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Master's Thesis
in the Department of Biomedical Science
Effects of dasatinib on skin
pigmentation
Graduate School of Ajou University
Major in Molecular Medicine
Effects of dasatinib on skin
pigmentation
Hee Young Kang, Advisor
I submit this thesis as the Master's thesis
in the Department of Biomedical Science.
August, 2020
Graduate School of Ajou University
The Master's thesis of Bogyeong Kang in Department
of Biomedical Science is hereby approved.
Thesis Defense Committee President Hee Young Kang
Tae Jun Park Jang Hee Kim
Graduate School of Ajou University
-ABSTRACT-Effects of dasatinib on skin pigmentation
Background Dasatinib is a small molecule tyrosine kinase inhibitor that has activity against Src and c-Kit tyrosine kinases. Cases of dasatinib on skin pigmentation were examined have been reported.
Objective The effects on dasatinib on skin pigmentation were examined. Methods The effects on pigmentation were investigated by measuring melanin contents, tyrosinase activity and MITF and tyrosinase expressions in B16 melanoma cells and normal human melanocytes. The pigmentation were also assessed in the ex vivo skin. To explain the mechanism of dasatinib, ERK signaling pathway were examined.
Results Dasatinib treatment increased melanin contents and tyrosinase activity and MITF expression, which eventually led to pigmentation in melanocytes. The stimulatory action of dasatinib in pigmentation was further shown in ex vivo cultured skin. Furthermore, the molecular mechanism underlying the melanogenic effect of dasatinib was associated with the ERK-dependent phosphorylation of CREB. The ERK inhibitor PD98059 not only inhibited the phosphorylation of CREB but also abrogated dasatinib-induced melanocyte differentiation.
Conclusion Dasatinib induced skin pigmentation. These data suggest that dasatinib is a potential therapeutic for hypopigmentary disorders.
TABLE OF CONTENTS
ABSTRACT
ⅰTABLE OF CONTENTS
ⅱLIST OF FIGURES
ⅳⅠ.INTRODUCTION
1Ⅱ. MATERIALS AND METHODS
41. Materials 4
2. Cell culture 4
3. Cell viability test 4
4. Apoptosis assay 5
5. Melanin content and tyrosinase activity 5
6. Real-time PCR analysis 6
7. Western blot analysis 6
8.
Ex vivo
skin organ culture and pigmentation assay 79. Enzyme-linked immunosorbent assay (ELISA) 7
10. Statistical analysis 8
Ⅲ. RESULTS
91. Dasatinib induces pigmentation of B16 melanoma cells. 9
2. Dasatinib induces the melanogenesis of normal human melanocytes
at subtoxic concentrations. 13
3. Dasatinib increases cutaneous pigmentation. 19
signaling pathway. 22
Ⅳ. DISCUSSION
31Ⅴ. REFERENCES
35LIST OF FIGURE
Figure 1. Structure of dasatinib and cytotoxicity of dasatinib in B16
melanoma cells. 10
Figure 2. Dasatinib induced melanogenesis in B16 melanoma cells. 11 Figure 3. Effects of dasatinib on the mRNA expression of pigmentation related genes in B16 melanoma cells. 12 Figure 4. Morphology of human melanocytes treated with dasatinib. 14 Figure 5. Cell viability of dasatinib-treated normal human melanocytes. 15 Figure 6. Sub-G1 cell population in dasatinib-treated human melanocytes was determined by FACS flow cytometry. 16 Figure 7. Dasatinib increased pigmentation in normal human melanocytes.
17 Figure 8. Dasatinib induced MITF, tyrosinase and TRP1 expression in
melanocytes. 18
Figure 9. Dasatinib increased pigmentation in cultured human skin. 20 Figure 10. Dasatinib induced expression of pigmentation in ex vivo skin. 21 Figure 11. Dasatinib induced ERK-CREB signaling pathway. 24 Figure 12. Dasatinib induced the degradation of phospho-Erk after 24h. 25 Figure 13. Effects of dasatinib on p38 and JNK phosphorylation in human
melanocytes. 26
Figure 14. PD98059 abolishes the dasatinib-induced phosphorylation of Erk
and CREB. 27
Figure 15. Dasatinib-induced melanocytes differentiation requires activation
of Erk. 28
Figure 17. Effects of dasatinib on cAMP formation. 28 Figure 18. A hypothetical mechanisms underlying the dasatinib melanogenic
Ⅰ. INTRODUCTION
Melanocytes are derived from neural crest that synthesize melanin pigments. Fully differentiated melanocytes are characterized by pigmentation and well-developed dendrites. The process of producing pigments is called melanogenesis. The understanding of the mechanisms of melanogenesis helps us to explain the pigmentation defects (hypo or hyper-pigmentary disorders) allows the development of potential therapeutic strategies.
