Fig 1.
(A) The size distribution and (B) particle concentration of fractions 7, 8, and 9 were analyzed by NTA. (C) FDNV morphology was observed under TEM at 100,000X (scale bar = 200 nm) and 300,000X (scale bar = 100 nm). (D) The zeta potential of isolated FDNVs was measured using a Zetasizer. (E) Distribution of tentatively identified metabolites in FDNVs using LC-MS/MS. Data are represented as means ± SD of three independent experiments in duplicate; ns = not significant. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA).
Table 1.
Size of FDNVs in fractions 7, 8 and 9 using nanoparticle tracking analysis (n = 3).
Fig 2.
Cytotoxic effects of FDNVs and fingerroot extract on colorectal cancer and normal colon epithelial cell lines.
Cell viabilities of (A) HT-29, (B) HCT116, and (C) CCD 841 CoN cells were determined using MTT assay after treatment with FDNVs and fingerroot extract for 24, 48, and 72 h. Data are represented as means ± SD of three independent experiments in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA).
Table 2.
IC50 values of FDNVs and fingerroot extract against colorectal cancer cells.
Fig 3.
Internalization of FDNVs in colorectal cancer and normal colon epithelial cells.
(A) HT-29, (B) HCT116, and (C) CCD 841 CoN cells were incubated with PKH67-labeled FDNVs (green) for 24 h. Cells were also stained with DAPI (blue) and Phalloidin (red) to label the nucleus and actin filaments, respectively. (D) The green fluorescence intensity of FDNVs was determined using ImageJ software. The values are shown as means ± SD of three independent experiments in duplicate. ***P < 0.001 (one-way ANOVA). Scale bar = 10 μm.
Fig 4.
The inhibition of FDNVs internalization in colorectal cells.
(A) HT-29 and HCT116 cells were pretreated with chlorpromazine, amiloride, and filipin for 1 h and then incubated with PKH67-label FDNVs (green) for an additional 3 h in the presence of the inhibitors. (B) HT-29 and HCT116 cells were pretreated with cytochalasin D for 1 h and then incubated with PKH67-label FDNVs for an additional 2 h in the presence of the inhibitor. Cells were then fixed and stained with DAPI (blue). The green fluorescence intensity of FDNVs was determined by ImageJ software. Bar graphs show the uptake efficiency of FDNVs in HT-29 and HCT116 cells. The values are presented as means ± SD of three independent experiments in duplicate. ***P < 0.001 (one-way ANOVA). Scale bar = 10 μm.
Fig 5.
Representative FACS quantitative analyses showing FDNVs-induced apoptosis.
(A) HT-29, (B) HCT116, and (C) CCD 841 CoN cells were treated with FDNVs at indicated concentrations for 48 h. The apoptosis induction of FDNVs-treated cells was examined using flow cytometry-based Annexin V staining. Data were analyzed using Kaluza analysis software and shown as means ± SD of three independent experiments in duplicate. *P < 0.05, **P < 0.01 and ***P < 0.001 (one-way ANOVA).
Fig 6.
Effect of FDNVs on apoptosis-related genes expression.
(A) HT-29 and (B) HCT116 cells were treated with FDNVs for 24 h. The expression of target genes was determined using quantitative RT-PCR. The relative quantitation of each gene was normalized to the constitutive expression of GADPH. The results are mean ± SD of three independent experiments in duplicate and presented as fold change. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with untreated cells (one-way ANOVA).
Fig 7.
Induction of intracellular ROS and GSH in FDNVs-treated cells.
(A) HT-29, (B) HCT116, and (C) CCD 841 CoN cells were treated with FDNVs for 6 h. Cells with 200 μM H2O2 for 3 h were used as a positive control. Intracellular ROS and GSH were determined using CM-H2DCFDA and Glutathione assay kit, respectively. The levels of intracellular ROS are represented as a ratio to untreated control. The levels of intracellular GSH are represented as mean ± SD. All experiments were performed in three independent experiments in duplicate: ns = not significant. *P < 0.05, **P < 0.01 and ***P < 0.001 (one-way ANOVA).