International Journal of Myeloma 4(3): 13–20, 2014
©日本骨髄腫学会

ORIGINAL

Shikonin, a naphthoquinone compound, rapidly inducescell death of human myeloma cells via productionof reactive oxygen species
Takahiro KIMURA1, Tomonori NAKAZATO1,2 and Masahiro KIZAKI3
Shikonin is a naphthoquinone derivative extracted from a traditional Chinese medical herb, Lithospermum­erythrorhizon. It has been found to posses a variety of biological activities including strong wound healing, antibacterial, anti-inflammatory, and anti-tumor effects. Recently, it has been reported that shikonin induces cell deathof various cancer cells. However, the mechanisms responsible for these activities remain unknown. In addition, no
studies have addressed the effects of shikonin on multiple myeloma cells. In this study, we investigated the effectsof shikonin on cell proliferation, cell cycle progression, and mechanism of apoptotic cell death in human multiple
myeloma cells. Shikonin markedly suppressed the cellular growth of various myeloma cell lines via inductionof apoptosis. Shikonin-induced cell death was in association with the loss of mitochondrial transmembrane potentials (ΔΨm), the release of cytocrome c and Smac/DIABLO. Elevation of intracellular oxygen species (ROS) production was shown during shikonin-induced cell death in myeloma cells. Shikonin markedly inhibited IL-6-induced
phosphorylation of STAT3 in myeloma cells. The antioxidants, NAC, ameliorated shikonin-induced cell death andinhibition of STAT3 phosphorylation in myeloma cells. Our results indicate that shikonin induced cell death inmyeloma cells, and that this was mediated through inhibition of mitochondrial damage and IL-6/STAT pathways by
production of ROS.
Key words:

shikonin, cell death, caspase, STAT3, multiple myeloma, reactive oxygen species (ROS)

Introduction

prolonged use and repeated disease relapse may lead to thedevelopment of drug resistance in myeloma cells 5). Therefore,

Multiple myeloma is a plasma cell malignancy that often
remains fatal despite the use of high dose chemotherapy with1)

novel effective and less toxic therapeutic strategies are desiredin order to improve clinical outcomes.

hematopoietic stem cell transplantation . Recently, novel

Shikonin is a natural naphthoquinone derivative com-

agents such as thalidomide, lenalidomide, and the proteasome

pound that is present in the root tissues of Lithospermum

inhibitor bortezomib have been introduced for the treatment

erythrorhizon. This herb has previously been reported to haveof this disease and have remarkably improved patient out-

many medicinal properties including anti-bacterial, anti-. However, adverse events and complications of these

inflammatory­, wound-healing, and anti-tumor effects 6). Several

agents are often problematic in the clinical setting. In addition,

studies have shown that shikonin induces p53-mediated cell

come

2–4)

cycle arrest and cell death in various human malignant cancer
Received: November 17, 2013, accepted: January 21, 2014
1
Division of Hematology, Department of Internal Medicine andPathology, Keio University School of Medicine
2
Department of Hematology, Yokohama Municipal Citizen’s Hospital3
Department of Hematology, Saitama Medical Center, Saitama
Medical University
Corresponding author: Masahiro KIZAKI, MD
Department of Hematology, Saitama Medical Center, Saitama
Medical University, 1981 Kamoda, Kawagoe, Saitama 350-8550, Japan
TEL & FAX: 81-49-228-3837
E-mail: makizaki@saitama-med.ac.jp

cells, inhibits epidermal growth factor receptor signaling inhuman epidermoid carcinoma cells, and induces cell deathin HL60 human myeloid leukemia cell line 7–9). However, themechanisms re­sponsible for these activities of shikonin­remain unknown.
It has been suggested that the production of reactive oxygen species (ROS) is a common mechanism in one of the representative pathways of apoptotic cell death 10). These oxidant
and their products are capable of depleting glutathione (GSH)or damaging the cellular antioxidant defense system, and they
can directly induce cell death 11). Recently, we and other groups

International Journal of Myeloma

vol. 4 no. 3 (2014)13

KIMURA et al.

have found that intracellular production of ROS is important inRhodamine 123 (Sigma) at 37°C for 30 min. Rhodamine 123

inducing apoptotic cell death in human myeloma cells 12,13).

intensity was determined by flow cytometry as a measure

In the present study, we conducted an in vitro study toof ∆Ψm.

assess the effects of shikonin on myeloma cell proliferation,
Cell cycle analysis

and the underlying mechanisms.

