Antitumor activity of MEK and PI3K inhibitors against malignant pleural mesothelioma cells in vitro and in vivo.
ABSTRACT Malignant pleural mesothelioma (MPM) is an aggressive malignancy for which there is no approved targeted therapy. We examined the therapeutic efficacy of the mitogen-activated protein kinase kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) inhibitors against human MPM cell lines both in vitro and orthotopically inoculated into severe combined immunodeficient (SCID) mice. In addition, the molecular mechanisms of these agents were confirmed in vitro and in vivo. The MEK or the PI3K inhibitor suppressed MPM cell growth in vitro in a dose-dependent manner via induction of G1 cell cycle arrest and apoptosis. In addition, combined use of the MEK and PI3K inhibitors showed an additive or synergistic inhibitory effect on MPM cell growth compared to treatment with either individual drug. Treatment with MEK or PI3K inhibitor suppressed the production of thoracic tumors and pleural effusion and prolonged the survival time of EHMES-10 cell-bearing SCID mice. The combination therapy more effectively prolonged the survival time compared to treatment with either individual drug. Immunohistochemical and western blot analysis of thoracic tumors suggested that these agents induced cell cycle arrest, apoptosis and inhibition of tumor angiogenesis. Our results suggest that a combination of MEK and PI3K inhibitors is a promising therapeutic strategy for MPM.
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ABSTRACT: Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, is reported to exert anti-tumor effects by upregulating the expression of the natural killer group 2D (NKG2D) ligands on tumor cells; however, the mechanisms vary in different tumor types, and the effect and mechanism of action of VPA in pancreatic cancer cells are unknown.BMC Cancer 05/2014; 14(1):370. · 3.33 Impact Factor
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ABSTRACT: Malignant mesothelioma is a deadly tumor whose diagnosis and treatment remain very challenging. There is an urgent need to advance our understanding of mesothelioma biology and to identify new molecular markers for improving management of patients. CD157 is a membrane glycoprotein linked to ovarian cancer progression and mesenchymal differentiation. The common embryonic origin of ovarian epithelial cells and mesothelial cells and the evident similarities between ovarian and mesothelial cancer prompted us to investigate the biological role and clinical significance of CD157 in malignant pleural mesothelioma (MPM). CD157 mRNA and protein were detected in four of nine MPM cell lines of diverse histotype and in 85.2% of MPM surgical tissue samples (32/37 epithelioid; 37/44 biphasic). CD157 expression correlated with clinical aggressiveness in biphasic MPM. Indeed, high CD157 was a negative prognostic factor and an independent predictor of poor survival for patients with biphasic MPM by multivariate survival analysis (HR = 2.433, 95% CI 1.120-5.284; p = 0.025). In mesothelioma cell lines, CD157 gain (in CD157-negative cells) or knockdown (in CD157-positive cells) affected cell growth, migration, invasion and tumorigenicity, most notably in biphasic MPM cell lines. In these cells, CD157 expression was associated with increased activation of the mTOR signaling pathway, resulting in decreased platinum sensitivity. Moreover, a trend towards reduced survival was observed in patients with biphasic MPM receiving postoperative platinum-based chemotherapy. These findings indicate that CD157 is implicated in multiple aspects of MPM progression and suggest that CD157 expression could be used to stratify patients into different prognostic groups or to select patients that might benefit from particular chemotherapeutic approach.Oncotarget 07/2014; · 6.64 Impact Factor
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ABSTRACT: Malignant pleural mesothelioma (MPM) is an aggressive disease with a poor prognosis. Studies have shown that both MET and its key downstream intracellular signaling partners, PI3K and mTOR, are overexpressed in MPM. Here we determined the combinatorial therapeutic efficacy of a new generation small molecule inhibitor of MET, ARQ 197, and dual PI3K/mTOR inhibitors NVP-BEZ235 and GDC-0980 in mesothelioma cell and mouse xenograft models. Cell viability results show that mesothelioma cell lines were sensitive to ARQ 197, NVP-BEZ235 and GDC-0980 inhibitors. The combined use of ARQ 197 with either NVP-BEZ235 or GDC-0980, was synergistic (CI<1). Significant delay in wound healing was observed with ARQ 197 (p<0.001) with no added advantage of combining it with either NVP-BEZ235 or GDC-0980. ARQ 197 alone mainly induced apoptosis (20±2.36%) that was preceded by suppression of MAPK activity, while all the three suppressed cell cycle progression. Both GDC-0980 and NVP-BEZ235 strongly inhibited activities of PI3K and mTOR as evidenced from the phosphorylation status of AKT and S6 kinase. The above observation was further substantiated by the finding that a majority of the MPM archival samples tested revealed highly active AKT. While the single use of ARQ 197 and GDC-0980 inhibited significantly the growth of MPM xenografts (p<0.05, p<0.001 respectively) in mice, the combination of the above two drugs was highly synergistic (p<0.001). Our results suggest that the combined use of ARQ 197/NVP-BEZ235 and ARQ 197/GDC-0980 is far more effective than the use of the drugs singly in suppressing MPM tumor growth and motility and therefore merit further translational studies.PLoS ONE 01/2014; 9(9):e105919. · 3.53 Impact Factor
INTERNATIONAL JOURNAL OF ONCOLOGY 41: 449-456, 2012
Abstract. Malignant pleural mesothelioma (MPM) is an aggres-
sive malignancy for which there is no approved targeted therapy.
