30. May 2010 by admin.

In my presentation Towards a Systems Theory of Aging I argue that the two theories Programmed epigenomic changes and Decline in functioning of the stem cell supply chain are complimentary and equivalent and have the potential for providing a framework for an overall systems view of aging that knits together a large collection of traditional special theories of aging.  In this post, I review some research that is relevant to this assertion, especially with respect to the relationships between the Programmed epigenomic changes theories and the aging theories 6. Chronic Inflammation, 7. Immune System Compromise, 8. Neurological Degeneration, 10. Susceptibility to Cancers, and 11. Susceptibility to Cardiovascular Disease.   

The 2010 publication Epigenetics in atherosclerosis and inflammation is a review study.  “Atherosclerosis is a multifactorial disease with a severe burden on western society. Recent insights into the pathogenesis of atherosclerosis underscore the importance of chronic inflammation in both the initiation and progression of vascular remodelling. — Besides genetic factors also epigenetic mechanisms play an essential and fundamental role in the transcriptional control of gene expression.  –. The concept of epigenetic regulation is gradually being recognized as an important factor in the pathogenesis of atherosclerosis. Recent research provides an essential link between inflammation and reprogramming of the epigenome.” The Programmed epigenomic changes theory of aging asserts that age-related reprogramming of the epigenome increases susceptibility to inflammation and inflammation-related diseases. 

The 2008 publication Epigenetic regulation of gene expression in the inflammatory response and relevance to common diseases highlights the same points, extending their scope to autoimmune diseases and cancers. “It is clear that the epigenetic state is a central regulator of cellular development and activation. Emerging evidence suggests a key role for epigenetics in human pathologies, including in inflammatory and neoplastic disorders. The epigenome is influenced by environmental factors throughout life. Nutritional factors can have profound effects on the expression of specific genes by epigenetic modification, and these may be passed on to subsequent generations with potentially detrimental effects. Many cancers are associated with altered epigenetic profiles, leading to altered expression of the genes involved in cell growth or differentiation. Autoimmune and neoplastic diseases increase in frequency with increasing age, with epigenetic dysregulation proposed as a potential explanation. In support of this hypothesis, studies in monozygotic twins revealed increasing epigenetic differences with age. Differences in methylation status of CpG sites, monoallelic silencing, and other epigenetic regulatory mechanisms have been observed in key inflammatory response genes. The importance of the epigenome in the pathogenesis of common human diseases is likely to be as significant as that of traditional genetic mutations.”A number of studies have been concerned with identifying epigenomic changes associated with particular cancers.  Although they are often highly technical, they show that complicated epigenomic/epigenetic changes are involved in cancer processes. 

 The 2007 publication Epigenetic profiling of multidrug-resistant human MCF-7 breast adenocarcinoma cells reveals novel hyper- and hypomethylated targets is an example.  “Presently, two hypotheses, genetic and epigenetic, have been proposed to explain mechanisms of acquired cancer drug resistance. In the present study, we examined the alterations in epigenetic mechanisms in the drug-resistant MCF-7 human breast cancer cells induced by doxorubicin (DOX) and cisplatin (cisDDP), two chemotherapeutic drugs with different modes of action. Despite this difference, both of the drug-resistant cell lines displayed similar pronounced changes in the global epigenetic landscape showing loss of global DNA methylation, loss of histone H4 lysine 20 trimethylation, increased phosporylation of histone H3 serine 10, and diminished expression of Suv4-20h2 histone methyltransferase compared with parental MCF-7 cells. In addition to global epigenetic changes, the MCF-7/DOX and MCF-7/cisDDP drug-resistant cells are characterized by extensive alterations in region-specific DNA methylation, as indicated by the appearance of the number of differentially methylated DNA genes. A detailed analysis of hypo- and hypermethylated DNA sequences revealed that the acquisition of drug-resistant phenotype of MCF-7 cells to DOX and cisDDP, in addition to specific alterations induced by a particular drug only, was characterized by three major common mechanisms: dysfunction of genes involved in estrogen metabolism (sulfatase 2 and estrogen receptor alpha), apoptosis (p73, alpha-tubulin, BCL2-antagonist of cell death, tissue transglutaminase 2 and forkhead box protein K1), and cell-cell contact (leptin, stromal cell-derived factor receptor 1, activin A receptor E-cadherin) and showed that two opposing hypo- and hypermethylation processes may enhance and complement each other in the disruption of these pathways. These results provided evidence that epigenetic changes are an important feature of cancer cells with acquired drug-resistant phenotype and may be a crucial contributing factor to its development. Finally, deregulation of similar pathways may explain the existence and provide mechanism of cross-resistance of cancer cells to different types of chemotherapeutic agents.”

The 2008 publication Epigenetic mapping and functional analysis in a breast cancer metastasis model using whole-genome promoter tiling microarrays states “Breast cancer metastasis is a complex, multi-step biological process. Genetic mutations along with epigenetic alterations in the form of DNA methylation patterns and histone modifications contribute to metastasis-related gene expression changes and genomic instability. — . RESULTS: We integrated data from the tiling microarrays with targets identified by Ingenuity Pathways Analysis software and observed epigenetic variations in genes implicated in epithelial-mesenchymal transition and with tumor cell migration. We identified widespread genomic hypermethylation and hypomethylation events in these cells and we confirmed functional associations between methylation status and expression of the CDH1, CST6, EGFR, SNAI2 and ZEB2 genes by quantitative real-time PCR. Our data also suggest that the complex genomic reorganization present in cancer cells may be superimposed over promoter-specific methylation events that are responsible for gene-specific expression changes. CONCLUSION: This is the first whole-genome approach to identify genome-wide and gene-specific epigenetic alterations, and the functional consequences of these changes, in the context of breast cancer metastasis to lymph nodes. This approach allows the development of epigenetic signatures of metastasis to be used concurrently with genomic signatures to improve mapping of the evolving molecular landscape of metastasis and to permit translational approaches to target epigenetically regulated molecular pathways related to metastatic progression.”

The 2009 publication  Pituitary tumours: all silent on the epigenetics front states “Investigation of the epigenome of sporadic pituitary tumours is providing a more detailed understanding of aberrations that characterise this tumour type. Early studies, in this and other tumour types adopted candidate-gene approaches to characterise CpG island methylation as a mechanism responsible for or associated with gene silencing. However, more recently, investigators have adopted approaches that do not require a priori knowledge of the gene and transcript, as example differential display techniques, and also genome-wide, array-based approaches, to ‘uncover’ or ‘unmask’ silenced genes. Furthermore, through use of chromatin immunoprecipitation as a selective enrichment technique; we are now beginning to identify modifications that target the underlying histones themselves and that have roles in gene-silencing events. Collectively, these studies provided convincing evidence that change to the tumour epigenome are not simply epiphenomena but have functional consequences in the context of pituitary tumour evolution. Our ability to perform these types of studies has been and is increasingly reliant upon technological advances in the genomics and epigenomics arena. In this context, other more recent advances and developing technologies, and, in particular, next generation or flow cell re-sequencing techniques offer exciting opportunities for our future studies of this tumour type.”Relating to almost all areas of medicine, starting in the early-2000s more and more research publications have been appearing pointing out the importance of epigenomic regulation in disease etiology and progression. 

 For example, the 2008 review publication Epigenetic Regulation of Vascular Endothelial Gene Expression “Epigenetics has emerged as an increasingly powerful paradigm to understand complex non-Mendelian diseases. For example, epigenetics provides a newer perspective for understanding how gene expression is perturbed in prevalent diseases of the human vascular system characterized by a dysfunctional endothelium.⁴” 

The 2009 publication Epigenetics and periodontal disease: future perspectives states “Periodontitis is a multifactorial infection characterized by inflammation and destruction of tooth supporting tissues, as a result of the response of a susceptible host to bacterial challenge. Studies have demonstrated that epigenetic events are able to influence the production of cytokines, contributing to the development of inflammatory diseases. Epigenetic events act through the remodeling of chromatin and can selectively activate or inactivate genes, determining their expression. The epigenetic process, by inducing a change in cytokine profile, may subsequently influence the pathogenesis and determine the outcome of many infectious diseases. These findings may have relevance for inflammatory diseases in which the expression of cytokines is unregulated. The purpose of this review is to show evidence that supports the hypothesis that epigenetic alterations, such as hyper and hypomethylation, of cytokine genes, could help to understand the mechanisms related to periodontal disease activity. Therefore, epigenetics may have future impact on diagnosis and/or therapeutics of periodontal disease.“

The 2009 publication Epigenetic mechanisms that underpin metabolic and cardiovascular diseases relates to other critical disease fronts and focuses on lifelong changes in the epigenome and their affect on disease susceptibilities:

·     “Developmental plasticity enables an organism to respond to environmental cues and adjust its phenotypic development to match its environment.

·     Developmental plasticity is effected, at least in part, by epigenetic changes that are established in early life and modulate gene expression during development and maturity.

·     In mammals, the window during which the epigenome is susceptible to nutritional cues extends from conception to at least weaning.

·     Mismatch between the early and mature environments may result in inappropriate patterns of epigenetic changes and gene expression that increase subsequent susceptibility to metabolic and cardiovascular diseases.

·     The available evidence suggests that interventions to prevent metabolic and cardiovascular diseases should focus on the prenatal and early postnatal periods.”   

