Journal of Cancer Therapy, 2011, 2, 77-90
doi:10.4236/jct.2011.22009 Published Online June 2011 (http://www.SciRP.org/journal/jct)
77
Induction of Mitochondrial Pathways and
Endoplasmic Reticulum Stress for Increasing
Apoptosis in Ectopic and Orthotopic
Neuroblastoma Xenografts
Surajit Karmakar1, Subhasree Roy Choudhury1, Naren Lal Banik2, Swapan Kumar Ray1
1Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine, Columbia, USA;
2Department of Neurosciences, Medical University of South Carolina, Charleston, USA.
Received February 26th, 2011; revised April 25th, 2011; accepted May 4th, 2011.
ABSTRACT
Cancers are characterized by deregulation of multiple signaling pathways and thus monotherapies are hardly effective.
Neuroblastoma, which often occurs in adrenal glands, is the most common childhood malignancy. Malignant neuro-
blastoma resists traditional treatments and further studies are needed for effective therapeutic interventions. We evalu-
ated synergistic efficacy of N-(4-hydroxyphenyl) retinamide (4-HPR) and genistein (GST) for induction of apoptosis in
human malignant neuroblastoma SH-SY5Y and SK-N-BE2 cells in culture and activation of multiple pathways for in-
creasing apoptosis in ectopic and orthotopic neuroblastoma xenografts in nude mice. Combination of 4-HPR and GST
synergistically reduced cell viability, caused subG1 accumulation, increased caspase-3 activity for apoptosis in vitro
and reduced tumor growth in vivo. Western blotting indicated that combination therapy down regulated Id2 to induce
differentiation, increased pro-apoptotic Bax and decreased anti-apoptotic Bcl-2 leading to an increase in Bax:Bcl-2
ratio, increased mitochondrial Bax level, caused mitochondrial release of Smac/Diablo, down regulation of the bacu-
lovirus inhibitor-of-apoptosis repeat containing (BIRC) proteins such as BIRC-2 and BIRC-3, and activation of calpain
and caspase-3 in SH-SY5Y xenografts. Accumulation of apoptosis-inducing-factor (AIF) in cytosol and increase in
caspase-4 activation suggested involvement of mitochondrial pathway and endoplasmic reticulum (ER) stress, respec-
tively, for apoptosis in SH-SY5Y xenografts. In situ immunofluorescent labelings of SH-SY5Y and SK-N-BE2 xenograft
sections showed overexpression of calpain, caspase-12, and caspase-3, and AIF, suggesting induction of mitochondrial
caspase-dependent and caspase-independent pathways for apoptosis. Collectively, synergistic effects of 4-HPR and
GST induced mitochondrial pathways and also ER stress for increasing apoptosis in ectopic and orthotopic neuroblas-
toma xenografts in nude mice.
Keywords: Apoptosis, Endoplasmic Reticulum Stress, Genistein, N-(4-Hydroxyphenyl) Retinamide, Neuroblastoma
1. Introduction
Retinoid controls a wide range of biological phenomena
including differentiation, proliferation, and apoptosis in a
variety of cancers in humans [1]. Fenretinide or
N-(4-hydroxyphenyl) retinamide (4-HPR), a synthetic
retinoid, has shown the most promising broad spectrum
anti-cancer efficacy against a variety of in vitro and in
vivo animal studies [2], and chemopreventive clinical
trials [3]. Notably, 4-HPR showed low toxicity and an-
ti-tumor efficacy even in all-trans retinoic acid (ATRA)
or 13-cis retinoic acid (13-CRA) resistant cancer cells;
and several in vitro studies reported that dose-dependent
anti-tumor efficacy of 4-HPR was mainly due to growth
arrest and apoptosis in neuroblastoma [3,4]. Treatment of
neuroblastoma patients with high dose of 4-HPR accu-
mulated a plasma 4-HPR concentration that was able to
induce apoptosis in neuroblastoma cells [5]. Interestingly,
4-HPR in comparatively low concentrations induces cel-
lular differentiation due to differential expression of a
variety of key proteins [6]. Thus, therapeutic potential of
low dose of 4-HPR in combination with other therapeutic
agent needs to be explored for induction of apoptosis in
various cancers including neuroblastoma.

Induction of Mitochondrial Pathways and Endoplasmic Reticulum Stress for Increasing Apoptosis in
78
Ectopic and Orthotopic Neuroblastoma Xenografts
Development of therapeutic agents from dietary phy-
tochemicals is very promising to fight various human
cancers [7]. Genistein (GST), an isoflavonoid primarily
derived from soybean, has been reported to inhibit the
growth of various cancer cells through alterations in cell
signaling pathways, cell cycle, and apoptosis [8]. GST
induced apoptosis through endoplasmic reticulum stress
and mitochondrial damage in hepatoma Hep3B cells [9]
and Ca2+-mediated calpain/caspase-12-dependent apop-
tosis in breast cancer MCF-7 cells [10]. GST potentially
inhibited cellular proliferation in 5 neuroblastoma N2A,
JC, SK-N-SH, MSN, and Lan5 cell lines through induc-
tion of apoptosis [11]. We previously reported that GST
induced activation of calpain and caspases for apoptosis
in human neuroblastoma SH-SY5Y cells [12]. These
studies suggest that GST could be a strong anti-cancer
agent for treating neuroblastoma in vivo as well.
