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ORIGINAL PAPER
Valproic Acid Induced Differentiation and Potentiated Efficacyof Taxol and Nanotaxol for Controlling Growth of HumanGlioblastoma LN18 and T98G Cells
Subhasree Roy Choudhury • Surajit Karmakar •
Naren L. Banik • Swapan K. Ray
Received: 29 April 2011 / Revised: 11 July 2011 / Accepted: 14 July 2011 / Published online: 24 July 2011
� Springer Science+Business Media, LLC 2011
Abstract Glioblastoma shows poor response to current
therapies and warrants new therapeutic strategies. We
examined the efficacy of combination of valproic acid
(VPA) and taxol (TX) or nanotaxol (NTX) in human
glioblastoma LN18 and T98G cell lines. Cell differentia-
tion was manifested in changes in morphological features
and biochemical markers. Cell growth was controlled with
down regulation of vascular endothelial growth factor
(VEGF), epidermal growth factor receptor (EGFR), nuclear
factor-kappa B (NF-jB), phospho-Akt (p-Akt), and multi-
drug resistance (MDR) marker, indicating suppression of
angiogenic, survival, and multi-drug resistance pathways.
Cell cycle analysis showed that combination therapy (VPA
and TX or NTX) increased the apoptotic sub G1 population
and apoptosis was further confirmed by Annexin V-FITC/
PI binding assay and scanning electron microscopy.
Combination therapy caused activation of caspase-8 and
cleavage of Bid to tBid and increased Bax:Bcl-2 ratio and
mitochondrial release of cytochrome c and apoptosis-
inducing factor (AIF). Upregulation of calpain and casp-
ases (caspase-9 and caspase-3) and substrate degradation
were also detected in course of apoptosis. The combination
of VPA and NTX most effectively controlled the growth of
LN18 and T98G cells. Therefore, this combination of drugs
can be used as an effective treatment for controlling growth
of human glioblastoma cells.
Keywords Apoptosis � Glioblastoma � Nanotaxol �Taxol � Valproic acid
Introduction
Glioblastoma is the most common and deadliest malignancy,
accounting for nearly 40% of primary brain tumors in adults
with a mean survival of less than 1 year following diagnosis
[1]. It is a heterogenous and highly vascularized brain tumor
characterized by poorly differentiated neoplastic astrocytes
that avoid apoptosis due to P-glycoprotein (P-gp) mediated
multi-drug resistance [2]. Development of new therapeutic
strategies that induce differentiation and apoptosis, provide
anti-angiogenic activity, and overcome multi-drug resis-
tance is urgently warranted.
Valproic acid (VPA), a histone deacetylase inhibitor,
has been shown to induce differentiation and anti-angio-
genic and anti-proliferative activities in several cancers,
including both P-gp positive and negative cell lines [3].
The multimodal efficacy of VPA with other anti-cancer
drugs could be useful for successful treatment of glio-
blastoma. While the anti-cancer efficacy of taxol (TX) is
well established in ovarian, head-and-neck, bladder, breast,
and lung cancers [4], recently TX has also been shown to
provide potent therapeutic effects in experimental brain
tumor [5, 6].
Because of its low aqueous solubility, TX is less per-
meable to the blood–brain-barrier (BBB) [7]. Besides, the
commercial formulation of 6 mg/ml (7 mM) TX in 1:1
mixture of the solvents Cremophor EL� (CrEL, poly-
oxyethylated castor oil) and ethanol has systemic toxicity,
side effects, and short time stability, and is associated with
hypersensitivity reactions [8]. TX is a substrate for the P-gp
S. Roy Choudhury � S. Karmakar � S. K. Ray (&)
Department of Pathology, Microbiology, and Immunology,
University of South Carolina School of Medicine, Columbia,
SC 29209, USA
e-mail: [email protected]
N. L. Banik
Department of Neurosciences, Medical University
of South Carolina, Charleston, SC 29425, USA
123
Neurochem Res (2011) 36:2292–2305
DOI 10.1007/s11064-011-0554-7
[9] and co-administration of the P-gp inhibitor PSC833
[10] or HM30181A [11] with TX has shown significant
therapeutic efficacy in brain tumor.
Many attempts have been made to reformulate TX-
bound nanoparticles, which are natural or synthetic poly-
mers of lipids or metal containing vehicles of dimension
\100 nm and hold promise as the effective drug delivery
carriers across the BBB [12]. ABI-007, also known as
‘Abraxane’, is a Cremophor-free, albumin-bound 130-nm
nanoparticle form of TX that has shown anti-cancer
activity in human lung (H522), breast (MX-1), ovarian
(SK-OV-3), prostate (PC-3), and colon (HT29) tumor xe-
nografts [13]. The delivery of TX as Abraxane has been
found to overcome insoluble nature and solvent related
toxicities of TX [14]. The clinical potency of Abraxane in
metastatic breast cancer [15] and advanced non-small cell
lung cancer patients is well established [14], but the ther-
apeutic potency of Abraxane or nanotaxol (NTX) in glio-
blastoma has not yet been explored.
In this study, we investigated the anti-tumor efficacy of
VPA, TX, or NTX alone and in combination in multi-drug
resistant glioblastoma LN18 (with overexpression of P-gp)
and T98G (with less expression of P-gp) cell lines [2, 16–
19]. The combination of VPA and TX or NTX was found
to markedly down regulate several survival signals and
angiogenesis markers, induce differentiation, and activate
mitochondria-dependent pathways for apoptosis in both
LN18 and T98G cell lines.
