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 , Among them, bone pain and hypercalcemia result from, first, the activation of osteoclasts (OCs) through release of OC-stimulating factors from myeloma cells , 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 . 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 . It has also been reported that BPs induce the apoptosis of macrophages  and breast cancer cells .
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).
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.
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).
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).
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.
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.
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).
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.
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).
BPs have been widely used as a standardized supportive therapy to reduce SREs in myeloma patients through the suppression of OCs . 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 . 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 . It was reported that YM-175 has a stronger inhibitory effect on sterol biosynthesis than pamidronate . YM-175 has also been shown to inhibit sterol biosynthesis in J774 mouse macrophage cells .
Macrophages and DCs have important roles in human immunity. Macrophages arise by differentiation from monocytes . 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.
We thank R. Izawa, H. Ozaki, and M. Takahashi for their technical assistances.