Cancer Carcinogenesis arisesfrom genetic and epigenetic alteration of the molecular pathways responsiblefor regulating properties of normal cells such as proliferation,differentiation, cell death or motility 1.
Normal cells regulate thoseproperties to keep cells in control and maintain cellular integrity. Whenseveral mutations build up, normal cell function is lost and the cells maydevelop multiple hallmarks of cancer. The six hallmarks characterizing cancerare sustaining proliferating signal, uncontrolled growth, resisting apoptosis,enabling unlimited replication, inducing angiogenesis, and enabling metastasis1. The hallmarks are known to be enabled through genomic instability causingmutations, and inflammation that accelerate tumorigenesis 1. Moreover, reprogrammingof energy metabolism and evading immune destruction are important players inthe development of cancer 1. The mutations in one or more tumour suppressorgenes and proto-oncogenes give variations in cancer malignancy 2.
A chief hallmark ofcancer is the ability to sustain proliferative signals 1. Normal cellscontrol the growth promoting signals to maintain the normal cell function andintegrity 1. The growth factor signals play important role in cell regulationby allowing cells to proceed through the cell cycle from the resting phase, G03. However, cancer cells deregulate these signals and derail from theiroriginal functional purpose 1. Cancer cells can achieve deregulation throughseveral ways, including producing growth factor ligands themselves, which inturn activate the proliferative signaling 4. Moreover, cancer cells mayinduce surrounding normal cells to produce growth factors 4.
Since the growthfactors require binding of receptors to activate the signal, normal cellshaving limited number of receptors on surface are the limiting factors ofproliferative signaling. However, cancer cells may also increase the growthfactor receptors on cell surface or alter the receptor structure to make themhypersensitive or independent of growth factor ligands 1. Various types ofgrowth factors and growth factor receptors are involved in the growth of cancercells 5. The epidermal growth factor receptor (EGFR) and the EGF-family ofpeptide growth factors play a central role in various types of cancers 5. Theactivating mutations caused by deletion of exon 19 and a single-pointsubstitution in exon 21 constitute about 90% of all EGFR activating mutations9. The activation of EGFR receptors leads to activation in several downstreampathways that regulate cell proliferation 1.
EGFR ErbB family of receptortyrosine 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 hasextracellular ligand-binding domain, hydrophobic transmembrane domain andintracellular tyrosine kinase domain 5. The extracellular domain binds to thegrowth factor ligand resulting in receptor dimerization 6.
Unligated EGFR canalso dimerize and lead to the activation of downstream pathways, but it is10-times slower than ligand-stimulated 7. Even in the absence of ligands, theEGFR fluctuate between monomer and dimer states, but the ligands are requiredfor downstream signaling 8. The formation of dimers can be either homo orhetero, both leading to activation of intrinsic tyrosine kinase domain 5.When one of the ligands, extracellular growth factor (EGF), binds to thereceptors, phosphorylation occurs at specific tyrosine residues within thecytoplasmic tail 5. Intracellular proteins containing Src homology 2 (SH2)and phosphotyrosine binding (PTB) domains bind to phosphorylated tyrosineresidue leading to activation of several intracellular signaling pathways 5.
Activationof EGFR leads to downstream activation of PI3K/AKT/mTOR and Ras/MAPK signalingpathways that contribute in cancerous properties. Overexpression of EGFR isobserved in ovarian 10, glioblastoma 11, lung 12, neck 13, and breast14 cancer cells among others. Thus, making EGFR an attractive target foranticancer therapies.PI3K/Akt/mTOR Pathway One of the downstreampathways of EGFR is the PI3K/AKT/mTOR pathway that controls cell proliferation,prolonged survival, and ability of metastasis 15.
Phosphatidylinositol3-kinases (PI3Ks) are the lipid kinases that translate extracellular signalinto intracellular signals, leading to multiple downstream pathways 16. Eightmammalian PI3K enzymes can be grouped into three classes based on theirstructures 17. Class I can be further divided into two classes where class IAPI3Ks are heterodimers of a p110 catalytic subunit and a p85 regulatory subunit17. Three isoforms of p110 are present in class IA which are p110?, p110? andp110? encoded by PIK3CA, PIK3CB, and PIK3CD 16. These isoforms associatewith five isoforms of p85 encoded by PIK3R15. Class IB PI3Ks are heterodimersof a p110? catalytic subunit and a p101 or p87 regulatory isoform 18. The IAsubclass is most frequently activated in cancer 17. Moreover, p110? and p110?are largely restricted to leukocytes 15.
