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Research Newsletter-Fall/Winter 2013

SELECTED RECENT PUBLICATIONS BY LPI SCIENTISTS

Summarized by Stephen Lawson, LPI Administrative Officer

LPI scientists in boldface.


Healthy Aging Program


GUO C, ROSOHA E, LOWRY MB, BORREGAARD N, and GOMBART AF. Curcumin induces human cathelicidin antimicrobial peptide gene expression through a vitamin D receptor-independent pathway. J. Nutr. Biochem. 24:754-759, 2013

Vitamin D induces the synthesis of an antimicrobial peptide in the body called cathelicidin, which attacks pathogenic microbes. In cell-culture studies, the authors found that polyunsaturated fatty acids did not induce cathelicidin but that curcumin—derived from the spice turmeric—did, although not as strongly as vitamin D. This induction was independent of the vitamin D receptor, and its mechanism is currently being investigated in the Gombart lab.

GUO C, SINNOTT B, NIU B, LOWRY MB, FANTACONE ML, and GOMBART AF. Synergistic induction of human cathelicidin antimicrobial peptide gene expression by vitamin D and stilbenoids. Mol. Nutr. Food Res. 1-9, 2013

Using human cells in culture, the authors screened 446 molecules being investigated in human clinical trials to find ones that affect the regulation of the cathelicidin antimicrobial peptide gene. Two candidates induced cathelicidin: the stilbenoid resveratrol in red grapes and wine and pterostilbene, found in blueberries. Each compound also acted synergistically with vitamin D to further increase cathelicidin levels. More research is necessary to determine if these effects can be achieved in humans.

CAMPBELL Y, FANTACONE ML, and GOMBART AF. Regulation of antimicrobial peptide gene expression by nutrients and by-products of microbial metabolism. Eur. J. Nutr. 51:899-907, 2012

In this paper, the authors review the effect of nutrients and by-products of gut microbes on the regulation of antimicrobial peptides like defensin and cathelicidin synthesized in immune and epithelial cells. Vitamins A and D upregulate genes for the production of antimicrobial peptides important in innate immunity. Additionally, butyrate, a fatty acid formed in the gut by the bacterial metabolism of fiber, effectively increases antimicrobial peptide synthesis, especially cathelicidin. In rabbits, butyrate-induced stimulation of cathelicidin has been shown to ameliorate intestinal shigella infection. Bile acids also increase cathelicidin, possibly explaining the sterility of the bile duct. Sulforaphane, found in cruciferous vegetables like broccoli, has been shown to induce defensins in cultured human colon cells.

DIXON BM, BARKER T, MCKINNON T, CUOMO J, FREI B, BORREGAARD N, and GOMBART AF. Positive correlation between circulating cathelicidin antimicrobial peptide (hCAP18/LL-37) and 25-hydroxyvitamin D levels in healthy adults. BMC Res. Notes 5:575-579, 2012

Vitamin D strongly induces the antimicrobial peptide cathelicidin. The authors measured blood levels of vitamin D and cathelicidin in 19 healthy, middle-aged men and women. Serum levels of vitamin D up to 32 ng/mL were positively correlated with increasing concentrations of cathelicidin, although there was no correlation at serum levels of vitamin D above 32 ng/mL. This study suggests that protection against infection or sepsis may be increased by vitamin D supplementation or sun exposure that raises serum levels of vitamin D to 32 ng/mL.

FOK WC, ZHANG Y, SALMON AB, BHATTACHARYA A, GUNDA R, JONES D, WARD W, FISHER K, RICHARDSON A, and PEREZ VI. Short-term treatment with rapamycin and dietary restriction have overlapping and distinctive effects in young mice. J. Gerontol. A. Biol. Sci. Med. Sci. 68:108-116, 2013

Rapamycin is a natural antifungal, antibiotic substance found in the soil on Easter Island. Rapamycin and caloric restriction both increase the lifespan of mice by inhibiting cell growth and increasing autophagy (the degradation of dysfunctional cells). Unlike rapamycin, caloric restriction improved insulin sensitivity and glucose handling, attenuated weight gain, improved antioxidant mechanisms, and decreased adiposity in mice.

