Co-Inhibition of Kv1.3 Channel Activity by Selected Chalcones and Statins in a Model of Cancer Cell Line Jurkat T

TL;DR

This study shows that combining chalcones and statins can strongly inhibit a potassium channel in cancer cells, potentially leading to new cancer treatments.

Contribution

The study reveals that combining prenylated chalcones with statins enhances Kv1.3 channel inhibition in cancer cells.

Findings

Isobavachalcone almost completely and irreversibly inhibits Kv1.3 channels in Jurkat T cells.

Co-application of isobavachalcone with mevastatin or simvastatin significantly enhances channel inhibition.

Non-prenylated chalcones inhibit Kv1.3 but less effectively and in a reversible manner.

Abstract

Voltage-gated potassium channel Kv1.3 plays an important role in the regulation of survival and apoptosis in many cell types, including both normal and cancer cells. Inhibitors of these channels may potentially find clinical applications in the treatment of various diseases, including certain cancers characterized by the over-expression of Kv1.3. In this study, the effects of isobavachalcone (IBC) and two non-prenylated chalcones—2′-hydroxy-4,3′-dimethoxychalcone (HDC) and 2′-hydroxy-2-methoxychalcone (HMC)—on Kv1.3 channel activity were investigated in the Jurkat T cancer cell line using the whole-cell patch-clamp technique. The electrophysiological measurements were preceded by experiments assessing cell viability, and the patch-clamp data were consistent with results obtained from MTT-based assays. We observed an almost complete and irreversible inhibition of Kv1.3 in the presence of IBC. The non-prenylated chalcones also inhibited the channels, but with lower potency and in a reversible and incomplete manner. The inhibitory effect of IBC was significantly enhanced upon co-application with simvastatin (SIM) and mevastatin (MEV). In contrast, inhibition by the non-prenylated chalcones was significantly increased only in the presence of mevastatin, but not simvastatin. The channel inhibition may be related to the anti-proliferative and pro-apoptotic activities of these compounds in Kv1.3-expressing cancer cells. Altogether, our results indicate that both prenylated and non-prenylated chalcones, particularly in combination with statins, may represent biologically active scaffolds, warranting further optimization and preclinical evaluation.

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1. Introduction

Voltage-gated potassium channel Kv1.3 belongs to the group of Shaker-related mammalian channels. The channel is encoded by the KCNA3 gene. Kv1.3 channels are referred to as “delayed rectifier” channels, which open upon membrane depolarization and then undergo slow and complex inactivation [1]. The channel opening allows potassium ions to flow down their electrochemical gradient. Kv1.3 channels exhibit high selectivity toward potassium ions over other monovalent cations, except for rubidium [1].

Kv1.3 channels are widely expressed in various immune, neural, and peripheral tissues [1]. They are also found in intracellular membranes, notably the inner mitochondrial membrane (mitoKv1.3) [2]. Channel expression can be markedly altered in certain cancers [3,4,5,6,7,8,9].

Activity of Kv1.3 channels plays an important role in many processes, including cell proliferation and programmed cell death (apoptosis) [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16]. Activity of the channels is inhibited by many different compounds [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16]. Inhibition of the channels may putatively be beneficial in the therapy of various diseases, including some cancers, with an over-expression of the Kv1.3 channel, such as melanoma, pancreatic ductal adenocarcinoma (PDAC), multiple myeloma, and B-type chronic lymphocytic leukemia (B-CLL) [3,4,5,6,7,8,9,10,11,12,13,14].

It was shown that some naturally occurring polycyclic compounds may effectively inhibit the Kv1.3 channel in cancer cells and significantly reduce the viability of these cells by an induction of the mitochondrial pathway of apoptosis [9,12,14]. Among these compounds are two flavonoid derivatives which belong to the group of chalcones: xanthohumol and licochalcone A [14,15]. It was also shown that the application of xanthohumol significantly reduces the viability of the Kv1.3 channel-expressing cancer cell line Jurkat via an induction of the mitochondrial pathway of apoptosis [12,14].

In our study, we focused on 1-[2,4-dihydroxy-3-(3-methyl-2-butenyl)-phenyl]-3-(4-hydroxyphenyl)-2-propen-1-one, known as isobavachalcone (IBC). The chemical structure of this compound is presented in Table 1. This plant-derived compound, firstly isolated from Psoralea corylifolia, shares multiple pharmacological activities, including anti-cancer, anti-microbial, anti-inflammatory, anti-oxidative, and neuroprotective [16,17].

