Shikonin Directly Targets Mitochondria and Causes Mitochondrial Dysfunction in Cancer Cells

Abstract

Chemotherapy is a mainstay of cancer treatment. Due to increased drug resistance and the severe side effects of currently used therapeutics, new candidate compounds are required for improvement of therapy success. Shikonin, a natural naphthoquinone, was used in traditional Chinese medicine for the treatment of different inflammatory diseases and recent studies revealed the anticancer activities of shikonin. We found that shikonin has strong cytotoxic effects on 15 cancer cell lines, including multidrug-resistant cell lines. Transcriptome-wide mRNA expression studies showed that shikonin induced genetic pathways regulating cell cycle, mitochondrial function, levels of reactive oxygen species, and cytoskeletal formation. Taking advantage of the inherent fluorescence of shikonin, we analyzed its uptake and distribution in live cells with high spatial and temporal resolution using flow cytometry and confocal microscopy. Shikonin was specifically accumulated in the mitochondria, and this accumulation was associated with a shikonin-dependent deregulation of cellular Ca(2+) and ROS levels. This deregulation led to a breakdown of the mitochondrial membrane potential, dysfunction of microtubules, cell-cycle arrest, and ultimately induction of apoptosis. Seeing as both the metabolism and the structure of mitochondria show marked differences between cancer cells and normal cells, shikonin is a promising candidate for the next generation of chemotherapy.

Full text

Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2012, Article ID 726025, 15 pages
doi:10.1155/2012/726025
Research Article
Shikonin Directly Targets Mitochondria and Causes
Mitochondrial Dysfunction in Cancer Cells
Benjamin Wiench,1Tolga Eichhorn,1Malte Paulsen,2and Thomas Efferth1
1Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University,
Staudinger Weg 5, 55128 Mainz, Germany
2Cytometry Core Facility, Institute of Molecular Biology, Ackermannweg 4, 55128 Mainz, Germany
Correspondence should be addressed to Thomas Efferth, efferth@uni-mainz.de
Received 10 July 2012; Accepted 7 September 2012
Academic Editor: Ke Liu
Copyright © 2012 Benjamin Wiench et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Chemotherapy is a mainstay of cancer treatment. Due to increased drug resistance and the severe side effects of currently used
therapeutics, new candidate compounds are required for improvement of therapy success. Shikonin, a natural naphthoquinone,
was used in traditional Chinese medicine for the treatment of different inflammatory diseases and recent studies revealed the
anticancer activities of shikonin. We found that shikonin has strong cytotoxic effects on 15 cancer cell lines, including multidrug-
resistant cell lines. Transcriptome-wide mRNA expression studies showed that shikonin induced genetic pathways regulating cell
cycle, mitochondrial function, levels of reactive oxygen species, and cytoskeletal formation. Taking advantage of the inherent
fluorescence of shikonin, we analyzed its uptake and distribution in live cells with high spatial and temporal resolution using
flow cytometry and confocal microscopy. Shikonin was specifically accumulated in the mitochondria, and this accumulation was
associated with a shikonin-dependent deregulation of cellular Ca2+ and ROS levels. This deregulation led to a breakdown of the
mitochondrial membrane potential, dysfunction of microtubules, cell-cycle arrest, and ultimately induction of apoptosis. Seeing as
both the metabolism and the structure of mitochondria show marked differences between cancer cells and normal cells, shikonin
is a promising candidate for the next generation of chemotherapy.
1. Introduction
Cancer is a leading cause of death worldwide and the global
burden of cancer continues to increase, largely because of the
growth and aging of the world population [1]. Resistance
to cell death and reprogramming of metabolic pathways
aretwohallmarksofhumancancercellsaswellasmajor
causes of chemotherapy inefficacy [2]. Mitochondria are
key structures for both these traits: (i) mitochondria are
crucial for cellular energy production and cell survival; (ii)
mitochondria are major regulators in the intrinsic apoptotic
pathway [3]. Mitochondrial membrane permeabilization
(MMP) and the subsequent release of mitochondrial death
effectors (e.g., cytochrome c) are key events for caspase
activation and apoptosis [4]. The induction of mitochondrial
apoptosis can be triggered by various intracellular stimuli
such as Ca2+ overload or high levels of reactive oxygen
species (ROS) [5]. Furthermore, both these stimuli reinforce
each other, leading to Ca2+/ROS-mediated mitochondrial
dysfunction [6].
In cancer cells, the structure and function of mito-
chondria differ significantly from normal eukaryotic cells
[7]. Cancer cells display decreased mitochondrial activity
and instead shift to aerobic glycolysis for ATP produc-
tion, a phenomenon known as the Warburg effect [8].
Cancer cells are often more resistant to activation of the
mitochondrial apoptotic pathway due to overexpression of
antiapoptotic Bcl-2 family proteins [9] or stabilization of
the mitochondrial membrane against apoptosis-associated
permeabilization [10]. Another trait associated with cancer
cells is elevated ROS levels, probably caused by mitochon-
drial dysfunction [11]. Therefore, it is conceivable that
cancer cells have a lower tolerance to further oxidative
insults induced by ROS-generating drugs [9,12]. Because

