Synergistic Interaction between 5-FU and an Analog of Sulforaphane-2-Oxohexyl Isothiocyanate-In an In Vitro Colon Cancer Model

Abstract

Combination therapy is based on the beneficial effects of pharmacodynamic interaction (synergistic or additive) between combined drugs or substances. A considerable group of candidates for combined treatments are natural compounds (e.g., isothiocyanates) and their analogs, which are tested in combination with anticancer drugs. We tested the anticancer effect of the combined treatment of isothiocyanate 2-oxohexyl isothiocyanate and 5-fluorouracil in colon and prostate cancer cell lines. The type of interaction was described using the Chou-Talalay method. The cytostatic and cytotoxic activities of the most promising combined treatments were investigated. In conclusion, we showed that combined treatment with 5-fluorouracil and 2-oxohexyl isothiocyanate acted synergistically in colon cancer. This activity is dependent on the cytostatic properties of the tested compounds and leads to the intensification of their individual cytotoxic activity. The apoptotic process is considered to be the main mechanism of cytotoxicity in this combined treatment.

Keywords: 5-fluorouracil; isothiocyanates; sulforaphane; synergistic effect.

Figure 1

Structural formulae of isothiocyanates: (A) sulforaphane; (B) 2-oxohexyl isothiocyanate.

Figure 2

Cell growth inhibition and interaction types. The changes in cell growth of cancer cell lines after the administration of compounds alone and in combination: 5-fluorouracil (5-FU) with 2-oxohexyl ITC (hex) in HT-29 (A), Caco-2 (B), PC-3 (C), and LNCaP (D) cell lines. * The cell growth values after the administration of a combination treatment were statistically significantly different from the cell growth values after administrations of 2-oxohexyl ITC and 5-FU alone; p < 0.05. Cell growth was determined using the MTT method. Combination treatment: the cells were incubated with 2-oxohexyl ITC for 24 h and then with 5-FU for 72 h. In individual administrations, one component of the combination was used. Combination index values (CI) depending on the fraction affected (fa) in HT-29 (E) and Caco-2 (F) colon cancer cell lines and in PC-3 (G) and LNCaP (H) prostate cancer cell lines for 5-FU administration with 2-oxohexyl ITC. CI > 1 = antagonism; CI < 1 = synergism; CI ± 1.0 = an additive effect. The Chou-Talalay method was used to determine the CI. The arrows indicate the type of interaction for fa = 0.75.

Figure 3

Morphological changes in the HT-29 (A), Caco-2 (B), and PC-3 (C) cells treated with 5-FU, hex, and the combined treatment (mx). The changes in distribution of the cell cycle phases in colon and prostate cancer cell lines when compared to the control after the administration of the combined treatment and the individual compounds in the HT-29 (D), Caco-2 (E), and PC-3 (F) cell lines. Cell cycle distribution was determined by flow cytometry. Combination treatment: the cells were incubated with 2-oxohexyl ITC for 24 h and then with 5-FU for 72 h. In individual administrations, one component of the combination was used. The changes of the living cells percentage in the cell culture after the administration of individual compounds and the combined treatment in the Caco-2 (G), HT-29 (H), and PC-3 (I) cell lines. Large black star—statistically significant difference between the percentage of living cells after the administration of the combined treatment and after administrations of 2-oxohexyl ITC and 5-FU alone. * Statistically significant difference from the control; p < 0.05. Cell viability was determined using the FDA/PI method using flow cytometry. Apoptosis detection in HT-29 colon cancer and Caco-2 cancer cell lines is indicated by arrows in graphs (G,H). Green—cell membrane stained with Annexin V-FITC. Red—nuclei stained with PI.

Figure 4

Apoptosis studies. (A) Caspase (Caspase 9, Caspase 8, and Caspase 3) activity/mg protein normalized to the control (ctrl) after the combined 5-FU and 2-oxohexyl ITC treatment, individual 5-FU treatment, individual hex treatment, and positive control treatment (ctrl+) after 24 (A) and 72 h incubation (B). * Statistically significant difference from the control; p < 0.05. Changes in the levels of apoptosis-related proteins obtained by Western blotting after the administration of individual compounds, the combined treatment of 5-FU with hex, and for the control cells (ctrl) after 24-h incubation (C) and after 72-h incubation (D). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as an internal control. The results of the bands of procaspase forms were quantified by densitometric analysis, and their intensity was normalized with respect to GAPDH.

Figure 5

Autophagy detection: visualization of autophagic vacuoles in cells after the combined treatment with 2-oxohexyl ITC (hex) and 5-FU (A). Protein expression of LC3-II. GAPDH was a loading control. Protein levels were evaluated by the Western blot method (B).