Sunitinib potentiates the cytotoxic effect of electrochemotherapy in pancreatic carcinoma cells

The human pancreatic cancer cell line BxPC-3 (ATCC^® CRL-1687™) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) in 2020 and cultured in Advanced RPMI-1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5% (v/v) fetal bovine serum (FBS, Gibco), GlutaMAX (100x, Gibco), and penicillin–streptomycin (100x, Sigma Aldrich, Merck, Darmstadt, Germany) in a 5% CO2 humidified atmosphere at 37°C. The cells were routinely tested for mycoplasma infection by a MycoAlertTM PLUS Mycoplasma Detection Kit (Lonza, Basel, Switzerland) and were mycoplasma free.

A stock solution of 3 mg/ml bleomycin (Bleomycin medac, Medac, Germany) was diluted in 0.9% NaCl saline (B. Braun Melsungen AG, Melsungen, Germany) to seven working solutions in the range from 7 μM to 7 x 10-6 μM.

Cisplatin Kabi 1 mg/mL (Fresenius Kabi, Bad Homburg, Germany) was diluted in 0.9% NaCl saline to 4 working solutions: 833.3, 83.33, 8.33 and 0.83 μM.

Suntinib (50 mg, Selleckchem, Houston, TX, USA) was first diluted in dimethyl sulfoxide (DMSO, Sigma Aldrich, D2650-100ML) to a concentration of 10 mM and further diluted in 0.9% NaCl saline to 7 working solutions: 100, 75, 50, 25, 10, 5 and 1 μM.

Cells were trypsinized, centrifuged and resuspended in electroporation buffer (EP buffer: 250 mM sucrose (Sigma Aldrich, S0389); 10 mM K₂HPO₄ (Sigma Aldrich; 2.5 mM KH₂PO₄ (Sigma Aldrich); 2 mM MgCl₂ x 6H₂O (Sigma Aldrich) to a concentration of 2.5 x10⁷ cells/ml. Each experimental group contained 10⁶ cells in 40 μL of EP buffer and 10 μL of bleomycin or cisplatin solution or 10 μL of saline in the case of controls (untreated control cells- Ctrl or electroporated cells -EP). The final concentrations of cytotoxic drugs were therefore 5 times lower than the working concentrations (1.4, 1.4 x 10^-1, 1.4 x 10^-2, 1.4 x 10^-2, 1.4 x 10^-3, 1.4 x 10^-4, 1.4 x 10^-5, 1.4 x 10^-6 μM for bleomycin and 166.7, 16.7, 1.7, and 0.17 μM for cisplatin). Fifty microliters of the mixture was pipetted between two electrodes (2 mm gap) and electroporated (8 pulses, 1300 V/cm, 100 μs duration at frequency 1 Hz) with an Electro Cell B10 electric pulse generator (LEROY Biotech, Saint-Orens-de-Gameville, France). The cell mixture was transferred into the wells of a 24-well ultralow attachment plate (Corning, Corning, NY, USA), and five minutes after pulse delivery, cell culture medium was added, and the cells were seeded for further assays. For the unelectroporated groups, 50 μL of the mixture was transferred into a 24-well ultralow attachment plate, and medium was added.

After ECT, 4 x 10³ or 2 x 10³ cells were seeded into the wells of 96-well plates for 3 or 7 days, respectively, in 7 technical replicates. Cells were incubated at 37°C in a 5% CO₂ humidified incubator until measurement. Cell survival was measured with a Presto Blue viability assay (Thermo Fisher Scientific) 3 and 7 days after the treatment. Presto Blue (10 μl/ well) was added to the cells, and 30 minutes thereafter, fluorescence intensity was measured with a Cytation 1 microplate reader (BioTek, Winooski, VT, USA). The viability of the cells after ECT was normalized to the control groups: untreated control cells (Ctrl) for the bleomycin groups and electroporated cells (EP) for the ECT bleomycin groups to eliminate the effect of pulses alone. The concentration that killed 50% of the cells, the EC₅₀ dose, was estimated directly from cell survival curves.

A monolayer of 80% confluent cells was trypsinized, centrifuged and counted. In each well of a 96-well plate, 4 x 103 or 2 x 103 cells were seeded in 90 μL for 3 or 7 days of incubation with sunitinib, respectively. Ten microliters of sunitinib was added to the cells to obtain final concentrations of 10, 7.5, 5, 2.5, 1, 0.5 and 0.1 μM sunitinib and incubated for 3 or 7 days in a 5% CO2 humidified atmosphere at 37°C. Then, the cell survival assay was performed as described above. The viability of the cells was normalized to that of untreated control cells (Ctrl).

