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Steroid hormones, including oestrogens, may influence cell migration during both wound healing and metastasis. More recently the adrenal precursor dehydroepiandrosterone (DHEA) has been shown to improve wound healing and cell migration. This project investigated the effect of oestradiol and DHEA alone or in combination with a specific aromatase inhibitor (Arimidex), on a human keratinocyte cell line NCTC 12544 using the scratch wound assay. Cell migration was measured over 6 different time points.
Significant increase in cell migration was observed with DHEA and the effect of DHEA was blocked by the aromatase inhibitor (Arimidex), which suggests that conversion to 17Î²-oestradiol is required.
Further understanding of the mode of action of DHEA may be crucial for developments in more suitable therapies; particularly in age related impaired wound healing.
This research was completed with the assistance of all the laboratory staff at the School of Life Sciences department, University of Bradford. I would like to thank Dr. M.J.Thornton for her guidance and encouragement that enabled me to carry out my research efficiently. Also, Dr A. Graham for her cell culture training, her confidence and support. Finally, I would like to thank all the technicians especially Nigel Clough and Andy Reed for their contributions in the cell culture hoods, trusting and providing us with equipment required for cell culture and for the collection of our results.
3Î²-hydroxysteroid dehydrogenase 3Î²-HSD
16Î±-hydroxylated E2 (oestriol) E3
17Î²-hydroxysteroid dehydrogenase 17Î²-HSD
B-Cell CLL/Lymphoma 2 Bcl-2
DST, DHEA sulphotransferase
Dulbecco's Modified Eagle Medium DMEM
Fetal Bovine Serum FBS Guanine nucleotide-binding protein G protein
Hormone replacement therapy HRT
Hydroxyl group OH
Mitogen-Activated Protein Kinase MAPK
Phosphate Buffered Saline PBS
Phosphatidylinositol 3 PI3
1.1 DHEA and wound healing
Dehydroepiandrosterone (DHEA) is a ubiquitous adrenal steroid hormone with immunomodulatory properties (Mills et al., 2005). In humans, DHEA and DHEA-sulphates (DHEA-S) are the most abundant steroid hormones in the bloodstream and healthy levels of DHEA allow a balance of other body hormones. DHEA is known as the "The Mother Hormone", as the body can convert DHEA into many different steroids it requires or may be lacking, provided the appropriate enzymes are present, to maintain a proper hormonal balance (figure 1). DHEA and DHEA-S are synthesised in the adrenal cortex and released into the bloodstream in response to the hormone adrenocorticotropin, secreted by the anterior pituitary gland. DHEA and DHEA-S circulate at very high concentrations in the blood of young adults. Plasma DHEA-S levels in adult men and women are some 100-500 times higher than those of testosterone and 1,000-10,000 times higher than those of 17Î²-oestradiol, thus providing a large reservoir of substrate for conversion to androgens and/or oestrogens in peripheral intracrine tissues including the skin.
Wound healing is delayed and impaired in the elderly, particularly in males who heal more slowly than females, with reduced matrix deposition and an increased inflammatory response.(Gilliver et al., 2006) This suggests that circulating levels of sex hormones, as well as cell ageing mechanisms, contribute to impaired wound healing in the elderly (Gilliver and Ashcroft, 2007). The significant declining levels of circulating DHEA and oestrogens may account for the delayed healing of cutaneous wounds in aged individuals (Mills et al., 2005). The elderly suffer from impaired healing of acute wounds, which are characterized by delayed closure, increased local inflammation, and excessive proteolytic activity, which can lead to chronic non healing wounds (Gilliver and Ashcroft, 2007). The conversion of DHEA locally to downstream steroid hormones leads to oestrogenic and/or androgenic effects and this may be important in age-related skin homeostasis (Mills et al., 2005). Systemic DHEA levels have been reported to be strongly associated with the protection against chronic venous ulceration in humans (Mills et al., 2005). DHEA accelerated healing in an impaired healing animal model (mice rendered hypogonadal), and was associated with an increased matrix deposition and dampening of the exaggerated inflammatory response (Mills et al., 2005). These effects were shown to be mediated by local conversion of DHEA to oestrogen, and modulated via the oestrogen receptor. In vitro studies suggest a direct effect on specific pro-inflammatory cytokine production by macrophages via mitogen activated protein kinase (MAPK) and phosphatidylinositol 3 (PI3) kinase pathways (Simoncini et al., 2003). Studies have shown that local injection of DHEA accelerates impaired healing in an ageing mouse colony (Mills et al., 2005). Suggestions have been made that exogenous application of DHEA accelerates impaired wound repair, results which may be applicable to the prophylaxis (Mills et al., 2005) and treatment of human impaired wound healing states; this is particularly interesting because mice do not produce DHEA and the application of an aromatase inhibitor blocked the effects of DHEA, indicating conversion to oestradiol.