We examined the therapeutic efficacy of the mitogen-activated
protein kinase kinase (MEK) and phosphatidylinositol 3-kinase
(PI3K) inhibitors against human MPM cell lines both in vitro
and orthotopically inoculated into severe combined immuno-
deficient (SCID) mice. In addition, the molecular mechanisms
of these agents were confirmed in vitro and in vivo. The MEK
or the PI3K inhibitor suppressed MPM cell growth in vitro in
a dose-dependent manner via induction of G1 cell cycle arrest
and apoptosis. In addition, combined use of the MEK and PI3K
inhibitors showed an additive or synergistic inhibitory effect
on MPM cell growth compared to treatment with either indi-
vidual drug. Treatment with MEK or PI3K inhibitor suppressed
the production of thoracic tumors and pleural effusion and
prolonged the survival time of EHMES-10 cell-bearing SCID
mice. The combination therapy more effectively prolonged the
survival time compared to treatment with either individual drug.
Immunohistochemical and western blot analysis of thoracic
tumors suggested that these agents induced cell cycle arrest,
apoptosis and inhibition of tumor angiogenesis. Our results
suggest that a combination of MEK and PI3K inhibitors is a
promising therapeutic strategy for MPM.
Malignant pleural mesothelioma (MPM) is an aggressive
neoplasm that arises from mesothelial cells. It was reported that
asbestos, iron, and simian virus 40 were linked to the etiology
of MPM (1-4). MPM was once considered a rare disease, but its
incidence is increasing worldwide (5).
Current aggressive multimodality therapy for MPM
(consisting of surgical resection, cytotoxic chemotherapy,
and radiation) offers survival benefits for only a small subset
of patients in early stages of the disease (6). Recently, the
multi-targeted antifolate pemetrexed has been approved as the
front-line agent in combination with cisplatin for the treatment
of MPM (7). However, most of the patients relapsed within a
year after starting treatments. Therefore, new and more effective
therapies are necessary to improve the prognosis of this disease.
The mitogen-activated protein (MAP) kinase kinase (MEK)-
extracellular signal-regulated kinase (ERK) pathway and the
phosphatidylinositol 3-kinase (PI3K)-Akt pathway play critical
roles in the regulation of cell proliferation, growth, differentia-
tion and survival (8-10). These pathways are activated in many
types of solid tumor models, including MPM (8,9,11-13). It
was also reported that inhibition of these pathways affected
the proliferation of MPM cell lines in vitro (14,15). However,
no report has demonstrated growth-inhibitory effects of these
agents on MPM cells in vivo.
In the present study, we examined whether the MEK or the
PI3K inhibitor affected the growth of MPM cells in vitro and
in vivo. Furthermore, we evaluated the possibility that combined
use of the MEK inhibitor and the PI3K inhibitor might enhance
Materials and methods
Cell cultures. The human mesothelioma cell line EHMES-10
was established from the pleural effusion of a patient with MPM
in our institution (16,17). MSTO211H was purchased from the
American Type Culture Collection (Manassas, VA, USA).
These cell lines were cultured in RPMI-1640 medium (Nikken
Bio Medical Laboratories, Kyoto, Japan) supplemented with
10% fetal bovine serum (Hyclone, Logan, UT, USA), penicillin
(100 U/ml) and streptomycin (50 mg/ml) in a 37˚C humidified
incubator with 5% carbon dioxide.
Reagents and inhibitors. MEK inhibitor U0126 and PI3K
inhibitor LY294002 were purchased from LC Laboratories
Antitumor activity of MEK and PI3K inhibitors against
malignant pleural mesothelioma cells in vitro and in vivo
SEIGO MIYOSHI1, HIRONOBU HAMADA1,2, NAOHIKO HAMAGUCHI1, AKI KATO1, HITOSHI KATAYAMA1,
KAZUNORI IRIFUNE1, RYOJI ITO1, TATSUHIKO MIYAZAKI3, TAKAFUMI OKURA1 and JITSUO HIGAKI1
1Department of Integrated Medicine and Informatics, Ehime University Graduate School of Medicine, Ehime;
2Department of Health and Sports Medical Sciences, Graduate School of Health Sciences, Hiroshima University, Hiroshima;
3Department of Pathogenomics, Ehime University Graduate School of Medicine, Ehime, Japan
Received February 6, 2012; Accepted April 17, 2012
Correspondence to: Dr Hironobu Hamada, Department of Health
and Sports Medical Sciences, Graduate School of Health Sciences,
Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553,
Key words: malignant pleural mesothelioma, mitogen-activated
protein kinase kinase inhibitor, phosphatidylinositol 3-kinase
inhibitor, cell cycle, apoptosis, angiogenesis
MIYOSHI et al: MEK AND PI3K INHIBITORS FOR MALIGNANT PLEURAL MESOTHELIOMA
(Woburn, MA, USA). For in vitro experiments, these agents were
dissolved in dimethylsulfoxide (DMSO) (Sigma-Aldrich Co.,
St. Louis, MO, USA) and were added to cells in medium with
a final DMSO concentration of 1.0%. For in vivo studies, these
agents were prepared as a suspension in a vehicle consisting of
40% DMSO in phosphate-buffered saline (PBS) (Wako Pure
Chemical Industries, Osaka, Japan). Rabbit polyclonal antibodies
against ERK1/2, phospho-ERK1/2, Akt, phospho-Akt, p27kip1,
cyclin E, cyclin D1, p70S6K, phospho-p70S6K, S6, phospho-
S6, p90 ribosomal S6 kinase (p90RSK), phospho-p90RSK,
glycogen synthase kinase-3β (GSK3β), phospho-GSK3β, Bad,
phospho-Bad, poly(ADP-ribose) polymerase (PARP), procas-
pase 3, hypoxia-inducible factor 1α (HIF1α), and β-actin were
purchased from Cell Signaling Technology (Danvers, MA,
USA). Rabbit polyclonal antibody against vascular endothelial
growth factor (VEGF) was purchased from Millipore Co.