Cells (1 × 105) were suspended in hypotonic solution (0.1%

Materials and Methods

Triton X-100, 1 mM Tris-HCl (pH 8.0), 3.4 mM sodium citrate,
and 0.1 mM EDTA) and stained with 50 μg/ml of PI. The DNA

Cells and cell culture

content was analyzed by flow cytometry. The population of

The human myeloma cell lines, IM9, RPMI8226, HS-Sultan
and U266, were cultured in RPMI1640 medium (GIBCO BRL,

cells in each cell cycle phase was determined using Mod FIT
software (Becton Dickinson).

Grand Island, NY) supplemented with 10% fetal bovine serum(FBS) (GIBCO) in a humidified atmosphere with 5% CO2. These

Caspase assays

cell lines were obtained from the Japan Cancer Research

The activation of caspase-3 was analyzed using a caspase-3

Resources Bank (Tokyo, Japan). The morphology was evaluatedassay kit from BD Biosciences (San Jose, CA). Briefly, the FITC-

on cytospin slide preparations with Giemsa staining, and the

conjugated antibody against the active form of caspase-3 pro-viability was assessed by trypan blue dye exclusion.

vided in the kit was used for FACS analysis according to the
manufacturer’s instructions (BD Biosciences).

Cell proliferation assay
Cell proliferation was measured using an MTS proliferation

Measurement of intracellular superoxide production

assay kit (Roche Molecular Biochemicals, Mannheim, Germany).

To assess the production of superoxide, control and shikonin-

Cells were plated on 96-well culture plates at 5 × 104 cells/ml intreated cells were incubated with 5 μM dehydroxyethidium

a total volume of 100 μM with the indicated reagents. After a(DHE) (Molecular Probes, Eugene, OR), which is oxidized to

2-day incubation, cellular proliferation was measured using

ethidium, a fluorescent intercalator, by cellular oxidants, par-the MTS assay. Mean and standard deviation were calculated

ticularly superoxide radicals. Cells (1 × 105) were stained withfrom triplicate experiments.

5 μM DHE for 30 min at 37°C, and were then washed andresuspended in PBS. The oxidative conversion of DHE to

Reagents

ethidium was measured by flow cytometry (Becton Dickinson).

Shikonin was purchased from BIOMOL International LP(Plymouth Meeting, PA) (Fig. 1A). N-acetyl-L-cystein (NAC) andCell lysate preparation and western blotting

L-buthionine sulfoximine (BSO) were obtained from Sigma

Cells were collected by centrifugation at 700 g for 10 min

Chemical Co. (St. Louis, MO). These agents were dissolved inand then the pellets were resuspended in lysis buffer (1%PBS.

NP-40, 1 mM phenylmethylsulfonyl fluoride (PMSF), 40 mM
Tris-HCl (pH 8.0), and 150 mM NaCl) at 4°C for 15 min. Mito-

Assays for apoptotic cell deathchondrial and cytosolic fractions were prepared with digitonin-

Cell death was determined by morphological change as wellnagarse treatment. Protein concentrations were determined

as by staining with annexin V-FITC and PI labeling. Apoptotic

using a protein assay DC system (Bio-Rad, Richmond, CA). Cell

cells were quantified by annexin V-FITC and PI double staining

lysates (20 μg protein per lane) were fractionated in 12.5%

using a staining kit purchased from PharMingen (San Diego,

SDS-polyacrylamide gel prior to transfer to the membranes

CA). In addition, induction of apoptosis was detected by DNA(Immobilon-P membranes, Millipore, Bedford, MA) using a

