Kinetin and N-6-Benzyladenine Influence on Cell Migration and Collagen Biosynthesis in Human Skin Fibroblasts
Corresponding author: Dr. Agata Jabłońska-Trypuć Bialystok University of Technology, Faculty of Civil Engineering and Environmental Engineering, Department of Sanitary Biology and Biotechnology, Poland. Tel +48 601653570, Fax +48 85 7469015,
E-mail: firstname.lastname@example.org Received: 02-02-2017
K – Kinetin;
BA – N-6-Benzyladenine;
PDE – Phosphodiesterase;
TNF – Tumor Necrosis Factor
Cytokinins are one of the major groups of plant hormones. Although their effect on the biochemical changes that occur in plant cells and their molecular mechanism of action were fairly well established, there is a significant lack of information considering their effect on the growth and metabolism of ani- mal and human cells . In our previous research we showed a positive influence of selected cytokinins – kinetin (K) and N-6-benzyladenine (BA) on oxidative stress parameters . Our recent findings also revealed that traumatic acid – a cyto- kinin, that in terms of chemical structure belongs to the group of fatty acids, enhances collagen biosynthesis and reduces oxi- dative stress in cultured human skin fibroblasts . Therefore we decided to investigate the effect of K and BA in previously established concentrations on cell migration and collagen, as a basic protein that builds the skin, content. A convenient test model for determining the influence of biologically active com- pounds on fundamental biochemical parameters and morpho- logical changes in the dermis is in vitro human skin fibroblasts culture. Since the cytokinins were not studied in detail as a potential compounds that act therapeutically on human skin, the aim of the present study was to evaluate their effect on the proliferative capacity of the main cells in the dermis – fibro- blasts, on the morphological changes taking place within them, on cells migration and on a number of biochemical parameters such as total protein and collagen content. Collagen is a prima- ry protein constituent of the skin . Many active substances used in therapeutic formulations and supplements have favor- able effects on metabolic processes of skin cells. Examples of such compounds are phytohormones, such as K and BA.
Due to the lack of data regarding the influence of selected cy- tokinins: K and BA on collagen biosynthesis in normal skin derived fibroblasts under physiological conditions, without any stress factors, we decided to study this effect. In our re- search we focused on the most appropriate research model: fibroblasts, which account for the largest number of cells in skin. In our experiments we used the tested compounds at concentrations established previously . In those previously selected concentrations we examined K and BA influence on total protein content, collagen content and on cell migration established in Wound Healing Assay.
Materials and Methods
Chemicals and Reagents
All the reagents: K, BA, phosphate-buffer saline (PBS), DMEM, FBS was purchased from Sigma (St. Louis, MO, USA). All other reagents and solvents were of analytical grade. Tissue culture dishes and flasks were purchased from Sarstedt.
K and BA influence on selected parameters was tested in nor- mal human skin fibroblasts in physiological conditions (from 10 to 15 passages). The fibroblast cells were provided by the Department of Cell Biology, Faculty of Biochemistry, Biophys- ics and Biotechnology of the Jagiellonian University in Krakow Poland. The number of a statement on ethical approval for the study from Bioethical Committee is R-I-002/42/2009. Fibro- blasts were maintained in DMEM supplemented with 10% FBS at 37°C in a humified atmosphere of 5% CO2 in air. Fibroblasts in density 1×105 cells/ml were incubated with or without the test compounds in tissue culture 6-well plates in 2 ml of cul- ture medium. Collagen content in cells and medium and total protein content were studied at K concentrations of 10-5 and 10-6M and BA concentrations of 10-6 and 10-7M.
Chemical Treatment of Cells
K and BA were stored in a refrigerator (below -4°C), protect- ed from light and was added to the cultured cells for a final concentration in the range of 10-5 to 10-6M for K and 10-6 to 10-7M for BA. The control cells were incubated without the test compound.
Estimation of Cell number
The number of fibroblasts was determined by direct counts of living and dead cells using the hemocytometer and Trypan Blue dye.
In all conducted assays, data were normalized to each other by measuring the total cell number to exclude the risk that vari- able cell numbers were lysed.
