Biological Effects of EF24, a Curcumin Derivative, Alone or Combined with Mitotane in Adrenocortical Tumor Cell Lines

Abstract

Background: Curcumin has numerous properties and is used in many preclinical conditions, including cancer. It has low bioavailability, while its derivative EF24 shows enhanced solubility. However, its effects have never been explored in adrenocortical tumor cell models. The efficacy of EF24 alone or combined with mitotane (reference drug for adrenocortical cancer) was evaluated in two adrenocortical tumor cell lines, SW13 and H295R.

Method and results: EF24 reduced cell viability with an IC50 (half maximal inhibitory concentration) of 6.5 ± 2.4 μM and 4.9 ± 2.8 μM for SW13 and H295R cells, respectively. Combination index (EF24 associated with mitotane) suggested an additivity effect in both cell lines. Cell cycle analysis revealed an increase in subG0/G1 phase, while motility assay showed a decrease in migratory cell capacity, and similarly, clonogenic assay indicated that EF24 could reduce colony numbers. Furthermore, Wnt/β-catenin, NF-κB, MAPK, and PI3k/Akt pathways were modulated by Western blot analysis when treating cells with EF24 alone or combined with mitotane. In addition, intracellular reactive oxygen species levels increased in both cell lines.

Conclusion: This work analyzed EF24 in adrenocortical tumor cell lines for the first time. These results suggest that EF24 could potentially impact on adrenocortical tumors, laying the foundation for further research in animal models.

Keywords: EF24; adrenocortical; curcumin; preclinical cell model.

Figure 1

Chemical structure of the two compounds used in subsequent experiments. (A) mitotane (1-(2-Chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane,1-Chloro-2-[2,2-dichloro-1-(4-chlorophenyl)ethyl]-benzene); (B) and EF24 ((3E,5E)-3,5-bis[(2-fluorophenyl)methylene]-4-piperidinone).

Figure 2

MTT and SRB assay for SW13 and H295R cells treated for 24 h and 72 h. (A) MTT test for EF24; (B) SRB assay for EF24; (C) MTT test for mitotane; (D) SRB assay for mitotane; (E) MTT test for combination index calculation in SW13 at 24 h; (F) MTT test for combination index calculation in H295R at 72 h. Different drug concentrations were used following a series of CI values generated by the CompuSyn 3.0.1 program. Experiments were performed in quadruplicate and repeated three times. Treatment vs. control: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Representative cell cycle analyses. SW13 cells treated at 24 h: (A) Control; (B) EF24 6.5 μM; (C) EF24+mitotane; (D) mitotane 8 μM. H295R cells treated at 72 h: (E) Control; (F) EF24 5 μM; (G) EF24 + mitotane; (H) mitotane 10 μM. Experiments were performed in triplicate.

Figure 4

Cell motility assay for SW13 and H295R cells treated with EF24, mitotane, and their combination at 24 h and 72 h, respectively. (A) Representative images of the wound healing assay (C = control, E = EF24, M = mitotane, E + M = EF24 + mitotane). (B,C) Quantification of cell motility for SW13 and H295R cells. Results are expressed as the percentage reduction of the initial scratch compared with the corresponding untreated cells. Data are shown as the means of nine measurements. Experiments were performed in triplicate. Treatment vs. control: * p < 0.05.

Figure 5

Cell morphology evaluated by Wright’s staining method after treatment with EF24, mitotane, and their combination in SW13 and H295R cells treated at 24 h and 72 h, respectively. The arrows show apoptotic (white) or necrotic cells (black). (C = control, E = EF24, M = mitotane, E + M = EF24 + mitotane).

Figure 6

Representative clonogenic assay in SW13 and H295R cells treated with EF24, mitotane, and their combination at 24 h and 72 h, respectively. (A) SW13 cells. (B) H295R cells. (C,D) Statistical analysis (C = control, E = EF24, M = mitotane, E + M = EF24 + mitotane). Treatment vs. control: * p < 0.05. Each experiment was performed in triplicate and repeated twice.

Figure 7

Representative Western blot analyses for adrenocortical tumor cells. (A) SW13 cells; (B) H295R cells; 1 and 5 = untreated control; 2 and 6 = EF24 at 6.5 μM for SW13 and 5 μM for H295R cells; 3 and 7 = mitotane 8 μM for SW13 cells and 10 μM for H295R cells; 4 and 8 = EF24 + mitotane. Experiments were performed in triplicate.

Figure 8

Histograms of intracellular levels of reactive oxygen species in SW13 (A) and H295R (B) cells after treatment with EF24, mitotane, and their combination. DCFH-DA = 2′,7′-dichlorofluorescein diacetate. NAC = N-acetil-cysteine. E = EF24. M = mitotane. E + M = EF24 + mitotane. Treatment vs. control: ** p < 0.01, *** p < 0.001. Experiments were performed in triplicate and repeated twice.