Uncoupling Protein 2 Inhibition Research Articles

Abstract 692: Uncoupling protein 2 (Ucp2) loss of function enhances mitochondrial ROS and sensitizes PDAC to radiation therapy

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a dependence on highly active mitochondria to grow and metastasize. Normally mitochondria exist in long networks due to mitochondrial fusion but in PDAC mitochondria are fragmented due to excessive fission. We previously found that forcing PDAC cells to increase mitochondrial fusion suppressed tumor growth and metastasis, leading to improved survival in multiple preclinical models. The tumor suppressive mechanisms of mitochondrial fusion remain unclear, and thus we explored the role of redox homeostasis through uncoupling protein 2 (UCP2), a metabolic protein normally upregulated in PDAC that was downregulated upon forced mitochondrial fusion. We hypothesize that UCP2 mediates the oncogenic effects of mitochondrial fragmentation in PDAC. Methods: We explored if Ucp2 is necessary and sufficient for inducing tumor suppression upon mitochondrial fusion. The effects of Ucp2 loss or overexpression were characterized by metabolic flux assays, proliferation, mitochondrial ROS (mROS), membrane potential, and morphology measurements. We also examined if Ucp2 recapitulates mitochondrial fusion-induced tumor suppression via pharmacological inhibition and genetic ablation and assessed the impact on mitochondrial health and cell growth. We utilized the LSL-KRASG12D; Trp53R172H/+; Ptf1⍺ Cre (KPC) cell line as our parental line for all genetic manipulations, with a nontargeting (sgGFP) KPC cell line as a control. Results: Murine PDAC cells with enhanced mitochondrial fusion reduced Ucp2 transcript levels by 70% and was correlated with 3.5-fold increase in mROS, 50% reduction in OXPHOS via oxygen consumption rate assay, and a 4-fold increase in membrane potential compared to controls. Ucp2 re-expression did not rescue these phenotypes, suggesting that Ucp2 alone may not be sufficient for tumor suppression. However, inhibition of Ucp2 in PDAC cells reduced proliferation by 70%, while inducing a 3-fold increase in mROS levels, which was recapitulated with Ucp2 abrogation, thus Ucp2 may be necessary for tumor suppression. Enhanced mROS via Ucp2 inhibition significantly increased radiosensitivity (p-value < 0.0001), suggesting that although it does not recapitulate the mitochondrial fusion phenotype, UCP2 may be a novel target for radiosensitization in PDAC. Conclusions: Mitochondrial fusion may not suppress tumor growth by altering redox homeostasis via UCP2, but forcing mitochondria to fuse elucidated the impacts of UCP2 downregulation on cell metabolism. Thus, we uncovered that UCP2 may be a novel target for radiosensitization. Further insight into UCP2’s role in redox balance, and how it is disrupted with UCP2 modulation enables development of novel therapeutic combinations with UCP2 inhibition that complement and potentially synergize with existing therapeutic strategies such as radiation. Citation Format: Emily G. Caggiano, Meifang Yu, Ariana Acevedo-Diaz, Cullen M. Taniguchi. Uncoupling protein 2 (Ucp2) loss of function enhances mitochondrial ROS and sensitizes PDAC to radiation therapy [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 692.

Mitochondria of lung venular capillaries mediate lung-liver cross talk in pneumonia.

Failure of the lung's endothelial barrier underlies lung injury, which causes the high mortality acute respiratory distress syndrome (ARDS). Multiple organ failure predisposes to the mortality, but mechanisms are poorly understood. Here, we show that mitochondrial uncoupling protein 2 (UCP2), a component of the mitochondrial inner membrane, plays a role in the barrier failure. Subsequent lung-liver cross talk mediated by neutrophil activation causes liver congestion. We intranasally instilled lipopolysaccharide (LPS). Then, we viewed the lung endothelium by real-time confocal imaging of the isolated, blood-perfused mouse lung. LPS caused alveolar-capillary transfer of reactive oxygen species and mitochondrial depolarization in lung venular capillaries. The mitochondrial depolarization was inhibited by transfection of alveolar Catalase and vascular knockdown of UCP2. LPS instillation caused lung injury as indicated by increases in bronchoalveolar lavage (BAL) protein content and extravascular lung water. LPS or Pseudomonas aeruginosa instillation also caused liver congestion, quantified by liver hemoglobin and plasma aspartate aminotransferase (AST) increases. Genetic inhibition of vascular UCP2 prevented both lung injury and liver congestion. Antibody-mediated neutrophil depletion blocked the liver responses, but not lung injury. Knockdown of lung vascular UCP2 mitigated P. aeruginosa-induced mortality. Together, these data suggest a mechanism in which bacterial pneumonia induces oxidative signaling to lung venular capillaries, known sites of inflammatory signaling in the lung microvasculature, depolarizing venular mitochondria. Successive activation of neutrophils induces liver congestion. We conclude that oxidant-induced UCP2 expression in lung venular capillaries causes a mechanistic sequence leading to liver congestion and mortality. Lung vascular UCP2 may present a therapeutic target in ARDS.NEW & NOTEWORTHY We report that mitochondrial injury in lung venular capillaries underlies barrier failure in pneumonia, and venular capillary uncoupling protein 2 (UCP2) causes neutrophil-mediated liver congestion. Using in situ imaging, we found that epithelial-endothelial transfer of H2O2 activates UCP2, depolarizing mitochondria in venular capillaries. The conceptual advance from our findings is that mitochondrial depolarization in lung capillaries mediates liver cross talk through circulating neutrophils. Pharmacologic blockade of UCP2 could be a therapeutic strategy for lung injury.

