PLAGL2-EGFR-HIF-1/2α Signaling Loop Promotes HCC Progression and Erlotinib Insensitivity.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide, hence a major public health threat. Pleomorphic adenoma gene like-2 (PLAGL2) has been reported to play a role in tumorigenesis. However, its precise function in HCC remains poorly understood. APPROACH AND RESULTS: In this study, we demonstrated that PLAGL2 was up-regulated in HCC compared with that of adjacent nontumorous tissues and also correlated with overall survival times. We further showed that PLAGL2 promoted HCC cell proliferation, migration, and invasion both in vitro and in vivo. PLAGL2 expression was positively correlated with epidermal growth factor receptor (EGFR) expression. Mechanistically, this study demonstrated that PLAGL2 functions as a transcriptional regulator of EGFR and promotes HCC cell proliferation, migration, and invasion through the EGFR-AKT pathway. Moreover, hypoxia was found to significantly induce high expression of PLAGL2, which promoted hypoxia inducible factor 1/2 alpha subunit (HIF1/2A) expression through EGFR. Therefore, this study demonstrated that a PLAGL2-EGFR-HIF1/2A signaling loop promotes HCC progression. More importantly, PLAGL2 expression reduced hepatoma cells' response to the anti-EGFR drug erlotinib. PLAGL2 knockdown enhanced the response to erlotinib. CONCLUSIONS: This study reveals the pivotal role of PLAGL2 in HCC cell proliferation, metastasis, and erlotinib insensitivity. This suggests that PLAGL2 can be a potential therapeutic target of HCC.

Reinvigorating exhausted CD8+ cytotoxic T lymphocytes in the tumor microenvironment and current strategies in cancer immunotherapy.

Immunotherapy has revolutionized the treatment of cancer in recent years and achieved overall success and long-term clinical benefit in patients with a wide variety of cancer types. However, there is still a large proportion of patients exhibiting limited or no responses to immunotherapeutic strategy, some of which were even observed with hyperprogressive disease. One major obstacle restricting the efficacy is that tumor-reactive CD8+ T cells, which are central for tumor control, undergo exhaustion, and lose their ability to eliminate cancer cells after infiltrating into the strongly immunosuppressive tumor microenvironment. Thus, as a potential therapeutic rationale in the development of cancer immunotherapy, targeting or reinvigorating exhausted CD8+ T cells has been attracting much interest. Hitherto, both intrinsic and extrinsic mechanisms that govern CD8+ T-cell exhaustion have been explored. Specifically, the transcriptional and epigenetic landscapes have been depicted utilizing single-cell RNA sequencing or mass cytometry (CyTOF). In addition, cellular metabolism dictating the tumor-infiltrating CD8+ T-cell fate is currently under investigation. A series of clinical trials are being carried out to further establish the current strategies targeting CD8+ T-cell exhaustion. Taken together, despite the proven benefit of immunotherapy in cancer patients, additional efforts are still needed to fully circumvent limitations of exhausted T cells in the treatment. In this review, we will focus on the current cellular and molecular understanding of metabolic changes, epigenetic remodeling, and transcriptional regulation in CD8+ T-cell exhaustion and describe hypothetical treatment approaches based on immunotherapy aiming at reinvigorating exhausted CD8+ T cells.

CircPTK2 (hsa_circ_0005273) as a novel therapeutic target for metastatic colorectal cancer.

