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The Human Tripeptide GHK-Cu in Prevention of Oxidative Stress and

Degenerative Conditions of Aging: Implications for Cognitive Health

Article  in  Oxidative Medicine and Cellular Longevity · May 2012

DOI: 10.1155/2012/324832 · Source: PubMed

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Hindawi Publishing Corporation
Oxidative Medicine and Cellular Longevity
Volume 2012, Article ID 324832, 8 pages
doi:10.1155/2012/324832

Review Article
The Human Tripeptide GHK-Cu in Prevention of
Oxidative Stress and Degenerative Conditions of Aging:
Implications for Cognitive Health

Loren Pickart, Jessica Michelle Vasquez-Soltero, and Anna Margolina

Skin Biology, Research & Development Department, 4122 Factoria Boulevard SE-Suite No. 200, Bellevue, WA 98006, USA

Correspondence should be addressed to Loren Pickart, [email protected]

Received 13 January 2012; Accepted 27 February 2012

Academic Editor: Marcos Dias Pereira

Copyright © 2012 Loren Pickart et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Oxidative stress, disrupted copper homeostasis, and neuroinflammation due to overproduction of proinflammatory cytokines are
considered leading causative factors in development of age-associated neurodegenerative conditions. Recently, a new mechanism
of aging—detrimental epigenetic modifications—has emerged. Thus, compounds that possess antioxidant, anti-inflammatory
activity as well as compounds capable of restoring copper balance and proper gene functioning may be able to prevent age-
associated cognitive decline and ward off many common neurodegenerative conditions. The aim of this paper is to bring attention
to a compound with a long history of safe use in wound healing and antiaging skin care. The human tripeptide GHK was discovered
in 1973 as an activity in human albumin that caused old human liver tissue to synthesize proteins like younger tissue. It has high
affinity for copper ions and easily forms a copper complex or GHK-Cu. In addition, GHK possesses a plethora of other regenerative
and protective actions including antioxidant, anti-inflammatory, and wound healing properties. Recent studies revealed its
ability to up- and downregulate a large number of human genes including those that are critical for neuronal development and
maintenance. We propose GHK tripeptide as a possible therapeutic agent against age-associated neurodegeneration and cognitive
decline.

1. Introduction gene expression by means of DNA methylation and histone

modification [3].
Today the most widespread neurological problems are Since the importance of oxidative stress in the
considered neurodegenerative diseases of aging such as development of age-related neurodegeneration is well
Alzheimer’s and Parkinson’s that rob people from their established, a number of antioxidant compounds are
golden years causing early debilitation and dependency considered promising in prevention of neurodegenerative
on special care. Despite all advances in neuroscience in disorders, including vitamin E, melatonin, green tea
recent decades, age-associated cognitive decline resulting polyphenols, resveratrol, and others [4]. However, so far the
from neurodegenerative processes in the brain remains a clinical studies produce mixed results with many promising
challenge for researchers and clinicians due to the highly approaches such as vitamin E therapy failing to slow down
complex nature of its pathogenesis. the progression of age-related neurodegenerative conditions
Currently, the key processes leading to neurodegenera- [5]. There are also studies investigating an effect of metal
tion are thought to be oxidative stress, disruption of transi- chelators and dietary approaches aimed at reducing intake
tional metal homeostasis, and neuroinflammation [1, 2]. of iron and copper [6]. Finally, researchers are investigating
There is also growing evidence that neurodegenerative diverse compounds capable of favorably altering gene
diseases such as Alzheimer’s and Parkinson’s may be caused expression (epigenetic modifiers), reversing effects of
by detrimental environmental and dietary factors that alter environmental perturbagens [7].
2 Oxidative Medicine and Cellular Longevity

