key: cord-0964032-hpkkk77t
authors: Teisseyre, Andrzej; Uryga, Anna; Michalak, Krystyna
title: Statins as inhibitors of voltage-gated potassium channels Kv1.3 in cancer cells
date: 2021-01-07
journal: J Mol Struct
DOI: 10.1016/j.molstruc.2021.129905
sha: 81205a6c63c5eb03fe41a7dad602b0b047fa7588
doc_id: 964032
cord_uid: hpkkk77t

Voltage-gated potassium channels are integral membrane proteins selectively permeable for potassium ions and activated upon change of membrane potential. Voltage-gated potassium channels of the Kv1.3 type were discovered both in plasma membrane and in inner mitochondrial membrane (mito Kv1.3 channels). For some time Kv1.3 channels located both in plasma membrane and in mitochondria are considered as a potentially new molecular target in several pathologies including some cancer disorders. Lipophilic non-toxic organic inhibitors of Kv1.3 channels may potentially find a clinical application to support therapy of some cancer diseases such as breast, pancreas and lung cancer, melanoma or chronic lymphocytic leukaemia (B-CLL). Inhibition of T lymphocyte Kv1.3 channels may be also important in treatment of chronic and acute respiratory diseases including severe pulmonary complication in corona virus disease Covid 19, however further studies are needed to confirm this supposition. Statins are small-molecule organic compounds, which are lipophilic and are widely used in treatment of hypercholesterolemia and atherosclerosis. Electrophysiological studies performed in our laboratory showed that statins: pravastatin, mevastatin and simvastatin are effective inhibitors of Kv1.3 channels in cancer cells of human T cell line Jurkat. We showed that application of the statins in the concentration range from 1.5 μM to 50 μM inhibited the channels in a concentration-dependent manner. The inhibitory effect was the most potent in case of simvastatin and the least potent in case of pravastatin. The inhibition was partially irreversible in case of simvastatin and fully reversible in case of pravastatin and mevastatin. It was accompanied by a significant acceleration of the current inactivation rate without any significant change of the activation rate. Mechanism of the inhibition is probably complex, including a direct interaction with the channel protein and perturbation of lipid bilayer structure, leading to stabilisation of the inactivated state of the channels.

Voltage-gated potassium channels of the Kv1.3 type are widely expressed in many types of cells, both normal and cancer [1, 2] . Activity of Kv1.3 channels plays important role, for example in setting resting membrane potential, cell proliferation, apoptosis and volume regulation [3, 4] . Kv1.3 channels are expressed both in the plasma membrane and in the inner mitochondrial membrane (mito Kv1.3 channels) [4, 5] . Specific blockers of Kv1.3 channels in human T lymphocytes potentially may be applied in selective immunosuppression [3, 5] and in treatment of chronic respiratory diseases [6] . Recently formulated hypothesis claims that inhibition of T lymphocyte Kv1.3 channel might suppress the "cytokine storm" in severe cases of COVID-19 disease and this could be a novel therapeutic strategy to combat the disease [7] . The hypothesis was supported by the fact that chloroquine, which reduces both the viral replication and production of cytokines by leukocytes, also inhibits Kv1.3 channels in T lymphocytes [8] and this inhibition may exert a "cytokine storm" -suppressing immunosuppressive effect [7] .

Several studies demonstrated an altered expression of Kv1.3 channels in some cancer disorders such as breast, colon, pancreas, smooth muscle, skeletal muscle, lung, kidney and prostate cancer [5, [9] [10] [11] . Inhibitors of Kv1.3 channels may potentially find a clinical application in therapy of some cancer disorders characterized by an over-expression of Kv1.3 channels, such as for example: breast and lung cancer, melanoma, pancreatic ductal adenocarcinoma or chronic lymphocytic leukemia [10 -12] . Among many inhibitors of Kv1.3 channels the most promising candidates for potential clinical application in cancer therapy could be lipophilic small-molecule organic compounds being able to simultaneously inhibit 6 cancer cell proliferation (by inhibition of plasma membrane Kv1.3 channels) and to induce selective cancer cell death (by inhibition of mito Kv1.3 channels) [11] .

