Tranilast inhibits the cell growth of normal human keratinocytes in vitro
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- Kubo, M., Zhao, Y. & Moriguchi, T. Arch Dermatol Res (2012) 304: 745. doi:10.1007/s00403-012-1291-8
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Tranilast is used clinically as a drug for hypertrophic scars or keloids. Recently, the roles of keratinocytes in the pathogenesis of those conditions have been noted. Therefore, we first examined the effect of tranilast on the cell growth of normal human keratinocytes. A cell growth assay demonstrated that the cell number significantly decreased during 48 h cultures with the addition of tranilast (5–400 μM) compared with a control (tranilast 0) in a dose-dependent manner. Morphologically, cell spreading was decreased and the cell body was elongated with higher concentrations (200–400 μM) of tranilast, and the cell area decreased significantly. The effect was not due to cytotoxicity. The inhibition of cell growth and the changes in cell morphology by the treatment of 100 μM tranilast reversed after the removal of the tranilast. Immunohistochemical staining revealed that F-actin and vinculin expression with tranilast-treated keratinocytes decreased significantly in a dose-dependent manner (100–400 μM). In addition, cell cycle examination showed that 400 μM of tranilast caused G0/G1 arrest with the keratinocytes. From these data we concluded that tranilast inhibited the growth of normal human keratinocytes, and one of its mechanisms may involve decreasing cell spreading by inhibition of F-actin fiber and focal contact formation with the cells.
KeywordsTranilastKeratinocytesGrowthCell spreadingG0/G1 arrest
Complete EpiLife growth medium
Tranilast, N-(3,4-dimethoxycinnamoyl)anthranilic acid, was developed in Japan as an anti-allergic drug. Initially, it was introduced for the treatment of allergic diseases such as atopic dermatitis, allergic rhinitis and asthma . Later, tranilast was also discovered to be useful for the treatment of hypertrophic scars and keloids [22, 25].
Pharmacological studies demonstrated that tranilast inhibits the degranulation of stimulated mast cells [1, 14], which functions as an anti-allergic action. In addition, many in vitro and some in vivo studies reported that tranilast exhibited direct actions on various parenchymal cells. Tranilast inhibited proliferation of the mouse keratinocyte cell line PAM212 , bovine retinal pigment epithelial cells , human fibroblasts [11, 13], human endothelial cells , human vascular smooth muscle cells [5, 15, 26], and rat mesangial cells . Tranilast has also been reported to have significantly reduced collagen synthesis [5, 28], gel contraction [13, 27], chemotaxis , cell migration , TGF-β1 secretion [19, 27], MMP-1 secretion , ECM production , and to have enhanced cytokine-induced nitric oxide production  with various parenchymal cells.
Epithelial–mesenchymal interactions are important for morphogenesis and homeostasis in the skin [3, 24], and recent studies have noted the roles of keratinocytes in the pathogenesis of hypertrophic scars or keloids [2, 6, 16].
Therefore, we first examined the effect of tranilast on the cell growth of normal human keratinocytes by a cell growth assay using the crystal violet staining method. We demonstrated that tranilast inhibited the growth of normal human keratinocytes in vitro. We also examined the mechanisms of the inhibition, and showed changes in cell morphology and the decrease of F-actin and vinclin expression with the cells for the first time.
Materials and methods
Tranilast (MW = 327.33) was kindly provided by Kissei Pharmaceutical Co., Ltd. (Matsumoto, Japan). A 20 mM stock solution of the tranilast was made by solubilizing it with 0.1 M potassium phosphate buffer (pH 7.2), protected from light, and stored at 4 °C.
Normal human keratinocyte cultures
Normal human keratinocytes from neonatal foreskins were purchased from Cascade Biologics (Portland, OR, USA). The cells were expanded in a serum-free, low calcium (0.06 mM) and animal product-free EpiLife growth medium. The complete EpiLife growth medium (CEGM) was EpiLife basal medium + human keratinocyte growth supplement-V2 (Cascade Biologics) + 100 U penicillin per ml and 100 μg streptomycin per ml from Sigma-Aldrich Co. (St. Lous, MO, USA). The third-passaged keratinocytes were trypsinized with 0.1 % trypsin (ICN Biomedicals Inc., Aurora, OH, USA) containing 0.02 % EDTA (Sigma-Aldrich) after washing twice with Hanks’ balanced salt solution without calcium and magnesium (Sigma-Aldrich). Trypsinization was stopped by the addition of two times the volume of 0.1 % soybean trypsin inhibitor (Sigma-Aldrich) to trypsin. The cells were frozen in the CEGM plus 10 % DMSO, and stored in liquid nitrogen for the following experiments.