The human copper-binding tripeptide glycyl-L-histidyl- H

L-lysine (GHK) is a compound with a long history of safe H
N
use in wound healing and antiaging skin care. Since its
discovery in 1973, almost four decades of extensive research
have established its diverse beneficial actions in many organs Cu O
N H
and tissues including nervous tissue, skin, intestine, bone,
O H
and blood vessels. The molecule has a high affinity for Cu (II) N
and forms the chelate GHK-Cu. The GHK copper complex N H
H H
(or GHK-Cu) has been proven to exhibit antioxidant, anti-
N N
inflammatory, regenerative, and wound healing actions [8]. H
Recent studies demonstrated that the GHK tripeptide up- H
and downregulates a large number of human genes, which O
may contribute to the pleiotropic health promoting effects O
H
of its copper complex [9]. We propose that the GHK-
Cu complex may act therapeutically against age-associated Figure 1: Molecular structure of the tripeptide GHK-Cu. In
neurodegeneration and cognitive decline. solution lysine carboxyl groups of neighboring complexes may
participate in a complex formation.

2. Copper Binding Properties of the Human

Tripeptide GHK versus GHK binding constant 16 log10 = 16.44). It has been
Human peptide GHK was isolated in 1973 as an activity established that copper (II) redox activity is silenced when
in human plasma that caused old human liver tissue to copper ions are complexed with the GHK tripeptide, which
synthesize proteins like younger tissue [10]. In human allows the delivery of nontoxic copper into the cell [12–14].
plasma GHK is present at about 200 micrograms/liter in
men of age 20–25 but declines to 80 micrograms/liter by 3. The Copper Paradox
age 60–80. Subsequent studies established this activity as a
tripeptide with an amino acid sequence glycyl-L-histidyl-L- The brain is exceptionally rich in copper, which plays an
lysine with a strong affinity for copper that readily formed important role in its physiology and pathology. Copper
the complex GHK-Cu. Since GHK-Cu promotes cell growth, deficiency caused by bariatric surgery or gastrointestinal
it was proposed that the GHK acts by delivering copper bleeding led to myelopathy (human swayback), paralysis,
required for the cellular functions into the cell in a form that blindness, and behavioral and cognitive changes [15–17].
is nontoxic and can be utilized by the cell [11]. Mice born and maintained on a copper-deficient diet had
The molecular structure of the GHK copper complex 80% reduction in brain copper level at 6–8 weeks and had
(GHK-Cu) has been extensively studied using X-ray crystal- neuronal and glial changes typical for neurodegenerative
lography, EPR spectroscopy, X-ray absorption spectroscopy, disorders [18].
and NMR spectroscopy as well as other methods such Despite unquestionable essentiality of copper, unreg-
as titration. In the GHK-Cu complex, the Cu (II) ion is ulated copper ions may increase oxidative damage. It is
coordinated by the nitrogen from the imidazole side chain known that amyloid precursor protein (APP) implicated in
of the histidine, another nitrogen from the alpha-amino development of Alzheimer’s disease (AD) can convert Cu
group of glycine, and the deprotonated amide nitrogen of (II) into Cu (I) potentially increasing oxidative damage [19].
the glycine-histidine peptide bond (Figure 1). Since such a However, it remains unclear whether copper accumulation
structure could not explain a high stability constant of the in senile plaques of AD patients is a cause or a consequence
GHK-Cu complex (log10 = 16.44 versus 8.68 of the GH of pathological processes observed in AD. According to
copper complex, which is similar to the GHK-Cu structure), Exley followed by Bolognin et al., only aluminum, but not
it was proposed that another amino group participates copper or iron, is capable of triggering amyloid precipitation
in the complex formation. According to the recent study and APP and tau181 protein overproduction [20, 21].
by Hureau et al., the Cu (II) is also coordinated by the The study by Kawahara et al. showed that copper and
oxygen from the carboxyl group of the lysine from the carnosine attenuate neurotoxicity of another compound
neighboring complex. Another carboxyl group of lysine involved in neurodegeneration—prion protein [22]. Bishop
from a neighboring complex provides the apical oxygen, and Robinson observed that amyloid beta protein may be
resulting in the square-planar pyramid configuration. Many neuroprotective when combined with copper. According to
researchers proposed that, at the physiological pH, GHK- these authors, amyloid beta injected simultaneously with
Cu complexes can form binary and ternary structures which copper was not toxic, while iron and zinc complexed with
may involve amino acid histidine and/or the copper binding amyloid beta were more toxic than amyloid beta alone [23].
region of the albumin molecule. Lau and Sarkar found Since copper accumulates in senile plaques of AD
also that GHK can easily obtain copper 2+ bound to other patients, some authors proposed dietary restriction of copper
molecules such as the high affinity copper transport site or intake of copper chelators as a preventive therapy for
on plasma albumin (albumin binding constant log10 = 16.2 the elderly. However, several studies demonstrated that AD
Oxidative Medicine and Cellular Longevity 3

