Effects of cucurbitacin I on in vitro proliferation of HaCaT cells and the expression of keratin 17, signal transducer and activator of transcription 3 and vascular endothelial growth factor

Abstract

Objective To evaluate effects of cucurbitacin I on in vitro proliferation of HaCaT cells and the expression of keratin 17 (K17) , signal transducer and activator of transcription 3 (STAT3) and vascular endothelial growth factor (VEGF) in cultured HaCaT cells. Methods In vitro cultured HaCaT cells were divided into 6 groups to be treated with cucurbitacin I at different concentrations of 0.0125, 0.025, 0.05 and 0.1 μmol/L (0.0125, 0.025, 0.05 and 0.1 μmol/L cucurbitacin I groups) , DMEM containing the same volume of DMSO as 0.1 μmol/L cucurbitacin I (DMSO group) , DMEM (negative control group) and 10 nmol/L calcipotriol (positive control group) , respectively. Cell counting kit-8 (CCK8) assay was performed to assess in vitro cellular proliferative activity after 12-, 24-, 36-hour treatment, reverse transcription (RT) -PCR to measure the mRNA expression of K17 and VEGF in HaCaT cells after 24-hour treatment, and Western blot analysis to determine the protein expression of K17, STAT3, phosphorylated-STAT3 (p-STAT3) and VEGF after 24-hour treatment. Statistical analysis was carried out by one-way analysis of variance (ANOVA) , repeated measures ANOVA, Student-Newman-Keuls (SNK) -q test and Pearson correlation analysis. Results The proliferative activity of HaCaT cells started to decrease after 12-hour treatment with cucurbitacin I at the concentration of 0.0125 μmol/L. When the concentration of cucurbitacin I increased up to 0.1 μmol/L, the cell proliferation rates were inhibited by 43.00% ± 2.11% and 48.98% ± 2.27% after 24-and 36-hour treatment respectively. Compared with the negative control group, cucurbitacin I at different concentrations all could inhibit in vitro proliferation of HaCaT cells (all P < 0.05) , and the inhibitory effects increased gradually with the increase of cucurbitacin I concentration and treatment duration. After 24-hour treatment, the mRNA expression of K17 and VEGF and the protein expression of K17, VEGF and P-STAT3 were significantly decreased along with the increase of concentration of cucurbitacin I (all P < 0.05) . Conclusion Cucurbitacin I can inhibit in vitro proliferation of HaCaT cells, and down-regulate the mRNA expression of K17 and VEGF and protein expression of K17, VEGF and P-STAT3. Key words: Cucurbitacins; Keratin-17; STAT3 transcription factor; Vascular endothelial growth factors; HaCaT cells