ApoE mimetic peptide targeted nanoparticles carrying a BRD4 inhibitor for treating Medulloblastoma in mice

Article abstract of DOI:10.1016/j.jconrel.2020.04.053

Treatment of medulloblastoma (MB) is challenging due to diverse genetic make-up, chemoresistance and inefficient drug transport across the blood brain barrier (BBB). Since hedgehog (Hh) signaling regulates cancer cell proliferation and tumorigenicity, Hh

Full text of DOI:10.1016/j.jconrel.2020.04.053

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
JournalofControlledRelease323(2020)463–474
Contents lists available at ScienceDirect
Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel
ApoE mimetic peptide targeted nanoparticles carrying a BRD4 inhibitor for
treating Medulloblastoma in mice
Qiyue Wanga, Virender Kumara, Feng Lina, Bharti Sethia, Donald W. Coulterb,
Timothy R. McGuirec, Ram I. Mahatoa,⁎
a Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USA
b Department of Pediatrics, University of Nebraska Medical Center, Omaha, NE 68198, USA
c Department of Pharmacy Practice, University of Nebraska Medical Center, Omaha, NE 68198, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Treatment of medulloblastoma (MB) is challenging due to diverse genetic make-up, chemoresistance and in-
efficient drug transport across the blood brain barrier (BBB). Since hedgehog (Hh) signaling regulates cancer cell
proliferation and tumorigenicity, Hh inhibitors have the potential to treat sonic Hh driven MB (SHH-MB), but
their repeated use develops chemoresistance due to mutations in smoothened (SMO). Herein, we aimed to
overcome these problems by modulating GLI transcription using JQ1, which is a small molecule BRD4 inhibitor.
JQ1 inhibited HD-MB03 and DAOY cell proliferation, with the IC50 of 402 and 4220 nM, respectively. JQ1
inhibited colony formation, but increased apoptosis in HD-MB03 and DAOY cells. Western blot analysis con-
firmed significant inhibition of GLI1 and c-MYC protein expression in DAOY and HD-MB03 cells, respectively.
JQ1 was encapsulated into apolipoprotein (ApoE) mimetic peptide decorated nanoparticles (ApoE-NPs), with
the mean particle size of 64 nm and drug loading of 10% (w/w). ApoE-NPs increased JQ1 concentration in the
tumor by 5 and 8 folds at 6 and 24 h after systemic administration into orthotopic MB tumor bearing NSG mice
compared to non-targeted JQ1 loaded NPs. Although there was also modest increase in JQ1 delivery to the liver,
there was no hepatotoxicity as evidenced by H&E staining and little increase in serum ALT and AST after
treatment with JQ1 loaded ApoE-NPs. There was also significant decrease in the orthotopic MB tumor burden
after systemic administration of JQ1 loaded ApoE- NPs at the dose of 10 mg/kg every 3rd day for a total of 8
injections. In conclusion, JQ1 loaded NPs have the potential to treat Group 3 and SHH driven MB in mice.
Medulloblastoma
ApoE mimetic peptide
JQ1
BRD4
Nanoparticles
Biodistribution
1. Introduction
prognosis, mutations in PTCH and SMO proteins, and amplification of
GLI1/2 gene. Group 3 MB is one of the most aggressive subgroups ac-
Medulloblastoma (MB) is an aggressively growing brain tumor in
children. It spreads through cerebrospinal fluid (CSF) and metastasizes
to different locations along the brain surface of the brain and spinal
cord [1]. The 5-year survival rate for children with average-risk is
70–80%, whereas high-risk accounts for 60–65% [2]. The standard
treatment of MB is the intensive adjuvant chemotherapy combined with
high dose irradiation of the brain and spinal cord, but this treatment
protocol has only 60% cure rate, and usually results in growth im-
pairment, endocrine disorders, and neurocognitive deficits [3,4].
Therefore, more effective, less toxic therapeutics are urgently needed
for MB, which is classified into four different subgroups, each with
different origins, pathogenesis, and potential therapy targets [5]. SHH-
MB subgroup represents ~30% of MB cases with an intermediate
count for approximately 25% of all MB with high metastatic potential
and poor prognosis [5].