BACKGROUND: As a novel class of noncoding RNAs, circRNAs have been recently identified to regulate tumorigenesis and aggressiveness. However, the function of circRNAs in colorectal cancer (CRC) metastasis remains unclear. We aimed to identify circRNAs that are upregulated in CRC tissues from patients and study their function in CRC metastasis. METHODS: We compared six pairs of CRC tissues and their matched adjacent non-tumor tissues by using circRNA microarray. We first evaluated the expression of circPTK2 (hsa_circ_0005273) in fresh tissues from CRC tumors and corresponding adjacent tissues by qPCR analysis. CircPTK2 expression levels in the tissue microarray with 5 years of survival information were determined by RNA-ISH analysis. Meanwhile, the expression levels of circulating circPTK2 were further analyzed according to the patients' clinical features. We analyzed cell apoptosis, colony formation, migration, and invasion in CRC cells. To further elucidate the effect of circPTK2 in CRC metastasis, we also conducted a colon cancer hepatic and pulmonary metastasis experiment. We used RNA biotin-labeled pull down and mass spectrometry to identify the target of circPTK2. We established a PDTX model to evaluate the effect of shRNA specifically targeting circPTK2 on tumor metastasis. RESULTS: We identified a novel circRNA, circPTK2, which is back-spliced of three exons (exons 27, 28 and 29) of PTK2 by using circRNA microarray, bioinformatics and functional studies. CircPTK2 was elevated in CRC tissues and positively associated with tumor growth and metastasis. CRC patients with increased circPTK2 expression were positively correlated with poorer survival rates. Furthermore, our studies showed that circPTK2 could promote EMT of CRC cells in vitro and in vivo by binding to vimentin protein on sites Ser38, Ser55 and Ser82. We further demonstrated the interaction of circPTK2 and vimentin mediated the regulation of CRC by knockdown or overexpression of vimentin. In addition, we revealed that tail vein injection of shRNA specifically targeting circPTK2 blunt tumor metastasis in a patient-derived CRC xenograft model. CONCLUSIONS: Collectively, these results demonstrate that circPTK2 exerts critical roles in CRC growth and metastasis and may serve as a potential therapeutic target for CRC metastasis, and also a promising biomarker for early diagnosis of metastasis.

Anidulafungin-Induced Suicidal Erythrocyte Death.

BACKGROUND/AIMS: The novel antifungal drug Anidulafungin is used for the treatment of diverse fungal infections including candidiasis and aspergillosis. The traditional antifungal drug amphotericin B has previously been shown to trigger eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, activated protein kinase C (PKC), casein kinase 1α or p38 kinase and activated caspases. Inhibitors of eryptosis include nitric oxide (NO). The present study explored, whether Anidulafungin induces eryptosis. METHODS: Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from DCFDA dependent fluorescence, and ceramide abundance at the erythrocyte surface utilizing specific antibodies. Hemolysis was quantified by measuring haemoglobin concentration in the supernatant. RESULTS: A 48 hours exposure of human erythrocytes to Anidulafungin (1.5 - 6 µg/ml) significantly increased hemolysis and the percentage of annexin-V-binding cells, and significantly decreased forward scatter. Anidulafungin (6 µg/ml) slightly, but significantly inceased Fluo3-fluorescence and the effect of Anidulafungin on annexin-V-binding was slightly, but significantly blunted by removal of extracellular Ca2+. The effect of Anidulafungin on annexin-V-binding was further significantly blunted by the p38 kinase inhibitor SB203580 (2 µM) and NO donor nitroprusside (1 µM). An increase of extracellular K+ concentration significantly blunted the effect of Anidulafungin on cell volume but not on annexin-V-binding. Anidulafungin rather decreased DCFDA fluorescence and the effect of Anidulafungin on annexin-V-binding was not significantly blunted by the antioxidant N-acetylcysteine (1 mM). Moreover, the effect of Anidulafungin on annexin-V-binding was not paralleled by significant increase of ceramide abundance and was not significantly blunted by PKC inhibitor staurosporine (1 µM), casein kinase 1α inhibitor D4476 (10 µM) or pancaspase inhibitor zVAD (10 µM). CONCLUSIONS: Anidulafungin triggers hemolysis and eryptosis with cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to Ca2+ entry and activation of p38 kinase.

Influence of γ-Secretase Inhibitor 24-Diamino-5-Phenylthiazole DAPT on Platelet Activation.