It was shown that IBC may exert anti-proliferative and pro-apoptotic effects on Kv1.3 channel-expressing cancer cell lines [16]. Our unpublished preliminary results also showed that IBC was an inhibitor of Kv1.3 channels in Jurkat T cells. However, the inhibitory effect on the channels was not studied in detail. These initial observations prompted us to investigate the effects of IBC more systematically. Consequently, we designed the present study to characterize its impact on Kv1.3 channel activity and explore potential interactions with statins. In particular, it was of interest to study whether the putative inhibitory effect on the channel was concentration-dependent and reversible.

Previous studies have shown that the anti-cancer activity of flavonoids may be augmented by statins, lipid-lowering drugs commonly used in hypercholesterolemia [18]. Statins are inhibitors of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase (HMGCR), which catalyzes the key step in the endogenous synthesis of cholesterol. Abnormality in the level of cholesterol has been positively correlated with the risk of cancer. Cholesterol influences the fluidity of lipid membranes and, as a consequence, cellular transport and the activity of proteins incorporated into the membranes. Also, an elevated level of cholesterol in mitochondria may cause alterations in mitochondrial function and physiology. An increase in mitochondrial cholesterol accumulation has been found as one of the factors involved in cancer development and cellular resistance to apoptotic death [19]. Due to the inhibition of HMGCR, the statins reduce the level of cholesterol but also the prenylation of proteins, which can lead to disturbances in the processes associated with cell life and death. It is widely known that statins exert anti-cancer effects against cancers derived from different origins [20].

Results of our previous studies provided evidence that the co-application of xanthohumol with some statins may lead to an additive inhibitory effect on the Kv1.3 channels [12,14]. Thus, it was of interest to investigate whether the co-application of IBC with the statins led to an analogous effect. Therefore, IBC was studied when applied alone and in combination with selected statins: simvastatin and mevastatin. The chemical structures of the studied compounds are presented in Table 1.

The prenyl group of polyphenol compounds has been found to be one factor associated with their increased anti-cancer activity [21]. Also, the results of our previous studies showed that the prenylation of flavonoids improves the ability of these compounds to inhibit Kv1.3 channels [9,12,14]. Thus, one of the main aims of our work was to compare the inhibitory effect of IBC with those exerted on the channel by two non-prenylated chalcones: 2′-hydroxy-4,3′-dimethoxychalcone (HDC) and 2′-hydroxy-2-methoxychalcone (HMC) (Table 1).

Both compounds share anti-cancer activities against cancer cells, including those expressing Kv1.3 channels [22]. The compounds were tested when applied alone and in combination with the statins: simvastatin and mevastatin.

Since Kv1.3 channels are predominantly and abundantly expressed in human leukemic cell line Jurkat T [23,24], these cells were used as a model system to study the influence of the selected compounds on the channel activity in cancer cells.

In this study, we examined the effects of IBC and non-prenylated chalcones—HMC and HDC—on Kv1.3 channels in Jurkat T cells. We tested these compounds both individually and in combination with simvastatin or mevastatin to investigate potential interactions. Electrophysiological recordings were conducted to assess channel activity, while MTT assays were used to evaluate cell viability, enabling us to explore the relationship between Kv1.3 inhibition and cellular metabolic activity.

2. Results

To assess the viability of Jurkat T cells exposed to flavonoids or statins, mitochondrial activity was measured. We used a method that is based on the ability of mitochondrial enzymes, mainly succinate dehydrogenase, to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MT—T) into insoluble formazan crystals. Due to the fact that mitochondrial enzymes are active only in live cells, the absorbance of dissolved formazan is proportional to the number of viable cells. The results demonstrated a concentration-dependent reduction in cell viability following incubation with each of the tested compounds (Figure 1A–C).

IBC exhibited greater potency in inhibiting cell growth (IC_50_ = 8.4) compared with the non-prenylated chalcones (IC_50_ for HMC = 39.2; IC_50_ for HDC = 30.1). Among the statins, SIM was a more effective inhibitor of cell viability (IC_50_ = 22.3) than MEV (IC_50_ = 29.9). Moreover, we checked the influence of solvent—DMSO—on the viability of Jurkat T cells. However, no reduction in cell viability was observed in the presence of DMSO at concentrations equivalent to those used as a solvent for chalcones or statins (Supplementary Figure S1). The primary aim of our study was to evaluate the potential of selected chalcones and statins to co-inhibit Kv1.3 channel activity. Therefore, as a first step, we examined the effects of selected compound combinations on cell growth. In these experiments, the compounds were applied at a 1:1 ratio, each at a concentration of 6 µM. We selected a concentration of 6 µM for the combination treatments because, as shown in Figure 1A–C, this concentration results in a moderate reduction in cell viability (~55–70% depending on the compound). Using this submaximal concentration allows us to evaluate potential synergistic or additive effects of the drug combinations while minimizing cytotoxicity that could mask specific interactions. Concentrations higher than 6 µM caused a steep decline in viability, particularly for IBC (Figure 1A), which could lead to excessive cell death and obscure the combinatorial effects we aim to study. Considering the cytotoxicity of the individual agents, this concentration was optimal for detecting biological effects of the compound combinations. Our results demonstrated additive interactions for the combinations of IBC with SIM or MEV (Figure 2A). Cell viability was reduced by more than 30% in the presence of IBC/SIM or IBC/MEV compared with IBC or the respective statins applied alone. Furthermore, both non-prenylated chalcones exerted stronger inhibitory effects on Jurkat T-cell growth when combined with MEV (Figure 2B). We observed statistically significant differences between the mitochondrial activity of cells treated with HMC, HDC, or MEV alone and the activity measured in cells incubated with the combinations HMC/MEV or HDC/MEV.