Studies performed during in our laboratory showed that to this group of compounds may also belong some plant-derived flavonoids and substituted stilbenes, which exert antiproliferative and pro-apoptotic effects on Kv1.3 channel-expressing cancer cells and combine high efficiency and specificity in cancer cell elimination with good bioavailability and low cytotoxicity [10, 11] . We showed that genistein, a plant-derived is flavone known as a potent protein tyrosine kinase (PTK) inhibitor, and a substituted stilbene -resveratrol, a natural anti-cancer agent present at highest concentrations in red grapes and wine, both are inhibitors of Kv1.3 channels in human T lymphocytes [11] . Moreover, two synthetic methoxy-derivatives of flavonoid naringenin (4', 7-dimethylether and 7-methylether) and one synthetic tetramethoxy-derivative of piceatannol also inhibit Kv1.3 channels in normal human T lymphocytes [11] . Another two flavonoids that inhibit Kv1.3 channels expressed both in normal human T lymphocytes and in Jurkat T cells are acacetin and chrysin [11] .

Studies performed recently in our laboratory provide evidence that a natural derivative of a flavonoid naringenin isolated from common hops (Humulus lupulus) -8prenylnaringenin (8-PN), a potent phytoestrogen present in beer, inhibits Kv1.3 channels both in normal human T lymphocytes and in human Jurkat T cells when applied at low micromolar concentrations [11] . Ability to inhibit Kv1.3 channels in cancer cells is shared by other hops-derived prenylated compounds such as: xanthohumol, a prenylated chalcone and two prenylflavanones -isoxanthohumol and 6-prenylnaringenin (6-PN) [11] . All these compounds inhibited Kv1.3 channels much more strongly than all non-prenylated plantderived compounds tested in our laboratory [11] . These results may confirm the hypothesis that the presence of prenyl group in a molecule is a factor that facilitates the inhibition of Kv1.3 channels by compounds from the groups of flavonoids and chalcones [11] .

Other small-molecule organic compounds applied in medical therapy are statins [13] .

These compounds are known as inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. This enzyme catalyzes reduction of HMG-CoA to L-mevalonate, a key intermediate in biosynthesis of cholesterol and many isoprenoid metabolites. Thus, inhibition of this reductase strongly inhibits biosynthesis of cholesterol and isoprenoid metabolites. Therefore, statins are widely applied in treatment of hypercholesterolemia and atherosclerosis [13] . Recently formulated hypothesis claims that statins might also be key therapeutic agents in therapy of severe COVID-19 cases [14] . This is due to the HMG-CoA reductase inhibition, which leads to depletion of cellular and plasma membrane cholesterol.

It was shown that this reduction of cholesterol content in cell membranes prevents SARS-CoV-2 virus entry into the host cell even if the viral spike protein is bound to the lipid raft ACE2 receptor [14] . Moreover, it was shown that statins -mevastatin and simvastatin exert antiproliferative, pro-apoptotic and reversing drug resistance effect in Kv1.3 channelexpressing human colon adenocarcinoma cell line LoVo and its doxorubicin-resistant subline LoVo/Dx [15] . Besides LoVo cells simvastatin also inhibits proliferation and induces apoptosis of other Kv1.3 channel-expressing cancer cells such as breast adenocarcinoma (MCF-7 and MDA-MB-231), leukaemia (Jurkat T and CEM) and promyelocytic leukaemia (HL60) [11, 13] cells. Similarly, to simvastatin also pravastatin inhibits proliferation and induces apoptosis of Kv1.3 channel-expressing leukemic Jurkat T and CEM cells [13] . It is therefore possible that antiproliferative and proapoptotic effects of simvastatin, mevastatin and pravastatin on cancer cells may be, at least partially, due to inhibition of Kv1.3 channels. However, a little is known about influence of statins on activity of Kv1.3 channels in cancer cells. Preliminary electrophysiological study applying the "patch-clamp" technique showed that pravastatin, lovastatin and simvastatin are inhibitors of Kv1.3 channels in nontumour murine thymocytes [16] . A more detailed study performed with lovastatin showed that this compound inhibits Kv1.3 channels expressed both in normal human T lymphocytes and in cancer cells of human Jurkat T cell line [17] . Studies performed recently by Wang and co-workers have shown that simvastatin inhibits Kv1.3 channels in human monocytic leukaemia THP-1 cells in a concentration-dependent manner [18] . However, these studies were performed applying a voltage step protocol, in which the membrane voltage is changed in a stepwise manner [18] . This is in contrast to a gradual change of the voltage that occurs [19, 20] these cells were used in our study as a model system of cancer cells. Obtained results provide evidence that all selected statins effectively inhibit Kv1.3 channel in Jurkat T cells.

