Combination of Goniothalamin and Sol-Gel-Derived Bioactive Glass 45S5 Enhances Growth Inhibitory Activity via Apoptosis Induction and Cell Cycle Arrest in Breast Cancer Cells MCF-7

Abstract

Background: Combination of natural products with chemically synthesised biomaterials as cancer therapy has attracted great interest lately. Hence, this study is aimed at investigating the combined effects of goniothalamin and bioactive glass 45S5 (GTN-BG) and evaluating their anticancer properties on human breast cancer cells MCF-7.

Methods: The BG 45S5 was prepared using the sol-gel process followed by characterisation using PSA, BET, SEM/EDS, XRD, and FTIR. The effects of GTN-BG on the proliferation of MCF-7 were assessed by MTT, PrestoBlue, and scratch wound assays. The cell cycle analysis, Annexin-FITC assay, and activation of caspase-3/7, caspase-8, and caspase-9 assays were determined to further explore its mechanism of action.

Results: The synthesised BG 45S5 was classified as a fine powder, having a rough surface, and contains mesopores of 12.6 nm. EDS analysis revealed that silica and calcium elements are the primary components of BG powders. Both crystalline and amorphous structures were detected with 73% and 27% similarity to Na₂Ca₂(Si₂O₇) and hydroxyapatite, respectively. The combination of GTN-BG was more potent than GTN in inhibiting the proliferation of MCF-7 cells. G0/G1 and G2/M phases of the cell cycle were arrested by GTN and GTN-BG. The percentage of viable cells in GTN-BG treatment was significantly lower than that in GTN. In terms of activation of initiator caspases for both extrinsic and intrinsic apoptosis pathways, caspase-8 and caspase-9 were found more effective in response to GTN-BG than GTN.

Conclusion: The anticancer effect of GTN in MCF-7 cells was improved when combined with BG. The findings provide significant insight into the mechanism of GTN-BG against MCF-7 cells, which can potentially be used as a novel anticancer therapeutic approach.

Figure 1

Scanning electron microscopy and SEM micrographs of the bioactive glass 45S from a lower to higher magnification (100 μm to 1 μm; a–d).

Figure 2

(a) EDS spectrum and (b) XRD pattern of the synthesised BG 45S5 with 73% of pattern similarity to Na₂Ca₂(Si₂O₇) and 27% to hydroxyapatite.

Figure 3

FTIR spectra of the sol-gel-derived BG 45S5.

Figure 4

The cytotoxicity of BG against (a) HMSC and (b) MCF-7 cells, at different concentrations from 0 to 1 mg/mL, was assessed by the MTT assay for 24-, 48-, and 72-hour incubation times.

Figure 5

(a) MCF-7 and (b) HMSC cells were treated with GTN at IC₅₀ and 0.5 mg/mL of BG. Cell viability was assessed via the MTT assay. All experiments were done in triplicate, and the data is represented by means and standard deviations. Comparisons between the groups of different treatments were done by using 1-way ANOVA, followed by Tukey's posttest for multiple comparisons. Not significant is denoted as ns; the significant difference in the treated cells as compared to untreated cells is marked as ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001, whereas the significant difference between GTN and GTN-BG treatments is represented as ⁺p < 0.05, ⁺⁺p < 0.01, and ⁺⁺⁺p < 0.001.

Figure 6

The inhibitory effects of GTN and GTN-BG at IC₂₅, IC₅₀, and IC₇₅ in MCF-7 following 24, 48, and 72 hours of incubation time. All experiments were done in triplicate, and the data is represented by means and standard deviations. The comparisons between the groups were done by using two-way ANOVA, followed by the Bonferroni posttest for multiple comparisons. Significant differences between the treated (DOX, GTN, and GTN-BG) and untreated (UT) groups were marked as ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001, and the significant differences between GTN and GTN-BG treatments were represented as ^∗p < 0.05, ⁺⁺p < 0.01, and ⁺⁺⁺p < 0.001.

Figure 7

(a) Confluent monolayer of MCF-7 cells was mechanically wounded (scratch assay), leaving two wound edges separated by a 700-800 μm wide void (gap). The proliferation of untreated MCF-7 cells and the cells treated with BG, GTN, GTN-BG, and DOX on the void was monitored using the IncuCyte ZOOM® system at 10x magnification. The time-lapse images were taken at 0, 6, 12, and 21 hours, which are represented as A, B, C, and D, respectively. (b) The real-time proliferation of MCF-7 cells on the gap and the percentage of wound confluence were measured by using the IncuCyte™ ZOOM Live Cell System (Essen BioScience, USA) for up to 12 hours.

Figure 8

DNA histogram of MCF-7 cells in (a) untreated, (b) BG-treated, (c) GTN-treated, (d) GTN-BG-treated, and (e) DOX-treated groups after 48 hours of exposure time. The bar graph shows the quantitative data based on DNA histograms. Comparisons between the different treatment groups were done by using 1-way ANOVA, followed by Dunnett's posttest to detect any significant differences between the treated and untreated cells (^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001).

Figure 9

The percentage of stained MCF-7 cells following Annexin V-FITC/PI analysis by using flow cytometry for 48 hours. All experiments were performed in triplicate, and the data is represented by means ± standard deviations. Comparisons between the different treatment groups were done by using 2-way ANOVA, followed by the Bonferroni posttest for multiple comparisons. The significant difference between the treated and untreated cells is marked as ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001, whereas the significant difference between GTN- and GTN-BG-treated cells is represented by ⁺p < 0.05, ⁺⁺p < 0.01, and ⁺⁺⁺p < 0.001.

Figure 10

Activation of caspase-8, caspase-9, and caspase-3/7 in MCF-7 cells after 48 hours. All experiments were performed in triplicate, and the data is represented by means ± standard deviations. Comparisons between the groups of different treatments were done by using 1-way ANOVA, followed by the Bonferroni posttest for multiple comparisons. The significant difference between the treated and untreated cells is marked as ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001, whereas the significant difference between GTN- and GTN-BG-treated cells is represented as ⁺p < 0.05, ⁺⁺p < 0.01, and ⁺⁺⁺p < 0.001.