30. October 2009 by admin.

The lowly naked mole rat is in the news again.  I talked about the little critter in my in my February 2009 post Animal models of aging – the African naked mole rat.  I said “This little critter is the size of a tiny mouse but lives about eight times longer.  Living up to 28 years, it is the longest-living rodent.  Its secret to longevity is not known but there are clues.  For example they are very cool, they can all but shut down their metabolism, and they spend a great deal of their life sleeping.  Surprisingly, the markers of oxidative damage in these tiny rats exceed those of mice when they are relatively young.   However the rate of accrual of oxidative damage in these rats does not appear to markedly ramp up with age as it does with mice.  They change very little as they age and females more than 20 years old can give birth.  It seems that the mole rat has a powerful long-lived antioxidant defense system which mice do not have.”

The news items that surfaced yesterday reported on work by Vera Gorbunova and her team at the University of Rochester and included a nice story in the New York  Times entitled The Life Span of a Rodent May Aid Human Health.  Even apart from their longevity these little naked mole rat critters are fascinating.  They cannot see and spend their entire lives underground.  They “live in large colonies, presided over by a queen, in which only the queen and a few privileged males breed while the rest of the colony—all members of the same family—work together to raise young and maintain the colony. Wild colonies range in size from 20 to 300 individuals, with an average colony consisting of 75 individuals(ref).”  They have a very hierarchical social system with the queen at the top, then her favorite mates, ranging down to the workers.  She is extremely bossy.  They dig extensive tunnels looking for tubers which they share for food with other members of their colony.   Besides dead reckoning, they use the earth’s magnetic field to help them navigate underground, at least for longer distances(ref).  A colony of naked mole rats can build a system of tunnels stretching up to two or three miles in cumulative length. ^“Ensconced in the arid soils of Africa, these three-inch-long creatures must continually dig tunnels in search of sporadic food supplies and evade the deadly jaws of snakes(ref).”  If you are interested in learning more about their lifestyle, you can check the article The Naked Truth about Mole-Rats.

Physically, the naked mole rats are quite unique.  Apparently they have no pain sensors on their skins and use their fang-like teeth to dig their tunnels. Their large, protruding teeth are used to dig, and their lips are sealed just behind the teeth to prevent soil from filling their mouths while digging(ref).” They look a little like tiny walruses.  “They have little hair (hence the common name) and wrinkled pink or yellowish skin. — The naked mole rat is well adapted for the limited availability of oxygen within the tunnels that are its habitat: its lungs are very small and its blood has a very strong affinity for oxygen, increasing the efficiency of oxygen uptake. It has a very low respiration and metabolic rate for an animal of its size, about 2/3 that of a mouse of the same size, thus using oxygen minimally. In long periods of hunger, such as a drought, its metabolic rate can be reduced by up to 25 percent(ref).”

“Some of the “hottest” research on naked mole rats today concerns senescence, or aging. Naked mole rats in the lab have reached up to 28 years of age. And it’s not just the controlled environments of their captivity that are doing this. –Braude has observed mole rats in the wild that are 17 years and older. But these are the breeders. Lab researchers didn’t realize that in the wild workers only live two or three years(ref).” 

Though there are many interesting things about these little beasties, let me get down to the questions of why they live so long and what might be the lessons for us.  To start, there are two clues from my previous post on them:

1.      Naked mole rats go inactive for periods and turn their metabolism way down.

2.     Naked mole rats have a powerful long-lived antioxidant defense system which mice do not have. 

The next point is based on the research reported yesterday:

3.     Naked mole rats never get cancers.

Despite their very long lives which provide plenty of time for their cells to grow cancerous, naked mole rat have never been found with tumors of any kind. A report just published in The Proceedings of the National Academy of Sciences, Hypersensitivity to contact inhibition provides a clue to cancer resistance of naked mole-rat, suggests why.   “Here we show that naked mole-rat fibroblasts display hypersensitivity to contact inhibition, a phenomenon we termed “early contact inhibition.” Contact inhibition is a key anticancer mechanism that arrests cell division when cells reach a high density. In cell culture, naked mole-rat fibroblasts arrest at a much lower density than those from a mouse. — We demonstrate that early contact inhibition requires the activity of p53 and pRb tumor suppressor pathways. Inactivation of both p53 and pRb attenuates early contact inhibition. Contact inhibition in human and mouse is triggered by the induction of p27/Kip1. In contrast, early contact inhibition in naked mole-rat is associated with the induction of p16(INK4a).” 

In essence, the observed results were that expression of p16(INK4a) “makes the cells “claustrophobic,” stopping the cells’ proliferation when too many of them crowd together, cutting off runaway growth before it can start. The effect of p16 is so pronounced that when researchers mutated the cells to induce a tumor, the cells’ growth barely changed, whereas regular mouse cells became fully cancerous(ref).”  [I have discussed  p16(INK4a) at some length in my treatise and in this blog(ref), a tumor suppressor protein also very active in humans.  But nowhere previously have I seen any discussion of p16(INK4a) making cells claustrophobic.  Apparently this does not happen in humans where weaker contact inhibition is instead triggered by p27/Kip1.]  In any event, whether or not contact resistance is the central element, naked mole rats have an incredibly effective defense against cancers.

Finally, to top it off, it is believed that

4.     Naked mole rats body cells express telomerase.

In 2006 the same lead researcher, Vera Gorbunova, studied small rodents to see how their telomerase expression varied.  She “investigated 15 rodents from across the globe to determine what level of telomerase activity each species expressed, to see if there were some correlation she could find. — The species ranged from tiny field mice to the 100-pound capybara from Brazil. Lifespans ranged from three years for the mice, to 23 or more for common backyard squirrels(ref).”  She found the smaller the critters the higher degree of expression of telomerase – presumably also in the naked mole rat.  Her results were published in the paper Telomerase activity coevolves with body mass, not lifespan where she concludes “Here we show that telomerase activity does not coevolve with lifespan but instead coevolves with body mass: larger rodents repress telomerase activity in somatic cells. These results suggest that large body mass presents a greater risk of cancer than long lifespan, and large animals evolve repression of telomerase activity to mitigate that risk.”  Of course, we are like very large rodents in the respect that telomerase activity in our somatic cells is very repressed.

These studies provide interesting insights and food for speculation.  First of all, for my readers who see telomerase activation as a one-track approach to life extension, telomerase expression by itself does not correlate with lifespan.  Second, it could well be that the message of the naked mole rat is that a combination of a very powerful anti-cancer defense with activated telomerase might lead to significantly greater longevity.  That, by the way, has been my personal view for some time now.  In my treatise I have written “I speculate that protection against carcinogenesis in the course of such telomerase stimulation can probably be achieved through strengthening of apoptotic mechanisms such as P53, P16 and P21. Credence is given to this view by a very recent finding that mice which possess extra copies of both telomerase-creating and antitumor genes live 26% to 40% longer than their normal cohorts(ref).”  Both the Lifestyle Regimen and the Supplement Regimen in my anti-aging firewall program suggest numerous provisions for the avoidance of cancers.  The treatise contains an extensive discussion of telomerase activation and the supplement regimen suggests use of a telomerase activator.

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29. October 2009 by admin.

MicroRNAs (miRNAs) are short (22 nucleotides, more or less) single-stranded RNA molecules which do not encode proteins.  Discovered in 1993 they are recently coming under intense research scrutiny because of the important roles they play in post-transcriptional regulation of gene expression.  According to a feature article published last year in Gen Tapping miRNA-Regulated Pathways, “miRNAs are master regulators of gene expression, according to William S. Marshall, Ph.D., president and CEO of miRagen Therapeutics. “You can have one microRNA that controls multiple genes and one gene that is controlled by multiple microRNAs.” They exert negative regulation and have been shown to control expression of entire signaling pathways(ref).”  “About 3% of human genes encode for miRNAs, and up to 30% of human protein coding genes may be regulated by miRNAs. MicroRNAs play a key role in diverse biological processes, including development, cell proliferation, differentiation, and apoptosis(ref).”

The number of discovered human miRNAs continues to grow.  “ – It can be argued that, based on computational analysis “there may be as many as 50,000 miRNAs in the human genome and each may have as many as a few thousand potential targets(ref).” “Approximately 400 to 500 miRNAs have been characterized in humans to date, according to Dr. Marshall, and 80–150 are typically expressed in any particular cell type(ref).”  That was written some 18 months ago.  I suspect that by now the number of characterized miRNAs is climbing up over a thousand.

