International Journal of Myeloma 6(1): 7–11, 2016
©Japanese Society of Myeloma

ORIGINAL

Susceptibility to bendamustine considerably varies amongmyeloma cells, but is enhanced in acidic conditions
Shingen NAKAMURA1, Hirokazu MIKI2, Asuka ODA1, Ryota AMACHI3,Jumpei TERAMACHI4, Kimiko SOGABE1, Hikaru FUJINO1, Tomoko MARUHASHI1,Shiro FUJII1, Kumiko KAGAWA1 and Masahiro ABE1
Myeloma (MM) cells and osteoclasts create a highly acidic milieu in MM bone lesions by proton produced byosteoclasts and lactate by MM cells. Although bendamustine exerts clinical activity against MM, the potential
mechanisms of action of bendamustine remain largely unknown. In the present study, we therefore exploredanti-MM activity of bendamustine with special reference to its effects on MM cells in acidic conditions.
Bendamustine at the range of 25 to 100 μM dose-dependently induced death in INA6 and TSPC-1 MM cells.
However, bendamustine did not impair the viability of RPMI8226, U266 and OPC cells even at 100 μM. In acidic
conditions at pH 6.8 or lower, the bendamustine’s cytotoxic effects were further enhanced in INA6 and TSPC-1 cells,and triggered in RPMI8226 and MM.1S cells which were resistant to bendamustine at pH 7.4. Intriguingly,
bendamustine reduced Pim-2 protein levels in MM cells and enhanced its anti-MM activity in combination with thePim inhibitor SMI-16a, preferentially in acidic conditions. These results demonstrate that anti-MM effects ofbendamustine are augmented in acidic conditions, but considerably vary among MM cells, and that Pim inhibition
further enhances the bendamustine’s anti-MM activity in acidic conditions.
Key words:

bendamusutine, myeloma bone lesion, acidic condition, Pim-2

Introduction

progression of bone disease, the bone marrow microenvironment is skewed by MM cells, which underlies the unique

Multiple myeloma (MM) is characterized by the accu­
mulation of neoplastic plasma cells in the bone marrow, andpathophysiology of MM and confers aggressiveness and drug
resistance in MM cells.

generates bone destruction through enhancement of osteo-

We previously demonstrated that osteoclasts are not only

clastogenesis and concomitant suppression of osteoblasticbone-resorbing cells but also potently support MM cell growthdifferentiation from bone marrow stromal cells. Along with theand survival 1,2). Osteoclasts produce multiple growth and survival factors for MM cells, including TNF family cytokines, BAFFand APRIL, thereby forming a vicious cycle between osteo­

Received: August 28, 2015, accepted: October 8, 2015
1
Department of Hematology, Endocrinology and Metabolism,
Tokushima University Graduate School, Tokushima, Japan
2
Division of Transfusion Medicine and Cell Therapy, Tokushima
University hospital, Tokushima, Japan3
Department of Orthodontics and Dentofacial Orthopedics,
Tokushima University Graduate School, Tokushima, Japan
4
Department of Histology and Oral Histology, Tokushima University
Graduate School, Tokushima, Japan
Corresponding author: Masahiro ABE
Department of Hematology, Endocrinology and Metabolism,
Tokushima University Graduate School, 3-18-15 Kuramoto-cho,
Tokushima 770-8503, Japan
E-mail: masabe@tokushima-u.ac.jp

clastogenesis and myeloma tumor progression 3). Osteoclasts
produce a large amount of protons, and MM cells produce­lactate through enhanced glycolysis; therefore, bone lesions
with an interaction between MM cells and acid-producing
osteoclasts create a highly acidic microenvironment. Tumoracidity has been known to confer drug resistance in cancers 4);
therefore, the development of novel therapies targeting MMcells in the pathologically skewed tumor microenvironment inacidic bone lesions is needed to further improve the outcomeof treatment for MM.
Bendamustine, a purine analogue/alkylator hybrid agent,
exerts anti-tumor activity against various human cancers,
including MM. However, the potential mechanisms of action ofInternational Journal of Myeloma

vol. 6 no. 1 (2016)7NAKAMURA et al.

