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Steriod hormones, including oestrogens, may influence cell migration during both wound healing and metastasis. Oestradiol is the naturally occurring biological active oestrogen, which improves wound healing by accelerating cell migration. Animal studies also indicate that selective oestrogen receptor modulators such as tamoxifen and raloxifene accelerate wound healing, although, another study in vitro reported that none of them modulate cell migration. Hence, in this study the role of oetrodial, tamoxifen, and raloxifene in stimulating keratinocyte cells (NCTC) migration were assesed at several concentrations (1nM10nM,100nM, 1000nM), using a scratch wound assay. Oestradiol substantially accelerate keratinocyte cell migration, whereas, neither tamoxifen nor raloxifene accelerated cell migration.
A comparison of SERMs in the modulation of cell migration
Cell migration plays a key role during wound healing process and it is a synchronized physiological process that results from a complex interaction among the site of cell attachment to the extracellular matrix, the proteins within the cell focal adhesion complexes, and the dynamics of filamentous-actin (F-actin) stress fibers(McLean et al 2005). However, cell migration is also required for cancer cell spreading, invasion, and metastasis. The expansion of focal contacts from the focal complexes, dynamic F-actin cytoskeleton remodelling, and the disassembly of cell adhesion sites lead to the generation of membrane protrusions and traction forces that eventually create cell movement(Zamir and Geiger 2001), and these changes can be induced by oestrodial and drugs such as selective oestrogen receptor modulators(Acconcia et al 2006).
Oestrogens naturally occur in our body and are the terminal ligand in the biosynthetic pathway of gonadal steroid hormones. Oestrogens are derived from androgens following the cleavage of the C-19 angular methyl group and the formation of aromatic A ring by aromatase complex. Oestrone is derived from androstenedione, where as oestradiol is formed from testosterone (Payne and Hale 2004). Oestrogens are found in both females and men. In females, oestrogen is produced in the ovary while in men oestrodial is produced in peripheral tissues by the action of aromatase on testosterone (Simpson 1998). Dehydroepiandrosterone (DHEA) synthesized in the adrenal zona reticlularis serves as the main precursor of active oestrogen in post-menopausal women, although, the production of DHEA also decreases with age (Parker et al 1997). Oestrogen acts via two intracellular proteins (ERα and ERβ) that belong to the nuclear superfamily (Moverare et al 2002). This signalling pathway is called Genomic signalling, and has slow effects. Like all intracellular steroid receptors, ERα and ERβ are ligand activated nuclear transcription factors that enhance target-gene transcription upon binding to chromatin. Activation of the target gene by 17β-estradiol activates both ERα and ERβ to increase transcriptional activities when dimmers of the liganded receptors bind to oestrogen response elements (ERE) which are specific DNA palindrome sequence located in the promoter region of oestrogen-regulated target genes (Klinge 2001). Activation of transcription also involves the recruitment of co-activators such as GRIP1 and SRC-1 and the histone acetyltransferases p300/CREB-binding protein and pCAF (Webb et al 2003). Coactivators are tissue specific and there is some evidence that ERα and ERβ vary in their requirement for coactivators in a cell and tissue dependent manner (Smith and O'Malley 2004). Oestrogen can also act via cell membrane forms of oestrogen receptors that are linked to cystolic signal transduction proteins, which generate different signalling pathways via second messengers (Nadal et al 2001). This signalling pathway is called non-genomic, and has very rapid effects. Oestrogen can activate second messengers such as adenlate cyclise and cAMP (Aronica et al 1994), phospholipase C (Lieberherr et al 1993), protein kinase C (Marino et al 2002) and mitogen-activated protein kinase (MAPK) (Shaul 1999; Russel et al 2000). Oestrogen can also activate voltage-gated ion channels (Pappas et al 1995; Razandi et al 1999; Nadal et al 2001), which causes an increase in levels of intracellular calcium (Benten et al 2001).
Both ERβ and ERα intracellular receptors are expressed in human skin, however, their expressions vary from one anatomical site to another. Thornton et al 2003 demonstrated that epidermal keratinocytes from the human scalp skin of both sexes express ERβ significantly. Nevertheless, other study by Nelson 2006 found that breast skin largely expresses ERα. Thus, suggesting differences between ERβ and ERα with respect to their tissue distribution. The action of oestradiol via cell membrane receptors stimulated proliferation of cultured human keratinocytes (Kanda and Watanabe 2004), furthermore, increased phosphorylation levels of ERK1 and ERK2 kinases was seen in human epidermal keratinocytes cultured with estradiol. Kanda and Watanabe (2003) demonstrated that oestrogens blocks apoptosis by increasing the expression of anti-apoptotic protein (bcl-2) in cultured epidermal keratinocytes, suggesting its protective function on the human epidermis.