Wound Healing Assay
Fibroblasts cells migration was assayed by using Wound Heal- ing Assay . Confluent cells in 35-mm-diameter dishes were damaged by scraping the monolayer with a sterile pipette tip (500µm in diameter). The cultured were washed twice with PBS to remove cellular debris and various concentrations of tested molecules were added to the respective wells. Wounds were photographed before and after 6, 12 and 24 hours in the presence of compounds using Nikon inverted microscope.
Total Protein Content in cells
After the homogenization of fibroblast cells and extraction in 0.1M NaOH at 4ºC total protein content was calculated. The concentration of proteins was determined spectrophotomet- rically as per Lowry et al . Folin phenol reagent with a pro- tein kit calibrated with bovine serum albumin as the standard was used in the experiment. The absorbance of the extracts was measured spectrophotometrically at 750 nm (Lowry et al,
Collagen content estimation method is based on the spectro- photometrical determination of Sirius red in saturated picric acid selective attachment to fibrillar collagens (types I to V), specially to the Gly-X-Y chain in helical structure . According to this assay soluble (i.e. culture medium) and insoluble colla- gen can be measured. A working solution of collagen (250 μg/ ml) was prepared by diluting stock with 0.5M acetic acid. Af- ter preparing collagen standards, the samples were prepared. If assaying culture medium the serum concentration must be no more than 5%. For assaying collagen in culture medium, 50 to 100μl aliquots of standards, blank (medium) and samples were placed in eppendorf tubes. Subsequently 1ml of the dye solution was added and mixed gently at room temperature for 30 min. The samples were centrifuged at 10.000g for 5 min. to pellet the collagen. The supernatant was removed without disturbing the pellet. 1000μl of 0.1M HCl was added to each tube to remove unbound dye. Afterwards samples were cen- trifuged at 10.000g for 5 min. to pellet the collagen and 1000μl 0.5M NaOH was added to each tube and tubes were vortexed to release the bound dye. The solutions were transferred to cuvettes and read at 540nm.When assaying collagen in cells, cell pellet was first extracted with 50μl of 0.5M acetic acid at 4°C for several hours to overnight. After all mentioned above procedures, solution was centrifuged at 2500g for 5 min. to re-pellet any cell debris and read at 540nm.
For parametric data one-way analysis of variance (ANOVA) fol- lowed Tuckey test was applied. Results from five independent experiments were expressed as mean ± standard deviation (SD) of mean for parametric data. Significance was considered when p≤0,05. Statistica 12.5 was used.
It was observed that N6-benzyladenine caused a significance decrease in living cell number. The slightest impact of the test- ed compound was observed at the concentrations of 10-6 and 10-7M. The number of cells was inversely proportional to the increase in N6-benzyladenine concentration, and it was the lowest at 10-4 M. Mitotic divisions were observed, therefore it is considered that N6-benzyladenine did not arrest cell divi- sion completely. However, the number of cells was significant- ly decreased compared to the control. The impact of N6-ben- zyladenine on cells proliferation is depicted in Figure1.
Kinetin influenced positively on cells division in cultured fibro- blasts. The most favorable concentrations of the second test-
ed compound were estimated to be 10-5 and 10-6 M. At 10-4 M concentration of K, a decreases compared to the control were observed, especially on days 3 ,4 and 5. The influence of K on cells proliferation is depicted in Figure 2.
Figure 1. The effect of various concentrations of BA on fibroblast cell
number during 5-day incubation (n=5).
Figure 2. The effect of various concentrations of K on fibroblast cell
number during 5-day incubation (n=5).
Having examined the cell number we chosen the two most preferred concentrations of N6-benzyladenine and kinetin at which the total living cell number was the highest among the cultures, and then tested the collagen content in cells and culture medium and Wound Healing Assay at each of the two concentrations.
Total protein content in cells
In the case of K an increase in the total protein content was ob- served on the first day at a concentration of 10-5M by 89.54% compared to the control and on the second day and at a con- centration of 10-6M by 46.4% compared to the untreated con- trol cells (Figure 3).
