Synergistic effect of magnetic nanoparticles of Fe(3)O(4) with gambogic acid on apoptosis of K562 leukemia cells

Abstract

Gambogic acid (GA) has a significant anticancer effect on a wide variety of solid tumors. Recently, many nanoparticles have been introduced as drug-delivery systems to enhance the efficiency of anticancer drug delivery. The aim of this study was to investigate the potential benefit of combination therapy with GA and magnetic nanoparticles of Fe(3)O(4) (MNPs-Fe(3)O(4)). The proliferation of K562 cells and their cytotoxicity were evaluated by MTT assay. Cell apoptosis was observed and analyzed by microscope and flow cytometry, respectively. Furthermore, real-time polymerase chain reaction and Western blotting analyses were performed to examine gene transcription and protein expression, respectively. The results showed that MNPs-Fe(3)O(4) dramatically enhanced GA-induced cytotoxicity and apoptosis in K562 cells. The typical morphological features of apoptosis treated with GA and MNPs-Fe(3)O(4) were observed under an optical microscope and a fluorescence microscope, respectively. The transcription of caspase-3 and bax gene in the group treated with GA and MNPs-Fe(3)O(4) was higher than that in the GA-alone group or MNPs-Fe(3)O(4)-alone group, but the transcription of bcl-2, nuclear factor-kappaB, and survivin degraded as did the expression of corresponding proteins in K562 cells. Our data suggests a potential clinical application of a combination of GA and MNPs-Fe(3)O(4) in leukemia therapy.

Keywords: K562 leukemia cells; apoptosis; gambogic acid; magnetic nanoparticles of Fe3O4; traditional Chinese medicine.

Figure 1

Effect of the different concentrations of gambogic acid (GA) on growth inhibition ratio of K562 cells by MTT assay.

Figure 2

Growth inhibition ratio of GA with or without MNPs-Fe₃O₄-treated K562 cells for 48 hours. Abbreviations: MNPs-Fe₃O₄, magnetic nanoparticles of Fe₃O₄; GA, gambogic acid.

Figure 3

Effect of MNPs-Fe₃O₄ on GA-induced apoptosis in K562 cells for 48 hours. A) Control; B) Incubated with 10 mg/L MNPs-Fe₃O₄; C) Incubated with 0.6 mg/L GA; D) Incubated with 0.6 mg/L GA and 10 mg/L MNPs-Fe₃O₄. Abbreviations: MNPs-Fe₃O₄, magnetic nanoparticles of Fe₃O₄; GA, gambogic acid.

Figure 4

Morphological features of K562 cells after treatment for 48 hours by optical microscope (1000x, Wright staining). A) Control; B) Incubated with 0.6 mg/L GA and 10 mg/L MNPs-Fe₃O₄; C) Incubated with 6 mg/L GA. Abbreviations: MNPs-Fe₃O₄, magnetic nanoparticles of Fe₃O₄; GA, gambogic acid.

Figure 5

Nucleolus morphological changes of K562 cells after different treatment for 48 hours under fluorescence microscope (400x, DAPI staining). A) Control; B) Incubated with 0.6 mg/L GA; C) Incubated with 0.6 mg/L GA and 10 mg/L MNPs-Fe₃O₄. Abbreviations: MNPs-Fe₃O₄, magnetic nanoparticles of Fe₃O₄; GA, gambogic acid.

Figure 6

Transcription of caspase-3, bax, bcl-2, NF-κB, and survivin in K562 cells after treatment of GA with or without MNPs-Fe₃O₄ for 48 hours. A) Control; B) Incubated with 10 mg/L MNPs-Fe₃O₄; C) Incubated with 0.6 mg/L GA; D) Incubated with 0.6 mg/L GA and 10 mg/L MNPs-Fe₃O₄. Notes: *,**P < 0.05, synergia of GA with MNPs-Fe₃O₄ compared to GA alone; #,##,***P < 0.05, synergia of GA with MNPs-Fe₃O₄ compared to GA alone. Abbreviations: MNPs-Fe₃O₄, magnetic nanoparticles of Fe₃O₄; GA, gambogic acid; NF-κB, nuclear factor-kappaB.

Figure 7

Expression of caspase-3, bax, bcl-2, NF-κB, and survivin protein in K562 cells by western blot after treatment of GA and/or MNPs-Fe₃O₄ for 48 hours. Line 1: Control; Line 2: Incubated with 10 mg/L MNPs-Fe₃O₄; Line 3: Incubated with 0.6 mg/L GA; Line 4: Incubated with 0.6 mg/L GA and 10 mg/L MNPs-Fe₃O₄. Abbreviations: MNPs-Fe₃O₄, magnetic nanoparticles of Fe₃O₄; GA, gambogic acid; NF-κB, nuclear factor-kappaB.