS). The quantitative analysis of the extracellular vesicles (EVs) indicates that the isolated EVs were predominantly exosomes. It was further supported by the detection of exosomal markers, and the absence of large EV-related protein in the isolated EVs. The exosomes were readily taken up by primary HBMVECs. HIV-exosomes induce cellular and mitochondrial superoxide production but reduce mitochondrial membrane potential in HBMVECs. HIV-exosomes increase mitochondrial hyperfusion, possibly due to loss of phosphorylated dynamin-related protein 1 (p-DRP1). HIV-exosomes, containing the HIV-Tat protein, and viral Tat protein reduce the expression of p-DRP1 and p-eNOS, and accelerate brain endothelial dysfunction. Finally, exosomes isolated from HIV-1 infected primary human peripheral blood mononuclear cells (hPBMCs) produce more exosomes than uninfected controls and reduce both p-DRP1 and p-eNOS expressions in primary HBMVECs. Our novel findings reveal the significant role of HIV-exosomes on dysregulation of mitochondrial function, which induces adverse changes in the function of the brain microvascular endothelium.

Fenpropathrin induces degeneration of dopaminergic neurons via disruption of the mitochondrial quality control system

The synthetic pyrethroid derivative, fenpropathrin, is a widely used insecticide. However, a variety of toxic effects in mammals have been reported. In particular, fenpropathrin induces degeneration of dopaminergic neurons and parkinsonism. However, the mechanism of fenpropathrin-induced parkinsonism has remained unknown. In the present study, we investigated the toxic effects and underlying mechanisms of fenpropathrin on perturbing the dopaminergic system both in vivo and in vitro. We found that fenpropathrin induced cellular death of dopaminergic neurons in vivo. Furthermore, fenpropathrin increased the generation of reactive oxygen species, disrupted both mitochondrial function and dynamic networks, impaired synaptic communication, and promoted mitophagy in vitro. In mice, fenpropathrin was administered into the striatum via stereotaxic (ST) injections. ST-injected mice exhibited poor locomotor function at 24 weeks after the first ST injection and the number of tyrosine hydroxylase (TH)-positive cells and level of TH protein in the substantia nigra pars compacta were significantly decreased, as compared to these parameters in vehicle-treated mice. Taken together, our results demonstrate that exposure to fenpropathrin induces a loss of dopaminergic neurons and partially mimics the pathologic features of Parkinson’s disease. These findings suggest that fenpropathrin may induce neuronal degeneration via dysregulation of mitochondrial function and the mitochondrial quality control system.

Fanning the Flames of Inflammaging: Impact of Monocyte Metabolic Reprogramming.

Monocytes are circulating innate immune cells that are functionally dysregulated during aging. However, while metabolic regulation of innate immune cell function is now well-established, only a handful of studies have examined this in the context of aging. In a recent article published in Aging Cell, Saare et al. observe comprehensive metabolic reprogramming of otherwise unstimulated monocytes isolated from older adults. These cells display increased glucose uptake and dysregulation of mitochondrial function, concomitant with activation of signaling pathways contributing to increased inflammation. These findings suggest a mechanism whereby metabolic reprogramming in aged monocytes contributes to chronic low-grade inflammation and open new avenues of investigation into the biological underpinning of inflammaging.

Alterations of Mitochondria and Related Metabolic Pathways in Leukemia: A Narrative Review.

Dysregulation of mitochondrial function often precedes malignant transformation of hematopoietic stem cells (HSCs). Mitochondria have a direct role in the maintenance of HSC functions. For example, D-2-hydroxyglutarate, generated due to the activity of mutated mitochondrial isocitrate dehydrogenase (IDH), has been implicated in the pathogenesis of leukemia. Furthermore, disturbances in the fatty acid breakdown and pyruvate oxidation are often seen in leukemic cells. These and other abnormalities expedite leukemogenesis and chemoresistance of leukemic cells. However, it needs to be elucidated whether these aberrations are the result or cause of leukemogenesis. Accordingly, for this review, a search was carried out in PubMed and Google Scholar databases until June 2019 to assess the relationship between metabolic pathways in altered mitochondria and leukemia development. In the present review, an overview of mitochondria-related mechanisms and their abnormalities in leukemia is presented, with mitochondrial pathways and factors, such as mitophagy, intermediary metabolism enzymes, oncometabolites and reactive oxygen species' generation, discussed as potential diagnostic and therapeutic targets in leukemia.

