Ion Channel Drugs Suppress Cancer Phenotype in NG108-15 and U87 Cells: Toward Novel Electroceuticals for Glioblastoma

The combination of pantoprazole with lamotrigine at 100 µM, NS1643 at 20 µM, and rapamycin at 100 nM were the only combinations that showed significantly more efficacy than pantoprazole alone at reducing cell proliferation after 6 days of treatment, with the combination with rapamycin showing the most significant difference (Figure 2A). The reductions in cell number for these combinations compared to control were 85%, 86%, and 90%, respectively. The proportion of cells in G1 and early S only slightly increased for pantoprazole treatments in combination with lamotrigine and NS1643, but the combination with rapamycin did increase the G1 proportion (Figure 2B). Taken together, these data reveal that several FDA approved drugs for human use can be repurposed in combination and can significantly reduce cancer cell proliferation.

To understand whether our treatments have a persistent effect on the cells (stop the proliferation of the cells even after the drugs were withdrawn), we performed a recovery test (Figure 3). Cells were treated with the drugs for 6 days, and then the drugs were removed (demarcated by the dashed line), after which the cells were cultured in media containing no drugs for another 4 days.

Although the three combinations with pantoprazole showed significantly less cell proliferation after 6 days of treatment, the cells showed some recovery after treatment was removed (Figure 3). The slope of each combinatorial treatment from day 6 to day 10 was compared to pantoprazole alone. Pantoprazole in combination with retigabine was the only treatment combination that was significantly different, except the positive control, which showed no recovery, indicative of cells that had terminally differentiated. One thing to note was that the treatment of pantoprazole with retigabine, although not significantly different than pantoprazole at day 6, did show fewer cells than the treatment of pantoprazole with lamotrigine at day 10. Therefore, we decided to perform further analysis on this combination.

Many of the significant treatments in NG108-15 cells were also significantly effective in human glioblastoma U87 cells compared to the control after 6 days (Figure 4). Notably, NS1643 at 50 µM significantly decreased cell proliferation as compared to control by 1.7-fold but was much more effective when combined with pantoprazole, rapamycin, or temozolomide (TMZ) with a fold decrease to control of 3.3, 2.7, and 2.5 respectively. Pantoprazole also worked very well in this cell line with a significant percentage reduction in cells to control of 54% and showed very significant differences in cell proliferation as compared to control when combined with rapamycin, retigabine, NS1643, lamotrigine, and TMZ with a percent reduction in cells of 60%, 72%, 72%, 61%, and 61% respectively (Figure 5A). TMZ was very effective at reducing cell number as compared to control in U87 cells (43% decrease), but combinations with rapamycin, pantoprazole, or NS1643 significantly increased the effectiveness up to 55%, 61%, 61% reduction in cells compared to control respectively (Figure 5A). The cell cycle data in Figure 4B shows that some of the most effective combinations increased the G1 and early S proportion of cells but not all. Pantoprazole showed its characteristic increase in the early S proportion of cells and rapamycin, an increase in the G1 proportion as seen in the NG108-15 cells. TMZ and NS1643 treatment did not show a large change in the proportion of cells in each stage of the cell cycle as compared to control. TMZ in combination with rapamycin increased the proportion of cells in G1, and combination with NS1643 increased the proportion of cells in early S as compared to TMZ alone (Figure 5B). Pantoprazole combinations consistently showed a larger proportion of cells in early S as compared to TMZ alone, with a complimentary decrease of cells in late S, G2, and M (Figure 5B).

Unfortunately, we were not able to obtain an accurate proportion of cells in each cell cycle stage for the best treatment in U87 cells, pantoprazole with retigabine, due to an autofluorescent aggregation in the cytoplasm that obscured the FUCCI reporter signal.

