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Master's Thesis
in the Department of Biomedical Sciences
Senolytic Drugs are Potential Skin
Lightening Agent for Aging Pigmentation
Ajou University Graduate School Cancer Biology
Ji Hee Park
Senolytic Drugs are Potential Skin Lightening Agent for Aging Pigmentation
Tae Jun Park, Advisor
I submit this thesis as the
Master's thesis in the Department of Biomedical Sciences
February, 2021
Ajou University Graduate School
Cancer Biology Major
Ji Hee Park
The Master’s thesis of Ji Hee Park in the Department of Biomedical Sciences in hereby approved.
Thesis Defense Committee President
Tae Jun Park Seal Hee Young Kang Seal Jang-Hee Kim Seal
Ajou University Graduate School
December 22, 2020
– Abstract –
Senolytic Drugs are Potential Skin Lightening Agent for Aging Pigmentation
Cutaneous aging process is an important factor which modifies the pigmentary system.
Given that cellular senescence is a fundamental aging mechanism, senescent fibroblasts accumulated at the dermis are involved in melanocyte differentiation via stromal-epithelial interactions during aging. Both ABT263 and ABT737, which are well known senolytic drugs, led apoptosis of senescent cells accumulated in tissues. Senolytic drugs induce apoptosis only senescent, but not normal cells. In this study, they did not affect melanin synthesis of melanocytes, however, melanocytes’ pigmentation was decreased via regulation of senescent fibroblasts apoptosis. Therefore, senolytic drug, especially ABT263 and ABT737, induced apoptosis in senescent cell and regulated the skin pigmentation in the elderly. As a result, we improved skin pigmentation.
Key words: Senolytic drug, skin pigmentation, aging, senescent fibroblasts
TABLE OF CONTENTS
ABSTRACT‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ i TABLE OF CONTENTS ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ ii LIST OF FIGURES ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ iii I. INTRODUCTION ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 1 II. MATERIALS AND METHODS ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 2 1. Materials ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 2 2. Cell culture ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 2 3. In vitro model of senescent fibroblasts ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 2 4. Cell viability test ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 3 5. Apoptosis assay ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 3 6. Caspase 3/7 activity assay ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 3 7. Melanin content and tyrosinase activity assay ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 4 8. Real-time PCR analysis ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 4 9. Western blot analysis ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 5 10. Statistical analysis ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 5 III. RESULTS ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 6 1. Specificity of senolytic drugs for senescent fibroblasts of the skin ‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 6 2. ABT263/ABT737 decreased skin pigmentation in vitro ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 12 3. The elimination of senescent fibroblasts induces skin lightening ‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 20 IV. DISCUSSION & CONCLUSION ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 22 V. REFERENCES ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 23 국문요약 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 25
LIST OF FIGURES
▶ Figure 1. Effect of quercetin/dasatinib combination on skin senescent fibroblasts‧‧‧‧ 7
▶ Figure 2. Effect of ginkgo biloba extract on skin senescent fibroblasts ‧‧‧‧‧‧‧‧‧‧‧‧‧8
▶ Figure 3. Effect of resveratrol on skin senescent fibroblasts ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧9
▶ Figure 4. Effect of ABT263/ABT737 on skin senescent fibroblasts ‧‧‧‧‧‧‧‧‧‧‧‧‧‧10
▶ Figure 5. Effect of ABT263/ABT737 in melanocyte ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧13
▶ Figure 6. UVA-induced senescent (UVAiS) fibroblasts model ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧15
▶ Figure 7. ABT263 reduces skin pigmentation in vitro ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧16
▶ Figure 8. ABT737 reduces skin pigmentation in vitro ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧17
▶ Figure 9. Effect of UVA on melanocyte proliferation ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧18
▶ Figure 10. ABT263/ABT737 reduce skin pigmentation in vitro ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧19
▶ Figure 11. ABT263 decrease skin pigmentation ex vivo ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧21
I. INTRODUCTION
There is increasing evidence of a crucial role of senescent fibroblasts in skin pigmentation via secreting senescence-associated secretory phenotypes(SASPs) (Kovacs et al., 2010; Salducci et al., 2014). During the skin aging process, senescent fibroblasts are accumulated in dermal compartments (Mine et al., 2008; Coppé et al., 2010; Velarde et al., 2016; Ghosh et al., 2016). Skin lesions related with aging such as senile lentigo or melasma showed increased numbers of senescent fibroblasts (Yoon et al., 2018; Kim et al., 2019). Crosstalk between senescent fibroblasts and melanocytes plays an important role in the stimulation of melanogenesis and in subsequent aging-related pigmentation (Yoon et al., 2018). Up to date, several kinds of senescent cells targeting drugs inducing apoptosis were developed. However, these drugs worked on cells are working cell type dependent manner and the senolytic action mechanism are different among drugs (Kirkland, J. L. et al., 2017). Therefore, at first, we screened well known senolytic drugs for which drugs could target the skin dermis senescent fibroblasts.