Bromodomain and extra-terminal domain (BET) family proteins,
such as BRD2, BRD3, and BRD4 are related with recruited transcrip-
tional activators [6]. BRD4 promotes progression and regulation of cell
growth and transcription in neoplastic cell proliferation in different
cancer types including neuroblastoma and glioblastoma [7,8]. Cere-
bellar granule neuron precursors (CGNPs) undergo rapid SHH-depen-
dent expansion perinatally and excessive SHH pathway activity pro-
motes MB. Most common MYC oncogenes include c-MYC and MYCN,
which are at the crossroad of many biological pathways and processes
involved in neoplastic cell growth and proliferation, which are fre-
quently amplified in MB and are associated with a poor prognosis and
⁎ Corresponding author at: Department of Pharmaceutical Sciences, University of Nebraska Medical Center (UNMC), 986025 Nebraska Medical Center, Omaha, NE
68198-6025, USA.
E-mail address: ram.mahato@unmc.edu (R.I. Mahato).
https://doi.org/10.1016/j.jconrel.2020.04.053
Received 20 March 2020; Received in revised form 22 April 2020; Accepted 30 April 2020
Availableonline05May2020
0168-3659/©2020ElsevierB.V.Allrightsreserved.

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Q. Wang, et al.
JournalofControlledRelease323(2020)463–474
tumor recurrence [4,9].
purchased from (Roche, Indianapolis, IN). 3-(4,5-Dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma
Aldrich (St. Louis, MO). ApoE mimetic peptide (COG133) was pur-
chased from Adooq Bioscience (Irvine, CA).
Since Hh signaling pathway regulates cell growth and cancer stem
cell (CSC) proliferation, Hh inhibitors have the potential to treat SHH-
MB [10]. Unfortunately, their repeated use develops chemoresistance
because of mutations in SMO [11,12]. N-MYC is a downstream target of
Hh whose induction can drive CGNP proliferation even in the absence
of SHH signaling. Also, there is higher c-MYC expression in Group
3 MB. Herein, we treated SHH driven and Group 3 MB using JQ1, which
is a small molecule BRD4 inhibitor [13,14]. SHH-MB can be treated by
overcoming the problems created by SMO mutation because JQ1 can
effectively modulate GLI transcription through BRD4 inhibition [6].
JQ1 can also be used to treat Group 3 MB by inhibiting c-MYC gene
expression.
2.2. Cytotoxicity and colony formation assay
HD-MB03 and DAOY cells were cultured in 96-well plates at
5 × 103 per well in 100 μL DMEM and EMEM media under 5% CO2 at
37 °C in a 95% humidified atmosphere, respectively. After 48 h post-
treatment with various concentrations of JQ1, the cell viability was
assessed by MTT (0.5 mg/mL) assay. Results are presented as the
mean
S.D. For clonony formation assay, HD-MB03 and DAOY cells
Since JQ1 is highly hydrophobic, it needs to be encapsulated into
nanoparticles (NPs) for in vivo delivery to the tumor after systemic
administration to minimize undesired toxicity. Additionally, nano-
formulation ensures preferential accumulation of JQ1 in tumor cells via
the enhanced permeability and retention (EPR) effect [15]. We pre-
viously reported methoxy poly(ethylene glycol)-block-poly(2-methyl-2-
benzoxycarbonyl-propylene carbonate) (mPEG-PBC) conjugated poly-
carbonate-based polymer for drug delivery with high drug loading and
low toxicity [16–18]. However, the blood-brain barrier (BBB) regulates
drug transport to the brain. Successful treatment strategy of MB re-
quires drugs to pass the BBB, which is a huge challenge for the disease
treatment of central nervous system. Recently, use of noninvasive
routes shows their utility for drug to delivery to the brain. The nano-
platform modified with antibodies or protein fragments could directly
access into the brain via receptor-mediated endocytosis or transcytosis
[19,20].