The same combinations that were significantly better than pantoprazole in NG108-15 cells were also significant in U87 cells, along with the added combinations of TMZ or retigabine (Figure 5A). Pantoprazole in combination with rapamycin, lamotrigine, NS1643, TMZ or retigabine were all significantly better than pantoprazole alone. The most significant combinations were with NS1643 or retigabine, a 1.6-fold decrease than pantoprazole alone. The combination of pantoprazole with NS1643 was so effective that cutting the pantoprazole concentration by half and combining it with NS1643 at 50 µM was significantly more effective than pantoprazole at 100 µM alone (1.2-fold decrease). The characteristic increase in G1 when pantoprazole was used in NG108-15 cells was also seen in U87 cells. Additionally, when pantoprazole was combined with rapamycin or NS1643 an increase in the proportion of the cells in early S was seen, with a complementary decrease in late S, G2, and M (Figure 5B). Thus, we conclude that NS1643, retigabine, rapamycin, lamotrigine, and pantoprazole are also effective in a human glioblastoma cell line, and NS1643 or pantoprazole potentiate the action of the standard TMZ treatment.

The combination of pantoprazole with rapamycin shows a similar recovery slope after day 6 as pantoprazole alone (Figure 6). However, combinations of pantoprazole with retigabine or NS1643 at 50 µM show reduced recoveries compared to pantoprazole alone, although not significantly so. TMZ treatment alone only showed a slight increase in proliferation after treatment was removed, and NS1643 alone showed a high increase in proliferation. However, the combination of NS1643 with TMZ did not show an increase in proliferation after removal of treatment, but the recovery slope was not significantly different from TMZ alone. Surprisingly the positive control using cAMP in combination with rapamycin started proliferating after day 8, revealing that the bioelectric drugs have a more stable effect on the cells than even the powerful cAMP signal.

We performed electrophysiological recordings to determine the change in resting membrane potential of NG108-15 cells treated with the compounds that significantly reduced cell proliferation as compared to control immediately after application. We measured the baseline Vmem first and recorded the membrane potential continuously for 3 min upon compound treatment. We then calculated the difference in Vmem post treatment.

Rapamycin, retigabine, NS1643, TMZ, and lamotrigine all significantly hyperpolarized the cells as compared to the control (Figure 7). Retigabine, lamotrigine, and NS1643 are known hyperpolarizing agents, but TMZ has been shown to depolarize U373 glioma cells [93]—an effect opposite to what we observed in NG108-15 cells, possibly due to differential expression levels of KCa3.1 channels in these two cell lines (not confirmed). Rapamycin also hyperpolarized the NG108-15 cells, a novel effect suggesting that existing cancer drugs could have bioelectric mechanisms of action that are not yet recognized. Surprisingly, while pantoprazole did not have an immediate effect on the membrane potential of the cells, its combination with retigabine and rapamycin, which both individually hyperpolarize the cells, instead depolarized the cells. The combination of pantoprazole with lamotrigine and NS1643 did not observably change the membrane potential as compared to control.

We next wanted to ask whether, in addition to the effects on proliferation, our treatments also exerted a differentiating influence, which could be beneficial with respect to the future behavior of treated cells in vivo. Differentiation markers for neuronal lineage were used to stain NG108-15 cells incubated for six days with the most effective treatments observed in the proliferation data (Figure 8). We found that treatment with pantoprazole at 100 µM combined with NS1643 at 50 µM or with rapamycin at 100 nM consistently showed a significant increase in neuronal differentiation markers, including Microtubule Associated Protein 2 (MAP2) [94], Neuron-Specific Class III β-Tubulin (TujI) [95], Neuron-Specific Enolase (NSE) [96], and Neural Filament Medium Chain (NFM) [97]. The combination of pantoprazole with retigabine at 10 µM showed increases in all but MAP2. Combinations of pantoprazole with either NS1643 or retigabine showed a significant increase in neuronal markers compared to either drug alone. The combination of NS1643 with pantoprazole showed higher levels of both MAP2 and Tuj1, than for pantoprazole alone. Retigabine in combination with pantoprazole showed higher immunoreactivity of NFM as compared to pantoprazole alone. These observations suggest that these treatments are pushing the treated cells toward a more differentiated state.

