Effects of camptothecin on autophagy of human primary keratinocytes
10.3760/cma.j.issn.0412-4030.2018.07.007
- VernacularTitle:喜树碱对人原代角质形成细胞自噬的影响
- Author:
Yangyang HAO
1
,
2
;
Liangyu ZHANG
;
Xiang WANG
;
Yunfeng TONG
;
Ying SUN
;
Yang CHEN
Author Information
1. 313000浙江湖州,解放军第九八医院皮肤科
2. 湖州市第一人民医院皮肤科,313000
- Keywords:
Psoriasis;
Camptothecin;
Autophagy;
Cell proliferation;
Apoptosis;
Keratinocytes
- From:
Chinese Journal of Dermatology
2018;51(7):510-514
- CountryChina
- Language:Chinese
-
Abstract:
Objective To evaluate the effect of camptothecin on the autophagy of human primary keratinocytes (HPKs).Methods HPKs were isolated from foreskin tissues of healthy males by a two-step digestion method,and the third-passage cells were used for following experiments.These HPKs were randomly divided into several groups:experimental groups treated with camptothecin at concentrations of 200 nmol/L,2 and 6 μ mol/L separately,and a control group treated with 0.1％ dimethyl sulfoxide (DMSO).After 24-and 48-hour treatment,cell counting kit-8 (CCK-8)assay was conducted to estimate the proliferative activity of HPKs.Flow cytometry was performed to detect cell apoptosis after 24-hour treatment,and Western blot analysis to measure the expression of autophagy-associated proteins such as microtubule-associated protein 1 light chain-3 (LC3) and p62.Some other HPKs were treated with 2 μmol/L camptothecin for 24 hours.Indirect immunofluorescence assay was performed to observe changes in LC3 expression,and transmission electron microscopy to observe the ultrastructure of autophagosomes,so as to further validate the inductive effect of camptothecin on autophagy.Results The inhibitory effect of camptothecin on the proliferation of HPKs gradually increased along with the increase of camptothecin concentration,and there was a significant difference in the proliferation inhibition rates among the experimental groups and control group at 24 hours (F =152.9,P ＜ 0.01).Additionally,the proliferation inhibition rates were significantly higher in the 2-,6-μmol/L camptothecin groups than in the control group (t =12.09,18.76,both P ＜ 0.01),but there was no significantly difference between the 200-μmol/L camptothecin group and control group (t =2.24,P ＞ 0.05).At 48 hours,there was still a significant difference in the proliferation inhibition rates among the experimental groups and control group (F =123.8,P ＜ 0.01),and all the experimental groups showed increased proliferation inhibition rates compared with the control group (all P ＜ 0.01).At 24 hours,the cell apoptosis rates also significantly differed among the control group,200-nmoYL,2-μmol/L and 6-μmol/L camptothecin groups (2.30％ ± 1.68％,15.90％ ±2.14％,29.33％ ± 3.51％,35.28％ ± 3.05％,respectively;F =89.57,P ＜ 0.01),and all the three experimental groups showed higher cell apoptosis rates compared with the control group (all P ＜ 0.01).After 24-hour treatment with 2 or 6 μmol/L camptothecin,the protein expression of LC3]Ⅱ were significantly up-regulated,but the protein expression of p62 was significantly down-regulated.Indirect immunofluorescence assay showed that the percentage of autophagosome-positive cells was significantly higher in the 2-μmol/L camptothecin group than in the control group (60.16％ ± 8.78％ vs.38.96％ ±13.12％,t =3.003,P ＜ 0.05).After 24-hour treatment with 2 μmol/L camptothecin,autophagosomes and autolysosomes were observed in HPKs with a transmission electron microscope.Conclusion Camptothecin at concentrations of 2 and 6 μmol/L can increase the autophagy level in HPKs,meanwhile,inhibit cell proliferation and induce cell apoptosis.