MicroRNAs work by turning gene expression off.  “MicroRNAs downregulate gene expression either by degradation of messenger RNA through the RNA interference pathway or by inhibiting protein translation(ref).” “–more short regulatory RNAs were identified in almost all multicellular organisms, including flowering plants, worms, flies, fish, frogs, mammals [38, 40, 41, 48, 71], and in single cellular algae and DNA viruses [66, 75]. — Computational predictions of miRNA targets suggest that up to 30% of human protein coding genes may be regulated by miRNAs [46, 68]. This makes miRNAs one of the most abundant classes of regulatory genes in humans. MicroRNAs are now perceived as a key layer of post-transcriptional control within the networks of gene regulation(ref).”

MicroRNAs play many roles in organisms “The literature includes examples of miRNAs that function as oncogenes, with their overexpression contributing to tumorigenesis, and of others that act as tumor suppressors, which when downregulated contribute to cancer(ref).” A single miRNA may simultaneously impact in complicated ways on several different genes and gene-activation pathways.  “There is accumulating evidence that short RNAs can not only affect the levels of proteins, but that proteins may also affect the production of microRNAs(ref).”

Large complements of miRNAs are expressed in embryonic stem cells as pointed out in the publication MicroRNA discovery and profiling in human embryonic stem cells by deep sequencing of small RNA libraries.  “A custom data analysis pipeline delineated expression profiles for 191 previously annotated miRNAs, 13 novel miRNAs, and 56 candidate miRNAs.”  Further, interestingly, some of the miRNAs found in embryonic stem cells seem not to be found in other cells and seem to be regulated by many of the same transcription factors  used to revert somatic cells back to induced pluripotent stem status  “Finally, integration of our data with genome-wide chromatin immunoprecipitation data on OCT4, SOX2, and NANOG binding sites implicates these transcription factors in the regulation of nine of the novel/candidate miRNAs identified here(ref).”  miRNAs in embryonic stem cells seem to play important roles in cell differentiation.  Another study MicroRNA expression patterns and function in endodermal differentiation of human embryonic stem cells concludes “Our results demonstrate that expression of specific miRNAs correlates with that of specific genes upon differentiation, and highlight the potential role of miRNAs in endodermal differentiation of hESC.”

Dozens of new papers related to miRNAs have been published in the last few months, and the perceived regulatory importance of these little critters continues to grow.  “Thousands of miRNA genes have been found in diverse species, and many of them are highly conserved. With the miRNA roles identified in nearly all aspects of biological processes, evidence is mounting that miRNAs could represent a new layer of regulatory network, and their regulatory effect might be much more pervasive than previously suspected(ref).”

A number of the more-recent publications have focused on the roles of miRNAs in cancers(ref)(ref)(ref), in Alzheimer’s disease(ref)(ref), in herpes infections(ref), in coronary artery cell senescence(ref) and restenosis(ref) and in many other disease processes.  Examples of efforts by university research scientists and biotech companies to develop therapeutic products based on miRNA approaches are outlined in the Gen article.

Of special interest to me is yet-another view of aging in which miRNAs play the lead roles.  Quoting again from the Gen article, “Eugenia Wang, Ph.D., professor at the University of Louisville, has proposed that miRNAs have a critical role in “a universal or system-specific programmatic shift of signaling control” that occurs at mid-life and brings about a decline in cellular health status associated with aging, which may precipitate increased risk of late-life diseases. In her presentation, she will review the hypothesis that the changes in expression of most if not all aging-related genes are controlled by underlying hubs and the belief that miRNAs, acting as molecular master switches, are candidate hubs.” 

In fact the May 2009 issue of Current Genomics focuses on the roles of miRNAs in the aging process and Dr. Wang wrote the editorial Hot Topic: MicroRNA Regulation and its Biological Significance in Personalized Medicine and Aging. “This issue focuses on a discussion of microRNA’s post-transcriptional control of the aging process.”  The ground covered in that issue is large and interesting enough for me to make it the subject of a subsequent post. For the moment I will comment that Dr.  Wang’s view of aging appears to suggest specific mechanisms for the operation of the 13^th theory of aging covered in my treatise, Programmed Epigenomic Changes.  As I understand it, this view says that miRNAs are the signaling messengers and “hit men” for programmed aging, progressively and systematically switching off disease-protecting genes as an organism ages.  It is interesting that in the previous post Homicide by DNA methylation I outlined a view of a Russian scientist suggesting a different mechanism for operation of the Programmed Epigenomic Changes theory.  This view suggests that mutated genes due to progressive DNA methylation might be the cause for genomic and therefore somatic deterioration and loss of disease protection with age.  Of course, both the miRNA explanation and the DNA methylation explanations for aging could be compatible, and molecular links between the two sets of process could be discovered.  I plan to come back to this topic.

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26. October 2009 by admin.

A recent publication suggests that DNA methylation may be the cause of aging and death in higher organisms.  The May 2009 publication by Alexander L. Mazin from Lomonosov Moscow State University is entitled Suicidal function of DNA methylation in age-related genome disintegration and presents a very dark view of DNA methylation as possibly being at the heart of the aging process.

The 13^th theory of aging covered in my treatise is called Programmed Epigenomic Changes and envisages aging as a systematically articulated set of epigenomic changes, involving changes in DNA methylation in cells accumulated with aging.  The new Mazin review publication proposes a mechanism for such programmed aging, seeing DNA methylation as a wrecking-ball process that progressively introduces mutations in key genes, eventually wrecking the genome causing aging and death.

I introduced the topic of DNA methylation in this blog in the post Epigenetics, epigenomics and aging and have mentioned it in a number of subsequent posts.  Its role in aging is outlined in the post DNA methylation, personalized medicine and longevity.   As I said there, DNA methylation is a process by means of which sites adjacent to genes on chromosomes (promoter regions) are chemically methylated after a cycle of DNA replication(ref).  The methylation is passed on in the course of cell divisions and through generations of people.  The methylation pattern captures the ancestral history of the cell that is not in the genes themselves and is unique to every cell.  DNA methylation is thought to be one of the main ways epigenetic information is captured and passed on.

The Mazin paper states “This review will put forward the hypothesis that the host-defense role of DNA methylation in silencing and mutational destroying of retroviruses and other intragenomic parasites was extended during evolution to most host genes that have to be inactivated in differentiated somatic cells, where it acquired a new function in age-related self-destruction of the genome.”  He is saying that cell differentiation and specialization requires silencing of certain genes, which ones depending on the destination cell type, and that task – necessary for the development of complex organisms – is accomplished by DNA methylation.  Further, he is saying that DNA methylation does something else too, and that is eventually wrecking the genome by creating mutations in certain genes.  The paper goes on to say “The proposed model considers DNA methylation as the generator of 5mC > T transitions that induce 40–70% of all spontaneous somatic mutations of the multiple classes at CpG and CpNpG sites and flanking nucleotides in the p53, FIX, hprt, gpt human genes and some transgenes.”  “The accumulation of 5mC-dependent mutations explains: global changes in the structure of the vertebrate genome throughout evolution; the loss of most 5mC from the DNA of various species over their lifespan and the Hayflick limit of normal cells; the polymorphism of methylation sites, including asymmetric mCpNpN sites; cyclical changes of methylation and demethylation in genes. The suicidal function of methylation may be a special genetic mechanism for increasing DNA damage and the programmed genome disintegration responsible for cell apoptosis and organism aging and death.”

The theory is plausible.  DNA methylation is known to be capable of exercising mutagenic and epigenetic effects.  Multiple publications discuss mutations in relationship to methylation in CpG sites within genes(ref)(ref)(ref).    In a previous paper DNA Cytosine Methylation Produces CpG and CpNpG Hotspots for Various Types of Mutations in Human Genes Mazin stated “The evidence is presented that both CpG and CpNpG sites of DNA methylation and their 5`-, 3`-neighboring nucleotides are hotspots not only for 5mC>T transitions, but also for most types of mutations. 40-70% of all spontaneous mutations are found at these sites, and mutation frequencies at the hotspots are 10-40 times higher than the average for the genes studied. 52-77% of CpG sites could be lost because of relict germ-line 5mC>T substitutions, and 10-20% of somatic mutations result in the emergence of new sites of methylation in these genes. Various mutagenes induce significant changes in mutation spectra at sites of methylation. Thus, one of the basic functions of DNA methylation is mutation destruction of most host genes that responsible for human genetic diseases, aging, and cancer.”    