Materials and Methods
Reagents
VPA and primary b-actin IgG antibody (monoclonal clone
AC-15) were obtained from Sigma Chemical (St. Louis,
MO). TX (paclitaxel) was procured from Bristol-Myers
Squibb (Princeton, NJ). NTX (Abraxane) was obtained
from Abraxis Oncology (Schaumburg, IL). The colori-
metric MTT assay kit was procured from Millipore
Chemicon (Temecula, CA). AnnexinV-fluorescein isothi-
ocyanate (FITC)/propidium iodide (PI) kit for detection of
apoptosis was purchased from BD Biosciences (San Jose,
CA). Wright staining kit was bought from Fisher Scientific
(Middletown, VA). Other primary IgG antibodies were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Depending on the primary antibody, the secondary anti-
body used was either horseradish peroxidase (HRP)-con-
jugated goat anti-mouse IgG or HRP-conjugated goat anti-
rabbit IgG (ICN Biomedicals, Aurora, OH). Enhanced
Chemiluminescence (ECL) Plus reagent was purchased
from GE Healthcare Bio-Sciences (Piscataway, NJ).
Cell Culture and Treatments
Human glioblastoma LN18 and T98G cell lines were
purchased from the American Type Culture Collection
(ATCC, Manassas, VA). Both LN18 and T98G cells
were separately grown in monolayer to sub-confluency in
75-cm2 flasks containing 10 ml of DMEM and RPMI
1640 (Mediatech, Herndon, VA), respectively, and sup-
plemented with 10% fetal bovine serum (FBS) (Invitro-
gen, Carlsbad, CA), 1% penicillin, and 1% streptomycin
(GIBCO, Grand Island, NY) in a fully-humidified incu-
bator containing 5% CO2 and 95% air at 37�C. Cells
were allowed to starve for 24 h in RPMI 1640 or
DMEM with 2% FBS, 1% penicillin, and 1% strepto-
mycin and thereafter maintained in this low-serum con-
dition during the treatments. Freshly prepared stock
solutions of VPA, TX, and NTX were made in serum-
free medium just prior to treatment. Dose–response
studies were carried out to determine the suitable doses
of the drugs for the inhibition of cell growth, induction
of differentiation, and cell death. Cells were treated with
0.5, 1, 2, or 4 mM of VPA for 72 h followed by treat-
ment with either 50 nM TX or 50 nM NTX for 24 h
alone and in combination.
MTT Assay
The differential sensitivities of LN18 and T98G cells to
VPA, TX, or NTX either alone or in combination were
determined using the 3-[4,5-dimethylthiazole-2-yl]-2,5-
diphenyltetrazolium bromide (MTT)-based colorimetric
assay. We used different doses of VPA to induce differ-
entiation whereas a fix dose of either TX or NTX to induce
apoptosis. The rationale was to reduce cell proliferation
with induction of differentiation and increase apoptosis
using combination of drugs in glioblastoma cells. For MTT
assay, both cell lines were first exposed to 0.5, 1, 2, and
4 mM VPA for 72 h. At 48 h post-exposure of VPA, cells
were treated with 50 nM TX or NTX for another 24 h
alone and in combination with VPA (total treatment for
72 h). Cells were then exposed to MTT reagent for 4 h at
37�C after which isopropanol was used to dissolve the
formazan crystals. The optical density (OD) was recorded
at 570 nm in a microplate reader. Percent growth inhibition
was determined as [1-(OD of treated cells/OD of control
cells)]9 100. All experiments were performed in tripli-
cates. Cell viability data were analyzed using Compusyn
software (ComboSyn, Paramus, NJ) to generate a combi-
nation index (CI). Conventionally, CI [ 1 indicates
antagonism, CI = 1 indicates additive effect, and CI \ 1
indicates synergism [20].
Neurochem Res (2011) 36:2292–2305 2293
123
Wright Staining for Examination of Morphological
Features of Apoptosis
Both LN18 and T98G cells were treated with 2 mM VPA
for 72 h as monotherapy and at 48 h post-exposure of
VPA, 50 nM TX or NTX was added alone separately or in
combination with VPA for another 24 h (total treatment for
72 h). The cells (adherent and non-adherent) from each
treatment were centrifuged and sedimented on the culture
dish and washed twice with phosphate-buffered saline
(PBS, pH 7.4). The cells were fixed with 95% (v/v) ethanol
and dried before Wright staining. The morphology of
apoptotic cells were detected under the light microscope.
Cells were considered apoptotic with characteristic features
such as chromatin condensation, cell-volume shrinkage,
and membrane-bound apoptotic bodies [18].
Cell Cycle Analysis
LN18 and T98G cells were seeded in a 6-well plate at a
density of 1 9 105 cells per well and incubated for 24 h in
low-serum medium prior to addition of drugs. Cells were
untreated (control, CTL) and treated with 2 mM VPA for
72 h as monotherapy and at 48 h post-exposure of VPA,
50 nM TX or NTX was added alone separately or in
combination with VPA for another 24 h (total treatment for
72 h). Cells were trypsinized and harvested by centrifu-
gation. Flow cytometric analysis of cell cycle was per-
formed by staining of permeabilized cells with PI for DNA
content. Cells were re-suspended in PBS, fixed with 70%
ethanol, labeled with staining solution (0.05 mg/ml PI,
2 mg/ml RNase A, 0.1% TritonX-100 in PBS), and incu-
bated for 30 min at room temperature (RT) in darkness.
