International Journal of Myeloma 5(1): 1–7, 2015
©日本骨髄腫学会

ORIGINAL

Induction of endoplasmic reticulum stress by bortezomib
sensitizes myeloma cells to DR5-mediated cell death
Hirokazu MIKI1, Shingen NAKAMURA2, Asuka ODA2, Ryota AMACHI3, Keiichiro WATANABE3,
Derek HANSON2, Jumpei TERAMACHI4, Masahiro HIASA5, Hikaru YAGI2, Kimiko SOGABE2,
Mamiko TAKAHASHI2, Tomoko MARUHASHI2, Kengo UDAKA2, Takeshi HARADA2, Shiro FUJII2,
Ayako NAKANO6, Kumiko KAGAWA2, Masaki RI7, Shinsuke IIDA7, Shuji OZAKI8,
Toshio MATSUMOTO2,9 and Masahiro ABE2
TNF-related apoptosis-including ligand/Apo2 (TRAIL)-mediated immunotherapy is an attractive anti-tumor
modality with high tumor specificity. In order to improve its therapeutic efficacy, we further need to implement a
novel maneuver for sensitization of malignant cells to TRAIL. Bortezomib (BTZ), a novel anti-myeloma (MM) agent,potently induces endoplasmic reticulum (ER) stress to cause apoptosis. Here, we explored the roles of BTZ in thecytotoxicity of anti-TRAIL receptor agonistic antibodies against MM cells with special reference to ER stress. BTZ
enhanced the expression of death receptor 5 (DR5) but not DR4 in MM cells at surface protein as well as mRNAlevels. However, the DR5 expression was not affected by BTZ without ER stress induction in MM cells with a point
mutation in a BTZ-binding proteasome β5 subunit. Tunicamycin, an ER stress inducer, was able to enhance the DR5
expression even in the BTZ-resistant MM cells, suggesting the role of ER stress in up-regulation of DR5 expression.
Interestingly, BTZ facilitated extrinsic caspase-mediated apoptosis by anti-DR5 agonistic antibody in MM cells
along with reducing c-FLICE-like interleukin protein, a caspase 8 inhibitor. These results suggest that BTZ enhances
DR5 expression and its downstream apoptotic signaling through ER stress to sensitize MM cells to TRAIL-mediatedimmunotherapy.
Key words:

multiple myeloma, bortezomib, TRAIL, DR5, ER stressReceived: August 7, 2014, accepted: September 21, 2014
1
Division of Transfusion and Cell Therapy Medicine, Tokushima
University Hospital2
Department of Medicine and Bioregulatory Sciences, The Universityof Tokushima Graduate School of Health Biosciences3
Department of Orthodontics and Dentofacial Orthopedics, The
University of Tokushima Graduate School of Oral Science
4
Department of Histology and Oral Histology, The University ofTokushima Graduate School of Oral Science5
Department of Biomaterials and Bioengineering, The University ofTokushima Graduate School of Oral Science
6
Department of Internal Medicine, Naruto Hospital
7
Department of Hematology and Oncology, Nagoya City University
Graduate School of Medical Sciences
8
Department of Hematology, Tokushima Prefectural Central Hospital
9
Fujii Memorial Institute for Medical Reseach, The University ofTokushima
Corresponding author: Masahiro ABE, M.D.
Department of Medicine and Bioregulatory Sciences, The Universityof Tokushima Graduate School of Health Biosciences, 3-18-15
Kuramoto, Tokushima 770-8503, Japan
TEL: 81-88-633-7120, FAX: 81-88-633-7121
E-mail: masabe@tokushima-u.ac.jp

Introduction
Multiple myeloma (MM) still remains incurable because of
its resistance to chemotherapeutic drugs and an escape fromtumor immune surveillance, although new agents as well as
high dose chemotherapy followed by stem cell transplantation
have been introduced into a clinical practice with better
quality of therapeutic response and outcome. Therefore,
alternative approaches are needed to overcome drug resistanceto improve the therapeutic outcome in patients with MM.
Immunotherapies have been getting generally accepted
as attractive treatment options for yet incurable malignancies
by conventional chemotherapeutic agents. One such approachis a TNF-related apoptosis-including ligand/Apo2 (TRAIL)mediated immunotherapy 1–3). TRAIL binds to two different
proapoptotic receptors, death receptor 4 (DR4) and DR5.
Unlike Fas ligand and TNF-α, TRAIL is able to induce cell deathin malignant cells with marginally affecting normal tissues;
TRAIL-mediated immunotherapy is, therefore, regarded as an
attractive tumor-specific strategy against various cancers,
including MM 4–6).