The most recent post in this blog, The NRG1 Gene – an important new tumor suppressor gene, points to a specific mechanism of how DNA methylation can lead to cancers – through silencing the NRG1 tumor suppressor gene.  According to the Mazin hypothesis more than silencing of genes is involved through methylation – they are mutated, irreparably damaged.  Numerous other “housekeeping” genes are likewise affected by the methylation.  Such gene damage, according to my reading of the hypothesis, leads to multiple disease susceptibilities and degenerative processes associated with aging.

If the Mazin hypothesis is correct, the mutational damage due to DNA methylation is in the genes themselves and therefore cannot be corrected through demethylation or any form of epigenomic reversal.  It says you start out in life with mostly good genes and end up with enough bad ones to kill you.  Further old cells with such damage reverted to iPSC pluripotent status would continue to contain age-related mutational damage and, in that way, would be genetically different than embryonic stem cells were for the same individual.  Because the reverted cells would continue to contain the genetic seeds for age-related self-destruction, they could not be used to close the loop in the stem cycle supply chain as suggested in the blog post The stem cell supply chain – closing the loop for very long lives.  This is a rather gloomy outlook on life extension for it says that without constant gene-correction therapy to reverse the results of constant mutations due to DNA methylation, aging is indeed irreversible.

I would strongly prefer a scenario where our genes are more or less constant through life and aging mainly goes on in the epigenome where it might, in principle at least, be reversible.  We will have to see what happens if and when other researchers pay attention to Mazin’s hypothesis.  Meanwhile there is more that can be said about DNA methylation and you can probably expect another post on that topic soon.

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25. October 2009 by admin.

A couple of important tumor suppressor genes have figured heavily in my past writings and in anti-aging science discussions, P21 and P53.  Another tumor suppressor gene may now be coming onto center stage, NRG1.  

Paying attention to the general press 10 days ago, one would get the impression that the NRG1 gene was just discovered and represents a breakthrough in cancer genetics with headlines such as Breast cancer gene discovery ‘most important for 20 years, Gene discovery is the biggest cancer success in 20 years, and Scientists find gene that stops some cancers in their tracks.  “Scientists have found a faulty gene linked to half of all breast cancers which experts have hailed as the most important discovery in the disease since the 1970s(ref).”    

 Actually, the NRG1 gene has been known for some time and its role in cancers has been investigated since 1998.  A Google search on the gene, Neuroregulin 1 (NRG1), returns 112,000 entries.  It is hardly newly-discovered.  The evidence for the tumor-suppressing properties of NRG1 is not new either.  It has been steadily accumulating and NRG1’s exact role in many cancers is still not well understood. 

The NRG1 gene encodes the protein Neuregulin 1 which is also known as NRG1.    “It is known that an extraordinary variety of different isoforms are produced from the NRG1 gene by alternative splicing. These isoforms include heregulins (HRGs), glial growth factors (GGFs) and sensory and motor neuron-derived factor (SMDF)(ref).” “The neuregulins are receptor tyrosine kinase ligands that play a critical role in the development of the heart, nervous system, and breast(ref).”  “NRG1 encodes growth factors that bind to tyrosine kinases ErbB3 and ErbB4, and can both stimulate cell proliferation and apoptosis. NRG1 is also quite frequently broken by chromosome translocations(ref).”  Among its other activities, it has been thought that “Neuregulin 1 (NRG1) is a leading schizophrenia susceptibility gene(ref),” and it may be involved in bipolar disorders(ref).

The October 2009 publication that set off the recent flurry of press reports is The NRG1 gene is frequently silenced by methylation in breast cancers and is a strong candidate for the 8p tumour suppressor gene.  “We found that most breast cancer cell lines had reduced or undetectable expression of NRG1. This included cell lines that had translocation breaks in the gene. Similarly, expression in cancers was generally comparable to or less than that in various normal breast samples. Many non-expressing cell lines had extensive methylation of the CpG island at the principal transcription start site at exon 2 of NRG1. Expression was reactivated by demethylation. Many tumours also showed methylation, whereas normal mammary epithelial fragments had none. Lower NRG1 expression correlated with higher methylation. — The short arm of chromosome 8 is frequently lost in epithelial cancers, and NRG1 is the most centromeric gene that is always affected. NRG1 may therefore be the major tumour suppressor gene postulated to be on 8p: it is in the correct location, is antiproliferative and is silenced in many breast cancers.” 

I touched on the role of DNA methylation in my blog post  DNA methylation, personalized medicine and longevity  and in Epigenetics, epigenomics and aging  where I pointed out “Already, certain DNA methylation changes are known to be associated with aging and others associated with certain diseases like lupus and scleroderma.”    I subsequently added a candidate theory of aging to my Anti-Aging Firewalls treatise EPIGENOMIC CHANGES IN DNA METHYLATION AND HISTONE ACTYLATION.  What is important in the current discussion is that in many cancer lines the expression of the NRG1 gene is shut down by methylation, meaning that no NRG1 tumor-suppressor protein is produced.  

The recent publication includes several laboratory displays graphically relating NRG1 expression to methylation in breast cancer and normal cells and concludes “– we suggest that NRG1 may be the principal tumour suppressor gene that leads to the loss of 8p in many breast and other epithelial cancers. NRG1 expression seems to be silenced in most breast cancers compared with the main types of mammary epithelial cell—this could be because tumours arise from a specialized population in which NRG1 is normally silenced, but we prefer the interpretation that NRG1 is silenced by aberrant methylation or other—as yet unknown—events such as promoter mutation. Expression of NRG1 in mammary cells is antiproliferative to the cells that express it, and array-CGH identifies NRG1 as the gene most likely to be a principal 8p tumour suppressor.” 

Actually the central results of this study were communicated in a poster presentation with the same title in May 2008.”  The story was not picked up by the press until more than a year later.

It is interesting to look at the history of discovery of the tumor suppressing properties of NRG1.  I mention only a few of the many publications which are building blocks for the most-recent  study.  The first relevant publication article I have seen cited was a 1998 one Neu differentiation factor (NDF), a dominant oncogene, causes apoptosis in vitro and in vivo.  “– we find that tumors induced by NDF (NRG1) display extensive apoptosis in vivo. NDF is therefore an oncogene whose deregulation can induce transformation as well as apoptosis.”

The 2000 study Chromosome translocations in breast cancer with breakpoints at 8p12 studied breakpoints and translocations on chromosome 8p in several cancer lines, relevant to the later discovery that these breaks and translocations were in NRG1.  This is one of several prior and subsequent studies looking at chromosomal rearrangements on 8p in cancers(ref).  One 2000 study states “We conclude that chromosome 8p carries a tumour suppressor gene or genes, the loss of which results in growth advantage of breast tumour cells, especially in carriers of the BRCA2 999del5 mutation.”  The recent research strongly suggests that that gene is NRG1.

The discovery of the involvement of two key genes in breast cancer, BRCA1 and BRCA2 was very exciting but led to a hunt for additional genes that might be involved, such as pointed out in the 2002 review article Genes other than BRCA1 and BRCA2 involved in breast cancer susceptibility.

The 2004 publication A Recurrent Chromosome Breakpoint in Breast Cancer at the NRG1/Neuregulin 1/Heregulin Gene states “We previously reported that five breast cancer cell lines have chromosome translocations that break in the NRG1 gene and that could cause abnormal NRG1 expression. — Breaks in NRG1 were detected in 6% (19 of 323) of breast cancers and in some lung and ovarian cancers. In an unselected series of 213 cases with follow-up, breast cancers where the break was detected tended to be high-grade (65% grade III compared with 28% of negative cases). They were, like breast tumors in general, mainly ErbB2 low (11 of 13 were low) and estrogen receptor positive (11 of 13 positive).

The 2005 paper NRG1 gene rearrangements in clinical breast cancer: identification of an adjacent novel amplicon associated with poor prognosis did some applicable legwork looking at rearrangements of the NRG1 gene and how they are specifically implicated in breast carcinoma oncogenesis.

The 2005 study Activation of ErbB2 by Overexpression or by Transmembrane Neuregulin Results in Differential Signaling and Sensitivity to Herceptin looks at the use of herceptin as a cancer treatment targeting NRG1.  “Treatment with the anti-ErbB2 receptor antibody Herceptin had an inhibitory effect on proliferation only in cells expressing neuregulin but not on cells overexpressing ErbB2, and its inhibitory activity was accompanied by a decrease in p21. These results suggest that Herceptin may also be of help in the treatment of tumors in which neuregulin feeds the tumoral tissue.”

