THE IMPACT OF IYENGAR YOGA ON DEMANDS OF ILLNESS, COPING, AND. LYMPHOCYTE NF-κB ACTIVATION IN BREAST CANCER SURVIVORS

Transcription

1 THE IMPACT OF IYENGAR YOGA ON DEMANDS OF ILLNESS, COPING, AND LYMPHOCYTE NF-κB ACTIVATION IN BREAST CANCER SURVIVORS By PAMELA ELLEN SCHULTZ A thesis submitte in partial fulfillment of the requirements for the egree of MASTER OF SCIENCE IN EXERCISE SCIENCE WASHINGTON STATE UNIVERSITY SPOKANE Clinical an Experimental Exercise Science Grauate Program DECEMBER 2007

2 To the Faculty of Washington State University: The members of the Committee appointe to examine the thesis of PAMELA ELLEN SCHULTZ fin it satisfactory an recommen that it be accepte. Chair ii

3 ACKNOWLEDGEMENTS There are numerous iniviuals who have helpe make this research project possible an bring it to completion. First an foremost, I woul like to thank my avisor, Sally Blank, for the opportunity to be a part of this research project. I am grateful for the unening challenges that presente themselves, what I have learne in the process, an how I have grown both intellectually an professionally. Your guiance an mentorship has helpe mol the person I am prou to be toay. Thank you. I woul also like to thank my committee members Mel Haberman an Kenn Daratha. Your eication an support through this entire process has been increible. I have enjoye exploring an integrating into my final project, the psychosocial aspects of cancer survivorship an various ata mining techniques. I coul not have foun a better thesis committee to learn from. Thank you. I woul like to thank my fellow grauate stuents along with the many iniviuals in the Health Sciences Builing who so willingly an consistently provie me with support an encouragement to o my best work possible. An finally, Mo, Jui, Nicole, an my entire fabulous family my success is because you are a part of my life. Thank you. iii

4 THE IMPACT OF IYENGAR YOGA PRACTICE ON DEMANDS OF ILLNESS, COPING, LYMPHOCYTE NF-κB ACTIVATION IN BREAST CANCER SURVIVORS Abstract by Pamela Ellen Schultz, M.S. Washington State University Spokane December 2007 Chair: Sally E. Blank Breast cancer survivorship is associate with changes in physical, psychological, an social aspects of well-being. Such changes may require significant cognitive an emotional aaptation. Coping strategies whether positive or negative may preict how well a survivor is able to aapt an may affect how their boy respons to the illness. Integrative meicine often incorporates min-boy therapies, such as yoga, to increase overall wellbeing an physical health in breast cancer survivors. The present stuy was esigne to investigate the relationship between Iyengar yoga practice, illness-relate stressors an lymphocyte NF-κB activation in female breast cancer survivors. Females who were approximately 1 to 11 years post-iagnosis with stage I-IV breast cancer were ranomly assigne to either the yoga (Y, n = 13) or control (C, n = 11) group. The eightweek yoga intervention inclue three yoga sessions per week. Participants complete a Demans of Illness Inventory (DOII) an Revise Ways of Coping Checklist (RWCCL) prior to (T1) an following (T2) the intervention. Bloo samples were taken at T1 an T2 for use in in vitro unstimulate an stimulate lymphocyte NF-κB analyses. iv

5 Lymphocytes were stimulate with phytohemaglutinin (PHA), phorbol 12-myristate 12- acetate (PMA), an ionomycin. Nuclear extracts of stimulate cells were analyze for NF-κB activation by an electrophoretic mobility shift assay. Cytosolic an nuclear extracts from unstimulate an stimulate paire aliquots were analyze for cellular location of NF-κB by chemiluminescence. Data were analyze by inepenent an paire t-tests, an linear regression. A global measure of the DOII reveale a significant interaction between time an group effects (F = 5.275, p = 0.028) revealing ecrease DOII in the yoga group from T1 to T2. DOII was inversely correlate with overall active coping strategies (r = -0.23, p = 0.05) an count your blessings (r = -0.51, p <0.000). Yoga participation was associate with a tren for ecrease lymphocyte activation (p = 0.077). The ecrease change in lymphocyte NF-κB activation was correlate with increase use of passive coping strategies (r = 0.900, p = 0.03). The results support the hypothesis that yoga participation meiates perceive emans of illness associate with breast cancer survivorship an that lymphocyte NF-κB signaling pathways are involve in physiological changes that enhance psychosocial well-being. Key Wors: breast cancer, emans of illness, coping, yoga, integrative meicine, immune function, lymphocytes, NF-κB v

6 TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ABSTRACT LIST OF TABLES LIST OF FIGURES iii iv viii ix CHAPTER 1. INTRODUCTION 1 Statement of the Problem 4 Hypotheses 4 2. REVIEW OF LITERATURE 5 Breast Cancer Survivorship 5 Ajustment an Aaptation 6 Assessing Quality of Life 8 Integrative Cancer Care 10 Yoga Interventions for Cancer Survivors 11 Introuction to the Immune Response an Cancer 14 Nuclear Factor kappa B 16 Cell Signaling Pathways from Mitogen Stimulation 20 Gene Expression an NF-κB Regulation 23 Relevant Biological Assays 25 Summary MATERIALS AND METHODS 28

7 Sample Population 28 Lymphocyte Samples 30 Thawing an Washing Proceures 30 Cytosolic Extraction (Proceure A) 31 Nuclear Extraction (Proceure B) 32 Protein Extractions (Proceure C) 33 Dialysis of Samples 34 Protein Concentration 34 Determining Protein Concentration 35 Determination of NF-κB Activation 36 Determination of cellular NF-κB location 38 Psychosocial/Quality of Life Assessment 39 Statistical Analyses 40 REFERENCES 41 APPENDIX A. MANUSCRIPT 46 Title 47 Abstract 48 Introuction 50 Materials an Methos 52 Results 57 Discussion 59 Conclusions 63 vii