(Tokyo, Japan). Mouse monoclonal antibody against CD31/
platelet/endothelial cell adhesion molecule-1 was purchased
from BD Pharmingen (Tokyo, Japan) for in vivo immunohis-
tochemical study. Mouse monoclonal antibody against CD31
(PECAM-1) was purchased from Cell Signaling Technology for
in vivo western blot analysis. Horseradish peroxidase conjugated
goat anti-rabbit IgG and horse anti-mouse IgG were purchased
from Cell Signaling Technology.
Cell proliferation assay. The cell proliferation assay reagent
1,3-benzene disulfonate) (Roche Diagnostics GmbH,
Mannheim, Germany) was used to assess the effect of U0126
or LY294002 on cell growth. MPM cells (1x104 cells/well) were
plated in 96-well plates (Nunc, Roskilde, Denmark) and were
exposed to various concentrations of test agents dissolved in
DMSO. Controls received DMSO vehicle at a concentration
equal to that of drug treated cells. After drug treatment for 72 h,
10 µl of WST-1 reagent were added to each well. Absorbance
was measured at 450 nm with a reference wavelength at 690 nm
by an E max precision microplate reader (Molecular Devices,
Cell cycle analysis. MPM cells, treated with or without test
agents for 24 h, were trypsinized and collected, and the cell
nuclei were stained using the CycleTest Plus DNA Reagent Kit
(Becton-Dickinson, San Jose, CA, USA). Cells were subjected
to FACScan analysis, and cell cycle profiles were determined
using ModFitLT software (Becton-Dickinson, San Diego, CA,
USA). This analysis was carried out independently three times.
DNA fragmentation assay. We examined DNA fragmentation
to assess apoptosis in EHMES-10 or MSTO211H cells. Cells
were treated with either U0126 or LY294002 or a combination
of both for 24 h. DNA fragmentation was evaluated using the
Cell Death Detection ELISA kit (Roche Molecular Biochemical,
Indianapolis, IN, USA) as previously reported (18).
Western blot analysis. Cultured cells were treated with lysis
buffer [25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X,
50 mM NaF and 1 mM Na3VO4] containing proteinase
inhibitor cocktail (Roche Diagnostics GmbH). Tumor tissue
samples were homogenized in lysis buffer. Insoluble materials
were removed by centrifugation at 4˚C for 15 min 15,000 x g.
Protein concentration was determined using a Bio Rad Protein
Assay Kit (Bio Rad Laboratories, Hercules, CA, USA).
Proteins were separated on 7.5 to 15% polyacrylamide
gels (Bio Rad Laboratories). After electrophoresis, the protein
was transferred to a nitrocellulose membrane and detected by
immunoblotting using SNAP i.d. Protein Detection System
(Millipore Co.) as previously described (19). This analysis was
carried out independently three times.
Experimental animals. Male severe combined immunodefi-
cient (SCID) mice (six to eight weeks old) were obtained from
Clea Japan (Osaka, Japan), fed autoclaved standard pellets and
water, and maintained under specific pathogen-free conditions
throughout this study. All of the protocols involving SCID
mice were approved by the guidelines established by the Ehime
University Committee on Animal Care and Use.
Orthotopic implantation model. Cultured EHMES-10 cells
were harvested, washed twice and re-suspended in PBS. The
SCID mice were inoculated in the thoracic cavity with the tumor
cells (3x106 cells/mouse), as previously described (17,20). Seven
days after inoculation, mice were randomized into eight groups
(n=7 mice/group) to receive vehicle alone (DMSO + PBS),
U0126 alone (20, 30 and 40 mg/kg), LY294002 alone (12.5,
25 and 50 mg/kg) and a combination of U0126 (30 mg/kg)
and LY294002 (25 mg/kg). These agents were administrated
intraperitoneally twice a week. Mice were sacrificed on day 30
after tumor cell inoculation. The tumor tissue was excised and
weighed, and the volume of pleural effusion was measured.
We also measured the body weights and serum levels of total
protein (TP), blood urea nitrogen (BUN), creatinine (Cre),
aspartate amino transferase (AST) and alanine aminotrans-
ferase (ALT), and evaluated the degree of dermatopathy as a
measure of side effects.
Immunohistochemistry. Paraffin-embedded tissues were
subjected to immunohistochemistry with anti-phospho-
ERK1/2 monoclonal antibody, phospho-Akt monoclonal
antibody, or anti-p27kip1 monoclonal antibody. For in situ
apoptosis detection, we used terminal deoxynucleotidyl
transferase-mediated dUTP nick end labeling (TUNEL assay)
with the In situ Apoptosis Detection Kit (Takara Biomedicals,
Ohtsu, Japan). Frozen tissue sections were used for identifica-
tion of endothelial cells using rat anti-mouse CD31/platelet/
endothelial cell adhesion molecule-1 monoclonal antibody.
Immunohistochemical procedures were performed using the
Envision™ Systems (Dako, Glostrup, Denmark) method, as
previously described (21). Phospho-ERK1/2- or phospho-
Akt- or p27kip1-positive cells were visualized with Fuchsin+
substrate-chromogen (Dako). Antibodies against TUNEL
assay or CD31 localization were detected using a peroxidase
reaction with 3-diaminobenzidine (Dako).
Statistical analysis. In vitro study data are presented as
means ± SD, and were analyzed using ANOVA followed by
Dunnett's t-test. In vivo data were expressed as median values
and ranges. The Mann-Whitney U test was used to compare
groups. The Kaplan-Meier method was used to evaluate the
survival analysis and comparisons were made using a log-rank
test. Drug interactions were analyzed by the Chou and Talalay
INTERNATIONAL JOURNAL OF ONCOLOGY 41: 449-456, 2012
method using the CalcuSyn software program (version 2.0;
Biosoft, Cambridge, UK). The combination index (CI) was
simulated from each level of fractional affect. According to
this method, a CI<0.3, 0.3-0.7, 0.7-0.9, 0.9-1.1, 1.1-1.45, 1.45-3.3
and >3.3 indicates highly synergistic, synergistic, moderate to
slight synergistic, nearly additive, slight to moderate antago-
nistic, antagonistic and strong antagonistic, respectively.
