Effect of interaction of magnetic nanoparticles of Fe₃O₄ and artesunate on apoptosis of K562 cells

Ying Wang1 Department of Hematology, the 2nd Hospital of Hebei Medical University, People’s Republic of China

Yuxiang Han1 Department of Hematology, the 2nd Hospital of Hebei Medical University, People’s Republic of China

Yingying Yang1 Department of Hematology, the 2nd Hospital of Hebei Medical University, People’s Republic of China

Jingci Yang1 Department of Hematology, the 2nd Hospital of Hebei Medical University, People’s Republic of China

Xiaonan Guo1 Department of Hematology, the 2nd Hospital of Hebei Medical University, People’s Republic of China

Jingnan Zhang1 Department of Hematology, the 2nd Hospital of Hebei Medical University, People’s Republic of China

Ling Pan1 Department of Hematology, the 2nd Hospital of Hebei Medical University, People’s Republic of ChinaCorrespondence[email protected]

Guohua Xia2 Department of Hematology, the Affiliated Zhongda Hospital, Medical School, Southeast University, Nanjing, People’s Republic of China

Baoan Chen2 Department of Hematology, the Affiliated Zhongda Hospital, Medical School, Southeast University, Nanjing, People’s Republic of China

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Pages 1185-1192 | Published online: 09 Jun 2011

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Introduction
Materials and methods
Results
Discussion
Conclusion
Acknowledgements
References

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Abstract

The present study evaluated whether the magnetic nanoparticles of Fe₃O₄ (MNPs-Fe₃O₄) could enhance the activity of artesunate (ART), and to explore its potential mechanisms. Cytotoxicity of the copolymer of ART with MNPs-Fe₃O₄ on K562 cells was detected by MTT assay and the apoptosis rate of K562 cells was measured by flow cytometry. Protein expression levels of bcl-2, bax, bcl-rambo, caspase-3, and survivin in K562 cells were measured by Western blot. After being incubated with the copolymer of ART with MNPs-Fe₃O₄ for 48 hours, the growth inhibition rate of K562 cells was significantly increased compared with that of K562 cells treated with ART alone (P < 0.05), and the apoptosis rate of K562 cells was increased significantly compared with that of K562 cells treated with ART alone, suggesting that MNPs-Fe₃O₄ can enhance the activity of ART. Interestingly, the copolymer-induced cell death was attenuated by caspase inhibitor Z-VAD-FMK. Our results also showed that treatment with the copolymer of MNPs-Fe₃O₄ and ART increased the expression of bcl-2, bax, bcl-rambo, and caspase-3 proteins, and decreased the expression of survivin protein in K562 cells compared with ART treatment alone. These results suggest that MNPs-Fe₃O₄ can enhance ART-induced apoptosis, which may be related to the upregulation of bcl-rambo and downregulation of survivin.

Keywords:

magnetic nanoparticles
MNPs-Fe₃O₄
artesunate
K562 cells
apoptosis
bcl-rambo

Introduction

Artesunate (ART), a semi-synthetic derivative of artemisinin extracted from the Chinese herb Artemisia annua, is a safe and effective antimalarial drug. Recently, several studies showed that ART could inhibit the growth of various carcinoma cell lines^{Citation1–Citation3} and suppress tumor cell proliferation by inducing apoptosis,^Citation4 indicating that ART may be a promising novel candidate for leukemia therapy. However, a variety of lines of evidence indicate that ART inhibited cell proliferation in a dose-dependent manner, and a high concentration of ART induced embryo toxicity,^{Citation5–Citation7} reversible renal damage toxicity,^Citation8 and toxicity to bone development,^Citation9 which limit the usefulness of ART.