In order to study the inhibitory effects of the selected compounds on the activity of Kv1.3 channels in Jurkat T cells, whole-cell ramp currents were recorded under control conditions, upon application of the examined compounds and after wash-out of them. Upon application of the voltage ramp, the cell membrane was gradually depolarized from −100 mV to +40 mV. The ramps were applied every 30 s. Before the application of the ramp and during the time interval between successive amps, the examined cell was held at the holding potential of −90 mV (see Section 4). Figure 3 depicts examples of the whole-cell ramp currents recorded in a Jurkat T under control conditions, upon the application of IBC at a concentration of 3 μM and after wash-out of the compound. The figure depicts raw currents (without leak subtraction). The ramp current contained two components: a small linear one and a much bigger non-linear one. The linear current was an unspecific leak current, which was irrelevant for the study and was subtracted from the total current during the off-line analysis. Our previous studies have shown that the non-linear component is due to an activation of Kv1.3 channels [25]. Application of IBC significantly diminished the amplitude of the Kv1.3 current. Interestingly, the current did not fully recover after the wash-out of IBC. This indicates that IBC exerted an inhibitory effect on Kv1.3 channels and this effect was partially irreversible.

The inhibitory effect of IBC on Kv1.3 whole-cell currents in Jurkat T cells was studied at different concentrations of the compound (Figure 4). Figure 4 depicts relative peak currents upon the application of IBC at various concentrations ranging from 1.5 μM to 15 μM and after wash-out of the drug. Upon application of IBC at concentrations of 1.5 μM, 3 μM, 6 μM, 9 μM, and 15 μM, the relative peak currents were reduced to 0.55 ± 0.17 (n = 16), 0.40 ± 0.20 (n = 11), 0.34 ± 0.14 (n = 26), 0.10 ± 0.22 (n = 21), and 0.01 ± 0.02 (n = 12) of the control value, respectively. The reduction in the relative currents was statistically significant (p < 0.05) for all concentrations tested. After wash-out of the drug applied at above-mentioned concentrations, the relative peak currents recovered to 0.58 ± 0.25 (n = 12), 0.51 ± 0.20 (n = 4), 0.52 ± 0.25 (n = 15), 0.40 ± 0.19 (n = 7), and 0.04 ± 0.03 (n = 3) of the control value, respectively. The relative peak currents after wash-out of the drugs applied at all concentrations were significantly (p < 0.05) smaller than the control values.

The inhibitory effect of IBC on Kv1.3 currents in Jurkat T cells was statistically significant, dose-dependent, and almost complete at a concentration of 15 μM. Moreover, the effect was partially irreversible at concentrations up to 9 μM and almost fully irreversible at a concentration of 15 μM (Figure 4). The estimated value of the half-blocking concentration was 2 μM (Figure 4).

The next stage of our study was to investigate whether the co-application of IBC with simvastatin and mevastatin leads to an additive inhibitory effect on the channel, as has occurred, in most cases, upon the co-application of xanthohumol with mevastatin [12,14]. If the inhibitory effects upon the co-application of IBC with the statins were additive, the relative peak current upon co-application would be equal to the product of the multiplication of the currents recorded upon the application of each compound alone [12,14].

Figure 5A depicts relative peak currents upon the application of IBC and simvastatin alone and upon the co-application of IBC with simvastatin. The concentration of all the compounds was equal to 6 μM. Such a concentration was chosen because it was high enough to inhibit a majority of Kv1.3 channels, both in the case of IBC (this study) and simvastatin [12,14]. Upon the co-application of IBC with the statin, the relative peak current was equal to 0.16 ± 0.10 (n = 16) of the control value (Figure 5A, Table 2). This value was significantly (p < 0.05, one-way ANOVA) lower than the values upon application of IBC alone (0.34 ± 0.14, n = 26, this study) and simvastatin alone (0.50 ± 0.10, n = 16) [12] at the same concentration. On the other hand, relative peak currents upon application of IBC and simvastatin alone were not significantly (p > 0.05, one-way ANOVA) different from each other. The relative peak current upon co-application (0.16) was not significantly (p > 0.05) different than the product of the multiplication of the currents upon the application of each compound alone (0.34 × 0.5 = 0.17, Table 2). This may indicate that the inhibitory effects of IBC and simvastatin were additive. The relative peak current after the wash-out was equal to 0.41 ± 0.21 (n = 10) of the control value. This demonstrates that the inhibitory effect upon the co-application of IBC and simvastatin was partially irreversible.

