(526h) Caspase-Dependent HDAC4 Translocation Due to Microsecond Pulsed Electric Field (?sPEF) Exposure of Glioblastoma Cells

Electroporation-based technologies using microsecond pulsed electric field (µsPEF) exposures are established as laboratory and clinical tools that permeabilize cell membranes to induce a variety of desired bioeffects. When used for cancer treatment, PEF exposures specifically triggers cell death through caspase activation ^1-4. nsPEF exposure induces apoptosis in different cell types through intrinsic and extrinsic apoptosis pathways, which both lead to activation of different caspases ⁵.

Overexpression of the class IIa histone deacetylase 4 (HDAC4) has been reported for glioblastoma cells ^{6, 7}. Dephosphorylation of HDAC4 leads to import into the nucleus and easy access to specific transcription factors, enabling repression of genes. Nuclear accumulation of HDAC4 promotes neuronal cell apoptosis ⁸. In prior work, we have shown that µsPEF exposure can lead to nuclear accumulation of HDAC4 and a decrease in cell proliferation of U87-MG cells ⁹. In this study we demonstrate the role of caspases in HDAC4 translocation under standard culture conditions and following µsPEF exposure of glioblastoma cells. These results can help to better understand the effect of µsPEF exposure on mechanisms of epigenetic modification.

Materials & Methods

U87-MG cells were grown in T-75 flasks containing DMEM supplemented with 10 vol% fetal bovine serum (FBS, HyClone, SH30396.03, MA, USA) and 1 vol% antibiotic/antimycotic. To track HDAC4 localization, immunofluorescence assay (IFA) was performed 3 h after exposure using a HDAC4 polyclonal antibody (BioVision # 3604A-100, Milpitas, CA, USA) and a goat anti-rabbit IgG (H&L) (DyLight^® 488, NC, USA) as the secondary antibody and fluorescence marker.

Two kinds of custom buffer solutions were used throughout the experiments. Standard Outside Solution (SOS) consisted of 5 mM KCl, 2 mM CaCl₂, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2 mM MgCl₂, 10 mM Glucose, and 135 mM NaCl. Calcium-Free Standard Outside Solution (CAF) consisted of all the SOS components, except instead of CaCl₂, it contained 2 mM potassium ethylene glycol tetraacetic acid (K-EGTA). Solution pH was adjusted to 7.4 using NaOH.

A commercial electroporator (BTX Gemini X2 with PetriPulser electrode array and cuvette) was used to control square-wave, monopolar µsPEF duration (100 µs) and magnitude (1.45 kV/cm), while varying the number of pulses (0, 1, 10, 20 pulses, delivered at 1 Hz) to determine thresholds for HDAC4 translocation and proliferation reduction of U87-MG cells. For IFA, cells were cultured and exposed on glass-bottom petri dishes.

To identify the cause of cell death with respect to HDAC4 translocation and long-term effects of µsPEF exposure on HDAC4 localization, U87-MG cells were exposed to 10 consecutive pulses of 100 µs duration and 1.45 kV/cm at a repetition rate of 1 Hz. Then subcellular location of HDAC4 was acquired after 3h. To identify the role of caspase in HDAC4 translocation due to µsPEF exposure, caspases were inhibited by 50 µM Z-VAD-FMK (AAT Bioquest, 13300, CA, USA) with 1 h incubation. Each sample petri dish was filled with 1 mL of SOS or CAF containing inhibitor before pulse treatment. Then, cells were exposed to µsPEF. Samples were fixed with paraformaldehyde 3h after exposure, and the IFA protocol was performed as described above.

Results

Cells treated with the caspase inhibitor alone show significantly higher nuclear HDAC4 when compared to the untreated controls in CAF. The presence of extracellular Ca²⁺ neutralizes this effect, showing no significant changes between inhibitor-treated samples and controls (Fig. 1A). To determine whether caspases play a role in µsPEF exposure-induced HDAC4 nucleocytoplasmic shuttling, sham exposure controls are compared to 10 pulse exposures (Fig. 1B). The µsPEF exposure itself leads to nuclear import of HDAC4 in CAF. Caspase inhibition plus µsPEF exposure amplifies this trend in CAF. However, the presence of extracellular Ca²⁺ again does not show significant changes between shams and µsPEF exposures in SOS (Fig.1B). Comparing all samples receiving µsPEF exposure emphasizes the correlation between caspase activity and HDAC4 translocation (Fig. 1C). These results suggest that caspases at least partially determine nucleocytoplasmic shuttling of HDAC4 in CAF, whereas Ca²⁺ influx eliminates this effect by preventing nuclear accumulation of HDAC4.

Fig 1. The role of caspase activity in msPEF exposure-induced HDAC4 nucleocytoplasmic translocation in U87-MG cells is tested by pharmacological inhibition and shown by comparison of, A: Sham exposure controls, B: Sham controls vs. msPEF exposure, and C: msPEF exposures in solutions with and without extracellular Ca²⁺. Statistical significance is tested using two-way ANOVA with Tukey’s method, with symbols representing no significance (ns), P £ 0.0021 (**), P £ 0.0002 (***), and P £ 0.0001 (****).

References

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