Doxorubicin (DOX

Results and discussion for pathway maps by bioinformatics tools
INTRODUCTION
Doxorubicin (DOX, also named Adriamycin) is a commonly used chemotherapeutic agent for
treating hematologic and tumor malignancies including breast cancer, leukemia, and
sarcomas. The mechanism of the anti-tumor action of DOX is probably mediated through the
inhibition of the macromolecule synthesis resulting from its intercalation into the DNA
structure. Despite DOX is extensively used in clinical cancer chemotherapy, it is known to
induce organ toxicities in which cardio toxicity results in cardiomyopathy and sequent fatal
congestive heart failure (78) . In regard to the action of DOX causing cardiotoxicity, several
cellular mechanisms have been proposed to account for the pathogenesis of cardiomyopathy
induced by DOX. These include cardiac cell apoptosis (62; 70) , concomitant cardiac
fibrosis (57; 93) , altered cardiac energy metabolism (11; 56) , and changes in sarcolemmal
calcium transport and cardiac calcium handling (64; 83) . By further understanding the
underlying mechanisms of the DOX-induced cardiotoxicity, it is of hope new therapeutic
strategies can be developed to prevent or mitigate the detrimental effect of DOX on cardiac
function during chemotherapeutic treatment.
Animal
Male C57BL/6 mice obtained from the Laboratory Animal Services Centre were used. Mice
were housed in a temperature and humidity controlled environment and were exposed to a
12:12-hours light: dark cycle.
Experimental Protocol
Mice were randomly assigned to one of the following groups: Saline (n = 10) DOX (DOX,
doxorubicin; n = 11). Mice assigned to DOX group were exposed to a single intraperitoneal
(i.p.) injection of DOX at a dose of 25 mg/kg to induce cardiotoxicity as shown in a previous
study (77) . After the ten days experimental period, mice were euthanized by overdose of
ketamine and xylazine. Heart was immediately removed and washed with cold phosphate
buffered saline (PBS). Total mRNA was extracted from mice right ventricle in DOX and control
groups for microarray analysis. Significantly regulated genes were put into the Gene Ontology
for pathway analysis.

DISCUSSION OF PATHWAY MAPS INVOLVED IN DOX CARDIOTOXICITY
Doxorubicin-induced cardiomyopathy is mediated via various pathways that synergistically
work together leading to cardiotoxicity eventually resulting to congestive heart failure.
However, in these pathways there are both down regulation and up regulation of a variety of
genes that are expressed into decreased or increased levels of their target molecules
(Chandler, et al. 2007). The variations in levels of molecules that are expressed from the
genes that are either down regulated or up regulated by doxorubicin are the ones attributable
to the cardiotoxic effects of doxorubicin. However, these genes have extensive interactions
that constitute a pathway map where a multitude of genes are involved. However, in this
discussion a few pathway maps will be considered, and only a limited number of genes will be
focused on for discussion in each pathway map selected. The pathway maps to be
considered are discussed below:

  1. Cell adhesion_ECM remodeling
    Cell adhesion ECM remodelling is one of the most significant pathways involved in
    doxorubicin cardiomyopathy mediated by its cardiotoxic effects irrespective of its wide use in
    cancer chemotherapy where a number of genes are either up regulated or down regulated. In
    particular, Collagen I, Collagen III and Osteonectin are down regulated meaning they are
    expressed in lower levels than the physiological levels (Chandler, et al. 2007). However,
    Kallikrein 3 is up regulated through doxorubicin administration meaning expression levels are
    higher than physiological levels. The pathway map is shown below:
  2. Map: Cell adhesion_ECM remodeling
    ( TOC )