Melanogenesis is regulated under the influence of paracrine factors secreted by neighboring cells, such as keratinocyte, fibroblast and immune cells (Yuan et al., 2018). Many studies have highlighted interactions between melanocytes and their surrounding environment. For example, dermal endothelial cells play an inhibitory role on skin pigmentation by secreting copious amounts of TGFβ1 (Park JY et al., 2016). Fibroblast derived clusterin (CLU) inhibits pigmentation via TGF-β signaling pathway (Lee J et al., 2017). SDF1 (Stromal-derived factor 1: CXCL12, a kind of chemokine, has an inhibitory action of cAMP signaling that promotes melanin production, and thus has melanin production inhibitory action.) emitted by young fibroblasts. Senescence fibroblasts are present in the upper layer of the dermis reduced SDF1 expression level. Eliminating senescent fibroblast in the upper layer of the dermis by Radiofrequency may create an unaging fibroblast in the lower layer of the dermis and improve melasma (Yoon JE et al., 2018).
Diverse melanogenesis signaling pathways regulate microphthalmia-associated transcription factor (MITF), a master regulator of melanogenesis, which upregulates melanogenesis enzymes tyrosinase (TYR), tyrosine-related protein-1 (TRP-1) and TRP-2. MITF expression is mainly
induced by the cyclic adenosine monophosphate protein (cAMP) response element-binding protein (CREB), which is regulated by certain upstream signaling molecules, including PKA, mitogen-activated protein kinases (MAPKs), and AKT (Gonzalez GA et al., 1989;,). Many studies have highlighted regulating melanogenesis pathways by material from various sources. drug repurposing can help in translating the use of novel and potent melanogenesis regulators from bench to clinic expeditiously due to the already established safety and toxicology profile of the drug.
Tyrosine kinase inhibitors (TKIs) such as imatinib, sunitinib, dasatinib are used in the treatment of chronic myeloid leukemia (CML) (Garcia-Gutierrez et al., 2019). These drugs uncommonly showed side effect like hair or skin color changes (Ricci et al., 2016). Many TKIs have activity against c-Kit, a receptor tyrosine kinase which binds to a substance called stem cell factor (SCF), which causes certain types of blood cells to grow (Galanis et al., 2015). SCF/KIT pathway may function as a primary mechanism for regulating both proliferation and differentiation of melanocytes (Grichnik et al., 1998; Kim M et al., 2018).
Dasatinib is a multi-targeted tyrosine kinase inhibitor (TKI) and was initially developed as a Src-family kinase inhibitor, but it also inhibits BCR-ABL, eptinA2, platelet-derived growth factor receptor (PDGFR), and c-kit (Kantarjian et al., 2006). Recently, certain off-target effects of dasatinib, including cell differentiation, have attracted considerable interest (Steegmann JL et al., 2012; Fang Y et al., 2013; Johnson DE et al., 2008).
Adipogenic or osteoblastic differentiation–induction effects of dasatinib were also reported (Borriello A et al., 2011; Boufker H et al., 2010). Dasatinib also exhibits anti-inflammatory effects; i.e., it inhibits the functions of some inflammatory cells, including T lymphocytes(Silva AL et al., 2016). Interestingly, dasatinib has the capacity selectively to eliminate senescent cells through ephrin signaling pathways to delay aging (Kirkland JL et al., 2017; Zhu Y et al., 2015).
