ELUCIDATION OF THE ROLE OF NOTCH PATHWAY IN DOXORUBICIN-

INDUCED CARDIOTOXICITY

By

Hind Nasser Alotaibi

1. ABSTRACT
Doxorubicin (DOX) is one of the most effective chemotherapeutic drugs; however, its
incidence of cardiotoxicity compromises its therapeutic effect. Notch pathway has been
reported to contribute to DOX cancer resistance. Therefore; inhibitors of Notch pathway arise
as promising remedies to ameliorate DOX resistance. Notch pathway plays an essential role
in neonatal cardiomyocyte development; however, its role in adult cardiomyocyte and its role
in DOX cardiotoxicity are not identified. We hypothesize that Notch pathway has a
protective effect against DOX-induced cardiotoxicity, and interfering with the pathway by
using γ-secretase inhibitors (GSIs) such as BMS-906024 may exacerbate DOX-induced
cardiomyocyte damage. To examine this hypothesis, a series of experiments are designed
using different schedules of DOX, and different doses of BMS-906024. Parameters indicative
to DOX-induced myocardial damage, oxidative stress, DOX provoked inflammatory, and
apoptotic effect will be evaluated in rats. The Notch pathway related parameters will be
performed using quantitative real-time polymerase chain reaction (RT-PCR), Western
blotting, and immunohistochemistry. We believe our study will have a great clinical value by
highlighting the possible unwanted, synergic cardiotoxic effect of DOX and BMS-906024
combination therapy and suggesting the need for a tumor-targeted therapy for BMS-906024.

2. INTRODUCTION
Doxorubicin (DOX) is well known and highly effective anti-neoplastic agent. It is used
to treat many cancers, including solid tumors, leukemia, and lymphoma (1). The anticancer
effect of DOX is believed to be achieved through two major mechanisms; (i) intercalating
into deoxyribonucleic acid (DNA) and inhibition of topoisomerase-II-mediated DNA repair
and (ii) generation of free radicals leading to DNA, cellular membranes, and proteins damage
(2). Although DOX is currently one of the most effective chemotherapeutic agents, its
clinical use is limited by associated high-incidence cardiotoxicity which has irreversible
consequences (3). Although the pathogenesis of cardiotoxicity is not clear, a solid body of
evidence indicates that oxidative stress, inflammation and apoptosis are involved with
oxidative stress remains the cornerstone. Several observations suggest that DOX interact with
iron, inducing reactive oxygen species (ROS) production. ROS ultimately lead to oxidative
damage of cellular and mitochondrial membranes and cellular macromolecules with
consequent strong inflammatory response which enhances cardiac damage (4). Another
limitation to DOX use is the development of resistance in treated cancer cells. Drug

2
resistance has been linked to upregulation of efflux molecules; ABC transporters; and
activation of a variety of pathways including FOXO3a, PI3K/Akt, and NF-κB. Recently, the
Notch pathway has been found to play a role in DOX resistance (4).
The Notch signaling pathway is a highly conserved signal transduction mechanism. It
is essential to normal embryonic development, cellular proliferation, specification, and
differentiation (5). In mammals, there are four receptors; Notches 1~4, and five ligands;
Delta-like ligand 1 (DLL1), 3 (DLL3), 4 (DLL4), Jagged-1 (JAG1) and Jagged-2 (JAG2) (6).
Notch signaling is triggered by direct interaction of receptors with ligands expressed on
neighboring cells. This releases the Notch intracellular domain (NICD) from the membrane
after cleavage by gamma-secretase (7). NICD translocates into the nucleus, where it
modulates transcription in the receiving cells via recombinant signal binding protein 1 for Jκ
(RPB-Jκ) transcription factor. The most prominent Notch target genes are the hairy and
enhancer of split (Hes) and hairy and enhancer of split with YRPW motif (Hey) families,
which are negative regulators of transcription (Figure 1). While Hes genes are essential in
neural and endocrine functions, Hey genes play a critical role during the development of the
cardiovascular system. Other well-known Notch targets include cyclin D1, cyclin A and
transcription factors of the nuclear transcription factor-kB (NF-kB) family. The directly and
indirectly Notch-regulated genes and proteins are very large and still new targets are being
discovered. The result of Notch activation is cell context dependent and the output is strongly
affected by timing, duration, and dose of activation (8).
Notch pathway plays an essential role in the cardiovascular system; especially during
embryonic development. Notch pathway also plays an important role in preserving
endothelium integrity by protecting endothelial cells from apoptosis. However, the role of
Notch pathway in cardiomyocyte biology after birth is still not well characterized (8). In
addition, Notch pathway has been found to be activated in the majority of solid tumors and
leukemias and thus plays a significant role in cancer chemoresistance by inhibiting cancer
cells apoptosis induced by treatments (6). It has been found that Notch pathway was
dysregulated upon DOX treatment in cancer cells, and plays a significant role in DOX
resistance (9). Claims for the Co-administration of inhibitors of Notch pathway to minimize
DOX resistance are emerging (10).
However, with possible role of Notch pathway in cardiovascular system, and limited
knowledge about effect of DOX on Notch activity in cardiomyocytes; we hypothesize that
such combination exacerbates DOX cardiotoxic effects.