Fig. 1: The top scored map (map with the lowest pvalue) based on the enrichment distribution
sorted by ‘Statistically significant Maps’ set. Experimental data from all files is linked to and
visualized on the maps as thermometerlike figures. Upward thermometers have red color and
indicate upregulated signals and downward (blue) ones indicate downregulated expression
levels of the genes.
In pathway map illustrated in Fig.1 above indicate that there are four genes that are affected
by doxorubicin administration either through up regulation or down regulation and they include
Collagen I, Collagen III, Osteonectin and Kallikrein 3. The first three genes are down
regulated while the last one is up regulated and the pathways through which these genes
impart their effects in doxorubicin-induced cardiomyopathy are discussed below:
Collagen I
Collagen I gene is responsible for encoding collagen I which is the most abundant form in
human body. However, the transcription of collagen I gene is up regulated by stromelysin-2,
MMP-1, MMP-9 and MMP-13. Collagen I gene activation mediates it activity through integrin
outside-in signaling. For instance, MMP-9 and MMP-13 both of which activates collagen I
gene also activates collagen IV which leads to down regulation of alpha-1/beta-1 integrin as a
result of integrin outside-in signaling up regulation. MMP-9 which is responsible of collagen I
down regulation activation is also responsible for the down regulation of IL-8 and IL8RA
eventually leading to up regulation of chemotaxis in cardiomyocytes. Furthermore, MMP-9 up
regulates verscan gene leading to down regulation of CD44 which then down regulates
matrilysin (MMP-7) which subsequently down regulates HB-EGF. HB-EGF then down
regulates ErbB4 and EGFR both of which up regulates the activity of ERBB-family signaling.
In addition, EGFR also up regulates the activity of EGF signaling pathway.
Collagen III
Collagen III gene is responsible of encoding collagen III. Down regulation of collagen III gene
is mediated by MMP-1, MMP-13 and stromelysin-2. However, the down regulation of collagen
III which seem to act through similar mechanism with collagen I is centrally controlled by

MMP-13 which is up regulated by TIMP1, TIMP2 and TIMP3 while at the same time it is also
down regulated by stromelysin-1 and plasmin. However, MMP-2, MMP-9 and MMP-13 are
responsible of collagen IV up regulation which subsequently leads to down regulation of
alpha-1/beta-1 integrin eventually leading to up regulation of integrin outside-in signaling
through which it mediates its effects during doxorubicin-induced fibrosis which then results to
cardiomyopathy. MMP-13 which is responsible of collagen III up regulation is also responsible
of MMP-9 down regulation which then down regulates IL-8 and IL8RA eventually leading to
up regulation of chemotaxis in cardiomyocytes. Furthermore, MMP-9 up regulates verscan
gene leading to down regulation of CD44 which then down regulates HB-EGF. HB-EGF then
down regulates ErbB4 and EGFR both of which up regulates the activity of ERBB-family
signaling. In addition, EGFR also up regulates the activity of EGF signaling pathway.
Osteonectin
Osteonectin gene is another gene which is down regulated by Stromelysin-1 as a result of
doxorubicin administration. However, osteonectin which is secreted by osteoblasts shows
affinity for collagen as well as mediating functions such as collagen binding and cell-matrix
interactions. Moreover, osteonectin is also involved in increasing the production and activity of
matrix metalloproteinases (MMP), enzymes that play a crucial role in collagen cleavage
(Chandler, et al. 2007).
Kallikrein 3
Kallikrein 3 up regulation is another effect caused by doxorubicin administration in order to
mediate the action of doxorubicin in cardiac fibrosis one of the mechanisms responsible of
doxorubicin-induced cardiomyopathy. However, up regulation of kallikrein 3 gene caused by
doxorubicin administration is down regulated by kallikrein 2 and both kallikrein 2 and kallikrein
3 are responsible of up regulation of IBP4. Subsequently, IBP4 leads to up regulation of IGF-1
and IGF-2 both of which lead to down regulation of IGF-1 receptor eventually leading to up
regulation of the activity of IGF-1R signaling. These pathways are responsible of the
mechanisms through which kallikrein 3 mediates its effects in doxorubicin-induced
cardiomyopathy.