8 References 65 B. TABLES AND FIGURES 68 C. SELF ORGANIZING MAPS 78 viii

9 LIST OF TABLES Page Table 1. Demographic Characteristics of Intervention Groups 68 Table 2. Change in DOII from Pre- to Post-Intervention 69 Table 3. Frequency of Coping Strategies Pre- an Post-Intervention 70 ix

10 LIST OF FIGURES Page Figure 1. Interaction between Time an Group for Present DOI 72 Figure 2. EMSA for NF-κB etection 73 Figure 3. Activate Lymphocyte NF-κB Activation at T1 an T2 74 Figure 4. Percent Change of NF-κB Activation is correlate with Passive Coping Scores 75 x

11 CHAPTER ONE INTRODUCTION Breast cancer is the most common newly iagnose cancer among woman in the Unite States. 1 As breast cancer survival rates improve a greater number of women have to aapt to an live with the biopsychosocial impacts of cancer survivorship. Breast cancer inclues many psychological an social harships that are irectly relate to iagnosis, treatment, an survivorship. 2-4 Many of the concerns present at time of iagnosis an treatment persist throughout survivorship. 2 The relationships among psychosocial challenges of breast cancer survivorship an isease prognosis an progression remain uncertain. Several moels exist linking breast cancer survivors physical an psychological stress with coping responses For example, emotional an behavioral isengagement, low perceive control an fear of treatments are each associate with negative coping strategies such as avoiance an acceptance-resignation. 5, 9 On the other han, harmony an peace are associate with seeking support an active coping, an inversely associate with avoiance. 9 Ajustment an aaptation to breast cancer inclue processing a wie range of stressors into irecte thoughts an actions that reflect coping strategies. Instruments such as the Revise Ways of Coping Checklist measure the egree that various coping strategies are use by an iniviual Coping strategies are often classifie as active coping (positive in nature) an passive/emotion-focuse coping (more negative mine). 9 It is thought that ajustment through coping meiates the outcome of one s perceive stress level. 1

12 A survivor s quality of life (QOL) reflects their multiimensional perspective an attempt to restore normalcy, achieve wholeness, an re-establish a sense of purpose in life. 15 The Demans of Illness Inventory (DOII) was evelope to assess iniviual an family aaptation to chronic illness. 16, 17 Demans are typically conceptualize as harships or etrimental events that call for some type of coping an aaptive response. The DOII inclues a selection of QOL omains relate to cancer survivorship an a 17, 18 variety of chronic iseases such as breast cancer, iabetes, an colorectal cancer. The significance of QOL in cancer survivorship warrants integration of complementary therapies into mainstream meical care to provie aitional treatment for illness-relate symptoms. Cancer survivors often choose to use complementary therapies to alleviate feelings of istress, an not generally because of issatisfaction with traitional western meicine. 19 Min-boy therapy is a popular integrative moality use to inuce relaxation an improve emotional well-being through healing methos that enhance the min s interactions with boily function. 20 Yoga is regare as a promising metho for the treatment of stress-relate symptoms of chronic isease, incluing cancer because many yogic traitions integrate physical, mental, an spiritual elements within the practice. The results of several pilot stuies support ancient beliefs regaring the health benefits of yoga practice In aition to emotional well-being, yoga may have a positive effect on immune function in breast cancer survivors. Meical treatments an psychological aaptation can significantly influence immune parameters of breast cancer survivors. The immune system functions to maintain homeostasis within the boy. Homeostasis is threatene by cancer initiation, its progression, an meical cancer treatment therapies. Research investigating the role of 2

13 immunity in response to cancer-relate stressors is complicate by iniviual variability; no two iniviuals respon ientically to the same stressor, nor oes a like group of cancer survivors experience the same stressors to the same egree of potency. Nonetheless, it is acknowlege that immune cells play a part in tumor evelopment an isease progression. 24, 25 Innate an aaptive immune cells, incluing lymphocytes, can foster an environment of immunoserveillance an/or immunosubversion via multiple mechanisms incluing the secretion of soluble factors. 26 The transcription factor nuclear factor kappa B (NF-κB) serves as a primary cell signaling pathway an is central to the regulation of iverse cellular immunological processes. 27 NF-κB is a collection of imeric proteins sequestere in the cytoplasm as latent complexes boun to its inhibitor. 28, 29 NF-κB complexes are compose of homoan hetero-imers mae up of RelA/p65, c-rel, RelB, NF-κB1(p50/p105) an NF-κB2 (p52/p100). 28 Each family member contains an N-terminal 300-amino-aci omain (Rel homology omain) that is responsible for imerization an DNA bining. 30 Activation of NF-κB has been calle a sensor for smoke an stress signals as it results from a wie range of stimuli an is involve in gene expression for hunres of genes, incluing certain lymphocyte signaling pathways. 31 Mitogen stimulation is a metho use to bring about lymphocyte proliferation involving activation of NF-κB. In vitro, it is thought mitogen stimulation of lymphocytes mimics the response of certain antigens an is therefore a practical technique to stuy mechanisms involve in 32, 33 lymphocytes activation. 3

