Page 25 - A. Aging

Senolytic drug improve the skin pigmentation

1,2
1
1
Ji Hee Park , Jung Eun Yoon , Young-Kyoung Lee and Tae Jun Park 1,2,3
1 Department of Biochemistry and Molecular Biology, Ajou University School of Medicine;
2 Department of Biomedical Sciences, Ajou University Graduate School of Medicine;
3 Institute on Aging, Ajou University Medical Center, Suwon, 16499, Korea
BACKGROUND AIM
Cutaneous aging process is an important extrinsic factor which modifies the pigmentary Even in the skin pigment fields, the fact that senescent fibroblasts induce
system. Given that cellular senescence is a fundamental aging mechanism, we examined pigmentation is considered an important phenomenon. Therefore, the technology to
the role of senescent cells in aging pigmentation and found that senescent fibroblasts remove senescent fibroblasts has been thought to be an important issue in the field
accumulate at the sites of age-related pigmentation and alter melanocyte differentiation of skin pigmentation. We expected that senolytic drug, especially ABT263, induce
via stromal-epithelial interactions during aging. Senolytic drugs can induce apoptosis in apoptosis in senescent cell and that regulate the skin pigmentation.
senescent, but not in non-senescent cells. ABT263, one of the senolytic drug, it can induce
apoptosis in senescent cells which accumulate in many tissues that aging.
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 (M253) 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 48h, 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-methoxy psoralen (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.
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 SuperScript TM 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′.
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.
RESULTS
Figure 1. DMSO ABT263 2.5μM ABT737 2.5 μM

2 1.5
Sham 1.5 1
Sham Cell counting (Fold induction vs. DMSO) 0.5 1 PUVA Cell counting (Fold induction vs. DMSO) 0.5 ** **

0
DMSO ABT263 2.5μM ABT737 2.5μM N=3 0 DMSO ABT263 2.5μM ABT737 2.5μM N=3
PUVA (Cont) (Cont) (**p<0.05 vs. Cont)
Figure 1. ABT263 and ABT737, which were well known Bcl2 inhibitors could induced senescent
fibroblasts specific apoptosis concentration dependently.
Figure 2. Figure 3.
DMSO ABT263 2.5 μM ABT737 2.5 μM A B Sham PUVA
120 90 **
Melanocyte Sham, PUVA SA-β-Gal SA-β-Gal + (%) 60
30
N=3
Primary melanocytes 8% 85% 0
2 C Sham+DMSO Sham+ABT263 PUVA+DMSO PUVA+ABT263 Sham PUVA
(Fold induction vs. DMSO)
Cell counting 1

0 N=4
DMSO ABT263 2.5μM ABT737 2.5μM
Figure 2. The cells number data showed that ABT263 and ABT737 were not influence P=0.01 P=0.004 P=0.03 P=0.007
on human melanocytes viability 4 7
CONCLUSION REFERENCES 3 6 5
Recently, the studies of killing senescent Hall BM, Balan V, Gleiberman AS et al. Aging of MITF mRNA expression (Fold induction vs. Cont) 2 TYR mRNA expression (Fold induction vs. Cont) 4
cells is emerging as a major issue in mice is associated with p16(Ink4a)- and 3
beta-galactosidase-positive macrophage
suppressing aging. It has been reported accumulation that can be induced in young mice 1 2
that when p16 INK4A positive senescent cells by senescent cells. Aging (Albany NY) 2016; 8: 1
were eliminated from an old mouse, the 1294– 315. 0 N=4 0 N=4
Yoon JE, Kim Y, Kwon S, Kim M, Kim YH, Kim JH,
lifespan increased and the overall aging Park TJ, Kang HY. Senescent fibroblasts drive aging Sham+DMSO(Cont) Sham+ABT263 Sham+DMSO(Cont) Sham+ABT263
phenomenon was suppressed (B.M. Hall., pigmentation: A potential therapeutic target for
2016). Even in the skin pigment fields, the senile lentigo. Theranostics. 2018;8(17):4620-32. PUVA+DMSO PUVA+ABT263 PUVA+DMSO PUVA+ABT263
Kim Y, Kang B, Kim JC, Park TJ, Kang HY, Senescent
fact that senescent fibroblasts induce fibroblast-derived growth differentiation factor 15 Figure 3. (A) co-culture system using primary melanocytes and senescent fibroblasts (B) UVA-induced senescent (UVAiS) fibroblasts
pigmentation is considered an important (GDF15) induces skin pigmentation, The Journal of (C) mRNA expression levels of MITF and tyrosinase
phenomenon. Therefore, the technology to Investigative Dermatology. 2020.
Kang B, Kim Y, Park TJ, Kang HY. Dasatinib, a
remove senescent fibroblasts has been second-generation tyrosine kinase inhibitor, ACKNOWLEDGEMENTS
thought to be an important issue in the induces melanogenesis via ERK-CREB-MITF-
tyrosinase signaling in normal human melanocytes.
field of skin pigmentation. Biochem Biophys Res Commun. 2020 Mar This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health industry
19;523(4):1034-1039. Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HN14C0094).