Induction of apoptosis by HBI-8000 in adult T-cell leukemia/lymphoma is associated with activation of Bim and NLRP3

Abstract

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell malignancy caused by human T-cell lymphotropic virus 1. Treatment options for acute ATL patients include chemotherapy, stem cell transplantation, and recently the anti-chemokine (C-C motif) receptor 4 antibody, although most patients still have a poor prognosis and there is a clear need for additional options. HBI-8000 is a novel oral histone deacetylase inhibitor with proven efficacy for treatment of T-cell lymphomas that recently received approval in China. In the present study, we evaluated the effects of HBI-8000 on ATL-derived cell lines and primary cells obtained from Japanese ATL patients. In most cases HBI-8000 induced apoptosis in both primary ATL cells and cell lines. In addition, findings obtained with DNA microarray suggested Bim activation and, interestingly, the contribution of the NLR family, pyrin domain containing 3 (NLRP3) inflammasome pathway in HBI-8000-induced ATL cell death. Further investigations using siRNAs confirmed that Bim contributes to HBI-8000-induced apoptosis. Our results provide a rationale for a clinical investigation of the efficacy of HBI-8000 in patients with ATL. Although the role of NLRP3 inflammasome activation in ATL cell death remains to be verified, HBI-8000 may be part of a novel therapeutic strategy for cancer based on the NLRP3 pathway.

Keywords: Adult T-cell leukemia lymphoma; Bim; NLRP3; apoptosis; histone deacetylase inhibitors.

Figure 1

Apoptosis in adult T‐cell leukemia/lymphoma (ATL)‐derived cell lines induced by HBI‐8000. (a) ATL‐derived cell lines and Jurkat cells (2–5 × 10⁵/mL) were treated with either the vehicle or indicated concentrations of HBI‐8000 for 72 h, then cell viability was evaluated using MTS assay. All experiments were carried out in triplicate and the results are expressed as the mean ± SD. (b–d) ATL‐derived cell lines and Jurkat cells (2–5 × 10⁵/mL) were treated with either the vehicle or 4 μM HBI‐8000 for 48 h, and collected. (b) Annexin‐V/propidium iodide staining was carried out and the percentage of annexin‐V‐positive cells was evaluated. Experiments were carried out in triplicate and the results are expressed as the mean ± SD. (c) Cell cycle analysis. The percentage of cells in the sub‐G₁, G₁, S, and G₂/M phase was evaluated. Increased numbers of cells in the G₁ phase and those with G₁ arrest were observed among KK1 and SO4 cells treated with HBI‐8000. (d) Evaluation of loss of mitochondrial membrane potential. Cells were incubated with JC‐1 dye and analyzed using FCM to determine the percentage with low JC‐1 red fluorescence. Except for KK1 cells, most of the cell lines showed decreased mitochondrial membrane potential (ΔψM). (e) Activated (cleaved) caspase‐3 was evaluated using an Apopcyto caspase‐3 colorimetric assay kit. Values for fold induction of signaling (HBI‐8000‐treated cells/non‐treated cells) are indicated.

Figure 2

HBI‐8000 inhibits cell viability and induces apoptosis in primary adult T‐cell leukemia/lymphoma (ATL) cells. Cells (1 × 10⁶/mL) were treated with either vehicle or indicated concentrations of HBI‐8000. (a) After 72 h, cell viability was evaluated using MTS assay. All experiments were carried out in triplicate and the results are expressed as the mean ± SD. (b) After 48 h, annexin‐V/propidium iodide staining was carried out and the percentage of annexin‐V‐positive cells was evaluated. Experiments were carried out in triplicate and the results are expressed as the mean ± SD.

Figure 3

Analysis of acetylated histones, tumor suppressor genes, and antibody array in adult T‐cell leukemia/lymphoma (ATL) cell lines and primary ATL cells. (a–d) ATL cell lines (2–5 × 10⁵/mL), primary ATL cells, and normal CD4 lymphocytes (1 × 10⁶/mL) were treated with either the vehicle or the indicated concentrations of HBI‐8000 for 48 h, then collected. Whole‐cell lysates were prepared and Western blotting was carried out. In the membrane for CD4 lymphocytes, KOB cells treated with HBI‐8000 were used as positive control.

Figure 4

Analysis of intrinsic apoptotic and NLRP3 pathways in HBI‐8000‐induced cell death. (a, b) Adult T‐cell leukemia/lymphoma (ATL) cell lines (2–5 × 10⁵/mL), primary ATL cells, and normal CD4 lymphocytes (1 × 10⁶/mL) were treated with 4 or 20 μM HBI‐8000 for 24 h, and collected. Quantitative RT‐PCR assays for Bim, NLRP3, and porphobilinogen deaminase were carried out. Values for fold induction (HBI‐8000 treated/untreated cells) after calculation of the relative ratios as compared to porphobilinogen deaminase are indicated. Quantitative data for Bim in HBI‐8000‐treated KK1 cells and NLRP3 in HBI‐8000‐treated SO4 cells showed increases. However, the levels in non‐treated cells were lower than the detectable limit (star). Similarly, quantitative data for Bim in non‐treated LMWT5 cells, and NLRP3 in non‐treated KOB and KK1 cells showed quite low values. (c–f) Two ATL cell lines (2–5 × 10⁵/mL), three types of primary ATL cells, and normal CD4 lymphocytes (1 × 10⁶/mL) were treated for 48 and 24–48 h, respectively, with either the vehicle or the indicated concentrations of HBI‐8000. After collecting cells, Western blot analysis was carried out. In the membrane for CD4 lymphocytes, KOB cells treated with HBI‐8000 or THP‐1 cells were used as positive control. The antibody for procaspase‐1 used in this study is known to detect caspase‐1 isoforms (β, γ, δ) as well as caspase‐1α. Casp, caspase.

Figure 5

Knockdown experiments using siRNA of Bim and NLRP3 in HBI‐8000‐induced cell death. At 12 h after transfection, cells (1–2 × 10⁵/mL) were incubated for 24 h with either the vehicle or 1–2 μM HBI‐8000. (a) Cell viability relative to untreated cells in each experiment was evaluated using MTS assay. (b) The percentage of annexin‐V‐positive cells was evaluated by FCM. Results in 5 (a, b) are expressed as the mean ± SD for three independent experiments and were analyzed using Student's t‐test with Welch's correction (*P < 0.05, **P < 0.01) as compared with HBI8000‐treated si‐Control cells. (c) After transfection using si‐Control, siRNA#1, or #2, cells (1–2 × 10⁵/mL) were incubated for 24 h with either vehicle or 1–2 μM HBI‐8000. Cells were harvested and Western blot analysis was carried out. Representative results using siRNA#1 are shown.