6

fragmentation assay. Cells (1 × 10 ) were harvested and incu-

standard protocol. Antibody binding was detected by usingbated in a lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM EDTA,

an enhanced chemiluminescence kit for Western blotting

and 0.5% Triton-X) at 4°C. After centrifugation, supernatants

detection with hyper-ECL film (Amersham, Buckinghamshire,

were collected and incubated with RNase A (Sigma) at 50

UK). Blots were stained with Coomassie brilliant blue to

μg/ml and proteinase K (Sigma) for 1 h at 37°C. The DNA­confirm equal amounts of protein extract on each lane. The­samples were subjected to 2% agarose gel and were visual-following antibodies were used in this study: anti-caspase 3,

ized by ethidium bromide staining. The mitochondrial mem-

-cytochrome c (PharMingen, San Diego, CA), -Bcl-2, -Bcl-XL,

brane potential (ΔΨm) was determined by flow cytometry-β-actin (Santa Cruz Biotech, Santa Cruz, CA), -Bax, -Bid and(FACSCalibur; Becton Dickinson, San Jose, CA). Briefly, cells-Smac/DIABLO (MBL, Nagoya, Japan).

were washed twice with PBS and incubated with 1 μg/ml
14International Journal of Myeloma

vol. 4 no. 3 (2014)

Shikonin induces apoptosis of myeloma cells

Figure 1. Effects of shikonin on cell growth and apoptotic cell death in myeloma cells. (A) Chemical structure of shikonin. (B) Various human
myeloma cell lines (HS-Sultan, RPMI8226, IM9, and U266) were treated with various concentrations (0–1 μM) of shikonin for 24 h, and cellproliferation was determined by MTS assay. (C) Morphological changes characteristic of apoptotic cell death in RPMI8226 myeloma cells.
RPMI8226 cells were treated with 0.5 μM shikonin for 24 h, and cytospin slides were then prepared and stained with Giemsa. Original
magnification, ×1000. (D) Agarose gel electrophoresis demonstrating DNA fragmentation in RPMI8226 cells treated with 0.2 and 0.5 μM
shikonin for 6 h. (E) Cell cycle analysis. RPMI8226 cells were treated with 0.5 μM shikonin for the indicated times (0–12 h), and subsequently
stained with PI. DNA content refers to the population of apoptotic cells. Similar results were obtained in three independent experiments.

Resultsby shikonin by means of DNA ladder formation and annexin
V/PI staining. DNA ladder formation was confirmed at as early

Shikonin inhibited cellular proliferation of various humanas 6 h by electrophoresis of genomic DNA extracted frommyeloma cells

myeloma cells treated with 0.5 μM shikonin (Fig. 1D). RPMI8226

We first examined whether shikonin inhibited the growth ofand IM9 cells cultured with shikonin at various doses (0–1 μM)

various myeloma cells, including IM9, RPMI8226, HS-Sultan,

and for various durations (0–18 h) exposure were stained withand U266 cells. Shikonin inhibited the cellular growth of allannexin V/PI to detect externalization of phosphatidyl serine

myeloma cells in a dose (0–1 μM)- and time (0–24 h)-dependent

on the cell membrane. Numbers of annexin V-positive andmanner (Fig. 1B and data not shown). Cell growth was sup-PI-negative cells were increased as early as 6 h, indicating thatpressed from as early as 6 h, and the typical morphologicalshikonin rapidly induced cell death in myeloma cells (Fig. 2).

appearance of apoptotic cell death was observed, including
condensed chromatin and fragmented nuclei with apoptotic
bodies (Fig. 1C, D).

Shikonin decreased expression of caspase by myeloma cells
Caspases are believed to play a central role in mediating
various apoptotic responses. To address the apoptotic path-

Shikonin induced G1 cell cycle arrest and subsequent cell

way in shikonin-treated myeloma cells, we next examined thedeathactivation of caspases. To clarify the activation of caspase-3,

Cultivation with shikonin increased the population of cells

the percentage of cells expressing the active form of caspase-3

in the G0/G1 phase with a reduction of cells in the S phase

was analysed by FACS. After incubation with 0.5 μM shikonin(Fig. 1E). We confirmed the induction of apoptotic cell deathfor 6 h, the percentage of myeloma cells expressing the active

International Journal of Myeloma

vol. 4 no. 3 (2014)15

KIMURA et al.