2 Evidence-Based Complementary and Alternative Medicine
of the altered mitochondrial functions in neoplasia, direct
targeting of mitochondria in cancer cells has become an
attractive strategy in cancer chemotherapy over the last few
years. A direct induction of apoptosis in cancer cells via the
mitochondrial pathway allows one to circumvent upstream
signal transduction steps frequently impaired in human
cancers [12]. Thus, compounds targeting mitochondria may
help to improve the poor results of traditional therapies
and furthermore represent a promising approach for the
treatment of cancer cells resistant to standard chemotherapy
[5].
The naphthoquinone pigment shikonin is the most
important pharmacologically active substance in the dried
root of Lithospermum erythrorhizon. In traditional Chinese
medicine root extracts of Lithospermum erythrorhizon have
been used to treat macular eruption, measles, sore throat,
carbuncles, and burns [13]. The antitumor effect of shikonin
was first evidenced by its activity against murine sarcoma-
180 [14]. A clinical trial using shikonin in 19 cases of
late-stage lung cancer revealed that a shikonin-containing
mixture was safe and effective for the treatment of late-stage
cancer [15]. The mechanism by which shikonin triggers its
cytotoxic effect against malignant cells is controversial. A
very recent study showed that shikonin inhibits cancer cell
glycolysis by targeting tumor pyruvate kinaseM2 [16].
In this study, we show for the first time that the natural
compound shikonin directly targets mitochondria causing
mitochondrial dysfunction and ultimately apoptosis. We
confirmed a number of recent findings regarding the cellular
effects of shikonin, but our data suggests that most of
them are downstream events. The primary effect of shikonin
is the direct targeting of mitochondria, which causes a
dose-dependent overproduction of ROS and an increase
in intracellular calcium levels, leading to breakdown of
the mitochondrial membrane potential and induction of
the mitochondrial pathway of apoptosis. The increase in
intracellular ROS levels and the mitochondrial injury cause
other cellular effects such as oxidative DNA damage and
inhibition of cancer cell migration.
2. Materials and Methods
2.1. Chemicals. Shikonin was purchased from Enzo Life
Sciences (Lausen, Switzerland) and a 50 mM stock solution
was prepared by dissolving it in DMSO. Doxorubicin and
daunorubicin were provided by the University Medical
Center of the Johannes Gutenberg University (Mainz, Ger-
many) and dissolved in PBS (Invitrogen, Germany) at a
concentration of 10 mM. Geneticin was purchased from
Sigma-Aldrich (Munich, Germany) at a concentration of
50 mg/mL in sterile-filtered H2O.
2.2. Cell Cultures. U937, 789-O, MCF-7, SK-BR-3, SW1116,
HCT-116, SW680, Capan1, and SUIT-2 cell lines were
obtained from the German Cancer Research Center (DKFZ,
Heidelberg, Germany). The original source of these cell lines
is the American Type Culture Collection (ATCC, USA).
U937 and 786-O cells were maintained in complete RPMI
1640 medium with 2 mM L-glutamine (Invitrogen, Ger-
many) supplemented with 10% FBS (Invitrogen, Germany)
and 1% penicillin (100 U/mL)-streptomycin (100 μg/mL)
(Invitrogen, Germany). MCF-7, SKBR3, SW-1116, HCT-
116, SW680, Capan1, and SUIT-2 cells were cultured in com-
plete DMEM culture medium with GlutaMAX (Invitrogen,
Germany) supplemented with 10% FBS and 1% penicillin
(100 U/mL)-streptomycin (100 μg/mL).
CCRF-CEM and CEM/ADR5000 cells were kindly
provided by Dr. Axel Sauerbrey (University of Jena, Depart-
ment of Pediatrics, Jena, Germany) and HL-60 and HL60/AR
cells by Dr. J. Beck (University of Greifswald, Department
of Pediatrics, Greifswald, Germany). The original source of
these cell lines is the ATCC. Cells were cultured in complete
RPMI 1640 medium with 10% FBS and 1% penicillin-
streptomycin. To maintain the multidrug-resistance phe-
notypes, the MDR1-expressing CEM/ADR5000 cells were
treated with 5000 ng/mL doxorubicin and the MRP1-
expressing HL-60/AR subline was cultured in medium
containing 100 nM daunorubicin.
MDAMB231 pcDNA3 and MDAMB231BCRP breast
cancer cell lines transduced with control vector (MDA-MB-
231-pcDNA3) or with cDNA for the breast cancer-resistant
protein BCRP (MDA-MB-231-BCRP clone 23) were kindly
provided by Dr. Douglas D. Ross (University of Maryland,
Department of Medicine, Baltimore, Maryland). Both cell
lines were continuously treated with 800 ng/mL geneticin.
The multidrug resistance profile of these cell lines has been
reported [17].
The human osteosarcoma cell line U2OS is stably
transfected with a GFP fusion construct of α-tubulin.
The cell line was kindly provided by Joachim Hehl, Light
Microscopy Centre, ETH Zurich. Retinal pigment epithelial
(RPE-1) cells stably expressing GFP-EB3 were obtained
from Professor W. Krek, ETH Zurich [18]. U2OS-GFP-
αTubulin and RPE-1-GFP-EB3 cells were maintained in
DMEM medium containing 10% FBS (Invitrogen, Germany)
and 1% penicillin (100 U/mL)-streptomycin (100 μg/mL)
(Invitrogen, Germany) and were continuously treated with
250 μg/mL and 500 μg/mL geneticin, respectively.
All cell lines were maintained in a humidified environ-
ment at 37◦Cwith5%CO
2and subcultured twice per week.
All experiments were performed on cells in the logarithmic
growth phase.
2.3. Resazurin Reduction Assay. Resazurin reduction assay
[19] was performed to assess cytotoxicity of shikonin toward
various sensitive and resistant cancer cell lines. The assay
is based on reduction of the indicator dye, resazurin, to
the highly fluorescent resorufin by viable cells. Nonviable
cells rapidly lose the metabolic capacity to reduce resazurin
and thus produce no fluorescent signal. Briefly, adherent
cells were detached by treatment with 0.25% trypsin/EDTA
(Invitrogen, Germany) and an aliquot of 1 ×104cells was
placed in each well of a 96-well cell culture plate (Thermo
Scientific, Germany) in a total volume of 200 μL. Cells were
allowed to attach overnight and then were treated with
different concentrations of shikonin. For suspension cells,

Evidence-Based Complementary and Alternative Medicine 3
aliquots of 2 ×104cells per well were seeded in 96-well-
plates in a total volume of 100 μL. Shikonin was immediately
added in varying concentrations in an additional 100 μLof
culture medium to obtain a total volume of 200 μL/well.
After 24 h or 48 h, 20 μL resazurin (Sigma-Aldrich, Germany)
0.01% w/v in ddH2O was added to each well and the plates
were incubated at 37◦C for 4 h. Fluorescence was measured
on an Infinite M2000 Proplate reader (Tecan, Germany)
using an excitation wavelength of 544nm and an emission
wavelength of 590 nm. Each assay was done at least two times,
with six replicates each. The viability was evaluated based
on a comparison with untreated cells. IC50 values represent
the shikonin concentrations required to inhibit 50% of cell
proliferation and were calculated from a calibration curve by
linear regression using Microsoft Excel.
2.4. Microarray Gene Expression Profiling. To t al R NA f r om
U937 cells after 24 h of treatment with shikonin at IC50
concentration was isolated using RNeasy Kit from Qiagen
(Hilden, Germany) according to the manufacture’s instruc-
tion. The quality of total RNA was checked by gel analysis
using the total RNA Nanochip assay on an Agilent 2100
Bioanalyzer (Agilent Technologies GmbH, Berlin, Germany).
Only samples with RNA index values greater than 9.3
were selected for expression profiling. Biotin-labeled cRNA
samples for hybridization on Illumina Human Sentrix-HT12
Bead Chip arrays (Illumina, Inc.) were prepared according
to Illumina’s recommended sample labeling procedure based
on the modified Eberwine protocol [20]. Biotin-16-UTP
was purchased from Roche Applied Science (Penzberg,
Germany). The cRNA was column purified with TotalPrep
RNA Amplification Kit and eluted in 60μLofwater.Quality
of cRNA was controlled using the RNA Nanochip assay
on an Agilent 2100 Bioanalyzer and spectrophotometrically
quantified (NanoDrop). Hybridization was also performed
according the manufacturer’s recommendations. Microarray
scanning was done using a Beadstation array scanner, setting
adjusted to a scaling factor of 1 and PMT settings at 430. Data
was extracted for each bead individually, and outliers were
removed when the MAD (median absolute deviation) was
greater than 2.5. Data analysis was performed by normalizing
the signals using the quantile normalization algorithm
without background subtraction, and differentially regulated
genes were defined by calculating the standard deviation
differences of a given probe in a one-by-one comparison of
samples or groups. The expression data obtained was filtered
with Chipster data analysis platform. Filtered genes were fed
into Ingenuity Pathway Analysis software, which assigned
them to networks, functions, and pathways.
2.5. Real-Time Reverse Transcription-PCR. The same RNA
samples used in the microarray experiments were also used
for RT-PCR experiments. Total RNA samples were converted
to cDNA by reverse transcriptase (Invitrogen) with random
hexamer primers. Quantification of cDNA was performed by
real-time PCR using a Taq-polymerase master mix (Roche)
containing the fluorescent dye SYBR Green (Biozol) and
the CFX384 Real-Time PCR Detection System (Bio-Rad).
The efficiency of all primer pairs used for real-time PCR
expression was better than 90%. PCR was performed with
an initial denaturation at 95◦Cfor5minfollowedby50
cycles consisting of strand separation at 95◦Cfor30sand
annealing and extension at 60◦C for 40 s. After PCR product
amplification, melting curves were computed. Expression
levels were normalized to the transcription level of G6PD. All
samples were run in triplicates.
2.6. Fluorescence Scan. Shikonin was solved in aqueous
buffer at a final concentration of 50 μM.A2Dexcita-
tion (200–600 nm) versus emission (300–700 nm) scan was
performed on a Fluorolog-2 spectrofluorometer (Horiba,
Unterhaching).
2.7. Measurement of Cellular Uptake by Flow Cytometry.
Cellular drug uptake experiments were performed according
to a recently published protocol [21] using the intrinsic
fluorescence of shikonin. Briefly, 1 ×106U937 cells in
complete RPMI 1640 culture medium lacking phenol red
indicator were transferred into a 5 mL FACS tube (Becton-
Dickinson, Germany) in a total volume of 2 mL. The cells
were measured on an LSR-Fortessa FACS analyzer (Becton-
Dickinson, Germany) with 640 nm excitation (40 mW) and
detected using a 730/45 nm bandpass filter. The background
fluorescence of the cells was measured for 2 min and adjusted
to be 10×above the electronic noise of the analyzer to ensure
precise measurements even in samples with large cellular-
based coefficients of variation (CV). Dead cells were excluded
by FSC/SSC gating. After 2 min of recording, the FACS tube
was removed from the flow cytometer without stopping the
recording and shikonin was added immediately in the desired
concentration. The cell suspension containing the drug was
gently but thoroughly mixed and reinserted into the flow
cytometer within 30 sec after removing it from the machine.
The measurements continued to 20 min total time at a flow
rate of 50–70 cells per second while recording the area signal
of all significant channels. Cytographs were analyzed using
FlowJo software (Celeza, Switzerland).
2.8. Cellular Localization via Confocal Microscopy. For intra-
cellular localization studies of shikonin, cells were stained
with MitoTracker Green FM (Invitrogen, Germany). This
fluorescent dye passively diffuses across the plasma mem-
brane and accumulates in active mitochondria. Briefly,
4×104U937cellswereplacedineachwellofasterileibiTreat
μ-slide (ibidi, Germany) pretreated with polyethylenimine
for sufficient cell adhesion or 2 ×104SKBR3 cells were
seeded in untreated ibiTreat μ-slides and allowed to attach
overnight. According to the manufacturer’s protocol, cells
were incubated with 100 nM MitoTracker Green FM for
45 min at 37◦C in the dark. After incubation, the staining
solutionwasremovedandthecellswereresuspendedinfresh
medium containing 25 μM shikonin. Live cell imaging was
performed on a Leica TCS SP5 confocal microscope with
a40
×/1.30 oil objective (Leica, Germany). The microscope
was controlled by LAS AF software (Leica, Germany).
A 561 nm laser was used for excitation of shikonin, and

