PHD Thesis

1. CHRONIC FEAR, STRESS, ANXIETY AND DEPPRESION RELATION TO CANCER INCIDENCE IN HUMANS BY SOMAYEH ZAMINPIRA AND SORUSH NIKNAMIAN University of Cambridge Cellular and Molecular Biology Department Cambridge United Kingdom 1

2. Table of Contents 1.PREFACE Page # 3 2.ABSTRACT 8 3.CHAPTER ONE: EMHC HYPOTHESIS OF CANCER 11 4.CHAPTER TWO: CHRONIC FEAR RELATION TO CANCER 24 5.CHAPTER THREE: STRESS, ANXIETY, FEAR AND DEPPRESION RELATION TO CANCER 34 6.CHAPTER FOUR: THE IMPACT OF SEROTONIN ON THE CAUSE AND TREATMENT OF CANCER 47 7.CHAPTER FIVE: AGRICULTURAL REVOLUTION AND THE EPIDEMIC OF CANCER 57 8.CHAPTER SIX: CANCER INCIDENCE IN WILD ANIMALS AND THE REJECTION OF PETO’S PARADOX THEORY. 72 9.CHAPTER SEVEN: HOW BUTTERFLY EFFECT IN THEORETICAL PHYSICS EXPLAINS THE MAIN CAUSE OF CANCER. 82 10. CHAPTER EIGHT - CONCLUDING REMARKS 94 11. REFERENCES 98 2

3. PREFACEE Cancer disease has become the first cause of death in the United States and world- wide. Most Researchers estimate that 595,690 of American people will die from cancer at the end of the year 2017. That means 1,600 deaths/day approximately [1]. Cancer in modern societies is commonly treated with the combination of organ surgery, chemotherapy and radiotherapy which has many side effects and the high possibility of metastasis. Many kinds of diet strategies have been experimented. However, none of them have been particularly effective. According to Otto Warburg hypothesis in the 1930’s, the cause of cancer is the change in the metabolism of mitochondrion in human cells. Low oxygen in tissues in combination with high blood glucose will change the cell respiration from aerobic to anaerobic which leads to fermentation type of respiration. [2] Professor Thomas N. Seyfried in 2012 proposed cancer as a metabolic-mitochondrial disease. He has mentioned that all cancer cells’ mitochondria are damaged and do not work properly. [3] Mitochondrion is a microorganism living inside our cells and produce energy in the form of Adenosine Triphosphate (ATP) from fats, glucose and glutamine. In order to produce ATP, mitochondria need oxygen through the krebs cycle which is called “Aerobic Respiration”. On the other hand, when the amounts of oxygen go under 30% in the tissues and cells, mitochondria cannot respire properly which causes the increase in the amounts of Reactive Oxygen Species (ROS) or better called “Inflammation” inside the cells and causes damage to the mitochondria. According to Professor Thomas N. Seyfried, the main cause of cancer is the increase in ROS inside normal cells. [Thomas N. Seyfried et al., 2012] In 2017 we hypothesized with the sponsorship of the Weston A. Price Foundation (WAPF), that cancer is an Evolutionary Metabolic Disease which we proposed in the 4th International Conference in Medicine and Neurological Diseases which held in Fulda/Germany. Evolutionary Metabolic Hypothesis of Cancer (EMHC) The first living cells on Earth are thought to have arisen more than 3.5 × 109 years ago, when the Earth was not more than about 109 years old. The environment lacked oxygen but was presumably rich in geochemically produced organic molecules, and some of the earliest metabolic pathways for producing ATP may have resembled 3

4. present-day forms of fermentation. In the process of fermentation, ATP is made by a phosphorylation event that harnesses the energy released when a hydrogen-rich organic molecule, such as glucose, is partly oxidized. The electrons lost from the oxidized organic molecules are transferred via NADH or NADPH to a different organic molecule or to a different part of the same molecule, which thereby becomes more reduced. At the end of the fermentation process, one or more of the organic molecules produced are excreted into the medium as metabolic waste products. Others, such as pyruvate, are retained by the cell for biosynthesis. The excreted end- products are different in different organisms, but they tend to be organic acids. Among the most important of such products in bacterial cells are lactic acid which also accumulates in anaerobic mammalian glycolysis, and formic, acetic, propionic, butyric, and succinic acids. The first cell on the earth before the entrance of the bacteria did contain nucleus and used the fermentation process to produce ATP for its energy. Then an aerobic proteo-bacterium enters the eukaryote either as a prey or a parasite and manages to avoid digestion. It then became an endosymbiont. As we observe, the fermentation process used the glucose or even glutamine to produce ATP, but the aerobic process used the glucose, fat and protein to produce more ATP than the previous one. The symbio-genesis of the mitochondria is based on the natural selection of Charles Darwin. Based on Otto Warburg Hypothesis, in nearly all cancer cells, the mitochondrion is shut down or are defected and the cancer cell do not use its mitochondrion to produce ATP. This process of adaptation is based on Lamarckian Hypothesis of Evolution and the normal cells goes back to the most primitive time of evolution to protect itself from apoptosis and uses the fermentation process like the first living cells 1.5 billion years ago. Therefore, cancer is an evolutionary metabolic disease which uses Glucose and Glutamine as the main food to produce ATP and Lactic Acid. In another study, we hypothesized that increasing the ROS in a cell can cause damage to the DNA of the mitochondrion and also Nucleus DNA, but another reason behind turning the normal cell into cancer cell is the chaos caused by the increasing of inflammation inside each cell and increasing the intracellular ROS. These chaos causes some abnormal messaging between the DNA of the nucleus to stop the apoptosis and turning the oxidative phosphorylation to the fermentation in cytosol. Normally by damaging to the mitochondria, the cell should apoptosis. however; the nucleus sends wrong messages to stop the apoptosis and do fermentation process in cytosol to survive the cell. Even some normal left mitochondria would be shut down and stop the oxidative phosphorylation. This is the main and the real reason how increasing intracellular inflammation. [5] With all the information above, the basic way to stop respiration of cancer cells is to stop feeding them. The main source of fuel in cancer cells are Glucose and Glutamine. The basic diet for weakening or even killing these cells is Ketogenic Diet (KD). Ketogenic diet is a kind of regime which uses high fat content and low carbohydrate. 4

5. This diet changes the metabolic state into the condition called Ketosis. After several days, fat becomes your body’s primary energy source which causes an increase in the levels of compounds which is called "ketones" in the blood [6]. In general, a ketogenic diet used for weight loss is about 60-75% of calories as fat, with 15-30% of calories from protein and 5-10% of calories from carbs. However, when a ketogenic diet is being used therapeutically for the treatment of cancer, the fat content may be significantly higher that is up to 90% of calories, and the protein content lower [7]. Presently, limited researches do seem to show that a ketogenic diet may reduce tumor size and rate of progression in certain cancers. One of the few documented published case studies was performed on a 65-year-old woman with brain cancer. Following surgery, she received a ketogenic diet. Meanwhile, the tumor’s progression slowed. But, 10 weeks after returning to a normal diet, she experienced a significant increase in tumor growth [8]. Similar case reports examined the reactions to a ketogenic diet in two young girls who were undergoing treatment for advanced brain cancer. Researchers observed that glucose uptake was decreased in the tumors of both patients. One of the girls reported that her quality of life had improved and remained on the diet for 12 months. During that time her cancer showed no further progression [9]. One study monitored tumor growth in response to a high-carb versus a ketogenic diet in 27 patients with cancer of the digestive tract. Tumor growth increased by 32.2% in patients who received the high-carb diet, but decreased by 24.3% in the patients on the ketogenic diet [10, 11]. In another study, three out of five patients on a ketogenic diet combined with radiation or chemotherapy experienced complete treatment. More interesting, the other two participants found the disease progressed after they stopped the ketogenic diet [12]. We had organized a research in the Violet Cancer Institute (VCI) on 54 patients with 6 different types of cancer. The aim of this research is to figure out the effectiveness of the Sorush Cancer Treatment Protocol (SCTP) which is based on the Evolutionary Metabolic Hypothesis of Cancer (EMHC) and introducing the Specific Ketogenic Diet (SKD) plus Intravenous Ozone Therapy (IOT) in Phase (1) on 54 cancer patients (Ozone has been found to be an extremely safe medical therapy, free from side effects. In a 1980 study done by the German medical society for ozone therapy, 644 therapists were pulled regarding their 384,775 patients, comprising a total of 5,979,238 ozone treatments administered. There were only 40 cases of side effects noted out of this number which represents the incredibly low rate of 0.000007% and only 4 fatalities. Ozone has thus proven to be the safest medical therapy ever devised. [16]), and combination of Hyperbaric Oxygen Therapy with vitamin/mineral and herbal supplementation beside the SKD and IOT in Phase (2) of this research on the remained 31 cancer patients. Based on the researches from 1928-2016 and the experimentation of cancer treatments and protocols on cancer patients, we have reached a treatment and decided to test it on 5

6. 54 voluntaries cancer patients in the first stage of their disease. In this treatment we used a 5-day water fasting state, the Specific Ketogenic Diet (SKD) designed by ourselves and Intravenous Ozone Therapy (IOT) in the duration of 90 days (Phase 1) and another 90 days (Phase 2) with the entrance of Hyperbaric Oxygen Therapy (HBO2T) and several supplements which we have been effective in previous studies on cancer patients. We have used the measurement of saliva PH, the MRI device and statistical methods to test the shrinkage of the tumors. After Phase (1) of this research on 54 patients the average percentage decrease in the tumors was 58% and after Phase (2) on 31 remained cancer patients the average percentage decrease in the tumors was 98.8%. The average saliva PH in the fasting state of the cancer patients improved from acidic to alkaline as well. In conclusion, we have reached an effective cancer treatment based on SCTP by the usage of SKD, IOT, HBO2T and several supplements. There was an obvious improvement of cancer tumor decrease, lifestyle, saliva PH and we did not observe any side effects and cachexia in any of the patients. [4] TUMOR SIZE AVERAGE BEFORE THE EXPERIMENT, AFTER PHASE (1) AND PHASE (2) 17 2.21 0 20 18 18 16.5 16 14.5 14 12 11.16 10 8 8 8 6 6 4.5 4.12 4 3.68 2 0.21 0.14 0 0 0 0 Breast Cancer Colorectal Cancer Kidney Cancer Liver Cancer Lung Cancer Figure (2): The blue line shows the tumor size before the experiment begins. The orange line shows the tumor size after Phase (1) of the treatment. The white line shows the shrinkage and improvement of the cancer patients tumors after 180 days. We named this methodology used in this treatment the Sorush Cancer Treatment Protocol (SCTP). This protocol includes: 5-day water fasting, SKD, IOT, HBO2T, natural Vitamin/Mineral Supplements, Herbal Supplements, 3000-5000 mg ascorbic 6

7. acid, probiotic foods and cottage cheese mixed with flaxseed oil. The total calorie intake by the patients should be 1200-1500 Cal/Day. The dietary restrictions in this protocol are: Dairy, Industrial Vegetable oils, Margarines, Alcohol, Gluten, Soy Products (Miso and Natto are exceptional), Artificial Sweeteners as well as Stevia, Fruit juices and any types of sugar including Honey. The duration of the SCTP should not exceed 6 months to reduce the possibilities of ketoacidosis occurrence in patients. SKD 5-day Water Fasting IOT SCTP Herbal Supplements HBO2T Vitamin/Mineral Figure (6): SCTP procedure in brief The most important note in this protocol is that we banned the consumption of industrial vegetable oils and margarine. In our research which we published with the help of Miriam Kalamian EdM, MS, CNS, we concluded that Omega-6 Linoleic acid from vegetable oils increases oxidative stress in the body of humans, contributing to endothelial dysfunction and heart disease. The consumption of these harmful oils which are high in mega-6 polyunsaturated fats results in changing the structure of cell membrane which contribute to increasing inflammation and the incidence of cancer. [13] We replaced MCT by Extra Virgin Coconut Oil which we found to have more advantages in the Ketogenic Diet for the treatment of cancer. [14] Furthermore; we have prohibited the consumption of any soy products in our protocol since there 7

