ABSTRACT
Objective To evaluate effects of camptothecin on the autophagy of HaCaT cells.Methods Some cultured HaCaT cells were divided into several groups to be treated with camptothecin at concentrations of 5,10,25,50,100 and 200 nmol/L,and 0.1％ dimethyl sulfoxide (DMSO) (control group),respectively.Cell counting kit-8 (CCK-8) assay was conducted to estimate the proliferative activity of HaCaT cells after 24-and 48-hour treatment,flow cytometry to evaluate cell apoptosis after 24-hour treatment,and Western blot analysis to measure the expression of autophagy-related proteins microtubuleassociated protein 1 light chain 3 (LC3) and p62.Some HaCaT cells were divided into 2 groups to be treated with 10 nmol/L camptothecin and 0.1％ DMSO for 24 hours,respectively.Then,indirect immunofluorescence assay (IFA) was performed to determine the LC3 expression.Results Camptothecin at low concentrations of 5 and 10 nmol/L had no significant effects on the proliferation and apoptosis of HaCaT cells.Compared with the control group,the cellular proliferative rates were significantly inhibited by (31.23 ± 1.00)％,(54.21 ± 8.10)％ and (66.75 ± 10.70)％ in the 50-,100-and 200-nmol/L camptothecin groups after 24-hour treatment respectively,and by (25.81 ± 5.99)％,(44.35 ± 5.32)％,(65.81 ± 8.28)％ and (73.23 ± 9.59)％ in the 25-,50-,100-and 200-nmol/L camptothecin groups after 48-hour treatment respectively (all P ＜ 0.001).After 24-hour treatment,the apoptosis rates were significantly higher in the 50-,100-and 200-nnol/L camptothecin groups (14.46％ ± 2.38％,19.15％ ± 1.59％,29.88％ ± 1.37％,respectively) than in the control group (3.80％ ± 0.13％,all P ＜ 0.001).After 24-hour treatment with 5 and 10 nmol/L camptothecin,the protein expression of LC3 Ⅱ was significantly up-regulated,while p62 protein expression was significantly down-regulated:IFA showed that the percentage of autophagosome-positive cells was significantly higher in the 10-nmol/L camptothecin group than in the control group after 24-hour treatment (36.67％ ± 4.55％ vs.6.23％ ± 0.92％,t =6.546,P =0.003).Conclusions Camptothecin at low concentrations of 5 and 10 nmol/L can induce autophagy of HaCaT cells,but has no obvious effects on cell proliferation and apoptosis.Camptothecin at concentrations of 50,100 and 200 nmol/L can inhibit cell proliferation,promote cell apoptosis,and decrease autophagy levels.
ABSTRACT
Objective To estimate the effects of camptothecin (CPT) on the expression of hypoxia-inducible factor-1α (HIF-1α) in HaCaT cells under hypoxic conditions (2％ O2),and to explore the potential therapeutic mechanism of topical CPT for psoriasis.Methods Some HaCaT cells were classified into 6 groups:5 test groups cultured in Dulbecco's modified Eagle's medium (DMEM) with the presence of CPT at 12.5,25,50,100 and 200 nmol/L respectively,and 1 control group cultured in DMEM with the presence of dimethyl sulfoxide (DMSO).All the 6 groups of cells were cultured under normoxic conditions for 12,24,48 or 72 hours or under hypoxic conditions for 12 hours.Cell counting kit-8 (CCK-8) assay was conducted to estimate the proliferation of HaCaT cells after the normoxic culture,and Western blot to quantify the protein expression of HIF-1α after the hypoxic culture.Some HaCaT cells were classified into a normoxia group (21％ O2) and a hypoxia group (2％ O2),and each group was divided into a CPT (100 nmol/L)-treated subgroup and a non-intervention subgroup (treated with the vehicle).After 12-hour culture,real-time fluorescencebased quantitative PCR was performed to measure the mRNA expression of HIF-1α.Statistical analysis was carried out by using Levene'.s test,one-way analysis of variance,Dunnett-t test and factorial analysis with the SPSS16.0 software.Results After treatment with CPT at 12.5-200 nmol/L for 12-72 hours,the proliferation of HaCaT cells was inhibited in a concentration-and time-dependent manner.More concretely,the cell proliferation rates were inhibited by 17.66％ ± 6.46％,33.11％ ± 4.63％ and 56.31％ ± 1.69％ respectively in HaCaT cells after 12-hour treatment with 200 nmol/L CPT as well as 24-hour treatment with 100 and 200 nmol/L CPT compared with the control group at the corresponding time points (all P ＜ 0.05).The protein expression level of HIF-1 α was significantly decreased in HaCaT cells after 12-hour treatment with CPT at 12.5,25,50,100 and 200 nmol/L under hypoxic conditions compared with the control group (0.348 ± 0.065,0.261 ± 0.112,0.115 ± 0.043,0.045 ± 0.024 vs.1.445 ± 0.329,all P＜ 0.05).The mRNA expression level of HIF-1α (expressed as △Ct) in the CPT-treated subgroup and non-intervention subgroup was-5.575 ± 0.29 and -5.451 ± 0.21 respectively in the normoxia group,significantly higher than that in the hypoxia group (-6.543 ± 0.57 and -6.203 ± 0.31 respectively,F =29.856,P ＜ 0.05),while there was no significant difference between the CPT-treated and non-intervention subgroups (F =1.667,P ＞ 0.05).Conclusions CPT at 100 nmol/L could inhibit the expression of HIF-1α protein,but had no obvious effect on that of HIF-1α mRNA.