Inhibition of autophagy enhances the anticancer activity of silver nanoparticles

Figures & data

Scheme 1. Schematic illustration of the theory that Ag NPs induced cytoprotective autophagy and that inhibition of autophagy enhanced the antitumor efficacy of Ag NPs.

Figure 1. Characterization of Ag NPs. (A) XRD pattern of Ag NPs. (B) UV-Vis spectrum of Ag NPs. (C) TEM image of Ag NPs. Scale bar: 50 nm. (D) Fourier transform infrared spectra of PVP and Ag@PVP.

Figure 2. Ag NPs induce autophagosome accumulation. (A) Fluorescence microscopy images of HeLa EGFP-LC3 cells treated with PBS (control), 200 nM rapamycin (Rap) and 10 μg/mL Ag NPs for 4 h. Scale bar: 10 μm. (B) Time course of EGFP-LC3 dot formation in HeLa EGFP-LC3 cells treated with 10 μg/mL Ag NPs. Mean ± SEM, n = 3. (C) Dose-dependent EGFP-LC3 dot formation in HeLa EGFP-LC3 cells treated with Ag NPs for 8 h and 12 h. Mean ± SEM, n = 3. (D) Fluorescent colocalization between EGFP-LC3 dots and other autophagy-related markers: monodansylcadaverine (MDC) and LysoTracker Red (LT) in HeLa EGFP-LC3 cells treated with 10 μg/mL Ag NPs for 4 h. The right panel is a high magnification image of the indicated portion. Scale bar: 20 μm.

Figure 3. Additional autophagic features induced by Ag NPs. (A) Hela cells were treated with various doses of Ag NPs for 24 h and then subjected to western blotting with anti-LC3 antibody and anti-GAPDH antibody, whose detection served as loading control. (B) TEM of HeLa cells treated with PBS (control) or 10 μg/mL Ag NPs for 24 h. The right panel is a high magnification image of the indicated portion. Arrows indicate autophagosomes and autolysosomes. (C) Bio-TEM images of HeLa cells treated with 20 μg/mL Ag NPs for 24 h. The internalized Ag NPs were indicated by black dashed line. N, nuclei.

Figure 4. Ag NPs induced autophagy by enhancing autophagosome formation. (A) HeLa cells were treated with 20 ug/mL Ag NPs for 24 h in the presence or absence of bafilomycin A₁ (BFA). Endogenous LC3-II levels were detected by protein gel blotting with anti-LC3 antibodies and quantified by densitometric analysis relative to GAPDH. Mean ± SEM, n = 3. *P <0.05, ***P < 0.001. (B) HeLa cells were treated with 20 ug/mL Ag NPs for 24 h in the presence or absence of chloroquine (CQ). Endogenous LC3-II levels were detected by western blotting with anti-LC3 antibodies and quantified by densitometric analysis relative to GAPDH. Mean ± SEM, n = 3. *P <0.05, ***P < 0.001. (C) Western blotting of EGFP and GAPDH (served as loading control) in HeLa EGFP-LC3 cells treated with different concentrations of Ag NPs for 24 h. (D) HeLa cells were treated with 10 ug/mL Ag NPs for 1 h or HBSS (starvation) for 2 h. Endogenous SQSTM1 levels were detected by protein gel blotting with anti-SQSTM1 antibodies and quantified by densitometric analysis relative to GAPDH. Mean ± SEM, n = 3. ***P < 0.001.

Figure 5. Ag NPs induced autophagy in a PtdIns3K-dependent and MTOR-independent fashion. (A) Western blotting of LC3 in HeLa cells treated with PBS (control) or 10 μg/mL Ag NPs for 24 h in the presence or absence of 1 μM wortmannin (Wort). (B) HeLa cells treated with PBS (control) or 1 μM wortmanin for 24 h and analyzed by AKT and phospho-AKT western blotting. (C) HeLa cells treated with PBS (control), 200 nM rapamycin (Rap) or 10 μg/mL Ag NPs for 24 h, were analyzed for MTOR activity by protein gel blotting for levels of total (T) and phosphor (P)-MTOR and RPS6KB.

Figure 6. Ag NPs elicited significant cytotoxicity that was not likely due to released silver ions. (A) MTT cytotoxicity assay of HeLa cells exposed to varying concentrations of Ag NPs for 24 h. Mean ± SEM, n = 5. (B) MTT cytotoxicity assay of P-MEF and I-MEF cells exposed to varying concentrations of Ag NPs for 24 h. Mean ± SEM, n = 5. (C) ICP-MS assay of the free silver ions (Ag⁺) released from Ag NPs (10 μg/mL) in deionized water for 0 h or 24 h incubation. Mean ± SEM, n = 3. ***P < 0.001 comparing to the starting (0 h) measurement. (D) MTT cytotoxicity assay of HeLa cells exposed to varying concentrations of Ag⁺ for 24 h. Mean ± SEM, n = 5. (E) HeLa cells exposed to varying concentrations of Ag⁺ for 24 h. Endogenous LC3-II levels were detected by western blotting with anti-LC3 antibodies and quantified by densitometric analysis relative to GAPDH. Mean ± SEM, n = 3, *P < 0.05, **P < 0.01, compared to the group without Ag⁺.

