Cancer Therapy: Preclinical
Ingenuity Network-Assisted Transcription Profiling: Identification
of a New Pharmacologic Mechanism for MK886
¤ ez,3 Daniel Sackett,2 Huaitian Liu,4 Joanna Shih,5
Anatoly L. Mayburd,1 Alfredo Martln
1
1
Jordy Tauler, Ingalill Avis, and James L. Mulshine6
Abstract
The small molecular inhibitor MK886 is known to block 5-lipoxygenase-activating protein
ALOX5AP and shows antitumor activity in multiple human cell lines. The broad antitumor therapeutic window reported in vivo for MK886 in rodents supports further consideration of this structural class. Better understanding of the mode of action of the drug is important for application in
humans to take place. Affymetrix microarray study was conducted to explore MK886 pharmacologic mechanism. Ingenuity Pathway Analysis software was applied to validate the results at the
transcriptional level by putting them in the context of an experimental proteomic network. Genes
most affected by MK886 included actin B and focal adhesion components. A subsequent National Cancer Institute-60 panel study, RT-PCR validation followed by confocal microscopy, and
Western blotting also pointed to actin B down-regulation, filamentous actin loss, and disorganization of the transcription machinery. In agreement with these observations, MK886 was found to
enhance the effect of UV radiation in H720 lung cancer cell line. In light of the modification of
cytoskeleton and cell motility by lipid phosphoinositide 3-kinase products, MK886 interaction
with actin B might be biologically important. The low toxicity of MK886 in vivo was modeled
and explained by binding and transport by dietary lipids. The rate of lipid absorbance is generally
higher for tumors, suggesting a promise of a targeted liposome-based delivery system for this
drug. These results suggest a novel antitumor pharmacologic mechanism.
5-Lipoxygenase is the upstream enzyme in the leukotriene
pathway7 regulating chemotaxis (1), blood coagulation (2),
inflammation (3), pain sensation (4), and blood pressure (5).
It is also known to stimulate cancer growth (6 – 8). Interruption
of the 5-lipoxygenase pathway is shown to exert a marked
anticancer effect in a number of reports (9, 10). After activation
by external stimuli, 5-lipoxygenase localizes on the inner leaflet
of the nuclear membrane (11) depending on 5-lipoxygenaseactivating protein ALOX5AP (FLAP) located in the same
membrane. The drug compound MK886 (Biomol Research,
Authors’ Affiliations: 1Intervention Section, National Cancer Institute, NIH;
2
Laboratory of Integrative and Medical Biophysics, National Institute of Child
Health and Human Development, NIH, Bethesda, Maryland; 3 Department of
Neuroanatomy and Cell Biology, Instituto Cajal, Consejo Superior de
Investigaciones Cientificas, Madrid, Spain; 4Center for Bioinformatics/Science
Applications International Corporation and 5Biometric Research Branch, National
Cancer Institute, NIH, Rockville, Maryland; and 6Rush University Medical Center,
Chicago, Illinois
Received 10/4/05; revised 11/30/05; accepted 12/16/05.
Grant support: Ministry of Science and Education of Spain grant BFU200402838/BFI (A. Martl¤ nez) and the Intramural Research Program of the NIH,
National Cancer Institute.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Supplementary data for this article are available at Clinical Cancer Research
Online (http://clincancerres.aacrjournals.org/).
Requests for reprints: Anatoly L. Mayburd, Intervention Section, National Cancer
Institute, NIH, Bethesda, MD 20859. Phone: 301-402-3308; Fax: 301-435-8036;
E-mail: mayburna@ mail.nih.gov.
F 2006 American Association for Cancer Research.
doi: 10.1158/1078-0432.CCR-05-2149
Clin Cancer Res 2006;12(6) March 15, 2006
Plymouth Meeting, PA) is known to bind FLAP at 100 nmol/L
and was used in the pioneering work of FLAP isolation (12). In
knockout mice studies, the ablation of FLAP led to the
development of an atherosclerosis-resistant strain of mice
(13). The role of the pathway in the inflammatory response
leading to cardiovascular disease is being rapidly explored
(14, 15).
In preclinical studies, the drug is well tolerated in rodents at
high dosages of f800 Amol/L/kg (640 mg/kg; ref. 16). The
drug level sufficient to prevent cancer in vivo is 10 mg/kg in
rodents (17). A 64-fold difference between the in vivo tolerated
and cancer-inhibiting dosages makes this structural class
worthy of further exploration, with the goal of developing a
human version of the treatment.
MK886 has been studied using small-scale experiments, and
no consensus as to its prevailing mechanism of action has been
reached. MK886 has been reported to have an antitumor effect
caused by the mitochondrial permeability transition pore
opening (18). The synergy between g-linolenic acid effect and
MK886 action against a refractory pancreatic cancer line PANC1
is described by Harris et al. (19). Protection against MK886induced apoptosis is shown by the exogenous addition of a
5-lipoxygenase product, 5-hydroxyeicosatetraenoic acid and
thiols (20). The fatty acid oxidation via alternative pathways is
proposed as a cause of cell death (21). We have reported the
relative redistribution of bioactive peroxide products between
the 5- and 15-lipoxygenase pathways as well as the involvement
of peroxisome proliferator-activated receptor g (PPARg) as the
7
1820
http://cgap.nci.nih.gov/Pathways/Kegg/hsa00590.
www.aacrjournals.org
Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2006 American Association for Cancer
Research.
Ingenuity Network-Assisted Transcription Profiling
likely cause of MK886-mediated inhibitory effects (22).
Another study suggests that MK886 drug effect seems to be
independent of the presence of its target FLAP (23). These
varied findings point to a complex MK886 mechanism.