Immunometabolic modulation by targeting UCP2 and IL-17A boost anti-tumor immunity against pancreatic cancer

Abstract Metabolic reprograming of cytotoxic effectors by tumor microenvironment (TME) is one of the key mechanisms by which TME impairs the success of cancer immunotherapy. Therapeutic strategies that mitigate the immunosuppressive TME and selectively enhance bioenergetic fitness of cytotoxic effectors may enhance benefits of immunotherapy, particularly in TME-abundant pancreatic cancer. IL-17 is a key TME cytokine engaged in a feed-forward loop promoting the immunosuppressive milieu that facilitates cancer progression. Mitochondrial uncoupling protein 2 (UCP2) acts as an inhibitor of T cell metabolic fitness and directly promotes tumorigenesis. Therefore, we hypothesized that targeting IL-17 and UCP2 could act additionally to enhance antitumor immune response and directly inhibit pancreatic cancer growth. UCP2 deficiency regulated bioenergetic metabolism and mTOR signaling in cytotoxic T cells to enhance IFN-γ production and anti-tumor immune responses. UCP2 inhibition increased anti-tumor immunity in PBMCs and tumors of patients with PDAC. The dual inhibition of IL-17 and UCP2 boosted antitumor immunity and suppressed tumorigenesis in tumor-bearing mice. Combination of UCP2 and IL-17 blockade may offer additional complimentary and cooperative mechanisms of antitumor immunity against pancreatic cancer. 1. Ministry of Science and Technology (MOST 108-2320-B-039-024-MY3) 2. China Medical University Hospital (DMR-110-186) 3. Chinese Medicine Research Center, China Medical University from the Featured Areas Research 4. Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (CMRC-CHM-2)

Abstract 4830: Uncoupling protein 2 (UCP2) loss of function mediates PDAC tumor suppression by mitochondrial fusion

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a dependence on highly active mitochondria to grow and metastasize. We previously found that mitochondrial fusion suppressed PDAC growth and metastasis, leading to improved survival in a preclinical model. The tumor suppressive mechanisms of mitochondrial fusion remain unclear, and thus we explored the role of redox homeostasis through uncoupling protein 2 (UCP2). Methods/Results: Murine pancreatic cancer cells (KPC) with enhanced mitochondrial fusion (sgDrp1) exhibited higher levels of mitochondrial ROS compared to controls targeting GFP (sgGFP), suggesting altered redox homeostasis. We performed RNAseq on these two cell lines and found that sgDRP1 cells exhibited reduced levels of UCP2. The UCP2 protein is known to have indirect antioxidant effects by reducing mitochondrial reactive oxygen species via uncoupling of ATP production from the electron transport chain. We found that abrogating UCP2 in KPC cells reduced its capacity for OXPHOS. Moreover, rescue of UCP2 in sgDrp1 cells restored their mitochondrial activity. Conclusions: Mitochondrial fusion may suppress tumor growth and induce mitophagy by altering redox homeostasis. UCP2 may be a mediator of the tumor suppressive phenotype induced by mitochondrial fusion, thus linking mitochondrial dynamics and PDAC antioxidant strategies. Future Work: These results will be explored for their translation in vivo and the effects of pharmacological inhibition of UCP2, both in vitro and in vivo, via the specific inhibitor Genipin, will be explored. Citation Format: Ariana Carolina Acevedo-Diaz, Emily G. Caggiano, Meifang Yu, Cullen Taniguchi. Uncoupling protein 2 (UCP2) loss of function mediates PDAC tumor suppression by mitochondrial fusion. [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 4830.