bendamustine remain largely unknown. In the present study,Cytotoxicity assay (WST assay)

we explored the mechanisms of anti-MM activity by benda-

Cell viability was determined by Cell Counting Kit-8 assay

mustine with special reference to its effects on MM cells in(DOJINDO, Kumamoto, Japan) according to the manufacturer’s

acidic bone lesions, which confer drug resistance.

instructions. Briefly, cells were incubated in culture plateswith 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-di­Materials and Methodssulphophenyl)-2H-tetrazolium monosodium salt (WST-8) for
4 hours. The absorbance of each well was measured at 450 nm

Reagents

with a microtiter plate reader (Model 450 micro plate reader;

The following reagents were purchased from the indicated

Bio-Rad Laboratories, Hercules, CA).

manufacturers: bendamusutine and mouse polyclonal antiβ-actin antibody from Sigma (St. Louis, MO); rabbit anti-Pim-2

Results and Discussion

antibody from Cell Signaling Technology (Beverly, MA, USA);horseradish-peroxidase-conjugated goat anti-rabbit IgG andhorse anti-mouse IgG from Cell Signaling Technology (Beverly,Cytotoxic activity of bendamustine on MM cells
We first looked at the cytotoxic activity of bendamustineat 25 to 100 μM against different MM cell lines. BendamustineMA).

dose-dependently induced cell death in INA6 and TSPC-1 cells,Cells and cultures

and weakly in MM.1S cells (Fig. 1A). However, bendamustine

Human MM cell lines, RPMI8226 and U266, were obtained

did not impair the viability of RPMI8226, U266, and OPC cells

from American Type Culture Collection (ATCC) (Rockville, MD).

even at 100 μM. These results suggest that susceptibility toINA6 and MM.1S MM cell lines were kindly provided by Dr.

bendamustine significantly varied among MM cells.

Renate Burger (University of Kiel, Kiel, Germany) and Dr. Steven

Although bendamustine marginally induced death in U266

Rosen (Northwestern University, Chicago, IL), respectively.

cells in vitro (Fig. 1A), bendamustine has been reported to sup-TSPC-1 and OPC MM cell lines were established in our labora-press U266 cell growth in bone lesions in NOD/SCID/IL-2Rγctory 1). MM cells were cultured in RPMI1640 supplemented(null) (NOG) mice 5), suggesting dependence of bendamustine’s

with 5% fetal bovine serum, 2 mM of L-glutamine (Sigma), 100

cytotoxic activity on the microenvironment in bone lesions inU/mL of penicillin G and 100 mg/mL of streptomycin (Sigma).

MM. Because MM creates an acidic microenvironment in bone

pH values in culture media were adjusted by adding lactic acidlesions, we hypothesized that acidic conditions may affect(Wako, Osaka, Japan) or hydrochloride (Wako). IL-6-dependent

bendamustine’s anti-MM effects. To verify the hypothesis, weINA-6, TSPC-1 and OPC cells were cultured in the presence ofnext examined the cytotoxic effects of bendamustine on MM1 ng/ml of rhIL-6 (PEPROTECH EC, London, UK). Primary bonecells in acidic conditions. INA6 and TSPC-1 cells susceptible tomarrow stromal cells were isolated from fresh bone marrow

bendamustine at 50 μM albeit at pH 7.4 underwent cell death

aspirates from patients with MM, and cultured as previously

more preferentially in acidified conditions (Fig. 1B). Inter­1)

described . All procedures involving human specimens wereestingly, acidic conditions at pH 6.8 or lower were able to trig-

performed with written informed consent according to theger bendamustine’s cytotoxic activity against RPMI8226 andDeclaration of Helsinki and using a protocol approved by theMM.1S cells which were resistant to bendamustine at 50 μM

Institutional Review Board for human protection.

at pH 7.4. In contrast to bendamustine, the alkylator melphalan
exerted the cytotoxic activity in a manner independent ofWestern blot analysis

ambient pH values against INA6 and RPMI8226 cells which

Whole cell lysate was lysed in RIPA buffer. These lysates were

were susceptible and resistant to bendamustine, respectively

supplemented with 1 mM phenylmethylsulfonyl fluoride and(Fig. 1C). Cytotoxic activity of bendamustine was similarly

protease inhibitor cocktail solution (Sigma). Cell lysates andenhanced in acidic conditions created by hydrochloride com-conditioned media were electrophoresed in 10% SDS-PAGE

pared to those by lactic acid, which can negate the possibility

gel and blotted onto polyvinylidene difluoride membranes

that lactic acid itself affects the activity of bendamustine (Fig.