Figure 3. The effect of selected concentrations of K on total protein content of fibroblast cells during a 5-day incubation. Data are present- ed as the mean ±SD (n=5).
BA definitely influenced tested parameter less (Figure 4). The highest increase in protein content – by 38.46% relatively to control, was observed on the last day in concentrations of 10- 6M and by 84.73% relative to the control on day 1 at 10-7M. None of the tested cytokinins did not cause any decrease in total protein content below the level of control. All the tested compounds influence this parameter positively, but obtained results are statistically insignificant.
Figure 4. The effect of selected concentrations of BA on total protein content in fibroblast cells during 5-day incubation. Data are present- ed as the mean ±SD (n=5).
Collagen content in cells and medium
K definitely stimulates the biosynthesis and secretion of colla- gen into the culture medium (Figure 5.). At 10-5M concentra- tion of K on the first day of the experiment we observed a large increase in the content of the protein as much as 321.12% rel- atively to the control, and in day 4 by 345.18% compared to control, untreated cells. In contrast, at a concentration of 10-6M on day 4 the most intense increase in the content of the basic structural protein was observed.
Figure 5. The effect of selected concentrations of K on collagen con- tent in medium during a 5-day incubation. Data are presented as the mean ±SD, different letters (a, b) indicate statistical difference (≤0,05) estimated by Tuckey test (n=5).
It amounted 131.32% in comparison to the control. For BA a significant stimulation of collagen content in the medium was observed at 10-6M concentration (Figure 6.).
Figure 6. The effect of selected concentrations of BA on collagen con-
tent in medium during a 5-day incubation. Data are presented as the
mean ±SD, different letters (a, b) indicate statistical difference (≤0,05)
estimated by Tuckey test (n=5).
It was even by 265.7% relatively to control on day 4 and by 203.67% relatively to control on day 1. While at a concentra- tion of 10-7M we observed a decrease in collagen content by about 10% on each day of the experiment. The obtained re- sults regarding collagen content in medium under the influ- ence of selected cytokinins are statistically relevant.
In cultures stimulated with K the highest content of collagen in the cells was observed on the day 3 (Figure 7.). At a concentra- tion of 10-5M, it was by 157% and at 10-6M – by 66.34% com- pared to control. In day 4 a decrease in the content of structur- al protein in both concentrations was detected. It amounted by 12% at the 10-5M concentration and by 4.73% at the 10-6M concentration compared to the control.
Figure 7. The effect of selected concentrations of K on collagen con- tent in cells during a 5-day incubation. Data are presented as the mean ±SD (n=5).
BA caused high increase in collagen content in the cells on day 3 and on the last day of the experiment (Figure 8.). On day 3 at a concentration of 10-6M an increase in collagen content com- pared to the control was by 154%, and a concentration of 10- 7M – by 73.85%. On the last day of the study the amount of this protein increased by 86.86% at a concentration of 10-6M and by 76.6% at a concentration of 10-7M. BA influenced cells at a concentration of 10-7M showing a decrease in collagen content on day 4 (by 0.44%). The obtained results regarding collagen content in cells under the influence of selected cytokinins are statistically insignificant.
Figure 8. The effect of selected concentrations of BA on collagen content in cells during a 5-day incubation. Data are presented as the mean ±SD (n=5).
Wound Healing Assay
In the experiment with K at a concentration of 10-6M the quite fast movement of cells was observed. The slowest rate of cells migration was detected when they incubated with BA in a con- centration of 10-7M (Figure 9.).
Figure 9. Effect of K and BA in two selected concentrations on migra- tion (wound healing assay) in normal human dermal fibroblasts.
Figure 10. Effect of K 10-6 M concentrations on cells morphology.