The 2008 publication Epigenetic principles and mechanisms underlying nervous system functions in health and disease states “Epigenetics and epigenomic medicine encompass a new science of brain and behavior that are already providing unique insights into the mechanisms underlying brain development, evolution, neuronal and network plasticity and homeostasis, senescence, the etiology of diverse neurological diseases and neural regenerative processes. Epigenetic mechanisms include DNA methylation, histone modifications, nucleosome repositioning, higher order chromatin remodeling, non-coding RNAs, and RNA and DNA editing. RNA is centrally involved in directing these processes, implying that the transcriptional state of the cell is the primary determinant of epigenetic memory. This transcriptional state can be modified not only by internal and external cues affecting gene expression and post-transcriptional processing, but also by RNA and DNA editing through activity-dependent intracellular transport and modulation of RNAs and RNA regulatory supercomplexes, and through trans-neuronal and systemic trafficking of functional RNA subclasses. These integrated processes promote dynamic reorganization of nuclear architecture and the genomic landscape to modulate functional gene and neural networks with complex temporal and spatial trajectories. Epigenetics represents the long sought after molecular interface mediating gene-environmental interactions during critical periods throughout the lifecycle. The discipline of environmental epigenomics has begun to identify combinatorial profiles of environmental stressors modulating the latency, initiation and progression of specific neurological disorders, and more selective disease biomarkers and graded molecular responses to emerging therapeutic interventions. Pharmacoepigenomic therapies will promote accelerated recovery of impaired and seemingly irrevocably lost cognitive, behavioral, sensorimotor functions through epigenetic reprogramming of endogenous regional neural stem cell fate decisions, targeted tissue remodeling and restoration of neural network integrity, plasticity and connectivity.”

The above is just a small sample of the river of applicable literature but enough to make the point: The Programmed epigenomic changes theory of aging is critically implicated in a large number of disease and disease progression processes. It will clearly take a lot more research to establish that this theory is capable of explaining all the phenomena described by all the other theories of aging but I strongly suspect that this will in time happen.

Epigenomic interventions to deal with various diseases are now in the clinical trials phase.  Specifically, a number of histone deacetylase inhibitors are now in clinical trials, basically substances that keep apoptosis and other critical genes active.  Describing these will be the subject of a subsequent post.  I also hope to characterize some interesting research relating lifelong nutrition patterns to epigenomic changes.

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27. May 2010 by admin.

The p21 gene has long been known for its role in cell cycle arrest and apoptosis.  In case of DNA damage it signals to the p53 gene to initiate apoptosis of the cell, averting the possibility of tumorgenesis.  Very-recent research indicates something else – expression of P21 is part of what keeps us from growing new limbs or other body parts like salamanders or newts.  This blog post reviews the new research in the context of what has long been known about P21.

Some background on P21

P21 is a cell cycle regulator, specifically a CDK (cyclin-dependent kinase) inhibitor(ref).  It has long been known to impede stem cell differentiation and proper embryonic development.  For example, the 1996 publication Targeted in vivo expression of the cyclin-dependent kinase inhibitor p21 halts hepatocyte cell-cycle progression, postnatal liver development and regeneration states “The CDK inhibitor p21 (WAF-1/CIP-1/SDI-1) has been implicated in DNA damage-induced p53-mediated G1 arrest, as well as in physiological processes, such as cell differentiation and senescence, that do not involve p53 function. — These results provide the first in vivo evidence that appropriate p21 levels are critical in normal development and further implicate p21 in the control of multiple cell-cycle phases.”

P21 has been studied for its role in fibrosis and other lung diseases.  A 2004 publication is entitled Induction of CDK inhibitor p21 gene as a new therapeutic strategy against pulmonary fibrosis.  The 2008 publication P21 regulates TGF-beta1-induced pulmonary responses via a TNF-alpha-signaling pathway relates “Transforming growth factor (TGF)-beta(1) is an essential regulatory cytokine that has been implicated in the pathogenesis of diverse facets of the injury and repair responses in the lung. The types of responses that it elicits can be appreciated in studies from our laboratory that demonstrated that the transgenic (Tg) overexpression of TGF-beta(1) in the murine lung causes epithelial apoptosis followed by fibrosis, inflammation, and parenchymal destruction. Because a cyclin-dependent kinase inhibitor, p21, is a key regulator of apoptosis, we hypothesized that p21 plays an important role in the pathogenesis of TGF-beta(1)-induced tissue responses. — Collectively, our studies demonstrate that p21 regulates TGF-beta(1)-induced apoptosis, inflammation, fibrosis, and alveolar remodeling by interacting with TNF-alpha-signaling pathways.”

Expression of P21 is a barrier to stem cell differentiation.   The 2000 publication Hematopoietic Stem Cell Quiescence Maintained by p21^cip1/waf1 states “Therefore, p21 is the molecular switch governing the entry of stem cells into the cell cycle, and in its absence, increased cell cycling leads to stem cell exhaustion. Under conditions of stress, restricted cell cycling is crucial to prevent premature stem cell depletion and hematopoietic death.”  In the absence of P21, hematopoietic stem cells would not remain quiescent in their niches but would instead prematurely differentiate when stress occurs exhausting the pools of those cells and interrupting the normal functioning of the stem cell supply chain leading to premature death.  The 2009 paper Accelerating stem cell proliferation by down-regulation of cell cycle regulator p21 offers a consistent message.  “Inhibition of the cell cycle regulator p21 results in significant acceleration of mesenchymal stem cell proliferation without promoting spontaneous cellular differentiation.”

P21 is also implicated in active disease processes especially in certain cancers.  For example, the 2009 paper Cell-cycle restriction limits DNA damage and maintains self-renewal of leukemia stem cells argues that p21 gives cancer cells the chance to repair their DNA and keep living.  “Here we demonstrate that expression of the cell-cycle inhibitor p21 is indispensable for maintaining self-renewal of leukemia stem cells. Expression of leukemia-associated oncogenes in mouse haematopoietic stem cells (HSCs) induces DNA damage and activates a p21-dependent cellular response, which leads to reversible cell-cycle arrest and DNA repair.  Activated p21 is critical in preventing excess DNA-damage accumulation and functional exhaustion of leukemic stem cells. These data unravel the oncogenic potential of p21 and suggest that inhibition of DNA repair mechanisms might function as potent strategy for the eradication of the slowly proliferating leukemia stem cells.”

There is a lot more that can be said about P21 but the above is sufficient for this post.

P21 and organ renewal

Interesting news relating P21 to limb and appendage regeneration is reported in a March 2010 report Lack of p21 expression links cell cycle control and appendage regeneration in mice.  “Animals capable of regenerating multiple tissue types, organs, and appendages after injury are common yet sporadic and include some sponge, hydra, planarian, and salamander (i.e., newt and axolotl) species, but notably such regenerative capacity is rare in mammals. The adult MRL mouse strain is a rare exception to the rule that mammals do not regenerate appendage tissue. Certain commonalities, such as blastema formation and basement membrane breakdown at the wound site, suggest that MRL mice may share other features with classical regenerators.  As reported here, MRL fibroblast-like cells have a distinct cell-cycle (G2/M accumulation) phenotype and a heightened basal and wound site DNA damage/repair response that is also common to classical regenerators and mammalian embryonic stem cells.”  MRL mice do not express P21 and are like salamanders in an important respect.  When wounded “The super-healing mice form a “blastema”, a clump of immature cells that behave like stem cells, at the injury site. The blastema cells differentiate into the proper cells, leaving virtually no scar tissue or other trace of the injury. Such regenerative power is almost unknown in mammals. But it is common in amphibians such as the newt and axolotl, which can regrow entire limbs(ref).”  MRL mice are known to be able to at least partially regenerate digits and “have a far superior ability to regenerate cardiac tissue than do regular mice, and humans(ref).”  

The researchers discovered that the wound-injury response of P21-knockout mice was like that of MRL mice.  In response to a hole punched in a mouse’s ear, the MRL and P21- mice formed healing blastemas which closed up the holes like they were never there while ordinary P21+ mice formed scars and the holes remained open. 

What about the absence of the protective role of P21 in P21- and MRL mice?  Are they more cancer prone? “DNA damage is a hallmark of cancer, and regeneration in the healer mice and p21 knockout mice features an increase in DNA damage in the dividing cells at the blastema. — The link between regeneration and cancer notwithstanding, the researchers who originally created the p21 knockout mice haven’t found any evidence that the lack of a p21 gene leads to increased rates of cancer. — Along with the cell proliferation and DNA damage in the heightened regeneration, there’s an increased rate of apoptosis, which kills off cells too severely damaged to be repaired, —  “The combined effects of an increase in highly regenerative cells and apoptosis may allow the cells of these organisms to divide rapidly without going out of control and becoming cancerous,” Heber-Katz (an author of the study) said. “In fact, it is similar to what is seen in mammalian embryos, where p21 also happens to be inactive after DNA damage. The down regulation of p21 promotes the induced pluripotent state in mammalian cells, highlighting a correlation between stem cells, tissue regeneration, and the cell cycle(ref).”

