Electrochemotherapy with Calcium Chloride and 17β-Estradiol Modulated Viability and Apoptosis Pathway in Human Ovarian Cancer

Abstract

Estrogens (Es) play a significant role in the carcinogenesis and progression of ovarian malignancies. Depending on the concentration, Es may have a protective or toxic effect on cells. Moreover, they can directly or indirectly affect the activity of membrane ion channels. In the presented study, we investigated in vitro the effectiveness of the ovarian cancer cells (MDAH-2774) pre-incubation with 17β-estradiol (E₂; 10 µM) in the conventional chemotherapy (CT) and electrochemotherapy (ECT) with cisplatin or calcium chloride. We used three different protocols of electroporation including microseconds (µsEP) and nanoseconds (nsEP) range. The cytotoxic effect of the applied treatment was examined by the MTT assay. We used fluorescent staining and holotomographic imaging to observe morphological changes. The immunocytochemical staining evaluated the expression of the caspase-12. The electroporation process's effectiveness was analyzed by a flow cytometer using the Yo-Pro™-1 dye absorption assay. We found that pre-incubation of ovarian cancer cells with 17β-estradiol may effectively enhance the chemo- and electrochemotherapy with cisplatin and calcium chloride. At the same time, estradiol reduced the effectiveness of electroporation, which may indicate that the mechanism of increasing the effectiveness of ECT by E₂ is not related to the change of cell membrane permeability.

Keywords: 17β-estradiol; MDAH-2774; calcium electroporation; chemotherapy; electrochemotherapy; ovarian cancer.

Figure 1

MDAH-2774 cells viability measured by the MTT assay after 24 h and 72 h incubation in different 17β-estradiol concentrations. Notes: (mean ± SD) N = 3, * p < 0.05 compared to control, # p < 0.05 compared between different incubation times.

Figure 2

MDAH-2774 cells viability measured by the MTT assay after 24 h (a,b) and 72 h (c,d) incubation in increasing cisplatin (a,c) and CaCl₂ (b,d) concentrations. Notes: (mean ± SD) N = 3, * p < 0.05 compared to control, # p < 0.05 compared between different pre-incubation conditions.

Figure 3

MDAH-2774 cells viability measured by the MTT assay after (a) µEP and nsEP with (b) 25 µM cisplatin and (c) 2.5 mM CaCl₂. Notes: (mean ± SD) N = 3, * p < 0.05 compared to control, # p < 0.05 compared between different pre-incubation conditions; ps—pulses.

Figure 4

Caspase-12 immunocytochemical staining after exposure to cisplatin (5 µM and 25 µM) and CaCl₂ (1 mM and 2.5 mM) in the MDAH-2774 cell line.

Figure 5

Immunofluorescence staining of MDAH-2774 cells (60×) after 24 h incubation with cisplatin and CaCl₂. Alexa Fluor^TM 546 phalloidin used for actin filaments labeled. Scale bars correspond to 100 µm.

Figure 6

Immunofluorescence staining of MDAH-2774 cells (60×) after µEP and nsEP with the addition of cisplatin and CaCl₂. Alexa Fluor^TM 546 phalloidin used for actin filaments labeled. Scale bars represent 100 µm; ps—pulses.

Figure 7

Yo-Pro-1^TM fluorescent dye absorption in MDAH-2774 cells after µEP and nsEP; ps—pulses.

Figure 8

Holotomographic microscopy studies after 24 h with cisplatin and CaCl₂. Scale bars represent 20 µM.