Further experiments with combined therapy were performed with several doses of sunitinib. For combining ECT cisplatin with sunitinib, the 7-day observation period was selected, since the effect of ECT cisplatin was observed only after several days. Four concentrations of cisplatin (166.7, 16.7, 1.7, and 0.17 μM) and three concentrations of sunitinib were used: 5 μM sunitinib, which represents the approximation of the EC₅₀ dose, and one lower (2.5 μM) and one higher (7.5 μM) tested dose. For combining ECT bleomycin with sunitinib, the 3-day observation period was selected, since bleomycin effects are observed after several hours. Five final concentrations of bleomycin were tested (0.14 - 1.4 x 10^-5 μM) in combination with three final concentrations of sunitinib 7.5, 5 and 2.5 μM. After ECT treatment, 4 x 10³ or 2 x 10³ cells in 90 μL were seeded in 96-well plates for 3 or 7 days, respectively. Ten microliters of sunitinib solution was added to the wells, and the cells were incubated at 37°C in a 5% CO₂ humidified incubator for 3 or 7 days in a 5% CO₂ humidified atmosphere at 37°C. Then, the cell survival assay was performed as described above. The viability of the cells was normalized to control groups: cells with added sunitinib for bleomycin + sunitinib groups and electroporated cells with added sunitinib for ECT bleomycin + sunitinib groups to eliminate the effect of pulses and sunitinib. The combination effects (additivity, synergism, and antagonism) of the treatments were calculated by the Chou-Talalay computational method by using CompuSyn software (ComboSyn, Inc, Paramus, NJ, USA).20 Combination indexes below 1 were considered synergistic.

Due to the minimal or no effect of bleomycin or cisplatin alone on cell viability, cell death kinetics were determined only in cells treated with EP and ECT. Cell death was determined through kinetic evaluation of diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific) uptake, determining increased cell membrane permeability as a marker of cell death. Briefly, ECT with 0.14 μM bleomycin was evaluated alone or combined with 2.5, 5 and 7.5 μM sunitinib. Afterwards, 5 x 10³ control or EP-treated cells and 25 x 10³ ECT-treated cells were seeded in each well of a black 96-well plate. Different cell plating densities were used because of prominent cell death that follows instantly after ECT. These dead cells do not attach to the plate and represent a limitation for the usage of imaging techniques. Cells were incubated for 2 hours to allow cells to attach. Thereafter, medium with non-attached dead cells was removed for cell imaging purposes, and 200 μL of fresh phenol-free medium containing the appropriate concentration of sunitinib and 0.5 μM DAPI was added. Brightfield and fluorescence images (one image per well) of cells were captured every 4 h for 48 hours with Cytation 1 (BioTek) with a 4-x objective. For each time point, the number of DAPI-positive nuclei (representing dead cells) was determined with Gen 5 Data analysis software (BioTek).

For the determination of receptor tyrosine kinases, 90% confluent cells in T25 flasks were harvested, and RNA was extracted using a peqGOLD Total RNA Kit (VWR, West Chester, PA, USA) according to the manufacturer’s instructions. RNA concentrations were quantified by spectrophotometry at 260 nm (Cytation 1), and purity was determined by measuring the ratio of absorbance at A260 nm/280 nm and A260 nm/230 nm. Afterwards, 2000 ng of total RNA was reverse transcribed into cDNA using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. After reverse transcription, 10 ng of cDNA template was used in 20 μL of RT-qPCRs using predesigned primers: Hs.PT.58.45689824 (KIT), Hs.PT.58.19454325 (FLT3), Hs.PT.58.4318912 (RET), Hs.PT.58.3285240 (KDR), Hs.PT.58.22892761 (PDGFR), Hs.PT.58.40906831 (FLT1) or custom made primers: F-AGGTGATGGAAGAAGTGGTG, R-AGGATTTGGTGTGAGCGATC (Glucuronidase Beta; GUSB) (Integrated DNA Technologies, Coralville, IA, USA) and PowerUp™ SYBR™ Green Master Mix (Thermo Fisher Scientific,). The RT-qPCRs were run until the 40^th cycle with the following cycling conditions: 2 min at 50°C, 2 min at 95°C, 40 cycles of 15 s at 95°C, and 1 min at 60°C; for the melting curve determination, 15 s at 95°C, 1 min at 60°C, and 15 s at 95°C. The results were analyzed on a QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific). Ct values above the 35^th cycle were considered undetermined. Relative expression was calculated by normalization to the expression of the housekeeping gene GUSB using ΔCt method.21

The values in this study are presented as the mean (AM) ± standard error of the mean (SEM). Comparisons between two groups were performed using unpaired two-tailed Student’s t test. The comparison of means of more than two groups was statistically evaluated by one-way analysis of variance (one-way ANOVA) followed by Dunnett’s or Tukey’s multiple comparisons test. A p value of <0.05 was considered to be statistically significant. A sample size (n) for each experiment represents biological replicates and is presented in each figure legend. For statistical analysis and preparation of graphs, GraphPad Prism 9 (La Jolla, CA, USA) was used. The combination effects (additivity, synergism, and antagonism) of the treatments were calculated by the Chou-Talalay computational method by using CompuSyn software).20 Combination indexes below 1 were considered synergistic.