Staining cells treated for 6 days with S100 calcium-binding protein B (S100B) [98] and Glial Fibrillary Acidic Protein (GFAP) [99] markers revealed differentiation of NG108-15 cells towards an astrocytic/oligodendrocytic or astrocytic lineage, respectively (Figure 9A,B). Both markers were significantly upregulated in all combination treatment groups as well as in pantoprazole alone. The combination of NS1643 with pantoprazole showed significantly higher S100B and GFAP immunoreactivity than with pantoprazole alone. All treatments but NS1643 alone showed a significant increase in CREB, known to play an important role in driving differentiation [16,100,101,102,103] (Figure 9C). Pantoprazole alone, along with its combinations, also showed an increase in connexin 43 (Cx43) expression, a known marker for glioblastoma differentiation [104].

We also sought to determine whether our treatments may induce senescence, as this would be an important outcome with respect to the course of the malignancy in vivo. Treatments of NG108-15 cells with pantoprazole alone or in combination with NS1643, retigabine, and rapamycin all showed a very significant increase in a senescence-associated beta-galactosidase activity stain [105] (Figure 10A). To explain this increase, we also looked at the p27Kip1 level, which is known to inhibit the cell cycle and cause senescence [106,107,108,109,110,111,112,113] (Figure 10E). Immunoreactivity levels of p27Kip1 in all combined treatments except for pantoprazole with rapamycin were elevated. To confirm the senescence phenotype, we also looked at the size of the nuclei, which were significantly larger in the pantoprazole, NS1643, and pantoprazole in combination with NS1643, retigabine, or rapamycin-treated groups (Figure 10F). We also tested the cells for cleaved caspase 3 (Figure 10D), a marker of apoptosis and LC3B (Figure 10C), a marker of autophagy. We found that levels for both these markers were under 1.4% and 2.5%, respectively, after 6 days of treatment which may indicate a small percentage of cells that did not senesce due to the highly heterogeneous nature of these cancer cells. Although low, there was a significant increase in cleaved caspase 3 staining for cells treated with pantoprazole or pantoprazole with NS1643, retigabine, or rapamycin. Surprisingly, we observed that treatment with retigabine or NS1643 decreased the cleaved caspase 3 positive cells significantly as compared to the control. In addition, the LC3B stain showed an increase in cells treated with pantoprazole and pantoprazole with NS1643, although not significant with this assay.

However, there was a significant increase in bright LC3B puncta in cells that were treated with any combination containing rapamycin, as was expected (Figure 10C). Proliferation in NG108-15 cells as measured by BrdU incorporation showed a significant decrease with treatments of pantoprazole alone, retigabine alone, and combinations of pantoprazole with NS1643, retigabine, and rapamycin after 6 days (Figure 10B), agreeing with the live cell counts obtained in Figure 1A. Thus, we conclude that the combinatorial treatments that resulted in the lowest cellular proliferation also showed increased markers for senescence, some increase in autophagy, and did not show a large fraction of apoptotic cells.

U87 cells showed a significant increase in neuronal markers for the combination treatments with TMZ as compared to control (Figure 11). NFM and NSE, similar to what was seen in NG108-15 cells, were also increased in cells treated with pantoprazole or combination treatments with pantoprazole and NS1643 or retigabine (Figure 11C). TujI was only highly elevated in the U87 cells treated with NS1643 and TMZ (Figure 11B). MAP2 showed high levels of immunoreactivity when cells were treated with combinations of TMZ and NS1643 or pantoprazole (Figure 11A). It should be noted that cell staining was highly heterogeneous, with some cells staining more brightly than others with the same treatment.

Astrocytic differentiation markers were increased in treatments with pantoprazole and pantoprazole in combination with NS1643, retigabine, rapamycin, and TMZ, as well as NS1643 in combination with TMZ (Figure 12). Vimentin, a known marker for astrocytes [114,115], was significantly increased when cells were treated with pantoprazole in combination with NS1643, TMZ, and rapamycin and with NS1643 in combination with TMZ (Figure 12A). CREB (Figure 12B), a known marker for differentiation and S100B (Figure 12C) and GFAP (Figure 12D), markers for astrocytic differentiation, also were increased by these same treatments (Figure 12B). These results agreed well with what was found in NG108-15 cells.

U87 cells showed increases in O4 (Figure 13A) for all treatments except those containing retigabine. Sox10 levels (Figure 13B) also increased for all treatments, excluding retigabine alone. When taken together, the above data suggest that the differentiating activity of these treatments are not specific to one species or one type of cell line.