Neuroblastoma is the most frequent extracranial child-
hood solid tumor derived from sympathetic nervous sys-
tem mostly affecting adrenal gland and it also usually
metastasizes in other body parts including chest, neck,
lymph nodes, pelvis, liver, and bone [13,14]. The current
therapy for this childhood malignancy comprises surgery,
radiation, and chemotherapy; but in most cases of neuro-
blastoma, therapeutic inefficacy leading to poor clinical
outcome may render in excess of 15% of all pediatric
cancer related deaths in children [15,16]. Thus, innova-
tive therapeutic approach is urgently warranted for suc-
cessful treatment of neuroblastoma. Neuroblastoma
shows a complex clinical as well as biological heteroge-
neity [13]. In this investigation, we explored the efficacy
of the combination of 4-HPR (for induction of differen-
tiation) and GST (for induction of apoptosis) in human
neuroblastoma cells and in ectopic SH-SY5Y and or-
thotopic SK-N-BE2 xenografts in athymic nude mice.
Our data showed that combination of 4-HPR and GST
caused more anti-tumor efficacy than monotherapy and
they worked synergistically to activate multiple molecular
mechanisms for increasing apoptosis in human neuro-
blastoma cells and in preclinical ectopic SH-SY5Y and
orthotopic SK-N-BE2 xenografts in athymic nude mice.
2. Materials and Methods
2.1. Human Neuroblastoma Cell Lines and
Culture Conditions
Human neuroblastoma SH-SY5Y and SK-N-BE2 cell
lines were purchased from the American Type Culture
Collection (ATCC, Manassas, VA). Cells were grown in
75-cm2 flasks containing 10 ml of 1xRPMI 1640 sup-
plemented with 10% fetal bovine serum (FBS) and 1%
penicillin and 1% streptomycin in a fully-humidified
incubator containing 5% CO2 at 37˚C. Cell lines were
serially passaged following trypsinization using a tryp-
sin/EDTA solution.
2.2. MTT Assay
The 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay was used to determine the sensitivi-
ties of human neuroblastoma SK-N-BE2 and SH-SY5Y
cell lines to 4-HPR (0.25, 0.5, and 1 μM for 72 h) and
GST (50, 100, and 200 μM for 24 h) alone or in combi-
nation. Cell viability data were analyzed on the basis of
the multiple drug-effect equation (classical isobologram)
using the Compusyn software (ComboSyn, Paramus, NJ).
The effect (synergistic, additive, or antagonistic) of the
combination of drugs (4-HPR and GST) was expressed
as the combination index (CI), where CI < 1 indicated
synergism, CI = 1 indicated additive effect, and CI >1
indicated antagonism [17].
2.3. Flow Cytometric Analysis of Cell Cycle
Following treatment, cells were collected by trypsiniza-
tion and centrifugation, then washed with PBS, and fixed
with 70% ethanol. Cells were labeled with propidium
iodide (PI) solution (0.05 mg/ml PI, 2 mg/ml RNase A,
0.01% Triton X-100 in PBS) and incubated for 30 min at
room temperature in darkness. Cell cycle (DNA content)
was analyzed using an Epics XL-MCL Flow Cytometer
(Beckman Coulter, Fullerton, CA) [17].
2.4. Colorimetric Assay for Determination of
Caspase-3 Activity
Caspase-3 activity in the lysates of cells or xenografts
was measured using the commercially available cas-
pase-3 colorimetric assay kit (Sigma, St. Louis, MO).
2.5. Annexin V-FITC/PI Staining and Flow
Cytometry for Determination of Apoptosis
Cells from single and combination treatments were har-
vested and processed with an Annexin V-fluorescein
isothiocyanate (FITC)/propidium iodide (PI) staining kit
(BD Biosciences, San Jose, CA) for flow cytometric
analysis for quantitative determination of apoptosis using
an Epics XL-MCL Flow Cytometer (Beckman Coulter,
Fullerton, CA). Further, we used 100 μM z-DEVD-fmk
(a caspase-3-specific inhibitor) to determine the in-
volvement of caspase-3 in apoptosis in cells following
combination therapy [17].