3
 Figure1. Modified schematic illustration of the Notch signaling pathway (11).

Research objectives
The aim of this study is to elucidate the role of Notch pathway in DOX- induced
cardiotoxicity and the impact of blocking Notch pathways using a γ-seretase inhibitor (BMS-
906024) on DOX- cardiotoxicity using rat model, exploring the following objectives:
a) Investigate the potential role of Notch pathway in DOX- induced cardiotoxicity by
measuring expression of Notch1 receptor, Hes1, Hey1, and NICD in rat
cardiomyocytes.
b) Study the effect of BMS-906024 on DOX-induced cardiotoxicity by measuring
cardiotoxicity markers, oxidative stress related parameters, antiapoptotic parameters,
and histopathological examination in rats.

3. RESEARCH STRAEGY
3.1 SIGNIFICANCE
Notch pathway plays a significant role in cardiovascular system. Notch signaling is
critical in mammalian cardiogenesis and mutations in this signaling pathway are linked to
human congenital heart disease (11). Stem cells precursors of cardiomyocytes express high
levels of Notch1 receptor and active Notch signaling is required for their proliferation. The
role of Notch pathway in cardiomyocyte biology after birth is still not well characterized.
Neonatal rat cardiomyocytes were isolated at birth express high levels of Notch1 and are
actively proliferating, but after many passages in culture, Notch1 expression becomes
undetectable and these cells lose their proliferative ability (12). However, some studies have
shown that Notch1 was reactivated in cardiomyocytes in ischemic heart and reduced
apoptosis by activating Akt pathway (13). Similarly, Notch signaling was absent under
normal physiological conditions in adult rat myocardium, but it become transiently

4
reactivated in cardiomyocytes, following a myocardial infarct. These studies indicate a role
for Notch pathway in the repair of damaged myocardium. In agreement with this hypothesis,
infarction size, fibrosis, and hypertrophic response were increased in transgenic mice
overexpressing the Notch ligand Jagged1 on cardiomyocytes, compared to wild type animals.
Among the molecular mechanisms identified to explain Notch protective activity on the
damaged myocardium is increased cardiomyocyte survival, stimulation of angiogenesis and
augmented number of cardiac precursor cells (14). Moreover, expression of Notch signaling
components has been observed in myocardium biopsies from heart failure patients. In
addition, Notch plays an important role in preserving endothelium integrity by protecting
endothelial cells from apoptosis induced by conditions such as inflammation, and ischemia
(15).
In contrast, Campa et al, 2008 have shown that forced activation of Notch in mature
cardiomyocytes is associated with cell cycle progression block and apoptosis, indicating that
a prolonged and uncontrolled Notch activation can be lethal for these cells. In conclusion,
active Notch signaling is required for proliferation of cardiac stem cells and survival of
cardiomyocytes, but timing and dosing of activation have to be tightly controlled to avoid
negative effects on cell survival (8). Several Notch inhibitors are being now under
investigation. They act by inhibiting gamma secretase enzyme which is responsible for the
cleavage of NICD and activationg Notch signaling. Therefore, γ-secretase inhibitors (GSI)
are considered Notch1 blockers. γ-secretase has now been proposed to be a therapeutic target
in various cancers, immunologic disorders, vasculitis, diabetic nephropathy, ischemic
reperfusion injury in the kidney, ischemic stroke, hearing loss, and fibrosis. It is also likely
that additional disease indications may emerge. Currently, a main focus of the repurposing of
GSIs has been in cancer with multiple human trials underway. GSIs may be used as
monotherapy and combination therapies with other agents are being explored. Combination
of GSIs with DOX in breast cancer trial enhanced DOX sensitivity (10). For example of a γ-
secretase inhibitor (GSI) with anti-notch activity, BMS-906024 which is consider as potent
notch inhibitor for the treatment of leukemia and solid tumors (16).
Considering the role played by Notch in cardiomyocyte survival, our study will
provide new insight on the consequences of the use of BMS-906024 as an additive agent to
DOX treatment which may worsen the cardiotoxic effect of DOX, suggesting that BMS-
906024 when it is given with DOX it should be injected directly to the tumor or by using
tumor-targeted delivery formulation to avoid the unwanted effect on the heart.

