Effects of Green Silver Nanoparticles on Apoptosis and Oxidative Stress in Normal and Cancerous Human Hepatic Cells in vitro

Figures & data

Table 1 Anticancer Properties of Green Synthesized AgNPs

Biological Entity Used for Reduction	Cancer Cells Under Study	Reference
Cleome viscosa fruit extract	Lung (A549) and ovarian (PA1) cancer cell lines	^Citation8
Annona muricata leaf extract	Human fibroblasts isolated from dermis	^Citation9
N,N,N-trimethyl chitosan chloride and polyelectrolyte complex	Colon cancer cell lines (HCT116) and Mammalian cell lines (African green monkey kidney cell lines (VERO cells)	^Citation10
Rheum Rhabarbarum fresh stem extract	Cervical carcinoma HeLa cell line	^Citation11
Matricaria chamomilla	A549 lung cancer cells	^Citation12
Zataria multiflora leaf extract	Cervical carcinoma cells (HeLa cell line)	^Citation13
Phoenix dactylifera hair-root extract	Human breast cancer (MCF7 cell line)	^Citation14

Table 2 DLS Measurements of Nanoparticle Size and Zeta Potential to Compare the Dispersion Characteristics of Particles in Simplified and in vitro Biological Representative Media

Nano Powder	Dispersion Media	DLS Mean ± S.E. (nm)	Zeta Potential ± S.E. (mV)
gAgNPs ≥100 nm	Milli-Q-H2O	122± 2.0	−11.4± 2.6
	DMEM/un-supplemented media	160± 3	−9.1 ± 2.4

Notes: Data represent the mean ± standard error of the mean (S.E) of three-independent experiments, carried out at 24°C, with each experimental mean generated from triplicate measurement of each sample (n = 3).

Figure 1 (A) TEM image of green silver nanoparticles (gAgNPs) (B) Distribution green silver nanoparticles (gAgNPs) size (%).

Figure 2 Cytotoxicity of gAgNPs on CHANG and HuH-7 cells for 24 hrs, as evaluated by (A). MTT (B). LDH assays. Each value represents the mean ±SE of three experiments. n= 3, *p < 0.05, ** p < 0.01 vs control.

Figure 3 DCF Fluorescence intensity of CHANG and HuH-7 cells after exposure to NPs for 24 hr (A) Control CHANG cells (B) CHANG cells at 40 µg/mL gAgNPs exposure (C) CHANG cells at 100 µg/mL gAgNPs exposure (D) Control HuH-7 cells (E) HuH-7 cells at 40 µg/mL gAgNPs exposure (F) HuH-7 cells at 100 µg/mL gAgNPs exposure (G) % ROS generation due to NPs exposure to cells. Each value represents the mean ±SE of three experiments. *p< 0.05 and **p< 0.01vs. control. Scale bar is 200 µm.

Figure 4 After exposure of gAgNPs on CHANG and HuH-7 cells for 24 hrs (A). Levels of LPO (B). GSH in cells. Each value represents the mean ±SE of three experiments. *p < 0.05 and**p < 0.01 vs control.

Figure 5 Photomicrograph of JC-1 fluorescence intensity in CHANG cells (A) and HuH-7 cells (B) after exposure to gAgNPs (C). JC-1 fluorescence ratio as marker of MTP loss. Each value represents the mean ±SE of three experiments. *p < 0.05 and **p < 0.01 vs control. Scale bar is 20 µm.

Figure 6 Chromosomal condensation (A). Control CHANG cells (B). CHANG cells exposed at 20 µg/mL gAgNPs (C). CHANG cells exposed at 40 µg/mL gAgNPs (D). Control HuH-7 cells (E). HuH-7 cells exposed at 20 µg/mL gAgNPs (F). HuH-7 cells exposed at 40 µg/mL gAgNPs and (G) Induction of caspase-3 activity in CHANG and HuH-7 cells after exposure to gAgNPs for 24 hrs. Each value represents the mean ±SE of three experiments. *p < 0.05 and **p < 0.01 vs control. Arrow indicates fragmented chromosome. Scale bar is 200 µm.

Figure 7 The fluorescence scatter diagram after exposure to gAgNPs (40 µg/mL) to cells (A). Control CHANG cells (B). CHANG cells exposed at 40 µg/mL gAgNPs (C). Control HuH-7 cells (D). HuH-7 cells exposed at 40 µg/mL gAgNPs and (E) Induction of early and late apoptotic cells (%) after exposure to gAgNPs (40 µg/mL) for 24 hrs. Each value represents the mean ±SE of three experiments. *p < 0.05 vs control. FL-1 Height and FL-2 Height represents Annexin-V-FITC and PI staining cells, respectively. LL – living cells (Annexin-V^–/PI^–stained), LR – Early apoptotic cells (Annexin-V⁺/PI^–stained), UL – Necrotic cells (Annexin-V^–/PI⁺ stained), UL – Late apoptotic cells (Annexin-V⁺ PI⁺ stained).

Figure 8 Genotoxic effect of gAgNPs exposure to CHANG and HuH-7 cells (A). Tail DNA (%) (B). Olive tail moment. DNA damage photomicrograph of cells due to gAgNPs exposure (C). Control CHANG cells (D). CHANG cells exposed at 40 µg/mL gAgNPs (E). Control HuH-7 cells (F). HuH-7 cells exposed at 40 µg/mL gAgNPs. Each value represents the mean ±SE of three experiments. *p < 0.05 and **p < 0.01 vs control. Scale bar is 50 µm.

Figure 9 Expression of pro and anti-apoptotic proteins as evaluated by immuno blotting for 24 hrs (A) CHANG cells (B) HuH-7 Cells (C) Densitometry analysis of relative protein level of Bax/Bcl2 ratio. Each value represents the mean ±SE of three experiments. n= 3, ** p < 0.01 vs control.

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