Differences between groups are considered statistically
significant at P<0.05.
Growth inhibition of MPM cells by U0126 and/or LY294002
treatment. The effects of U0126 or LY294002 at concentrations
ranging from 20 to 200 µM on the proliferation of EHMES-10 or
MSTO211H cells were determined with the WST-1 assay. Each
agent inhibited MPM cell growth in a dose-dependent manner
(Fig. 1A). The IC50 values for U0126 and LY294002 against
EHMES-10 cells were 66.8 µM and 20.7 µM, respectively.
Moreover, the IC50 values for U0126 and LY294002 against
MSTO211H cells were 39.0 µM and 29.9 µM, respectively.
We evaluated the effect of combining treatments with
U0126 and LY294002. The ratio of IC50 values for U0126 and
LY294002 against EHMES-10 cells was approximately 3:1
while the ratio was 4:3 against MSTO211H cells. Therefore, the
two MPM cell lines were exposed to varying concentrations
of U0126 and LY294002 at fixed ratios of 3:1 or 4:3, as appro-
priate. Cell viability was then assessed by the WST-1 assay. The
averaged CIs for EHMES-10 cells and MSTO211H cells were
1.017 and 0.54, which indicates a nearly additive effect and a
synergistic effect, respectively (Fig. 1B).
Induced G1 cell cycle arrest of MPM cells after treatment with
U0126 and/or LY294002. To investigate the mechanisms of
growth inhibition of MPM cells by U0126 or LY294002 treat-
ment, we performed cell cycle analysis of EHMES-10 cells
or MSTO211H cells treated with 80 µM U0126 and/or 80 µM
LY294002. Treatment with U0126 or LY294002 for 24 h
significantly increased the G1-phase populations compared to
control in both MPM cell lines (all, P<0.05) (Fig. 2A). In addi-
tion, U0126 alone and LY294002 alone significantly increased
the percentage of MPM cells in the sub-G1 phase, indicative
of cell apoptosis, compared to control (all, P<0.05). Combining
treatment with U0126 with that of LY294002 led to a signifi-
cant increase in the sub-G1 phase population in both cell lines
compared to control or individual drugs (all, P<0.01).
We also analyzed the expression of cell cycle regulatory
proteins after treatment with U0126 and/or LY294002 in both
EHMES-10 cells and MSTO211H cells. Both agents increased
p27kip1 expression and decreased cyclin E expression in
both cell lines (Fig. 2B). A decrease of cyclin D1 expression
was observed in treatment with either U0126 or LY294002
in EHMES-10 cells, and following treatment with U0126 in
Figure 1. In vitro responses of EHMES-10 cells and MSTO211H cells to MEK and/or PI3K inhibitors. (A) The effects of U0126 or LY294002 on the prolifera-
tion of MPM cells. MPM cells were treated with U0126 or LY294002 for 72 h, and cell viability was determined with the WST-1 assay. (B) Analysis of the
combined treatment of MPM cells with U0126 and LY294002. Inhibitors were used in combination in a fixed dose ratio for 72 h, and cell viability was assessed
with the WST-1 assay. The fractional effect versus combination index (Fa-CI) curve was calculated with CalcuSyn software.
MIYOSHI et al: MEK AND PI3K INHIBITORS FOR MALIGNANT PLEURAL MESOTHELIOMA
Induction of apoptosis by U0126 and/or LY294002 treatment.
We assessed the ability of U0126 and LY294002 to induce apop-
tosis in MPM cells. DNA fragmentation analysis showed that
treatment with U0126 or LY294002 alone induced apoptosis in
EHMES-10 cells and in MSTO211H cells in a dose-dependent
manner. Furthermore, the combined treatment with 80 µM
U0126 and 80 µM LY294002 significantly increased the number
of apoptotic cells compared to control and to treatment with
U0126 alone in EHMES-10 cells (all, P<0.01) and compared to
control and treatment with the individual drug in MSTO211H
cells (all, P<0.01) (Fig. 2C). Western blot analysis showed that
treatments with U0126 and/or LY294002 increased the level of
89 kDa cleaved PARP and decreased the levels of phospho-Bad
and procaspase 3 in both cell lines (Fig. 2B).
Signaling alterations induced by treatment with U0126 and/or
LY294002. To investigate the effects of U0126 and LY294002
on intercellular signaling, MPM cells were treated with U0126
or LY294002 or a combination of both. As shown in Fig. 2B,
U0126 blocked the phosphorylation of ERK1/2 and p90RSK,
and decreased HIF1α and VEGF expression in EHMES-10
cells. On the other hand, treatment with LY294002 suppressed
the phosphorylation of Akt, p70S6K, S6 and GSK3β, and
inhibited HIF1α and VEGF expression in EHMES-10 cells.
Use of the combination treatment inhibited the phosphoryla-
tion of all of the above proteins and decreased the level of
HIF1α and VEGF in EHMES-10 cells. Signaling alterations in
MSTO211H cells tended to be similar to those in EHMES-10
cells, except for phospho-GSK3β alteration.
Antitumor activity of U0126 and/or LY294002 in EHMES-10
cell xenografts. To assess the in vivo therapeutic efficacy of
U0126 and/or LY294002, SCID mice bearing EHMES-10
xenografts were treated with vehicle, U0126, LY294002,
or a combination of U0126 and LY294002 as described in
Materials and methods. Administration of 30 or 40 mg/kg
of U0126, or 50 mg/kg of LY294002, or use of combined
therapy with 30 mg/kg of U0126 and 25 mg/kg of LY294002
significantly prolonged the survival time of EHMES-10
cell-bearing SCID mice compared to the control group (all,
P<0.01) (Fig. 3A-C). The combination therapy more effec-
tively prolonged the survival time compared to treatment with
either individual drug, although statistical significance was not
obtained (Fig. 3C).
