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Introduction

Betulinic acid (BTA) is a carboxylic derivative of betulin, a naturally occurring triterpene predominantly found in the birch bark and other plants (1). It exhibits broad-spectrum biological activity even at low concentrations, such as anti-bacterial, anti-inflammatory, anti-herpes simplex virus-1 or anti-malarial (2–4). Previous studies have indicated the potential use of BTA as a new anticancer drug (5,6). It has been reported to induce apoptosis in various human cancer cell lines (7–10). This process occurs independently of the cascades that mediate programed cell death and without the activation of p53 protein, which is responsible for the promotion of apoptosis in cancer cells (11–13). A very important BTA feature is its lack of cytotoxicity against normal cells (14); therefore, it is hypothesized that the use of BTA in cancer treatment may protect patients from the adverse effects of many standard cytostatic drugs (such as cisplatin). However, one of the drawbacks of BTA therapeutic use is its low solubility. One method to overcome this limitation could be structural modification as indicated by several studies (3,4,15). Another solution may be a physical method, such as electroporation, which allows the flow of molecules into the cell.

Electroporation (EP) is a technique that enables the formation of unstable and hydrophilic pores in cell membranes following exposure to high-intensity short electrical pulses that induce the formation of breaks in membranes, through which macromolecules can enter from the intercellular space. In addition, drugs can also penetrate the cell through the pores created by EP. EP has not been fully explored yet. The ‘pores’ created in EP are unstable, form quickly and disappear within a few seconds to several minutes after the exposure of the cell to the electric field (16,17).

The combination of EP and chemotherapy (CT) is termed electrochemotherapy (ECT). It allows for the delivery of drugs directly into the cell (18). When cytotoxic agents are poorly transported into the cell, the use of ECT enables the passage of cytostatic drugs, enhancing the local treatment of cancer and potentially reducing the side effects of systemic CT by reducing the required doses of drugs. ECT is much faster and more efficient than CT alone, which is crucial for the treatment of patients with cancer. In particular, it may be beneficial for the treatment of drug-resistant tumors in cases when the efficiency of the working dose has been substantially reduced, for example by multidrug resistance mechanisms (16,18). Furthermore, electrical pulses cause decreased blood perfusion in vessels surrounding a tumor and can prolong contact of drugs with cancer cells (16,19). ECT is a very promising method of treatment for superficially located tumors. In some cases, ECT limits the necessity for surgical intervention and saves the organ (19). Currently, in many European countries, bleomycin and cisplatin (CP) are the only cytostatic drugs that are clinically approved for use in ECT protocols (16,18). Therefore, exploration of a less toxic, natural-origin drugs (such as BTA) for use in ECT is highly desirable.

Recent studies have demonstrated that elevated levels of heat shock protein (HSPs), which are ubiquitous intracellular ‘stress proteins’ or molecular chaperones (20–22), can increase the aggressiveness of cancer, or alter the response to chemo- or radiotherapy (23). HSPs are large and heterogeneous molecules involved in a multitude of housekeeping functions within a cell (24–26). Under physiological conditions, HSPs have an important role in stabilizing and maintaining the conformational structure of a protein (20,25,27). Transcription of genes encoding HSPs may be activated by various stimuli, including physical (temperature and radiation), chemical (toxic compounds), and biological factors (cytokines, oxygen-free radicals, and infections) (28). Under cellular stress, HSPs bind to proteins with abnormal structure, thereby preventing the formation of aggregates and allowing the refolding of denatured proteins (25). Additionally, HSPs are indirectly involved in silencing or decreasing the effects of stress factors (20). Among the HSP family, HSP27 and HSP70 are reported to be involved in neoplastic processes, with expression of HSP27 and HSP70 increased in various cancer cell lines (29). These two chaperone proteins inhibit programed cell death, thus supporting tumor development and promoting CT resistance. HSP70 and HSP27 have dual effects on cancer cells; they suppress anticancer mechanisms and also promote the expression of genes responsible for metastases. On the contrary, HSP70 and HSP27 can activate immune pathways that target cancer cells (30,31). HSP70 has an important role in the maintenance of cellular homeostasis. Overexpression of HSP70, and HSP72 in particular, may occur in different types of cancers, Alzheimer disease and various kidney diseases (28,32). HSP27 is member of the small HSP subfamily, associated with a variety of signaling pathways that are critical for cellular functions (33). Among the other roles, small HSPs are involved in the antioxidant defense system within cells (34). HSPs accomplish this via two mechanisms: Indirectly, in which HSPs increase the cellular glutathione level; and directly way, in which HSPs neutralize protein oxidation. Increased expression of HSP27 contributes to resistance to CT and is associated with poor prognosis (35,36). Therefore, HSPs have a potential role in the treatment efficacy among different types of cancers (23,37,38).