Oleanolic acid alleviates oxidative stress in Alzheimer's disease by regulating stanniocalcin-1 and uncoupling protein-2 signalling.

Oxidative stress is thought to play an important role in the occurrence and development of Alzheimer's disease (AD) and antioxidants may delay or even treat AD. Oleanolic acid (OA) exhibits antioxidant properties against many diseases. However, its effects on oxidative stress in AD remain unclear. Here, we explored the role and mechanism of action of OA in N2a/APP695swe cells exposed to oxidative stress. The cells were incubated with different concentrations of OA (0, 5, 8, 10, 15, and 25μmol/L) for 24hours. Higher concentrations of OA (10, 15, and 25μmol/L) significantly suppressed the apoptosis, caspase-3 activity, reactive oxygen species level, and β amyloid (Aβ) content and increased the viability of these cells. OA (10μmol/L) also increased the expression of stanniocalcin-1 (STC-1) and uncoupling protein-2 (UCP2) in N2a/APP695swe cells. STC-1 interference markedly reversed the effect of OA on UCP2, indicating that OA may regulate UCP2 expression in N2a/APP695swe cells via STC-1. Moreover, UCP2 inhibition significantly reversed the OA-mediated effects on cell viability, caspase-3 activity, reactive oxygen species, and Aβ level. Thus, OA regulates UCP2 expression via STC-1 to alleviate oxidative stress and Aβ level in N2a/APP695swe cells.

Molecular Mechanisms Responsible for Pharmacological Effects of Genipin on Mitochondrial Proteins

Genipin, a natural compound from Gardenia jasminoides, is a well-known compound in Chinese medicine that is used for the treatment of cancer, inflammation, and diabetes. The use of genipin in classical medicine is hindered because of its unknown molecular mechanisms of action apart from its strong cross-linking ability. Genipin is increasingly applied as a specific inhibitor of proton transport mediated by mitochondrial uncoupling protein 2 (UCP2). However, its specificity for UCP2 is questionable, and the underlying mechanism behind its action is unknown. Here, we investigated the effect of genipin in different systems, including neuroblastoma cells, isolated mitochondria, isolated mitochondrial proteins, and planar lipid bilayer membranes reconstituted with recombinant proteins. We revealed that genipin activated dicarboxylate carrier and decreased the activity of UCP1, UCP3, and complex III of the respiratory chain alongside with UCP2 inhibition. Based on competitive inhibition experiments, the use of amino acid blockers, and site-directed mutagenesis of UCP1, we propose a mechanism of genipin’s action on UCPs. At low concentrations, genipin binds to arginine residues located in the UCP funnel, which leads to a decrease in UCP’s proton transporting function in the presence of long chain fatty acids. At concentrations above 200 μM, the inhibitory action of genipin on UCPs is overlaid by increased nonspecific membrane conductance due to the formation of protein-genipin aggregates. Understanding the concentration-dependent mechanism of genipin action in cells will allow its targeted application as a drug in the above-mentioned diseases.

UCP2 alleviates tubular epithelial cell apoptosis in lipopolysaccharide-induced acute kidney injury by decreasing ROS production

Uncoupling protein 2 (UCP2), an anion transporter, modulates the production of mitochondrial reactive oxygen species (ROS) and plays an important role in protecting against cell apoptosis. However, the role of UCP2 in sepsis-associated AKI remains unclear. In the present study, we investigated the role of UCP2 in LPS-induced AKI in vitro and in vivo. UCP2 expression was increased in tubular epithelial cells (TECs) treated with LPS. Accordingly, UCP2 expression was distinctly upregulated in renal tissues from the animals with LPS-induced AKI. Furthermore, UCP2 silencing dramatically aggravated LPS-induced apoptosis, accompanied by increased ROS production in renal tubular epithelial cell. Additionally, the inhibition of UCP2 by genipin, a specific UCP2 inhibitor, exacerbated the kidney injury of animals with LPS-induced AKI. Moreover, NAC (N-acetylcysteine), a potent ROS scavenger, obviously suppressed apoptosis induced by UCP2 silencing, which suggests that the increased ROS levels were associated with tubular epithelial cell apoptosis induced by UCP2 silencing. Therefore, UCP2 exerts a protective effect on the LPS-induced apoptosis of tubular epithelial cells by reducing excess ROS production. In conclusion, our findings highlight the renoprotective actions of UCP2 on inhibiting the production of apoptotic factors and oxidative stress to improve tubular cell survival in the LPS-induced AKI model.

Uncoupling Protein 2 Drives Myocardial Dysfunction in Murine Models of Septic Shock.