Hormones, in terms of chemical structure, constitute a very diverse group of organic compounds responsible for the prop- er conduct of biochemical and physiological processes within cells and tissues. However, at a certain age a significant decline in the production of hormones by the human organism, espe- cially in women, is observed. It can have a meaning impact on the skin physiological condition and to a certain extent on the functioning of the whole organism. Many studies have shown that the effect of topical application of preparations contain- ing hormones is not limited to the application place, but may cause many side effects in relation to the whole organism [8,9]. Therefore, in many countries, estrogenic hormones of animal origin usage in preparations without a prescription is prohib- ited. Phytohormones – hormones of a plant origin, similarly to human hormones, are compounds with high biological ac- tivity. They are chemical signals produced by certain tissues and transported with plant juice to areas in which they initiate growth and development processes. Since some of them are similar in their chemical structure and operation to the human hormones, including estrogen, there is the possibility of using
them in medicine. Unlike the human hormones, their plant origin equivalents are characterized by a broad spectrum of activity. The same compound can affect a variety of metabol- ic processes. The nature and effect of the hormone is largely dependent on: its concentration, the site of its action and its interactions with other phytohormones . Specific impact of phytohormones on receptors differs from the human hor- mones activity. The have also a very potent antioxidant prop- erties. They act as a free radicals scavengers and inhibit cell mutations having anti-cancer properties. All those beneficial effects of plant hormones on the human body indicate that there is a clear need to study the impact of successive groups of these compounds on the human organism. Thus far, the exact influence that have some of the plant-derived substances, e.g. cytokinins especially K and BA on human cells is not known.
Conducted study revealed a significant increase in cells pro- liferation under the influence of K, whereas BA caused a de- crease in the total cell number. By using plant model it was shown that K activates cells divisions through cyclin D – CycD3 pathway and influences the MAP kinases pathway [11, 12]. In similar way it may affect the mitotic division of fibroblasts. Kowalska E. (1992) revealed that K cause an increase in the total DNA content in fibroblasts nucleus . An increase in nucleic acids content due to intensive biosynthesis of import- ant macromolecules in the S phase of the cell cycle. The impos- sibility of the transition from the G1 to the S phase is one of the markers of cellular aging [14, 15]. K may be a factors that causes and increase in DNA content and therefore, through un- locking of the repression of a certain genes, enables cells re-en- ter the cell cycle. On the other hand BA has less impact on cells proliferation than kinetin and obtained results even revealed its inhibitory effect, which is in consistent with the literature and our previous results [2, 16, 17]. Tests performed on tumor cells, in particular myeloid leukemia cell line, showed that BA analogues inhibit CDK and therefore slow cell proliferation. At two concentrations: 10-6 M and 10-7 M, observed decrease in living cell number was the least, and these two concentrations were chosen for further experiments.
We observed that K treated cells are not subject to morpholog- ical changes associated with aging as fast as control, untreated cells (Figure 10). Normal human dermal fibroblasts, derived from adult donors, cultured in vitro are characterized by an elongated, fusiform shape. They are thin, long and arranged in parallel to each other in a regular confluent monolayer. With time the cells become heterogenous, large, flattened, amor- phous and filled with residual lysosomal corpuscles and often contain more than one nucleus. It has been observed, which is in consistent with literature reports, that the cells treated with K did not pass the described morphological changes associated with aging to such an extent as the cells in the control culture [18, 19]. Our study showed that the cells even in the last day of the experiment maintain the characteristic fusiform shape, are arranged in parallel to each other and form monolayer. K also
affects the rate of migration of fibroblasts, as demonstrated in the wound assay test. After creating a confluent layer, cells in- habit a place that has illustrated interrupted tissue. Under the influence of K at a concentration of 10-6M, it was observed that after about 24 hours, “wound” is almost entirely covered with cells. K is more active in stimulation of cells migration than BA. Our study also demonstrated that K has more stimulating ef- fect than BA on cells proliferation and total protein content, especially during the first days of experiment. The highest activity of K was observed at 10-5M concentration increas- ing the protein content by about 89,54% relative to control, while at a concentration 10-6M an increase by 46.4% relative to the control on the first day of the experiment was noticed. Literature data also confirm the increase of the protein con- tent in fibroblast cultures treated with K [12,19]. K impact on the content of collagen in the culture medium and in the cells was not previously tested, and there is no data in the lit- erature on the subject. Our study indicates that the hormone enhances the biosynthesis of described protein increasing its content. Particularly, it stimulates the biosynthesis of collagen at a concentration of 10-5M, increasing its amount in both the culture medium and the cells. An intense increase in the colla- gen quantity in the medium within the first two days of the ex- periment was observed. Subsequently increases and decreases in collagen content in the culture medium and the cells were observed, which suggests that the collagen has been extensive- ly synthesized and secreted by the fibroblasts to the culture medium. Barciszewski J. et al (1999) pointed out that K can act on the levels of transcription, translation, post-translational metabolism, and it is not known at which level it can stimulate the biosynthesis of collagen . Our results confirm that K beneficially affects the basic components of the extracellular matrix that forming the dermis. Kimura T. et al. (2004) has suggested that K treated skin exhibited significantly improved morphological organization of fibroblasts as compared to the untreated control . Prior to stimulation with K within the dermis thickening of collagen fibers that having an irregular layout was visible. After 50 days of K treatment, collagen fibers recovered an appropriate thickness and density of the weave and the natural way of spreading in the connective tissue. Im- proving the quality of extracellular matrix components such as collagen and elastin fibers was observed as a result of K treat- ment of the skin. The experiments were conducted on the Mex- ican hairless dogs .