8. is many evidences show the relation between the consumption of Soy and cancer incidences specially breast cancer. [15] ACKNOWLEDGEMENTS We would like to mention that the sponsor for all the researches of us, are Weston A. Price Foundation (WAPF) and Violet Cancer Institute (VCI). We would also like to thank specially Professor Thomas N. Seyfried Ph.D. and Professor Stephanie Seneff Ph.D. for their supportive help in the researches, Professor Dominic D’Agostino Ph.D. and Professor Angela Poff Ph.D. for their informative help. ABSTRACT This thesis introduces a hypothesis which is based on the Warburg Hypothesis [M1] and also lends further support to the metabolic theory of cancer put forth by Prof. Thomas N. Seyfried. As nearly all cancer cells have defective mitochondria they are unable to produce energy from oxidative phosphorylation. Instead, they generate ATP primarily from fermentation of glucose in the cytosol. We have done a three months’ study based on the Special Keto-Diet plus Ozone therapy for each patients with 6 different cancer metastatic tumors at the Violet Cancer Institute (VCI) in Iran/Tehran. The results were the total significant reduction in cancer tumors and we conclude that the special Keto-Diet plus Ozone therapy would be one of the best treatment method for reducing the cancer tumor size and the possibility of the metastasis of malignant cancer tumors. Our latest study is put forth the correctness of the cancer as an Evolutionary Plus Metabolic disease. Understanding the significance of this landmark hypothesis offers oncologists and cancer biologists insight into evolutionary aspects of this disease and new directions for research and treatment strategies based on cancer cell vulnerabilities. Fear is a chain reaction in the brain that happens when one encounters a potentially harmful stimulus. receives information from many parts interprets this information to generate the emotion of fear. When the amygdala generates a fear hypothalamus. The hypothalamus then sends of the body to trigger a fight‐or‐flight response. are secreted by the adrenal gland. The effect of adrenaline increasing heart rate, hypocapnia and decreases blood flow to the brain. The effect of cortisol is increasing levels by converting stored glycogen and fats into blood sugar. It also suppresses The amygdala is the part of the brain that of the brain and emotion, it sends impulses the impulses to many different parts Fear (epinephrine) hormones is blood glucose 8

9. the immune system and causes inflammation. The prime cause of cancer is increasing the amounts of ROS in healthy cells. The aim of this review is to show the effect of chronic fear on the cause of cancer in humans by reviewing related clinical studies and biochemistry of fear and cancer. The role of fear, adrenaline and cortisol in causing the hypoxia in tissues is mentioned in this thesis. Anxiety is an emotion characterized by an unpleasant state of inner turmoil, often accompanied by nervous behavior, such as pacing back and forth, somatic complaints, and rumination. Depression is a state of low mood and aversion to activity that can affect a person's thoughts, behavior, feelings, and sense of well-being. Fear in human beings may occur in response to a specific stimulus occurring in the present, or in anticipation or expectation of a future threat perceived as a risk to body or life. In psychology, stress is a feeling of strain and pressure. Also this is one type of psychological pain. Cancer as mentioned by Drs. Zaminpira and Niknamian, is an Evolutionary Metabolic Disease (EMHC) which is caused by increasing the amounts of Reactive Oxygen Species (ROS) through the Butterfly Effect (BE) inside human eukaryotic cells. Therefore; increasing inflammation is a promising factor in the cause of cancer. The aim of this review and meta-analysis is to find the link between the depression, stress, fear and anxiety and the possibility of causing cancer. These emotional states have been observed in cancer patients as well. Anxiety and Fear are the two main emotional states which are the side effects of cancer disease, and also, high amounts of emotional stress and depression have been discussed in this review to raise the possibility in causing cancer. In animal cells, mitochondria are unique organelles in that they contain a genome of their own. There are small circular chromosomes in each mitochondrion that have genes for some of the mitochondrial proteins. But the mitochondrial chromosomes do not have genes for all the proteins found in mitochondria. The genes for the remaining proteins are found in the cellular genome which is found in the nucleus. So to get some functional mitochondria requires gene expression of both nuclear and mitochondrial genes. By the Evolutionary Metabolic Hypothesis of Cancer (EMHC), the main reason behind the cause of cancer, is increasing the amounts of Reactive Oxygen Species and intracellular inflammation which cause damage to the mitochondria. Increasing the inflammation will cause chaos in normal cells and causes the nucleus to send wrong messages instead of apoptosis, that means turning the oxidative phosphorylation into fermentation in cytosol. Therefore, by three-year study over cancer and normal cells, we have come to the conclusion that the real reason behind the cause of cancer is the increasing of ROS above the normal limits that causes butterfly effect inside the normal cells. This thesis goes through many researches based on the effectiveness of the neurotransmitter serotonin on cancer cells and also the impact of SSRIs (selective serotonin reuptake inhibitors) drugs on the cause of certain types of 9

10. cancers. Serotonin has been shown to be a mitogenic factor for a wide range of normal and tumor cells. Serotonin exhibits a growth stimulatory effect in aggressive cancers and carcinoids usually through 5- HT1 and 5-HT2 receptors. In contrast, low doses of serotonin can inhibit tumor growth via the decrease of blood supply to the tumor, suggesting that the role of serotonin on tumor growth is concentration- dependent. Serum serotonin level was found to be suitable for prognosis evaluation of urothelial carcinoma in the urinary bladder, adenocarcinoma of the prostate and renal cell carcinoma. The mechanism that connect serotonin with tumor evolution seem to be related to the serotonin impact on tumor associated macrophages. Cancer affects all animals containing eukaryote cells as well. Less is known about the cancers that affect wild animals, since they move around and may not be easily observed for a long period of time. This review about cancers in wild animals contains useful data for the study of human cancers as well. Certain cancers in dinosaurs show that this metabolic disease is primitive and may have been around since the beginning of the multicellular organisms. This data also shows there has been some cancer types in naked mole rats and wild sharks as well. Nowadays, Tasmanian Devils are plagued by an infectious cancer known as Tasmanian devil facial tumor disease (DFTD). Since the emergence of the disease in 1996, the population has declined by more than 60 percent. This type of cancer has an allograft transmission. It seems earthworms contain an anti-cancer agent which could be of great interests in the treatment of cancer. In the discussion part of our review we have discussed how Peto’s Paradox theory of cancer is not true and we have mentioned many data of the cancer incidences in whales and elephants. This thesis also goes deeply through the nutrition of the first primate and analyses the nutrition in the line of human evolution. Simply dividing the shifts in the nutrition through the human evolution, it is obvious that some elements in the diet are more important than the others. Since the beginning of the Neolithic, the ratio of plant-to-animal foods in the diet has sharply increased from an average of probably 65% to 35% during Paleolithic times to as high as 90% to 10% since the advent of agriculture. The changes in diet from hunter-gatherer times to agricultural times have been almost all detrimental, although there is some evidence indicating that at least some genetic adaptation to the Neolithic has begun taking place in the approximately 10,000 years since it began. With the much heavier reliance on starchy foods that became the staples of the diet, tooth decay, malnutrition, and rates of infectious disease increased dramatically over Paleolithic times, further exacerbated by crowding leading to even higher rates of communicable infections. This evidences shows that the immune system of human beings became weaker and many metabolic diseases including cancer has become epidemic. The height of humans has decreased in the agricultural revolution as well. 10

11. CHAPTER ONE Evolutionary Metabolic Hypothesis of Cancer (EMHC) Introduction Warburg effect in Plant physiology The Warburg Effect (WE), is the decline in the photosynthesis rate by high oxygen concentrations. [1][2] Oxygen is an inhibitor of the carbon dioxide fixation by RuBisCO. Which initiates photosynthesis. Oxygen stimulates photo-respiration, which decreases photosynthetic result. These two mechanisms working together, are responsible for the Warburg Effect (WE). [3] Warburg Effect in Oncology The Warburg effect (WE) in oncology, says that cancer cells produce energy by a high rate of glycolysis followed by lactic acid fermentation in the cytosol, [4][5] rather than by a comparatively low rate of glycolysis followed by oxidation of pyruvate in mitochondria. [6][7][8] The latter process is aerobic and uses oxygen. Malignant tumor cells have glycolytic rates 200 times higher than normal tissues of origin. This happens even if oxygen is plentiful and present as well. Otto Heinrich Warburg proved this change in metabolism is the prime cause of cancer. [9] 11

12. Picture [1]: The differences between cancer cells and normal cells as the explanation of the Warburg Effect (WE). Warburg Hypothesis of Cancer (WHC) The Warburg Hypothesis of Cancer (WHC), explains that the real cause of tumorigenesis is an insufficient cellular respiration caused by insult to mitochondria. [10] Warburg Effect (WE) explains the observation that cancer cells, grown in-vitro, goes through the process of glucose fermentation even when enough oxygen is present for cell respiration. The Warburg Hypothesis of Cancer (WHC) shows that the Warburg Effect (WE) was the prime cause of cancer. The current popular opinion is that cancer cells ferment glucose and sugar molecules while keeping up the same level of respiration that was present before the process of carcinogenesis, and therefore, the Warburg effect would be defined as the observation that cancer cells exhibit glycolysis with lactic acid production and mitochondrial respiration even if the enough oxygen is present. [11] Warburg Hypothesis of Cancer (WHC) was discovered by the Nobel prize winner, Otto Heinrich Warburg in 1924. [12] He hypothesized that cancer, malignant growth, and tumor growth are caused by the fact that tumor cells mainly generate energy as the form of adenosine triphosphate (ATP) by glycolysis. This is in contrast to normal cells, which produces energy from oxidative breakdown of pyruvate. Therefore, based on Warburg discovery, the cause of cancer cells should be interpreted as stemming from a lowering of mitochondrial respiration. Warburg reported a fundamental difference between normal and cancerous cells to be the ratio of glycolysis to respiration. This observation is also known as the Warburg Effect 12

13. (WE). Cancer is caused by mutations and altered gene expression, in a process called malignant transformation, resulting in an uncontrolled growth of cells.[13][14] The metabolic differences observed by Warburg adapts cancer cells to the hypoxic conditions inside solid tumors, and results largely from the same mutations in oncogenes and tumor suppressor genes that cause the other abnormal characteristics of cancer cells.[15] Hence, the metabolic change observed by Warburg is not so much the cause of cancer, as he claimed, but rather, it is one of the characteristic effects of cancer-causing mutations. The prime cause of cancer is when the oxygen respiration changes in normal body cells by a fermentation of sugar. [16] MATERIALS AND METHODS We have done the treatment based on the special model of ketogenic diet and ozone therapy on the 54 cancer patients, including; Liver cancer, Colorectal cancer, kidney cancer, brain metastatic tumors, breast cancer, lung cancer. This was a double blind controlled study which has done at the Violet Cancer Institute (VCI) in Iran/Tehran. 10 patients with the liver cancer, 5 patients with the kidney cancer, 11 with brain metastatic tumors, 18 patients with the breast cancer tumors, 5 patients with the lung cancer and 5 patients with colorectal cancer. Table (1) The methodology of this study was based on 5 days of water fasting to make the normal cells go to the catabolism state, after 5 days we started the special Keto- 13

14. Diet the 80 percent saturated fat including MCT and animal and coconut saturated fats. 15 percent protein powder with the lowest glutamine which we have produced at the Violet laboratory, and 5 percent complex carbohydrates with the highest fiber. The Ozone therapy method was three days per week on each patient with 5% Ozone in 100 Mole Oxygen. After 3 months of this study the average results of the reduction in cancer tumors by MRI device were: 1.45 Percent decrease in tumor size in lung cancer tumors. 2.25 percent decrease in tumor size in colorectal cancer tumors. 3.75 percent decrease in tumor size in breast cancer tumors. 4.62 percent decrease in tumor size in liver cancer tumors. 5.54 percent decrease in tumor size in kidney cancer tumors. 6.87 percent decrease in tumor size in brain cancer tumors. Percentage of Reduction in Cancer Tumors 100 80 60 40 20 0 LIVER KIDNEY BRAIN BREAST LUNG COLORECTAL Table (2) Metabolic Mathematical Model as a Proof of EMHC Hypothesis To properly understand or derive a mathematical model for cancer growth, we should understand the process of the ontogenetic development of an organism. This process is fueled by metabolism and follows a certain pattern which occurs primarily 14