4 Evidence-Based Complementary and Alternative Medicine
the emitted signal was detected at 680–780 nm. MitoTracker
Green FM was excited with a 488 nm laser and detected
at 500–549 nm. Analysis and averaging of images were
performed with LAS AF software; further image processing
was carried out with Adobe Photoshop.
2.9. Analysis of Mitochondrial Membrane Potential. The
effects of shikonin on the mitochondrial membrane potential
were analyzed by JC-1 (Biomol, Germany) staining. JC-1
is a dye that can selectively enter into mitochondria and
exhibits an intense red fluorescence in healthy mitochondria
with normal membrane potentials. In cells with reduced
mitochondrial membrane potential, the red fluorescence
disappears. Briefly, 1 ×106U937 cells were incubated with
JC-1 staining solution according to the manufacturer’s
protocol for 30 min. Stained cells were treated with different
concentrations of shikonin or DMSO (solvent control) for
6 h. Subsequently, cells were measured in an LSR-Fortessa
FACS analyzer (Becton-Dickinson, Germany). For each
sample, 1 ×104cells were counted. The JC-1 signal was
measured with 561 nm excitation (150 mW) and detected
using a 586/15 nm bandpass filter. The shikonin signal was
analyzed with 640 nm excitation (40 mW) and detected using
a 730/45 nm bandpass filter. All parameters were plotted
on a logarithmic scale. Cytographs were analyzed using
FlowJo software (Celeza, Switzerland). All experiments were
performed at least in triplicate.
2.10. Measurement of Reactive Oxygen Species by Flow Cytom-
etry. 2,7-Dichlorodihydrofluorescein diacetate (H2DCFH-
DA) (Sigma-Aldrich, Germany) is a probe used for the
highly sensitive and quantifiable detection of reactive oxygen
species (ROS). The nonfluorescent H2DCFH-DA diffuses
into the cells and is cleaved by cytoplasmic esterases into
2,7-dichlorodihydrofluorescein (H2DCF) which is unable
to diffuse back out of the cells. In the presence of hydrogen
peroxide, H2DCF is oxidized to the fluorescent molecule
dichlorofluorescein (DCF) by peroxidases. The fluorescent
signal emanating from DCF can be measured and quan-
tified by flow cytometry, thus providing an indication
of intracellular ROS concentration [22,23]. Briefly, 2 ×
106U937 cells were resuspended in PBS and incubated with
2μMH
2DCFH-DA for 20 min in the dark. Subsequently,
cells were washed with PBS and resuspended in RPMI
1640 culture medium containing different concentrations of
shikonin or DMSO (solvent control). After 1 h of incubation,
cells were washed and suspended in PBS. Subsequently
cellsweremeasuredinaFACSCaliburflowcytometer
(Becton-Dickinson, Germany). For each sample 1 ×104
cells were counted. DCF was measured at 488 nm excitation
(25 mW) and detected using a 530/30nm bandpass filter. All
parameters were plotted on a logarithmic scale. Cytographs
were analyzed using FlowJo software (Celeza, Switzerland).
All experiments were performed at least in triplicate.
For the measurement of real-time kinetics of ROS
induction, U937 cells were stained with H2DCFH-DA as
described above. The cells were measured on an LSR-Fortessa
FACS analyzer (Becton-Dickinson, Germany) with 488 nm
excitation (100 mW) for DCF and 649 nm excitation for
shikonin. DCF and shikonin fluorescence were detected
using a 530/30 nm and a 730/45 nm bandpass filter, respec-
tively. After 2 min of recording, 0.6 μM shikonin was added to
the cell suspension and the tube was immediately reinserted
into the flow cytometer. The measurement continued up
to 1 h total time at a flow rate of 50–70 cells per second.
Cytographs were analyzed using FlowJo software (Celeza,
Switzerland).
2.11. DNA Damage Detection and Quantification by Alkaline
Elution Assay. The alkaline elution assay was originally
described by Kohn et al. [24] and modified by Epe et al. [25].
In this study, the assay was used to quantify different types
of DNA modifications generated in U937 cells after shikonin
treatment. Briefly, 106cells were treated with 0.3 μM
shikonin (IC50)for3hand6horDMSO(solventcontrol).
After incubation, cells were lysed on a polycarbonate filter
(2 mm pore size) by pumping a lysis solution (100 mM
glycine, 20 mM Na2EDTA, 2% SDS, 500 mg/L proteinase
K, pH 10.0) through the filter for 90 min at 25◦C. After
washing, the DNA remaining on the filter was incubated
with the repair endonuclease Fpg protein (1 μg/mL) for
30 min at 37◦C to detect DNA modifications sensitive to
oxidative stress. To quantify DNA single-strand breaks, the
incubation was also carried out without endonuclease. After
Fpg endonuclease incubation the DNA was eluted with an
alkaline solution at 25◦C and its elution rate determined. The
slopes of elution curves obtained with γ-irradiated cells were
used for calibration (6 Gγ=1 single-strand break/106bp).
2.12. Measurement of Intracellular Calcium Signaling. For
analysis of intracellular calcium signaling after shikonin
treatment, the chemical calcium indicator indo-1
(Invitrogen, Germany) was used. The dye can be excited
in the UV range at ∼350 nm and peak emission occurs
at ∼405 nm and ∼485 nm in the Ca2+ bound and free
states, respectively [26]. In this way, a relatively accurate
measurement of the intracellular Ca2+ concentration
by a fluorometric ratio technique is possible. Briefly,
2×106U937 cells in 2 mL 1640 RPMI culture medium
without phenol red indicator were incubated with 1 μM
indo-1 for 30 min at 37◦C in the dark. Following incubation,
cells were centrifuged and resuspended in fresh culture
medium.Subsequently,cellsweremeasuredonanLSR-
Fortessa FACS analyzer (Becton-Dickinson, Germany)
with 355 nm excitation (20 mW) and detected using
405/20 nm and 530/30 nm bandpass filters. The ratio of
both signals (405/20 nm/530/30 nm) was used as an index
for intracellular calcium concentration. After 2 min, the
FACS tube was removed from the flow cytometer without
stopping the recording and shikonin was added immediately
in the desired concentration. The cell suspension containing
the drug was mixed and reinserted into the flow cytometer
within 30 sec after being removed. The measurements
continued up to 60 min total time at a flow rate of 100–
150 cells per second while recording the area signal of
all significant channels. Cytographs were analyzed using