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MIKI et al.

However, weak expression of the TRAIL receptors as well as(PBMCs) were isolated from fresh peripheral blood fromthe suppression of their downstream pro-apoptotic signaling

healthy donors 10). Primary MM cells were purified from boneoften cause malignant cell resistance to TRAIL; and sensitiza-

marrow mononuclear cells (BMMCs) using CD138 microbeads

tion of malignant cells to TRAIL has become a major issue inand a magnetic cell sorting system (Miltenyi Biotec, Auburn,the TRAIL-mediated immunotherapy. To restore the sensitivity

CA). Human MM cell lines, RPMI 8226, KMS-11 and U266, wereto TRAIL, we need to develop novel therapeutic maneuvers to

obtained from the American Type Culture Collection (ATCC,

up-regulate surface TRAIL receptors along with stimulation ofManassas, VA). The human MM cell line OPC was establishedDR-mediated pro-apoptotic signaling.

in our laboratory 11). The human MM cell line INA-6 was kindly

The proteasome inhibitor bortezomib (BTZ) is widely usedprovided by Dr. Renate Burger (University of Kiel, Kiel, Ger-in treatment of MM with improved response rates in patientsmany). The human MM cell lines OPM-2 was purchased from7)with both relapsed/refractory and newly diagnosed MM . BTZ

the German Collection of Microorganisms and Cell Culturesinduces misfolded protein accumulation in MM cells followed(Braunschweig, Germany). BTZ-resistant MM cell lines with a

8,9)

.

point mutation in the β5 subunit of a 26S proteasome, KMS-11/

However, the effects of ER stress induced by BTZ on TRAIL-

BTZ and OPM-2/BTZ, were kindly provided from Kyowa Hakko

mediated MM cell death are largely unknown. In the present

Kirin Co. Ltd. (Tokyo, Japan) 12). Cells were cultured in RPMI1640

study, we therefore aimed to clarify the role of BTZ on TRAIL(Sigma) supplemented with 5% FCS (Life Technologies, Grand

receptor editing and TRAIL-mediated cell death in MM cells

island, NY), penicillin (100 units/mL), and streptomycin (100with special reference to ER stress. We demonstrated here that

μg/mL) at 37°C in a humidified atmosphere with 5% CO2.

by endoplasmic reticulum (ER) stress-associated apoptosis

BTZ enhanced the surface expression of DR5 but not DR4 in
MM cells and its downstream apoptotic signaling through theinduction of ER stress to sensitize MM cells to an anti-DR5

Flow cytometry
Cell preparation and staining for flow cytometry were
performed as described previously 13). Briefly, cells were incu-

agonistic antibody.

bated in 100 μl PBS with 2% human γ-globulin with saturating

Materials and Methods

concentrations of different FITC-conjugated monoclonal
antibodies on ice for 40 minutes. They were then washed

Reagents

and analyzed by flow cytometry using EPICS-Profile (Coulter

Bortezomib was purchased from Millenium Pharmaceuticals,

Elecronics, Hialeah, FL).

Inc. (Cambrigde, MA, USA). Rabbit monoclonal antibodies
against caspase 9, caspase 3, cleaved caspase 3, poly (ADP-

Cell viability and apoptosis assay

ribose) polymerase (PARP), and mouse monoclonal antibodies

MM cells were incubated with various concentrations of BTZ

against activating transcription factor 4 (ATF4) and C/EBP-

with or without TRAIL agonistic antibody at 37°C for 48 hours.

homologous protein (CHOP) were purchased from Cell Sig-

Viable cell numbers were measured by a cell proliferation

naling Technology Japan (Tokyo, Japan). Mouse monoclonal

assay using 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-­antibodies against caspase 8, c-FLICE-like interleukin protein(2,4-disulfophenyl)-2H-tetrazolium (WST-8; Kishida Chemical,(c-FLIP) and β-actin were obtained from Medical and Biotech-

Osaka, Japan). Apoptosis in MM cells was evaluated by stain-

nological Laboratories (Nagoya, Japan), Santa Cruz Biotechnol-

ing the cells with an annexinV-FITC and propidium iodide

ogy (Santa Cruz, CA), Abcam (Cambridge, UK) and Sigma (Saintlabeling kit (MEBCYTO Apoptosis Kit; MBL, Nagano, Japan)