The line of research was conducted in the Wistar Institute in the laboratory of Dr. Ellen Heber-Katz.  Dr. Heber Katz has been involved in highly related research since the 1990s.  In fact they accidentally discovered the regenerative capability of MRL mice back in the mid 90s when they punched holes in the ears of some of these animals to identify them, a standard laboratory procedure.  In normal mice the holes stay permanently open.  In the MRL mice the holes healed closed without a trace.  This set Dr. Katz and her group off on a chain of discoveries based on working with normal and super-healer mice.  In 1998 the group reported on multiple regions in 5 chromosomes connected with wound healing.  The latest discovery is that reported here(ref).The hope is that it may ultimately be possible to regenerate damaged or missing organs, possibly even fingers or limbs in human beings through temporary inactivation of P21. “If humans can be induced to heal like these healer mice, it would be possible to repair skin wounds without scarring, and to induce regrowth of cartilage. Internal healing could be improved, including that of damage from heart attacks, as a 2001 study in PNAS said(ref).”  Spinal cord tissue injury is another potential area of application.

On a personal note, I have been concerned about loss of nerve connections in two fingers and spinal cord tissue damage.  See my posts Nerve regeneration and Spinal cord injury pain – a personal story and a new paradigm.  And I have been looking forward to the still far-off days when these parts of me can be regenerated.  I believe the current research about P21 moves us a step further up the long ladder leading to practical regenerative medicine.

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26. May 2010 by admin.

I have finished drafting the Power Point presentation for the Society’s 39^th annual meeting in Portland Oregon early next month.   I am still polishing up the presentation, fixing mysteriously broken links, adding citations, etc., but I have put it online where it can be previewed by my readers at Towards a Systems Theory of Aging.  The conclusion of the presentation is “The two theories Programmed epigenomic changes and Decline in functioning of the stem cell supply chain are complimentary and equivalent and reflect cutting-edge developments in molecular and cell biology and genomics.  They have the potential for providing a framework for an overall systems view of aging that knits together a large collection of traditional special theories of aging.”  Expect minor changes and improvements between now and the time of the conference. 

With this done, I am happily getting back to my regular blogging activities.  As always, any and all comments are most welcome.

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21. May 2010 by admin.

Readers will notice that blog postings have been coming less frequently recently in recent weeks.  This situation is likely to last until June 7 or so.  A number of temporary professional and personal factors have been conspiring to consume my time making it difficult for me to do the usual careful research that goes into preparing a blog entry.  These include preparing my presentation Towards A Systems View Of Aging for the American Aging Society to be given in Portland Oregon at their annual meeting coming up early in June, unbelievable preparations for an immense wedding for my son Joe that will take place tomorrow, opening up my summer island home on Lake Winnipesaukee, updating  my article Protection Against Radiation - - The Second Line Of Defense to create a version that has been accepted for publication, deworming virus-infested computers, birthdays, anniversaries and other celebrations, handling a number of other family matters, and regularly exercising.  I am fortunate to have a large extended family and very close friends, and I enjoy a rich family and personal life.  All this contributes to my enjoyment of life and my longevity.

The message is that I am passionately committed to the work of this blog and will soon be back at it.   Usage, incidentally, has been continuing to increase about 20% per month.  I am grateful to the increasing number of readers who contribute highly useful comments and for the rest of you readers who are finding value here..

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15. May 2010 by admin.

Melanoma is the most serious form of skin cancer. It is a cancer “–  of melanocytes which are found predominantly in skin but also in the bowel and the eye (see uveal melanoma).  – Melanocytes are normally present in skin, being responsible for the production of the dark pigment melanin.^[2] Despite many years of intensive laboratory and clinical research, the greatest chance of cure is in the early surgical resection of thin tumors(ref).”  In 2009, 68,720 new cases of melanoma were reported in the US and the disease led to 8,650 deaths(ref). “According to a WHO report about 48,000 melanoma related deaths occur worldwide per year(ref).”^[6] 

Melanoma is also among the most-studied cancers.  The US government clinical trials database shows 910 clinical trials for melanoma in various stages.  This blog entry reports a selection of recent (2010) published research findings.

An April 2010 e-publication Vaccination with autologous dendritic cells pulsed with multiple tumor antigens for treatment of patients with malignant melanoma: results from a phase I/II trial provides a testimony to the relative inefficacy of a high-technology vaccine in the face of progressive malignant melanoma.  See the earlier blog post Dendritic cell cancer immunotherapy.  According to the new publication “Dendritic cells are regarded as the most effective antigen presenting cells and coordinators of the immune response and therefore suitable as vaccine basis. Here we present results from a clinical study in which patients with malignant melanoma (MM) with verified progressive disease received vaccination with autologous monocyte-derived mature dendritic cells (DC) pulsed with p53, survivin and telomerase-derived peptides (HLA-A2(+) patients) or with autologous/allogeneic tumor lysate (HLA-A2(-) patients) in combination with low-dose interleukin (IL)-2 and interferon (IFN)-alpha2b. Results: Of 46 patients who initiated treatment, 10 stopped treatment within 1-4 weeks because of rapid disease progression and deterioration. After 8 weeks, 36 patients were evaluable: no patient had an objective response, 11 patients had stable disease (SD); six had continued SD after 4 months, and three patients had prolonged SD for more than 6 months. The mean overall survival time was 9 months, with a significantly longer survival (18.4 months) of patients who attained SD compared with patients with progressive disease (PD) (5 months). Induction of antigen-specific T-cell responses was analyzed by multidimensional encoding of T cells using HLA-A2 major histocompatibility complex (MHC) multimers. Immune responses against five high-affinity vaccine peptides were detectable in the peripheral blood of six out of 10 analyzed HLA-A2(+) patients. There was no observed correlation between the induction of immune responses and disease stabilization. A significant lower blood level of regulatory T cells (CD25(high) CD4 T cells) was demonstrable after six vaccinations in patients with SD compared with PD. Conclusions: Vaccination was feasible and safe.  Treatment-associated SD was observed in 24% of the patients. SD correlated with prolonged survival suggesting a clinical benefit. Differences in the level of regulatory T cells among SD and PD patients could indicate a significant role of these immune suppressive cells”  I decode this to mean that about a quarter of the treated patients extended their mean survival time from 9 months to 18.4 months.  While this is only a Phase I/II study, it casts doubt on whether dendritic cell cancer immunotherapy, a major focus of current cancer research, will provide a magic bullet against aggressive cancers.

A study published this week in the journal Cell A Temporarily Distinct Subpopulation of Slow-Cycling Melanoma Cells Is Required for Continuous Tumor Growth may help explain several key aspects of melanoma, such as its aggressiveness.  The study suggests that there are two important temporary subpopulations of melanoma cells, both tumorigenic but different in their differentiation and replication potentials.  There is at any time a relatively small slowly-dividing population of cells characterized by the JARID1B demethylase biomarker.  This population is stem-cell-like and necessary for continuous tumor growth.  The JARID1B- cells, on the other hand, divide very rapidly but are insufficient for continuous tumor maintenance.  Moreover, JARID1B- cells can transform into being JARID1B+ cells and the other way around.  “Melanomas are highly heterogeneous tumors, but the biological significance of their different subpopulations is not clear. Using the H3K4 demethylase JARID1B (KDM5B/PLU-1/RBP2-H1) as a biomarker, we have characterized a small subpopulation of slow-cycling melanoma cells that cycle with doubling times of >4 weeks within the rapidly proliferating main population. Isolated JARID1B-positive melanoma cells give rise to a highly proliferative progeny. Knockdown of JARID1B leads to an initial acceleration of tumor growth followed by exhaustion which suggests that the JARID1B-positive subpopulation is essential for continuous tumor growth. Expression of JARID1B is dynamically regulated and does not follow a hierarchical cancer stem cell model because JARID1B-negative cells can become positive and even single melanoma cells irrespective of selection are tumorigenic. These results suggest a new understanding of melanoma heterogeneity with tumor maintenance as a dynamic process mediated by a temporarily distinct subpopulation.  º The H3K4 demethylase JARID1B marks a subpopulation of slow-cycling melanoma cells.  º The JARID1B+ subpopulation is required for continuous tumor maintenance .  º Cells can lose or gain JARID1B expression and do not follow a stem cell hierarchy.  º Tumor initiation is not necessarily linked with tumor maintenance.”  A possible implication if the study is that an effective therapy regimen for melanoma will have to target both the fast (JARID1B-) and slow-growing (JARID1B+) cell populations if it is to be effective.  The study is also important for pointing out how the JARID1B- cells can convert themselves into the stem-cell-like JARID1B+ cells, an effect not observed in other cancers.   For background relating to cancer stem cells you can check out the earlier blog entries On cancer stem cells, Big pharma is targeting cancer stem cells, Update on cancer stem cells,  and News on disabling cancer stem cells.