Simvastatin was the most potent channel inhibitor, whereas pravastatin was the weakest one.

The human leukemic T cell line, Jurkat (clone E6-1), was purchased from American Type . Such a low calcium concentration was applied to prevent the activation of calcium-activated K + channels K Ca 2.2 abundantly expressed in Jurkat T cells [22] . The chemicals were purchased from the Polish Chemical Company (POCH, Gliwice, Poland), except of HEPES and EGTA that were purchased from SIGMA. The examined statins were purchased from Alexis Biochemicals (Lausen, Switzerland).

Dishes with cells were placed under an inverted Olympus IMT-2 microscope. Solutions containing tested compounds were applied using eight-channel gravitation perfusion system (ALA Scientific Instruments, Farmingdale, NY, USA). Pipettes were pulled from a borosilicate glass (Hilgenberg, Germany) and fire-polished before the experiment. The pipette resistance was in the range of 3-5 MΩ.

Whole-cell potassium currents in TL were recorded applying the patch-clamp technique [23] . The currents were recorded using an EPC-7 Amplifier (HEKA, Germany), low-pass filtered at 3 kHz, digitised using a CED Micro 1401 analogue-to-digital converter (Cambridge, UK) with a sampling rate of 10 kHz. The influence of selected compounds on the activity of the channels was studied by applying the voltage ramp protocol. Voltage ramps gradually depolarising cell membranes from -100 mV up to +40 mV were applied every 30 s; the ramp duration was 340 ms and holding potential -90 mV (see below). Upon application of the voltage ramp protocol, potassium currents in Jurkat T cells could be stably recorded for at least 20 minutes after "break-in" to the whole-cell configuration. During the off-line analysis the value of Kv1.3 current at the end of a voltage ramp (+40 mV) was calculated. For this purpose, the leak current estimated at +40 mV was subtracted from the total ramp current recorded at this voltage.

In order to study the influence of selected compounds on the channel activation and inactivation kinetics in more detail another protocol of depolarising voltage stimuli was applied. This protocol contained a sequence of depolarising voltage steps from the holding potential of -90 mV to +60 mV (500 ms step duration) applied every 30 s (see below).

All experiments were carried out at room temperature (22-24 o C).

Unless otherwise stated the data are presented as mean  standard deviation.

The inhibition of the channel is presented in terms of a relative current recorded upon application of the studied compounds, defined as I/I contr ; where: I -Kv1. 3 This figure depicts the raw currents (without leak subtraction) that were recorded applying the voltage ramp protocol. The evoked currents contained two components: small linear and much bigger non-linear. The linear current was the unspecific leak current (reversal potential equal to 0 mV), whereas the non-linear component was due to activation of Kv1.3 channels [22, 24] .

Apparently, application of the statins significantly diminished the amplitude of Kv1.3 current. The reduction of the amplitude was more potent in case of simvastatin and mevastatin than in case of pravastatin, although the later compound was applied at higher concentration ( Figure 2B-D) . Interestingly, the current did not recover completely after wash-out of simvastatin ( Figure 2B ). This indicates that the inhibitory effect of simvastatin was partially irreversible. On the other hand, the currents recovered completely after washout of mevastatin and pravastatin ( Figure 2C and 2D ). This indicates that the inhibitory effect of the compounds was reversible. after wash-out of the compounds.

Apparently, the inactivation was much more rapid in the presence of the statins than under control conditions. Interestingly, inactivation was still markedly accelerated after wash-out of simvastatin ( Figure 5B ). This indicates that acceleration of inactivation by simvastatin was partially irreversible. On the other hand, inactivation acceleration was completely removed after wash-out of mevastatin and pravastatin ( Figure 5C and 5D ). This indicates that acceleration of inactivation upon application of these compounds was reversible. Figure 6C ).