15. through cell division. mathematical model based on energy conservation was derived to model such growth and shows that regardless of the different masses and development times, all taxons share a common growth pattern [35]. It may be possible that cancer growth may be modeled in the same way. As the total energy that goes into the development of the organism either goes into the maintenance of existing tissue or the creation of new tissue, we can express this as B is the energy that an organism uses while at rest. The variables, Bc and Nc are the metabolic rate for an individual cell and the number of cells in a particular organism respectively; the NcBc term represents the energy to maintain existing tissue. Ecis the energy needed to create new tissue from an individual cell. We assume that variables Ec, Bc and mc all remain constant during an organism’s growth and is pertinent to a particular type of organism. Thus the total mass of an organism, m, can be determined from the mass of an individual cell and the number of cells, m = mcNc. By differentiating and substituting this into the equation, the result is: Given that where B0 depends on a particular taxon, As the B0, mc and Ec terms are constant, we can express the above equation succinctly as where a ≡ B0mc/Ec and b ≡ Bc/Ec. 15

16. The 3/4 exponent is roughly the same for all organisms, whether they be mammals, birds, fish or plants. Thus the exponent describes the overall allometry of B from birth to maturity. As there is a tendency for natural selection to optimize energy transport, this has led to the evolution of a fractal-like distribution network. This exponent is related to the scaling in the total number, Nt of capillaries [35] [36]. As we already know, the total number of cells is related to the organism’s mass. This exponent has the profound implication in that it sets limits on the growth of an organism. Thus the point in which an organism stops growing, i.e. dm/dt = 0, we see that where M is the symptotic maximum body size. Thus the variation on M among different species within a taxon is determined entirely by the cellular metabolic rate, Bc, which scales to M-1/4. As cancer growth follows the same principles, blood and nutrients enter into and feed a tumor, we expect the same scaling principles to apply and thus by using this Universal Law for ontogenetic growth we hope to derive a similar universal law for cancer growth. As B0, mc and Ec are approximately constant, a is independent of M and b = a/M1/4. We can thus rewrite this equation as by Solving this differential equation, the result is: m0 is the mass of the organism at birth (t = 0). As a and b can be determined from fundamental parameters of a cell, a universal equation has been derived. We can see from the figures below that all growth curves follow the same path. We can infer that if similar considerations are made for cancerous cells, a similar growth curve will be obtained. 16

17. DISCUSSIONS AND RESULTS Introduction to the Evolutionary Metabolic Hypothesis of Cancer (EMHC) The first living cells on Earth are thought to have arisen more than 3.5 × 109 years ago, when the Earth was not more than about 109 years old. The environment lacked oxygen but was presumably rich in geochemically produced organic molecules, and some of the earliest metabolic pathways for producing ATP may have resembled present-day forms of fermentation. In the process of fermentation, ATP is made by a phosphorylation event that harnesses the energy released when a hydrogen-rich organic molecule, such as glucose, is partly oxidized. The electrons lost from the oxidized organic molecules are transferred via NADH or NADPH to a different organic molecule or to a different part of the same molecule, which thereby becomes more reduced. At the end of the fermentation process, one or more of the organic 17

18. molecules produced are excreted into the medium as metabolic waste products. Others, such as pyruvate, are retained by the cell for biosynthesis. The excreted end- products are different in different organisms, but they tend to be organic acids. Among the most important of such products in bacterial cells are lactic acid which also accumulates in anaerobic mammalian glycolysis, and formic, acetic, propionic, butyric, and succinic acids. [30] dFigure [2]: Symbio-genesis of mitochondria. [31] As we can see in the figure [2], the first cell on the earth before the entrance of the bacteria did contain nucleus and used the fermentation process to produce ATP for its energy. Then an aerobic proteo-bacterium enters the eukaryote either as a prey or 18

19. a parasite and manages to avoid digestion. It then became an endosymbiont. As we observe, the fermentation process used the glucose or even glutamine to produce ATP, but the aerobic process used the glucose, fat and protein to produce more ATP than the previous one. The symbio-genesis of the mitochondria is based on the natural selection of Charles Darwin. Based on Otto Warburg Hypothesis and Seyfried Metabolic Hypothesis of cancer, in nearly all cancer cells, the mitochondrion is shut down or are defected and the cancer cell do not use its mitochondrion to produce ATP. [32] This process of adaptation is based on Lamarckian Hypothesis of Evolution and the normal cells goes back to the most primitive time of evolution to protect itself from apoptosis and uses the fermentation process like the first living cells 1.5 billion years ago. Therefore, cancer is an evolutionary metabolic disease which uses glucose as the main food to produce ATP and Lactic Acid. The prime cause of cancer is the abundance of Reactive Oxygen Species produced by mitochondria that is a threat to the living normal cell and causes mitochondrial damage mainly in its cristae. [S. Niknamian et al 2016.] Research Studies as the Proof of Cancer as a Metabolic Disease In a study by Michael Ristow and colleagues, colon cancer lines were modified to overexpress frataxin. The results of their research shows that an increase in oxidative metabolism induced by mitochondrial frataxin may inhibit cancer growth in mammals. [17] Studies published since 2005 have shown that the Warburg Effect (WE) may lead to a promising approach in the treatment of solid tumors. Alpha-cyano-4- hydroxycinnamic acid (ACCA, CHCA), a small-molecule inhibitor of mono- carboxylate transporters (MCTs) which prevent lactic acid build up in tumors, has been successfully used as a metabolic target in brain tumor pre-clinical research. [18][19][20][21] Higher affinity MCT inhibitors have been developed by Astra- Zeneca.[22] The chemical dichloro-acetic acid (DCA), which promotes respiration and the activity of mitochondria, has also been shown to kill cancer cells in-vitro and in some animals. [23] Our body, kills damaged cells by apoptosis most of the time, a process that involves mitochondria and is a self-destruction, Therefore, this mechanism fails in Malignant Cancer Cells (MCC) where the mitochondria are shut 19

20. down or is damaged. The reactivation of mitochondria in cancer cells restarts their apoptosis program.[24] Besides human research at the University of Alberta led by Dr Evangelos Michelakis, other glycolytic inhibitors besides DCA that hold promise include Bromo-pyruvic acid, being studied at The University of Texas M. D. Anderson Cancer Center (ACC), 2-deoxyglucose (2-DG) at Emory University School of Medicine (EUSM), and lactate dehydrogenase A at Johns Hopkins University School of Medicine. [25] Cancer as a metabolic disease Impaired cellular metabolism of energy is the characteristic of nearly all cancers regardless of cellular or tissue origin. Normal cells derive most of their energy from oxidative phosphorylation, Therefore, most cancer cells become dependent on substrate level phosphorylation to meet their energy needs. Many Evidences are reviewed in the support of a general hypothesis that genomic instability and essentially all hallmarks of cancer, including aerobic glycolysis which is called Warburg Effect (WE), can be linked to impaired mitochondrial function and energy metabolism. [26] Oncogenic Paradox High variety of things from viruses, bacteria to radiation, chemicals and oxidation can damage DNA and cause mutations. [One Renegade Cell: How Cancer Begins, Robert A. Weinberg] There are hundreds of thousands of significant mutations are associated with tumors. One single colon cancer cell can contain 11,000 mutations. [27] The number and type of mutations found in cancer cells are so serious that they would cause a healthy embryo to spontaneously abort. The transformation of a healthy cell into a cancerous cell malignant transformation happens in the very same special way every time. There is not one example of a mutation that causes the same type of cancer. Even those mutations most strongly associated with certain cancers only cause cancer in certain humans. Cancer cells within the same tumor can have different mutation patterns. Mutated genes thought to be strongly associated with cancer oncogenes, sometimes do promote tumor growth, but sometimes they inhibit tumor growth, and sometimes they do both. If one transplants mutated cancer cell DNA into a healthy cell, the healthy cell almost never becomes cancerous. Only 2 out of 24 experiments were successful in transformingnormal cells into cancer cells. This result is against the mutation theory of cancer, which explains why mutation theory of cancer is not right. [28][29] Damaged Mitochondria and Cancer 20

21. Billions of years ago, before plants took hold on the planet, earth’s atmosphere had very little oxygen and living creatures used fermentation to generate energy. Organisms were very simple, without sophisticated controls to help them decide when to reproduce. They just reproduced as fast as they possibly could. [26] Mitochondria appeared about 1.5 billion years ago, about a billion years after oxygen became available, and probably already had the ability to switch back and forth between fermentation and respiration, depending on how much oxygen was around. Many cells will simply extinct if their mitochondria are damaged, but if the damage is not too sudden or severe, some cells will be able to adapt and survive by switching back to fermentation to make energy.[27] Mitochondrial damage unlocks an ancient toolkit of pre-existing adaptations that allow cells to survive in low-oxygen environments. Mitochondria are so good at producing energy that their arrival on the evolutionary scene is thought to be largely responsible for the increase in complexity of living things. Building and supporting elaborate new creatures with specialized organs and capabilities takes a lot of energy. If an organism does not constantly pour energy into a living thing to maintain its form and function, it will gradually succumb to entropy or chaos. For cells, this means regressing which means DNA becomes unstable. [28] Cells lose their unique shapes, become disorganized and start reproducing uncontrollably. Any number of environmental hazards can damage mitochondria. These are the same kinds of things thought typically as damaging DNA and causing cancer. However, the previous lines in this research article proposed that damaged DNA is not the primary cause of cancer. It is the mitochondria that are responsible. Mitochondria take care of cells and DNA. Mitochondrial damage happens first, and then genetic instability follows. Even though there is plenty of oxygen around, damaged mitochondria have no choice but to resort to fermentation, which is primitive and wasteful. Cells cannot stay in shape and under control under these circumstances. They may be able to live, but it will not be normal. Cells with damaged mitochondria, only if they survive, are at high risk for becoming cancerous. [29] Cancer and Sugar Nearly all tumors depend heavily on glucose for survival, which is how PET scans are able to find many tumors hiding in normal tissues. PET scans follow radioactive glucose as it travels through the bloodstream. Radio-labeled glucose accumulates in tumor tissue more than in the normal tissues surrounding it, and lights up on the scan. There is a strong connection between high blood sugar or hyperglycemia, diabetes, and cancer. It is well-documented that the growth of brain tumors is more accelerated and prognosis is worse in animals and humans 21

22. with higher blood glucose levels. [30] Hyperglycemia is directly linked with poor prognosis in humans with malignant brain cancer and is connected to the rapid growth of most malignant cancers. High blood glucose raises insulin levels, which stimulates cancer cells to take in and use more glucose. [31] This makes it easier for cancer cells to nourish themselves. Insulin also turns up the activity of the fermentation pathway, and fermentation leads to additional cellular damage. High blood glucose also raises levels of another circulating hormone called IGF-I or Insulin-like Growth Factor I. Cancer cells with receptors on their surfaces for this hormone grow more rapidly. IGF-I turns on a chemical pathway that drives tumor cell growth. Which is the PI3K-Akt-HIF-1 alpha pathway. This pathway sets the stage for cells to multiply, escape apoptosis, and recruit their own angiogenesis. Angiogenesis is required for tumors to grow beyond 2 millimeters in size. The genes for this growth pathway are also turned up by the fermentation process. More glucose means more fermentation and more insulin and more IGF-I mean more tumor growth. Briefly, cancer is a disease of growth, and insulin is the mother of all growth hormones. [32] Regardless of which type of cancer one may have, what grade or stage it might be, or which mutations or genetic markers it might have, the hallmark of all cancer cells is damaged mitochondria. Cancer is not a collection of unrelated diseases that each need to be treated individually, cancer is one disease which is a mitochondrial disease, and diseased mitochondria prefer glucose and glutamine for fuel. Healthy cells with healthy mitochondria are flexible and can adapt to just about any fuel source, but not cancer cells. In fact, the majority of cells function best when they burn fat for energy. Cancer cells are bad at burning fat, because fat burning requires respiration, which requires healthy mitochondria. [33] Cancer Treatment Based on Evolutionary Metabolic Hypothesis of Cancer If food is restricted enough to lower blood glucose, then insulin and IGF-1 levels will also be lower, quieting the tumor driving genes and pathways which is described in previous lines. This means that fermentation becomes harder for tumors to recruit new blood vessels, and tumor growth slows. Under low blood glucose levels, glucagon comes in. this is the opposite of insulin hormone. Glucagon stimulates fat burning, which raises ketones and fatty acids in the blood. Ketones and fatty acids are just breakdown products of fats. [34] Ketone bodies and fatty acids cannot be fermented, thus, cancer cells cannot utilize them for fuel. Glucose restriction stresses cancer cells. However, most healthy cells prefer to use fatty acids and ketones for energy. Glucose restriction is good for healthy cells. Glucagon also keeps blood. sugar from dropping too low by turning on a process in the liver called gluconeogenesis. This is why humans never need to eat any carbohydrates. Humans 22