patients have reduced, not elevated, brain and cerebrospinal activity and can trap copper, rendering brain tissue copper
fluid copper level [24, 25]. Currently, many authors suggest deficient. Transgenic mice overexpressing APP had reduced
mild copper deficiency as a causative factor in AD and SOD1 activity in the brain. SOD1 activity was restored by
possibly other neurodegenerative disorders [26, 27]. In copper supplementation [35]. It has been shown that GHK-
a placebo-controlled, double-blinded, randomized clinical Cu increases the level of antioxidant enzymes and SOD
trial, oral copper supplementation (8 mg/day) in 68 AD activity, supposedly by supplying copper necessary for its
patients had a beneficial effect on relevant AD biochemical function [36].
markers. The authors concluded that long-term intake of GHK-Cu also reduces oxidative damage by modulating
copper can be excluded as a causative factor in AD and iron levels. The presence of iron complexes in damaged
may in fact be protective [28]. In addition, several studies tissues is detrimental to wound healing, due to the increased
revealed molecular mechanisms underlying beneficial effects lipid peroxidation in the presence of iron ions, as well as
of copper in AD, such as an inhibition of beta-amyloid microbial infection mediated by iron. Pickart demonstrated
peptide production [29]. that GHK-Cu inhibited lipid peroxidation if the iron source
Due to high importance of copper in the brain was ferritin. It was proposed that GHK-Cu binds to the
metabolism and a possible role of copper deficiency in the channels of ferritin involved in iron release and physically
development of Alzheimer’s and other neurodegenerative prevents the release of Fe (II). Thus, GHK-Cu exhibits
disorders, limiting of the dietary copper intake in elderly may antioxidant function in wounds by inhibiting ferritin iron
actually increase their chance of developing neurodegener- release in damaged tissues, preventing inflammation and
ative disorders. Another option is to use compounds that microbial infections [37]. GHK-Cu produced a 75% reduc-
can form nontoxic complexes with Cu (II), preventing its tion of gastric mucosa homogenates of lipid peroxidation
accumulation in senile plaques and increasing its bioavail- in the range 10–100 mM suggesting that copper-peptide
ability. According to Narahara et al., β-citryl-L-glutamate— complexes are able to effectively neutralize damaging oxygen-
a compound that is abundant in the developing brain— derived free radicals [38].
has SOD-like activity when complexed with copper and GHK (in this experiment the peptide alone, not its
may be neuroprotective [30]. Rózga and Bal propose that copper complex, was used) has been proven to quench
human serum albumin (HSA) may be neuroprotective due alpha,beta-4-hydroxy-trans-2-nonenal—a toxic product of
to its ability to bind both copper and amyloid beta protein fatty acids’ lipid peroxidation that play important role in
[31]. Perrone at al. reported copper transfer from amyloid the pathogenesis of several age-related conditions including
beta to a copper binding domain of HSA that is similar Alzheimer’s disease, neuropathy, and retinopathy [39]. GHK
to the tripeptide GHK’s structure with the same copper- peptide was also able to quench acrolein—another toxic
binding histidine residue (DAHK) [32]. Thus GHK peptide, product of lipid peroxidation involved in the development
a natural copper-binding and copper-regulating molecule of many age-related degenerative disorders. The authors
with well-established regenerative and protective actions proposed that GHK may be used for prevention of some age-
in different organs and tissues, should be considered a related pathologies, including Alzheimer’s disease [40].
promising therapeutic agent in preventing and correcting Inflammation and oxidative damage due to the over-
copper imbalance in neurodegenerative disorders. production of proinflammatory cytokines play an important
role in the development of AD and other neurodegenerative
conditions [41]. In 2001 McCormack et al. established
4. Antioxidant and Anti-Inflammatory that GHK-Cu decreased proinflammatory cytokine TGF-
Properties of GHK-Cu beta in human fibroblast culture [42]. In 2003 Canapp et al.
demonstrated that GHK-Cu improved healing of ischemic
The brain’s high metabolic activity results in elevated oxygen wounds and suppresses inflammation by lowering the level
consumption and constant production of reactive oxygen of acute-phase inflammatory cytokines such as TGF-beta and
species (ROS) in mitochondria. At the same time, the TNF-alpha [43].
brain tissue is rich in unsaturated fatty acids and transition
metal ions yet has relatively fewer antioxidants comparing 5. GHK Stimulates Blood Vessel Growth
to other organs creating favorable conditions for oxidative
damage. Since the blood-brain barrier prevents many dietary Vascular factors play an important role in the development
antioxidants from entering the brain, it largely relies on of many neurodegenerative diseases of aging. Since the brain
endogenous antioxidants such as Cu- and Zn-dependent is a highly metabolically active organ and requires constant
superoxide dismutase (Cu, Zn SOD1). This enzyme requires supply of oxygen and nutrients, a well-developed, adequate
metal ions copper and zinc in order to be active. Hence, vascular network is essential for its health [44].
copper deficiency can lead to reduced SOD activity and From wound healing studies, it is known that GHK-Cu
increased oxidative brain damage [33]. When pregnant rats helps reestablish blood flow into damaged tissues through
were kept on a copper-deficient diet, the embryos displayed a mixture of three actions: angiogenesis (new blood-vessel
low SOD activity, increased super oxide anion radical level, formation), anticoagulation, and vasodilation. GHK-Cu
and higher incidence of DNA damage and malformations increases the expression of basic fibroblast growth factor
[34]. Amyloid precursor protein (APP) that is implicated in and vascular endothelial growth factor, both of which aid
Alzheimer’s disease (AD) development has copper binding blood vessel formation [45]. In addition GHK-Cu’s ability
4 Oxidative Medicine and Cellular Longevity

