Anti-Leukemic Effects of Idesia polycarpa Maxim Branch on Human B-Cell Acute Lymphoblastic Leukemia Cells

Abstract

Patients with pediatric B-cell acute lymphoblastic leukemia (B-ALL) have a high survival rate, yet the prognosis of adults and patients with relapsed/refractory disease is relatively poor. Therefore, it is imperative to develop new therapeutic strategies. Here, we screened 100 plant extracts from South Korean Flora and investigated their anti-leukemic effect using CCRF-SB cells as a B-ALL model. The top cytotoxic extract identified in this screening was the Idesia polycarpa Maxim. branch (IMB), which efficiently inhibited the survival and proliferation of CCRF-SB cells, while having minimal to no impact on normal murine bone marrow cells. Mechanistically, the IMB-induced proapoptotic effect involves the increase of caspase 3/7 activity, which was shown to be associated with the disruption of the mitochondrial membrane potential (MMP) through the reduction in antiapoptotic Bcl-2 family expression. IMB also promoted the differentiation of CCRF-SB cells via the upregulation of the expression of differentiation-related genes, PAX5 and IKZF1. Given that resistance to glucocorticoid (GC) is often found in patients with relapsed/refractory ALL, we investigated whether IMB could restore GC sensitivity. IMB synergized GC to enhance apoptotic rate by increasing GC receptor expression and downmodulating mTOR and MAPK signals in CCRF-SB B-ALL cells. These results suggest that IMB has the potential to be a novel candidate for the treatment of B-ALL.

Keywords: B-cell acute lymphoblastic leukemia; Idesia polycarpa Maxim branch; anti-leukemic effect; differentiation; glucocorticoid resistance.

Figure 1

IMB inhibited cell growth in CCRF cells. (A) ALL cell line (CCRF-SB) or normal bone marrow cells (BM) was exposed to IMB (0, 60, 80, or 100 µg/mL) for 24 h and the MTS assay was performed to measure cell viability. N.S: not significant. (B) CCRF-SB cells were treated with IMB (0, 10, 20, 30, 40, or 50 µg/mL) for 120 h. Cell number was measured using the trypan blue assay. (C) CCRF-SB cells were treated with IMB (0, 40, or 50 µg/mL) for 24 h. The images display a representative experiment from three independent experiments. (D) CCRF-SB cells were cultured with IMB (50 µg/mL) for 24 h and mRNA expression of cell cycle-related genes was analyzed by RT-PCR. (E) CCRF-SB cells were treated with increasing concentrations of IMB (0, 60, 80, or 100 µg/mL) for 24 h, followed by PI staining, and then, the apoptotic cells were examined by flow cytometry. (F) To examine the effect of long-term treatment of IMB on CCRF-SB, CCRF-SB cells were treated with vehicle (DMSO) or IMB (15 µg/mL) for 2 weeks in methylcellulose and the number and size of the clones were measured. The colony images were representative of three independent experiments. Statistical significance was measured using the two-tailed Mann–Whitney test or the two-tailed one-way ANOVA test (* p < 0.05).

Figure 2

IMB has low toxicity to normal cells. (A) After a vehicle (100 µL of dimethyl sulfoxide, DMSO) or IMB (100 mg kg⁻¹) was injected into athymic nude mice intraperitoneally daily for 21 days, the ratio of leukemic cells in bone marrow, spleen, or peripheral blood was measured by flow cytometry to confirm the toxicity of IMB to nude mice. (B) For histological analysis of the mouse heart, kidney, liver, lung, and spleen, H&E staining was performed. (C) Mouse bodyweights were measured every other day for 21 days while the injection was administered. Statistical significance was measured using the two-tailed one-way ANOVA test.

Figure 3

IMB reduced antiapoptotic protein expression and disrupted mitochondrial membrane potential (MMP). (A) The caspase-3/7 activity was observed using ELISA-based bioluminescence assays after treatment with vehicle (DMSO) or IMB (100 µg/mL) for 24 h in CCRF-SB cells. (B) Western blot was performed to determine the effect of IMB on the antiapoptotic proteins Mcl-1 and Bcl-2 in CCRF-SB cells. β-actin was used as a loading control. (C) CCRF-SB cells were cultured with vehicle (DMSO) or IMB (100 µg/mL) for 24 h and stained with JC-1, followed by observing MMP using a fluorescence microscope. Statistical significance was measured using the two-tailed Mann–Whitney test (* p < 0.05).

Figure 4

IMB affects the phenotypic differentiation of CCRF-SB cells. (A) CCRF-SB cells were treated with vehicle (DMSO) or IMB (40 µg/mL) for 120 h and Giemsa staining was performed. The images display a representative experiment from three independent experiments. (B) CCRF-SB cells were cultured with IMB (40 µg/mL) for 72 h and mRNA expression of differentiation-related genes (PAX5 and IKZF1) was analyzed by RT-PCR. Statistical significance was measured using the two-tailed Mann–Whitney test (* p < 0.05).

Figure 5

Cotreatment with IMB and dexamethasone (Dex) overcame glucocorticoid resistance. (A) CCRF-SB cells were exposed to IMB (0, 40, 50, or 60 µg/mL) and/or Dex (0, 10, 100, or 1000 nM) for 48 h or 72 h, and the MTS assay was performed to measure cell viability. (B) CCRF-SB cells were treated with IMB for 48 h or 72 h, followed by mRNA expression analysis of the glucocorticoid receptor alpha (GRα) gene by RT-PCR. (C) Western blotting was performed to determine the expressions of p-Akt, p-4EBP1, Bcl-2, Mcl-1, and p-Erk1/2 in CCRF-SB cells. β-actin was used as a loading control. Statistical significance was measured using the two-tailed Mann–Whitney test or the two-tailed one-way ANOVA test (* p < 0.05).