Reversal of multidrug resistance by magnetic Fe₃O₄ nanoparticle copolymerizating daunorubicin and MDR1 shRNA expression vector in leukemia cells

Figures & data

Figure 1 Construction of PGC silencer-U6-neo-GFP-shRNA expression system.

Notes: U6 Promoter: 6077–6422; shRNA: 9–55; CMV promoter: 105–704; GFP: 709–1428; SV40 Promoter: 2098–2475; Neomycin: 2503–3297; pUC ori: 3534–4702; Ampicillin: 4805–5659.

Figure 2 Sequence of shRNA/MDR1.

Notes: A) PGY1–1; B) PGY1–2; C) PGY1–3.

Figure 3 Morphology of K562/A02 cells after transfected for 48 hours under fluorescence microscope (400× SYBR^® Green I staining).

Figure 4 Effects of shRNA expression vectors on MDR1 mRNA and its protein in K562/A02 cells after treatment for 48 hours. A) Transcription of MDR1 mRNA detected by quantitative real-time PCR described before; B) Expression of P-gp determined by Western-blot analysis described before.

Notes: 1. Negative control (PGC silencer-U6-neo-GFP empty plasmid); 2. Positive control (PGC silencer-U6-neo-GFP GAPDH plasmid); 3. PGY1–1; 4. PGY1–2; 5. PGY1–3. *P < 0.05, when compared to positive control group; P < 0.05, the group of transfected cells with PGY1–2 was less than that of transfected cells with PGY1–1 or PGY1–3.

Table 1 The cytotoxicity of DNR, MNP (Fe₃O₄) with DNR, PGY1–2 with DNR, MNP (Fe₃O₄) and PGY1–2 with DNR on K562/A02 for 48 hours determined by MTT assay (mean ± SD)

Groups	IC50 of DNR (μg/mL)
DNR 14.82 ± 0.96
10 μ g/mL MNP (Fe3O4) + DNR	4.75 ± 0.59 (3.15)*
1.3 μg/mL PGY1–2 + DNR	1.08 ± 0.37 (13.7) *
10 μg/mL MNP (Fe3O4) + 1.3 μg/mL PGY1–2 + DNR	0.97 ± 0.41 (15.3) *

Notes: The values in parentheses were fold reversal (FR) calculated as follows: FR = IC_{50 DNR alone}/IC_{50 DNR + agent}.

* P < 0.05, compared with DNR group.

Abbreviations: DNR, daunorubicin; FR, fold reversal; IC₅₀, inhibitory concentration at 50%; MNP (Fe₃O₄), magnetic nanoparticles of Fe₃O₄;rate; SD, standard deviation.

Figure 5 Effects of MNP (Fe₃O₄) and/or PGY1–2 on MDR1 mRNA in K562 cells and K562/A02 cells after treatment for 48 hours by Quantitative real-time PCR analysis.

Notes: 1. K562/A02-untreated; 2. K562/A02 treated with 1.0 μg/mL DNR; 3. K562/A02 treated with 10 μg/mL MNP (Fe₃O₄); 4. K562/A02 treated with PGY1–2; 5. K562/A02 treated with 10 μg/mL MNP(Fe₃O₄) and 1.0 μg/mL DNR; 6. K562/A02 treated with PGY1–2 and 1.0 μg/mL DNR; 7. K562/A02 treated with 10 μg/mL MNP (Fe₃O₄), PGY1–2 and 1.0 μg/mL DNR; 8. K562-untreated. *P < 0.05, when compared to K562/A02 incubated with 1.0 μg/mL DNR; **P < 0.05, when compared to K562/A02 incubated with 1.0 μg/mL DNR in the presence of PGY1–2 or MNP (Fe₃O₄).

Abbreviations: MNP (Fe₃O₄), magnetic nanoparticles of Fe₃O₄; DNR, daunorubicin.

Figure 6 Effects of MNP (Fe₃O₄) and/or PGY1–2 with DNR on P-gp in K562 cells and K562/A02 cells for 48 hours by Western-blot assay.

Notes: 1. K562-untreated; 2. K562/A02-untreated; 3. K562/A02 treated with 1.0 μg/mL DNR; 4. K562/A02 treated with 10 μg/ml MNP (Fe₃O₄); 5. K562/A02 treated with PGY1-2; 6. K562/A02 treated with 10 μg/ml MNP (Fe₃O₄) and 1.0 μg/ml DNR; 7. K562/A02 treated with PGY1–2 and 1.0 μg/ml DNR; 8. K562/A02 treated with 10 μg/ml MNP (Fe₃O₄), PGY1–2 and 1.0 μg/ml DNR.

Abbreviations: MNP (Fe₃O₄), magnetic nanoparticles of Fe₃O₄; DNR, daunorubicin.

Figure 7 Intracellular accumulation of DNR in K562/A02 cells after treatment with different agent for 48 hours.

Notes: A) control (K562/A02); B) K562/A02 incubated with 1.0 μg/mL DNR; C) K562/A02 incubated with 1.0 μg/mL DNR and 10 μg/mL MNP (Fe₃O₄); D) K562/A02 incubated with 1.0 μg/mL DNR and PGY1–2; E) K562/A02 incubated with 1.0 μg/mL DNR, 10 μg/mL MNP (Fe₃O₄) and PGY1–2.

Abbreviations: MNP (Fe₃O₄), magnetic nanoparticles of Fe₃O₄; DNR, daunorubicin.