BACKGROUND/AIMS: DAPT (24-diamino-5-phenylthiazole) inhibits γ-secretase, which cleaves the signaling molecule CD44, a negative regulator of platelet activation and apoptosis. CD44 is a co-receptor for macrophage migration inhibitory factor (MIF) an anti-apoptotic pro-inflammatory cytokine expressed and released from blood platelets. Whether DAPT influences platelet function, remained, however, elusive. Activators of platelets include collagen related peptide (CRP). The present study thus explored whether DAPT modifies the stimulating effect of CRP on platelet function. METHODS: Platelets isolated from wild-type mice were exposed for 30 minutes to DAPT (10 µM). Flow cytometry was employed to estimate Orai1 abundance with specific antibodies, cytosolic Ca(2+)-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from α(IIb)ß3 integrin abundance, generation of reactive oxygen species (ROS) from DCFDA fluorescence, mitochondrial transmembrane potential from TMRE fluorescence, phospholipid scrambling of the cell membrane from annexin-V-binding, relative platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. RESULTS: Exposure of platelets to 2-5 µg/ml CRP was followed by significant increase of Orai1 abundance, [Ca2+]i, and P-selectin abundance, as well as by α(IIb)ß3 integrin activation, ROS generation, mitochondrial depolarization, enhanced annexin-V-binding, decreased cell volume, and aggregation. All CRP induced effects were significantly blunted in the presence of DAPT. CONCLUSIONS: The γ-secretase inhibitor DAPT counteracts agonist induced platelet activation, apoptosis and aggregation.

Effect of Lysosomotropic Polyamineoxidase Inhibitor MDL-72527 on Platelet Activation.

BACKGROUND/AIMS: The polyamine oxidase inhibitor MDL-72527 (N1,N4-bis(2,3-butadienyl)-1,4-butanediamine) were expected to increase the abundance of spermine, a powerful inhibitor of platelet activation. Nothing is known, however, on the sensitivity of platelet function and survival to MDL-72527 exposure. The present study thus explored whether MDL-72527 modifies function and survival of platelets without and with platelet activation by collagen related peptide (CRP). METHODS: Platelets isolated from wild-type mice were exposed for 30 minutes to MDL-72527 (100 µM) with or without subsequent activation with CRP (2-5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbß3 integrin abundance, generation of reactive oxygen species (ROS) from DCFDA fluorescence, phospholipid scrambling of the cell membrane from annexin-V-binding, platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. RESULTS: In the absence of CRP, exposure of platelets to MDL-72527 did not significantly modify [Ca2+]i, P-selectin abundance, αIIbß3 integrin abundance, ROS, annexin-V-binding, and forward scatter. The addition of 2-5 µg/ml CRP was followed by significant increase of [Ca2+]i, P-selectin abundance, αIIbß3 integrin activation, ROS abundance, annexin-V-binding, and aggregation as well as a significant decrease of forward scatter, all effects significantly blunted or virtually abolished in the presence of MDL-72527. CONCLUSIONS: MDL-72527 is a powerful inhibitor of platelet activation, apoptosis and aggregation.

Conjugated bilirubin triggers anemia by inducing erythrocyte death.

UNLABELLED: Hepatic failure is commonly associated with anemia, which may result from gastrointestinal bleeding, vitamin deficiency, or liver-damaging diseases, such as infection and alcohol intoxication. At least in theory, anemia during hepatic failure may result from accelerated clearance of circulating erythrocytes. Here we show that bile duct ligation (BDL) in mice leads to severe anemia despite increased reticulocyte numbers. Bilirubin stimulated suicidal death of human erythrocytes. Mechanistically, bilirubin triggered rapid Ca(2+) influx, sphingomyelinase activation, formation of ceramide, and subsequent translocation of phosphatidylserine to the erythrocyte surface. Consistent with our in vitro and in vivo findings, incubation of erythrocytes in serum from patients with liver disease induced suicidal death of erythrocytes in relation to their plasma bilirubin concentration. Consistently, patients with hyperbilirubinemia had significantly lower erythrocyte and significantly higher reticulocyte counts compared to patients with low bilirubin levels. CONCLUSION: Bilirubin triggers suicidal erythrocyte death, thus contributing to anemia during liver disease.

Stimulation of Suicidal Erythrocyte Death by the Antimalarial Drug Mefloquine.

