Aurantoside C Targets and Induces Apoptosis in Triple Negative Breast Cancer Cells

Abstract

Triple negative breast cancer (TNBC) is a subtype of breast cancers that currently lacks effective targeted therapy. In this study, we found that aurantoside C (C828), isolated from the marine sponge Manihinea lynbeazleyae collected from Western Australia, exhibited higher cytotoxic activities in TNBC cells compared with non-TNBC (luminal and normal-like) cells. The cytotoxic effect of C828 was associated to the accumulation of cell at S-phase, resulting in the decline of cyclin D1, cyclin E1, CDK4, and CDK6, and an increase in p21. We also found that C828 inhibited the phosphorylation of Akt/mTOR and NF-kB pathways and increased the phosphorylation of p38 MAPK and SAPK/JNK pathways, leading to apoptosis in TNBC cells. These effects of C828 were not observed in non-TNBC cells at the concentrations that were cytotoxic to TNBC cells. When compared to the cytotoxic effect with the chemotherapeutic drugs doxorubicin and cisplatin, C828 was found to be 20 times and 35 times more potent than doxorubicin and cisplatin, respectively. These results indicate that C828 could be a promising lead for developing new anticancer agents that target TNBC cells.

Keywords: apoptosis; aurantoside C; cell cycle analysis; marine sponge; triple negative breast cancer.

Figure 1

The crude extracts of the sponge Manihinea lynbeazleyae collected off the coast of Western Australia was screened for cytotoxic effect in TNBC and non-TNBC cells and the chemical structure of the bioactive compound isolated. (A) Percentage of cell viability in TNBC SUM159PT cells, and non-TNBC MCF7 and MCF10A cells after treatment with crude solvent extracts of sponge M. lynbeazleyae for 24 h. CellTiter-Glo^® was used to measure cell viability. Three independent experiments were performed, each of them done in triplicates. One way ANOVA with Tukey’s posthoc test was used for statistical analysis **** p < 0.0001, and ns = not significant. (B) Chemical structure of Aurantoside C (C828) isolated as the bioactive compound.

Figure 2

Comparison of the cell viability of Aurantoside C (C828) in TNBC cells and non-TNBC (luminal and normal-like) cells. (A) Dose response curve showing the cytotoxic effect of C828 after 24 h of treatment in a range of human TNBC, luminal and normal-like breast epithelial cells. (B) The dose-response curves of C828 compared with chemotherapeutic drugs, cisplatin and doxorubicin on SUM159PT cells after 24 h of treatment. Three independent experiments were performed, each of them done in triplicates. The graphs plotted are the representative graphs and the results represent IC₅₀ ± SD from three independent experiments. One way ANOVA with Tukey’s post hoc test was used for statistical analysis **** p < 0.0001, and *** p < 0.001.

Figure 3

C828 increase DNA content at S-phase of the cycle in TNBC cell lines but not in non-TNBC cells. Effect of C828 on cell cycle distribution in (A) SUM159PT cells, (B) MCF7, and (C) MCF10A cells. Cells were treated with vehicle, 1 μM and 5 μM of C828 for 24 h. 0.1% DMSO diluted in media was used as a vehicle control. The distribution of cells in different phases of the cycle was measured by flow cytometry and data analyzed by FlowJo. Three independent experiments were performed, each of them done in triplicates. The fractions of cells in each phase of the cycle is shown in bar diagram as the mean ± S.D. One way ANOVA, with Tukey’s post hoc test was used for statistical analysis **** p < 0.0001, * p < 0.1 and ns = not significant.

Figure 4

C828 inhibits cell cycle related proteins in TNBC cell lines. Effect of C828 on cell cycle related proteins in (A) SUM159PT, (B) MCF7, and (C) MCF10A cells. Cells were treated with 0.01, 0.1, 0.5, 1 and 5 μM of C828 for 24 h. Cells treated with 0.1% DMSO diluted in media were used as a vehicle control. Whole cell lysates were isolated, quantification of proteins was done and immunoblotted with antibodies specific for cyclin D1, CDK4, CDK6, cyclinE1, CDK2, and p21. α-tubulin was used as a loading control for each experiment. ImageJ was used to quantify each blot. The blots were normalized against tubulin and then against control protein (when C828 was not added). Two independent experiments were performed. The histogram below each Western blot represents the average normalized values of the band from each experiment. One way ANOVA with Tukey’s posthoc test was used for statistical analysis **** p < 0.0001, *** p < 0.001, ** p < 0.01, and ns = not significant.

Figure 5

Effect of C828 in the phosphorylation of Akt/mTOR and NF-κB pathways in TNBC and non-TNBC cells. (A) SUM159PT cells, (B) MCF7 cells, and (C) MCF10A cells were treated with 0, 0.01, 0.1, 0.5, 1, and 5 μM of C828 for 24 h. Whole cell lysates were isolated, quantification of proteins was done and immunoblotted with antibodies specific for p-Akt (Ser473), p-Akt (Thr308), p-mTOR (Ser2448) and p-p65 (Ser536). α-tubulin was used as a loading control for each experiment. ImageJ was used to quantify each blot. The blots were normalized against tubulin and then against control protein (when C828 was not added). Two independent experiments were performed. The histogram below each western blot represents the average normalized values of the band from each experiment. One way ANOVA with Tukey’s post hoc test was used for statistical analysis **** p < 0.0001, and ns = not significant.

Figure 6

C828 activates phosphorylation of MAPK pathways in TNBC cells. (A) SUM159PT cells, (B) MCF7 cells, and (C) MCF10A cells were treated with 0, 0.01, 0.1, 0.5, 1, and 5 μM of C828 for 24 h. Whole cell lysates were isolated, quantification of proteins was done and immunoblotted with antibodies specific for p-p38 MAPK and p-SAPK/JNK. α-tubulin was used as a loading control for each experiment. ImageJ was used to quantify each blot. The blots were normalized against tubulin and then against control protein (when C828 was not added). Two independent experiments were performed. The histogram below each western blot represents the average normalized values of the band from each experiment. One way ANOVA with Tukey’s post hoc test was used for statistical analysis **** p < 0.0001, ** p < 0.01, and ns = not significant.

Figure 7

C828 induces apoptotic cell death in TNBC cells. (A) SUM159PT cells, (B) MCF7 cells, and (C) MCF10A cells were treated with vehicle control and 0.5 μM, 1 μM, and 5 μM of C828, respectively, for 24 h. Treated cells were stained using Annexin-V FITC and PI and analyzed by flow cytometry and data were processed using Flow Jo 10. Experiments were performed in triplicates and the bar diagram represents the percentage of apoptotic cells (early and late apoptosis appearing in the right lower and upper quadrants). One way ANOVA with Tukey’s post hoc test was used for statistical analysis **** p < 0.0001, * p < 0.1, and ns = not significant.

Figure 8

C828 selectively induces apoptotic cell death in TNBC cells. (A) SUM159PT cells, (B) MCF7 cells, and (C) MCF10A cells were treated for 24 h with vehicle control and 0.5 μM and 1 μM of C828 respectively and analysed by immunofluorescence assay. Cells were stained with anti-cleaved caspase-3 antibody (green) and counterstained with Hoechst 33258 (blue). Image J software was used to quantify the cells that are shown in bar diagram. Experiments were performed in triplicates. One way ANOVA with Tukey’s post hoc test was used for statistical analysis **** p < 0.0001, * p < 0.1, and ns = not significant.