Benefits

 Targeted therapy is gaining importance due to its specificity towards cancer cells while sparing
toxicity to off-target cells
 Chemotherapy is limited due to lack of selectivity for tumor cells over normal cells resulting in
insufficient drug concentrations in tumors, systemic toxicity and the appearance of drug-
resistant tumor cells
 Targeted therapy aims at delivering drugs to particular genes or proteins that are specific to
cancer cells or the tissue environment that promotes cancer growth. Effectiveness of the
therapy lies in targeted release of therapeutics at the disease site while minimizing the off-target
side effects caused to normal tissues
 Monoclonal antibody bind to targeted antigens with exceptional specificity
 They are used in targeted therapy for the delivery of active therapeutics [10], prodrug activation
enzymes [11, 12] and chemotherapy toxins [13-15]. Monoclonal antibodies block a specific
target on the outside of cancer cells or in the tissue surrounding it. Monoclonal antibodies are
used to deliver chemotherapeutic drugs and radioactive substances, directly to cancer cells

 Grade 1-2 toxicities, often chronic and responsible for adversely affecting the quality of a
patient’s life, were not reported
3
 targeted therapies interfere with specific proteins involved in tumorigenesis. Rather than using
broad base cancer treatments, focusing on specific molecular changes which are unique to a
particular cancer, targeted cancer therapies may be more therapeutically beneficial for many
cancer types, including lung, colorectal, breast, lymphoma and leukemia
 Chemotherapeutic drugs target rapidly dividing cells, such as cancer cells; however, these drugs
also target certain normal cells, such as intestinal epithelium
 Like conventional chemotherapy, targeted cancer therapies use compounds that inhibit cancer
growth and metastasis. However, the mechanisms of action for targeted cancer therapies differ
from standard chemotherapy. As the name suggests, targeted therapies interfere with specific
proteins involved in tumorigenesis. Rather than using broad base cancer treatments, focusing on
specific molecular changes which are unique to a particular cancer, targeted cancer therapies
may be more therapeutically beneficial for many cancer types, including lung, colorectal, breast,
lymphoma and leukemia
 radiation therapy, which targets all cells within the localized tumor microenvironment, or
chemotherapy, which targets rapidly growing and dividing cells
 Both of these traditional methods are crude in their approach to targeting cancer cells, as there
are many non-cancerous cells that are destroyed during these treatments
 it is common for cancer cells that are not targeted and killed by the two traditional methods to
acquire resistance to the treatment and ultimately result in a more aggressive cancer
 With these traditional cancer treatments, the patient usually experiences severe side effects,
such as pain, nausea, diarrhea, cardiotoxicity, hair loss, darkened or dry skin and depression of
the immune system [10-12]. These side effects are a result of the lack of specificity of the
traditional cancer treatments, as cancerous cells are not the only type of quickly dividing cells
within the body. Other cell types with high rates of division are those present in bone marrow,
the digestive tract and hair follicles, leading to the side effects listed above
 Chemotherapy interfere with cell division or DNA synthesis
 traditional cancer treatments described above have significant side effects, in part due to their
lack of specificity
 the effects of both the small molecule inhibitors and monoclonal antibodies are easily reversed
once the drug is finished being administered to the patients
 specificity of the treatment that allows for the targeted cancer therapy drugs to be more precise
and display fewer side effects than the traditional cancer treatments, which result in damage
upon the immune system and other organ systems of the body
 targeted therapies should be more efficacious and less detrimental to normal tissue and cells
than today’s current treatment regimens
 Patients are able to live longer, have a better quality of life, have a higher survival rate and
decreased side effects because of these novel cancer treatment drugs that are being developed
4
 Surgery is ineffective in patients with advanced tumor pathology or whenever the surgical act
failed to remove all the tumor mass
 Chemotherapy causes toxicity to normal tissues and the development of drug resistance
mechanisms by tumor cells represented important obstacles to overcome
 In particular, unlike the classic chemotherapy approach, which acts on both normal cells and
cancer cells, the targeted therapy intervenes on altered key oncogenes or tumor suppressor
genes involved in tumor promotion. By this way, these new selective inhibitors are able to affect
only altered cancer cells with minor side effects toward the normal cells
5
 main disadvantage relies on their relative non-selectivity in targeting rapidly dividing non-
cancerous cells such as hair follicles, bone marrow and gastrointestinal epithelial cells. This
commonly manifests as serious adverse effects on patients such as hair loss, anemia, infections
(due to low white blood cell count), infertility, nausea and vomiting. As a result, the effective
therapeutic dose is unattained and the efficacy of conventional chemotherapeutic agents is
compromised
 This is commonly experienced in the clinic, when a chemotherapy regimen is administered for a
delimited period, but the dose has to be reduced or treatment is postponed as a safety
precaution despite tumor responsiveness [5, 6]. Furthermore, many conventional
chemotherapeutics do not accumulate in the tumor mass at effective therapeutic
concentrations, thereby cannot effectively inhibit their proliferation and metastasis. This is
particularly true at the core micro-regions of tumors; due to the disorganized intratumoral
vasculature and high interstitial fluid pressure as a consequence of aberrant angiogenesis and
poor lymphatic drainage. Some types of cancers such as brain gliomas, are also difficult to reach
with traditional chemotherapeutics, as they are unable to penetrate the blood brain barrier.