Mucocutaneous side effects of dasatinib represent the most frequent non-hematological adverse events (Grasso V et al., 2014). In particular, cases of diffuse cutaneous hyperpigmentation or hypopigmentation were observed during dasatinib treatments (Boudadi K et al., 2014; Alharbi B et al., 2018; Lin YC et al.,2012). Because long-lasting treatments are likely with TKIs, knowledge about their effects on normal cells is crucial.
In this study, we examined the effects of dasatinib on melanocyte biology and skin pigmentation to elucidate the mechanisms potentially responsible for skin pigmentary changes caused by dasatinib and to provide a proof of concept of the repurposing approach of dasatinib in skin pigmentary diseases.
Ⅱ. MATERIALS AND METHODS
1. Materials
Dasatinib was purchased from Cayman Chemical (Ann Arbor, MI) and the Erk inhibitor PD98059 was bought from Cell Signaling Technology (Danvers, MA).
2. Cell Culture
The murine B16 melanoma cells were cultured in DMEM containing 10% heat-activated fetal bovine serum (FBS, Gibco-BRL, Bethesda, MD) and 1% antibiotics (Gibco, Waltham, MA). Primary human melanocytes were isolated from human foreskins (average age 18 years) obtained during surgery (IRB No. AJIRB-BMR-SMP-17-438). These cells were cultured in F12 medium supplemented with 10% fetal bovine, 24 μg/mL 3-isobutyl-1-methylxanthine, 80 nM 12-O-tetradecanoyl phorbol 13-acetate (TPA), 1.2 ng/mL basic fibroblast growth factor (bFGF), and 0.1 μg/mL cholera toxin (all from Sigma-Aldrich, St. Louis, MO). For the experiments, melanocytes were used at passages from 2 to 7 and maintained in MCDB-153 (Sigma-Aldrich) containing 4% heat-inactivated FBS, 1% penicillin/streptomycin, 0.6 ng/mL bFGF, 5 μg/mL insulin, 0.1 μg/mL α-tocopherol and 1 μg/mL apo-transferrin.
3. Cell viability test
The murine B16 melanoma cells were seeded (0.7x103/well) in24-well plates and incubated 37 ℃. Following 24h, Cells were treated with dasatinib for 3days. Also, Primary human melanocytes (1x104/well) were seeded in24-well plates and treated with dasatinib (1~200 nM) for 5 days. Then, the cells were washed with PBS and replaced with fresh medium containing 10% methylthiazolyldiphenyl-tetrazolium bromide (MTT) solution. Cell viability was determined by measuring the optical density at 570 nm using an ELISA reader.
4. Apoptosis assay
Melanocytes were treated with dasatinib (100 or 200 nM). Three days later, floating cells were fixed and the DNA was stained with 50 μL of 100 μg/mL RNase (Sigma-Aldrich) and 200 μL of propidium iodide (50 μg/mL). After staining, the sub-G1 population was analyzed by flow cytometry using FACS Diva software on a FACS Canto™ II system (BD Biosciences, San Jose, CA).
5. Melanin content and tyrosinase activity
Cells were lysed with 0.1 M phosphate buffer (pH 6.8) containing 1% Triton X-100 and a protease inhibitor cocktail (Roche, Basel, Switzerland). The protein concentration in the supernatants was measured using a Lowry assay system. Pellets were solubilized in 100 μL of 1 N sodium hydroxide (NaOH) for 3 h at 60 °C and the absorbance was measured at a
wavelength of 490 nm. The melanin content was calculated from a standard curve using synthetic melanin (Sigma-Aldrich). For the assay of the tyrosinase activity, each sample was incubated with 2 mM L-DOPA (Sigma-Aldrich) in 0.1 M phosphate buffer (pH 6.8) for 90 min at 37 °C. After incubation, the tyrosinase activity was measured at a wavelength of 490 nm.
