Suppression of colon carcinoma cell growth by polyphenols

C54

[Aims] Flavonoids, including luteolin, are polyphenolic compounds and display a wide range of pharmacologic properties, including anti-inflammatory and anticancer effects. We have previously reported that an ethanol extract of Vitex agnus-castus fruits (Vitex) which include luteolin suppressed cell growth of several human cancer cell lines including colon carcinoma cell line (COLO 201) through apoptosis. In this study, we extended our previous study in order to characterize apoptosis pathways, and to conduct in vivo efficacy study of Vitex in a xenograft model. [Methods] COLO 201 cells were treated with Vitex for 48 h with or without several inhibitors including caspase inhibitors. DNA fragmentation rate was analyzed on an agarose gel electrophoresis. Levels of mRNA expression were detected by RT-PCR and fluorometric assay was performed for caspase activity. COLO 201 cells (5 x 10⁶ cells/0.2 ml of PBS) were injected once to 7-week old nude mice subcutaneously at scapulae. Mice were received intraperitoneal daily injections of Vitex (1 mg in PBS) for additional 6 weeks. Histological studies were also conducted. [Results] DNA fragmentation was observed from 24 h after Vitex-treatment. Vitex-induced apoptosis was not inhibited with an antioxidant, NAC. Vitex-induced apoptosis was inhibited pan-caspase inhibitor or JNK inhibitor, but not p38 inhibitor. Caspase-9 and caspase-3 were activated from 6 h and 12 h, respectivery, after Vitex-treatment but caspase-8 was not activated. Furthermore, endoplasmic reticulum (ER) stress related mRNA levels, including heme oxigenase (HO)-1, C/EBP homologous protein (CHOP) and glucose-regulated protein (GRP) 78 mRNA, were increased from 3 h after Vitex-treatment. Tumor of COLO 201 xenografts was growth without Vitex. However, tumor growth was suppressed with Vitex about 50% on tumor volume and weight after 7-week Vitex treatment. [Discussion] Vitex-induced apoptosis in COLO 201 cells was not mediated through oxidative stress. Vitex-treatment was induced caspase-9 and -3 activation, but not caspase-8, suggesting that this apoptosis was used for mitochondrial pathway, but not death receptor pathway. JNK activation, but not p38, was involved in the apoptosis induction. Results indicated ER-stress mediated apoptosis induction. Thus, these data suggested that Vitex was induced apoptosis in COLO 201 cells through JNK to mitochondria signaling cascade, which resulted in the induction of ER stress in vitro. In nude mice xenograft model, Vitex significantly suppressed the tumor growth, suggesting that Vitex display antitumor effect in vivo. It should be noted that V. agnus-castus fruits extract has been used as a folk medicine for the treatment of menstrual disorder and related hormone problems in European countries. It has also been clinically used for PMS treatment as well. Our in vitro and in vivo results together with clinical application results suggest that Vitex may have a potential as a new supplemental compound for colon cancer treatment that may improve QOL of patients by reducing adverse effects of current chemotherapy drugs.