5
3.2 APPROACH
3.2.1 Materials and kits

DOX and BMS 906024 authentic powders will be purchased from Sigma-Aldrich
(MO, USA). Troponin-I (cTnI) kits will be provided from Biosource Europe (S.A., Belgium).
Creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) will be purchased from
Randox Laboratory Ltd (Crumlin, UK). Glutathione peroxidase (GPx), superoxide dismutase
(SOD), and catalase (CAT) kits will be provided from Cayman (MI, USA). Enzyme-linked
immunosorbent assay (ELISA) kits for the determination of tumor necrosis factor alpha
(TNF-α) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), bcl-2–
associated X protein (Bax), and b-cell lymphoma 2 (BCL-2) will be purchased from R&D Co
(Minneapolis, USA). For immunohistochemistry, the primary antibodies for alpha-smooth
muscle actin (α-SMA), NICD, inducible nitric oxide synthase (iNOS), and Hes1 proteins will
be purchased from Biovision (Ca, USA); Santa Cruz Biotechnology (Ca, USA) and pierce
antibody product (IL, USA), respectively; For western blot; the primary antibodies for
Notch1, and Hey1 will be purchased from Santa Cruz Biotechnology (CA, USA) and
secondary antibodies will be provided from Sigma-Aldrich (MO, USA). For real time
polymerase chain reaction (RT-PCR) RNeasy Plus Mini Kit reagents will be purchased from
Qiagen China Co. (Shanghai, China), TaqMan Reverse Transcription Reagents, and SYBR
Green PCR Master Mix from Applied Biosystems Life Technologies (CA, USA).

3.2.2 Animals
Wistar albino rats (male) weighing (200-220) g will be purchased from Experimental
Animal Care Center, College of science, King Saud University. Animals will be kept in
special cages and maintained under a constant lighting cycle (12 h-light and 12 h-dark) with
air conditioning at 20+2 °C and 65% humidity for about one week before the experiment for
acclimatization. The experimental protocol will be conducted according to the Local Ethics
Committees at King Saud University. We already have applied for an ethical approval from
Scientific Research Ethics Committee at King Saud University.