6. Real-time PCR analysis
Total RNA was extracted using an RNeasy Mini kit (Qiagen, Valencia, CA) and the first-strand cDNA was synthesized using the SuperScriptTMIII reverse transcriptase kit (Invitrogen, Waltham,MA). Quantitative real-time PCR was performed using SYBR Green Super Mix (Bio-Rad, Hercules,CA). The primer sequences used were as follows: human MITF:5‘-AGAACAGCAACGCGCAAAGAAC-3’,5‘-TGATGATCCGATTCACC AAATCTG-3’, human Tyrosinase: 5’-CACCACTTGGCCTCAATTC-3’, 5‘-AAAGCCAAACTTGCAGTTTCCAC-3’, human 18S: 5‘-CGGCTACCACATCCAAGGAA-3’,5‘-GCTGGAATTACCGCGGCT-3’, mouseMITF: 5‘-CTCAGTAGCAGCCTATT-3’, 5‘-TCTTCCTTCACTCCAGA-3’ mouse Tyrosinase: 5‘-TTAAGCTGACAAGGGTCTT-3’, 5’-ACATACCTTGAACCGCTAGA-3’; and mouse GAPDH: 5‘-GAGTACGTCGTCGAGTCCA-3’, 5’-ATGGCA TGGACTGTGGTCA-3’.
7. Western blot analysis
The cells were directly lysed in 100 μl of a 2X SDS-sample buffer. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA). The antibody against MITF was purchased from Abcam (Cambridge, UK), and the antibodies against tyrosinase, TRP1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), p-p38 and p38α/β were from Santa Cruz Biotechnology (Dallas, TX). The antibodies for p-Erk1/2, Erk1/2, p-SAPK/JNK, SAPK/JNK, p-STAT1 (Y701), STAT1, p-STAT3 (Y705), p-STAT3 (S727), STAT3, p-CREB and CREB were purchased from Cell Signaling Technology (Danvers, MA).
8.
Ex vivo
skin organ culture and pigmentation assayFive human skin samples (average age of 30) were obtained during surgical procedures. Informed written consent was obtained from each patient before the skin biopsy, and this study was approved by the institutional review board of Ajou University Hospital (AJIRB-BMR-SMP-17-438). A sterilized stainless steel grid was placed on a culture dish containing MCDB-153 supplemented with 4% FBS. The skin specimens were placed on the stainless steel grid and maintained in an incubator at 37 °C with 5% CO2. After three days, the specimens were fixed in 10% formalin and embedded in paraffin. Melanin pigments were perceived by Fontana-Masson staining. An image analysis was performed with ImageProPlus4.5 (Media Cybernetics Co., Rockville, MD) and the pigmented area relative to the epidermal area (PA/EA) was measured.
Immunohistochemical staining was conducted using antibodies against MITF (1:20 dilution; Abcam) and tyrosinase (1:300 dilution; Invitrogen). MITF expression levels were counted as number of positive cells per one millimeter of basal layer (cells/mm) and tyrosinase was measured as the stained area per epidermal area (SA/EA).
9. Enzyme-linked immunosorbent assay (ELISA)
1.5 × 106 Melanocytes were treated with 200 nM dasatinib for 2 hr, and then analyzed cAMP levels in the cell lysates. cAMP ELISA kits (R&D System, Minneapolis, MN) used according to the manufacturer’s instructions.
10. Statistical analysis
Data were presented as the mean ± standard deviation (SD) of more than three independent experiments. Statistical analyses were performed using the Mann-Whitney U test (SPSS 25.0; IBM Corp., Armonk, NY). A value of p < 0.05 was considered significant.
Ⅲ. RESULTS
1. Dasatinib induces pigmentation of B16 melanoma cells
We first investigated cytotoxicity effect of dasatinib on B16 melanoma cells. As a result, there was no cytotoxicity of dasatinib at the indicated concentrations (Figure 1). When B16 melanoma cells were treated with dasatinib, pellet color of lysed cells were highly increased. Melanin levels and tyrosinase activity were significantly higher in the treated cells with dasatinib (Figure 2). In addition, the mRNA level of melanogenesis-associated genes, microphthalmia-associated transcription factor (MITF) and tyrosinase were significantly upregulated (Figure 3).