Abstract 432: Rapamycin Can Ameliorate Antiretroviral Drug Mediated Cardiotoxicity

Introduction: In current therapy, due to application of combined antiretroviral drugs (cART), HIV related mortality significantly reduced, and AIDS became a chronic disease. Although, cART treatment increases life expectancy of AIDS patients, growing AIDS population suffer due to several organ diseases and heart failure is one of the leading causes of comorbidity and mortality. Hypothesis: We tested the hypothesis that application of cART induces cardiotoxicity through inhibition of cellular protein quality control (PQC) and dysregulation of mitochondrial function. Method and results: Cellular protein quality control was analyzed using cardiac tissue samples from HIV+ patients treated with cART and healthy donors. Western blot shows that there is upregulation of autophagy marker protein LC3 II in ART treated heart compared to healthy donor. Additionally, we found that there are abnormalities in the mitochondrial oxidative phosphorylation complexes (OXOPHOS) in the ART treated patient’s heart, compared to healthy donor heart. Using neonatal rat ventricular cardiomyocytes, we tested the role of ART in cellular PQC and mitochondrial function. Our data shows that cardiomyocytes treated with the cART (Ritonavir+ Atazanavir+ abacavir+ Lamivudine) have increased expression of autophagy proteins LC3 II and P62 and higher level of pELF2α. These suggest that, the cardiomyocytes treated with ART have dysregulation of autophagy and increased ER stress. Further, we found that cardiomyocytes treated with cART have high level of mitochondrial superoxide and decreased mitochondrial membrane potential. Mitochondrial function detected in whole cells shows that in ART treated cardiomyocytes there is significantly low OCR compared to control cells. Interestingly, application of pharmacological drug rapamycin can improve the cellular PQC and mitochondrial function in ART treated cells. Conclusion: ART treatment induces cardiotoxicity through inhibition of cellular PQC and dysregulation of mitochondrial function and rapamycin might be important therapeutic drug to reduce the ART mediated cardiotoxicity.

Cinnamaldehyde in flavored e-cigarette liquids temporarily suppresses bronchial epithelial cell ciliary motility by dysregulation of mitochondrial function.

Aldehydes in cigarette smoke (CS) impair mitochondrial function and reduce ciliary beat frequency (CBF), leading to diminished mucociliary clearance (MCC). However, the effects of aldehyde e-cigarette flavorings on CBF are unknown. The purpose of this study was to investigate whether cinnamaldehyde, a flavoring agent commonly used in e-cigarettes, disrupts mitochondrial function and impairs CBF on well-differentiated human bronchial epithelial (hBE) cells. To this end, hBE cells were exposed to diluted cinnamon-flavored e-liquids and vaped aerosol and assessed for changes in CBF. hBE cells were subsequently exposed to various concentrations of cinnamaldehyde to establish a dose-response relationship for effects on CBF. Changes in mitochondrial oxidative phosphorylation and glycolysis were evaluated by Seahorse Extracellular Flux Analyzer, and adenine nucleotide levels were quantified by HPLC. Both cinnamaldehyde-containing e-liquid and vaped aerosol rapidly yet transiently suppressed CBF, and exposure to cinnamaldehyde alone recapitulated this effect. Cinnamaldehyde impaired mitochondrial respiration and glycolysis in a dose-dependent manner, and intracellular ATP levels were significantly but temporarily reduced following exposure. Addition of nicotine had no effect on the cinnamaldehyde-induced suppression of CBF or mitochondrial function. These data indicate that cinnamaldehyde rapidly disrupts mitochondrial function, inhibits bioenergetic processes, and reduces ATP levels, which correlates with impaired CBF. Because normal ciliary motility and MCC are essential respiratory defenses, inhalation of cinnamaldehyde may increase the risk of respiratory infections in e-cigarette users.

Stephanthraniline A suppresses proliferation of HCT116 human colon cancer cells through induction of caspase-dependent apoptosis, dysregulation of mitochondrial function, cell cycle arrest and regulation of Akt/p38 signaling pathways.