ECT is effective in various cutaneous cancer types, including in melanoma treatment, and in cancer of internal organs, such as colorectal metastases (39). Therefore, in the present study, cell lines from melanoma and ovarian metastases of colonic carcinoma were used as a model to investigate the ECT approach in vitro. The aim of this study was to examine the efficacy of BTA as a novel natural-origin compound that can be used for ECT. Cisplatin was also used with EP as the ‘gold standard’ cytostatic drug. Whether HSPs can be used as biomarkers of the therapeutic effects in cancer cells in the response to stress induced by ECT was also investigated.

Materials and methods

Cell culture

Two metastatic human cancer cell lines were used, SW626 and Me45. SW626 cells (American Type Culture Collection, Manassas, VA, USA) are derived from an ovarian metastasis of colon adenocarcinoma. Me45 cells are a metastatic human pigmented malignant melanoma cell line was a kind gift from Professor Z. Krawczyk, established in the Department of Experimental and Clinical Radiobiology, Center of Oncology (Gliwice, Poland) (40). Both cell lines were cultured in polystyrene flasks as a monolayer in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and 50 µg/ml streptomycin (Sigma-Aldrich; Merck KGaA) at 37°C in a humidified atmosphere with 5% CO2. Cells were harvested by trypsinization (0.25% trypsin and 0.02% EDTA).

MTT cell viability assay

MTT assay (Sigma-Aldrich; Merck KGaA) was performed to determine the cell viability. Briefly, 1×104 cells/well were seeded into 96-well plates and cultured overnight. The cells were incubated with selected concentrations of drugs (0–50 µM) and with or without EP. MTT assay was performed at selected time points after the incubation (24, 48, and 72 h) and according to the manufacturer's protocol. Results were determined using a multiwell scanning spectrophotometer at 570 nm (EnSpire Multimode Plate Reader; PerkinElmer, Inc., Waltham, MA, USA). Cell viability is expressed as normalized percentage of treated cells compared to untreated control cells.

Chemotherapeutic compounds

In this study, two different cytostatic agents were selected. BTA was purchased from Sigma-Aldrich (Merck KGaA), as a naturally derived compound with a potential use in CT, and CP (Sigma-Aldrich; Merck KGaA), as a standard cytostatic drug. Briefly, the cells were incubated with drugs for 24, 48 and 72 h at concentrations ranging from 1–50 µM. For further studies, concentrations were selected according to the results obtained from the MTT assay.

EP protocol

EP of cells was performed using an ECM 830 device (BTX; Harvard Apparatus, Holliston, MA, USA). To test the viability of cells following EP, cells were suspended in EP buffer (pH 7.4; 10 mM phosphate buffer, 1 mM MgCl2 and 250 mM sucrose) with a low electrical conductivity and placed in cuvettes with parallel electrodes (gap of 4 mm). The experiment was performed according to the following selected parameters: A series of eight electric pulses of 800–2,000 V/cm, 100 µsec long with 1 sec intervals. Conditions were selected based on previous studies (41,42). After pulsing, the cells were incubated for 10 min at 37°C to enable the resealing of the cell membrane. Subsequently, the cells were tested for viability using the MTT test or by performing immunocytochemical (ICC) analysis. For morphological studies, the cells were seeded on slides 1 day before or were suspended in EP buffer immediately before EP. The adhered cells were electroporated using the Petri Pulser™ (BTX; Harvard Apparatus) consisting of 13 gold-plated electrodes spaced 2 mm apart. Microscopic images were collected after a specified time: Immediately (15 sec) or 10 min after EP, using a camera connected to an inverted microscope (Olympus CX41; Olympus Corporation, Tokyo, Japan).