Cardiac dysfunction is a major component of sepsis-induced multiorgan failure in critical care units. Uncoupling protein 2 (UCP2) involves immune response, regulation of oxidative stress, and maintenance of mitochondrial membrane potential as well as energy production. However, whether and how UCP2 plays roles in the development of septic cardiac dysfunction are largely unknown. Here, intraperitoneal injection of LPS significantly activated UCP2 expression accompanied by a significant decrease of cardiac function and caused a significantly lower survival rate in mice. Of note, knockdown of UCP2 through a cardiotropic adenoassociated viral vector carrying a short hairpin RNA (shRNA) specifically targeting the UCP2 evoked resistance to LPS-triggered septic cardiac dysfunction and lethality in vivo. Moreover, UCP2 deficiency ameliorated the reduced levels of intracellular ATP in the LPS-challenged heart tissues and preserved mitochondrial membrane potential loss in primary adult mouse cardiomyocytes in LPS-challenged animals. Mechanistically, we confirmed that the inhibition of UCP2 promoted autophagy in response to LPS, as shown by an increase in LC3II and a decrease in p62. At last, the autophagy inhibitor 3-MA abolished UCP2 knockdown-afforded cardioprotective effects. Those results indicate that UCP2 drives septic cardiac dysfunction and that the targeted induction of UCP2-mediated autophagy may have important therapeutic potential.

Impact of the inhibition of UCP2 on the activity and presence of Amylin in RIN‐5F cells

Amylin is a fibrillogenic protein co‐secreted with insulin from pancreatic beta cells. Amylin has been shown to function in metabolism through its influence on glucose homeostasis, which includes sending satiety signals to the brain, inhibiting glucagon secretion and delaying gastric emptying. In addition to amylin's direct role on metabolism it has also been proposed to have an impact on amyloid beta, a major component of the amyloid plaques found in Alzheimer's disease. Alzheimer's disease brains have been shown to have amyloid plaque formation and decreased expression of Uncoupling Protein 2 (UCP2), which is a protein that also has a role in metabolism. Traditionally, uncoupling proteins have been known to function as mitochondrial transporter proteins that create proton leaks across the inner mitochondrial membrane resulting in the dissipation of energy in the form of heat. Knowing that UCP2 is expressed in pancreatic beta cells and has been shown to have diminished levels in Alzheimer's disease brains the focus of this study was to examine the influence of UCP2 on the presence and expression of amylin in RIN‐5F cells (a pancreatic beta cell line). siRNA UCP2 constructs were created and immunocytochemistry experiments and ELISA assays were conducted to examine the presence of amylin and the activity level of amylin, respectively. Initial studies suggest that inhibition of UCP2 may impact the activity level and presence of amylin.Support or Funding InformationTameka Clemons at Spelman College Department of Chemistry and BiochemistryThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fatty Acid‐Induced Hepatocellular Carcinoma Growth is Mediated by Decreasing Mitochondrial H2O2 Emission Coupled to Increased Glutathione Levels

Rationale and HypothesisHigh fat diets are associated with increased hepatocellular carcinoma (HCC) risk, as well as increased HCC growth rates. Accelerated HCC growth in response to fatty acid challenges has been attributed to a variety of signalling pathways, but the role of metabolic and redox flexibility in mediating a pro‐growth environment in this context has not been examined. Here we examined the direct effect of fatty acid challenges on HCC growth in relation to altered metabolic and redox homeostasis.Experimental approachThe HCC cell line HepG2 was incubated with 0μM, 50μM and 100μM palmitoylcarnitine (PCarn) for up to 48 hrs. We measured clonogenic survival, mitochondrial H2O2 emission and glutathione following PCarn incubations with and without the glutathione depleting agent buthionine sulfoximine (BSO) and inhibition of uncoupling protein‐2 (UCP2) activity by genipin.Results100μM PCarn increased clonogenic survival in HepG2 by 8% more than control (p<0.05) at 48 hrs which represents a marked early effect considering the population doubling time of HepG2 cells is ~48 hrs. This was associated with an increase in both reduced and oxidized glutathione at both 24 and 48 hrs (p<0.05). Depleting glutathione with BSO prevented PCarn‐stimulated growth (p<0.05). In a separate experiment, acute incubations of 100μM PCarn increased H2O2 emission within the first 10 minutes (p<0.05) followed by a decrease in H2O2 at 1 hr that remained lower at 24 hrs (p<0.05). The acute increase in H2O2 followed by a more chronic depression in H2O2 suggested PCarn might have triggered a compensatory mechanism. In support of this notion, inhibition of UCP2 with genipin sensitized HepG2 cells to PCarn‐induced decreases in clonogenic survival (p<0.05) without a change in UCP2 protein content.ConclusionCollectively, this data suggests that PCarn‐induced HCC growth is in part attributed to elevated glutathione. Increases in glutathione may be a result of UCP2 rapidly attenuating PCarn‐induced H2O2 emission which may permit greater glutathione synthetic rates and creation of a pro‐growth redox environment.Support or Funding InformationFunding was provided to C.G.R.P. by National Science and Engineering Research Council (#436138‐2013) with infrastructure supported by Canada Foundation for Innovation, Ontario Research Fund and the James H. Cummings Foundation. P. C. T. was supported by an NSERC CGS‐D scholarship.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Matrine attenuates oxidative stress and cardiomyocyte apoptosis in doxorubicin-induced cardiotoxicity via maintaining AMPKα/UCP2 pathway

