The Efficacy of Electrochemotherapy with Dacarbazine on Melanoma Cells

Abstract

Electrochemotherapy (ECT) involves locally applying electrical pulses to permeabilize cell membranes, using electroporation (EP). This process enhances the uptake of low-permeant chemotherapeutic agents, consequently amplifying their cytotoxic effects. In melanoma treatment, dacarbazine (DTIC) is a cornerstone, but it faces limitations because of poor cell membrane penetration, necessitating the use of high doses, which, in turn, leads to increased side effects. In our study, we investigated the effects of DTIC and EP, both individually and in combination, on the melanoma cell line (SK-MEL-30) as well as human dermal fibroblasts (HDF) using in vitro assays. First, the effects of different DTIC concentrations on the viability of SK-MEL-30 and HDF cells were determined, revealing that DTIC was more effective against melanoma cells at lower concentrations, whereas its cytotoxicity at 1000 μM was similar in both cell types. Next, an ideal electric field strength of 1500 V/cm achieved a balance between permeability (84%) and melanoma cell viability (79%), paving the way for effective ECT. The combined DTIC-EP (ECT) application reduced IC₅₀ values by 2.2-fold in SK-MEL-30 cells and 2.7-fold in HDF cells compared with DTIC alone. In conclusion, ECT not only increased DTIC’s cytotoxicity against melanoma cells but also affected healthy fibroblasts. These findings emphasize the need for cautious, targeted ECT management in melanoma therapy.

Introduction

Melanoma, responsible for over 80% of skin cancer-related deaths, is the most aggressive and poorly prognosed form of skin cancer.¹ The incidence of melanoma has risen in recent years, primarily because of increased exposure to ultraviolet radiation and sunlight, posing a serious threat to human health and life.² Surgical excision is the gold standard of therapy for malignant melanoma, especially nonmetastatic or early-stage cases.³ However, when melanoma reaches an advanced metastatic stage, surgery alone is insufficient, necessitating the use of chemotherapeutic agents.⁴ Unfortunately, patients with advanced melanoma exhibit a poor prognosis with a median survival time of only 6–9 months,⁵ as it is highly resistant to conventional chemotherapy.⁶

Dacarbazine (DTIC), an alkylating agent, is currently used as a first-line chemotherapeutic agent for treating metastatic melanoma.⁷ Alkylating agents were the first antineoplastic drugs used in cancer therapy and remain one of the most commonly used anticancer compounds.⁸ However, melanoma and other solid cancers like glioblastoma often exhibit resistance to DNA alkylating agent-based chemotherapies, including DTIC and temozolomide (TMZ).⁹ This resistance, associated with increased expression of the DNA repair protein methylguanine-DNA methyltransferase, is frequently encountered in melanoma.¹⁰ However, clinical trials show that DTIC has moderate efficacy against tumors.¹¹ Also, its therapeutic efficiency is restricted by nonspecific toxicity to normal cells, short half-life, poor solubility in water, and the resistance mechanism observed in melanoma cells.¹² Despite these limitations, DTIC remains one of the main chemotherapeutics for metastatic melanoma,¹³ and research on combination therapy strategies is underway.

In recent years, electroporation (EP) or electropermeabilization has emerged as a potent technique to enhance cell membrane permeability using high-voltage and short-duration electrical pulses.¹⁴ This technique can be used either alone, known as irreversible EP, or in combination with anticancer agents, referred to as electrochemotherapy (ECT), and it has demonstrated effectiveness in treating various types of cancers.¹⁵ For instance, ECT can be applied to research on treatment for various cancers,¹⁶ such as colon cancer,¹⁷ prostate cancer,¹⁸ and, especially, melanoma.¹⁹ ECT comprises the concurrent application of a cytotoxic agent possessing limited tissue penetration and reversible EP. This technique offers a localized and nonthermal treatment modality.²⁰ However, it is crucial to adjust the EP parameters before the application of ECT as reversible EP attempts to permeabilize the cells while maintaining their viability. When applied at high extracellular drug concentrations, ECT enhances the uptake of poorly permeant chemotherapeutic drugs into cells, resulting in effective antitumor activity.²¹