14 Statement of the Problem Breast cancer survivors eal with a multitue of stressors that cause many to seek complementary therapies in hopes of increasing their quality of life. Diagnosis, treatment an the many illness-relate stressors can have a cumulative etrimental effect on immune function of survivors. As survivors seek out avice for appropriate complementary therapies, western meicine is only beginning to acknowlege an explore the benefits of these various methos, incluing yoga. The influence of Iyengar yoga practice on immune function is incompletely unerstoo. To ate, the effects of Iyengar yoga practice on the activation of transcription factor NF-κB in lymphocytes from breast cancer survivors is unknown. The purposes of this stuy were: 1) to examine the impact of Iyengar yoga on the activation of NF-κB in PHA/PMA/ionomycin stimulate peripheral bloo lymphocytes from women with stage I-IV breast cancer; 2) to examine the effect of Iyengar yoga on psychosocial stressors, as measure by RWCCL an DOII, in women with stage I-IV breast cancer; 3) to examine associations among NF-κB activation an psychosocial meiators an outcomes as measure by RWCCL an DOII. Hypotheses The hypotheses of this stuy were: as compare with wait-liste controls, breast cancer survivors who participate in yoga will have 1) altere NF-kB activation in stimulate lymphocytes; an 2) increase quality of life with reuce cancer-relate symptoms as etermine by the RWCCL an DOII. 4

15 CHAPTER TWO LITERATURE REVIEW Breast Cancer Survivorship Accoring to the American Cancer Society, a woman in the Unite States has a 12.5% chance (1 in 8) of eveloping breast cancer in her lifetime. 1 Female breast cancer comprises the largest group of cancer survivors an is the most common site for newly iagnose cancer cases among woman in the Unite States. 1 Death rates from breast cancer however have ecline by an average 2.3% per year since 1990, largely attribute to earlier etection through screening, increase awareness, an improve treatment. 1 As survival rates improve however, a greater number of women have to aapt to an live with the biopsychosocial impacts of cancer survivorship. Quality of life (QOL) concerns of breast cancer survivors often fit into one of the following omains: physical, emotional, social, sexual, an spiritual well-being. Although new issues may emerge, long-term cancer survivors are concerne with many of the same issues as at the time of iagnosis an treatment. 2 Breast cancer survivorship inclues experiences such as pain, fatigue, sexual isruption, appearance an boy-image concerns, emotional istress, epression, worries about insurance, worry for the future health of one s chilren, worry of recurrence, late effects of cancer treatment, evelopment of secon cancers, feelings of powerlessness, isappointment, futility, meaninglessness, remorse, eath anxiety, an questions regaring purpose in life. 2-4 The significance of unerstaning QOL an then minimizing the number of stressors that are part of chronic illness was acknowlege early by Hans Selye (mi 5

16 1900 s) in his work efining the effects of stress an the resulting general aaptation synrome (GAS). 34 Selye s GAS is a framework aroun which psychological factors can an o elicit a physiologic stress response in iniviuals with chronic physical illness. 34 Specifically, Selye examine the neuroenocrine effects an conceptualize the boy s response to stress as consisting of three phases relate to aaptation. 35 He propose that ifferent stressor stimuli of equal magnitue potency o not necessarily cause the same synrome in ifferent iniviuals, an that the same egree of stress inuce by the same stimulus may provoke ifferent lesions in ifferent iniviuals. 34 Simply state an applicable to breast cancer, survivors may share organ vulnerability, however each survivor iffers in his/her response to the same biopsychosocial stressor. An iniviual s relationship between psychological stress an illness may be meiate through means of ajustment, perceptions of stress, an personal/social resources. Ajustment an Aaptation to Breast Cancer Survivorship The effectiveness an means by which one copes with the iagnosis an/or isease-relate stressors of breast cancer often efines how well or poorly he/she has ajuste or continues to aapt to the isease. Coping is efine as ongoing cognitive an behavioral efforts to manage specific external an/or internal emans that are appraise as taxing or exceeing the resources of the person ; or simply cognitive or behavioral efforts to manage psychological stress. 36 Through a variety of ientifie coping strategies, researchers examine how iniviuals process their thoughts an actions leaing to aaptation outcomes. 6

17 Coping through cognitive, emotional, or behavioral isengagement is suggeste to be etrimental to long-term ajustment to breast cancer, while coping through active acceptance, seeking social support, emotional expression, an other approach-oriente coping strategies preicts iminishe istress over time. 5-8, 37 Negative strategies may interfere greatest by preventing aaptive thoughts an behaviors. Survivors who use avoiance as a coping strategy or feel resigne to their fate may be less likely to use active coping strategies. 9, 38 Feelings of harmony an peace preict less frequent use of enial/avoiance as enial an avoiance were foun to be inversely associate to seeking support an active coping, respectively. 9 Women who are more frequent users of active coping/social support when ealing with their cancer iagnosis report greater inner peace later in life. 9 It is believe that means of active coping with social support may help survivors fin meaning through fostering engagement an emotional expression to others while also increasing feelings of self-efficacy an personal control. 9 Perceive control, incluing a combination of coping strategies, self-efficacy, an personal beliefs about control, has a strong relationship to aaptation to illness in breast cancer patients. 10 Low perceive control correlates with uncertainty, fear of treatments, an overall poorer aaptation to illness. 10 Among patients with a low sense of control, active coping strategies are often not evelope. 10 Instea, coping strategies such as avoiance, acceptance-resignation, anxious preoccupation, an/or fatalism are more common an highly correlate with symptoms of anxiety an epression. 10 Patients with high perceive control on the other han is associate with problem-focuse coping an show reuce anxiety an epression. 7 7