DNA content was then analyzed using an Epics XL-MCL
Flow Cytometer (Beckman Coulter, Fullerton, CA), as we
reported recently [21].
Flow Cytometric Analysis of Apoptosis
The percentage of cells undergoing apoptosis was quanta-
tively determined by Annexin V-FITC/PI binding assay.
LN18 and T98G cells were seeded in 6-well plate at a
density of 1 9 105 cells per well and incubated for 24 h in
low-serum medium prior to addition of drugs. Cells were
untreated (CTL) and treated with 2 mM VPA for 72 h as
monotherapy and at 48 h post-exposure of VPA, 50 nM
TX or NTX was added alone separately or in combination
with VPA for another 24 h (total treatment for 72 h). After
treatments, both adherent and non-adherent cells were
harvested and double-stained using the Annexin V-FITC/PI
staining kit according to the manufacturer’s protocol. Cells
were washed with cold PBS, resuspended in 19 binding
buffer (0.1 M HEPES/NaOH, pH 7.4, 1.4 M NaCl, 25 mM
CaCl2), stained with Annexin V-FITC/PI, and incubated
for 15 min at RT in the darkness. Cells were analyzed for
Annexin V-stained apoptotic population using an Epics
XL-MCL Flow Cytometer (Beckman Coulter, Fullerton,
CA), as per our previous reports [20, 21].
Morphological Analysis by Scanning Electron
Microscopy
Both LN18 and T98G cells were grown to sub-confluency
on 13-mm round cover slip and incubated in low-serum
medium for 24 h before addition of drugs. Cells were either
untreated (CTL) and treated with 2 mM VPA for 72 h as
monotherapy and at 48 h post-exposure of VPA, 50 nM
TX or NTX was added alone separately or in combination
with VPA for another 24 h (total treatment for 72 h). After
the treatments, cells were fixed with 2.5% glutaraldehyde
in 4% paraformaldehyde in phosphate buffer (pH 7.4) for
1 h at RT. Cells were post-fixed in 1.0% osmium tetroxide
(OsO4) for 10 min and washed in 0.1 M phosphate buffer
(pH 7.2). Dehydration was done in ascending concentration
of ethanol. Cover slips were dried with the critical point
apparatus using liquid CO2 as transition fluid. The speci-
mens were cold sputter coated with gold and observed in a
JEOL 6,300 V Scanning Electron Microscope (SEM) at an
accelerating voltage of 15 kV, as we reported earlier [21].
Protein Extraction and Western Blotting
For Western blotting, total proteins were extracted fol-
lowing homogenization of cells, quantitated spectrophoto-
metrically, denatured in boiling water for 5 min, and
resolved by SDS-polyacryl amide gel electrophoresis and
then electroblotted to the polyvinylidene (PVDF) mem-
branes. The blots were incubated with a primary IgG
antibody followed by incubation with an alkaline HRP-
conjuaged secondary IgG antibody. Subsequently, specific
protein bands were detected by HRP/H2O2-catalyzed oxi-
dation of luminol in alkaline conditions using the ECL
system and autoradiography [18, 22].
Statistical Analysis
Results were analyzed using Minitab� 15 Statistical Soft-
ware (Minitab, State College, PA). Data were expressed as
mean ± standard error of mean (SEM) of separate exper-
iments (n C 3) and compared by one-way analysis of
variance (ANOVA) followed by Fisher’s post hoc test.
Significant difference between CTL and a treatment was
indicated by *P \ 0.05, or **P \ 0.001; and between TX
and VPA ? TX, or NTX and VPA ? NTX was indicated
by #P \ 0.05.
2294 Neurochem Res (2011) 36:2292–2305
123
Results
Treatments Decreased Cell Viability
The growth inhibitory effect of VPA, TX, and NTX either
alone or in combination on LN18 and T98G cells were
determined (Fig. 1). VPA monotherapy was used at dif-
ferent concentrations such as 0.5, 1, 2, and 4 mM for 72 h
while 50 nM TX or 50 nM NTX was used alone for 24 h.
For combination studies, the anti-proliferative effect was
determined by treating both cell lines with VPA (0.5, 1, 2,
and 4 mM) for 48 h and then either 50 nM TX or 50 nM
NTX was added in presence of VPA for another 24 h (total
treatment for 72 h). VPA inhibited cell growth in both cell
lines (Fig. 1a). Treatment with VPA dose-dependently
decreased the cell viability in LN18 and T98G cells. The
percentages of cell viability in LN18 and T98G cells after
treatment with 50 nM TX or NTX for 24 h were also
decreased (Fig. 1a). Most importantly, treatment with
VPA ? TX, or VPA ? NTX showed significant reduction
in cell viability when compared with TX or NTX mono-
therapy (Fig. 1a).
We used a variety of doses of VPA but a fixed dose of
either TX or NTX and found that higher doses of combi-
nation of drugs showed more cell death than lower doses.
But our aim was to use particular doses of the drugs, which
would induce cellular differentiation, inhibit cell prolifer-
ation, and arrest cell cycle to trigger apoptosis. VPA
(2 mM) and TX or NTX (50 nM) alone showed the best
efficacy, VPA (2 mM) ? TX (50 nM) produced the best
synergistic efficacy (CI = 0.791 in LN18 cells and
CI = 0.98 in T98G cells), and VPA (2 mM) ? NTX (50
nM) also showed the best synergistic efficacy (CI = 0.45
in LN18 cells and CI = 0.49 in T98G cells) (Fig. 1a) and
therefore, these treatments were finally selected for further
studies for inducing differentiation and apoptosis.