Figure 7. Inhibition of autophagy by wortmannin enhanced cytotoxicity of Ag NPs in HeLa cells. (A) Cell viability of HeLa cells treated with PBS (control) or 10 μg/mL Ag NPs for 24 h together with 1 μM wortmannin or not. Mean ± SEM, n = 5. ***P < 0.001 comparing to the control group. (B) ANXA5-FITC PtdIns assay of HeLa cells treated as (A) for 24 h. Shown were the relative percentage of live (lower-left quadrant), early apoptotic (lower-right quadrant), and late apoptotic and necrotic (upper-right quadrant) cells. (C) Representative fluorescence pictures (the right panel) and the cell death rate of HeLa cells treated with PBS (control) or 10 μg/mL Ag NPs for 20 h in the presence or absence of wortmannin. Cell death was assessed by Hoechst 33342 PI staining and expressed as the percentage of PtdIns-stained cells. Mean ± SEM, n = 3. ***P < 0.001. (D) Western blotting of CASP3 in HeLa cells treated with PBS (control) or 10 μg/mL Ag NPs for 24 h in the presence or absence of wortmannin.

Figure 8. Inhibition of autophagy by ATG5 siRNA treatment enhanced cytotoxicity of Ag NPs in HeLa cells. (A) Western blotting of LC3 and ATG5 in HeLa cells transfected with ATG5 siRNA or control siRNA for 48 h. (B) Cell viability of HeLa cells treated with 10 μg/mL Ag NPs for 24 h after transfection with ATG5 siRNA or control siRNA for 48 h. Mean ± SEM, n = 5. ***P < 0.001 comparing to the control siRNA group. (C) ANXA5-FITC PI assay of HeLa cells treated with 10 μg/mL Ag NPs for 24 h after transfection with ATG5 siRNA or control siRNA for 48 h. (D) Cell death rate of HeLa cells treated with PBS (control) or 10 μg/mL Ag NPs for 20 h after transfection with ATG5 siRNA or control siRNA for 48 h. Mean ± SEM, n = 3. ***P < 0.001. (E) Western blotting of CASP3 and cleaved-CASP3 in HeLa cells treated with 10 μg/mL Ag NPs for 24 h after transfection with ATG5 siRNA or control siRNA for 48 h.

Figure 9. Autophagy-inducing ability and synergetic killing effect of Ag NPs with wortmannin in B16 cells. (A) TEM of B16 cells treated with PBS (control) or 50 μg/mL Ag NPs for 24 h. The right panel is a high magnification image of the indicated portion. Arrows indicate autophagosomes and autolysosomes. (B) Western blotting of LC3 in B16 cells treated with PBS (control) or 50 μg/mL Ag NPs for 24 h in the presence or absence of 1 μM wortmannin. (C) MTT assay of B16 cells treated with PBS (control) or 50 μg/mL Ag NPs for 24 h in the presence or absence of 1 μM wortmannin. Mean±SEM, n = 5. ***P < 0.001 comparing to the control group. (D) ANXA5-FITC PtdIns assay of B16 cells treated as in (C) for 24 h. Shown are the relative percentages of live (lower-left quadrant), early apoptotic (lower-right quadrant), and late apoptotic and necrotic (upper-right quadrant) cells.

Figure 10. Inhibition of autophagy enhanced the antitumor effect of Ag NPs in a mouse model. (A and B) Synergetic tumor-shrinking effect of wortmannin and Ag NPs in mice with melanoma. The change of tumor volume (A) and weight ratio of tumor tissues (B) from C57BL/6 mice treated with normal saline (control), 25 nmol/kg wortmannin(s.c.), 1.5 mg/kg Ag NPs (s.c.), 1.5 mg/kg Ag NPs plus 25 nmol/kg wortmannin. Mean ± SEM, n = 4. **P <0.01, ***P < 0.001 comparing to the control group. (C) TUNEL staining (red) of sections from tumor in (B) was performed to show apoptotic cells. Nuclei were stained with DAPI (blue). (D) Western blotting of LC3 was performed to show autophagy level of B16 melanoma treated with saline, wortmannin, Ag NPs or Ag NPs plus wortmannin.