The April 2010 publication Melanoma: Stem cells, sun exposure and hallmarks for carcinogenesis, molecular concepts and future clinical implications reports on a review study, the purpose of which was to identify molecular biomarkers for melanoma.  A vivid view is provided of how melanoma originates: “RESULTS: Melanocyte precursors undergo several genome changes -UV-induced or not- which could be either mutations or epigenetic. These changes provide stem cells with abilities to self-invoke growth signals, to suppress antigrowth signals, to avoid apoptosis, to replicate without limit, to invade, proliferate and sustain angiogenesis. Melanocyte stem cells are able to progressively collect these changes in their genome. These new potential functions, drive melanocyte precursors to the epidermis were they proliferate and might cause benign nevi. In the epidermis, they are still capable of acquiring new traits via changes to their genome. With time, such changes could add up to transform a melanocyte precursor to a malignant melanoma stem cell. CONCLUSIONS: Melanoma cannot be considered a “black box” for researchers anymore. Current trends in the diagnosis and prognosis of melanoma are to individualize treatment based on molecular biomarkers. Pharmacogenomics constitute a promising field with regard to melanoma patients’ treatment. Finally, development of novel monoclonal antibodies is expected to complement melanoma patient care while a number of investigational vaccines could find their way into everyday oncology practice.”  I find it interesting here that the genome changes leading up to a cancer could be epigenetic as well as mutations in the genome.  Until recently the conventional wisdom has been that cancers are caused only by gene mutations. 

The January 2010 review publication Molecular cytogenetics of cutaneous melanocytic lesions - diagnostic, prognostic and therapeutic aspects is concerned with cytogenetic alterations in melanocytic tumours.  The focus in this paper is on molecular biology and genetic changes rather than on cancer stem cells.  “This review demonstrates that at present cytogenetics has mainly increased our understanding of the pathogenesis of melanocytic tumours, with an important role for activation of the mitogen-activated protein kinase (MAPK) signaling pathway in the initiation of melanocytic tumours. Mutations in BRAF (in common naevocellular naevi), NRAS (congenital naevi), HRAS (Spitz naevi) and GNAQ (blue naevi) can all cause MAPK activation. All these mutations seem early events in the development of melanocytic tumours, but by themselves are insufficient to cause progression towards melanoma. Additional molecular alterations are implicated in progression towards melanoma, with different genetic alterations in melanomas at different sites and with varying levels of sun exposure. This genetic heterogeneity in distinct types of naevi and melanomas can be used for the development of molecular tests for diagnostic purposes. However, at the moment only few molecular tests have become of diagnostic value and are performed in daily routine practice. This is caused by lack of large prospective studies on the diagnostic value of molecular tests including follow-up, and by the low prevalence of certain molecular alterations. For the future we foresee an increasing role for cytogenetics in the treatment of melanoma patients with the increasing availability of targeted therapy. Potential targets for metastatic melanoma include genes involved in the MAPK pathway, such as BRAF and RAS. More recently, KIT has emerged as a potential target in melanoma patients. These targeted treatments all need careful evaluation, but might be a promising adjunct for treatment of metastatic melanoma patients, in which other therapies have not brought important survival advantages yet.”  As pointed out in the previously-cited paper, there is a need for identifying biomarkers and developing better molecular tests related to them.

One potentially useful biomarker for melanoma is Nestin, as pointed out in the January 2010 publication Stem cell marker nestin expression in peripheral blood of patients with melanoma.  “There is continued interest in markers indicative of circulating melanoma cells. Nestin is a neuroepithelial intermediate filament protein that was found to be expressed in melanoma and in various cancer stem cells. Objective: We investigated expression of nestin in peripheral blood of melanoma patients. Patients/Methods: We analyzed nestin expression by flow cytometry and by quantitative reverse transcription polymerase chain reaction (qRT-PCR) both in tissues (n=23) and blood samples (n=102) from patients with AJCC stage III-IV melanoma. Forty-six negative controls were also added. Results: Flow cytometry did not reveal nestin expressing cells in peripheral blood of healthy volunteers. In melanoma patients, however, nestin protein was expressed in a proportion of melanoma cells enriched from peripheral blood by immunomagnetic sorting. In melanoma tissue samples a significant correlation was found between mRNAs encoding for nestin and tyrosinase (p=0.001) and Mart-1 (p=0.002), whereas in blood a significant correlation was only observed for tyrosinase (p=0.015), but not for Mart-1 (p=0.53). Nestin expression was higher in stage IV patients compared to stage III/IV with no evidence of disease (NED), positively correlated to tumour burden, and positively correlated to expression of tyrosinase and Mart-1. Conclusions: Nestin showed to be an additional marker of interest for circulating melanoma cells.”

The February 2010 publication Prognostic significance of the hair follicle stem cell marker nestin in patients with malignant melanoma also looked at the use of Nestin as a predictive biomarker, this time related to prognosis of outcome in melanoma patients. “Nestin is an intermediate filament protein, and serves as a hair follicle stem cell and neural stem cell marker. Recent studies have suggested that nestin expression is also important for tumorigenesis. Previous reports from our laboratory have revealed that nestin is a marker of HMB-45-negative melanoma cells in dermal invasive lesions of nodular malignant melanoma. The present study examines nestin expression in malignant melanoma and investigates the relationship between nestin expression and prognosis in patients.  We immunohistochemically stained 78 formalin-fixed and paraffin-embedded malignant melanomas for nestin, HMB-45 and S100 reactivity. We found that nestin, HMB-45 and S100 protein were detected in 56.5%, 88.4% and 100% of malignant melanomas, respectively. The 5-year survival rate of stage I and II nestin-positive cases was significantly decreased compared to the nestin-negative cases (p < 0.05). In addition, the 5-year survival rate exceeded 80% in nestin-negative malignant melanomas at all stages of tumor development. We conclude that nestin expression may be a predictor of poor prognosis in patients with malignant melanoma.”

Similar points are made in the January 2010 paper The stem cell marker nestin predicts poor prognosis in human melanoma.  “These results suggest that nestin expression in both tumoral and endothelial cells may be considered an important early prognostic marker in melanoma.”

Collectively, these papers indicate the importance of developing biomarker tests for melanoma, and the emergence of Nestin as such a biomarker.  I note that the emphasis on discovering reliable predictive biomarkers applies to diseases across the spectrum.  See the recent blog entry Harnessing the engines of finance and commerce for life-extension .  The discovery of disease biomarkers is identified there as central to a new paradigm in medicine, Personalized Predictive Preventative Participatory Medicine.

There is much more going on in the world of Melanoma research and I will pick this thread up again in a later blog entry.  I will also discuss research relating various supplements to melanomas. 

As a final note, by far the most effective protection against melanomas and other skin cancers is UV avoidance.  Wear sunhats, clothes, sunglasses and sunscreen protective against both UVa and UVb when outdoors in the summer and at high altitudes, and stay away from tanning beds.

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12. May 2010 by admin.

This blog entry is about three recent research results where the outcomes were the opposite of what might have been expected. 

1.     Chocolate consumption and depression are correlated 

The April 2919 publication Chocolate and Depressive Symptoms in a Cross-sectional Analysis indicates a surprising relationship between chocolate consumption and mood, namely that consumption and depression seem to be correlated.  “A sample of 1018 adults (694 men and 324 women) from San Diego, California, without diabetes or known coronary artery disease was studied in a cross-sectional analysis. The 931 subjects who were not using antidepressant medications and provided chocolate consumption information were the focus of the analysis. Mood was assessed using the Center for Epidemiologic Studies Depression Scale (CES-D). Cut points signaling a positive depression screen result (CES-D score, 16) and probable major depression (CES-D score, 22) were used. Chocolate servings per week were provided by 1009 subjects. — Results  Those screening positive for possible depression (CES-D score 16) had higher chocolate consumption (8.4   servings per month) than those not screening positive (5.4 servings per month) (P = .004); those with still higher CES-D scores ( 22) had still higher chocolate consumption (11.8 servings per month) (P value for trend, <.01). These associations extended to both men and women. These findings did not appear to be explained by a general increase in fat, carbohydrate, or energy intake.” 

The study does not indicate a causal connection or, if one exists, direction of the cause.  Does eating chocolate induce depression or do depressed people eat more chocolate?  If it is the first case, the result is the opposite of what I would have thought.  In the second case, eating chocolate while feeling depressed seems completely understandable to me since I am a chocolate-eater.  I have mentioned health benefits of consuming chocolate in several blog entries.  For example, see Health and longevity benefits of dark chocolate. 

2.     Cognition and HIV HAART therapy

A rather interesting recent result is that cognition improves in AIDS patients when HAART therapy is discontinued.  HAART stands for highly active antiretroviral therapy and is normally administered as a cocktail of multiple antiretroviral drugs combined into a single pill.  For a long time it has been thought that anti-retroviral therapy in AIDS patients improves cognitive functioning.  For example. the 1999 publication Positive and sustained effects of highly active antiretroviral therapy on HIV-1-associated neurocognitive impairment reports for a sample of 26 patients” “Conclusion: HAART produces a positive and sustained effect on neurocognitive impairment in HIV-infected patients. A reduction of plasma viral load was associated with the regression of neuropsychological test abnormalities.” The recent result associated with discontinuation of HAART therapy again is the opposite of what was expected. 

The April 2010 publication Neurocognitive effects of treatment interruption in stable HIV-positive patients in an observational cohort reports “Methods: Neurocognitive function was assessed as part of ACTG 5170, a multicenter, prospective observational study of HIV-infected subjects who elected to discontinue ART. Eligible subjects had CD4 count >350 cells/mm3, had HIV RNA viral load <55,000 cp/mL, and were on ART ( 2 drugs) for 6 months. Subjects stopped ART at study entry and were followed for 96 weeks with a neurocognitive examination. — Results: A total of 167 subjects enrolled with a median nadir CD4 of 436 cells/mm3 and 4.5 median years on ART. Significant improvements in mean neuropsychological scores of 0.22, 0.39, 0.53, and 0.74 were found at weeks 24, 48, 72, and 96 (all p < 0.001). In the 46 subjects who restarted ART prior to week 96, no significant changes in neurocognitive function were observed. — Conclusion: Subjects with preserved immune function found that neurocognition improved significantly following antiretroviral treatment (ART) discontinuation. The balance between the neurocognitive cost of untreated HIV viremia and the possible toxicities of ART require consideration.”   

