YM-175 induces apoptosis of human native monocyte-lineage cells via inhibition of prenylation

Authors

  • Akiyoshi Miwa,

    1. Department of Hematology, National Center for Global Health and Medicine, Shinjyuku-ku, Tokyo 162-8655, Japan
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  • Naoki Takezako,

    Corresponding author
    1. Department of Biochemistry, Jichi Medical University, Shimotsuke-shi, Tochigi 329-0498, Japan
    2. Department of Hematology, National Hospital Organization Disaster Medical Center of Japan, Tachikawa-shi, Tokyo 190-0014, Japan
    3. Department of Medical Informatics, National Hospital Organization Disaster Medical Center of Japan, Tachikawa-shi, Tokyo 190-0014, Japan
    • Department of Medical Informatics, National Hospital Organization Disaster Medical Center of Japan, 3256 Midori-cho, Tachikawa City, Tokyo 190-0014, Japan
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  • Hiroko Hayakawa,

    1. Department of Biochemistry, Jichi Medical University, Shimotsuke-shi, Tochigi 329-0498, Japan
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  • Morisada Hayakawa,

    1. Department of Biochemistry, Jichi Medical University, Shimotsuke-shi, Tochigi 329-0498, Japan
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  • Shin-ichi Tominaga,

    1. Department of Biochemistry, Jichi Medical University, Shimotsuke-shi, Tochigi 329-0498, Japan
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  • Ken Yanagisawa

    1. Department of Biochemistry, Jichi Medical University, Shimotsuke-shi, Tochigi 329-0498, Japan
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Abstract

Nitrogen-containing bisphosphonates (NCBPs) have been widely used as standard supportive therapy to reduce skeletal-related events (SREs) in myeloma patients through suppression of osteoclast activity. In various prospective randomized trials that were performed following preliminary reports concerning efficacy, NCBPs have shown a significant beneficial effect on myeloma bone disease through both suppression of bone resorption and direct antimyeloma activity. Thus, NCBPs have an influence on many types of human cells. In this study, we examined the effect of an NCBP (YM-175) on an apoptosis of a monocytic cell line and of human native monocytes/macrophages and dendritic cells (DCs). We confirmed that monocytes, monocyte-derived macrophages, DCs, and a monoblastic cell line (THP-1) showed dose-dependent and time-dependent apoptosis related to the activation of caspases after exposure to YM-175 at concentrations below that at which the apoptosis of myeloma cell lines was induced. Such apoptosis of monocytic cells was suppressed by the addition of farnesol or geranylgeraniol. These findings suggest that the inhibition of monocyte-lineage cells or DCs by NCBPs might interfere with phagocytic activity or pathogen-presenting activity. Am. J. Hematol. 2012. © 2012 Wiley Periodicals, Inc.

Introduction

Multiple myeloma is a lethal disease that involves plasma-cell proliferation frequently associated with osteolytic lesions. Most patients die despite the use of several innovative treatment modalities, and the percentage of those who live for 10 years is limited [1, 2]. Patients have various degrees of cytopenia, significant bone pain, hypercalcemia, and renal failure [3, 4], which result in substantial morbidity [5], Among them, bone pain and hypercalcemia result from, first, the activation of osteoclasts (OCs) through release of OC-stimulating factors from myeloma cells [6], and second, the suppression of osteoblasts through release of osteoblast inhibitory factors from myeloma cells such as DKK-1.

Bisphosphonates (BPs) have been widely used as a standard supportive therapy to reduce skeletal-related events (SREs) in myeloma patients through suppression of OC activity [7]. Several reports have suggested that BP treatment may be associated with an increase in survival, raising the possibility that these compounds might have a direct effect on tumor cells [8, 9]. YM-175 (incadronate) has a direct effect on human myeloma cell lines in vitro [8]. It has also been reported that BPs induce the apoptosis of macrophages [10] and breast cancer cells [11].

Dendritic cells (DCs) are antigen-presenting cells for cytotoxic T lymphocytes (CTLs). Myeloma cells are highly sensitive to antigen-specific CTLs, which are activated when tumor-specific antigens are presented by DCs [12, 13]. In addition, DCs are generated from monocytic cells [14, 15]. Therefore, we speculated that YM-175 could have an effect on monocytes, macrophages, and DCs, which play an important role in human immunity. The present study was performed to investigate the apoptotic effect of YM-175, a nitrogen-containing bisphosphonate (NCBP), on a monocytic-cell line and human peripheral monocytic cells.

