ONCOLOGY REPORTS 27: 39-44, 2012
Abstract. Skeletal complications represent major clinical
problems in multiple myeloma (MM). MM cells are known
to induce differentiation of osteoclasts and inhibit osteoblasts.
Receptor activator of nuclear factor-κB ligand (RANKL) and
osteoprotegerin (OPG) are key molecules for osteoclastogen-
esis. Although OPG interacts with tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL), the contribution of TRAIL
to skeletal-related events (SRE) remains a matter of debate. In
the present study, we examined the role of TRAIL in MM bone
lesions. Myeloma cells were purified from 56 MM patients by
CD138-immunomagnetic beads. TRAIL, DKK-1 and MIP1α
RNA expression in purified MM cells was analyzed by real-
time PCR. Immunohistochemistry of TRAIL was performed
on paraffin-embedded plasmacytoma tissue sections. The
concentration of TRAIL in the serum and bone marrow plasma
from MM patients was analyzed by ELISA. TRAIL expres-
sion was significantly higher in MM cells than in plasma cells
from patients with monoclonal gammopathy of undetermined
significance (MGUS). TRAIL staining was detected in the
cytoplasm of myeloma cells. TRAIL expression in MM cells
correlated with bone marrow plasma TRAIL concentration.
TRAIL expression had a positive correlation with osteolytic
markers, such as serum calcium and urinary deoxypyridinoline.
These results suggest that TRAIL, produced from myeloma
cells, may play an important role in bone resorption of MM
patients. Inhibition of this pathway may lead to development of
a new therapeutic approach preventing bone resorption in MM.
Multiple myeloma (MM) is a B-cell neoplasia characterized
by clonal expansion of malignant plasma cells in the bone
marrow. Fractures, skeletal-related plasmacytoma, bone pain
and hypercalcemia are major skeletal-related events (SRE)
in MM patients, which develop through an MM-inflicted
imbalance between osteoclastogenesis and osteoblastogenesis.
Osteoclastogenesis is known to be induced by the receptor
activator of nuclear factor-κβ ligand (RANKL) (1) and is
inhibited by osteoprotegerin (OPG) (2). OPG is also known
to act as a soluble inhibitor of tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL) (3). TRAIL, also known
as APO2L, is one of the tumor necrosis factor superfamily
members and is a potent stimulator of apoptosis (4). Since
OPG is an inhibitor of RANKL, a possible role of TRAIL as
an osteoclast inducer has been suggested (5,6). Some reports
have indicated a direct induction of osteoclast formation by
TRAIL (7). Moreover, a contribution of TRAIL to bone lesion
formation by inducing apoptosis of osteoblasts has also been
reported (8,9). However, the association of TRAIL with bone
lesions in MM remains a matter of debate.
In the present study, we investigated the expression of
TRAIL in purified MM cells and examined its association with
SRE. The concentrations of TRAIL were also determined in
the peripheral blood and bone marrow. Analyses were carried
out to compare the significance of TRAIL relative to other
SRE-related biomarkers, namely Dickkopf-1 (DKK-1) (10) and
macrophage inflammatory protein-1α (MIP1α) (11).
Materials and methods
Cell culture. Human myeloma cell lines, KMS-12-BM (12),
KMS-12-PE (12), U266 (13) and RPMI-8226 (14) were
cultured in RPMI-1640 containing 10% fetal bovine serum at
37˚C under 5% CO2.
Patients. The subjects of the present study consisted of
56 patients with MM and 12 patients with monoclonal
gammopathy of undetermined significance (MGUS). Clinical
specimens were obtained from the MM and MGUS patients
at the Department of Hematology, Kumamoto University
Hospital. Written informed consent was obtained from all
participants according to the Declaration of Helsinki. The
clinical data of the patients (radiological findings, hemograms,
blood chemistry and urinary deoxypyridinoline) were also
TRAIL produced from multiple myeloma cells
is associated with osteolytic markers
YAWARA KAWANO1, SHIKIKO UENO1, MASAHIRO ABE2, YOSHITAKA KIKUKAWA1,
HIROMICHI YUKI1, KENICHI IYAMA3, YUTAKA OKUNO1, HIROAKI MITSUYA1 and HIROYUKI HATA1
1Department of Hematology, Kumamoto University School of Medicine, Kumamoto; 2Department of Medicine and
Bioregulatory Sciences, The University of Tokushima Graduate School of Medical Sciences, Tokushima;
3Department of Surgical Pathology, Kumamoto University Hospital, Kumamoto, Japan
Received July 11, 2011; Accepted August 24, 2011
Correspondence to: Dr Hiroyuki Hata, Department of Hematology,
Kumamoto University Hospital, 1-1-1 Honjo, Kumamoto City,
Kumamoto 860-8556, Japan
Key words: multiple myeloma, TRAIL, bone resorption
KAWANO et al: TRAIL PRODUCING MYELOMA
obtained. The characteristics of the 56 MM patients are
summarized in Table I.