Cell growth assay
For the assays, 3 × 103 viable normal human keratinocytes (fifth passaged) in 100 μl of the CEGM were plated to each well of a 96-well flat-bottomed tissue-culture treated plate (Corning Inc., NY, USA), and were cultured for 24 h. Then, 100 μl of CEGM containing various concentrations (0, 2, 10, 20, 50, 100, 200, 400, 800 μM) of tranilast or buffer (containing the same volume of 20 mM potassium phosphate buffer for making a 800 μM tranilast containing medium) were added to the wells. The final concentrations of tranilast in each well were, therefore, 0, 1, 5, 10, 25, 50, 100, 200, 400 μM. The final concentration of the buffer control was the same as in that of 400 μM tranilast (we called this Buf. 400). After further 48 h culturing, 100 μl of the medium was removed from each well, and the cells were fixed adding 100 μl of 2 % glutaraldehyde for 10 min at 25 °C. Then the plates were gently washed with PBS three times followed by washing five times with distilled water, dried, and stained with 0.1 % crystal violet in 0.2 M 2-(N-Morpholino)ethanesulfonic acid (MES) (Sigma-Aldrich), pH 6.0 for 20 min at 25 °C with agitation. The plates were again washed five times in distilled water, and dried. Stained cells were observed under a phase contrast microscope (Nikon, ECLIPSE TE2000-U, Tokyo, Japan), then photographed by a CCD camera (Keyence, VB-7000, Osaka, Japan), and were solubilized with 10 % acetic acid by agitating for 15 min at 25 °C. The optical density (OD) of the solution was measured on a Microplate Reader (Corona Electric Co. Ltd., MTP-120, Hitachinaka, Ibaraki, Japan) at wavebands of 590–450 nm. The results were presented as the mean ± standard deviation from four samples for each condition assayed.
Measurement of cell area
Using the photographs with the CV-stained specimen of the growth assay, the cell area with tranilast-treated cells (100, 200, 400 μM) and the control cells (tranilast 0, Buf. 400) was measured for 200 cells each using software of IPLab (Solution Systems Inc., Funabashi, Chiba, Japan).
Trypan blue dye exclusion test
4 × 105 viable keratinocytes (fifth passaged) were plated in each of five 100 mm dishes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), and cultured for 48 h. Then the medium was changed to CEGM with or without tranilast (0, 100, 200, 400 μM) or a medium containing buffer only (Buf. 400), and cultured for a further 48 h. After the trypsinization, the cell viability was examined by the trypan blue dye exclusion test.
LDH release assay
Lactate dehydrogenase (LDH) in the supernatant with the cultures of normal human keratinocytes with or without addition of tranilast was measured using the CytoTox 96® Non-Radioactive Cytotoxicity Assay kit (Promega Corporation, Madison, WI, USA) according to the manufacturer’s protocol. The culture conditions were the same as in the cell growth assay.
Recovery of cell growth after removal of tranilast
2 × 105 viable cells (fifth passaged) in 2 ml of CEGM were plated into each of 21 60 mm dishes (Becton, Dickinson and Company), and cultured for 24 h. Three dishes were used for a 1-day cell count. The rest of the dishes were divided into two groups (nine dishes each). Two milliliters of CEGM with 200 μM tranilast was added to one group of nine dishes (the final concentration of tranilast was 100 μM), and 2 ml of CEGM without tranilast was added to the other group of nine dishes. Then they were cultured for a further 48 h. At 3 days, three dishes of each group were used for the cell count. The rest of the dishes (12 dishes) were further divided into four groups as described “Results”. Four milliliters of CEGM with or without 100 μM tranilast was added after the dishes were washed one time with CEGM. They were further cultured for 48 h, and the cell numbers at 5 days were counted using a hemocytometer.
Recovery of cell morphology after removal of tranilast
The recovery of cell morphology after the removal of tranilast was also observed as follows: 5 × 105 keratinocytes (sixth passaged) in 2.5 ml CEGM were plated into each of 10 60 mm dishes, and cultured for 24 h. Then, to each two dishes, 2.5 ml of the CEGM with or without tranilast (0, 200, 400, 800 μΜ) or the medium containing buffer only (Buf. 800) were added, and cultured for a further 48 h. At 3 days, the dishes were divided into two groups. Five milliliters of CEGM with or without tranilast (0, 100, 200, 400 μM, Buf. 400) was added to one group of dishes (five dishes) after being washed one time with CEGM. To the other group of dishes, the medium of all the dishes was changed to 5 ml of CEGM. All were cultured for a further 48 h. The cells were observed under a phase contrast microscope (Nikon Corporation, ECLIPSE TE2000-U, Tokyo, Japan), and photographed by a CCD camera (Keyence Corporation, VB-7000, Osaka, Japan) at 3 and 5 days.