FRANCO MC, YE Y, REFAKIS CA, FELDMAN JL, STOKES AL, BASSO M, MELERO FERNANDEZ DE MERA RM, SPARROW NA, CALINGASAN NY, KIAEI M, RHOADS TW, MA TC, GRUMET M, BARNES S, BEAL MF, BECKMAN JS, Mehl R, and Estevez AG. Nitration of Hsp90 induces cell death. Proc. Natl. Acad. Sci. USA 110:1102-1111, 2013

Photo of Zhen Yu

Peroxynitrite is a potent oxidant formed from the reaction between superoxide and nitric oxide that can damage proteins, leading to the death of motor neurons, through a process called nitration. The heat shock protein Hsp90 is synthesized in cells to help fold and stabilize more than 200 proteins involved in cell survival and death. In this study, the authors showed that nitration of a single amino acid (tyrosine) in Hsp90 changes it from a pro-survival protein to a mediator of motor neuron death. Nitrated Hsp90 was found in ALS patients and in cases of spinal cord injury, further implicating peroxynitrite in neuronal cell death and damage.

KEITH DJ, BUTLER JA, BEMER B, DIXON B, JOHNSON S, GARRARD M, SUDAKIN DL, CHRISTENSEN JM, PEREIRA C, and HAGEN TM. Age and gender dependent bioavailability of R- and R,S-α-lipoic acid: A pilot study. Pharmacol. Res. 66:199- 206, 2012

R-alpha lipoic acid is a naturally occurring substance synthesized in the body and present in food like spinach and broccoli. Lipoic acid is an antioxidant and plays a role in the cell’s energy metabolism in mitochondria. In old rats, lipoic acid supplementation increases other antioxidant levels and protects against age-related stress. It detoxifies heavy metals, ameliorates inflammation, and has been used in people to treat diabetic neuropathy. Commercially made lipoic acid is a racemic mixture of the R and S forms. In this study, the authors gave 500 mg of either R-alpha-lipoic acid or R,S-alpha-lipoic acid to 10 young adults (18-45 years old) and nine elderly adults (over 75 years old) to determine how much of the different forms was absorbed into the blood stream. Absorption was quite variable. Generally, bioavailability of both forms was about the same in older and younger adults.

KYME P, THOENNISSEN NH, TSENG CW, THOENNISSEN GB, WOLF AJ, SHIMADA K, KRUG UO, LEE K, MULLER-TIDOW C, BERDEL WE, HARDY WD, GOMBART AF, KOEFFLER HP, and LIU GY. C/EBPε mediates nicotinamide-enhanced clearance of Staphylococcus aureus in mice. J. Clin. Invest. 122:3316-3329, 2012

The authors found that vitamin B3 (niacin), when injected into mice infected with the bacterium Staphylococcus aureus, enhanced the killing of S. aureus up to 1,000-fold. Pretreatment of human blood with B3 greatly reduced the survival of added S. aureus. B3 had no effect in mice without neutrophils, suggesting that these immune cells are crucial for the antibacterial effect. In contrast to mice, B3 stimulated the production of an antimicrobial peptide in human neutrophils. These results suggest that safely achievable levels of B3 may have therapeutic value in treating certain infections.

WONG CP, MAGNUSSON KR, and HO E. Increased inflammatory response in aged mice is associated with age-related zinc deficiency and zinc transporter dysregulation. J. Nutr. Biochem. 24:353-359, 2013

Aging is associated with a decline in the performance of the immune system. The authors used cell cultures and mice to study the effect of zinc on immunity and found that age-related zinc deficiency in immune cells and dysregulation of zinc transporters impaired immune function and increased inflammation. Epigenetic influences, especially the under- or over-methylation of DNA, also contributed to the age-related decline in zinc status and enhanced inflammation. Zinc supplementation of old mice consuming a zinc-adequate diet dramatically reduced serum levels of inflammatory markers like IL-6 and TNF-α.