Several studies reported that oestrogens play a vital role in regulating skin preservation and turn over (Hardman et al 2008). Oestrogens sustain dermal thickness, promote preservation of extracellular matrix collagen levels, and skin structural integrity (Brincat et al 1987). The decline in oestrogen level, which is normally seen in postmenopausal women, has remarkable effects on skin and cutaneous healing response. In postmenopausal women impaired wound healing is related with increased inflammation, dysregulated protease activity, and decreased matrix deposition. Delays in wound healing lead to local infection, a transition toward a chronic and non-healing wound. A sudden onset of skin aging is also associated with reduced levels of oestrogen in postmenopausal women, which results in increased number and depth of wrinkles, increased skin dryness, and loss of skin elasticity (Brincat 2000). Oestrogen replacement therapies have been shown to effectively improve wound healing, increase epidermal thickness and keratinocyte proliferation (Son et al 2005), increase skin collagen (Varila et al 1995), decrease facial wrinkling and increase skin rigidity (Wolff et al 2005), and increase elasticity (Sator et al 1987) . However, recent studies have suggested that oestrogen therapy may increase the risk of cardiovascular events (Grady et al 2002), endometrial and breast cancer (ossouw et al 2002).
Recent evidence suggests that selective oestrogen receptor modulators such as raloxifene and tamoxifen may improve wound healing in both in vivo and in vitro models of wound healing. Matthew and colleagues (2008) using ovariectomized female mice representing a validated model of age- associated delayed wound healing demonstrated that tamoxifen and raloxifene accelerate cutaneous wound healing. Another study in vitro on mechanically wounded adult human dermal fibroblasts also reported that both tamoxifen and raloxifene stimulated proliferation, although neither modulated migration, suggesting agonist action in human dermal fibroblasts (Stevenson et al 2009).
SERMs are a new class drugs that have ether oestrogenic (i.e. agonist) or anti oestrogenic (i.e. antagonist) depending on the target tissue; thus they have tissue specific effects (Jordan et al 2007). Both ERα and ERβ receptors are expressed across ranges of tissues and they show similar affinity in binding 17-β oestradiol. These differences of ERs in tissues together with other proteins such as, co-activator, co-repressor, and promoter levels determine the effect of one SERM. Current SERMs such as raloxifene and tamoxifen generally act as agonists in liver, in bone by preventing bone resorption, and on the cardiovascular system by reducing the markers of cardiovascular risk such as LDL. They are often antagonists in tissues such as breast and brain, but show a mixed response in the uterus (Jordan 1998; Diel 2002). For example, tamoxifen (non-steriodal a triphenylene) blocks the action of oestrogen in breast cancer cells; conversely, in uterine cells it acts as an oestrogen agonist, and therefore increases the risk of endometrial cancer (Stygar et al 2003). Unlike tamoxifen, Raloxifene (derived from a benzothiophene series of anti-oestrogen) acts as antagonists in both tissues and can be used to treat breast cancer without increased risk of endometrial cancer associated with tamoxifene treatment. (Cummings et al 1999). Raloxifene is now approved for the prevention and treatment of osteoporosis in postmenopausal women (reviewed in Neven and Vergotta 2001).
Although little is known about the effect of SERMs on the skin, some studies report that tamoxifen may improve wound healing and improve dermal scarring. A study by Acconcia et al (2006) found that both oestrogen and tamoxifen induce cytoskeleton remodelling and migration in endometrial cancer cells, cell migration is vital for wound healing process. Another study by Surazynski et al (2003) compared the effect of both 17β-oestradiol and raloxifene on collagen biosynthesis, and found that raloxifene has a stronger positive stimulatory effect than 17β-oestradiol.