A 2006 poster publication The NEUREGULIN1 gene and breast cancer is interesting in that it telegraphs the essence of the heralded 2009 results.  “Our current work shows that NRG1 expression is silenced in many breast cancer cell lines (17 out of 23 lines), as compared with normal breast cell lines. Western blotting experiments also indicate that NRG1 is downregulated at the protein level. To investigate whether NRG1 maybe repressed by epigenetic mechanisms, we examined DNA methylation at a CpG island present in the promoter and the first exon of the gene using bisulphite sequencing. This region is heavily methylated in 76.5% (13/17) of breast cancer cell lines that have no NRG1 expression. In contrast, the region is relatively unmethylated in normal breast lines, and in cancer cell lines expressing NRG1. Treatment of cancer cell lines with 5-aza-2-deoxycytidine, which abolished DNA methylation, activated the expression of NRG1 by 7–100 times. — These results suggest that DNA methylation is a key mechanism that silences NRG1 expression in breast cancer cells, and our current view is that NRG1 could be the long-sought tumour suppressor on 8p, with the translocations either inactivating the gene or producing aberrant transcripts.” 

Paul Edwards, a molecular biologist at Cambridge University, and his team were involved in this 2006 as well as the 2008 and 2009 reporting.  And there are a number of other studies over the years related to NRG1 and cancers.  A point I am making is that “breakthrough genetic discoveries” reported in the popular press are often the results of years of hard study by many people.  I do believe the latest results by Edward’s team represents solid and important progress.  However, we still only partially understand the role of NRG1 in other cancers and the likely role of NRG1 as a key tumor suppressor gene must be confirmed.   Despite sensational reporting, progress is achieved through many small incremental steps and there is far yet to go.

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22. October 2009 by admin.

I am partial to an occasional slice of pizza despite my health-driven dietary compulsions. I love to sprinkle generous amounts of oregano on the slices and have often wondered what the health properties of that pungent herb are.  I recently learned that oregano is rich in rosmarinic acid and therefore, as my grandmother used to say “it is good for you.”  I review some of the things the literature says about rosmarinic acid here.

 “Rosmarinic acid, C₁₈H₁₆O₈, is a natural polyphenol antioxidant carboxylic acid found in many Lamiaceae herbs used commonly as culinary herbs such as lemon balm, rosemary, oregano, sage, thyme and peppermint^[1] (ref).”  It has anti-inflammatory, anti-bacterial, anti-viral, radio-protective, photo-protective, anti-allergenic and anti-oxidant properties.  It is somewhat structurally related to curcumin and resveratrol and is an ester of caffeic acid.  (See the blog entry Phytochemicals – focus on caffeic acid.)

Rosmarinic acid induces apoptosis in some cancer cell lines.  The 2009 paper Salvia Fruticosa, Salvia Officinalis, and Rosmarinic Acid Induce Apoptosis and Inhibit Proliferation of Human Colorectal Cell Lines: The Role in MAPK/ERK Pathway states “Aromatic plants of the genus Salvia (sage) have been attributed many medicinal properties, which include anticancer activity. In the present study, the antiproliferative and proapoptotic effects of water extracts of Salvia fruticosa (SF) and Salvia officinalis (SO) and of their main phenolic compound rosmarinic acid (RA) were evaluated in two human colon carcinoma-derived cell lines, HCT15 and CO115, which have different mutations in the MAPK/ERK and PI3K/Akt signaling pathways. These pathways are commonly altered in CRC, leading to increased proliferation and inhibition of apoptosis. Our results show that SF, SO, and RA induce apoptosis in both cell lines, whereas cell proliferation was inhibited by the two sage extracts only in HCT15. SO, SF, and RA inhibited ERK phosphorylation in HCT15 and had no effects on Akt phosphorylation in CO115 cells. The activity of sage extracts seems to be due, at least in part, to the inhibition of MAPK/ERK pathway.”

Rosmarinic acid has anti-oxidative activity.  Several publications point to this effect such as the 2008 paper Antioxidant activities of rosemary (Rosmarinus Officinalis L.) extract, blackseed (Nigella sativa L.) essential oil, carnosic acid, rosmarinic acid and sesamol.   The 2007 study Water and methanolic extracts of Salvia officinalis protect HepG2 cells from t-BHP induced oxidative damage states  “The most abundant phenolic compounds present in the extracts were rosmarinic acid and luteolin-7-glucoside. Both extracts, when co-incubated with the toxicant, protected significantly HepG2 cells against cell death. The methanolic extract, with a higher content of phenolic compounds than the water extract, conferred better protection in this in vitro model of oxidative stress with liver cells.”  A 2006 study Phenolic compounds protect HepG2 cells from oxidative damage: relevance of glutathione levels compared the antioxidant capabilities of several polyphenols  “If the effects of quercetin are given the reference value 1, the compounds rank in the following order according to inhibition of cell death: luteolin (4.0) > quercetin (1.0) > rosmarinic acid (0.34) > luteolin-7-glucoside (0.30) > caffeic acid (0.21). The results underscore the importance of the compound’s lipophilicity in addition to its antioxidant potential for its biological activity.”

Rosemary and oregano my offer several health benefits associated with rosmarinic acid content.  The 2006 publication Antioxidant and antimicrobial activities of rosemary extracts linked to their polyphenol composition concludes “Carnosic acid and rosmarinic acid may be the main bioactive antimicrobial compounds present in rosemary extracts. From a practical point of view, rosemary extract may be a good candidate for functional foods as well as for pharmaceutical plant-based products.”   The 1999 publication Pharmacology of rosemary (Rosmarinus officinalis Linn.) and its therapeutic potentials “Rosmarinic acid is well absorbed from gastrointestinal tract and from the skin. It increases the production of prostaglandin E2 and reduces the production of leukotriene B4 in human polymorphonuclear leucocytes, and inhibits the complement system. It is concluded that rosemary and its constituents especially caffeic acid derivatives such as rosmarinic acid have a therapeutic potential in treatment or prevention of bronchial asthma, spasmogenic disorders, peptic ulcer, inflammatory diseases, hepatotoxicity, atherosclerosis, ischaemic heart disease, cataract, cancer and poor sperm motility.”

A 2007study The effects of essential oils and aqueous tea infusions of oregano (Origanum vulgare L. spp. hirtum), thyme (Thymus vulgaris L.) and wild thyme (Thymus serpyllum L.) on the copper-induced oxidation of human low-density lipoproteins looked at “the antioxidative capacity effect of essential oils and aqueous tea infusions obtained from oregano, thyme and wild thyme on the oxidation susceptibility of low-density lipoproteins (LDL).  – The strong protective effect of aqueous tea infusions is proposed to be the consequence of large amounts of polyphenols, namely rosmarinic acid and flavonoids (quercetin, eriocitrin, luteolin-7-O-glucoside, apigenin-7-O-glucoside, luteolin, apigenin), with the most pronounced effect in the case of oregano.”  This is interesting and would seem to be a good argument for drinking oregano tea(ref), something I have never quite done.

Both water-soluble oil-soluble extracts of rosemary are commercially available.  The 2009 publication In vitro antimicrobial and antioxidant activity of commercial rosemary extract formulations concludes “Reducing power and free radical scavenging effectiveness was higher in water-soluble formulations, according to their higher total phenolic content, but in an aqueous emulsion system of linoleic acid, they exhibited lower antioxidant activity. This correlated well with the higher efficiency of antimicrobial activity of oil-soluble formulations, despite the lower total phenolic content of these extracts.”

The list of research showing potential benefits of rosmarinic acid and the herbs containing it seems to grow proportionally to the effort I have been putting into searching the literature on it.  For example Rosmarinic acid, a photo-protective agent against UV and other ionizing radiations, Radioprotective-antimutagenic effects of rosemary phenolics against chromosomal damage induced in human lymphocytes by gamma-rays  Antiviral and Anti-Inflammatory Effects of Rosmarinic Acid in an Experimental Murine Model of Japanese Encephalitis, Perilla-derived Rosmarinic Acid’s Effectiveness Against Hay Fever Confirmed, and Evaluation of clonal herbs of Lamiaceae species for management of diabetes and hypertension

I have written about several other phytochemicals in this blog and in my treatise ANTI-AGING FIREWALLS THE SCIENCE AND TECHNOLOGY OF LONGEVITY.  See, for example, yesterday’s post Nrf2 and cancer chemoprevention by phytochemicals. Who would think that, after a bout of dental x-rays, eating a slice of pizza with a generous layer of oregano sprinkled on it could be a good thing to do from a radioprotective viewpoint?

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21. October 2009 by admin.

