Paclitaxel-Fe3O4 nanoparticles inhibit growth of CD138(-) CD34(-) tumor stem-like cells in multiple myeloma-bearing mice

Abstract

Background: There is growing evidence that CD138(-) CD34(-) cells may actually be tumor stem cells responsible for initiation and relapse of multiple myeloma. However, effective drugs targeted at CD138(-) CD34(-) tumor stem cells are yet to be developed. The purpose of this study was to investigate the inhibitory effect of paclitaxel-loaded Fe3O4 nanoparticles (PTX-NPs) on CD138(-) CD34(-) tumor stem cells in multiple myeloma-bearing mice.

Methods: CD138(-) CD34(-) cells were isolated from a human U266 multiple myeloma cell line using an immune magnetic bead sorting method and then subcutaneously injected into mice with nonobese diabetic/severe combined immunodeficiency to develop a multiple myeloma-bearing mouse model. The mice were treated with Fe3O4 nanoparticles 2 mg/kg, paclitaxel 4.8 mg/kg, and PTX-NPs 0.64 mg/kg for 2 weeks. Tumor growth, pathological changes, serum and urinary interleukin-6 levels, and molecular expression of caspase-3, caspase-8, and caspase-9 were evaluated.

Results: CD138(-) CD34(-) cells were found to have tumor stem cell characteristics. All the mice developed tumors in 40 days after injection of 1 × 10(6) CD138(-) CD34(-) tumor stem cells. Tumor growth in mice treated with PTX-NPs was significantly inhibited compared with the controls (P < 0.005), and the groups that received nanoparticles alone (P < 0.005) or paclitaxel alone (P < 0.05). In addition, the PTX-NPs markedly inhibited interleukin-6 secretion, increased caspase-8, caspase-9, and caspase-3 expression, and induced apoptosis of tumor cells in the treated mice.

Conclusion: PTX-NPs proved to be a potent anticancer treatment strategy that may contribute to targeted therapy for multiple myeloma tumor stem cells in future clinical trials.

Keywords: Fe3O4 nanoparticles; multiple myeloma; paclitaxel; tumor stem cells.

Figure 1

CD138⁻ CD34⁻ cell characteristics. (A) Proliferation potency in common medium. (B) Clonogenicity capability in soft agar medium with 15 day culture. (C) Clone formation rate. (D) Survival rate after culture of cells with vincristine 5 μg in 96-well plate for 72 hours. (E) Cell migration rate. (F) Cell invasion rate. (G) Images (arrows) of tumors in NOD/SCID mice injected with 1 × 10⁶ human U266 CD138⁻ CD34⁻ multiple myeloma cells and non-CD138⁻ CD34⁻ cells after 70 days. Note: **P < 0.01. Abbreviations: NPs, Fe₃O₄ nanoparticles; PTX, paclitaxel; PTX-NPs, paclitaxel-loaded Fe₃O₄ nanoparticles.

Figure 2

Transmission electron microscopic images of PTX-NPs and paclitaxel release from these particles. (A) Nanoparticles in the transmission electron microscopic images were 7.02 nm in size. (B) Mean hydrodynamic diameter of 65.1 nm for reconstituted PTX-NPs measured by dynamic light scattering. (C) Kinetics of paclitaxel release from PTX-NPs. (D) Stability of drug-loaded nanoparticles. (E) Schematic representation of PTX-NPs. Abbreviation: PTX-NPs, paclitaxel-loaded Fe₃O₄ nanoparticles.

Figure 3

Relative tumor volumes and tumor images. (A) In around 40 days, palpable tumors in the NOD/SCID mice inoculated with 1 × 10⁶ U266 CD138⁻ CD34⁻ cells were treated with normal saline, Fe₃O₄ nanoparticles alone (2 mg/kg), paclitaxel alone (4.8 mg/kg), and PTX-NPs 0.64 mg/kg, respectively, according to a schedule of once a week, 0.2 mL on each occasion for 2 weeks. (B) Tumor sizes for mice treated with various agents. (C) The PTX-NPs group mice had a longer survival time compared with the other groups. Notes: *P < 0.05; **P < 0.01; ***P < 0.005. Abbreviations: ns, no statistical significance; NPs, Fe₃O₄ nanoparticles; PTX, paclitaxel; PTX-NPs, paclitaxel-loaded Fe₃O₄ nanoparticles.

Figure 4

IL-6 concentration detected by enzyme-linked immunosorbent assay. (A) Serum IL-6 concentration. (B) Urinary IL-6 concentration. Note: *P < 0.05; **P < 0.01. Abbreviations: IL-6, interleukin-6; NPs, Fe₃O₄ nanoparticles; PTX, paclitaxel; PTX-NPs, paclitaxel-loaded Fe₃O₄ nanoparticles.

Figure 5

Representative histopathologic changes in tumor tissue in mice after treatment (400×). (A) Hematoxylin and eosin staining, (B) TUNEL staining by immunohistochemistry, (C) Bcl-2 staining by immunohistochemistry, and (D) Bax staining by immunohistochemistry. Notes: *P < 0.05; **P < 0.01; ***P < 0.005. Abbreviations: ns, no statistical significance; NPs, Fe₃O₄ nanoparticles; PTX, paclitaxel; PTX-NPs, paclitaxel-loaded Fe₃O₄ nanoparticles; HE, hematoxylin and eosin; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

Figure 6

Expression of apoptosis-related proteins in multiple myeloma-bearing mouse model after treatment. (A) After treatment, mouse tumor tissues were homogenized and analyzed for expressions of caspase-8, caspase-9, and caspase-3, by Western blotting. GAPDH served as a loading control. (B) Relative intensity of protein expression. Notes: *P < 0.05; **P < 0.01; ***P < 0.005.