Role of NKCC1 Activity in Glioma K+ Homeostasis and Cell Growth: New Insights With the Bumetanide-Derivative STS66

Abstract

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.

NKCC1, an Elusive Molecular Target in Brain Development: Making Sense of the Existing Data

“…During the second postnatal week in rats and mice [4] and around the time of full-term birth in humans [8,9], KCC2 is upregulated, leading to a decrease in neuronal intracellular chloride concentration ([Cl − ] i ) and a shift towards more hyperpolarizing GABAergic responses. NKCC1 is currently gaining increased attention as a possible therapeutic target for treating a myriad of CNS disorders [10], and the search for brain-permeable and specific NKCC1 drugs is heating up [11][12][13]. However, a major caveat here is that our understanding of the spatiotemporal expression patterns of NKCC1 in the brain is still in its infancy.…”

“…For rat and human in situ probes and PCR fragments, the corresponding mouse nucleotide sequences are indicated. (10) Mouse in situ probe nt 615-1206[68], (11) rat in situ probe nt 883-1376[66],(12) rat PCR amplicon nt 938-1468[69], (13) rat in situ probe nt 946-983[65],(14,15) rat in situ probes 988-1179 and nt 988-2190 of rat NKCC1 [70], (16) rat PCR fragment nt 1033-1435 amplified with single-cell RT-PCR [71], (17) exon 4 and 5 of human NKCC1 targeted with qRT-PCR [72], (18) rat amplicon nt 1650-1751 amplified by RT-PCR…”

Pharmacological tools to target NKCC1 in brain disorders

“…Finally, NKCC1 expression was detected in several human glioma cell lines [98][99][100], with high expression associated with poor prognosis of mesenchymal glioblastoma multiforme [11,12] (Table 1). Inhibition of NKCC1 by bumetanide reduced glioma cell migration in vitro and/or invasion of peritumor tissue in vivo, and promoted apoptosis of tumor cells [11,[98][99][100] (Table 2).…”

All-atom Simulations Uncover the Molecular Terms of NKCC1 Transport Mechanism

“…10 Withal, a growing body of evidences links NKCC1 expression to tumor infiltration and progression in multiple cancer types (lung adenocarcinoma, prostate cancer, esophageal squamous carcinoma, brain tumors). 11 In this scenario, NKCC1 represents an appealing pharmacological target to treat a variety of diseases associated with Cldis-homeostasis. Indeed, marketed drugs targeting NKCC1, such as the loop diuretics furosemide and bumetanide, which are commonly prescribed for pulmonary edema, 12 also exhibit anti-seizure, 13 anti-epileptic, 14 antiparkinsonian 15 and anti-tumor activity.…”

“…Indeed, marketed drugs targeting NKCC1, such as the loop diuretics furosemide and bumetanide, which are commonly prescribed for pulmonary edema, 12 also exhibit anti-seizure, 13 anti-epileptic, 14 antiparkinsonian 15 and anti-tumor activity. 11 Nevertheless, a poor selectivity jeopardizes their prolonged use for chronic patients. 16 The NKCC1 structure exhibits a pseudo-symmetric topology composed by two inverted repeats of five transmembrane helices forming a central binding cavity.…”