6
3.2.3 Experimental design
3.2.3.1 : Optimization of the model of Dox-induced cardiotoxicity
Two schedules of Dox-induced cardiotoxicity; a single dose schedule adopted from
Matawy et al, 2014 (18), and three doses schedule adopted from Arola et al, 2000 (19) will
be compared. Three groups of animals will be used; six animals per group as following:
Group I: Control will receive normal saline.
Group II: Will receive 3 doses of DOX at 1st, 3rd and 5th day (5 mg/Kg) (19).
Group III: Will receive a single dose of DOX (15 mg/Kg) (18).
Two days after DOX treatment, animals will be scarified; serum and heart will be
obtained. CK-MB and LDH will be assayed to evaluate cardiotoxicity. Model that induces
cardiotoxicity will be used for further experiments.
3.2.3.2 : Dose-response curve for selected γ-seretase inhibitor; BMS-906024
Six groups of animals; 6 animals per group; will be used as following:
Group I: Control will receive only vehicle; normal saline with 0.5% DMSO.
Group II: DOX; dose and schedule selected based on previous experiments.
Group III: Will receive BMS-906024 0.5 mg/kg/d for 14 days followed by DOX.
Group IV: Will receive BMS-906024 1 mg/kg/d for 14 days followed by DOX.
Group V: Will receive BMS-906024 2 mg/kg/d for 14 days followed by DOX.
Group VI: Will receive BMS-906024 4 mg/kg/ d for 14 days followed by DOX.
Two days after last DOX dose, animals will be sacrificed; serum and hearts will be
obtained. LDH and CK-MB will be used to compare cardiotoxicity in different groups. Dose
that shows a greater significant difference from DOX will be selected for further experiments.
3.2.3.3: Experimental study of the effect of DOX and BMS-906024 on cardiotoxicity
and Notch pathway
Rats will be randomly divided into five groups; 6 rats in each as following:
Group I: Control rat will receive only the drug vehicle.
Group II: DOX group will be treated with DOX to induced cardiotoxicity; dose will be
selected based on experiments in 3.2.3.1.
Group III: DOX + γ-secretase inhibitor; BMS-906024; rats will be treated for 14 days with
BMS-906024; dose will be selected based on experiments in 3.2.3.2. DOX will be injected
I.P. according to selected protocol from previous experiment.
Group IV: BMS-906024 rats; will be treated with BMS-906024 only for 14 days; dose will
be selected based on previous experiment.

7
Group V: Dexrazoxane + DOX rats; will be treated with Dexrazoxane (60 mg/kg) for 14
days; on the 15th DOX will be injected(1).

8
3.2.4 Collection of blood sample
At the end of the study, all rats will be re-weighed, anesthetized, and sacrificed by
decapitation. Trunk blood samples will be collected, allowed coagulating, centrifuged at 3000
rotation per minute (rpm) at 4°C for 30 minutes to obtain the serum. The serum samples will
be divided into aliquots and stored at -80°C until biochemical analysis (20).

3.2.5 Separation of heart tissue

The hearts will be rapidly removed, rinsed with ice-cold normal saline and weighed.
The ratio of heart weight to body weight (HW/BW) will be calculated. Half of at least three
heart tissues from each group will be fixed in 10% formalin for histopathological and
immunohistochemical studies. The remaining excised heart tissues will be frozen in liquid
nitrogen and will be stored at -80°C for further biochemical studies (20).

3.2.6 Histopathology studies

Small pieces of heart ventricle tissues from all treatment groups will be fixed with
10% buffered formalin and then embedded into paraffin and cut into 5 μm sections. The
sections will then be stained with routine hematoxylin and eosin (H&E) for histopathological
changes such as inflammation, necrosis, fibrosis, or hypertrophy as described earlier (21).

3.2.7 Determination of cardiac injury biomarkers

Serum level of cardiac troponin-I (cTnI), as an indicator for cardiotoxicity and
hypertrophy, will be determined using rat troponin-I ELISA kit from MyBioSource,Inc. (San
Diego, CA) according to manufacturer’s instructions.

3.2.8 Evaluation of the parameters related to the antioxidant activity

Glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD)
activity will be estimated colorimetrically according to manufacturer’s instructions.

3.2.9 Evaluation of the anti-inflammatory parameters

NF-κB and TNF-α level will be examined by using ELISA kits obtained from
DuoSet, R&D Systems; (MN, USA) according to manufacturer’s instructions.

9
3.2.10 Evaluation of apoptosis-related parameters
BCL-2, BAX protein level in heart tissues by ELISA kits according to the
manufacturer's instructions. DNA fragmentation will be quantified by measuring the
cytosolic nucleosomes using a Cell Death ELISA kit from Roche Diagnostics GmbH
(Indianapolis, USA).