We also evaluated the effect of U0126 and/or LY294002
on the production of thoracic tumors and pleural effusion in
Figure 2. The mechanisms by which U0126 or LY294002 inhibited growth of EHMES-10 cells and MSTO211H cells in vitro. (A) The effects of U0126 and/
or LY294002 on the cell cycle profile. After treatment with 80 µM U0126 and/or 80 µM LY294002 for 24 h, MPM cells were collected, fixed, strained with
propidium iodide and analyzed by flow cytometry. Data shown are representative of three independent experiments. *P<0.05, compared to control; #P<0.01,
compared to control; +P<0.01, compared to individual drug. (B) Effects of U0126 and/or LY294002 on the expression of phospho-extracellular signal-regulated
kinase (ERK)1/2 (p-ERK1/2), phospho-Akt (p-Akt), p27kip1, cyclin E, cyclin D1, phospho-p70S6K (p-p70S6K), phospho-S6 (p-S6), phospho-p90 ribosomal
S6 kinase (p90RSK) (p-p90RSK), phospho-glycogen synthase kinase-3β (GSK3β) (p-GSK3β), phospho-Bad (p-Bad), poly (ADP-ribose) polymerase (PARP),
procaspase 3, hypoxia-inducible factor 1α (HIF1α) and vascular endothelial growth factor (VEGF). Tumor cells were treated with or without U0126 (80 µM),
and/or LY294002 (80 µM) for 24 h. Then, cells were lysed, and the indicated proteins were detected by immunoblotting. Data shown are representative of three
independent experiments. (C) Cytoplasmic histone-associated DNA fragments determined by ELISA-based quantification. MPM cells were treated with U0126
or LY294002 or a combination of 80 µM U0126 and 80 µM LY294002 for 24 h. C, control; U, U0126; LY, LY294002; COM, combination of 80 µM U0126 and
80 µM LY294002; *P<0.01, compared to control; #P<0.01, compared to treatment with 80 µM U0126 alone; +P<0.01, compared to the individual drug.
INTERNATIONAL JOURNAL OF ONCOLOGY 41: 449-456, 2012
EHMES-10 cell-bearing SCID mice (Fig. 3D, Table I). U0126
and/or LY294002 significantly inhibited tumor growth and
pleural effusion production compared to control (all, P<0.05).
The combination therapy more effectively inhibited tumor
growth compared to treatments with individual drugs, although
statistical significance was not obtained.
Figure 3. Effects of U0126 or LY294002 or the combination of U1026 and LY294002 on severe combined immunodeficiency (SCID) mice bearing EHMES-10
cells. (A-C) Survival times of EHMES-10 cell-bearing SCID mice treated with U0126, LY294002, or the combination of U0126 and LY294002. EHMES-10 cells
(3x106) were inoculated into the thoracic cavity of SCID mice. Seven days after inoculation, SCID mice were randomized into eight groups (n=7 mice/group)
to receive vehicle (DMSO + PBS), U0126 (20, 30 and 40 mg/kg), or LY294002 (12.5, 25 and 50 mg/kg), or a combination of U0126 (30 mg/kg) and LY294002
(25 mg/kg). U, U0126; LY, LY294002; *P<0.01, compared to control. (D) Formation of thoracic tumors and pleural effusion by EHMES-10 cells with or without
test agents. Mice were sacrificed on day 30 and thoracic tumors and pleural effusions were evaluated. Arrowheads, pleural effusions; arrows, thoracic tumors.
Table I. Effects of U0126, LY294002 and combination therapy on thoracic tumor and pleural effusion produced by MPM cells
in SCID mice.
Thoracic tumor Pleural effusion
Incidence Weight (mg) Volume (µl)
Control 5/5 487.7 (162.8-907.1) 5/5 150 (50.0-280.0)
Combination 3/5 0/5
EHMES-10 cells were inoculated into thoracic cavity of severe combined immunodeficiency mice on day 0, and the mice were treated with
U0126 (30 or 40 mg/kg) or LY294002 (25 or 50 mg/kg) or combination with 30 mg/kg U0126 and 25 mg/kg LY294002 i.p. on twice/week. Mice
were sacrificed on day 30 and thoracic tumor and pleural effusion were evaluated. Data are median values (ranges). U, U0126; LY, LY294002;
aP<0.05 compared to control.
MIYOSHI et al: MEK AND PI3K INHIBITORS FOR MALIGNANT PLEURAL MESOTHELIOMA
Immunohistochemical staining and western blot analysis to
clarify the antitumor mechanisms of U0126 and/or LY294002
in vivo. Immunohistochemical analysis showed that treatment
with U0126 or with LY294002 reduced phospho-ERK1/2-
positive tumor cells or phospho-Akt-positive tumor cells,
respectively (Fig. 4A). Furthermore, treatment with an individual
drug increased the number of p27kip1-positive tumor cells and
TUNEL assay-positive tumor cells, and decreased the number
of CD31-positive endothelial cells. The combination therapy
group showed decreased phospho-ERK1/2 and phospho-Akt
activities and CD31-positive endothelial cells, and increased
p27kip1-positive tumor cells and TUNEL assay-positive tumor
Figure 4. Histological and western blot analysis of thoracic tumors produced by EHMES-10 cells. SCID mice bearing EHMES-10 cells were treated
with 30 mg/kg U0126, 25 mg/kg LY294002 and a combination of 30 mg/kg U0126 and 25 mg/kg LY294002. Mice were sacrificed on day 30 after tumor cell
inoculation. (A) Thoracic tumors were analyzed by H&E and immunohistochemistry of phospho-extracellular signal-regulated kinase (ERK)1/2 (p-ERK1/2),
phospho-Akt (p-Akt), p27kip1, TUNEL and CD31. Magnification, x400. (B) Western blots analysis showing modulation of phospho-extracellular signal-regulated
kinase (ERK)1/2 (p-ERK1/2), phospho-Akt (p-Akt), cyclin E, cyclin D1, p27kip1, poly(ADP-ribose) polymerase (PARP), procaspase 3, hypoxia-inducible factor
1α (HIF1α), vascular endothelial growth factor (VEGF) and CD31 after treatment with U0126 and/or LY294002.