BACKGROUND: The antimalarial drug mefloquine has previously been shown to stimulate apoptosis of nucleated cells. Similar to apoptosis, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include oxidative stress, increase of cytosolic Ca2+-activity ([Ca2+]i), and ceramide. METHODS: Phosphatidylserine abundance at the cell surface was estimated from annexin V binding, cell volume from forward scatter, reactive oxidant species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, [Ca2+]i from Fluo3- fluorescence, and ceramide abundance from specific antibody binding. RESULTS: A 48 h treatment of human erythrocytes with mefloquine significantly increased the percentage of annexin-V-binding cells (≥5 µg/ml), significantly decreased forward scatter (≥5 µg/ml), significantly increased ROS abundance (5 µg/ml), significantly increased [Ca2+]i (7.5 µg/ml) and significantly increased ceramide abundance (10 µg/ml). The up-regulation of annexin- V-binding following mefloquine treatment was significantly blunted but not abolished by removal of extracellular Ca2+. Even in the absence of extracellular Ca2+, mefloquine significantly increased annexin-V-binding. CONCLUSIONS: Mefloquine treatment leads to erythrocyte shrinkage and erythrocyte membrane scrambling, effects at least partially due to induction of oxidative stress, increase of [Ca2+]i and up-regulation of ceramide abundance.

Rapid Upregulation of Orai1 Abundance in the Plasma Membrane of Platelets Following Activation with Thrombin and Collagen Related Peptide.

BACKGROUND: Blood platelets accomplish primary hemostasis following vascular injury and contribute to the orchestration of occlusive vascular disease. Platelets are activated by an increase of cytosolic Ca2+-activity ([Ca2+]i), which is accomplished by Ca2+-release from intracellular stores and subsequent store operated Ca2+ entry (SOCE) through Ca2+ release activated Ca2+ channel moiety Orai1. Powerful activators of platelets include thrombin and collagen related peptide (CRP), which are in part effective by activation of small G- protein Rac1. The present study explored the influence of thrombin and CRP on Orai1 protein abundance and cytosolic Ca2+-activity ([Ca2+]i) in platelets drawn from wild type mice. METHODS: Orai1 protein surface abundance was quantified utilizing CF™488A conjugated antibodies, and [Ca2+]i was determined with Fluo3-fluorescence. RESULTS: In resting platelets, Orai1 protein abundance and [Ca2+]i were low. Thrombin (0.02 U/ml) and CRP (5ug/ml) within 2 min increased [Ca2+]i and Orai1 protein abundance at the platelet surface. [Ca2+]i was further increased by Ca2+ ionophore ionomycin (1 µM) and by store depletion with the sarcoendoplasmatic Ca2+ ATPase inhibitor thapsigargin (1 µM). However, Orai1 protein abundance at the platelet surface was not significantly affected by ionomycin and only slightly increased by thapsigargin. The effect of thrombin and CRP on Orai1 abundance and [Ca2+]i was significantly blunted by Rac1 inhibitor NSC23766 (50 µM). CONCLUSION: The increase of [Ca2+]i following stimulation of platelets with thrombin and collagen related peptide is potentiated by ultrarapid Rac1 sensitive translocation of Orai1 into the cell membrane.

Involvement of Ca2+ Activated Cl- Channel Ano6 in Platelet Activation and Apoptosis.