to stimulate synthesis of collagen and elastin is useful in In 2009, a group of researchers from the Seoul National
restoring integrity of blood vessel walls. University (Republic of Korea) demonstrated that GHK-Cu
Sage et al. observed that endothelial cells at the site of in concentrations of 0.1–10 microM increases expression
an injury produce a protein called SPARC that contains of integrins and p63. Since these molecules are considered
GHK sequence. SPARC protein turned out to be abundant proliferative markers of epidermal stem cells, the authors
in all tissues that undergo rapid remodeling such as skin concluded that GHK-Cu helps to maintain an active prolif-
or embryonic tissues. When the tissue is damaged, tissue erative state of epidermal stem cells [59].
proteases break down SPARC, releasing an array of GHK and Iorio et al. used a repository of transcriptional responses
GHK-containing copper-binding peptides, which stimulate to compounds, the Connectivity Map (cMap) [60], and
cell proliferation and new vessels growth. When blood flow MANTRA software (http://mantra.tigem.it/) to explore net-
is sufficiently restored, SPARC inhibits cell proliferation and works of compounds producing similar transcriptional
growth, controlling new vessels progression [46]. responses. GHK, as one of the compounds studied, increased
mRNA production in 268 genes while suppressing 167 [61].
6. GHK Increases Neurotrophins Also, Hong et al. used genome-wide profiling to identify
genetic biomarkers (genetic signature) for metastasis-prone
Among compounds that have a protective effect and can colorectal cancer as well as their perturbagens—substances
reduce oxidative damage are some neurotrophic factors such that modulated their expression. GHK suppressed RNA pro-
as brain-derived neurotrophic factor (BDNF) [47]. There duction in 70% of 54 human genes overexpressed in patients
is evidence that GHK increases production of neurotrophic with an aggressive metastatic form of colon cancer and was
factors. Both Sensenbrenner et al. and Lindner et al. found active at a low nontoxic 1 micromolar concentration [62].
that GHK stimulates the outgrowth of cultured nerves [48,
49]. Ahmed et al. established that nerve stubs placed in a
collagen tube impregnated with GHK (used without copper) 8. The Connectivity Map and
had an increased production of nerve growth factor and the GHK-Affected Genes
the neurotrophins NT-3 and NT-4 increased migration of
cells into collagen tube and sped up the regeneration of Our own studies using the Broad Institute’s Connectivity
nerve fibers. In addition, GHK also increased axon count and Map (cMap) showed that GHK activates numerous genes
proliferation of the Schwann cells compared to the control involved in nervous system physiology, development, and
group [50]. maintenance. The Connectivity Map holds three GHK gene
expression profiles created using the GeneChip HT Human
Genome U133A Array. Two of the profiles emerged from
7. GHK as a Gene Regulator the treatment of the PC3 cell line; the other profile came
Epigenetic modification of gene expression is currently from the cell line MCF7. All cell lines were treated with GHK
considered a link between the environment, aging, and at 1 micromolar. By selecting all three gly-his-lys instances
neurodegeneration. It has been shown that some dietary we determined which genes are affected. Most interesting
and environmental factors, such as certain toxins, may result are the genes that are remarkably up- and downregulated.
in abnormal DNA methylation and histone modification, Using the cMap one can enter amplitude thresholds for
altering gene activity. Recent studies revealed that some both up- and downregulated genes in order to find the
well-known antioxidant and anti-inflammatory substances genes whose expression are significantly altered. With an up
such as plant flavonoids may counteract these deleterious gene amplitude threshold of 0.40 (equivalent to a 1.5-fold
changes by modulating activity of certain genes, reducing induction) and a down gene amplitude threshold of −0.40
glial inflammation and inhibiting production of neurotoxins (equivalent to a 1.5-fold repression), we discover that 76
[51, 52]. genes lay above the up threshold and 6 lay below the down
First evidence that GHK-Cu may regulate an activity of threshold.
certain genes came from wound healing and skin remodeling In the cMap genes are represented by probe set IDs
studies. By 1983, Pickart had established that GHK-Cu accel- in the tag lists, which identify the up- and downregu-
erates wound healing and contraction, improves the take lated genes. Running the probe set IDs in the tag lists
of transplanted skin, and also possesses anti-inflammatory through the “Batch query” in the NetAffx Analysis Center
actions [53, 54]. (http://www.affymetrix.com/), annotations for each probe
Subsequent studies directed by Maquart et al. (France) set are retrieved. Reviewing the annotations we observe
demonstrated that GHK-Cu at a very low, nontoxic con- that 5 genes associated with nerves are stimulated (mRNA
centration (1–10 nanomolar) stimulated both the synthesis expression is increased) by GHK while none are suppressed
and breakdown of collagen and glycosaminoglycans [55, 56]. (mRNA expression is decreased). These 5 genes are listed in
It modulated an expression of both metalloproteinases and Table 1 along with their corresponding average fold change.
their inhibitors (TIMP-1 and TIMP-2), improving wound
healing and facilitating skin remodeling processes [57]. In 9. GHK May Reverse Gene Silencing
2000 the same group demonstrated that GHK-Cu increased
mRNA for collagen, dermatan sulfate, chondroitin sulfate, Epigenetic silencing of certain genes is currently con-
and a small proteoglycan decorin [58]. sidered the main reason for age-associated increase in
Oxidative Medicine and Cellular Longevity 5