A cluster of research reports has appeared during the last few years looking at  mechanisms through which substances rich in phytochemicals (e.g. coffee, chocolate, turmeric, olive oil, broccoli, red hot peppers, green tea, garlic, blueberries, rosemary, oregano, sage) are cancer-preventative. While these foods have been studied for many years a new focal point has been moving to center stage - study of what these substances are doing in terms of gene expression as a key to understanding their therapeutic value.  The 2005 paper Dietary cancer-chemopreventive compounds: from signaling and gene expression to pharmacological effects articulates this emerging viewpoint.  “The process of cancer development (carcinogenesis leading to advanced metastasized cancers) in humans generally takes many years through initiation, promotion and progression. Because advanced metastasized cancers are almost impossible to treat, cancer chemoprevention for the control and containment of early cancer development is highly desirable. Recent studies have provided strong evidence that many daily-consumed dietary compounds possess cancer-protective properties that might interrupt the carcinogenesis process. These properties include the induction of cellular defense detoxifying and antioxidant enzymes, which can protect against cellular damage caused by environmental carcinogens or endogenously generated reactive oxygen species. These compounds can also affect cell-death signaling pathways, which could prevent the proliferation of tumor cells.”

One master activator of antioxidant and anticancer genes appears to be Nuclear factor-erythroid-2-related factor 2 (Nrf2). The sequence of events involved in phytochemical chemoprevention mediated by Nrf2 is complex and is summarized in the 2008 publication Nrf2 as a master redox switch in turning on the cellular signaling involved in the induction of cytoprotective genes by some chemopreventive phytochemicals.  “A wide array of dietary phytochemicals have been reported to induce the expression of enzymes involved in both cellular antioxidant defenses and elimination/inactivation of electrophilic carcinogens. Induction of such cytoprotective enzymes by edible phytochemicals largely accounts for their cancer chemopreventive and chemoprotective activities.” For those of you who have a taste for molecular biology, that document goes on to explain “Nuclear factor-erythroid-2-related factor 2 (Nrf2) plays a crucial role in the coordinated induction of those genes encoding many stress-responsive and cytoptotective enzymes and related proteins. These include NAD(P)H:quinone oxidoreductase-1, heme oxygenase-1, glutamate cysteine ligase, glutathione S-transferase, glutathione peroxidase, thioredoxin, etc. In resting cells, Nrf2 is sequestered in the cytoplasm as an inactive complex with the repressor Kelch-like ECH-associated protein 1 (Keap1). The release of Nrf2 from its repressor is most likely to be achieved by alterations in the structure of Keap1. Keap1 contains several reactive cysteine residues that function as sensors of cellular redox changes. Oxidation or covalent modification of some of these critical cysteine thiols would stabilize Nrf2, thereby facilitating nuclear accumulation of Nrf2. After translocation into nucleus, Nrf2 forms a heterodimer with other transcription factors, such as small Maf, which in turn binds to the 5′-upstream CIS-acting regulatory sequence, termed antioxidant response elements (ARE) or electrophile response elements (EpRE), located in the promoter region of genes encoding various antioxidant and phase 2 detoxifying enzymes. Certain dietary chemopreventive agents target Keap1 by oxidizing or chemically modifying one or more of its specific cysteine thiols, thereby stabilizing Nrf2. In addition, phosphorylation of specific serine or threonine residues present in Nrf2 by upstream kinases may also facilitate the nuclear localization of Nrf2. Multiple mechanisms of Nrf2 activation by signals mediated by one or more of the upstream kinases, such as mitogen-activated protein kinases, phosphatidylionositol-3-kinase/Akt, protein kinase C, and casein kinase-2 have recently been proposed.”

Two signaling pathways frequently mentioned in this blog, the MAPK/ERK and PI3K/Akt pathways, appeared to be involved as pointed out in the 2009 publication Salvia Fruticosa, Salvia Officinalis, and Rosmarinic Acid Induce Apoptosis and Inhibit Proliferation of Human Colorectal Cell Lines: The Role in MAPK/ERK Pathway. “In the present study, the antiproliferative and proapoptotic effects of water extracts of Salvia fruticosa (SF) and Salvia officinalis (SO) and of their main phenolic compound rosmarinic acid (RA) were evaluated in two human colon carcinoma-derived cell lines, HCT15 and CO115, which have different mutations in the MAPK/ERK and PI3K/Akt signalling pathways. — Our results show that SF, SO, and RA induce apoptosis in both cell lines, whereas cell proliferation was inhibited by the two sage extracts only in HCT15. SO, SF, and RA inhibited ERK phosphorylation in HCT15 and had no effects on Akt phosphorylation in CO115 cells. The activity of sage extracts seems to be due, at least in part, to the inhibition of MAPK/ERK pathway.” 

Phytochemical cancer chemoprevention may involve a number of additional pathways besides Nrf2 .  A number of phytosubstances are powerful anti-inflammatories for example and this may play a role in their control of cancer.  Bromelain, ginger, curcumin, aswagandah and boswellia are in this category.  Inhibition of TNFalpha-activated NF-kappaB signaling may also play an important role in preventing cancer activation as pointed out in my treatise.  A number of herbal substances that are NF-kappaB inhibitors are in my combined anti-aging firewall. 

The use of herbal substances for cancer chemoprevention is receiving a lot of attention, especially now that the chains of molecular activities initiated by these traditional substances are starting to be understood.  The 2007 publication Chemopreventive herbal anti-oxidants: current status and future perspectives states “Cancer chemoprevention is fast becoming a lucrative approach for controlling cancer. Carcinogenesis being a complex multi-step, multi-factorial process, a number of chemopreventive interventions can be employed. These strategies are generally directed against two broad events of carcinogenesis viz., initiation and promotion/progression. Anti-initiation interventions principally involve inhibition of carcinogen activation, scavenging of free radicals and reactive carcinogen metabolites along with enhanced detoxification of carcinogens by modulating cellular metabolism. Anti-promotion strategies involve attenuation of enhanced cellular proliferation along with induction of cellular apoptosis and differentiation. Dietary agents or herbal anti-oxidants due to low toxicity and relative safety are promising chemopreventive agents.” 

Other publications on cancer chemoprevention include Comprehensive review of cancer chemopreventive agents evaluated in experimental carcinogenesis models and clinical trials, Chemopreventive effects of natural dietary compounds on cancer development,  Organosulfur compounds in cancer chemoprevention, Cancer prevention by natural compounds, Cruciferous vegetables and cancer prevention, Cruciferous vegetables and human cancer risk: epidemiologic evidence and mechanistic basis, Cruciferous vegetables: cancer protective mechanisms of glucosinolate hydrolysis products and selenium. Following the hyperlinks to and from these publications will lead to many more.Those of you familiar with my treatise ANTI-AGING FIREWALLS - THE SCIENCE AND TECHNOLOGY OF LONGEVITY  know that I suggest consumption of phytochemical-rich foods like blueberries, walnuts, chocolate and green tea and suggests taking a substantial number of phytosubstance supplements including resveratrol, curcumin, boswellia, ashwagandah, pycnogenol, green tea extract, olive leaf extract, lycopene, allicin and  OPC grape seed extract.  The blog post Phytochemicals – focus on caffeic acid  looks at one important phytochemical in depth, the one that is in coffee. And the post Health and longevity benefits of dark chocolate looks at another of my favorite phytosubstances.

Another action of Nrf2 is protection of arteries from fluid sheer stress generated by blood flow.  The 2009 publication Regulation of shear-induced nuclear translocation of the Nrf2 transcription factor in endothelial cells describes how sheer stress induces nuclear translocation of Nrf2 which restores laminar flow.  The paper concludes “Our data suggest that the atheroprotective effect of laminar flow is partially attributed to Nrf2 activation which results in ARE-mediated gene transcriptions, such as HO-1 expression, that are beneficial to the cardiovascular system.”  Several other papers have been written on this effect  such as the 2007 paper Shear stress stabilizes NF-E2-related factor 2 and induces antioxidant genes in endothelial cells: role of reactive oxygen/nitrogen species.

For a personal story from my childhood involving two phytosubstances and cancer, see the blog post Red wine, hot peppers and my uncle Gigi.

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20. October 2009 by admin.