3.2.11 Evaluation of the parameters related to Notch signaling pathway

Notch1 and Hey1 protein expression will assess by western blot. For extraction of
total protein, tissue homogenate will be mixed with HEPES (pH 7.9) containing 420 mM
NaCl, 1.5 mM MgCl2, 0.1 mM EGTA, 0.5 mM DTT, 5% glycerol, and Halt protease
inhibitor cocktail for 1 h at 4°C , then centrifuged at 15,000 g for 15 min at 4°C. Then the
supernatant will be assayed for protein concentration using the Direct Detect milipore which
is used for Western blot analyses. A 20 µg aliquot of each protein sample will be separated
on a 4 to 11% Tris-glycine gradient gel, and then transferred to nitrocellulose membranes.
Then block of the membranes with 5% skim milk in TBS-T (Trypsin buffered saline, 1%
Tween-20) buffer for 60 min. The Notch1, and Hey1 antibodies will be added at
concentration (1 1000) and the blots to be incubated for 1 hour in room temperature. Then
washing for 10 minutes in TBS-T will be performed for three times, and then incubation will
be carried out for 1 hour with the secondary horseradish-peroxidase-conjugated goat anti-
mouse antibodies. Chemiluminescence (Sigma) detection kits will be used to detect the blots.
Then the chemiluminescence will be captured on Image Quit (22).
Hes -1 and Hey-1 gene expression will be measured by RT-PCR. For isolation of total
ribonucleic acid (RNA) from frozen tissues the Trizol reagent Invitrogen (Carlsbad, CA,
USA) will be used according to the manufacturer’s instructions. Three micrograms of total
RNA will be reverse transcribed (RT) into complementary DNA (cDNA) using a kit. After
reverse transcription, cDNA will be used for semi-quantitative PCR by sets of specific
primers.

3.2.12. Determination of smooth muscle α-actin, NICD, iNOS, and Hes1 using
immunohistochemistry
Heart sections will be incubated overnight at 4°C with either anti-smooth muscle α-
actin (α-SMA), anti-NICD antibody, anti Hes1 antibody, and anti-iNOS antibody. Detection
of the primary antibody will be carried out by using secondary antibodies. Incubation with (3,

10
3′-diaminobenzidine tetrachloride), heart tissues will develop the color and then visualized by
light microscope (24).

3.2.13 Statistical analysis

Data will be presented as the mean ± SE. One way analysis of variance (ANOVA)
followed by Tukey Kraimer Multiple Comparison Test is going to be used to compare
differences between the means of the different groups. A P value of 0.05 or less will be set as
target level of significance.

4. TIMELINE

Task/Month 1 2 3 4 5 6 7 8 9 10 11 12
1. Experimental work. √ √ √
2. Measurement of √ √
cardiotoxicity parameters; CK-MB,
LDH, and cTnI.
3. Measurement of CAT, SOD, √
and GPx.
4. Histopathological examination √
by H&E.
5. Measurement of ELISA of √
NF-κB, TNF-α, BAX, and BCL-2.
6. Immunohistochemistry of √ √ √ √
Hes1, iNOS, NICD, and α-SMA.
7. RT-PCR of Hey-1, and Hes-1. √ √ √ √
8. Western blot of Notch1, and √ √
Hey1.
9. Data analysis. √ √
10. Writing the thesis. √ √