Myeloma cell purification. After isolation of mononuclear
cells from bone marrow samples using Ficoll-Paque Plus (GE
Healthcare, Uppsala, Sweden), myeloma cells were purified
using CD138-immunomagnetic beads (Miltenyi Biotech,
Paris, France) as previously described (15).
cDNA synthesis, RT-PCR and real-time PCR. RNA was
extracted from purified myeloma cells using the TRIzol reagent
(Invitrogen, Carlsbad, CA). cDNA synthesis was performed
using a SuperScript Ⅲ First-Strand Synthesis system for
RT-PCR (Invitrogen) according to the manufacturer's protocol.
The expression of TRAIL was determined using semi-
quantitative RT-PCR. PCR primers for TRAIL (16) and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (17)
were previously reported. The PCR conditions used were as
follows: initial incubation at 94˚C for 3 min, followed by 35
cycles of 94˚C for 60 sec, 64˚C for 60 sec and 72˚C for 60 sec.
Quantitative Taqman PCR was performed using Assay-
on-Demand primers and the Taqman Universal PCR Master
Mix reagent (Applied Biosystems, Foster City, CA). Real-time
RT-PCR was performed using an ABI Prism 7700 Sequence
Detection System (Applied Biosystems). The ΔΔCT method
was utilized to analyze the relative changes in gene expression.
The expression levels of β-actin were used to normalize the
relative expression levels of TRAIL, MIP1α and DKK-1. An
MM cell line, KMS-12-BM, was used as a positive control for
TRAIL and MIP1α. The U266 cell line was used as a positive
control for DKK-1. The expression of each positive control was
defined as 100.
ELISA analysis. Among the 56 MM patients, whose TRAIL
mRNA expressions were analyzed using real-time PCR,
serum samples were available for 31 patients. The TRAIL
concentrations in the serum samples from these MM patients
and 7 healthy donors were analyzed using a TRAIL/APO2L
ELISA kit (Diaclone, Besançon, France) according to the
manufacturer's instructions. We also analyzed the TRAIL
concentrations in bone marrow plasma samples from 12 MM
Immunohistochemistry. Immunohistochemistry was
performed on paraffin-embedded plasmacytoma tissue
sections using a rabbit polyclonal anti-TRAIL primary anti-
body (clone ab2056; Abcam, Cambridge, UK) according to the
Statistical analysis. The expression levels of TRAIL among
the 2 groups were compared by the Mann-Whitney U test.
Correlations among the patient characteristics, gene expres-
sions and TRAIL concentrations were analyzed by Spearman's
Table I. Patient characteristics.
Gender (male/female) 30/26
Age, years 44-82 (mean 63.5)
IgG, immunoglobulin G; IgA, immunoglobulin A; BJP, Bence-Jones
protein. DS stage; Durie-Salmon stage.
Figure 1. TRAIL mRNA expression in myeloma cells. (A) TRAIL mRNA
expression in myeloma cell lines. The results of RT-PCR amplifications are
shown at the bottom. The data obtained by the real-time PCR and regular
PCR are correlated. (B) Expression of TRAIL in purified MM cells. The
expression levels vary among the cases. (C) TRAIL expression in myeloma
cells from MM and MGUS patients. The TRAIL expression levels are greater
in MM patients (n=56) than in MGUS patients (n=12) (*p=0.0002, Mann-
Whitney U test). Horizontal bars indicate the median.
ONCOLOGY REPORTS 27: 39-44, 2012
correlation analysis. Values of p<0.05 were considered to
indicate statistical significance.
Production of TRAIL by myeloma cells. TRAIL mRNA
expression was detected at various levels in myeloma cell
lines (Fig. 1A) and purified primary myeloma cells (Fig. 1B).
The levels of TRAIL expression were significantly higher in
myeloma cells from MM patients than in plasma cells from
MGUS patients (p<0.01; Fig. 1C). TRAIL staining was detected
in the cytoplasm of myeloma cells in paraffin-embedded plas-
macytoma tissues (Fig. 2). These results indicate that TRAIL
is produced by myeloma cells at both the mRNA and protein
TRAIL concentrations in the peripheral blood and bone
marrow plasma. The serum TRAIL concentrations of 31
myeloma patients and 7 healthy donors were analyzed using
ELISA. The serum TRAIL concentrations of the MM patients
did not significantly differ from those of the healthy donors
(p=0.51). No correlation was observed between the TRAIL
expression in myeloma cells and the serum TRAIL concentra-
tion (rs=0.18, p=0.28).