Figure 2. Detection of apoptotic cells by annexin
V-FITC and PI double staining. RPMI8226 and IM9cells were cultured with various concentrations(0–1 μM) of shikonin for indicated times (0–18 h),
stained with annexin V-FITC and PI labeling, andanalyzed using flow cytometry. Three independ­ent experiments were performed and all gave
similar results.

Figure 3. Effects of shikonin on caspase-3 activity andmitochondrial pathway via the mitochondrial
membrane potential (ΔΨm). (A) Detection of theactive form of caspase-3 by FACS. PRPMI8226 andIM9 cells were treated with 0.5 or 1 μM shikoninfor indicated times (0–12 h). The percentage ofcells expressing the active form of caspase-3 wasanalyzed using the specific antibody by FACS. (B)ΔΨm as estimated by Rhodamine 123 intensity
on flow cytometric analysis. RPMI8226 and IM9
myeloma cells were cultured with various concentrations (0–0.5 μM) of shikonin for 6 h, andRhodamine 123 fluorescence was analyzed byflow cytometry. (C) Western blot analysis of
mitochondrial apoptogenic proteins in shikonintreated RPMI8226 and IM9 cells. Cells were incubated with 1 μM shikonin for various times (0–6 h).
The cytosolic and mitochondrial proteins wereanalyzed by Western blotting with anti-cytochromec, Smac/DIABLO, and AIF antibodies. These findings indicated that shikonin-induced cell deathoccurred via the mitochondrial pathway.

16

International Journal of Myeloma

vol. 4 no. 3 (2014)

Shikonin induces apoptosis of myeloma cells

Figure 4. Expression of apoptotic cell deathassociated proteins. RPMI8226 and IM9cells were treated with 0.5 μM shikonin for
the indicated time. Cell lysates (20 μg eachlane) were fractionated on 12.5% SDSpolyacrylamide gels and analyzed byWestern blotting with antibodies against
p53, Mcl-1, BCL-2, Bax and β-actin proteins.
Protein levels of caspase-3 were detectedby Western blot analysis using antibody
against caspase-3. Shikonin-induced processing of caspase-3 (32 kDa) was indicatedby the appearance of a 17 kDa cleaved
active form.

form of caspase-3 were increased (Fig. 3A). In response to

trast, shikonin did not modulate the levels of anti-apoptotic

shikonin, the expression levels of caspase-3 in RPMI8226 andprotein Bcl-2 in myeloma cells (Fig. 4).

IM9 cells were decreased (Fig. 4), indicating that shikonininduced cell death in myeloma cells is mediated through the

Shikonin inhibited IL-6-inducible STAT3 phosphorylation

activation of caspase-3.

in myeloma cells
Numerous reports suggest that IL-6 promotes survival

Shikonin-induced death signaling is mediated through theand proliferation of multiple myeloma cells through themitochondrial pathway

phosphorylation of STAT3. We therefore examined whether

Recent studies have suggested that mitochondria play an

shikonin could inhibit IL-6-induced STAT3 phosphorylation.

essential role in death signal transduction 14,15). Mitochondrial

As shown in Fig. 5, IL-6-induced STAT3 phosphorylation waschanges, including permeability transition pore opening andclearly inhibited by shikonin in a time-dependent manner.

the collapse of the mitochondrial ΔΨm, result in the release ofcytochrome c into the cytosol, which subsequently causes cell

Shikonin-induced cell death was triggered by ROS

death by the activation of caspases. After treatment with 0.5

production

μM shikonin for 3 h, low Rh123 staining in myeloma cells indi-

To determine the intracellular concentration of ROS, IM9cated an increase in the loss of mitochondrial ΔΨm (Fig. 3B).