In the realm of ECT for metastatic melanoma, the two most commonly used chemotherapeutic agents are bleomycin and cisplatin.²² Over the past decade, bleomycin-ECT has gained recognition as a treatment approach for locally advanced metastatic melanoma.^23,24 Furthermore, the toxicity of cisplatin can be enhanced when administered in conjunction with EP.²⁵ These antineoplastic drugs have been shown to be suitable for the efficient elimination of melanoma. By considerably increasing the efficacy of chemotherapeutic agents compared with standard chemotherapy, ECT presents an opportunity to reduce drug doses and their associated side effects. Despite these achievements, it is worth noting that DTIC remains the primary chemotherapeutic agent for metastatic melanoma. Surprisingly, there is a lack of research assessing the efficacy of DTIC-EP (ECT) on human dermal healthy fibroblast cells and human melanoma cell lines. Therefore, the primary objective of this study is to evaluate the cytotoxic effects of DTIC, both alone and in combination with EP, using an in vitro experimental model that mimics clinical scenarios in that it involves melanoma and dermal fibroblast cells.

Materials and Methods

Cell culture

Human melanoma cell lines (SK-MEL-30, SAP Institute, Ankara, Turkey) and human dermal fibroblast (HDF, PCS-201-010, Manassas, VA, USA) cells were cultured in Dulbecco’s modified Eagle medium. The culture medium was supplemented with 10% (v/v) fetal bovine serum (FBS), 1% l-glutamine, and 1% penicillin–streptomycin. Incubation was carried out at 37°C in a humidified atmosphere with 5% CO₂.

DTIC treatment

DTIC (D2390, Sigma) was prepared as a 10 mM stock solution in 0.9% NaCl and stored in aliquots at −20°C. These aliquots were further diluted in the cell culture medium. HDF and SK-MEL-30 cells were seeded at a density of 10⁵ cells/well in 96-well plates and incubated overnight. Subsequently, the cells were treated with varying concentrations of DTIC (0–1000 μM) for 24 h. Following incubation, the cytotoxicity was assessed based on a cell viability analysis using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Setting the critical electric field parameters for the effective electroporation protocol

This experimental step aims to optimize the EP settings before proceeding with ECT (DTIC-EP) treatments. To achieve this, we examined the impact of various electric field strengths (0–2000 V/cm) on the viability and permeability of melanoma and dermal fibroblast cells. We administered EP using the ECM 830 Square Wave Electroporation System (BTX, Harvard Apparatus) with eight square wave pulses (0–2000 V/cm, 100 μs, 1 Hz). Following the EP treatments, we assessed cytotoxic effects through the MTT assay, whereas permeability effects were evaluated using propidium iodide (PI, ABP Biosciences) dye in both qualitative and quantitative analyses.

Electroporation analysis using flow cytometry and fluorescence microscopy

PI is a small fluorescent compound that has an affinity for DNA, but it is unable to pass through cells possessing intact plasma membranes.²⁶ Hence, the membrane-impermeable fluorescent dye PI was used as an indicator molecule in flow cytometry and fluorescence microscopy to investigate the effects of different electric field intensities on membrane permeabilization. Flow cytometry analysis was conducted using a FACScan instrument (Becton-Dickinson) to quantify the percentage of PI-positive cells. A cell suspension (100 μL) containing 1 × 10⁶ cells/mL was loaded into EP cuvettes with 4 mm gaps. Subsequently, before applying the EP, 10 μL of PI was added to the culture medium in a final concentration of 40 μM. After the EP, the cells were left for incubation at room temperature for 10 min to allow for the PI uptake.²⁷ Control groups were also set up in the cuvettes under identical conditions, except that EP was not applied. Following these procedures, the cells were transferred onto glass coverslips, and their fluorescent images (PI emits red fluorescence, Ex/Em = 535/617 nm) were immediately captured using fluorescence microscopy (Leica DMI 4000B). Hence, using both flow cytometry and fluorescence microscopy analyses facilitated a comprehensive evaluation of the electric field’s impact on membrane permeability.