Cardiovascular and Metabolic Diseases


FREI B, BIRLOUEZ-ARAGON I, and LYKKESFELDT J. Authors’ perspective: What is the optimum intake of vitamin C in humans? Crit. Rev. Food Sci. Nutr. 52:815-829, 2012

Linus Pauling’s favorite molecule—vitamin C—is required to prevent scurvy but has other important effects: enhancing the absorption of nonheme iron, scavenging free radicals as the premier antioxidant, helping to synthesize neurotransmitters and carnitine, and detoxifying histamine. The authors reviewed the evidence for vitamin C’s role in the primary prevention of heart disease, stroke, and cancer, concluding that 200 mg/day may be optimum. Vitamin C reduces hypertension, improves endothelial function or arterial relaxation, and decreases chronic inflammation, a hallmark of chronic diseases. They note that standard clinical trials designed to assess drug efficacy aren’t appropriate to study micronutrients like vitamin C for many reasons, including the lack of a true placebo group. Instead, they argue that the optimum intake of vitamin C can be deduced from all the available evidence from clinical trials and observational studies, as well as based on biological plausibility.

DEPNER CM, PHILBRICK KA, and JUMP DB. Docosahexaenoic acid attenuates hepatic inflammation, oxidative stress, and fibrosis without decreasing hepatosteatosis in a Ldlr(-/-) mouse model of Western diet-induced nonalcoholic steatohepatitis. J. Nutr. 143:315-323, 2013

Nonalcoholic steatohepatitis (NASH), marked by liver damage, inflammation, oxidative stress, and fibrosis, is associated with obesity. Using a mouse model of NASH induced by feeding a Western-type diet high in fat and carbohydrates, the authors showed that supplementation with physiologically relevant doses of docosahexaenoic acid (DHA), an omega-3 fatty acid found in fatty fish, attenuated all of the markers of NASH. DHA was more effective than eicosapentaenoic acid (EPA), which is a precursor for DHA synthesis in the body.

WEI H, ZHANG W, MCMILLEN TS, LEBOEUF RC, and FREI B. Copper chelation by tetrathiomolybdate inhibits vascular inflammation and atherosclerotic lesion development in apolipoprotein E-deficient mice. Atherosclerosis 223:306-313, 2012

Copper is an essential nutrient, like iron, that has been implicated in the inflammatory process of atherosclerosis. Adhesion molecules synthesized by surface cells of blood vessels and arteries can cause white blood cells called monocytes to adhere to these surface cells. The monocytes then migrate into the intima and become macrophages that engulf oxidized cholesterol, leading to the formation of “foam” cells, a hallmark of atherosclerotic plaque formation. Tetrathiomolybdate (TTM) is a small molecule containing sulfur and molybdenum that has a strong binding affinity for copper. The authors showed that treating mice that develop atherosclerosis with TTM dramatically reduced copper levels in the aorta and heart, and, consequently, inhibited atherosclerosis. TTM did not affect iron homeostasis or reduce oxidative stress; rather, it chelated and removed copper, thereby limiting vascular inflammation.

MILLER GW, ULATOWSKI L, LABUT EM, LEBOLD KM, MANOR D, ATKINSON J, BARTON CL, TANGUAY RL, and TRABER MG. The α-tocopherol transfer protein is essential for vertebrate embryogenesis. PLoS ONE 7:e47402, 2012

The alpha-tocopherol transfer protein (TTP) recognizes only the alpha-tocopherol form of vitamin E for distribution to tissues. TTP is present in zebrafish, which are a good model to study embryogenesis and fetal development. Inhibition of TTP in zebrafish embryos results in severe malformations of the head and eyes, suggesting that TTP plays a crucial role in delivering vitamin E necessary for the proper development of the vertebrate central nervous system.

Photo of LeeCole Legette

MICHELS AJ, HAGEN TM, and FREI B. Human genetic variation influences vitamin C homeostasis by altering vitamin C transport and antioxidant enzyme function. Ann. Rev. Nutr. 33:45-70, 2013

The absorption of vitamin C from the gut into the blood stream is regulated by vitamin C transport molecules on the cells that line the intestine. Other factors, such as oxidative stress, inflammation, and enzyme activity that uses up vitamin C, also influence vitamin C levels in blood and tissues. This paper reviews the subtle differences in genes that code for the vitamin C transporters. These differences, called singlenucleotide polymorphisms (SNPs), result in different vitamin C dynamics among people. When possible, these SNPs should be taken into consideration when selecting populations to study the effect of vitamin C on chronic disease risk.