Table 1
Gene products and functions (from GENE database;
Probe set ID Gene symbol Fold change∗
http://www.ncbi.nlm.nih.gov/gene)
Sigma nonopioid intracellular receptor 1, plays an important role in the
cellular functions of various tissues associated with the endocrine,
214484 s at SIGMAR1 2.46
immune, and nervous systems. Mutations are implicated in early-onset
dementia and neurodegeneration.
Laforin, a dual-specificity phosphatase associates with polyribosomes.
205231 s at EPM2A This gene is defective in a neurodegenerative disorder associated with 2.32
epileptic seizures (Lafora disease). Possibly a repair enzyme.
Apoptosis inhibitory protein; functions include suppression of neuronal
204860 s at NAIP 2.13
apoptosis
Fibroblast growth factor receptor 2, influences mitogenesis and
208229 at FGFR2 1.9
differentiation, important in embryonic brain development.
Slit homolog 2, neuronal repellent factor, nervous system development;
209897 s at SLIT2 downregulation of this gene by neuronal differentiation factor promotes 1.67
tumor growth in neuroblastomas [63].
∗
This represents the average fold change of gene expression of the three GHK instances profiled in the cMap. Furthermore, it should be noted that all instances
were performed using doses of GHK at 1 micromolar. Peak cellular responses to GHK have been recorded at 1 nanomolar; higher doses can reduce the cellular
response [56].