Electroporation with DTIC (ECT) protocol

Following the optimization of the EP application based on its effects on cell permeability and viability, we established the ECT protocol. Briefly, 100 L of cell suspensions at 1 × 10⁶ cells/mL (SK-MEL-30 and HDF) were loaded into cuvettes. Various concentrations of DTIC (0–1000 μM) were subsequently added to the cell suspensions in the EP cuvettes. Electric pulses consisting of eight square waves (1 Hz, 100 μs) were administered to SK-MEL-30 and HDF cells at a field strength of 1500 V/cm (a critical value determined through EP optimization). After the treatments, the cells were seeded in 96-well plates at a density of 10⁵ cells/well and allowed to incubate overnight. Subsequently, we assessed the efficacy of ECT by measuring cell viability using the MTT assay. As a control group, cells were exposed to DTIC alone except for voltage application.

Cell viability

The assessment of cytotoxic effects induced by DTIC, EP alone, and ECT on both cell lines was conducted using the MTT assay. Following the treatment, the cells were seeded at a density of 10⁵ cells/well in 96-well plates and incubated overnight in a humidified atmosphere (37°C, 5% CO₂). After incubation, 10 μL of MTT solution was added to each well, followed by a further 4 h incubation at 37°C. Subsequently, the culture medium was removed, and 100 μL of dimethyl sulfoxide was added to each well. The absorbance at 570 nm (with a reference wavelength of 690 nm) was then measured using a microplate reader (TECAN-Sunrise ELISA Reader).

Statistical analysis

All data were presented as the mean ± standard deviation (SD) based on four independent replicates (n = 4). The Student’s t-test was used to determine the statistical significance. The IC₅₀, which represents a 50% inhibition of cell growth, was calculated using a nonlinear regression curve fitting with GraphPad Prism 9.5.0 (GraphPad Inc.). Statistical significance was denoted by a p-value less than 0.05, with significance levels depicted as follows: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Results

Effects of DTIC on the viability of HDF and SK-MEL-30 cells

We evaluated the cytotoxicity of DTIC on HDF and SK-MEL-30 cells at various concentrations using the MTT assay after a 24 h incubation period (Fig. 1). HDF cell viability remained relatively stable at approximately 84% up to DTIC doses of 500 μM but declined to 48% at 1000 μM. Conversely, SK-MEL-30 cell viability was approximately 56% at DTIC doses up to 500 μM but dropped to 49% at 1000 μM. The IC₅₀ values for DTIC in HDF and SK-MEL-30 cells were determined as 1094 μM and 1095 μM, respectively, at the 24th h (Table 1).

FIG. 1.

In vitro cytotoxicity of dacarbazine after 24 h treatments on human dermal fibroblast (HDF) cells and human melanoma cell lines (SK-MEL-30). Cell viability assay was performed by MTT. The differences between HDF and SK-MEL-30 cells at the same dose were compared statistically. Values represent mean ± standard deviation (n = 4), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Table 1.

The IC₅₀ Values of Dacarbazine, Electroporation, and the Combination of DTIC with EP (ECT)

Treatments	SK-MEL-30 (IC50)	HDF cells (IC50)
DTIC	1094 μM	1095 μM
EP	1815 V/cm	2014 V/cm
DTIC + EP (ECT)	491.5 μM	401.3 μM

DTIC, dacarbazine; ECT, electrochemotherapy; EP, electroporation; HDF, human dermal fibroblast.