For a great many years the medical establishment warned everyone of dire consequences that could result from taking large doses of vitamin D.  The daily maximum of 400iu was the strict acceptable limit to avoid vitamin D toxicity.  In recent years the consensus of the medical establishment has flipped as a result of many studies showing dire consequences of too little vitamin D and strong benefits of taking it as a supplement.  As I state in my treatise “A recent eight-year study of 3,258 men and women  indicates that the higher the blood level of Vitamin D, the less is the chance of dying from heart disease - and the less the chance of dying from a number of other diseases as well.”  – “Today there are over 80 population and laboratory studies indicating that vitamin D can reduce incidences of and mortality due to multiple kinds of cancer with reductions of 50% or more in some cases.  The biological impact of this substance is far from simple; it activates 200 or more human genes and has differential affects in regulating cancer cells with respect to cell proliferation, apoptosis and differentiation.  It also regulates angiogenesis.  Several studies of nursing home and residential care residents show that people taking vitamin D supplements suffer fewer falls – the reduction being between 23% to 53%.  And I have already mentioned Vitamin D’s role with respect to reducing heart disease fatalities.   Daily doses of 1,000 or 2,000iu are now thought to be harmless and often recommended for older people.“  Advising people to take even larger doses is not unusual now.

There is a possibility the viewpoint may be flipping again, at least for patients with certain genetic defects.  Mouse studies suggest that excess levels of vitamin D under certain situations may lead to premature aging.   The concern is current.  The 2009 publication Vitamin D and aging states “Recent studies using genetically modified mice, such as FGF23-/- and Klotho-/- mice that exhibit altered mineral homeostasis due to a high vitamin D activity showed features of premature aging that include retarded growth, osteoporosis, atherosclerosis, ectopic calcification, immunological deficiency, skin and general organ atrophy, hypogonadism and short lifespan. The phenotype reversed by normalizing vitamin D and/or mineral homeostasis.” 

The story is also summarized in the abstract of the 2006 publication Hypervitaminosis D and premature aging: lessons learned from Fgf23 and Klotho mutant mice. “The essential role of low levels of vitamin D during aging is well documented. However, possible effects of high levels of vitamin D on the aging process are not yet clear. Recent in vivo genetic-manipulation studies have shown increased serum level of vitamin D and altered mineral-ion homeostasis in mice that lack either fibroblast growth factor 23 (Fgf23) or Klotho (Kl) genes. These mice develop identical phenotypes consistent with premature aging. Elimination or reduction of vitamin-D activity from Fgf23 and Kl mutant mice, either by dietary restriction or genetic manipulation could rescue premature aging-like features and ectopic calcifications, resulting in prolonged survival of both mutants. Such in vivo experimental studies indicated that excessive vitamin-D activity and altered mineral-ion homeostasis could accelerate the aging process.”  

So, these studies have led me to inquire about what is involved and what is the danger of taking 2000 to 4000iu of vitamin D3 a day as I have personally been doing.  My bottom line is that you should be particularly concerned if you are a Klotho-knockout mouse, a FGF23 knockout mouse or a vitamin D receptor knockout mouse.  If you happen to be in one of the first two categories,  you may be able to live longer by immediately ceasing to take vitamin D supplements.  Also, you could have a Hypervitaminosis D problem if your natural FGF23 or Klotho expression is sub-par.

My remarks are based on the fact that the vitamin D research is based on experiments with Klotho knockout and FGF23 knockout (FGF23-/- and Klotho-/-) mice.   Conceivably, similar D hypervitaminosis could happen in people with Klotho or FGF23 deficiencies.  See the recent post Klotho anti-aging gene in the news.  I have reviewed the following papers which describe mouse studies on laboratory animals with the characteristics in parentheses:

The 1997 paper Mutation of the mouse Klotho gene leads to a syndrome resembling ageing (mutated Klotho gene)

The 2002 publication Association of human aging with a functional variant of Klotho (mutated Klotho gene)

The 2004 paper Klotho is a serum factor related to human aging (FGF23 null mice)

The 2006 publication Premature aging-like phenotype in fibroblast growth factor 23 null mice is a vitamin D-mediated process (FGF23 null mice)

The 2006 paper Genetic ablation of vitamin D activation pathway reverses biochemical and skeletal anomalies in Fgf-23-null animals (FGF23 null mice)

The 2006 publication Hypervitaminosis D and premature aging: lessons learned from Fgf23 and Klotho mutant mice (FGF23 null or Klotho null mice)

The 2007 paper Premature aging in Klotho mutant mice: cause or consequence? (FGF23 null or Klotho null mice)

The 2008 paper FGF-23-Klotho signaling stimulates proliferation and prevents vitamin D-induced apoptosis (FGF23 null or Klotho null mice)

The 2009 publication Vitamin D and aging (FGF23 null or Klotho null mice) 

All these studies related to Hypervitaminosis D were based on working with mice in which either Klotho or FGF23 or both were knocked out.  It seems that when the transduction pathways initiated by FGF23 and Klotho are working well, apoptosis caused by excessive systemic vitamin D and resulting tissue atrophy is avoided.    

Another  2009 study is of particular interest because the mice were ones with the vitamin D receptor knocked out instead of Klotho or FGF23. The publication Premature aging in vitamin D receptor mutant mice  (vitamin D receptor (VDR) knockout mice)  states “Overall, VDR KO mice showed several aging related phenotypes, including poorer survival, early alopecia, thickened skin, enlarged sebaceous glands and development of epidermal cysts.”  “Since the phenotype of aged VDR knockout mice is similar to mouse models with hypervitaminosis D(3), our study suggests that VDR genetic ablation promotes premature aging in mice, and that vitamin D(3) homeostasis regulates physiological aging.”   

Knocking out VDR seems capable of producing the same pro-aging affect as hypervitaminosis D(3).  I believe the conclusion that “vitamin D(3) homeostasis regulates physiological aging” could be of profound importance if it is born out. 

Since I have normal Klotho and FGF23 expression as far as I know, I am not personally worried about Hypervitaminosis D at the moment.

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19. October 2009 by admin.

Since its discovery in 1997 the Klotho gene has been known to be involved with longevity.  I came across a recent news article describing research linking expression of the gene to reduction in hypertension, and this led me to look into what is known about the gene.  As reported here, Klotho appears to have a lot of interesting properties.

“The Klotho gene codes for a transmembrane protein that, in addition to other effects, provides some control over the sensitivity of the organism to insulin and appears to be involved in aging. Its discovery was documented in 1997 by Kuro-o et al.^[1] The name of the gene comes from Klotho or Clotho, one of the Moirae, or Fates, in Greek mythology(ref).”  In mice at least the Klotho protein acts like a hormone.  It circulates through the blood and binds to cells.

The newly-reported publication is Klotho Gene Delivery Prevents the Progression of Spontaneous Hypertension and Renal Damage.  “Researchers, led by principal investigator Zhongjie Sun, tested the effect of an anti-aging gene called Klotho on reducing hypertension. They found that by increasing the expression of the gene in laboratory models, they not only stopped blood pressure from continuing to rise, but succeeded in lowering it. Perhaps most impressive was the complete reversal of kidney damage, which is associated with prolonged high blood pressure and often leads to kidney failure(ref).”  The experiment was on laboratory rats where the gene was delivered by a virus vector.  “This is the first study showing that a decline in Klotho protein level may be involved in the progression of hypertension and kidney damage, Sun said. With age, the Klotho level decreases while the prevalence of hypertension increases. —  Researchers used one injection of the Klotho gene in hypertensive research models and were able to markedly reduce blood pressure by the second week. It continued to decline steadily for the length of the project – 12 weeks. The Klotho gene was delivered with a safe viral vector that is currently used for gene therapy. The virus is already approved by the U.S. Food and Drug Administration for use in humans(ref).”  Of course, it is hoped that the research will at some point lead to a Klotho therapy for humans for hypertension and associated kidney damage.

Studies relating Klotho to aging started to appear soon after its discovery.  Most of these are based on working with rats or mice.  Some of the earlier publications related deficiency of Klotho to classical indicators of aging.  The 1977 publication Mutation of the mouse Klotho gene leads to a syndrome resembling ageing states “A defect in Klotho gene expression in the mouse results in a syndrome that resembles human ageing, including a short lifespan, infertility, arteriosclerosis, skin atrophy, osteoporosis and emphysema. —  The Klotho gene product may function as part of a signaling pathway that regulates ageing in vivo and morbidity in age-related diseases.” 

The 2002 publication Association of human aging with a functional variant of Klotho concludes “These results suggest that the KL-VS allele influences the trafficking and catalytic activity of Klotho, and that variation in Klotho function contributes to heterogeneity in the onset and severity of human age-related phenotypes.”  The study suggested that one of the aging mechanisms that may be accelerated by insufficient expression of Klotho is the buildup of advanced glycation endproducts (AGEs). “Multiple models of aging invoke accelerated or excessive posttranslational modification of proteins including glycation. Resultant advanced glycation end-products (AGEs) elicit a wide range of responses that have been proposed to contribute to many age related phenotypes, including atherosclerosis, Alzheimer’s disease, diabetic complications, and microvascular changes (29). It is possible that the proposed glycosidase activity of Klotho retards the accumulation of AGEs.” Note that Tissue Glycation is an important theory of aging discussed in my treatise.