Apolipoprotein E (ApoE) is a chimera peptide composed of fats and
proteins that binds to very low-density lipoprotein (VLDL) receptor for
targeting NPs to the brain. However, ApoE molecular size is too big for
accumulating therapeutic concentration in the brain. Therefore, we will
decorate NP surface with ApoE mimetic peptide and facilitate JQ1 into
brain with this ApoE mimetic peptide conjugated polymeric NPs. In this
study, we determined the effect of JQ1 on orthotopic Group 3 MB
bearing mice after systemic administration of ApoE mimetic peptide
conjugated NPs carrying JQ1. Our previously reported PEG-PBC was
synthesized and modified by ApoE-mimetic peptide COG133 to prepare
JQ1 loaded NPs for brain targeting through low-density lipoprotein
receptor (LDLR) mediated endocytosis.
were seeded at 250 cells/well into 6-well plates and allowed to attach
for 24 h. Cells were then treated with JQ1 at the concentrations of 0, 2
and 4 μM for DAOY cells and 0, 400 and 800 nM for HD-MB03 cells.
After a 7 day-incubation, colonies in each well were fixed by 10%
formalin, stained with 0.5% crystal violet solution and visualized under
a microscope. Then, the crystal violet was dissolved in 1.5 mL of 10%
acetic acid solution and the optical density (OD) was measured at
590 nm. Each group was performed in triplicates.
2.3. Cycle and apoptosis analysis
HD-MB03 and DAOY cells pretreated with JQ1 concentrations of 0,
200 and 400 nM and 0, 2 and 4 μM, respectively for 48 h were har-
vested and fixed in 70% ethanol at 4 °C for 2 h. 1 × 106 cells were
stained with 50 mg/mL propidium iodide (PI) for 30 min at room
temperature in dark. Cell cycle distribution was analyzed by flow cy-
tometry using BD Calibur flow cytometer (BD Biosciences, CA). For the
apoptosis evaluation, 1 × 106 HD-MB03 and DAOY cells pretreated
with JQ1 for 48 h were harvested and stained by Alexa Fluor 647-
Annexin V and PI at room temperature for 30 min in dark. The apop-
tosis was analyzed by flow cytometry using a BD LSRII flow cytometer
(BD Biosciences, CA).
2.4. C-MYC and GLI1 expression by real-time PCR and western blot
analysis
To determine c-MYC and GLI1 expression at mRNA and protein le-
vels by real time RT-PCR and Western blot analysis after treating
3 × 105 HD-MB03 and DAOY cells per well in 6-well plates with
400 nM and 4 μM of JQ1 for 48 h, respectively. For real-time PCR
analysis, treated HD-MB03 and DAOY cells were washed, lysed, and the
total RNA was extracted using RNeasy mini kit. The concentration and
purity of total RNA were determined by UV spectrophotometer at 260
and 280 nm. Multiscribe reverse transcription kit was employed to
convert total RNA to cDNA per manufacturer's instruction (Applied
Biosystems). SYBR Green-I dye universal PCR master was used to run
RT-PCR on a LightCycler 480 Instrument (Roche) using primer as fol-
lows. Human GLI1: forward, 5′-CCA ACT CCA CAG GCA TAC AGG AT-
3′; reverse, 5′-CAC AGA TTC AGG CTC ACG CTT C-3′. Human c-MYC:
forward, 5′-CTG CGA CGA GGA GGA GAA CT −3′; reverse, 5′-GGC AGC
AGC TCG AAT TTC TT-3′. Human GAPDH was used as a housekeeping
gene: forward, 5′-ACC ACA GTC CAT GCC ATC AC -3′; reverse, 5′-TCC
ACC ACC CTG TTG CTG TA-3′.