Determination of effective electric field strength

We attempted to determine the ideal electric field strength before testing the effects of ECT on both melanoma and healthy cells. This was obtained by applying various electric fields (0–2000 V/cm, 100 μs, and 1 Hz) to SK-MEL-30 and HDF cells. To measure permeabilization efficiency, we used the PI method, and the results were subsequently analyzed using flow cytometry (Fig. 2). As depicted in Figure 2, the increase in external electric field strength consistently correlated with enhanced cell permeabilization and an elevated uptake of PI. Furthermore, we evaluated the impact of EP on cell viability using the MTT assay (Fig. 2). For both cells, a clear trend emerged: an increase in electric field strength was consistently associated with enhanced permeability while concurrently resulting in reduced cell viability. For melanoma cells, an electric field strength of 1500 V/cm yielded a permeabilization rate of 84% and corresponding cell viability of 79% (Fig. 2a). Subsequently, to emulate potential clinical applications, we adopted 1500 V/cm from melanoma cells as a reference, with average values of 95% for permeability and 71% for viability observed in fibroblast cells at 1500 V/cm (Fig. 2b).

FIG. 2.

Effect of electroporation (EP) alone on viability and permeability of (a) human melanoma cells (SK-MEL-30) and (b) healthy human dermal fibroblast (HDF) cells at varying field strengths (0–2000 V/cm, 100 μs, ×8 pulses, 1 Hz). Viability and permeability were measured by MTT and flow cytometry (PI uptake), respectively. PI, propidium iodide.

Fluorescence microscopy was additionally used to visualize the electropermeabilization of SK-MEL-30 and HDF cells, focusing on the uptake of PI, as illustrated in Figure 3. The analysis of PI’s fluorescent intensity within the cells revealed a direct correlation between the electric field’s strength and the number of permeabilized cells (Fig. 3).

FIG. 3.

Fluorescence images of human melanoma cells (SK-MEL-30) and human dermal fibroblast (HDF) cells were obtained immediately after electroporation (0–1500 V/cm) using fluorescence microscopy (scale bar, 100 μM). As an indicator fluorescence dye, propidium iodide (PI) emits red fluorescence with an Ex/Em wavelength of 535/617 nm.

Application of DTIC with electroporation (ECT)

The application of DTIC combined with EP, a process known as ECT, was performed with the effective electric field strength obtained from Figure 2. For melanoma cells, we administered 1500 V/cm alongside DTIC, using varying DTIC concentrations ranging from 0 to 1000 μM (Fig. 4a). This analysis showed the effect of DTIC-EP on melanoma cell viability compared with DTIC alone. At lower doses, there was no significant difference in melanoma cell viability between ECT and DTIC alone. However, it emerged that ECT at 750 and 1000 μM was more effective as compared with the cells treated with only DTIC (Fig. 4a). A parallel procedure was replicated with human fibroblast cells, applying DTIC combined with 1500 V/cm electric field intensity across a range of concentrations (0–1000 μM) (Fig. 4b). In fibroblast cells, ECT led to a significant reduction in cell viability in comparison with DTIC alone, particularly at concentrations ranging from 200 to 1000 μM. Notably, IC₅₀ values for ECT were determined as 491.5 μM for melanoma cells and 401.3 μM for healthy skin fibroblast cells (Table 1).

FIG. 4.

Comparing dacarbazine electroporation (DTIC-EP, electrochemotherapy [ECT]) and dacarbazine (DTIC) alone for in vitro cytotoxicity on (a) human melanoma cell lines (SK-ML-30) and (b) human dermal fibroblast (HDF) cells. Cell viability was performed by MTT assay 24 h after the treatment. The differences between DTIC and DTIC + EP at the same dose were compared statistically. Values represent mean ± standard deviation (n = 4), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Discussion