Adrenal precursors DHEA and DHEA-sulphate (DHEA-S) synthesize a large amount of androgens locally (figure 1) in peripheral target tissues (Labrie et al., 2006). Androgens regulate hair growth and sebum production and the skin is an important target organ for androgens (Gilliver et al., 2003) and there is evidence from previous studies to suggest that androgens influence the wound healing process negatively (Ashcroft and Mills, 2002). From the in vitro studies and limited number of in vivo studies so far undertaken it appears that androgens negatively influence all phases of wound healing from initial clot formation to long-term wound remodelling
Cutaneous wound healing is a complex process involving several overlapping phases (Singer and Clark, 1999). There are a number of steps involved in the re-establishment of the skin barrier leading up to wound healing. Firstly, hemostasis (blood clot formation) and inflammation occurs; the inflammatory response causes the proliferation and migration of dermal and epidermal cells to re-establish the skin barrier and to replace damaged tissue (re-epithelialisation) via wound closure (Hackam et al., 2002); and finally tissue remodelling and differentiation for the recovery and restoration of the skin tissue (Hackam et al., 2002).
1.2 DHEA in the presence of aromatase inhibitors (Arimidex)
Aromatase inhibitors (such as Arimidex) may block the beneficial effects DHEA of cutaneous wound healing and this has been demonstrated on a mouse model (Gilliver et al., 2007) but to date there have been no investigations into the effects of aromatase inhibitors on the cutaneous wound healing process in humans. Therefore, post menopausal patients who take aromatase inhibitors as an adjunct to breast cancer therapy may be at increased risk of delayed wound healing (Howgate et al., 2009). Administration of oestradiol and DHEA reverses age-related chronic wound healing impairment via hormone replacement therapy (HRT) in postmenopausal women significantly (Gilliver and Ashcroft, 2007). The administration of 17Î²-oestradiol, either systemically or topically, has been shown to reverse the fundamental repair defects observed in postmenopausal women (Gilliver and Ashcroft, 2007). By contrast, androgenic hormones retard repair and interfere with the accumulation of the structural proteins that reconstitute the damaged dermis (Gilliver and Ashcroft, 2007).
1.3 DHEA and the skin
There are a number of layers of specialised epithelial cells, keratinocytes, which make up the human epidermis. Normal homoeostasis of the epidermis requires that the balance between keratinocyte proliferation and terminal differentiation be tightly regulated (Lock and Hotchin, 2009). DHEA has been shown to have a protective role against apoptosis in keratinocytes using non-cancerous immortalized human HaCaT cells (Alexaki et al., 2009). It does so by transmitting signals via specific G protein-coupled, membrane binding sites and inhibiting apoptosis, through prevention of mitochondrial disruption and altered balance of Bcl-2 proteins (Alexaki et al., 2009).
Human keratinocytes are a major component of the epidermis, comprising 95% of the cells; they are specialised cells that synthesise keratin. The keratinocytes proliferation and migration can be mediated by the activation of the non-genomic pathway via the 17Î²-oestradiol isoforms (Thornton, 2005).