Figure 5B depicts relative peak currents upon application of IBC and mevastatin alone and upon co-application of IBC with mevastatin, at the same concentration equal to 6 μM. Upon co-application of IBC with the statin, the relative peak current was equal to 0.16 ± 0.13 (n = 36) of the control value (Figure 5B). This value was significantly (p < 0.05, one-way ANOVA) lower than the values upon application of IBC alone (see above) and mevastatin alone (0.41 ± 0.11, n = 21) [12], at the same concentration. On the other hand, relative peak currents upon application of IBC and mevastatin alone were not significantly (p > 0.05, one-way ANOVA) different from each other. The relative peak current upon co-application (0.16) was not significantly (p > 0.05) different than the product of the multiplication of the currents upon the application of each compound alone (0.34 × 0.41 = 0.14, Table 2). This indicates that the inhibitory effects of IBC and mevastatin were additive. The relative peak current after the wash-out was equal to 0.48 ± 0.30 (n = 19) of the control value. Evidently, the inhibitory effect upon co-application of IBC and mevastatin was partially irreversible.

Results of our previous studies provided evidence that the presence of a prenyl group in a molecule facilitates the inhibition of the Kv1.3 channel in cancer cells [9,12,14]. Therefore, it was of interest to compare the inhibitory effect of IBC, which is a prenylated chalcone, with the effect exerted by non-prenylated chalcones: HDC and HMC (Table 1). Figure 6 depicts whole-cell potassium currents recorded in a Jurkat T cell applying a voltage ramp (Figure 6A) under control conditions, upon application of HDC at a concentration of 6 μM and after wash-out of the drug (Figure 6B). Figure 6C depicts the same currents recorded under control conditions, upon application of HMC at a concentration of 6 μM and after wash-out of the drug. Remarkably, the application of both compounds significantly reduced the amplitude of Kv1.3 currents. However, in contrast to what was observed in the case of the application of IBC, the currents completely recovered after wash-out of the compounds. Thus, in contrast to IBC, the inhibitory effects exerted on the channel by both non-prenylated chalcones were reversible.

The application of HDC at a concentration of 6 μM reduced the relative peak currents to 0.37 ± 0.16 (n = 13) of the control value (Figure 7). This value was markedly higher than the currents recorded upon the application of IBC at the same concentration (Figure 4). When the concentration of HDC was increased to 30 μM, the relative peak current was further reduced to 0.15 ± 0.10 (n = 2) of the control value (Figure 7). The reduction in relative peak currents was statistically significant (p < 0.05) for both concentrations tested (Figure 7).

The application of HMC at a concentration of 6 μM reduced the relative peak currents to 0.57 ± 0.22 (n = 14) of the control value (Figure 7). This value was markedly higher than the currents recorded upon the application of IBC at the same concentration (Figure 4). In contrast to what was observed in the case of HDC, an increase in the HMC concentration to 30 μM did not further reduce the relative peak current. Instead, the current was slightly increased to 0.65 ± 0.28 (n = 9) of the control value (Figure 7). Thus, the inhibitory effect of HMC on the channels was concentration-independent. The reduction in relative peak currents was statistically significant (p < 0.05) for both concentrations tested (Figure 7).

Obtained data provided evidence that the channel inhibition upon application of both non-prenylated chalcones was not complete, even at a concentration of 30 μM. On the other hand, the inhibitory effect of IBC on the channel was almost complete at a concentration of 15 μM (Figure 4). These results indicate that both non-prenylated chalcones were less potent inhibitors of the channel than IBC was.

It was shown that the co-application of IBC with the statins simvastatin and mevastatin led to an additive inhibitory effect on Kv1.3 currents (see above). Therefore, it was of interest to study whether the co-application of HDC and HMC with simvastatin and mevastatin led to a similar effect. Surprisingly, the co-application of both non-prenylated chalcones at a concentration of 6 μM with the same concentration of simvastatin did not exert any inhibitory effect on the current. On the other hand, the co-application of both chalcones with the same concentration of mevastatin produced an additive inhibitory effect on the currents (see below).