Research articles implicate low levels of Klotho expression with endothelial dysfunction, pulmonary emphysema,  impairment of osteoblast and osteoclast differentiation, cognition impairment and other disease processes in mouse models.

The 2004 paper Klotho is a serum factor related to human aging looks at Klotho protein in human serum in 112 individuals.  “ The population aged from 0 to 91 years screened by ELISA revealed that the level of serum KL declined while age increased, though each individual level was variable and that the trend of decreasing in serum KL had no difference in sex. – Conclusion: Our data suggest that KL is a serum factor related to human aging.”

Klotho’s actions and the channels it works through are complex.  From the 2006 paper Toward a better understanding of Klotho: “Suggested functions of Klotho are (i) a fundamental regulator of calcium homeostasis, namely, a cofactor for the fibroblast growth factor (FGF) receptor 1c in FGF23 signaling and a regulator of parathyroid hormone secretion; (ii) a hormone that interferes with the intracellular signaling of insulin and insulin-like growth factor-1 (IGF-1); and (iii) a beta-glucuronidase that activates the transient receptor potential ion channel TRPV5 by trimming its sugar moiety.”   

As to how Klotho may impact on longevity: a) I have already mentioned its actions in averting tissue glycation, b) the IGF-1 pathway has long been known to be associated with longevity and is that affected by calorie restriction, and c) Klotho promotes the body’s antioxidant defenses.

Klotho expression is also important for averting premature aging due to overexpression of Vitamin D.  The mechanism is associated with its function in regulating FGF23.  (YES my reader friends, in animal model experiments, overexpression of vitamin D leads to premature aging.  I will cover that issue in a separate blog post).  The 2002 publication Klotho, a gene related to a syndrome resembling human premature aging, functions in a negative regulatory circuit of vitamin D endocrine system states “These observations suggest that Klotho may participate in a negative regulatory circuit of the vitamin D endocrine system, through the regulation of 1alpha-hydroxylase gene expression.” The 2008 paper FGF-23-Klotho signaling stimulates proliferation and prevents vitamin D-induced apoptosis states “We show that the signal transduction pathways initiated by FGF-23-Klotho prevent tissue atrophy by stimulating proliferation and preventing apoptosis caused by excessive systemic vitamin D. Because serum levels of active vitamin D are greatly increased upon genetic ablation of Fgf-23 or Klotho, we find that these molecules have a dual role in suppression of apoptotic actions of vitamin D through both negative regulation of 1alpha-hydroxylase expression and phosphoinositide-3 kinase-dependent inhibition of caspase activity.”

A number of other papers also deal with the involvement of Klotho and FGF23 in vitamin D mediated premature aging, for example Premature aging-like phenotype in fibroblast growth factor 23 null mice is a vitamin D-mediated process concludes “our data support a new model of interactions among Fgf-23, vitamin D, and Klotho, a gene described as being associated with premature aging process.”  

Besides Klotho defects being implicated in disease processes there is some evidence that overexpression of Klotho could be life-extending.  For example, the 2005 paper Suppression of Aging in Mice by the Hormone Klotho states “Here, we show that overexpression of Klotho in mice extends life span. Klotho protein functions as a circulating hormone that binds to a cell-surface receptor and represses intracellular signals of insulin and insulin-like growth factor 1 (IGF1), an evolutionarily conserved mechanism for extending life span. Alleviation of aging-like phenotypes in Klotho-deficient mice was observed by perturbing insulin and IGF1 signaling, suggesting that Klotho-mediated inhibition of insulin and IGF1 signaling contributes to its anti-aging properties. Klotho protein may function as an anti-aging hormone in mammals.” The point is reinforced in several other publications.

The link of Klotho to insulin is further reinforced by a 2007 study that involved comparing the expression of genes in young and old brains(ref).  “They observed that the levels of Klotho in the brain showed a striking decrease with aging. The association between Klotho and aging prompted Abraham’s group to investigate the regulation of Klotho further. These studies lead to the observation that secretion of Klotho is regulated by insulin. — To their surprise, they found that insulin, a hormone usually associated with diabetes, increases significantly the levels of secreted Klotho. The reason this finding is important is because excess insulin has been previously implicated in a biochemical pathway that is associated with a decreased life span and elevated oxidative stress. — In addition, this observation provides a potentially pivotal link between Klotho and sugar metabolism, and raises an intriguing relationship between Klotho and type II diabetes, commonly known as late onset diabetes. The authors are proposing a novel mechanism of action for Klotho whereby insulin increases Klotho secretion, i.e., activity, and in turn, the secreted Klotho inhibits insulin’s actions in the cell, which are known to be detrimental when insulin is in excess(ref).”

One viewpoint is that Klotho derives much of its anti-aging capability from the protein acting “by increasing the cell’s ability to detoxify harmful reactive oxygen species (ref).  “Using cultured cells and transgenic mice, the researchers showed that Klotho increases resistance to oxidative stress.   “Increased longevity is always associated with increased resistance to oxidative stress,” explains Kuro-o (the man who discovered Klotho). “Oxidative stress causes the accumulation of oxidative damage to important biological macromolecules such as DNA, lipids, and proteins that would result in functional deterioration of the cell, which eventually causes aging(ref).”  Of course, this viewpoint is consistent with the classical Oxidative Damage theory of aging.

A 2007 report is entitled Obesity May Be Associated With A Relative Of Anti-aging Gene, Klotho.  Differences in how to interpret results with Klotho knockout mice has also engendered controversy about its anti-aging capacities such as described in a blog article I found entitled Controversial Klotho in cancer.

Despite that it has been studied for over a dozen years, the exact anti-aging mechanisms of Klotho still are not clearly identified.  The 2008 paper Klotho as a regulator of oxidative stress and senescence states “The Klotho gene functions as an aging-suppressor gene that extends life span when overexpressed and accelerates aging-like phenotypes when disrupted in mice. — The secreted Klotho protein can regulate multiple growth factor signaling pathways, including insulin/IGF-1 and Wnt, and the activity of multiple ion channels. Klotho protein also protects cells and tissues from oxidative stress, yet the precise mechanism underlying these activities remains to be determined. Thus, understanding of Klotho protein function is expected to provide new insights into the molecular basis for aging, phosphate/vitamin D metabolism, cancer and stem cell biology.”                          

As time goes on we will doubtlessly be hearing more about Klotho.

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17. October 2009 by admin.

An interesting report came to my attention relating to telomeres in pythons, and this set me off for the umpteenth time pursuing further research and thoughts about cell senescence, telomere lengths and telomerase. I share that all here.

First of all, about water pythons (Liasis fuscus), snakes that grow to about 2 meters in length.  They are found in flood plains in Australia and New Guinea and are relatively long-lived.  The just-released study report Short Telomeres in Hatchling Snakes: Erythrocyte Telomere Dynamics and Longevity in Tropical Pythons starts out with  “In the present study we explore whether age- and sex-specific telomere dynamics affect life span in a long-lived snake, the water python (Liasis fuscus).”  “– Erythrocyte TL (telomere length) was measured using the Telo TAGGG TL Assay Kit (Roche). In contrast to other vertebrates, TL of hatchling pythons was significantly shorter than that of older snakes. However, during their first year of life hatchling TL increased substantially. While TL of older snakes decreased with age, we did not observe any correlation between TL and age in cross-sectional sampling. In older snakes, female TL was longer than that of males. When using recapture as a proxy for survival, our results do not support that longer telomeres resulted in an increased water python survival/longevity.” – “In fish high telomerase activity has been observed in somatic cells exhibiting high proliferation rates. Hatchling pythons show similar high somatic cell proliferation rates. Thus, the increase in TL of this group may have been caused by increased telomerase activity. In older humans female TL is longer than that of males. This has been suggested to be caused by high estrogen levels that stimulate increased telomerase activity. Thus, high estrogen levels may also have caused the longer telomeres in female pythons. The lack of correlation between TL and age among old snakes and the fact that longer telomeres did not appear to affect python survival do not support that erythrocyte telomere dynamics has a major impact on water python longevity.” 