The skin fibroblast cells and human embryonic stem cells can also express both 17Î²-oestradiol membrane receptors (Randall, 2007). Studies have suggested treatment with oestradiol can control differentiation of human embryonic stem cells into a series of cell types to induce gene expressions of those involved in differentiation (Charlet et al., 2008). Two 17Î²-oestradiol pathways mechanisms exist (Kelly and Levin, 2001). Firstly the classical (i.e genomic) pathway which is induced through ligand activated intracellular receptors in order to regulate transcription of genes via protein to protein directly interacting on the DNA and binding to specific co activators or repressors. Secondly, the non-genomic pathway which is mediated by signalling responses binding directly to oestradiol (Kelly and Levin, 2001).
1.4 Cell migration and wound assay
Embryonic development, homeostasis and wound healing, immune responses, and cancer metastasis are examples of biological processes in which cell migration has a vital role (Nor and Polverini, 1999). The wound healing assay is a simple, inexpensive method which allows studies of directional cell migration in vitro mimicking cell migration during wound healing in vivo (Rodriguez et al., 2005). However, the in vitro method is only two dimensional and therefore three dimensional methods in vivo would be required to mimic more closely the processes occurring in vivo. This method involves creating a "wound" in a cell monolayer, in this case the keratinocyte monolayer, and capturing images at the beginning and at regular intervals during cell migration until the wound closes. These images are then compared to allow quantification of the migration rate of the cells.
The aim of this study was to investigate the effects of oestradiol and DHEA alone and in combination with an aromatase inhibitor on the migration of cultured adult human keratinocyte cell line (NCTC), following in vitro mechanical wounding. The secondary stage (re-epithelialisation) of hemostasis will be the main aim, since the rate of migration of human keratinocytes in vitro following mechanical wounding will be established in response to incubation with DHEA.
Figure 1. Flow diagram representing the complexity of androgen conversion in peripheral cells, adapted from Schmidt et al., 2005. The major precursors DHEA-S, DHEA, and Androstenedione (ASD), are converted to downstream metabolites via various enzyme pathways once they have entered the peripheral cell.
Materials and Methods
All materials and equipment used, together with the supplier and manufacturer information can be found in tables shown in Appendix 1.
2.1 The effect of DHEA on the migration of established human transformed cell line keratinocyte; NCTC 12544, following mechanical wounding in vitro
A scratch wound assay adapted from Morales et al., 1995 was used to assess the migration of the established human transformed cell line keratinocyte; (NCTC 12544) in response to DHEA and oestradiol. The NCTC 12544 cell line was provided by Dr. M.J Thornton and were trypsinised passaged to establish subcultures. Once the cells were confluent, the trypsin was removed and 10% DMEM growth medium supplemented with 10% FBS (50 ml FBS, with 500 ml DMEM), was seeded into the flask. 5 ml of glutamine and 5 ml of Penicillin (to improve sterility) were also added to the suspension. Haemocytometry was used to estimate the number of cells per suspension and the total number of viable cells present in the suspension was calculated.
Cells were seeded into 35mm dishes at a density of 35,000 cells per dish. 2ml DMEM growth medium supplemented with 10% FBS (50 ml FBS with 500 ml DMEM), Penicillin and glutamine was added to each dish. The cells were incubated at 5% CO2 and 37ï‚°C until confluent, which took approximately 3 to 4 days, with a media change of 2% low serum media (10 ml FBS with 500 ml DMEM) every 2 to 3 days. The medium was then removed once the cells were â‰¥ 95 % confluent (check under inverted Nikon eclipse TS100 microscope, magnification Ã-10) and the cells were washed two times with PBS to remove any debris. Once washed, the cells were mechanically wounded using a standard paperclip (Figure 2B). A template of the dish (Figure 1A) was designed prior to wounding, and this template was used to scratch the cells along a standard diameter of the wells. The use of a paperclip was adapted from a previous study (Morales et al., 1995).
