ORIGINAL RESEARCH ARTICLES Crocin Triggers the Apoptosis Through Increasing the Bax/Bcl-2 Ratio and Caspase Activation in Human Gastric Adenocarcinoma, AGS, Cells Reyhane Hoshyar, 1 S. Zahra Bathaie, 1 and Majid Sadeghizadeh 2 We previously showed the anticancer property of crocin, a carotenoid isolated and purified from saffron against chemical-induced gastric and breast cancer in rats. In this study, the mechanism of crocin action was investigated in the gastric adenocarcinoma (AGS) cells in comparison with human normal fibroblast skin cells (HFSF-PI3). Crocin revealed a dose- and time-dependent cytotoxic effect against an AGS cell line, as determined by 3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Crocin-induced apoptosis was evidenced by flow cytometry and measuring caspase activity. The increased sub-G1 population and activated caspases in the treated AGS cells confirmed its anticancer effect. Expression of both Bax and Bcl-2 was determined using a semiquantitative reverse transcriptase–polymerase chain reaction and Western blot in these cells before and after treatment with crocin. Apoptosis was significantly stimulated as indicated by increasing the Bax/Bcl-2 ratio after crocin treatment. All of the above-mentioned parameters remained normal in HFSF-PI3 treated with crocin. These data are providing insight into the molecular mechanisms underlying the crocin-induced apoptosis in the AGS cells, rendering it as the potential anticancer agent. Introduction G astric cancer, a malignant epithelial tumor, origina- tes from the glandular epithelium of the gastric mu- cosa. Current therapies for gastric cancer consist of surgery, chemotherapy, and radiation (Watanabe et al., 2010). How- ever, these therapies are often ineffective. Therefore, the de- sign of more effective drugs with fewer side effects or complementary treatment to enhance the quality of life of these patients is essential. In the last two decades, botanicals with effective anticancer activity have been studied (Garodia et al., 2007). Saffron, Crocus Sativus L., is a plant of the iris family (Iridaceae); the stigmas of its flower contain various chemical constituents, including carotenoids, anthocyanin, monoterpene aldehydes, and flavonoids (Abdullaev and Espinosa-Aguirre, 2004). Saffron has shown therapeutic ef- fects in various diseases that have been extensively reviewed by us (Bathaie and Mousavi, 2010; Mousavi and Bathaie, 2011). Crocin, the main constituent of saffron extract, is one of the few water-soluble carotenoids in nature. Crocin acts as an antioxidant by removing free radicals that lead to protect cells and tissues against oxidants (Soeda et al., 2007). This component not only possesses the antitumor activity, but also has been used as a chemopreventive compound in both in vivo and in vitro conditions (Escribano et al., 1996; Zhao et al., 2008; Sajjadi et al., 2011). Recently, our team has investigated the mechanism of crocin action in the term of its interaction with high- molecular-weight DNA, different oligonucleotides (Bathaie et al., 2007; Hoshyar et al., 2008; Hoshyar et al., 2012), his- tone H1, and H1–DNA complex (as a model of chromatin) (Ashrafi et al., 2005). Our results showed that crocin bound to DNA through the minor groove and induced the B-to-C- DNA transition, which was accompanied by DNA precip- itation at higher concentrations of crocin (Bathaie et al., 2007). In addition, the great potential of this ligand to treat the rat models of both chemical-induced breast and gastric cancers was shown in our laboratory [manuscripts under preparation]. However, the exact mechanism of crocin ac- tion is not known, yet. Furthermore, according to our lit- erature review, the effect of crocin on human gastric cancer cell lines has not been evaluated, yet, so that the aim of the present study was to investigate the inhibitory effect of crocin on the proliferation and induction of apoptosis in the human adenocarcinoma gastric cell line, AGS, in compari- son with the normal human fibroblast cells HFSF-PI3. In addition, the molecular mechanism of crocin action was investigated. Therefore, the impact of crocin on Bax 1 Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran. 2 Department of Genetics, Faculty of Bio Sciences, Tarbiat Modares University, Tehran, Iran. DNA AND CELL BIOLOGY Volume 32, Number 2, 2013 ª Mary Ann Liebert, Inc. Pp. 50–57 DOI: 10.1089/dna.2012.1866 50