ANTI-CANCER AND CHEMOPREVENTIVE EFFICACY OF GRAPE SEED EXTRACT AGAINST HEAD AND NECK SQUAMOUS CELL CARCINOMA
THROUGH CELLULAR METABOLISM by
SANGEETA SHROTRIYA M.S., Seoul National University, 2007
B.Pharm, University of Dhaka, 2000
A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment
of the requirements of the degree of Doctor of Philosophy
Toxicology Program 2013
ii This thesis for the Doctor of Philosophy degree by
Sangeeta Shrotriya has been approved for the
Toxicology Program by
Vasilis Vasiliou, Chair Rajesh Agarwal, Advisor
David Ross Robert Sclafani
Gagan Deep
iii Shrotriya, Sangeeta (Ph.D., Toxicology)
Anti-cancer and Chemopreventive Efficacy of Grape Seed Extract against Head and Neck Squamous Cell Carcinoma through Cellular Metabolism
Thesis directed by Professor Rajesh Agarwal
ABSTRACT
Grape seed extract (GSE), rich in proanthocyandins, has been proven to possess anti-cancer and chemopreventive efficacy in various organs associated malignancies. However, the detailed mechanistic insight for its efficacy against head and neck
squamous cell carcinoma (HNSCC) are largely unknown. Therefore, the proposed study was designed to thoroughly evaluate a) the molecular mechanism involved in cell growth inhibition and cell death following GSE treatment, b) to determine the molecular
mechanism involved in GSE-mediated metabolic oxidative stress and modulation of cellular metabolism, c) to assess the chemopreventive efficacy of GSE in long-term 4-nitroquinoline-1-oxide (4NQO)-induced oral carcinogenesis mouse model in HNSCC. Our study for the first time revealed that GSE selectively arrested HNSCC cells at G2/M phase of cell cycle by activating DNA damage check-point kinases, decreasing the expression of DNA repair enzymes expression, and facilitating apoptotic cell death. GSE-induced increased accumulation of intra-cellular reactive oxygen species (ROS) was identified as a major mechanism involved in cell viability, DNA damage and apoptosis of HNSCC cells. These preliminary observations laid a foundation for second part of the proposed study, where we investigated the mechanism involved in accumulation of
iv intracellular ROS in HNSCC cells. We have demonstrated that GSE inhibited the activity of mitochondrial complex III and modulated the cellular metabolism in HNSCC.
In recent years, growing evidence suggest that one of the key factor in poor prognosis of head and neck cancer is due to the diagnosis of the disease at advanced stages, therefore, targeting this cancer at earlier stage of disease development is believed to provide better clinical outcome. Therefore, on the third part of this study, we have examined the efficacy of GSE in tumor progression using 4NQO-induced oral
tumorigenesis model. Our findings revealed that GSE remarkably inhibited the tumor progression of premalignant lesions to papilloma by modulating the various molecules deregulated in HNSCCs. Together, results from the current study clearly exhibit that GSE have multiple cellular targets, further supporting its translational potential in intervention and prevention of HNSCC.
The form and content of this abstract are approved. I recommend its publication. Approved: Rajesh Agarwal
v
This dissertation is dedicated to my wonderful parents, my husband, my son and my siblings
vi ACKNOWLEDGMENTS
I would like to thank my advisor Dr. Rajesh Agarwal for providing me with an opportunity to work under his guidance and freedom to pursue my ideas. His constant supervision has helped me to develop as a critical independent researcher over the years. I further would like to thank my committee members including my thesis committee chair, Dr. Vasilis Vasiliou, and members Dr. David Ross, Dr. Robert Sclafani, Dr. Gagan Deep for their scientific input. I am grateful to late committee member Dr. Alvin M. Malkinson who has inspired me to keep learning throughout the study period.
Sincere thanks to all of the past and present colleagues in Dr. Agarwal’s
laboratory and Dr. Chapla Agarwal who have created friendly, productive and supportive environment. My scientific research motive came to a success with the help of great number of people who need to be acknowledged for their support. My thanks to Dr. Manish Patel for allowing me to use XF24 analyzer, and Pamela Lopert and Shane D Rowley for helping during this study. Special thanks to Dr. David Orlicky for being such an incredible teacher and assisting me to understand histopathology. My sincere thanks to Dr. David Siegel for his training on confocal microscopy, he is the best.
Graduate school has overall been an incredible experience having such caring friend as you all are (Swetha and family, Gaurav and family, Shikha, Bipesh, Molly, Stephnie). I am grateful to all of you for patiently listening to my frustrations, giving me the warmth and affection of friendship and in too many ways to list.
Finally, I am grateful to my family included my parents, sisters, brother, parents-in-law, sister-parents-in-law, brother-in-laws, nieces and nephews who supported me to excel in life regardless of any odds. Thanks to my son, Aaron who has been an excellent spirit and
vii has given a new meaning to life. Last, but not the least, I am indebted to my husband, Deepak, for his untiring patience, unconditional love and support, that helped me to stay grounded during the graduate school career regardless of what we went through together.
viii TABLE OF CONTENTS
CHAPTER
I. INTRODUCTION……….……….1
Cancer: initiation, promotion and progression………...1
Molecular and genetics changes during carcinogenesis………...2
Head and neck squamous cell carcinoma (HNSCC)………...3
Incidence, epidemiology and risk factors………...3
Major molecular and genetic predispositions in HNSCC …………...7
General principles of HNSCC management ……….…...10
Surgery and/or radiation therapy...10
Chemotherapy ………..11
Multimodal therapy………...12
Molecular and targeted therapy…..………...………....12
Existing management of HNSCC: limitation and prospects……….16
Chemoprevention……….………..17
Chemoprevention in head and neck cancer………...18
Clinical studies: single and combination of chemopreventive agents…..………...20
Grape Seed Extract (GSE)………..………...24
GSE: source, chemical components and chemical structure……….24
Bioavailability of active components of GSE………...28
Prospective chemopreventive agent-GSE……….29
GSE: anti-cancer and chemopreventive efficacy………..30
GSE: clinical studies……….40
Summary………...44
Objective and specific aims of this study ………46
Innovativeness of the study………..47
ix II. THE IMPORTANCE OF REACTIVE OXYGEN SPECIES IN
GSE-MEDIATED CELL GROWTH INHIBITION AND INDUCTION
OF CELL DEATH IN HEAD AND NECK CANCER CELLS………49
Introduction ………...49
Materials and methods ………...53
Results ……….………..57
GSE strongly inhibits growth, and causes G2/M arrest and apoptotic death in HNSCC cells………...57
GSE activates DNA damage sensor kinases and associated cellular check-points as well as caspases in HNSCC cells………...61
GSE causes DNA damage and decreases the level of repair enzymes in HNSCC cells ………..63
GSE induces sub-nuclear phosho-H2A.X foci formation, without the formation of Rad51 and Brca1 repair foci in HNSCC cells……….……..66
GSE-induced DNA damage and apoptotic cell death is through an increased intra-cellular ROS level in HNSCC cells………...69
GSE suppresses human HNSCC cell as xenograft growth in nude mice………...………...70
Effect of GSE on proliferation, apoptosis, and DNA damage biomarkers in FaDu xenografts...73
Discussion ………...79
III. GSE TARGETS CELLULAR REDOX BALANCE AND BIOENERGETICS TO INDUCED AUTOPHAGY AND APOPTOSIS IN HUMAN HEAD AND NECK SQUAMOUS CARCINOMA CELLS………...84