18 Lazarus an Folkman s theory of Stress an Coping inclues eight coping strategies that iniviually an/or in combination, affect aaptation outcomes (quality of life). 39 Classifie as either problem-focuse (active) coping or emotion-focuse (passive) coping, the eight strategies inclue: confrontive, istancing, self-controlling, seeking social support, accepting responsibility, escape-avoiance, planful problem solving, an positive reappraisal. 39 Derive from this theory, the Ways of Coping Checklist was create as an instrument to measure a variety of coping methos in response to a general range of stressors. 12, 14 Multiple versions of this instrument have been create incluing the original 64-item inex as well as a shorter revise 42-item, 4-point Likert-type rating scale , 39 Total scores are calculate by summing the rating for all items. Raw scores may be calculate to represent overall active an passive coping strategies as well as specific iniviual coping strategies incorporate into the questionnaire. 13 Although not an all inclusive list, specific coping strategies incorporate into the various versions inclue problem-focuse, seeking social support, blames self, wishful thinking, blames others, count your blessings, avoiance, an religiosity. Determining relationships between coping strategies an aaptation to illness allows clinicians to tailor appropriate interventions to breast cancer survivors so that quality of life can be enhance. Assessing Quality of Life of Breast Cancer Survivors Tools create to measure QOL allow researchers an/or clinicians to examine the impact of various interventions an treatments on overall well-being an health. QOL is best assesse from a multiimensional perspective as survivors attempt to restore normalcy, achieve wholeness, an re-establish a sense of purpose in life. 15 Numerous 8

19 instruments have been evelope to quantify the QOL an the psychosocial impact of chronic illness however the value of a given instrument varies with iniviual nee. The usefulness of a given instrument may be questionable if the scope of the tool is restricte to a single isease population, a limite time of illness trajectory, or measures only a single construct. 17 The Demans of Illness Inventory (DOII) was evelope in theory aroun iniviual an family aaptation to chronic illness. 16, 17 Demans of illness are thought not to be ientical to illness stressors, harships, concerns, or problems, but instea appraise as ifficulties an challenges. 16 Demans are typically conceptualize as harships or etrimental events that call for some type of coping an aaptive response. 17 It is appropriate then to consier emans as an outcome of ajustment by which coping strategies may meiate. Specific areas of interest within the DOII inclue personal meaning (priorities, values, an goals that change with illness; uncertainty, mortality; family susceptibility), social relationships (changes in social activities; helping others unerstan or accept the illness; overprotective responses), self-image (changes in physical appearance, feelings of attractiveness; impact of isfiguring surgery or treatment), monitoring treatment an symptoms (vigilance to new boily sensations; preoccupation with symptoms; fears of recurrence, unetecte metastasis, or progressive nature of the isease), an treatment issues (accommoation to regimen; treatment evaluation; relationship with proviers). 17 An early version of the DOII consiste of a 125 item instrument that also integrate topics relate to physical symptoms (somatic responses of illness an anxiety) an family 9

20 functioning (general systems theory aspects of ecision making, aaptation, integration; partner caretaking, care of chilren; work or job situation). Integrative Cancer Care for Breast Cancer Survivors Integrative cancer care refers to complementary an/or alternative therapies implemente as part of a treatment program to improve survival, survivorship an QOL of cancer patients. Integrative cancer therapies possess potential to contribute to improving survival as well as quality of life of cancer patients if they are integrate into a comprehensive an scientifically base program aapte to the nees of the patient s isease, stage, an biological an social iniviuality. 40 Integrative meicine is commonly practice as a means to specifically reuce the symptom of psychological istress, an not reportely use for reasons of issatisfaction with traitional meical care. 19 Among breast cancer survivors, use of complementary therapies inclues reasons such as to assist the boy s natural forces to heal, to boost the immune system, to increase quality of life, an to gain a feeling of control of the cancer. 41 Breast cancer survivors that use complementary an alternative meicines reporte less severe anxiety an epression symptoms as compare with survivors given treatment as usual. 41 Min-boy therapy is a popular integrative moality use to inuce relaxation an improve emotional well-being through healing methos that enhance the min s interactions with boily function. 20 Breast cancer survivor participation in minfulnessbase stress reuction (MBSR) programs is associate with improvements in quality of life (QOL), moo states, stress symptoms, an immune parameters such as circulating lymphocyte number an in vitro cytokine prouction. 42 Stress management techniques 10

21 incluing cognitive-behavioral interventions an relaxation techniques have been use for a variety of intervention motives. 43 The effectiveness of a specific intervention is often epenent on the health-outcome measure assesse. A combination of cognitivebehavioral (enhancing psychological outcomes) an muscle relaxation techniques (enhancing physiological outcomes) appears to be more effective than using a single technique in improving psychological an/or physiological health. 43 Yoga Interventions for Cancer Survivors Develope as part of traitional Inian meicine, yoga was formulate by ancient civilizations as a metho to unite all the movements one nees for physical health with breathing an meitation practices that ensure peace of min. 44 Derive from the root yug (to join), the wor yoga has come to escribe a metho of iscipline: to join the boy to the min an together join to the self (soul); the union between the iniviual self an the transcenental self. 45 Yoga combines muscle relaxation, meitation an physical training while focusing on asanas (postures) an pranayama (controlling the breath). Yoga is regare as a promising metho for the treatment of stress-relate symptoms because of its emphasis on integrating physical, mental, an spiritual elements. A stuy examining the effect of a cognitive-behavioral therapy (n = 19) compare to a Kunalini yoga program (n = 18) over a 4-month perio showe that both interventions resulte in significant improvements of various psychological an physiological outcomes, incluing stress behavior, anger, exhaustion, QOL, heart rate, bloo pressure, an circulating catecholamine an cortisol concentrations. 46 The purpose of the stuy was to investigate stress management techniques an inclue assume healthy aults (male 11