Immunohistochemical examination for F-actin and vinculin expression
Then 3 × 104 of viable normal human keratinocytes (fifth passaged) in 200 μl of CEGM were plated in each well of a Lab-Tek 8 Chamber Slide (Nalge Nunc International, Rochester, NY, USA), and cultured for 24 h. Next, 200 μl of the CEGM with or without tranilast (tranilast 0, 200, 400, 800 μΜ) or the medium containing buffer only (Buf. 800) was added and cultured for a further 48 h. The cells were fixed with 2 % paraformaldehyde/0.1 M phosphate buffer (PB) for 10 min at RT, and washed three times in PBS cotaining 0.02 % sodium azide (PBS/azide). Cells were incubated with rhodamine-conjugated phalloidin (×1,000 dilution in PBS containing 0.1 % DMSO) (Sigma-Aldrich) for 40 min, washed four times in PBS/azide; and then again were incubated with anti-vinculin monoclonal antibody (×50 dilution) (Sigma-Aldrich) for 30 min and washed three times in PBS/Azide, followed by incubation with FITC conjugated anti-mouse IgG (×100 dilution) (Dako Denmark A/S, Glostrup, Denmark). All the antibody incubation was done in a moist and dark box at 25 °C. Lastly, cells were washed three times in PBS/azide, mounted in VECTASHIELD (Vector Laboratories Inc., Burlingame, CA, USA), and observed under a Laser Scanning Microscope (Olympus Corp., FV300, Tokyo, Japan).
Cell cycle examination
3 × 105 viable keratinocytes (fifth passaged) were plated in each of five 100 mm dishes, and cultured for 48 h. Then the medium was changed to complete EpiLife growth medium with or without tranilast (0, 100, 200, 400 μM) or the medium containing buffer only (Buf. 400), and cultured for a further 48 h. Cells were trypsinized, and the cell numbers were assessed. The cells were fixed with 50 % methanol at 25 °C for 10 min, centrifuged at 4,000 rpm for 5 min, washed once with PBS/0.02 % EDTA, and treated with 100 μg/ml RNAase (Sigma-Aldrich)/PBS + 0.02 % EDTA for 30 min at 37 °C. Then, the cells were stained with 50 μg/ml propidium iodide (Sigma-Aldrich) for 10 min at 25 °C and in dark. Propidium iodide-stained keratinocytes were examined for the cell cycle by a FACS Caliber flow cytometer and analyzed with ModFit LT (BD Biosciences, San Jose, Ca, USA).
Statistical comparison of multiple means was done by one-way analysis of variance (ANOVA) followed by the Fisher test on a StatView 5.0 (SAS Institute Inc., Cary, NC, USA). A comparison of two means was done by the Student’s unpaired t test. A p value of <0.05 was considered to be significant.
Tranilast inhibited the cell growth of normal human keratinocytes
Tranilast caused changes in cell morphology
Cell area decreased significantly at the higher concentrations of tranilast
Cell area measured by IPLab decreased significantly at higher concentrations (200, 400 μM) of tranilast (ANOVA, Fisher test). The decrease in cell area was in a dose-dependent manner (Fig. 2b), while, a buffer control (Buf. 400) showed no change in the cell area compared with the control (tranilast 0) (Fig. 2b).
Tranilast was not cytotoxic for the keratinocytes
Next, we examined the mechanisms of the inhibition of cell growth with the tranilast-treated keratinocytes. First, we examined the cytotoxicity of tranilast by two methods–a trypan blue dye exclusion test and an LDH release assay. The trypan blue dye exclusion test showed no change in the cell viability of the keratinocytes treated with 100, 200, 400 μM for 48 h compared with the controls (tranilast 0, Buf. 400). All of the viability was above 90 % (data not shown). There was no increase in LDH in the supernatant of the tranilast-treated keratinocytes at any concentrations (1–400 μM) for 48 h treatment compared with the controls (tranilast 0, Buf. 400) (data not shown). Therefore, neither test showed cytotoxicity with the tranilast-treated keratinocytes under the conditions tested.
Inhibition of cell growth with tranilast-treated keratinocytes was reversible
Changes in cell morphology with tranilast-treated keratinocytes were reversible
Expression of F-actin and vinculin decreased with the tranilast-treated keratinocytes
Cell cycle arrested at G0/G1 with the 400 μM tranilast-treated keratinocytes
For the first time, we demonstrated that tranilast inhibited the cell growth of normal human keratinocytes, although there was one report that described the inhibitory effect of tranilast on cell growth with the mouse keratinocyte cell line (PAM212) . It has also been reported that tranilast inhibited the cell growth of various parenchymal cells such as bovine retinal pigment epithelial cells , human fibroblasts [11, 13], human microvascular endothelial cells , human vascular smooth muscle cells [5, 15, 26], and rat mesangial cells  in vitro.