It is interesting that the scores continued to improve during the 96 week period following discontinuation of the HAART therapy.  An important point is that the typical 2010 HAART therapy is different than the 1999 therapy and more effective in its anti-viral effects. So, it might be that one or more of the drugs in the 2010 HAART therapy that were not in the 1999 therapy are responsible for cognitive impairment.

3.     Vitamin-B therapy does not help diabetic nephropathy

We supplement-oriented types often have a default assumption that vitamin therapies are likely to have positive effects.     Specifically, B vitamins have been thought to be useful for treating neurological pathologies.  Not necessarily so, according to the April 2010 publication Effect of B-Vitamin Therapy on Progression of Diabetic Nephropathy.  “Context  Hyperhomocysteinemia is frequently observed in patients with diabetic nephropathy. B-vitamin therapy (folic acid, vitamin B₆, and vitamin B₁₂) has been shown to lower the plasma concentration of homocysteine.  Objective  To determine whether B-vitamin therapy can slow progression of diabetic nephropathy and prevent vascular complications.  Design, Setting, and Participants  A multicenter, randomized, double-blind, placebo-controlled trial (Diabetic Intervention with Vitamins to Improve Nephropathy [DIVINe]) at 5 university medical centers in Canada conducted between May 2001 and July 2007 of 238 participants who had type 1 or 2 diabetes and a clinical diagnosis of diabetic nephropathy.  Intervention  Single tablet of B vitamins containing folic acid (2.5 mg/d), vitamin B₆ (25 mg/d), and vitamin B₁₂ (1 mg/d), or matching placebo.  Main Outcome Measures  Change in radionuclide glomerular filtration rate (GFR) between baseline and 36 months. Secondary outcomes were dialysis and a composite of myocardial infarction, stroke, revascularization, and all-cause mortality. Plasma total homocysteine was also measured.  Results  The mean (SD) follow-up during the trial was 31.9 (14.4) months. At 36 months, radionuclide GFR decreased by a mean (SE) of 16.5 (1.7) mL/min/1.73 m² in the B-vitamin group compared with 10.7 (1.7) mL/min/1.73 m² in the placebo group (mean difference, –5.8; 95% confidence interval [CI], –10.6 to –1.1; P = .02). There was no difference in requirement of dialysis (hazard ratio [HR], 1.1; 95% CI, 0.4-2.6; P = .88). The composite outcome occurred more often in the B-vitamin group (HR, 2.0; 95% CI, 1.0-4.0; P = .04). Plasma total homocysteine decreased by a mean (SE) of 2.2 (0.4) µmol/L at 36 months in the B-vitamin group compared with a mean (SE) increase of 2.6 (0.4) µmol/L in the placebo group (mean difference, –4.8; 95% CI, –6.1 to –3.7; P < .001, in favor of B vitamins).  Conclusion  Among patients with diabetic nephropathy, high doses of B vitamins compared with placebo resulted in a greater decrease in GFR and an increase in vascular events.” GFR stands for  glomerular filtration rate, the best test to measure level of kidney function and determine the stage of  a kidney disease.  Lower is worse and scores below 15 indicate a pathology.  The B-vitamins did lower homocysteine but they also significantly lowered GFR indicating an overall negative effect in the population studied.

A few observations with respect to the life sciences and longevity:

·        Reasonable conjectures and conclusions might not be valid.

·        What we know we don’t know expands faster than what we know.  Uncertainty expands faster than the certainty. 

·         We have to live with that growing uncertainty.  For example, does eating chocolate cause depression or is that a good thing to do if you have depression?  In the past this was not something to be concerned about.. 

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6. May 2010 by admin.

I have posted several blog entries related to Alzheimer’s Disease (AD), including  New views of Alzheimer’s disease and new approaches to treating it,  The social cost of Alzheimer’s disease and late-life dementia, Diet and cognition, Warding off Alzheimer’s Disease and things in my diet,  and a short post Deconstructing Alzheimer’s Disease – role of mitochondria.  The purpose of this blog entry is to report on some additional mostly-recent research findings, focusing particularly on dietary substances and supplements and their impacts on AD.  Topics covered are research related to 1.  Relationship of SIRT1 and taking resveratrol to AD, 2. Dietary substances and supplements and AD, 3. Clinical trials of dietary substances for AD, and 4. Curcumin and AD. 

1.      SIRT1 and Resveratrol and Alzheimer’s Disease 

I have discussed the SIRT1 gene and protein in numerous contexts in this blog.  In the blog entry SIRT1, mTOR, NF-kappaB and resveratrol I mentioned links between SIRT1 activation, mTOR signaling suppression, and inhibition of NF-kappaB.  And I discussed how some researchers think activation of SIRT1 might confer a strong a therapeutic effect for control of Alzheimer’s disease.  Several review articles published in the last couple of years articulate that hypothesis and suggest a potential role for resveratrol in controlling AD.  These articles include the March 2010 e-publication Resveratrol as a Therapeutic Agent for Neurodegenerative Diseases, the 2009 publication Resveratrol and neurodegenerative diseases: activation of SIRT1 as the potential pathway towards neuroprotection and the 2008 publication Modulation of sirtuins: new targets for antiageing. “ — increasing SIRT1 has been found to protect cells against amyloid-beta-induced ROS production and DNA damage, thereby reducing apoptotic death in vitro. Moreover, it has been demonstrated that Alzheimer’s and Huntington’s disease neurons are rescued by the over-expression of SIRT1, induced by either caloric restriction or administration of resveratrol, a potential activator of this enzyme. The therapeutic use of resveratrol (a polyphenol present in red wines) and other related compounds, which utilize SIRT1 pathway modulators, in treating aging-related brain disorders will be discussed in this review(ref).”   

While these and other review articulate this hypothesis (that resveratrol and SIRT1 activation could be useful in controlling AD), current experimental evidence to this effect seems to be scarce.  There were earlier experiments indicating that resveratrol is neuroprotective against beta-amyloid-induced neurotoxicity in rat neurons(ref), but I am unaware of any tests of the effects of taking resveratrol on warding off or treating human Alzheimer’s disease.  Personally, I think resveratrol is a great supplement but the hypothesis that resveratrol and SIRT1 activation could be useful in controlling AD remains an untested conjecture as far as I know.  Theoretical reasons for taking resveratrol may be strong but clinical data on effectiveness in AD is nonexistent. 

2.     Alzheimer’s Disease, dietary substances and supplements

A large number publications based on in-vitro and mouse studies suggest that polyphenols occurring in common dietary substances and supplements may be useful for treating or preventing the onset of Alzheimer’s or other neurodegenerative diseases.  Examples are:

·          (2010) l-theanine protects the APP (Swedish mutation) transgenic SH-SY5Y cell against glutamate-induced excitotoxicity via inhibition of the NMDA receptor pathway. 

·         (2010) [Review on the neuroprotective effects of green tea polyphenols for the treatment of neurodegenerative diseases] 

·         (2010) Fish oil enhances anti-amyloidogenic properties of green tea EGCG in Tg2576 mice.

·         (2010) Nanolipidic particles improve the bioavailability and alpha-secretase inducing ability of epigallocatechin-3-gallate (EGCG) for the treatment of Alzheimer’s disease.

·          (2010)  Naturally occurring phytochemicals for the prevention of Alzheimer’s disease.

·         The 2009 study Cinnamon extract inhibits tau aggregation associated with Alzheimer’s disease in vitro reports “An aqueous extract of Ceylon cinnamon (C. zeylanicum) is found to inhibit tau aggregation and filament formation, hallmarks of Alzheimer’s disease (AD). The extract can also promote complete disassembly of recombinant tau filaments and cause substantial alteration of the morphology of paired-helical filaments isolated from AD brain.”

·         (2009) Natural Compound In Extra-Virgin Olive Oil — Oleocanthal — May Help Prevent, Treat Alzheimer’s 

·         (2009)  l-Theanine, an amino acid in green tea, attenuates beta-amyloid-induced cognitive dysfunction and neurotoxicity: reduction in oxidative damage and inactivation of ERK/p38 kinase and NF-kappaB pathways.

·         (2009) Neuroprotective molecular mechanisms of (-)-epigallocatechin-3-gallate: a reflective outcome of its antioxidant, iron chelating and neuritogenic properties. 

·         (2009) Green tea (-)-epigallocatechin-3-gallate inhibits beta-amyloid-induced cognitive dysfunction through modification of secretase activity via inhibition of ERK and NF-kappaB pathways in mice.

·         (2009) Green tea polyphenol (-)-epigallocatechin-3-gallate enhances the inhibitory effect of huperzine A on acetylcholinesterase by increasing the affinity with serum albumin.

·         (2009) Neuroprotective effect of epigallocatechin-3-gallate against beta-amyloid-induced oxidative and nitrosative cell death via augmentation of antioxidant defense capacity.