Materials and Methods

Chemicals and antibodies

YM-175 (incadronate, cycloheptylaminomethylene-1, and 1-BP) was obtained from Astellas Pharma (Tokyo, Japan), and pamidronate disodium salt hydrate, zoledronic acid monohydrate, 12-O-tetradecanoylphorbol 13-acetate, phorbol ester (PMA), geranylgeraniol (GGOH), and farnesol (FOH) were purchased from Sigma-Aldrich (St. Louis, MO). We used the following antibodies: anticaspase-3 antibody (BioVision, Lyon, France), anticaspase-4 antibody (MBL, Nagoya, Japan), anticaspase-7 antibody (BioVision), anti-Rap1A-antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Rap1-antibody (Santa Cruz Biotechnology), horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Bio-Rad, Hercules, CA), HRP-conjugated anti-goat IgG antibody (ZYMED Laboratories, South San Francisco, CA), and HRP-conjugated anti-rabbit IgG antibody (Amersham Biosciences, Piscataway, NJ).

Cell culture

A human monocytic leukemia cell line, THP-1, and human myeloma cell lines, ARH-77 and RPMI8226, were obtained from the Cell Resource Center for Biomedical Research, Tohoku University. These cells were cultured in RPMI1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Sigma-Aldrich).

Human peripheral blood mononuclear cells derived CD14-positive monocytes were purchased from Cambrex Bio Science (Walkersville, MD). Human CD14-positive monocytes were cultured in Macrophage-SFM medium (GIBCO, Grand Island, NY) supplemented with 50 ng/ml recombinant GM-CSF (rGM-CSF; R&D Systems, Minneapolis, MD). CD14-positive human monocytes were cultured for 12 hr and differentiated into DCs by the addition of 50 ng/ml lipopolysaccharide derived from Escherichia coli strain 055: B5 (Sigma-Aldrich), as described previously [14, 15]. Using FACS analysis, these human DCs were assessed for CD1a, 80, 83, 86, and 209 positivity. CD14-positive human monocytes were cultured for 24 hr and then differentiated into the macrophages by the addition of PMA (10 ng/ml). Using FACS analysis, these human macrophages were assessed for CD11c and CD32 positivity.

FACS analysis

Cell-surface staining used direct immunofluorescence (FACScan; Becton Dickinson), and the samples were analyzed using Cell Quest software (Becton Dickinson). Staining was performed with the following FITC- and phycoerythrin (PE)-labeled monoclonal antibodies: PE-CD1a, PE-CD11c, FITC-CD32, FITC-CD80, FITC-CD86, FITC-CD209, FITC-mouse IgG1 (all from Pharmingen, San Diego, CA); FITC-CD14, FITC-HLA-DR, PE-mouse IgG1 (all from Becton Dickinson); PE-CD83 (from Coulter/Immunotech, Miami, FL); and FITC-mouse IgG2 (both from Sigma, St Louis, MO). Primary antibodies were directed toward a panel of cell-surface markers and compared to the appropriate isotype-matched controls.

Assays of apoptosis

The effects of YM-175 on monocytic cells were assessed using the Vybrant® Apoptosis Assay Kit #2 (Molecular Probes, Eugene, OR) following the manufacturer's recommendations. Briefly, 1 × 105/ml (cell lines) or 1 × 106/ml (CD14-positive monocytes and DCs) were incubated in a 96-well microculture plate (CellSeed, Tokyo, Japan) in the absence or presence of various concentrations of YM-175. After the indicated periods, the cells were washed twice and analyzed by flow cytometry.

Western blot analysis

Protein samples were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Following electrophoresis, the proteins were blotted onto a polyvinylidene difluoride membrane (Millipore Co.). For the detection of caspases, the membranes were reacted with primary antibody against the indicated caspase at room temperature. Then the membranes were left for 1 hr to react with anti-mouse IgG (Bio-Rad, Hercules, CA) or anti-goat IgG antibody (ZYMED Laboratories, South San Francisco, CA) conjugated with HRP, as the secondary antibody. Bands were visualized on X-ray films using an ECL system (Amersham Biosciences).