22 an female) who were employees of a large financial company. 46 Preliminary evience also supports the feasibility an efficacy of select yoga interventions as therapeutic moalities for cancer survivors, incluing breast cancer. Yoga practices taught in the Iyengar, Tibetan, an Hatha traitions are associate with moest improvements in sleep quality, moo, stress, cancer-relate symptoms, an overall quality of life among cancer 47, 48 survivors. Breast cancer survivors may also benefit from yoga taught as an active practice, i.e., physically challenging asanas. This hypothesis is base on observations by Holmes et al. 49 inicating that participation in regular moerate-intensity physical activity reuce risk of eath from breast cancer. Documenting physiological benefits of yoga intervention for breast cancer survivors is important because yogic traitions iffer in their approach to the yoga asanas. Iyengar yoga, base on the teachings of B.K.S. Iyengar, consists of an orerly an progressive metho of a series of postures that are ajuste to meet the nees an physical conitions of the stuent. 45 Iyengar yoga incorporates the use of props (e.g., chairs, belts, blankets, blocks) to assist the practitioners in assuming the posture without strain. Accoring to Iyengar traition 45, specific anatomical guielines irect the execution of the asanas an pranayama, an specific postures are ajuste so that various organs, joints, an muscles are properly positione to facilitate physiologic changes. A pilot stuy investigating the effect of Iyengar yoga on the emotional states of healthy aults reporte improve psychological well-being following a single yoga class. 23 Increases in positive moos, ecreases in negative moos, an increases in energy levels resulte regarless of the asanas practice. Specific poses affecte moo ifferently, with back bens associate with 12

23 greater increases in positive moos an particularly so for those who were relatively hostile or epresse. 23 Results from aitional pilot stuies examining the physical an psychological benefits of Iyengar yoga for breast cancer survivors commonly reveal a positive impact 22, 50 on moo, quality of life an stress levels. As healthy iniviuals experience similar benefits, it is unknown whether or not breast cancer survivors who may begin at a less optimal physiological an psychological status achieve any greater benefit. 50 However, improvements in illness-relate symptoms incluing relieve joint aches an shouler stiffness, improve boy posture an boy image, an increase feelings of relaxation in their aily lives were reporte following an eight week Iyengar yoga intervention. 22 Repeate finings support yoga s efficacy for potential to provie both physical an 21-23, 47, 50 psychological benefits to breast cancer survivors. Ancient eastern yogic philosophy an health benefits of yoga practice are rapily gaining acceptance in western meicine. Scientists within the fiel of psychoneuroimmunology are now working towar a greater unerstaning of the physiological process involve in min-boy practices. Simply state, psychoneuroimmunology is the scientific stuy of how the nervous system an the min influence the immune system. As a quickly eveloping fiel of stuy, it is promising that future research will reveal the mechanisms behin Patanjali s classical Yoga-Sutra (c. 200 C.E) an the role they play in the healthy as well as the isease. 51 Central to this effort is a eveloping framework for biological signaling pathways pertinent to immune function. 13

24 Introuction to the Immune Response an Cancer The human immune response is compose of a large variety of cells an meiators which interact in a ynamic network to protect the boy. Depening on past exposure an antigen specificity, the immune response is commonly ivie into two subtypes: innate an aaptive immunity. 24 Recognition of a foreign pathogen elicits a response that at the cellular, tissue, an organism levels results in clearance of the pathogen or foreign matter if eeme harmful. 24, 26 Innate immunity is also thought of as the first line of efense, proviing an immeiate response to invaing or infectious pathogens or amage cells. 26 Aaptive immunity, also known as acquire immunity, facilitates memory-recognition an increase states of immune response to subsequent exposure with the same antigen. 26 Soluble factors such as complement, antiboies an cytokines secrete from immune cells meiate crosstalk between innate an aaptive immune cells. 24 Innate immunity typically involves an acute inflammatory response which then serves to activate an regulate aaptive immunity. 24 A ysregulate aaptive immune response such as the case in chronic inflammatory conitions may have the reverse effect an in turn meiate a chronic innate response often resulting in tissue amage. 26 Chronically unbalance interactions between innate an aaptive immune cells leas to chronic versus acute inflammation, which can lea to increase risk of cancer evelopment an growth, 24 with lymphocytes playing a prominent role in immune response an inflammation. 52 Lymphocyte subsets incluing B cells, T cells an natural killer (NK) cells are all involve in immunosurveillance an the crosstalk between innate an aaptive immunity. In terms of immunosurveillance, for example, CD8+ cytotoxic T lymphocytes recognize 14

25 an kill tumor cells by secreting perforin which bins to major histocompatibility complex (MHC) class I receptors on tumor cells an inuces apoptosis. 53 Tumor angiogenesis provies a means of growth for the carcinogenic tissue. In aition, they secrete the anti-angiogenic cytokine interferon-γ (INF-γ). 53 Activate CD4+ T cells participate in converting MHC class II macrophages, which secrete interleukin (IL) -10, into INF-γ secreting macrophages. The CD4+ T h 1 cells also secrete INF-γ while CD4+ T h 2 cells prouce IL-4 which inirectly blocks tumor-angiogenesis. 53 Activate NK cells are involve in tumor lysis an apoptosis irectly via a perforin-epenent manner an inirectly through cell-cell contact, for example, with enritic cells in the presence of IL-4 an IL Regulate NK cell activity thus plays a key role in killing carcinogenic cells. As the immune system functions to maintain homeostasis, it is propose that cancer cells may escape innate an aaptive immune responses via immunoselection (selection of non-immunogenic tumor-cell-variants) or immunosubversion (active suppression of the immune response). 53 Finke et al. 25 hypothesize that two conitions exist enabling tumor cells to escape immunosurveillance an essentially thrive in inflammatory environments. The first conition occurs when lymphocytes within a microenvironment are functionally impaire. 25 The secon conition requires inflammatory-cell-erive meiators secrete from activate fibroblasts, enothelial cells, an lymphocytes in response to tumor progression in the microenvironment. Lymphocytes can also secrete matrix metalloproteinases, a key component to the extracellular matrix an tumor growth. 54 In aition, tumor microenvironments are rich in lymphocyte-erive cytokines, namely tumor necrosis factor -α (TNF α), transforming 15

