Potassium channels and TRPV1 modulators on SU-DIPG-36 and SU-DIPG-50 cells: in vitro effects and channel currents characterization

Diffuse intrinsic pontine glioma (DIPG) is a rare and fatal brainstem tumour in pediatrics. We studied the role of K+ and TRPV1 channels in this disease, because of the involvement of these channels in neoplastic growth. Specifically, our study concerns H3.1K27M mutated cells (SU-DIPG-36) and H3.3K27M mutated cells (SU-DIPG-50). In vitro cell viability experiments with crystal violet and CCK-8 assays were performed using potassium and TRPV1 channel modulators on both cell lines; the most effective were repaglinide (REPA) and glibenclamide (GLIB) after 6-72 hours of incubation time. Based on IC50 values, the potassium channel modulators effectiveness scale for DIPG-36 cells was REPA CV>REPA CCK-8>GLIB CCK-8, both after 48 and 72 hours of incubation: the most effective drug was repaglinide, which had the lowest IC50 using crystal violet assay, with a value of 3.222x10−6 M after 48 hours of incubation and 3.217x10−6 M after 72 hours of incubation. Also, on DIPG-50 cells, the effectiveness scale after 48 hours of incubation was GLIB CCK-8 (IC50 = 1.312x10−7M) >REPA CCK-8>GLIB CV>REPA CV. After 72 hours of incubation the effectiveness scale was REPA CCK-8 (IC50 = 1.288x10−7M>REPA CV> GLIB CV>GLIB CCK-8. Moreover, we carried out a clonogenic test using repaglinide (100 μM) on SU-DIPG-36 cells, which confirmed efficacy. About TRPV1 channel modulators, capsaicin (CAPS)(100 μM) showed cytotoxicity, with the major effect on DIPG-50 cells after 72 hours of incubation of −34.3%; these effects may be the result of a mechanism of cell apoptosis developed due to this agonist. Patch-clamp experiments showed that all DIPG-36 cells were sensitive to BaCl2 (10 mM) - TEA (5 mM). On DIPG-36 cells (N = 7 cells) the application of glibenclamide (10μM) reduced control currents at +80 mV (Vm) (834.525 ± 176.20 pA) and at −80 mV (Vm) (-201.198 ± 46.23 pA) by −27.58% and -9.06%, respectively, so this shows contribution of K-ATP channels. On 4 cells, capsazepine (1 μM), reduced control currents at +80 mV (Vm) (904.54 ± 241.29 pA) of −16.43%; instead, at +180 mV (Vm), control currents (4069.52 ± 1049.74 pA) were reduced of -31.39%; this was further reduced by ruthenium red (10 μM), so this implies TRPV1 channel is functionally active in these cells. Instead, DIPG-50 were less sensitive to BaCl2(10 mM)-TEA (5 mM) or not responsive. On DIPG-50 (N = 5 cells), repaglinide (100 μM) at +60 mV (Vm) reduced control currents (178.5079 ± 96.62 pA), with a percentage of −27.63%; at −80 mV (Vm), reduction of the control current (−60.26 ± 15.23 pA) was of -15.03%. The application of diazoxide (250 μM) did not further activate the currents. Repaglinide and glibenclamide were therefore the most potent antiproliferative drugs on both cells, so potassium ion channels can be investigated as a new biological target for future DIPG therapy; also TRPV1 channels modulators have also been shown to have interesting effects, which should be further investigated.