Introduction ……….………..84
Material and methods……….88
Results ………...……….…….………..95
GSE causes generation of mitochondrial ROS in HNSCC cells………..……….….………..95
x GSE inhibits electron transport chain (ETC) complex III, but not complex I
activity in HNSCC cells…..………...97
GSE disrupts cellular redox defense resulting in oxidative stress in HNSCC cells……….…….…...98
GSE disrupts mitochondrial membrane potential of HNSCC cells …….. ………....100
Effect of GSE on bioenergetics in HNSCC cells ………...101
GSE activates AMPK and inhibits mTOR signaling molecules in HNSCC cells ………..….…………...107
GSE induces autophagy in HNSCC cells………...109
Effect of GSE on the expression of phospho-AMPK and p62 in FaDu xenograft tissue………....114
Discussion ……….…….……….115
IV. THE CHEMOPREVENTIVE POTENTIAL OF THE GRAPE SEED EXTRACT IN SUPPRESSING 4-NITROQUINOLINE 1-OXIDE INDUCED PROGRESSION OF PREMALIGNANT LESIONS OF TONGUE CARCINOGENESIS IN C57BL/6 MICE……...121
Introduction ………...………...121
Materials and methods ………...………125
Results ……….………...………128
General observation after 16 weeks of 4NQO exposure ………128
Effect of GSE on 4NQO-induced tongue pre-neoplastic and neoplastic lesions ……….……….………..131
Effect of GSE feeding on proliferation and apoptosis in tongue tissues………...133
Effect of GSE on the expression of phospho-EGFR in tongue tissues………..……….138
Effect of GSE on the expression of phospho-AMPK and p62 in tongue tissues………...138
xi V. SUMMARY DISCUSSION...…..……….147 REFERENCES ……….………151 APPENDIX
xii LIST OF TABLES
TABLE
1.1 Global and USA statistics: estimated incidence and mortality in both sex
for the year 2013……….…….4 1.2 Risk factors associated with HNSCC development….………...……….6 1.3 Selective clinical chemopreventive trials in HNSCC patients………...26
xiii LIST OF FIGURES
FIGURE
1.1 Schematic diagram illustrating primary, secondary and tertiary
chemoprevention at various stages of carcinogenesis ………..………19 1.2 Chemical structures of different components of grape seed extract …...………25 1.3 Molecular bases for the cancer preventive and therapeutic efficacy
of grape seed extract proanthocyanidins………45 2.1 Effect of GSE on cell growth and cell death in HNSCC cells………..…...58 2.2 Effect of GSE on live and dead cell number of NHEK cells ………59 2.3 Effect of GSE on cell cycle distribution in both Detroit 562 and FaDu
cells.………..…...60 2.4 Effect of GSE on the apoptosis in HNSCC cells ………..60
2.5 Effect of GSE on the expression of DNA damage sensors, and checkpoint
signaling in HNSCC cells ………..…. .62 2.6 Effect of GSE on the expression of protein associated with the apoptosis in
HNSCC cells ………...64 2.7 Effect of GSE on the expression of DNA damage
molecules in HNSCC cells……….65 2.8 Effect of GSE on DNA damage in HNSCC cells as
measured by comet assay ………..………....65 2.9 Effect of GSE on the expression of DNA damage repair
molecules in HNSCC cells ……..………..67 2.10 Effect of GSE on phospho-H2A.X foci formation in HNSCC cells …..………...68 2.11 Effect of GSE on Brca1 foci formation in HNSCC cells ……..…...68 2.12 Effect of GSE on Rad51 foci formation in HNSCC cells ……..…...69
xiv 2.13 Effect of GSE on intra-cellular accumulation of ROS in
HNSCC cells ………...71 2.14 NAC reversed increased accumulation of intracellular ROS
generation by GSE in HNSCC cells ………72 2.15 Treatment with NAC reverses GSE-induced decreased cell
number and cell death in both HNSCC cells ………...72 2.16 Treatment with NAC reverses GSE-induced DNA damage
and apoptosis in both HNSCC cells ………73 2.17 Effect of dietary feeding of 0.2% GSE in HNSCC Detroit 562
and FaDu tumor volume in athymic nude mice. ………..75 2.18 Effect of dietary feeding of 0.2% GSE in HNSCC
Detroit 562 and FaDu tumor weight in athymic nude mice ………76 2.19 Effect of GSE on proliferation marker in FaDu tumor
xenograft model.. ………...77 2.20 Effect of GSE on apoptosis marker in FaDu tumor
xenograft model………78 2.21 Effect of GSE on DNA damage marker in FaDu tumor
xenograft model ………...78 3.1 GSE inhibits complex III activity and enhances mitochondrial ROS
generation……….………96 3.2 PEG-SOD treatment compromises GSE-induced mitochondrial ROS
generation and apoptosis in HNSCC cells………....97 3.3 GSE treatment did not affect complex I activity and SOD addition
did not significantly GSE-mediated inhibition of complex
III activity in HNSCC cells………..99 3.4 GSE targets intracellular antioxidants………... ……….100 3.5 GSE disrupts mitochondrial membrane potential in HNSCC cells………...…..101 3.6 GSE modulates oxidative phosphorylation in HNSCC cells………..104 3.7 GSE treatment decreases proton leak and respiratory
xv 3.8 GSE treatment inhibits glycolytic rates in HNSCC cells………...………106 3.9 GSE decreases NADH/NAD+ in HNSCC cells ………...107 3.10 GSE treatment activates AMPK in HNSCC cells………...108 3.11 GSE inhibited Akt, mTOR and its downstream signaling
molecules in HNSCC cells………...110 3.12 GSE induces autophagy in both HNSCC cells………...112 3.13 GSE-induced apoptotic cell death was enhanced by
autophagic inhibitors in FaDu cells………113 3.14 GSE-induced autophagy and apoptosis are independent
pathways in FaDu cells………...114 3.15 Effect of dietary feeding of GSE on the expression of
phospho-AMPK in FaDu cells xenograft tissue……….117 3.16 Effect of dietary feeding of GSE on the expression of
p62 in FaDu cells xenograft tissue……….117 4.1 Structure of 4NQO and its mutagenic metabolites……….123 4.2 Molecular alteration at various stages of 4NQO induced oral
carcinogenesis process ………..124 4.3 Experimental protocol for chemopreventive studies of GSE on
4NQO –induced oral carcinogenesis model………...129 4.4 Effect of GSE on body weight, water, and diet consumption..………..130 4.5 Effect of GSE on 4NQO-induced pre-neoplastic and neoplastic
lesions ….………..……….132 4.6 A detailed histopathological evidences of normal mucosa, epithelial
hyperplasia and dysplasia lesions from 4NQO, and 4NQO+GSE
treated group tongue tissues………...134 4.7 Representative images of papilloma from 4NQO group tongue tissue
histopathology analysis ………. 135 4.8 Effect of GSE on cell proliferation in 4NQO-induced oral
xvi 4.9 Effect of GSE on apoptosis in 4NQO-induced oral carcinogenesis model……137 4.10 Effect of GSE on the expression of phospho-EGFR in 4NQO-induced
oral carcinogenesis model……….…..140 4.11 Effect of GSE in the expression of phospho-AMPK in 4NQO-induced
oral carcinogenesis model………...141 4.12 Effect of GSE in the expression of p62 in 4NQO-induced oral
carcinogenesis mode………...142 5.1 Schematic representation illustrating the different cellular
xvii LIST OF ABBREVIATIONS
ACC Acetyl-CoA Carboxylase
AICR American institute of cancer research
AMPK Adenosine monophosphate protein kinase
AMPK AMP-activated protein kinase
ARE Antioxidant response element
ATCC American type culture collection
Atg Autophagy specific gene
ATM Ataxia telangiectasiamutated
ATR Ataxia telangiectasia-Rad3-related
Brca1 Breast cancer gene 1
BrdU 5-bromo-2’-deoxyuridine
Cdc25C Cell division cycle 25C
CDKs Cyclin dependent kinases
Chk1/2 Checkpoint kinase 1/2
CREB1 Cyclic AMP-response element binding
protein 1 CYP450 Cytochrome P450s DAB: 3,30-diaminobenzidine DAPI 4′, 6′-diamidino-2-phenylindole DCF-DA 2′, 7′-dichlorofluorescein DHE Dihydroeithidium DiOC6 (3) 3, 3' dihexyloxacarbocyanine
DMEM Modified Eagle’s medium
DMSO Dimethyl sulfoxide
FACS Fluorescence activated cell sorting
ECL Enhanced chemiluminescence
ECAR Extracellular acidification rate
EF2 Elongation factor 2
xviii
EGFR Epidermal growth factor receptor
EM Electron microscope
EMT Epithelial mesenchymal transition
ER Endoplasmic reticulum
ETC Electron transport chain
FBS Fetal bovine serum
FDA Food and drug administration