DTIC stands as the sole Food and Drug Administration–approved first-line chemotherapeutic agent for metastatic melanoma,²⁸ but its clinical utility faces significant constraints because of issues like poor solubility, instability, and severe toxicity to normal cells, particularly in melanoma therapy.²⁹ The metabolic activity of DTIC can be observed within a relatively short time frame. Breithaupt et al. found that the plasma disappearance of DTIC was biphasic, with a terminal half-life of roughly 41.4 min. In addition, the plasma decay of the main metabolite, 5-aminoimidazole-4-carboxamide (AIC), had a half-life ranging from 43 to 116 min.³⁰ In patients with cancer, DTIC exhibited a mean half-life of 2 h, whereas its metabolite AIC, detectable in plasma upon DTIC infusion, displayed a mean half-life of 3.25 h. The amount of AIC excreted in urine increased with increasing DTIC dose, indicating rapid metabolic activity.³¹ In evaluating the effect of DTIC alone on melanoma cell viability, we observed a reduction of 20% at 50 μM and 50% at dosages exceeding 500 μM after 24 h, as depicted in Figure 1. A parallel observation by Mantso et al. demonstrated that DTIC (30 μM) exhibited the same pattern of effects on melanoma cells over 24, 48, and 72 h at 37°C, displaying similar levels of cytotoxicity: approximately 80% for 24 h and 48 h and around 65% for 72 h.³² In another study, DTIC (1800 and 2000 μg/mL) showed similar cytotoxicity after 24 and 48 h in B16F10 melanoma cells.³³ Our results are consistent with previous studies of the results of MTT analysis.

We investigated the impact of various electric field strengths (0–2000 V/cm) on the viability and permeability of melanoma and dermal fibroblast cells using the EP system. The results showed that an increase in electric field strength was consistently associated with enhanced permeability while resulting in reduced cell viability for both cell types (Fig. 2). We also used fluorescence microscopy to visualize the electropermeabilization of the cells, focusing on the uptake of PI (Fig. 3). The effectiveness of EP depends on achieving a refined balance between cellular permeability and viability.³⁴ Therefore, we sought to identify the optimal electric field strength before assessing the effects of ECT (EP with DTIC) on both malignant and healthy cells to approximate clinical scenarios. The optimal electric field was found to be 1500 V/cm, which allowed for permeability (84%) without causing excessive cell death (viability: 79%) for melanoma cell lines (Fig. 2a). After determining a reference value of 1500 V/cm for melanoma cells, its corresponding fibroblast cell viability (71%) and permeability (95%) were also examined (Fig. 2b). Alkis et al. observed that T98G and U118G glioblastoma cell lines exhibited 80% cell viability and favorable permeability at 1000 V/cm and 1250 V/cm, respectively.³⁵ Similarly, human colorectal cancer cells (Caco-2) demonstrated optimal response to EP at 1250 V/cm, yielding permeability and cell viability values of 45% and 20%, respectively.³⁴

The size and shape of cells have a significant impact on their response to electric fields. The larger the cells, the more sensitive to electric pulses both in terms of permeability and, as a direct consequence, loss in viability.³⁶ A study showed that fibroblast spheroids with smaller sizes were less sensitive to calcium EP compared with cancer cell spheroids.³⁷ However, based on the research conducted by Gibot and Rols, it was found that small human HCT-116 colorectal spheroids are more sensitive to electric field pulses than larger ones.³⁸ Furthermore, normal cells have been shown to exhibit more effective membrane repair compared with cancer cell lines, resulting in higher viability after the plasma membrane EP.³⁹ Consistent with this situation, our experiment found that a 2000 V/cm electric field reduces fibroblast cell viability to 40% and melanoma cell viability to 20%. However, Skolucka et al. showed that human fibroblasts are more sensitive to electric fields than melanoma cells.⁴⁰ These findings underscore the variability in permeability and viability values across different cell types. Following the determination of the 1500 V/cm value and observation of its effects, the ECT method was established.

In attempts to enhance DTIC efficacy, DTIC is often combined with various therapeutic agents, including antimitotic compounds and DNA alkylating agents.⁴¹ Nevertheless, the emergence of resistance mechanisms, particularly against DNA alkylating agents such as DTIC and TMZ, poses a substantial challenge in the therapeutic management of melanoma.⁴² For instance, Zheng et al. reported that continuous, low-dose, or long-term treatment of alkylating agents to cancer cells supported the resistance mechanism.⁴³ Owing to this, a critical factor in enhancing the effectiveness of the therapy may be the burst release of alkylating chemicals to the targeted tumor site without developing resistance mechanisms.⁹ In other words, the design of the therapy that will ensure the sufficient therapeutic concentration of the DTIC at the tumor site may be critical in melanoma therapy. One of the best examples of this scenario is ECT, which is a promising method of enhancing the cytotoxic effects of chemotherapeutics via increased cell membrane permeability with reversible EP.²⁰ It is crucial to recognize that DTIC remains the primary chemotherapeutic agent used for the management of metastatic melanoma. Remarkably, there is an absence of research on the effectiveness of DTIC-EP (ECT) on human melanoma cell lines and HDF cells. Therefore, the main objective of this study is to evaluate the effects of DTIC on melanoma and fibroblast cells, both alone and in combination with EP, using an in vitro experimental model.