To minimize side effects associated with dosage during chemotherapy, a promising approach is to combine a conventional chemotherapy with new strategies to maximize efficacy of tumor treatment through inducing cell apoptosis. Recent studies demonstrate that nanotechnologies and nanoparticles, which have unique peculiarities such as stable biomolecular absorption, high surface energy, and small sizes comparable to those of biomolecules, are becoming a focus for human medical application, including the delivery of antineoplastic agents to cells and tissues.^{Citation10,Citation11} We have recently reported that MNPs-Fe₃O₄, as an anticancer drug deliverer, could enhance the sensitivity of anticancer drugs and reverse the drug resistance of tumor cells.^{Citation12,Citation13}

Apoptosis is the main mechanism of cell death, and is mediated by a cell-intrinsic suicide program, with the relative balance of pro- and anti-apoptotic signaling pathways determining the fate of the cell. There are two main pathways in mammals. One is the death receptor signaling pathway,^Citation14 which is regulated by the IAP family including mainly X-IAP, c-IAP1, c-IAP2, KIAP, and survivin. Another pathway is the mitochondrial signaling pathway, in which bcl-2 plays an important role and bcl-2/bax is the key point of cell apoptosis.^Citation15 Recently, bcl-rambo, a novel member of bcl-2 family, has been shown to induce apoptosis independent of both bcl-2/bax ratio and the death receptor pathway, but this effect can be specifically blocked by the IAP family.^Citation16 The final executor of apoptosis is the caspase family, particularly caspase-3, which is the central proteases for apoptosis.^Citation17

Accordingly, the copolymer of ART with MNPs-Fe₃O₄ may provide a novel clinical candidate for the treatment of leukemia. Here, the sensitivity of K562 cells to this copolymer and its possible underlying mechanisms were investigated.

Materials and methods

Main materials

The following materials were purchased: 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) (Ameresco, Solon, OH); Z-VAD-FMK (Beyotime Biotechnology); RPMI 1640 medium and fetal calf serum (GIBCO, USA); artesunate (Guilin Pharmaceutical Co., Ltd., China); Annexin V-FITC (Biosea Biotechnology Co., Ltd., Beijing, China); total protein extraction kit P1250 (Applygen Technologies Inc., Beijing, China); BCA protein assay kit (Biosynthesis Biotechnology Co., Ltd., Beijing, China); anti-bcl-2, anti-bax, anti-survivin (Bioworld), anti-bcl-rambo (Biovision), anti-active caspase-3 (Cell Signaling), anti-GAPDH (Santa Cruz, CA), and goat-antirabbit HRP-IgG (Santa Cruz, CA); Western chemiluminescent HRP substrate (Millipore Corporation, Billerica, MA). MNPs-Fe₃O₄ was kindly supplied by the state Key Laboratory of Bioelectronics, Southeast University, Nanjing, China, and was well distributed in 0.01 M phosphate-buffered saline (PBS) by ultrasound to obtain MNPs-Fe₃O₄ colloidal suspension. ART at different concentrations (12.5, 25, 50, 75, and 100 μM) was conjugated with MNPs-Fe₃O₄, at the molar ratios of 25:1, 50:1, and 100:1 by mechanical absorption polymerization at 4°C for 12 hours, as previously reported.^{Citation18,Citation19}

Cell line and cell culture

K562 cells, derived from a patient with chronic myeloid leukemia in blast crisis, had been constantly preserved in our laboratory, and were cultured in a flask in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified 5% CO₂ incubator.

Cytotoxicity assays

K562 cells (2 × 10⁴ cells per well) in logarithmic growth phase were cultured in 96-well flat-bottomed plates in a triplicate pattern and treated with different concentrations of ART, MNPs-Fe₃O₄, or the copolymer of ART with MNPs-Fe₃O₄ in the presence or absence of 40 μM Z-VAD-FMK for 48 hours. MTT (20 μL, 5 mg/mL) was added to each well and incubated for 4 hours. Then, 200 μL of dimethylsulfoxide was added to each well and the plate was vortexed for 10 minutes at 37°C. Finally, the optical density value (A) of each well was measured at a measurement wavelength of 540 nm using a plate reader (Model 550, BIO-RAD, Japan). Cell growth inhibition ratio was calculated as (1 − A₅₄₀ of experimental well/A₅₄₀ of blank control well) × 100%. Each assay was repeated at least 3 times.