These factors play a paramount role in drug resistance [7]. Chemotherapeutic agents with
inadequate bioavailability and pharmacokinetic profiles are more inclined to metabolism and
excretion before reaching cancerous cells [8]. Cancerous cells not killed during treatment are
likely to acquire resistance and eventually lead to a more aggressive form of tumors with high
probability of organ damage and death
 Targeted cancer therapy has proven more effective than conventional chemotherapeutics as the
maximum tolerated dose is higher so patients are able to tolerate therapeutic doses with less
severe adverse effects. This resulted in improved patient response rates and survival
6
 improve outcomes and survival rates since targeted therapies directly target the mutations in
your unique form of cancer.  Therefore, targeted therapy only impacts the cancer cells in your
body.  Your treatment doesn’t harm the healthy cells in your body.  Targeted therapy reduces
the risk of cancer recurrence.
 Fewer and less severe side effects Targeted therapies signicantly reduce the severity and
frequency of side effects that you experience.  This is because standard chemotherapy attacks
all the growth cells in your body (and why hair loss is common during standard chemotherapy).
 You receive less of the drug overall than you would with standard chemotherapy.  Targeted
therapy does not have a damaging impact on your body long-term, compared to standard
treatment.
7
 precisionlized oncology has helped improve treatment outcomes and quality of life compared to
traditional chemotherapy
 treatment-related adverse reactions are dramatically strong therefore being feared by patients
 The molecularly targeted therapies with less adverse reactions have prolonged disease control,
and ultimately improved long-term survival outcomes
8
 Targeted therapies are generally better tolerated than traditional chemotherapy
 targeted therapies provide treatment options for some patients who may not otherwise be
candidates for anticancer therapy. For instance, nonsmall cell lung cancer and non-Hodgkin’s
lymphoma primarily affect elderly patients, many of whom have medical comorbidities that limit
the use of standard chemotherapy. Targeted therapies such as erlotinib and rituximab are often
less toxic and better tolerated than traditional chemotherapy, offering these patients additional
treatment options
11
 tailor diagnosis and treatment to each patient's individual biologic profile, while minimizing
exposure to unnecessary or ineffective therapies
12
 New cancer targeted therapies that make use therapeutic antibodies or small molecules have
made treatment more tumor specific and less toxic
 surgery nor radiation or the two in combination could adequately control the metastastic cancer
and that, for treatment to be effective, therapy needed to reach every organ of the body
 Antibody-targeted therapy is more effective as their own role as anticancer agents, their ability
to target tumors also enables them to improve as the selectivity of other types of anti-cancer
agents, some of which cannot be applied effectively alone
 high levels of selective toxicity cannot be achieved with anticancer chemotherapeutics because
of the lack of unique molecular targets that would distinguish them from normal cells. This can
lead to increased toxicities against normal tissues, including bone marrow, gastrointestinal tract
and hair follicle tissues. Furthermore, trying to avoid the side effects that occur as a result of
toxicities to normal tissues, we often give sub-optimal doses of anticancer chemotherapeutics,
resulting in the eventual failure of therapy, which is often accompanied by the development of
drug resistance and metastatic disease
 The selective toxicity of an anticancer drug can be increased by either increasing the amount of
the drug that reaches the cancer tissue or by decreasing the concentration of drug that reaches
at the normal tissues. Therefore, ligand-targeted therapy makes possible tumor specificity and
limited toxicity and shows promise in the development of novel therapies for cancer. Ligand-
 targeted therapy can carry higher doses of a drug to the tumor tissue and may overcome
obstacles presented by cytotoxic chemotherapy
13
 Long-term tumor remission can be achieved by immune checkpoint inhibitors, which is
confirmed by improvements in overall survival (OS) and progressionfree survival (PFS)
14
 Chemotherapy uses cytotoxic drugs to not only kill rapid dividing cancer cell but also to destroy
normal cells. One of the advantages of molecular targeted therapy is its ability to deliver drug
effectively with high specificity, while being less toxic compared to conventional chemotherapy
 molecular targeted therapy has been used to successfully improve patient survival in diseases
that are relatively insensitive to chemotherapy such as non-small cell lung cancer (NSCLC).
Radiation therapy alongside lung surgery is the treatment of choice if diagnosed at early stage.
However, approximately 75% of newly diagnosed non-small cell lung cancer (NSCLC) patients are
usually at the advanced stages (stage III or IV)
17
 The disadvantages of radiation therapy include: damage to surrounding tissues (e.g. lung, heart),
depending on how close they are to the tumor inability to kill tumor cells that cannot be seen on
imaging scans and are therefore not always included on the 3D models used to plan the
radiation  This can include cancer in near-by or cancer that has spread to distant locations
( disease). inability to kill the all cancer cells in tumors. This is more likely with large tumors.