Stevenson et al (2009) assessed the effect of tamoxifen and raloxifene on the migration and proliferation of adult human dermal fibroblasts in vitro by scratch wound assay, and their findings demonstrated that both tamoxifen and raloxifene modulate cell proliferation, but not cell migration. The skin is a complex organ with numerous tissues and cells, each with diverse intensities and ratios of oestrogen receptor (ER) expression. Hence, this finding may not represent the effect of tamoxifen and raloxifene in other cells such as keratinocytes and endothelial cells. Further studies are needed to see whether tamoxifen and raloxifene produce the same effect, using the same method (scratch wound assay), but different cells. This study is designed to compare the effects of 17β-oestradiol, tamoxifen, and raloxifene on the cell migration of adult human keratinocytes following mechanical wounding in vitro.
The aim of this study is to compare the effects of 17beta oestradiol with two different SERMs (tamoxifen and raloxifene) on cell migration using wound scratch assay over different period times in vitro.
passaging and seeding of keratinocyte cells (NCTC) into 6 culture wells
Keratinocyte Cells (NCTC) were provided by Dr Thornton M J, in a T75 flask. Once confluent, the cells were removed from the flask into suspension with the aid of trypsin .The medium was removed from the flask, and cells were washed twice with PBS to remove any traces of serum. The cells were detached from the flask surface by adding 3ml trypsin for about 1 minute. After incubation at 37°C for about for 4 minute, 24ml DMEM was added to the flask to make cell suspension. 50ul from the suspension were taken, and used to estimate the number of cells by counting on a haemocytometer. The Cells were counted three times (3x50ul), and the average number in the cell suspension determined (see appendix1). Then, the cells were seeded into culture well (35mm) at a density of 30,000 cells per well in growth media. Cells were maintained in the dish for about 3 days at 37°C with 2ml of DMEM growth medium supplemented with FBS, streptomycin and penicillin until confluent.
Wound scratch assay
Once cells reached confluence the medium was removed, and the wells were washed twice with PBS, to remove the serum. Then cells were starved by incubating them at 37°C with 2ml of phenol-free, serum-free DMEM overnight. The cells were washed twice with PBS. The Cells were wounded with a sterile standard paperclip by one perpendicular linear scratch, creating a wound across the diameter of wells according the template designed earlier.
Time course assay
following scratching, the cells were washed twice with PBS to remove detached cells and debris. 2ml of phenol red-free, serum-free DMEM supplemented with either absolute ethanol vehicle control (0.0001%), 1nM, 10nM, 100nM, or 100nM 17¢-oestradiol, 1nM, 10nM, 100nM, 1000nM tamoxifen, or 1nM, 10nm, 100nM, and 1000nM raloxifene was added to individual wells in triplicate. The cells were incubated at 37°C for 48 hours, with photographs taken at each time point (0hr, 3hr, 6hr, 24hr and 48hr) were measured at six fixed points. The average distance between the wound edges for each individual group (derived from a total of 18 measurements, n=6 x3 wells) was calculated for the different time points and from this the mean migratory distance for each time point was ascertained (see appendix 3). The magnified (x140) migratory distance in centimeter (cm) was converted to a real migratory distance in millimeter (mm), then, migratory distance in mm converted in a distance in micrometer (um) as shown in appendix 3).
The raw data showing the measured wound edges and migration distances in the presence of ether control (0.0001%) or oestradiol, tamoxifen, and raloxifene, at different time points can be found in the tables in appendix3.
2.1 The effect of β-oestradiol on keratinocyte cell (NCTC) migration
β-oestradiol accelerate keratinocyte cell (NCTC) migration (appendix3, table ). Migration in the presence of absolute ethanol vehicle control (0.0001%), 1nM β-oestradiol, 10nM β-oestradiol, 100nM β-oestradiol or 1000nM β-oestradiol at fixed time point of 0hr, 3hr, 6hr, 24hr, and 48hr, at six fixed points per well, each in triplicate. Significant increase in migration was seen with 10nM β-oestradiol, 100nM β-oestradiol and 1000nM β-oestradiol (appendix3, table). Migration in the presence of 10nM and 100nM β-oestradiol (fig 1,2) than seen with 1000nm. No result was obtained for 1nM oestradial due to contamination of cells and as there was no opportunity to repeat the experiment.
2.2, The effect of tamoxifen and raloxifene in keratinocyte cell line (NCTC)
Neither tamoxifen nor raloxifene accelerate keratinocyte cell line migration.
