Anticancer Potential of Biogenic Silver Nanoparticles: A Mechanistic Study

Abstract

The continuous loss of human life due to the paucity of effective drugs against different forms of cancer demands a better/noble therapeutic approach. One possible way could be the use of nanostructures-based treatment methods. In the current piece of work, we have synthesized silver nanoparticles (AgNPs) using plant (Heliotropiumbacciferum) extract using AgNO3 as starting materials. The size, shape, and structure of synthesized AgNPs were confirmed by various spectroscopy and microscopic techniques. The average size of biosynthesized AgNPs was found to be in the range of 15 nm. The anticancer potential of these AgNPs was evaluated by a battery of tests such as MTT, scratch, and comet assays in breast (MCF-7) and colorectal (HCT-116) cancer models. The toxicity of AgNPs towards cancer cells was confirmed by the expression pattern of apoptotic (p53, Bax, caspase-3) and antiapoptotic (BCl-2) genes by RT-PCR. The cell viability assay showed an IC₅₀ value of 5.44 and 9.54 µg/mL for AgNPs in MCF-7 and HCT-116 cell lines respectively. We also observed cell migration inhibiting potential of AgNPs in a concentration-dependent manner in MCF-7 cell lines. A tremendous rise (150-250%) in the production of ROS was observed as a result of AgNPs treatment compared with control. Moreover, the RT-PCR results indicated the difference in expression levels of pro/antiapoptotic proteins in both cancer cells. All these results indicate that cell death observed by us is mediated by ROS production, which might have altered the cellular redox status. Collectively, we report the antimetastasis potential of biogenic synthesized AgNPs against breast and colorectal cancers. The biogenic synthesis of AgNPs seems to be a promising anticancer therapy with greater efficacy against the studied cell lines.

Keywords: ROS; RT-PCR; cytotoxicity; scratch assay; silver nanoparticles.

Figure 1

The solution of AgNO₃ (A)_, after reaction with plant extract (B), synthesized silver nanoparticles (AgNPs) (C).

Figure 2

(A): Scanning electron microscopy (SEM) image of biogenic silver nanoparticles (size ~15 nm), (B) Transmission electron microscopy (TEM) image of prepared silver nanoparticles from the extract (average size ~15 nm). (C) Fourier-transform infrared (FTIR) spectra of silver nanoparticles showing the functional groups in used chemicals. (D) X-ray diffraction spectrum of biosynthesized silver nanoparticles.

Figure 3

Cell viability measurement by MTT-based colorimetric method. Cells were treated with different concentrations of AgNPs for 24 h. The results represent the means of three separate experiments for each MCF-7 (A) and HCT-116 (B) cell lines and error bars represent the standard error of the mean. Treated groups showed statistically significant differences from the control group by ANOVA (p < 0.05). IC₅₀ values were measured by prism software (Inset).

Figure 4

Morphological changes in MCF-7 and HCT-116 cells treated with increasing concentration of AgNPs after 24 h. Leica microscope having 10× magnification was used to capture the images.

Figure 5

Scratch was made onto a monolayer of each cell line and treated by 2–10 µg/mL of AgNPs (indicated appropriately in the figure) for different time points. Comparison of migration in both control and test was made by taking images at different time intervals (0, 24, and 48 h). Results are represented by marking the scratch with parallel lines and visually displaying the number of cells migrated to the scratch area denoting metastasis inhibition.

Figure 6

Comet assay to assess DNA damage induced by AgNPs in cancer cells. The results demonstrated significant DNA damage by AgNPs (pictograph, A) and as evident by the tail length of DNA in cancer cells (B).

Figure 7

Oxidative stress response of AgNPs towards MCF-7 and HCT-116. ROS generation in AgNPs treated MCF-7 (A) and HCT-116 (B) cells. Relative fluorescence of DCF was measured using a spectrofluorometer with excitation at 485 and emission at 530 nm. The results represent the means of three separate experiments, ad error bars represent the standard error of the mean. Treated groups showed statistically significant differences from the control group by ANOVA (p < 0.05). Image of the cells showing fluorescence was captured by a Leica microscope (20 × magnification).

Figure 8

Effect of AgNPs on mRNA expression level of apoptotic markers in cancer cells. Cells were treated with different concentration of AgNPs for 24 h. Nanostructure-induced alterations in mRNA expression levels are expressed as fold change in relative quantity with those of control cells. CT values for other genes were calculated in reference to GAPDH control. (A) Breast cancer cells (MCF-7) were treated with 5 µg/mL and 10 µg/mL silver nanoparticles, while (B) HCT-116 cells were incubated with 10 and 20 µg/mL for 24 h.