FCCP Carbonyl cyanide p-trifluromethoxy phenyl
hydrazone
FGF Fibroblast growth factor
FITC Fluorescein isothiocyanate
GLUTs Glucose transporters
GR Glucocorticoid receptor
GSE Grape seed extract
GSP Grape seed proanthocyanidins
GSPE Grape seed proanthocyanidins extract
GSH Gluthathione
H&E Hematoxylin and eosin
HDAC Histone deacetylase
HGF Hepatocyte growth factors
HNSCC Head and neck cancer
HPV Human papilloma virus
HRR Homologous recombination repair
IFN-α Interferon alpha
IGFBP-3 Insulin-like growth factor binding protein 3
IGF1R Insulin-like growth factor 1 receptor
IHC Immunohistochemistry
IL-8 Interleukin-8
KRS Krebs-ringer bicarbonate solution
LC3 Light chain-3
xix
MMC Mitomycin C
MMP Mitochondrial membrane potential
MMPs metalloproteinases
MRC Mitochondrial reserve capacity
mTOR Mammalian target of rapamycin;
NAC N-acetylcysteine
NAD Nicotinamide adenine dinucleotide oxidized
NADH Nicotinamide adenine dinucleotide reduced
NCI National cancer institute
NF-kB Nuclear factor kappa B
NHEJ Non-homologous end joining
NHEK Normal human epidermal keratinocytes
NNK 4-methylnitrosamine-1(3-pyridyl)-1-butanone
NSCLC Non-small cell lung carcinoma
OPCs Oligomeric proanthocyanidins
OCR Oxygen consumption rate
PARP Poly (ADP-ribose) polymerases
PCNA Proliferating cell nuclear antigen
PI Propidium iodide
PIN Prostatic intraepithelial neoplasia
PMSF Phenylmethylsulfonyl fluoride
PSA Prostate specific antigen
ROS Reactive oxygen species
RT Radiotherapy
SCC Squamous cell carcinoma
SOD Superoxide dismutase
SPT Secondary primary tumor
STAT3 Signal transducer and activator of transcription 3
TEM Transmission electron microscopy
xx
TKIs Tyrosine kinase inhibitors
TPA 12-O-tetradecanoylphorbol-13-acetate
TRAMP Transgenic adenocarcinoma of the mouse prostate
TUNEL Terminal deoxynucleotidyl transferase dUTP nick
end labeling
VEGF Vascular endothelial growth factor
uPA Urokinase plasminogen activator
2-DG 2-Deoxyglucose
3-MA 3-Methyladenosine
4E-BP1 Eukaryotic translation initiation factor 4E-binding
protein 1
4HPR 4-Hydroxylcarbophenylretinamide-4
5-FU 5-Flurouracil
4NQO 4-Nitroquinoline 1-oxide
1 CHAPTER I
INTRODUCTION
Cancer is a chronic dreaded disease and is one of the major causes of mortality worldwide. According to the 2008 report of the International Agency for Research on Cancer (IARC), every year there are about 12.7 million new cancer cases and 7.6 million cancer-related deaths around the world [1]. In the United States alone, approximately 1.67 million new cancer cases and 0.58 million deaths are projected to occur from cancer in the year 2013 [2]. To fight this deadly disease, continuous efforts have been made in the field of intervention; however, due to increased life expectancy, exponential
population growth, and increased prevalence of risk-associated behaviors, these efforts have unfortunately yield minimal improvements. Rather, it is expected that the global burden of cancer will continue to rise in the years to come. Hence, there is an urgent need to develop newer tools for the prevention, intervention, and treatment of cancer.
Cancer: Initiation, Promotion and Progression
Cancer is a multistage and polygenic disease. The multistage model of
carcinogenesis is distinctly divided into initiation, promotion and progression stages as first described by Armitage and Doll in 1954 [3]. After repeated exposure to mutagens, normal cells undergo genetic alterations constituting initiation stage. During tumor promotion, genetic and epigenetic changes further accumulate in cells triggering
uncontrolled cell proliferation. Subsequently, cellular and micro-environmental dynamics further drive tumor cells towards advanced stages leading to invasiveness and metastasis
2 during tumor progression [4, 5]. This multistage model of carcinogenesis is now well documented experimentally and remains widely accepted.
Molecular and Genetic Changes during Carcinogenesis
During carcinogenesis (a multistage process), tumor cells develop genetic and epigenetic changes at the molecular level acquiring a paradoxical state of dysfunctional self-sufficiency in promoting tumor growth and metastasis with sustained heterogeneity [6, 7]. Common genetic alterations resulting in tumorigenesis are seen in three types of genes: oncogenes, tumor-suppressor genes and stability genes [8-10]. Oncogenes are functionally heterogeneous group of genes, which are constitutively active in most of the cancer cells. These genes positively regulate complex cascades/mechanisms within the cancer cell which additionally promote cell growth [11]. In contrast, mutations in tumor-suppressor genes lead to reduction in the target genes expression and/or their activities. Such inactivation results from either missense mutations at sites that are necessary for tumor-suppressor activity, or mutations that lead to the formation of truncated proteins or epigenetic silencing of these genes [12]. It is believed that the major anomalies in the genome leading to tumorigenesis result from mutations in tumor suppressor genes [13]. The third types of genes, termed stability genes include those involved in DNA damage repair that stabilizes the genome. These genes do not directly regulate proliferation, but instead maintain genomic stability [13]. Together, mutations in these genes functionally integrate to confer certain hallmark characteristics to cancer cells. These characteristics include sustained proliferation, immortality, evading tumor suppressors activity,
resistance to programmed cell death, angiogenesis, and activating invasion and metastasis [14]. In addition to these well-established hallmarks and the gene deregulation that results
3 in them, the last few decades have seen progress in the field of the cancer research
revealing more distinctive features of cancer cells including reprogramming energy metabolism, evading immune surveillance, and remodeling the tumor microenvironment [6]. Recognition of all these hallmarks of cancer cells is believed to make a momentous difference while designing and developing new means to treat cancer.
Head and Neck Squamous Cell Carcinoma (HNSCC)
HNSCC is the cancer of multiple organs; oral cavity, oropharynx, nasopharynx, hyphopharynx, and the larynx. Squamous cell carcinoma (SCC) constitutes more than 90% of malignant tumors of the head and neck region. Other head and neck malignancy tumor type include minor gland carcinomas, and lymphomas accounting about ≤ 5% of all oral cancers. Apart from being anatomically complex, HNSCC is heterogeneous in nature. The heterogeneity in HNSCC was highlighted by Slaughter and colleagues with the concept of “field cancerization” explaining multifocal characteristics and the
possibility of developing secondary primary tumors in HNSCC patients post therapy [15]. Collectively, HNSCC is an intricate disease with several risk factors, demographic
inconsistency, and differences based upon ethnicity, gender, and age.
Incidence, Epidemiology and Risk Factors
HNSCC is a devastating disease worldwide accounting for 650,000 new cases and 350,000 deaths every year [16]. In 2013 in the United States alone, approximately 53,640 new cases and 11,520 deaths are projected to occur [2]. Globally, this cancer carries a mortality rate of ≥ 50% which has not changed in the past few decades. Moreover, the incidences of human papillomavirus (HPV)-associated oropharyngeal cancer is
4 colon cancer (1999-2008), and breast cancer (1999-2004)] around the world [17]. The incidence and mortality statistics of head and neck cancer as well as cancer overall from around the world and from USA are summarized in Table 1.1 [2].