Louis, MO), respectively. FITC-conjugated mouse monoclonalaccording to the manufacturer’s instruction.

antibodies against human DR4 and DR5 were from Biolegend(San Diego, CA). Horseradish peroxidase-conjugated goat

Western blot analysis

anti-mouse antibody was from Invitrogen Life Technologies

Cells were collected and lysed in a lysis buffer (Cell Signal-(Carlsbad, CA). The human monoclonal anti-DR5 agonistic

ing, Beverly, MA) supplemented with 1 mM phenylmethylsul-

antibody R2-E11 was a kind gift from from Kyowa Hakko Kirin

fonyl fluoride and protease inhibitor cocktail solution (Sigma).

Co. Ltd. (Tokyo, Japan).

The cell lysates were subjected to SDS-PAGE on a 10% polyacrylamide gel, and then transferred to polyvinylidene difluo-

Cells and cultures

ride membranes (Millpore, Billerica, MA). The membranes were

The use of human samples was approved by the Institu-

blocked with 5% non-fat dry milk in TBS with 0.01% Tween 20

tional Review Board at University of Tokushima (Tokushima,

for 1 hour at room temperature and incubated for 16 hours at

Japan), and informed consent was obtained according to the

4°C with the primary antibodies. After washing, a secondary

Declaration of Helsinki. Peripheral blood mononuclear cells

horseradish peroxidase-conjugated antibody was added2International Journal of Myeloma

vol. 5 no. 1 (2015)

TRAIL-mediated immunotherapy with bortezomib

Figure 1. Up-regulation of DR5 expression in MM cells by BTZ. (A) MM cell lines, primary MM cells, and peripheral blood mononuclear cells(PBMCs) were incubated with BTZ at 10 nM for 24 hours, and the surface expression of DR5 was analyzed by flow cytometry. (B) RPMI 8226,
INA-6 and OPM-2 cells were incubated with BTZ at 10 nM for different periods as indicated. DR4 and DR5 mRNA expression was determinedby real time RT-PCR. GAPDH was used as an internal control. *P < 0.05.

and the membranes were developed using the enhancedof the ABI 7300 (Applied Biosystems, Foster City, CA, USA).

chemiluminescence plus Western blotting detection system

Primers used were as follows: DR4 sense 5’-AAGTTTGTCGTC(American Biosciences, Piscataway, NJ).

GTCGGGGTCCT-3’ and antisense 5’-GGTGGACACACTCTCCCA­
AAGGGC-3’, DR5 sense 5’-TCTCCTGAGATGTGCCGGAAGTGCC-3’

Quantitative real-time PCR

and antisense 5’-GCTGGGACTTCCCCACTGTGCTTT-3’, GAPDH

Cells were harvested and total RNA was extracted from cells

sense 5’-AATCCCATCACCATCTTCCA-3’ and antisense 5’-TGGAC­

using TRIZOL reagent (Invitrogen). Equal amounts of total RNA

TCCACGACGTACTCA-3’. Products were run on 2% agarose gelswere subjected to reverse transcription using Superscript II

containing ethidium bromide.

(Invitrogen). Real-time PCR was performed using Platinium
SYBR Green qPCR SuperMix UDG with Rox (Invitrogen) with

Statistical analysis

the following amplification program: one cycle of 50°C for

Comparisons between experimental data were performed

2 minutes and 95°C for 2 minutes and 40 cycles of 95°C for

by one-way analysis of variance (ANOVA) or one-sided, paired

15 seconds and 60°C for 30 seconds. The reaction was followed

t-test. P below .05 was considered statistically significant.

by a melting curve protocol according to the specifications

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MIKI et al.

Figure 2. Induction of caspase activation and cell death in MM cells. (A) RPMI 8226 and KMS-11 cells were treated with BTZ at the indicatedconcentrations or the anti-DR5 agonistic antibody R2-E11 at 100 ng/mL alone or both in combination for 24 hours. The protein levels of
c-FLIP, caspase 8, caspase 3, and PARP were analyzed by Western blotting. β-actin was used as a protein loading control. (B) RPMI 8226, INA6
and OPC cells were treated with BTZ or the anti-DR5 agonistic antibody R2-E11 at 100 ng/mL alone or both in combination for 48 hours. Cell
viability was analyzed by WST-8 assay. *P < 0.05.