BACKGROUND/AIMS: The ubiquitously expressed Ca2+ Activated Cl- Channel Ano6 participates in the stimulation of cell membrane scrambling. Defective Ano6 underlies the Scott syndrome, an inherited bleeding disorder with impaired scrambling of plasma membrane phospholipids. At least in theory, the bleeding disorder of Scott syndrome may result from impaired platelet function. Activators of platelets include thrombin and collagen related peptide (CRP), which trigger increase of cytosolic Ca2+-activity ([Ca2+]i), production of reactive oxygen species (ROS), degranulation, integrin activation, as well as cell shrinkage and phospholipid scrambling of the cell membrane. The present study thus explored whether Ano6 modifies activation-induced alterations of cytosolic Ca2+-activity ([Ca2+]i), degranulation (P-selectin exposure), integrin activation, phosphatidylserine exposure on the platelet surface and platelet volume. METHODS: Platelets from mice lacking Ano6 (ano6-/-) were compared to platelets from corresponding wild-type mice (ano6+/+). [Ca2+]i was estimated from Fluo-3 fluorescence, ROS from DCFDA fluorescence, degranulation from P-selectin abundance, integrin activation from αIIbß3-integrin abundance, phosphatidylserine abundance from annexin-V-binding, and cell volume from forward scatter. RESULTS: Platelet number in blood was slightly higher in ano6-/- mice than in ano6+/+ mice. Without activation [Ca2+]i and volume were similar in ano6-/- and ano6+/+ platelets as well as ROS abundance, P-selectin abundance, αIIbß3 integrin activation, and phosphatidylserine exposure were negligible in both genotypes. Thrombin (0.01 U/ml) and CRP (2 or 5 µg/ml) increased [Ca2+]i, ROS abundance, platelet degranulation, αIIbß3 integrin activation, and triggered annexin-V-binding as well as cell shrinkage, all effects less pronounced in ano6-/- than in ano6+/+ platelets. CONCLUSIONS: Genetic knockout of Ano6 blunts thrombin- and CRP-induced activation and apoptosis of blood platelets.

Enhanced suicidal erythrocyte death contributing to anemia in the elderly.

BACKGROUND/AIMS: Anemia, a common condition in the elderly, could result from impaired formation and/or from accelerated loss of circulating erythrocytes. The latter could result from premature suicidal erythrocyte death or eryptosis characterized by phosphatidylserine (PS) exposure at the erythrocyte surface. Triggers of eryptosis include increased cytosolic Ca(2+)-concentration ([Ca(2+)]i), oxidative stress and ceramide. The present study explored whether eryptosis is altered in elderly individuals and, if so, to identify underlying mechanisms. METHODS: Blood was drawn from healthy young (n=11, age 31.3 ± 1.7 years) and elderly (n=16, age 88.6 ± 0.9 years) individuals. PS exposure was estimated from annexin V-binding, cell volume from forward scatter, [Ca(2+)]i from Fluo3-fluorescence, reactive oxygen species (ROS) from 2',7'dichlorodihydrofluorescein fluorescence, reduced glutathione (GSH) from mercury orange fluorescence and ceramide from FITC-conjugated antibody binding in flow cytometry. Measurements were made in erythrocytes from freshly drawn blood and in erythrocytes exposed in vitro for 24 h to plasma from young or elderly individuals. RESULTS: Elderly individuals suffered from severe anemia (hemoglobin 10.5 ± 0.3 g/100 ml) despite enhanced number of reticulocytes (2.3 ± 0.2%). The percentage of PS-exposing erythrocytes was significantly higher in the elderly (2.5 ± 0.2%) than in the young volunteers (1.3 ± 0.1%). The increase in PS exposure was paralleled by significant increase of ROS and significantly decreased levels of reduced GSH. Erythrocyte [Ca(2+)]i, and ceramide abundance tended to be higher in the elderly, differences, however, not reaching statistical significance. CONCLUSIONS: The anemia of elderly individuals is mainly if not exclusively due to enhanced eryptosis, resulting at least in part from GSH deficiency and increased oxidative stress.

Up-regulation of Orai1 expression and store operated Ca(2+) entry following activation of membrane androgen receptors in MCF-7 breast tumor cells.