Figure 8 depicts the relative peak currents upon the application of HDC (A) and HMC (B) alone, mevastatin alone, and the currents upon the co-application of HDC (A) and HMC (B) with mevastatin, at the same concentration equal to 6 μM. Upon the co-application of HDC and HMC with the statin, the relative peak current was equal to 0.19 ± 0.10 (n = 11) of the control value (Figure 8A) and 0.25 ± 0.10 (n = 15) of the control value (Figure 8B), respectively. This value was significantly (p < 0.05, one-way ANOVA) lower than the values upon application of both chalcones alone (see above) and mevastatin alone (see above), at the same concentration. On the other hand, relative peak currents upon application of HDC and mevastatin alone (Figure 8A) and HMC and mevastatin alone (Figure 8B) were not significantly (p > 0.05, one-way ANOVA) different from each other. The relative peak currents upon co-applications (see above) were not significantly (p > 0.05) different than the products of multiplication of the currents upon the application of each compound alone (0.37 × 0.41 = 0.15 for HDC and 0.57 × 0.41 = 0.23 for HMC, respectively, Table 2). This may indicate that the inhibitory effects of both non-prenylated chalcones and mevastatin were additive.

An isobolographic analysis to detect the existence of putative synergy between the statins and the chalcone derivative was performed. For pure compounds and their combinations, dose and effect data were obtained from the relative peak currents. For all combinations studied, the CI values of chalcone derivatives with mevastatin were clearly below 1, suggesting synergy between these compounds. In the case of a combination with simvastatin, synergy was observed only in the presence of IBC. The highest CI value, still below one, was recorded for the combination of HMC with mevastatin (Table 3).

3. Discussion

The main aim of our study was to investigate the influence of selected chalcones (HMC, HDC, and IBC) and statins (SIM and MEV) on the activity of Kv1.3 channels in Jurkat cells. Kv1.3 is a well-established molecular target in T lymphocytes and plays a critical role in the regulation of immune cell activation and proliferation, making it a promising target in autoimmune and inflammatory disorders [6].

In order to determine appropriate concentrations for electrophysiological experiments, the cytotoxic effects of all tested compounds were first evaluated using a mitochondrial activity assay (Figure 1 and Figure 2). As shown in Figure 1A–C, all chalcones and statins reduced Jurkat cell viability in a concentration-dependent manner. Notably, the prenylated chalcone IBC exhibited significantly higher cytotoxic potency compared with the non-prenylated analogues HMC and HDC. This observation is consistent with previous studies demonstrating that prenylation increases the biological activity of flavonoids by enhancing their lipophilicity and affinity for cellular membranes, which may facilitate intracellular accumulation and target engagement [26,27]. Similar effects of prenylation on cytotoxic and anti-proliferative activity have been reported for other chalcone derivatives in cancer and immune cell models [28,29].

Among the statins, SIM showed stronger inhibitory effects on Jurkat cell viability than MEV (Figure 1C), which may be attributed to its higher lipophilicity and improved membrane permeability, consistent with previous reports demonstrating that lipophilic statins such as simvastatin enter cells primarily via passive diffusion and exert more pronounced cytotoxic effects relative to hydrophilic counterparts due to enhanced cellular uptake and interaction with intracellular targets [30].

Furthermore, combination treatments revealed that IBC combined with either statin produced significantly stronger reductions in mitochondrial activity than single-agent treatments (Figure 2A). Such additive effects may reflect the simultaneous modulation of distinct cellular pathways, including ion channel function and cholesterol-dependent membrane organization, both of which are known to influence T-cell homeostasis [31,32,33].

Importantly, the patterns observed in the viability assays were consistent with electrophysiological measurements of Kv1.3 inhibition (Figure 4, Figure 5, Figure 7 and Figure 8), suggesting that the metabolic effects detected at the mitochondrial level correlate with functional suppression of potassium channel activity. This concordance supports the interpretation that the observed additive interactions between chalcones and statins are, at least in part, mediated through Kv1.3-dependent mechanisms.

Results of patch-clamp recordings have shown that the application of a prenylated chalcone IBC inhibited the activity of the Kv1.3 channel in Jurkat T cells. The inhibitory effect occurred in a concentration-dependent manner, with the estimated value of half-blocking concentration equal to 2 μM (Figure 4). The inhibitory effect of IBC was almost complete and irreversible at a concentration of 15 μM. The inhibitory effect was additive when IBC was co-applied with simvastatin and mevastatin (Figure 5). Non-prenylated chalcones HDC and HMC also inhibited the channels, but less potently than IBC did (Figure 7). The inhibitory effects of both non-prenylated chalcones were additive when co-applied with mevastatin (Figure 8).

