Biophysical Characterization and Expression Analysis of Kv1.3 Potassium Channel in Primary Human Leukemic B Cells

Abstract

Background/Aims: Pharmacological inhibition of the potassium channel Kv1.3 has been shown to selectively kill B cells from patients with chronic lymphocytic leukemia (B-CLL). Here we aimed to biophysically characterize and compare Kv1.3 channel activity in B cells isolated either from healthy subjects or patients and investigated the mechanism accounting for the increased protein expression in B-CLL cells. Methods: Kv1.3 activity was measured by patch clamp, while expression of the channel protein was assessed by Western blot and FACS analysis. B-CLL cells were co-cultured with mesenchymal stromal cells (MSC) and Kv1.3 inhibitor-induced apoptosis was assessed. Results: We demonstrate that Kv1.3 is highly expressed and is more active at resting membrane potential in human B-CLL cells than in healthy cells. Channel expression in pathologic cells decreased by the B-RAF kinase inhibitor PLX-4720, while it increased with Doxazosin, an α1-adrenoceptor antagonist. Kv1.3 inhibitors induced death in B-CLL cells also when co-cultured with MSC. Conclusion: Our results contribute to the characterization of B-CLL cells, as it shows that upregulation of Kv1.3 in pathologic B lymphocytes is linked to the oncogenic B-RAF signalling. We also conclude that Kv1.3 inhibitors represent a valuable tool to induce apoptosis of B-CLL cells even in the presence of MSC.

Thymoquinone Rescues T Lymphocytes from Gamma Irradiation-Induced Apoptosis and Exhaustion by Modulating Pro-Inflammatory Cytokine Levels and PD-1, Bax, and Bcl-2 Signaling

“…Consistent with these findings, it has been reported that cell apoptosis induced by radiation is regulated by a complex balance between pro-apoptotic factors, including caspase-3, c-caspase-3 and Bax, and anti-apoptotic factors, including the Bcl-2 family [71,72]. Recently, it has been shown that induction of apoptosis in T cells was associated with imbalance activation of caspase and Bcl-2 [73][74][75]. In the present study, we observed that TQ reduced radiation-induced caspase-3 and Bax activation and enhanced Bcl-2 expression.…”

Tumor-reducing effect of the clinically used drug clofazimine in a SCID mouse model of pancreatic ductal adenocarcinoma

“…In healthy individuals Kv1.3 is prevalently expressed mainly in the CNS and in immune cells [14]. Aberrant (mostly high) expression of Kv1.3 has instead been observed in different types of tumors [13, 15], such as melanoma [16], prostate [17], breast [18], B-cell lymphoma [19], chronic lymphocytic leukemia (B-CLL) [20, 21] gastric [22], pancreatic tumor [23] and in lung cancer [24]. Overexpression of Kv1.3, like that of other Kv channels, in cancer cells could give an advantage to cancer cells enhancing tumorigenic processes such as proliferation, cell migration and metastasis [10].…”

Impact of intracellular ion channels on cancer development and progression

“…Furthermore, Kv1.3 displays an altered expression level in various cancers (Arcangeli et al 2009 ; Comes et al 2015 ; Bielanska et al 2009 ), such as lymphoma (Alizadeh et al 2000 ), melanoma (Artym and Petty 2002 ), glioma (Preussat et al 2003 ), breast (Abdul et al 2003 ; Jang et al 2009 ), prostate (Abdul and Hoosein 2006 ), gastric (Lan et al 2005 ) and colon cancer (Abdul and Hoosein 2002 ). Kv1.3 was also shown to be present in the mitochondrial inner membrane; in particular, it has been shown to be expressed in the prostate and breast cancer cell lines PC3 and MCF-7, respectively, and lymphoma and leukemia cells in the mitochondria (Leanza et al 2013b ; Szabo et al 2015 ; Gulbins et al 2010 ).…”

“…We then provided evidence that pharmacological inhibition of mitoKv1.3 by membrane permeant blockers, the psoralens Psora-4 and PAP-1, and the riminophenazine clofazimine, is sufficient to trigger apoptotic cell death in cancer cells but not in healthy ones (Leanza et al 2012a , 2013b , 2014a , Szabo et al 2015 ). Inhibition of mitoKv1.3 induced a block of the potassium flux into the mitochondria inducing mitochondrial membrane hyperpolarization followed by reduction of the respiratory chain complexes, which causes increased ROS production.…”

“…Importantly, these effects were also obtained with the B-CLL cells that are mutated in p53, or overexpressing anti-apoptotic Bcl-2, and were independent of the currently used prognostic factors (ZAP70, CD38 and hypersomatic mutation) (Leanza et al 2013b ). Moreover, these compounds killed primary tumor B-cells even when they were co-cultured together with mesenchymal stromal cells, which mimic the lymph node micro-environment, favoring the pathological B-cells' survival (Szabo et al 2015 ). The selectivity versus cancer cells, according to our experiment-based model, is due to a synergistic effect of mitoKv1.3 inhibition and an increase of ROS: reactive oxygen species are often altered in tumor cells leading them to more easily reach the critical threshold necessary to trigger cell death upon exogenous oxidative insult (Ralph et al 2010 ).…”