myeloma cells were cultured with DHE, and the fluorescence

The loss of ΔΨm appeared in parallel with the activation of

was measured by flow cytometry (Fig. 6A). Treatment with

caspase-3, as well as with induction of cell death. In addition,

shikonin 1 μM caused notable ROS production and cell death inshikonin induced a substantial release of various mitochon-

myeloma cells. Shikonin-induced ROS production in myeloma

drial apoptogenic proteins (e.g., cytochrome c, Smac/DIABLO

cells was completely blocked by the antioxidant NAC (Fig. 6A,

and AIF) from the mitochondria into the cytosol in myelomaB). BSO, an inhibitor of glutathione (GSH) synthesis, is reported

cells (Fig. 3C). These findings suggest that mitochondrial dys-

to increase ROS levels, accompanied by a reduction in GSH 16).

function caused the release of cytochrome c, Smac/DIABLO

As shown in Fig. 6C, BSO enhances shikonin-induced cell deathand AIF into the cytosol, and that caspase-3 was then acti-

in myeloma cells via production of intracellular ROS. Interest-vated, thereby propagating the death signal.

ingly, NAC decreased the phosphorylation of STAT3 in IL-6/
shikonin-treated myeloma cells (Fig. 6D). These results indicate

Shikonin down-regulated the expression of Mcl-1 andthat the modulation of molecules involved in the redox system

up-regulated that of Bax and p53

may determine the sensitivity of myeloma cells to shikonin.

To investigate the molecular mechanisms of shikonininduced cell death in myeloma cells, the expression of several

Discussion

cell death-associated proteins was examined (Fig. 4). Mcl-1, a
critical survival factor for myeloma cells, was down-regulated

Shikonin is a naphthoquinone compound extracted fromin shikonin-treated myeloma cells. The pro-apoptotic protein

Lithospermum erythrorhizon. It has been shown to have a

Bax and the tumor suppressor, p53, were up-regulated. In con-variety of biological activities such as anti-bacterial, anti-International Journal of Myeloma

vol. 4 no. 3 (2014)17

KIMURA et al.

Figure 5. Shikonin induced cell death through by blocking the JAK/STAT signaling pathway. RPMI8226 cells were cultured with 10 mg/ml IL-6 +/– 0.5 μM of shikonin for theindicated time. RPMI8226 cells were treated with 10 ng/ml IL-6 for various times (0–6h), and cell lysates (20 μg/lane) were fractionated on 12.5% SDS-polyacrylamidegels and Western blotting with antibodies against phospho-STAT3 and STAT was
the performed (upper panel). RPMI8226 cells were then pretreated with 0.5 μM of
shikonin for the indicated time, and cells were stimulated with IL-6 (10 ng/ml) for 15
min. Western blotting was performed to assess the phosphorylated states or the
expression of STAT3 was performed (lower panel).

Figure 6. ROS generation by shikonin in myeloma cells. (A) To determine the intracellularconcentration of ROS, IM9 myeloma cells were cultured with DHE, and the fluorescence
was meas­ured by flow cytometry. DHE-derived fluorescence in untreated IM-9 cells and incultures treated for 3 h with 1 μM shikonin is shown. Treatment with 10 mM of antioxidant
NAC completely inhibited production of intracellular ROS in IM9 cells. (B) The antioxidant,
NAC, blocked shikonin-induced cell death in IM9 cells. IM9 cells were treated with various
concentrations (0–0.5 μM) of shikonin for 24 h following preincubation with 5 mM NAC.
Annexin V-FITC/PI cells were measured using a FACSCalibur flow cytometer. (C) BSO to
decrease intracellular levels of reduced GSH enhanced shikonin-induced cell death in
IM9 cells. Cells were pretreated with 100 μM BSO for 4 h, and were then treated with orwithout 0.2 and 0.5 μM shikonin for 24 h. Annexin V-FITC/PI cells were measured using a
FACSCalibur flow cytometer. (D) Effects of the antioxidant NAC on the phosphorylation of
IL-6/shikonin-treated RPMI8226 cells. Cells were treated with 5 mM NAC, and thencultured with shikonin and IL-6 for the indicated time. Western blotting to assess thephosphorylation stated and performed.

