Blockade of the forward Na⁺/Ca²⁺ exchanger suppresses the growth of glioblastoma cells through Ca²⁺‐mediated cell death

Figures

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  Figure 1. Recording and blockade of the Na+/Ca2+ exchanger (NCX) currents in glioblastoma cells. (a) Human glioblastoma U87 cells were held at −50 mV; the membrane currents associated with NCX were recorded by a voltage ramp from +60 to −120 mV. Ni2+ (10 mM) was used to block the NCX currents. (b, c) NCX currents recorded before and after application of bepridil (0.1–100 μM) or KB‐R7943 (0.1–100 μM). (d–f) The currents recorded before and after application of the reverse NCX blockers SEA0400 (0.1–100 μM), SN‐6 (0.1–30 μM), and YM244769 (0.1–30 μM). The reverse NCX current was measured at +50 mV, and forward NCX current was measured at −110 mV

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  Figure 2. Effect of the Na+/Ca2+ exchanger (NCX) blockers on viability of human glioblastoma cell lines. (a, b) Viability of adult glioblastoma cell lines (U87 and U251) after exposure to NCX blockers bepridil, KB‐R7943, SEA0400, SN‐6, or YM244769 at 0–100 μM for 72 hr. n = 5 independent tests per group. (c) Viability of paediatric glioblastoma cell line SF188 after 72‐hr incubation with the same NCX blockers at 0–100 μM. n = 5 independent tests per group. (d) For U87, U251, and SF188 cell lines, concentration of the NCX blockers required to produce half maximal inhibition (IC50: μM, mean ± SEM) of cell viability. IC50 was obtained by fitting the normalized concentration–response relationship to the equation: V/V0 = 1/{1 + [C/IC50]n}, where V 0 and V are the cell viability measured in control and in the presence of a blocker, C is the concentration of the blocker, and n is the Hill coefficient. n = 5 independent cultures in each group

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  Figure 3. Effect of bepridil and KB‐R7943 on the level of [Ca2+]i measured by Ca2+ imaging. (a) Flua‐4 AM‐loaded U87 cells were imaged and perfused with bepridil (25 or 50 μM) in the HEPES‐buffered solution containing 2‐mM Ca2+. (b) Ca2+ imaging of U87 cells before and after perfusion with KB‐R7943 (25 or 50 μM). (c) In the Ca2+‐free solution, Ca2+ imaging of U87 cells before and after perfusion with bepridil or KB‐R7943. (d) Ca2+ imaging before and after perfusion with bepridil or KB‐R7943, followed by application of 5‐μM thapsigargin. (e) U87 cells were pretreated with a Ca2+ chelator BAPTA‐AM (20 μM) for 2 hr and then imaged and perfused with bepridil or KB‐R7943. The traces are representative of three separate experiments, and n = 12–18 cells were imaged per experiment

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  Figure 4. Bepridil caused cell cycle arrest and apoptosis in glioblastoma cells. (a, b) Cell cycle assay of U87 cells incubated with bepridil (25 μM) for 6, 12, 18, and 24 hr. Cell fractions in each phase were from cell cycle analysis; bepridil increased the proportion of cells in G0/G1 phase and reduced cell fraction in S phase in a time‐dependent manner. (c) Cells were pretreated with BAPTA‐AM (10 μM) for 18 hr and then incubated with bepridil (25 μM) for 18 hr. BAPTA‐AM alone did not incur cell fraction changes but significantly attenuated bepridil‐induced cell cycle arrest. *P < .05, G0/G1 phase of BAPTA‐AM + bepridil versus bepridil alone; n = 5 independent experiments. (d, e) U87 cells were treated with bepridil (25 μM) for 6, 12, 24, and 48 hr and then analysed with Annexin V–FITC/PI apoptosis assay. Bepridil incurred a time‐dependent increase of apoptotic cells (Annexin V+). (f) Cells were pretreated with BAPTA‐AM (10 μM) for 18 hr and then incubated with bepridil (25 μM) for 24 hr. BAPTA‐AM significantly suppressed bepridil‐induced apoptosis. n = 5 independent experiments; *P < .05, apoptotic cell fraction in BAPTA‐AM + bepridil versus bepridil alone, with the parametric one‐way…

