In the present study, we have shown that combinatorial treatment with low doses of I3C and silibinin afforded remarkable cancer preventive activities in vitro and in vivo. Exposure of A549 cells and H460 cells to lower concentrations of I3C and silibinin caused strong antiproliferative and apoptotic effects, whereas treatment with I3C or silibinin alone did not show any activity. Similarly, in tumor bioassays with A/J mice, NNK-induced lung tumors were more effectively inhibited by I3C plus silibinin than by the single agent.
In our earlier studies, using NNK plus benzo[a
]pyrene models of lung tumorigenesis, we showed that dietary administration of I3C (112 μmol/g diet) to chemically treated A/J mice dramatically reduced the multiplicity of lung tumors by ~85% (19
). Upon extending our studies to a vinyl carbamate model, we observed a complete abolition by I3C of larger tumors and adenocarcinoma (20
). Despite these strong chemopreventive activities of I3C, mice fed on this compound had consistently lower body weight (~10% reduction) although food consumption was not affected. This could be a problem for the development of I3C as a chemopreventive agent since promising agents should be free from adverse effects. To overcome this problem, we have been testing, using in vitro
models, the antiproliferative and apoptotic effects of combinations of I3C and other chemopreventive agents (isothiocyanates, myo-inositol, inositol hexaphosphate, deguelin, I3C and diindolylmethane). Of the different combinations, I3C plus silibinin showed the strongest growth suppression and apoptotic effects. Interestingly, the concentrations of I3C and silibinin in the mixture required to induce antiproliferative effects were ~10-fold and at least 2-fold lower, respectively, than the concentration of the individual agent required to induce the same effects. Nakamura et al.
) studied apoptosis induction by combinations of I3C and genistein in human colon cancer cells. Although combinatorial treatment caused synergistic effects, the concentration of I3C used in this assay was exceedingly higher (300 μM) than that used in our study. Generally, I3C possesses a poor biological activity under in vitro
test conditions. Previous studies showed that, irrespective of the cell line used, under in vitro
conditions, I3C induces significant antiproliferative and apoptotic effects in cancer cells only at concentrations exceeding 200 μM (26
). In the present study, although silibinin caused moderate inhibition of cell growth at a concentration of 50 μM, the same concentration failed to induce apoptosis. Indeed, earlier reports on the effect of silibinin against non-small cell lung cancer cell lines showed that the major biological effect of silibinin in non-small cell lung cancer cells is growth inhibition and that cell death is not the prime reason for the reduction in cell number (27
When extrapolated on the basis of body surface area, which is considered to be the best approach for the translation of doses from animals to humans (28
), the dose of I3C used in this study is comparable with the amount of I3C (800 mg/person, orally) given in a phase I trial and was found to be devoid of adverse effects (29
). However, it is not possible to compare the effective concentrations of I3C or its metabolites under in vitro
and in vivo
conditions as the compound is unstable and the breakdown products formed under in vitro
conditions are different qualitatively and quantitatively from those reported in studies with mice (13
). On the basis of an earlier report where administration of 0.1% silibinin to mice in the diet lead to a plasma silibinin level of 15–30 μM (31
), the levels of silibinin achievable in the plasma, from the silibinin dose given to the mice, are expected to be equivalent to or more than the silibinin concentration (50 μM) used for the combinatorial treatment in the in vitro
studies. However, silibinin at this concentration failed to modulate growth or survival of A549 and H460 cells, whereas it inhibited NNK-induced lung tumorigenesis. This could be related to differences in the biology of the target cells (full-blown cancer cells for in vitro
studies versus mouse lung cells at early stage of NNK-induced tumorigenesis) or the frequency of administration of the agent (short term therapy in in vitro
models versus chronic dosing in the animal study).
In non-small cell lung cancer as well as NNK-induced mouse lung tumors, activating K-RAS mutations are very common (32
). Activation of K-RAS leads to stimulation of the Raf/mitogen-activated ERK kinase/ERK and PI3-kinase/Akt pathways, which have many redundant functions in tumor growth and survival. Therefore, simultaneous inhibition of both pathways is considered to be critical for the effective treatment of lung cancer harboring K-RAS activation. Using a mouse lung cancer model driven by mutant K-RAS, Engelman et al.
) showed that lung tumors regress completely only when mice were treated with a combination of PI3K/mTOR inhibitor and mitogen-activated ERK kinase inhibitor. In the present study, we showed that combinatorial treatment with I3C and silibinin effectively abrogated activation of Akt and ERK in A549 and H460 cells as well as in mouse lung tumor tissues. In tumor tissues, we also observed reduction in the level of cyclin D1, a key regulator of G1
/S transition and a downstream effector of both Akt and ERK, and PARP cleavage, a marker for apoptosis. At this point, it is not clear if I3C and silibinin target separated pathways or have a dual effect on both Akt and ERK activation. However, the strong antiproliferative and apoptotic effects in A549 and H460 cells and the efficacy against NNK-induced lung adenocarcinoma in A/J mice by I3C plus silibinin could be related to modulation of Akt and ERK activation. However, like most naturally occurring agents, I3C and silibinin are known to affect several targets (12
), and, therefore, inhibition by the agents of other lung cancer-related signaling pathways could not be excluded.
In conclusion, in the present study, we showed the strong antitumor activities of low doses of a combination of I3C and silibinin in cell line and animal models of lung cancer. In the mouse study, combinatorial treatment did not cause any adverse effects, which is one of the desirable properties of promising chemopreventive agents. Moreover, the dose levels of I3C and silibinin used in the present study are similar to the amounts of the compounds used in human clinical trials. Therefore, our findings could be used as the basis for the clinical trial of a combination of I3C and silibinin for the prevention of lung cancer in current as well as former smokers.