To determine the TRAIL concentrations in the bone
marrow microenvironment, bone marrow plasma samples
from 12 MM patients were analyzed. The bone marrow plasma
TRAIL concentration showed a significant correlation with
the TRAIL expression in MM cells (rs=0.63, p<0.05; Fig. 3),
indicating that TRAIL may be functional in the bone marrow
Correlation between TRAIL and bone resorption. We further
examined the associations of TRAIL with various factors
involved in bone resorption, such as the serum calcium and
urinary deoxypyridinoline/creatinine (U-DPD) levels. Among
the 56 MM patients, the serum calcium levels of 51 patients who
were not previously treated by bisphosphonates were evaluated.
There was a positive correlation between TRAIL expression
and the serum calcium levels (rs=0.28, p=0.045; Fig. 4A). Since
U-DPD is reportedly correlated with the extent of bone disease
in MM (18), U-DPD was measured in 22 patients. A positive
correlation was also observed between TRAIL expression and
the U-DPD level (rs=0.51, p=0.02; Fig. 4B).
To evaluate the role of TRAIL in MM skeletal lesions, the
existence of bone-associated plasmacytomas, radiation therapy
or surgery for skeletal lesions were surveyed in our cohort. As
shown in Fig. 4C and D, TRAIL expression was significantly
higher in MM patients with bone-associated plasmacytomas
(p<0.05) and radiation therapy or surgery for skeletal lesions
Since DKK-1 and MIP1α are well-known factors that regu-
late the inhibition of osteoblast differentiation and induction of
osteoclast differentiation, respectively, we further compared
the expressions of TRAIL, DKK-1 and MIP1α in MM cells
from 56 MM patients. DKK-1 expression did not correlate
with serum calcium level (rs=0.18, p=0.21) or U-DPD levels
(rs=0.23, p=0.26). MIP1α expression correlated with serum
calcium levels (rs=0.36, p=0.01) and U-DPD level (rs=0.48,
p=0.028) but no significant differences were observed between
MIP1α expression and the existence of bone-associated
plasmacytomas (p=0.64) or with therapy for skeletal lesions
(p=0.59). Interestingly, TRAIL significantly correlated to all
of these parameters; patients with bone-associated plasma-
cytomas (p<0.05), radiation therapy or surgery for skeletal
lesions (p<0.01), serum calcium level (rs=0.28, p=0.045)
Figure 2. TRAIL production in MM cells. Two representative cases are shown. Immunohistochemical staining reveals the existence of TRAIL in the cyto-
plasm. Original magnification, x1,000.
Figure 3. Correlation between TRAIL expression and TRAIL concentra-
tion in bone marrow plasma (rs=0.63, p<0.05, n=12, Spearman's correlation
KAWANO et al: TRAIL PRODUCING MYELOMA
and U-DPD (rs=0.51, p=0.02). These analyses revealed that
TRAIL was superior to DKK-1 or MIP1α for predicting bone-
related problems. The data are summarized in Table II.
Since a previous report indicated that TRAIL is a major
pathogenic mechanism for anemia in MM patients (19), we
evaluated the association between TRAIL expression levels
and hemoglobin levels. However, no association was observed
between these two factors (rs=0.0017, p=0.99). The number
of platelets showed no correlation with TRAIL expression
Changes in TRAIL expression during the clinical course of
three patients. We carried out analyses during the clinical
course of three patients to examine whether TRAIL expres-
sion changed in association with the disease status (Fig. 5). In
cases 1 and 2, TRAIL expression increased ~10-fold. In case 1,
bone-associated plasmacytoma requiring irradiation appeared.
Moreover, osteolytic regions increased despite chemotherapy.
In case 2, back pain and U-DPD markedly increased although
no fractures, osteolytic lesions or plasmacytoma were detected
by regular examinations. Case 3, who had hypercalcemia on
admission which improved after chemotherapy, showed an
~100-fold decrease in TRAIL expression. These results suggest
that serum calcium or urine DPD levels vary according to the
Our analyses indicate the production of TRAIL by myeloma
cells at the mRNA and protein levels. Although there are only
Figure 4. TRAIL expression and SRE. (A) Correlation between TRAIL expression and the serum calcium levels (rs=0.28, p<0.05). (B) Correlation between
TRAIL expression and the U-DPD levels (rs=0.51, p<0.05). (C) Higher TRAIL expression level in MM patients with bone-associated plasmacytomas
(Cytoma+) than in patients without bone-associated plasmacytomas (Cytoma-) (xp=0.045). (D) Higher TRAIL expression level in MM patients with radiation
therapy or surgery for skeletal lesions (Treatment+) than patients without radiation therapy or surgery for skeletal lesions (Treatment-) (#p=0.004). Horizontal
bars indicate the median.