26 growth factor -β (TBF β), IL-1, an IL-6, which are all meiators of cancer evelopment. 24 Furthermore, chronically activate innate immune cells may inirectly suppress anti-tumor aaptive immunosurveillance. 24 Nuclear Factor kappa B (NF-κB) Transcription factor nuclear factor kappa B (NF-κB) is central to the regulation of iverse biological processes. 27 Within various cancer cells as well as lymphocytes, NFκB serves as a primary cell signaling pathway. The mammalian NF-κB family consists of a collection of imeric proteins sequestere in the cytoplasm as latent complexes boun to a member of the IκB (inhibitor of NF-κB) protein family. 28, 29 Dimeric proteins existing as both homo- an heteroimers which make up the NF-κB complexes inclue RelA (p65), c-rel, RelB, NF-κB1(p50/p105) an NF-κB2 (p52/p100). 28 Each family member contains an N-terminal 300-amino-aci omain (Rel homology omain) that is responsible for imerization, interaction with IκB s, an DNA bining to κb promoter or enhancer regions of more than 400 ifferent target genes. 30 In aition, RelA, c-rel an RelB also contain C-terminal transcription activation omains (TADs) which enable activation of gene expression. 30 Unless boun to a protein containing a TAD (such as Bcl-3), p50 an p52 homoimers are incapable of gene transcription, an their bining to κb sites in unstimulate cells serves to repress gene expression. 30 The IκB family, consisting of IκBα, IκBβ, IκBε, IκBγ, Bcl-3, an two precursor proteins: p100 an p105, are preominately locate in the cytoplasm. 28, 29 Differing from the other IκB family members, Bcl-3 functions as a co-activator promoting activation at the site of DNA bining within the nucleus

27 Activation of NF-κB can occur by a iverse array of stimuli. Inflammatory cytokines, mitogens, receptor ligans, growth factors, oncogenes, bacterial proucts, chemicals, an cellular stress are all capable of inucing NF-κB activation an subsequently eliciting a complex range of responses 28, 31, 55 as epicte in Figure 1 below. 56 General receptors responsible for initiating cell signaling pathways inclue B cell receptors, T cell receptors, Toll-like receptors (TLR), IL-1 receptors, an the TNFreceptor (TNFR) superfamily of molecules (molecules other than TNF-α). 57 Figure

28 NF-κB activation pathways are generally classifie as either canonical (classical) or non-canonical (alternative) epening on whether activation involves IκB egraation or p100 processing. 58, 59 A thir pathway leaing to NF-κB activation is sometimes referre to as the atypical pathway. 60 Although similar to the classical pathway in IκB egraation, the atypical pathway iffers in the essential kinases which lea to inhibitor egraation. Ultimately however, activate NF-κB imers are liberate from their boun inhibitor an translocate to the nucleus where NF-κB is further involve in specific gene expression activity. The canonical, or classical, pathway is initiate by various inflammatory stimuli, incluing extracellular pro-inflammatory cytokines such as tumor necrosis factor (TNF)- α an IL-1, engagement of the T cell receptor (TCR) or exposure to bacterial proucts such as lipopolysaccharie (LPS). 60 Bining leas to sequential recruitment of various aaptor molecules to the cell membrane within the intracellular compartment which activate the IκB kinase (IKK) complex. 61 The three core subunits that make up the IKK complex consist of IKKα (also known as IKK1), IKKβ (IKK2), an IKKγ (NEMO, NFκB essential moulator). The canonical pathway is characterize by IKKβ as being the preominant IκB kinase leaing to the rapi phosphorylation of the IκBα at Ser32 an Ser 36 an subsequent ubiquitin-inuce egreation by the 26S proteasome. 60 The egraation of IκB exposes the nuclear localization signal of the NF-κB family protein, leaing to its nuclear translocation. 62 The non-canonical pathway operates primarily in B lymphocytes an is stimulate through the TNFR superfamily, incluing receptors for B-cell activating factor (BAFF), lymphotoxins an CD40 ligan (CD40L). 58 This pathway is consiere NEMO- 18

29 inepenent an controls the activation of complexes that consist of p100. Following the recruitment of aaptor proteins, NF-κB inucing kinase (NIK) is activate an responsible for the phosphorylation of the p100 NF-κB subunit by IKKα. Processing of p100, via ubiquitination by the 26S proteasome results in the formation of p52 an allows p52-containing immers to translocate to the nuclues. 28 RelB-p52 heteroimers are frequently activate as a consequence of non-canonical pathway activation an have a high affinity for a istinct subset of κb elements which may lea to the regulation of a istinct subset of NF-κB genes. 60 Figure 2 illustrates the primary ifference between the canonical an non-canonical pathways, involving iffering IKK/IκB/NF-κB interactions. Figure 2. Canonical Pathway Non-Canonical Pathway TCR TNFR IL-1R TLR BCR TNF-SF NEMO IKKα IKKβ IKKα IKKα P IκBα p50 REL-A REL-B P p100 p50 REL-A REL-B p52 p50 REL-A REL-B p52 Atypical pathways leaing to NF-κB activation are believe to be IKK inepenent. Tyrosine-kinase-epenent pathways escribe as phosphorylating IκBα at Tyr42 may result from stimuli such as hypoxia an hyrogen-peroxie. 60 Treatment of 19