Class II and III both contain asingle catalytic subunit 17. In the absence ofactivating signals, p85 interacts with p110 to inhibit kinase activity 17.When activating signals are present, p85-p110 heterodimer is recruited to theplasma membrane to interact with RTK phosphotyrosine residues and SH2 domainson p85 17. The binding of heterodimer to the phosphotyrosine residue willrelease p110 catalytic subunit from inhibition and lead to the activation ofPI3K 17.
Moreover, activation of PI3K can be stimulated by activated Raswhich binds to p110 17. The activated PI3K phosphorylates phosphatidylinositol4,5-biphosphate (PIP2) on the 3′ – OH position to produce phosphatidylinositol3,4,5-triphosphate (PIP3) 19. The lipid product, PIP3, act as a secondary messengerby binding proteins containing pleckstrin homology (PH) domains 19. PIP3brings two PH domain containing kinases called phosphoinositide-dependentkinase 1 (PDK1) and protein kinase B (PKB) or Akt near each other which willlead to downstream activation 19.
Inactivation of PIP3 is carried out by thephosphatase and tensin homolog protein (PTEN) or inositol polyphosphate,4-phosphatase type II (INPP4B) 17. PTEN dephosphorylates PIP3 to PIP2 andINPP4B dephosphorylates PIP2 to phosphatidylinositol-4-phosphate (PIP) 16. Other thanoverexpression of EGFR activation, aberrant activation of PI3K signaling incancer can be due to several different mechanisms such as loss of function ofPTEN, mutation of PIK3CA, oractivation by Ras 16. Loss of PTENon 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 asovarian and breast cancer 23, and squamous lung cancer 24. Most of themutations in PIK3CA result in helicaldomain and kinase domain of p110? 17. The mutation in helical reducesinhibition of p110? by p85, while the mutation in kinase domain increaseinteraction of p110? with lipid membranes 17. In other class I catalyticisoforms, only few cases are found in cancer 17.
Mutation of PIK3CB was detected in a breast cancer25. Mutations in PIK3R1 coding forp85 regulatory isoforms have been identified in multiple cancers 17. Reducedexpression of p85 increased the activation of PI3K and mutation in theinter-SH2 domain that makes contact with p110 hindered the inhibition of p110by p85 17. In other classes of PI3K, few reported cases of mutation in classII is present, but the functional consequence is not fully understood 17. Activation of Akt leadsto multiple cellular processes such as apoptosis, cell proliferation, and cellmigrations. Akt is a serine/threonine-specific protein kinase with threeisoforms; Akt1, Akt2, and Akt3 26.
The isoforms are present throughout bothnormal and cancer tissues 27. PH domain of Akt binds to PIP3 to bephosphorylated by PDK1 at Thr308 18. Second site, Ser473, is phosphorylatedby PDK2 28. Mammalian target of rapamycin complex 2 (mTORC2) as well asmitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK2) functionallyact as PDK2 28, 29. Phosphorylation of both sites lead to full activation ofAkt and its downstream pathways 27. After the phosphorylation Akt is able totranslocate to the nucleus from the cytoplasm which in turn affecttranscription regulators 30. Activation of Akt inhibits proapoptotic proteinsof the B-cell leukemia/lymphoma-2 (BCL-2) family, stimulates glycolysis tosupply ATP, transcribes factors forkhead box O (FoxO) and nuclear factor-kappaB(NF-kB) to transcribe antiapoptotic genes, and increases mouse double minute 2homolog (mdm2) to regulate p53 30.
In many human cancers, amplifiedactivation of Akt is reported 31, 32. Mammalian target ofrapamycin (mTOR) is a serine/threonine kinase that plays critical role in cellmetabolism, growth, proliferation and survival 33. mTOR is located downstreamof Akt and for mTOR to be activated, multiple molecules are required to form acomplex 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 mTORC1is PI3K-Akt activity 17. While regulators for mTORC2 is unclear, someevidences show direct association with ribosome is required for mTORC2activation 35. mTORC1 consists of mTOR, regulatory-associated protein of mTOR(Raptor); mammalian lethal with Sec13 protein8 (mLST8), proline-rich Aktsubstrate 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 regulatemTOR since mTOR is the catalytic subunit of the complex 33.