It was previously demonstrated that BA at concentrations of 10-6M and 10-7M acts relatively the least on the suppression of the fibroblasts division and therefore these concentrations were used for study the effect of BA on other biochemical pa- rameters such as antioxidant enzyme activity, lipid peroxida- tion, glutathione and sulfhydryl groups content in the cells . It is possible that the inhibition of cell divisions in fibroblasts culture during the incubation with BA occurs similarly as in cancer cells, i.e. BA strongly and specifically inhibits the activ-
ity of important protein kinases CDK. These enzymes have a significant participation in the interphase and in mitosis . Although BA inhibits cell proliferation in culture and few fi- broblasts in the phase of mitosis was observed, but in terms of morphology hormone-stimulated cells maintained their characteristic elongated shape of the cells in the early growth phase. This is in accordance with Johnson G.S. et al. (1974) data revealing that N-6 adenine derivatives influence the shape of the cells causing their elongation . Further, the compounds which include the BA cause a significant slowdown in cell movement and increasing intensity of their adhesion to the substrate. This is in accordance with our results, which indi- cate that at the concentration of 10-7M BA the least stimulated cell migration tested in wound assay. In our study we did not check the level of intracellular cAMP and therefore we do not know whether BA changes its content in the cells or acts com- pletely independently of cAMP. However it can influence pro- tein kinases activity, change the distribution of Ca2+, Na+, K+ or through the direct impact on microtubules and microfilaments
– change the shape of fibroblasts. According to Boichot E. et al (2000) a group of BA derivatives has proved, inhibitory prop- erties towards phosphodiesterase (PDE) . PDE belongs to the family of common in mammalian tissues enzymes that are involved in signal transduction pathway through the hydroly- sis of cAMP and cGMP. Because PDEs activity is essential in the process of inflammation, their inhibition by BA or its deriva- tives may be useful in the treatment of inflammatory diseases. As a result of PDE inhibition the synthesis of tumor necrosis factor TNF-α is also halted. It should also be mentioned that TNF-α is a key factor involved in inflammatory disorders de- velopment. PDE inhibitors, which are also anti-TNF-α-factors are used in the treatment of diseases related to inflammation,
e.g. atopy, rheumatoid arthritis, and neurological diseases and many others [23 – 25]. BA which is probable PDEs inhibitor meets the requirements of anti-inflammatory drug and there- fore can be used in the treatment of inflammatory disorders.