2.6. Human Neuroblastoma Ectopic and
Orthotopic Xenografts in Athymic Nude Mice
Six-week-old female athymic nude (nu/nu) mice were
obtained from Charles River Laboratories (Wilmington,
MA). All animal studies were conducted following

Induction of Mitochondrial Pathways and Endoplasmic Reticulum Stress for Increasing Apoptosis in 79
Ectopic and Orthotopic Neuroblastoma Xenografts
guidelines of the NIH Animal Advisory Committee and
also approved by our Institutional Animal Care and Use
Committee (IACUC). For developing ectopic human
neuroblastoma xenografts, SH-SY5Y cells (6 × 106) in
100 µl of (1:1) mixture with Matrigel (BD Biosciences,
San Jose, CA) were implanted by subcutaneous (s.c.)
injection on the flank of each mouse under isoflurane
anesthesia condition. Palpable xenografts were devel-
oped within 6 - 8 days. Tumors were measured using an
external caliper and volume was calculated by the for-
mula: 4/3 × (length/2) × (width/2)2. For developing
orthotopic human neuroblastoma xenografts, nude mice
were anaesthetized using ketamine (50 mg/kg) and xy-
lazine (35 mg/kg) intraperitoneally (i.p.). After a midline
incision, a total of 6 × 105 SK-N-BE2 neuroblastoma
cells in 15 μl of 1xRPMI 1640 were injected in the cap-
sule of the left adrenal gland using a 22 gauge needle
attached to a Hamilton syringe. After implantation of
cells, abdominal wall and skin were closed with a 5-0
LOOK® suture (Surgical Specialties, Reading, PA) and
antiseptic-analgesic ointments were applied and animals
were kept in a pathogen-free animal facility [18]. Ani-
mals with 3 weeks-old neuroblastoma xenografts were
randomly divided into four groups: control (CTL),
4-HPR, GST, and 4-HPR + GST. Animals in control
group did not receive any therapy. Each animal in other
group received i.p. a daily dose of 4-HPR (20 µg/kg/day),
GST (2 mg/kg/day), or 4-HPR (20 µg/kg/day) + 4 h later
GST (2 mg/kg/day) for 8 days.
2.7. Histological Examination of Tumor
Xenografts
After completion of treatment schedule, mice were sacri-
ficed, both ectopic and orthotopic neuroblastoma xeno-
grafts were surgically collected. After washing with PBS,
pH 7.4, tumors were either immediately frozen in liquid
nitrogen and strored at –80˚C for future use or immedi-
ately frozen (–80˚C) in Optima Cutting Temperature
media (Fisher Scientific, Suwanee, GA) and 5 µm sec-
tions were cut with a cryostat. These sections were
stained for routine histological examinations [19].
2.8. In Situ Immunofluorescent Labeling for
Detection of Pro-Apoptotic Protein
Expression
For in situ immunofluorescent labelings, samples were
fixed to slides using 95% (v/v) ethanol for 10 min and
rinsed twice for 10 min each in PBS, pH 7.4. Fixation
and all subsequent steps were conducted at room tem-
perature. Sections were blocked with 2% (v/v) horse and
goat sera (Sigma, St. Louis, MO) for 30 min. Samples
were probed with (1:100) primary IgG antibody for 1 h
and slides were rinsed twice in PBS, pH 7.4, for 5 min
each followed by incubation with (1:75) secondary Texas
Red-conjugated anti-rabbit IgG antibody (Vector Labo-
ratories, Burlingame, CA) for 30 min. Slides were then
rinsed twice in PBS, pH 7.4, for 5 min and in distilled
water for 5 min. The slides were immediately mounted
with VectaShield (Vector Laboratories) and viewed
promptly under a fluorescence microscope at 200x mag-
nification (Olympus, Japan), and digital pictures were
taken with Image-Pro Plus software (Media Cybernetics,
Silver Spring, MD), as we described recently [20].
2.9. Combined Terminal dUTP Nick-End
Labeling (TUNEL) and Double
Immunofluorescent Stainings for Detection
of Apoptosis and Upregulation of a
Pro-Apoptotic Protein
Briefly, xenograft tissues were sectioned, fixed in 95%
(v/v) ethanol for 10 min and then post-fixed in 4% me-
thanol-free formaldehyde for 10 min. The sections were
saturated with TdT equilibration buffer (Promega, Mad-
ison, WI, USA) (50 μl/slide) for 5 min and then replaced
with TUNEL reaction mixture (50 μl/slide) and were
incubated for 1 h at 37˚C in an OmniSlide Thermal Cy-
cler (Hypaid, UK). Following the TUNEL reaction,
slides were immuno-stained with primary IgG antibody
for specific protein and then incubated with flourescein-
ated secondary antibodies and images were taken [20].
2.10. Western Blotting and Detection of Specific
Protein Bands
Western blotting was used for analyzing specific proteins
[20]. Briefly, total protein samples from tumor tissue
were extracted, measured spectrophotometrically and
denatured. Samples were loaded onto the SDS–polya-
crylamide gradient (4% - 20%) gels (Bio-Rad, Hercules,
CA), resolved by electrophoresis, and electroblotted to
membranes. The blots were incubated with a primary
IgG antibody followed by incubation with an alkaline
horseradish peroxidase (HRP)-conjugated secondary IgG
antibody. Specific protein bands on the blots were de-
tected by HRP/H2O2 catalyzed oxidation of luminol in
alkaline condition using enhanced chemiluminescence
(ECL) system (GE Healthcare Bio-Sciences, Piscataway,
NJ) followed by autoradiography. Autoradiograms were
scanned using Photoshop software (Adobe Systems, Se-
attle, WA), and OD of each band was determined using
NIH Image software.