The inhibition of cell growth with normal human keratinocytes by tranilast was dose-dependent (up to 400 μM tranilast) and statistically significant with over 5 μM of tranilast compared with that of a control (tranilast 0). Previous reports also demonstrated that the inhibition of cell growth with tranilast was dose-dependent [4, 5, 11–13, 15, 26] with various cell types examined up to 300 μM.
We examined the mechanisms of the inhibition of cell growth with the normal human keratinocytes by tranilast. Tranilast did not have a cytotoxic effect on the keratinocytes with 48 h treatment. Other reports also described no cytotoxicity of tranilast for various cell types [4, 5, 11, 12, 15, 26, 27] examined by a trypan blue dye exclusion test or an LDH release assay.
In fact, in our study, cell growth quickly recovered after the removal of 100 μM tranilast. Previous report also demonstrated the recovery of cell growth with PAM212 cells  after the removal of tranilast.
Most importantly, this study was the first to demonstrate changes in the cell morphology of keratinocytes treated with tranilast. Namely, cell spreading was reduced and the cell body was elongated in a dose-dependent manner. A statistically significant decrease in the cell area at high concentrations of tranilast (200, 400 μM) was demonstrated. No other reports described the changes in cell morphology. Rather, they noted that there were no morphological changes following treatment with tranilast even at 300 μM [23, 26].
In addition, an immunohistochemical study demonstrated a significant reduction in F-actin and vinculin expression with those cells. Vinculin is one of the components of focal contacts . Since focal contacts are associated with bundles of actin microfilaments , focal contacts apparently play an important role in cell spreading and migration . Therefore, we thought that the decrease in F-actin fiber and focal contact formation might be responsible for the decrease in cell areas treated with higher concentrations of tranilast. The precise molecular mechanisms, however, have not been clarified in this study.
We also examined cell cycles with tranilast-treated keratinocytes, and demonstrated that treatment with 400 μM tranilast caused G0/G1 arrest of the cells. Other reports also described tranilast induced G0/G1 arrest with leiomyoma cells , human coronary smooth muscle cells , and human aortic smooth muscle cells . Shime et al.  and Kusama et al.  reported that arrest of the cell cycle at the G0/G1 phase was through the suppression of CDK2 activity via an induction of p21waf1 and p53. Shime et al.  stated that further studies are necessary to clarify whether or not the induction of p53 and p21waf1 is a primary mechanism underlying the inhibitory effect of tranilast. In addition, Miyazawa et al.  reported that the inhibitory effect of tranilast on PDGF-BB induced proliferation is due to S-phase blockage and may be, at least in part, involved in the direct suppression of c-myc gene expression.
The relationship between the cell shape (cell spreading) and the cell cycle were reported in several papers [7, 9, 21]. Huang et al.  demonstrated that cells prevented from cell spreading failed to progress through G1 to the S-phase. They also reported that a similar block in cell cycle progression was induced before this same shape-sensitive restriction point by disrupting the actin network using cytochalasin or by inhibiting cytoskeletal tension generation using an inhibitor of actomyosin interactions .
From these data we concluded that tranilast inhibited the cell growth of normal human keratinocytes in vitro in a dose-dependent manner. One of its mechanisms may involve a decrease in the spreading of the cells by inhibition of F-actin fiber and focal contact formation with the cells. This ultimately caused G0/G1 arrest of the cell cycle with the tranilast-treated keratinocytes. The complete molecular mechanisms underlying those phenomena, however, should be clarified in the future.
We hypothesize that the inhibition of the cell growth of human keratinocytes by tranilast is beneficial for the treatment of hypertrophic scars or keloids, because there have been reports indicating that keratinocytes overlying hypertrophic scars or keloids play a role in the development of pathological fibrosis by influencing the behavior of dermal fibroblasts [2, 6, 16] through paracrine and double paracrine effects. Funayama et al.  reported that keloid-derived fibroblasts showed a greater proliferation and minimal apoptosis when they were co-cultured with normal skin-or keloid-derived keratinocytes utilizing a serum-free indirect co-culture system (most significantly in the latter). They also demonstrated that extracellular signal-regulated kinase and c-Jun N-terminal kinase phosphorylation and expression of Bcl-2 and transforming growth factor-β1 were all significantly upregulated in fibroblasts (normal skin-derived and keloid-derived) when they were co-cultured with keloid- and normal skin-derived keratinocytes (most significantly with the co-culture of keloid-derived fibroblasts and keloid-derived keratinocytes). The hypothesis should be proved in the future.
The authors are grateful to Professor Hironobu Katsuyama for his contribution in statistical analysis of the data. We wish to thank Kissei Pharmaceutical Co. for providing the tranilast used in this study.
Conflict of interest
The authors declare that we have no conflict of interest.