tumorogenesis, oxidative stress, and inflammation. It is easily passing through the lipids of the epidermal barrier
commonly accepted that human health is at its best until [66, 67]. The peptide could be administered intravenously
approximately the age of 20–25. It then begins to decline in or orally when encapsulated into liposomes. Strong systemic
later years. Recent genetic studies demonstrated accumula- wound healing was induced in pigs at about 1.1 mg GHK-
tion of altered gene products in various tissues starting from Cu per kilogram body weight which would correspond to
the age of 20–25 [64]. In later decades of life, the genes about 75 mgs in humans. This is about 300-fold below GHK-
produce less regenerative proteins but more inflammatory Cu’s toxic action (lowering of blood pressure). Much lower
and oncogenic genes. The key enzymes implicated in gene dosages may also be effective since GHK-Cu’s actions on cells
silencing are the family of histone deacetylase proteins generally occur at a 1 nanomolar concentration [68].
(HDACs). Inhibitors of selected HDACs possess neuropro-
tective and neuroregenerative properties in animal models 11. Conclusion
of brain diseases and have been suggested as promising
therapeutic drugs [65]. Results from the Broad Institute’s The multifaceted nature of age-associated cognitive decline
Connectivity Map and ChemBank found that GHK is a calls for complex approaches that address all key factors
strong inhibitor of several HDACs. Although the data was involved in the development of neurodegenerative disor-
collected for GHK without copper, we cannot exclude the ders such as oxidative stress, neuroinflammation, disrupted
possibility that the actual gene regulator was the GHK bioavailability of copper, impaired circulation, and altered
copper complex formed in the culture media, since, as it gene expression.
was mentioned above, GHK can easily obtain copper from The human tripeptide GHK has a long history of safe
other biological molecules such as albumin. It is also possible use in wound healing and skin care; it is naturally occurring,
that GHK and GHK-Cu have complimentary effects on gene nontoxic, and is active at a very low nanomolar concentra-
activity. At present, it is not always possible to track gene tion. It readily forms complexes with copper, regulating its
effects to its protein product; however, we may conclude from metabolism and improving its bioavailability. It possesses
all experimental data on GHK-Cu effects that the reversing of antioxidant, anti-inflammatory, and regenerative properties,
gene silencing by GHK has protective and health-promoting improves circulation, supports stem cell functions, and
benefits. promotes nerve outgrown and synthesis of neurotrophic
factors. Recent studies demonstrated its ability to regulate a
10. Therapeutic Administration of GHK-Cu large number of human genes. At 1 micromolar it was able
to suppress 70% of genes overexpressed in metastatic colon
It is possible that administration of GHK-Cu could be used cancer. It upregulates p63 and integrins in epidermal stem
as a preventive and regenerative therapy for senescent or cells, increases collagen, glycosaminoglycans, and decorin
damaged brain tissue. Using GHK-Cu has an advantage over expression. Our studies with the Broad Institute’s Connec-
using just GHK, since it alleviates copper deficiency without tivity Map revealed its ability to regulate a large number of
the risk of oxidative damage. Even though it is yet not clear human genes including those that are involved in nervous
whether or not the GHK-Cu peptide can pass the blood- system physiology, development, and maintenance.
brain barrier, there is a high possibility that it will do so, Even though it is not always possible to distinguish
since GHK-Cu has a very high uptake into human skin, between activity of GHK peptide and its copper complex
6 Oxidative Medicine and Cellular Longevity

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