Evidence-Based Complementary and Alternative Medicine 5
FlowJo software (Celeza, Switzerland). The Ca2+ exchanger
ionomycin (Sigma-Aldrich, Germany) raises intracellular
calcium levels and served as positive control at a final
concentration of 0.4 μM. Cells treated with DMSO served as
a solvent control.
2.13. Cell Cycle Analysis. For cell-cycle analysis, 1 ×106U937
cellsweretreatedwithdifferent concentrations of shikonin
for 24 h. Following incubation, cells were washed in PBS and
fixed in ice-cold 95% ethanol (Sigma-Aldrich, Germany).
After washing in PBS, cells were incubated with 10 μg/mL
RNase A (Applichem Lifescience, Germany) and 50 μg/mL
propidium iodide (PI, Sigma-Aldrich, Germany) in PBS
for 1 h in the dark. Cells were measured on an LSR-
Fortessa FACS analyzer (Becton-Dickinson, Germany). 1 ×
104cellswerecountedforeachsample.PIwasmeasured
with 488 nm excitation (100 mW) and detected using a
610/20 nm bandpass filter. Cytographs were analyzed using
FlowJo software (Celeza, Switzerland). All experiments were
performed at least in triplicate.
2.14. Caspase-Glo 3/7 and Caspase-Glo 9 Assay. The influ-
ence of shikonin on caspase 3/7 and caspase 9 activity in
U937 leukemia cells was detected using Caspase-Glo 3/7
and Caspase-Glo 9 Assay kits (Promega, Germany). Cells
cultured in RPMI 1640 medium were seeded in 96-well
plates and treated with different concentrations of shikonin
or DMSO (solvent control). After 6h of treatment, cellular
caspase 3/7 or caspase 9 activity was determined according
to the manufacturer’s protocol. Luminescence was measured
using the Infinite M2000 Proplate reader (Tecan, Germany).
Caspase activity was expressed as percentage of the untreated
control.
2.15. Scratch Migration Assay. The scratch migration assay is
a well-developed method to investigate drug effects on cell
migration in vitro [27]. Briefly, 1 ×106SKBR3 cells were
seeded in each well of a 6-well plate and allowed to grow
to a confluent monolayer. The cell monolayer was carefully
scraped with a sterile p200 pipet tip to create a scratch.
Subsequently, cells were washed with PBS and treated
with DMEM culture medium containing different subtoxic
concentrations of shikonin or DMSO (solvent control).
Images of the scratches were taken every 3 h using a phase
contrast microscope (Optika, Italy) at 10x magnification.
Data analysis was performed with TScratch software [28].
2.16. Imaging of the Structure and Dynamics of the Micro-
tubule Cytoskeleton by Confocal Microscopy. Live cell imaging
was performed on a Leica TCS SP5 confocal microscope with
a 40x/1.30 oil objective. The microscope was controlled by
LAS AF software. A 488 nm laser was used for excitation of
GFP, and the emitted signal was detected at 500–549nm.
Shikonin was excited with a 561 nm laser and the emitted flu-
orescence was detected at 680–780 nm. Analysis and averag-
ing of images were performed with LAS AF software; further
image processing was carried out with Adobe Photoshop.
2×104U2OS-GFP-αTubulin or RPE-1-GFP-EB3 cells
were seeded in each well of a sterile ibi Treat μ-slide (ibidi,
Germany) and cells were allowed to attach overnight. Cells
were treated with 25 μM shikonin and subsequently analyzed
by confocal microscopy. Each experiment was repeated at
least three times and representative images and videos were
selected.
3. Results
3.1. Cytotoxic EffectofShikoninonCancerCells. To investi-
gate the effect of shikonin against various types of cancer,
a panel of 15 sensitive and multidrug resistant cancer cell
lines was treated with shikonin for 24 or 48 h. The results
are summarized in Tabl e 1 . Shikonin inhibited proliferation
by 50% in nearly all cancer cell lines at concentrations
below 10 μM after 24 h. Only the pancreatic carcinoma cell
line SUIT2 was more resistant to shikonin; it showed IC50
values of 12.9 and 18.5 μM after 24 and 48 h of treatment,
respectively. Interestingly, all five tested leukemia cell lines
had IC50 values below 1 μM, and the most sensitive cell
line was the histiocytic leukemia cell line U937, which
was subsequently used for gene expression analysis under
shikonin treatment.
Besides non-MDR cell lines like U937, shikonin appeared
to be highly effective against three known multidrug-
resistant cancer cell lines: CEM/ADR5000, HL-60/AR, and
MDAMB231/BCRP (Tab le 1 ). Similarly to their sensitive
counterparts, these cell lines showed no or negligible resis-
tance against shikonin.
3.2. Gene Expression Profiling Identifies Novel Molecular Key
Players and Genetic Networks. We performed gene expres-
sion analysis to identify possible targets and mechanisms
of shikonin’s anticancer activities in histocytic leukemia
U937 cells. U937 cells were treated with 0.3 μM(IC
50)
shikonin or DMSO solvent control for 24h before total
RNA was isolated for a whole human genome mRNA gene
expression microarray. Bioinformatic analysis identified 683
genes significantly deregulated (P<0.01) after shikonin
treatment. For a number of genes, the expression array
was validated using RT-qPCR, which yielded comparable
results (See Table S1 in Supplementary Material available
online at doi: 10.1155/2012/726025). Using the Ingenuity
Pathway Analysis tool, we correlated the deregulated genes
with 71 biological functions (or diseases) including cell death
and cell cycle, cancer, cellular movement and cell mor-
phology, inflammatory response, protein folding, synthesis
and posttranscriptional modification, energy production
and cellular growth, and DNA repair and free radical
scavenging. Furthermore, four genetic networks were found
to be significantly deregulated in U937 cells after shikonin
treatment and were correlated to discrete cellular functions
like DNA repair, energy production, cell morphology, and
cellular development. Subsequent analysis of the microar-
ray showed that a whole subset of genes responsible for
mitochondrial function was deregulated. Combining the
information obtained from Ingenuity Pathway Analysis and