For Western blot analysis, total protein was isolated from HD-MB03
and DAOY cell lysates by incubating with RIPA lysis buffer and cen-
trifuged by 10,000 ×g for 10 min at 4 °C. Protein concentration was
determined using BCA™ protein assay kit (Thermo Scientific). Equal
amounts of protein were separated in 4–15% mini PROTEAN poly-
acrylamide gel followed by transferring to polyvinylidene fluoride
(PVDF) (Life Technologies, Carlsbad, CA) membranes by iBlot gel
transfer system. Membranes were blocked with 1 × blocking buffer (LI-
COR Biosciences, Lincoln, NE) for 2 h. Membranes were incubated with
primary antibodies (1:1000 dilution) for c-MYC (10828–1-AP)
2. Materials and methods
2.1. Materials
JQ1 was purchased from MedChem Express (Princeton, NJ).
Dulbecco's Modified Eagle Medium (DMEM), Eagle's Minimum
Essential Medium (EMEM), high glucose medium, Dulbecco's phosphate
buffered saline (DPBS), and 0.25% trypsin were purchased from
Hyclone (Logan, UT). Fetal bovine serum (FBS), antibiotic-antimycotic
for cell culture, halt protease and phosphatase inhibitor cocktail
(100×), Pierce BCA protein assay kit, and HEPES buffer were pur-
chased from Millipore Sigma (St. Louis, MO). Human c-MYC primary
antibody was purchased from Proteintech (Manchester, UK). Human
GLI1 primary antibody, human MYCN primary antibody and GAPDH
primary antibody was purchased from Santa Cruz Biotechnology
(Dallas, TX). Human MB cell lines, such as DAOY and HD-MB03 cells
were purchased from American Type Culture Collection (ATCC)
(Manassas, VA), and were cultured in Eagle's Minimum Essential
Medium (EMEM) and DMEM, respectively, containing 10% FBS and 1%
penicillin/streptomycin in a humidified 37 °C incubator supplemented
with 5% CO2. Total RNA isolation kit was obtained from Qiagen
(Gaithersburg, MD). TaqMan reverse transcription reagent kit was
purchased
from
Life
Technologies
(Grand
Island,
NY).
Radioimmunoprecipitation assay (RIPA) buffer and SYBR green-1 were
464

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Q. Wang, et al.
JournalofControlledRelease323(2020)463–474
EE (%) = Amount of JQ1 encapsulated/Amount of JQ1 added × 100%
(Proteintech), GLI1 (sc-20,687), n-MYC (sc-53,993) and GAPDH (sc-
365,062) (Santa Cruz Biotechnology, Inc.) for overnight. The next day,
membranes were incubated with horseradish peroxidase-conjugated
secondary antibodies (anti-mouse, sc-2055, anti-rabbit, sc-2054; and
anti-goat, sc-2056; Santa Cruz Biotechnology, Dallas, TX). Specific
protein bands were visualized by incubating the membrane with lu-
minal reagent (sc-2048; Santa Cruz Biotechnology), and the images
were recorded with FUJIFILM-LAS-4000 luminescent image analyzer
(FUJIFILM Medical Systems Inc., USA). To ascertain comparative ex-
pression and equal loading of the protein samples, the membrane
stained earlier was stripped and re-probed with GAPDH antibody.
(1)
DL (%) = Amount of JQ1 encapsulated/Amount of polymer × 100%
(2)
To evaluate JQ1 in vitro release, formulations were prepared and
transferred into a dialysis bag with cutoff 1000 Da followed by placed
in pH 7.4 PBS solution. At regular time intervals, 1 mL release media
was collected and replaced by equal volume of fresh media. JQ1 con-
centration in release media was measured by HPLC-PAD as described
above.
2.5. Synthesis and characterization of ApoE mimetic peptide conjugated
PEG-PBC polymer
2.7. Cellular uptake of targeted nanoparticles
To measure the cellular uptake of NPs, HD-MB03 and DAOY cells
were seeded in the 6 well plate with density of 2 × 105 cells/well and
allowed to attach overnight. Culture medium was replaced with FBS-
free medium and coumarin 6 loaded ApoE-NPs or non-targeted NPs (1%
loading) were added with concentration of 100 μg/mL and plates were
keeping culture for 4 h. MB cells were then collected and analyzed
using flow cytometry.