Table II. Summary of gene expressions and bone resorption-related parameters.
Parameters DKK-1 MIP1α TRAIL
MIP1α, macrophage inflammatory protein-1α; DKK-1, Dickkopf-1; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; Cytoma,
bone-associated plasmacytoma; intervention, radiation therapy or surgery for skeletal lesions; Ca, serum calcium level; U-DPD, urinary
deoxypyridinoline/creatinine. Results showing p<0.05 are indicated in bold.
ONCOLOGY REPORTS 27: 39-44, 2012
a few reports showing TRAIL production by MM cells, our
finding is compatible with a previous report showing expres-
sion of TRAIL in myeloma cells based on analysis of the gene
expression profile. Jourdan et al reported that TRAIL was
one of the major up-regulated genes among the 58 anti- and
pro-apoptotic proteins in purified myeloma cells (20). TRAIL
was also one of the up-regulated genes in a hyperdiploid
(HY) group, as well as DKK-1, in a University of Arkansas
microarray analysis (21). The HOVON-65/GMMG-HD4 clas-
sification added three new classifications to the University of
Arkansas classification (22). In this classification, TRAIL was
reported to be one of the down-regulated genes in the MAF/
MAFB (MF) cluster, which showed the lowest incidence of
bone lesions. These reports also support our findings.
Although the serum TRAIL concentrations were not corre-
lated with TRAIL mRNA expression levels in myeloma cells,
the bone marrow plasma TRAIL concentrations were corre-
lated with TRAIL mRNA expression levels in myeloma cells.
These data strongly suggest that TRAIL should be maintained
at higher concentrations in the bone marrow microenviron-
On the other hand, the DKK-1 expression in MM cells did
not show an association with the bone resorption in our cohort.
Since DKK-1 suppresses osteoblast differentiation (10), rather
than manipulating osteoclastogenesis, DKK-1 failed to show
correlations with serum calcium and U-DPD levels, which
directly reflect osteoclast activation rather than osteoblast
activation. On the other hand, correlations of MIP1α, a known
direct osteoclast stimulatory factor in MM (11), with the serum
calcium and U-DPD levels were observed. These results indi-
cate that DKK-1 itself may not solely serve as a predictor of
TRAIL was the only factor examined that had a positive
correlation with hypercalcemia, U-DPD, bone-associated
plasmacytomas and therapy against skeletal lesions. These
findings strongly suggest that TRAIL is a potent inducer of
bone resorption. There have been some reports supporting
this hypothesis (7). Vitovski et al showed that TRAIL inhibits
OPG-mediated inhibition of osteoclastogenesis in vitro (5).
Coluccci et al suggested that MM T cells support osteoclast
formation through OPG/TRAIL interactions (6). It was also
reported that TRAIL can induce osteoclast differentiation
from precursor cells, indicating a direct association of TRAIL
(7). A recent report suggests that TRAIL down-regulates
the release of OPG by stroma cells (23). Our preliminary
study showed that TRAIL at 1-100 ng/ml did not affect the
viability of osteoclasts generated from human PBMCs as
well as RANKL-stimulated RAW264.7 pre-osteoclastic cells
(data not shown) in contrast to the induction of apoptosis in
osteoblasts by TRAIL as previously reported (8,9). However,
the mechanisms involved in the regulation of osteoclasto-
genesis by TRAIL are unknown. The issue of whether TRAIL
stimulates osteoclasts directly or indirectly as a consequence
of inhibiting OPG or both is still a matter of debate.
TRAIL has been recognized as an apoptosis-inducing
cytokine (24). The mechanism of MM cell survival from
the TRAIL produced by themselves is still unknown. There
is a report that c-FLIP is produced in MM cells when MM
cells adhere to stromal cells and is responsible for protecting
MM cells against TRAIL (25). These findings may explain
how MM cells survive in the bone marrow microenvironment
containing high concentrations of TRAIL.
Taken together, TRAIL may be a key molecule for inducing
SRE in MM patients. Our findings may lead to the development
Figure 5. Changes of parameters during the clinical course. Expression of TRAIL mRNA, serum calcium concentration and urine DPD levels at pre and post
treatment are shown. Cases 1 and 2 showed elevation of all these parameters while improvement of parameters was found in case 3.
KAWANO et al: TRAIL PRODUCING MYELOMA Download full-text
of new strategies targeting TRAIL to reduce bone resorption
in MM patients.
This work was supported by National Cancer Research and
Development Fund in Japan.
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