2.11. Colorimetric Assay for Determination of
Caspase-4 Activity
Caspase-4 activity in tumor lysates was measured using

Induction of Mitochondrial Pathways and Endoplasmic Reticulum Stress for Increasing Apoptosis in
Ectopic and Orthotopic Neuroblastoma Xenografts
80
treatments (Figure 1). Changes in cell viability in the commercially available caspase-4 activity colorimet-
ric assay kit (BioVision, Mountain View, CA). were determined by the MTT assay (Figure 1(a)). We
used 4-HPR or GST alone as monotherapy and also
4-HPR + GST as combination therapy. Notably, 4-HPR
(0.5 μM for 72 h) + GST (100 μM for 24 h) and 4-HPR (1
μM for 72 h) + GST (200 μM for 24 h) caused most sig-
nificant decrease in cell viability when compared with
CTL and other treatment groups (Figure 1(a)). Data were
analyzed to generate the combination index (CI) of the
drugs. The effect of 4-HPR + GST was found to produce
CI < 1 in neuroblastoma SK-N-BE2 (CI = 0.78) and
SH-SY5Y (CI = 0.95) cells, indicating synergistic action
of the combination of drugs. Finally, 4-HPR (0.5 μM for
72 h), GST (100 μM for 24 h), and 4-HPR (0.50 μM for
72 h) + GST (100 μM for 24 h) were selected as effective
treatments and used in all subsequent cell culture studies.
2.12. Statistical Analysis
Data were analyzed using the StatView software (Abacus
Concepts, Berkeley, CA), expressed as arbitrary units ±
standard error of mean (SEM) of separate experiments (n
≥ 3), and compared by one-way analysis of variance
(ANOVA) followed by the Fisher post hoc test. A sig-
nificant difference between control and treatment was
indicated by * (p < 0.05) or ** (p < 0.001).
3. Results
3.1. Changes in Cell Viability
We used human malignant neuroblastoma SK-N-BE2 and
SH-SY5Y cells in cultures to determine the effects of
Figure 1. Effects of treatments on human malignant neuroblastoma SK-N-BE2 and SH-SY5Y cells. (a) Changes in cell viabil-
ity in neuroblastoma cells as determined by MTT assay. Treatments: 1. control (CTL); 2. 4-HPR (0.25, 0.5, and 1 μM for 72 h); 3.
GST (50, 100, and 200 μM for 24 h); 4. 4-HPR (0.25 μM for 72 h) + GST (50 μM for 24 h); 5. 4-HPR (0.5 μM for 72 h) + GST
(100 μM for 24 h); and 6. 4-HPR (1 μM for 72 h) + GST (200 μM for 24 h). Finally, 4-HPR (0.5 μM for 72 h) + GST (100 μM
for 24 h) was selected for all other cell culture studies. (b) Cell cycle analysis by flow cytometry in SK-N-BE2 and SH-SY5Y
cells. Combination therapy induced apoptotic subG1 accumulations.

Induction of Mitochondrial Pathways and Endoplasmic Reticulum Stress for Increasing Apoptosis in 81
Ectopic and Orthotopic Neuroblastoma Xenografts
3.2. Cell Cycle Analysis
We found treatments (4-HPR, GST, and 4-HPR + GST)
significantly increased apoptotic subG1 phase (10, 18,
and 28%, respectively) in SK-N-BE2 cells (Figure 1(b)).
In case of SH-SY5Y cells (Figure 1(b)), 4-HPR treat-
ment showed non-significant change, while GST (35%)
and 4-HPR + GST (51%) treatments exhibited significant
increases in apoptotic subG1 phase, compared with un-
treated cells (Figure 1(b)).
3.3. Increase in Caspase-3 Activity
We determined the caspase-3 activity in apoptosis and
amounts of apoptotic cells following treatments (Figure
2). Increases in caspase-3 activity in course of apoptosis
in both neuroblastoma cell lines were measured by a co-
lorimetric assay (Figure 2(a)). The caspase-3 activity in
cell lysate was most significantly found in both cell lines
after treatment with 4-HPR + GST, compared with un-
treated cells (Figure 2(a)).
Figure 2. Increases in caspase-3 activity and apoptotic populations in SK-N-BE2 and SH-SY5Y cells. Treatments: CTL,
4-HPR (0.5 μM for 72 h), GST (100 μM for 24 h), and 4-HPR (0.5 μM for 72 h) + GST (100 μM for 24 h). (a) Determination
of caspase-3 activity using a colorimetric assay. (b) Annexin V-FITC/PI staining and flow cytometric determination of apop-
tosis. Treatment with 4-HPR + GST effectively controlled growth of neuroblastoma cells due to increase in caspase-3 activity
for apoptosis.