  1. Immune response_IL 13 signaling via JAK STAT
    Cardiac inflammation is one of the most significant mechanisms underlying the development
    of cardiomyopathy. Therefore, cardiac inflammation in doxorubicin-induced cardiomyopathy
    there are several inflammatory markers that have been used to diagnose, evaluate and
    monitor cardiac inflammation such as those shown in Fig. 2 below (Chandler, et al. 2007).
    However, such molecular markers are expressed from various genes that are either up
    regulated or down regulated by doxorubicin in doxorubicin-induced cardiomyopathy. In this
    pathway map, three key genes such as: MMP-8, RNS8 and COL1A whose expression
    products mediate cardiomyocytes inflammation in cardiotoxicity induced by doxorubicin
    eventually leading to cardiomyopathy and congestive heart failure are discussed. The

respective pathways through which these genes impart there effects are highlighted and
discussed in order to provide an overview of the entire pathway map involved in cardiac
inflammation during doxorubicin-induced cardiomyopathy.

  1. Map: Immune response_IL13 signaling via JAKSTAT
    ( TOC )

Fig. 2: The second scored map (map with the third lowest pvalue) based on the enrichment
distribution sorted by ‘Statistically significant Maps’ set. Experimental data from all files is
linked to and visualized on the maps as thermometerlike figures. Upward thermometers have
red color and indicate upregulated signals and downward (blue) ones indicate downregulated
expression levels of the genes.
In pathway map illustrated in Fig.2 above indicate that there are three genes that are affected
by doxorubicin administration either through up regulation or down regulation and they include
MMP-8, RSNB and COL1A. The first two genes are up regulated while the last one is down
regulated and the pathways through which these genes impart their effects in doxorubicin-
induced cardiomyopathy are discussed below:
MMP-8
MMP-8 gene which is responsible of encoding matrix metalloproteinase-8 is one of the most

important genes that mediate the effects of doxorubicin in doxorubicin-induced
cardiomyopathy. However, MMP-8 which also acts as a fibrosis factor belong to the group of
enzymes known as matrix metalloproteinase (MMP) which has a considerable number of
isoforms which are mostly involved in collagen cleavage in many connective tissues and they
are encoded by MMP genes which are localized at chromosome 11q22.3 (Chandler, et al.,
1997). Up regulation of MMP-8 leads to down regulated of IL13RA2. However, IL13RA2 up
regulates IL-13. This occurs through cleavage of IL13RA2 leading to sequestration of IL-13
and subsequent inhibition of IL-13 signaling. Moreover, the generated IL-13 leads to down
regulation of INOS which eventually results to inhibition of NO synthesis and signaling.
Subsequently, this leads to inhibition of NO mediated signal transduction.
RSN8
RSN8 gene is another gene which is up regulated by doxorubicin during doxorubicin-induced
cardiomyopathy. However, the doxorubicin mediated up regulation of RSN8 is at the centre of
STAT 6 gene which leads to eventual up regulation of RSN8 gene whose expression products
are responsible for activation of myofibroblast differentiation. However, STAT 6 is up regulated
by SHP-1 leading to mediation of activation of multiple molecules all of which eventually lead
to inflammation activation.
COL1A
COL1A gene which is located on chromosome 2 is responsible for encoding of collagen
alpha-1(I) chain, a protein that is found in many connective tissues. However, administration
of doxorubicin leads to down regulation of COL1A which mediates fibrosis in doxorubicin-
induced cardiomyopathy. The down regulation of COL1A in doxorubicin-induced
cardiomyopathy is centrally mediated by STAT 6. However, SHP-1 leads to up regulation of
STAT 6 which is then responsible of down regulation of COL1A gene leading to mediation of
fibrosis in doxorubicin-induced cardiomyopathy.