We tested U87 cells for the same proliferation, senescence, autophagy, and apoptosis markers that we had tested in NG108-15 cells and found similar results (Figure 14). All treatments showed an increase in senescence markers, with the most significant being the combination of pantoprazole with NS1643, TMZ, and the combination of NS1643 and TMZ (Figure 14A). The size of the nuclei for the combination treatments with the highest levels of senescent cells was also significantly larger than the control, confirming this phenotype (Figure 14F). We also looked at p27Kip1 levels and saw similar changes to those observed in NG108-15 cells (Figure 10E). All the treatments with pantoprazole showed an increase in p27Kip1 levels, and NS1643 in combination with TMZ showed the highest increase. Cleaved caspase 3 (Figure 14E) and LC3B puncta (Figure 14C) positive cells indicated very low non-significant levels of expression, indicating that the cells were not undergoing a high level of apoptosis or autophagy relative to control, respectively. BrdU incorporation decreased in all treatments that showed lower proliferation (Figure 14B). These data show that the low proliferation of the U87 cells treated with the most successful drug combinations was driven by increased levels of senescence.

We used the voltage dye DiBAC4(3) [116,117] to measure the resting membrane potential of six-day-treated U87 cells (Figure 15B). We found that NS1643 alone and in combination with pantoprazole showed a significant hyperpolarization as compared to control. To investigate further, we used pHRodo Green to see if the internal pH of the cells was being changed in response to treatment (Figure 15C). We saw a dramatic increase in pH with all the treatments that incorporated NS1643, and we saw a slight increase in pH for pantoprazole with retigabine. In addition to the cytoplasmic pH, we wanted to test the lysosomal pH due to reports that pantoprazole de-acidifies the lumen of the lysosome [118,119]. The dye, LysoSensor Green (Figure 15A), showed a dramatic alkalization of the lysosomes when treated by NS1643 alone or in combination. Pantoprazole did not show an alkalinization of the lysosome in the U87 cells, in agreement with what has previously been reported when pantoprazole is delivered in neutral cell culture media at pH 7.4 [120]. In fact, pantoprazole and its combination with TMZ showed a significant increase in lysosomes, which also agrees with that same study performed in neutral pH media [120]. In addition to the dyes, we also tested the ratio of cytoplasmic to nuclear YAP (Figure 15E). Pantoprazole has been found to decrease YAP activity in ovarian cancer and in the liver [121,122]. Since YAP has been found to be a master regulator of the cell cycle, especially in cancer, we were interested to see the effect our treatments would have on this protein [122,123,124,125]. We found that pantoprazole alone did not have a significant effect on the cytoplasmic to nuclear ratio of YAP, but when combined with NS1643 or TMZ, it showed a significant decrease indicative of less YAP in the nucleus as compared to the cytoplasm.

This significant decrease in the YAP nucleus to cytoplasmic ratio was also evident for the NS1643 in combination with TMZ treatment. When taken together, these data indicate that NS1643, in combination or alone, can increase cytoplasmic calcium levels, increase cytoplasmic pH, and increase lysosomal pH, but a significant decrease in the YAP nuclear to cytoplasmic ratio is only seen when it is in combination with pantoprazole or TMZ.

To determine whether the effects we observed were specific for cancer cells and could be expected to be usable in vivo without harming native neurons, we tested these compounds on human induced pluripotent stem cells derived from fibroblasts and made to commit to a neuronal stem cell lineage. These hiNSCs were differentiated for 7 days in neuronal media and then treated with electroceuticals for 3 days. The short treatment time was necessary to be able to perform a Live/Dead assay without too much cell detachment.

Toxicity analysis by Live/Dead stain in human neurons shows that only three out of 24 treatments showed significant toxicity (Figure 16A). Pantoprazole showed a slight increase in toxicity that was significant compared to control, and pantoprazole with lamotrigine also showed a slight increase but was more significant than pantoprazole alone when compared to the control. NS1643 at 50 µM in combination with TMZ showed the most toxicity when compared to control. However, the difference between the control toxicity and the most toxic combination of NS1643 at 50 µM and TMZ was still only 5.7% higher than control. A senescence assay was also performed on the neurons under all treatments tested in the Live/Dead stain, but no significant differences were found, and all senescence levels were all under 1.75%, a stark contrast to the greater than 50% senescence levels in NG108-15 and U87 cells after 6 days of treatment (Figure 10A and Figure 14A).

