Calcium-independent disruption of microtubule dynamics by nanosecond pulsed electric fields in U87 human glioblastoma cells
Résumé
High powered, nanosecond duration, pulsed electric fields (nsPEF) cause cell death by a mechanism that is not fully understood and have been proposed as a targeted cancer therapy. Numerous chemotherapeutics work by disrupting microtubules. As microtubules are affected by electrical fields, this study looks at the possibility of disrupting them electrically with nsPEF. Human glioblastoma cells (U87-MG) treated with 100, 10 ns, 44 kV/cm pulses at a frequency of 10 Hz showed a breakdown of their interphase microtubule network that was accompanied by a reduction in the number of growing microtubules. This effect is temporally linked to loss of mitochondrial membrane potential and independent of cellular swelling and calcium influx, two factors that disrupt microtubule growth dynamics. Super-resolution microscopy revealed microtubule buckling and breaking as a result of nsPEF application, suggesting that nsPEF may act directly on microtubules. Traditional cytotoxic chemotherapies are associated with severe side-effects and new generation targeted therapies can fail due to drug resistance. High powered, nanosecond duration pulsed electric fields (nsPEF) have been proposed as a minimal side-effect, electrical cancer therapy that is unlikely to result in resistance. Glioblastoma multiforme (GBM) is an incurable brain cancer showing resistance to surgery, radiotherapy and chemotherapy 1. The need for an effective treatment for GBM, and its previously demonstrated sensitivity to pulsed electric fields 2 , makes it of interest for targeting by nsPEF. Studies have demonstrated that nsPEF induce cell death by apoptosis and necrosis in vitro and reduce the size of tumours both in animal models and in humans 3–8. The effect of nsPEF on cells is characterized by nanopora-tion of the plasma membrane 3,9–12 , rapid phosphatidylserine externalisation 6,13,14 , transient spikes in intracellular calcium concentration that are proportional to pulse intensity 3,15–17 , loss of mitochondrial membrane potential (Δ Ψ m) 18,19 and cellular swelling and blebbing 12,20,21. Apoptosis following nsPEF treatment can be either dependent or independent of caspase activation 6,7,13. Whilst nsPEF induced apoptotic death has been well studied, the mechanism whereby nsPEF triggers apoptosis remains unclear. Microtubules are hollow, cylindrical, structures composed of repeating α and β heterodimers of the protein tubulin. Forming part of the cell cytoskeleton, microtubules are highly dynamic structures subject to constant lengthening and shortening. In interphase cells they are nucleated in microtubule-organizing centres and grow out towards the cell periphery. Depolymerisation of the interphase microtubule network is an intrinsic, early event in the execution phase of normally occurring apoptosis, aiding phagocyte attachment 22 and the release of microtubule sequestering proapoptotic proteins 23. Due to the polarity of their protein structure and charge, it has been shown in vitro that purified microtubules will align with an electric field 24,25 and that, in cells, electric fields can disrupt their polymerization 26. Given these properties, we hypothesized that nsPEF might have a direct effect on microtubules.
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