BACKGROUND: Membrane androgen receptors (mAR) are functionally expressed in a variety of tumor-cells including the breast tumor-cell line MCF-7. They are specifically activated by testosterone albumin conjugates (TAC). The mAR sensitive signaling includes activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) and reorganization of the actin filament network. Signaling of tumor-cells may further involve up-regulation of pore forming Ca(2+) channel protein Orai1, which accomplishes store operated Ca(2+) entry (SOCE). This study explored the regulation of Orai1 abundance and SOCE by mAR. METHODS: Actin filaments were visualized utilizing confocal microscopy, Rac1 activity using GST-GBD assay, Orai1 transcript levels by RT-PCR and total protein abundance by western blotting, Orai1 abundance at the cell surface by confocal microscopy and FACS-analysis, cytosolic Ca(2+) activity ([Ca(2+)]i) utilizing Fura-2-fluorescence, and SOCE from increase of [Ca(2+)]i following readdition of Ca(2+) after store depletion with thapsigargin (1 µM). RESULTS: TAC treatment of MCF-7 cells was followed by Rac1 activation, actin polymerization, transient increase of Orai1transcript levels and protein abundance, and transient increase of SOCE. The transient increase of Orai1 protein abundance was abrogated by Rac1 inhibitor NSC23766 (50 µM) and by prevention of actin reorganization with cytochalasin B (1 µM). CONCLUSIONS: mAR sensitive Rac1 activation and actin reorganization contribute to the regulation of Orai1 protein abundance and SOCE.

Up-regulation of Na(+)-coupled glucose transporter SGLT1 by caveolin-1.

The Na(+)-coupled glucose transporter SGLT1 (SLC5A1) accomplishes concentrative cellular glucose uptake even at low extracellular glucose concentrations. The carrier is expressed in renal proximal tubules, small intestine and a variety of nonpolarized cells including several tumor cells. The present study explored whether SGLT1 activity is regulated by caveolin-1, which is known to regulate the insertion of several ion channels and carriers in the cell membrane. To this end, SGLT1 was expressed in Xenopus oocytes with or without additional expression of caveolin-1 and electrogenic glucose transport determined by dual electrode voltage clamp experiments. In SGLT1-expressing oocytes, but not in oocytes injected with water or caveolin-1 alone, the addition of glucose to the extracellular bath generated an inward current (Ig), which was increased following coexpression of caveolin-1. Kinetic analysis revealed that caveolin-1 increased maximal Ig without significantly modifying the glucose concentration required to trigger half maximal Ig (KM). According to chemiluminescence and confocal microscopy, caveolin-1 increased SGLT1 protein abundance in the cell membrane. Inhibition of SGLT1 insertion by brefeldin A (5µM) resulted in a decline of Ig, which was similar in the absence and presence of caveolin-1. In conclusion, caveolin-1 up-regulates SGLT1 activity by increasing carrier protein abundance in the cell membrane, an effect presumably due to stimulation of carrier protein insertion into the cell membrane.

Regulation of the voltage gated K channel Kv1.3 by recombinant human klotho protein.

BACKGROUND/AIMS: Klotho, a protein mainly produced in the kidney and released into circulating blood, contributes to the negative regulation of 1,25(OH)2D3 formation and is thus a powerful regulator of mineral metabolism. As ß-glucuronidase, alpha Klotho protein further regulates the stability of several carriers and channels in the plasma membrane and thus regulates channel and transporter activity. Accordingly, alpha Klotho protein participates in the regulation of diverse functions seemingly unrelated to mineral metabolism including lymphocyte function. The present study explored the impact of alpha Klotho protein on the voltage gated K+ channel Kv1.3. METHODS: cRNA encoding Kv1.3 (KCNA3) was injected into Xenopus oocytes and depolarization induced outward current in Kv1.3 expressing Xenopus oocytes determined utilizing dual electrode voltage clamp. Experiments were performed without or with prior treatment with recombinant human Klotho protein (50 ng/ml, 24 hours) in the absence or presence of a ß-glucuronidase inhibitor D-saccharic acid-1,4-lactone (DSAL, 10 µM). Moreover, the voltage gated K+ current was determined in Jcam lymphoma cells by whole cell patch clamp following 24 hours incubation without or with recombinant human Klotho protein (50 ng/ml, 24 hours). Kv1.3 protein abundance in Jcam cells was determined utilising fluorescent antibodies in flow cytometry. RESULTS: In Kv1.3 expressing Xenopus oocytes the Kv1.3 currents and the protein abundance of Kv1.3 were both significantly enhanced after treatment with recombinant human Klotho protein (50 ng/ml, 24 hours), an effect reversed by presence of DSAL. Moreover, treatment with recombinant human Klotho protein increased Kv currents and Kv1.3 protein abundance in Jcam cells. CONCLUSION: Alpha Klotho protein enhances Kv1.3 channel abundance and Kv1.3 currents in the plasma membrane, an effect depending on its ß-glucuronidase activity.