In general, HNSCC is more common among men as compared to women the ratio ranging from 2:1 to 4:1; however, this gender based prevalence of HNSCC has changed recently, with the increase in number of female smokers over the years [18].
Incidence and mortality rates of HNSCC vary demographically as well, with 2/3rd of these malignancies occurring in developing countries. The incidence of laryngeal and hypopharyngeal SCCs are higher in Southern and Central Europe, and some parts of South America, while incidences of these SCCs are lowest in South-East Asia and Central Africa [19]. The oral and oropharyngeal SCCs are widespread among both men and women throughout South Asia, whereas nasopharyngeal cancer is much more frequent in Southern China, Hong Kong, and Northern Africa. Furthermore, there are notable disparities in incidence among different ethnicities and races [20]. The incidence
Table 1.1: Global and USA statistics: estimated incidence and mortality in both sexes for the year 2013
Deaths New cases
7.6 million 12.7 million
Worldwide
Head and neck cancer statistics
0.58 million 1.6 million
United States of America
53,640 650,000
11,520 United States of America
350,000 Worldwide Overall statistics of cancer Deaths New cases 7.6 million 12.7 million Worldwide
Head and neck cancer statistics
0.58 million 1.6 million
United States of America
53,640 650,000
11,520 United States of America
350,000 Worldwide
Overall statistics of cancer
5 of laryngeal cancer is higher in the African Americans populations as compared to Caucasian, Asian, and Hispanic groups, along with corresponding lower survival rate, the reason for which being largely unknown [21, 22].
Several behavioral, environmental, viral and genetic factors have been associated with the development of HNSCC and are listed in Table 1.2. More than 70% of head and neck cancers are due to tobacco and alcohol consumption [23]. Tobacco smoking, and consumption of smokeless tobacco products, such as chewing tobacco or snuff, is
causally related to cancer at many of the specific sites within this group [24, 25]. Several studies have also indicated that risk is substantially greater with increasing number and duration of cigarettes smoked, and decreased with longer duration since quitting [23, 25]. It has been reported that risks, particularly of laryngeal and pharyngeal cancers, are also increased in those who have never smoked themselves, but have had prolonged exposure to involuntary smoking (passive smoking) at home or at work [25]. Likewise, a coherent correlation with alcohol intake and HNSCC development has also been established. As compared to non or occasional drinkers, light drinkers have a modestly increased risk of oral and pharyngeal cancers, while in heavy drinkers this risk increases by 5-fold for oral cancers and 7-fold for pharyngeal cancers [26].
For individuals using both tobacco and alcohol, these risk factors appear to have synergistic effects, accelerating the risk of both oral and pharyngeal cancer by nearly 35 fold [26]. Though tobacco and alcohol users are considered the highest risk individuals, the history of exposure is not considered adequate to determine HNSCC progression with accuracy. Under such circumstances, genetic variations have also been implicated to play
6 Table 1.2: Risk factors associated with HNSCC development
Risk factors type Associated risk factors Organs associated in head and neck region
Ref Behavioral Factors Alcohol Oral cavity
Nasopharyngeal, and Oropharyngeal
[26]
Tobacco and related products (cigarette, cigar, pipe), betel quid, areca nut and gutka chewing
Lips, oral and buccal cavity
Nasopharyngeal Oropharyngeal
[27]
Viral Factor Human papilloma virus
(HPV) Oral cavity Oropharyngeal [28, 29] Epstein-Barr virus [30] Nasopharyngeal
Hepatitis C, human herpesvirus-8, and human immunodeficiency virus
Oral and buccal cavity Oropharyngeal Environmental/
Occupational Factors
Exposure to environmental carcinogens including asbestos, pesticides, and polycyclic aromatics hydrocarbons, mustard gas, construction and metallurgy, etc
Nasal cavity,
Paranasal cavity, and Nasopharyngeal
[31]
Genetic Factors
Fanconi anemia,
Dyskeratosis congenita Oral cavity and Pharyngeal [32]
Other Factors Passive smoking, Sniffing Laryngeal and pharyngeal
[31, 33, 34] Diet (Vitamin A and iron
deficiencies, preserved meat with a high content of nitrites etc)
Oral and laryngeal
a major role in individuals at risk [27, 35]. Furthermore, a strong connection exists between Epstein-Barr virus exposure and the development of nasopharyngeal cancer [28, 29]. Various studies support the etiological role of HPV in oropharyngeal cancer [28, 36]. A case-control study with oropharyngeal cancer patients has identified HPV DNA type 16 in 72% of cases, and the HPV-status of a tumor as a strong prognostic factor [29, 37].
7 Over the years, the occurrence of oropharyngeal cancer (mostly HPV+ cases) has been growing in younger populations, raising more questions and concern [36, 38]. Such a rise could be a result of changes in life style and sexual behavior. Other minor risk factors associated with HNSCC are poor diet, poor dental hygiene, gastro-esophageal reflux, marijuana exposure, and various inherited syndromes [31]. Hence, HNSCC is
characterized as cancer with multifactorial etiopathologies involving diverse anatomical structures and huge demographic variability, thus, demanding more defined therapeutics
or preventive approaches to improve clinical outcomes.
Major Molecular and Genetic Predispositions in HNSCC
Molecular and genetic analysis have revealed that multiple pathways are compromised in head and neck cancer [33]. Pathways that are critically altered include protein 53 (P53), epidermal growth factor receptor [39], signal transducer and activator of transcription [40], vascular endothelial growth factor (VEGF), and mammalian target of rapamycin (mTOR), which have also been identified as potential therapeutic targets [41, 42].
The genes p53 and Rb are highly deregulated in head and neck cancer [43]. Somatic mutation at the codon 238-248 of p53 is more prevalent (60-80% of cases) and correlated directly with patient‟s survival rate [43-46]. The loss of chromosome 9p21, 17q13 loci results in inactivating tumor suppressor genes including p16 and p53,
respectively [33]. Beside this, overexpression of dominant negative p53 and cyclin D1, as well as increased telomerase activity (≥ 80%), together confer deregulated cell cycle and resistance to DNA damage stimulators in these cancer types [47]. Furthermore, HPV encodes viral proteins E6 and E7 that bind and inactivate p53 and Rb, respectively,
8 together, disrupting the cell cycle regulation in HPV+ tumors [48]. Numerous
epidemiological and laboratory evidence suggest that HPV is an independent risk factor, and is strongly linked with a subtype of HNSCC (oropharyngeal cancer) [49]. Most HPV+ tumors are observed among young non-smokers and non-drinkers, and usually present at advanced stage at the time of diagnosis [50, 51]. Fortunately, HPV positivity status is considered as a marker with better clinical response to therapy. In fact, vaccines have been developed against HPV.
Similarly, a clinical study with HNSCC patients (n=37) and healthy individuals (n=35) showed that tumor cells are more sensitive to irradiation-mediated DNA damage and displayed impaired DNA repair, indicating the crucial role of DNA repair mechanism in HNSCC treatment [52]. Another study with archival human head and neck cancer tumors specimens revealed that Ku80, a DNA repair protein, was overexpressed, thus indicating this pathway as an attractive therapeutic target [53].