Our drugs were selected based on their predicted effects on Vmem, which has been shown in amphibian models in vivo to prevent and reverse tumorigenesis and metastatic behavior [34,47]. Indeed, a number of drugs with bioelectric targets, such as ivermectin (a chloride channel drug) [126,127,128,129], salinomycin and monensin (ionophores) [126,130], a variety of potassium channel drugs [25,55,131,132,133,134], and drugs targeting proton pumps [135] have been discovered to have anti-cancer activity in various screens [26,136,137,138,139]. Thus, our combinations of compounds represent novel entries to the field of electroceuticals: the repurposing of known ion channel-targeting drugs to manipulate complex cell outcomes [140,141]. This approach has already been used for the design of interventions to repair birth defects of the brain [142,143]. It is likely that a better understanding of the control of cell behavior, alone and in tissues, will enable much more precisely targeted electroceutical interventions in cancer as part of the goal of normalizing cells as an alternative to traditional chemotherapy [144].

The NG108-15 hybrid cell line used in this study shows cancer stem cell characteristics, can be easily transfected and selected and has been used to study neuronal differentiation for many years. To find treatments that would be robust, we screened all our compounds in high serum media, which is usually prohibitive for NG108-15 differentiation [90,145,146]. In addition, the high serum media provided an abundance of growth factors that have been shown to be secreted in the peripheral zone of resected GBM tumors and are thought to drive the migration and proliferation of GBM stem cells in the area [10]. The best performing novel combinations were pantoprazole with retigabine, lamotrigine, NS1643, or rapamycin and reduced proliferation when compared to the control by 80%, 85%, 86%, and 90%, respectively. FUCCI analysis showed that the cell cycle proportion was dominated by cells in either G1 or early S. All these compounds are already FDA approved for other conditions except for NS1643.

We then used the best performing novel combinations to inform a screen of compounds on a human glioblastoma cell line, U87 (ATCC). Our proliferation assays showed 13 drug or drug combinations that reduced proliferation compared to control more significantly than the leading glioblastoma treatment, TMZ. Of these 13, the top four treatments were combinations of pantoprazole or NS1643 with TMZ, and the combination of pantoprazole with retigabine or NS1643, which reduced proliferation when compared to control by 62%, 63%, 71%, and 74%, respectively. FUCCI analysis showed that the cell cycle proportion was again dominated by cells in either G1 or early S.

Differentiation therapy for GBM is an alternative treatment strategy that could possibly overcome the issue of reoccurrence after resection of the tumor [15]. For differentiation therapy to be successful, treatment needs to be effective at clinically relevant concentrations, and differentiation needs to be permanent, without cell cycle re-entry after treatment is stopped. This study showed that hyperpolarizing compounds in combination with pantoprazole can be used to arrest the cell cycle of proliferating cells and drive them towards partial differentiation and senescence.

NG108-15 cells treated with the most successful drug combinations showed neuronal, astrocytic, and oligodendrocyte differentiation markers as well as senescence markers. The mixed nature of differentiation markers in NG108-15 cells has been reported previously [147] and might be due to their hybrid neuroblastoma/glioma status. Many of the combinations of drugs we tested in NG108-15 cells were also successful in U87 cells and resulted in dramatically less proliferation, less progression through the cell cycle and a significant increase in differentiation markers, which need the cell cycle to be arrested long enough to accumulate [148]. The mixed nature of the differentiation markers that were elevated in U87 cells shows that this line can differentiate into the neuron, astrocyte, and oligodendrocyte lineages, in agreement with past studies [149,150]. It is important to note that many of the differentiation markers were heterogeneously expressed and that there is a possibility that in each sample, there exist a multitude of differentiated phenotypes instead of just one type. Connexin 43 was also upregulated in treated NG108-15 cells, showing that this marker of cell-to-cell communication was increased as cells went to a more differentiated state, as supported by a previous study [104]. Interestingly, both NS1643 and retigabine are known to work on KCNQ2/3 channels, which are important for neuronal differentiation [151], and NS1643 activates hERG channels which have also been shown to be important in neuronal differentiation [65].