At time point 0 (t=0hr) the scratches were photographed using an inverted Nikon eclipse TS100 microscope. At time points 0, 3, 6, 24, 36, 48 and 72 hours and varying concentrations of 17ï¢-oestradiol, DHEA and DHEA in combination with Arimidex (1nM, 10nM, 100nM and 1000nM), photographs of the distance between the wounded cell edges (migrating cells) were taken. Photographs of the central part of the wound were taken at each time point. The distance between the wound edges per well was measured using a standard template (Figure 2C) and measuring how much of the wound had closed at 6 fixed points (1 cm apart) along the length of the wound. The average distance between the wound edges in each well at varying concentrations (mean derived from 6 per well, 3 wells per concentration measurements) was calculated for individual time points and from this the mean migratory distance for each time point was calculated (Appendix 2). The mean migratory distance was then converted into a distance in micrometres (ïm).
2.2 Migration of human cell line keratinocytes (NCTC 12544), in the presence of 17ï¢ï€oestradiol
2.2.1 Dose response effects
Migration assays were carried out on human cell line keratinocytes (NCTC 12544) derived from skin to compare the effects of a variety of concentrations of 17ï¢-oestradiol (1nM, 10nM, 100nM and 1000nM). 2ml of phenol red-free, serum-free DMEM supplemented with eitherabsolute ethanol vehicle control (0.0001%), 1nM 17ï¢-oestradiol, 10nM 17ï¢-oestradiol, 100nM 17ï¢-oestradiol or 1000nM 17ï¢-oestradiol was added to individual dishes in triplicate. Cell migration was assessed after fixed time points, as discussed in 2.1. A stock solution of 17ï¢-oestradiol was required to be serial diluted to achieve the varying concentrations. Migrating cells were photographed as described in section 5.1.
2.3 Migration of human keratinocytes in the presence of DHEA alone and in combination with an aromatase inhibitor
Human keratinocytes (NCTC 12544) derived from skin were prepared and wounded for migration as described in 2.1. 2ml of experimental growth medium were added to each dish in triplicate containing the following;
Absolute ethanol vehicle control (0.0001%)
17ï¢-oestradiol (1nM, 10nM, 100nM and 1000nM)
DHEA (1nM, 10nM, 100nM and 1000nM)
DHEA in the presence of Arimidex (1nM, 10nM, 100nM and 1000nM)
The concentration of DHEA and Arimidex were chosen as such due to a previous study (Stevenson et al., 2009). The cells were then cultured for 48 hours and the migratory distance was measured at fixed time points at varying concentrations as described in 2.1.
For each parameter, the data was calculated in triplicates. Data are presented as the triplicate mean (n=3) Â±SEM. Significance was accepted at *P < 0.05, paired Student's t-test.
Figure 2. Template used in triplicates for migration assays A pre designed template of a well of the 6 - well plate (A) was used to create a constant scratch creating a wound in the cells using the firm tip of a standard paperclip (B). A second template (C) was constructed to allow successful and accurate readings of all the wells as the distance between each reading line was 1 cm apart on the template and this was measured at single fixed time points in each dish for each concentration.
3.1 DHEA accelerates migration of human keratinocytes, following mechanical wounding in vitro, and is blocked by an aromatase inhibitor
A time course assay was carried out to observe the effects of 17Î²-oestrodiol, DHEA and DHEA in the presence the aromatase inhibitor, Arimidex in vitro migration of keratinocyte cells using a scratch wound assay. Four different doses of each hormone were evaluated at 1nm, 10nm, 100nm and 1000nm.
A significant increase in migration was seen with DHEA (p<0.05) at all four concentrations. No effect observed at 3 hours, but can be observed at 6, 24, 36 and 48 hours. Incubation of DHEA in combination with Arimidex completely reverses (p<0.05) the effect at each time point and concentration.
Appendix 2 outlines an example of the raw data collected from the photographs at each time point for each varying concentration, and the calculations carried out for each set of results per well.
Figure 3. A scratch wound assay was used to measure the migration of cultured keratinocytes over a series of time points. (A) 0 hours showing the template used to measure the same 6 points at each time point. (B) 3 hours, cells can be seen migrating into the 'wound' (arrows). (C) 6 hours, arrows show migrating cells. Rounded cells suggest they are dividing. (D) 48 hours, the gap was significantly closed. Migrating cells can be observed by an altered morphology (arrows) having a more dendritic appearance magnification Ã-100.