ECT in vitro

The effect of ECT with CP and BTA in comparison to untreated control cells was analyzed in SW626 and Me45 cancer cell lines. Safe EP parameters and non-cytotoxic concentrations of drugs were selected (50 µM CP; 20 µM BTA for Me45 and 1 µM BTA for SW626). The cells were prepared for the experiment as described in the EP protocol and were subjected to electric pulses, following suspension in cuvettes containing CP or BTA in EP buffer. After 10 min recovery time at 37°C, cells were resuspended in culture medium and subjected to the same procedures described in EP protocol.

ICC avidin-biotin complex (ABC) analysis

After CT, EP or ECT cells were plated (2×103) into 10-well microscopic slides (Thermo Fisher Scientific, Inc.). After 24, 48 or 72 h, the cells were fixed with 4% formalin for 10 min at room temperature. Blocking was performed using PBS with 5% fetal bovine serum for 1 h at room temperature and incubated overnight at 4°C with rabbit monoclonal antibodies against HSP27 (G3.1; cat. no. sc-59562) or HSP70 (3A3; cat. no. sc-32239; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) using 1:200 dilution in antibody diluent (EMD Millipore, Billerica, MA, USA). After 24 h, the slides were incubated with biotinylated secondary anti-rabbit antibody (DAKO LSAB 2 kit; cat. no. K0675; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) for 30 min at room temperature. The ICC assay was completed using the peroxidase ABC method according to the manufacturer's protocol (DAKO LSAB 2 kit). Briefly, slides were incubated with horseradish peroxidase-conjugated streptavidin for 10 min, followed by 5 min incubation with diaminobenzidine at room temperature. All slides were counterstained with Mayer's hematoxylin for 1 min at room temperature (Sigma-Aldrich; Merck KGaA). Blinded samples were analyzed using an upright microscope equipped with a 40 × objective (Olympus BX51; Olympus Corporation). The expression was determined semi-quantitatively by counting the percentage of positively stained cells in randomly selected fields (from a total of 100 cells per sample, with a minimum four fields analyzed). The intensity of staining was evaluated as follows: (−) negative, (+) weak, (++) moderate, and (+++) strong.

Statistical analysis

For the ECT experiments, as there were two factors affecting the proliferation of cells (drug and electric pulses), two-way analysis of variance and Tukey's multiple comparison test in Prism software (v.7.0; GraphPad Software, Inc., La Jolla, CA, USA) was applied to verify the statistical difference between experimental groups. Data are expressed as the mean ± standard deviation (n=3). P<0.05 was considered to indicate a statistically significant difference.

Results

Cytotoxicity analysis

In Me45 cells, the reduction in cell survival following treatment with CP and BTA for 24 and 48 h was ≤30% less cells compared with the control group (Fig. 1A and B). Some values were comparable or higher than the control cells. BTA had the most potent negative impact on Me45 cells at concentrations >10 µM for 72 h (Fig. 1A); at this concentration, BTA caused ~70% decrease in the cell viability. The cytotoxic activity of CP (Fig. 1B) was minimal at lower concentrations (≤10 µM), and a higher decrease (~35%) was detected after the longest incubation duration (72 h).

Figure 1. (A and B) Me45 and (C and D) SW626 cell survival rates after 24, 48 and 72 h of incubation with BTA or CP. Data are presented as the mean ± standard deviation (n≥3). Statistical significance in comparison with the control groups (0 µM) was determined using two-way analysis of variance with Dunnett's multiple comparisons test method. *P≤0.05; **P≤0.001; ***P≤0.0001; ****P≤0.00001 vs. 0 µM at each time point. BTA, betulinic acid; CP, cisplatin.

In the SW626 cell line, the low cell viability was obtained after 24 h incubation with BTA (Fig. 1C). Compared with the control cells, the survival rate was ~15 and ~20% following treatment with BTA at 20 and 50 µM, respectively, for 24 h. After incubation for 48 and 72 h, there was an increase viability of cells compared with 24 h. This indicates that not all cells were affected by BTA and were still able to proliferate. For cells incubated for 72 h with BTA, the survival rate was ~50% regardless of the concentration used. SW626 cells exhibited limited sensitivity to CP (Fig. 1D) at all tested concentrations and all time points (≤30% reduction in viability).