It is worth noting that in our study, we observed slightly higher viability of SK-MEL-30 cells treated with ECT (DTIC-EP) compared with those treated with DTIC alone (50–300 μM), but this difference was not statistically significant (Fig. 4a). The reason for this could be attributed to the unique characteristics of ECT and its interaction with the EP process. Nonetheless, we found that ECT at 750 and 1000 μM was more effective than cells treated with only DTIC (Fig. 4a). EP proved an effective delivery method to increase the intracellular concentration of several chemotherapeutic drugs, such as bleomycin, cisplatin, melphalan, methotrexate, and doxorubicin.⁴⁴ Among these, only bleomycin and cisplatin were identified as potential candidates for ECT of patients with cancer. When ECT is combined with bleomycin, the cytotoxicity of bleomycin can be significantly increased, with enhancements ranging from 300- to 700-fold.²⁴ Similarly, when ECT is applied in combination with cisplatin, the toxicity of cisplatin is increased by a factor of 2.3–8.²⁵ These drugs have been found to be effective in combination with ECT because of their synergistic effects with the electric field. However, the efficacy of ECT in combination with different drugs can vary depending on the specific drug and cancer type. Not all drugs will necessarily show increased toxicity when combined with ECT. For instance, carboplatin and vincristine showed varying degrees of in vitro potentiation, whereas drugs like oxaliplatin, paclitaxel, and gemcitabine did not show in vitro potentiation.⁴⁴ The complexity of the response to EP in combination with specific drugs becomes evident from these findings, underscoring the dependence on the characteristics of each drug and its interactions with the EP process.

In our study, the combined application of DTIC-EP (ECT) resulted in a 2.2-fold decrease in IC₅₀ values in SK-MEL-30 cells and a 2.7-fold decrease in HDF cells, in comparison with the use of DTIC alone (Table 1). This result could be related to the difference in EP susceptibility exhibited by cancer and normal cells.⁴⁵ Furthermore, Kim et al. investigated the influence of physicochemical factors on the EP efficiency of lung cancer cells and normal lung cells. Their findings revealed that the EP efficiency, as indicated by PI dye uptake, was lower in cancer cells compared with normal cells, and this difference was attributed to physicochemical factors such as membrane stiffness, transmembrane potential, and cell size.⁴⁶ These insights emphasize that EP is affected by many biophysical factors, highlighting the need for tailored approaches in ECT.

Conclusions

In summary, melanoma remains a highly challenging and deadly form of skin cancer, necessitating improved therapeutic approaches. Our research highlights the potential of ECT to amplify the cytotoxic effects of DTIC, an alkylating chemotherapeutic agent commonly used in melanoma treatment. EP-mediated delivery of DTIC, ECT, represents a promising strategy to enhance chemotherapy efficacy while reducing its associated toxicities. It offers a targeted approach that enhances drug delivery to tumor cells, minimizing collateral damage to healthy tissue. However, our study’s results revealed that DTIC’s cytotoxic effects on healthy cells increased by EP, so ECT applications should be conducted with sensitivity and locally. Overall, understanding the cytotoxic effects of this approach on both malignant and healthy cells is crucial for advancing its clinical applications and potentially improving the outcomes for patients with metastatic melanoma.

Author Disclosure Statement

The authors declare no conflict of interest.

Funding Information

The research was supported by the Scientific Research Projects (01/2015-03) at Gazi University. The authors express their gratitude for the assistance provided by Dr. Ongun Onaran, Dr. Sanem Ozcelik, and Dr. Mehmed Zahid Tuysuz.