30 ultraviolet (UV) light or expression of the HER2 oncogene in breast cancer cells can result in the irect phosphorylation of IκBα by casein kinase-ii (CKII) at sites in its C- terminal omain. 60, 63 In both cases, IκB is either egraate or isassociate from NF-κB allowing for translocation to the nucleus an transcriptional activity. Cell Signaling Pathways from Mitogen Stimulation Just as NF-κB activation results from a vast array of stimuli, there are also numerous signaling pathways which link cellular stimulation to the IKK/IκB/NF-κB moule. Mitogen stimulation involves funamental signal transuction pathways leaing to NF-kB activation in lymphocytes. A mitogen is a chemical or protein that can mimic actions of antigen an initiate cell mitosis, resulting in cell proliferation. As lymphocytes respon to antigen an mitogen stimuli similarly, in vitro mitogen stimulation provies the means to assess potential lymphocyte response to isease or other stressor. 32 Phytohemagglutinin (PHA) an phorbol-12 myristate 13-acetate (PMA) plus ionomycin are common mitogens involve in lymphocyte activation. Although PHA, PMA an ionomycin signal transuction pathways iffer slightly in their primary course of action, it is believe that each is associate with protein kinase C (PKC) isoforms, which in turn 64, 65 activate the IKK/IκB/NF-κB cascae of events. Phytohemagglutinin has a high affinity for T lymphocyte surface receptors, namely the IL-2R. 66 Activation of T cells via PHA results in cellular proliferation an increase T cell expression of IL-2 an its receptor, involve in cyclic activation. 67 Interestingly, IL-2 is a principle (autocrine an paracrine) cytokine in T cell activation an IL-2 synthesis is epenent on NF-κB activation. 65 NK cell activity also involves 20

31 NF-κB activity inuce by IL-2R signaling. 68 Exact mechanisms linking IL-2R an NFκB activation continue to be investigate, however it is establishe that transuction involves upstream protein tyrosine kinases (PTK) that lea to PKC activation an NF-κB nuclear translocation. 65 Lymphocyte proliferation is also epenent on NF-κB activation however inepenent of IL-2 prouction an the IL-2 receptor. 69 A commonly route mitogen stimulation pathway involves the TCR complex (CD3/CD4) an CD28 in T cells an the BCR in B cells. Stimulate TCR/CD28 elicits the recruitment of PTKs as well as other aaptor proteins to the phosphorylate TCR. This action sequentially generates phospholipase C (PLC)-γ1 an inositol-3 phosphate (IP3) activity which results in the increase of intracellular calcium an iacylglycerol which stimulates PKCs. 70 PKC isoforms may iffer between T an B cells 70 an epenency of calcium for activation, 71 however similarly, ownstream of PKC activation, activation of the IKK complex is linke to the Carma1/Bcl10/MALT1 complex an NF-κB nuclear translocation. 70 Plasmatic membrane content of DAG an cytosolic free calcium concentration are important secon messengers which serve to activate PKC. 64 The phorbol ester, PMA, has the ability to substitute for iacylglycerol (DAG), bypassing cell-surface receptors an calcium signaling to irectly activate PKC an activation of NF-κB. 67 Ionomycin, a calcium ionophore, causes an increase in intracellular calcium; thus also inucing activation an proliferation of lymphocytes via NF-κB signaling pathways. 72 Typically, PMA is use in conjunction with ionomycin for the inuction of NF-κB activation in lymphocytes where calcium is require for activation of PKC. 67 Global lymphocyte stimulation using the combination of PHA, PMA, an ionomycin, integrates multiple 21

32 segments an pathways leaing to NF-κB activation via various lymphocyte subtypes an cell-surface receptors. Illustrate in Figure 3 below, PHA/PMA/ionomycin signaling as iscusse above proves itself as an integrative network which commonly leas to NF-κB activation. Demonstration of ifferences in responsiveness of peripheral bloo lymphocytes to mitogen stimulation (in vitro) can facilitate a greater unerstaning of lymphocyte response to therapeutic intervention via ientification of signaling pathways. Signal transuction leaing to NF-κB nuclear translocation however only precees events within the nucleus, such as gene transcription, which establishes the final cellular response. 64, 73, 74 Figure 3. Ionomycin Ca ++ ionophore TCR CD3/CD4 PHA CD28 IL-2R PMA IP 3 Aapter Proteins PLC-γ1 PTK PTK Ca ++ DAG IP 3 -K CN PKC NEMO-IKK Malt1/Bcl-10/CARMA1 IκB NK-κB NK-κB 22

33 Gene Expression an NF-κB Regulation Gene expression associate with activate NF-κB an NF-κB-meiate transcription can be categorize into four groups base on function: inflammatory response an immuno-regulatory functions, negative regulators of NF-κB, apoptotic functions, an positive regulators of cell cycle. 28 The inflammatory response associate with NF-κB activation inclues pro-inflammatory cytokines (e.g., TNF-α, IL-1, IL-6), chemokines an their receptors (e.g., MIP-1α; monocyte chemoattractant protein-1α), an ahesion molecules (e.g., VCAM-1, ICAM-1, an ELAM-1; vascular cell ahesion molecule-1, intercellular cell ahesion molecule-1, enothelial-leukocyte ahesion molecule-1). 28 Bining of TNF-α an IL-1 to their receptors strongly activates NF-κB an may generate a chronic inflammatory response if left unchecke. 30, 61 For autocrine circuits such as TNF-α/IL-1, numerous mechanisms exist to ensure appropriate control of NF-κB transcription factor activity. In aition to its irect relationship with inflammation, regulation of cell cycle an survival implicates the importance of NF-κB in cell-to-cell communication among immune cells as well as neoplastic cells. Regulation of NF-κB activity exists at many levels. Beginning with the cell surface receptor, inhibition within a particular NF-κB signaling pathway may occur anywhere from receptor recognition to upstream an ownstream signaling of IκB ubiquitination, selective interaction with co-activator an co-repressor proteins, promoter regions, post-translational moification of NF-κB an histone moifications. 28 Following receptor recognition, NF-κB activation is tightly regulate at the level of the inhibitor. For example, inflammatory signalsomes (e.g. TNF, IL-1, various TLR ligans) are associate with a variety of kinase pathways which lea to a coorinate 23