Bleomycin alone did not significantly affect cell survival, either after 3 days or after 7 days. (Figure 1A, B). Electroporation of cells significantly increased the cytotoxic effect of bleomycin. A 50% decrease in viability was observed at bleomycin concentrations higher than 1.4 x10^-3 μM after 3 days of incubation. More precisely, the EC₅₀ for ECT with bleomycin was 6.1 x10^-3 μM. ECT at the two highest concentrations, 0.14 μM and 1.4 μM, killed 90% of cells (Figure 1A). After 7 days of incubation, the EC₅₀ dose for ECT with bleomycin could not be determined, since the lowest tested dose of 1.4 x10^-6 killed more than 50% of the cells. Bleomycin doses higher than 0.014 μM killed 90% of cells after 7 days of incubation (Figure 1B).

Figure 1

When cisplatin was used as a cytotoxic drug in ECT, a significantly lower survival fraction of BxPC-3 cells was observed only when using the highest tested cisplatin concentration, 166.67 μM, 3 days after the treatment (Figure 1C). The EC₅₀ dose for ECT with cisplatin after 3 days of incubation was 60 μM. After 7 days of incubation, cisplatin alone without electroporation at a concentration of 166.67 μM killed approximately 50% of the cells. However, when cells were treated with ECT, a significant decrease in cell viability was observed at a 1.67 μM cisplatin dose. The EC₅₀ for ECT with cisplatin after 7 days of incubation was 6 μM (Figure 1D).

First, the expression of potential targets of the tyrosine kinase inhibitor sunitinib was determined in BxPC-3 pancreatic carcinoma cells. All of the tyrosine-kinase receptors that are known targets of sunitinib were expressed in BxPC-3 cells, with FLT3, PDGFR and VEGFR1 being the most highly expressed among them (Figure 2A). After confirmation of sunitinib targets in BxPC-3 pancreatic carcinoma cells, they were treated with different concentrations of sunitinib and incubated for 3 or 7 days to confirm and determine its cytotoxicity. The survival of cells after incubation with lower concentrations of sunitinib, 0.1 μM, 0.5 μM and 1.0 μM, was not significantly reduced compared to control cells (Figure 2 B, C). When the cells were treated with higher concentrations, i.e., > 1.0 μM, survival was significantly reduced, especially after 7 days of incubation (Figure 2C). The EC₅₀ values for sunitinib after 3 days of incubation were 8.1 μM and 5 μM after 7 days of incubation.

Figure 2

For combined treatment, five bleomycin concentrations and three sunitinib concentrations, 2.5, 5.0 and 7.5 μM, were tested. The cytotoxic effect was determined 3 days after the treatment due to the fast-cytotoxic action of bleomycin. Compared to ECT with bleomycin in monotherapy, the combination with sunitinib had an increased cytotoxicity, but only at the highest used concentration of bleomycin 0.14 μM (Figure 3A). The synergistic effect of ECT with 0.14 μM bleomycin and sunitinib was confirmed by the Chou-Talalay method with combination indexes of 0.11 and 0.07 for 5 and 7.5 μM sunitinib, respectively.20 When lower doses of bleomycin were used in combination treatment, no significant changes in cell survival were observed compared to ECT in monotherapy (Figure 3A).

Figure 3

For combined treatment of ECT with cisplatin and sunitinib, four cisplatin concentrations and three sunitinib concentrations, 2.5, 5.0 and 7.5 μM, were used. The cytotoxic effect was evaluated 7 days after the treatment due to the slower action of cisplatin, which was observed in previous experiments (Figure 1C, D). Due to this later time point of evaluation compared to ECT with bleomycin, the combination with 7.5 μM sunitinib could not be tested, as this concentration of sunitinib alone was too cytotoxic (Figure 2C). Compared to ECT with cisplatin, the combination with sunitinib did not achieve higher cytotoxicity (Figure 3B). At cisplatin concentrations of 1.67 μM and 16.67 μM, the cytotoxic effect after combined therapy was even lower than that after monotherapy. This may indicate an antagonistic effect of combination therapy with 2.5 or 5.0 μM sunitinib at concentrations of 1.67 μM and 16.67 μM cisplatin; however, this could not be confirmed by the Chou-Talalay method.

Cell death was determined only after ECT with bleomycin, as it was more effective in the BxPC-3 pancreatic carcinoma cell line than ECT with cisplatin. The kinetic measurements of DAPI uptake by the permeabilized dead cells demonstrated that the majority of cells died soon after ECT, reaching significance at 8 hours after the treatment compared to control (Ctrl) and electroporated cells only (EP) (Figure 4A). The number of dead cells further increased until approximately 20 hours after the treatment, when it reached a plateau (Figure 4A).

Figure 4

The addition of sunitinib to control untreated cells (Ctrl) or electroporated cells only (EP) did not increase the numbers of dead cells in the first 48 hours, suggesting that cell death induced by sunitinib takes place after the first 48 hours (Figure 4 B, C). However, the combination of ECT with bleomycin and 5 or 7.5 μM sunitinib resulted in significantly higher numbers of dead cells compared to ECT alone (Figure 4D). However, the combination of ECT with bleomycin and 2.5 μM sunitinib did not result in a significant increase in the numbers of dead cells (Figure 4D). The time frame of cell death after combination treatment was similar to that after ECT monotherapy, reaching a plateau in the first 20 hours (Figure 4D).