6 Evidence-Based Complementary and Alternative Medicine
Tab l e 1: IC50 values (mean ±SEM) of shikonin for a panel of 15 different sensitive and resistant cancer cell lines after 24 and 48 h as assayed
by resazurin reduction assay.
Cell line Cancer type Shikonin
IC50 [μM] (24 h) IC50 [μM] (48 h)
U937 Histiocytic leukemia cell line 0.30 ±0.003 0.19 ±0.003
CCRF-CEM Acute lymphocytic leukemia cell line 0.37 ±0.01 0.24 ±0.001
CEM/ADR5000∗Acute lymphocytic leukemia cell line 0.36 ±0.05 0.38 ±0.01
HL-60 Acute myelocytic leukemia cell line 0.39 ±0.01 0.42 ±0.001
HL-60/AR∗Acute myelocytic leukemia cell line 0.95 ±0.003 0.47 ±0.001
MCF-7 Breast carcinoma cell line 9.05 ±0.08 10.51 ±0.03
SK-BR-3 Breast adenocarcinoma cell line 9.21 ±0.08 8.70 ±0.03
MDA-MB-231/pcDNA3 Breast carcinoma cell line 1.23 ±0.03 0.88 ±0.03
MDA-MB-231/BCRP∗Breast carcinoma cell line 2.61 ±0.06 1.48 ±0.04
786-O Kidney carcinoma cell line 9.44 ±0.13 8.03 ±0.06
SW-1116 Colorectal carcinoma (GIII) cell line 6.63 ±0.09 4.42 ±0.06
HCT-116 Colorectal carcinoma 4.74 ±0.07 8.54 ±0.01
SW680 Colorectal carcinoma 7.21 ±0.13 2.96 ±0.06
Capan1 Pancreas adenocarcinoma cell line 7.23 ±0.28 6.21 ±0.14
SUIT-2 Pancreatic carcinoma cell line 12.92 ±0.35 18.50 ±0.09
∗MDR cancer cell lines with various drug resistances.
our own in-depth analysis of the deregulated genes, we
were able to divide the major cellular functions affected by
shikonin into four categories: mitochondrial function, ROS
induction and DNA damage, apoptosis and cell cycle arrest,
and cytoskeleton and migration (Figure S1).
3.3. Mitochondrial Drug Accumulation and Breakdown
of the Mitochondrial Membrane Potential. Since shikonin
had a strong effect on gene expression associated with
mitochondrial function and metabolism, we proposed
that the mitochondrion itself is a possible target of the
compound. Shikonin has a specific inherent fluorescence
spectrum that has until now remained either unnoticed
or ignored. We measured a two-dimensional fluorescence
spectrum of emission versus excitation wavelengths for
shikonin and observed strong and specific fluorescence
in the visible spectrum (Figure 1(a)). We performed flow
cytometric cellular uptake assays based on the inherent
fluorescence of the molecule, which indicate that shikonin
enters the cells within 10 min and that the cellular concen-
tration increases with higher application doses (Figure 1(b)).
Washout experiments showed that shikonin remains stable
inside the cells for at least 20 min, suggesting that no direct
diffusion or transport out of the cell takes place (Figure 1(b),
lower right panel). We further exploited the inherent fluo-
rescence of shikonin to map its cellular localization in U937
and SK-BR-3 cells by confocal fluorescence microscopy.
This examination revealed tubular structures with strong
shikonin fluorescence, likely to be mitochondria, around
the nucleus. In order to validate this assumption, U937
cells were stained with the mitochondrial dye MitoTracker
Green and then incubated with a higher dose of shikonin
to enhance detection via confocal microscopy. As shown in
Figure 1, shikonin (Figure 1(c), middle position) was clearly
colocalized with the MitoTracker signal (Figure 1(c), left
position) only minutes after shikonin application, corrob-
orating the fact that shikonin accumulates directly in and
around the mitochondria after its rapid cellular uptake. For
improved visualization of the mitochondria, the experiment
was repeated with the adherent SKBR-3 breast cancer cell
line, resulting in a similar accumulation of shikonin in and
around the mitochondria (Figure 1(c)).
The localization of shikonin to the mitochondria nat-
urally led to the question of whether shikonin indeed
influences mitochondrial function as suggested by our
gene expression profiling results. We hypothesized that
shikonin negatively influences the mitochondrial membrane
potential. U937 cells were stained with JC-1, a marker
for intact mitochondrial membrane potential, and treated
with 0.3–1.2 μM shikonin for 6 h. Subsequently, the cellular
shikonin signal and the JC-1 signal were analyzed by flow
cytometry. The potential difference across the mitochondrial
membrane was significantly reduced in cells treated with
shikonin, as indicated by reduced red fluorescence of JC-1 in
these samples (Figure 1(d)). The mitochondrial membrane
potential was reduced more quickly and to a greater extent
in cells treated with increasing doses of shikonin (data not
shown). This suggests that shikonin itself acts to reduce the
membrane potential rather than interacting with or blocking
a specific target protein.
3.4. Induction of Reactive Oxygen Species and DNA Damage.
As we have shown, shikonin enters cells quickly, accumulates
in the mitochondria, and negatively influences the mito-
chondrial membrane potential. However, we wanted to know
whether the cytotoxicity of shikonin is mediated largely
by mitochondrial deregulation. Mitochondria are the most
significant source of cellular ROS [29]. Given our results,

Evidence-Based Complementary and Alternative Medicine 7
2D excitation/emission scan of shikonin Localization of shikonin in mitochondria
Mitochondrial membrane potentialReal-time shikonin uptake kinetic
Shikonin fluorescence
Time (s)
Shikonin fluorescence
Time (s)
Shikonin fluorescence
Time (s)
Shikonin fluorescence
Time (s)
Shikonin fluorescence
Emission
Excitation
SK-BR-3 U937
MitoTracker Shikonin Merge: MitoTracker + shikonin
Control
Shikonin fluorescence
Shikonin fluorescence
Shikonin fluorescence
0.15 μM shikonin 0.3 μM shikonin
0.6 μM shikonin 0.6 μM shikonin
after washout
200
250
300
350
400
450
500
550
600
300 350 400 450 500 550 600 650 700
Fluorescence intensity
min max
0
102
103
104
105
104
105
0300 600 900 1.2 K
0
102
101
101
102
102
103103
103
104
104
105
105
0300 600 900 1.2 K
0
102
103
104
105
0 300 600 900 1.2 K
0
102
103
104
105
0 300 600 900 1.2 K
101
101
102
102
103
103
104
104
105
105
101
101
102
102
103
103
104
104
105
105
101
101
102
102
103
103
104
104
105
105
Q1
0.010%
Q2
0.744%
Q4
1.19%
Q3
98.1%
Q1
5.47%
Q2
91.3%
Q1
0.01%
Q2
0.744%
Q4
0.279%
Q3
2.98%
Q1
15.4%
Q2
84.5%
Q4
0.021%
Q3
0.094%
Q1
59.5%
Q2
40.5%
Q4
0.00%
Q3
0.00%
(a) (c)
(b) (d)
0.3 μM shikonin
0.6 μM shikonin 1.2 μM shikonin
Mitochondrial membrane
potential (JC-1)
Mitochondrial membrane
potential (JC-1)
Figure 1: Shikonin accumulates in mitochondria and causes a breakdown of the mitochondrial membrane potential. (a) 2D excitation (200–
600 nm) versus emission (300–700 nm) fluorescence spectrum of 50 μM shikonin in aqueous buffer. (b) Real-time kinetics and quantification
of cellular shikonin uptake by flow cytometry. The inherent fluorescence of intracellular shikonin was measured at 640nm excitation with
a 730/45 nm bandpass filter. A dose-dependent increase of the cellular shikonin fluorescence was observed after treatment with increasing
concentrations (0.15, 0.3 and 0.6 μM) of shikonin. After 20min of incubation with 0.6 μM shikonin and subsequent washing of the cells,
shikonin’s fluorescence was still detectable in cells, indicating persistent intracellular accumulation of shikonin. (c) Shikonin localizes to
mitochondria. U937 or SK-BR-3 cells were stained with MitoTracker Green and subsequently treated with 25 μM shikonin. Cells were then
examined by confocal microscopy at an excitation wavelength of 488 nm and 561 nm and emission at 500–549 nm and 680–780 nm for
MitoTracker Green and shikonin, respectively. (d) Breakdown of the mitochondrial membrane potential. U937 cells were stained with JC-1,
which has a strong red fluorescence in healthy mitochondria. Shikonin induced a dose-dependent decrease of the red JC-1 fluorescence after
6 h of treatment with increasing concentrations of shikonin (0.3, 0.6, and 1.2 μM), indicating a breakdown of the mitochondrial membrane
potential.

