Effects of camptothecin on the expression of hypoxia-inducible factor-1α in HaCaT cells / 中华皮肤科杂志

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 groups5 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.