mLST8 issuggested to shuttle mTOR between the two mTOR complexes to keep them indynamic equilibrium 36. Akt activates mTOR through phosphorylating tuberoussclerosis complex 2 (TSC2) which prevents TSC1/TSC2 complex formation anddrives GTPase Rheb into the GTP-bound active state leading to activation ofmTORC1 at Ser2448 37. Moreover, Akt phosphorylates and inhibits PRAS40, whichis a negative regulator of mTORC1 38. Activated mTOR is then phosphorylatesp70S6 kinase (p70S6K) and eukaryotic translation initiation factor 4E-bindingprotein 1 (4EBP1) 39. Activation of mTOR is involved in some of the cancerhallmarks such as cell growth and metastasis as it is reported in malignantmelanoma 40.Ras/MAPK Pathway Rat sarcoma (Ras)family of proteins are important components of the large family of GTPase thatcycles through inactive GDP-bound state and active GTP-bound state 41.
Threehuman RAS genes encode for fourhighly related 188 to 189 amino acids Ras proteins (HRas, KRas4A, KRas4B andNRas) 41. Ras proteins are involved in intracellular pathways regulating cellproliferation, differentiation, invasion, adhesion, and apoptosis 41. WhenRTK dimerizes through the binding of a ligand, the receptor becomes activatedand autophosphorylated 41. The phosphorylated tyrosine residue in C-terminalregion in intracellular domain generates binding sites for proteins thatcontaining 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 Rasby catalyzing the GDP to GTP 42. Active Ras will recruit the serine/threonineprotein kinases c-Raf and B-Raf 42. The activated Raf isoforms willphosphorylate 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 isphosphorylated at Thr185 and Tyr187 42. The phosphorylation of both sites isrequired for ERK to be activated 42. When ERKs are activated, they willphosphorylate various downstream cytoplasmic and nuclear effector proteinsinvolved in cell growth, proliferation, survival, and motility 42.
The activatedRas can also activate PI3K-Akt pathway by activating p110 directly 43. On theother hand, there are evidence that the activation of the Ras/ERK may lead toapoptosis 42. Out of three RAS genes, K-RAS point mutation at exon 12 is the most common mutationinvolving RAS gene mutation 42.
K-RAS mutations are frequent inpancreatic, colorectal and lung cancers, while N-RAS mutation is common in melanoma and H-RAS mutation is common in salivary gland 42. The mutation ofRas leads to insensitive to inactivation by GTPase-activating proteins (GAP) andremain activated 42. Mutations in K-RASgene occur frequently in non-small cell lung cancer (NSCLC) and more frequentlyin adenocarcinoma 44. One of the NSCLC, NCI-H1299, is an adenocarcinomahaving NRAS mutation 44. While KRAS mutation is directly relatedtobacco carcinogen exposure, NRASmutation showed only 3 of 20 smokers showed similar mutation as KRAS mutation 44. Like other celllines harbouring KRAS mutation, NCI-H1299and other NRAS mutation harbouringcell lines show highly phosphorylated ERK1/2 44. Constant activation of Rasprotein will lead to activation of both PI3K/Akt and Ras/MAPK pathwayscontributing to proliferation, survival, migration and growth.
AMPK 5′-adenosinemonophosphate-activated protein kinase (AMPK) is a serine/threonine proteinkinase having central role in metabolic pathways, and protecting cells againstphysiological and pathological stress 45. When AMPK is activated, it blocksenergy expenditure and switches on metabolism to generate adenosinetriphosphate (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, theregulatory ? isoforms (?1-2) are encoded by PRKAB1-2,and ? isoforms (?1-3) are encoded by PRKAG1-346. The phosphorylation of Thr172 in the ? subunit is required for AMPKactivation 46. Moreover, the most commonly expressed isoforms are ?1, ?1 and?1 46. AMPK? subunit containscystathionine-?-synthase domain repeats giving potential adeninenucleotide-binding sites 47. Site 4 of the repeats is always bound to AMPmolecule while site 1 and 3 fight for AMP, ADP, or ATP 47. Binding of AMP onsite 3 appears to regulate the phosphorylation of Thr172 47.