To address the complexity of the proposed MK886 mechanism, we used a novel combinatorial research strategy. We used
the data from expression profiling, reduced first by t test, to select
for significantly affected genes and then reduced the output by an
automatic mechanism-proposing software Ingenuity Pathway
Analysis (Mountain View, CA). The National Cancer Institute
(NCI)-60 type of information-rich data uses the similarity of cell
growth inhibition profiles across multiple cell lines as the
measure of the similarity between the compared drug mechanisms (24). The coclustering profiles are assumed to represent
similar mechanism of action and we applied this tool as well.
By using microarrays followed by several layers of validating
tools, we attempted to identify additional biological target(s) of
MK886, thus suggesting a process to refine a lead compound
for relevant pharmacologic applications. The lipid-processing
pathways are prominent in the development of cancer and its
therapy. The attempts to affect these pathways pharmacologically may involve side effects based on binding to unintended
sites, and defining the actual antitumor mechanism can be
helpful in the efforts to understand potential complications.
The results of our current study points to a possible novel
cytotoxic anticancer mechanism.
Materials and Methods
Cell lines. Cell lines [A549, non – small cell bronchoepithelial
carcinoma, NCI-H720, atypical carcinoid, H157, Mus musculus (myeloma), H1299, non – small cell lung carcinoma], were procured from
American Type Culture Collection (ATCC, Manassas, VA). NCI-N417
small cell lung carcinoma was derived from NCI. The A549, H157,
N417, H1299, and H720 cells were grown in RPMI 1640 (Invitrogen,
Carlsbad, CA), supplemented with 5% FCS, 2 mmol/L glutamine and
penicillin/streptomycin (Invitrogen). Human osteosarcoma cells
(143.98.2; ATCC) was grown in DMEM supplemented with 10% fetal
bovine serum (FBS), penicillin (100 units/mL), and streptomycin
(100 Ag/mL). The malignant mouse astrocytoma cell line CT-2A was a
generous gift from Prof. T.N. Seyfried (Boston College, Chestnut Hill,
MA). Human umbilical vascular endothelial cells (HUVEC) and EBM2
medium supplemented with ‘‘bullet kit’’ were purchased at Cambrex
(East Rutherford, NJ) and HUVECs were grown according to the
supplied protocol. The immortalized mouse embryo fibroblast cell line
MEF8 was obtained by transforming primary fibroblasts isolated from
12-day-old embryos with the SV40 major T antigen. MEF8 and CT2A
cell lines were grown in a DMEM supplemented with 5% FCS
(Invitrogen) at 37jC with 5% CO2.
Real-time PCR. RNA isolation and quantification and quantitative
real-time PCR (RT-PCR) were conducted as described in ref. (22). The
gene sequences and the primer sequences (with melting temperatures)
used for RT-PCR are given in the supplementary file ‘‘RT-PCR primers.’’
Normalization was conducted using the average of the panel of nine
random genes, listed in the Supplementary Table S1.
Microarray data generation. Data sets in this report were generated
by treating A549 and H720 cell line with MK886 (Calbiochem, San
Diego, CA) at 1 Amol/L for 24 hours. Six Affymetrix Human Genome
U133 Plus 2.0 microarray chips (Affymetrix, Santa Clara, CA) were used
in triplicate for the treated and untreated classes according to the
protocol of the manufacturer available online.8 Briefly, total RNA was
isolated from the cancer cell lines using RNeasy mini-kit (Qiagen,
Chatsworth, CA). The quality of the RNA isolation was assessed by
A 260/A 280 (>1.9, <2.1) ratio as well as by 1% agarose gel electrophoresis
www.aacrjournals.org
(intact r-RNA bands). The first-strand cDNA, the double-strand cDNA,
and cRNA were synthesized, and cRNA was fragmented using the
protocol-recommended kits. All intermediate and final products were
tested on agarose gel to comply with the molecular mass distribution as
described in the protocol. The cRNA was mixed with internal controls
for hybridization and B2 oligonucleotide for automatic grid alignment,
the chip was prehybridized with 1 hybridization buffer for 10 minutes
at 45jC and then hybridized with the cRNA cocktail for 16 hours at
60 rpm in a rotating exposure chamber. Upon completion, the arrays
were washed, stained with streptavidin phycoerythrin, and scanned.
The data were submitted to Gene Expression Omnibus public
depository, according to the minimum information about a microarray
experiment checklist, series entry GSE3202.
Microarray data analysis. Microarray probe data were obtained with
Microarray Suit 5.0 (Mas 5.0; Affymetrix, Santa Clara, CA). The robust
multiarray average method and quantile normalization were used to
produce normalized probe set summary measures. The calculation was
implemented in the R package Affy of the Bioconductor software
project.9 Identification of probe sets that were differentially expressed
between the treatment and control group was done using two-sided
univariate t tests. Specifically, differentially expressed genes were
identified as those genes that were significant at the 0.001 level (P <
0.001) and that were at least 1.5-fold different in the mean expressions.
Pathway identification. The differentially expressed probe sets were
overlaid on a cellular pathway map in the Ingenuity Pathway Analysis
using resource database Knowledge Base (Winter 04 Release containing
20,000 genes). The resulting networks were represented in table and
graphic format.
Anoikis treatment. Poly-hydroxyethyl methacrylate (poly-HEMA,
Sigma, Saint Louis, MO) was solubilized in methanol (50 mg/mL) and
diluted in ethanol to a final concentration of 10 mg/mL. To prepare
poly-HEMA-coated dishes, 4 mL poly-HEMA solution were placed onto
100 mm dishes and dried in a tissue culture hood. The poly-HEMA
coating was repeated twice, followed by three washes with PBS. One
million five hundred thousand trypsinized A549 cells were plated onto
poly-HEMA-coated dishes for 12 hours in RPMI with 5% FBS. The cells
were resuspended after 12 hours and RNA extracted for RT-PCR analysis.