Results

YM-175 induces apoptosis of THP-1 cells

We first examined the effects of YM-175 on the apoptosis of myeloma cell lines. The cells cultured with YM-175 for 72 hr, and induced apoptosis was assessed by FACS analysis (Fig. 1).

Figure 1.

YM175 induces apoptosis in human myeloma cell lines. Myeloma cell lines [RPMI8266 (a, b) and ARH-77 (c, d)] were cultured for 72 hr with (b, d) or without (a, c) 450 μmol/l of YM175. Cultured cells were analyzed using FACS method. The lower right area indicates early apoptotic cells, and the upper right area indicates late apoptotic cells.

Although, the sensitivity to YM-175 varied among the myeloma cell lines and was low in the presence of high concentration (450 μmol/l).

We tested other BPs (pamidronate disodium salt hydrate and zoledronic acid monohydrate) and YM-175 to assess their effects on the apoptosis of a monocytic cell line (THP-1 cells). When cells were treated with YM175 or other BPs, YM-175 and the other BPs all had an apoptotic effect on THP-1 cells (Fig. 2A). We performed subsequent examinations using YM-175, based on this result. YM-175 induced apoptosis of THP-1 cells in a time-dependent manner (Fig. 2B). THP-1 cells were also treated with various concentrations of YM-175 for 72 hr (Fig. 3), and the dose dependency was also observed. Furthermore, THP-1 cells were reveled to be more susceptible to YM-175 than myeloma cell lines.

Figure 2.

Time course of apoptosis of the THP-1 human monocytic cell line caused by YM-175, zoledronic acid monohydrate, and pamidronate. A: THP-1 cells were treated with YM-175, zoledronic acid monohydrate, or pamidronate and subjected to FACS analysis after 48 hr [(a) no drugs, (b) YM-175(20 μmol/l), (c) zoledronic acid monohydrate (20 μmol/l), and (d) pamidronate (20 μmol/l)]. Early apoptotic cells are seen in the lower right area. B: THP-1 cells were treated with 450 μmol/l of YM175. Cultured cells were analyzed using FACS method at various period [(a) 0, (b) 6, (c) 12, (d) 24, (e) 36, (f) 48, and (g) 72 hr]. The lower right area indicates early apoptotic cells, and the upper right area indicates late apoptotic cells.

Figure 3.

YM175 induces apoptosis of THP-1 cells in a dose-dependent manner. THP-1 cells were treated with various concentrations of YM175 for 72 hr. Cultured cells were analyzed using FACS method [(a) 0, (b) 12.5, (c) 25, (d) 50, (e) 75, (f) 100, (g) 200, (h) 300, (i) 400, and (j) 450 μmol/l). The lower right area indicates early apoptotic cells, and the upper right area indicates late apoptotic cells.

YM-175 induces apoptosis in a caspase-dependent manner

To examine the pathway of apoptosis induced by YM-175, we carried out Western blot analysis using anticaspase antibodies at various periods (Fig. 4). The THP-1 cells were treated with YM-175 (450 μmol/l). The cleaved caspase-3 was detected at 12 hr and peaked after 60 hr (Fig. 4a). Caspase-7 activation began after 36 hr (Fig. 4b) and caspase-4 activation also began after 36 hr, and the activation was maximum at 60 hr (Fig. 4c). These results indicate that caspase activation plays a role in the YM-175-induced apoptosis observed in the THP-1 cells.

Figure 4.

YM-175 induces apoptosis in a caspase-dependent manner. Western blot analysis using anticaspase antibodies at various periods. The THP-1 cells were treated with YM-175 (450 μmol/l). Whole-cell extracts were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and immunoblots were probed with anticaspase-3 (Fig. 4a), anticaspase-7 (Fig. 4b), and anticaspase-4 (Fig. 4c).