Blockage of L2HGDH-mediated S-2HG catabolism orchestrates macrophage polarization to elicit antitumor immunity.

The high infiltration of tumor-associated macrophages (TAMs) in the immunosuppressive tumor microenvironment prominently attenuates the efficacy of immune checkpoint blockade (ICB) therapies, yet the underlying mechanisms are not fully understood. Here, we investigate the metabolic profile of TAMs and identify S-2-hydroxyglutarate (S-2HG) as a potential immunometabolite that shapes macrophages into an antitumoral phenotype. Blockage of L-2-hydroxyglutarate dehydrogenase (L2HGDH)-mediated S-2HG catabolism in macrophages promotes tumor regression. Mechanistically, based on its structural similarity to α-ketoglutarate (α-KG), S-2HG has the potential to block the enzymatic activity of 2-oxoglutarate-dependent dioxygenases (2-OGDDs), consequently reshaping chromatin accessibility. Moreover, S-2HG-treated macrophages enhance CD8+ T cell-mediated antitumor activity and sensitivity to anti-PD-1 therapy. Overall, our study uncovers the role of blockage of L2HGDH-mediated S-2HG catabolism in orchestrating macrophage antitumoral polarization and, further, provides the potential of repolarizing macrophages by S-2HG to overcome resistance to anti-PD-1 therapy.

Stress-induced epinephrine promotes hepatocellular carcinoma progression via the USP10-PLAGL2 signaling loop.

Hepatocellular carcinoma (HCC) is associated with a poor prognosis. Our previous study demonstrated that Pleomorphic adenoma gene like-2 (PLAGL2) was a potential therapeutic target in HCC. However, the mechanisms that lead to the upregulation of PLAGL2 in HCC remain unclear. The present study revealed that stress-induced epinephrine increased the expression of PLAGL2, thereby promoting the progression of HCC. Furthermore, PLAGL2 knockdown inhibited epinephrine-induced HCC development. Mechanistically, epinephrine upregulated ubiquitin-specific protease 10 (USP10) to stabilize PLAGL2 via the adrenergic ß-receptor-2-c-Myc (ADRB2-c-Myc) axis. Furthermore, PLAGL2 acted as a transcriptional regulator of USP10, forming a signaling loop. Taken together, these results reveal that stress-induced epinephrine activates the PLAGL2-USP10 signaling loop to enhance HCC progression. Furthermore, PLAGL2 plays a crucial role in psychological stress-mediated promotion of HCC progression.

Inhibition of colonic tumor growth by the selective SGK inhibitor EMD638683.

BACKGROUND: The serum and glucocorticoid inducible kinase SGK1, which was originally cloned from mammary tumor cells, is highly expressed in some but not all tumors. SGK1 confers survival to several tumor cells. Along those lines, the number of colonic tumors following chemical carcinogenesis was decreased in SGK1 knockout mice. Recently, a highly selective SGK inhibitor (EMD638683) has been developed. The present study explored whether EMD638683 affects survival of colon carcinoma cells in vitro and impacts on development of colonic tumors in vivo. METHODS: Colon carcinoma (Caco-2) cells were exposed to EMD638683 with or without exposure to radiation (3 Gray) and cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, mitochondrial potential from JC-9 fluorescence, caspase 3 activity from CaspGlow Fluorescein staining, DNA degradation from propidium iodide staining as well as late apoptosis from annexin-V FITC and propidium iodide double staining. In vivo tumor growth was determined in wild type mice subjected to chemical carcinogenesis (intraperitoneal injection of 20 mg/kg 1,2-dimethylhydrazine followed by three cycles of 30 g/L synthetic dextran sulfate sodium in drinking water for 7 days). RESULTS: EMD638683 treatment significantly augmented the radiation-induced decrease of forward scatter, increase of phosphatidylserine exposure, decrease of mitochondrial potential, increase of caspase 3 activity, increase of DNA fragmentation and increase of late apoptosis. The in vivo development of tumors following chemical carcinogenesis was significantly blunted by treatment with EMD638683. CONCLUSIONS: EMD638683 promotes radiation-induced suicidal death of colon tumor cells in vitro and decreases the number of colonic tumors following chemical carcinogenesis in vivo.