LEBOLD KM, LÖHR CV, BARTON CL, MILLER GW, LABUT EM, TANGUAY RL, and TRABER MG. Chronic vitamin E deficiency promotes vitamin C deficiency in zebrafish leading to degenerative myopathy and impaired swimming behavior. Comp. Biochem. Physiol. 157:382-389, 2013

As in humans, ascorbic acid and alpha-tocopherol are vitamins for zebrafish. The authors fed diets with or without vitamin E to zebrafish for a year, with decreasing amounts of vitamin C in the diets for the last 150 days. At the end of the experiment, swimming behavior in response to a stimulus was measured. The zebrafish fed the diet without vitamin E exhibited high levels of oxidative stress markers and sluggish swimming behavior associated with myopathy of skeletal muscles. They also had lower levels of vitamin C than the fish fed vitamin E. The low levels of dietary vitamin C were insufficient to recycle vitamin E from its oxidized to reduced state.

TRABER MG. Mechanisms for the prevention of vitamin E excess. J. Lipid Res. 54:2295-2306, 2013

Alpha-tocopherol is the only one of the eight forms of vitamin E that is recognized by the Institute of Medicine to meet vitamin E requirements in humans. Vitamin E is a fat-soluble antioxidant that protects polyunsaturated fatty acids from oxidation. Despite being fat-soluble, the alphatocopherol form of vitamin E does not accumulate in the liver to harmful levels. Alpha-tocopherol is selected by the alpha-tocopherol transfer protein—synthesized in the liver—for distribution to lipoproteins circulating in blood. Other forms of vitamin E are rapidly metabolized by cytochrome P450 enzymes in the liver, and the metabolites are excreted. Alpha-tocopherol does not accumulate in the liver because excessive amounts are either metabolized or excreted in bile.

JUMP DB, DEPNER CM, and TRIPATHY S. Omega-3 fatty acid supplementation and cardiovascular disease. J. Lipid Res. 53:2525-2545, 2012

Consumption of fatty fish containing omega-3 fatty acids has been recommended for many years to help prevent cardiovascular diseases. In this paper, the authors review the evidence for the effect of omega-3 fatty acids like docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on the primary and secondary (therapeutic) protection against cardiovascular diseases, as well as discuss the mechanisms that account for these effects. Omega-3 fatty acids reduce blood triglyceride levels, attenuate inflammation, and are anti-arrhythmic. DHA changes the fluidity of cell membranes and their cholesterol content. By improving mitochondrial function in cardiac cells, DHA has been found to help protect against cardiac arrhythmia, which is the main cause of sudden cardiac death. Omega-3 fatty acids have generally been shown to help prevent cardiovascular disease, whereas randomized clinical trials to test the effect on clinical events in patients with cardiovascular disease (secondary prevention) have had inconsistent results. DHA is poorly synthesized in the body from alpha-linolenic acid (ALA), the major source of omega-3 fatty acids in plants, algae, and yeast. ALA consumption does little to protect against cardiovascular disease.

Cancer Chemoprotection Program


PARASRAMKA MA, DASHWOOD WM, WANG, R, ABDELLI A, BAILEY GS, WILLIAMS DE, HO E, and DASHWOOD RH. MicroRNA profiling of carcinogeninduced rat colon tumors and the influence of dietary spinach. Mol. Nutr. Food Res. 56:1259-1269, 2012.

MicroRNAs are small pieces of RNA that affect translation, the process by which messenger RNA codes for proteins after being transcribed from DNA. Dysregulation of microRNAs is associated with cancer and other pathologies. In this year-long study, the authors investigated the role of microRNA dysregulation in the development of colon cancer in rats induced by exposure to heterocyclic amines (cooked-meat mutagens) found in proteinacious food cooked at high temperatures. After the exposure to heterocyclic amines for several weeks (postinitiation), rats that were fed a diet containing freeze-dried spinach for the remainder of the experiment had a lower incidence of colon tumors, which correlated with the partial reversal of microRNA dysregulation.