Oxidative stress and cardiomyocyte apoptosis are involved in the pathogenesis of doxorubicin (DOX)-induced cardiotoxicity. Matrine is well-known for its powerful anti-oxidant and anti-apoptotic capacities. Our present study aimed to investigate the effect of matrine on DOX-induced cardiotoxicity and try to unearth the underlying mechanisms. Mice were exposed with DOX to generate DOX-induced cardiotoxicity or normal saline as control. H9C2 cells were used to verify the effect of matrine in vitro. DOX injection triggered increased generation of reactive oxygen species (ROS) and excessive cardiomyocyte apoptosis, which were significantly mitigated by matrine. Mechanistically, we found that matrine ameliorated DOX-induced uncoupling protein 2 (UCP2) downregulation, and UCP2 inhibition by genipin could blunt the protective effect of matrine on DOX-induced oxidative stress and cardiomyocyte apoptosis. Besides, 5′-AMP-activated protein kinase α2 (Ampkα2) deficiency inhibited matrine-mediated UCP2 preservation and abolished the beneficial effect of matrine in mice. Besides, we observed that matrine incubation alleviated DOX-induced H9C2 cells apoptosis and oxidative stress level via activating AMPKα/UCP2, which were blunted by either AMPKα or UCP2 inhibition with genetic or pharmacological methods. Matrine attenuated oxidative stress and cardiomyocyte apoptosis in DOX-induced cardiotoxicity via maintaining AMPKα/UCP2 pathway, and it might be a promising therapeutic agent for the treatment of DOX-induced cardiotoxicity.

UCP2 Mitigates the Loss of Human Spermatozoa Motility by Promoting mROS Elimination

Background/Aims: To demonstrate the function of uncoupling protein 2 (UCP2) in the regulation of human spermatozoa motility. Methods: Semen samples were collected from donors with either normal spermatozoa motility (normospermia [NS]) or poor spermatozoa motility (asthenospermia [AS]). UCP2 protein in spermatozoawas quantified by Western blotting. The level of mitochondrial reactive oxygen species (mROS) was evaluated by MitoSOX Red. The activity of mitochondrial membrane potential (MMP) in spermatozoa was evaluated by a JC-1 assay and the ATP level was monitored by a luciferin-luciferase assay. Results: UCP2 was expressed in both NS and AS groups, with the former exhibiting a higher level than the latter. Immunofluorescence analysis shows that UCP2 is mainly located at the mid-region of human spermatozoa. The inhibition of UCP2 by a highly selective inhibitor, Genipin, results in not only impaired spermatozoa mobility (P<.05) but also an elevated level of mROS (P<.05), suggesting that UCP2 is involved in the maintenance of the spermatozoa mobility, which probably is achieved by promoting mROS elimination. Furthermore, H₂O₂ treatment of spermatozoa increases the mROS level coupled with the loss of spermatozoa mobility. Unexpectedly, this treatment also has a positive impact on the expression of UCP2 within a certain range of supplemental H₂O₂, indicating the moderate mROS level possibly serves as a feedback signal to stimulate the expression of UCP2. Finally, the treatment of spermatozoa by an ROS scavenger, N-acetyl-l-cysteine (NAC),decreases the level of mROS and increases the curvilinear velocity (VCL) of spermatozoa, but the UCP2 level is not affected. Conclusion: These results suggest an UCP2–mROS–motility regulatory system exists for maintaining spermatozoa mobility in humans. In such a system, UCP2 fulfills its function by promoting mROS elimination, and slightly over-produced mROS in turn serves as a signal to stimulates the expression of UCP2. This regulatory system represents a new potential target for the discovery of novel pharmaceuticals for the treatment of patients with low spermatozoa motility.