inflammatory, and anti-tumor effects 10). Shikonin ointment

and caspase-3 substrate cleavage in HL60 and K562 leukemic

also has the ability to accelerate wound healing 10). Recently,

cells 19). It is also reported to inhibit transcription factor IID

it has been reported that shikonin inhibited growth of osteo-binding to the TNF-α promoter and block TNF-α pre-mRNAsarcoma and Ewing sarcoma cells 17,18). In addition, shikoninsplicing, resulting in suppression of TNF-α 20,21).

has been associated with cell death and inhibition of angio-

Multiple myeloma is a plasma cell neoplasm derived fromgenesis in studies that showed increased DNA fragmentation

clonal B cell lineage cells. The development of new agents

18

International Journal of Myeloma

vol. 4 no. 3 (2014)

Shikonin induces apoptosis of myeloma cells

such as the proteasome inhibitor, bortezomib, and the

upstream mediator during shikonin-induced apoptotic cell

immunomodulatory drugs, thalidomide and lenalidomide,­

death in myeloma cells, as well as in leukemic cells as previ-

has led to improved outcomes in patients with multiple mye­ously reported 32). Previous investigations have reported that

22,23). However, a higher proportion of patients cannot

shikonin is an inhibitor of tumor proteasome activity in vitro

expect long-term remission due to drug-resistant disease,­and in vivo, and it might be one of the important molecularminimal residual disease, or complications such as severe in­mechanism in shikonin-induced anti-tumor effect 6). Althoughfections 1–4). Therefore, new potent therapeutic agents andspecific molecular targets for shikonin has not been dis­

substantial therapeutic advances are needed for the treat-covered, further studies to examine the accumulation ofment of multiple myeloma.

­various ubiquitinated proteins in shikonin-treated myelomaloma

In this study, we have demonstrated that shikonin rapidly

cells for assessing proteasome activity are also needed.

induces apoptotic cell death in various myeloma cell lines in

In conclusion, our results indicate that shikonin induces

association with the down-regulation of anti-apoptotic pro-apoptotic cell death of human multiple myeloma cells via pro-tein, Mcl-1 and up-regulation of pro-apoptotic protein Bax,duction of ROS, resulting in mitochondrial injury and caspasethe loss of mitochondrial membrane potentials (ΔΨm), theactivation in addition to inhibiting the phosphorylation of

release of mitochondrial apoptogenic proteins such as cyto-

STAT3. Natural compounds might have multiple targets forchrome c, Smac/DIABLO and AIF from mitochondria into thetheir biological effects on cancer cells. Therefore, shikonin has

cytosol, and the activation of caspase-3. Bax is a pro-apoptotic

a potential as a novel agent for the treatment of multiple

member of the Bcl-2 family that resides in the cytosol andmyeloma with less toxic effects than existing drugs.

24)translocates to mitochondria during induction of cell death .
Compared with chemosensitive myeloma cells, chemoresis-

Acknowledgements

tant myeloma cells are also reported to express higher levelsof the anti-apoptotic proteins, Bcl-2 and Bcl-xL 25,26). Previous

We thank Chika Nakabayashi for her helpful technicalstudies have found that shikonin inhibits the expression ofassistance. This study was supported in part by the Ministry of

Bcl-2 during induction of apoptotic cell death in myeloma

Education, Culture, Sports, Science and Technology of Japan

cells. Recent reports suggest that alterations in the ratio(KAKENHI 24591409).

between pro-apoptotic and anti-apoptotic members of the
Bcl-2 family, rather than the absolute expression level of any

Conflict of Interest

single Bcl-2 member, can determine apoptotic sensitivity,
which would interfere with the availability and translocation

The authors have no conflicts of interest.

of the Bax protein from the cytosol to mitochondria 27). We
also found that Smac, which stands for second mitochondria-