Fig. 1 VPA, TX, and NTX alone and combination therapy decreased
cell viability and increased apoptotic features in LN18 and T98G
cells. Treatments: 0 control (CTL), 1 CTL (untreated) or monotherapy
(0.5 mM VPA, and 50 nM TX or NTX) or combination therapy
(0.5 mM VPA followed by either 50 nM TX or 50 nM NTX), 2 CTL
(untreated) or monotherapy (1 mM VPA, and either 50 nM TX or
50 nM NTX) or combination therapy (1 mM VPA followed by either
50 nM TX or 50 nM NTX), 3 CTL (untreated) or monotherapy
(2 mM VPA, and either 50 nM TX or 50 nM NTX) or combination
therapy (2 mM VPA followed by either 50 nM TX or 50 nM NTX),
and 4 CTL (untreated) or monotherapy (4 mM VPA, and either
50 nM TX or 50 nM NTX) or combination therapy (4 mM VPA
followed by either 50 nM TX or 50 nM NTX). Treatment schedule
with monotherapy: VPA for 72 h, TX or NTX for 24 h; and with
combination therapy: VPA for 48 h and then TX or NTX in presence
of VPA for 24 h (total treatment for 72 h). a MTT assay was used to
assess cell viability following monotherapy and combination therapy.
b Wright staining to evaluate the morphological features of differ-
entiation and apoptosis
Neurochem Res (2011) 36:2292–2305 2295
123
Induction of Morphological and Biochemical Features
of Differentiation and Apoptosis
Wright staining revealed that exposure to VPA inhibited cell
proliferation and induced morphological features of differ-
entiation such as long and thin projections of the cells
(Fig. 1b). Western blotting showed the biochemical features
of astrocytic differentiation with upregulation of the glial
fibrillary acidic protein (GFAP) and down regulation of the
inhibitor of differentiation 2 (ID2) in both LN18 and T98G
cells (Fig. 2a, c, d). Uniform b-actin expression served as an
internal control. Increase in GFAP, an astrocyte-specific
intermediate filament protein, is a biochemical marker of
differentiation in glioblastomas. The treatment with VPA ?
TX caused upregulation of GFAP expression in T98G cells
only whereas VPA ? NTX significantly increased GFAP
expression in both LN18 and T98G cells, when compared
with control or monotherapy (Fig. 2a, c, d).
Inhibition of Angiogenic, Survival, and Multi-Drug
Resistance Markers
Vascular endothelial growth factor (VEGF) is a major
regulatory factor in tumor angiogenesis and predominantly
Fig. 2 Combination therapy
(either VPA ? TX or
VPA ? NTX) induced
differentiation and inhibited
angiogenic, survival, and multi-
drug resistance factors.
Treatments: control (CTL);
2 mM of VPA, 50 nM TX,
50 nM NTX, VPA
(2 mM) ? TX (50 nM), VPA
(2 mM) ? NTX (50 nM).
Treatment schedule with
monotherapy: VPA for 72 h,
TX or NTX for 24 h; and with
combination therapy: VPA for
48 h and then TX or NTX in
presence of VPA for 24 h (total
treatment for 72 h). a Western
blotting showed uniform level
of 42 kD b-actin, upregulation
of glial fibrillary acidic protein
(GFAP), and down regulation of
inhibitor of differentiation
factor 2 (ID2) following
combination therapy.
b Representative Western blots
showed levels of VEGF, EGFR,
NF-jB, total Akt, p-Akt, and
MDR (P-gp). c Densitometric
analysis of percent change in
protein levels in LN18 cells.
d Densitometric analysis of
percent change in protein levels
in T98G cells. Combination
therapy inhibited angiogenic,
survival, and multi-drug
resistance markers
2296 Neurochem Res (2011) 36:2292–2305
123
overexpressed in glioblastoma [23]. Western blotting was
used to examine any effect of the single or combinatorial
treatment on VEGF expression. Both single and combina-
tion therapies were found to potently inhibit VEGF
expression in both LN18 and T98G cells (Fig. 2b–d).
Another genetic alteration associated with glioblastoma
is amplification of epidermal growth factor receptor
(EGFR) gene, resulting in overexpression of EGFR protein.
EGFR amplification has been shown to increase glioblas-
toma proliferation and invasion [24]. We assessed expres-
sion of EGFR by Western blotting. Use of VPA ? TX was
found to significantly reduce EGFR expression whereas
VPA ? NTX totally abolished EGFR expression in both
LN18 and T98G cell lines (Fig. 2b), compared with all
other treatments (Fig. 2c, d).
The levels of several anti-apoptotic proteins were
determined by Western blotting (Fig. 2b). Treatment with
the combination of drugs (either VPA ? TX or VPA ?
NTX) showed dramatic down regulation of the survival
factors such as NF-jB, total-Akt, and p-Akt in both LN18
and T98G cell lines (Fig. 2b). Treatment with VPA ?
NTX most effectively inhibited expression of the survival
factors (e.g., NF-jB, total-Akt, and p-Akt) in both cell lines
(Fig. 2c, d).
Untreated LN18 cells (harboring overexpression of
P-gp) showed high level of P-gp expression. Treatment
with 2 mM VPA alone and especially combination of drugs
(either VPA ? TX or VPA ? NTX) remarkably down
regulated the P-gp expression in LN18 cell line (Fig. 2b, c).
No detectable level of P-gp was observed in untreated
T98G cells (harboring very little expression of P-gp). Use
of VPA ? NTX was more effective than that of
VPA ? TX in down regulating MDR expression.