Results and Discussion

BTZ enhances anti-DR5 agonistic antibody-mediatedactivation of the extrinsic apoptotic pathway and death in

BTZ up-regulates DR5 expression in MM cells

MM cells

We first examined whether BTZ affects the surface expres-

Because BTZ up-regulates DR5 expression in MM cells, wesion of TRAIL receptors, DR4 and DR5, on MM cells. BTZ at 10next looked at the effects of BTZ on anti-DR5 agonisticnM up-regulated the surface level of DR5 on primary MM cells

antibody-mediated activation of the extrinsic apoptotic

as well as all MM cell lines tested (Fig. 1A). However, BTZ did

pathway and death in MM cells. BTZ at 10 nM induced the

not up-regulate the surface level of DR4 on MM cells (data not

activation of caspase 8 and caspase 3, and the cleavage ofshown). Real time RT-PCR demonstrated BTZ increased thecaspase 3 and PARP along with decreasing c-FLIP protein

DR5 mRNA expression by BTZ in RPMI 8226, INA-6 and OPM-2

levels in RPMI 8226 and KMS-11 cells (Fig. 2A). Treatment

MM cells (Fig. 1B), suggesting the up-regulation of DR5 atwith the anti-DR5 agonistic antibody R2-E11 at 100 ng/mL intranscriptional levels.

combination with BTZ at 10 nM markedly reduced c-FLIP
protein levels and enhanced the activation of caspase 8 andthe cleavage of caspase 3 and PARP, although the anti-DR5

4International Journal of Myeloma

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TRAIL-mediated immunotherapy with bortezomib

Figure 3. Up-regulation of DR5 as well as ER stress by BTZ was absent in BTZ-resistant MM cells. (A) KMS-11 and OPM-2 cells, and their derived
BTZ-resistant cell lines with a point mutation in a β5 subunit (KMS-11/BTZ and OPM-2/BTZ cells, respectively) were treated with BTZ at 10 nM
for 24 hours. The surface expression of DR5 was analyzed by flow cytometry. (B) KMS-11 and KMS-11/BTZ cells were treated with BTZ at theindicated concentrations for 24 hours. The protein levels of c-FLIP, ATF4, CHOP, caspase 8, caspase 9, caspase 3, and PARP were analyzed byWestern blotting. β-actin was used as a protein loading control. (C) OPM-2 and OPM-2/BTZ cells were treated with BTZ or the anti-DR5
agonistic antibody R2-E11 at 500 ng/mL alone or both in combination for 48 hours, and stained with annexin V-FITC and propidium iodide(PI). Cells were then analyzed by flow cytometry to determine the percentage distribution of cells displaying annexin V staining (early
apoptosis) or both annexin V and PI staining (late apoptosis).

agonistic antibody R2-E11 alone showed only marginal effects

BTZ does not up-regulate DR5 expression in MM cells withon these caspase and PARP cleavage. Consistently, BTZ anda β5 subunit mutation

R2-E11 in combination cooperatively enhanced cell death in

To further clarify the role of ER stress induced by BTZ on DR5

RPMI 8226, INA6 and OPC cells, whereas BTZ or R2-E11 alone

expression and DR5-mediated cytotoxicity, we examined theat this experimental condition only partially induced MM celleffects of BTZ, using KMS-11 and OPM-2 cells with a point

death (Fig. 2B). These results suggest that BTZ potentiates

mutation in the β5 subunit of a 26S proteasome, KMS-11/

DR5-mediated activation of the extrinsic apoptotic pathway

BTZ and OPM-2/BTZ, respectively, which are resistant toand cell death in MM cells.

BTZ-induced cell death. Although BTZ up-regulated DR5
expression on parental KMS-11 and OPM-2 cells, the DR5
up-regulation by BTZ was completely absent in the BTZ-

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MIKI et al.

Figure 4. Induction of ER stress and DR5 in BTZ-resistant MM cells by tunicamycin. (A) KMS-11 and KMS-11/BTZ cells were incubated withtunicamycin at 1 μM for 24 hours. Cell lysates were then extracted, and the protein levels of ATF4 and CHOP were analyzed by Western
blotting. (B) KMS-11 and OPM-2 cells, and their derived BTZ-resistant cell lines (KMS-11/BTZ and OPM-2/BTZ cells, respectively) were treatedwith tunicamycin at 1 μM for 24 hours. The surface expression of DR5 was analyzed by flow cytometry. (C) KMS-11 and KMS-11/BTZ cells
were treated with tunicamycin at 1 μM or the anti-DR5 agonistic antibody R2-E11 at 100 ng/mL alone or both in combination for 48 hours.
Cell viability was analyzed by WST-8 assay.*P < 0.05.