Based on this background, we focused on targeting senescent fibroblasts in the dermis.
In our previous study, we observed de-pigmentation of skin as removing senescent cells by radio-frequency technique (Yoon et al., 2018). Senescent fibroblasts were replaced by young fibroblasts resulting in improved skin pigmentation (Yoon et al., 2018). However, this technique requires invasion which is not easy to access. Therefore, we tried to develop a novel and non-invasive technique to remove senescent fibroblasts from dermis of the elderly. The hypothesis is eliminating senescent fibroblasts inhibits aging related skin pigmentation.
II. MATERIALS AND METHODS
Materials
ABT263, ABT737 were purchased from Selleckchem (Houston, TX) and the Dasatinib was bought from Cayman Chemical (Ann Arbro, MI). Quercetin, Ginkgo biloba extract and resveratrol were purchased from Sigma-Aldrich (St. Louis, MO).
Cell Culture
Normal human fibroblasts were isolated from skin biopsies (AJIRB-BMR-SMP-17- 438). Fibroblasts at passages 3-7 were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco-BRL, Grand Island) supplemented with 10 % FBS. Primary human melanocytes were purchased from Thermo Fisher (#C1025C, Waltham, MA). These cells were cultured in Medium254 (M254) supplemented with Human Melanocyte Growth Supplement (HMGS) (all from Thermo Fisher). For the experiments, melanocytes were used at passages from 2 to 5 and maintained in F12 medium supplemented with 10% fetal bovine, 12 μg/mL 3-isobutyl-1-methylxanthine, 40 nM 12-O-tetradecanoyl phorbol 13- acetate (TPA), 0.6 ng/mL basic fibroblast growth factor (bFGF), and 0.05 μg/mL cholera toxin (all from Sigma-Aldrich). Primary melanocytes were seeded at the bottom of 6-well plate and fibroblasts were seeded into inserted Transwell chambers(SPL Life Sciences Co., Korea) in each case. After 48 h, the Transwell chambers were translocated into melanocytes seeded into 6-well plates and maintained for 5days.
In vitro Model of Senescent Fibroblasts
Primary fibroblasts were prepared and maintained in DMEM (Gibco-BRL) supplemented with 10 % FBS (Gibco-BRL). For senescence induction, fibroblasts were treated with 8-methoxypsoralen (8-MOP) at 25 ng/ml with fresh media for 16 h. The cells
were then irradiated with 5 J/cm2 UVA (wavelength 320-400 nm, maximum peak 350 nm) using a LZC-1 photoreactor system (Luzchem Research Inc. Ontario) and maintained in DMEM for 7 days.
Cell Viability Test
Normal human fibroblasts and senescent fibroblasts (1x105/well) were seeded in 24- well plates and after two days were treated with ABT263, ABT737 (0.5-2.5 μM), dasatinib (50-500 nM) combinated with quercetin (20 μM), Resveratrol (25-100 μM) and Ginkgo biloba extract (25-100 μg/ml) for three 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.
Apoptosis Assay
Normal human fibroblasts and senescent fibroblasts were treated with dasatinib (50-500 nM) combinated with quercetin (20 μM) and Resveratrol (25-100 μM). After three days, 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 FACSCanto™
II system (BD Biosciences, San Jose, CA).
Caspase 3/7 Activity Assay
Young or senescent fibroblasts were seeded in 24-well plates and after two days they were treated with ABT263, ABT737 (1-2.5 μM) for two days. Then, we collected cells from suspended culture media and analysed caspase 3/7 activity using a Synergy 2
luminometer (BioTek, Winooski, VT) according to the luciferase reporter assay system instructions (Promega, Madison, WI).