Amplification of several oncogenes (e.g. c-myc, Ras, erbB 1/2 ) has been
observed in HNSCC and has been associated with poor prognosis [54] Epidermal growth factor receptor [39], is one of the widely over-expressed oncogenes in HNSCC (80-100%). It has been shown to be associated with more aggressive phenotype, increased resistance to anti-EGFR treatment, and a poor clinical outcome [55, 56]. This surface receptor has been reported to regulate the activation of many downstream signaling pathways involved in cell proliferation, apoptosis, invasion and metastasis [55, 57, 58]. The most common mutant variant of EGFR in HNSCC is EGFRvIII (42% cases). This variant type is directly correlated with aggressive tumor growth and chemo-resistance to anti-tumor drugs, including monoclonal antibody against EGFR [33, 59]. Other growth
9 factors such as hepatocyte growth factor (HGF), MNNG HOS transforming gene (MET), transforming growth factor-alpha/beta (TGF-α/β), basic fibroblast growth factor (bFGF), interleukin 8 (IL-8), vascular endothelial growth factor (VFGF), metalloproteinases (MMPs), and signal transducers and activators of transcription 3 (STAT3) are also reported to play a critical role in the development of HNSCC [45]. Similarly, the insulin-like growth factor-1 receptor (IGF-1R), which activates Akt and MAPK, contribute to tumor progression by increasing the invasive and metastatic potentials of head and neck cancers [60]. Barnes et al have shown that IGF-1R forms heterodimer with EGFR, and thereby activates the EGFR pathway. Consistent with this, successful silencing of both of these receptors resulted in inhibition of cell growth and induction of apoptosis, indicating the therapeutic advantage of targeting both these pathways simultaneously [60].
The direct downstream target of tyrosine kinase receptors (EGFR, IGF-1R), and the PI3K/Akt signaling pathway is mammalian target of rapamycin (mTOR), which is found to be activated in 90-100% of HNSCC. mTOR is a complex of two components: mTORC1 and mTORC2. mTORC1 is regulated by multiple signals, such as growth factors, nutrients, energy status, oxygen and cellular stress [61], and it‟s activation directly regulates phosphorylation of p70S6K and 4EBP1, thus triggering protein synthesisinvolved in the cell cycle and ribosomal function [62]. Numerous components of the mTOR signaling pathways are reported to be deregulated in HNSCC [63].
Evaluation of mTOR protein expression in 25 laryngeal cancer patients treated with postoperative radiotherapy has demonstrated high expression in recurrent tumors, indicating an aggressive phenotype [64]. Likewise, elevated level of eIF4E is correlated with more aggressive form and recurrence in HNSCC patients [64, 65]. The active form
10 of S6 (phosphorylated) is shown to be elevated in cell lines, early dysplastic lesions and carcinomas [66]. Furthermore, mTOR acts as a central regulator of glucose and lipid metabolism [62, 67]. A preclinical study suggests that inhibition of mTOR severely impairs tumor cell survival and proliferation by affecting cellular metabolism [67]. Furthermore, it has also been reported that a combination of deficient mitochondria, over expression of glucose transporters (GLUTs), alterations of PI3K signaling, and
overexpression of mTOR pathways, together compromise cellular metabolism in head and neck cancer, and therefore, they might serve as potential therapeutic and preventive
targets for HNSCC [33, 68, 69].
General Principles of HNSCC Management
Despite the advances made in the field, successful clinical management of HNSCC still faces challenge due to biological, pathophysiological, and anatomical heterogeneity. Therefore, the selection of standard treatment modalities is based on a wide array of factors; the stage and site of the primary tumor, preservation of the
underlying anatomical structures and their function, patient and institutional preference, patient age and general health condition [70, 71].
Surgery and /or Radiation Therapy
Generally, early-stage (stage I and II) HNSCC is managed with the use of a single modality treatment (surgery or RT), while tumors at advanced stage are managed with multimodality therapy [72]. Surgery is the first line of treatment in patients diagnosed with a palpable tumor at stage I/II (applicable to 30-40% of HNSCC patients), under the condition where a clear margin can be set between the tumor and normal tissue. Adjuvant RT is given after surgery in case of lymph node involvement [73]. RT remains the
11 therapy of choice when preservation of organ functionality is important in oropharyngeal, hypopharyngeal and laryngeal cancers [74]. Both of these treatment modalities have similar clinical outcomes with the response rate ranging between 60% and 90% [75]. Although patients at an early stage (stage I/II) of disease appear to have excellent
prognosis after surgery or RT, the risk for recurrence and development of second primary tumors is not predictable, thereby, demanding close monitoring of patients and alternative treatment modalities. Patients at an advanced stage of malignancy are provided with supportive care followed by single and/or combination therapies as necessary [75].
Chemotherapy
Chemotherapeutic agents are used as single modality interventions as well as in combination with surgery or radiation in patients with recurrent and/or metastatic tumor. The most commonly used therapeutic agents include methotrexate, bleomycin, cisplatin and 5-flurouracil (5-FU), with improved clinical response in 15-30% of patients with no significant indication of superiority among each other [75]. Similarly, taxanes are also considered highly efficacious agents with 20%-43% response rates, but unfortunately, these rates are dependent on patient and tumor characteristics [75]. However, in recent years, there has been a shift in clinical practice to use these chemotherapeutic agents in combination and this has been reported to have a synergistic effect [75, 76]. The results from these combination studies are interesting in terms of higher instances of patient response, but are also associated with toxicity, and ultimately do little change the overall survival rate in patients [76].
12 Multimodal Therapy
Despite the usage of aggressive treatment modalities in the management of locally advanced disease, 20-30% patients still develop locoregional recurrence and distant metastasis [77, 78]. Only few cases of locoregional recurrence can be treated by surgery and/or re-irradiation under clinical setting. Chemotherapeutic agents alone or in
combinations with biological targets are the mainstay of treatment [75]. These combination therapies show clinical response in 20-40% of the patients without any remarkable change in overall survival, and in fact enhanced toxicity [79, 80]. However, new molecular targeted therapies are emerging with the promises to minimize toxicity and improve survival in head and neck cancer patients [30].
Molecular and Targeted Therapy
It has been described above that numerous molecular pathways are involved in promoting the growth and survival of tumor cells in HNSCC. Several of these molecular pathways are now being targeted with specific therapeutic agents to improve clinical outcomes. Such agents include inhibitors that target growth factors (EGF, VEGF, FGF, HGF etc) and their receptors [EGFR, VEGFR, FGFR, and c-Met etc], signal transduction pathways (mTOR), cyclooxygenase 2 (COX-2), protein degradation (protease inhibitors), and hypoxia (HIF-α) [33, 81].
EGFR inhibitors. EGFR and its ligands are upregulated in 80-100 % of HNSCC patients and are associated with poor prognosis [81, 82]. Two strategies are employed to inhibit EGFR signaling in clinical practices; use of chimeric human and/or mouse
antibodies (cetuximab, panituximab) that bind to the extracellular domain of the receptor, and use of small molecule tyrosine kinase inhibitors (TKIs) that interrupt EGFR signal
13 transduction by binding reversibly to the intracellular catalytic domain of EGFR TK (Gefititinib, Erlotinib) [83, 84]. Cetuximab is the first anti-EGFR monoclonal antibody approved by Food and Drug Administration (FDA) for use in advanced colon cancer patients, and is the only approved EGFR inhibitor for use in locally advanced head and neck cancer patients either alone or in combination [85]. In a study, cetuximab was used alone and/or in combination with platinum in platinum-refractory patients with 10-30% response rate, and 46-55% disease controlled in patients [84]. Many combination trials are being conducted using cetuximab with other chemotherapeutic agents or radiation in advanced HNSCC patients, and these studies have shown that cetuximab offers clinical benefit [85, 86]. Likewise, EGFR tyrosine kinase inhibitors (TKIs) are under
investigation in phase I and phase II head and neck cancer trials. They have shown limited efficacy as a single agent, however, response rates seem encouraging when used in combination with other conventional chemotherapy agents [87, 88]. The clinical trials studying the efficacy of erlotinib in patients with resected HNSCC are ongoing and are expected to define the efficacy and safety profile of erlotinib in these HNSCC patients (http://clinicaltrials.gov).
VEGF inhibitors. VEGF is overexpressed in HNSCC and has also been linked to the mechanism of EGFR resistance, tumor progression, and poor prognosis [89, 90]. Current anti-angiogenic therapies targeting VEGF and its receptors consist of monoclonal antibodies (for instance bevacizumab, ranibizumab) or small molecular inhibitor of TKs: lapatinib, sunitinib, sorafenib, axitinib and pazopanib. In a phase I/II trial study of
erlotinib in combination with bevacizimab (VEGF monoclonal antibody), it was reported that patients untreated with any other treatment modality responded to this combination
14 but patients treated earlier mostly remained unresponsive [91]. In another study, patients responded to combination therapy of permetrexed (suppresses DNA synthesis and folate metabolism) with bevacizumab, 59% patients had an improved disease state, but
experienced bleeding complications [87].