23. are always able to make all the glucose they need out of proteins and fats. [35] The brain cannot burn fatty acids but it can burn ketones, and under low glucose conditions, the brain gradually shifts from burning mostly glucose to burning mostly ketones. The brain may still require a small percentage of glucose to function at its best, but there is always enough glucose in the bloodstream because of glucagon, and most other organs will pass up glucose under these conditions in order to let the brain have first dibs. Cancer cells and healthy cells both have a molecule on their surfaces called GLUT-1. [36] This glucose transporter ushers glucose out of the bloodstream and into cells. Under low glucose conditions, healthy cells will create more of these transporters and display them on their surfaces so as to optimize their ability to obtain glucose. Cancer cells, which are damaged, and therefore less flexible and adaptable, are not able to do this. In fact, when glucose levels are low, cancer cells are even weaker than usual. Not only can they not raise their GLUT-1 levels, their GLUT-1 levels actually drop. This is one more way that glucose restriction impairs cancer cells. Even though there is always some glucose in the bloodstream because of gluconeogenesis, cancer cells are less able to access it than healthy cells because they are damaged. [37] When ketones are burned for energy instead of glucose, fewer reactive oxygen species (ROS) are generated. These are free radicals that cause oxidative damage. This means that shifting the body from being a carbohydrate-burning machine to becoming a fat-burning machine reduces oxidative damage, and therefore potentially reduces risk for numerous chronic diseases. Diets that raise blood levels of ketones are considered to be neuroprotective. That is they protect brain cells from harm. Glucose burning is neurotoxic and burning ketones instead simply restores the natural, healthy level of disease resistance humans inherited from their ancestors. One reason why ketogenic diet is under consideration for the treatment of so many neurological diseases, from autism to Alzheimer’s to multiple sclerosis and epilepsy to Parkinson’s Disease, is that the transition from glucose burning to ketone burning is powerfully anti-inflammatory. There is no drug therapy can target as many pro- inflammatory mechanisms in the microenvironment as can dietary energy restriction. Real progress in tumor management will be achieved once patients and the oncology community come to recognize this fact. In fact, it is inflammation which damages mitochondria and respiration, and thus, inflammation may be the true cause of cancer. [38] 23

24. CHAPTER TWO CHRONIC FEAR AND CANCER IN HUMANS INTRODUCTION Fear Fear is a feeling induced by perceived danger or threat that occurs in certain types of organisms, which causes a change in metabolic and organ functions and ultimately a change in behavior, such as fleeing, hiding, or freezing from perceived traumatic events. Fear in human beings may occur in response to a specific stimulus occurring in the present, or in anticipation or expectation of a future threat perceived as a risk to body or life. [1] The fear response arises from the perception of danger leading to confrontation with or escape from/avoiding the threat (also known as the fight-or-flight response), which in extreme cases of fear (horror and terror) can be a freeze response or paralysis. In humans and animals, fear is modulated by the process of cognition and learning. Thus fear is judged as rational or appropriate and irrational or inappropriate. An irrational fear is called a phobia. [2] Psychologists such as John B. Watson, Robert Plutchik, and Paul Ekman have suggested that there is only a small set of basic or innate emotions and that fear is one of them. This hypothesized set includes such emotions as acute stress reaction, anger, angst, anxiety, fright, horror, joy, panic, and sadness. Fear is closely related to, but should be distinguished from, the emotion anxiety, which occurs as the result of threats that are perceived to be uncontrollable or unavoidable. The fear response serves survival by generating appropriate behavioral responses, so it has been preserved throughout evolution. [3] Cancer The most important difference between normal cells and cancer cells is how they respire. Normal cells use the sophisticated process of respiration to efficiently turn any kind of nutrient that is fat, carbohydrate or protein into high amounts of energy in the form of ATP. This process requires oxygen and breaks food down completely into harmless carbon dioxide and water. Cancer cells use a primitive process of fermentation to inefficiently turn either glucose from carbohydrates or the amino acid glutamine from protein into small quantities of energy in the form of ATP. This process does not require oxygen, and only partially breaks down food molecules into 24

25. lactic acid and ammonia, which are toxic waste products. Nearly all researches from 1934 to 2016, mention that in all cancer cells there is some mitochondrial damages and abnormal deformations mostly in mitochondrial cristae. The prime cause of cancer is mitochondrial damage, which is caused by increasing the amount of ROS and inflammation inside or around eukaryotic cells. [4] Bohr Effect The Bohr Effect is a physiological phenomenon first described in 1904 by the Danish physiologist Christian Bohr, stating that hemoglobin's oxygen binding affinity is inversely related both to acidity and to the concentration of carbon dioxide. Since carbon dioxide reacts with water to form carbonic acid, an increase in CO2 results in a decrease in the blood pH, resulting in hemoglobin proteins releasing their load of oxygen. Conversely, a decrease in carbon dioxide provokes an increase in pH, which results in hemoglobin picking up more oxygen. The Bohr effect increases the efficiency of oxygen transportation through the blood. After hemoglobin binds to oxygen in the lungs due to the high oxygen concentrations, the Bohr effect facilitates its release in the tissues, particularly those tissues in most need of oxygen. [31-36] MATERIALS AND METHODS The brain structures that are the center of most neurobiological events associated with fear are the two amygdalae, located behind the pituitary gland. Each amygdala is part of a circuitry of fear learning. [5] They are essential for proper adaptation to stress and specific modulation of emotional learning memory. In the presence of a threatening stimulus, the amygdalae generate the secretion of hormones that influence fear and aggression. [6] Once a response to the stimulus in the form of fear or aggression commences, the amygdalae may elicit the release of hormones into the body to put the person into a state of alertness, in which they are ready to move, run, fight, etc. This defensive response is generally referred to in physiology as the fight- or-flight response regulated by the hypothalamus, part of the limbic system. [7] Once the person is in safe mode, meaning that there are no longer any potential threats surrounding them, the amygdalae will send this information to the medial prefrontal cortex (mPFC) where it is stored for similar future situations, which is known as memory consolidation. [8] Some of the hormones involved during the state of fight-or-flight include epinephrine, which regulates heart rate and metabolism as well as dilating blood vessels and air passages, norepinephrine increasing heart rate, blood flow to skeletal muscles and the release of glucose from energy stores, and cortisol which 25

26. increases blood sugar, increases circulating neutrophilic leukocytes, calcium amongst other things. [9,10] After a situation which incites fear occurs, the amygdalae and hippocampus record the event through synaptic plasticity. [11] The stimulation to the hippocampus will cause the individual to remember many details surrounding the situation. [12] Plasticity and memory formation in the amygdala are generated by activation of the neurons in the region. Experimental data supports the notion that synaptic plasticity of the neurons leading to the lateral amygdalae occurs with fear conditioning. [13] In some cases, this forms permanent fear responses such as posttraumatic stress disorder (PTSD) or a phobia. [14] MRI and fMRI scans have shown that the amygdalae in individuals diagnosed with such disorders including bipolar or panic disorder are larger and wired for a higher level of fear. [15] Pathogens can suppress amygdala activity. Rats infected with the toxoplasmosis parasite become less fearful of cats, sometimes even seeking out their urine-marked areas. This behavior often leads to them being eaten by cats. The parasite then reproduces within the body of the cat. There is evidence that the parasite concentrates itself in the amygdala of infected rats. [16] In a separate experiment, rats with lesions in the amygdala did not express fear or anxiety towards unwanted stimuli. These rats pulled on levers supplying food that sometimes sent out electrical shocks. While they learned to avoid pressing on them, they did not distance themselves from these shock-inducing levers. [17] Several brain structures other than the amygdalae have also been observed to be activated when individuals are presented with fearful vs. neutral faces, namely the occipitocerebellar regions including parietal / superior temporal gyri. Interestingly, fearful eyes, brows and mouth seem to separately reproduce these brain responses. Scientists from Zurich studies show that the hormone oxytocin related to stress and sex reduces activity in one’s brain fear center. [18,19] the fusiform gyrus and the inferior FEAR ADRENALINE CORTISOL Figure (1): Fear results in the secretion of adrenaline that causes the increase in cortisol hormone levels. 26

27. As shown in Figure (1), fear increases the secretion of the hormone adrenaline and this hormone causes the incline in cortisol levels in body. Fear and Inflammation Michopoulos V. et al stated that the study of inflammation in fear- and anxiety-based disorders has gained interest as growing literature indicates that pro-inflammatory markers can directly modulate affective behavior. Indeed, heightened concentrations of inflammatory signals, including cytokines and C-reactive protein, have been described in posttraumatic stress disorder (PTSD), generalized anxiety disorder (GAD), panic disorder (PD), and phobias (agoraphobia, social phobia, etc.). However, not all reports indicate a positive association between inflammation and fear- and anxiety-based symptoms, suggesting that other factors are important in future assessments of inflammation's role in the maintenance of these disorders (ie, sex, co-morbid conditions, types of trauma exposure, and behavioral sources of inflammation). The most parsimonious explanation of increased inflammation in PTSD, GAD, PD, and phobias is via the activation of the stress response and central and peripheral immune cells to release cytokines. Dysregulation of the stress axis in the face of increased sympathetic tone and decreased parasympathetic activity characteristic of anxiety disorders could further augment inflammation and contribute to increased symptoms by having direct effects on brain regions critical for the regulation of fear and anxiety (such as the prefrontal cortex, insula, amygdala, and hippocampus). Taken together, the available data suggest that targeting inflammation may serve as a potential therapeutic target for treating these fear- and anxiety-based disorders in the future. However, the field must continue to characterize the specific role pro-inflammatory signaling in the maintenance of these unique psychiatric conditions. [Michopoulos V. et al, 2016] Melamed S et al. concluded that based on evidence that psychological stress may induce a chronic inflammatory process, we hypothesized that the stress caused by chronic fear of terror may be associated with low-grade inflammation. This hypothesis was examined in employed men and women with the presence of low- grade inflammation measured by high sensitivity C-reactive protein (CRP). Apparently healthy employed adults (N = 1153) undergoing periodic health check- ups in a tertiary hospital in Israel completed a questionnaire. Fear of terror (scored 1-5) was assessed by three items measuring the extent to which respondents have deep concern for personal safety, elevated tension in crowded places, and fear of terror strikes causing harm to one's self or one's family members. The main outcome measure was the presence or absence of an elevated CRP level (>3.0 mg/L). Women scored significantly higher on fear of terror compared with men (M = 2.16 vs. M = 27