Cells migrating and dividing
Figure 4. The migration of cultured human keratinocytes in vitro. A scratch wound assay was used to measure the migration of wounded keratinocytes in response to vehicle control (pink), 17Î²-oestradiol (green), DHEA (blue) or DHEA in combination with Arimidex (red) supplemented with (A)1nM and (B) 10nM over a 48 hour period. Six time points per dish were assessed and performed in triplicates. Data are presented as triplicate mean Â± SEM. * p< 0.05; paired Student's t-test
Figure 5. The migration of cultured human keratinocytes in vitro. A scratch wound assay was used to measure the migration of wounded keratinocytes in response to vehicle control (pink), 17Î²-oestradiol (green), DHEA (blue) or DHEA in combination with Arimidex (red) supplemented with (A) 100nM and (B) 1ÂµM over a 48 hour period. Six time points per dish were assessed and performed in triplicates. Data are presented as triplicate mean Â± SEM. * p< 0.05; paired Student's t-test.
DHEA and DHEA-S are the most abundant circulating sex hormones in humans and are thought to act as adrenal precursors for androgens and oestrogens. Although in rodents DHEA improves cutaneous wound healing (Ashcroft et al., 2003), their effects on human skin requires further study. The previous in vivo study demonstrated that DHEA substantially accelerates wound healing primarily via its conversion to oestrogen and subsequent signalling through the oestrogen receptors (Howgate et al., 2009). Therefore the mode of action of DHEA in the healing wound appears to mimic that of oestrogen.
This study has compared the effects of 17Î²-oestradiol, DHEA and DHEA in combination with Arimidex on human keratinocyte cell line NCTC 12544, following in vitro mechanical wounding. Increased migration was observed in response to DHEA; significant acceleration was observed particularly between 6 to 48 hours. Cells incubated with DHEA in the presence of Arimidex had a similar level of cell migration to those incubated with absolute ethanol vehicle control (0.0001%) until time point 24 hours; from 24 to 48 hours an increase in migration of cells incubated with DHEA in combination with Arimidex compared to absolute ethanol vehicle control (0.0001%) was observed. A significant negative effect of cell migration was observed for cells incubated with DHEA in combination with Arimidex compared to those cells incubated with DHEA, as Arimidex significantly blocked the effects of DHEA on cell migration.
Keratinocyte migration increased in response to DHEA, and the effect was inhibited by the aromatase inhibitor Arimidex (Mills et al., 2005). This suggests that conversion to 17Î²-oestradiol is required, and corresponds with an in vivo study.
These results also correspond to a previous study (Stevenson et al., 2009), indicating that keratinocytes contain the necessary enzymes for conversion of DHEA into oestradiol. This suggests that the conversion of DHEA into oestradiol is important in the wound healing process and this is particularly important for further research into the treatment of acute and chronic wounds of the elderly. This is because DHEA in the healing wound appears to mimic the mode of action of oestrogen and therefore may prove to be a more suitable therapeutic candidate since it does not have the systemic side effects of oestrogen therapy.
4.2. Future Studies
This study investigated the effects of DHEA and DHEA in combination with Arimidex on a keratinocyte cell line; this study was therefore only limited to primary cells. To mimic more closely the in vivo effects, further studies need to be carried out using organ culture models. To differentiate and isolate cell proliferation and cell migration, independent proliferative assays in the presence of a proliferation marker or mitomycin C (MMC) need to be investigated. DHEA accelerates wound healing and Arimidex reduces the effects of DHEA, therefore the use of an oestrogen antagonist can be used in further studies to investigate why 17Î²-oestradiol does not have a positive or negative effect on cell migration. Finally, other agonists (SERMS) can be used in further studies to investigate the selective inhibition or stimulation of DHEA and the effects of the oestrogen receptor in combination with DHEA.