COX-2 inhibitors. COX-2 is highly expressed in head and neck cancer as compared to normal oral mucosa [33]. COX-2 has been targeted in oral cancer
chemoprevention studies either by COX-2 inhibitors (selective and non-selective) alone or in combination with EGFR inhibitors, and shown to reduce oral cancer incidence, invasiveness and cancer-associated mortality [92, 93]. COX-2 inhibition combined with radiotherapy, chemotherapy and/or other targeted therapy has shown benefit in 30% of patients [92, 94]. A recent study suggested that regular use of aspirin (a non-selective COX-2 inhibitor) is associated with 22% reduction in head and neck cancer risk [95]. This observation was further supported by a hospital based study where the effect of aspirin was more pronounced in cigarette smoking or alcohol consuming population, indicating the significance of this pathway in the intervention [96].
p53 inhibitors. p53 is one of the most commonly mutated genes in HNSCC [39]. Considerable efforts have been made to target non-functional p53 by gene transfer using viral carriers. In a Phase II multicenter study, patients (previously treated or with
recurrence) were treated with intra-tumoral Advexin, an adenovirus coding functional p53. The findings from this study revealed that some patients benefited from this
treatment with improvement in overall survival [97]. But, unfortunately, serious adverse effects; pain at injection site (45%), asthenia (13%) with change in response rate to treatment were also reported [97]. The ONYX-15 virus (targeted mutant p53) was
15 administrated as mouthwash to limited number of patients with premalignant and
dysplastic lesions. The result from this study suggested that premalignant lesions were decreased in one third of the patients, indicating its effectiveness [53].
HPV vaccines. Accumulating evidence suggest that HPV status is an important prognostic factor and the use of molecular targeted therapies have better outcome in HPV+ HNSCC patients [98-100]. The HNSCC patients positive for HPV have
remarkably better survival compared to HPV (-) patients [101]. The average survival of HPV (+) patients treated with concurrent therapy is 6 to 8 months, which is superior when compared to supportive care alone. In recent years, there has been rise in the incidence of oropharyngeal cancer (HPV positive), and the FDA has approved two HPV vaccines [Gardasil (HPV quadrivalent), Adervari (human HPV bivalent )] for use in females between age 9-26 years [102].
Others inhibitors. Additional oncogenic growth and/or survival pathways such as mTOR, IGF-1R, TGF-α/β are also considered potential targets for the treatment of HNSCC and studies using the agents against these molecular targets are underway [103]. Proteosomal inhibitors (Bortezomib), histone deacetylase (HDAC) inhibitors, signal transducers and activation of transcription inhibitors, and poly-ADP-ribose polymerase inhibitors (PARP) have shown some promising results in preclinical studies, and these inhibitors are being investigated in ongoing clinical studies [103-105]. Lately, a systemic targeted approach with radiolabelled antibodies has also been developed for imaging and therapeutic purposes in HNSCC [106].
16 Existing Management of HNSCC: Limitation and Prospects
The acute toxicity associated with conventional therapies includes mucositis, radiation dermatitis, loss of taste, hoarseness of voice, and weight loss [107]. Mucositis is observed in 80-90% of patients receiving chemo-radiotherapy and 30-40% of cases are severe, thereby causing difficulty in adhering to treatment [107]. During radiation therapy, loss of taste, mucosal atrophy, and skin discoloration are common adverse effects, as well as the development of resistance to radiation and chemo-radiotherapy [108]. Surgical treatment in HNSCC patients may result in some impairment of functional structures with numbness in the treated area, and loss of function of the trapeziuss muscles producing a painful shoulder condition [108]. These specific and non-specific systemic toxicities associated with conventional treatments prevent timely completion of therapy, affecting overall survival rates of patients [108]. Many
chemotherapeutic agents result in bone marrow suppression, leucopenia and anemia. The clinical usefulness of the common therapeutic agent cisplatin is diminished due to
nephrotoxicity, and/or neurotoxicity [103, 109]. Taxanes have good response rates, but exhibit poor response when used after failure of cisplatin combination [109, 110].
The EGFR inhibitor cetuximab is the only targeted therapy for HNSCC that has received approval from the FDA in the last decade. The overall results revealed that only 10-20% of patients benefited from cetuximab and remaining exhibiting intrinsic
resistance, thereby, limiting its clinical use [88, 103, 104]. Similarly, TKIs have shown promising outcome in HNSCC, but lack of sufficient data from phase III clinical studies limit their prospect into standard treatment regimen [111]. Anti-VEGF treatment
17 The results from some of these studies remain forthcoming; however others have failed to demonstrate much advantage in patient survival while carrying significant bleeding risks [91, 112, 113]. Similarly, recent studies with COX-2 inhibitors alone or in combination with chemotherapeutic or radiotherapy are still investigational [114]. In HNSCC, > 50% of patients that have undergone surgical resection of primary tumor develops loco-regional recurrence and the molecular genesis driving this phenomenon of secondary tumor development is largely unknown.
Hence, when patients have reached the limit of toxicity and tolerance, it seems very unlikely that standard treatment modalities will further improve the outcome. It is, thus, inevitable that additional alternative strategies are required in head and neck cancer either to advance the therapeutic index of conventional treatment modalities or adding ]non-toxic agents to make significant difference in clinical implications.
Chemoprevention
The term „chemoprevention‟ was coined by Sporn et al., in 1976 and has been defined as the use of both natural and/or synthetic agents to reverse, suppress, and /or prevent initiation, promotion and progression of tumorigenesis [115]. Broadly, chemoprevention has been categorized as primary, secondary, and tertiary
chemoprevention depending upon the applicability at different stages of tumorigenesis as illustrated in Figure 1.1. Primary chemoprevention is directed towards high-risk
individuals and implies to agents that prevent incidence of precursor lesions; secondary chemoprevention is focused on the regression of prevalent precursor lesions; and tertiary chemoprevention is aimed at preventing the recurrence of secondary primary tumors (SPTs) and/or the spreading of primary tumors to other organs [115, 116]. Although the
18 history of conceptual chemoprevention dates back several decades, it has only recently gained attention with the completion of successful clinical trials involving
chemopreventive agents in high-risk individuals, suggesting that chemoprevention could be an appealing alternative strategy conquering tumorigenesis [117]. For example, Tamoxifen was approved by the FDA to use in breast cancer, and dutasteride and
finasteride have shown chemopreventive potential in prostate cancer patients [116]. With this growing interest, the National Cancer Institute (NCI) has emphasized both preclinical studies as well as early phase I/II/III clinical trials with chemopreventive agents.
Currently, more than 400 potential chemopreventive agents are under clinical
investigation (NCI factsheet) [118]. Regardless of limited success in clinical settings, the beneficial values of these agents are greater and this field is continuously growing.
Chemoprevention in Head and Neck Cancer
In recent years, advances have been made in understanding the molecular
pathogenesis of cancers of epithelial origin including head and neck cancer, thus resulting in the development of two important aspects that provide a strong rationale for
chemoprevention studies in these cancers. These justifications include: a) concept of field cancerization, and b) multistage carcinogenesis. Slaughter and colleagues suggested the concept of field cancerization as the persistent exposure of surface epithelium to
chemical, viral and/or environmental carcinogens resulting in genetic variation at multifocal areas throughout the oral and orophyngeal mucosa [19]. After treatment for primary tumors, studies suggest that patients with precancerous lesions may develop secondary primary tumors-SPTs [119, 120]. The incidences of SPTs are believed to vary (9.4-14%), and are considered major threats to long term survival in HNSCC patients
19 [119, 120]. This concept of field cancerization has clearly explained the increased risk of SPTs and local recurrence of primary tumors in head and neck cancer patients. Hence, patients with SPTs warrant additional considerations during treatment and/or
chemoprevention. The molecular and pathological analysis of field cancerization has further highlighted the concept of multistage carcinogenesis in different cancers including head and neck cancers [119].