NS1643, a hERG channel opener and potassium modulator which also potentiates KCNQ2, KCNQ4, and KCNQ2/3 [85,152], was shown to hyperpolarize both cell lines that we tested and was one of the most successful single treatments. In combination with pantoprazole, it could arrest the NG108-15 cell cycle long enough to enable differentiation in a high serum medium and work well in U87 cells. Treatment with NS1643 in combination with pantoprazole showed a significant effect that was not seen with either treatment alone. Assays in U87 cells showed that this novel combinatorial treatment caused the pH of the cytoplasm and lysosomes to alkalize drastically and that cytoplasmic calcium increased significantly along with an increase in the cell cycle inhibitor p27Kip1 and an increase in senescence-associated beta-galactosidase positive cells. In addition, combination treatments with NS1643 also resulted in a decrease in YAP nuclear to cytoplasmic ratio, elevated levels of which are known to accelerate cancer cell cycle progression, proliferation, therapy resistance, and metastasis [124,125,153]. Thus, both YAP translocation and increased p27Kip1 levels were found in the cells that showed the most senescence, confirming what other studies have reported: that both proteins are involved in senescence [111,154]. Proliferation under these senescence-inducing treatments was reduced dramatically, and U87 cells did not re-enter the cell cycle up to 4 days after treatment was removed. The combination of NS1643 with TMZ was also very effective at reducing proliferation in U87 cells, although according to our voltage dye assays, it did not hyperpolarize U87 cells. However, the cells did significantly express a variety of differentiation markers after 6 days of treatment.

The cells treated with NS1643 and TMZ also showed, similar to the other NS1643 containing treatments, an increase in lysosomal and cytoplasmic pH, an increase in p27kip1, and a significant decrease in the YAP nuclear to cytoplasmic ratio. It is possible that the voltage dye did not correctly report the membrane potential with this combination due to an interaction with the dye molecule itself, as has been reported for other compounds [155,156].

In agreement with our results, NS1643 has also been shown to induce senescence in melanoma and breast cancer [55,131,157]. This senescent phenotype is thought to occur through elevated internal calcium levels (a response to hyperpolarization), which trigger the activation of calcineurin, which in turn dephosphorylates NFAT and results in its translocation to the nucleus [134]. In the nucleus, it is possible that translocated NFAT and activated calcineurin could be increasing the expression of p21WAF1/CIP1, also found caused by NS1643 [134], by a mechanism similar to that found in differentiating keratinocytes where calcineurin increases Sp1/Sp3-dependent transcription and p21 promoter activity in combination with NFAT [158]. That same study performed on keratinocytes showed that calcineurin inactivation resulted in less p27kip1 as well [158]. Interestingly, mitochondrial ROS production has been found to be increased by calcineurin activation in neurons [159], and ROS levels were found elevated in breast cancer cells treated with NS1643 [131]. Elevated ROS levels have been found to decrease the proteasome function [160] and could possibly increase p27kip1 levels through decreased ubiquitination and degradation [161]. Currently, no publications have shown the efficacy of NS1643 on glioblastoma or its efficacy in combination with rapamycin, pantoprazole, or TMZ.

Another successful combination included the KCNQ2-5/Kv7.2–7.5 channel opener, and FDAapproved epilepsy treatment, retigabine [87]. Although the application of retigabine alone hyperpolarized NG108-15 cells only slightly and reduced proliferation only marginally by itself, its ability to stop NG108-15 proliferation was increased most significantly with pantoprazole. Pantoprazole, a proton-pump inhibitor, worked well on its own at reducing proliferation in these two cell lines. However, when treatment was removed, the cells immediately re-entered the cell cycle, illustrating that treatment with pantoprazole alone does not arrest the cell cycle long enough to allow for terminal differentiation. When pantoprazole was combined with retigabine, a synergistic effect was achieved that resulted in fewer cells re-entering the cell cycle after treatment was removed. Interestingly we saw a depolarization of the NG108-15 cells when they were treated with the combination, but no change was seen in the membrane potential of the U87 cells.