UCP2 inhibition induces ROS/Akt/mTOR axis: Role of GAPDH nuclear translocation in genipin/everolimus anticancer synergism

Several studies indicate that mitochondrial uncoupling protein 2 (UCP2) plays a pivotal role in cancer development by decreasing reactive oxygen species (ROS) produced by mitochondrial metabolism and by sustaining chemoresistance to a plethora of anticancer drugs. Here, we demonstrate that inhibition of UCP2 triggers Akt/mTOR pathway in a ROS-dependent mechanism in pancreatic adenocarcinoma cells. This event reduces the antiproliferative outcome of UCP2 inhibition by genipin, creating the conditions for the synergistic counteraction of cancer cell growth with the mTOR inhibitor everolimus. Inhibition of pancreatic adenocarcinoma cell growth and induction of apoptosis by genipin and everolimus treatment are functionally related to nuclear translocation of the cytosolic glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The synthetic compound (S)-benzyl-2-amino-2-(S)-3-bromo-4,5-dihydroisoxazol-5-yl-acetate (AXP3009), which binds GAPDH at its redox-sensitive Cys152, restores cell viability affected by the combined treatment with genipin and everolimus, suggesting a role for ROS production in the nuclear translocation of GAPDH. Caspase-mediated apoptosis by genipin and everolimus is further potentiated by the autophagy inhibitor 3-methyladenine revealing a protective role for Beclin1-mediated autophagy induced by the treatment. Mice xenograft of pancreatic adenocarcinoma further confirmed the antiproliferative outcome of drug combination without toxic effects for animals. Tumor masses from mice injected with UCP2 and mTOR inhibitors revealed a strong reduction in tumor volume and number of mitosis associated with a marked GAPDH nuclear positivity. Altogether, these results reveal novel mechanisms through which UCP2 promotes cancer cell proliferation and support the combined inhibition of UCP2 and of Akt/mTOR pathway as a novel therapeutic strategy in the treatment of pancreatic adenocarcinoma.

Abstract 3561: UCP2’s tumor-promoting role via regulating lipid signaling and PLCγ -1 activity

Abstract Mitochondrial abnormalities are long suspected to play important roles in tumor cell differentiation, proliferation and metabolism. Dysfunction of mitochondrial-dependent processes appear to be key features of cancerous cells. Among many such abnormal features of cancerous cells, a mitochondrial uncoupling protein 2 (UCP2) is shown to be up-regulated in various aggressive human cancers. Uncoupling proteins are a family of mitochondrial proteins present in the inner mitochondrial membrane whose physiological role is to decrease membrane potential and reactive oxygen species (ROS) production. UCP2 over-expression recently been proposed as a novel survival mechanism for cancer cells. However, till date, the exact role of UCP2 in cancer remains inconclusive. We recently reported that UCP2 appears to be a key regulator of cell proliferation, cell cycle and cell death during skin tumorigenesis. Using JB6P+ cells that overexpress UCP2, we showed that UCP2 expression correlated closely with cell proliferation and cell transformation. Moreover, inhibition of UCP2, decreased colony formation and 3D culture growth. Since UCP2 is the crucial player in the regulation of reactive oxygen species, and mitochondrial bioenergetics, we wanted to dissect out the role of UCP2 in redox regulation and tumor promotion. Our data demonstrated that UCP2 differentially regulated ROS. UCP2 upregulation, decreased superoxide production, whereas increased hydrogen peroxide production with a concomitant increase in manganese superoxide dismutase (MnSOD) expression and activity. Furthermore, hydrogen peroxide was responsible for induction of lipid peroxidation and PLCγ-1 activation in UCP2 overexpressed cells. Moreover, PLCγ-1 activation enhanced tumorigenecity. Strikingly, pharmacological and siRNA mediated inhibition of PLCγ-1 markedly reduced colony formation and 3D growth in vitro. Lastly, restricting hydrogen peroxide production with hydrogen peroxide scavenger catalase, suppressed lipid peroxidation and dampened PLCγ-1 activity. Taken together, our data suggest that hydrogen peroxide might be the mediator of UCP2’s tumor promoting role, and pharmacological disruption of PLCγ-1, and/or hydrogen peroxide may have clinical utility for UCP2-overexpressed cancers. This research was funded by NCI and Feist-Wellier Cancer Center of LSUHSC-S Citation Format: Annapoorna Sreedhar, Yunfeng Zhao. UCP2’s tumor-promoting role via regulating lipid signaling and PLCγ -1 activity [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 3561. doi:10.1158/1538-7445.AM2017-3561