Our previous study has shown that a prenylated chalcone, xanthohumol, inhibited the activity of the Kv1.3 channel in Jurkat T cells in a concentration-dependent manner [9,14]. The value of half-blocking concentration was about 3 μM, which was similar to the value obtained in this study for IBC. However, in contrast to what was observed in the case of IBC, the inhibitory effect of xanthohumol was not complete and fully reversible, even at a concentration of 30 μM [14]. Moreover, upon the co-application of xanthohumol with the statins, an additive inhibitory effect was observed only in the case of the co-application with mevastatin [12,14]. These results may indicate that the inhibitory effect of xanthohumol on the Kv1.3 channel was weaker than the effect observed for IBC in this study.

On the other hand, results obtained by Phan and co-workers have shown that the application of licochalcone A, another plant-derived chalcone, exerted a potent inhibitory effect on Kv1.3 channels expressed in Jurkat T cells [15]. The inhibitory effect occurred in a concentration-dependent manner with the half-blocking concentration value of 0.83 μM. A complete inhibition occurred at a concentration of 10 μM [15]. These results demonstrate that licochalcone A is a stronger inhibitor of the Kv1.3 channel in Jurkat T cells than IBC is. The inhibitory effect of licochalcone A on Kv1.3 channels in Jurkat T cells is reversible [15].

Our study demonstrated that the inhibitory effect of IBC on Kv1.3 channels is partially irreversible. A partially irreversible inhibitory effect on the Kv1.3 channel was also observed in the case of application of simvastatin [34,35,36,37]. In this case, it was shown that the inhibitory effect on the Kv1.3 channel was probably complex and included both reversible specific interactions with the channel protein and partially irreversible perturbations in the structure of the lipid bilayer [34]. In the case of the application of simvastatin, the channel inhibition was accompanied by a significant acceleration of the inactivation rate of the currents [34,35,36,37]. Such an acceleration was not observed for IBC in this study. Thus, the mechanism of the inhibitory effect of IBC is probably different from that of simvastatin; however, it may also be complex and include both channel-specific and non-specific interactions. More research studies, including the docking analysis, are necessary to further elucidate the mechanism of block of the Kv1.3 channel by IBC.

The channel inhibition also occurred upon the application of non-prenylated chalcones HDC and HMC. In the case of HDC, the inhibitory effect was weakly concentration-dependent and incomplete at a concentration of 30 μM. In the case of the application of HMC, the inhibition was concentration-independent and incomplete at a concentration of 30 μM. Moreover, an additive inhibitory effect was only observed when both compounds were co-applied with mevastatin, but not upon co-application with simvastatin. These results might indicate that the presence of a prenyl group is the factor that facilitates the inhibition of the Kv1.3 channel by the examined chalcones. Such a facilitation of the channel block was also observed in the group of flavonoids [9,14].

The results of the present study are consistent with our previous observations demonstrating that co-application of chalcones with the statins simvastatin or mevastatin produces an additive inhibitory effect on Kv1.3 channel activity [12,14]. Notably, the inhibitory effect was more pronounced when chalcones were co-applied with mevastatin, in agreement with our earlier findings [12,14]. Although simvastatin and mevastatin are structurally closely related, they differ in key physicochemical and pharmacokinetic properties, including lipophilicity, cellular uptake, and intracellular processing. These differences may affect membrane composition and the local functional microenvironment of Kv1.3 channels, thereby providing a plausible explanation for the observed differences in channel inhibition. However, a definitive mechanistic explanation would require additional targeted studies beyond the scope of the present work.

The present study demonstrates that the tested chalcones and statins functionally inhibit Kv1.3 channel activity, as evidenced by electrophysiological recordings. However, based on the current data, it cannot be unequivocally concluded whether the observed inhibition results exclusively from direct interactions of the compounds with the Kv1.3 channel protein or whether it is partially mediated by indirect mechanisms affecting processes that regulate channel function. Given the lipophilic nature of the investigated compounds, it is plausible that they may alter membrane properties, lipid microdomains, or intracellular signalling pathways that secondarily influence Kv1.3 channel activity. Such indirect effects have previously been reported for other small-molecule Kv1.3 inhibitors, including statins, which have been shown to exert both channel-specific and membrane-mediated effects [37]. Therefore, the inhibitory action observed in this study is likely to be complex and may involve a combination of direct channel blockade and indirect modulation of the channel’s functional microenvironment. Further studies, including molecular docking analyses and experiments addressing membrane and signalling contributions, will be required to delineate the precise mechanisms underlying Kv1.3 channel inhibition by the tested compounds. Moreover, the inhibition of the Kv1.3 channel may be related to the anti-cancer activities of IBC and other chalcones tested in this study [16,17,22]. It is known that some small-molecule lipophilic inhibitors of the Kv1.3 channel may simultaneously inhibit the proliferation of Kv1.3 channel-expressing cancer cells and induce the apoptosis of these cells via an activation of the mitochondrial pathway of this process [3,4,5,6,7,8,9,10,11,12,13,14]. The inhibition of proliferation is related to an inhibition of the Kv1.3 channel in the plasma membrane, whereas an induction of the mitochondrial pathway of apoptosis is a result of an inhibition of the mitoKv1.3 channel [3,4,5,6,7,8,9,10,11,12,13,14]. Importantly, these compounds may selectively eliminate Kv1.3 channel-expressing cancer cells, while sparing the normal ones [3,4,5,6,7,8,9,10,11,12,13,14]. Therefore, the compounds may putatively be applied to support chemotherapy of some cancer disorders, with an over-expression of the Kv1.3 channel, such as melanoma, pancreatic ductal adenocarcinoma (PDAC), multiple myeloma, and B-type chronic lymphocytic leukemia (B-CLL) [3,4,5,6,7,8,9,10,11,12,13,14].