Filamentous actin labeling for flow cytometry and confocal microscopy
study. The cells were trypsinized, washed in PBS and fixed in 4%
immunohistochemistry grade formalin for 10 minutes at room
temperature. Cells were permeabilized with 0.5% Triton X-100 for 5
minutes at room temperature and washed with PBS. The rhodamine
phalloidin conjugate (Cytoskeleton, Inc., Denver, CO) was diluted in
methanol and used according to the protocol of the manufacturer. For
confocal microscopy, cells were seeded onto glass slides and exposed to
a regular medium (control) or to 10 Amol/L MK886 for 20 hours. After
treatment, cells were fixed in 4% paraformaldehyde for 10 minutes,
permeabilized in 1% Triton X-100 in PBS for 10 minutes, and exposed
to Bodipy-phallacidin (Molecular Probes, Eugene, OR) at a dilution
1:200 in PBS for 1 hour. Slides were observed with a confocal
microscope (Leitz DM IRB, Berlin, Germany).
G-actin polymerization in vitro
Spin-down assay. Rabbit skeletal muscle actin and pyrene muscle
actin were procured from Cytoskeleton. The actin solution (0.25 mg/
mL) was redistributed into the Eppendorf tubes, 120 AL in each, mixed
with MK886 and other drugs, and left to incubate on ice for 15 minutes.
After incubation, the 20 polymerization buffer (KMEI), prepared
according to the protocol of the manufacturer, was added to initiate
polymerization. After 15 minutes, the tubes were transferred to a cooled
table top centrifuge and spun at 14,000 rpm (25,000 g). The resulting
molecular weight gradient of different filamentous actin (F-actin)
polymerized products was sampled (10 AL of total 120 AL) from the top
8
9
1821
http://www.affymetrix.com/support/technical/technotesmain.affx.
http://www.bioconductor.org.
Clin Cancer Res 2006;12(6) March 15, 2006
Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2006 American Association for Cancer
Research.
Cancer Therapy: Preclinical
Table 1. Layout of the gene ontologies
Gene ontology
DNA binding
RNA binding
Purine nucleotide binding
Transition metal ion binding
Electrochemical potential driven transporter activity
Phosphotransferase activity
Hydrolase activity
Peptidase activity
Protein kinase activity
Unlassified
No. genes,
response
No. genes,
Unigene
Fraction of genes,
response
Fraction of genes,
Unigene
Enrichment
9
8
8
5
4
4
3
3
3
66
1,977
2,210
701
543
4,871
2,965
3,048
1,109
7,324
31,061
0.079646
0.070796
0.070796
0.044248
0.035398
0.035398
0.026549
0.026549
0.026549
0.584071
0.035424
0.039599
0.012561
0.00973
0.08728
0.053128
0.054615
0.019871
0.131233
0.556559
2.2
1.8
5.6
4.5
0.40
0.66
0.48
1.33
0.2
1.05
NOTE: The 97 responses that pass the primary filtering are distributed by functions as indicated. This distribution was compared with the original one, computed by
querying Unigene clusters database of Entrez with the same or the equivalent keywords. The functional enrichment coefficient was computed by relating the fractions
in the microarray output and the general gene population.
and the protein concentration analyzed by the Lowry assay (25) in
triplicates using a 1 mL cuvette.
Viscosity shift assay. Cannon-Manning Semi-Micro Viscosimeter
(Cannon Instrument, Co., State College, PA) was used to assess the
viscosity shift upon actin polymerization in the presence and in the
absence of the compounds of interest. Briefly, the time interval elapsing
while the fixed volume of a liquid passes between the upper and lower
marks is directly proportional to viscosity (in quadruplicates).
Pyrene actin fluorescence shift. The pyrene-labeled actin (Cytoskeleton) was mixed at a ratio of 1:5 with unlabeled actin, spun at 10,000 g
to remove polymerized actin, and polymerized in the cuvette of a
fluorimeter (ISS PC1 spectrofluorometer, Champaign, IL) according to
the protocol of the manufacturer. The time course of the reaction was
monitored at the excitation wavelength 360 nm and emission at 400 nm.
Combined MK886 and UV radiation exposure. The A549 and H720
cell lines were seeded in the Costar 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) plates at the density of 2 104
cells per well (H720 that forms clumps was seeded based on total
protein, equal to the A549 protein content). The cells were exposed to
0.75 Amol/L MK886 serum-free for 24 hours. After the exposure to
MK886, the cells were irradiated by UV at 302 nm in Multilimage Light
Cabinet (Imgen Technologies, Alexandria, VA) at 960 Wt/m2 irradiation
density for the indicated periods of time. The formal analysis of synergy
was conducted as in ref. (26).
Bioavailability studies. Liposin II (10%) is a soluble, nonpyrogenic
triglyceride emulsion supplied as a 10% solution containing 5%
safflower oil and 5% soybean oil (neutral glycolipids), 2.5% glycerin
in H2O for injections, and 1.2% egg phosphatides as an emulsifier
(Abbott Laboratories, Chicago, IL). Liposin II was added to the medium
at 0.01% and cell culture survival was assessed by MTT. The experiment
was conducted in triplicate.
Supplementary Data. Supplementary Data are available on the
AACR website under the name ‘‘MK886 data’’ and contain the
supporting information describing microarray data, RT-PCR primers
and results, and NCI-60 panel profiling.