·         (2009) Botanical phenolics and brain health.

·         (2009) Natural antioxidants protect neurons in Alzheimer’s disease and Parkinson’s disease.

·         (2008) Simultaneous manipulation of multiple brain targets by green tea catechins: a potential neuroprotective strategy for Alzheimer and Parkinson diseases.

·          (2008) Cell signaling pathways and iron chelation in the neurorestorative activity of green tea polyphenols: special reference to epigallocatechin gallate (EGCG).

·          (2008) Lipoic acid as an anti-inflammatory and neuroprotective treatment for Alzheimer’s disease. 

·         (2008) Targeting multiple neurodegenerative diseases etiologies with multimodal-acting green tea catechins.

·         (2008) Green tea epigallocatechin-3-gallate (EGCG) reduces beta-amyloid mediated cognitive impairment and modulates tau pathology in Alzheimer transgenic mice.

·         (2008) Green tea catechins prevent cognitive deficits caused by Abeta1-40 in rats. 

·         (2008) Epicatechin gallate increases glutamate uptake and S100B secretion in C6 cell lineage. 

·         (2008) Neuroprotective effects of (-)-epigallocatechin-3-gallate (EGCG) on paraquat-induced apoptosis in PC12 cells. 

Again, the clinical evidence for the effectiveness of most of these substances against Alzheimer’s disease seems to range from being very limited to nonexistent.  Pharmaceutical companies are understandably only willing to spend millions or hundreds of millions of dollars on clinical trials of substances that they own the rights to – and that excludes foods and plant-based supplements.  However, some public agencies have been stepping in to fill the gaps. 

3.     Clinical Trials of dietary substances for AD

The clinicaltrials.gov database shows some 781 clinical trials for AD of which approximately 100 are ongoing.  I went only through the first 300 trials listed to come up with the following related to dietary substances or supplements.  

The Department of Veterans Affairs is currently sponsoring two such trials now in the recruiting phase, A Single Center, Multi-site, Randomized, Double-blind, Placebo-controlled Trial of Resveratrol With Glucose and Malate (RGM) to Slow the Progression of Alzheimer’s Disease.  And A Randomized, Clinical Trial of Vitamin E and Memantine in Alzheimer’s Disease (TEAM-AD)   Another clinical trial Lipoic Acid and Omega-3 Fatty Acids for Alzheimer’s Disease sponsored by the Oregon Health and Science University is not yet recruiting.  The clinical trial Lutein and Alzheimer’s Disease Study (LAD) sponsored by the Oregon Health and Science University is listed as still in the recruiting phase.

A clinical study Prevention of Alzheimer’s Disease by Vitamin E and Selenium (PREADVISE) sponsored by the National Institute on Aging (NIA) is ongoing.

4.     Completed clinical trials of dietary substances for AD

A clinical trial VITAL - VITamins to Slow ALzheimer’s Disease (Homocysteine Study) sponsored by the National Institute on Aging has been completed.  The substances tested were Folate, Vitamin B6 and Vitamin B12.  The 18-month double blind study involved in 409 participants in 38 study locations and was designed to measure differences in decline of cognitive functioning.  The study results, reported in the publication High-dose B vitamin supplementation and cognitive decline in Alzheimer disease: a randomized controlled trial, were negative. “A higher quantity of adverse events involving depression was observed in the group treated with vitamin supplements. CONCLUSION: This regimen of high-dose B vitamin supplements does not slow cognitive decline in individuals with mild to moderate AD.” 

Another completed clinical trial is OmegAD (Omega-3 and Alzheimer’s Disease), sponsored by Karolinska University Hospital.  The randomized double-blind trial involved omega-3 fatty acid treatment of 174 patients with mild to moderate Alzheimer’s Disease .  The results were underwhelming as reported in the 2006 paper Omega-3 fatty acid treatment in 174 patients with mild to moderate Alzheimer disease: OmegAD study: a randomized double-blind trial.  “CONCLUSIONS: Administration of omega-3 fatty acid in patients with mild to moderate AD did not delay the rate of cognitive decline according to the MMSE or the cognitive portion of the Alzheimer Disease Assessment Scale. However, positive effects were observed in a small group of patients with very mild AD.” 

A clinical trial Long-term Use of Galantamine Versus Nootropics (Memory Enhancing Drugs) in Patients With Alzheimer’s Dementia Under Conditions of Daily Routine sponsored by Janssen-Cilag G.m.b.H has been completed but no results of it have yet been published.  The nootropics were evaluated secondarily, galantamine being the main focus of the study.  The nootropics included ginkgo biloba, nicergoline, and piracetam. 

A clinical trial Effect of Panax Ginseng on the Cognitive Performance in Alzheimer’s Disease sponsored by the Seoul National University Hospital has been completed.  The results are published in the 2008 publication Panax ginseng enhances cognitive performance in Alzheimer disease.  “Consecutive AD patients were randomly assigned to the ginseng (n=58) or the control group (n=39), and the ginseng group was treated with Panax ginseng powder (4.5 g/d) for 12 weeks. Cognitive performances were monitored using the mini-mental state examination (MMSE) and Alzheimer disease assessment scale (ADAS) during 12 weeks of the ginseng treatment and at 12 weeks after the ginseng discontinuation. MMSE and ADAS scales showed no baseline difference between the groups. After ginseng treatment, the cognitive subscale of ADAS and the MMSE score began to show improvements and continued up to 12 weeks (P=0.029 and P=0.009 vs. baseline, respectively). After discontinuing ginseng, the improved ADAS and MMSE scores declined to the levels of the control group. These results suggest that Panax ginseng is clinically effective in the cognitive performance of AD patients.” 

A number of clinical trials are shown as completed but no publications report on their results.A competed pilot clinical trial Alzheimer’s Disease: Potential Benefit of Isoflavones shows no publication history up to this point. A clinical trial Fish Oil and Alpha Lipoic Acid in Treating Alzheimer’s Disease Phases I and II sponsored by the Oregon Health and Science University appears to be completed in 2007 but no associated publication is listed.  The study Curcumin in Patients With Mild to Moderate Alzheimer’s Disease sponsored by the John Douglas French Foundation is shown as completed but I could find no publication reporting on it. 

Please note that the above is only a sample of the available clinical trial information.  I reviewed only 300 out of 781 studies listed.  Assuming the above-mentioned trials are representative, I am struck by how thin our clinical trial information is with respect to the effectiveness of supplements against Alzheimer’s disease 

4.     Curcumin and Alzheimer’s Disease

There seems to be a considerable literature with respect to curcumin and AD(ref).  While I have not independently verified the statements and citations therein, the October 2, 2007  item Turmeric and Alzheimer ’s disease by Jacob Schor, ND appears to me to be particularly informative.  He makes a compelling case for why curcumin may be helpful in prevention of AD. He points out that the incidence of AD in India where curcumin is commonly found in foods is a quarter that in the US. (Note that some of this might be explainable by differing demographic factors like life expectancy.)  As I have been pointing out, we lack the information that could be provided by a large and well-designed clinical trial of curcumin in AD.  According to the Pauling Foundation web site: “In Alzheimer’s disease, a peptide called amyloid beta forms aggregates (oligomers), which accumulate in the brain and form deposits known as amyloid plaques (72). Inflammation and oxidative damage are also associated with the progression of Alzheimer’s disease (73). Curcumin has been found to inhibit amyloid beta oligomer formation in vitro (74). When injected peripherally, curcumin was found to cross the blood brain barrier in an animal model of Alzheimer’s disease (74). In animal models of Alzheimer’s disease, dietary curcumin has decreased biomarkers of inflammation and oxidative damage, amyloid plaque burden in the brain, and amyloid beta-induced memory deficits (74-77). It is not known whether curcumin taken orally can cross the blood brain barrier or inhibit the progression of Alzheimer’s disease in humans. As a result of the promising findings in animal models, clinical trials of oral curcumin supplementation in patients with early Alzheimer’s disease are under way (59, 78). The results of a 6-month trial in 27 patients with Alzheimer’s disease found that oral supplementation with up to 4 g/day of curcumin was safe (4). Larger controlled trials are needed to determine whether or not oral curcumin supplementation is efficacious in Alzheimer’s disease.” 

Please see the medical disclaimer for this blog.

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3. May 2010 by admin.

As a kid in a traditional Italian family, I was raised on olive oil.  And I now consume generous quantities of extra-virgin olive oil (EVOO) just about every day.  For one thing, I love its taste.  I am so hooked on EVOO that I shudder when a friend serves me a wonderful meal with a great salad, but sets out bottles of commercial salad dressing instead of dressing it with EVOO.  I am particularly bothered by bottled dressings with big labels saying MADE WITH REAL ITALIAN OLIVE OIL and showing ladies in colorful dresses carrying baskets of olives while the very fine print on the back of the bottle lists the amount of olive oil as only 15%, and it’s not even extra-virgin oil.  I have always known that olive oil is good for me.  But for most of my life I could not respond intelligently if somebody asked me “why?”  I thought here, as a break from heavy stuff in molecular biology, I would addresses that question and review some of the research on olive oil.  I particularly focus on EVOO. 