11
5. REFERENCES
1. Octavia Y, Tocchetti CG, Gabrielson KL, et al. Doxorubicin-induced
cardiomyopathy: from molecular mechanisms to therapeutic strategies. Journal of molecular
and cellular cardiology. 2012;52(6):1213-25.
2. Thorn CF, Oshiro C, Marsh S, et al. Doxorubicin pathways: pharmacodynamics and
adverse effects. Pharmacogenetics and genomics. 2011;21(7):440.
3. Polegato BF, Minicucci MF, Azevedo PS, et al. Acute doxorubicin-induced
cardiotoxicity is associated with matrix metalloproteinase-2 alterations in rats. Cellular
physiology and biochemistry. 2015;35(5):1924-33.
4. Sims JT, Ganguly SS, Bennett H, et al Imatinib reverses doxorubicin resistance by
affecting activation of STAT3-dependent NF-κB and HSP27/p38/AKT pathways and by
inhibiting ABCB1. Plos one. 2013;8(1):e55509.
5. Chen Y, Zheng S, Qi D, et al. Inhibition of Notch signaling by a γ-secretase inhibitor
attenuates hepatic fibrosis in rats. Plos one. 2012;7(10):e46512.
6. Yuan X, Wu H, Xu H.Notch signaling: an emerging therapeutic target for cancer
treatment. Cancer letters. 2015;369(1):20-7.
7. Aithal MG, Rajeswari N. Role of Notch signalling pathway in cancer and its
association with DNA methylation. Journal of genetics. 2013;92(3):667-75.
8. Campa, V. M., et al. Notch activates cell cycle reentry and progression in quiescent
cardiomyocytes. Journal of cell biology.2008;183(1): 129-141.
9. Mei H, Yu L, Ji P. Doxorubicin activates the Notch signaling pathway in
osteosarcoma. Oncology letters. 2015;9(6):2905-9.
10. Wang Z, Li Y, Ahmad A. Targeting Notch signaling pathway to overcome drug
resistance for cancer therapy. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer.
2010;1806(2):258-67.
11. Sandberg WJ. Activation of Notch signaling in cardiomyocytes during post-infarction
remodeling. Scandinavian Cardiovascular Journal. 2010;44(6):359-66.
12. Gude NA, Emmanuel G, Wu W. Activation of Notch-mediated protective signaling in
the myocardium. Circulation research. 2008;102(9):1025-35.
13. Nemir M, Metrich M, Plaisance I, et al. The Notch pathway controls fibrotic and
regenerative repair in the adult heart. European heart journal. 2012;35(32):2174-85.
14. Gude N, Sussman M. Notch signaling and cardiac repair. Journal of molecular and
cellular cardiology. 2012;52(6):1226-32.

12
15. Chanoit G, Lee S, Xi J, et al. Exogenous zinc protects cardiac cells from reperfusion
injury by targeting mitochondrial permeability transition pore through inactivation of
glycogen synthase kinase-3β. American journal of physiology-heart and circulatory
physiology.2008;295(3):H1227-H33.
16. Gavai AV, Quesnelle C, Norris D, et al. Discovery of clinical candidate BMS-906024:
a potent pan-notch inhibitor for the treatment of leukemia and solid tumors. ACS medicinal
chemistry letters.2015;6(5):523-7.
17. Gu J-W,Rizzo P,Pannuti A, et al. Notch signals in the endothelium and cancer stem-
like cells: opportunities for cancer therapy. Vascular cell.2012; 4(1): 7.
18. Mantawy EM, El-Bakly WM, Esmat A, et al. Chrysin alleviates acute doxorubicin
cardiotoxicity in rats via suppression of oxidative stress, inflammation and apoptosis.
European journal of pharmacology. 2014;728:107-18.
19. Arola OJ, Saraste A, Pulkki K, et al. Acute doxorubicin cardiotoxicity involves
cardiomyocyte apoptosis. Cancer research.2000;60(7):1789-92.
20. Yao, Q.-y., et al. Inhibition by curcumin of multiple sites of the transforming growth
factor-beta1 signalling pathway ameliorates the progression of liver fibrosis induced by
carbon tetrachloride in rats. BMC complementary and alternative medicine.2012; 12(1):
15620.
21. Kapoor P, Deshmukh R. VEGF: A critical driver for angiogenesis and subsequent
tumor growth: An IHC study. Jourmal of oral maxillofac pathology.2010;16(3):330-7.
22. Aithal, M. G. and N. Rajeswari. Role of Notch signalling pathway in cancer and its
association with DNA methylation. Journal of genetics. 2013;92(3): 667-675.
23. Kumar, G. M., et al. Extraction of RNA from fresh, frozen, and lyophilized tuber and
root tissues. Journal of agricultural and food chemistry.2007; 55(5): 1674-1678.
24. Zhou H-M, Zhong M-L, Zhang Y-F, et al. Natakalim improves post-infarction left
ventricular remodeling by restoring the coordinated balance between endothelial function and
cardiac hypertrophy. International journal of molecular medicine. 2014;34(5):1209-18.

13