Antiproliferative Effects of Crocin in HepG2 Cells by Telomerase Inhibition and hTERT Down-Regulation

Abstract

Crocin, the main pigment of Crocus sativus L., has been shown to have antiproliferative effects on cancercells, but the involved mechanisms are only poor understood. This study focused on probable effect of crocin onthe immortality of hepatic cancer cells. Cytotoxicity of crocin (IC50 3 mg/ml) in hepatocarcinoma HepG2 cellswas determined after 48 h by neutral red uptake assay and MTT test. Immortality was investigated throughquantification of relative telomerase activity with a quantitative real-time PCR-based telomerase repeatamplification protocol (qTRAP). Telomerase activity in 0.5 μg protein extract of HepG2 cells treated with 3mg/ml crocin was reduced to about 51% as compared to untreated control cells. Two mechanisms of inhibition,i.e. interaction of crocin with telomeric quadruplex sequences and down regulation of hTERT expression, wereexamined using FRET analysis to measure melting temperature of a synthetic telomeric oligonucleotide in thepresence of crocin and quantitative real-time RT-PCR, respectively. No significant changes were observed in theTm telomeric oligonucleotides, while the relative expression level of the catalytic subunit of telomerase (hTERT)gene showed a 60% decrease as compared to untreated control cells. In conclusion, telomerase activity of HepG2cells decreases after treatment with crocin, which is probably caused by down-regulation of the expression ofthe catalytic subunit of the enzyme.