YM-175 prevents the prenylation of Rap1A

NCBPs inhibit farnesyl diphosphate (FPP) synthase, an enzyme in the mevalonate pathway. YM-175 also inhibits the synthesis of FPP and its derivative, geranylgeranyl diphosphate (GGPP) in the same manner as other NCBP. FPP and GGPP are essential for the posttranslational prenylation and thus proper functioning of small GTP-binding proteins (such as Ras, Rho, and Rap1A). Therefore, we examined the effects of YM-175 on the prenylation of Rap1A on behalf of small GTP-binding proteins.

After the pretreatment of THP-1 cells with YM-175, the cells were harvested at various periods for the analysis of prenylation of Rap1A, and we confirmed that the inhibition of prenylation began after 12 hr (Fig. 5A).

Figure 5.

YM175 prevents the prenylation of RAP1A, and prenylation of Rap1A was remarkably recovered by preincubation with FOH and GGOH in dose-dependent manner. A: Cell lysates from THP-1 treat with 450 μmol/l of YM175 for 0–72 hr were subjected to Western blot analysis using antibodies for the unprenylated RAP1A and total RAP1A. B: THP-1 cells were pretreated with FOH at the indicated concentration for 1 hr and then incubated with or without YM-175 for 48 hr. Representative immunoblots (10 μg protein/lane) are shown. C: THP-1 cells were pretreated with geranylgeraniol (GGOH) at the indicated concentration for 1 hr and then incubated with or without YM-175 for 48 hr. Representative immunoblots (10 μg protein/lane) are shown.

Next, we examined the addition of cell-permeable precursors of FPP and GGPP; FOH and GGOH. Western blot analysis revealed that prenylation of Rap1A was remarkably recovered by preincubation with FOH (Fig. 5B) and GGOH (Fig. 5C) in dose-dependent manner. FACS analysis also revealed that apoptosis of THP-1 cells was prevented by the addition of GGOH (Fig. 6A) or FOH (Fig. 6B). These findings suggest that YM-175 inhibits the prenylation of small GTP-binding proteins and leads the THP-1 cells to apoptosis.

Figure 6.

Apoptosis of THP-1 cells is prevented by the addition of GGOH or FOH: FACS analysis. A: FACS analysis of THP-1 cells treated in one of the following ways: cultured in medium only (a), cultured with GGOH (10 μmol/l) (b), cultured with YM-175 (450 μmol/l) (c), cultured with YM-175 (450 μmol/l) plus GGOH (1 μmol/l) (d), cultured with YM-175 (450 μmol/l) plus GGOH (5 μmol/l) (e), and cultured with YM-175 (450 μmol/l) plus GGOH (10 μmol/l) (f). B: FACS analysis of THP-1 cells treated in one of the following ways: cultured in medium only (a), cultured with FOH (10 μmol/l) (b), cultured with YM-175 (450 μmol/l) (c), cultured with YM-175 (450 μmol/l) plus FOH (1 μmol/l) (d), cultured with YM-175 (450 μmol/l) plus FOH (5 μmol/l) (e), and cultured with YM-175 (450 μmol/l) plus FOH (10 μmol/l) (f).

YM-175 induces apoptosis of native human cells via the prevention of the prenylation

Because THP-1 cells are malignant transformed cells, these results should not be directly linked to the roles played by YM-175 in patients. To clarify the effects of YM-175 on human native monocytic cells, we examined them by FACS analysis. First of all, we established a serum-free system, whereby human CD14+ peripheral blood monocytes were induced to rapid differentiation into DCs as described previously [13, 14] (data not shown). Subsequently, we revealed that YM-175 induced apoptosis to DCs derived from human peripheral blood, and it was prevented by the addition of FOH and/or GGOH (Fig. 7). FACS analysis revealed that apoptosis was also induced to human CD14+ peripheral blood monocytes by YM-175, and it was again prevented by the addition of FOH and/or GGOH (Fig. 8).

Figure 7.

YM-175 induces apoptosis of native human DC via the prevention of the prenylation. Human CD14+ peripheral blood monocytes were induced to rapid differentiation into dendritic cells (DCs) under a serum-free condition. FACS analysis of DCs derived from human peripheral blood that were treated in one of the following ways: cultured in medium only (a), cultured with YM-175 (450 μmol/l) (b), cultured with YM-175 (450 μmol/l) plus GGOH (10 μmol/l) (c), and cultured with YM-175 (450 μmol/l) plus FOH (10 μmol/l) (d), and cultured with YM-175 (450 μmol/l) plus GGOH (10 μmol/l) and FOH (10 μmol/l) (e).

Figure 8.

YM-175 induces apoptosis of native human CD14+ peripheral blood monocytes via the prevention of the prenylation. FACS analysis of human CD14+ peripheral blood was treated in one of the following ways: cultured in medium only (a), cultured with YM-175 (450 μmol/l) (b), cultured with YM-175 (450 μmol/l) plus GGOH (10 μmol/l) (c), cultured with YM-175 (450 μmol/l) plus FOH (10 μmol/l) (d), and cultured with YM-175 (450 μmol/l) plus GGOH (10 μmol/l) and FOH (10 μmol/l) (e).

Discussion

BPs have been widely used as a standardized supportive therapy to reduce SREs in myeloma patients through the suppression of OCs [7]. The mechanism of OC-suppressive effects is related mainly with the specific suppression of FPP synthase catalyzing the conversion of geranyl diphosphate to FPP [15, 16]. In addition, suppression of another enzyme GGPP has been reported [17]. The suppression of FPP synthase and GGPP synthase is linked with decreased prenylation of intracellular small G-proteins [15, 16, 18]. It was confirmed that small G-protein is responsible for cell growth, antiapoptotic effects, and survival of OCs [15–21]. Therefore, when prenylation of small G-proteins is disturbed by BPs, the function of OCs might be severely affected.

Through various prospective randomized trials following preliminary reports concerning the efficacy, BPs have shown lots of significant beneficial effects on myeloma bone disease via suppression of bone resorption [22–25]. Following these studies, the administration of BPs has been listed as one of the standardized supportive therapies in myeloma guidelines presented successively by several study groups [26–30].

NCBPs induce apoptosis of human myeloma cells and have antitumor activity [8, 9, 31–33]. Some studies have demonstrated the accumulation of unprenylated Rap1A, indicating the uptake of BPs by nonskeletal tumors and inhibition of farnesyl pyrophosphate synthase [33, 34]. These studies have provided evidence that NCBPs show direct antitumor activity against myeloma cells [33, 34]. YM-175 is one of the NCBPs, and it inhibits enzymes involved in the synthesis of farnesyl pyrophosphate from mevalonate [35]. It was reported that YM-175 has a stronger inhibitory effect on sterol biosynthesis than pamidronate [35]. YM-175 has also been shown to inhibit sterol biosynthesis in J774 mouse macrophage cells [35].

Macrophages and DCs have important roles in human immunity. Macrophages arise by differentiation from monocytes [36]. They phagocytose pathogens and present antigens from the pathogens to T cells. DCs also arise from monocytes [14, 15] and act as antigen-presenting cells for CTLs [37, 38]. The present study demonstrated that CD14-positive peripheral blood monocytes, macrophages differentiated from monocytes by phorbol ester, CD209-positive cells or DCs induced by cytokines, and a monoblastic cell line (THP-1) showed a dose-dependent and time-dependent increase of apoptosis due to caspase activation after exposure to NCBPs at concentrations lower than those inducing apoptosis of myeloma cell lines. These findings suggest that NCBPs could potentially suppress the presentation of antigens by monocytes and DCs.

Furthermore, recent studies have detected unusual or characteristic adverse events associated with the administration of NCBPs, such as osteonecrosis of the jaw (ONJ) [39–41]. The precise mechanism of ONJ has not been elucidated, even though various factors and conditions have been analyzed to assess their roles in this devastating adverse event. Several studies have found that actinomycetes, which are phagocyted by macrophages, exist in the focal lesions of ONJ patients [42, 43]. These findings may indicate that apoptosis of monocyte-lineage cells mediated via the loss of prenylation of small G-proteins could contribute to ONJ.

Further studies need to be done to confirm the influence of apoptosis of monocyte-lineage cells (macrophages and DCs) on human immunity and immunotherapy for myeloma, such as studies in animal models or using PBMCs from myeloma patients.

Acknowledgements

We thank R. Izawa, H. Ozaki, and M. Takahashi for their technical assistances.

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