Botanicals have provided parent structures for many of the current efficacious chemotherapeutic agents (25
). There needs to be a continued effort to tap this resource in order to discover active botanical components or metabolites with novel, potent, and distinct anticancer actions (27
). The identification of non-toxic chemo-adjuvant treatments derived from herbal medicines remains a potentially very productive area of research to advance cancer chemoprevention.
Previous ginseng anticancer evaluations largely focused on the herb’s parent compounds, i.e., the ginsenosides Rb1 and Rg1. Unfortunately these parent compounds have exhibited only limited anticancer activities. Our group observed that after steaming treatment, the chemical composition of ginseng was changed, and the antiproliferative activity increased significantly. Steaming of American ginseng augmented ginsenoside Rg3 content and increased the ginseng’s antiproliferative potential. Although Rg3 exhibited more antiproliferative activity than its parent compound Rb1, its potency was still low, with an IC50
> 100 μM for colorectal cancer cells (29
). Identification of more potent ginseng-derived anticancer compounds is challenging, as ginseng contains a diverse number of compounds and potential metabolites, and many appear to possess anticancer activities.
The common method of ginseng administration is the oral route. Orally administered ginsenosides are poorly absorbed, and some appear to require bacterial metabolism to be absorbed and biologically active (30
). After ginseng ingestion, C-K is a major metabolite reaching the systemic circulation (14
). In natural products research, many previous studies employed primarily reductionist approaches in screening compounds for bioactivity, and often only parent compounds were investigated. For ginseng studies, saponin bioavailability is an important issue linked to the compound’s pharmacological effects, and it seems to have been overlooked.
In this study, we observed that the Rb1 metabolite C-K possesses very significant anti-colorectal cancer activities compared to its parent compound Rb1. The IC50
for C-K for inhibiting colon cancer cell proliferation and inducing apoptosis was 30 μM – 50 μM, suggesting that its antiproliferative effect is greater than that of Rg3, the compound derived from Rb1 by steaming treatment. We also observed significant antitumor activity of C-K, but not of Rb1, against cancer cells, but not against non-cancer cells. The microbe requirements for generation of gut microbiota-derived ginseng metabolites with respect to colorectal cancer chemoprevention will necessitate further study. Functional profiles of enteric microbiota with this activity can be studied in a number of ways, including establishing “humanized” enteric microbiota or transfer of defined microbiota using germ-free animal models (31
The mechanisms mediating the colon cancer chemopreventive effects of C-K are largely unknown. Because inhibition of cell cycle progression and induction of apoptosis are important mechanisms mediating the effects of many anticancer agents, in this study we evaluated the effects of C-K on the cell cycle and apoptosis. C-K increased the fraction of colon cancer cells in the G1 phase, with SW480 cells showing greater G1 arrest. We also observed that C-K markedly induced colon cancer cell apoptosis, and that SW-480 cells again appeared to be more sensitive to C-K. Comparing C-K effects on cell cycle and apoptosis, we showed that induction of apoptosis appeared to be greater than cell cycle slowing. This result suggests that the cancer cell growth inhibitory effect of C-K was predominantly mediated by induction of apoptosis.
The two cell lines used in this study have varied p53 expression. HCT-116 is p53 wild type, while SW-480 cells contain a p53 mutation. Cancer cells with p53 mutations are resistant to many chemotherapeutic agents. Interestingly, we observed that C-K caused greater apoptosis induction abilities in a p53 mutant cell line (SW-480) than in a p53 wild type line (HCT-116), suggesting that C-K might prove useful in p53-mutated colon cancers.
To further explore the mechanism mediating C-K-induced enhanced apoptosis, we assayed the activities of caspases 3, 8 and 9. Since our flow cytometry experiments showed that the apoptotic effects of C-K were greater in SW-480 than HCT-116 cells, we examined SW-480 cells for caspase activity. At concentrations of 40 and 50 μM, C-K significantly up-regulated the activities of these caspases, especially caspases 8 and 9. Our docking analysis further suggested interaction sites between C-K and caspases 8 and 9. These caspases are situated at critical points in apoptotic pathways, and our studies suggest that C-K may enhance apoptosis by direct physical interactions with these enzymes.
In summary, we have demonstrated that C-K exerts potent antiproliferative and pro-apoptotic effects in colon cancer cells. This compound requires biotransformation of ginseng by bacteria, revealing a potentially important role for bacteria in mediating the chemopreventive effects of naturally occurring substances. Future studies to determine the pathways that mediate C-K-induced G1 cell cycle slowing and caspase activation are warranted. Investigation of the colonic microbiome requirements and elucidation of potential differences in human hosts (e.g., ethic differences) with respect to C- K generation will also be important. Finally, animal studies demonstrating that C-K exerts a chemopreventive effect in colonic tumorigenesis should be undertaken to assess the potential of this agent in future human trials.