It is known that IBC exerts anti-proliferative and pro-apoptotic effects on various cancer cell lines, including those expressing Kv1.3 channels, such as myeloid leukemia HL60, K562, MOLM-13 cells, colorectal cancer SW480 cells, breast cancer MCF-7 and MDA-DB-231 cell lines, lung cancer A549 cells, and prostate cancer PC-3 and LNCaP cell lines [16,17,22]. Non-prenylated chalcones also exert anti-proliferative and pro-apoptotic effects on Kv1.3 channel-expressing cancer cell lines, including Jurkat T cells [22]. It may be possible that these effects are related to channel inhibition by the compounds. More studies are necessary to elucidate the relationship between the channel inhibition and death of Kv1.3 channel-expressing cancer cells. Nevertheless, results of this study may indicate that chalcones, both prenylated and non-prenylated ones, may putatively be applied to support chemotherapy of the above-mentioned cancers.

The main advantages of these compounds are good bioavailability and low toxicity for normal cells. The main disadvantage is the fact that these compounds have to be applied at high doses, which may not be achievable under clinical conditions or may lead to unwanted side effects. In order to reduce the required therapeutic dose and probability of side effects, it may be advantageous to co-apply the chalcones with the statins. It was shown that the co-application of flavonoids (6-hydroxyflavone, 7-hydroxyflavone, and baicalein) with the statins simvastatin and mevastatin significantly improved anti-cancer activities of these compounds, due to a synergistic action [18]. The flavonoids co-applied with the statins may effectively eliminate even multidrug-resistant colorectal cancer cells [30]. Such a synergistic anti-cancer action may also occur upon the co-application of the chalcones with the statins. An alternative way to facilitate the anti-cancer activity of the chalcones is co-application with anti-cancer drugs, such as gemcitabine and abraxane, in the case of therapy of pancreatic ductal adenocarcinoma [38]. Although significant biological effects were observed at micromolar concentrations in vitro, it should be noted that such concentrations may not be readily achievable or sustainable in vivo due to pharmacokinetic limitations, including absorption, distribution, metabolism, and clearance. Therefore, the present results should be interpreted as a proof-of-concept, demonstrating biological activity at the cellular level rather than as direct evidence of therapeutic potential.

4. Materials and Methods

4.1. Chalcones and Statins

IBC was purchased from Alexis Biochemicals (Lausen, Switzerland) and both studied statins were from Sigma-Aldrich (St. Louis, MO, USA). HDC and HMC were synthesized, as described in previous studies [39], and obtained from the Department of Food Chemistry and Biocatalysis (Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland). Stock solutions of the compounds were prepared in DMSO (30 mM) for further investigations.

4.2. Cell Culture and Solutions

The human leukemic T-cell line, Jurkat (clone E6-1), was purchased from the American Type Culture Collection (Manassas, VA, USA). The Jurkat cells were grown in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% heat-inactivated FBS, 10 mM HEPES, and 2 mM glutamate. Cells were grown on culture plates at 37 °C in a 5% CO2-humidified incubator. In order to prepare the cells for “patch-clamp” experiments, cell-containing dishes were perfused by the external solution, containing 150 mM NaCl, 4.5 mM KCl, 1 mM CaCl_2_, 1 mM MgCl_2_, 10 mM HEPES, and 10 mM glucose, and pH of the solution = 7.35 (adjusted with NaOH). The internal (pipette) solution contained 150 mM KCl, 1 mM CaCl_2_, 2 mM MgCl_2_, 10 mM HEPES, and 10 mM EGTA, and pH of the solution = 7.2 (adjusted with KOH). The concentration of free calcium ions in the internal solution was below 100 nM (assuming the dissociation constant for EGTA at pH = 7.2 of 10–7 M) [40]. The intracellular calcium concentration was kept at such a low level in order to prevent the activation of calcium-activated K+ channels KCa2.2, which are abundantly expressed in Jurkat T cells [40,41]. The chemicals were purchased from the Polish Chemical Company (POCH, Gliwice, Poland), except for HEPES and EGTA, which were purchased from Sigma-Aldrich (St. Louis, MO, USA). The examined chalcones were purchased from Alexis Biochemicals (Lausen, Switzerland).