34 IKK activation an egraation/synthesis of IκB proteins. 75 The pathway leaing to IKK activation etermines the IKK/IκB/NF-κB signaling moule, the nature of the NF-κB response an the kinetics of NF-κB activation. 75 IκB proteins may intrinsically control intensity, uration an specificity of the NF-κB response. 28 The IκBα subunit is responsible for a negative feeback loop uring short term stimulation that rives oscillations in NF-κB translocation an gene transcription events. 76 Gene transcription of IκBα is regulate itself by NF-κB activation. Present in the nucleus before shuttling back to the cytoplasm, IκBα can bin to RelA an p50 subunits an prevent DNA bining. 76 Contrary however, neither IκBβ nor IκBγ gene expression is not regulate by NF-κB an uring sustaine NF-κB stimulation, these subunits act to stabilize the NF-κB response. 76 Activation of NF-κB leaing to gene transcription is further regulate at the site of DNA bining. The specific κb sequence etermines DNA bining affinity of the NFκB complexes as well as the ability of the NF-κB immers to interact prouctively with cofactors. 28 A ynamic environmental transcription factor profile within the nucleus establishes combinatorial control of target gene transcription. 75 Synergistic interactions often occur between NF-κB an partner transcription factors which are time-epenent an tissue specific. 75 Specific κb site sequences not only coe for specific NF-κB combinations, but also require particular allosteric conformational changes of NF-κB imers in orer to elicit gene transcription. 75 Transcriptional regulation in response to the 75, 77 cellular milieu meiates evelopmental, homeostatic an pathological events. Further regulation of NF-κB-DNA bining involves the unerstaning of epigenetic control, chromatic structure an histone patterns. Histones consist of the core nucleosomal proteins associate with gene transcription. Genes that are actively 24

35 transcribe or represse are associate with specific sets of histone moifications, also known as the histone coe. 75 Histone moification may result from acetylation, phosphorylation, ubiquitylation, glycosylation, or sumoylation, an provies the basis for spatial an time-epenent NF-κB-cofactor-DNA transcription. 75 For example, LPS stimulation results in biphasic recruitment of NF-κB to promoter regions as etermine by histone acetylation. 28, 78 As etermine in macrophages with an inflammatory stimuli, hypoacetylate promoter regions exhibite elaye NF-κB recruitment an activation of transcription compare to regions with constitutive acetylation. 78 In this moel, the iffering acetylation moification at promoter regions emonstrates a means by which various target genes can regulate the kinetics of NF-κB activation following upstream signaling pathways. Relevant Biological Assays A practical metho for the etermination of NF-kB activation inclues nuclear protein extraction from peripheral bloo lymphocytes followe by protein labeling an electrophoretic-inuce protein separation. For example, sample protein labele with a specific NF-kB/Rel oligo (single DNA stran allowing for a DNA-protein complex to form) prior to a electrophoretic-inuce shift, allows for visualization techniques incluing chemiluminescence an fluorescence staining. 79, 80 The electrophoretic mobility shift assay (EMSA) 79 is a technique base on the observation that the migration of DNA through a nonenaturing polyacrylamie gel is hinere when protein is boun to it. Analysis of EMSA involves the ientification an quantification of bans that have migrate a istance corresponing to the molecular weight of the complex. 81 Two-color 25

36 fluorescence EMSA visualization involves a short incubation perio using a SYBR green stain (staining for DNA), followe by a longer incubation using a SYPRO re stain (to visualize protein). The location of the DNA-protein interactions is then etermine by a visualize overlaying of the gel images in which the complex, staine both green an re, appears yellow. 80 Bans ientifie as yellow via image analysis can then be quantifie to numerically represent activate protein, that which is actively able to bin to DNA. This metho oes not require raioactive isotopic labeling, pre-labeling or seconary etection methos an therefore may serve to as a novel technique that is both straightforwar an accurate. In aition to EMSA, an aitional traitional metho for etermination an quantification of transcription factor proteins consists of a chemilumiscence ELISA assay. This metho, following cytosolic an nuclear protein extraction, involves incubating the sample protein with a primary antiboy which targets NF-κB with high affinity, an a seconary antiboy which bins to the primary an acts as a fluorescent tag. 82 A fluorescence spectrometer is use to capture an quantitate the fluorescence emitte from the excite fluorophores once expose to light. 82 This metho is very sensitive to environmental conitions an quantity of protein. However, with aequate protein, this technique serves as a promising metho for quantifying total transcription factor present in extracte protein. 26

37 Summary Diagnosis of breast cancer can have significant physical an psychological consequences that last the uration of one s lifetime. Survivorship is a unique experience for each iniviual as no two people process stressful stimuli in the same manner. Active coping strategies often meiate an improve quality of life relate to fewer reporte emans of illness while passive coping acts in contrast. Integrative meical care provies a means for survivors to alleviate isease-associate physical ailments as well as illness-relate stressors. Various pilot stuies have establishe baseline ata supporting numerous health benefits of Iyengar yoga practice among breast cancer survivors which inclue improve physical an emotional well-being. Psychological stress can have a suppressive effect on immune function an may impact survival of those living with a chronic isease, such as cancer. Necessary components of a healthy immune system inclue prouction an regulation of cytokines, acute phase proteins, an ahesion molecules, all of which require gene expression meiate by NF-kB. As an inflammatory transcription factor, NF-kB is often use as a biological measure of cellular stress. To ata, no stuy has focuse on the impact of Iyengar yoga on lymphocyte NF-κB activation an examine meiators of psychosocial stress simultaneously in breast cancer survivors. 27