Photo of a laboratory

CLARKE JD, RIEDL K, BELLA D, SCHWARTZ SJ, STEVENS JF, and HO E. Comparison of isothiocyanate metabolite levels and histone deacetylase activity in human subjects consuming broccoli sprouts or broccoli supplement. J. Agric. Food Chem. 59:10955-10963, 2011

DNA in cells is wrapped around proteins called histones. When chemical acetyl groups are added to histones (acetylation), genes are turned on; when acetyl groups are removed (deacetylation), genes are turned off. Inhibitors of deacetylation cause tumor-suppressor genes to remain active. Glucosinolates are phytochemicals found only in cruciferous vegetables like broccoli. When such vegetables are chopped or chewed, an enzyme called myrosinase is released that converts the glucosinolates into a number of compounds, including an isothiocyanate called sulforaphane, which inhibits histone deacetylation. Gut bacteria also synthesize myrosinase. In this paper, the authors measured the amount of glucosinolate metabolites like sulforaphane in the urine of subjects consuming broccoli sprouts or a broccoli supplement and also assessed histone deacetylase activity in blood cells. Consuming broccoli sprouts was much more effective in elevating sulforaphane levels in urine and in decreasing histone deacetylase activity in blood cells, probably because of the absence of myrosinase in the broccoli supplement.

CLARKE JD, HSU A, YU Z, DASHWOOD RH, and HO E. Differential effects of sulforaphane on histone deacetylases, cell cycle arrest and apoptosis in normal prostate cells versus hyperplastic and cancerous prostate cells. Mol. Nutr. Food Res. 55:999-1009, 2011

Sulforaphane is an isothiocyanate present in cruciferous vegetables that has previously been identified as an anticancer phytochemical due to its influence on phase 2 enzymes that modify xenobiotics and toxins for excretion from the body. More recently, sulforaphane has been found to act as a histone deacetylase inhibitor. In this role, sulforaphane helps to preserve the acetylation of histones surrounding DNA, leading to the activation of tumor-suppressor genes. The authors exposed normal, benign hyperplastic, and cancerous prostate cells in vitro to sulforaphane, which acted as a histone deacetylase inhibitor and induced cell-cycle arrest and apoptosis (programmed cell death) in the hyperplastic and cancerous cells but not in the normal cells.

RAJENDRAN P, HO E, WILLIAMS DE, and DASHWOOD RH. Dietary phytochemicals, HDAC inhibition, and DNA damage/repair defects in cancer cells. Clin. Epigenetics 3:4, 2011

In this review, the authors discuss the role of phytochemicals in cancer prevention, focusing on several mechanisms by which these phytochemicals lead to the impairment or death of cancer cells. Tea polyphenols, curcumin in the spice turmeric, indole-3-carbinol and isothiocyanates like sulforaphane in cruciferous vegetables, selenium, allium in garlic, genistein in soy, and quercetin in apples and onions can affect gene expression by altering the activity of histones, which are molecules around which DNA is wound. By inhibiting a chemical modification called histone deacetylation, these phytochemicals can turn on tumor-suppressor genes and affect DNA damage status. They can cause breaks in DNA through the generation of reactive oxygen species—easily repaired in normal cells but not in cancer cells. Most can aid in DNA repair in normal cells, and many, such as curcumin, resveratrol in grapes and wine, sulforaphane, and quercetin, can inhibit DNA damage repair in cancer cells, leading to cell death.

SHOREY LE, MADEEN EP, ATWELL LL, HO E, LÖHR CV, PEREIRA CB, DASHWOOD RH, and WILLIAMS DE. Differential modulation of dibenzo[def,p]chrysene transplacental carcinogenesis: Maternal diets rich in indole-3-carbinol versus sulforaphane. Toxicol. Appl. Pharmacol. 270:60-69, 2013