28. 1.68, respectively; p <.0001). Most of the study participants who scored high (4 or 5) on fear of terror, reported having experienced this feeling for 1 year or more. In women only, there was a positive association between fear of terror and risk of elevated CRP level (adjusted OR = 1.7, 95% CI 1.2-2.4) in a multivariate model adjusting for generalized anxiety, depressive symptoms, and potentially confounding demographic and biomedical variables. Chronic fear of terror in women, but not in men, is associated with elevated CRP levels, which suggests the presence of low-grade inflammation and a potential risk of cardiovascular disease. [Melamed S. et al., 2015] In conclusion, fear and anxiety increase the inflammation and suppress the immune system in human body. Secretion of cortisol causes the decrease in immune system response throughout the body. Fear, Hypocapnia, Blood Glucose and Hypoxia Chronic fear causes the secretion of cortisol in the body which increases the blood glucose levels. The research studies below show that high blood glucose is linked with the hypoxia in tissues through the Bohr Effect. Adrenaline hormone causes hypocapnia and decreases the blood flow to the brain. Therefore; hypoxia in tissues specifically in the brain is the result of the chronic fear. Study done in 2010 by Heinis and Simon: When cultured in collagen, embryonic pancreatic cells were hypoxic and expressed HIF1alpha and rare beta-cells differentiated. In pancreata cultured on filter (normoxia), HIF1alpha expression decreased and numerous beta-cells developed. During pancreas development, HIF1alpha levels were elevated at early stages and decreased with time. To determine the effect of pO2 on beta-cell differentiation, pancreata were cultured in collagen at increasing concentrations of O2. Such conditions repressed HIF1alpha expression, fostered development of Ngn3-positive endocrine progenitors, and induced beta-cell differentiation by O2 in a dose-dependent manner. By contrast, forced expression of HIF1alpha in normoxia using DMOG repressed Ngn3 expression and blocked beta-cell development. Finally, hypoxia requires hairy and enhancer of split (HES)1 expression to repress beta-cell differentiation. These data demonstrate that beta-cell differentiation is controlled by pO2 through HIF1alpha. Modifying pO2 should now be tested in protocols aiming to differentiate beta-cells from embryonic stem cells. [22] One study by Cheng et al in 2010 demonstrated that Hypoxia-inducible factor- 1alpha (HIF-1alpha) is a transcription factor that regulates cellular stress responses. 28

29. While the levels of HIF-1alpha protein are tightly regulated, recent studies suggest that it can be active under normoxic conditions. We hypothesized that HIF-1alpha is required for normal beta cell function and reserve and that dysregulation may contribute to the pathogenesis of type 2 diabetes (T2D). Increasing HIF-1alpha levels markedly increased expression of ARNT and other genes in human T2D islets and improved their function. [23] Regazzetti et al in 2009 showed that in both human and murine adipocytes, hypoxia inhibits insulin signaling as revealed by a decrease in the phosphorylation of insulin receptor. In 3T3-L1 adipocytes, this inhibition of insulin receptor phosphorylation is followed by a decrease in the phosphorylation state of protein kinase B and AS160, as well as an inhibition of glucose transport in response to insulin. These processes were reversible under normoxic conditions. The mechanism of inhibition seems independent of protein tyrosine phosphatase activities. Overexpression of HIF- 1alpha or -2alpha or activation of HIF transcription factor with CoCl(2) mimicked the effect of hypoxia on insulin signaling, whereas downregulation of HIF-1alpha and -2alpha by small interfering RNA inhibited it. We have demonstrated that hypoxia creates a state of insulin resistance in adipocytes that is dependent upon HIF transcription factor expression. Hypoxia could be envisioned as a new mechanism that participates in insulin resistance in adipose tissue of obese patients. [24] Halberg et al in 2009 demostrated that Adipose tissue can undergo rapid expansion during times of excess caloric intake. Like a rapidly expanding tumor mass, obese adipose tissue becomes hypoxic due to the inability of the vasculature to keep pace with tissue growth. Consequently, during the early stages of obesity, hypoxic conditions cause an increase in the level of hypoxia-inducible factor 1alpha (HIF1alpha) expression. Using a transgenic model of overexpression of a constitutively active form of HIF1alpha, we determined that HIF1alpha fails to induce the expected proangiogenic response. In contrast, we observed that HIF1alpha initiates adipose tissue fibrosis, with an associated increase in local inflammation. "Trichrome- and picrosirius red-positive streaks," enriched in fibrillar collagens, are a hallmark of adipose tissue suffering from the early stages of hypoxia-induced fibrosis. Lysyl oxidase (LOX) is a transcriptional target of HIF1alpha and acts by cross-linking collagen I and III to form the fibrillar collagen fibers. Inhibition of LOX activity by beta-aminoproprionitrile treatment results in a significant improvement in several metabolic parameters and further reduces local adipose tissue inflammation. Collectively, our observations are consistent with a model in which adipose tissue hypoxia serves as an early upstream initiator for adipose tissue dysfunction by inducing a local state of fibrosis. [25] 29

30. Glaasford et al in 2007 showed that Apelin, a novel peptide with significant cardioactive properties, is upregulated by insulin in adipocytes. However, the mechanism by which insulin promotes apelin production is unknown. Hypoxia- inducible factor-1 (HIF-1), a heterodimeric transcription factor involved in the angiogenic and metabolic responses to tissue hypoxia, has been shown to be activated by insulin in various settings. We therefore hypothesized that HIF-1 regulates insulin-mediated apelin expression in adipocytes. 3T3-L1 cells were differentiated into adipocytes in culture. For experiments, serum-starved 3T3-L1 cells were exposed to insulin and/or a 1% O (2) environment. Apelin expression was assessed using quantitative real-time PCR and ELISA. To directly assess the role of HIF-1 in apelin production, we differentiated mouse embryonic fibroblasts (MEFs) containing a targeted deletion of the HIF-1alpha gene into adipocytes and measured their response to insulin and hypoxia. Apelin expression in mature 3T3-L1 adipocytes was increased significantly by insulin and was attenuated by pharmacological inhibition of insulin signaling. Exposure of cells to either hypoxia or the chemical HIF activators dimethyloxaloylglycine (DMOG) resulted in significant upregulation of apelin, consistent with a role for HIF in apelin induction. Moreover, hypoxia-, CoCl(2)-, DMOG-, and insulin-induced apelin expression were all attenuated in differentiated HIF-1alpha-deficient MEFs. In summary, in cultured 3T3-L1 adipocytes and differentiated MEFs, HIF-1 appears to be involved in hypoxia- and insulin-induced apelin expression. [26] cobalt chloride (CoCl(2)) and Chen et al in 2006 demostrated that Low plasma levels of adiponectin (hypoadiponectinemia) and elevated circulating concentrations of plasminogen activator inhibitor (PAI)-1 are causally associated with obesity-related insulin resistance and cardiovascular disease. However, the mechanism that mediates the aberrant production of these two adipokines in obesity remains poorly understood. In this study, we investigated the effects of hypoxia and reactive oxygen species (ROS) on production of adiponectin and PAI-1 in 3T3-L1 adipocytes. Quantitative PCR and immunoassays showed that ambient hypoxia markedly suppressed adiponectin mRNA expression and its protein secretion, and increased PAI-1 production in mature adipocytes. Dimethyloxallyl glycine, a stabilizer of hypoxia- inducible factor 1alpha (HIF-1alpha), mimicked the hypoxia-mediated modulations of these two adipokines. Hypoxia caused a modest elevation of ROS in adipocytes. However, ablation of intracellular ROS by antioxidants failed to alleviate hypoxia- induced aberrant production of adiponectin and PAI-1. On the other hand, the antioxidants could reverse hydrogen peroxide (H2O2)-induced dysregulation of adiponectin and PAI-1 production. H2O2 treatment decreased the expression levels of peroxisome proliferator-activated receptor gamma (PPARgamma) and 30

31. CCAAT/enhancer binding protein (C/EBPalpha), but had no effect on HIF-1alpha, whereas hypoxia stabilized HIF-1alpha and decreased expression of C/EBPalpha, but not PPARgamma. Taken together, these data suggest that hypoxia and ROS decrease adiponectin production and augment PAI-1 expression in adipocytes via distinct signaling pathways. These effects may contribute to hypoadiponectinemia and elevated PAI-1 levels in obesity, type 2 diabetes, and cardiovascular diseases. [27] Moritz and Meier et al in 2002 showed that to become insulin independent, patients with type 1 diabetes mellitus require transplantation of at least two donor pancreata because of massive beta-cell loss in the early post-transplantation period. Many studies describing the introduction of new immunosuppressive protocols have shown that this loss is due to not only immunological events but also non- immunological factors. To test to what extent hypoxia may contribute to early graft loss, we analyzed the occurrence of apoptotic events and the expression of hypoxia- inducible factor 1 (HIF-1), a heterodimeric transcription factor consisting of an oxygen-dependent alpha subunit and a constitutive beta subunit. Histological analysis of human and rat islets revealed nuclear pyknosis as early as 6 h after hypoxic exposure (1% O2). Moreover, immune-reactivity to activated caspase-3 was observed in the core region of isolated human islets. Of note, both of these markers of apoptosis topographically overlap with HIF-1alpha immune-reactivity. HIF- 1alpha mRNA was detected in islets from human and rat as well as in several murine beta-cell lines. When exposed to hypoxia, mouse insulinoma cells (MIN6) had an increased HIF-1alpha protein level, whereas its mRNA level did not alter. In conclusion, our data provide convincing evidence that reduced oxygenation is an important cause of beta-cell loss and suggest that HIF-1alpha protein level is an indicator for hypoxic regions undergoing apoptotic cell death. These observations suggest that gene expression under the control of HIF-1 represents a potential therapeutic tool for improving engraftment of transplanted islets. [28] As a result, adrenaline hormone causes hypocapnia and decreases the blood flow to the brain as well. Therefore; hypoxia in tissues specifically in the brain is the result of the chronic fear. Hypoxia, Inflammation and Cancer The most important fundamental difference between normal cells and cancer cells is how they make energy. Normal cells use the sophisticated process of respiration to efficiently turn fat, carbohydrate, or protein into high amounts of energy. This process requires oxygen and breaks food down completely into carbon dioxide and 31

32. water. Cancer cells use a primitive process called fermentation to inefficiently turn either glucose which is primarily from carbohydrates or the amino acid glutamine which is from protein into small amounts of energy. The most important findings from these researches are that fats cannot be fermented. This process does not require oxygen, and only partially breaks down food molecules into lactic acid and ammonia, which are toxic waste products. Normal cells sometimes have to change to fermentation process if they are temporarily experiencing an oxygen shortage. However, no cell in its right condition would ever choose to use fermentation when there is enough oxygen. It doesn’t produce nearly as much energy and creates toxic byproducts. Briefly, fermentation is primitive and wasteful. Cancer cells use fermentation even when there’s plenty of oxygen around, which is the explanation of the Warburg Effect, considered the metabolic signature of cancer cells. If a cell turning glucose into lactic acid when there is oxygen available, this would be a cancer cell. [29] Hypoxia as well as inflammation in tissues and normal cells increase the amounts of ROS in cells which is the prime cause of cancer. [30] DISCUSSION From all aspects which is written in the materials and methods, chronic fear makes the adrenal glands to secrete adrenaline hormone which increases the amounts of cortisol latterly. Cortisol hormone causes inflammation increasing blood glucose levels and suppressing the immune system response. [Michopoulos V. et al, 2016] Adrenaline on the other hand, causes hypocapnia and hyperventilation which through Bohr Effect causes hypoxia which leads to the incline in the amounts of ROS in tissues. Adrenaline also decreases the blood flow to the brain which decreases the amounts of oxygen in the brain. [22-28] Figure (2): The impact of cortisol and adrenaline hormones on causing cancer 32

33. Fear and Evolution From an evolutionary different adaptations that have been useful in our evolutionary past. They may have developed during different time periods. Some fears, such as fear of heights, may be common to all mammals and developed during the mesozoic period. Other fears, such as fear of snakes, may be common to all simians and developed during the cenozoic time period. Still others, such as fear of mice and insects, may be unique to humans and developed during the paleolithic and neolithic time periods (when mice and insects become important carriers of infectious diseases and harmful for crops and stored foods). Fear is high only if the observed risk and seriousness both are high, and it is low if risk or seriousness is low. [20,21] psychology perspective, different fears may be INCREASING BLOOD GLUCOSE LEVEL HYPOXIA IN TISSUES INFLAMMATION CHRONIC FEAR INCREASING INTRACELLULAR ROS SUPPRESSING THE IMMUNE SYSTEM HYPOCAPNIA Figure (3): Side effects of the chronic fear HYPOXIA INREASING INTRACELLULAR ROS SUPPRESSED IMMUNE SYSTEM CANCER Figure (4): The reason of cancer incidence from chronic fear 33