Table II. Side effects of treatment with U0126 or LY294002 or combination therapy after 30 days.
Weight (g) TP (g/dl) BUN (mg/dl) Cre (mg/dl) AST (IU/l) ALT (IU/l)
Mice were treated with U0126 (30 or 40 mg/kg) or LY294002 (25 or 50 mg/kg) or combination with 30 mg/kg U0126 and 25 mg/kg LY294002
i.p. twice/week, and the body weights, serum levels of total protein (TP), blood urea nitrogen (BUN), creatinine (Cre), aspartate amino trans-
ferase (AST) and alanine aminotransferase (ALT) were evaluated at the end of therapy on day 30. Data are median values (ranges). U, U0126;
LY, LY294002; aP<0.05 compared to control.
INTERNATIONAL JOURNAL OF ONCOLOGY 41: 449-456, 2012
cells. The effect of the inhibitors delivered in combination was
more pronounced than the drugs applied individually in the
analyses of p27kip1 and TUNEL assay.
Western blot analysis showed that treatment with U0126,
LY294002 and a combination of these agents inhibited ERK1/2
phosphorylation, Akt phosphorylation, and the phosphorylation
of ERK1/2 and Akt, respectively (Fig. 4B). In treatment with
U0126, LY294002 and the combination, we observed inhibited
expression of cyclin E, cyclin D1, procaspase 3, HIF1α, VEGF,
and CD31, and increased expression of p27kip1 and 89 kDa
Side effects of treatment with U0126 and/or LY294002. To
examine the side effects of treatment with U0126 and/or
LY294002, the body weights and serum levels of TP, BUN, Cre,
AST and ALT were determined at the end of therapy on day 30
(Table II). Dermatopathy was also evaluated during the treat-
ment with these agents. Side effects were not observed after the
administration of these agents.
The Ras pathway is one of the most frequently deregulated
pathways in cancer (22). Ras signals through multiple effector
pathways, including the RAF/MEK/ERK and PI3K/Akt
signaling cascades. A previous study reported that these
pathways were frequently activated in MPM (13). Therefore,
downregulation of these pathways might contribute to the inhi-
bition of tumor development and progression. In this study, we
showed that treatment with MEK and PI3K inhibitors, U0126
and LY294002, inhibited MPM cell growth via cell cycle arrest,
apoptosis, and inhibition of tumor angiogenesis in vitro and
in vivo. In addition, each drug prolonged the survival time of
SCID mice bearing EHMES-10 cells. When drugs were applied
in combination, survival times were longer than those achieved
with the individual treatments.
Treatment with a MEK inhibitor or a PI3K inhibitor has
been shown to inhibit the growth of many types of cancer cells,
including MPM cell lines, via induction of cell cycle arrest
and apoptosis (14,15,23-25). In the present study, MEK and
PI3K inhibitors suppressed growth of MPM cells in a dose-
dependent manner. Flow cytometric analysis showed that the
treatment with MEK or PI3K inhibitors achieved G1 cell cycle
arrest of MPM cells. DNA fragmentation analysis showed
apoptosis of MPM cells following treatment with these agents.
It was reported that treatment of MPM with a MEK inhibitor
induced p27kip1 upregulation (26). A PI3K inhibitor induced
p27kip1 upregulation and inhibition of phosphorylation of
p70S6K and S6 (14,26). The present study showed similar
results. In addition, treatment of MPM cells with a MEK
inhibitor downregulated cyclin E and cyclin D1, and inhibited
phosphorylation of p90RSK and Bad. Inhibition of cyclin E
and phospho-Bad expression was observed in treatment with a
PI3K inhibitor for MPM cells. Treatment of EHMES-10 cells
with a PI3K inhibitor reduced cyclin D1 and phospho-GSK3β
Our study demonstrated the efficacy and the mechanisms
of action of a MEK inhibitor and a PI3K inhibitor for MPM not
only in vitro but also in in vivo experiments. All previous reports
of MEK inhibitors and PI3K inhibitors for MPM cells have been
limited to showing the inhibitory effects and mechanisms in vitro
(14,26). Our study showed that treatment with a MEK inhibitor
or a PI3K inhibitor prolonged the survival time of EHMES-10
cells-bearing SCID mice. Tumor weight and pleural effusion
at day 30 were reduced by these treatments. Furthermore, the
immunohistochemical and western blot analyses of thoracic
tumors suggested that the MEK and the PI3K inhibitors induced
cell cycle arrest and cell apoptosis, which was compatible with
the results of the in vitro study.
Treatments with the MEK inhibitor or the PI3K inhibitor
might be associated with inhibition of angiogenesis in MPM
cells. More than 60% of patients with MPM commonly present
with a pleural effusion associated with breathlessness, often
accompanied by chest wall pain, which compromises their
quality of life (27). Angiogenesis has significant effects on the
development of a pleural effusion and ascites (28,29). It was
also reported that treatment with a MEK or a PI3K inhibitor
suppressed proangiogenic cytokine production in melanoma
and MPM cells (15,23). Our study showed that these agents
significantly inhibited pleural effusion production and CD31
protein expression and decreased CD31-positive endothelial
cells compared to controls. In addition, western blot analysis
showed that treatment of these agents decreased the expression
of HIF1α and VEGF, both of which play an essential role in
tumor angiogenesis and progression, in vitro and in vivo.
