Green-Synthesized Silver Nanoparticles Induced Apoptotic Cell Death in MCF-7 Breast Cancer Cells by Generating Reactive Oxygen Species and Activating Caspase 3 and 9 Enzyme Activities

Abstract

Silver nanoparticles are among the most significant diagnostic and therapeutic agents in the field of nanomedicines. In the current study, the green chemistry approach was made to optimize a cost-effective synthesis protocol for silver nanoparticles from the aqueous extract of the important anticancer plant Fagonia indica. We investigated the anticancer potential and possible involvement of AgNPs in apoptosis. The biosynthesized AgNPs are stable (zeta potential, -16.3 mV) and spherical with a crystal size range from 10 to 60 nm. The MTT cell viability assay shows concentration-dependent inhibition of the growth of Michigan Cancer Foundation-7 (MCF-7) cells (IC₅₀, 12.35 μg/mL). In addition, the fluorescent microscopic analysis shows activation of caspases 3 and 9 by AgNPs that cause morphological changes (AO/EB assay) in the cell membrane and cause nuclear condensation (DAPI assay) that eventually lead to apoptotic cell death (Annexin V/PI assay). It was also observed that AgNPs generate reactive oxygen species (ROS) that modulate oxidative stress in MCF-7 cells. This is the first study that reports the synthesis of a silver nanoparticle mediated by Fagonia indica extract and evaluation of the cellular and molecular mechanism of apoptosis.

Figure 1

Proposed mechanism of silver ion reduction by plant metabolite into silver nanoparticles.

Figure 2

Variation in color intensity of green silver nanoparticles mediated at different AgNO₃ concentrations by leaf extracts combined at the ratio of 1 to 10 (extract : AgNO₃).

Figure 3

Optimization of different parameters for bioinspired synthesis of silver nanoparticles. UV-vis spectrum of AgNPs mediated by leaf aqueous extracts of Fagonia indica: (a) effect of AgNO₃ concertation, (b) effect of extract and AgNO₃ (1 mM) ratios on the synthesis of green nanoparticles, (c) effect of temperature, and (d) duration of time for synthesis of silver nanoparticles at different time intervals.

Figure 4

X-ray diffraction (XRD) pattern of green-synthesized AgNPs showing Bragg reflection at angle 2 theta.

Figure 5

DLS analysis of green-synthesized nanoparticles in (a) zeta size and (b) zeta potential.

Figure 6

Morphology of AgNPs. SEM micrograph at the scale of 200 nm shows spherical nanoparticles.

Figure 7

Cytotoxicity of extract and AgNPs in MCF-7 cells. Values are the average ± standard deviation of three experiments conducted in duplicates: (a) percent growth inhibition; (b) IC₅₀ concentration.

Figure 8

Morphological observation of MCF-7 cells treated with extract and AgNPs. (a) Acridine orange-ethidium bromide (AO/EB) staining. Green indicates viable cells, and reddish/orange staining of the cells indicates apoptotic cells. (b) Morphological changes in the nuclei of MCF-7 cells after treatment with extract and AgNPs induced apoptosis. The changes were observed with DAPI nuclear staining of the treated cells.

Figure 9

Flow cytometry analysis of MCF-7 cells by double-labelling with Annexin V and PI dyes. The figure shows the early apoptotic, late apoptotic, live, and dead cells given in each quadrant of the untreated growth control cell compared to AgNP-treated cells.

Figure 10

Proposed mechanism of apoptosis induced by caspases and reactive oxygen species.

Figure 11

Quantification of caspase 3 and caspase 9 activity in MCF-7 cells exposed to 12.35 μg/mL AgNPs and 25.09 μg/mL extract.

Figure 12

Effects of extract and AgNP exposition on ROS generation in MCF-7 cells. (a) Extent of H₂O₂ generation at different time intervals in MCF-7 cells stained with a DCFDH fluorescent probe. (b) Standard curve of H₂O₂.