4.3. MTT Assay

The effects of the tested compounds on Jurkat T-cell viability were assessed using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide—Supplementary Figure S2). Jurkat cells were seeded at a density of 3 × 10^4^ onto a 96-well plate. Stock solutions of the compounds were diluted in culture medium. The diluted solutions were then applied to the cells to achieve final concentrations of 1.5, 3, 6, 15, or 30 µM in each well. In the case of non-prenylated chalcones, the concentration range was extended to 50 µM. The cells were incubated with the compounds for 48 h in a humidified atmosphere of 5% CO_2_ at 37 °C. Then, MTT labelling reagent (5 mg/mL) was added to each well, and the plates were incubated for an additional 4 h. The resulting formazan crystals were then dissolved in dimethyl sulfoxide (DMSO). Optical density (OD) was measured at 570 nm using a Tecan Infinite M200 spectrophotometer (Tecan Group Ltd., Männedorf, Switzerland). Viability of treated cells was calculated relative to the control cells, which were assumed to have 100% viability. The measured parameters were expressed as mean values ± standard deviation (SD) from three independent experiments (n = 3). Statistical significance was evaluated using Student’s t-test (* p < 0.05) with Statistica 10 software (Tibco Software Inc., Palo Alto, CA, USA).

4.4. Patch-Clamp Recording

Dishes with cells were placed under an inverted Olympus IMT-2 microscope (Hachioji-shi, Japan). The external solution with and without the selected compounds was applied by the eight-channel gravitation perfusion system (ALA Scientific Instruments, Farmingdale, NY, USA). Pipettes were pulled from a borosilicate glass (Hilgenberg, Bolanden, Germany). The pipette resistance was higher than 3 MΩ.

Whole-cell potassium currents in Jurkat T cells were recorded by applying the patch-clamp technique [42]. The currents were recorded using an EPC-7 Amplifier (HEKA, Reutlingen, Germany), low-pass filtered at 3 kHz, digitized 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 depolarizing cell membranes from −100 mV (start of the ramp) up to +40 mV (end of the ramp, the peak value), were applied every 30 s; the ramp duration was 340 ms and the holding potential was −90 mV. Upon application of the voltage ramp protocol, potassium currents in Jurkat T cells could be stably recorded for at least 20 min after “break-in” to the whole-cell configuration. During the off-line analysis, the value of the Kv1.3 current at the end of a voltage ramp (+40 mV-peak current) was calculated. For this purpose, the leak current estimated at +40 mV was subtracted from the total ramp current recorded at this voltage.

All experiments were carried out at room temperature (22–24 °C).

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

4.5. Isobolographic Analysis

Isobolographic analysis to detect the existence of putative synergy between the statins and the chalcone derivative was performed via CompuSyn software (1.0 version), based on the model of Chou and Martin [43], yielded combination index values (CI) for statin: chalcone mixtures. CI values were calculated. CI = 1 indicates an additive effect, CI < 1—synergism, and CI > 1—antagonism.

4.6. Patch-Clamp Data Analysis

The inhibition of the channel is presented in terms of a relative peak current (Irel), defined as I/Icontr, where I—Kv1.3 current recorded upon application of an examined compound at +40 mV (the peak value), and Icontr—Kv1.3 current recorded on the same cell at +40 mV under control conditions. Unless otherwise stated, statistical analysis was performed applying Student’s unpaired t-test. The results were considered statistically significant when p < 0.05.

5. Conclusions

Given the involvement of multiple signalling pathways in cancer progression, combination-based therapeutic strategies represent a promising approach for further optimization of anti-cancer treatment. Such a “dual therapy” represents a theoretical strategy that could allow for dose reduction of individual agents. Inhibition of Kv1.3 channels in cancer cells may constitute one of the mechanisms contributing to reduced cell proliferation and the induction of apoptosis. Our in vitro studies using Jurkat cells demonstrated that IBC and non-prenylated chalcones inhibit Kv1.3 channel activity both when applied alone and in combination with selected statins. Notably, exposure to mevastatin enhanced the Kv1.3 inhibitory potential of all chalcones examined. Further studies are warranted to elucidate additional mechanisms underlying the observed effects of these drug combinations. Future experiments should include pharmacokinetic profiling, toxicity assessment, and validation in relevant animal models before any conclusions regarding therapeutic applicability can be drawn.