Flow Cytometric Analysis of Cell Cycle
Flow cytometric analysis was performed to examine the
cell cycle distribution patterns in LN18 and T98G cells
following treatments (Fig. 3). Flow cytometric data
revealed that treatment with VPA caused a small increase
in sub G1 phase, a decrease in G0/G1 phase, and an
increase in G2/M population in both cell lines. Treatment
with TX showed a significant increase in sub G1 phase and
down regulated G0/G1 population with a significant
increase in G2/M phase in both LN18 and T98G cell lines.
A significant increase in sub G1 phase and a decrease in
both G0/G1 and G2/M populations in both cell lines
(Fig. 3a) were seen due to treatment with NTX. Combi-
nation therapy (either VPA ? TX or VPA ? NTX) most
effectively increased the sub G1 apoptotic phase. Notably,
the use of VPA ? NTX caused more apoptosis than the use
of VPA ? TX in both cell lines (Fig. 3b).
Fig. 3 Cell cycle analysis by
flow cytometry. Histograms
display DNA content (x axis, PI
fluorescence) versus counts
(y axis). Treatments: control
(CTL); 2 mM VPA, 50 nM TX,
50 nM NTX, VPA
(2 mM) ? TX (50 nM), VPA
(2 mM) ? NTX (50 nM).
Treatment schedule with
monotherapy: VPA for 72 h,
TX or NTX for 24 h; and with
combination therapy: VPA for
48 h and then TX or NTX in
presence of VPA for 24 h (total
treatment for 72 h). a Cell cycle
phase distribution was shown by
histograms. b Bar diagram
representation of cells in sub G1
phase
Neurochem Res (2011) 36:2292–2305 2297
123
Flow Cytometric Detection of Apoptosis by Annexin
V-FITC/PI Binding Assay
Annexin V-FITC/PI double-labeling technique was used to
understand the nature of cell death (apoptotic or necrotic)
after the treatments (Fig. 4). Monotherapy with VPA, TX,
and NTX induced apoptosis in LN18 and T98G cells
(Fig. 4a). But the combination therapy showed a significant
increase in apoptosis, when compared with either treatment
alone. Flow cytometric data revealed that combination
therapy (either VPA ? TX or VPA ? NTX) most signifi-
cantly increased apoptosis in both glioblastoma cell lines
(Fig. 4b). However, use of VPA ? NTX showed greater
induction of apoptosis than the use of VPA ? TX in both
cell lines. Annexin V-FITC/PI staining confirmed the
induction of cell death through phosphatidylserine exter-
nalization and indicated the mode of cell death was
apoptosis and not necrosis.
Scanning Electron Microscopy to Detect Fine
Morphological Changes
Scanning electron microscopy of both LN18 and T98G
cells after treatment with VPA showed heterogeneity of
cytoplasmic prolongations and microextensions, including
blebs and filopodia formation (Fig. 5). On the other hand,
combination therapy (either VPA ? TX or VPA ? NTX)
most effectively induced certain features of apoptosis
Fig. 4 Flow cytometric detection of apoptosis. Treatments: control
(CTL); 2 mM VPA, 50 nM TX, 50 nM NTX, VPA (2 mM) ? TX
(50 nM), VPA (2 mM) ? NTX (50 nM). Treatment schedule with
monotherapy: VPA for 72 h, TX or NTX for 24 h; and with
combination therapy: VPA for 48 h and then TX or NTX in presence
of VPA for 24 h (total treatment for 72 h). a Double parameter dot-
plots show FITC-fluorescence (x axis) versus PI-fluorescence (y axis).
Quadrants: lower left, normal live cells (PI negative and Annexin V
negative); lower right, early apoptotic cell (PI negative and Annexin
V positve); upper right, late apoptotic or necrotic cells (PI positive
and Annexin V positive); and upper left, mechanically injured cells
(PI positive and Annexin V negative). b Bar diagram representation
of apoptosis. Combination therapy significantly induced apoptosis in
both LN18 and T98G cells
2298 Neurochem Res (2011) 36:2292–2305
123
including membrane blebbing, perforations, loss of mem-
brane integrity, nuclear fragmentations, cell shrinkage, and
formation of apoptotic bodies (Fig. 5).
Activation of Caspase-8 and Proteolytic Cleavage
of Bid to tBid
In order to determine the changes in apoptosis related
proteins in both LN18 and T98G cells following treat-
ments, we performed Western blotting (Fig. 6). Uniform
b-actin expression served as a loading control. Compared
with control and monotherapy, treatment with VPA ?
NTX caused substantial increase in the 20 kD active cas-
pase-8 fragment in both cell lines. Active caspase-8
cleaved Bid as a substrate for generation of tBid (Fig. 6a)
and its translocation to mitochondria for induction of cell
death. Thus, this combination therapy was capable of
inducing apoptosis partly via extrinsic pathway in both
LN18 and T98G cell lines.
Induction of Apoptosis with an Increase in Bax:Bcl-2
Ratio
The Bcl-2 family of proteins plays a key role in regulation
of mitochondrial permeability during apoptosis via intrinsic
pathway. The levels of expression of pro-apoptotic protein
Bax and anti-apoptotic protein Bcl-2 were examined by
Western blotting to determine the changes in Bax:Bcl-2
ratio (Fig. 6b). Expression of b-actin, considered as a
loading control, was uniform in all treatment groups. Cells
treated with TX, NTX, and VPA ? TX showed an increase
in Bax expression and decrease in Bcl-2 expression in both
cell lines (Fig. 6c). Use of VPA ? NTX most dramatically
increased Bax expression and decreased Bcl-2 expression,
when compared with control or single treatments. Combi-
nation therapy significantly increased the Bax:Bcl-2 ratio in
both cell lines. An increase in the Bax:Bcl-2 ratio can
trigger release of several pro-apoptotic molecules from
mitochondria for activation of intrinsic caspase cascade for
apoptosis.