References

derived activator of caspases, was released from mitochondria
to the cytosol during shikonin-induced cell death. Smac binds
to XIAP and eliminates its inhibitory effect on caspase-9.
Various anti-myeloma agents trigger the loss of mitochondrialΔΨm and the release of mitochondrial apoptogenic proteins
cytochrome c and Smac/DIABLO 24,28).
Elevation of intracellular ROS production was also demonstrated during shikonin-induced cell death in myeloma celllines. Various studies have shown that stress-induced changes
in mitochondrial transmembrane potential correlate with anincrease in ROS and the release of mitochondrial cytochromec and Smac/DIABLO. The role of ROS in mediating apoptotic
cell death in various cancer cells is well established 29,30). The
generation of ROS has also been linked to the release of Smacor cytochrome c from mitochondria to the cytosol during
apoptosis 31). The present study found that the antioxidant,
NAC, significantly blocked ROS production and shikonininduced cell death via inhibition of phosphorylation of STAT3
in myeloma cells. These results suggest that ROS is a key an1) Kyle RA, Rajkumar SV. Multiple myeloma. Blood. 2008; 111: 2962–
72.
2) Palumbo A, Anderson K. Multiple myeloma. N Engl J Med. 2011;364: 1046–60.
3) Boyd KD, Pawlyn C, Morgan GJ, Davies FE. Understanding themolecular biology of myeloma and its therapeutic implications.
Expert Rev Hematol. 2012; 5: 603–17.
4) Watanabe R, Tokuhira M, Kizaki M. Current approaches for thetreatment of multiple myeloma. Int J Hematol. 2013; 97: 333–44.
5) Kumar S, Lee JH, Morgan G, Richardson PG, Crowley J, Haessler J,
et al. Risk of progression and survival in multiple myeloma
relapsing after therapy with IMiDs and bortezomib: A multicenterinternational myeloma working group study. Leukemia. 2012; 26:
149–57.
6) Yang H, Zhou P, Huang H, Chen D, Ma N, Cui QC, et al. Shikoninexerts antitumor activity via proteasome inhibition and cell death
induction in vitro and in vivo. Int J Cancer. 2009; 124: 2450–9.
7) Wu Z, Wu LI, Li LH, Tashiro S, Onodera S, Ikejima T. Shikonin
regulates HeLa cell death via caspase-3 activation and blockageof DNA synthesis. J Asian Nat Prod Res. 2004; 6: 155–66.
8) Yeh CC, Kuo HM, Li TM, Lin JP, Yu FS, Lu HF, et al. Shikonin-induced

International Journal of Myeloma

vol. 4 no. 3 (2014)

19

KIMURA et al.

apoptosis involves caspase-3 activity in a human bladder cell line(T24). In Vivo. 2007; 21: 1011–9.
9) Yoon Y, Kim YO, Lim NY, Jeon WK, Sung HJ. Shikonin, an ingredientof Lithospermum erythorhizon induced apoptosis in HL60 humanpromyelocytic leukemia cell line. Planta Med. 1999; 65: 532–5.
10) Hampton MB, Fadeel B, Orrenius S. Redox regulation of the
caspases during apoptosis. Ann NY Acad Sci. 1998; 854: 328–35.
11) Troyano A, Fernandez C, Sancho P, de Blas E, Aller P. Effect of
glutathione depletion on antitumor drug toxicity (apoptosis
and necrosis) in U937 human promyelocyticcells. J Biol Chem.
2001; 276: 47107–15.
12) Nakazato T, Ito K, Ikeda Y, Kizaki M. Green tea component, catechin, induces apoptosis of human malignant B cells via production of reactive oxygen species. Clin Cancer Res. 2005; 11: 6040–9.
13) Baysan A, Yel L, Gollapudi S, Su H, Gupta S. Arsenic trioxide
induces apoptosis via the mitochondrial pathway by upregulating the expression of Bax and Bim in human B cells. Int J Oncol.
2007; 30: 313–8.
14) Green DR, Reed JC. Mitochondria and apoptosis. Science. 1998;
281: 1309–12.
15) Gross A, McDonnell JM, Korsmeyer SJ. Bcl-2 family members andthe mitochondria in apoptosis. Genes Dev. 1999; 13: 1899–911.
16) Bailey HH. L-S, R-buthionine sulfoximine: historical development
and clinical issues. Chem Biol Interact. 1998; 111–112: 239–54.
17) Chang IC, Huang YJ, Chiang TI, Yeh CW, Hsu LS. Shikonin induces
apoptosis through reactive oxygen species/extracellular signalregulated kinase pathway in osteosarcoma. Biol Pharm Bull. 2010;
33: 816–24.
18) Chen C, Shanmugasundaram K, Rigby AC, Kung AL. Shikonin, anatural product from the root of Lithospermum erythrorhizon, is acytotoxic DNA-binding agent. Eur J Pharma Sci. 2013; 49: 18–26.
19) Han W, Xie J, Fang Y, Wang Z, Pan H. Nec-1 enhances shikonininduced apoptosis in leukemia cells by inhibition of RIP-1 and
ERK1/2. Int J Mol Sci. 2013; 13: 7212–25.
20) Staniforth V, Wang SY, Shyur LF, Yang NS. Shikonin, phyto­
compounds from Lithospermum erythrorhizon, inhibits the20