inability to relieve mass eect in certain parts of the body (e.g. brain). This can lead to the need
for surgery. poor killing of cancer cells in areas that do not have a good supply of oxygen (e.g. in
an area after surgery; in a limb with a poor blood supply) increased incidence in wound
complication and poor healing (e.g. if surgery is used after radiation; or in parts without good
circulation) inconvenience of radiation therapy (e.g. in some cases it must be delivered daily, 5
days per week, for 1-2 months)
 The disadvantages of surgery include: inability to kill microscopic disease around the edges of
the tumor may leave tumor cells in the patient after surgery. the patient must be able to tolerate
the surgery and anesthesia (i.e. have minimal medical problems, have good lung function, not be
on certain medications) some damage to nearby normal tissues (e.g. removing ribs or normal
lung tissue to reach a lung tumor) complications from surgery (e.g. infection, and others that are
site-specic) inability to remove cancer in other parts of the body (i.e. metastatic disease) inability
to safely remove kill cells in certain parts of the body where radiation therapy may have less side
eects (e.g. in certain types of brain tumors) removal of an organ which may aect the patients
quality of life (e.g. breast, larynx, bowel) inability of a surgeon to discern cancer cells from
normal cells with the naked eye (particularly after chemotherapy or radiation have been
delivered to the site)
 The advantages of systemic therapy include: the ability to kill many cancer cells throughout the
entire body (including cancer cells in the main tumor, and other tumors in the body) work
together with radiation therapy (i.e. can kill more cells together than either therapy could do
alone ability to kill microscopic disease at the edge of the main tumor that may not be seen by
the naked eye of a surgeon (thereby decreasing the chance that therecancer cells will be left
behind at the time of surgery) tailoring of the systemic treatment for each patient (e.g. specic
hormonal therapies for breast cancers; targeted therapies for lung cancers), a backbone of
personalized medicine preservation of an organ (e.g. not removing a breast, larynx, or part of
the gastrointestinal tract, which would have signicant negative impact on a patients quality of
life)
Bevacizumab
 18
 Vascular endothelial growth factor A (VEGF) is a potent proangiogenic growth factor that
stimulates the proliferation, migration, and survival of endothelial cells
 Bevacizumab (Avastin; Genentech, Inc., South San Francisco, CA) is a humanized anti-VEGF
monoclonal IgG1 antibody (molecular weight, 149 kDa)
 it is approved for the treatment of advanced colorectal cancer (CRC),
 , colorless solution administered i.v
 Cancer cells and tissues often have high metabolic rates; this may result in a demand for oxygen
and nutrients exceeding the supply. As a result, these tissues are characterized by the presence
of hypoxia, which is also the primary factor controlling angiogenesis. Under hypoxic conditions,
hypoxia inducible factor (HIF) binds to the hypoxia response element present in the VEGF gene,
thus inducing the transcription of VEGF protein [18]. Circulating VEGF binds to VEGF receptor
(VEGFR)-1 and VEGFR-2 and to its coreceptors neuropilin (NRP)-1 and NRP-2 with high binding
affinity [19]. These receptors are expressed on the surface of endothelial cells, and they play a
critical role in the development of angiogenesis by stimulating the recruitment and proliferation
of endothelial cells
 Bevacizumab acts by selectively binding circulating VEGF, thereby inhibiting the binding of VEGF
to its cell surface receptors. This inhibition leads to a reduction in microvascular growth of tumor
blood vessels and thus limits the blood supply to tumor tissues. These effects also lower tissue
interstitial pressure, increase vascular permeability, may increase delivery of chemotherapeutic
agents, and favor apoptosis of tumor endothelial cells
19
 angiogenic response to VEGF is mainly mediated via activation of VEGFR-2, expressed primarily
in endothelial cells. VEGFR-2 activation initiates several intricate signaling paths, which
eventually lead to different endothelial responses: cell survival, proliferation, migration, invasion
into the surrounding tissue, vascular permeability and vascular inflammation [25]. These
responses involve (i) a number of pivotal effectors such as phosphoinositide 3 kinase (PI3K),
phospholipase Cγ (PLCγ), SRC, focal adhesion kinase (FAK) and Rho family of GTPases; (ii) several
multifunctional docking proteins and adaptors; and (iii) VEGFR-2 partners such as Neuropilin 1
(NRP1), integrins and vascular endothelial (VE)-cadherin
 VEGFR-2 is a tyrosine kinase receptor (RTK). Binding of VEGF to VEGFR-2 promotes receptor
dimerization, allowing trans/autophosphorylation of intracellular tyrosine residues. Among the
19 tyrosine residues present in the intracellular domain of VEGFR-2, there are five major
phosphorylation sites: Y951, Y1054, Y1059, Y1175 and Y1214.
 The Y1054 and Y1059 are located in the kinase domain activation loop, and their
phosphorylation is critical for receptor catalytic activity
 Y951 is located in the kinase insert domain, and its phosphorylation serves as a binding site for T
cell-specific adaptor (TSAD) also known as VEGFR-2-associated protein (VRAP)
 The Y1175 and Y1214 are located in the carboxy-terminal domain. Phosphorylation of Y1175
creates a binding site for PLCγ, p85 subunit of PI3K [30], the adaptor proteins SHB [31] and SCK
[32]. This residue is well-recognized as a critical mediator of VEGFR-2 signaling. Phosphorylated
Y1214 has been described to bind the adaptor protein NCK
 Activated VEGFR-2 directly recruits PLCγ, which is in turn phosphorylated [38, 39].
Phosphorylated PLCγ hydrolyzes the membrane phospholipid phosphatidylinositol-4,5-
biphosphate (PIP2), generating diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). This
latter mobilizes Ca2+ from the endoplasmic reticulum, thus leading to an increase in intracellular
Ca2+. IP3-mediated Ca2+ increase supports DAG-induced activation of PKC, from which it is
 triggered the RAF1-MEK-ERK1/2 mitogen-activated protein (MAP) kinase cascade, that is, the
best characterized pathway propagated downstream to VEGF/VEGFR-2/PLCγ axis resulting, in
particular, in endothelial cell proliferation trough the ERK1/2-dependent regulation of gene
transcription
 VEGF-induced activation of PKC also results in activation of protein kinase D (PKD), found to
influence ERK1/2 activation and cell proliferation [43]. In response to VEGF, gene repressive
action of histone deacetylases (HDAC) 5 and 7 in endothelial cells is overcome by the PLCγPKC-
PKD pathway-dependent HDAC 5 and 7 phosphorylation and nuclear export, resulting in the
regulation of gene transcription, cell proliferation and migration [44, 45]. Moreover, VEGF-
induced PKC-dependent activation of PKD has also been reported to induce HSP27
phosphorylation and mediate cell migration
 In endothelial cells, VEGF stimulates receptor-mediated activation of PLCγ leading to an increase
in intracellular Ca2+, calcineurin activation and NFAT nuclear translocation that in turn leads to
the transactivation of genes that are essential for angiogenesis [49, 50], such as COX-2 resulting
in synthesis of PGE2, a mediator of endothelial cell migration and tube formation
 VEGF/VEGFR-2 axis activates PI3K. The activated PI3K converts the plasma membrane lipid PIP2
to phosphatidylinositol-3,4,5-trisphosphate (PIP3), and signaling proteins with pleckstrin
homology (PH) domains accumulate at site of PI3K activation by directly binding to PIP3 such as
serine-threonine kinases AKT and phosphoinositide-dependent kinase 1 (PDK1). PI3K/AKT
pathway is considered the main mechanism by which VEGF induces endothelial cell survival. AKT
mediates both short- and long-term cell survival effects by inhibiting (through direct
phosphorylation) pro-apoptotic proteins such as BAD, caspase 9 and forkhead transcription
factors, and by upregulating anti-apoptotic proteins such as BCL-2. VEGF also induces expression
of the anti-apoptotic proteins BCL-2 and A1 in endothelial cells. induction of the IAP family anti-
apoptotic proteins survivin and XIAP—which inhibit caspase 3, 7 and 9—by VEGF in endothelial
cells
 VEGFR-2 activates RAC through the SRC-dependent phosphorylation of the nucleotide-exchange
factor VAV2 and that RAC activation, in turn, promotes the p21-activated kinase (PAK)- mediated
phosphorylation of VE-cadherin resulting in the recruitment of beta-arrestin 2 to phosphorylated
VE-cadherin, thereby promoting its internalization into clathrin-coated vesicles and the
consequent disassembly of intercellular junctions. rapid endocytosis of VE-cadherin disrupts
endothelial barrier function
 20
 In normal cells, the EGFR signaling cascade begins with ligand activation of EGFR (Figure 1). Up to
eleven ligands can bind the ErbB family of receptors, including EGF and transforming growth
factoralpha [1]. Ligand binding induces dimerization of the receptor with formation of
homodimers and heterodimers, which leads to the activation of tyrosine kinase. The intracellular
tyrosine kinase residues then become autophosphorylated, inducing activation of multiple signal
transduction pathways. Two main intracellular pathways activated by EGFR are the
mitogenactivated protein kinase (MAPK) pathway and the phosphatidylinositol 3-kinase- (PI3K-)
protein kinase B (AKT) pathway. These pathways lead to the activation of various transcription
factors that then impact cellular responses such as proliferation, migration, differentiation, and
apoptosis
 EGFR has been reported to be overexpressed in anywhere from 25% to 82% of colorectal
cancers
 23
5FU
 Antimetabolite drugs work by inhibiting essential biosynthetic processes, or by being
incorporated into macromolecules, such as DNA and RNA, and inhibiting their normal function.
The fluoropyrimidine 5-fluorouracil (5-FU) does both
 The mechanism of cytotoxicity of 5-FU has been ascribed to the misincorporation of
fluoronucleotides into RNA and DNA and to the inhibition of the nucleotide synthetic enzyme
thymidylate synthase (TS)
 t. 5-FU-based chemotherapy improves overall and disease-free survival of patients with resected
stage III colorectal cancer2 . Nonetheless, response rates for 5-FU-based chemotherapy as a
first-line treatment for advanced colorectal cancer are only 10–15%
 5-FU is an analogue of uracil with a fluorine atom at the C-5 position in place of hydrogen (FIG.
1). It rapidly enters the cell using the same facilitated transport mechanism as uracil6 . 5-FU is
converted intracellularly to several active metabolites: fluorodeoxyuridine monophosphate
(FdUMP), fluorodeoxyuridine triphosphate (FdUTP) and fluorouridine triphosphate (FUTP) (FIG.
1) — these active metabolites disrupt RNA synthesis and the action of TS
 TS catalyses the reductive methylation of deoxyuridine monophosphate (dUMP) to
deoxythymidine monophosphate (dTMP), with the reduced FOLATE 5,10-
methylenetetrahydrofolate (CH2 THF) as the methyl donor (FIG. 2). This reaction provides the
sole de novo source of thymidylate, which is necessary for DNA replication and repair. The 36-
kDa TS protein functions as a dimer, both subunits of which contain a nucleotide-binding site
and a binding site for CH2 THF. The 5-FU metabolite FdUMP binds to the nucleotide-binding site
of TS, forming a stable TERNARY COMPLEX with the enzyme and CH2 THF, thereby blocking
binding of the normal substrate dUMP and inhibiting dTMP synthesis
 Depletion of dTMP results in subsequent depletion of deoxythymidine triphosphate (dTTP),
which induces perturbations in the levels of the other deoxynucleotides (dATP, dGTP and dCTP)
through various feedback mechanisms10. Deoxynucleotide pool imbalances (in particular, the
dATP/dTTP ratio) are thought to severely disrupt DNA synthesis and repair, resulting in lethal
DNA damage
 TS inhibition results in accumulation of dUMP, which might subsequently lead to increased levels
of deoxyuridine triphosphate (dUTP)13,14. Both dUTP and the 5-FU metabolite FdUTP can be
misincorporated into DNA. Repair of uracil and 5-FU-containing DNA by the nucleotide excision
repair enzyme uracil-DNA-glycosylase (UDG)15 is futile in the presence of high (F)dUTP/dTTP
ratios and only results in further falsenucleotide incorporation. These futile cycles of
misincorporation, excision and repair eventually lead to DNA strand breaks and cell death
 The 5-FU metabolite FUTP is extensively incorporated into RNA, disrupting normal RNA
processing and function. 5-FU misincorporation can result in toxicity to RNA at several levels. It
not only inhibits the processing of pre-rRNA into mature rRNA21,22, but also disrupts post-
transcriptional modification of tRNAs23,24 and the assembly and activity of snRNA/protein
complexes, thereby inhibiting splicing of pre-mRNA25,26. In addition, rRNA, tRNA and snRNA all
contain the modified base pseudouridine, and 5-FU has been shown to inhibit the post-
transcriptional conversion of uridine to pseudouridine in these RNA species
 Leucovorin (vitamin given together with 5fu).High intracellular levels of the reduced folate CH2
THF are necessary for optimal binding of FdUMP to TS. LV enters the cell via the reduced folate
carrier and is anabolized to CH2 THF, which is then polyglutamated by folylpolyglutamate
synthetase. POLYGLUTAMATION not only increases the cellular retention of CH2 THF, but also
enhances the stabilization of its ternary complex with TS and FdUMP.
 24

 5-FU is a heterocyclic aromatic organic compound with a structure similar to that of the
pyrimidine molecules of DNA and RNA; it is an analogue of uracil with a fluorine atom at the
C-5 position in place of hydrogen
 In mammalian cells, 5-FU is converted to fluorodeoxyuridine monophosphate (FdUMP),
which forms a stable complex with thymidylate synthase (TS), and thus inhibits
deoxythymidine monophosphate (dTMP) production. dTMP is essential for DNA replication
and repair and its depletion therefore causes cytotoxicity
 TS, an essential enzyme for catalyzing the biosynthesis of thymidylate, is implicated in the
regulation of protein synthesis and apoptotic processes [12,13]. TS catalyzes the methylation
of deoxyuridine monophosphate (dUMP) to dTMP, for which 5,10-
methylenetetrahydrofolate (CH2THF) is the methyl donor, and finally provides with the
reaction thymidylate to maintain DNA replication and repai
 Reduction of dTMP leads to downstream depletion of deoxythymidine triphosphate (dTTP),
which induces perturbations in the levels of the other deoxynucleotides (dATP, dGTP and
dCTP). Finally the imbalances (the ATP/dTTP ratio specifically) are thought to severely
disrupt DNA synthesis and repair, resulting in lethal DNA damage
 Accumulation of dUMP, which might subsequently lead to increased levels of deoxyuri-dine
triphosphate (dUTP), can be misincorporated Molecules 2008, 13 1553 into DNA, and FdUTP,
the metabolic product of 5-FU has the same action [19]. Furthermore, repairing enzyme
 uracil-DNA-glycosy-lase (UDG) is suggested to be useless in the presence of high (F)
dUTP/dTTP ratios and only results in further false DNA repair
 5-FU is a pyrimidine analogue that can be misincorporated into RNA and DNA in place of
uracil or thymine. The interference with the normal biosynthesis or function of nucleic acids
is therefore another possible mechanism of action for 5-FU. 5-FU can be misincorporated
into DNA of drugtreated cells, and accumulation of 5-FU in the genome, rather than uracil
excision, is correlated with 5- FU cytotoxicity in mammalian cells
 5-FU inhibits premRNA splicing, through its effect on pseudouridylation of U2 snRNA
 The action of 5-FU is not only inhibiting the processing of pre-rRNA into mature rRNA, but
also disrupting post-transcriptional modification of tRNAs and the assembly activity of
snRNA/protein complexes, thereby inhibiting splicing of pre-mRNA [28]. 5-FU-containing
RNA can also inhibit pseudouridylation, the most abundant post-transcriptional modification
of noncoding RNA
25
 5-FU exerts anti-proliferative effects through the inhibition of thymidylate synthase (TS),
which decreases thymidylate levels and increases uracil incorporation into DNA [14]. In the
reaction catalyzed by TS, the cofactor N5,N10-methylene tetrahydrofolate (CH2H4PteGlu) is
the methyl and electron donor and also rate limiting in the reaction because its intracellular
concentrations are lower than dUMP. The active 5-FU metabolite, FdUMP, forms a stable
ternary complex with the active site cysteine of TS and CH2H4PteGlu, thereby suicide
inhibiting dTMP synthesis [15]. Therefore, Leucovorin (folinic acid, LV) is administered
clinically in combination with 5-FU to enhance its therapeutic effect, because LV is readily
converted to CH2H4PteGlu
 5-FU exerts anti-proliferative effects through the inhibition of thymidylate synthase (TS),
which decreases thymidylate levels and increases uracil incorporation into DNA [14]. In the
reaction catalyzed by TS, the cofactor N5,N10-methylene tetrahydrofolate (CH2H4PteGlu) is
the methyl and electron donor and also rate limiting in the reaction because its intracellular
concentrations are lower than dUMP. The active 5-FU metabolite, FdUMP, forms a stable
ternary complex with the active site cysteine of TS and CH2H4PteGlu, thereby suicide
inhibiting dTMP synthesis [15]. Therefore, Leucovorin (folinic acid, LV) is administered
clinically in combination with 5-FU to enhance its therapeutic effect, because LV is readily
converted to CH2H4PteGlu
 5-FU can also be directly incorporated into RNA and DNA and alter transcription and
replication, respectively
28
Doxorubicin
 There are two proposed mechanisms by which doxorubicin acts in the cancer cell (i)
intercalation into DNA and disruption of topoisomerase-II-mediated DNA repair and (ii)
generation of free radicals and their damage to cellular membranes, DNA and proteins
 doxorubicin is oxidized to semiquinone, an unstable metabolite, which is converted back to
doxorubicin in a process that releases reactive oxygen species. Reactive oxygen species can
lead to lipid peroxidation and membrane damage, DNA damage, oxidative stress, and
triggers apoptotic pathways of cell death
 doxorubicin can enter the nucleus and poison topoisomerase-II, also resulting in DNA
damage and cell death
29
 Doxorubicin is an antibiotic derived from the Streptomyces peucetius bacterium
 The primary mechanism of action of doxorubicin involves the drug’s ability to intercalate
within DNA base pairs, causing breakage of DNA strands and inhibition of both DNA and RNA
synthesis. Doxorubicin inhibits the enzyme topoisomerase II, causing DNA damage and
induction of apoptosis. When combined with iron, doxorubicin also causes free radical-
mediated oxidative damage to DNA, further limiting DNA synthesis
30
 Doxorubicin (also called adriamycin) belongs to a class of compounds with similar structures,
called anthracyclines. Like daunorubicin, the first anthracycline compound to be described,
doxorubicin was isolated from Streptomyces peucetius, a soil bacterium
 doxorubicin-mediated cell death, including topoisomerase II poisoning, DNA adduct
formation, oxidative stress, and ceramide overproduction
 Anthracycline drugs such as doxorubicin are mostly planar molecules that preferentially
intercalate between neighboring DNA base pairs, anchored on one side by one or more
sugar moieties that sit in the DNA minor groove. When DNA is topologically constrained, as
in the case of plasmid circles, the strand separation that occurs during intercalation unwinds
the double helix and produces DNA supercoils, resulting in increased torsional stress.
torsioninduced nucleosome destabilization is emerging as a significant molecular mechanism
for the action of doxorubicin and related anthracycline drugs
 The most parsimonious model for doxorubicin action involves topoisomerase II poisoning,
resulting in double-strand DNA breaks and cell death at clinically relevant drug
concentrations. The enzyme binds DNA supercoils and entangled DNA, breaks both strands
of one DNA duplex, passes the other duplex through the resulting gap and reseals the break.
This process results in the release of torsional stress formed during biological processes such
as DNA replication and transcription (discussed below) [12]. In addition, topoisomerase II is
essential for decatenation of DNA during mitosis, and deficiency in topoisomerase II
prevents normal cytokinesis resulting in cell death.
 As a DNA intercalator, doxorubicin prefers the intercalation site containing adjacent GC base
pairs, probably due to specific hydrogen-bond formation between doxorubicin and guanine
(Fig. 1a) [21–23]. Formation of doxorubicin-DNA adducts has been shown to activate DNA
damage responses and induce cell death independent of topoisomerase II
 The quinone structure of doxorubicin can be oxidized to a semiquinone radical through
addition of one electron, mediated by a number of NAD(P)H-oxidoreductases [5, 34].
Semiquinone radicals quickly react with oxygen to generate superoxide and hydrogen
peroxide causing DNA damage. Additionally, doxorubicin is an iron chelator and the
doxorubicin-iron complex catalyzes the conversion of hydrogen peroxide to highly reactive
hydroxyl radicals [35]. Thus, doxorubicin-induced release of free radicals may cause
oxidative stress, resulting in DNA damage and cell death
31
 Antitumour antibiotics (anthracyclines) interfere with the enzymes involved in DNA
replication and are capable of inflicting their action regardless of what cell cycle phase the
cell is in, though the preference would be in mitotic cells
 Its primary action is to inhibit topoisomerase I and II and intercalating into DNA to interfere
with its uncoiling, ultimately inducing programmed cell death
 Doxorubicin is also known to intercalate itself into the DNA, with the inhibition of both DNA
and RNA polymerase, ultimately ceasing DNA replication and RNA transcription
 doxorubicin enters the cell through diffusion using its higher affinity to bind to the
cytoplasm’s proteasome. A doxorubicin proteasome complex is formed when doxorubicin
 binds to the proteosome’s 20S subunit, where it is then translocated through the nuclear
pore complexes into the nucleus. Doxorubicin has a higher affinity for nuclear DNA over the
proteasome it is attached to, allowing it to dissociate itself from the proteasome and bind to
the DNA.[8] Other doxorubicin actions include free radical generation which causes further
DNA damage, inhibition of macromolecule production, DNA unwinding/separation and
increase in alkylation
 doxorubicin can affect the cell membrane directly by binding to plasma proteins causing
enzymatic electron reduction of doxorubicin. This can cause the formation of highly reactive
species of hydroxyl free radicals
 Results have suggested also that doxorubicin results in autophagy, being cytoprotective as a
response to DNA damage. Nuclear enzyme (poly (ADP-ribose) polymerase-1) (PARP-1)
activation is a vital event that decides whether the cell will undergo autophagy
 Genotoxic stress can cause PARP-1 to hyperactivate, which in turn depletes both NAD+ and
ATP. The cell will then experience energy failure that would be irreversible, resulting in cell
death
 doxorubicin hyperactivates PARP-1
 doxorubicin induces the activation of AMPK via ROS-dependent LKB1 activation, allowing it
to act as the upstream signal. Doxorubicin-induced ROS production can occur in multiple
ways. The first involves triggering a Fenton-type reaction by chelating intracellular iron,
producing hydroxyl radicals that are highly reactive. Other reactions involve‘redox cyclers’
that react with flavoprotein reductase to generate superoxide when molecular oxygen is
present. In detail AMPK modulates cell death by activating JNK kinase

Herceptin Action • Downregulation of ERBB2 (Her-2) receptor resulting in decreased

receptor availability • Inhibition of Her family dimerisation – blocks signaling • Inhibition of
PI3K/AKT pathway and MAPK • Induction of p27 with G1 arrest • Antibody-dependent cell-
mediated cytotoxicity

32

Trastuzumab

 0verexpression of human epidermal growth factor receptor type 2 (HER2, also referred
to as HER2/neu or ErbB-2), a 185-kD receptor first described more than two decades
ago,1 occurs in 20 to 30% of invasive breast carcinomas
 In general, patients with breast-cancer cells that overexpress this receptor or that have a
high copy number of its gene have decreased overall survival and may have differential
responses to a variety of chemotherapeutic and hormonal agents
 One such medication is trastuzumab (Herceptin, Genentech), a humanized monoclonal
antibody. Trastuzumab binds to the extracellular juxtamembrane domain of HER2 and
inhibits the proliferation and survival of HER2-dependent tumors. It is approved by the
Food and Drug Administration (FDA) for patients with invasive breast cancers that
overexpress HER2
 HER1, HER2, HER3, and HER4 (also called epidermal growth factor receptors ErbB-1,
ErbB-2, ErB-3, and ErB-4, respectively) are transmembrane tyrosine kinase receptors
with partial homology that normally regulate cell growth and survival, as well as
adhesion, migration, differentiation, and other cellular responses
 Each of these receptors consists of an extracellular binding domain, a transmembrane
lipophilic segment, and (except for HER3) a functional intracellular tyrosine kinase
 domain. The tyrosine kinase domains are activated by both homodimerization and
heterodimerization, generally induced by ligand binding. In contrast to the extracellular
domains of the three other HER receptors, the extracellular domain of HER2 can adopt a
fixed conformation resembling a ligand-activated state, permitting it to dimerize in the
absence of a ligand.8 Receptor overexpression or mutation can also induce
dimerization.9 Once activated, the signal-transduction cascades of these receptors
promote cellular proliferation and survival.10 In addition, cleavage of the extracellular
domain of HER2 leaves a signaling remnant (p95) at the cell membrane
 HER2 signaling promotes cell proliferation through the RAS–MAPK pathway and inhibits
cell death through the phosphatidylinositol 3'-kinase–AKT–mammalian target of
rapamycin (mTOR) pathway
 Trastuzumab consists of two antigen-specific sites that bind to the juxtamembrane
portion of the extracellular domain of the HER2 receptor and that prevent the activation
of its intracellular tyrosine kinase
 prevention of HER2-receptor dimerization, increased endocytotic destruction of the
receptor, inhibition of shedding of the extracellular domain, and immune activation
 trastuzumab recruits immune effector cells that are responsible for antibody-dependent
cytotoxicity via Fc receptor
 trastuzumab has been reported to increase tumor infiltration by lymphoid cells and
modulation of in vitro antibody-dependent cytotoxicity
33
 The overexpression or gene amplification of HER2 has been found in about 20–30% of
breast cancers, which is classified as the HER2- positive subtype
 This dimerization results in autophosphorylation and/or transphosphorylation of specific
tyrosine residues in EGFR intracellular domains, which in turn leads to the activation of
the Ras/Raf/mitogen-activated protein kinase, the phosphoinositide 3-kinase/Akt, and
the phospholipase Cγ (PLCγ)/protein kinase C (PKC) pathways
 HER2 dimerization promotes the mislocalization and rapid degradation of cell cycle
inhibitor p27Kip1 protein leading to cell cycle progression
 Trastuzumab (or Herceptin) was developed by Genentech Inc (San Francisco, CA, USA) as
a recombinant humanized monoclonal antibody directed against the extracellular
domain IV of HER2
 Trastuzumab has been proposed to trigger HER2 internalization and degradation
through promoting the activity of tyrosine kinase – ubiquitin ligase c-Cbl (Klapper et al.,
2000). It was observed that the binding of trastuzumab to HER2 recruits cCbl to its
docking site, Tyr1112 where c-Cbl ubiquitinates HER2 and leads to its degradation
 As an antibody, one of the major mechanisms of trastuzumab is to attract immune cells
to tumor sites that overexpress HER2, by a mechanism called antibody-dependent
cellular cytotoxicity (ADCC). natural killer cells could target HER2-overexpressing cells
coated with trastuzumab via a CD16-mediatedADCC mechanism(Fc gamma receptors)
 The most well known effect of trastuzumab is the inhibition of the MAPK and PI3K/Akt
pathways (Figure 1), which leads to an increase in cell cycle arrest, and the suppression
of cell growth and proliferation. It is widely accepted that by interfering with the
dimerization of HER2, trastuzumab inhibits HER2 activation and suppresses Akt
phosphorylation
 Other groups critically showed that trastuzumab, by binding to HER2, can block tyrosine
kinase Src signaling and thus, increases PTEN level and activity. This also results in the
suppression of PI3K/Akt signaling and reduction in cell growth and survival
 trastuzumab was demonstrated to induce cell cycle arrest by restoring p27 and
suppressing CDK2 activity in BT474 and SKBR3 breast cancer cells

34

 The HER2 monomeric protein has three major regions—the extracellular amino-terminal
region comprised of four domains (domains I–IV), the hydrophobic transmembrane
domain, and the carboxy-terminal kinase domain comprised of the juxtamembrane
domain, tyrosine kinase, and C-terminal tail with autophosphorylation sites
 Kinase signaling can then stimulate autophosphorylation and downstream signaling,
primarily through the PI3K-Akt-mTOR and Ras-Raf-MEK-Erk1/2 pathways. Activation of
these pathways promotes proliferation, evasion of apoptosis, angiogenesis, and
invasion, leading to tumor growth and progression
 a PI3Ks are heterodimers of a p85 regulatory subunit and p110 catalytic subunit [27].
Upon growth factor stimulation, p85 binds to membrane bound receptors or adaptor
proteins, thus recruiting PI3K to the cell membrane. The p110 catalytic portion of PI3K is
then activated by membrane receptors, and phosphorylates PIP2 to PIP3, activating
kinase events downstream. The lipid phosphatase PTEN dephosphorylates PIP3 to create
PIP2, counteracting the kinase function of PI3K
 trastuzumab downregulates total levels of HER2 on the cell surface [45, 46], which may
ultimately reduce downstream PI3K and MAPK signaling
 Trastuzumab has been shown to block cleavage of the extracellular domain of HER2,
thus, preventing formation of the constitutively active membrane-bound 95-kiloDalton
HER2 protein called p95 HER2 [46, 47]. Trastuzumab may also selectively inhibit HER2-
HER3 heterodimerization [46, 48], reducing PI3K signaling
 Induction of cell cycle arrest by trastuzumab is mediated by p27kip1 and inhibition of
cdk2 activity
 trastuzumab appears to reduce angiogenesis, which may result in increased permeability
of blood vessels [51], potentially increasing delivery of drugs to the tumor. Finally, HER2-
positive cells that become “coated” in trastuzumab will be recognized by specific
immune cells, causing antibody-dependent cellular cytotoxicity (ADCC), or lysis of the
antibody-bound cells
35
 trastuzumab (Herceptin) is humanized monoclonal antibody that interferes with the
HER2 receptor. It is currently the only FDA-approved therapeutic antibody for HER2-
positive breast cancer
 HER are cell membrane-bound glycoproteins, comprising 4 distinct receptors: HER1,
HER2, HER3, and HER4.1-3 These receptors are divided into 3 regions: an extracellular
ligand binding region, an intracellular region with tyrosine kinase activity, and a region
that spans the cell membrane and anchors the receptor to the cell. Ligand binding to the
extracellular domain promotes formation of dimmers, homodimers (between
monomers of same receptor), or heterodimers (between the bound receptor and other
members of the HER family), activating tyrosine kinase, and triggering a cascade of
complex cell biochemistry that regulates various cell functions such as cell proliferation,
angiogenesis, apoptosis, adhesion, and motility
 Overexpressed HER2 sends signals without mitogen arriving and binding to any receptor.
These signals promote invasion, survival, and angiogenesis of tumoral cells
 Trastuzumab binds to domain IV of the extracellular segment of the HER2.4 This process
prevents dimerization, causing cell arrest during the G1 phase
36
 HER2 signaling reduces expression of the proteins cyclin D and c-myc, which sequester
the cyclin dependent kinase (cdk) inhibitor p27kip1, such that it is not available to inhibit
cdk2 activity and ultimately results in increased cellular proliferation
 Overexpression of HER2, with subsequent constitutive kinase activation, is found in
approximately 20– 30% of human breast cancers, primarily owing to gene amplification,
and is associated with reduced disease free and overall survival
 Another mechanism that appears to be employed by trastuzumab is inhibition of
angiogenesis. Reduced microvessel density in vivo (Izumi et al., 2002; Klos et al., 2003;
Wen et al., 2006) and reduced endothelial cell migration in vitro (Klos et al., 2003) have
been observed in response to trastuzumab. Trastuzumabtreated mouse mammary
tumors showed reduced expression of VEGF, transforming growth factor-a, Ang-1 and
PAI-1, which normally act to promote angiogenesis
 trastuzumab is its ability to block cleavage of the HER2 extracellular domain (ECD). In
addition to the full-length 185 kDa HER2 receptor, a 95-kDa Nterminally truncated
membrane-associated HER2 fragment (p95) with increased kinase activity (Christianson
et al., 1998) can sometimes be found on the cell surface. Along with the presence of
p95, a 110-kDa ECD is released and detected in cell culture media of HER2-
overexpressing cells
 39
 Erk activate MEF2, c-Fos, Fra 1, Sap-1, c-Myc and NF-κB that regulate the cell
proliferation, survival, motility and angiogenesis
40

41
 Differences between targeted and classical therapy

 Drugs that block only the mutant form of the protein but do not interfere with the
activity of the normal version would only affect cancer cells, and would leave healthy
cells untouched.
The specificity of current drugs does have one drawback. Blocking a single pathway in a
cancer cell may be enough to slow it down, but it often does not inhibit the cancer
enough to kill it. Targeted therapy does not inflict dna damage thus reducing the risk of
tumour becoming more aggressive.
 Targeted therapies act on specific molecular targets that are associated with cancer,
whereas most standard chemotherapies act on all rapidly dividing normal and cancerous
cells.
 Targeted therapies are deliberately chosen or designed to interact with their target,
whereas many standard chemotherapies were identified because they kill cells. 
 Targeted therapies are often cytostatic (that is, they block tumor cell proliferation), whereas
standard chemotherapy agents are cytotoxic (that is, they kill tumor cells).