Activation of Mitochondrial Pathway of Apoptosis
An alteration in the permeability of the outer mitochondrial
membrane causes the release of various pro-apoptotic
molecules, including cytochrome c and AIF, which pro-
mote the mitochondrial pathway of apoptosis (Fig. 7). We
used b-actin as cytosolic loading control and expression
was almost uniform in all treatment groups. Cells treated
with TX, NTX, VPA ? TX, or VPA ? NTX showed an
increase in cytosolic levels of cytochrome c in both LN18
and T98G cell lines (Fig. 7a). Single treatment with VPA
showed upregulation of cytosolic cytochrome c in T98G
cells but not in LN18 cells (Fig. 7a). Mitochondrial release
of cytochrome c is a well known pre-requisite for forma-
tion of apoptosome and activation of caspases for apopto-
sis. Treatment with NTX alone or VPA ? NTX generated
the cytosolic cleaved AIF fragments in both LN18 and
T98G cells (Fig. 7a) and thereby facilitated apoptosis.
Upregulation of Cysteine Proteases
Western blotting was used to determine expression of
proteases such as calpain and caspase-9 in both LN18 and
T98G cells following single (VPA, TX, and NTX) and
combination (either VPA ? TX or VPA ? NTX) treat-
ments (Fig. 7b). Level of b-actin expression remained
uniform in all treatments and was used as an internal
control. Single or combination therapy increased expres-
sion of calpain, a Ca2?-dependent cysteine protease, and
also expression and activation of caspase-9 in both glio-
blastoma cell lines (Fig. 7b). Further, activation of caspase-
Fig. 5 Scanning electron photomicrographs of LN18 and T98G cells.
Treatments: control (CTL); 2 mM VPA, 50 nM TX, 50 nM NTX,
VPA (2 mM) ? TX (50 nM), VPA (2 mM) ? NTX (50 nM). Treat-
ment schedule with monotherapy: VPA for 72 h, TX or NTX for
24 h; and with combination therapy: VPA for 48 h and then TX or
NTX in presence of VPA for 24 h (total treatment for 72 h). Without
any treatment (CTL) both cell lines showed normal morphology; VPA
treatment induced differentiation with filopodia and extensive cyto-
plasmic prolongation; TX, VPA ? TX, NTX, and VPA ? NTX
treatments induced apoptotic features such as membrane blebbing,
perforation, and disruption of membrane integrity (magnification
93000)
Neurochem Res (2011) 36:2292–2305 2299
123
3 was detected following combination therapy in both cell
lines (Fig. 7b). Caspase-3, the major executioner caspase
[25], causes cleavage of cellular substrates to promote
morphological and biochemical features of apoptosis.
Increase in Proteolysis
Calpain and caspase-3 can be simultaneously activated
for apoptosis in glioblastoma cells [18]. We found the
increases in calpain and caspase-3 activities in the forma-
tion of calpain-specific 145 kD spectrin breakdown prod-
ucts (SBDP) and caspase-3 specific 120 kD SBDP,
respectively, in both LN18 and T98G cells following
combination therapy (Fig. 7c).
The increase in caspase-3 activity was further examined
in the cleavage of inhibitor of caspase-3 activated DNase
(ICAD) (Fig. 7c) that would then release CAD from the
CAD/ICAD complex followed by CAD translocation to the
nucleus for DNA fragmentation. Our results showed that
single therapy (VPA, TX, and NTX) or combination ther-
apy (either VPA ? TX or VPA ? NTX) markedly caused
cleavage of ICAD in LN18 and T98G cells (Fig. 7c). Thus,
degradation of ICAD further confirmed an increase in
caspase-3 activity in apoptosis.
Taken together, the results of our investigation showed
induction of differentiation, suppression of angiogenesis,
survival, and multi-drug resistance markers, activation of
proteolytic pathways, and cleavage of specific substrates
for apoptosis in both LN18 and T98G cells following
combination therapy (either VPA ? TX or VPA ? NTX).
Based on our experimental results, we provided a sche-
matic model to show the sequence of molecular events in
these pathways after the treatments of human glioblastoma
cells (Fig. 8).
Fig. 6 Western blotting to
examine activation of extrinsic
pathway and Bax:Bcl-2 ratio in
LN18 and T98G cells.
Treatments: control (CTL);
2 mM VPA, 50 nM TX, 50 nM
NTX, VPA (2 mM) ? TX
(50 nM), VPA (2 mM) ? NTX
(50 nM). Treatment schedule
with monotherapy: VPA for
72 h, TX or NTX for 24 h; and
with combination therapy: VPA
for 48 h and then TX or NTX in
presence of VPA for 24 h (total
treatment for 72 h).
a Representative Western blots
showed expression of 42 kD
b-actin, activation of caspase-8
in the generation of 20 kD
active fragment, and 22 kD Bid
cleavage to 15 kD tBid.