Effect of EP on cell morphology

The effect of the electrical pulse on SW626 and Me45 cancer cells was observed and recorded using a standard microscope equipped with a camera (Fig. 2). The pores were formed in the cancer cell membrane by stimulation with the high electric field (1,200 V/cm). Cytoplasmic outflow was also observed as ‘bubbles’ at 10 min after EP (Fig. 2C and D). In addition, to induce and observe visible changes in cell morphology of the cells adhered to the plate after EP, the intensity of the electric field was increased to 3,000 V/cm (Fig. 2E and F).

Figure 2. Impact of electroporation on the cell morphology. (A) SW626 cells in suspension 15 sec after electroporation with 1,200 V/cm electric intensity; (B) Me45 cell in suspension 15 sec after electroporation with 1,200 V/cm electric intensity; (C) SW626 cells in suspension ~10 min after electroporation with 1,200 V/cm electric intensity; (D) Me45 cells in suspension ~10 min after electroporation with 800 V/cm electric intensity; (E) SW626 cells adhered to the substrate after electroporation with 3,000 V/cm electric intensity; (F) Me45 cells adhered to the substrate after electroporation with 2,600 V/cm electric intensity. White arrows indicate characteristic ‘bubbles’ and cytoplasm outflow.

ECT

Based on the results of the cytotoxicity and the EP analysis, the effective concentrations of drugs and parameters of EP were selected. Both cell lines were electroporated using the standard parameters (8 pulses, 100 µs pulse duration, 1 Hz frequency). Two values of electric field strength (800 and 1,200 V/cm) with limited toxic effect were selected for subsequent experiments (Fig. 3). The selection was also based on our previous studies (43,44) and according to standard ECT procedures where the European Standard Operating Procedures of Electrochemotherapy protocol is used. However, the results indicate that selected electric field values had relatively low lethality. Only in melanoma cells was observed a 20% decrease of cell viability observed after electro-pulsation without any drug. This demonstrated that the selected strengths of the electric field were sufficient for cell permeabilization, enabling the chemotherapeutic agent to enter the cells. Electric field strength >800 V/cm caused a significant decrease in cell viability in both cell lines.

Figure 3. Dependence of SW626 and Me45 cell survival rate on electric field intensity compared with the untreated cells (0 V/cm). Data are presented as the mean ± standard deviation (n≥3); *P≤0.05; ****P≤0.00001 vs. 0 V/cm as assessed by two-way analysis of variance with Dunnett's multiple comparisons test.

In Me45 cells, CP at 50 µM and BTA at 20 µM, and three different incubation times (24, 48 and 72 h) were used (Fig. 4A-C). EP at 800 and 1,200 V/cm intensity caused an increase in cell death induced by BTA at 24 and 48 h (Fig. 4A and B), compared with BTA alone. The presence of CP in the EP buffer caused a greater reduction in the cell viability than that of BTA in Me45 cells. As the intensity of the electrical pulses increased, there the drug-induced cytotoxicity was increased the Me45 cells. ECT with BTA had a most significant effect than EP or CT alone.

Figure 4. ECT with CP and BTA expressed as a percentage of control cell survival rate in melanoma cells (Me45) after (A) 24 h, (B) 48 h, (C) 72 h, and in ovarian metastatic cells (SW626) after (D) 24 h, (E) 48 h, (F) 72 h of incubation after ECT. All data are presented as mean ± standard deviation (n≥6). **P≤0.001; ***P≤0.0001 vs. 0 V/cm. ECT, electrochemotherapy; CP, cisplatin; BTA, betulinic acid.

In SW626 cells, two sublethal concentrations of cytostatic drugs (50 µM CP and 1 µM BTA) and three different incubation times (24, 48 and 72 h) were used (Fig. 4D-F). According to the results, ECT slightly improved the efficiency of the applied drug. The viability was decreased by 30% below the level obtained for CP alone at the same concentration. The 1,200 V/cm electric field caused a decrease in the cell viability compared with drug treatment alone after 24 h (Fig. 4D). No significant increase in the cytotoxic effect of was observed after 48 h. A slight decrease of ~20% was induced by ECT compared with CP and BTA alone after 72 h incubation (Fig. 4E and F).