Upregulation of store operated Ca channel Orai1, stimulation of Ca(2+) entry and triggering of cell membrane scrambling in platelets by mineralocorticoid DOCA.

BACKGROUND/AIMS: Mineralocorticoid excess leads to vascular injury, which is partially due to hypertension but in addition involves increased concentration of cytosolic Ca(2+) concentration in platelets, key players in the pathophysiology of occlusive vascular disease. Mineralocorticoids are in part effective by rapid nongenomic mechanisms including phosphatidylinositide-3-kinase (PI3K) signaling, which involves activation of the serum & glucocorticoid inducible kinase (SGK) isoforms. SGK1 has in turn been shown to participate in the regulation of the pore forming Ca(2+) channel protein Orai1 in platelets. Orai1 accomplishes entry of Ca(2+), which is in turn known to trigger cell membrane scrambling. Platelets lack nuclei but are able to express protein by translation, which is stimulated by PI3K signaling. The present study explored whether the mineralocorticoid desoxycorticosterone acetate (DOCA) influences platelet Orai1 protein abundance, cytosolic Ca(2+)-activity ([Ca(2+)]i), phosphatidylserine abundance at the cell surface and/or cell volume. METHODS: Orai1 protein abundance was estimated utilizing CF™488A conjugated antibodies, [Ca(2+)]i utilizing Fluo3-fluorescence, phosphatidylserine abundance utilizing FITC-labelled annexin V, and cell volume utilizing forward scatter in flow cytometry. RESULTS: DOCA (10 µg/ml) treatment of murine platelets was followed by a significant increase of Orai1 protein abundance, [Ca(2+)]i, percentage of phosphatidylserine exposing platelets and platelet swelling. The effect on [Ca(2+)]i, phosphatidylserine abundance and cell volume were completely abrogated by addition of the specific SGK inhibitor EMD638683 (50 µM) CONCLUSIONS: The mineralocorticoid DOCA upregulates Orai1 protein abundance in the cell membrane, thus increasing [Ca(2+)]i and triggering phosphatidylserine abundance, effects paralleled by platelet swelling.

Stimulation of suicidal erythrocyte death by ipratropium bromide.

BACKGROUND/AIMS: Ipratropium bromide, an anticholinergic agent widely used in obstructive lung disease, has previously been shown to trigger suicidal death of nucleated cells or apoptosis. Despite their lack of mitochondria and nuclei, key organelles in the execution of apoptosis, erythrocytes may similarly undergo suicidal cell death, which is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine-exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)). The present study explored whether ipratropium bromide triggers eryptosis. METHODS: [Ca Ca(2+)](i) was estimated utilizing Fluo3 fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, and hemolysis from hemoglobin release. RESULTS: A 48 h exposure to ipratropium bromide (1 nM) significantly increased [Ca(2+)](i), decreased forward scatter and increased annexin-V-binding. Ipratropium bromide treatment was followed by slight but significant increase of hemolysis. Removal of extracellular Ca(2+) or inhibition of Ca(2+) permeable cation channels with amiloride (1 mM) virtually abolished cell membrane scrambling. Ca(2+) ionophore ionomycin (1 µM, 30 min) increased the percentage of phosphatidylserine exposing erythrocytes to similarly high levels in the absence and presence of ipratropium bromide (1 nM). CONCLUSIONS: Ipratropium bromide triggers suicidal erythrocyte death or eryptosis, an effect mainly due to stimulation of Ca(2+)-entry.