(Millipore, Bedford, MA). After blocking with 5% non-fat dry

1D). Thus, anti-MM effects of bendamustine appear to bemilk, the membranes were incubated with primary antibodies

uniquely induced and increased in acidic conditions.

overnight at 4°C, followed by washing and addition of a horseradish-conjugated secondary antibody for 1 hour. The protein

Bendamustine reduces Pim-2 protein expression in MM cells

bands were visualized with an Enhanced Chemiluminescence

The serine/threonine kinase Pim-2 has been demonstrated

Plus Western Blotting Detection System (Amersham Biosciences,

to be constitutively over-expressed in MM cells 6–8), and further

Piscataway, NJ).

up-regulated in MM cells by IL-6 and/or TNF family cytokines

8International Journal of Myeloma

vol. 6 no. 1 (2016)

Susceptibility to bendamustine considerably varies among myeloma cells, but is enhanced in acidic conditions

Figure 1. Cytotoxic activity of bendamustine against MM cells. (A)Cytotoxic activity of bendamustine on MM cell lines. MM celllines as indicated were cultured in the presence or absence ofbendamustine (benda) at the indicated concentrations. rh IL-6
was added at 1 ng/ml in cultures with IL-6-dependent INA6,TSPC-1 and OPC cell lines. (B) Anti-MM activity of bendamustinein acidic conditions. INA6, TSPC-1, MM.1S and RPMI8226 cells
were cultured at the indicated pH values in the presence (graybar) or absence (black bar) of bendamustine at 50 μM. (C) Cytotoxic activity of melphalan on MM cells. INA6 and RPMI8226
cells were cultured at the indicated pH values in the presence(gray bar) or absence (black bar) of melphalan at 50 μM. pH­values of culture media were adjusted by addition of lactic acid.
(D) Cytotoxic activity of bendamustine in media acidified by­lactic acid or hydrochloride. INA6 and RPMI8226 cells were cultured in media acidified by lactic acid or hydrochloride as indicated. Cell viability was determined after culturing for 24 hours,
using a WST assay. The results were expressed as percent ratiosof baseline without bendamustine or melphalan at pH 7.4.

such as TNF-α, BAFF and APRIL produced by bone marrow6)

activity is dependent on its protein levels 10). IL-6 is amongstromal cells as well as osteoclasts . Pim-2 plays a critical role

predominant enhancers of Pim-2 expression in MM cells

in MM cell survival and is regarded as an important thera-­elaborated by bone marrow stromal cells 6).

6–9). Pim-2 is autophosphorylated and con-

We found that treatment with bendamustine at 50 μM, thestitutively active without post-translational modification; itsconcentration to induce their cell death as shown in Figure 1B,

peutic target in MMInternational Journal of Myeloma

vol. 6 no. 1 (2016)

9

NAKAMURA et al.