Olive oil and the Mediterranean Diet

There is a two-part generic argument often heard for olive oil.  The first part is that a Mediterranean Diet contributes significantly to health and longevity.  The second part is that olive oil is an essential component of a Mediterranean diet and therefore must be one of the key “good for you” components.  There seems to be good research evidence for the first-part argument as outlined in my August 2009 blog post Recent research on the Mediterranean diet.  The second part of the argument by itself does not meet the “beyond a reasonable doubt” test needed to convict somebody in a court trial.  What if something else in the Mediterranean Diet is providing most of the benefits, like the tomatoes?  The rest of this blog entry will establish the value of EVOO beyond a reasonable doubt.

Olive oil and heart disease risk

A good place to start is with a carefully controlled and fairly-large international 2006 study that  directly relates phenolic content of olive oils to familiar lipid levels like HDL and triglycerides:  The effect of polyphenols in olive oil on heart disease risk factors: a randomized trial “BACKGROUND: Virgin olive oils are richer in phenolic content than refined olive oil. — OBJECTIVE: To evaluate whether the phenolic content of olive oil further benefits plasma lipid levels and lipid oxidative damage compared with monounsaturated acid content. DESIGN: Randomized, crossover, controlled trial. SETTING: 6 research centers from 5 European countries. PARTICIPANTS: 200 healthy male volunteers. MEASUREMENTS: Glucose levels, plasma lipid levels, oxidative damage to lipid levels, and endogenous and exogenous antioxidants at baseline and before and after each intervention. INTERVENTION: In a crossover study, participants were randomly assigned to 3 sequences of daily administration of 25 mL of 3 olive oils. Olive oils had low (2.7 mg/kg of olive oil), medium (164 mg/kg), or high (366 mg/kg) phenolic content but were otherwise similar. Intervention periods were 3 weeks preceded by 2-week washout periods. RESULTS: A linear increase in high-density lipoprotein (HDL) cholesterol levels was observed for low-, medium-, and high-polyphenol olive oil: mean change, 0.025 mmol/L (95% CI, 0.003 to 0.05 mmol/L), 0.032 mmol/L (CI, 0.005 to 0.05 mmol/L), and 0.045 mmol/L (CI, 0.02 to 0.06 mmol/L), respectively. Total cholesterol-HDL cholesterol ratio decreased linearly with the phenolic content of the olive oil. Triglyceride levels decreased by an average of 0.05 mmol/L for all olive oils. Oxidative stress markers decreased linearly with increasing phenolic content. Mean changes for oxidized low-density lipoprotein levels were 1.21 U/L (CI, -0.8 to 3.6 U/L), -1.48 U/L (-3.6 to 0.6 U/L), and -3.21 U/L (-5.1 to -0.8 U/L) for the low-, medium-, and high-polyphenol olive oil, respectively. LIMITATIONS: The olive oil may have interacted with other dietary components, participants’ dietary intake was self-reported, and the intervention periods were short. CONCLUSIONS: Olive oil is more than a monounsaturated fat. Its phenolic content can also provide benefits for plasma lipid levels and oxidative damage. International Standard Randomised Controlled Trial number: ISRCTN09220811”.  So, in only three weeks cholesterol and triglyceride scores improved in the olive oil takers and the scores increased most markedly in those taking the olive oil with the most phenolic content, i.e., the extra-virgin olive oil. And the result is not just because the olive oil is a “good fat.”

The 2007 publication Changes in the phenolic content of low density lipoprotein after olive oil consumption in men. A randomized crossover controlled trial reports on a trial cohort of 30 men, and my impression is that this cohort may have been part of the larger cohort of the first study mentioned above.  The writeup has a somewhat different focus, however, focusing on the antioxidant properties of virgin olive oil. “Olive oil decreases the risk of CVD (cardiovascular disease). This effect may be due to the fatty acid profile of the oil, but it may also be due to its antioxidant content which differs depending on the type of olive oil. In this study, the concentrations of oleic acid and antioxidants (phenolic compounds and vitamin E) in plasma and LDL were compared after consumption of three similar olive oils, but with differences in their phenolic content. Thirty healthy volunteers participated in a placebo-controlled, double-blind, crossover, randomized supplementation trial. Virgin, common, and refined olive oils were administered during three periods of 3 weeks separated by a 2-week washout period. Participants were requested to ingest a daily dose of 25 ml raw olive oil, distributed over the three meals of the day, during intervention periods. All three olive oils caused an increase in plasma and LDL oleic acid (P < 0.05) content. Olive oils rich in phenolic compounds led to an increase in phenolic compounds in LDL (P < 0.005). The concentration of phenolic compounds in LDL was directly correlated with the phenolic concentration in the olive oils. The increase in the phenolic content of LDL could account for the increase of the resistance of LDL to oxidation, and the decrease of the in vivo oxidized LDL, observed in the frame of this trial. Our results support the hypothesis that a daily intake of virgin olive oil promotes protective LDL changes ahead of its oxidation.”

Coming back to the Mediterranean Diet, the 2005 review article The phenolic compounds of olive oil: structure, biological activity and beneficial effects on human health relates the phenolic content of olive oil to the diet’s salutary benefits. “The Mediterranean diet is rich in vegetables, cereals, fruit, fish, milk, wine and olive oil and has salutary biological functions. Epidemiological studies have shown a lower incidence of atherosclerosis, cardiovascular diseases and certain kinds of cancer in the Mediterranean area. Olive oil is the main source of fat, and the Mediterranean diet’s healthy effects can in particular be attributed not only to the high relationship between unsaturated and saturated fatty acids in olive oil but also to the antioxidant property of its phenolic compounds. The main phenolic compounds, hydroxytyrosol and oleuropein, which give extra-virgin olive oil its bitter, pungent taste, have powerful antioxidant activity both in vivo and in vitro.”

Again, the messages appear to be that it is the phenolic ingredients in olive oil that are important, that their antioxidant activities are important and that the most healthful olive oil is the one with the most concentration of the polyphenols, namely first-press extra-virgin olive oil (EVOO). 

Further, though olive oil seems to be a simple substance the biochemical activities of olive oil polyphenols is not simple, as discussed in the 2008 paper Nutritional benefit of olive oil: the biological effects of hydroxytyrosol and its arylating quinone adducts.  “A unique characteristic of olive oil is its enrichment in oleuropein, a member of the secoiridoid family, which hydrolyzes to the catechol hydroxytyrosol and functions as a hydrophilic phenolic antioxidant that is oxidized to its catechol quinone during redox cycling. Little effort has been spent on exploring the biological properties of the catechol hydroxytyrosol quinone, a strong arylating electrophile that forms Michael adducts with thiol nucleophiles in glutathione and proteins. This study compares the chemical and biological characteristics of hydroxytyrosol with those of the tocopherol family in which Michael adducts of arylating desmethyltocopherol quinones have been identified and correlated with biologic properties including cytotoxicity and induction of endoplasmic reticulum stress. It is noted that hydroxytyrosol and desmethyltocopherols share many similarities, suggesting that Michael adduct formation by an arylating quinone electrophile may contribute to the biological properties of both families, including the unique nutritional benefit of olive oil.” 

The 2007 document The olive oil antioxidant hydroxytyrosol efficiently protects against the oxidative stress-induced impairment of the NObullet response of isolated rat aorta reports “Moreover, hydroxytyrosol was found to be a potent OH(*) scavenger, which can be attributed to its catechol moiety. Because of its amphiphilic characteristics (octanol-water partitioning coefficient = 1.1), hydroxytyrosol will readily cross membranes and provide protection in the cytosol and membranes, including the water-lipid interface. The present study provides a molecular basis for the contribution of hydroxytyrosol to the benefits of the Mediterranean diet.”

A number of other published studies confirm these messages like the 2004 publication  Effects of differing phenolic content in dietary olive oils on lipids and LDL oxidation–a randomized controlled trial and the 2010 publication Biological activities of phenolic compounds present in virgin olive oil, the 2005 report International conference on the healthy effect of virgin olive oil, the 2009 report Chemistry and health of olive oil phenolics, and a number of others. 

Olive oil and cancers

A number of studies relate the effects of the active polyphenols in olive oil to killing (induction of apoptosis in) cancer cells.  For example the 2009 study Anti-proliferative and apoptotic effects of oleuropein and hydroxytyrosol on human breast cancer MCF-7 cells reports “Olive oil intake has been shown to induce significant levels of apoptosis in various cancer cells. These anti-cancer properties are thought to be mediated by phenolic compounds present in olive. These beneficial health effects of olive have been attributed, at least in part, to the presence of oleuropein and hydroxytyrosol. In this study, oleuropein and hydroxytyrosol, major phenolic compound of olive oil, was studied for its effects on growth in MCF-7 human breast cancer cells using assays for proliferation (MTT assay), cell viability (Guava ViaCount assay), cell apoptosis, cellcycle (flow cytometry). Oleuropein or hydroxytyrosol decreased cell viability, inhibited cell proliferation, and induced cell apoptosis in MCF-7 cells. Result of MTT assay showed that 200 mug/mL of oleuropein or 50 mug/mL of hydroxytyrosol remarkably reduced cell viability of MCF-7 cells. Oleuropein or hydroxytyrosol decrease of the number of MCF-7 cells by inhibiting the rate of cell proliferation and inducing cell apoptosis. Also hydroxytyrosol and oleuropein exhibited statistically significant block of G(1) to S phase transition manifested by the increase of cell number in G(0)/G(1) phase.”