  1. Inflammation Amphoterin signaling
    Amphoterin is at the centre of the inflammation pathway map which mediates cardiotoxicity in
    doxorubicin-induced cardiomyopathy. However, Amphoterin receptor referred to as receptor
    for advanced glycation end products (RAGE), is a cell surface molecules which belong to the
    family of multi-ligand immunoglobulin and it is involved in interacting with distinct molecules
    that facilitate inflammation (Chandler, et al. 2007). Inflammation is one of the important
    mechanisms through which cardiotoxicity in doxorubicin-induced cardiomyopathy occurs.
    However, inflammation Amphoterin signalling pathway map is mediated by the binding of
    various ligands, particularly the Amphoterin to RAGE which leads to the triggering of the
    activation of key cell signalling pathways, such as NF-kappaB, MAP kinases, p21ras and
    cdc42/rac, thereby leading to the reprogramming of cellular properties (Chandler, et al. 2007).
    It is important to note that in the inflammation Amphoterin signalling, RAGE acts as the
    central cell surface receptor for Amphoterin. Indeed, the binding of Amphoterin to RAGE
    mediates a multitude of biochemical inflammation pathways in cardiomyocytes during

doxorubicin-induced cardiomyopathy eventually leading to congestive heart failure. However,
this pathway map showing process network in inflammation Amphoterin signaling during
doxorubicin-induced cardiomyopathy involves mediation of effects on Calgranulin A and
Calgranulin B genes. This is attributable to the fact that doxorubicin directly influence these
two genes Calgranulin A and Calgranulin B that subsequently lead to a multitude of molecular
pathways involving up regulation and down regulation of many genes and their respective
expression products.

  1. Process network: Inflammation Amphoterin signaling

Fig. 3: The process network above show an illustration of genes that are up regulated and
down regulated by doxorubicin in cardiotoxicity.
In the process network shown in Fig.3 above is an outline of inflammation Amphoterin
signaling pathway map where two main genes such as: Calgranulin A and Calgranulin B are
highlighted. For instance, in doxorubicin-induced cardiomyopathy it has been observed that

Calgranulin B has effect on NF-kB while a complex of Calgranulin A and Calgranulin B is
believed to act on RAGE leading to activation of p38 MAPK.
Calgranulin A
Calgranulin A is one of the most important molecules that mediate the inflammation
Amphoterin pathway. This molecule is also involved in acting on many other molecules on
which it leads to their up regulation or down regulation. For instance, Calgranulin A binds to
RAGE which also binds to many other molecules. The binding of Calgranulin to RAGE leads
to activation of p38 MAPK which then activates IKK (cat.) followed by activation of I-kB.
Subsequently, I-kB acts on NF-Kb leading to activation of IL-6 and E-selectin. In addition, p38
MAPK which is activated by Calgranulin A leads to activation of SP1 which then activates
ITGAM and ITGB2. Eventually, both ITGAM and ITGB2 lead to activation of alpha-M/ beta-2
integrin. However, RAGE which is activated by Calgranulin A leads to direct activation of
alpha-M/ beta-2 integrin. Moreover, a complex of both Calgranulin A and Calgranulin B also
binds to RAGE leading to activation of p38 MAPK which then activates IKK (cat.) followed by
activation of I-kB. Subsequently, I-kB acts on NF-Kb leading to activation of IL-6 and E-
selectin as observed in Calgranulin A alone.
Calgranulin B
Calgranulin B is another important molecule that mediates the inflammation Amphoterin
pathway. Calgranulin B is also involved in acting on many other molecules on which it leads to
their up regulation or down regulation. However, in the inflammation Amphoterin pathway
Calgranulin B has effect on NF-kB which then leads to several other pathways. NF-kB
activation leads to direct activation of TNF-alpha. NF-kB also acts on IL-12 which leads to
activation of alpha-M/ beta-2 integrin. In addition, activation of NF-Kb by Calgranulin B leads
to activation of SP1 which then activates ITGAM and ITGB2. Eventually, both ITGAM and
ITGB2 lead to activation of alpha-M/ beta-2 integrin.

References
Chandler, S., Miller, K. M., Clements, J. M., et al. (1997). Matrix metalloproteinases, tumor
necrosis factor and multiple sclerosis: an overview. Journal of Neuroimmunology,
72(2), 155–61.
Kuivaniemi, H., Tromp, G. & Prockop, D. J. (1991). Genetic causes of aortic aneurysms:
Unlearning at least part of what the textbooks say. Journal of Clinical Investigations,
88(5), 1441–4.

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