Results
Defining the optimal exposure. To study the MK886
mechanism at a level of exposure that minimizes nonspecific
effects, we evaluated a range of drug concentrations and
intervals of exposure. The optimal time point likely took place
at 1 mmol/L MK886 at 24 hours in serum-free conditions,
Clin Cancer Res 2006;12(6) March 15, 2006
because the effect of the drug was still reversible, as opposed to
longer exposures, and enabled us to focus on relatively specific
upstream events (see Supplementary Data).
Microarray and pathway analysis results. Six Affymetrix
U133 Plus 2.0 human arrays were hybridized, scanned, and 25
probe sets were identified as differentially expressed genes
between MK886 and control at the 0.001 level and with at least
1.5-fold mean difference in H720 cells. Overall, 97 probes
showed the P values <0.001. Weakness of microarray result
called for additional ways to ensure validity of the biological
information extracted in these experiments. The differentially
expressed probes were subjected to verification by RT-PCR and
yielded 21 probes showing the change in the same direction
corresponding to false detection rate of 16% (Supplementary
Table S2). The results of Gene Ontology analysis are given in
Table 1, and the results of the network reconstruction by
Ingenuity in Fig. 1 (see also Supplementary Figure S2,
annotation to the network).
Participation of genes in Ingenuity clusters further decreased
the P value of detection due to lower probability of random
detection if the responses formed proteomic connections. Even
weak responses, but relating to confirmed protein-protein
interactions, thus become more meaningful. The Gene Ontology
analysis inquired into the functions of all significantly affected
genes, regardless of their mutual relationships. Both analyses (by
Ontology and by Ingenuity) were concordant in the finding that
DNA and RNA binding, recombination, and repair was the
dominant category among the known affected functions. Assuming that the choice of dosage and time point led to detection of
relatively upstream events, the study focused first on the highest
Ingenuity score cluster containing the most statistically robust
candidates for hypothesis building. Actin B was among the genes
constituting network 1 (Supplementary Table S1) and was
significantly up-regulated in this treatment (3- to 4-fold) at the
level of transcription. Among the other fundamental cytoskeleton components, villin 210 is thought to be the interface
10
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retrieve&
dopt=full_report&list_uids=7430.
1822
www.aacrjournals.org
Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2006 American Association for Cancer
Research.
Ingenuity Network-Assisted Transcription Profiling
Fig. 1. Connectivity map of the responses
by Ingenuity Pathway assistant analysis. A
more detailed legend to the connectivity
map in Fig. 2 can be found in Supplementary
Fig. S2, explaining the symbols.
component between cytoskeleton, plasma membrane, and focal
adhesion kinase – associated cytoskeleton components.
The Ingenuity Pathway analysis11 suggested putative downstream targets responding to the potential cytoskeleton disruption by MK886. The binding partners of actin B that responded to
MK886 treatment included SWI/SNF-related matrix-associated
actin-dependent regulator of chromatin a4 (SMARCA4 12).
According to the connectivity diagram in Fig. 1, SMARCA4
interacts with E2F transcription factor 1, playing multiple crucial
roles in DNA-damage response, apoptosis, and the cell cycle.13
The up-regulation of SMARCA4 observed in these experiments
may be related to E2F1-mediated down-regulation of its nearest
neighbor partners, defined by the Ingenuity-derived connections
(Fig. 1), including a number of vital DNA processing proteins.
The link was supported by finding of another E2F transcription
factor family tumor-suppressor member E2F transcription
factor 4 in the network 2 (Supplementary Table S1).
As shown in Fig. 1, these downstream partner genes included
mutS2 homologue (MSH2) gene involved in mismatch repair.
Another partner, Asp-Glu-Ala-Asp/His box polypeptide 11
(DDX11), is a member of the DEAD box protein family of
RNA helicases.14 Still, another SMARCA4 down-regulated
‘‘client’’ was high-mobility group box 1, implicated in
chromatin structural modulation. 15 Other downstream
SMARCA4 targets included suppressor of RNA polymerase B
homologue SURB7.16 All these findings were pointing to a
novel link between cytoskeleton modulation, chromatin
remodeling, and regulation of expression.
NCI-60 panel analysis. The microarray results were validated
by the NCI-60 panel of activity profiles (24).17 The primary
biological activity data for MK886 (code number S736463) and
the results of the analysis are in the corresponding Supplementary Data. The data modestly pointed to coclustering of MK886
with genotoxic agents despite chemical stability of MK886
structure. This finding corroborated the results suggested by
Ingenuity clustering.
RT-PCR validation of microarray data. The gene identities,
their network affiliations, and the values of differential
14
11
http://www.ingenuity.com/.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retrieve&
dopt=Graphics&list_uids=6597.
13
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retrieve&
dopt=Graphics&list_uids=1869.
12
www.aacrjournals.org
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retrieve&
dopt=Graphics&list_uids=1663.
15
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retrieve&
dopt=Graphics&list_uids=3146.
16
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retrieve&
dopt=Graphics&list_uids=9412.
17
http://spheroid.ncifcrf.gov/scripts/.
1823
Clin Cancer Res 2006;12(6) March 15, 2006
Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2006 American Association for Cancer
Research.
Cancer Therapy: Preclinical
expression by microarray and RT-PCR are presented in the
Supplementary Table S2. The similarity of transcription profiles
was previously shown to indicate the similarity of the
mechanism of action for the compared agents (27) and thus
gave the rationale to our approach. Figure 2A presents a
comparison of H720-MK886-treated transcription profiles
generated, respectively, by RT-PCR and microarray data,
indicating similarity for the entire profile. Comparison of
anoikis-treated A549 and MK886-treated H720 transcription
profiles indicated an even greater similarity than in the above
case (Fig. 2A) and suggesting that MK886 mechanism is also
related to cytoskeleton alteration because its signature showed
similarity to transcription profiling signature of anoikis.