Moreover, AMPenhances 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 inactiveconformation 48. When AMP binds to AMPK? subunit, conformation change releasekinase domain of AMPK? from AID and allow activation of AMPK 48. Two upstreamkinases, LKB1 and calcium/calmodulin-dependent protein kinase kinase (CaMKK?),activate AMPK by phosphorylating Thr172 48. LKB1 forms heterotrimer withSte20 Related Adaptor (STRAD) and scaffolding protein Mouse protein-25 (MO25),allowing the complex to act as a regulator kinase to phosphorylate AMPK? Thr17245. However, CaMMK? phosphorylates AMPK without the metabolic stress signal45. In hypothalamus, neurons, and T lymphocytes, CaMKK? activate AMPK whencellular Ca2+ level is increased 48.
Mutation in LKB1 gene significantly reducedphosphorylation of AMPK which is found in lung and cervical cancers 49. ActivatedAMPK inhibits mTORC1 activation to conserve energy 45. AMPK phosphorylatesTSC2 on Thr1227 and Ser1345 leading to inactivation of Rheb by converting itinto GDP-bound confirmation 45.
Moreover, AMPK can phosphorylate and inhibitRaptor which prevents mTOR from phosphorylating downstream pathways 45. AMPKalso cause G1 cell cycle arrest by activating p53 50. Activation of AMPK inmelanoma 51, leukemia 52, breast cancer 53 and other types of cancersshowed anticancer effects such as inhibition of metastasis, inducing apoptosis,or inhibit cell growth. Tumour Suppressor p53 Tumour suppressorprotein p53 is encoded by TP53 whichis located in chromosome 17 54. p53 is expressed in normal cells and isactivated when intrinsic or extrinsic stress is picked up by the cells 55.Where stress signals are absent, p53 levels are regulated through degradationby murine double minute 2 (MDM-2) 55. MDM-2 and p53 act as a negativefeedback loop; MDM-2 level will be increased by p53 while MDM-2 inhibits p53activity 55.
Intrinsic and extrinsic stresses such as gamma or UVirradiation, alkylation of bases, depurination of DNA, or reaction withoxidative free radicals causing DNA damage leads to the activation of p53 55.Each different stress gives arise to specific modification in p53 proteinleading to specific downstream targets 55. The modifications of p53 increasethe half-life from few minutes to hours, and allow the modified p53 to bind tospecific DNA sequences and promote gene regulation 55. In some cells, p53protein induces PTEN that induce Akt activation which leads to activation ofMDM-2 resulting in p53 inhibition 55. In cancer cells, high level of Aktactivation is observed 31, 32 which in turn lead to inhibition of p53expression through activation of MDM-2. Phosphorylation of Ser33 and Ser46 ofp53 is induced by p38 MAP kinase (MAPK) 55.
p38 MAPK is one of the downstreamtarget of activated Ras pathway 55. This indicates activated Ras pathway canlead to p53 activation and downstream pathway leading to apoptosis of cells.AMPK is also known to activate p53 through phosphorylating MDM-4 on Ser342leading to inhibition of p53 ubiquitylation which enhances stabilization of p5356. The activation of AMPK activates p53 to induce cell cycle checkpoint andtherefore 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. Somecancer shows frameshift or nonsense mutation leading to the loss of p53expression in the cell 59.
However, missense mutation allows p53 to beexpressed in tumour cells, but they show diminished or loss of wild-typefunction of p53 59. NCI-H1299 harbours homozygous partial deletion of TP53 and as a result, they do notexpress p53 60. Since p53 regulates critical features of cell regulation suchas apoptosis, checkpoints for cell cycle, aid in DNA repair processes, amongothers, many anticancer treatments aim to restore the function of p53 or itsrelated pathways. Natural Compounds Historically, manypharmaceutical agents have been discovered by screening natural products fromplants.
Etoposide is derived from mandrake plant, Podophyllum peltatum 61 and pacilitaxel & docetaxel arederived from wood and bark of pacific yew, Taxusbrevifolia 62. These agents are successfully employed in cancertreatments. Several plant derived chemicals such as metformin 63, resveratrol64, and rosemary extract 65 were found to have anticancer effects. Polyphenolsderived from plants such as resveratrol and curcumin are known to showanticancer effects in vivo and in vitro 66. Anticancer Effects of Rosemary Extract (RE) Rosemary(Rosmarinus Officinalis L.
) has highcontent of polyphenolic compounds such as carnosic acid, rosmarinic acid, andcarnosol 65. Rosemary extract has been found to have anticancer effect onseveral cancer types including results from our own lab 65. Recently, areview of anticancer effect of rosemary extract was published by the member ofour lab 65. Findings of more recent researches are added to the table and aresorted by cancer cell type and