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  Figure 5. KB‐R7943 caused cell cycle arrest and apoptosis in glioblastoma cells. (a) U87 cells were incubated with 0.1% DMSO (vol/vol), BAPTA‐AM (10 μM), KB‐R7943 (25 μM), and BAPTA‐AM + KB‐R7943 for 18 hr and then underwent cell cycle analysis. (b) U87 cells underwent an apoptosis assay after incubation with 0.1% DMSO, 10‐μM BAPTA‐AM, 25‐μM KB‐R7943, and BAPTA‐AM + KB‐R7943 for 20 hr. (c) Cell fractions in G0/G1, S, and G2/M phases of each group. KB‐R7943‐induced G0/G1 arrest was not rescued by BAPTA‐AM. n = 5 independent experiments; data were analysed by one‐way ANOVA. (d) Percentage of apoptotic cells in each group. KB‐R7943‐induced apoptosis was not attenuated by BAPTA‐AM. n = 5 independent experiments; data were analysed by one‐way ANOVA

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  Figure 6. Bepridil induced activation of p38‐MAPK and JNK signalling. (a) U87 cells were treated with bepridil at 25 μM for 10, 30, 60, and 90 min. The phosphorylated and total ERK, p38‐MAPK, and JNK were detected by Western blot. Tubulin was an internal control. (b) Cells were pretreated with BAPTA‐AM (10 μM) for 18 hr and then incubated with bepridil (25 μM) for 30 min. Activity of the p38‐MAPK and JNK signalling was enhanced by bepridil but was attenuated in the presence of BAPTA‐AM

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  Figure 7. Involvement of MAPK signalling in bepridil‐induced cell cycle arrest and apoptosis. (a) Top panel, U87 cells were treated with DMSO (0.1/%, vol/vol), CC‐930 (10 μM), and PD169316 (10 μM) for 18 hr. Lower panel, cells were treated with bepridil (25 μM) alone or pretreated with CC‐930 (10 μM) and PD169316 (10 μM) for 2 hr and then incubated with bepridil for 18 hr. (b) Cell fractions in G0/G1, S, and G2/M phases of each group. Bepridil‐induced G0/G1 cell cycle arrest was rescued by PD169316 but not CC‐930. *P < .05, cell fraction in G0/G1 phase of PD169316 + bepridil‐treated group versus bepridil‐treated group. n = 5 independent experiments; data were analysed by one‐way ANOVA. (c, d) CC‐930 (10 μM) or PD169316 (10 μM) alone did not increase apoptosis in U87 cells. Cells were pretreated with CC‐930 or PD169316 for 2 hr and then incubated with bepridil (25 μM) for 24 hr; only CC‐930 significantly suppressed bepridil‐induced apoptosis. *P < .05, apoptotic cell fraction in CC‐930 + bepridil group versus bepridil group. n = 5 independent experiments; data were analysed by one‐way ANOVA

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  Figure 8. Impact of bepridil on human astrocytes (HA) and the Na+/Ca2+ exchanger (NCX) isoforms in each cell line. (a) Viability of U87, U251, and SF188 cells and HA after exposure to bepridil (25 μM, 48 hr); n = 5 independent tests in each group. (b) The bands of NCX1, NCX2, and NCX3 isoforms detected by Western blot analysis in the sample of HA and glioblastoma cell lines U87, U118, A172, U251, and SF188. (c) Representative recording of the NCX currents in HA and glioblastoma cell lines. (d) Viability of U87, U251, and SF188 cells and HA after exposure to KB‐R7943 (25 μM, 48 hr). n = 5 independent tests in each group

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  Figure 9. Bepridil crosses the BBB and inhibits growth of brain‐grafted glioblastoma. (a) Dotted box indicates the precursor to product ions (Q1 → Q3) selected for bepridil during quantitative optimization: 367.1 → 84.3 and 367.1 → 184.3 in positive ion mode. (b) Typical chromatogram of brain homogenate sample collected at 2 hr following systemic administration of bepridil (50 mg·kg−1, p.o.). (c, d) Bioluminescent images of U87‐luciferase glioblastoma cells implanted into the right cerebral striatum of nude mice. Images were taken by an IVIS Spectrum CT Imaging System before and after the period of treatment with bepridil or vehicle as described in methods. (e) Tumour size determined by bioluminescence and caliper measurement in each group before and after treatment. *P < .05, comparison between the two groups; n = 5 mice per group, analysed by Student's parametric unpaired t test