Thomas Erker

Introduction: Na⁺-K⁺-2Cl⁻ cotransporter isoform 1 (NKCC1) is important in regulating intracellular K⁺ and Cl⁻ homeostasis and cell volume. In this study, we investigated a role of NKCC1 in regulating glioma K⁺ influx and proliferation in response to apoptosis inducing chemotherapeutic drug temozolomide (TMZ). The efficacy of a new bumetanide (BMT)-derivative NKCC1 inhibitor STS66 [3-(butylamino)-2-phenoxy-5-[(2, 2, 2-trifluoroethylamino) methyl] benzenesulfonamide] in blocking NKCC1 activity was compared with well-established NKCC1 inhibitor BMT. Methods: NKCC1 activity in cultured mouse GL26 and SB28-GFP glioma cells was measured by Rb⁺ (K⁺) influx. The WNK1-SPAK/OSR1-NKCC1 signaling and AKT/ERK-mTOR signaling protein expression and activation were assessed by immunoblotting. Cell growth was determined by bromodeoxyuridine (BrdU) incorporation assay, MTT proliferation assay, and cell cycle analysis. Impact of STS66 and BMT on cell Rb⁺ influx and growth was measured in glioma cells treated with or without TMZ. Results: Rb⁺ influx assay showed that 10 μM BMT markedly decreased the total Rb⁺ influx and no additional inhibition detected at >10 μM BMT. In contrast, the maximum effects of STS66 on Rb⁺ influx inhibition were at 40–60 μM. Both BMT and STS66 reduced TMZ-mediated NKCC1 activation and protein upregulation. Glioma cell growth can be reduced by STS66. The most robust inhibition of glioma growth, cell cycle, and AKT/ERK signaling was achieved by the TMZ + STS66 treatment. Conclusion: The new BMT-derivative NKCC1 inhibitor STS66 is more effective than BMT in reducing glioma cell growth in part by inhibiting NKCC1-mediated K⁺ influx. TMZ + STS66 combination treatment reduces glioma cell growth via inhibiting cell cycle and AKT-ERK signaling.

Background and Purpose—: Inhibition of brain NKCC1 (Na ⁺ -K ⁺ -Cl ⁻ cotransporter 1) with bumetanide (BMT) is of interest in ischemic stroke therapy. However, its poor brain penetration limits the application. In this study, we investigated the efficacy of 2 novel NKCC1 inhibitors, a lipophilic BMT prodrug STS5 (2-(Dimethylamino)ethyl 3-(butylamino)-4-phenoxy-5-sulfamoyl-benzoate;hydrochloride) and a novel NKCC1 inhibitor STS66 (3-(Butylamino)-2-phenoxy-5-[(2,2,2-trifluoroethylamino)methyl]benzenesulfonamide), on reducing ischemic brain injury. Methods—: Large-vessel transient ischemic stroke in normotensive C57BL/6J mice was induced with 50-min occlusion of the middle cerebral artery and reperfusion. Focal, permanent ischemic stroke in angiotensin II (Ang II)–induced hypertensive C57BL/6J mice was induced by permanent occlusion of distal branches of middle cerebral artery. A total of 206 mice were randomly assigned to receive vehicle DMSO, BMT, STS5, or STS66. Results—: Poststroke BMT, STS5, or STS66 treatment significantly decreased infarct volume and cerebral swelling by ≈40% to 50% in normotensive mice after transient middle cerebral artery occlusion, but STS66-treated mice displayed better survival and sensorimotor functional recovery. STS5 treatment increased the mortality. Ang II–induced hypertensive mice exhibited increased phosphorylatory activation of SPAK (Ste20-related proline alanine-rich kinase) and NKCC1, as well as worsened infarct and neurological deficit after permanent distal middle cerebral artery occlusion. Conclusions—: The novel NKCC1 inhibitor STS66 is superior to BMT and STS5 in reducing ischemic infarction, swelling, and neurological deficits in large-vessel transient ischemic stroke, as well as in permanent focal ischemic stroke with hypertension comorbidity.

Resveratrol, a natural polyphenol, possesses many beneficial health properties but its therapeutic application is limited due to its low water solubility and instability against oxidative processes. To improve the stability and lipophilicity of the natural compound, we synthesized a resveratrol prodrug, termed FEHH4-1. In the present study, we compared the antiproliferative and pro-apoptotic effects of resveratrol with FEHH4-1 on Jurkat T-cells. Cell proliferation and viability were monitored by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay, annexin-V/7-amino-actinomycin D staining and western blot. To induce interleukin-2 (IL2) expression, cells were stimulated with phorbol 12-myristate 13-acetate/phytohemagglutinin. IL2 production was quantified by enzyme-linked immunosorbent assay. IL2 promoter activity was studied by a Jurkat T-cell line containing an IL2 promoter luciferase reporter construct. Both polyphenols inhibited proliferation, induced apoptotic cell death and blocked IL2 synthesis in Jurkat T-cells. Most importantly, FEHH4-1 was three-to four-times more potent than resveratrol. FEHH4-1 had improved antiproliferative and pro-apoptotic potential against Jurkat T-cells compared to resveratrol.

Acute hematological diseases (leukemias and aggressive lymphomas) can be cured in approximately half of the patients, while the other patients die from their disease. Chronic leukemias and indolent lymphomas can be well controlled for years in most cases. However, the cure rate of these patients is low and the course of the disease is characterized by frequent recurrence. Therefore, novel agents for monotherapies or combination therapies still need to be explored. The presented study describes the identification of the chalcone derivative 15 on different types of human malignant cells of the lymphoid and myeloid lineage. Further experiments performed with compound 15 on peripheral blood mononuclear cells (PBMCs) of chronic lymphocytic leukemia (CLL) patients clearly stated a higher cytotoxicity in PBMC from CLL patients compared to healthy donors (HD). The newly identified chalcone derivative 15 showed a higher therapeutic potential than fludarabine, a drug already in use in lymphoma treatment.