Melanin Content and Tyrosinase Activity
Melanocytes 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 495 nm.
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 SuperScriptTM III reverse transcriptase kit (Invitrogen, Waltham, MA). Quantitative real-time PCR was performed using SYBR Green SuperMix (Bio-Rad, Hercules, CA). The primer sequences used were as follows:
human MITF; 5′-AGAACAGCAACGCGCAAAGAAC-3′, 5′-
TGATGATCCGATTCACCAAATCTG-3′, human Tyrosinase; 5’-
CACCACTTGGCCTCAATTC-3’, 5′-AAAGCCAAACTTGCAGTTTCCAC-3′, and human 18S; 5′-CGGCTACCACATCCAAGGAA-3′, 5′-GCTGGAATTACCGCGGCT-3′.
Western Blot Analysis
Cells were lysed in RIPA buffer (1% NP-40, 150 mM NaCl, 10 mM Tris-HCl at pH 8.0, 1 mM EDTA) with a complete protease inhibitor (Sigma-Aldrich). The proteins were separated by SDS-polyacrylamide gel and transferred to PVDF membranes (Millipore, Billerica, MA). The antibody against MITF was purchased from Abcam (Cambridge, UK), and the antibodies against tyrosinase was from Santa Cruz Biotechnology (Dallas, TX) and Actin was from Abclone (Woburn, MA).
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
Specificity of Senolytic Drugs for Senescent Fibroblasts of the Skin
Firstly, the efficacy of senolytic drugs on dermal senescent fibroblasts was screened.
Quercetin/dasatinib combination was not effective on dermal senescent fibroblasts. Rather the drug combination induced cell cycle arrest in dermal young fibroblasts (Figure 1).
When we treated resveratrol or gingko biloba extract to young and senescent fibroblasts, these drugs were not effective (Figure 2, 3). Moreover, resveratrol induced young fibroblasts apoptosis (Figure 3). However, interestingly, ABT263 and ABT737, which were well known Bcl2 inhibitors (Tse, C. et al., 2008; Oltersdorf, T. et al., 2005), could induce specific apoptosis of senescent fibroblasts depending on drug concentration (Figure 4). The cell number was decreased by ABT263 or ABT737 treatment in senescent fibroblasts. Furthermore, caspase 3/7 activity was increased after treatment of ABT263 or ABT737 in senescent fibroblasts but not in young fibroblasts (Figure 4D). These data suggested that not all senolytic drugs but ABT263 and ABT737 could induce apoptosis of senescent fibroblasts and can be used as potential drugs for skin whitening.
(A)
(B) (C)
(D)
Figure 1. Effect of Quercetin/dasatinib Combination on Skin Senescent Fibroblasts. (A) Old fibroblasts and young fibroblasts were treated with the indicated concentrations of quercetin/dasatinib combination for three days. (B) Cell counting. (C) MTT assay. (D) FACS
(A)
(B) (C)
Figure 2. Effect of Ginkgo Biloba Extract on Skin Senescent Fibroblasts. (A) Old fibroblasts and young fibroblasts were treated with the indicated ginkgo biloba extract for three days. (B) Cell counting. (C) MTT assay. Bar = 100 μm.
(A)
(B) (C)
(D)
Figure 3. Effect of Resveratrol on Skin Senescent Fibroblasts. (A) Old fibroblasts and young fibroblasts were treated with the indicated resveratrol for three days. (B) Cell counting. (C) MTT assay. (D) FACS analysis. Percentages indicated the sub-G1 fraction. Bar = 100 μm.
(A)
(B)
(C)
(D)
Figure 4. Effect of ABT263/ABT737 on Skin Senescent Fibroblasts. (A) Old fibroblasts and young fibroblasts were treated with the indicated concentrations of ABT263/ABT737 for three days.
(B) Cell counting. (C) MTT assay. (D) Caspase 3/7 activity of ABT263(left), ABT737(right). 12 h (upper), 24 h (bottom). Bar = 100 μm. Data are presented as the mean ±SD (**p < 0.01 vs. Cont,
#p < 0.001 vs. Cont).