Combination therapy with the MEK and PI3K inhibitors
might be more rational than an individual drug for MPM. It was
reported that antitumor activity of a MEK inhibitor or a PI3K
inhibitor induces activation of the other pathway (30). Moreover,
several reports demonstrated that inhibition of both cascades
results in greater antitumor activity (15,23). In the present study,
the combination therapy with MEK and PI3K inhibitors was
also more effective compared to that of individual drugs both
in vitro and in vivo.
Treatment with a MEK inhibitor and a PI3K inhibitor
might be well tolerated. For example, treatment with the
MEK inhibitor CI-1040 caused only mild or moderate toxici-
ties such as diarrhea, nausea, asthenia, rash, and anorexia in
patients with advanced non-small cell lung, breast, colon, and
pancreatic cancers (31). In addition, Hu et al reported that
daily intraperitoneal administration of LY294002 at a dose
of 100 mg/kg caused body weight loss and dry skin in mice
with ovarian cancer (32). However, in the following study, side
effects were not shown by reduction of LY294002 adminis-
tration to three days per week (33). In the present study, side
effects were not observed using a combination of MEK and
In conclusion, our study demonstrates that in MPM cells
our selected MEK and PI3K inhibitors functioned via cell cycle
arrest, induction of apoptosis, and inhibition of tumor angiogen-
esis, both in vivo and in vitro. In addition, combining the MEK
inhibitor with the PI3K inhibitor had additive or synergistic
effects in vitro. Combination therapy with MEK and PI3K
inhibitors may represent a promising novel therapeutic strategy
in the treatment of MPM.
The authors thank Mr. K. Kameda (Ehime University, Ehime,
Japan) for technical assistance with this study.
MIYOSHI et al: MEK AND PI3K INHIBITORS FOR MALIGNANT PLEURAL MESOTHELIOMA
1. Broaddus VC: Asbestos, the mesothelial cell and malignancy: a
matter of life or death. Am J Respir Cell Mol Biol 17: 657-659,
2. Morinaga K, Kishimoto T, Sakatani M, Akira M, Yokoyama K
and Sera Y: Asbestos-related lung cancer and mesothelioma in
Japan. Ind Health 39: 65-74, 2001.
3. Dufresne A, Bégin R, Churg A and Massé S: Mineral fiber
content of lungs in patients with mesothelioma seeking
compensation in Quebec. Am J Respir Crit Care Med 153:
4. Light RW (ed): Primary tumors of the pleura: malignant meso-
theliomas, solitary fibrous tumors, body cavity lymphoma, and
pyothorax-associated lymphoma. In: Pleural Diseases. 4th edn.
Lippincott Williams and Wilkins, Philadelphia, PA, pp135-150,
5. Britton M: The epidemiology of mesothelioma. Semin Oncol
29: 18-25, 2002.
6. Zellos L and Sugarbaker DJ: Current surgical management of
malignant pleural mesothelioma. Curr Oncol Rep 4: 354-360,
7. Vogelzang NJ, Rusthoven JJ, Symanowski J, Denham C,
Kaukel E, Ruffie P, Gatzemeier U, Boyer M, Emri S,
Manegold C, Niyikiza C and Paoletti P: Phase III study of
pemetrexed in combination with cisplatin versus cisplatin alone
in patients with malignant pleural mesothelioma. J Clin Oncol
21: 2636-2644, 2003.
8. Manning BD and Cantley LC: AKT/PKB signaling: navigating
downstream. Cell 129: 1261-1274, 2007.
9. Marte BM and Downward J: PKB/Akt: connecting phos-
phoinositide 3-kinase to cell survival and beyond. Trends
Biochem Sci 22: 355-358, 1997.
10. Roberts PJ and Der CJ: Targeting the Raf-MEK-ERK mitogen
activated protein kinase cascade for the treatment of cancer.
Oncogene 26: 3291-3310, 2007.
11. Sebolt-Leopold JS, Dudley DT, Herrera R, Van Becelaere K,
Wiland A, Gowan RC, Tecle H, Barrett SD, Bridges A,
Przybranowski S, Leopold WR and Saltiel AR: Blockade of
the MAP kinase pathway suppresses growth of colon tumors
in vivo. Nat Med 5: 810-816, 1999.
12. Hoshino R, Chatani Y, Yamori T, Tsuruo T, Oka H, Yoshida O,
Shimada Y, Ari-i S, Wada H, Fujimoto J and Kohno M:
Constitutive activation of the 41-/43-kDa mitogen-activated
protein kinase signaling pathway in human tumors. Oncogene
18: 813-822, 1999.
13. de Melo M, Gerbase MW, Curran J and Pache JC: Phosphorylated
extracellular signal-regulated kinases are significantly
increased in malignant mesothelioma. J Histochem Cytochem
54: 855-861, 2006.
14. Altomare DA, You H, Xiao GH, Ramos-Nino ME, Skele KL,
De Rienzo A, Jhanwar SC, Mossman BT, Kane AB and
Testa JR: Human and mouse mesotheliomas exhibit elevated
AKT/PKB activity, which can be targeted pharmacologically
to inhibit tumor cell growth. Oncogene 24: 6080-6089, 2005.
15. Cole GW, Alleva AM, Zuo JT, Sehgal SS, Yeow WS,
Schrump DS and Nguyen DM: Suppression of pro-metastasis
phenotypes expression in malignant pleural mesothelioma by
the PI3K inhibitor LY294002 or the MEK inhibitor U0126.