2. Dasatinib induces the melanogenesis of normal human melanocytes at subtoxic concentrations.
The levels of cytotoxicity of dasatinib on the cells were examined first. Normal human melanocytes were treated with different concentrations (1 nM ~ 2 μM) of dasatinib and the cell viability was analyzed. The dasatinib concentration used was chosen on the basis of the dosage usually employed in studies with other cellular models (Futosi et al., 2012 ; Lin et al., 2012) and considering the likely plasma dasatinib levels (14.6 ng/mL) of treated patients (Luo et al., 2006). The dasatinib treatment did not result in any significant changes of the cell morphology (Figure 4) and cell viability at concentrations below 200 nM (Figure 5). However, high concentrations of up to 2 μM of dasatinib exhibited significantly decreased cell viability of normal human melanocytes. Given that 200 nM of dasatinib had no effects on apoptosis, an amount of 200 nM or less was established as the optimal concentration here with which to explore the biological function of dasatinib on pigmentation (Figure 6). When normal human melanocytes were treated with a non-cytotoxic concentration of dasatinib (1 ~ 200 nM), the melanin content and tyrosinase activity were increased (Figure 7). The increased melanin content was caused by the increased mRNA and protein expression levels of melanogenesis-associated proteins, MITF and tyrosinase (Figure 8). This finding indicates the capacity of dasatinib to induce melanocyte differentiation.
3. Dasatinib cutaneous pigmentation.
The effect of dasatinib on pigmentation was also examined in cultured human skin. Ex vivo human skin was maintained in the presence of 100 nM dasatinib. After three days, the skin showed significantly increased pigmentation in the presence of dasatinib compared to a control skin sample, as shown by Fontana-Masson staining (Figure 9). An image analysis showed an increase in the pigmented area/epidermal area (PA/EA) ratio in the dasatinib-treated skin compared with the control skin (0.054±0.018 vs. 0.092±0.023, n=5, p < 0.01). For a more in-depth examination of the melanogenesis changes, immunostaining for melanocyte-specific markers of MITF and tyrosinase was performed. As shown in Figure 10 , we found increased expression levels of MITF and tyrosinase in dasatinib-treated skin compared to the control skin, suggesting increased melanogenesis in dasatinib-treated skin (MITF: 2.53±1.03 vs. 5.42±1.83, n=4, p < 0.05, tyrosinase; 0.024±0.02 vs. 0.049±0.03, n=4, p < 0.05). Taken together, these results indicate that dasatinib has a stimulatory effect on skin pigmentation via the stimulation of MITF-tyrosinase expression.
4. Dasatinib stimulates melanogensis through the ERK-CREB signaling
Previous studies have demonstrated that the differentiation-induction effect of dasatinib depends on ERK and AKT signaling (Fang Y et al., 2013; Futosi K et al., 2012). To address whether there is an association between dasatinib-stimulated melanogenesis and the ERK and AKT signaling pathways, the effects of dasatinib on ERK and AKT phosphorylation were examined. The phosphorylation of ERK1/2 was detected at 1h and increased to a high level at a time point of 3h of the dasatinib treatment. In contrast, dasatinib did not affect AKT phosphorylation in melanocytes (Figure 11). However, the phosphorylation of ERK detected after 24h and reduced in normal human melanocytes treated dasatinib. These results are inferred that degradation to phospho-Erk after 24h. Overall mean terminal half-life of dasatinib is 3-5 hours (Hochhaus A et al., 2013). And also, dasatinib increased cAMP formation in melanocytes (Figure 17). Consistent with this, increased CREB phosphorylation was also observed as early as 1 h and then remained at a high level for at least 3 h (Figure 11). Furthermore, pretreatment with PD98059 20 μM, a specific inhibitor of the Erk pathway, recovered the phosphorylation of CREB (Figure 14) and the melanogenic effects of dasatinib on melanocytes the MITF and tyrosinase mRNA and protein levels were downregulated in the presence of PD98059 (Figure 15).
It was noted that PD98059 only modestly rescued dasatinib induced pCREB and MITF and tyrosinase levels, suggesting additional mechanisms
and JNK, suggesting they might also be contributes to the dasatinib’s differentiating effect on melanocytes. Previous studies have shown that STAT1 was phosphorylated at serine site by several kinases including MAPKs (Kovarik P et al., 1999) and dasatinib induces myeloid cell differentiation through the activation of STAT 1, which is dependent on MEK/ERK activation (Fang Y et al., 2013). We therefore examined the STATs signaling in dasatinib-treated melanocytes. As shown in Figure 16 , 200 nM dasatinib treatment led to an increased phosphorylation of STAT1 (Y701) and STAT3 (S727 and Y705) after 2 h in melanocytes.
