Received: 12 May, 2010. Accepted: 7 July, 2011. Original Research Paper International Journal of Biomedical and Pharmaceutical Sciences ©2012 Global Science Books Ginsenoside Compound K Induces Cell Cycle Arrest and Apoptosis in Human Colon Cancer Cells Yun Ju Jeong 1* • Hyun Ju You 1 • Geun Eog Ji 1,2 1 Department of Food and Nutrition, Research Institute of Human Ecology, Seoul National University, Shillim-dong, Kwanak-ku, Seoul 151-742, Korea 2 Research Institute, BIFIDO Co., Ltd., 688-1, Sang-oan-ri, Hongcheon-gun, Gangwon-do 250-804, Korea Corresponding author: * geji@snu.ac.kr ABSTRACT Compound K (CK) is one of the principal metabolites of ginseng in human body which has been reported to exert diverse pharmaceutical activities including anticarcinogenic and antitumor effects on different lineages of cancer cells. However, the effect and mechanism of CK on colon cancer cells are not fully understood. In the present study the screening process was conducted with 12 different ginsenosides and metabolites, which showed CK was the most potent growth inhibitory compound against HT-29 colon cancer cells. The IC 50 value of CK was 12.7 M at 72 h. Cellular responses and growth pattern was analyzed simultaneously after CK treatment by Real Time Cellular Analysis (RTCA) method. CK treatment at specific concentration and time-point represented characteristic cytostatic stage in growth profile of HT-29 cells, and flow cytometric analysis showed that CK induced G 1 phase arrest in cell cycle distribution followed by apoptosis. The G 1 phase arrest was accompanied by down-regulation of cyclin D3, CDK6, and up-regulation of p21 WAF-1/CIP1 , and apoptosis was evidenced by inactivation of p-Bcl-2 and p-Akt. These results demonstrated that CK caused growth inhibition of HT-29 cells by blocking cells in G 1 phase and inducing apoptosis. _____________________________________________________________________________________________________________ Keywords: ginsenoside, G 1 arrest, CK, compound K,HT-29, Colon cancer Abbreviations: CDK, cyclin dependent kinase; CI, cell index; CK, compound K; PD, protopanaxadiol; PT, protopanaxatriol; RTCA, real time cell analysis INTRODUCTION Ginseng (the root of Panax ginseng C. A. MEYER; family Araliaceae) has been widely used as a traditional medicine and reported to have various pharmaceutical activities in- cluding hypoglycemic (Attele et al. 2002), hepatoprotective (Lee et al. 2005), antiallergic (Choo et al. 2003) and anti- carcinogenic effects (Choo et al. 2008; Park et al. 2009). Ginsenosides belonging to the dammarane triterpene sapo- nin in ginseng are the principal components responsible for these biological activities, and so far more than 30 ginseno- sides are identified from ginseng extract and its metabolites. Several studies have shown the structure-activity relationship of ginsenosides on diverse physiological acti- vities (Liu et al. 2003; Zhou et al. 2006). Ginsenosides are generally divided into two categories, protopanaxadiol (PD) and protopanaxatriol (PT), based on hydroxyl groups attached to the ginsenoside skeleton (Shibata 2001). PD- type ginsenosides such as Rg3, Rh2, Compound K (CK) and PPD have been reported to show potent antitumor effect resulting from apoptosis on several cancer cell lines (Rg3 against HT-29 human colon cancer cell line (Lee et al. 2009) and B16 murine melanoma cell line (Chen et al. 2008); Rh2 against Caco-2 colon cancer cell line (Popovich and Kitts 2004); CK against HL-60 human leukemia cell line (Cho et al. 2009) and PPD against human fibrosarcoma HT1080 cell line (Li et al. 2006)). Major ginsenosides with various sugar moieties such as Rb1, Rb2, Rc, Rd, Re and Rg1 comprise more than 80% of total ginsenosides (Son et al. 2008). However, ginsenoside metabolites with less sugar moiety had tendency to show greater cytotoxic and anti- tumor effect compared to the ginsenosides with more sugar residues (Zhou et al. 2008; Noh et al. 2009). Sugar moieties of ginsenosides are removed during fer- mentation, resulting in the conversion of glycones to agly- cones (Chi and Ji 2005; Chi et al. 2005). Absorption of gin- senosides in human body mainly takes place in the intes- tinal tract after microbial biotransformation of glycone gin- senosides such as Rb1, Rb2, Rc, Re and Rd to their meta- bolites such as Rh1, Rh2 and CK (Tawab et al. 2003). Since the intestinal microbial composition differ individually, the degree of absorption and biological effects of ginsenosides were considered different depending on the composition of the individual’s intestinal microbial flora (Tawab et al. 2003). In the lumen of colon, ingested ginsenosides are pre- sent as mixture of their intact forms and hydrolyzed forms. Therefore, we firstly screened 12 major ginsenosides in gin- seng and their metabolites together using colon cancer cell line and normal colon cell line to find out the most effective growth inhibitory compound against colon cancer cells and to investigate the structure-activity relationship between major ginsenosides and metabolites. CK is the minor ginsenoside in ginseng extract and metabolized by hydrolyzing sugar moieties of the major ginsenosides such as Rb1, Rb2, Rc and Rd in colonic envi- ronment. Among various ginsenosides, CK has received attention for its several pharmaceutical activities such as en- hancing insulin secretion (Han et al. 2007), hypoglycemic (Yoon et al. 2007), hepatoprotective (Lee et al. 2005), anti- pruritic (Shin and Kim 2005) and anticarcinogenic (Cho et al. 2009) effect. However, the mechanism of anticarcino- genic effect of CK in colon cancer cells is not fully under- stood. In the present study, by using non-invasive real-time cellular analysis (RTCA) method, we monitored the cellular status and growth pattern of HT-29 colon cancer cells simultaneously for 72 h after the treatment of different con- centrations of CK, and found out the characteristic pattern in growth profile at specific concentration and time point of CK treatment. Based on these findings including the screen- ing process which demonstrated that CK was the most ®