Carboxyl
poly(ethylene
glycol)-block-poly(2-methyl-2-benzox-
ycarbonyl-propylene carbonate) (HOOC-PEG-PBC) was synthesized as
described previously [21]. Briefly, 2, 2-bis(hydroxymethyl)propionic
acid was stirred with benzyl bromide overnight to produce a white
solid, benzyl 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate. Tri-
phosgene was added to a round-bottom flask containing benzyl 2, 2-bis
(methylol)propionate and reacted for 3 h, yielding another white solid,
2-methyl-2-benzyloxycarbonyl-propylene carbonate (MBC). HOOC-
PEG-PBC was synthesized by 1, 8-diazabicyclo[5.4.0]undec-7-ene
(DBU) catalyzed ring-opening polymerization between HOOC-PEG-OH
and MBC. Finally, the amidation between HOOC-PEG-PBC
(0.044 mmol, 600 mg) and COG133 (0.048 mmol, 100 mg) afforded
COG133 conjugated polymer COG133-PEG-PBC in the presence of 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (0.66 mmol,
126.7 mg), N-hydroxysuccinimide (NHS) (0.33 mmol, 37.9 mg) and
trimethylamine (1.32 mmol, 132 mg). The chemical structure of
COG133-PEG-PBC was characterized by Proton Nuclear Magnetic Re-
sonance (1H NMR) and gel permeation chromatography (GPC) for de-
termining the molecular weight.
2.8. ApoE targeted drug delivery to brain tumor of orthotopic MB mouse
model
All the procedures involving animals were approved by the
Institutional Animal Care and Use Committee (IACUC) of the University
of Nebraska Medical Center and meet all the federal guidelines con-
cerning animal experimentation to ensure proper care and use of la-
boratory animals for research. HD-MB03 cells were transduced with
GFP and luciferase encoded lentivirus, sorted for GFP expression to
obtain a luciferase-expressing population (> 95%) and then expanded
in DMEM medium.
Luciferase expressing HD-MB03 cells were collected, washed, and
resuspended in PBS. A small hole, which situated 1 mm posterior of
lambdoid suture of the scalp over the cerebellum and 1 mm lateral of
sagittal suture, was drilled into the skull bone on anesthetized NSG
mice (females, 6–8 week old). 1 × 105 cells were stereotactically in-
jected 2 mm deep into cerebellum using Hamilton syringe.
2.6. Preparation and characterization of ApoE peptide conjugated JQ1
loaded nanoparticles
JQ1 loaded COG133 conjugated NPs (ApoE-NPs) were prepared by
mixing methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-
propylenecarbonate)-graft-dodecanol (mPEG-b-PCD) and COG133-PEG-
PBC at 70:30 w/w in acetone, followed by nanoprecipitation. Briefly,
3 mg COG133-PEG-PBC polymer, 7 mg mPEG-b-PCD and 1 mg of JQ1
were dissolved in 100 μL acetone followed by slowly injected into 1 mL
of PBS under magnetic stirring (1300 rpm) for 10 min at room tem-
perature. The solution was evaporated under reduced pressure to re-
move acetone and then filtered through 0.22 μm filter (Millipore) to
obtain the formulation. Mean particle size and size distribution were
measured by dynamic light scattering (DLS) using a Malvern Zetasizer
(Worcestershire, United Kingdom). To determine the surface mor-
phology and particle size of NPs by Atomic Force Microscopy (AFM),
the samples were diluted in PBS, deposited on mica modified with 1-(3-
aminopropyl)-silatrane (APS), rinsed with de-ionized (DI) water, and
dried with argon. Images were collected with MultiMode Nanoscope IV
system (Bruker Instruments, Santa Barbara, CA) in tapping mode at
ambient conditions. Silicon probes RTESPA-300 (Bruker Nano Inc., CA)
with a resonance frequency of ~300 kHz and a spring constant of
~40 N/m were used for imaging at scanning rate for about 2.0 Hz.
Images were processed using FemtoScan software (Advanced
Technologies Center, Moscow, Russia).