Taken together, these results suggest that the activation of ERK/CREB signaling is related to dasatinib-induced melanogenesis in normal human melanocytes.
Ⅳ. DISCUSSION
Melanocytes function in melanin production, distribution and transfer to keratinocytes to maintain constitutive skin color and protect skin from various internal and external stimuli by programmed melanogneic process called melanogenesis. Melanogenesis can be significantly influenced by complex interactions between melanocytes and their surrounding environment, including diverse types of cells (D'Mello SA et al., 2016). In human skin, regulating pigmentation is a important goal by medical and cosmetic reasons for many researchers.
Many studies have highlighted regulating melanogenesis by effective modulators. Early studies focused on inhibiting related-tyrosine (otherwise called dopachrome tautomerase (DCT) which an important protein for melanin synthesis. Hydroquinone(HQ) is the representative materials as a tyrosinase inhibitor. It is effective inhibited melanin synthesis (Inoue Y et al., 2013). However, HQ affects not only the formation, melanization, and degradation of melanosomes, but that it affects also the membraneous structures of melanocytes and eventually causes necrosis of whole melanocytes (O'Donoghue JL et al., 2006). But recently studies tried to regulate intraceulluar melanogenesis signaling pathways. Furthermore, many researchers studied paracrine factors from complex interations between melanocytes and neighboring cells (Yoon JE et al., 2018; Kim M et al.,
The present study demonstrated for the first time the capacity of dasatinib to induce melanocyte differentiation, as evidenced by a stimulatory effect of dasatinib on melanogenesis, which is an indication of melanocyte differentiation. Dasatinib had no effects on the proliferation and apoptosis of normal human melanocytes at concentrations lower than 200 nM. In contrast, the melanin content; tyrosinase activity levels; and the expressions of MITF, TRP1, and tyrosinase increase significantly after the dasatinib treatment. Additionally, an ex vivo skin culture system confirmed stimulatory action of dasatinib on skin pigmentation. These findings demonstrate that dasatinib induces melanocyte differentiation and functions as a melanogenic stimulator.
The findings here indicate that the biological function of dasatinib on inducing melanocyte differentiation is mediated by the activation of ERK and CREB. In our study, ERK phosphorylation was associated with increased cAMP and p-CREB expression levels and melanogenesis-associated signaling, including MITF expression and melanin production, in dasatinib-treated cells, which were ablated through preincubation in an ERK inhibitor. This indicates that ERK/CREB signaling may contribute to dasatinib-induced melanogenesis.
It has been known that the activation of MAPKs, a family of serine/threonine kinases, including p38, Erk, and JNK, is crucial during the melanogenesis of melanocytes, resulting in the upregulation of MITF and tyrosinase (Chung YC et al., 2017; Huang HC et al., 2016). The activation of Erk1/2 was also observed in a-melanocyte stimulating hormone-induced melanogenesis (Yanase H et al., 2001) and during UV irradiation induced
melanogenesis (Regazzetti C et al.,2018). In our study, MAPK pathway, Erk, p38 and JNK phosphorylation was associated with increased p-CREB expression levels and melanogenesis-associated signaling, including MITF expression and melanin production, in dasatinib-treated cells, which were ablated through preincubation in an Erk inhibitor.
Several mechanisms have been proposed to explain the differentiation-inducing effects of dasatinib on cells (Congleton J et al., 2012; Heo SK et al., 2014). The inhibition of Src-family kinases (Boufke H et al., 2010) or c-kit (Garcia-Gomez A et al., 2012) has been suggested as a significant mediator of dasatinib-induced cell differentiation. The activation of MEK/Erk cascades has also been found to contribute to dasatinib-induced cell differentiation, in accordance with our findings (Fang Y et al., 2013; Heo SK et al. 2014).
