S100A8 and S100A9 Promote Apoptosis of Chronic Eosinophilic Leukemia Cells

Abstract

S100A8 and S100A9 function as essential factors in inflammation and also exert antitumor or tumorigenic activity depending on the type of cancer. Chronic eosinophilic leukemia (CEL) is a rare hematological malignancy having elevated levels of eosinophils and characterized by the presence of the FIP1L1-PDGFRA fusion gene. In this study, we examined the pro-apoptotic mechanisms of S100A8 and S100A9 in FIP1L1-PDGFRα+ eosinophilic cells and hypereosinophilic patient cells. S100A8 and S100A9 induce apoptosis of the FIP1L1-PDGFRα+ EoL-1 cells via TLR4. The surface TLR4 expression increased after exposure to S100A8 and S100A9 although total TLR4 expression decreased. S100A8 and S100A9 suppressed the FIP1L1-PDGFRα-mediated signaling pathway by downregulating FIP1L1-PDGFRα mRNA and protein expression and triggered cell apoptosis by regulating caspase 9/3 pathway and Bcl family proteins. S100A8 and S100A9 also induced apoptosis of imatinib-resistant EoL-1 cells (EoL-1-IR). S100A8 and S100A9 blocked tumor progression of xenografted EoL-1 and EoL-1-IR cells in NOD-SCID mice and evoked apoptosis of eosinophils derived from hypereosinophilic syndrome as well as chronic eosinophilic leukemia. These findings may contribute to a progressive understanding of S100A8 and S100A9 in the pathogenic and therapeutic mechanism of hematological malignancy.

Keywords: FIP1L1-PDGFRα; S100 protein; TLR4; chronic eosinophilic leukemia; imatinib resistance.

Figure 1

S100A8 and S100A9 induce EoL-1 cell apoptosis via TLR4. (A–D) EoL-1, HL-60, K562, U937, Jurkat cells (A,B), eosinophils, neutrophils, lymphocytes, and monocytes isolated from normal subjects (C,D) were incubated for 24, 48, or 72 h in the absence (Con) or presence of indicated concentrations of S100A8 (A,C) and S100A9 (B,D) (3 < n < 5). (E) EoL-1 cells were pretreated with anti-S100A8 (A8), anti-S100A9 (A9), or rabbit control (Ro) antibodies (2 μg/mL) and subsequently incubated with S100A8 or S100A9 (10 μg/mL) (n = 4). (F) EoL-1 cells were pretreated with or without 1 μM TLR4 inhibitor (TLR4i) and polymyxin B (10 μg/mL) for 1 h, after which the cells were incubated for 72 h in the absence or presence of S100A8 and S100A9 (50 μg/mL) (3 < n < 5). Data are presented relative to the S100A8- or S100A9-treated group, which is set at 100% (E,F). Data are expressed as the means ± SD. *p < 0.05 indicates a significant difference between the S100A8- or S100A9-treated group and stimulator-treated groups. (G) EoL-1 cells were treated with indicated concentrations of LPS and MPLA for 24, 48, and 72 h (n = 3). Apoptosis was analyzed by measuring the binding of annexin V-FITC and PI. (H) Flow cytometry was applied to determine TLR4 expression in EoL-1, HL-60, K562, U937, Jurkat cells, and normal eosinophils, neutrophils, lymphocytes, and monocytes without stimulators (3 < n < 5). Baseline fluorescence was obtained by incubating normal rabbit antibodies and was set at 1. (I, J) EoL-1 cells were incubated for 24, 48, and 72 h in the absence (Con) or presence of S100A8 and S100A9 (10 μg/mL) (n = 3). TLR4 expression was detected using Western blotting (I) and flow cytometry (J). Data are expressed as the means ± SD. *p < 0.05 and **p < 0.01 indicate a significant difference between the control and stimulator-treated groups.

Figure 2

S100A8 and S100A9 suppress downstream signal of FIP1L1-PDGFRα by decreasing its expression. (A) EoL-1 cells were incubated with S100A8 and S100A9 (10 μg/mL) for 24, 48, and 72 h. After cell lysis, phosphorylation and non-phosphorylation of PDGFRα, STAT3, AKT, ERK1/2, p38 MAPK, and JNK were detected in the lysates by applying Western blotting. (B,C) EoL-1 cells were incubated with S100A8 and S100A9 (10 μg/mL) for the indicated time. After cell lysis, lysates were collected for detecting phosphorylation and non-phosphorylation of PDGFRα using Western blotting (B). Total RNAs from EoL-1 cells were collected, and RT-PCR was conducted for detection of PDGFRα (C). β-actin was used as an internal control. **p < 0.01 indicates a significant difference between the control and stimulator-treated groups.