Figure 2B shows RT-PCR validation of the prediction based
on the sum of high-throughput methods. The changes in the
transcript levels of DNA processing enzymes TOPO2A and
TBP were expected, in accordance with the cytoskeletontranscription linkage hypothesis, and were observed. The
transcript levels were affected 3- to 4-fold and in paradoxically
opposite directions, being increased in A549 and decreased in
H720 cell lines. The 20-fold increase in DDX11 transcript level
in H720 and 7-fold increase of the same transcript in A549
Fig. 2. RT-PCR validation of microarray results. A, comparison of differential
expression results, obtained by RT-PCR and microarray methods in different cell
lines. In A549,12 hours under anoikis treatment, studied by RT-PCR (gray); in H720,
24 hours under 1.0 Amol/L MK886 treatment, studied by microarray (black); in
H720, 24 hours under 1.5 Amol/L MK886 treatment, studied by RT-PCR (white).
The identities of the genes in the profile follow the order given in Supplementary
Table S2. B, the RT-PCR study of the selected genes, predicted to be involved in
cytoskeleton regulation of global transcription according to the present model. Black
columns, results in A549; gray columns, results in H720 (24 hours exposure, in
duplicates). Treated by 1 Amol/L MK886, or by 100 nmol/L Taxol, 24 hours,
serum-free.
Clin Cancer Res 2006;12(6) March 15, 2006
treated by 100 nmol/L Taxol provides more evidence of the link
between the cytoskeleton and DNA/RNA processing machinery.
Involvement of TBP in the studied mechanism required the
use of random gene panel for RT-PCR normalization. Although
cumbersome, this approach allowed to avoid the bias caused by
participation of the designated housekeeping genes in the
studied mechanism.
Validation of microarray results at protein level. MK886
treatment of A549 caused a dramatic decrease in F-actin level in
a certain fraction of cell populations, detected by confocal
microscopy. Interestingly (Fig. 3), immortalized but nonmalignant murine cells (MEF-8) were more resistant to the drug
compared with the purely malignant (CT-2A). Western blotting
also confirmed actin down-regulation in multiple cell lines (in
Supplementary Data).
Flow cytometry. The flow cytometry measurement of the
extent of F-actin depolymerization suggested that at 1 Amol/L
MK886 and 15 hours postexposure, the depolymerization
already developed, whereas proteolytic activation and cell
inhibition were still reversible (in Supplementary Data). This
result also suggested the importance of cytoskeleton alteration.
MK886 affects actin polymerization in vitro. To further
evaluate the possibility of cytoskeleton involvement, we
explored if actin directly interacts with MK886. Figure 4A
presents the results of the spin-down assay in the presence of
several pharmacologic agents. It can be noted that all tested
agents affected the residual concentration of actin monomer
after polymerization. The biggest effect was achieved in the
cytocholasin D control (Alexis, San Diego, CA) that is used
normally as actin destabilization control. MK886 showed
a significant effect as well. The effects of ciglitazone and
AA861 [2,3,5-trimethyl-6(12-hydroxy-5,10-dodecadynyl)-1,4benzochinone] from Biomol (Plymouth Meeting, PA) were
modest but detectable. Figure 4B presents the results of a time
course following pyrene-actin fluorescence enhancement. In
this experiment, no significant effect of MK886 was determined
at 2 and 6 Amol/L concentrations. However, at a higher level
(18 Amol/L), MK886 showed interference, suggesting an effect
on the fraction of the buried pyrene groups. In a control
experiment, the actin and KMEI buffer were diluted to the same
extent that was expected after an 18 Amol/L MK886 addition.
The difference observed between this control and the actual
addition of 18 Amol/L MK886 was significant. Figure 4C
presents the shifts in viscosity after polymerization buffer
was added to actin preincubated with drugs or with a solvent
control. It is known that shifting viscosity indicates the change
in the extent of actin polymerization and formation of smaller
aggregates in vitro following the interaction with MK886. The
strongest effect upon the viscosity increment was observed with
the cytocholasin D. However, as in the spin-down assay, the
presence of cytocholasin D was unable to completely block
the polymerization. The cytocholasin D effect was followed by
the effects of ciglitazon, MK886, and AA861.
Combined MK886 and UV radiation exposure. The MK886induced down-regulation of the components of UV damage
repair machinery prompted us to test its combination with UV
in search of possible therapeutic applications, e.g., sensitization for radiotherapy. Figure 5 shows the UV dose responses in
the presence and the absence of a relatively small MK886
dosage, exerting a small effect by itself. Our data indicate a
synergistic effect for H720 cell line (FICI = 0.25). However, the
1824
www.aacrjournals.org
Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2006 American Association for Cancer
Research.
Ingenuity Network-Assisted Transcription Profiling
Fig. 3. Confocal microscopy and Western blotting validation
of microarray results. A, confocal microscopy of murine malignant
(CT-2A), murine immortalized (MEF-8), and human malignant
(A549) cell lines treated by MK886 and stained for F-actin.
effect for A549 was only slightly superadditive according to the
formal analysis (FICI = 0.95). This experiment suggested that
for some cell lines, cytoskeleton alteration may enhance DNA
damage.
Bioavailability studies. What makes MK886 cancer selective?
Bioavailability of MK886 was modeled in vitro by creating a
system containing an emulsified lipid mixture (liposin II) and
serum components, mimicking the conditions in the blood
Fig. 4. Interference of the drugs with
G-actin polymerization in vitro. A, the results
of spin-down assay. The ordinate represents
the concentration of G-actin in equilibrium
with F-actin. Arrows, experimental
conditions: 10 Amol/L G-actin in G-buffer
being tested for stability (A); G-actin +
KMEI + 5 Amol/L cytocholasin D (CYTD ;
B); G-actin + KMEI + 5 Amol/L MK886 (C).