Induction of Mitochondrial Pathways and Endoplasmic Reticulum Stress for Increasing Apoptosis in
82
Ectopic and Orthotopic Neuroblastoma Xenografts
3.4. Increase in Apoptosis Was Associated with
Upregulation of Caspase-3
Our Annexin V-FITC/PI staining and flow cytometric
studies showed the percentages of Annexin V-positive/
PI-negative populations (early apoptotic) in neuroblas-
toma SK-N-BE2 (CTL, 2%; 4-HPR, 8%; GST, 20%; and
4-HPR + GST, 32%) and SH-SY5Y (CTL, 2%; 4-HPR,
3%; GST, 32%; and 4-HPR + GST, 47%) cells (Figure
2(b)). Pre-treatment with 100 μM z-DEVD-fmk (a cas-
pase-3-specific inhibitor) significantly reduced the capa-
bility of combination therapy for induction of apoptosis
to 5.28% in SK-N-BE2 cells and to 18.45% in SH-SY5Y
cells, indicating involvement of caspase-3 in induction of
apoptosis in both cell lines (Figure 2(b)).
3.5. Histological Evaluation of Efficacy of
Combination Therapy
We treated ectopic SH-SY5Y xenografts in nude mice
(Figure 3). As seen in the animals (Figure 3(a)), treat-
ments caused tumor regression. Following treatments,
the tumors were collected surgically to measure tumor
volume (Figure 3(b)). Treatment with 4-HPR + GST
caused more regression of SH-SY5Y tumors than either
drug alone. Further, we performed hematoxylin and eo-
sin (H&E) staining of tumor sections and histological
examination at 200x magnification under the light mi-
croscope (Figure 3(c)), showing maximum cell death
after combination therapy.
Similarly, the efficacy of combination therapy was
evaluated in orthotopic SK-N-BE2 xenografts in nude
mice (Figure 4). Following 4-HPR or GST monotherapy
and 4-HPR + GST combination therapy of orthotopic
SK-N-BE2 xenografts (Figure 4(a)), tumor sections
from each group were subjected to H&E staining fol-
lowed by histological examination under the light mi-
croscope at 200x magnification (Figure 4(b)). In or-
thotopic xenografts also, combination therapy caused
maximum cell death.
Examination of sections of all neuroblastoma ectopic
SH-SY5Y xenografts and orthotopic SK-N-BE2 xeno-
grafts after H&E staining revealed that untreated tumors
maintained characteristic growth of neuroblastoma, 4-HPR
alone inhibited tumor cell proliferation and caused neu-
ronal differentiation, GST alone induced apoptosis, and
4-HPR + GST combination therapy dramatically in-
creased the amounts of apoptosis.
3.6. Combination Therapy down Regulated Id2
and Increased Bax:Bcl-2 Ratio in SH-SY5Y
Xenografts
We performed Western blotting (Figure 5) and found
Figure 3. Efficacy of combination therapy in ectopic SH-
SY5Y xenografts. (a) Ectopic SH-SY5Y xenografts in nude
mice. Treatments (8 days): control (CTL), 4-HPR (20
µg/kg/day), GST (2 mg/kg/day), and 4-HPR (20 µg/kg/day)
plus 4 h later GST (2 mg/kg/day). (b) The representative
tumors after treatments. (c) Histological features (at 200x
magnification) of SH-SY5Y xenografts after treatments.
that treatment with 4-HPR + GST down regulated in-
hibitor-of-differentiation 2 (Id2), and changed the levels
of expression of total Bax and Bcl-2 (Figure 5(a)) re-
sulting in significant increase in the Bax:Bcl-2 ratio,
compared with CTL xenograft (Figure 5(b)). The eleva-
tion of Bax:Bcl-2 ratio could make cells fully committed
to apoptosis due to mitochondrial release of several
pro-apoptotic molecules. Further, we isolated the mito-
chondrial fractions to analyze the change in mitochon-
drial Bax level and monitored the COX-4 level as mito-
chondrial internal control (Figure 5(c)). We found that
treatment with 4-HPR + GST caused more increase in
mitochondrial Bax level in the SH-SY5Y xenografts than
either therapy alone (Figure 5(d)).

Induction of Mitochondrial Pathways and Endoplasmic Reticulum Stress for Increasing Apoptosis in 83
Ectopic and Orthotopic Neuroblastoma Xenografts
Figure 4. Efficacy of combination therapy in orthotopic
SK-N-BE2 xenografts. (a) Orthotopic SK-N-BE2 xeno-
grafts in nude mice. Treatments (8 days): CTL, 4-HPR
(20 µg/kg/day), GST (2 mg/kg/day), and 4-HPR (20
µg/kg/day) + 4 h later GST (2 mg/kg/day). Inset: arrow-
head indicated location of orthotopic SK-N-BE xenograft
on the adrenal gland. Arrow indicated location of tumor
on the adrenal gland in animal. (b) Histological features
(at 200x magnification) of orthotopic SK-N-BE2 xeno-
grafts following treatments.