Migration in the presence of absolute ethanol vehicle control (0.0001%), 1nM tamoxifen or raloxifene, 10nM tamoxifen or raloxifene, 100nM tamoxifen or raloxifene or 1000nM tamoxifen or raloxifene was assessed at fixed time point of 0hr, 3hr, 6hr, 24hr, and 48hr, at six fixed points per well, each in triplicate (section 2). No accelerated migration was observed in the presence of both tamoxifen and raloxifene, in all cases (fig, appendix3 table). No result was obtained for 1nM tamoxifen and raloxifene due to contamination of cells and as there was no opportunity to repeat the experiment.
The overall aim of this study is to compare the effect of oestrogen, tamoxifen, and raloxifene, in cell migration in vitro using scratch wound assay.
In the past, the role of oestradiol, tamoxifene , and raloxifen in cell wound healing was demonstrated by animals studies(Matthew J et al 2008) and in vitro (by scratch wound assay). Oestrogen naturally occurs in the body and it plays a key role in regulating cell growth, proliferation, and migration. In mechanically wounded human dermal fibroblast cells, 17oestradiol accelerated cell migration, although, no increase in cell proliferation was observed (Stevenson et al 2009). However, oestradiol stimulated proliferation of cultured human keratinocytes (Kanda and Watanabe 2004). Furthermore, in endometrial cancer cells, both oestrogen and tamoxifen induce cytoskeletal remodelling by triggering rapid activation of ERK1/2, c-Src, and focal adhesion kinase signalling pathways and filamentous actin cytoskeltal changes, which eventually lead to cell migration (Acconcia et al 2006).
Both tamoxifen and raloxifene are synthetic drugs that act through oestrogen receptors. Recent evidence suggest that selective oestrogen receptor modulators such as raloxifene and tamoxifen may improve wound healing in both in vivo and in vitro models of wound healing. In ovariectomized female mice, both tamoxifen and raloxifene accelerate cutaneous wound healing (Matthew J et al 2008). However, neither tamoxifen nor raloxifene modulated migration of wounded human dermal fibroblast cell line(Stevenson et al 2009). To determine the effect of oestradiol, tamoxifen, and raloxifene on keratinocyte cell line (NCTC) migration a scratch wound assay was performed. Increased migration was observed in the presence of 10nM, 1000nm, and 1000nM β-oestradiol. significant, higher migration occurred in response to 10nM and 100nM than 1000nM β-oestradiol. No results was obtained for 1nM oestradiol, tamoxifen , and raloxifene, this is due to contamination of cells and limited time to repeat the experiment. In contrast, neither raloxifen nor tamoxifene accelerated wounded keratinocyte cell migration at any concentration, no significant difference from control (fig.).
Cell migration is crucial during wound healing, and it is used by cancer cells to invade neighbouring tissue and metastasis into other body parts. The development of focal contact from focal adhesion complexes, dynamic F-actin cytoskeleton remodelling, and the disassembly of cell adhesion sites eventually lead to the generation of membrane protrusions and traction forces that allow the cell to move(Zamir and Geiger 2001). This cascade of events results changes in cell morphology. Change in keratinocyte cell shape was observed throughout the experiment (48hr) in the presence oestradiol, tamoxifen, and raloxifene. In some cases, cells adopted a spindle shape, which may indicate that improved wound healing was mainly due to cell migration. However, in other cases a circular cell morphology was observed, may indicate that cell proliferation taking place , hence improved wound healing may be due to increased cell proliferation since the increase in number of cells can fill up the wound edges.
Both tamoxifen and raloxifene stimulated wounded fibroblast proliferation (Stevenson et al 2009), and the role of oestradiol in stimulating proliferation was demonstrated by several studies both in vitro and in vivo. Unfortunately, this study was restricted to cell migration; therefore, it is unclear if cell proliferation contributed to improved wound healing, especially in the presence of oestradiol. This could have been resolved by blocking cell proliferation with mitomycin C.