Mitochondrial Uncoupling Protein 2 in human cumulus cells is associated with regulating autophagy and apoptosis, maintaining gap junction integrity and progesterone synthesis

To explore the roles of mitochondrial Uncoupling Protein 2 (UCP2) in cumulus cells (CCs), human CCs were cultured in vitro, and the UCP2 was inhibited by treatment with Genipin, a special UCP inhibitor, or by RNA interference targeting UCP2. No significant differences in adenosine triphosphate levels and the ratio of ADP/ATP were observed after UCP2 inhibition. UCP2 inhibition caused a significant increase in cellular oxidative damage, which was reflected in alterations to several key parameters, including reactive oxygen species (ROS) and lipid peroxidation levels and the ratio of reduced GSH to GSSG. UCP2 blocking resulted in an obvious increase in active Caspase-3, accompanied by the decline of proactive Caspase-3 and a significant increase in the LC3-II/LC3-I ratio, suggesting that UCP2 inhibition triggered cellular apoptosis and autophagy. The mRNA and protein expression of connexin 43 (Cx43), a gap junction channel protein, were significantly reduced after treatment with Genipin or siRNA. The progesterone level in the culture medium was also significantly decreased after UCP2 inhibition. Our data indicated that UCP2 plays highly important roles in mediating ROS production and regulating apoptosis and autophagy, as well as maintaining gap junction integrity and progesterone synthesis, which suggests that UCP2 is involved in the regulation of follicle development and early embryo implantation and implies that it might serve as a potential biomarker for oocyte quality and competency.

The UCP2-related mitochondrial pathway participates in rhein-induced apoptosis in HK-2 cells.

Rhein is one of the main active compounds in total rhubarb anthraquinones (TRAs) that were reported to cause nephrotoxicity. This paper explored the mechanism of how rhein induced apoptosis in human renal proximal tubular epithelial cells (HK-2 cells). In this study, rhein was found to induce apoptosis in HK-2 cells according to the results of annexin V/PI staining assay. The underlying mechanisms were investigated, and the mitochondria-mediated pathway was found to be critical. A series of related biological events were explored, including the disruption of mitochondrial membrane potential (MMP), the decrease of the ATP level, the release of cytochrome c (Cyt-c) from the mitochondrion to the cytosol, and down-regulation of Bcl-2 and up-regulation of Bax. Furthermore, rhein significantly increased the levels of ROS and inhibited the expression of mitochondrial uncoupling protein 2 (UCP2). UCP2 inhibition dramatically boosted oxidative stress and exacerbated rhein-induced apoptosis, whereas co-incubation with an ROS scavenger N-acetylcysteine (NAC) could decrease rhein-induced apoptosis. In conclusion, our results have demonstrated that rhein induced apoptosis in HK-2 cells via the UCP2-related mitochondrial pathway and rhein might be a weak inhibitor of UCP2. Our findings provide new evidence that UCP2 plays an important role in the mitochondrial apoptotic pathway.

Uncoupling protein 2 regulates metabolic reprogramming and fate of antigen-stimulated CD8+ T cells.

Adoptive cell therapy (ACT) employing ex vivo-generated tumor antigen-specific CD8+ T cells shows tumor efficacy when the transferred cells possess both effector and memory functions. New strategies based on understanding of mechanisms that balance CD8+ T cell differentiation toward effector and memory responses are highly desirable. Emerging information confirms a central role for antigen-induced metabolic reprogramming in CD8+ T cell differentiation and clonal expansion. The mitochondrial protein uncoupling protein 2 (UCP2) is induced by antigen stimulation of CD8+ T cells; however, its role in metabolic reprogramming underlying differentiation and clonal expansion has not been reported. Employing genetic (siRNA) and pharmacologic (Genipin) approaches, we note that antigen-induced UCP2 expression reduces glycolysis, fatty acid synthesis and production of reactive oxygen species to balance differentiation with survival of effector CD8+ T cells. Inhibition of UCP2 promotes CD8+ T cell terminal differentiation into short-lived effector cells (CD62L(lo)KLRG1(Hi)IFNγ(Hi)) that undergo clonal contraction. These findings are the first to reveal a role for antigen-induced UCP2 expression in balancing CD8+ T cell differentiation and survival. Targeting UCP2 to regulate metabolic reprogramming of CD8+ T cells is an attractive new approach to augment efficacy of tumor therapy by ACT.

The Hydroxyl at Position C1 of Genipin Is the Active Inhibitory Group that Affects Mitochondrial Uncoupling Protein 2 in Panc-1 Cells.