34. CHAPTER THREE STRESS, ANXIETY, FEAR AND DEPRESSION RELATION TO CANCER INTRODUCTION Depression, Anxiety, Stress and Fear Depression is a state of low mood and aversion to activity that can affect a person's thoughts, behavior, feelings, and sense of well-being. A depressed mood is a normal temporary reaction to life events such as loss of a loved one. It is also a symptom of some physical diseases and a side effect of some drugs and medical treatments. Depressed mood is also a symptom of some mood disorders such as major depressive disorder or dysthymia. [1] Anxiety is an emotion characterized by an unpleasant state of inner turmoil, often accompanied by nervous behavior, such as pacing back and forth, somatic complaints, and rumination. [2] It is the subjectively unpleasant feelings of dread over anticipated events, such as the feeling of imminent death. [3] Anxiety is not the same as fear, which is a response to a real or perceived immediate threat, whereas anxiety is the expectation of future threat. [4] Anxiety is a feeling of uneasiness and worry, usually generalized and unfocused as an overreaction to a situation that is only subjectively seen as menacing. It is often accompanied by muscular tension, restlessness, fatigue and problems in concentration. Anxiety can be appropriate, but when experienced regularly the individual may suffer from an anxiety disorder. [5] In psychology, stress is a feeling of strain and pressure. Also this is one type of psychological pain. [6] Small amounts of stress may be desired, beneficial, and even healthy. Positive stress helps improve athletic performance. It also plays a factor in motivation, adaptation, and reaction to the environment. Excessive amounts of stress, however, may lead to bodily harm. Stress can increase the risk of strokes, heart attacks, ulcers, dwarfism, and mental illnesses such as depression. [7] Fear is a feeling induced by perceived danger or threat that occurs in certain types of organisms, which causes a change in metabolic and organ functions and ultimately a change in behavior, such as fleeing, hiding, or freezing from perceived traumatic events. Fear in human beings may occur in response to a specific stimulus occurring in the present, or in anticipation or expectation of a 34

35. future threat perceived as a risk to body or life. The fear response arises from the perception of danger leading to confrontation with or escape from/avoiding the threat (also known as the fight-or-flight response), which in extreme cases of fear (horror and terror) can be a freeze response or paralysis. [8] In humans and animals, fear is modulated by the process of cognition and learning. Thus fear is judged as rational or appropriate and irrational or inappropriate. An irrational fear is called a phobia. Psychologists such as John B. Watson, Robert Plutchik, and Paul Ekman have suggested that there is only a small set of basic or innate emotions and that fear is one of them. This hypothesized set includes such emotions as acute stress reaction, anger, angst, anxiety, fright, horror, joy, panic, and sadness. Fear is closely related to, but should be distinguished from, the emotion anxiety, which occurs as the result of threats that are perceived to be uncontrollable or unavoidable. The fear response serves survival by generating appropriate behavioral responses, so it has been preserved throughout evolution. [9] Cortisol and Epinephrine Cortisol is a steroid hormone, in the glucocorticoid class of hormones. It is produced in humans by the zona fasciculata of the adrenal cortex within the adrenal gland. [10] It is released in response to stress and low blood-glucose concentration. It functions to increase blood sugar through gluconeogenesis, to suppress the immune system, and to aid in the metabolism of fat, protein, and carbohydrates. [11] Epinephrine, also known as adrenalin or adrenaline, is a hormone, neurotransmitter, and medication. [12,13] Epinephrine is normally produced by both the adrenal glands and certain neurons. It plays an important role in the fight-or-flight response by increasing blood flow to muscles, output of the heart, pupil dilation, and blood sugar. [14,15] It does this by binding to alpha and beta receptors. It is found in many animals and some single cell organisms. [16,17] The Immune System, Cytokines, and Inflammation mounting a rapid innate immune system and inflammatory response to a specific trigger and then down-regulating the response once a pathogen has been cleared are critical for resolving infection, repairing tissue damage, and returning the body to a state of homeostasis (Kushner, 1982; Medzhitov, 2008). Recently, however, evidence has accumulated showing that when activation of the inflammatory response is altered or prolonged, it can actually cause more damage to a host than the pathogen itself (Barton, 2008). Indeed, it is now widely recognized that chronic inflammation plays a role in several major diseases including asthma, arthritis, 35

36. diabetes, obesity, atherosclerosis, certain cancers, and Alzheimer’s disease (Couzin- Frankel, 2010). One factor that can alter adaptive innate immune system responding and prolong inflammation is stress (Segerstrom & Miller, 2004; Steptoe et al., 2007). In this review, therefore, we consider how stress influences the regulation of inflammation in a way that may be relevant for depression. the immune system plays a critical role in keeping the body biologically healthy, especially during times of physical injury, wounding, and infection. A key component of this system is the inflammatory response, which is mediated by pro- and anti-inflammatory cytokines that identify, neutralize, and eliminate foreign pathogens such as bacteria and viruses. Inflammation is regulated most proximally by the expression of immune response genes including IL1B, IL6, and TNF. When activated, these genes promote the secretion of pro-inflammatory cytokines that mediate systemic inflammation. Inflammation is also regulated more distally by processes occurring in the brain, which detects social-environmental cues indicating possible danger. This neuro-inflammatory link is highly adaptive insofar as it can activate the CTRA before a physical injury or bacterial infection takes place. A downside of central regulation of systemic inflammation, however, is that it gives social, symbolic, and anticipated threats—including those that have not yet happened or that may never actually occur—the ability to activate the CTRA in the absence of actual physical threat. Under normal conditions, the SNS up-regulates CTRA-related inflammatory activity via stimulation of β-adrenergic receptors, and the HPA axis downregulates CTRA-related inflammatory activity via the production of cortisol. However, under conditions of prolonged actual or perceived threat, or possibly during acute stressors indicating social threat or physical danger, glucocorticoid resistance can develop, leading to excessive inflammation that increases a person’s risk for several disorders including depression, especially if activation of these pathways is prolonged. MATERIALS AND METHODS Cortisol can weaken the activity of the immune system. It prevents proliferation of T-cells by rendering the interleukin-2 producer T-cells unresponsive to interleukin- 1 (IL-1), and unable to produce the T-cell growth factor (IL-2). Cortisol also has a negative-feedback effect on interleukin-1. [18] 36

37. Though IL-1 is useful in combating some diseases, endotoxic bacteria have gained an advantage by forcing the hypothalamus to increase cortisol levels (forcing the secretion of corticotropin-releasing hormone, thus antagonizing IL-1). The suppressor cells are not affected by glucosteroid response-modifying factor, [19] so the effective setpoint for the immune cells may be even higher than the setpoint for physiological processes (reflecting leukocyte redistribution to lymph nodes, bone marrow, and skin). Rapid administration of corticosterone (the endogenous type I and type II receptor agonist) or RU28362 (a specific type II receptor agonist) to adrenalectomized animals induced changes in leukocyte distribution. Natural killer cells are affected by cortisol. [20] Cortisol stimulates many copper enzymes (often to 50% of their total potential), probably to increase copper availability for immune purposes. This includes lysyl oxidase, an enzyme that cross-links collagen, and elastin. Especially valuable for immune response is cortisol's stimulation of the superoxide dismutase, [21] since this copper enzyme is almost certainly used by the body to permit superoxides to poison bacteria. Cortisol counteracts insulin, contributes to hyperglycemia-causing hepatic gluconeogenesis[22] and inhibits the peripheral use of glucose (insulin resistance) by decreasing the translocation of glucose transporters (especially GLUT4) to the cell membrane. [23] However, cortisol increases glycogen synthesis (glycogenesis) in the liver. [24] The permissive effect of cortisol on insulin action in liver glycogenesis is observed in hepatocyte culture in the laboratory, although the mechanism for this is unknown. [25,26] As a hormone, epinephrine acts on nearly all body tissues. Its actions vary by tissue type and tissue expression of adrenergic receptors. For example, high levels of epinephrine causes smooth muscle relaxation in the airways but causes contraction of the smooth muscle that lines most arterioles. Epinephrine acts by binding to a variety of adrenergic receptors. Epinephrine is a nonselective agonist of all adrenergic receptors, including the major subtypes α1, α2, β1, β2, and β3. [27] Epinephrine's binding to these receptors triggers a number of metabolic changes. Binding to α-adrenergic receptors inhibits insulin secretion by the pancreas, stimulates glycogenolysis in the liver and muscle, [28] and stimulates glycolysis and inhibits insulin-mediated glycogenesis in muscle. [29,30] β adrenergic receptor binding triggers glucagon secretion in the pancreas, increased adrenocorticotropic hormone (ACTH) secretion by the pituitary gland, and increased lipolysis by adipose tissue. Together, these effects lead to increased blood glucose 37

38. and fatty acids, providing substrates for energy production within cells throughout the body. [31] Its actions are to increase peripheral resistance via α1 receptor-dependent vasoconstriction and to increase cardiac output via its binding to β1 receptors. The goal of reducing peripheral circulation is to increase coronary and cerebral perfusion pressures and therefore increase oxygen exchange at the cellular level. While epinephrine does increase aortic, cerebral, and carotid circulation pressure, it lowers carotid blood flow and end-tidal CO2 or ETCO2 levels. It appears that epinephrine may be improving macrocirculation at the expense of the capillary beds where actual perfusion is taking place. [32] George M. Slavich and Michael R. Irwin in 2014 concluded that we know a lot about the adverse social-environmental conditions that typically precipitate depression and about cognitive and emotional processes that mediate these effects. With the advent of new neuroimaging, immunological, and genome-wide profiling techniques, we are now poised to go one step deeper and elucidate the full set of biological mechanisms that link stress with depression. Inflammation is undoubtedly a key player in this link. As we have discussed, two general phenomena are consistent with the hypothesis that stress-related increases in inflammation are involved in depression. First, a large number of naturalistic and laboratory-based experimental studies have shown that stress is a potent activator of inflammation, and second, it is now well known that vaccinations and immunological challenges that up-regulate inflammatory activity evoke depressive-like behaviors in rodents and clinically significant episodes of depression in at least some people. In addition, these challenges have been shown to up-regulate peripheral and central cytokine production and to alter metabolic and neural activity in brain regions that have been implicated in depression. Many questions remain unanswered regarding these effects, including whether inflammation is necessary or sufficient for all cases of MDD. Nonetheless, based on existing data, we conclude that stress likely increases risk for depression in a substantial number of people by up-regulating inflammatory activity and by altering social, cognitive, and affective processes that are known to promote this disorder. These insights are important because they can help update contemporary theories of depression with information about biological mechanisms that are involved in the pathogenesis of MDD. For the potential of these insights to be fully realized, they will need to be translated into new strategies for modifying processes that promote depression (Sanislow et al., 2010). At a very general level, such processes include neurocognitive mechanisms like negative cognitive 38