For quantification of JQ1 concentration in the brain and other major
organs, mice bearing orthotopic Group 3 MB were injected with JQ1
loaded ApoE mimetic peptide conjugated and non-targeted NPs. At 6
and 24 h post injection, blood was withdrawn via cardiac puncture
before mice were euthanized and major organs including the brain were
collected, weighed, and homogenized for extraction of JQ1 from the
tissue samples. Drug concentrations in the plasma and tissues were
measured using LC-MS/MS (4000 QTRAP, AB, Sciex Inc.). Briefly,
100 mg of tissue samples were homogenized in 5 times HPLC grade
water and spiked with OTX015 as an internal standard (IS) [22]. Sub-
sequently, 1.0 mL of acetonitrile was added, followed by vortexing and
high-speed centrifugation. The supernatant was evaporated to dryness,
and the residue were reconstituted with 100 μL of acetonitrile: water
(60:40, v/v). LC-MS/MS data acquisition will be performed by using
Analyst® software on a QTRAP 4000 mass spectrometer. The mass
spectrometer will be operated in the positively selected reaction mon-
itoring (SRM) for JQ1 (m/z 401.0) and internal standard (m/z 383.0).
2.9. Organ histology and liver injury markers
The encapsulation efficiency (EE) and drug loading (DL) was mea-
sured by HPLC after dissolving NPs using acetonitrile under the fol-
lowing conditions: mobile phase: Acetonitrile: water (80:20), C18
column (250 × 4.6 mm, 5 μm, Alltech, Deerfield, IL), flow rate 1 mL/
min) and column temperature was 25 °C. Drug loading and en-
capsulation efficiency were calculated using the following formulas:
Tissues of major organs such as liver, lung, kidney, heart and spleen
isolated and cut into pieces and were fixed in 10% paraformaldehyde
solution overnight and then embedded in paraffin. Sections of 5-mm
thickness were stained with hematoxylin and eosin (H&E) by using
standard protocols. Sections were scanned at 40× with the iScan HT
Slide Scanner (Ventana Medical Systems, Tucson, AZ), and
465

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Q. Wang, et al.
JournalofControlledRelease323(2020)463–474
representative views of sections are shown. Liver injury markers such as
alanine aminotransferase (ALT) and aspartate aminotransferase (AST)
levels in the serum were measured using the ALT and AST assay kits
(Universal Biologicals Ltd. UK).
JQ1 could significantly inhibit colony formation at 24 h post treatment
in both DAOY and HD-MB03 cells, with higher effect in inhibiting
colony formation in HD-MB03 cells compared to DAOY cells. These
results suggest that JQ1 is more effective in Group 3 MB.
2.10. Antitumor effect of ApoE targeted JQ1 loaded nanoparticles in
3.2. JQ1 downregulated c-MYC and GLI1 expression
orthotopic MB mice
To confirm whether JQ1 can be used to treat both Group 3 and SHH
driven MB, we determined the effect on c-MYC and GLI1 expression at
mRNA and protein levels after incubating HD-MB03 and DAOY cells
with 400 nM and 4 μM of JQ1 for 48 h, respectively. As shown in
Fig. 2A, there was significant decrease in c-MYC and GLI1 mRNA ex-
pression compared to those of un-treated control cells. Similarly, the
expression of c-MYC and GLI1 at protein level in both DAOY and HD-
MB03 cells was inhibited upon treatment with JQ1 for 48 h determined
by Western blot analysis. There was also significant inhibition of n-
MYC, which is downstream signals of GLI1, expression at protein level
in both these cell lines (Fig. 2B and S1).
Orthotopic MB tumor was generated in NSG mice using stably lu-
ciferase expressing HD-MB03 cells. When the bioluminescence reached
2 × 106 p/s/cm2/sr, mice were divided into 3 groups (n = 5): i) control
group, ii) non-targeted NPs loaded with JQ1, and iii) ApoE-NPs loaded
with JQ1. Formulations were injected via tail vein at the dose of 10 mg/
kg every third day for four weeks. Mice were imaged by IVIS imaging
system to monitor tumor growth. At the end of studies, mice were sa-
crificed and main organs were removed, fixed with 4% formaldehyde
solution and embedded in paraffin. Effectiveness of the proposed
therapy was determined by measuring apoptosis and metastasis. The
slices tissues were stained by hematoxylin and eosin (H&E). Brain tis-
sues were further stained with c-MYC, Ki67 and caspase-3 on MB
samples by immunohistochemistry.