Anticancer Res 26: 809-822, 2006.
16. Yokoyama A, Kohno N, Fujino S, Hamada H, Inoue Y, Fujioka S
and Hiwada K: Origin of heterogeneity of interleukin-6 (IL-6)
levels in malignant pleural effusions. Oncol Rep 1: 507-511,
17. Nakataki E, Yano S, Matsumori Y, Goto H, Kakiuchi S,
Muguruma H, Bando Y, Uehara H, Hamada H, Kito K,
Yokoyama A and Sone S: Novel orthotopic implantation of
human malignant pleural mesothelioma (EHMES-10 cells)
highly expressing vascular endothelial growth factor and its
receptor. Cancer Sci 97: 183-191, 2006.
18. Rak-Mardyla A and Gregoraszczuk EL: ERK 1/2 and PI-3
kinase pathways as a potential mechanism of ghrelin action on
cell proliferation and apoptosis in the porcine ovarian follicular
cells. J Physiol Pharmacol 61: 451-458, 2010.
19. Ciofani G, Ricotti L, Danti S, Moscato S, Nesti C,
D'Alessandro D, Dinucci D, Chiellini F, Pietrabissa A, Petrini M
and Menciassi A: Investigation of interactions between poly-L-
lysine-coated boron nitride nanotubes and C2C12 cells: up-take,
cytocompatibility, and differentiation. Int J Nanomedicine 5:
20. Hamaguchi N, Hamada H, Miyoshi S, Irifune K, Ito R,
Miyazaki T and Higaki J: In vitro and in vivo therapeutic
efficacy of the PPAR-γ agonist troglitazone in combination
with cisplatin against malignant pleural mesothelioma cell
growth. Cancer Sci 101: 1955-1964, 2010.
21. Soga Y, Komori H, Miyazaki T, Arita N, Terada M, Kamada K,
Tanaka Y, Fujino T, Hiasa Y, Matsuura B, Onji M and Nose M:
Toll-like receptor 3 signaling induces chronic pancreatitis
through the Fas/Fas ligand-mediated cytotoxicity. Tohoku J
Exp Med 217: 175-184, 2009.
22. Wee S, Jagani Z, Xiang KX, Loo A, Dorsch M, Yao YM,
Sellers WR, Lengauer C and Stegmeier F: PI3K pathway activa-
tion mediates resistance to MEK inhibitors in KRAS mutant
cancers. Cancer Res 69: 4286-4293, 2009.
23. Bedogni B, Welford SM, Kwan AC, Ranger-Moore J, Saboda K
and Powell MB: Inhibition of phosphatidylinositol-3-kinase and
mitogen-activated protein kinase kinase 1/2 prevents melanoma
development and promotes melanoma regression in the trans-
genic TPRas mouse model. Mol Cancer Ther 5: 3071-3077,
24. Furuya F, Lu C, Willingham MC and Cheng SY: Inhibition of
phosphatidylinositol 3-kinase delays tumor progression and
blocks metastatic spread in a mouse model of thyroid cancer.
Carcinogenesis 28: 2451-2458, 2007.
25. McCubrey JA, Steelman LS, Abrams SL, Lee JT, Chang F,
Bertrand FE, Navolanic PM, Terrian DM, Franklin RA,
D'Assoro AB, Salisbury JL, Mazzarino MC, Stivala F and
Libra M: Roles of the RAF/MEK/ERK and PI3K/PTEN/AKT
pathways in malignant transformation and drug resistance. Adv
Enzyme Regul 46: 249-279, 2006.
26. Mukohara T, Civiello G, Johnson BE and Janne PA: Therapeutic
targeting of multiple signaling pathways in malignant pleural
mesothelioma. Oncology 68: 500-510, 2005.
27. Robinson BW and Lake RA: Advances in malignant mesothe-
lioma. N Engl J Med 353: 1591-603, 2005.
28. Li Q, Yano S, Ogino H, Wang W, Uehara H, Nishioka Y and
Sone S: The therapeutic efficacy of anti vascular endothelial
growth factor antibody, bevacizumab, and pemetrexed against
orthotopically implanted human pleural mesothelioma cells in
severe combined immunodeficient mice. Clin Cancer Res 13:
29. Yano S, Shinohara H, Herbst RS, Kuniyasu H, Bucana CD,
Ellis LM and Fidler IJ: Production of experimental malignant
pleural effusions is dependent on invasion of the pleura and
expression of vascular endothelial growth factor/vascular
permeability factor by human lung cancer cells. Am J Pathol
157: 1893-1903, 2000.
30. Rexer BN, Ghosh R and Arteaga CL: Inhibition of PI3K and
MEK: It is all about combinations and biomarkers. Clin Cancer
Res 15: 4518-4520, 2009.
31. Rinehart J, Adjei AA, Lorusso PM, Waterhouse D, Hecht JR,
Natale RB, Hamid O, Varterasian M, Asbury P, Kaldjian EP,
Gulyas S, Mitchell DY, Herrera R, Sebolt-Leopold JS and
Meyer MB: Multicenter phase II study of the oral MEK
inhibitor, CI-1040, in patients with advanced non-small-cell
lung, breast, colon, and pancreatic cancer. J Clin Oncol 22:
32. Hu L, Zaloudek C, Mills GB, Gray J and Jaffe RB: In vivo and
in vitro ovarian carcinoma growth inhibition by a phosphati-
dylinositol 3-kinase inhibitor (LY294002). Clin Cancer Res 6:
33. Hu L, Hofmann J, Lu Y, Mills GB and Jaffe RB: Inhibition of
phosphatidylinositol 3'-kinase increases efficacy of paclitaxel
in in vitro and in vivo ovarian cancer models. Cancer Res 62: