Cancer [4]. Moreover, cancer cells may induce surrounding

Cancer

            Carcinogenesis arises
from genetic and epigenetic alteration of the molecular pathways responsible
for regulating properties of normal cells such as proliferation,
differentiation, cell death or motility 1. Normal cells regulate those
properties to keep cells in control and maintain cellular integrity. When
several mutations build up, normal cell function is lost and the cells may
develop multiple hallmarks of cancer. The six hallmarks characterizing cancer
are sustaining proliferating signal, uncontrolled growth, resisting apoptosis,
enabling unlimited replication, inducing angiogenesis, and enabling metastasis
1. The hallmarks are known to be enabled through genomic instability causing
mutations, and inflammation that accelerate tumorigenesis 1. Moreover, reprogramming
of energy metabolism and evading immune destruction are important players in
the development of cancer 1. The mutations in one or more tumour suppressor
genes and proto-oncogenes give variations in cancer malignancy 2.

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            A chief hallmark of
cancer is the ability to sustain proliferative signals 1. Normal cells
control the growth promoting signals to maintain the normal cell function and
integrity 1. The growth factor signals play important role in cell regulation
by allowing cells to proceed through the cell cycle from the resting phase, G0
3. However, cancer cells deregulate these signals and derail from their
original functional purpose 1. Cancer cells can achieve deregulation through
several ways, including producing growth factor ligands themselves, which in
turn activate the proliferative signaling 4. Moreover, cancer cells may
induce surrounding normal cells to produce growth factors 4. Since the growth
factors require binding of receptors to activate the signal, normal cells
having limited number of receptors on surface are the limiting factors of
proliferative signaling. However, cancer cells may also increase the growth
factor receptors on cell surface or alter the receptor structure to make them
hypersensitive or independent of growth factor ligands 1. Various types of
growth factors and growth factor receptors are involved in the growth of cancer
cells 5. The epidermal growth factor receptor (EGFR) and the EGF-family of
peptide growth factors play a central role in various types of cancers 5. The
activating mutations caused by deletion of exon 19 and a single-point
substitution in exon 21 constitute about 90% of all EGFR activating mutations
9. The activation of EGFR receptors leads to activation in several downstream
pathways that regulate cell proliferation 1.

EGFR

            ErbB family of receptor
tyrosine kinases (RTK) is comprised of four receptors: EGFR (ErbB-1/HER1),
ErbB-2 (neu, HER2), ErbB-3 (HER3) and ErbB-4 (HER4) 5. The ErbB family has
extracellular ligand-binding domain, hydrophobic transmembrane domain and
intracellular tyrosine kinase domain 5. The extracellular domain binds to the
growth factor ligand resulting in receptor dimerization 6. Unligated EGFR can
also dimerize and lead to the activation of downstream pathways, but it is
10-times slower than ligand-stimulated 7. Even in the absence of ligands, the
EGFR fluctuate between monomer and dimer states, but the ligands are required
for downstream signaling 8. The formation of dimers can be either homo or
hetero, both leading to activation of intrinsic tyrosine kinase domain 5.
When one of the ligands, extracellular growth factor (EGF), binds to the
receptors, phosphorylation occurs at specific tyrosine residues within the
cytoplasmic tail 5. Intracellular proteins containing Src homology 2 (SH2)
and phosphotyrosine binding (PTB) domains bind to phosphorylated tyrosine
residue leading to activation of several intracellular signaling pathways 5. Activation
of EGFR leads to downstream activation of PI3K/AKT/mTOR and Ras/MAPK signaling
pathways that contribute in cancerous properties. Overexpression of EGFR is
observed in ovarian 10, glioblastoma 11, lung 12, neck 13, and breast
14 cancer cells among others. Thus, making EGFR an attractive target for
anticancer therapies.

PI3K/Akt/mTOR Pathway

            One of the downstream
pathways of EGFR is the PI3K/AKT/mTOR pathway that controls cell proliferation,
prolonged survival, and ability of metastasis 15. Phosphatidylinositol
3-kinases (PI3Ks) are the lipid kinases that translate extracellular signal
into intracellular signals, leading to multiple downstream pathways 16. Eight
mammalian PI3K enzymes can be grouped into three classes based on their
structures 17. Class I can be further divided into two classes where class IA
PI3Ks are heterodimers of a p110 catalytic subunit and a p85 regulatory subunit
17. Three isoforms of p110 are present in class IA which are p110?, p110? and
p110? encoded by PIK3CA, PIK3CB, and PIK3CD 16. These isoforms associate
with five isoforms of p85 encoded by PIK3R
15. Class IB PI3Ks are heterodimers
of a p110? catalytic subunit and a p101 or p87 regulatory isoform 18. The IA
subclass is most frequently activated in cancer 17. Moreover, p110? and p110?
are largely restricted to leukocytes 15. Class II and III both contain a
single catalytic subunit 17.

            In the absence of
activating signals, p85 interacts with p110 to inhibit kinase activity 17.
When activating signals are present, p85-p110 heterodimer is recruited to the
plasma membrane to interact with RTK phosphotyrosine residues and SH2 domains
on p85 17. The binding of heterodimer to the phosphotyrosine residue will
release p110 catalytic subunit from inhibition and lead to the activation of
PI3K 17. Moreover, activation of PI3K can be stimulated by activated Ras
which binds to p110 17. The activated PI3K phosphorylates phosphatidylinositol
4,5-biphosphate (PIP2) on the 3′ – OH position to produce phosphatidylinositol
3,4,5-triphosphate (PIP3) 19. The lipid product, PIP3, act as a secondary messenger
by binding proteins containing pleckstrin homology (PH) domains 19. PIP3
brings two PH domain containing kinases called phosphoinositide-dependent
kinase 1 (PDK1) and protein kinase B (PKB) or Akt near each other which will
lead to downstream activation 19. Inactivation of PIP3 is carried out by the
phosphatase and tensin homolog protein (PTEN) or inositol polyphosphate,
4-phosphatase type II (INPP4B) 17. PTEN dephosphorylates PIP3 to PIP2 and
INPP4B dephosphorylates PIP2 to phosphatidylinositol-4-phosphate (PIP) 16.

            Other than
overexpression of EGFR activation, aberrant activation of PI3K signaling in
cancer can be due to several different mechanisms such as loss of function of
PTEN, mutation of PIK3CA, or
activation by Ras 16. Loss of PTEN
on chromosome 10q is found in many types of cancers such as breast cancer 20,
melanoma 21, and prostate cancer 22. Mutation or amplification of PIK3CA is common in cancer cells such as
ovarian and breast cancer 23, and squamous lung cancer 24. Most of the
mutations in PIK3CA result in helical
domain and kinase domain of p110? 17. The mutation in helical reduces
inhibition of p110? by p85, while the mutation in kinase domain increase
interaction of p110? with lipid membranes 17. In other class I catalytic
isoforms, only few cases are found in cancer 17. Mutation of PIK3CB was detected in a breast cancer
25. Mutations in PIK3R1 coding for
p85 regulatory isoforms have been identified in multiple cancers 17. Reduced
expression of p85 increased the activation of PI3K and mutation in the
inter-SH2 domain that makes contact with p110 hindered the inhibition of p110
by p85 17. In other classes of PI3K, few reported cases of mutation in class
II is present, but the functional consequence is not fully understood 17.

            Activation of Akt leads
to multiple cellular processes such as apoptosis, cell proliferation, and cell
migrations. Akt is a serine/threonine-specific protein kinase with three
isoforms; Akt1, Akt2, and Akt3 26. The isoforms are present throughout both
normal and cancer tissues 27. PH domain of Akt binds to PIP3 to be
phosphorylated by PDK1 at Thr308 18. Second site, Ser473, is phosphorylated
by PDK2 28. Mammalian target of rapamycin complex 2 (mTORC2) as well as
mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK2) functionally
act as PDK2 28, 29. Phosphorylation of both sites lead to full activation of
Akt and its downstream pathways 27. After the phosphorylation Akt is able to
translocate to the nucleus from the cytoplasm which in turn affect
transcription regulators 30. Activation of Akt inhibits proapoptotic proteins
of the B-cell leukemia/lymphoma-2 (BCL-2) family, stimulates glycolysis to
supply ATP, transcribes factors forkhead box O (FoxO) and nuclear factor-kappaB
(NF-kB) to transcribe antiapoptotic genes, and increases mouse double minute 2
homolog (mdm2) to regulate p53 30. In many human cancers, amplified
activation of Akt is reported 31, 32.

            Mammalian target of
rapamycin (mTOR) is a serine/threonine kinase that plays critical role in cell
metabolism, growth, proliferation and survival 33. mTOR is located downstream
of Akt and for mTOR to be activated, multiple molecules are required to form a
complex 33. mTOR forms two multi-protein complexes which are mTOR complex 1
(mTORC1) and mTOR complex 2 (mTORC2) 33. One of the upstream regulators of mTORC1
is PI3K-Akt activity 17. While regulators for mTORC2 is unclear, some
evidences show direct association with ribosome is required for mTORC2
activation 35. mTORC1 consists of mTOR, regulatory-associated protein of mTOR
(Raptor); mammalian lethal with Sec13 protein8 (mLST8), proline-rich Akt
substrate 40kDA (PRAS40), and DEP domain-containing mTOR-interacting protein
(Deptor) 34. Raptor recruits other substrates to form mTORC1. Moreover,
Raptor positively regulates mTOR while PRAS40 and Deptor negatively regulate
mTOR since mTOR is the catalytic subunit of the complex 33. mLST8 is
suggested to shuttle mTOR between the two mTOR complexes to keep them in
dynamic equilibrium 36. Akt activates mTOR through phosphorylating tuberous
sclerosis complex 2 (TSC2) which prevents TSC1/TSC2 complex formation and
drives GTPase Rheb into the GTP-bound active state leading to activation of
mTORC1 at Ser2448 37. Moreover, Akt phosphorylates and inhibits PRAS40, which
is a negative regulator of mTORC1 38. Activated mTOR is then phosphorylates
p70S6 kinase (p70S6K) and eukaryotic translation initiation factor 4E-binding
protein 1 (4EBP1) 39. Activation of mTOR is involved in some of the cancer
hallmarks such as cell growth and metastasis as it is reported in malignant
melanoma 40.

Ras/MAPK Pathway

            Rat sarcoma (Ras)
family of proteins are important components of the large family of GTPase that
cycles through inactive GDP-bound state and active GTP-bound state 41. Three
human RAS genes encode for four
highly related 188 to 189 amino acids Ras proteins (HRas, KRas4A, KRas4B and
NRas) 41. Ras proteins are involved in intracellular pathways regulating cell
proliferation, differentiation, invasion, adhesion, and apoptosis 41. When
RTK dimerizes through the binding of a ligand, the receptor becomes activated
and autophosphorylated 41. The phosphorylated tyrosine residue in C-terminal
region in intracellular domain generates binding sites for proteins that
containing SH2 domains such as growth factor receptor-bound protein 2 (Grb2)
41. Grb2 then recruits guanine nucleotide exchange factor sons of sevenless
(SOS) at the plasma membrane which will in turn activate the membrane-bound Ras
by catalyzing the GDP to GTP 42. Active Ras will recruit the serine/threonine
protein kinases c-Raf and B-Raf 42. The activated Raf isoforms will
phosphorylate mitogen-activated protein kinase kinase-1 (MEK1) and MEK2 42.
MEK1/2 will then phosphorylate extracellular signal-regulated kinase 1 (ERK1)
and ERK2 42. ERK1 is phosphorylated at Thr202 and Tyr204 while ERK2 is
phosphorylated at Thr185 and Tyr187 42. The phosphorylation of both sites is
required for ERK to be activated 42. When ERKs are activated, they will
phosphorylate various downstream cytoplasmic and nuclear effector proteins
involved in cell growth, proliferation, survival, and motility 42. The activated
Ras can also activate PI3K-Akt pathway by activating p110 directly 43. On the
other hand, there are evidence that the activation of the Ras/ERK may lead to
apoptosis 42.

            Out of three RAS genes, K-RAS point mutation at exon 12 is the most common mutation
involving RAS gene mutation 42. K-RAS mutations are frequent in
pancreatic, colorectal and lung cancers, while N-RAS mutation is common in melanoma and H-RAS mutation is common in salivary gland 42. The mutation of
Ras leads to insensitive to inactivation by GTPase-activating proteins (GAP) and
remain activated 42. Mutations in K-RAS
gene occur frequently in non-small cell lung cancer (NSCLC) and more frequently
in adenocarcinoma 44. One of the NSCLC, NCI-H1299, is an adenocarcinoma
having NRAS mutation 44. While KRAS mutation is directly related
tobacco carcinogen exposure, NRAS
mutation showed only 3 of 20 smokers showed similar mutation as KRAS mutation 44. Like other cell
lines harbouring KRAS mutation, NCI-H1299
and other NRAS mutation harbouring
cell lines show highly phosphorylated ERK1/2 44. Constant activation of Ras
protein will lead to activation of both PI3K/Akt and Ras/MAPK pathways
contributing to proliferation, survival, migration and growth.

 

AMPK

            5′-adenosine
monophosphate-activated protein kinase (AMPK) is a serine/threonine protein
kinase having central role in metabolic pathways, and protecting cells against
physiological and pathological stress 45. When AMPK is activated, it blocks
energy expenditure and switches on metabolism to generate adenosine
triphosphate (ATP) 46. AMPK is a heterotrimeric complex consist of catalytic
? subunit, and regulatory ? and ? subunits 46. The catalytic ? isoforms
(?1-2) are encoded by PRKAA1-2, the
regulatory ? isoforms (?1-2) are encoded by PRKAB1-2,
and ? isoforms (?1-3) are encoded by PRKAG1-3
46. The phosphorylation of Thr172 in the ? subunit is required for AMPK
activation 46. Moreover, the most commonly expressed isoforms are ?1, ?1 and
?1 46.

            AMPK? subunit contains
cystathionine-?-synthase domain repeats giving potential adenine
nucleotide-binding sites 47. Site 4 of the repeats is always bound to AMP
molecule while site 1 and 3 fight for AMP, ADP, or ATP 47. Binding of AMP on
site 3 appears to regulate the phosphorylation of Thr172 47. Moreover, AMP
enhances liver kinase B1 (LKB1) dependent Thr172 phosphorylation 48. AMPK?
subunit contains N-terminal kinase domain followed by autoinhibitory domain
(AID) 48. AID interacts with kinase domain to keep AMPK in inactive
conformation 48. When AMP binds to AMPK? subunit, conformation change release
kinase domain of AMPK? from AID and allow activation of AMPK 48. Two upstream
kinases, LKB1 and calcium/calmodulin-dependent protein kinase kinase (CaMKK?),
activate AMPK by phosphorylating Thr172 48. LKB1 forms heterotrimer with
Ste20 Related Adaptor (STRAD) and scaffolding protein Mouse protein-25 (MO25),
allowing the complex to act as a regulator kinase to phosphorylate AMPK? Thr172
45. However, CaMMK? phosphorylates AMPK without the metabolic stress signal
45. In hypothalamus, neurons, and T lymphocytes, CaMKK? activate AMPK when
cellular Ca2+ level is increased 48. Mutation in LKB1 gene significantly reduced
phosphorylation of AMPK which is found in lung and cervical cancers 49. Activated
AMPK inhibits mTORC1 activation to conserve energy 45. AMPK phosphorylates
TSC2 on Thr1227 and Ser1345 leading to inactivation of Rheb by converting it
into GDP-bound confirmation 45. Moreover, AMPK can phosphorylate and inhibit
Raptor which prevents mTOR from phosphorylating downstream pathways 45. AMPK
also cause G1 cell cycle arrest by activating p53 50. Activation of AMPK in
melanoma 51, leukemia 52, breast cancer 53 and other types of cancers
showed anticancer effects such as inhibition of metastasis, inducing apoptosis,
or inhibit cell growth.

Tumour Suppressor p53

            Tumour suppressor
protein p53 is encoded by TP53 which
is located in chromosome 17 54. p53 is expressed in normal cells and is
activated when intrinsic or extrinsic stress is picked up by the cells 55.
Where stress signals are absent, p53 levels are regulated through degradation
by murine double minute 2 (MDM-2) 55. MDM-2 and p53 act as a negative
feedback loop; MDM-2 level will be increased by p53 while MDM-2 inhibits p53
activity 55. Intrinsic and extrinsic stresses such as gamma or UV
irradiation, alkylation of bases, depurination of DNA, or reaction with
oxidative free radicals causing DNA damage leads to the activation of p53 55.
Each different stress gives arise to specific modification in p53 protein
leading to specific downstream targets 55. The modifications of p53 increase
the half-life from few minutes to hours, and allow the modified p53 to bind to
specific DNA sequences and promote gene regulation 55. In some cells, p53
protein induces PTEN that induce Akt activation which leads to activation of
MDM-2 resulting in p53 inhibition 55. In cancer cells, high level of Akt
activation is observed 31, 32 which in turn lead to inhibition of p53
expression through activation of MDM-2. Phosphorylation of Ser33 and Ser46 of
p53 is induced by p38 MAP kinase (MAPK) 55. p38 MAPK is one of the downstream
target of activated Ras pathway 55. This indicates activated Ras pathway can
lead to p53 activation and downstream pathway leading to apoptosis of cells.
AMPK is also known to activate p53 through phosphorylating MDM-4 on Ser342
leading to inhibition of p53 ubiquitylation which enhances stabilization of p53
56. The activation of AMPK activates p53 to induce cell cycle checkpoint and
therefore induce apoptosis in cancer cells like glioblastoma cells 57. Moreover,
AMPK is seen to phosphorylate p53 at Ser15 directly 58.

            TP53 is the most commonly mutated gene in human cancer 59. Some
cancer shows frameshift or nonsense mutation leading to the loss of p53
expression in the cell 59. However, missense mutation allows p53 to be
expressed in tumour cells, but they show diminished or loss of wild-type
function of p53 59. NCI-H1299 harbours homozygous partial deletion of TP53 and as a result, they do not
express p53 60. Since p53 regulates critical features of cell regulation such
as apoptosis, checkpoints for cell cycle, aid in DNA repair processes, among
others, many anticancer treatments aim to restore the function of p53 or its
related pathways.  

Natural Compounds

            Historically, many
pharmaceutical agents have been discovered by screening natural products from
plants. Etoposide is derived from mandrake plant, Podophyllum peltatum 61 and pacilitaxel & docetaxel are
derived from wood and bark of pacific yew, Taxus
brevifolia 62. These agents are successfully employed in cancer
treatments. Several plant derived chemicals such as metformin 63, resveratrol
64, and rosemary extract 65 were found to have anticancer effects. Polyphenols
derived from plants such as resveratrol and curcumin are known to show
anticancer effects in vivo and in vitro 66.

Anticancer Effects of Rosemary Extract (RE)

            Rosemary
(Rosmarinus Officinalis L.) has high
content of polyphenolic compounds such as carnosic acid, rosmarinic acid, and
carnosol 65. Rosemary extract has been found to have anticancer effect on
several cancer types including results from our own lab 65. Recently, a
review of anticancer effect of rosemary extract was published by the member of
our lab 65. Findings of more recent researches are added to the table and are
sorted by cancer cell type and