Apoptosis assay by flow cytometry

Based on the results of cytotoxicity assay, the best combination condition of ART with MNPs-Fe₃O₄ (100:1 M/M) was used to measure the apoptosis rate. Briefly, after being treated with ART, MNPs-Fe₃O₄ or the copolymer of MNPs-Fe₃O₄ and ART for 48 hours, K562 cells were collected, washed twice with ice-cold PBS, and then cells was suspended in 200 μL of binding buffer and 10 μL of Annexin V-FITC for 15 minutes in the dark. Thereafter, 300 μL of binding buffer and 5 μL of propidium iodide were added to each sample. Finally, the cells were analyzed using BD FACSDiva flow cytometry (BD FACS Canto^™ II) with Cell Quest software.

Western blot analysis

After K562 cells were treated as mentioned above, total protein was extracted and quantified according to the manufacturer’s protocol. Each equal amount of protein was loaded on sodium dodecyl sulfate polyacrylamide gel, run at 100 volts for 2 hours, and then the protein was transferred to a polyvinylidene fluoride membrane. The membranes were blocked in 5% fat-free milk at room temperature for 2 hours, and the blots were stained with specific primary antibodies, including anti-bcl-2, anti-bax, anti-survivin, anti-bcl-rambo, anti-active caspase-3, and anti-GAPDH antibodies overnight at 4°C. Then, the membranes were washed and incubated with goat-antirabbit HRP-IgG for 1 hour at room temperature, and imaged with a chemiluminescent substrate. Thereafter, the bound immunoglobulins were removed from the membranes by washing twice with restore western blot stripping buffer, and the signal was detected using the ChemiDoc XRs+ (BIO-RAD) with the enhanced chemiluminescence and analyzed by the Image Lab (ECL). GAPDH was used as the internal control.

Statistical analysis

All experiments were repeated 3 times. Data are presented as mean ± standard deviation and analyzed using the SPSS (Release13.0; SPSS Inc, Chicago, IL). One-way analysis of variance (ANOVA), 2-sample t-test, or rank test was performed to illustrate the significant difference between the treated groups and the control. The difference was considered statistically significant at P < 0.05.

Results

Inhibition of cell growth

When K562 cells were treated with the copolymer of ART and MNPs-Fe₃O₄ at the molar ratio of 100:1, the cell viability was decreased significantly compared with that of K562 cells treated with ART alone (0.47 ± 0.11 vs 1.10 ± 0.02) (P < 0.05). Although the cell viability of K562 cells was reduced to 0.66 ±0.03 at a molar ratio of 25:1 and 0.73 ±0.04 at a molar ratio of 50:1, there were no significant differences between the copolymer and ART alone group (P > 0.05), suggesting that the molar ratio of 100:1 was the best condition for copolymerization.

Moreover, when ART at the concentrations of 75, or 100 μM was combined with MNPs-Fe₃O₄ (M/M = 100:1) cell proliferation was remarkably inhibited, especially at the concentration of 100 μM, compared with ART alone (P < 0.05) (); however there was no significant difference at the concentrations of 12.5, 25, and 50 μM (P > 0.05). The cell growth inhibition in the MNPs-Fe₃O₄ group was less than that in the ART group with or without MNPs-Fe₃O₄. In addition, it was noted that MNPs-Fe₃O₄ alone could slightly inhibit cell proliferation when its concentration was from 0.125 μM to 4 μM, but there was no significant difference (P > 0.05).

Figure 1 The inhibition ratio of K562 treated with ART or the copolymer of ART with MNPs-Fe₃O₄ (100:1) for 48 hours.

Note: *P < 0.05, compared with the ART alone.