(a)
(b)
(c)
(d)
Figure 5. Western blotting for biochemical feature of dif-
ferentiation and determination of Bax:Bcl-2 ratio and mito-
chondrial Bax level in SH-SY5Y xenografts. Treatments (8
days): CTL, 4-HPR (20 µg/kg/day), GST (2 mg/kg/day),
and 4-HPR (20 µg/kg/day) + 4 h later GST (2 mg/kg/day).
(a) Representative Western blots (n ≥ 3) to show expres-
sion of 42 kDa β-actin, 16 kDa Id2, 21 and 24 kDa Bax,
26 kDa Bcl-2. (b) Densitometric analysis to show change in
the Bax:Bcl-2 ratio. (c) Representative Western blots (n ≥
3) to show expression of 17 kDa COX-4, 21 and 24 kDa
Bax in mitochondrial fraction. (d) Densitometric analysis
to show relative changes in the mitochondrial Bax level
following treatments.
3.7. Combination Therapy Caused
Mitochondrial Release of Pro-Apoptotic
Molecules and Down Regulation of BIRC
Proteins for Activation of Cysteine Proteases
in SH-SY5Y Xenografts
Therapeutic treatment may cause mitochondrial release
of pro-apoptotic molecules [21]. We performed Western
blotting to examine mitochondrial release of Smac/Diablo
and levels of BIRC proteins and cysteine proteases in
SH-SY5Y xenografts (Figure 6). Compared with CTL or
4-HPR alone or GST alone, 4-HPR + GST highly in-
duced mitochondrial release of pro-apoptotic protein 25
kDa Smac/Diablo into the cytosol (Figure 6(a)) and lev-
els of the anti-apoptotic proteins 72 kDa BIRC-2
(cIAP-1), 68 kDa BIRC-3 (cIAP-2) in the cytosol (Fig-
ure 6(a)). We also found that combination therapy in-
creased expression of 80 kDa calpain, 20 kDa caspase-3,
and mitochondrial 67 kDa apoptosis-inducing factor
(AIF) into the cytosol (Figure 6(a)). Further, we used
Western blotting and colorimetric assay and found in-
creased caspase-4 activation and total caspase-4 activity,
respectively, indicating increase in ER stress, for apop-
tosis in the SH-SY5Y xenografts after treatment with
4-HPR + GST (Figure 6(b)).
3.8. Combination Therapy Caused Upregulation
of Calpain and Caspase-12 for Apoptosis in
Ectopic and Orthotopic Neuroblastoma
Xenografts
We performed the single immunofluorescent (SIF) and

Induction of Mitochondrial Pathways and Endoplasmic Reticulum Stress for Increasing Apoptosis in
84
Ectopic and Orthotopic Neuroblastoma Xenografts
Figure 6. Activation of caspase-dependent and caspase-
independent pathways for apoptosis in SH-SY5Y xeno-
grafts. Treatments (8 days): CTL, 4-HPR (20 µg/kg/day),
GST (2 mg/kg/day), and 4-HPR (20 µg/kg/day) + 4 h later
GST (2 mg/kg/day). (a) Representative Western blots (n ≥ 3)
to show expression of 42 kDa β-actin, 25 kDa Smac/Diablo,
72 kDa BIRC-2, 68 kDa BIRC-3, 80 kDa Calpain, 20 kDa
and 12 kDa active caspase-3, and 67 kDa AIF. (b) Western
blotting showed active 20 kDa caspase-4 fragment. Bar
diagram to show changes in caspase-4 activity as deter-
mined by a colorimetric assay.
double immunofluorescent (DIF) stainings of the xeno-
graft sections after the treatments (Figure 7). The SIF
staining showed that compared with CTL or monother-
apy, 4-HPR + GST combination therapy very signifi-
cantly increased expression of calpain and caspase-12 in
ectopic SH-SY5Y xenografts (Figure 7(a)) as well as in
orthotopic SK-N-BE2 xenografts (Figure 7(b)). Subse-
quently, the DIF staining showed highly remarkable
overexpression of calpain and caspase-12 in association
with increase in DNA fragmentation in ectopic SH-SY5Y
xenografts (Figure 7(a)) and orthotopic SK-N-BE2
xenografts (Figure 7(b)) following 4-HPR + GST com-
bination therapy.
3.9. Combination Therapy Induced Activation
of Caspase-Dependent and
Caspase-Independent Pathways for
Apoptosis
To explore the downstream executioner steps, we exam-
ined the caspase-3 and AIF levels in all treatment groups
from both SH-SY5Y and SK-N-BE2 xenografts (Figure
8). Following the SIF staining, we found significant in-
creases in caspase-3 and AIF expression in ectopic
SH-SY5Y xenografts (Figure 8(a)) as well as in or-
thotopic SK-N-BE2 xenografts (Figure 8(b)) due to
4-HPR + GST combination therapy. The DIF staining
showed that significant increases in caspase-3 and AIF
expressions were associated with DNA fragmentation in
both ectopic and orthotopic xenografts following the
4-HPR + GST combination therapy (Figure 8(b)). These
studies confirmed the involvement of caspase-dependent
pathway along with non-caspase pathway for induction
of apoptosis in both ectopic SH-SY5Y and orthotopic
SK-N-BE2 xenografts after the 4-HPR + GST combina-
tion therapy.