3.3. Cell cycle arrest and apoptosis
Cell cycle arrest was determined by propidium iodide (PI) staining
after incubating DAOY and HD-MB03 cells with JQ1 for 48 h. For DAOY
cells, results showed that treatment with JQ1 at 4 μM increased cell
arrest in G1 phase compared to control group (66.9% vs. 42.2%),
whereas small number of cells were arrested in S-phase (16.2%) and G2
phase (16.8%). Treatment of HD-MB03 cells with JQ1 at 400 nM caused
69.9% cell arrest in G1 phase from 50.1% in the control group, whereas
only 13.1% of these cells were arrested in S-phase, and 17.0%, re-
spectively in G2 phase (Fig. 3A and B). Further, apoptotic cell percen-
tage was determined using a flow cytometer after staining with Alexa
Fluor 647-Annexin V and PI. For DAOY cells, apoptotic cell population
was significantly increased when the cells were treated with JQ1 at
4 μM compared to the control group (25.5% vs. 8.5%, respectively).
Further, the apoptotic cell population increased to 26.6% from 8.26%,
respectively when HD-MB03 cells were treated with 400 nM JQ1
(Fig. 3A and B).
2.11. Statistical analysis
Data were expressed as mean
S.D. Differences between groups
were analyzed by one-way analysis of variance (ANOVA) and Tukey
multiple comparisons tests. *p < .05 was considered significant, and
⁎⁎p < .01, ⁎⁎⁎p < .001 were considered highly significant.
3. Results
3.1. Anticancer activity and inhibition of colony formation
Cytotoxicity of JQ1 on MB cell lines was determined in HD-MB03
and DAOY cells by incubating these cells at different drug concentra-
tions. As shown in Fig. 1A, there was dose-dependent cell killing, with
the IC50 values of 402 nM and 4.22 μM in HD-MB03 and DAOY cells,
respectively. The inhibitory effect of JQ1 on tumorigenic potential in
MB cells was determined by colony formation assay by incubating these
cells at two different concentrations: 2 and 4 μM for DAOY cells, but
only 400 and 800 nM for HD-MB03 cells. JQ1 greatly reduced colony
formation in a dose dependent manner (Fig. 1B). As shown in Fig. 1C,
3.4. Copolymer synthesis, formulation and characterization of JQ1 loaded
nanoparticles
COG133-PEG-b-PBC copolymer was synthesized as shown in
120
C)
120
A)
DAOY cell
DAOY
B)
Control
JQ1 2µM
JQ1 4µM
100
80
60
40
20
0
90
60
30
0
**
**
4
DAOY
IC50 4.22µM
Control
2
1
2
3
4
5
JQ1 (µM)
Log10 Conc. (nM)
JQ1 400nM
Control
JQ1 800nM
120
100
80
60
40
20
0
120
90
60
30
0
HD-MB03
HD-MB03 cell
**
HD-MB03
**
IC50 402nM
0
1
2
3
4
Control
400
JQ1 (nM)
800
Log10 Conc. (nM)
Fig. 1. A) Cytotoxic effect of JQ1 on HD-MB03 and DAOY cells. IC50 was calculated to be 402 nM and 4.22 μM in HD-MB03 and DAOY cells, respectively. B) Effect of
JQ1 on colony formation in HD-MB03 and DAOY cell, while DAOY cells were treated at the doses of 0, 2 and 4 μM, we treated HD-MB03 cells with 0, 400 and 800 nM
of JQ1 for 24 h. C) Effect of JQ1 on colony formation by quantified crystal violet. **, p < .01 (comparison between control vs JQ1 treated). (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
466