Densitometric analysis of
percent changes in protein
levels in LN18 and T98G cells.
b Representative Western blots
showed expression of 21 kD
Bax, and 26 kD Bcl-2.
c Densitometric analysis
showed the Bax:Bcl-2 ratio
2300 Neurochem Res (2011) 36:2292–2305
123
Discussion
In this investigation, we explored the efficacy of the
combination therapy (either VPA ? TX or VPA ? NTX)
in two multi-drug resistant glioblastoma cell lines LN18
(increased P-gp expression) [2] and T98G (reduced P-gp
expression) [16, 17]. This is the first report on a novel
combination therapy (either VPA ? TX or VPA ? NTX)
for human glioblastoma LN18 and T98G cells. Earlier
findings reported that VPA reduced cell proliferation,
induced cytotoxicity in human glioblastoma cells, and
increased GFAP expression for astrocytic differentiation
[26]. To observe the therapeutic effects, most of the pre-
clinical cell culture studies used a high dose of VPA that
could develop new derivatives of VPA having more potent
activity in lower dose than the parent VPA. The current
study for the first time demonstrated that either VPA ? TX
or VPA ? NTX down regulated ID2 expression in both
cell lines. The ID family of helix-loop-helix proteins is
involved in cellular proliferation and angiogenesis and
Fig. 7 Western blotting for
determination of mitochondrial
release of pro-apoptotic factors,
activation of proteases, and
substrate cleavage in LN18 and
T98G cells. Treatments: control
(CTL); 2 mM VPA, 50 nM TX,
50 nM NTX, VPA
(2 mM) ? TX (50 nM), VPA
(2 mM) ? NTX (50 nM).
Treatment schedule with
monotherapy: VPA for 72 h,
TX or NTX for 24 h; and with
combination therapy: VPA for
48 h and then TX or NTX in
presence of VPA for 24 h (total
treatment for 72 h).
a Representative Western blots
showed levels of 42 kD b-actin,
15 kD cytochrome c, and 67 kD
AIF. Densitometric analysis of
percent changes in protein
levels in LN18 and T98G cells.
b Representative Western blots
showed levels of 80 kD calpain,
35 kD caspase-9 active
fragment, and 20 kD caspase-3
active fragment. Densitometric
analysis of percent changes in
protein levels in LN18 and
T98G cells. c Representative
Western blots showed levels of
145 SBDP, 120 kD SBDP, and
40 kD ICAD fragment.
Combination therapy (either
VPA ? TX or VPA ? NTX)
activated calpain and caspase
mediated proteolysis leading to
apoptosis in LN18 and T98G
cells
Neurochem Res (2011) 36:2292–2305 2301
123
inhibition of ID protein indicates anti-proliferative and
anti-angiogenic activities [27].
The VEGF family and its receptors are known to act as the
central signaling pathway in glioblastoma angiogenesis [23].
Amplification of EGFR gene often occurs in glioblastoma
resulting in overexpression of EGFR, a transmembrane
tyrosine kinase receptor responsible for glioblastoma pro-
liferation and invasion [24]. Our combination therapy using
VPA ? TX or VPA ? NTX showed potent anti-angiogenic
activity by down regulating expression of VEGF and EGFR.
VPA is a potent inhibitor of tumor angiogenesis and caused
significant decrease in VEGF expression in colon adeno-
carcinoma cell line [28]. TX down regulates VEGF expres-
sion in LN18 cells [29] and other glioblastoma U138MG and
U251MG cells [30]. Our combination therapy showed the
anti-angiogenic potential by down regulating expression of
VEGF and EGFR in glioblastoma cells. A previous study
reported synergistic efficacy of combination of pegylated-
interferon-alpha (PEG-IFN-a) and TX in glioblastoma
U87MG cells due to inhibition of proliferation and angio-
genesis [31]. Similarly, a Phase II clinical trial of TX and
topotecan with filgrastim (also known as G-CSF) showed
therapeutic effects in glioblastoma patients [32], indicating
importance of TX in the combination therapy to control
growth of glioblastoma.
Aberrant or overexpressed Akt signaling is a major
event in glioblastoma. Phosphorylation of Akt (p-Akt)
results in activation of Akt kinase activity, which has high
potential to deregulate cell cycle, induce cell proliferation,
avoid apoptosis, and stimulate cell survival through
upregulation of NF-jB [33]. So, NF-jB down regulation
leads to apoptosis [34]. Combination therapy using
VPA ? TX and especially VPA ? NTX showed down
regulation of survival signals and anti-apoptotic factors due
to suppressed expression of total-Akt, p-Akt, and NF-jB in
both LN18 and T98G cells. TX in combination with all-
trans retinoic acid decreased expression of p-Akt and NF-
jB to promote apoptosis in U87MG xenografts [6],
implicating the importance of combination therapy for
suppression of survival pathways in glioblastoma.
Down regulation of P-gp expression was observed in both
LN18 and T98G cells following combination therapy, indi-
cating VPA could work as an inhibitor of P-gp to facilitate
TX and NTX induced cytotoxicity by overcoming multi-
drug resistance. Our study is in agreement with the previous
reports that demonstrate Elacridar [35] and Valspodar [10],
the strong inhibitors of P-gp, are highly effective in
increasing the delivery of TX across the BBB and signifi-
cantly reducing tumor volume in glioblastoma xenografts.
Recently, an oral combination therapy using the P-gp
inhibitor HM30181A and TX significantly regressed tumor
volume by inhibiting TX efflux in animal model of glio-
blastoma [11], thereby lending support to the possibility of
either VPA ? TX or VPA ? NTX as the potential combi-
nation therapy for treatment of glioblastoma.