4. Discussion
In this investigation, we found that 4-HPR + GST wor-
ked synergistically to significantly reduce the viability of
neuroblastoma SH-SY5Y cells (N-Myc expression with-
out N-myc gene amplification) and SK-N-BE2 cells
(N-Myc overexpression with N-myc gene amplification)
[22] and induce caspase-3 activity for apoptosis. Previ-
ous studies reported that 4-HPR [3,23] or GST [12] in-
duced apoptosis in a variety of cell lines including neu-
ronblastoma [4,12] with activation of caspase-3. The
synergistic efficacy of 4-HPR + GST activated multiple
pathways for increasing apoptosis, as suggested by mo-
lecular and histological observations, in two preclinical
neuroblastoma animal models such as ectopic SH-SY5Y
and orthotopic SK-N-BE2 xenografts.
We found that 4-HPR + GST caused down regulation
of Id2, a biochemical marker well known to antagonize
differentiation [24], indicating induction of molecular
mechanism of differentiation in SH-SY5Y xenografts.
The increases in Bax:Bcl-2 ratio and translocation of Bax
to mitochondria could be the key events for apoptosis in
neuroblastoma cells [12,25] and also well correlated

Induction of Mitochondrial Pathways and Endoplasmic Reticulum Stress for Increasing Apoptosis in
Ectopic and Orthotopic Neuroblastoma Xenografts
85
Figure 7. In situ stainings for examination of upregulation of calpain and caspase-12 in association with DNA fragmenta-
tion in the sections of neuroblastoma xenografts. Treatments (8 days): CTL, 4-HPR (20 µg/kg/day), GST (2 mg/kg/day),
and 4-HPR (20 µg/kg/day) + 4 h later GST (2 mg/kg/day). (a) The SIF and DIF stainings of the sections of ectopic
SH-SY5Y xenografts. (b) The SIF and DIF stainings of the sections of orthotopic SK-N-BE2 xenografts. In the sections (at
200x magnification), calpain or caspase-12 is stained in green and DNA fragmentation (TUNEL labeling) is stained in red.
In the bar diagrams, x-axis denotes 1 = CTL, 2 = 4-HPR, 3 = GST, and 4 = 4-HPR + GST.

Induction of Mitochondrial Pathways and Endoplasmic Reticulum Stress for Increasing Apoptosis in
86
Ectopic and Orthotopic Neuroblastoma Xenografts
Figure 8. In situ stainings for examination of upregulation of caspase-3 and AIF in association with DNA fragmentation in
the sections of neuroblastoma xenografts. Treatments (8 days): CTL, 4-HPR (20 µg/kg/day), GST (2 mg/kg/day), and
4-HPR (20 µg/kg/day) + 4 h later GST (2 mg/kg/day). (a) The SIF and DIF stainings of the sections of ectopic SH-SY5Y
xenografts. (b) The SIF and DIF stainings of the sections of orthotopic SK-N-BE2 xenografts. In the sections (at 200x
magnification), caspase-3 or AIF is stained in green and DNA fragmentation (TUNEL labeling) is stained in red. In the
bar diagrams, x-axis denotes 1 = CTL, 2 = 4-HPR, 3 = GST, and 4 = 4-HPR + GST.

Induction of Mitochondrial Pathways and Endoplasmic Reticulum Stress for Increasing Apoptosis in
Ectopic and Orthotopic Neuroblastoma Xenografts
87
following combination therapy with 4-HPR and l-threo-
dihydrosphingosine (safingol) in SK-N-MC cells [26].
Similarly, GST induced apoptosis through alterations in
Bax and Bcl-2 levels in breast [27] and head and neck
squamous cell carcinoma cell lines [28]. Thus, it would
be highly plaussible that combination of 4-HPR and GST
increased Bax:Bcl-2 ratio to trigger the mitochondrial
release of the pro-apoptotic molecule Smac/Diablo to
down regulate the anti-apoptotic BIRC proteins and ac-
tivate the downstream apoptotic pathways.