Figure 1.1: Schematic diagram illustrating primary, secondary and tertiary chemoprevention at various stages of carcinogenesis.
It is proposed that repeated exposure to risk factors accumulate somatic mutations over time, resulting in phenotypic progression from normal hyperplasia dysplasia carcinoma in situ invasive carcinoma in lieu of different stages of tumor initiation,
promotion and progression as illustrated in Figure 1.1. Therefore, the primary objective of chemoprevention in HNSCC is to intervene in these processes in multistage
carcinogenesis.
Pavia and colleagues performed meta-analysis of 16 studies (15 cases control studies, and 1 cohort study) to evaluate the role of fruits and vegetables intake in the risk of oral cancer development [121]. The investigators reported that oral cancer was reduced by 50% with the intake of a reasonable amount of fruits and vegetables [121].
INITIATION PROMOTION PROGRESSION
Primary prevention
Primary prevention High risk individuals
(smokers, alcoholics) precancerous lesions Individuals with Individuals with multiple tumors
Primary/Secondary Primary/Secondary prevention prevention Tertiary Tertiary prevention prevention Patients with SPTs or metastasis
INITIATION PROMOTION PROGRESSION
INITIATION PROMOTION PROGRESSION
Primary prevention
Primary prevention High risk individuals
(smokers, alcoholics) precancerous lesions Individuals with Individuals with multiple tumors
Primary/Secondary Primary/Secondary prevention prevention Tertiary Tertiary prevention prevention Patients with SPTs or metastasis
20 Chemopreventive efficacies of dietary components (such as anthocyans) on oral cancer are based on the evidence that consumption of fruits and vegetables is closely linked with a decreased risk of this cancer [121-123]. These observations are consistent with the recommended guidelines of the American Cancer Society [124] and the American Institute of Cancer Research (AICR) emphasizing the importance of increased intake of fruits and vegetables for cancer prevention [125, 126]. Despite the fact that
epidemiological and clinical based efficacy studies suggest the beneficial effect of dietary factors, still lacking are the definitive markers to evaluate the efficacy of these agents as well as to identify the risk population. Nevertheless, promising characteristics of
chemopreventive agents including non-toxicity, chemical diversity, multiple targets, affordability and availability around the globe, which together make chemoprevention an exciting area of research for years to come in different malignancies including head and neck cancer.
Clinical Studies: Single or Combination of Chemopreventive Agents
In head and neck cancer, retinoids or synthetic derivatives of retinoids are the most widely studied chemopreventive agents focused on primary and secondary chemoprevention. Moreover, vitamin E, β-carotene, selenium and other antioxidants alone or in combination with retinoids or their derivatives have also been studied for their effect on the premalignant lesions of the oral cavity or the oropharynx [127-129].
Epidemiological studies have suggested an increased risk of HNSCC in individuals with vitamin A deficiency [130, 131]. In addition, retinoids are crucial regulators of
physiological processes such as cell differentiation and apoptosis, which further assist in rationalizing their common use as chemopreventive agents in different malignancies
21 including head and neck cancer [131]. In 1986, Hong et al., conducted
placebo-controlled study on 13-cis-retinoic acid (13-c-RA) (dose 1-2 mg/kg body weight/day) in 44 patients with oral leukoplakia and found that leukoplakia was decreased by 10% in placebo group as compared to 67% in treatment group. There were, however, few limitations of this study including toxicity and disease relapse after discontinuation of therapy [132]. In a follow up to the previous study, authors used a high dose of isotretinoin (1.5 mg/kg body weight/day) for three months in patients (n=70) with leukoplakia; in the second phase, 55 patients who responded to first phase therapy were randomly assigned with a low maintenance dose of isotretinoin (0.5mg/kg /day) or β-carotene (30 mg per day) for nine months. The findings from this study revealed that patients given the maintenance dose of isotretinoin responded to therapy better than β-carotene and with minimal side effects [133]. It was speculated that isotretinoin possibly reversed the expression of nuclear retinoid receptor-β in dysplastic cells re-establishing the effect of the normal growth and differentiation in premalignant lesions [134, 135]. In one phase III clinical study conducted by the Radiation Therapy Oncology Group
(RTOG), 1190 patients with stage I and II HNSCC were randomly treated with either isotretinion (30 mg/kg/day) or placebo; however, patients did not show any significant benefit in terms of development of SPTs and overall survival rate [134]. The findings from this study did not comply with other studies where retinoids exhibited beneficial effects on premalignant lesions [128]. In another clinical study in leukoplakia patients, Vitamin A (200,000 IU/day for 6 weeks) was given to users of tobacco or betel nut, and results revealed that new keratosis with atypia was suppressed by 100% compared to placebo control (20%) [136]. Similarly, the European Study on chemoprevention with
22 Vitamin A and N-acetyl cysteine (EUROSAN) in the multicenter trial included 2,592 patients with HNSCC and lung cancers (previously treated), and were given placebo or Vitamin A (300,000 IU/day -1st year followed by 150,000 IU/day in 2nd year) plus N-acetyl Cysteine (60 mg/day for 2 years). No significant difference in incidence, survival and incidence of SPTs between placebo and treatment groups was observed after 49 months follow up study [137].
A placebo controlled trial, focused on evaluating the chemopreventive potential of β-carotene (30mg/day), showed that patients with oral leukoplakia (n=50) were benefited as compared to placebo patients (n=50) [138, 139]. However, further studies with larger patient population are required to draw conclusive results. In a Physician‟s health study, 22,071 healthy male physicians from the United States were enrolled for a randomized, double blind, placebo-controlled study. The physicians were divided into two groups: 11,036 physicians were randomly were assigned to receive β-carotene (50 mg every other day) and 11,035 were assigned to receive placebo. At the end of the study, after 13 years, no significant difference on the incidence of cancer, cardiovascular disease, and/or overall mortality was found regardless of their smoking history [140]. In another, double blind, randomized, placebo-controlled trial with α-tocopherol plus β-carotene, 540 patients of stage I/II HNSCC treated previously with radiation were given α-tocopherol (400 IU/day) plus β-carotene (30 mg/day) for 3 years. The result from this study showed that patients receiving supplements benefited early on, but not following discontinuation of the therapy and SPTs developed overtime [141]. In a study using the retinoid analog – N-4(-hydroxycarbophenyl retinamide)-[4-HPR], in patients with oral leukoplakia after surgical excision, patients benefited [142].
23 Similarly, α-tocopherol was evaluated in combination with other biological agents such as Interferon- α (IFN- α) and 13-c-RA. In this study, patients previously treated for locally advanced HNSCC were given a combination of 13-c-RA (50 mg/twice daily), IFN- α (3X106 U/m2, 3 times/week), and α-tocopherol (1,200 IU/day) for 12 months. The findings from this study demonstrated that combination of supplements was well
tolerated by patients and > 95% patients were overall disease free [143]. Patients with head and neck cancer have been treated with combination of chemopreventive agents or with other molecular targeted agents previously for better clinical outcomes. In a study, HNSCC patients receiving treatment for stage III/IV cancer were supplemented with sodium selenite (200 µg/day). The results showed that immunity was boosted in selenium treated patients compared to patients given placebo. However, the findings from this study are yet to be established in larger patients populations [144]. Some selective clinical studies with different chemopreventive agents are summarized in Table 1.3. Furthermore, natural compounds including green tea extracts, curcumin, β-carotene, pomegranate, resveratrol, and soybeans have shown promising effects in preclinical studies and are being investigated as prospective chemopreventive agents alone or in combination in head and neck cancer [145-147].