Figure 3

S100A8 and S100A9 are required for the mitochondrial apoptosis pathway. (A) EoL-1 cells were incubated with S100A8 and S100A9 (10 μg/mL) for 24, 48, and 72 h. After cell lysis, the pro-caspase 9, cleaved caspase 9, pro-caspase 3, caspase 3, cleaved caspase 3, AIF, Mcl-1, Bcl-2, Bax, phospho-Bad, and Bad in the lysates were detected by Western blotting. (B,C) EoL-1 cells were pretreated with or without 0.5 μM MG132 (B) and 10 μM z-DEVD-fmk (C) for 1 h, followed by incubation with S100A8 and S100A9 (10 μg/mL) for 48 h. Levels of Mcl-1 in the cell lysates were detected by Western blotting. β-actin was used as an internal control. (D) EoL-1 cells were incubated with S100A8 and S100A9 (10 μg/mL) for 24, 48, and 72 h, and supernatants were collected at appropriate time points. EoL-1 cells were incubated with S100A8 and S100A9 (10 μg/mL) for 24, 48, and 72 h in the absence or presence of the supernatant (n = 3). Apoptosis was analyzed by measuring the binding of annexin V-FITC and PI. *p < 0.05 and **p < 0.01 indicate a significant difference between the control and stimulator-treated groups.

Figure 4

S100A8 and S100A9 trigger apoptotic effects on EoL-1-IR cells. (A) EoL-1 and EoL-1-IR cells were incubated for 24, 48, and 72 h in the absence (Con) or presence of imatinib at the indicated concentration. Apoptosis was analyzed by measuring the binding of annexin V-FITC and PI. (B) EoL-1 and EoL-1-1R cells were incubated for 72 h in the absence or presence of imatinib at the indicated concentration. Following cell lysis, phosphorylation and non-phosphorylation of PDGFRα, STAT3, AKT, ERK1/2, p38 MAPK, and JNK in the lysates were detected by Western blotting. (C,D) EoL-1 and EoL-1-IR cells were incubated for 24, 48, and 72 h in the absence (Con) or presence of indicated concentrations of S100A8, S100A9 (C), LPS (100 ng/mL) and MPLA (100 ng/mL) (D) (n = 3). Apoptosis was analyzed by measuring the binding of annexin V-FITC and PI. (E,F) EoL-1-IR cells were incubated for 24, 48, and 72 h in the absence (Con) or presence of S100A8 and S100A9 (10 μg/mL), and TLR4 expression was detected using Western blotting (E) and flow cytometry (F). Data are expressed as the means ± SD. *p < 0.05 and **p < 0.01 indicate a significant difference between the control and stimulator-treated groups. (G,H) EoL-1 cells were incubated with S100A8 and S100A9 (10 μg/mL) for 72 h. After cell lysis, the indicated protein expression in the lysates was detected by Western blotting. β-actin was used as an internal control.

Figure 5

S100A8 and S100A9 suppress tumorigenesis induced by xenograft of EoL-1 and EoL-1-1R in NOD-SCID mice. (A–F) Five-week-old female NOD-SCID (NOD.CB17-Prkdcscid/J) mice were divided into six groups (n = 5 per a group) as described in the materials and methods: untreated, control, S100A8 treatment (0.5 and 1 mg doses), and S100A9 treatment (0.5 and 1 mg doses). The control, S100A8 treatment, and S1009 treatment groups were subcutaneously inoculated in the shoulder with EoL-1 cells (2 × 10⁷/100 μL) (A–C) or EoL-1-IR cells (1 × 10⁷/100 μL) (D–F). Treatment groups were subsequently administered subcutaneous doses of 0.5 or 1 mg S100A8 or S100A9 at days 5 and 7 after cell inoculation. The untreated group was injected with vehicle (PBS). Tumor volume (day 1–11) and tumor weight (day 11) were measured (A,B,D,E). Tumor mass was examined for detecting phospho-PDGFRα, PDGFRα, and Ki-67, applying immunohistochemistry and hematoxylin and eosin stain (Scale bar: 200 μm) (C,F), or subjected to Western blotting to determine phosphorylation and nonphosphorylation of PDGFRα, STAT3, AKT, ERK1/2, p38 MAPK, and JNK (G). β-actin was used as the internal control. *p < 0.05 indicates a significant difference between the control and stimulator-treated groups.

Figure 6

S100A8 and S100A9 elicit apoptosis of eosinophils isolated from CEL and HES patients. (A–D) Eosinophils were isolated from CEL (n = 1) (A), HES (n = 2) (B), and reactive eosinophilia subjects (n = 5) (C), and polymorphonuclear cells and mononuclear cells were separated from AML subjects (n = 5) (D). Eosinophils from CEL, HES, and AML were incubated for 24, 48, or 72 h, and the cells from reactive eosinopohilia were incubated for 48 h with the indicated concentrations of S100A8 and S100A9. Apoptosis was analyzed by measuring the binding of annexin V-FITC and PI. Data are expressed as the means ± SD. *p < 0.05 indicates a significant difference between the control and stimulator-treated groups.

Figure 7

Proposed apoptotic mechanism due to S100A8 and S100A9 in CEL eosinophils. S100A8 and S100A9 trigger apoptosis of chronic eosinophilic leukemia by suppressing FIP1L1-PDGFRα+-mediated proliferation by downregulating the mRNA and protein expression and by inducing mitochondrial-associated apoptosis via TLR4.