Iron Oxide Nanoparticles Combined with Cytosine Arabinoside Show Anti-Leukemia Stem Cell Effects on Acute Myeloid Leukemia by Regulating Reactive Oxygen Species

Abstract

Background and aim: Acute myeloid leukemia (AML), initiated and maintained by leukemia stem cells (LSCs), is often relapsed or refractory to therapy. The present study aimed at assessing the effects of nanozyme-like Fe₃O₄ nanoparticles (IONPs) combined with cytosine arabinoside (Ara-C) on LSCs in vitro and in vivo.

Methods: The CD34⁺CD38^-LSCs, isolated from human AML cell line KG1a by a magnetic activated cell sorting method, were treated with Ara-C, IONPs, and Ara-C+ IONPs respectively in vitro. The cellular proliferation, apoptosis, reactive oxygen species (ROS), and the related molecular expression levels in LSCs were analyzed using flow cytometry, RT-qPCR, and Western blot. The nonobese diabetic/severe combined immune deficiency mice were transplanted with LSCs or non-LSCs via tail vein, and then the mice were treated with Ara-C, IONPs and IONPs plus Ara-C, respectively. The therapeutic effects on the AML bearing mice were further evaluated.

Results: LSCs indicated stronger cellular proliferation, more clone formation, and more robust resistance to Ara-C than non-LSCs. Compared with LSCs treated with Ara-C alone, LSCs treated with IONPs plus Ara-C showed a significant increase in apoptosis and ROS levels that might be regulated by nanozyme-like IONPs via improving the expression of pro-oxidation molecule gp91-phox but decreasing the expression of antioxidation molecule superoxide dismutase 1. The in vivo results suggested that, compared with the AML bearing mice treated with Ara-C alone, the mice treated with IONPs plus Ara-C markedly reduced the abnormal leukocyte numbers in peripheral blood and bone marrow and significantly extended the survival of AML bearing mice.

Conclusion: IONPs combined with Ara-C showed the effectiveness on reducing AML burden in the mice engrafted with LSCs and extending mouse survival by increasing LSC's ROS level to induce LSC apoptosis. Our findings suggest that targeting LSCs could control the AML relapse by using IONPs plus Ara-C.

Keywords: Fe3O4 nanoparticles; acute myeloid leukemia; cytosine arabinoside; leukemia stem cells; reactive oxygen species.

Figure 1

Isolation and identification of LSCs. (A) FCM analysis of CD34⁺CD38^–cells in HL-60 cells (0.912%), KG1 cells (7.30%), and KG1a cells (32.9%). Isolation of CD34⁺CD38^–cells by magnetic activated cell sorting method from KG1a cell line and the purity of CD34⁺CD38^–cells (94.3%) was identified by FCM. (B) Cellular viability assay for LSCs and Non-LSCs incubated with various concentration of Ara-C (μM). (C) Cell proliferation assay for LSCs and Non-LSCs in vitro. (D) Clone assay for LSCs and Non-LSCs in the soft agar media. The black arrows represent the positive clones. (E) Statistical analysis of clone formation rate. **p <0.01 and ***p<0.001 were calculated by t test, referring to the statistically significant difference as compared to respective group.

Figure 2

FCM analysis of levels of apoptosis and ROS in LSCs and Non-LSCs treated with different agents. (A) IC₅₀ assay for KG1a cells analyzed by FCM and the cytotoxicity of IONPs to KG1a cells incubated with various concentration of IONPs (μg/mL). (B) Statistical analysis of the apoptosis rate. (C) FCM analysis of apoptosis rates of LSCs and Non-LSCs incubated with various agents. (D) Statistical analysis of apoptosis rates. (E) The cleaved caspase-3 expression analyzed by Western blot. (F) FCM analysis of ROS levels in LSCs and Non-LSCs treated with different agents. (G) Statistical analysis of ROS level in LSCs and Non-LSCs. *p <0.05, **p <0.01 and ***p < 0.001 were calculated by t test, referring to the statistically significant difference as compared to respective group.

Figure 3

Analysis of the expression levels of gp91-phox and SOD1. (A and B) The expression levels of gp91-phox and SOD1 analyzed by Western Blot in LSCs and Non-LSCs treated with different agents. (C–G) Semi-quantification of the expression levels of gp91-phox, SOD1, CD38, DEPTOR, and IFITM3 detected by RT-qPCR in LSCs and Non-LSCs treated with different agents. *p <0.05, **p <0.01 and ***p < 0.001 were calculated by t test, referring to the statistically significant difference as compared to respective group.

Figure 4

Assessment of AML xenograft model in NOD/SCID mice. (A) Weight of mouse body weights once every 10 days in mice injected with 5×10⁵ CD34⁺CD38^–AML cells or PBS through tail veins. (B) WBC counts of peripheral blood once every 10 days in mice injected with 5×10⁵ CD34⁺CD38^–AML cells or PBS. (C) HGB level of peripheral blood once every 10 days in mice injected with 5×10⁵ CD34⁺CD38^– AML cells or PBS. (D) Representative images of the size of spleens taken from the mice injected with 5×10⁵ CD34⁺CD38^–AML cells or PBS 30 days later. (E) The statistical analysis of spleen index in AML xenograft mice and control mice. (F) Representative images of AML cell shape (Wright-giemsa staining) on the smears of peripheral blood (PB, top, arrow, magnification × 1000) and juvenile cell shape (Wright-giemsa staining) on the smears of bone morrow (BM, bottom, arrow, magnification × 1000) in AML xenograft mice and control mice. (G) Statistical analysis of juvenile cell percentage in BM. *p <0.05, **p <0.01 and ***p < 0.001 were calculated by t test, referring to the statistically significant difference as compared to respective group.

Figure 5

Detection of WBC counts and HGB level in AML xenograft model after treated with different agents. (A) Dynamic state detection of WBC counts in AML bearing mice injected with Non-LSCs or LSCs after treated with different agents. (B) Dynamic state detection of HGB level in AML bearing mice injected with Non-LSCs or LSCs after treated with different agents. All the data represent as mean ± S.D. (n = 6); *P < 0.05, ** P < 0.01 and *** P < 0.001 were calculated by t test, referring to the statistically significant difference as compared to respective group.

Figure 6

Analysis of therapeutic effects of different agents on AML-bearing mice. (A and B) Representative images of blood cell shape (A) and AML cell shape (B) (Wright-giemsa staining, arrow, magnification × 400) on the smears of peripheral blood samples collected from the mice injected with LSCs after treated with different agents. (C and D) Representative images of juvenile cell shape (Wright-giemsa staining, arrow, magnification × 1000) on the smears of bone morrow (BM) in Non-LSCs (C) and in LSCs (D) xenograft mice after treated with different agents. (E) Statistical analysis of juvenile cell percentage of BM in mice after treated with different agents. (F) FCM analysis of the CD45⁺cells of bone marrow cell populations in mice treated with various agents. ***p<0.001 was calculated by t test, referring to the statistically significant difference as compared to respective group.