resistant KMS-11/BTZ and OPM-2/BTZ cells (Fig. 3A). Treat-tion of ER stress and thereby DR5 up-regulation appears toment with BTZ at 10 nM or more increased ATF4 and CHOP

be responsible for the enhancement of anti-MM effects of theprotein levels along with the activation of caspase 8, caspasecombinatory treatment with an anti-DR5 agonistic antibody

9 and caspase 3, and the cleavage of PARP in KMS-11 cells

and BTZ.

(Fig. 3B). However, these effects of BTZ were not observed in
the BTZ-resistant KMS-11/BTZ cells. Consistently, BTZ did

DR5 expression is up-regulated in the BTZ-resistant MM

not induce apoptosis in the OPM-2/BTZ cells (Fig. 3C). The

cells under ER stress by tunicamycin

cytotoxic effects of the anti-DR5 agonistic antibody R2-E11

In order to further clarify the relationship between ER stresswere equally observed in the parental and mutated OPM-2

and the up-regulation of DR5, we looked at the effects of ER

cells. However, the enhancement of cell death by R2-E11 instress induced by the ER stress inducer tunucamycin on DR5

combination with BTZ was only observed in the parental

expression and death in the parental and β5 subunit-mutated

OPM-2 cells but not in the mutated ones. Therefore, the induc-MM cells. Tunicamycin at 1 μM was able to increase ATF4 and6International Journal of Myeloma

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TRAIL-mediated immunotherapy with bortezomib

Conflicts of Interest Disclosures

CHOP protein levels in BTZ-resistant β5 subunit-mutated
KMS-11/BTZ cells as well as their parental cells (Fig. 4A), andup-regulate the DR5 expression on the surface of both theparental and mutated KMS-11 and OPM-2 cells (Fig. 4B). Consistent with the up-regulation of the surface DR5 expression,tunicamycin at 1 μM was able to induce significant cytotoxic
effects equally on both KMS-11 cells and bortezomib-resistant
KMS-11/BTZ cells in combination with R2-E11 at 100 ng/mL,
although tunicamycin or R2-E11 alone only minimally affected
the viability of these cells in this experimental condition (Fig.
4C). These results further corroborated the critical role of ER
stress in the up-regulation of DR5 in MM cells and anti-DR5
agonistic antibody-mediated MM cell death.
Because DR5 has been demonstrated to be one of the target
genes of CHOP 14,15) and ATF3 16) induced downstream of ATF4,
the up-regulation of DR5 in MM cells by BTZ is suggested atleast in part due to the induction of ATF4 through ER stress.
Collectively, BTZ enhances DR5 expression and its downstream
apoptotic signaling through ER stress to sensitize MM cells
to TRAIL-mediated immunotherapy. Furthermore, BTZ also
induces death receptor-independent apoptosis as a result ofexcessive ER stress, which may cooperatively enhance MM cell
death in combination with anti-DR5 agonistic antibody.
We have previously reported that MM cells post-translationally down-modulate the cell surface expression of DR4 but not
DR5 through ectodomain shedding by endogenous TNF-αconverting enzyme (TACE), and that TACE inhibition is able
to restore cell surface DR4 levels and the susceptibility of MM
cells to TRAIL or an agonistic antibody against DR4 17). TACEmediated shedding appears to be an important mechanism
for the reduction of surface DR4 levels on MM cells, which may
blunt TRAIL-mediated apoptosis by surrounding immune cells
expressing TRAIL to protect MM cells. Thus, DR4 and DR5
editing and expression on the surface of MM cells appear to
be differentially regulated. BTZ and TACE inhibitors seem good
options to revitalize TRAIL-mediated immunotherapy whose
therapeutic efficacy has been limited as a single treatment
modality. The combination of TRAIL-mediated immunotherapy
with BTZ and/or TACE inhibitors is warranted for further study
in patients with MM.

Acknowledgments
This work was supported in part by JSPS KAKENHI grant
numbers 23591390, 25860789 and 26461422. The funders had
no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors
thank Kyowa Hakko Kirin Co. Ltd. (Tokyo, Japan) for providing
the human monoclonal anti-DR5 agonistic antibody R2-E11
and BTZ-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ.

The authors declare no competing financial interests related
to this work.

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