Levels of expression of BIRC-2 (cIAP-1) and BIRC-3
(cIAP-2) are often stimulated in cancers and cause sup-
pression of caspase activation for inhibition of apoptosis
[29] and can act as oncogenes leading to cell survival,
transformation, and resistance to radiation and chemo-
therapies [30]. Smac/Diablo acts as an indirect activator
of caspases by inhibition of the BIRC proteins [21] to
facilitate the caspase-dependent pathway of apoptosis
following treatment with combination of 4-HPR and
GST in ectopic SH-SY5Y xenografts. Treatment with
this combination therapy increased activation of the
Ca2+-dependent cysteine protease calpain, which could
play a significant role in cell death with alternations in
expression of the Bcl-2 family members leading to an
increase in Bax:Bcl-2 ratio [31] and Bax translocation to
mitochondria for release of Smac/Diablo [32]. Calpain
can also activate caspase-3 for cytoskeletal brakedown
and apoptosis in cancers including neuroblastoma
[12,25]. Our finding is consistent with previous studies
demonstrating that increases in calpain [33] and cas-
pase-3 [34] activation play important role in induction of
apoptosis in SH-SY5Y xenografts after treatment with
4-HPR + GST.
Loss of mitochondrial membrane integrity can release
AIF to induce caspase-independent apoptosis [35] that
we also found following 4-HPR + GST therapy in ec-
topic SH-SY5Y xenografts. Calpain has been strongly
associated with mitochondrial release of AIF and trans-
location to the nucleus [36] to cause caspase-independent
nuclear DNA fragmentation. The increase in caspase-4
activation and activity following 4-HPR + GST combi-
nation therapy indicated involvement of ER stress in
induction of apoptosis that could be due to 4-HPR [37]
or GST [9] monotherapy or due to their combined effects.
Recent studies demonstrate that 4-HPR induces the gen-
eration of reactive oxygen species (ROS) for mitochon-
drial damage leading to apoptosis in six neuroblastoma
cell lines [38] and also ER stress induces cell death in
neuroectodermal tumor cells [39]. Studies in oral squa-
mous cell carcinoma (OSCC) cell line demonstrated that
combination of cetuximab and GST caused dose-de-
pendent significant decrease in cell proliferation when
compared with either drug alone [40]. Our observations
suggested that 4-HPR + GST combination therapy trig-
gered mitochondrial caspase-dependent and caspase-in-
dependent pathways and also ER stress for apoptosis in
SH-SY5Y xenografts. In line with our observation, a
recent investigation reported that combination of borte-
zomib and 4-HPR increased apoptosis through activation
of the ER stress in neuroblastoma cells when compared
with either monotherapy [41]. Also, increased survival
time in tumor-bearing mice and histological evaluation
proved that combination of bortezomib and 4-HPR in-
creased anti-tumor and anti-angiogenic mechanisms [41].
Similarly, combination of cetuximab and GST signifi-
cantly inhibited tumor growth and caused a substantial
delay in tumor growth in animal models of OSCC while
no delay in tumor growth was observed using either
monotherapy alone [40]. Thus, understanding the com-
plex regulation of cell death pathways through mito-
chondrial damage or ER stress in response to 4-HPR +
GST combination therapy has the potential to reveal
novel therapeutic targets in neuroblastoma, as we ob-
served in present preclinical models.
We used the SIF staining to examine a specific protein
expression and the DIF staining to examine a specific
protein expression in association with DNA fragmenta-
tion in course of apoptosis in both neuroblastoma ectopic
SH-SY5Y and orthotopic SK-N-BE2 xenografts. Our
results indicated significant involvement of both calpain
and caspase-12 overexpression in apoptosis in both neu-
roblastoma xenografts. We also found significant upre-
gulation of caspase-3 and AIF for apoptosis in both neu-
roblastoma xenografts. Calpain is known to cause ER
stress and activation of caspase-12 for apoptosis [42].
Active caspase-12 is able to directly activate caspase-9
and caspase-3 for inducing apoptosis [43,44]. Our ob-
servation is correlated with previous studies showing that
GST increased intracellular Ca2+ concentration due to
release from ER Ca2+ storage to activate the Ca2+-de-
pendent cysteine protease calpain and the ER protease
caspase-12 for apoptosis in breast cancer cells [10]. We
previously reported that GST caused increase in intra-
cellular free Ca2+ for activation of calpain and caspase-12
leading to activation of caspase-3 in SH-SY5Y cells for
apoptosis [12]. Activation of calpain could cause mito-
chondrial release of AIF to promote caspase-independent
apoptosis.
In conclusion, our investigation showed that 4-HPR +
GST worked synergistically for increasing apoptosis in
human malignant neuroblastoma SH-SY5Y and SK-N-BE2
cells and induced multiple molecular mechanisms for
increasing apoptosis in ectopic SH-SY5Y xenografts as
well as in orthotopic SK-N-BE2 xenografts. The results

Induction of Mitochondrial Pathways and Endoplasmic Reticulum Stress for Increasing Apoptosis in
88
Ectopic and Orthotopic Neuroblastoma Xenografts
from our investigation strongly suggest that combination
of 4-HPR and GST should be explored as a promising
therapeutic strategy for the treatment of malignant neu-
roblastoma in humans in the near future.
5. Acknowledgements
This investigation was supported in part by the grant R01
NS-57811 from the National Institutes of Health (Be-
thesda, MD) to S. K. R.
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