suppressed Pim-2 expression preferentially in acidic conditions
in INA6 and TSPC-1cells which were susceptible to bendamustine. The suppressive effects by bendamustine under acidic
conditions were more clearly observed in cocultures with bonemarrow stromal cells or in the presence of IL-6 in these MMcells whose Pim-2 expression was potently up-regulated (Fig.
2A). RPMI8226 cells were resistant to bendamustine at pH 7.4,
but became susceptible in acidic conditions (Fig. 1B). Bendamustine at 50 and 100 μM was also able to dose-dependently
decrease Pim-2 expression upregulated in RPMI8226 cells­under cocultures with bone marrow stromal cells in acidic
conditions (Fig. 2A). Therefore, acidic conditions are sug­gested to trigger or induce bendamustine’s cytotoxic activity
in parallel with suppression of Pim-2 expression in MM cells.
However, ­although the expression of Pim-2 was weak in
INA6 and TSPC-1 MM cells in the absence of BMSCs or IL-6
(Fig. 2A), acidic con­ditions still enhanced the killing effectof bendamustine on these MM cells (Fig. 1B). There may bemechanisms of acid-induced­cytotoxic effects of bendamustineother than Pim-2 reduction.
Combinatory effects of bendamustine and Pim inhibition
Because bendamustine suppresses Pim-2 expression in MM
cells in acidic conditions, and because Pim-2 is a critical antiapoptotic mediator in MM, we next examined the role ofadditional inhibition of Pim-2 activity in acid-triggered
bendamustine’s anti-MM activity. Inhibition of Pim-2 activity
by the Pim inhibitor SMI-16a even at suboptimal concentrations enhanced bendamustine’s cytotoxic effects at pH 6.4 on
RPMI8226 and U266 cells which were resistant to bendamustine at pH 7.4 (Fig. 2B). These results suggest acid-triggered
bendamustine’s anti-MM activity via curtailing Pim-2 expression and potentiation of bendamustine’s therapeutic efficacy
against MM in combination with Pim inhibitors in acidic MMbone lesions.
The present study demonstrates that sensitivity to bendamustine considerably varies among MM cells, but anti-MM
activity of bendamustine is augmented in acidic conditions

Figure 2. Bendamustine’s anti-MM activity via curtailing Pim-2expression. (A) Pim-2 protein expression in MM cells upon treatment with bendamustine. INA6 and TSPC-1 cells were cultured
alone or cocultured with bone marrow stromal cells (BMSC), orcultured in the presence of rh IL-6 at 1 ng/ml. Bendamustine(benda) was added at 50 μM. RPMI8226 cells were cultured
alone or cocultured with bone marrow stromal cells without orwith bendamustine at 50 or 100 μM. pH values of culture media
were adjusted by addition of lactic acid. After culturing for 24hours, cell lysates were harvested and subjected to Western
blotting to determine Pim-2 protein levels. (B) Combinatory
treatment with bendamustine and Pim inhibition in an acidiccondition. RPMI8226 and U266 cells resistant to bendamustine(benda) were cultured at pH 6.4 with bendamustine at 50 μM or
the Pim inhibitor SMI-16a at suboptimal concentrations, 12.5
and 25 μM in RPMI8226 and U266 cells, respectively, alone orboth in combination. Cell viability was determined after culturing for 48 hours, using a WST assay. The results were expressedas per cent ratios from baseline.

at least in part through down-regulation of Pim-2, which isessential for MM cell survival, and that Pim inhibition further

address precise mechanisms responsible for determining sus-enhances the bendamustine’s anti-MM activity in acidic con­ceptibility of MM cells to bendamustine for better use of thisditions. Acidic microenvironments are generally accepted tointeresting anti-tumor agent.

confer drug resistance and immune evasion capability in cancers 4). However, bendamustine appears to rather preferentiallyAcknowledgments

kill MM cells in acidic microenvironments, which can hardly betargeted with other currently available anti-MM agents. From

This work was supported in part by Grants-in-aid for Scien-the present study, combinatory treatment of bendamustinetific Research (C) to M.A., Grant-in-Aid for Young Scientists (B)and Pim inhibitors may become a novel therapeutic option

to R.A., S.N. and H.M. and a National Cancer Center Researchtargeting MM cells in acidic bone lesions. However, we need toand Development Fund (21-8-5) to M.A. from the Ministry of10International Journal of Myeloma

vol. 6 no. 1 (2016)

Susceptibility to bendamustine considerably varies among myeloma cells, but is enhanced in acidic conditions

Health, Labor and Welfare of Japan. The funders had no role instudy design, data collection and analysis, decision to publish,
or preparation of the manuscript.

Conflicts of Interest Disclosures
The authors declare no competing financial interests related
to this work.

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