Electrochemotherapy with bleomycin is effective in BRAF mutated melanoma cells and interacts with BRAF inhibitors

Human melanoma cell line SK-MEL-28 (American Type Culture Collection (ATCC), Manassas, VA, USA) with BRAF V600E mutation was cultivated in an Advanced MEM medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 5% fetal bovine serum (FBS, Gibco), 10 mM L-glutamine (GlutaMAX, Gibco), 100 U/ml penicillin (Grünenthal, Aachen, Germany) and 50 pg/μl gentamicin (Krka, Novo mesto, Slovenia) in a 5% CO₂ humidified incubator at 37°C.

Human melanoma cell line CHL-1 (ATCC) without BRAF V600 mutations was cultured in an Advanced RPMI 1640 medium (Gibco), supplemented with 5% FBS, 10 mM L-glutamine, 100 U/ml penicillin and 50 pg/ml gentamicin in a 5% CO ₂humidified incubator at 37°C.

Melanoma cells were grown as a monolayer until they reached at least 80% confluence. The medium was removed and the cells were washed with phosphate-buffered saline (PBS, Merck Millipore, Darmstadt, Germany). After that, cells were detached from the surface with 0.25% trypsin/EDTA in Hank’s buffer (Gibco). After detachment, trypsin was inactivated with an equal amount of cell culture medium with FBS and cells were collected and centrifuged. For electroporation, 88 μl of cell suspension (25 × 106 cells/ml) was prepared in electroporation buffer (125 mM sucrose, 10 mM K₂HPO₄, 2.5 mM KH₂PO₄, 2 mM MgCl₂ × 6H₂0) at 4°C. Cell suspension was mixed with 22 pl of different stock concentrations of bleomycin (Bleomycin medac, Medac, Wedel, Germany) to reach a final concentration of 1.4 × 10⁻¹² M, 1.4 × 10⁻¹¹ M, 1.4 × 10⁻¹⁰ M, 1.4 × 10⁻⁹ M, 1.4 × 10⁻⁸ M, 1.4 × 10⁻⁷ M, 1.4 × 10⁻⁶ M. Out of 110 pl of mixture, 50 pl served as a control for bleomycin treatment and other 50 pl was pipetted between two stainless steel parallel plate electrodes (2 mm apart) and 8 square-wave electric pulses (amplitude over distance ratio of 1300 V/ cm, duration of 100 ps and frequency of 1 Hz) were applied. Electric pulses were generated with the electric pulse generator GT-01 (Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia). The cells were incubated 5 min after electroporation at room temperature, and then cell culture medium was added. Afterwards, clonogenic assay was performed. Experimental groups were denoted: BLM (different concentrations of bleomycin); BLM + EP (ECT with bleomycin of different concentrations). Each group was normalized to the control group of same treatment regimen with 0 M bleomycin (Ctrl, EP).

After the treatment, cells were plated in 6 cm petri dishes with 4 ml of culture medium for clonogenic assay. The number of plated cells was in a range of 300-4000 cells, based on predicted cytotoxicity of the treatment. Colonies were formed after 12 and 8 days for SK-MEL-28 and CHL-1, respectively. After the colonies were formed, they were fixed and stained with crystal violet solution (Sigma-Aldrich, St. Louis, MO, USA) and counted. The colonies containing less than 50 cells were disregarded. Plating efficiency was calculated for each experimental group as the ratio between counted colonies and the number of plated cells. Plating efficiency of each treatment group was normalized to the untreated cells group, representing cell survival. IC₉₀ value was determined (drug concentration required to reduce cell survival for 90%).

Cell lines were tested for their sensitivity to vemurafenib treatment in order to confirm the selectivity of vemurafenib on BRAF V600E mutated cells and to determine the optimal concentration for the combination treatment. Cells were detached from cell culture dishes and prepared for clonogenic assay. Vemurafenib (MedChem Express, Monmouth Junction, NJ, USA) was obtained in 10 mM DMSO solution and was added to 6 cm culture dishes with 4 ml of culture medium to reach a final concentration of 0.5 pM, 2.5 pM, 5 pM and 10 pM. The final amount of DMSO in the cell culture medium represented 20000x — 1000x dilution and was not toxic to the cells. Afterwards, cells were cultured in a 5% CO₂ humidified incubator at 37°C until the formation of colonies, which were counted and analyzed as described. Each group was normalized to the control group with 0 pM vemurafenib.

ECT was performed as described. After that, cells were plated in 6 cm petri dishes for clonogenic assay and vemurafenib was added in each dish to reach a final concentration of 0.5 pM. Afterwards, cells were cultured in a 5% CO₂ humidified incubator at 37°C until the formation of colonies, which were counted and analyzed as described. Experimental groups were denoted: BLM (different concentrations of bleomycin); BLM + EP (ECT with bleomycin of different concentrations); BLM + VMF (bleomycin of different concentrations and addition of 0.5 pM vemurafenib), BLM + EP + VMF (ECT with bleomycin of different concentrations and addition of 0.5 pM vemurafenib). Each group was normalized to the control group of same treatment regimen with 0 M bleomycin (Ctrl, EP, Ctrl + VMF, EP + VMF).

The mode of interaction between the treatments with independent mode of action was calculated at the level of IC₉₀ by the method developed by Spector.13 For the analysis and graphical representation, SigmaPlot Software (version 12.0, Systat Software, London, UK) was used.

Vemurafenib treatment was tested for selective cytotoxicity to BRAF V600E mutated cells. Selectivity of BRAF inhibitor vemurafenib was confirmed on SK-MEL-28 BRAF V600E mutated melanoma cells. Cell survival was reduced with increasing vemurafenib concentration with IC₉₀ 5 pM (Figure 1A). In contrast, vemurafenib treatment did not reduce the survival of BRAF V600E non-mutated cell line CHL-1. The cell survival was higher than 94%, also with the highest concentration of vemurafenib used (Figure 1B). These results also provided bases for the combined vemurafenib and ECT treatment of cells, the concentration of 0.5 pM was selected, which reduced cell survival to 50%.

Figure 1

The effectiveness of ECT was tested on BRAF V600E mutated melanoma cells (SK-MEL-28) and non-mutated melanoma cells (CHL-1). ECT effectively reduced survival of both cell lines with IC₉₀ 3.8 × 10⁻¹⁰ M and 7.7 × 10⁻¹⁰ M for SK-MEL-28 and CHL-1, respectively. ECT was even more effective on BRAF V600E mutated SK-MEL-28 cells that required 2 times lower concentration of BLM at IC₉₀, compared to non-mutated cells CHL-1 (Figure 2), confirming that ECT is an effective method for treatment of also BRAF V600E mutated melanoma cells.

Figure 2

Concomitant treatment with ECT and vemurafenib was simulated in vitro on cells in the way, that ECT treated cells were seeded into dishes containing 0.5 μM vemurafenib. The vemurafenib treatment decreased survival of SK-MEL-28 cells for 50%. If the effect of the vemurafenib was eliminated (normalized to control groups with added vemurafenib for groups BLM + VMF and BLM + EP + VMF), as shown in Figure 3, then an increased effectiveness of ECT was observed on BRAF mutated SK-MEL-28 cells. A 4.5 times lower concentration of BLM was needed at the IC₉₀ for cells treated with vemurafenib (BLM + EP + VMF; IC₉₀ value 8.5 × 10⁻¹¹ M) compared to cells without vemurafenib treatment (BLM + EP; IC₉₀ value 3.8 × 10⁻¹⁰ M). The potentiation was more than additive (Figure 3), in fact, according to the method developed by Spector et al. was synergistic.13

Figure 3