ICC for HSPs

Various studies have indicated that HSPs have an important role in cancer progression (14). ICC analysis was used to detect expression of the chosen HSPs in the selected cancer cell lines after in vitro CT and ECT. The experiments were conducted with the same parameters as in the previous experiments and were observed using a standard upright microscope. HSPs were visualized with various intensities depending on the cell line. Tables I and II, and Fig. 5 present semi-quantitative values related to the intensities HSP27 and HSP70, and the number of stained cells. For SW626 cells, the highest number of positive cells and the most intense HSP staining (both HSP27 and HSP70) was observed after 72 h with ECT (Fig. 5B) in particular for CP treatment. In the case of SW626 cells whose appearances exhibited blebbing and affected morphology, HSPs were located inside the nucleus to a considerable extent. The intracellular localization of HSPs was distributed among the cytoplasm and nuclear envelope. The location of both HSPs suggests induction of apoptosis, for example by triggering endogenic apoptosis with the mitochondrial contribution. The relatively high intensity of HSPs in numerous SW626 cells was also observed after the exposure of the cells to ECT without BTA or CP (Fig. 5D). In SW626 cells, an intense positive immunoreaction of both HSPs was observed at 24 h post-ECT with BTA and even more so for CP, which suggests that shock caused by the electric field combined with drug administration had a substantial effect on the expression of HSPs. After 48 h incubation of ECT treated cells, the amount and intensity of both HSPs considerably decreased, whereas after 72 h incubation, they were found to be increased again. Melanoma cells also indicated an increased immunostaining in particular after exposition to the strongest electroporation parameters and 72 h (for EP-BTA and EP-CP), however the reaction was not as strong as in case of SW626 cells. This may result from an individual HSPs level for each cell line.

Figure 5. Evaluation of immunocytochemistry reaction with HSP27 antibody in (A) Me45 cells and (B) SW626 cancer cells, after electrochemotherapy with CP (50 µM) or BTA (50 µM) combined with pulsed electric field of 800 V/cm, eight pulses of 100 µs, after 24, 48 and 72 h. Immunoreaction of HSP27 and HSP70 of representative control (C) Me45 cells and (D) SW626 metastatic cells after 48 h. Selected controls represent immunoreaction after exposition to BTA (20 µM), CP (50 µM) or electroporation alone. CP, cisplatin; BTA, betulinic acid.

Table I. Evaluation of immunocytochemical reaction with HSP27 antibody in SW626 and Me45 cancer cell lines following ECT with CP or BTA.

Table I.

Evaluation of immunocytochemical reaction with HSP27 antibody in SW626 and Me45 cancer cell lines following ECT with CP or BTA.

A, SW626
		24 h		48 h		72 h
Drug	EP (V/cm)	I	%	I	%	I	%
Control cells	0	++	99
CP (50 µM)	0	++	94	+	35	++	97
	800	++	92	+	50	+++	100
	1,200	++	100	+	75	+++	100
BTA (1 µM)	0	+	85	+	40	++	98
	800	++	90	+	80	+++	100
	1,200	++	100	+	83	+++	100
B, Me45
		24 h		48 h		72 h
Drug	EP (V/cm)	I	%	I	%	I	%
Control cells	0	−	0	−	0	−	0
CP (50 µM)	0	−	0	−	0	−	0
	800	+++	100	+	100	++	100
	1,200	+++	100	+	100	++	100
BTA (20 µM)	0	−	0	−	0	−	0
	800	+++	100	+	100	++	100
	1,200	+++	100	+	100	++	100

[i] The expression of HSP27 was determined semi-quantitatively by counting percentage of positively stained cells in randomly selected fields (from a total of 100 cells per sample). I was evaluated as: (−) negative, (+) weak, (++) moderate and (+++) strong; % indicates the percent of positively stained cells. HSP27, heat shock protein 27; ECT, electrochemotherapy; EP, electroporation; BTA, betulinic acid; CP, cisplatin; I, the intensity of reaction.

Table II. Evaluation of immunocytochemical reaction with HSP70 antibody in SW626 and Me45 cancer cell lines following ECT with CP or BTA.

Table II.

Evaluation of immunocytochemical reaction with HSP70 antibody in SW626 and Me45 cancer cell lines following ECT with CP or BTA.