Stephanthraniline A (STA) is a C21 steroidal aglycone isolated from the stem of Stephanotis mucronata (Blanco) Merr. that exerts growth inhibition in human colon cancer cells. However, the intracellular molecular mechanisms whereby this occurs have not been well characterized. In this study, we found that STA significantly inhibits the growth of HCT116 colon cancer cells in a time- and concentration-dependent manner. The inhibitory effect of STA on cell growth was related to the induction of apoptosis. Activated caspase-3, caspase-8 and caspase-9, along with a decreased Bcl-2/Bcl-x ratio and loss of mitochondrial membrane potential (Δψm), were observed in response to STA treatment. Furthermore, treatment of HCT116 cells with STA resulted in G0/G1 phase cell cycle arrest accompanied by decreased mRNA levels of cyclin-dependent kinase 4 (CDK4), p21 and c-myc. Additionally, the inhibition of Akt signaling and activation of p38 signaling were observed after treatment with STA in HCT116 cells. These findings indicate that STA inhibits HCT116 cell growth by promoting apoptosis, the dysregulation of mitochondrial function, and cell cycle arrest.

Cinnamaldehyde in flavored e-cigarette liquids temporarily suppresses bronchial epithelial cell ciliary motility by dysregulation of mitochondrial function

Cinnamaldehyde in flavored e-cigarette liquids temporarily suppresses bronchial epithelial cell ciliary motility by dysregulation of mitochondrial function

Dysregulation of mitochondrial function and biogenesis modulators in adipose tissue of obese children.

We aimed to evaluate mitochondrial biogenesis (MB), structure, metabolism and dysfunction in abdominal adipose tissue from male pediatric patients with obesity. Samples were collected from five children with obesity (percentile ⩾95) and five eutrophic boys (percentile ⩾5/⩽85) (8-12 years old) following parental informed consent. We analyzed the expression of key genes involved in MB (sirtuin-1 (SIRT1), peroxisome proliferator-activated receptor-γ (PPARγ), PPARγ coactivator-1α (PGC1α), nuclear respiratory factors 1 and 2 (NRF1, NRF2) and mitochondrial transcription factor A (TFAM) and surrogates for mitochondrial function/structure/metabolism (porin, TOMM20, complex I and V, UCP1, UCP2, SIRT3, SOD2) by western blot. Citrate synthase (CS), complex I (CI) activity, adenosine triphosphate (ATP) levels, mitochondrial DNA (mtDNA) content and oxidative stress end points were also determined. Most MB proteins were significantly decreased in samples from children with obesity except complex I, V and superoxide dismutase-2 (SOD2). Similarly, CS and CI activity showed a significant reduction, as well as ATP levels and mtDNA content. PPARγ, PGC1α, complex I and V and SOD2 were hyperacetylated compared with lean samples. Concurrently, in samples from children with obesity, we found decreased SOD2 activity and redox state imbalance highlighted by decreased reduced glutathione/oxidized glutathione (GSH/GSSG) ratio and significant increases in protein carbonylation. Adipose tissue from children with obesity demonstrates a dysregulation of key modulators of MB and organelle structure, and displays hyperacetylation of key proteins and altered expression of upstream regulators of cell metabolism.

Abstract 4391: Identification of novel cancer driver genes in hepatocellular carcinoma cells by RNAi screening method

Abstract Recent studies revealed that EpCAM is one of the molecular markers for hepatic cancer initiating cells. However, the mechanism how the EpCAM expression in the liver cancer cell is regulated still remains to be delineated. In this study, we identified candidate genes critical for the maintenance of EpCAM positive hepatocellular carcinoma (HCC) cells using a global RNAi screening method. HCC cells were infected with pools of ~200,000 lentiviral shRNA library, then they were sorted to EpCAM positive or negative group. The abundance of shRNA in each group was analyzed using a microarray hybridization method. As a result, 76 candidate shRNAs with significantly different expression between EpCAM positive and negative populations were identified by class comparison analysis. Unsupervised clustering analysis of these genes in 247 HCC samples clearly separated EpCAM positive HCC cases from negative cases. Furthermore, we found that the EpCAM-dependent gene signature can predict survival in epithelial-like HCC cases. We focused on shRNA for PMPCB gene, which has an important role for processing mitochondrial protein, since it ranks as the top differentially altered shRNA-targeted gene between EpCAM positive and negative cells. PMPCB shRNA efficiently reduced EpCAM positive population and effectively inhibited cell proliferation both in vitro and in vivo. Interestingly, PMPCB shRNA specifically induced cell death in EpCAM positive HCC cells, but not in EpCAM negative cells. These data suggest that EpCAM positive cells functionally depend on PMPCB expression. On the other hand, we confirmed that PMPCB shRNA induced physiological dysregulation of mitochondrial function, leading to the increase of reactive oxygen species (ROS) production. We found that PMPCB shRNA significantly reduced TCF4 activity as well as the expression level of c-myc and cyclin D1, which are the downstream molecules of Wnt/β-catenin pathway. Although total β-catenin levels in the cytosol and nuclei fractions were not affected by PMPCB knockdown, overexpression of constitutively activated mutant β-catenin partially rescued the cells from PMPCB-induced apoptosis. Forkhead box O (FOXO) is known to be induced by the ROS production, bind β-catenin and decrease TCF activity, leading to the induction of apoptosis. We found that the nuclear translocation of FOXO family molecules, such as FOXO1 and FOXO3, were facilitated by PMPCB shRNA in EpCAM positive cells. These results suggest that the activation of FOXOs induced by PMPCB knockdown-mediated ROS production suppressed the activity of Wnt/β-catenin pathway, leading to the death of EpCAM positive HCC cells. In conclusion, we discovered a novel candidate target molecule of EpCAM positive HCC cells using a genome-wide RNAi screening method. Our study shed light on the mechanisms how EpCAM positive HCC cells are maintained and identified a new target molecule that has therapeutic vulnerability in EpCAM positive HCC cells. Citation Format: Atsushi Takai, Hien Dang, Naoki Oishi, Xin Wei Wang. Identification of novel cancer driver genes in hepatocellular carcinoma cells by RNAi screening method [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4391. doi:10.1158/1538-7445.AM2017-4391