Treating pregnant mice with the carcinogen dibenzo[def,p]chrysene (DBC), an environmental polycyclic aromatic hydrocarbon produced by the burning of fossil fuels, causes the offspring to develop aggressive lymphoma and lung and liver tumors. The authors showed in previous studies that supplementing the pregnant mice exposed to DBC with indole-3-carbinol (I3C), a phytochemical found in cruciferous vegetables, protected against mortality from lymphoma in the offspring and decreased the number of lung tumors. In the present study, the authors compared the effects of feeding freeze-dried broccoli sprout powder containing I3C, purified supplements of I3C, or sulforaphane (an isothiocyanate) to the pregnant mice. Surprisingly, maternal consumption of broccoli powder or sulforaphane increased morbidity in the offspring and did not inhibit tumorigenesis caused by exposure to DBC. Administering I3C with sulforaphane nullified the deleterious effects of sulforaphane alone. The sulforaphane dose in these experiments greatly exceeded the amount humans typically consume in broccoli, and the results may be explained by a sensitivity of the fetus to non-physiologically high concentrations of sulforaphane. More research is under way to understand these complex mechanisms.

HO E, BEAVER LM, WILLIAMS DE, and DASHWOOD RH. Dietary factors and epigenetic regulation for prostate cancer prevention. Adv. Nutr. 2:497-510, 2011

The authors discuss the importance of nutrient-gene interactions in cancer, especially those involving epigenetic regulation of gene activity. DNA methylation and histone modifications affect how genes are silenced or activated. For example, most DNA in prostate cancer cells is hypomethylated, while some specific genes are hypermethylated. This methylation activity influences how cancer progresses. The prevention or progression of prostate cancer may be influenced by dietary factors like B vitamins (folate and vitamins B6 and B12), the amino acid methionine, soy isoflavones, tea polyphenols, isothiocyanates (sulforaphane from cruciferous vegetables), selenium, and zinc, all of which influence methylation in desirable ways. Histone acetylation, an epigenetic event in which acetyl chemical groups are added to the histones that regulate DNA activity, is also associated with cancer chemoprotection. Histone deacetylase inhibitors, including fiber, garlic compounds, sulforaphane and indole-3-carbinol from cruciferous vegetables, selenium, curcumin from the spice turmeric, and tea polyphenols, prevent the removal of acetyl groups from histones, leading to apoptosis (programmed cell death) of cancer cells and the activation of tumor-suppressor genes. Much more research needs to be done, especially concerning dose, interactions, metabolism, timing, and tissue specificity.

Photo of a laboratory

SAUD SM, YOUNG MR, JONES-HALL YL, ILEVA L, EVBUOMWAN MO, WISE J, COLBURN NH, KIM YS, and BOBE G. Chemopreventive activity of plant flavonoid isorhamnetin in colorectal cancer is mediated by oncogenic Src and β-catenin. Cancer Res. 73:5473-5484, 2013

Isorhamnetin is a flavonol found in onions and almonds. It is similar in structure to the flavonol quercetin and can be formed from the metabolism of quercetin in the body. An association between the consumption of dietary isorhamnetin and a reduced risk of colorectal cancer recurrence was observed in the Polyp Prevention Trial, which was a four-year, randomized, nutritional intervention study with 1,905 subjects. In the present study, mice were treated with a chemical carcinogen (azoxymethane) and a colonic irritant to cause colorectal tumors. After that treatment, groups were given diets supplemented with isorhamnetin or the flavonols quercetin, rutin, or myricetin. The mice fed the isorhamnetin-supplemented diet had fewer tumors, smaller tumors, and a much higher survival rate than the control mice. Isorhamnetin was more effective than the other flavonols and also decreased inflammation, inhibited cell proliferation, and inhibited Src oncogene activity.

MENTOR MARCEL RA, BOBE G, SARDO C, WANG L-S, KUO C-T, STONER G, and COLBURN NH. Plasma cytokines as potential response indicators to dietary freezedried black raspberries in colorectal cancer patients. Nutr. Cancer 64:820-825, 2012

Twenty-four patients with colorectal cancer were enrolled in this clinical study to determine the effect of consuming a slurry of freeze-dried black raspberry powder in water three times a day for about three weeks, while receiving no other therapy. Plasma samples and biopsies of normal and cancer tissue in the colon were taken periodically and assayed for markers of inflammation, apoptosis (programmed cell death), cell proliferation, and angiogenesis (blood vessel formation). The consumption of black raspberry slurry improved these parameters and time-dependently decreased interleukin 8, which is a cytokine (cell-signaling molecule) associated with inflammation.


Last updated October 2013