8 Evidence-Based Complementary and Alternative Medicine
particularly those on shikonin’s effect on the mitochondrial
membrane potential, we analyzed cellular ROS levels after
shikonin treatment by H2DCFH-DA staining and flow
cytometry. Indeed, we observed a clear dose-dependent
increase in cellular ROS levels after very short incubation
periods (1 h) with shikonin (Figures 2(a) and 2(b)) confirm-
ing previous reports [30]. ROS levels after treatment with
0.6 μM shikonin are comparable to those after incubation
with 50 μMH
2O2, our positive control. Thus, shikonin is
indeed a potent ROS inducer (Figure 2(a)). We performed
real-time measurements of shikonin uptake and ROS induc-
tion to correlate the kinetics of these two processes. We
observed increased ROS production shortly after cellular
shikonin uptake, and ROS levels continuously increased for
at least 1 h after exposure to shikonin (Figure 2(c)). The
kinetics of ROS induction suggest that it is a primary and
direct effect of shikonin itself and not a downstream effect
mediated indirectly. Subsequent literature research supports
our conclusion, revealing that naphthoquinones other than
shikonin have been previously implicated in ROS induction
via a futile redox cycle in isolated mitochondria [31].
Abnormal accumulation of ROS is likely to give rise to
oxidative stress and cause DNA damage by modifications
such as 7,8-dihydro-8-oxoguanine (8-oxoG) [32]. Since
our results of the gene expression profiling also indicated
DNA damage after shikonin treatment, we anticipated
that shikonin-induced ROS was the cause of oxidative
DNA damage. The alkaline elution technique was used
to quantify DNA modifications sensitive to Fpg protein,
a repair endonuclease that recognizes and nicks the DNA
at sites of oxidative purine modifications such as 8-oxoG.
Untreated U937 control cells showed few single-strand
breaks, but had a mildly increased level of 0.3 oxidative DNA
modifications/106bp (Figure 2(d)). This elevated level of
oxidative DNA damage corroborates previous findings show-
ing an increased amount of oxidative stress and DNA damage
in leukemia cells [33]. Upon treatment of U937 cells with
0.3 μM shikonin, there was a 1.6-fold and 2.3-fold increase
in the amount of oxidative DNA modifications after 3 and
6 h treatment, respectively (Figure 2(d)). Interestingly, there
was no significant generation of single-strand DNA breaks
observed in cells treated with 0.3 μM shikonin during the
short incubation periods (3 and 6 h). This observation agrees
with a model in which shikonin causes oxidative DNA dam-
age mediated primarily by inducing ROS (Figures 2(a)–2(c)).
3.5. Induction of Intracellular Calcium Signaling. Increased
intracellular ROS levels are known to disturb cellular calcium
signaling [6]. Since we found several genes involved in
calcium homeostasis strongly deregulated after shikonin
treatment (e.g., genes coding for the calcium binding
proteins S100A8 and S100A9), we analyzed the effect of
shikonin on the intracellular calcium concentration [Ca2+]i
by indo-1 staining. In contrast to ionomycin, a molecule
used to increase [Ca2+]i, treatment with shikonin caused a
very short and weak decrease in free [Ca2+]ithat quickly
reversed into a slow but continuous increase in [Ca2+]iin
U937 cells (Figure 2(e)). After 30 min of treatment with
shikonin, the concentration of intracellular calcium is more
than 1.2-fold higher than that in the DMSO treated control
cells. Furthermore, the effect is stable for at least 1 h.
Interestingly, different concentrations of shikonin (0.6 and
1.2 μM) appear to have the same effect, and no obvious dose
dependency could be detected. The slow release of [Ca2+]i
after treatment with shikonin suggests that shikonin does not
interact directly with calcium storage regulators or serve as
a calcium ionophore/shuttler as does ionomycin. Instead, it
is likely that shikonin induces release of [Ca2+]iby the well-
known ROS-calcium signaling pathway [6], which would
also explain the rather slow kinetics of calcium signaling
observed (Figures 2(c) and 2(e)).
3.6. Long-Term Treatment with Shikonin Induces Cell-Cycle
Arrest and the Mitochondrial Pathway of Apoptosis. Given the
effects of shikonin on ROS generation, oxidative genotoxic
stress induction and calcium signaling, we further explored
the long-term effects of shikonin on the cell cycle. Flow
cytometric cell-cycle analysis was performed on U937 cells
after 24 h of treatment with different concentrations of
shikonin (Figure 3(a)). Shikonin significantly increased the
percentage of cells in the sub-G1 phase in a dose-dependent
manner (Figures 3(a) and 3(b)), representing an increase in
cell death, possibly by apoptosis [34]. However, the amount
of cells in the G1 and S phase was less affected than the G2
cell population at 0.3 μM(IC
50) shikonin (Figure 3(b)). At
this concentration, the population of cells in the G2 phase
was decreased by nearly half, indicating that, although the
cells were still able to enter S phase to some extent, they
were significantly stalled in the S phase. Interestingly, the
number of cells in the S phase was increased at subtoxic
concentrations of 0.1 μM shikonin, suggesting that very low
doses of shikonin might activate signaling processes driving
cell proliferation (data not shown). In summary, effective
doses of shikonin seem to cause an arrest of cells in the
G1 and S phase, preventing them from entering G2 phase.
Ultimately, this arrest leads to a strong increase in the
number of apoptotic cells.
Since shikonin accumulates in the mitochondria and
disrupts the mitochondrial membrane potential, we hypoth-
esized that induction of cell death in shikonin-treated cells
may be due to activation of the mitochondrial or intrinsic
apoptotic pathway leading to caspase 9 activation. Caspase
9 is activated after the release of cytochrome c from the
mitochondria, and once activated it cleaves and activates
the effector caspases 3 and 7, which mediate apoptotic cell
death [4,35]. We analyzed the activation of caspases 3, 7,
and 9 in U937 cells after 6 h of treatment with different
concentrations of shikonin. We observed a significant dose-
dependent increase in the activity of all three caspases in
shikonin-treated samples, consistent with the hypothesis
that shikonin activates the intrinsic pathway of apoptosis
(Figure 3(c)).
3.7. Inhibition of Cancer Cell Migration and Microtubule
Dynamics. The initial microarray experiment identified a
set of genes sensitive to shikonin and correlated with

