A novel curcumin-like dienone induces apoptosis in triple-negative breast cancer cells

Abstract

Purpose: According to the World Health Organization (WHO), breast cancer is the most common cancer affecting women worldwide. In the USA ~12.3 % of all women are expected to be diagnosed with various types of breast cancer, exhibiting varying degrees of therapeutic response rates. Therefore, the identification of novel anti-breast cancer drugs is of paramount importance.

Methods: The 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore was incorporated into a number of cytotoxins. Three of the resulting dienones, 2a, 2b and 2c, were tested for their anti-neoplastic potencies in a variety of human breast cancer-derived cell lines, including the triple negative MDA-MB-231 cell line and its metastatic variant, using a live-cell bio-imaging method. Special emphasis was put on dienone 2c, since its anti-cancer activity and its mode of inflicting cell death have so far not been reported.

Results: We found that all three dienones exhibited potent cytotoxicities towards the breast cancer-derived cell lines tested, whereas significantly lower toxicities were observed towards the non-cancerous human breast cell line MCF-10A. The dienones 2b and 2c exhibited the greatest selective cytotoxicity at submicromolar concentration levels. We found that these two dienones induced phosphatidylserine externalization in MDA-MB-231 cells in a concentration-dependent manner, suggesting that their cytotoxic effect might be mediated by apoptosis. This possibility was confirmed by our observation that the dienone 2c can induce mitochondrial depolarization, caspase-3 activation, cell cycle disruption and DNA fragmentation in MDA-MB-231 cells.

Conclusion: Our findings indicate that dienone 2c uses the mitochondrial/intrinsic pathway to inflict apoptosis in triple negative MDA-MB-231 breast cancer-derived cells. This observation warrants further assessment of dienone 2c as a potential anti-breast cancer drug.

Keywords: Anti-cancer drug discovery; Apoptosis; Caspase-3; Cell cycle; Curcumin analogues; DNA fragmentation; Mitochondrial depolarization.

Fig. 1

a General structures of series 1 and 2 of cytotoxins containing the 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore. b Synthesis of 3,5-bis(benzylidene)-1-[4-(2-diethylaminoethyloxy) phenylcarbonyl]-4-piperidone hydrochloride hemihydrate, dienone 2a, 1-[4-(2-dimethylaminoethoxy)phenylcarbonyl]-3,5-bis(4-methylbenzylidene)-4-piperidone hydrochloride hemihydrate, dienone 2b and 3,5-bis(benzylidene)-1-[3-(2-dimethylaminoethoxy)phenylcarbonyl]-4-piperidone hydrochloride, dienone 2c. c Structure of curcumin

Fig. 2

Representative overlay of live-cell images of MB-231 cells stained with Hoechst and propidium iodide (PI) used to determine CC₅₀ values. (a) Nuclei of untreated cells that serve as a negative control. (b) Cells treated with the pro-apoptotic agent H₂ O₂ (100 μM), resulted in a significant increase in PI-positive cells (dying cells; magenta color due to overlay of blue and red dyes). (c) Cells treated with dienone 2c (0.8 μM) displaying an increase in dying cells as in (b). Nuclei exhibiting a blue signal only are considered to be living cells

Fig. 3

Dienones 2b and 2c induce PS externalization in a concentration-dependent manner in MDA-MB-231 cells after a 48 h treatment. PS externalization was measured by flow cytometry. The total percentages of apoptotic cells (y-axis) are expressed as the sum of the percentages of early and late stages of apoptosis (green bars), as determined by the percentage of annexin V-FITC positive cells. (a and b) Cells exposed to the dienones that were only PI positive are considered to be necrotic (black bars). Each bar represents the average of three independent measurements and the error bars represent their corresponding standard deviations. The following controls were included: cells exposed to 100 μM etoposide as a positive control for apoptosis; cells treated with 0.4 % v/v DMSO as a solvent control; untreated cells as a negative control. Approximately 3000 events (cells) were collected and analyzed per sample using CXP software (Beckman Coulter)

Fig. 4

The cytotoxicity of dienone 2c is mediated by ΔΨm changes in MDA-MB-231 cells in a dose-dependent manner. After 8 h of treatment with 10 and 25 μM dienone 2c, changes in ΔΨm were monitored by JC-1 staining and flow cytometry. Dissipation of ΔΨm causes the JC-1 reagent to emit a green fluorescence signal, and a red signal when mitochondrial membranes are polarized. The average percentages of cells with depolarized membranes are shown in (a). Untreated and 1 % DMSO (0.4 % v/v) treated cells were used as controls. Statistical significance was determined using the two-tailed Student’s paired t-test of dienone 2c treated with untreated (*) and DMSO treated cells (‡). P < 0.001. Representative flow cytometric dot plots used to determine the percentages of cells with depolarized mitochondria are depicted in panels b–e

Fig. 5

Dienone 2c induces caspase-3 activation in a concentration-dependent manner in MDA-MB-231 cells. Intracellular active caspase-3 was detected after a 8 h incubation with dienone 2c using a NucView 488 fluorogenic substrate in conjunction with flow cytometry. The percentage of active caspase 3-positive cells, exhibiting a green fluorescence signal, are indicated on the y-axis, whereas on the x-axis the different treatments are indicated (a). Each bar represents the average of three independent measurements, and error bars correspond to the standard deviation of the mean. Representative dot plots used to obtain the percentages of cells positive to the NucView caspase-3/7 substrate are indicated on the y-axis (FL2 detector), whereas the FL1 detector measurements are depicted on the x-axis (b–f). After exposure to 10 μM (b) and 25 μM (c) dienone 2c for 8 h, the cells were incubated with the caspase-3 substrate, as detailed in Material and Methods. Solvent control (0.4 % DMSO; e) and untreated cells (f) were included as controls. Approximately 10,000 events were collected and analyzed per sample using CXP software (Beckman Coulter). Images of a representative cell captured in the green fluorescence channel (d) and the merged image of both the green and the differential interference contrast (DIC) channels (g) are depicted. In the merged image (g), the black arrow indicates the presence of a bleb, a morphological feature of apoptosis. The top right cell is negative for the NucView caspase-3/7 substrate

Fig. 6

Dienone 2c disturbs the cell cycle profile of breast cancer-derived cells and induces DNA fragmentation associated with reduced G0/G1 and S phase values in a dose-dependent manner. Cells were harvested, permeabilized, DAPI stained and analyzed by flow cytometry. (a–d) The percentages of cells in each phase are plotted along the y-axis and the different treatments are graphed along the x-axis. Each bar represents the average of four independent replicates, and the error bars represent the corresponding standard deviations. Several controls were included (purple bars): solvent controls (0.1 or 0.3 % v/v DMSO), a reference drug (4 μM camptothecin, CPT) and untreated cells. Significant differences between 1 and 3 μM dienone 2c treated cells compared to their corresponding 0.1 % or 0.3 % v/v DMSO treated cells (*) and untreated cells (‡) were P < 0.05. (e–i) Representative flow cytometric histograms employed to quantify the distributions of the cell cycle phases. Gates in the flow cytometric histograms (panels e–i) are from left to right: sub-G0/G1 (hypodiploid; counted as apoptotic subpopulation), G0/G1 (diploid), S (hyperdiploid) and G2/M (tetraploid)

Fig. 7

Schematic diagram depicting the suggested mechanisms involved in dienone 2c-induced apoptosis in MDA-M B-231 cells. Dienone 2c elicits the following pro-apoptotic biochemical phases: phosphatidylserine externalization, mitochondrial depolarization, caspase-3 activation, DNA fragmentation and cell death, which are indicative of early, middle and late apoptotic stages, respectively