ABT263/ABT737 Decreased Skin Pigmentation in vitro
To investigate the ABT263 and ABT737 as potential possible drugs for improving skin pigmentation, we investigated the effectiveness of ABT263 and ABT737 on primary human melanocytes. The number of cells and MTT assay data showed that these drugs did not influence on human melanocytes viability (Figure 5A, 5B). Furthermore, the expression level of melanin synthesis related genes such as MITF and tyrosinase in melanocytes was not significantly changed by ABT263 or ABT737 treatment in vitro (Figure 5C). These data showed that senolytic drugs ABT263 and ABT737 did not influence on primary melanocytes. Next, we evaluated ABT263 and ABT737 as potential drugs for improving skin pigmentation. We established co-culture system using primary melanocytes and senescent fibroblasts (Figure 7A). UVA-induced senescent (UVAiS) fibroblasts were established and the melanocytes were co-cultured with senescent fibroblasts using trans-well plates (Figure 6). The melanin content and tyrosinase activity in primary melanocytes were significantly increased cultured with senescent fibroblasts compared to a control (sham-irradiated fibroblasts) which was consistent with previous report (Kim et al., 2020). In addition, the mRNA and protein expression levels of MITF and tyrosinase were significantly upregulated (Figure 7, 8). Melanocyte proliferation was unaffected by the senescent fibroblasts (Figure 9). The effect of senolytic drugs ABT263 or ABT737 on senescent fibroblasts in pigmentation was further evaluated using trans-well study. The melanin content and tyrosinase activity were significantly down regulated with senolytic drugs treatment (Figure 10). These data suggest that ABT263 and ABT737 are potential drugs for improving skin pigmentation
(A)
(B)
(C)
Figure 5. Effect of ABT263/ABT737 in Melanocyte. (A) Human melanocytes were treated ABT263/ABT737 for three days and cell counting. (B) MTT assay of ABT263(left) and ABT737(right). (C) Real-time PCR analysis of MITF, tyrosinase (TYR) mRNA levels. Bar = 100 μm. N.S. indicates no significance.
Figure 6. UVA-induced Senescent (UVAiS) Fibroblasts Model. Primary human fibroblasts were irradiated UVA (5 J/𝑐𝑚2) and cultured for seven days. Cellular senescence was analyzed according to Senescence-associated beta-galactosidase (SA-β-Gal) staining and the number of positive fibroblast (arrow) was presented as a percentage(bottom). Data are presented as the mean ±SD (#p
< 0.001 vs. Cont, p value, t-test)
(A)
(B)
(C)
Figure 7. ABT263 reduces Skin Pigmentation in vitro. (A) Scheme of the co-culture model.
Melanocytes were co-cultured with young (Sham) or senescent fibroblasts (UVA). (B) The mRNA expression levels of MITF, tyrosinase (TYR) treated ABT263 for three days were analyzed by real- time PCR. (C) Protein expression levels. Bar = 100 μm. Data are presented as the mean ±SD (p value, t-test)
(A)
(B) (C)
Figure 8. ABT737 reduces Skin Pigmentation in vitro. (A) Melanocytes were co-cultured with young (Sham) or senescent fibroblasts (UVA) treated ABT737 for three days. (B) The mRNA expression level of MITF was analyzed by real-time PCR. (C) Protein expression levels. Bar = 100 μm. Data are presented as the mean ±SD (p value, t-test)
Figure 9. Effect of UVA on Melanocyte Proliferation. Melanocytes were co-cultured with young (sham) or senescent fibroblasts (UVA) for three days. Cell counting of melanocytes.
(A)
(B)
Figure 10. ABT263/ABT737 reduce Skin Pigmentation in vitro. (A) Melanin content (left) and TYR activity (right) treated ABT263 for five days were measured. (B) Melanin content treated ABT737 for five days were measured. Data are presented as the mean ±SD (p value, t-test)
The Elimination of Senescent Fibroblasts Induces Skin Lightening
To further investigate the impact of ABT263 on aging-related pigmentation, we applied ABT263 in aging related pigmented skin for targeting of senescent cells. Biopsied skins of the elderly with increased pigmentation were ex vivo cultured with ABT263 for three days and the skin samples were analyzed (Figure 11A). The elimination of the senescent fibroblasts by ABT263 treatment was accompanied by skin lightening. The Fontana- Masson staining showed a significant decrease in epidermal pigmentation by ABT263 treatment (Figure 11B). These findings suggest that aging related pigmented skin can be improved by removing senescent fibroblasts by senolytic drugs.
