Nanosecond Pulsed Electric Fields Enhance the Anti-tumour Effects of the mTOR Inhibitor Everolimus against Melanoma

Abstract

The PI3K/mTOR/AKT pathway is activated in most melanomas, but mTOR inhibitors used singly have limited activity against advanced melanomas. The application of nanosecond pulsed electric fields (nsPEFs) is a promising cancer therapy approach. In this study, we evaluated the synergistic anti-tumour efficacy of the mTOR inhibitor everolimus in conjunction with nsPEFs against melanoma. The combined treatment of nsPEFs and everolimus gradually decreased cell growth concurrent with nsPEF intensity. nsPEFs alone or combined with everolimus could promote melanoma cell apoptosis, accompanied with a loss in cellular mitochondrial membrane potential and an increase in Ca²⁺ levels. In vivo experiments showed that a combination of the mTOR inhibitor everolimus and nsPEFs improved the inhibitory effect, and all skin lesions caused by nsPEFs healed in 1 week without any observed adverse effect. Combination treatment induced caspase-dependent apoptosis through the upregulation of the pro-apoptotic factor Bax and downregulation of the anti-apoptotic factor Bcl-2. Everolimus and nsPEFs synergistically inhibited angiogenesis by decreasing the expression of vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), and CD34. Our findings indicate that nsPEFs in combination with an mTOR inhibitor can be used as a potential treatment approach for advanced melanoma.

Figure 1. nsPEF treatment inhibited melanoma cell growth in vivo and in vitro.

Effects of nsPEFs with high energy input in (a) A375 and (b) A875 cells. Pulse duration, 100 ns; electric field strength, 20–30 kV/cm; number of pulses delivered according to different energy levels, 10–100. The energy input of nsPEFs was calculated as energy input = (E² × D² × W × N)/(R × M), where E is the electric field strength (20–30 kV/cm); D, gap between electrodes (here, 2 mm); W, pulse duration (here, 100 ns); N, number of pulses (10–100 pulses); R, resistance in the cuvette with cells and suspending medium; and M, mass of the suspension in the cuvette. The specific parameters are listed in Supplementary Table S1. (c) Typical longitudinal monitoring of fluorescence images of tumour-bearing mice before and after nsPEF treatment. GFP activity in mice was detected by IVIS 200 before nsPEF treatment (day 0), as well as 1, 2, 4, and 8 days after nsPEF treatment. (d) Fluorescence intensities were quantified in photons per second. Ratios of mean ± SD (n = 5) were obtained. (e) Surface view after nsPEF treatment. The experiments were repeated thrice and yielded similar results. *P < 0.05, **P < 0.01 compared to control.

Figure 2. nsPEFs and everolimus synergistically inhibited melanoma cell growth in vitro.

(a) and (b) Were effects of everolimus on the proliferation of melanoma cells in vitro. A375 and A875 cells were treated with elevated concentrations of everolimus for 24 h, 48 h, and 72 h. Cell viabilities were determined by CellTiter-Glo analysis. (c) and (d) Cells were first treated with nsPEFs, followed by incubation with everolimus for 48 h; or treated with everolimus for 48 h prior to nsPEF treatment. Synergism quotients (SQ) are shown for both treatment orders. Mean ± SD values of triplicate samples were determined. *P < 0.05 compared to control, ^#P < 0.05 compared to everolimus single agent treatment.

Figure 3. nsPEFs and everolimus induced melanoma cell apoptosis, cellular mitochondrial membrane potential (ΔΨm) loss and Ca²⁺ increase.

(a) A375 cells were treated with everolimus for 48 h, followed by nsPEF treatment. Cell apoptosis was evaluated by FITC-Annexin V and PI staining using flow cytometry. The experiments repeated 3 times yielded similar results. Cells after nsPEF treatment were incubated with JC-1 or Fluo-3/AM at 37 °C for 30 min. (b) Loss of ΔΨm was detected using the fluorescent probe JC-1. Cells treated with 100 nM valinomycin were used as the positive control for decreased ΔΨm. (c) Intracellular Ca²⁺ level was quantified with the fluorescent dye Fluo-3/AM. Cells treated with 20 μM ionomycin were used as the positive control for intracellular Ca²⁺ level detection. The experiments repeated thrice yielded similar results. Values are means ± SD (n = 6). *P < 0.05 compared to control, ^#P < 0.05 compared to everolimus single agent treatment.

Figure 4. nsPEFs and everolimus synergistically suppressed tumour growth in vivo.

(a) Tumour volume change in the 4 groups after treatment. Tumour size was measured every 3–4 days after treatment. Values are means ± SEM (n = 5 mice per group). (b) Tumours derived from 4 groups 30 days after subcutaneous injection.

Figure 5. nsPEFs and everolimus induced cell apoptosis and decreased angiogenesis in vivo.

Immunohistochemical staining of the apoptosis markers Bax, Bcl-2, cleaved caspase-3, and cleaved caspase-6, and angiogenesis-associated markers VEGF, VEGFR, and CD34 (original magnification, ×200).

Figure 6. Schematic diagram of the nsPEF generator.