Evidence-Based Complementary and Alternative Medicine 9
ROS induction Life ROS induction kinetic
DNA damage
Quantification of ROS induction
ROS content (DCF)
Counts
Shikonin fluorescence
ROS content (DCF)
Time (s)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Fpg
0
1
2
3
4
5
6
0 0.3 0.6
ROS induction (fold increase)
U937 Control
Time (s)
0
Counts
0
100101102103104
100101102103104
NoH2DCFH-DA
H2O250 μM
Control
Shikonin 0.3 μM
Shikonin 0.6 μM
Shikonin (μM)
DNA modifications/106bp
∗
∗
∗
0
102
103
104
105
0 1 K2 K3 K4 K5 K
Control
SSB
0.3 μM
Shikonin(3 h)
0.3 μM
Shikonin(6 h)
Intracellular Ca2+
Ionomycin 0.4 μM
Shikonin 0.6 μM
Shikonin 1.2 μM
Changesin [Ca2+]i
0.5
1
1.5
2
2.5
0 1 K2 K3 K4 K5 K
1
1.5
(a)
(b)
(c)
(d)
(e)
Figure 2: Induction of ROS, oxidative DNA damage, and elevated intracellular Ca2+ levels by shikonin. (a) Flow cytometric analysis of ROS
levels after treatment with different concentrations of shikonin for 1 h or 50 μMH
2O2for 15 min in living U937 cells. Cells were stained
with H2DCFH-DA and measured at 488 nm excitation and detected using a 530/30 nm bandpass filter. (b) Statistical quantification of ROS
induction after shikonin treatment in U937 cells. Data points represent mean (fold change) ±SEM of at least three independent experiments.
(c) ROS induction kinetics in live cells. U937 cells were stained with H2DCFH-DA and ROS induction was measured by flow cytometry.
After 2 min, shikonin was added to the cells and measurement was continued for 1 h. Shikonin was excited at 640 nm and detected with
a 730/45 nm bandpass filter. DCF was excited with a 488 nm laser and detected using a 530/30 nm bandpass filter. (d) Induction of DNA
damage by shikonin measured using alkaline elution technique. Columns indicate the number of DNA single-strand breaks (SSB) and of Fpg-
sensitive modifications (oxidative DNA damage) after shikonin treatment. Data points represent mean ±SEM of at least three independent
experiments. (e) Real-time kinetics of intracellular Ca2+ levels after treatment with different concentrations of shikonin or ionomycin in
U937 cells. Cells were stained with indo-1 and [Ca2+]iwas measured by flow cytometry. After 2 min, shikonin was added to the cells and
measurement was continued for 1 h. Indo-1 was excited with a 355 nm laser and the ratio of the signals detected using a 405/20 nm filter
and a 530/30 filter (405/20 nm/530/30nm) was used as an index for intracellular calcium concentration (∗significant difference according to
Student’s t-test, P<0.05).

10 Evidence-Based Complementary and Alternative Medicine
DNA content (PI)
Counts
Cell-cycle analysis
0
500
1000
1500
050 K 100 K 150 K 200 K 250 K 050 K 100 K 150 K 200 K 250 K 050 K 100 K 150 K 200 K 250 K
0
200
400
600
800
0
100
200
300
Sub-G1
G1 Control
G2/M
S
0.3 μM 0.6 μM
(a)
Cell cycle quantification
0
20
40
60
80
100
120
0 0.3 0.6
Cell-cycle distribution of U937 cells (%)
Sub-G1
G1
S
G2
Shikonin (μM)
(b)
Caspase activity
0
200
400
600
800
1000
1200
0 0.3 0.6
Caspase activity (% control)
Caspase 9
Caspase 3/7
Shikonin (μM, 6 h)
∗
∗
∗
∗
(c)
Figure 3: Shikonin induces cell cycle arrest and apoptosis in U937 cells. (a) Typical DNA content histograms of U937 cells treated with
increasing concentrations of shikonin for 24 h. (b) Statistical analysis of cell cycle distribution of U937 cells after treatment with different
concentrations of shikonin for 24 h. Data points are means of at least three independent experiments. (c) Enzymatic activity of caspase 3/7
and caspase 9 after 6 h of shikonin treatment in U937 cells. The caspase activity (mean ±SD of at least three experiments) is expressed as
percentage relative to the untreated control (∗significant difference according to Student’s t-test, P<0.05).
cytoskeleton formation, cellular movement, and morphol-
ogy. We speculated that shikonin negatively influences cell
motility and performed scratch migration assays using the
highly metastatic breast cancer cell line SKBR-3 to investigate
the effect of shikonin on cancer cell migration (Figures 4(a)
and 4(b)). Experiments with SKBR3 cells treated with DMSO
solvent control demonstrated complete scratch closure in
most cases after 12 h. However, cells treated with 1.2 μM
shikonin (IC50/8) showed a considerably delayed closure of
the scratch; after 12 h only 55% of the initial scratch width
was closed. At a concentration of 2.3 μM shikonin (IC50 /4),
only 22% of the initial scratch width was recolonized
after 12 h. These results strongly indicate that shikonin
inhibits migration of SK-BR-3 breast cancer cells at sub-toxic
concentrations.
Microtubules are indispensable for the directional migra-
tion of cells [36]. Since our gene expression profiling showed
a high number of deregulated genes associated with the
microtubule cytoskeleton, we presumed that shikonin’s effect
on microtubules was responsible for its ability to inhibit
breast cancer cell migration. We therefore treated U2OS-
GFP-αTubulin cells with 25 μM shikonin and analyzed the
drug uptake as well as the direct effect of shikonin on the
microtubule cytoskeleton in real-time using high-resolution
confocal microscopy (Video S1). We observed rapid accu-
mulation of shikonin within the cells’ mitochondria, but
we detected no colocalization of shikonin with tubulin
filaments. However, with increasing cellular concentrations
of shikonin, the number of distinct tubulin filaments
decreased and the tubulin staining became progressively

Evidence-Based Complementary and Alternative Medicine 11
Control
Cell migration
1.2 μM 2.3 μM
0 h
6 h
12 h
(a)
Cell migration
0
10
20
30
40
50
60
70
80
90
100
612
Closure of the scratch (%)
Time after scratch initiation (h)
Control
1.2 μM
2.3 μM
∗
∗
∗
∗
(b)
Microtubule cytoskeleton
αtubulin αtubulin +shikonin
U2OS-GFP-αtubulin
(c)
EB3 EB3 + shikonin
RPE-1-GFP-EB3
(d)
Figure 4: Shikonin inhibits cancer cell migration and affects microtubule structure and dynamics. (a) Typical pictures at 0, 6, and 12h of
scratch migration assays using SK-BR-3 cells treated with different effective, but in this time-frame subtoxic, concentrations of shikonin. (b)
Statistical quantification of the scratch migration assay. Data points represent the mean ±SEM of at least three independent experiments.
(c) Live imaging of U2OS-GFP-αTubulin cells stably transfected with a GFP fusion construct of α-tubulin and treated with 25 μM shikonin.
With increasing cellular concentrations of shikonin, the number of distinct tubulin filaments decreased and the tubulin staining became
progressively diffuse. (d) Live imaging of RPE-1-GFP-EB3 cells stably expressing GFP-EB3 and treated with 25 μM shikonin. Shikonin caused
a slowdown and finally a complete disappearance of EB3 particles within 3 min after application, indicating disrupted microtubule formation
(∗significant difference according to Student’s t-test, P<0.05).