Although limited progress has been made in head and neck cancer
chemoprevention, the field still remains viable though with challenges of identifying safe and effective agents that can be used in high risk individuals as well as cancer patients. Other hurdles in the current chemoprevention studies are the lack of intervention models that consider all the risk factors, and molecular alterations associated with the
24 and create a better platform for intervention studies, providing better clinical outcomes in future.
Grape Seed Extract (GSE)
GSE: Source, Chemical Components and Chemical Structures
History of usage of grapes dates back thousands of years, however, grape seeds were not considered beneficial until 1970 when the French Professor Masquelier discovered that grape seed extract (GSE) is a powerful antioxidant [148]. This dietary supplement, rich in proanthocyanidins, is typically manufactured from the seeds of vitis
vinefera L. variety of grapes, and these seeds are generally the waste products from the
wine and grape juice manufacturing industries.
The basic structure of flavonoids includes phenyl benzopyran ring structure with C6-C3-C6 skeleton denoted by the A,C,B rings and where ring A is fused to the
heterocyclic benzopyran C, which is then fused to B-ring [149]. The substitutions by various functional groups in these rings give rise to different sub classes of flavonoids; flavonols, flavones, flavanones, isoflavones, flavan-3-ol [149]. Typically, GSE contains >85-90% flavonoid polyphenols [150]. It is rich in monomeric flavan-3-ols including (+)-catechin, (-)-(+)-catechin, (+)-epi(+)-catechin, and (-) - epi(+)-catechin, and other complex
oligomeric and polymeric proanthocyanidins [151, 152]. Proanthocyandins (dimers, trimers and tetramers) in GSE are formed by the condensation of the catechin, epicatechin and gallic acid subunits [152, 153]. The molecular structures of some components in GSE are shown in Figure 1.2.
25 Figure 1. 2: Chemical structures of different components of grape
Table 1.3: Selective clinical chemopreventive trials in HNSCC patients Primary, secondary and tertiary chemoprevention
Compounds Treatment scheme Type of
prevention Study sample Results Limitation of the study Ref Food and vegetables consumption
At least 5 fruits in a day Oral cancer Meta analysis
Positive NA
[121-123] Isotretinoin 1-2 mg/day vs. placebo for 3
months
Leukoplakia 44 Positive Toxicity and relapse
[132] Vitamin A 200,000 IU/day for
6 weeks Well-differentiated leucopenia in tobacco and betel nut chewers 54 [N=21 treatmen t, N=33 placebo] Positive NA [136] Isotretinoin and β-carotene
1.5 mg/kg/day for 3 months followed by maintenance of Isotretinoin (0.5mg/kg/day) or β-carotene (30 mg/day) Leukoplakia 70 Positive NA [133] β-carotene + Ascorbic acid + α-tocopherol 360 mg/day -β-carotene, 1000 mg/day is ascorbic acid, 800 IU/day of α-tocopherol for 9 months
Oral lesions 79 Positive NA [138]
Fenretinide 200 mg/day for 51 weeks Oral
Leukoplakia
137 Positive Less
toxicity
[142] Celecoxib 100 or 200 mg/twice a day for
12 weeks Oral Leukoplakia 40 Negative - [155]
Table 1.3: Contd...
Compounds Treatment scheme Type of
prevention
Study sample
Results Limitation of the study
Ref
Isotrenin 0.5 mg/day SPT 103 Positive High toxicity [128]
Selenium 200 µg/day Stage I/II post
operative/ post surgery
Positive NA [144]
α-tocopherol plus
β-carotene Radiation followed by α-tocopherol (400 IU/day) plus β-carotene (30 mg/day) for 3 years.
Stage I/II 540 Positive earlier in the treatment, but negative later Relapsed after discontinuation of the therapy [141]
Isotretinoin 30 mg/day for 3 years Stage I/II 1190 Negative NA [134]
β-carotene 50mg/day, 7.5 years Lung and HNSCC post therapy
264 Negative NA [156]
13-c-RA, IFN- α plus α-tocopherol
13-c-RA (50 mg/twice daily) and IFN-α (3X106 U/m2) → 3
times/weeks, and α-tocopherol (1200 IU/day) for 12 months
Locally advanced HNSCC N=45 (stage III-24%, stage IV-76%)
Positive Mild to moderate –
mucocutaneous hyperglyceridemia, hematological problems
[143]
SPT: secondary primary tumor; HNSCC: head and neck squamous cell carcinoma; IU: international units; 13-c-RA: 13-cis-retinoic acid;
IFN- α: interferon alpha; U: units; NA: not available
28 Bioavailability of Active Components of GSE
Numerous epidemiological and laboratory based studies have highlighted great health benefits with the use of polyphenolic compounds, however, the bioavailability of these compounds has remained nebulous. Hence, the following section will provide information about the bioavailability of these compounds based on preclinical and clinical models. Generally, monomeric flavanols are absorbed by the small intestine at different rates but the absorption of oligomeric forms has yet to be elucidated [157, 158].
Past studies conducted have shown that following oral administration of GSE, oligomeric flavanols and proanthocyanidins are fragmented into monomeric subunits by colonic microflora and rapidly transformed into glucuronidated, methylated and sulfated metabolites as detected in the plasma and/or urine [158, 159]. A preclinical study using Sprague Dawley rats evaluated the bioavailability of grape derived polyphenols (1 mg/kg body weight), and identified (+)-catechin glucuronide (C-G), (-) - epicatechin
glucuronide (EC-G), methyl-(+)-catechin-glucuronide (M-C-G), and methyl-(-) – epicatechin-glucuronide (M-EC-G) to be the major metabolites present in plasma [158]. The maximum level of these metabolites was reached at 3 h after oral administration, and the concentration slowly declined after 4 h. The investigator could not detect parent monomeric flavanols, dimers, trimers or tetramers in this study [158]. In contrast, another study designed to evaluate the absorption and metabolism of epicatechin, purified
proanthocyanidin dimers B2, A1 [epicatechin-(2-O-7, 4-8)-catechin] and A2 [epicatechin-(2-O-7, 4-8)-epicatechin], trimers, tetramers has shown that
proanthocyanidin dimers A1 and A2 were absorbed from the rat small intestine [160]. In a study by Ferruzzi and colleagues, where they administrated GSPE (grape seed
29 proanthocyanidins extract) in a mouse model of Alzheimer‟s disease, it was revealed that gallic acid, catechin and epicatechin accumulated in the brain tissues [161].
A study by Shrestha et al., has shown that after oral dosing of 3,3″-di-O-galloyl ester of procyanidin B2 (B2G2), epicatechin (EC) and 3-O-galloyl-epicatechin (ECG), B2G2 was absorbed and was intact when analyzed in liver microsomes unlike other flavanol monomers [162]. A study conducted in healthy subjects indicated that orally given (-) - epicatechin (50 or 500 mg/kg) was absorbed by the intestinal tract and (-) - epicatechin metabolites (glucuronidated and/or methylated) appeared in the urine [163]. In another study, Sano and colleagues administrated 2 g of proanthocyandins rich GSE to 4 healthy individuals and detected proanthocyanidins B1 in human serum 2 h after intake [164]. Yamakoshi et al., studied the safety of proanthocynidins present in grape seed extract and found that the LD50 (median lethal dose) was greater than 4 g/kg in male and female rats, demonstrating a lack of toxicity and supporting its safety in various products [165].
These studies in both animals and humans suggest that monomeric flavanols are absorbed as parent compounds and are rapidly metabolized to (+)-catechin glucuronide (C-G), (-) - epicatechin glucuronide (EC-G), methyl-(+)-catechin-glucuronide (M-C-G), and methyl-(-) – epicatechin-glucuronide (M-EC-G); whereas the absorption of
proanthocyanidins is reported to be lower. The more detailed mechanism of the absorption and metabolism of proanthocyanidins remains investigational.
Prospective Chemopreventive Agent- GSE
The use of grapes for health benefits has a long history in human civilization. Egyptians consumed grapes almost 6,000 years ago, and several ancient Greek