A, SW626
		24 h		48 h		72 h
Drug	EP (V/cm)	I	%	I	%	I	%
Control cells	0	++	100	++	100	++	100
CP (50 µM)	0	++	98	+	55	++	98
	800	++	100	+	78	+++	100
	1,200	++	100	+	81	++	100
BTA (1 µM)	0	+++	100	+/++	56	+/++	100
	800	++	100	++	80	++/+++	100
	1,200	++	100	++	87	++/+++	98
B, Me45
		24 h		48 h		72 h
Drug	EP (V/cm)	I	%	I	%	I	%
Control cells	0	−	0	−	0	−	0
CP (50 µM)	0	−	0	−	0	−	0
	800	−	0	−	0	−	0
	1,200	++	97	++	99	++	100
BTA (20 µM)	0	−	0	−	0	−	0
	800	+ (pattern distribution)	9	+	98	++	100
	1,200	+	92	+	89	+	100

[i] The expression of HSP70 was determined semi-quantitatively by counting percentage of positively stained cells in randomly selected fields (from a total of 100 cells per sample). I was evaluated as: (−) negative, (+) weak, (++) moderate and (+++) strong; % indicates the percent of positively stained cells. HSP70, heat shock protein 70; ECT, electrochemotherapy; EP, electroporation; BTA, betulinic acid; CP, cisplatin; I, the intensity of reaction.

In Me45 cells, the positive immunoreaction was obtained for HSP27 only (Fig. 5C). The color of the reaction was only visible in cells after ECT (Fig. 5A). In the control and samples untreated with EP, there was no positive staining of cells. In addition, after exposure of cells to 1,200 V/cm intensity, the cells had shrunk, the cell membrane appeared to lose its continuity and there was an even distribution of HSP27 in the cytoplasm. The intensity of reactions in Me45 cells varied depending on the length of incubation time post-ECT. The most intense HSP27 ICC staining was obtained after 24 h incubation with CP and BTA. This indicates a strong protective response in cells caused by environmental stress. After 48 h of incubation, the staining reaction decreased, and then increased again after 72 h with BTA and CP.

Discussion

The results in Me45 cells confirm the utility of EP technique. It significantly enhanced the cytotoxic effect of CP and to some extent enhanced the effect of BTA. SW626 cells were less susceptible to EP, thus we suppose this cell line may be EP-resistant. ECT-CP exhibited significant and ECT-BTA exhibited a less significant effect at the longest incubation time (72 h) in Me45 melanoma cells. In the case of the SW626 metastatic cell line the anticancer effect of ECT was not predominant. Thus, the data confirm that the use of EP is dependent on the tumor type (18).

CP has been the most effective drug used in the treatment of cancer in the past decades (41,42). Despite this, there are many types of cancers that are resistant to CP treatment, and this phenomena is not only dependent on drug biodistribution in the cell, but also involves many complex resistance mechanisms (43,44). This was also confirmed by the results of the present study. Both selected cell lines had low sensitivity to treatment with CP alone, and the viability was maintained in the control level after 24 h. A significant decrease of cell viability was exhibited after 48 and 72 h (~30%) or the highest concentrations. The mechanism of resistance to platinum compounds is achieved by reduced formation of cytotoxic platinum-DNA adducts, decreased drug accumulation, and increased inactivation of the drug by cellular proteins and non-protein thiol groups (41). Numerous studies reported that the use of EP in the treatment of cancer may counterbalance drug resistance phenomena (16,43,45). Previous studies have demonstrated that after ECT with CP, the viability of CP-resistant cells (OvBH-1 and SKOV-3) was decreased significantly compared with CP used alone (46). A recent study has also indicated the advantages of EP in the treatment of neuroblastoma cells, indicating that CP cytotoxicity was potentiated after exposure of cells to high intensity electric pulses (47). However, certain cell lines remain resistant to CT after EP treatment (48). In the present study, CP alone affected the viability of SW626 cells to a certain extent, and the use of EP significantly supported this effect. Regardless of the use of EP, CP caused a decrease in cell viability by up to 20%, even after 72 h incubation.