Flow cytometric analysis of the cell cycle revealed that
monotherapy with VPA or TX caused accumulation of
cells in sub G1 and G2/M phases in both LN18 and T98G
cells. These findings are in line with earlier studies showing
that VPA in LN18 and U251MG cell lines [26] and TX in
T98G and LN229 cell lines caused G2/M arrest [36]. Our
combination therapy using VPA ? TX and especially
VPA ? NTX showed an immense increase in sub G1
phase and distinctly induction of apoptosis. The maximum
AnnexinV-FITC binding without PI staining in both cell
lines following combination therapy suggested that the
mode of cell death was apoptosis and not necrosis. No
report on effects of NTX in glioblastoma is yet available,
but a recent report on the combination of TX and ceramide
in nanoemulsion formulation showed significant apoptosis
in glioblastoma U118MG cell line [37]. Our scanning
electron microscopy identified ultrastuctural features of
differentiation with extensive cytoplasmic prolongation
and filopodia formation in both LN18 and T98G cells due
Fig. 8 Schematic presentation of molecular components for apopto-
sis in human glioblastoma cells. Extrinsic death receptor and intrinsic
mitochondrial (both caspase-dependent and caspase-independent)
pathways were involved in apoptosis in human glioblastoma LN18
and T98G cells. Combination therapy with VPA ? TX and most
remarkably with VPA ? NTX induced differentiation, inhibited
angiogenic and survival factors, down regulated multi-drug resis-
tance, and causes activation of calpain and caspase-3 for induction of
apoptosis
2302 Neurochem Res (2011) 36:2292–2305
123
to VPA treatment, supporting a previous finding where
VPA treatment induced stellate and flattened morphologi-
cal appearances in glioblastoma cells [26]. The combina-
tion therapy (either VPA ? TX or VPA ? NTX) showed
characteristic features of apoptosis including membrane
asymmetry and blebbing, cell shrinkage, and apoptotic
body formation in both LN18 and T98G cells.
Treatment with either VPA ? TX or VPA ? NTX was
found to activate caspase-8 for Bid cleavage, indicating
activation of extrinsic or death receptor pathway for
apoptosis. Earlier studies reported that VPA alone [38] and
in combination with IFN-c [39] induced apoptosis through
activation of caspase-8, Bid cleavage, and alterations in
expression of Bax and Bcl-2. TX alone [6, 30, 40] or in
combination with all-trans retinoic acid [41] caused cas-
pase-8 activation for Bid cleavage to generate tBid and
trigger apoptosis. Caspase-8 mediated cleavage of Bid
provides a link between death receptor and mitochondrial
pathways of apoptosis. An increase in the Bax:Bcl-2 ratio
is a key determinant step for inducing the release of several
pro-apoptotic molecules from the mitochondria. In our
current study, VPA ? TX and more importantly VPA ?
NTX caused significant upregulation of Bax with con-
comitant down regulation of Bcl-2 in both LN18 and T98G
cells to cause an increase in the Bax:Bcl-2 ratio, which
appeared in line with previous studies where TX induced
apoptosis by phosphorylation and inactivation of Bcl-2 [42]
and caused upregulation of Bax and down regulation of
Bcl-2 in LN18 cells [29] and also in U138MG and
U251MG cells [30].
Translocation of tBid and upregulation of Bax are
associated with mitochondrial release of cytochrome c in
the cytosol [18], which is a prerequisite for formation of
apoptosome leading to activation of caspase-9 and caspase-
3 for apoptosis [43]. Previously, TX alone or in combina-
tion with all-trans retinoic acid was reported to dramati-
cally increase the level of cytosolic cytochrome c in LN18
cells as well as in T98G and U87MG xenografts [1, 6, 29].
Mitochondria can also induce caspase-independent apop-
tosis through release of AIF into the cytosol [44]. In this
study, we showed that combination therapy with
VPA ? TX and more importantly VPA ? NTX increased
the expression of active fragment of AIF in the cytosol.
Both tBid and Bax translocation to mitochondria can
change mitochondrial outer membrane permeabilization,
resulting in the release of cytochrome c in the cytosol for
activation of caspases. AIF is a mitochondrial membrane
integral protein that can be cleaved by active calpain for
formation of a soluble apoptogenic form to exert caspase-
independent apoptosis [45].
In our study, VPA ? NTX was more effective than
VPA ? TX to increase the expression of caspase-9, cal-
pain, and caspase-3. Calpain and caspase-3 activities
(shown in the formation of 145 kD SBDP and 120 kD
SBDP, respectively) and also caspase-3 mediated ICAD
cleavage were found in both LN18 and T98G cell lines.
The activation of these proteases and their substrate
cleavage leading to apoptosis were previously reported
in glioblastoma LN18 [26, 29, 46], U138MG and U251MG
cells [30], and T98G and U87MG xenografts [1, 6, 29].
Taken together, results from the current investigation
showed that VPA ? TX and most notably VPA ? NTX
induced differentiation, suppressed angiogenic and survival
factors, inhibited multi-drug resistance, and activated cal-
pain and caspase proteolytic cascades for induction of
apoptosis in human glioblastoma cells (Fig. 8). Thus, the
combination of VPA ? NTX could be further explored as
a potential therapeutic strategy for treatment of glioblas-
toma in preclinical as well as in clinical settings in the near
future.
Acknowledgments This investigation was supported in part by the
NS-57811 and NS-62327 grants from the National Institutes of Health
and the SCIRF-11-002 grant from the State of South Carolina.
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