Genipin (GNP) effectively inhibits uncoupling protein 2 (UCP2), which regulates the leakage of protons across the inner mitochondrial membrane. UCP2 inhibition may induce pancreatic adenocarcinoma cell death by increasing reactive oxygen species (ROS) levels. In this study, the hydroxyls at positions C10 (10-OH) and C1 (1-OH) of GNP were hypothesized to be the active groups that cause these inhibitory effects. Four GNP derivatives in which the hydroxyl at position C10 or C1 was replaced with other chemical groups were synthesized and isolated. Differences in the inhibitory effects of GNP and its four derivatives on pancreatic carcinoma cell (Panc-1) proliferation were assessed. The effects of GNP and its derivatives on apoptosis, UCP2 inhibition and ROS production were also studied to explore the relationship between GNP’s activity and its structure. The derivatives with 1-OH substitutions, geniposide (1-GNP1) and 1-ethyl-genipin (1-GNP2) lacked cytotoxic effects, while the other derivatives that retained 1-OH, 10-piv-genipin (10-GNP1) and 10-acetic acid-genipin (10-GNP2) exerted biological effects similar to those of GNP, even in the absence of 10-OH. Thus, 1-OH is the key functional group in the structure of GNP that is responsible for GNP’s apoptotic effects. These cytotoxic effects involve the induction of Panc-1 cell apoptosis through UCP2 inhibition and subsequent ROS production.

Altered expression of uncoupling protein 2 in GLP-1-producing cells after chronic high glucose exposure: implications for the pathogenesis of diabetes mellitus.

Glucagon-like peptide-1 (GLP-1) is a gut L-cell hormone that enhances glucose-stimulated insulin secretion. Several approaches that prevent GLP-1 degradation or activate the GLP-1 receptor are being used to treat type 2 diabetes mellitus (T2DM) patients. In T2DM, GLP-1 secretion has been suggested to be impaired, and this defect appears to be a consequence rather than a cause of impaired glucose homeostasis. However, although defective GLP-1 secretion has been correlated with insulin resistance, little is known about the direct effects of chronic high glucose concentrations, which are typical in diabetes patients, on GLP-1-secreting cell function. In the present study, we demonstrate that glucotoxicity directly affects GLP-1 secretion in GLUTag cells chronically exposed to high glucose. Our results indicate that this abnormality is associated with a decrease in ATP production due to the elevated expression of mitochondrial uncoupling protein 2 (UCP2). Furthermore, UCP2 inhibition using small interfering RNA (siRNA) and the application of glibenclamide, an ATP-sensitive potassium (KATP(+)) channel blocker, reverse the GLP-1 secretion defect induced by chronic high-glucose treatment. These results show that glucotoxicity diminishes the secretory responsiveness of GLP-1-secreting cells to acute glucose stimulation. We conclude that the loss of the incretin effect, as observed in T2DM patients, could at least partially depend on hyperglycemia, which is typical in diabetes patients. Such an understanding may not only provide new insight into diabetes complications but also ultimately contribute to the identification of novel molecular targets within intestinal L-cells for controlling and improving endogenous GLP-1 secretion.

Overexpression of uncoupling protein-2 in cancer: metabolic and heat changes, inhibition and effects on drug resistance.

This paper deals with the role of uncoupling protein-2 (UCP2) in cancer. UCP2 is overexpressed in cancer. This overexpression results in uncoupling of mitochondrial oxidative phosphorylation and a shift in production of ATP from mitochondrial oxidative phosphorylation to cytosolic aerobic glycolysis. UCP2 overexpression results in the following changes. Mitochondrial membrane potential (Δψ(m)) is decreased and lactate accumulates. There is a diminished production of reactive oxygen species and apoptosis is inhibited post-exposure to chemotherapeutic agents. There is an increase in heat and entropy production and a departure from the stationary state of non-cancerous tissue. Uncoupling of oxidative phosphorylation may also be caused by protonophores and non-steroidal anti-inflammatory drugs. UCP2 requires activation by superoxide and lipid peroxidation derivatives. As vitamin E inhibits lipid peroxidation, it might be expected that vitamin E would act as a chemotherapeutic agent against cancer. A recent study has shown that vitamin E and another anti-oxidant accelerate cancer progression. UCP2 is inhibited by genipin, chromane compounds and short interfering RNAs (siRNA). Genipin, chromanes and siRNA are taken up by both cancer and non-cancerous cells. Targeting the uptake of these agents by cancer cells by the enhanced permeability and retention effect is considered. Inhibition of UCP2 enhances the action of several anti-cancer agents.