12 Evidence-Based Complementary and Alternative Medicine
more diffuse (Figure 4(c)). Due to highly specific accumu-
lation of shikonin in and around the mitochondria, we
concluded that the disassembly of the tubulin network is
an indirect downstream effect of shikonin. The tubulin
filament disassembly could be a consequence of the reduced
amount of ATP generated in the mitochondria by oxidative
respiration or of the deregulated calcium signaling.
The microtubule-associated End binding protein-3
(EB3) binds to growing microtubule plus ends. The GFP-EB3
fusion proteins generate a punctuate pattern of EB3-GFP
comets throughout the cell and serve as an elegant marker
for visualizing microtubule growth events and dynamics [18,
37]. In order to examine variations in microtubule dynamics
after shikonin treatment, RPE-1-GFP-EB3 cells were treated
with 25 μM shikonin and drug uptake as well as effects on
EB3-GFP particle dynamics was analyzed in real-time exper-
iments using high-resolution confocal microscopy (Video
S2). As expected, there was no colocalization of shikonin
with EB3 comets. However, shikonin treatment did cause
a strong slowdown and finally a complete disappearance of
EB3 particles within 3 min of application. EB3-GFP particles
are known to disappear when microtubule growth is paused
or switches from a state of growth into a state of shrinkage
[18]. Furthermore, microtubule formation is dependent on
sufficient amounts of ATP [38] and is sensitive to changes in
calcium levels [39], both of which are significantly affected
by shikonin.
4. Discussion
We showed that shikonin has a strong cytotoxic effect on
a wide variety of cancer cell lines, especially different types
of leukemia and several known MDR cell lines. Microarray-
based gene expression analysis of U937 leukemia cells sug-
gested that the cytotoxicity of shikonin is based on the dis-
ruption of normal mitochondrial function, overproduction
of ROS, inhibition of cytoskeleton formation, and finally
induction of cell-cycle arrest and apoptosis. We were able
to validate all of these effects using in vitro cell culture
experiments exploiting the specific natural fluorescence of
shikonin and thereby identify the possible primary cellular
mechanism of shikonin’s cytotoxicity. We support our claim
with the finding that shikonin immediately accumulates in
the mitochondria of cancer cells and disrupts mitochondrial
function, as evidenced by the loss of mitochondrial mem-
brane potential. It was recently shown that shikonin induces
ROS and apoptosis in cancer cells [30] and our results
fully concur with this assertion. However, we would suggest
that other previously described mechanisms of action for
shikonin such as the induction of necroptosis [40], inhibition
of topoisomerase II activity [41], downregulation of NFκB
signaling [42], cell-cycle arrest through upregulation of p53
and downregulation of cyclin-dependent protein kinase 4
[43], inhibition of proteasome function [44], inhibition
of tumor necrosis factor alpha [45], deregulation of cal-
cium signaling, and microtubule disintegration are actually
downstream effects mediated (in most cases) not by a
direct interaction of shikonin with the suggested targets
but rather by the direct generation of ROS, the subsequent
dysregulation of mitochondria and induction of oxidative
damage.
A recent study showed that shikonin interferes with
cancer cells’ energy generation by targeting tumor pyruvate
kinase-M2 and thereby inhibiting glycolysis [16]. Our study
confirms that shikonin treatment causes reduced energy
production in cancer cells by affecting the mitochondrial
membrane potential, but the observed effects of shikonin on
ROS and mitochondrial function are not likely to be purely
based on blocking glycolysis. If glycolysis is unable to serve
as a source of acetyl-CoA for energy generation, cells can
compensate by shift to other metabolic pathways such as
fatty acid oxidation [46] or glutamine utilization [47]. Our
data does not exclude the possibility that shikonin has an
effect on pyruvate kinase-M2, but the direct targeting of
mitochondria and the complete loss of the mitochondrial
membrane potential as well as the rapid induction of ROS
make the electron chain the more likely target of shikonin.
Shikonin can be categorized as a mitocan [48], a class of
compounds that act by interfering with energy-generating
mitochondrial processes, which in turn leads to ROS
accumulation, mitochondrial destabilization, and induction
of apoptosis [12]. Shikonin itself is a naphthoquinone
derivative, and various substituted naphthoquinones have
been shown to be capable of redox cycling in isolated mito-
chondria [31]. During this process, reductive enzymes, for
example, mitochondrial NADH-ubiquinone oxidoreductase
(complex 1), metabolize quinones to unstable semiquinones
through one-electron reduction reactions [49]. When molec-
ular oxygen is present, such semiquinones enter into a redox
cycle leading to reformation of the original quinone, with the
associated generation of reactive oxygen species. Ultimately,
this cycle results in excessive ROS accumulation, depolar-
ization of the mitochondrial membrane, and induction of
apoptosis [50]. Due to the quinone structure of shikonin
and its accumulation in the mitochondria, we believe that
the ROS induction caused by shikonin is also based on such
a futile mitochondrial redox cycling. The elevated levels of
ROS strain the mitochondria, leading to a breakdown of
the mitochondrial membrane potential and finally to the
release of proapoptotic compounds and thus the activation
of caspases involved in the intrinsic pathway of apoptosis.
The oxidative DNA damage detected is also a consequence of
the elevated ROS production and could likely be the trigger
of the observed cell-cycle arrest [51].
Besides inducing ROS, some quinones have been shown
to cause release of calcium from isolated mitochondria
[31]. This is consistent with the elevated levels of [Ca2+]i
observed after shikonin treatment. We showed that shikonin,
in contrast to ionomycin, caused a slow and continu-
ous increase in intracellular calcium concentrations. This
suggests that shikonin does not shuttle extracellular or
intracellular stored calcium actively, but rather causes a
calcium release from calcium stores or other organelles,
for example, mitochondria, by an indirect mode such as
via ROS signaling pathway [6]. Nevertheless, elevated levels
of [Ca2+]iand ROS together appreciably disturb normal
calcium signaling [6]. Increased calcium levels promote

Evidence-Based Complementary and Alternative Medicine 13
the disassembly of microtubules by direct destabilization of
growing microtubule ends [39], which is in accordance with
our findings that shikonin inhibits cancer cell migration
by the disruption of microtubule cytoskeleton dynamics.
Indeed, shikonin treatment results in a complete inhibition
of EB3 protein dynamics and a loss of distinct microtubule
filaments, suggesting that the ATP shortage and deregulation
of calcium levels are dually destructive. These findings
motivate further investigations on the effect of shikonin in
the treatment of highly invasive cancer types.
Many established anticancer agents affect upstream sig-
naling pathways that ultimately converge on mitochondria
as regulators of cell death and survival [52]. These signaling
pathways are often deregulated in human cancers, and for
this reason many MDR phenotypes are resistant to classical
anticancer agents [2]. Thus, compounds that directly target
mitochondria can bypass deregulated upstream signaling
events and thereby circumvent the resistance mechanisms of
cancer cells [5]. However due to the basic mode of action
it is likely that shikonin also has an effect on noncancer
cells. Yet, shikonin bypasses resistances of known MDR
cell types and this makes further research on better and
more direct application methods an interesting project.
Numerous animal studies showed that the therapeutic effects
of shikonin apparently predominate the side effects [13,53]
and a clinical trial with shikonin showed that it can be
utilized in therapy [15]. Future studies should concentrate
on the reduction of side effects by chemical derivatization or
tissue targeted application.
In summary, our results indicate that shikonin accumu-
lates in the mitochondria of cancer cells, disrupts mitochon-
drial function, and finally causes apoptosis. As mitochondria
generate the majority of the cellular ATP supply and also
regulate the cell death machinery, they are promising targets
for cancer therapy. Hence, shikonin may have potential for
cancer treatment.
Abbreviations
8-oxoG: 7,8-Dihydro-8-oxoguanine
[Ca2+]i: Intracellular calcium concentration
CV: Coefficient of variation
DCF: Dichlorofluorescein
FACS: Fluorescence-activated cell sorting
FBS: Fetal bovine serum
Fpg: Formamidopyrimidine DNA glycosylase
GFP: Green fluorescent protein
H2DCF: 2,7-Dichlorodihydrofluorescein
H2DCFH-DA: 2,7-Dichlorodihydrofluorescein diacetate
JC-1: 5,5,6,6-Tetrachloro-1,1,3,3-tetraethyl-
benzimidazolylcarbocyanine
iodide
MDR: Multiple drug resistance
MMP: Mitochondrial membrane permeabilization
PBS: Phosphate buffered saline
PI: Propidium iodide
ROS: Reactive oxygen species
SEM: Standard error of the mean
SD: Standard deviation
SSB: Single strand breaks.
Disclosure
This work was funded by the Landesstiftung Rheinland-
Pfalz; the Institute of Molecular Biology gGmbH (IMB) and
the associated Core-Facilities are funded by the Boehringer-
Ingelheim Fund. The funders had no role in study design,
data collection and analysis, decision to publish, or prepara-
tion of the paper.
Conflict of Interests
The authors declare that there are no significant competing
financial, professional, or personal interests that might have
influenced the performance or presentation of the work
described in this paper.
Acknowledgments
The authors thank Andreas Vonderheit and Nadja Hellmann
for their great support on confocal microscopy and fluo-
rescence measurements. They are indebted to Karen Duffy
and Martha Paluschinski for their support in the laboratory
Karen Duffy (Cornell University, Ithaca, NY, USA) read and
corrected the paper.
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