(A)
(B)
Figure 11. ABT263 decrease Skin Pigmentation ex vivo. (A) Ex vivo culture model. (B) Pigmentation of ex vivo human skin visualized with Fontana-Masson staining. Bar = 100 μm. The pigmented area/epidermal area (PA/EA) ratio was measured by an image analysis.
IV. DISCUSSION & CONCLUSION
Recently, eliminating senescent cells is an emerging issue in suppressing aging process of the skin. Based on the previous research, senescent fibroblasts induce hyper- pigmentation of the aged skin. Efforts to remove senescent these fibroblasts are being proposed from several research fields. For instance, senescent cells can be removed by radiofrequency, or specific peptides (Kim et al., 2018; Zhu, Y. et al., 2015). It is possible to kill senescent cells in direct manner using radiofrequency, however, this method is quite invasive to be applied to patients. Furthermore, in the case of using the peptide, it is not fully effective to pass through the skin barrier of keratinocytes. Considering the safety and efficiency, senolytic drugs take advantages.
There are several kinds of senolytic drugs being developed. However, senolytic drugs have different effects depending on the cell types. It was revealed that dasatinib and quercetin, which are most popular senolytic drugs, did not show enough effect on senescent fibroblasts, Rather, cell proliferation was inhibited even in young fibroblasts by G1 cell cycle arrest. Furthermore, in the previous study, dasatinib induced melanogenesis in melanocytes (Kang et al., 2020). When dasatinib and quercetin were treated, apoptosis of senescent fibroblasts was not induced. Rather, resveratrol induces the death of young fibroblasts (Min et al., 2013). In contrast, ABT263 and ABT737 had less effects on young fibroblasts and killed senescent fibroblasts efficiently. These drugs are a Bcl2 inhibitor, which inhibits apoptosis. The data showed that ABT drugs increase the activity of caspase3/7 (Kobbe, C., 2019; Tse, C. et al., 2008; Kline MP et al., 2007). Moreover, ABT- type drugs have no effect on melanocyte, effective pigmentation inhibitory effect can be achieved. In conclusion, ABT-type drugs, especially ABT263, remove senescent fibroblast and improve skin pigmentation without using invasive method. Furthermore, it was confirmed that it can be used in actual skin pigmentation lesion. In conclusion, ABT263 and ABT737 are both effective senolytic drugs which can improve age-related skin hyper pigmentation by selective killing of senescent fibrblasts.
V. REFERENCES
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Br J Dermatol 2010; 163: 1020-7.
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국문요약
피부 노화 섬유아세포에서 특이 세놀리틱스 효능 및 항노화 미백 효능
피부의 노화 과정은 색소 체계를 변형시키는 중요한 외적 요인이다. 세포 노화가 근본 적인 노화 메커니즘이다. 노화 섬유아세포들이 노화 관련 색소 침착 부위에 축적되고 노화 중에 간질 상피 상호 작용을 통해 멜라닌세포 분화를 변화시킨다. 세놀리틱스 약 물은 노화성에서는 세포 사멸을 유발할 수 있지만 비노화성 세포에서는 그렇지 않다.
세놀리틱스 약물로 잘 알려진 ABT263과 ABT737은 노화성이 많은 조직에 축적되며 노 화성 세포에 사멸을 유도할 수 있다. ABT263과 ABT737은 멜라노사이트에서 멜라닌 합 성에 관여하지 않았지만, 노화 섬유아세포 사멸의 조절하면서 멜라노사이트의 색소 침 착을 감소시켰다. 우리는 다양한 세놀리틱스 약물 중에 특히 ABT263과 ABT737이 노화 섬유아세포에서 사멸을 유도하고 피부 색소를 조절하는 것을 밝혀내었다.
핵심어: 세놀리틱스 약물, 피부 색소 침착, 노화, 노화 섬유아세포