39. appraisals and neural sensitivity to social threat, which have been associated with inflammation and depression (Masten et al., 2011; Monroe, Slavich, Torres, & Gotlib, 2007b; Rueggeberg, Wrosch, Miller, & McDade, 2012); immunological processes such as preclinical levels of inflammation, which could presage the development of chronic inflammation and disease (G. E. Miller et al., 2011); and psychosocial factors such as parental behaviors, which have been found to influence the effects of social-environmental adversity on proinflammatory signaling (Chen, Miller, Kobor, & Cole, 2011; see also Carroll et al., 2013). The hope is that by targeting these and other dynamics, we may one day be able to reduce the prevalence of depression and the substantial financial burden and personal suffering associated with this common and costly disorder. [33] Sheldon Cohen et al. in 2012 did a research about stress and disease promotion. In Cohen's first study, after completing an intensive stress interview, 276 healthy adults were exposed to a virus that causes the common cold and monitored in quarantine for five days for signs of infection and illness. Here, Cohen found that experiencing a prolonged stressful event was associated with the inability of immune cells to respond to hormonal signals that normally regulate inflammation. In turn, those with the inability to regulate the inflammatory response were more likely to develop colds when exposed to the virus. In the second study, 79 healthy participants were assessed for their ability to regulate the inflammatory response and then exposed to a cold virus and monitored for the production of pro-inflammatory cytokines, the chemical messengers that trigger inflammation. He found that those who were less able to regulate the inflammatory response as assessed before being exposed to the virus produced more of these inflammation-inducing chemical messengers when they were infected. The immune system's ability to regulate inflammation predicts who will develop a cold, but more importantly it provides an explanation of how stress can promote disease, when under stress, cells of the immune system are unable to respond to hormonal control, and consequently, produce levels of inflammation that promote disease. Because inflammation plays a role in many diseases such as cardiovascular, asthma and autoimmune disorders, this model suggests why stress impacts them as well. [34] Michopoulos V. et al. in 2004 concluded that the study of inflammation in fear- and anxiety-based disorders has gained interest as growing literature indicates that pro- inflammatory markers can directly modulate affective behavior. Indeed, heightened concentrations of inflammatory signals, including cytokines and C-reactive protein, have been described in posttraumatic stress disorder (PTSD), generalized anxiety disorder (GAD), panic disorder (PD), and phobias (agoraphobia, social phobia, etc.). 39

40. However, not all reports indicate a positive association between inflammation and fear- and anxiety-based symptoms, suggesting that other factors are important in future assessments of inflammation's role in the maintenance of these disorders (ie, sex, co-morbid conditions, types of trauma exposure, and behavioral sources of inflammation). The most parsimonious explanation of increased inflammation in PTSD, GAD, PD, and phobias is via the activation of the stress response and central and peripheral immune cells to release cytokines. Dysregulation of the stress axis in the face of increased sympathetic tone and decreased parasympathetic activity characteristic of anxiety disorders could further augment inflammation and contribute to increased symptoms by having direct effects on brain regions critical for the regulation of fear and anxiety (such as the prefrontal cortex, insula, amygdala, and hippocampus). Taken together, the available data suggest that targeting inflammation may serve as a potential therapeutic target for treating these fear- and anxiety-based disorders in the future. However, the field must continue to characterize the specific role pro-inflammatory signaling in the maintenance of these unique psychiatric conditions. [35] Based on evidence that psychological stress may induce a chronic inflammatory process, we hypothesized that the stress caused by chronic fear of terror may be associated with low-grade inflammation. This hypothesis was examined in employed men and women with the presence of low-grade inflammation measured by high sensitivity C-reactive protein (CRP). Apparently healthy employed adults (N = 1153) undergoing periodic health check-ups in a tertiary hospital in Israel completed a questionnaire. Fear of terror (scored 1-5) was assessed by three items measuring the extent to which respondents have deep concern for personal safety, elevated tension in crowded places, and fear of terror strikes causing harm to one's self or one's family members. The main outcome measure was the presence or absence of an elevated CRP level (>3.0 mg/L). Women scored significantly higher on fear of terror compared with men (M = 2.16 vs. M = 1.68, respectively; p <.0001). Most of the study participants who scored high (4 or 5) on fear of terror, reported having experienced this feeling for 1 year or more. In women only, there was a positive association between fear of terror and risk of elevated CRP level (adjusted OR = 1.7, 95% CI 1.2-2.4) in a multivariate model adjusting for generalized anxiety, depressive symptoms, and potentially confounding demographic and biomedical variables. Chronic fear of terror in women, but not in men, is associated with elevated CRP levels, which suggests the presence of low-grade inflammation and a potential risk of cardiovascular disease. [36] Vignes M. et al. and Bouayed J, et al. summarized the data to support a link between oxidative stress and anxiety. While all of the data demonstrate that there is a link 40

41. between oxidative stress and high-anxiety-related behavior, a cause-effect relationship has yet to be completely established. Some of these studies suggest that oxidative stress causes anxiety-related behaviors but do not explain the underlying mechanisms. While there are some limits in the approach to establish the anxiogenic effect of oxidative stress, the available data are consistent this causal relationship. The potential causal role of oxidative stress on anxiety may generate interest in antioxidants. Masood et al. were able to show that oxidative stress-related anxiety can be reversed in mice upon inhibition of NADPH oxidase or phosphodiesterase-2, enzyme that is indirectly implicated in oxidative stress mechanisms. Surprisingly, they found that diazepam, which is a well-known anxiolytic, does not fully reverse the oxidative stress-related anxiety. These results point to a possible use for antioxidants in the prevention or reduction of high anxiety. Recent work has shown that some dietary polyphenols have both anxiolytic and antioxidant effects, which may be beneficial to anxious subjects. [37,38] It is well known that low/moderate concentrations of reactive oxygen species (ROS) affect a great number of physiological functions. [39] However, when ROS concentration exceeds the anti- oxidative capacity of an organism, animal cells enter a state termed oxidative stress, in which the excess ROS induces oxidative damage on cellular components. [40] As a result, oxidative stress has been implicated in a large range of diseases, including cancer. [41,42] The brain is highly vulnerable to oxidative stress due to its high O2 consumption, its modest antioxidant defenses and its lipid-rich constitution. [43,44] Human brain utilizes 20% of oxygen consumed by the body even though this organ constitutes only about 2% of the body weight. [45] When the production of oxygen- derived metabolites prevails over the brain defense systems, however, oxidative damage to nucleic acids, proteins and neuronal membrane lipids, which are rich in highly polyunsaturated fatty acids, can occur. [46] In presence of oxidative stress, the lipid-rich constitution of brain favors lipid peroxidation that results in decrease in membrane fluidity and damage in membrane proteins inactivating receptors, enzymes and ion channels. As a result, oxidative stress can alter neurotransmission, neuronal function and overall brain activity. [47] Oxidative stress has been associated with several diseases which are specific for nervous system impairment including neurodegenerative diseases and neuropsychiatric diseases, such as schizophrenia and major depressive disorder. [42-44] The intrinsic oxidative vulnerability of the brain has led some authors to suggest that oxidative damage may be a plausible pathogenic factor for certain neurological diseases including neuropsychiatric disorders. [40-46] 41

42. Stress and Illness including Cancer There is likely a connection between stress and illness. Theories of the stress–illness link suggest that both acute and chronic stress can cause illness, and several studies found such a link. [48] According to these theories, both kinds of stress can lead to changes in behavior and in physiology. Behavioral changes can be smoking and eating habits and physical activity. Physiological changes can be changes in sympathetic activation or hypothalamic pituitary adrenocorticoid activation, and immunological function. [49] However, there is much variability in the link between stress and illness. [50] Stress can make the individual more susceptible to physical illnesses like the common cold. [51] Stressful events, such as job changes, may result in insomnia, impaired sleeping, and health complaints. [52] Research indicates the type of stressor (whether it's acute or chronic) and individual characteristics such as age and physical well-being before the onset of the stressor can combine to determine the effect of stress on an individual. An individual's personality characteristics (such as level of neuroticism), [53] genetics, and childhood experiences with major stressors and traumas [54] may also dictate their response to stressors. Chronic stress and a lack of coping resources available or used by an individual can often lead to the development as depression and anxiety . [55] This is particularly true regarding chronic stressors. These are stressors that may not be as intense as an acute stressor like a natural disaster or a major accident, but they persist over longer periods of time. These types of stressors tend to have a more negative impact on health because they are sustained and thus require the body's physiological response to occur daily. This depletes the body's energy more quickly and usually occurs over long periods of time, especially when these microstressors cannot be avoided (i.e. stress of living in a dangerous neighborhood). See allostatic load for further discussion of the biological process by which chronic stress may affect the body. For example, studies have found that caregivers, particularly those of dementia patients, have higher levels of depression and slightly worse physical health than noncaregivers. [56] Studies have also shown that perceived chronic stress and the hostility associated with Type A personalities are often associated with much higher risks of cardiovascular disease. This occurs because of the compromised immune system as well as the high levels of arousal in the sympathetic nervous system that occur as part of the body's physiological response to stressful events. [57] However, it is possible for individuals to exhibit hardiness – a term referring to the ability to be both chronically stressed and healthy. [58] Many psychologists are currently of psychological issues such 42

43. interested in studying the factors that allow hardy individuals to cope with stress and evade most health and illness problems associated with high levels of stress. Stress can be associated with psychological disorders such as delusions, [59] general anxiety disorder, depression, and post-traumatic stress disorder. However, everyone experiences some level of stress, and diagnosis of stress disorders can only be performed by a licensed practitioner. According to a 2016 review article, pathological anxiety and chronic stress lead to structural degeneration and impaired functioning of the hippocampus. [60] It has long been believed that negative affective states, such as feelings of anxiety and depression, could influence the pathogenesis of physical disease, which in turn, have direct effects on biological process that could result in increased risk of disease in the end. However, studies done by the University of Wisconsin-Madison and other places have shown this to be partly untrue; although stress seems to increase the risk of reported poor health, the perception that stress is harmful increases the risk even further. [61,62] For example, when humans are under chronic stress, permanent changes in their physiological, emotional, and behavioral responses are most likely to occur. [63] Such changes could lead to disease. Chronic stress results from stressful events that persist over a relatively long period of time, such as caring for a spouse with dementia, or results from brief focal events that continue to be experienced as overwhelming even long after they are over, such as experiencing a sexual assault. Experiments show that when healthy human individuals are exposed to acute laboratory stressors, they show an adaptive enhancement of some markers of natural immunity but a general suppression of functions of specific immunity. By comparison, when healthy human individuals are exposed to real-life chronic stress, this stress is associated with a biphasic immune response where partial suppression of cellular and humoral function coincides with low-grade, nonspecific inflammation. Even though psychological stress is often connected with illness or disease, most healthy individuals can still remain disease-free after confronting chronic stressful events. Also, people who do not believe that stress will affect their health do not have an increased risk of illness, disease, or death. This suggests that there are individual differences in vulnerability to the potential pathogenic effects of stress; individual differences in vulnerability arise due to both genetic and psychological factors. In addition, the age at which the stress is experienced can dictate its effect on health. Research suggests chronic stress at a young age can have lifelong impacts on the biological, psychological, and behavioral responses to stress later in life. [64] As stress has a physical effect on the body, some individuals may not distinguish this from other more serious illnesses. Individuals experiencing stress are less likely 43

44. to see medical care for a symptom if the symptom is ambiguous (e.g. headache) and they are currently experiencing stress. If the symptom is unambiguous however (e.g. a breast lump), and the onset of the stressor is recent, individuals are motivated to seek care as usual. [65] In animals, stress contributes to the initiation, growth, and metastasis of select tumors, but studies that try to link stress and cancer incidence in humans have had mixed results. This can be due to practical difficulties in designing and implementing adequate studies. [66] DISCUSSION Fear hormones are secreted by the adrenal gland, an endocrine gland located on top of the kidneys. [67] The fear hormones circulate through the bloodstream to all cells of the body. [68] The effect of adrenaline is similar to the effect of the sympathetic nerve action. [69] Adrenaline increases heart rate, increases breathing rate, dilates blood vessels to the lungs and muscles. [70] Adrenaline also decreases blood flow to the brain and decreases digestion. Cortisol increases blood sugar level by converting stored glycogen and fats into blood sugar. Cortisol also suppresses the immune response and inflammation. Fear hormones result in a longer lasting and more widespread fight‐or‐flight response than the effects of the nervous system. [71- 73] Fear hormone action explains why one may feel the fight‐or‐flight response even after he/she realize there really is no danger. Daily life can involve many stimuli that are perceived as threatening. [74-76] Problems at work or at school, money or social problems, and medical problems can trigger a chronic (long term) fight‐or‐flight response. Even anticipating or worrying about things that might happen in the future can trigger the same response as actually experiencing it. Chronic stress occurs when the fight‐or‐flight response does not shut down to allow for the proper balance between fear and relaxation. Stress can increase a person’s risk of health problems. [77-80] The fight‐or‐flight response uses calories so the urge to eat makes sense after running. But, eating in response to daily stresses can lead to weight gain and obesity. In addition, stress increases cortisol levels causing elevated blood sugar levels that can lead to both weight gain and diabetes. When the fight‐or‐flight response causes blood pressure and heart rate to remain high, it puts extra strain on blood vessel walls. As a result, the linings of blood vessels can become damaged and the amounts of oxygen in blood can become lesser than normal. [81,82] An interruption of blood flow to the heart can lead to a heart attack. Blood vessels in the brain can also be blocked, resulting in brain‐damaging strokes. People suffering from stress secrete cortisol at much higher rates than normal people. There is evidence that abnormally high cortisol levels may actually be the initial trigger for 44

