Effective combination of cold physical plasma and chemotherapy against Ewing sarcoma cells in vitro

Abstract

Ewing's sarcoma (ES) is the second most common bone tumor in children and adolescents and is highly malignant. Although the new chemotherapy has significantly improved the survival rate for ES from about 10 to 75%, the survival rate for metastatic tumors remains around 30%. This treatment is often associated with various side effects that contribute to the suffering of the patients. Cold physical plasma (CPP), whether used alone or in combination with current chemotherapy, is considered a promising adjunctive tool in cancer treatment. This study aims to investigate the synergistic effects of CPP in combination with cytostatic chemotherapeutic agents that are not part of current ES therapy. Two different ES cell lines, RD-ES and A673, were treated with the determined IC₂₀ concentrations of the chemotherapeutic agents cisplatin and methotrexate (MTX) in combination with CPP. The effects on population doubling, cell viability, and apoptotic processes within these cell lines were assessed. This combination therapy has led to a reduction of population doubling and cell viability, as well as an increase in apoptotic activity in cells compared to CPP monotherapy. The results of this study provide evidence that combining CPP with non-common chemotherapy drugs such as MTX and CIS in the treatment of ES enhances the anticancer effects of these drugs. These findings open up new possibilities for the effective use of these drugs against ES.

Keywords: Chemotherapy; Cisplatin; Cold physical plasma; Ewing sarcoma; Methotrexate.

Figure 1

Inhibitory effect of cisplatin and methotrexate on Ewing’s sarcoma cells. The Ewing’s sarcoma cell lines A673 (A–C) and RD-ES (D–F) were treated with cisplatin and methotrexate (MTX). After 72 h, the cell viability was determined, and the IC₂₀ was calculated. Individual values are shown with a dose–response curve and the IC₂₀ calculated from this (A,B,D,E) as well as mean values of the calculated IC₂₀ (C,F). The mean values were examined with the t-test for significant differences, which were presented as follows: ****p < 0.0001.

Figure 2

Effect of a combined treatment of ES cell lines with CPP and chemotherapeutics. The Ewings sarcoma cell lines A673 (A,B) and RD-ES (C,D) were treated with CPP combined with the chemotherapeutic agents cisplatin and methotrexate (MTX). Cell viability was determined after 72 h. Shown are individual values with dose–response curves.

Figure 3

Effects of CPP treatment combined with chemotherapeutic agents on cell population doubling. A673 cells (A,B) and RD-ES cells (C,D) were treated with CPP and a combination of CPP and cytostatics. The number of living cells was determined after 120 h of incubation and the population of doublings was calculated. Mean values are shown. Significant differences were determined using ANOVA and Tukey's multiple comparisons test and are reported as follows (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

Figure 4

Apoptosis induction by the combination of CPP and cytostatic agents. To evaluate the apoptotic effect of combination therapy of the cell lines A673 and RD-ES Caspase 3/7 activity assay and TUNEL assay were used. The relative Caspase 3/7 signals and TUNEL signals of cells treated with CPP and combination therapy were normalized to the relative Caspase 3/7 signals and TUNEL signals of cells treated with Argon (control). The mean values ± SD are depicted in the graphs and were assessed for statistically significant differences using paired t-tests (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Figure 5

DNA damage marker formation upon CPP and chemotherapeutics treatment. A673 cells were stained 24 h after treatment with CPP, CIS, MTX, or combinations of these (A) to investigate the formation of nuclear pATM and yH2AX foci (B). Images were quantified by calculating the mean fluorescence intensity (MFI) of pATM (C) and yH2AX (D) in all detected nuclei of treated cells against untreated controls. The mean values ± SD are depicted in the graphs and were assessed for statistically significant differences using one-way ANOVA and Šídák's multiple comparisons test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).