The concentration of cAMP in the cell changes very rapidly in response to extracellular signals, which may be an increased level of benzyladenine. BA and its derivatives specifically in- hibit the activity of PDE, particularly PDE4, that may cause an accumulation of cAMP in the cell. cAMP activates cyclic AMP
-dependent protein kinase A, which catalyzes the phosphory- lation of serine and threonine residues of specific, intracellular proteins. BA changes the activity and may even affect gene ex- pression. Regulatory proteins produced as a result of the in- creased activity of the enzymes that are activated by protein kinases, may affect protein synthesis in the cell. However, the most recent literature data indicates that BA activates protein kinase A, but by cAMP-independent mechanism, possibly asso- ciated with other cell signaling pathways, for example associ- ated with TGF-β . This is consistent with the results of our studies, which indicate BA activating effect on protein synthe- sis, and even on the biosynthesis of the basic structural protein
– collagen. BA stimulates protein biosynthesis throughout the duration of the experiment. There were no decreases of test- ed protein content below the level of control. The most inten- sively phytohormone acts on day 1 when it raises the protein content by 35.74% at a concentration of 10-6M and by about 84.73% at a concentration of 10-7M relatively to the control. The study of collagen secreted by the cells into the culture me- dium also shows the strong stimulating effect of BA at a con- centration of 10-6M, in particular on the first and on the last day of the experiment. On day 3 there is a noticeable decline, but still the content of the tested structural protein is above the level of control. On the same day it showed a large increase in collagen content in the cells, while on the first and fourth day of the experiment the amount of collagen was much smaller than on the day 3. Thus it can be concluded that BA by protein kinase A activation, stimulates the biosynthesis and secretion of collagen into the culture medium in the first two days of ex- periment, followed by increasing the synthesis of this protein in the cells (on the third day), and then synthesized collagen is secreted into the medium on day fourth and fifth, causing a large increase in its content in the medium, up to 265.7%. In contrast, BA at a concentration of 10-7M causes a slight de- crease in collagen amount in the culture medium, which is maintained throughout the duration of the 5-day experiment at a level of about 10%. In fibroblasts under the influence of BA at a concentration of 10-7M an increase in collagen content on the first, third and fifth day was observed, and on the second and fourth day a slight decrease in the content of the protein of interest was noticed. These data seem to support the hypothe- sis of Swaney J.S. et al. (2005), that high levels of cAMP inhibits the biosynthesis of collagen, which could mean that activation of collagen biosynthesis by BA occurs actually on the cAMP – independent pathway, conditioned only by the activation of protein kinase A, which occurs through the interaction with TGF-β, which is a collagen biosynthesis stimulating factor . TGF-β activates a protein kinase A through the regulation of the protein complex Smad3/4 [28,29].
All tested in this study phytohormones have beneficial effect on metabolism, migration and proliferation of cells in the dermis. Examined compounds stimulate the biosynthesis of protein, collagen and positively affect the movement ability of fibroblasts. It is believed that K applied to the skin probably removes and reduces the damage caused by excessive UV ra- diation exposure. The fibers of collagen and elastin restore the proper structure and also excessive pigmentation of the skin is reduced. In addition, K diminishes TEWL and allows for a better functioning of the skin barrier layer. It is effective at low doses, as it was confirmed in our investigations on fibroblasts, but its effectiveness is highly dependent on time of application to the skin. It was also shown that the compound has no aller- genic properties and can be used even in the case of sensitive skin [30 – 34]. Well known and confirmed is the action of K, especially its ribosides forms on tumor cells where it stimu-
lated apoptosis. K nucleosides and K ribonucleosides have a stimulating effect on apoptosis of cancer cells while not dam- aging normal cells . Hence it can be concluded, based on the outcome of our research that K can be widely used in the treatment and prevention of a number of diseases especially related to the skin.
In contrast, BA, which derivatives have proven anticarcinogen- ic properties can be used as both a therapeutic agent in inflam- matory diseases and skin disorders related to the pigmenta- tion. There is a correlation between carcinogenesis in humans and plants. Factors that play an important role in a regulation of plant development and plant cells differentiation can also affect the differentiation of healthy and pathologically changed human cells by signal transducing system and therefore may be clinically useful in the treatment of various disease states. Their effectiveness demonstrated in this study in relation to the skin cells may indicate their potential therapeutic rele- vance.
This research was supported by The University of Medical Science in Białystok. We would like to thank the Authorities of The University of Medical Science in Białystok for their financial support of this project.
The authors report no conflicts of interest.
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