Since the inactivation rate of the Kv1.3 currents was significantly higher after exposure to the statins, it was of interest to study the influence of the compound also on the channel activation kinetics. Figure 7 The inhibitory effect of the two statins (simvastatin and mevastatin) on Kv1.3 channels was stronger than inhibition caused by the most of the non-prenylated flavonoids and stilbenes that were studied earlier in our laboratory [11] . We compared these two groups of compounds because both statins and flavonoids influence cholesterol synthesis pathway. M, for the peak and end-of-pulse current, respectively [17] . Zhao and co-workers showed that lovastatin shared the binding site with other inhibitors of Kv1.3 channels, such as verapamil and internally applied tetraethyl-ammonium (TEA) [17] . Both compounds inhibit Kv1.3 channels by direct interaction with the channel protein via the "open channel block" mechanism [25] . The "open-channel block" mechanism was originally proposed by Armstrong (1966) for blocking of voltage gated potassium channels by internally applied quaternary ammonium ions (QA) [26] . According to the "open channel block" mechanism, the molecule of inhibitor blocks the channel from the inner side of membrane by interaction with binding site in the central channel cavity while the channel is open [26] . The "open channel block" mechanism is revealed by an observation of significant acceleration of current inactivation without a change in the activation rate [26] . It is known that extracellularly applied small-molecule lipophilic compounds, such as verapamil, can diffuse through the cell membrane and inhibit Kv1.3 channel from the inner side [25, 27] . To the group of small-molecule lipophilic compounds belong also simvastatin and lovastatin [28] .

Mevastatin is structurally related to simvastatin (see above). According to the "open channel block" mechanism, the inhibitory effect should be reversible. The inhibitory effect is reversible in case of mevastatin; however, in case of simvastatin the inhibitory effect is partially irreversible at concentrations of 15 M and 30 M. This is in accordance to what was observed by Kazama and co-workers, who reported irreversible inhibition of Kv1.3 channels in murine thymocytes by simvastatin applied at 10 M concentration [16] . Such partial irreversibility could not be explained by the "open channel block" mechanism. This may be due to irreversible perturbations in structure of membrane lipid bilayer. Kazama and co-workers showed that application of simvastatin at 10 M concentration caused a significant and irreversible decrease of the membrane capacitance *16+. This was probably due to irreversible increase of membrane thickness, which was a consequence of accumulation of the drug in the plasma membrane *16+. Accumulated drug molecules may directly or indirectly interact with channel protein irreversibly reducing whole-cell peak current and accelerating channel inactivation *16+. This may indicate that the inhibitory effect of simvastatin on Kv1.3 channels probably occurs via a complex mechanism including both direct interaction with the channel protein via the "open channel block" mechanism and interactions of simvastatin with cell membrane leading to perturbations of lipid bilayer structure due to accumulation of simvastatin molecules in the plasma membrane.

Kv1.3 channels in human monocytic leukemia THP-1 cells in a concentration-dependent manner [18] . The estimated EC 50 value was 8.75±1.25 µM *18+, which was higher than that reported in our study (4.36±0.3 µM). This is not surprising, because such a difference may be due to application of different experimental protocol -voltage steps [18] different from the voltage ramp sequence applied in our studies. However, unexpectedly, in a marked contrast to our results, no significant acceleration of the Kv1.3-current inactivation upon application of simvastatin was observed [18] . The reason for such a discrepancy is not clear.

In contrast to simvastatin and mevastatin, the inhibitory effect of pravastatin on Kv1.3 channels is relatively small. This was in accordance to what was reported by Kazama and coworkers *16+. Moreover, in contrast to what was observed in case of simvastatin and mevastatin, an application of pravastatin at concentrations up to 50 M did not significantly accelerate inactivation of the currents (Figure 6) . Such a small effect may be explained by relatively small lipophilicity of pravastatin. It was shown that the octanol-water partition coefficient (P o/w ) value at physiological pH (7.4) is equal to 0.210.01 and 655 for pravastatin and simvastatin, respectively [28] . It means that pravastatin is 310 times less lipophilic than simvastatin. The mechanism of inhibitory effect of pravastatin on Kv1.3 channels remains to be elucidated. It is probably different than in case of simvastatin and 

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