Parkinson's Disease: From Pathogenesis to Pharmacogenomics.

Parkinson’s disease (PD) is the second most important age-related neurodegenerative disorder in developed societies, after Alzheimer’s disease, with a prevalence ranging from 41 per 100,000 in the fourth decade of life to over 1900 per 100,000 in people over 80 years of age. As a movement disorder, the PD phenotype is characterized by rigidity, resting tremor, and bradykinesia. Parkinson’s disease -related neurodegeneration is likely to occur several decades before the onset of the motor symptoms. Potential risk factors include environmental toxins, drugs, pesticides, brain microtrauma, focal cerebrovascular damage, and genomic defects. Parkinson’s disease neuropathology is characterized by a selective loss of dopaminergic neurons in the substantia nigra pars compacta, with widespread involvement of other central nervous system (CNS) structures and peripheral tissues. Pathogenic mechanisms associated with genomic, epigenetic and environmental factors lead to conformational changes and deposits of key proteins due to abnormalities in the ubiquitin–proteasome system together with dysregulation of mitochondrial function and oxidative stress. Conventional pharmacological treatments for PD are dopamine precursors (levodopa, l-DOPA, l-3,4 dihidroxifenilalanina), and other symptomatic treatments including dopamine agonists (amantadine, apomorphine, bromocriptine, cabergoline, lisuride, pergolide, pramipexole, ropinirole, rotigotine), monoamine oxidase (MAO) inhibitors (selegiline, rasagiline), and catechol-O-methyltransferase (COMT) inhibitors (entacapone, tolcapone). The chronic administration of antiparkinsonian drugs currently induces the “wearing-off phenomenon”, with additional psychomotor and autonomic complications. In order to minimize these clinical complications, novel compounds have been developed. Novel drugs and bioproducts for the treatment of PD should address dopaminergic neuroprotection to reduce premature neurodegeneration in addition to enhancing dopaminergic neurotransmission. Since biochemical changes and therapeutic outcomes are highly dependent upon the genomic profiles of PD patients, personalized treatments should rely on pharmacogenetic procedures to optimize therapeutics.

Antiproliferative activity of Alisol B in MDA-MB-231 cells is mediated by apoptosis, dysregulation of mitochondrial functions, cell cycle arrest and generation of reactive oxygen species.

Previous studies have demonstrated that Alisol B has inhibitory activity in cancer cells. However, the exact mechanism through which inhibition is achieved is still poorly understood. In the present study, the authors examined the effects of Alisol B in human breast cancer cells. Alisol B showed significant anticancer activity in MDA-MB-231 cells. The results demonstrated that the cytotoxicity induced by Alisol B was mediated by induction of apoptosis, decrease in mitochondrial membrane potential, cell cycle arrest, activation of caspases and accumulation of ROS (reactive oxygen species) level. Interestingly, pretreatment of cells with the general caspase inhibitor z-VAD-FMK significantly prevented Alisol B-induced apoptosis. Furthermore, western blot analysis revealed the upregulation of p-p38 and downregulation of p-AKT, p-p65 and p-mTOR. Taken together, the above results suggest that Alisol B suppresses the growth of MDA-MB-231 cells mainly through induction of apoptosis; this outcome may represent the major mechanism of Alisol B-mediated apoptosis.