45. depression in some individuals. High cortisol levels also result in sleep deprivation. Stress also affects the function of the immune system, the body’s natural means of fighting off infection. Stressed individuals produce lower levels of antibodies when exposed to pathogens. They also produce higher levels of cytokines, inflammation triggering chemicals secreted when fighting infections. Excessive inflammation is thought to increase the risks for heart disease, diabetes, and cancer. Feeling stressed mentally and physically may have serious health consequences. [83,84] Organ Effects Heart Increases heart rate; contractility; conduction across AV node Lungs Increases respiratory rate (Hypocapnia); bronchodilation Systemic Vasoconstriction and vasodilation Liver Stimulates glycogenolysis Systemic Triggers lipolysis Brain Decreasing Blood Flow to the Brain Table (1): Physiologic responses to epinephrine by organ a large literature exists demonstrating that major life events, especially those involving interpersonal loss and social rejection, are a key proximal risk factor for MDD. As it turns out, these stressors have been implicated not just in the development of depression but in the onset, exacerbation, or progression of a variety of health problems. These conditions include several that, like depression, are thought (or known) to be mediated at least in part by inflammation, such as asthma, rheumatoid arthritis, cardiovascular disease, chronic pain, and certain cancers (Bower, Crosswell, & Slavich, 2014; Chrousos, 2009; Cohen, Janicki-Deverts, & 45

46. Miller, 2007; Cutolo & Straub, 2006; Kivimäki et al., 2006; Loeser & Melzack, 1999; Lutgendorf et al., 2012, 2013; A. H. Miller, 2008; Reiche, Nunes, & Morimoto, 2004; Steptoe & Kivimäki, 2012; Walker, Littlejohn, McMurray, & Cutolo, 1999; R. J. Wright, 2011). As a result, we turn now to the question of whether stress is associated with inflammation. 46

47. CHAPTER FOUR THE IMPACT OF SEROTONIN IN THE CAUSE AND TREATMENT OF CANCER INTRODUCTION Carcinoid syndrome Carcinoid syndrome is a paraneoplastic syndrome comprising the signs and symptoms that occur secondary to carcinoid tumors. The syndrome includes flushing and diarrhea, and less frequently, heart failure, emesis and bronchoconstriction. [1] It is caused by endogenous secretion of mainly serotonin and kallikrein. The carcinoid syndrome occurs in approximately 5% of carcinoid tumors [2] and becomes manifest when vasoactive substances from the tumors enter the systemic circulation escaping hepatic degradation. Interestingly, if the primary tumor is from the GI tract (hence releasing serotonin into the hepatic portal circulation), carcinoid syndrome generally does not occur until the disease is so advanced that it overwhelms the liver's ability to metabolize the released serotonin. [3] Carcinoid tumors produce the vasoactive substance, serotonin. [4] It is commonly, but incorrectly, thought that serotonin is the cause of the flushing. The flushing results from secretion of kallikrein, the enzyme that catalyzes the conversion of kininogen to lysyl-bradykinin. The latter is further converted to bradykinin, one of the most powerful vasodilators known. Other components of the carcinoid syndrome are diarrhea (probably caused by the increased serotonin, which greatly increases peristalsis, leaving less time for fluid absorption), [5] a pellagra-like syndrome (probably caused by diversion of large amounts of tryptophan from synthesis of the vitamin B3 niacin, which is needed for NAD production, to the synthesis of serotonin and other 5-hydroxyindoles), fibrotic lesions of the endocardium, particularly on the right side of the heart resulting in insufficiency of the tricuspid valve and, less frequently, the pulmonary valve and, uncommonly, bronchoconstriction. [6] The pathogenesis of the cardiac lesions and the bronchoconstriction is unknown, but the former probably involves activation of serotonin 5-HT2B receptors by serotonin. [7] When the primary tumor is in the gastrointestinal tract, as it is in the great majority of cases, the serotonin and kallikrein are inactivated in the liver; manifestations of carcinoid syndrome do not occur until there are metastases to the liver or when the 47

48. cancer is accompanied by liver failure (cirrhosis). [8] Carcinoid tumors arising in the bronchi may be associated with manifestations of carcinoid syndrome without liver metastases because their biologically active products reach the systemic circulation before passing through the liver and being metabolized. In most patients, there is an increased urinary excretion of 5-HIAA (5-hydroxyindoleacetic acid), a degradation product of serotonin. The biology of these tumors is interesting as it differs from many other tumor types. Ongoing research on the biology of these tumors may reveal new mechanisms for tumor development. [9] [10] [11] Localization of tumors Tumor localization may be extremely difficult. Barium swallow and follow-up examination of the intestine may occasionally show the tumor. Capsule video endoscopy has recently been used to localize the tumor. Often laparotomy is the definitive way to localize the tumor. Another form of localizing a tumor is the Octreo-scan. A tracer agent of Indium 111 is injected into a vein where then the tumors absorb the radionuclide Indium 111 and become visible on the scanner. Only the tumors absorb the somatostatin agent Indium 111 making the scan highly effective. [12] [13] Disease progression is difficult to ascertain because the disease can metastasize anywhere in the body and can be too small to identify with any current technology. Markers of the condition such as chromo-granin-A are imperfect indicators of disease progression. Prognosis varies from individual to individual. It ranges from a 95% 5-year survival for localized disease to an 80% 5-year survival for those with liver metastases. The average survival time from the start of octreotide treatment has increased to about 12 years. [14] [15] [16] Serotonin Serotonin or 5-hydroxytryptamine (5-HT) is a monoamine neurotransmitter. Biochemically derived from tryptophan,[17] serotonin is primarily found in the gastrointestinal tract (GI tract), blood platelets, and the central nervous system (CNS) of animals, including humans. It is popularly thought to be a contributor to feelings of well-being and happiness. [18] Approximately 90% of the human body's total serotonin is located in the enterochromaffin cells in the GI tract, where it is used to regulate intestinal movements. [19] [20] 48

49. The serotonin is secreted luminally and basolaterally which leads to increased serotonin uptake by circulating platelets and activation after stimulation, [21] [22] which gives increased stimulation of myenteric neurons and gastrointestinal motility. The remainder is synthesized in serotonergic neurons of the CNS, where it has various functions. [23] These include the regulation of mood, appetite, and sleep. [24] Serotonin also has some cognitive functions, including memory and learning. [25] Modulation of serotonin at synapses is thought to be a major action of several classes of pharmacological antidepressants.Serotonin secreted from the enterochromaffin cells eventually finds its way out of tissues into the blood. [26] There, it is actively taken up by blood platelets, which store it. When the platelets bind to a clot, they release serotonin, where it serves as a vasoconstrictor and helps to regulate hemostasis and blood clotting. Serotonin is also a growth factor for some types of cells, which may give it a role in wound healing. [27] There are various serotonin receptors.Serotonin is metabolized mainly to 5-HIAA, chiefly by the liver. Metabolism involves first oxidation by monoamine oxidase to the corresponding aldehyde. This is followed by oxidation by aldehyde dehydrogenase to 5-HIAA, the indole acetic acid derivative. [28] The latter is then excreted by the kidneys.In addition to animals, serotonin is found in fungi and plants. Serotonin's presence in insect venoms and plant spines serves to cause pain, which is a side-effect of serotonin injection. [29] Serotonin is produced by pathogenic amoebae, and its effect on the gut causes diarrhea. Its widespread presence in many seeds and fruits may serve to stimulate the digestive tract into expelling the seeds. [30] Cellular effects of serotonin The 5-HT receptors, the receptors for serotonin, are located on the cell membrane of nerve cells and other cell types in animals, and mediate the effects of serotonin as the endogenous ligand and of a broad range of pharmaceutical and hallucinogenic drugs. Except for the 5-HT3 receptor, a ligand-gated ion channel, all other 5-HT receptors are G-protein-coupled receptors (also called seven-transmembrane, or heptahelical receptors) that activate an intracellular second messenger cascade. [31] [32] [33] Serotonergic action is terminated primarily via uptake of 5-HT from the synapse. This is accomplished through the specific monoamine transporter for 5-HT, SERT, on the presynaptic neuron. Various agents can inhibit 5-HT reuptake, including cocaine, dextromethorphan (an antitussive), tricyclic antidepressants and selective serotonin reuptake inhibitors (SSRIs). A 2006 study conducted by the University of Washington suggested that a newly discovered monoamine transporter, known as PMAT, may account for a significant percentage of 5-HT clearance. [34] [35] 49

50. Contrasting with the high-affinity SERT, the PMAT has been identified as a low- affinity transporter, with an apparent Km of 114 micromoles/l for serotonin; approximately 230 times higher than that of SERT. [36] However, the PMAT, despite its relatively low serotonergic affinity, has a considerably higher transport 'capacity' than SERT, "resulting in roughly comparable uptake efficiencies to SERT in heterologous expression systems." [37] The study also suggests some SSRIs, such as fluoxetine and sertraline anti-depressants, inhibit PMAT but at IC50 values which surpass the therapeutic plasma concentrations by up to four orders of magnitude. [38] Therefore, SSRI monotherapy is "ineffective" in PMAT inhibition. At present, no known pharmaceuticals are known to appreciably inhibit PMAT at normal therapeutic doses. The PMAT also suggestively transports dopamine and norepinephrine, albeit at Km values even higher than that of 5-HT (330–15,000 μ- moles/L). [39] [40] [41] Biochemical mechanisms In animals including humans, serotonin is synthesized from the amino acid L- tryptophan by a short metabolic pathway consisting of three enzymes: tryptophan hydroxylase (TPH), aromatic amino acid decarboxylase (DDC) and pyridoxal phosphate. The TPH-mediated reaction is the rate-limiting step in the pathway. TPH has been shown to exist in two forms: TPH1, found in several tissues, and TPH2, which is a neuron-specific isoform. [42] [43] Serotonin can be synthesized from tryptophan in the lab using Aspergillus niger and Psilocybe coprophila as catalysts. The first phase to 5-hydroxytryptophan would require letting tryptophan sit in ethanol and water for 7 days, then mixing in enough HCl (or other acid) to bring the pH to 3, and then adding NaOH to make a pH of 13 for 1 hour. Asperigillus niger would be the catalyst for this first phase. The second phase to synthesizing tryptophan itself from the 5-hydroxytryptophan intermediate would require adding ethanol and water, and letting sit for 30 days this time. The next two steps would be the same as the first phase: adding HCl to make the pH = 3, and then adding NaOH to make the pH very basic at 13 for 1 hour. This phase uses the Psilocybe coprophila as the catalyst for the reaction. [44] Serotonin taken orally does not pass into the serotonergic pathways of the central nervous system, because it does not cross the blood–brain barrier. [45] However, tryptophan and its metabolite 5-hydroxytryptophan (5-HTP), from which serotonin is synthesized, does cross the blood–brain barrier. These agents are available as dietary supplements, and may be effective serotonergic agents. One product of serotonin breakdown is 5- hydroxyindoleacetic acid (5-HIAA), which is excreted in the urine. Serotonin and 5- 50