OK.  Taken at face value, the study says to forget about the Telomere shortening theory of aging at least as far as these snakes are concerned.  What set me off thinking in the report, however, was the statement “However, other studies have demonstrated that TL does not affect survival among old humans. Furthermore, replicative senescence has been shown to be induced by changes in the protected status of the telomeres rather than the loss of TL.”  This is the opposite of the party line believed by most people doing telomerase activation as an anti-aging measure.  So I decided to pursue the author’s citations to check out these assertions. 

The 2005 study Telomere length in white blood cells is not associated with morbidity or mortality in the oldest old: a population-based study which studied 598 participants concludes “Telomere length at baseline was not predictive for mortality (P > 0.40 for all-cause, cardiovascular causes, cancer or infectious diseases, Cox regression for gender-adjusted tertiles of telomere length) or for the incidence of dementia (P = 0.78). Longitudinally, telomere length was highly unstable in a large fraction of participants. We conclude that blood monocyte telomere length is not a predictive indicator for age-related morbidity and mortality at ages over 85 years, possibly because of a high degree of telomere length instability in this group.” 

Another 2006 study No association between telomere length and survival among the elderly and oldest old looks at a different population and confirms the result. “Telomere length was measured as mean terminal restriction fragment length on blood cells from 812 persons, age 73 to 101 years, who participated in population-based surveys in 1997-1998. Among the participants were 652 twins. The participants were followed up through the Danish Civil Registration system until January 2005, at which time 412 (51%) were dead. RESULTS: Univariate Cox regression analyses revealed that longer telomeres were associated with better survival (hazard ratios = 0.89 [95% confidence interval = 0.76-1.04] per 1 kb in males and 0.79 [0.72-0.88] per 1 kb in females, respectively). However, including age in the analyses changed the estimates to 0.97 (0.83-1.14) and 0.93 (0.85-1.03), respectively.”  – “CONCLUSION: This longitudinal study of the elderly and oldest old does not support the hypothesis that telomere length is a predictor for remaining lifespan once age is controlled for.”  

A number of other recent studies also question the relationship of telomere lengths to mortality.  “The question remains as to whether telomere dynamics is a determinant or merely a predictor of human biological age over and above chronological ageing. Although several reports have suggested a link between telomere attrition and ageing phenotypes and disorders, both reference values and a complete set of determinants are missing(ref).”  What is happening to the Telomere shortening theory of aging?  In all fairness there are a number of other studies that relate telomere shortening to a number of disease processes. 

Another report cited in the python paper is the 2002 study Senescence Induced by Altered Telomere State, Not Telomere Loss  which seems to be another attack on those pursuing longevity through telomerase activation.  “Here, we report that overexpression of TRF2, a telomeric DNA binding protein, increased the rate of telomere shortening in primary cells without accelerating senescence. TRF2 reduced the senescence setpoint, defined as telomere length at senescence, from 7 to 4 kilobases. TRF2 protected critically short telomeres from fusion and repressed chromosome-end fusions in presenescent cultures, which explains the ability of TRF2 to delay senescence. Thus, replicative senescence is induced by a change in the protected status of shortened telomeres rather than by a complete loss of telomeric DNA.”  Google shows this study is cited by 403 others. 

Many of these studies deal with telomerase binding proteins and the very complex processes involved in telomere elongation.  While there seems consensus on the importance of telomere states I am not at all clear that the simplistic conclusion of the title of this report is widely endorsed.  Many publications express statements such as “loss of telomere integrity is a major trigger for the onset of premature senescence under mild chronic oxidative stress(ref).”  Note that in my treatise I have expanded the 12^th theory of aging to go beyond simple telomere shortening to read Telomere Shortening and Damage.

My own thoughts

1.      Cell senescence is a bad thing and is postponed or avoided under healthy conditions even in cells that have replicated many times by cell signaling that either a) naturally activates telomerase at the last minute, or b) activates telomerase binding factors to delay senescence such as suggested above, or c) triggers apoptosis.

2.     Loss of cells that have replicated many times due to healthy apoptosis need not affect health as long as there is a ready and able contingent of progenitor and stem cells to replace them (re the stem cell supply chain).

3.     Telomerase activation by using exogenous telomerase activators may or may not work to extend telomeres of somatic cells, depending on telomere binding factor complexities.  It may or may not work to thwart cell senescence.  The complex natural mechanisms that control telomere lengths may work to subvert telomere extension for many classes of cells.  Have you noticed that now, in year 2 of people striving to lengthen their telomeres via TA-2, astragaloside, etc. the almost complete absence of published reports of people that have demonstrably lengthened their telomeres? 

4.     Even if the average lengths of somatic cell telomeres can be increased by telomerase activation, this by itself may or may not have an effect on human longevity. (This statement could get me burned at the virtual stake in certain longevity circles, except for the following statement.)

5.     Telomerase activation may nonetheless have a strong positive effect in supporting healthy operation of the stem cell supply chain and may therefore be worth doing despite its effects on telomere lengths. See the blog post Revisiting telomere shortening yet-again.

For reference purposes, a list of my previous writings related to telomerase can be found in this post .

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17. October 2009 by admin.

I have written about cancer stem cells several times in this blog, but many oncologists and cancer researchers still see cancer stem cells mainly as hypothetical entities whose relevance if not very-existence is questionable.  A recent article in Gen points out that a number of pharmaceutical companies are betting big on cancer therapies based on going after cancer stem cells.

As I wrote in my July 2009 post On cancer stem cells, most cancer therapies are based on killing cancer cells – as many cells as possible.  But cancers frequently and persistently recur after bouts of radiation or chemotherapy.  The culprit is thought to be cancer stem cells, where any surviving ones simply go about making new cancer cells.  A new therapeutic concept is therefore to focus on killing the cancer stem cells.  “While normal stem cells are essential for development, play a key role in tissue maintenance, and aid in repair, cancer stem cells are believed responsible for tumorigenesis, metastases, and cancer recurrence(ref).”  I reported further research regarding cancer stem cells in my August 2009 blog post Update on cancer stem cells.

It turns out the way to do this is to target the same signaling pathways involved in the proliferation and differentiation of normal stem cells, pathways I have discussed previously in this blog. 

Notch is one such pathway which I discussed in the post On cancer stem cells.  As reported in Gen: “Different points in the (Notch) pathway have been targeted for drug development. OncoMed Pharmaceuticals’ OMP-21M18 is an antibody that blocks signals by binding to Delta-like ligand. The drug, which is in a clinical trial involving patients with advanced solid tumors, is part of a $1.4 billion collaboration with GlaxoSmithKline.  Merck and Roche have inhibitors to γ-secretase that cleaves the Notch receptor releasing the Notch intracellular domain, a transcription factor. Both companies’ drugs are in early testing against solid tumors. Finally, Trojantec is targeting the Notch pathway with a truncated version of Mastermind, a coactivator involved in chromatin-specific transcription. The drug may prove useful against tumors that overexpress Notch signaling components(ref).”  The role of Notch signaling in stem cell proliferation and differentiation was touched on in my blog post Niche, Notch and nudge.

PI3K/Akt is another pathway being targeted.  “The PI3K/Akt pathway’s importance in cancer is partly attributable to PI3K’s (phosphatidylinositol 3-kinase’s) association with oncogenic growth factor receptors, notably for epidermal growth factor, platelet-derived growth factor, and mesenchymal transition factor. The pathway is also prone to mutations associated with oncogenesis, including changes in the catalytic subunit of PI3K that occur in prostate, breast, endometrium, urinary tract, and colon cancers. — Similarly, mutations of the lipid phosphatase PTEN that normally serves to deactivate the PI3K/Akt pathway are found in cancers of the endometrium, brain, skin, and prostate, while mutations in the protein kinase Akt, which is downstream of PI3K, are overexpressed in head and neck squamous cell carcinoma, and in pancreatic and ovarian cancers. Eight drugs targeting the PI3K/Akt pathway are in clinical trials(ref).”  I have mentioned the P13K/Akt/mTOR and its relationship to stem cells in several posts including More mTOR links to aging theories.

The Hedgehog signaling pathway is another one being targeted by new drugs in the pipeline.   “The Hedgehog pathway provides an intercellular regulatory mechanism that serves essential functions in the normal proliferation and differentiation of stem cells. Mutations in this pathway figure in basal cell carcinoma, medulloblastoma, and other malignancies. Three drugs that interfere with hedgehog signaling are in clinical trials—two, Infinity Pharmaceuticals’ IPI-926 and Genentech/Curis’ GDC-0449, are derivatives of cyclopamine, which has been studied extensively(ref).”

Heavy players in the pharma industry are betting big on new therapies for going after cancer stem cells.  Perhaps more cancer researchers should start watching where the “smart money” is flowing.

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