G-actin + KMEI + 5 Amol/L ciglitazon
(CIGL ; D); actin + KMEI + 5 Amol/L AA861
(E). G-actin + polymerization buffer
(KMEI) and no drugs, in triplicates (F).
B, pyrene-actin fluorescence shift at
different conditions and time points.
10 Amol/L of actin, the concentration of
drugs are in Amol/L. *, dilution control for
the MK886 exposure at 18 Amol/L.
C, viscosity shift at different conditions.
The primary time units of viscosity
correspond to the time that takes a fixed
volume of the solution to flow between
the upper and lower marks of the kinematic
viscosimeter. Distilled water (A), 40 Amol/L
G-actin (B), 40 Amol/L G-actin + KMEI (C),
40 Amol/L G-actin + KMEI + 10 Amol/L
MK886 (D), 40 Amol/L G-actin + KMEI +
10 Amol/L ciglitazone (E), 40 Amol/L
G-actin + KMEI + 10 Amol/L AA861 (F),
40 Amol/L G-actin + KMEI + 10 Amol/L
cytocholasin D (G). The experiment was
conducted at 23jC in triplicates.
www.aacrjournals.org
1825
Clin Cancer Res 2006;12(6) March 15, 2006
Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2006 American Association for Cancer
Research.
Cancer Therapy: Preclinical
Fig. 5. Combined exposure of H720 and
A549 cell lines to MK886 and UV radiation.
A, survival of H720 after 24 hours with
0.75 Amol/L MK886 serum-free, followed
by UV exposure given in seconds.
Squares, MK886 only; diamonds, UV only;
triangles, MK886 and UV combined.
B, the same for A549 cell line.
stream. Figure 6 presents the survival data for A549, suggesting
a very significant protection against MK886 in the combined
presence of serum albumin and lipid emulsion, exceeding the
protection shown by the single components.
Discussion
In our previous works (22, 26), we showed biochemically
that MK886 causes nuclear translocation of PPARg. This
transcription factor is thought to be a tumor suppressor, and
this translocation occurs within the first 2 hours postexposure.
As a result, a potential tumor suppressor might accumulate in
the nucleus, initiating the first step of an apoptotic cascade. In
addition, according to Fig. 1, MK886 may act via cytoskeletoninduced alteration of chromatin structure leading to repression of vital pathways that maintain DNA integrity and enable
replication. Another possibility stems from the known affinity
purification technique for actin B, using immobilized DNaseI
as a bait (28). The sequestering of the potentially dangerous
enzyme depends on the state of actin polymerization
and proteolytic activation, converting DNaseI to its catalytically active form, able to cleave chromatin. This indirect
genotoxicity is potentially supported by NCI-60 profiling data.
Similar report connecting cytoskeleton and genotoxicity exists
for ocadaic acid, a marine-derived product of lipid nature
(29). On the other hand, actin depolymerization by cytocholasin D does not lead to the same consequences. This outcome
indicates that cytoskeleton alteration has to be a corroborating
factor in a more complex mechanism.
A broader picture in literature points to the role of
cytoskeleton alteration in the synergistic interactions with the
agents directly or indirectly targeting DNA. In our study,
MK886 have shown a synergistic interaction with UV damage
in H720 cell line. The comparison with A549 is difficult due to
a very different initial sensitivity of these cell lines to UV
damage. Even greater synergistic effects are described for
cytocholasin D combined with actinomycin D or etoposide
or mitomycin C (30). MK886 is not unique in this aspect and
our study partially explains these effects.
It seems that a broad array of sites being more sensitive in
cancer than in normal cells is targeted in MK886 mechanism of
action. The products of 5-lipoxygenase inhibited by MK886 are
known stimulators of cancer growth by autocrine and paracrine
pathways. The loss of this stimulation is relatively disadvantageous for cancer (9, 10). The close structural analogue of FLAP
is microsomal glutathione transferase and MK886 is likely to
suppress the protective glutathione conjugation in the cancer
Clin Cancer Res 2006;12(6) March 15, 2006
cells that show the increased free radical production and
dependence on such a protection (20). The MK886 effect on
mitochondria is also cancer specific due to a lower level of pH
that destabilizes mitochondria (31). It is to this list of damaging
factors that cytoskeleton alteration is added in case of MK886.
In such a context, the additional component makes the drug
effect self-synergistic, possibly explaining a cooperative dose
response curve of MK886 (serum-free; Fig. 6). By contrast, a low
dose of cytocholasin D affecting cytoskeleton alone, in the
absence of corroborative factors, may not suffice to trigger the
massive apoptosis observed for MK886.
To understand why the rodents display such a tolerance to a
drug with such a broad mechanism, we conducted a
bioavailability study modeling the major factors influencing
MK886 biodistribution. Figure 6 shows a very significant
protection of A549 against MK886 in the presence of both
serum albumin and emulsified fat, simulating a natural
concentration buffer. Absorption of the emulsified lipids
together with bound MK886 may lead to the conditions when
tumor is at a selective disadvantage. Multiple studies suggest
increased lipid absorption by tumors compared with normal
cells (32, 33). This difference might arise due to a more
permeable vasculature in tumor sites, overexpression of
scavenger receptors in cancer, more intense phagocytosis in
tumor cells, and greater reliance on anaerobic sources of energy.
As a result, the influx of the drug in cancer may exceed the
Fig. 6. Survival of A549 exposed at different concentrations of MK886 in the
presence and in the absence of protective agents. TIS, only serum-free medium;
FBS, 2.5% serum inTIS; LN, 0.01% liposin inTIS; FBS + LN, 2.5% FBS and 0.01%
liposin combined. The experiment was conducted in triplicate.