International Journal of Myeloma

vol. 4 no. 3 (2014)transcriptional activation of human tumor necrosis factor α­promoter in vivo. J Biol Chem. 2004; 279: 5877–85.
21) Chiu SC, Yang NS. Inhibition of tumor necrosis factor-α throughselective blockade of pre-mRNA splicing by shikonin. Mol
Pharmacol. 2007; 71: 1640–5.
22) Hideshima T, Mitsiades C, Tonon G, Richardson PG, Anderson KC.
Understanding multiple myeloma pathogenesis in the bonemarrow to identify new therapeutic targets. Nat Rev Cancer. 2007;
7: 585–98.
23) Mahindra A, Laubach J, Raje N, Munshi N, Richardson PG,
Anderson K. Latest advances and current challenges in the treatment of multiple myeloma. Nat Rev Clin Oncol. 2012; 9: 135–43.
24) Chauhan D, Hideshima T, Anderson KC. Apaf-1/cytochrome
c-independent and Smac-dependent induction of apoptosis in
multiple myeloma (MM) cells. J Biol Chem. 2001; 276: 24453–6.
25) Chauhan D, Hideshima T, Anderson KC. Apoptotic signaling in
multiple myeloma: therapeutic implications. Int J Hematol. 2002;
78: 114–20.
26) Tu Y, Renner S, Xu F, Fleishman A, Taylor J, Weisz J, et al. Bcl-X
expression in multiple myeloma: possible indicator of chemo­
resistance. Cancer Res. 1998; 58: 256–62.
27) Gross A, McDonnel JM, Korsmeyer SJ. Bcl-2 family members andthe mitochondria in apoptosis. Genes Dev. 1999; 13: 1899–911.
28) Chauhan D, Pandey P, Ogata A, Teoh G, Krett N, Halgren R, et al.
Cytochrome c-dependent and independent induction of apoptosis in multiple myeloma cells. J Biol Chem. 1997; 272: 29995–7.
29) Cai J, Jones DP. Mitochondrial redox signaling during apoptosis.
J Bioenerg Biomembr. 1999; 31: 327–34.
30) Simon HU, Haj-Yehia A, Levi-Schaffer F. Role of reactive oxygen
species (ROS) in apoptosis induction. Apoptosis. 2000; 5: 415–8.
31) Dvorakova K, Waltmire CN, Payne CM, Tome ME, Briehl MM, Dorr
RT. Induction of mitochondrial changes in myeloma cells by
imexon. Blood. 2001; 97: 3544–51.
32) Mao X, Yu CR, Li WH, Li WX. Induction of apoptosis by shikoninthrough a ROS/JNK-mediated process in Bcr/Abl-positive chronic
myelogenous leukemia (CML) cells. Cell Res. 2008; 18: 879–88.