The 2009 study Extra-virgin olive oil polyphenols inhibit HER2 (erbB-2)-induced malignant transformation in human breast epithelial cells: relationship between the chemical structures of extra-virgin olive oil secoiridoids and lignans and their inhibitory activities on the tyrosine kinase activity of HER2 reports “Extra-virgin olive oil (EVOO - the juice of the olive obtained solely by pressing and consumed without any further refining process) is unique among other vegetable oils because of the high level of naturally occurring phenolic compounds. We explored the ability of EVOO polyphenols to modulate HER2 tyrosine kinase receptor-induced in vitro transformed phenotype in human breast epithelial cells. — EVOO polyphenols induced strong tumoricidal effects by selectively triggering high levels of apoptotic cell death in HER2-positive MCF10A/HER2 cells but not in MCF10A/pBABE matched control cells. EVOO lignans and secoiridoids prevented HER2-induced in vitro transformed phenotype as they inhibited colony formation of MCF10A/HER2 cells in soft-agar. Our current findings not only molecularly support recent epidemiological evidence revealing that EVOO-related anti-breast cancer effects primarily affect the occurrence of breast tumors over-expressing the type I receptor tyrosine kinase HER2 but further suggest that the stereochemistry of EVOO-derived lignans and secoiridoids might provide an excellent and safe platform for the design of new HER2 targeted anti-breast cancer drugs.”

The 2008 paper Analyzing effects of extra-virgin olive oil polyphenols on breast cancer-associated fatty acid synthase protein expression using reverse-phase protein microarrays is another of several more relating EVOO to breast cancer. “These findings reveal for the first time that phenolic fractions, directly extracted from EVOO, may induce anti-cancer effects by suppressing the expression of the lipogenic enzyme FASN in HER2-overexpressing breast carcinoma cells, thus offering a previously unrecognized mechanism for EVOO-related cancer preventive effects.”

The 2008 document tabAnti-HER2 (erbB-2) oncogene effects of phenolic compounds directly isolated from commercial Extra-Virgin Olive Oil (EVOO) relates to the same theme.  “Among the fractions mainly containing the single phenols hydroxytyrosol and tyrosol, the polyphenol acid elenolic acid, the lignans (+)-pinoresinol and 1-(+)-acetoxypinoresinol, and the secoiridoids deacetoxy oleuropein aglycone, ligstroside aglycone, and oleuropein aglycone, all the major EVOO polyphenols (i.e. secoiridoids and lignans) were found to induce strong tumoricidal effects within a micromolar range by selectively triggering high levels of apoptotic cell death in HER2-overexpressors. Small interfering RNA-induced depletion of HER2 protein and lapatinib-induced blockade of HER2 tyrosine kinase activity both significantly prevented EVOO polyphenols-induced cytotoxicity. EVOO polyphenols drastically depleted HER2 protein and reduced HER2 tyrosine autophosphorylation in a dose- and time-dependent manner. EVOO polyphenols-induced HER2 downregulation occurred regardless the molecular mechanism contributing to HER2 overexpression (i.e. naturally by gene amplification and ectopically driven by a viral promoter). Pre-treatment with the proteasome inhibitor MG132 prevented EVOO polyphenols-induced HER2 depletion. CONCLUSION: The ability of EVOO-derived polyphenols to inhibit HER2 activity by promoting the proteasomal degradation of the HER2 protein itself, together with the fact that humans have safely been ingesting secoiridoids and lignans as long as they have been consuming olives and OO, support the notion that the stereochemistry of these phytochemicals might provide an excellent and safe platform for the design of new HER2-targeting agents.”   

It is interesting to me that the emphasis in the last-mentioned study and in several other studies seems to be not on promoting the use of EVOO as an anti-cancer health measure but rather on identifying biochemical pathways on which to base new drug developments.  Perhaps this is because the point of departure of most of these studies is acknowledging the health benefits of a Mediterranean diet and consuming lots of olive oil.  And, perhaps cynically, I wonder if it reflects research funding sources for whom finding a new blockbuster drug may be more important than public health. 

A 2010 study publication Extra-virgin olive oil-enriched diet modulates DSS-colitis-associated colon carcinogenesis in mice reports “RESULTS: Disease activity index (DAI) was significantly higher on SFO (sunflower oil) vs. EVOO diet at the end of the experimental period. EVOO-fed mice showed less incidence and multiplicity of tumors than in those SFO-fed mice. beta-catenin immunostaining was limited to cell membranes in control groups, whereas translocation from the cell membrane to the cytoplasm and/or nucleus was showed in DSS-treated groups and its expression was higher in SFO-fed animals. Cytokine production was significantly enhanced in SFO-fed mice, while this increase was not significant in EVOO-fed mice. Conversely, cyclooxigenase-2 (COX-2) and inducible nitric oxidase synthase (iNOS) expression were significantly lower in the animal group fed with EVOO than in the SFO group. CONCLUSIONS: These results confirm that EVOO diet has protective/preventive effect in the UC-associated CRC. This beneficial effect was correlated with a better DAI, a minor number of dysplastic lesions, a lower beta-catenin immunoreactivity, a proinflammatory cytokine levels reduction, a non modification of p53 expression and, COX-2 and iNOS reduction in the colonic tissue.”   

Olive oil and inflammation

A study in done back 2001 Protective effects upon experimental inflammation models of a polyphenol-supplemented virgin olive oil diet again demonstrated a dose-dependent effect of olive oil polyphenols in protecting rats from induced inflammation damage. “CONCLUSIONS: This study demonstrates that virgin olive oil with a higher content of polyphenolic compounds, similar to that of extra virgin olive oil, shows protective effects in both models of inflammation and improves the disease associated loss of weight. This supplementation also augmented the effects of drug therapy.”   

Synergy of olive oil with other supplements  

A 2006 study Intestinal anti-inflammatory activity of combined quercitrin and dietary olive oil supplemented with fish oil, rich in EPA and DHA (n-3) polyunsaturated fatty acids, in rats with DSS-induced colitis found synergy between administration of fish oil, quercetin and olive oil in a rat model of colitis.  “In addition, a complete restoration of colonic glutathione content, which was depleted as a consequence of the colonic insult, was obtained in rats treated with QR plus FO diet; this content was even higher than that obtained when colitic rats were treated with FO diet alone. When compared with the control colitic group, the combined treatment was also associated with a lower colonic nitric oxide synthase and cyclooxygenase-2 expression as well as with a significant reduction in different colonic proinflammatory mediators assayed, i.e. leukotriene B(4), tumor necrosis factor alpha and interleukin 1beta, showing a significantly greater inhibitory effect of the latter in comparison with rats receiving FO diet without the flavonoids (quercetin). CONCLUSIONS: These results support the potential synergism between the administration of the flavonoid and the incorporation of olive oil and n-3 PUFA to the diet for the treatment of these intestinal inflammatory disorders.”

EVOO and gene expression

Finally, I want to mention that new studies are starting to look at the effects of EVOO on gene expression.  For example, the 2010 publication Gene expression changes in mononuclear cells from patients with metabolic syndrome after acute intake of phenol-rich virgin olive oil.  “BACKGROUND: Previous studies have shown that acute intake of high-phenol virgin olive oil reduces pro-inflammatory, pro-oxidant and pro-thrombotic markers compared with low phenols virgin olive oil, but it remains unclear if the effects attributed to its phenolic fraction are exerted at the transcriptional level in vivo. To achieve this goal, we aimed at identifying in humans those genes which undergo expression changes mediated by virgin olive oil phenolic compounds. RESULTS: Postprandial gene expression microarray analysis was performed on peripheral blood mononuclear cells at the postprandial period. Two virgin olive oil-based breakfasts with high (398 ppm) and low (70 ppm) content of phenolic compounds were administered to 20 patients with metabolic syndrome following a double-blinded random crossover design. To eliminate the potential effect that might exist in their usual dietary habits, all subjects followed a similar low-fat, carbohydrate rich diet during the study period. Microarray analysis identified 98 differentially expressed genes (79 underexpressed and 19 overexpressed) when comparing the intake of phenol-rich olive oil with the low-phenol olive oil. Many of those genes are linked to obesity, dyslipemia and type 2 diabetes mellitus. Among these, several genes are involved in inflammatory processes mediated by transcription factor NF-kappa B, activator protein-1 transcription factor complex AP-1, cytokines, mitogen-activated protein kinases MAPKs or arachidonic acid pathways. CONCLUSION: This study shows that intake of a breakfast based in virgin olive oil rich in phenol compounds is able to repress the in vivo expression of several pro-inflammatory genes, thereby switching the activity of peripheral blood mononuclear cells to a less deleterious inflammatory profile. These results provide at least a partial molecular basis for the reduced risk of cardiovascular disease observed in Mediterranean countries, where virgin olive oil represents the main source of dietary fat.”   

I could continue this blog entry citing more and more studies but it should be clear by now that the old Italian folklore about the health value of olive oil is right-on and the pungent extra-virgin variety is by far the best.  What I have to figure out now based on the final study quoted is how can I work extra virgin olive oil into my regular daily breakfast?  I want to do that without adding carbs and confounding tastes like blueberries and EVOO.  I will need to do some experimenting.  Perhaps they will go together fine.

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