1826
www.aacrjournals.org
Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2006 American Association for Cancer
Research.
Ingenuity Network-Assisted Transcription Profiling
influx in normal cells, selectively affecting the latter under equal
other conditions. The novel experimental drug delivery systems
use specifically designed liposomes for that purpose (34). In
case of MK886, its hydrophobic character might enable its
tumor-specific delivery via a targeted liposome delivery system.
Figure 6 shows a steep dose response to MK886, indicating that
even a small relative dose accumulation in a tumor site may
cause a disproportional therapeutic effect. We find that these
features of MK886 mechanism and biodistribution recommend
it for future exploration in murine models of cancer.
The known fact of cytoskeleton modification by phosphoinositide-3-kinase products (35) points to a biologically
intended role of lipid binding to actin or its protein partners,
exploited in MK886 and ciglitazone antitumor mechanism.
Catalano et al. (8) described inhibition of p53 nuclear
translocation by 5-lipoxygenase products. It would be interesting to explore the role of natural 5-lipoxygenase and cyclooxygenase-2 products along the lines in the present report.
score higher than other responses, and relation of this effect to
anoikis was suggested by the RT-PCR experiment. This result
was confirmed more directly by interference of the drug with
actin polymerization. The link between cytoskeleton disruption
and DNA processing deregulation was modestly confirmed by
NCI-60 data and more reliably by RT-PCR and Western
blotting. A novel antitumor mechanism was proposed based
on these findings, addressing previously unknown aspects of
the action and the safety of known pharmacologic agents. A
mechanistic explanation was proposed for the augmentation of
DNA-directed toxicity by cytoskeleton alteration, observed in a
number of reports. Application of Inegnuity software in this
project enabled us to formulate a mechanistic hypothesis based
on relatively small-scale microarray study. Our work suggested
utility of this algorithm for other similar studies, aiming to
translate microarray results into a mechanistic hypothesis,
leading to the insights into therapy.
Acknowledgments
Conclusions
This study found that cytoskeleton and focal adhesion
functions responded to MK886 treatment with the Ingenuity
We thank Dr. W. Tellford (NCI Flow Cytometry core facility) and Dr. R.H.
Shoemaker (ScreeningTechnologies Branch, NCI) for technical assistance, and the
Fellows Editorial Board of NIH for their help with the review of the manuscript.
References
1. Pace E, Profita M, Melis M, et al. LTB4 is present in
exudative pleural effusions and contributes actively to
neutrophil recruitment in the inflamed pleural space.
Clin Exp Immunol 2004;135:519 ^ 27.
2. Calzada C,Vericel E, Mitel B, Lagarde M. Stimulation
of platelet aggregation in response to arachidonic acid
hydroperoxide via phospholipase activation. Lipids
1999;34 Suppl:S295.
3. Busse W, Kraft M. Cysteinyl leukotrienes in allergic
inflammation: strategic target for therapy. Chest
2005;127:1312 ^ 26.
4. CunhaJM, Sachs D, Canetti CA, Poole S, Ferreira SH,
Cunha FQ. The critical role of leukotriene B4 in antigen-induced mechanical hyperalgesia in immunised
rats. Br J Pharmacol 2003;139:1135 ^ 45.
5. JonesJE,WalkerJL, SongY, et al. Effect of 5-lipoxygenase on the development of pulmonary hypertensionin
rats. Am J Physiol Heart Circ Physiol 2004;286:
H1775 ^ 84.
6. Chen X,Wang S,Wu N, et al. Overexpression of 5-lipoxygenase in rat and human esophageal adenocarcinoma and inhibitory effects of zileuton and celecoxib
on carcinogenesis. Clin Cancer Res 2004;10:6703 ^ 9.
7. Runarsson G, Liu A, Mahshid Y, et al. Leukotriene B4
plays a pivotal role in CD40-dependent activation of
chronic B lymphocy tic leukemia cells. Blood
2005;105:1274 ^ 9.
8. Catalano A, Caprari P, Soddu S, Procopio A, Romano
M. 5-Lipoxygenase antagonizes genotoxic stress-induced apoptosis by altering p53 nuclear trafficking.
FASEB J 2004;18:1740 ^ 2.
9. Avis IM, Jett M, Boyle T, et al. Growth control of
lung cancer by interruption of 5-lipoxygenase-mediated growth factor signaling. J Clin Invest 1996;
97:806 ^ 13.
10. Matuyama M,Yoshimura R, Mitsuhashi M, et al. Lipoxygenase inhibitors attenuate growth of human renal
cell carcinoma and induce apoptosis through arachidonic acid pathway. Oncol Rep 2005;14:73 ^ 9.
11. Werz O, Bürkert E, Samuelsson B, Steinhilber D. Activation of 5-lipoxygenase by cell stress is calcium independent in human polymorphonuclear leukocytes.
Blood 2002;99:1044 ^ 5.
12. Dixon RA, Diehl RE, Opas E, et al. Requirement of a
5-lipoxygenase-activating protein for leukotriene synthesis. Nature 1990;343:282 ^ 4.
www.aacrjournals.org
13. Wickelgren I. Heart disease. Gene suggests asthma
drugs may ease cardiovascular inflammation. Science
2004;303:941.
14. Manev R, Manev H. 5-Lipoxygenase as a putative
link between cardiovascular and psychiatric disorders.
Crit Rev Neurobiol 2004;16:181 ^ 6.
