Effect Of Camptothecin On The Expression Of HIF-1α And CCL20 In HaCaT Cell Under Hypoxia Inducible

Objective: To investigate the effects of Camptothecin (CPT) on proliferation, apoptosis in Keratinocyte HaCaT cells under normoxia; observed the expression of HIF-1α and CCL20 in HaCaT cells under Hypoxia by Camptothecin (CPT) at nanomolar concentration. Discuss the potentially therapeutic mechanism of topical CPT in treating psoriasis.Methods:①HaCaT cells were divided five groups and treated with different nanomolar (12.5,25,50,100,200nmol/L) of CPT for12h,24h,48h and 72h, cell growth were evaluated by CCK-8. After HaCaT cells were exposed to different nanomolar CPT for 24h, cell apoptosis was analyzed by flow cytometry, and Caspase-3, Bcl-2 proteins were detected by Western blot. ②The five groups were cultured in hypoxia for 12h, Western blot was used to detect hypoxia-inducible factor-la (HIF-la) protein and the secretion of CCL20 was detected by ELISA Kit. ③HaCaT cells were cultured respectively in normoxia and hypoxia condition for 12h without CPT treated, the CCL20mRNA expression was detected by Real-time PCR. HaCaT cells were divided into the normoxia-cultured groups(21%O2) and the hypoxia-cultured groups(2%O2), furthermore in each group the cells were divided into the CPT-treated(100nmol/L) group and the non-CPT-treated group (the vehicle control group), after cultured for 12h, the HIF-lamRAN expression was detected by Real-time PCR.④ Statistical analysis was done by Levene’s Test, ANOVA analysis, Dunnett-t test, SNK-q test, independent-t test and factorial analysis using the SPSS 16.0 software.Result:1. Within the investigated time ranges, CPT of 12.5,25,50,100,200nmol/L induced growth inhibition in the time- and concentration-dependent manners. Compared with the vehicle control cell, a significant decrease was observed in the proliferative activity in HaCaT cell treated with CPT of 200nmol/L for 12 hour and 100,200nmol/L for 24 hours (17.66±6.46,33.11%±4.63%,56.31%±1.69%, P<0.05). Flow cytometry analysis demonstrated both 100 and 200nmol/L CPT could induce apoptosis in 24 hour. Caspase-3 protein was up-regulated in a concentration dependent manner; on the other hand Bcl-2 protein was down-regulated.2.Hypoxia inducible for 12 hours, CPT of 25,50,100,200nmol/L significantly decreased HIF-la protein compared with the vehicle control group (0.348±0.065, 0.261±0.112,0.261±0.112 and 0.045±0.024 vsl.445±0.329, P<0.05). And CPT of 100, 200nmol/L significantly decreased the secretion of CCL20 (1×105cells) compared with the vehicle control group. (64.35±19.70)pg/ml、(74.35±23.85) pg/ml vs (112.18±28.66) pg/ml, P<0.05.3. There were significantly difference in the expression levels of CCL20mRNA (given in △ CT value)of between the normoxia group and the hypoxia group (-15.19±0.13 vs-13.70±0.10 t=-14.430,P=0.001). In the normoxia group (21%O2), The expression levels of HIF-lamRNA (given in △ CT value) of CPT-treated cell and non-CPT-treated cell were-5.575±0.29 and -5.451±0.21 respectively, In the hypoxia group (2%O2), The expression levels of HIF-lamRNA of CPT-treated cell and non-CPT-treated cell were-6.543±0.57 and -6.203±0.31 respectively. The HIF-lamRNA levels were significantly lower in hypoxia-treated cell than in normoxia-treated cell (F=29.856, P=0.000). whenever in normoxic or hypoxic state, there were no significantly difference in the expression levels of HIF-lamRNA between CPT-treated cell and non-CPT-treated cell (F=1.667, P=0.213)Conclusion:Low concentration of CPT can induce HaCaT Cell apoptosis, and inhibit cell proliferation in a time and concentration-dependent manner. Low concentration of CPT can reduce the expression of HIF-1α protein without interfering with HIF-1αmRNA level in HaCaT cell under Hypoxia Inducible; the expression level of CCL20mRNA is up-regulate in the hypoxia condition, CPT can reduce the secretion of CCL20 in a certain concentration.