Synthesis of Curcumin Derivatives and Analysis of Their Antitumor Effects in Triple Negative Breast Cancer (TNBC) Cell Lines

Abstract

We analyzed antitumor effects of a series of curcumin analogues. Some of them were obtained by reaction of substitution involving the two phenolic OH groups of curcumin while the analogues with a substituent at C-4 was prepared following an original procedure that regards the condensation of benzenesulfenic acid onto the nucleophilic central carbon of the curcumin skeleton. We analyzed cytotoxic effects of such derivatives on two TNBC (triple negative breast cancer) cell lines, SUM 149 and MDA-MB-231, but only three of them showed an IC₅₀ in a lower micromolar range with respect to curcumin. We also focused on these three derivatives that in both cell lines exhibited a higher or at least equivalent pro-apoptotic effect than curcumin. The analysis of molecular mechanisms of action of the curcumin derivatives under study has highlighted that they decreased NF-κB transcriptional factor activity, and consequently the expression of some NF-κB targets. Our data confirmed once again that curcumin may represent a very good lead compound to design analogues with higher antitumor capacities and able to overcome drug resistance with respect to conventional ones, even in tumors difficult to treat as TNBC.

Keywords: NF-κB inhibition; antioxidant activity; antiproliferative activity; pro-apoptotic activity; prooxidant activity; sulfenic acid.

Figure 1

Structural skeletons of curcumin analogues 1–5.

Scheme 1

Synthetic procedures for curcumin analogues 3–5.

Figure 2

Cytotoxic activity of curcumin and its derivatives 1–3 on SUM 149 cells. Cell viability was assessed by MTS assay. Data are expressed as the mean of at least three different experiments performed in triplicate. Different letters represent significant differences in cytotoxic activity among the concentration (Tukey test, p < 0.05).

Figure 3

Cytotoxic activity of curcumin and its derivatives 1–3 on MDA-MB-231 cells. Cell viability was assessed by MTS assay. Data are expressed as mean of at least three different experiments performed in triplicate. Different letters represent significant differences in cytotoxic activity among the concentration (Tukey test, p < 0.05).

Figure 4

Representative example of flow cytometry analysis with propidium iodide. SUM 149 cells were treated with curcumin and its analogues at 15 μM for 24 h. Numbers in the panels indicate the % of the events in the preG₀–G₁ position.

Figure 5

Representative example of flow cytometry analysis with propidium iodide. MDA-MB-231 cells were treated with curcumin and its analogues at 25 μM for 24 h. Numbers in the panels indicate the % of the events in the preG₀–G₁ position.

Figure 6

NF-κB (p65 subunit) DNA binding capacity in nuclear extracts of SUM 149 cells. The cells were treated for 24 h with curcumin and derivatives 1–3. Results (mean ± standard error of two experiments carried out in duplicate) are expressed as arbitrary units/μg protein of cells nuclear extracts. Different letters (a and b) in the column of the cell lines and treatments represent significant differences among the different treatments. * Differences when treatments are compared to the control, p < 0.01.

Figure 7

NF-κB (p65 subunit) DNA binding capacity in nuclear extracts of MDA-MB-231 cells. The cells were treated for 8 h with curcumin and derivatives 1–3. Results (mean ± standard error of two experiments carried out in duplicate) are expressed as arbitrary units/μg protein of cells nuclear extracts. Different letters (a and b) in the column of the cell lines and treatments represent significant differences among the different treatments. Differences when treatments are compared to the control: ** p < 0.05, * p < 0.01.

Figure 8

Western blot analysis of the levels of Survivin and Bcl-2 in SUM 149 cells treated for 24 h with Compound 3. On the left the results are expressed as mean ± standard error (SE) of two different experiments; on the right, the results of a representative experiment. * Differences when treatments are compared to the control, p < 0.01.

Figure 9

Western blot analysis of the levels of IAP1 in MDA-MB-231 cells treated for 8 h with Compound 1. On the left are the results expressed as mean ± standard error (SE) of two different experiments; on the right, the results of a representative experiment. * Differences when treatments are compared to the control, p < 0.01.

Figure 10

Structure of Compound 3.

Figure 11

¹H NMR spectrum of Compound 3 in CDCl₃ as solvent.

Figure 12

¹³C NMR spectrum of Compound 3 in CDCl₃ as solvent.

Figure 13

Structure of Compounds 4 and 5.

Figure 14

¹H NMR spectrum of Compound 5 in CDCl₃ as solvent.

Figure 15

¹³C NMR spectrum of Compound 5 in CDCl₃ as solvent.

Figure 16

¹H NMR spectrum of Compound 4 in CDCl₃ as solvent.

Figure 17

¹³C NMR spectrum of Compound 4 in CDCl₃ as solvent.