RELAZIONE ATTIVITA’ SCIENTIFICA all'ESTERO
Dott.ssa Barbara Adinolfi
Università di Pisa
Report about the second part of Dr. Barbara Adinolfi’s scientific activity in Denmark (September 2012 – February 2013)
Institution of origin
Department of Pharmacy, University of Pisa, Pisa, Italy.
Host institution
Department of Biomedical Sciences, Faculty of Health Sciences, Panum Institute,University of Copenhagen Denmark.
Title
Role of voltage-activated (Kv) and calcium-activated (KCa) potassium channels as therapeutic targets in human cutaneous melanoma
Background
Data from the literature show that potassium (K+) channels are involved in the control of cell proliferation, differentiation, and apoptosis [1]; furthermore, their expression is altered in many human cancers [1-5]. For these reasons, K+ channels might represent an unexpected but promising targets for the development of novel anticancer strategies [6]. Several K+ channels subtypes have been characterized in human melanoma cells, although few evidences have been provided regarding their expression and function in this specific tumor type. In particular, inwardly rectifying K+ channel (KIR) and calcium-activated K+ (KCa) channel have been characterized in the following human melanoma cell lines: SK MEL 28 [7], C8161, C832C, C8146, SK28 [8] and IGR1, IPC298 and IGR39 [9,10]. The expression and function of the HERG protein have been also investigated in human melanoma cells [9,11-13]. In these papers, the authors clearly demonstrated that specific blockers and openers for different K+ channels were able to inhibit cell-cycle progression and migration, in the micromolar concentration range, suggesting a possible therapeutic potential of these compounds in the advanced phases of the disease .
Aim of the study
This project was aimed at investigating the role of specific Kv and KCa channel subtypes in the human A375 cutaneous melanoma cell line.
Phase 1
In the first part of the study we characterized the expression of the following receptor subtypes: voltage-gated (Kv) channels, HERG1, Eag1 and KCa channels, IK and SK1-3. Furthermore, we assessed the effect of selective K+ channel subtype blockers and openers on A375 cell proliferation. Briefly, our findings showed that the subtypes of potassium channels under the study are expressed in A375 melanoma cells and they may constitute new therapeutic targets in the treatment of cutaneous melanoma, as a concentration-dependent A375 cell viability reduction was observed after treatment with K channel modulators.
Phase 2
In the second part of the project, we performed electrophysiological measurements by using the Patch Clamp technique, to better characterize the functional role of the K+ channel subtypes in A375 melanoma cells. This phase of the study was performed in collaboration with researchers operating in the Department of Biomedical Sciences of the Panum Institute (University of Copenhagen) that have a long-time expertise in the field of ion channels, in particular K+ channels. They are specialized in the study of the molecular, cellular and in vivo function of potassium channels by using various electrophysiological techniques including Voltage Clamp, Two-Electrode Voltage Clamp (TEVC) and Patch Clamp.
Materials and Methods
All the experiments were performed at room temperature applying the whole-cell version of the patch clamp technique. Up to now, the experimental procedure has been optimized for the IK and SK1-3 calcium activated potassium channels. For each experiment, a cover slip (3.5 mm diameter) containing the cells was positioned in a perfusion chamber (volume ≈ 15 µL) and superfused at 1 mL/min with an extracellular solution. The extracellular solution had the following composition: 140 mM NaCl, 4 mM KCl, 0.1 mM CaCl2, 3 mM MgCl2 and 10 mM HEPES (pH, 7.4). The patch pipette contained: 144 mM KCl, 10 mM EGTA, 7.625 mM CaCl2, 1.205 mM MgCl2 and 10 mM HEPES (pH, 7.2). The free Ca2+ and Mg2+ concentration was calculated as 300 nM and 1 mM, respectively. All salts and buffer substances were of analytical grade or higher and obtained from commercial dealers. The electrodes were allowed to stabilize 1-2 min before equalization and giga-sealing and after break-through to the whole-cell configuration the pipette/cytoplasm were allowed to equilibrate for 5-8 min in order to obtain a stable baseline current before addition of compounds (K+ channel modulators). The series resistance (Rs, 80% compensation) and cell capacitance were monitored before each stimulus and only experiments with stable read-outs and initial Rs values below 5 MΩ were used. The holding potential was −90 mV and voltage ramps going from −120 to +30 mV during 200 ms were applied every 5 s. The positive modulators of hIK and hSK channels, NS309 (3,10,20 and 30nM) and 1-EBIO (up to 10µM), have been tested and the whole-cell current (nA) measured every 5 min. The effects of the combination of each opener with the IK blocker, Clotrimazole or TRAM-34 (30-300-3000nM), and with the SK blocker, Apamin (3-30-300nM), were also evaluated.
Results
The KCa positive modulators, NS309 and 1-EBIO, showed a concentration-dependent increase of hIK /SK current. Defining the current shortly before the individual application as 100%, the current was increased to 210% by NS309 and to 300% by 1-EBIO. Both the compounds were fully reversible upon washout of the compounds. Also the specific IK blockers, Clotrimazole and TRAM-34, and the SK blocker, Apamin, were tested on the NS309/1-EBIO-modulated channels. After the initial equilibration with the pipette solution, the current was further increased by application of the positive modulator at the appropriate concentration (30 nM). After a current plateau was reached, increasing concentrations of Apamin (hSK) and Clotrimazole or TRAM-34 (hIK) were co-administered with the K-channel opener. The blockers were able to block the modulated currents completely.
Conclusions
These findings demonstrate that functional IK and SK-channels are expressed in human A375 melanoma cells. Overall, our data supports the role of K+ channel subtypes in the control of cell proliferation in human malignant melanoma cells that may represent potential targets for novel therapeutic interventions.
References
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