The cAMP is a crucial signaling regulating pigmentation. cAMP leads to activation of PKA, followed by phosphorylation of CREB trancription factor. Of note, nilotinib, another second-generation tyrosine kinase inhibitor, induces the pigmentation of human melanoma cells via the phosphorylation of CREB, activating PKA signaling (Kim KI et al., 2018). Considering that Src-family kinases have been reported to activate the MEK/Erk pathway, these unexpected results may be explained by the fact that dasatinib is a weak RAF inhibitor and that the binding of a weak RAF inhibitor leads to the paradoxical activation of RAF followed by the activation of Erk (Packer LM et al., 2011). Dasatinib or nilotinib driving paradoxical Erk pathway
cAMP may be responsible for dasatinib-induced pigmentation.
In our study, we observed phosphorylation of STAT1 and STAT3 increased with 200 nM dasatinib (figure 16). Dasatinib induces myeloid cell differentiation through the activation of STAT 1, which is dependent on MEK/Erk activation (Fang Y et al., 2013). UVB irradiation stimulates basic fibroblast growth factor (FGF2) protein secretion from human keratinocytes to induce PAX3 (paired box 3) transcription through activation of STAT3 in melanocytes. PAX3 is regulated by STAT3 protein at the level of transcriptional (Dong L et al., 2012). This is estimated to induce differentiation of melanocyte. Considering the complex mechanisms involved in Erk-STAT pathway activation, further studies may be necessary to gain a better understand of the effects of the dasatinib-induced activation of Erk –STAT signaling. It should be noted that as a multi-kinase inhibitor, the mechanism of dasatinib-induced melanogenesis could not be summarized simply. In fact, there may be undefined mechanisms involved in the melanogenic activity of dasatinib.
In conclusion, these results demonstrated for the first time the differentiation-inducing effects of dasatinib in melanocytes, which depends on the activation of ERK-CREB-MITF-tyrosinase signaling cascades. Our results may provide a molecular theoretical basis for the future development of pigmentation-targeting treatment strategies.
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-국문요약-피부 색소 형성에 다사티닙의 효과
피부 색소 조절은 매우 중요하다. 우리는 이전 연구들을 통해서 타이로신 카이네이즈 억제제들로 치료를 한 환자들 중에 피부나 머리카락 색의 변화를 관찰하였다. 또한 이마티닙, 닐로티닙은 세포 실험에서도 그 변화를 관찰하여 기전연구가 되어있다. 이에 본 연구에서는 타이로신 카이네이즈 억제제 중 하 나인 다사티닙이 피부 색소형성에 미치는 영향을 연구하였다. 방법: 배양된 멜라닌 형성 세포들과 정상 사람 피부조직에서 다사티닙이 색소형성을 확인하였다. 다사티닙을 처리한 후 멜라닌 세포에서의 멜라닌 양, 티로시나아제 활성과 MITF, 티로시나아제의 mRNA양과 단백질 발현량을 관찰 하였다. 또한 다사티닙을 처리한 정상 사람 피부조직에서의 피부 색소 변화를 관찰하였다. 그리고 멜라닌 형성 세포에서의 다사티닙이 멜라닌 형성에 있어서 멜라닌 색소에서의 신호전달 기전을 확인해보았다. 결과: 배양된 멜라닌 형성세포와 정상 사람 피부조직에서 다사티닙은 색소 침착을 유발하는 것을 관찰하였다. 다사티닙을 처리한 멜라닌 형성세포의 티로 시나에제 활성, 멜라닌 양이 증가하였으며, MITF와 티로시나아제의 mRNA 양 과 단백질 발현량이 증가하였다. 또한 다사티닙을 처리한 정상 사람 피부 조직 에서 피부 색소가 증가하는 것을 관찰하였고 색소침착 관련인자인 MITF와 티 로시나아제 또한 발현을 증가하였다. 다사티닙은 Erk를 발현시켜 CREB의 활 성화시키는 신호 전달 기전을 통해 MITF와 티로시나에게의 발현을 증가 시켜 색소 형성 과정에 영향을 미침을 확인하였다. 또한 본 연구에서는 p38, JNK, c-AMP, STAT단백질의 발현도 다사티닙에 의해 높아지고 이는 멜라닌형성세포의 분화를 유도하는 것을 확인하였다.
결론: 다사티닙은 멜라닌 세포내에서 Erk-CREB 신호전달을 중심으로 멜 라닌 형성을 자극하며, 이는 잠재적 저색소 질환의 치료제로서 역할을 할 것으 로 생각된다.