Oestrogen act via two intracellular proteins (ERα and ERβ) that belong to the nuclear superfamily (Moverare et al 2002). In addition to the male and female reproductive tissues, ERα and ERβ are expressed in other tissues such as bone, brain, lung, bladder, thymus, pituitary, hypothalamus, heart, kidney, adrenal, the cardiovascular system and the skin including the hair follicle (reviewed in Thornton 2002, 2005). Like all intracellular steroid receptors, ERα and ERβ are ligand activated nuclear transcription factors that enhance target-gene transcription upon binding to chromatin. Activation of the target gene by 17β-estradiol activates both ERα and ERβ to increase transcriptional activities when dimmers of the liganded receptors bind to oestrogen response elements (ERE) which are specific DNA palindrome sequence located in the promoter region of oestrogen-regulated target genes (Klinge 2001). This signalling pathway is called Genomic signalling, and has slow effects. Oestrogens also act via cell membrane forms of oestrogen receptors that are coupled to cytosolic signal transduction proteins, which initiate different signalling cascades via conventional second messengers (Nadal et al 2001). Oestrogen can activate second messengers such as adenlate cyclise and cAMP (Aronica et al 1994), phospholipase C (Lieberherr et al 1993), protein kinase C (Marino et al 2002) and mitogen-activated protein kinase (MAPK) (Shaul 1999; Russel et al 2000). Oestrogen can also activate voltage-gated ion channels (Pappas et al 1995; Razandi et al 1999; Nadal et al 2001), which causes an increase in levels of intracellular calcium (Benten et al 2001). Unlike genomic signalling, the action of oestrogen through the membrane receptors (also called non-genomic signalling) has much faster effect.
Throughout the experiment, early change in keratinocyte cell migration was observed in response to 17βoesrtadial (i.e. 3hr after wounding) (fig.), and this may indicate that oestrogen was acting through membrane receptors (non-genomic signalling) rather than via ERα and ERβ proteins. Further study is required to determine whether the changes occurred in keratinocte cell migration in response to 17βoestradial were modulated via genomic or non-genomic pathways. This can be done by blocking G protein-coupled receptors or second messengers with a toxin, for example, by targeting G protein-coupled receptors with pertussis toxin or a Src inhibitor can also block cell migration.
Acconcia and colleagues (2006) reported that both oestrogen and tamoxifen induced rapid activation of C-Src and FAK signalling pathways, which eventually contributed to cytoskeleton remodelling and migration in endometrial cancer cells, suggesting non-genomic signalling regulated change in cell migration. This finding concurs with our finding on the effect of oestrogen in keratinoctye cell migration, however, their finding also contradicts with our finding on tamoxifen's effect on migration of keratinocyte since no migration was observed in the presence of tamoxifen, in all any concentration used.
SERMs are distinguished from oestrogens by their ability to function as both agonist and antagonist in different cell types (Jordan, 2004).Both tamoxifen and raloxifene can increase the levels of expression of ERα and ERβ (Haczynski et al 2004). Raloxifene showed a stronger positive stimulatory effect on collagen biosynthesis than 17βoesradial (Surazynski et al 2003).
raloxifene and tamoxifen generally act as agonists in liver, in bone, and on the cardiovascular system. They are often antagonists in tissues such as breast and brain, but show a mixed response in the uterus (Jordan 1998; Diel 2002). For example, tamoxifen blocks the action of oestrogen in breast cancer cells; conversely, in uterine cells it acts as an oestrogen agonist, and therefore increases the risk of endometrial cancer (Stygar et al 2003). Unlike tamoxifen, Raloxifene acts as antagonists in both tissues and can be used to treat breast cancer without increased risk of endometrial cancer associated with tamoxifene treatment. (Cummings et al 1999).
Re-epithelialisation of wounded skin is necessary for wound closure and restoration of barrier function and requires directional keratinocyte migration towards the centre of the wound (Christine E et al 2005). Following wounding of keratinocyte cell lines, oestradiol accelerated wound closure by increasing cell migration, suggesting the importance of oestradiol in re-epithelisation of keratinocytes. Neither tamoxifen nor raloxifene accelerated keratincyte cell migration, at any concentration used. Hence, both tamoxifen and raloxifene would not be suitable to improve re-epithelisation of keratinocyte as a substitute to oestrogen.
Unfortunately, this study was restricted to measuring changes in cell migration, therefore, it do not attempt to distinguish whether improved wound closure of wounded keratinocyte cell in response to oestradiol was due to cell migration or proliferation. Hence, another study that address this issue may be required, mitomycin C is used in several studies to block cell proliferation. In addition, any change in proliferation could be determined using tritiated thymidine assay in which change in DNA synthesis is measured.
This study does not address the issue whether changes in keratinocyte cell migrations were modulated via genomic or non-genomic pathways. This could be done by blocking G protein-coupled receptors or second messengers with a toxin, for example, by targeting G protein-coupled receptors with pertussis toxin or a Src inhibitor can also block cell migration.