15. Zhao L, Funk CD. Lipoxygenase pathways in atherosclerosis. Trends Cardiovasc Med 2004;14:191 ^ 5.
16. Ishii K, Motoyoshi S, Kawata J, Nakagawa H, Takeyama K. A useful method for differential evaluation of
anti-inflammatory effects due to cyclooxygenase and
5-lipoxygenase inhibitions in mice. Jpn J Pharmacol
1994;65:297 ^ 303.
17. Schuller HM, Zhang L, Weddle DL, Castonguay A,
Walker K, Miller MS. The cyclooxygenase inhibitor
ibuprofen and the FLAP inhibitor MK886 inhibit pancreatic carcinogenesis induced in hamsters by transplacental exposure to ethanol and the tobacco
carcinogen NNK. J Cancer Res Clin Oncol 2002;
128:525 ^ 32.
18. Gugliuccu A, Ranzato L, Scorrano L, et al. Mitochondria are direct targets of the lipoxygenase inhibitor MK886. A strategy for cell killing by combined
treatment with MK886 and cyclooxygenase inhibitors.
J Biol Chem 2002;277:31789 ^ 95.
19. Harris JE, Alferai WA, Meng J, Andersen KM. Fivelipoxygenase inhibitors reduce Panc-1survival: synergism of MK886 with g-linolenic acid. Adv Exp Med
Biol 1999;469:505 ^ 10.
20. Ghosh J, Mayers CE. Inhibition of arachidonate 5lipoxygenase triggers massive apoptosis in human
prostate cancer cells. Proc Natl Acad Sci U S A
1998;95:13182 ^ 7.
21. La E, Kern JC, Atarod EB, Kehrer JP. Fatty acid
release and oxidation are factors in lipoxygenase inhibitor-induced apoptosis. Toxicol Lett 2003;138:
193 ^ 20.
22. Avis I, Hong SH, Martinez A, et al. Five-lipoxygenase inhibitors can mediate apoptosis in human breast
cancer cell lines through complex eicosanoid interactions. FASEB J 2001;15:2007 ^ 9.
23. Datta K, Biswal SS, Kehrer JP. The 5-lipoxygenaseactivating protein (FLAP) inhibitor, MK886, induces
apoptosis independently of FLAP. Biochem J 1999;
340:371 ^ 5.
24. Weinstein JN, MyersTG, O’Connor PM, et al. An in-
1827
formation-intensive approach to the molecular pharmacology of cancer. Science 1997;275:343 ^ 9.
25. Lowry OH, Rosbrough NJ, FarrAL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol
Chem 1951;193:265.
26. Avis I, Martinez A, Tauler J, et al. Inhibitors of the
arachidonic acid pathway and peroxisome proliferator-activated receptor ligands have superadditive
effects on lung cancer growth inhibition. Cancer Res
2005;65:4181 ^ 90.
27. Semizarov D, Frost L, Sathy A, Kroeger P, Hlabert
DN, Fesik SW. Specificity of short interfering RNA determined through gene expression signatures. Proc
Natl Acad Sci U S A 2003;100:6347 ^ 52.
28. Ciszak L, Krawczenko A, Polzar B, Mannherz HG,
Malicka-Blaszkiewicz M. Carp liver actin: isolation, polymerization and interaction with deoxyribonuclease I
(DNase I). Biochim Biophys Acta 1999;1451:141 ^ 52.
29. Carvalho Pinto-Silva CR, Ferreira JF, Costa RH, Belli
Filho P, Creppy EE, Matias WG. Micronucleus induction in mussels exposed to okadaic acid. Toxicon
2003;41:93 ^ 7.
30. Shang X, ShionoY, FujitaY, Oka S,Yamazaki Y. Synergistic enhancement of apoptosis by DNA- and cytoskeleton damaging agents: a basis for combination
chemotherapy of cancer. Anticancer Res 2001;21:
2585 ^ 9.
31. KristianT, Bernardi P, Siesjo BK. Acidosis promotes
the permeability transition in energized mitochondria:
implications for reperfusion injury. J Neurotrauma
2001;18:1059 ^ 74.
32. Goncalves RP, Rodrigues DG, Maranhao RC. Uptake of high density lipoprotein (HDL) cholesteryl
esters by human acute leukemia cells. Leuk Res
2005;29:955 ^ 8.
33. Ito I, Began G, Mohiuddin I, Saeki T, et al. Increased
uptake of liposomal-DNA complexes by lung metastases following intravenous administration. Mol Ther
2003;7:409 ^ 18.
34. Hrzenjak A, Reicher H,WinterspergerA, et al. Inhibition of lung carcinoma cell growth by high density lipoprotein-associated a-tocopheryl-succinate. Cell
Mol Life Sci 2004;61:1520 ^ 31.
35. FraleyTS,TranTC, Corgan AM, et al. Phosphoinositide binding inhibits a-actinin bundling activity. J Biol
Chem 2003;278:24039 ^ 45.
Clin Cancer Res 2006;12(6) March 15, 2006
Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2006 American Association for Cancer
Research.
Ingenuity Network-Assisted Transcription Profiling:
Identification of a New Pharmacologic Mechanism for MK886
Anatoly L. Mayburd, Alfredo Martlínez, Daniel Sackett, et al.
Clin Cancer Res 2006;12:1820-1827.
Updated version
Supplementary
Material
Cited articles
Citing articles
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://clincancerres.aacrjournals.org/content/12/6/1820
Access the most recent supplemental material at:
http://clincancerres.aacrjournals.org/content/suppl/2006/03/24/12.6.1820.DC1
This article cites 35 articles, 14 of which you can access for free at:
http://clincancerres.aacrjournals.org/content/12/6/1820.full#ref-list-1
This article has been cited by 9 HighWire-hosted articles. Access the articles at:
http://clincancerres.aacrjournals.org/content/12/6/1820.full#related-urls
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2006 American Association for Cancer
Research.