BTA has been reported to decrease the growth and survival rate of several types of cancer (49,50). The effect is associated with the ability of BTA to induce programed cell death in tumor cells by triggering the mitochondrial apoptotic pathway and inhibition of multiple pro-oncogenic factors (13,51,52). The present study is the first to use BTA in ECT, which may overcome difficulty in BTA penetration through cells membranes. The effect of BTA in ECT was evaluated in two cell lines. In Me45 cells, the application of electrical pulses significantly increased the cytotoxic effect of BTA. Experiments on SW626 cells also confirmed the anticancer properties of BTA at low concentrations using EP, and significantly reduced cell survival, but with less effect than in melanoma cells. A closer examination on the effect of EP on the compound itself may be crucial. The data confirms the differences in ECT sensitivities between the two cell lines (53). One of the strategies to increase hydrosolubility and improve the anticancer properties of BTA is to use derivatives or analogs of BTA (e.g. with a triazole group added) (54). Another method to increase the toxic effect on cancer cells may be combination therapy. In recent studies, it was proposed that combining BTA with different active compounds, such as gemcitabine (55) or sorafenib (56), may increases the anticancer effects. In certain of these cases, the application of EP with CP may reduce the dose of drugs, which may minimize side effects.

HSPs expression in cells subjected to ECT does not clearly indicate whether a tumor cells will enter the apoptotic pathway or protect themselves. Despite this, HSP27 was detected in both cell lines following ECT. Upregulation of HSP27 has been reported in multiple types of malignancy, including ovarian carcinoma and melanoma. Along HSPs have been implicated in oncogenesis and CT resistance (36). The presence of HSP27 indicates activation of anti-apoptotic defense mechanisms, whereas the lack of HSP70 suggests the opposite. The current results indicate that EP enhanced HSP27 in both cell lines at all time point, but HSP70 only in SW626 cells. Other researchers have also demonstrated that EP induces the expression of HSP70 to a certain extent, as a result of environmental stress (57). However, another study demonstrated HSP70 induction may depend on the cell line (58). The data of the present study indicate that chemotherapeutic protocols may modulate expression of HSP27 and HSP70 in tumor tissues. Vargas-Roig et al (59) observed that after chemotherapy, nuclear HSP27 and HSP70 expression was increased, and HSP70 and heat shock cognate 70 cytoplasmic expression decreased in patients with breast cancer (59). Arts et al (60) reported that HSP27 expression was negative before and positive after chemotherapy in only 2/30 paired samples, whereas hsp27 expression was positive before and negative after chemotherapy in 5/30 samples. In general, elevated levels of HSPs are associated with drug resistance and poor prognosis (61). Therefore, the presence of these two proteins (HSP27 and HSP70) in untreated SW626 cells indicates higher resistance to the applied treatment. Untreated Me45 cells did not express HSPs; thus, they were more sensitive to ECT. This indicates stronger intracellular defense mechanisms of ovarian cancer cells.

Additionally, different cell lines may exhibit variation in their tolerance to electric fields. The effect of electric pulses depends on the size, density and shape of the cell (62). A recent study also reported the differences between cell lines in the kinetics of membrane resealing; this process determines how fast the electropores in membranes are closed following exposure to electric pulses (63). It has been reported that pores in the membrane of various tumor malignant cell lines reseal much faster (up to 300%) than in normal cell lines. Furthermore, a strong correlation between the resealing response of cancer cells and their resistance to standard drugs, such as CP, was reported. These properties may enhance or limit the efficiency of EP in cancer cells. Thus, further studies are required to assess the efficiency of this treatment modality.

In summary, the present findings indicate that ECT protocols are highly variable depending on the type of cancer cells. Ovarian metastatic SW626 cells were marginally more sensitive to standard therapy with CP then Me45 melanoma cells. Additionally, BTA, a natural compound, exhibited potent cytotoxic effects in SW626 cells. The application of EP enhanced the effects of BTA in Me45 melanoma cells, and may applied instead of CP. The next stages of research should focus on further characterization of the action of BTA on tumor cells. Furthermore, as therapies with natural compounds appears to be safe and cause less side effects than standard cytostatics, further research will aim to expand the pool of test compounds with anticancer properties that can be enhanced by EP.

Acknowledgements

Not applicable.

Funding

The present study was supported by the Wroclaw Medical University Statutory Funds ST.E130.16.060 (PI: M. Zalewski).

Availability of data and materials

The datasets used in this study are available from the corresponding author upon reasonable request.

Authors' contributions

JS, MK, JZ, MZ, JM and JK participated in the design of the study, data interpretation, and manuscript drafting. JM, JKut, and ACh performed the experiments. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

References