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Carcinogenesis. Apr 2011; 32(4): 561–567.
Published online Jan 27, 2011. doi:  10.1093/carcin/bgr010
PMCID: PMC3066421
Enhanced inhibition of lung adenocarcinoma by combinatorial treatment with indole-3-carbinol and silibinin in A/J mice
Abaineh Dagne,1 Tamene Melkamu,2 Melissa M. Schutten,1,2 Xuemin Qian,1 Pramod Upadhyaya,1 Xianghua Luo,1,3 and Fekadu Kassie1,2*
1Masonic Cancer Center
2College of Veterinary Medicine
3School of Public Health, University of Minnesota, Mayo Mail Code 806, 420 Delaware Street SE, Minnesota, MN 55455, USA
*To whom correspondence should be addressed. Tel: +1 612 625 9637; Fax: +1 612 626 5135; Email: kassi012/at/umn.edu
Received November 5, 2010; Revised December 23, 2010; Accepted January 10, 2011.
In earlier studies, we demonstrated the efficacy of indole-3-carbinol (I3C) against lung adenocarcinoma in A/J mice. However, these effects were accompanied by reductions in body weight gain. We therefore assessed if combinations of low doses of I3C with silibinin could inhibit lung tumorigenesis without causing undesirable side effects. In in vitro assays with A549 and H460 lung cancer cells, exposure of the cells to a mixture of low concentrations of I3C (50 μM) plus silibinin (50 μM) for 72 h caused inhibition of cell growth and extracellular signal-regulated kinase (ERK) and Akt activation and induction of apoptosis, whereas the individual agents did not have any effect. In mice pretreated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and given I3C (10 μmol/g diet) plus silibinin (7 μmol/g diet), multiplicities of tumors on the surface of the lung and adenocarcinoma were reduced by 60 and 95%, respectively. The individual effects of I3C and silibinin were relatively weaker: 43 and 36% reductions, respectively, in the multiplicity of tumors on the surface of the lung and 83 and 50% reductions, respectively, in the number of adenocarcinoma. Also, the expression of phospho-Akt, phospho-ERK and cyclin D1 and poly (ADP-ribose) polymerase cleavage were strongly modulated by I3C plus silibinin than by I3C or silibinin alone, suggesting that the chemopreventive activities of the mixture could be mediated, at least partly, via modulation of the level of these proteins. Taken together, our findings showed that mixtures of I3C and silibinin are more potent than the individual compounds for the chemoprevention of lung cancer in A/J mice.
Lung cancer is the leading cause of cancer death in the USA and worldwide (1), which indicates the urgent need to develop novel preventive and therapeutic approaches against this disease. One potential approach to reduce the mortality and morbidity of lung cancer is the use of chemopreventive agents. Chemoprevention has been found to be effective in populations at high risk for prostate, colon and breast cancers (24). Many of these strategies could be applicable for the prevention of lung cancer. In particular, the use of combinations of agents to target several pathways is highly warranted for the prevention of lung cancer owing to the multiple genetic and epigenetic changes induced by tobacco smoke (5,6), the main cause of lung cancer.
Accumulating data from in vitro and in vivo cancer models show that edible and medicinal plants contain cancer preventive compounds. Among these plant-derived anticancer agents are indole-3-carbinol (I3C) and silibinin. I3C is a glucobrassicin derivative present in commonly consumed cruciferous vegetables such as cabbage, cauliflower, broccoli and Brussels sprouts. It is released from cruciferous vegetables by the action of myrosinase, an enzyme present in these vegetables but released only when the plant tissue is damaged upon chewing or maceration (7). I3C inhibits tumorigenesis in a variety of animal cancer models, including cancers of the mammary gland, uterus, stomach, colon, lung and liver, by modulating carcinogen metabolism, inhibiting tumor cell proliferation, induction of apoptosis, inhibition of tumor angiogenesis and invasion (812). In the acidic milieu of the stomach, I3C undergoes condensation reactions to give rise to several products, the predominant one being 3,3-diindolylmethane (13). 3,3-Diindolylmethane is considered to be responsible for many of the physiological effects of I3C under in vivo conditions (14).
Silibinin is a polyphenolic flavonoid isolated from the milk thistle, Silybum marianum L. Gaertn (15). It exhibited antitumor activities in animal models of skin, prostate, lung, liver and colon cancer through cell cycle arrest, downregulation of anti-apoptotic gene products, inhibition of cell-survival kinases and inhibition of inflammatory transcription factors, invasion, angiogenesis and metastasis (1618). Silibinin is also a strong antioxidant, radical scavenger and a potent antihepatotoxic drug (15).
Although I3C possesses strong efficacy toward chemically induced lung tumorigenesis, these effects are associated with a reduction in body weight gain (19,20). Since chemopreventive agents should be not only effective but also free of adverse effects, we proposed a new approach to develop I3C as a lung cancer chemopreventive agent. We hypothesized that combinations of low doses of I3C and silibinin, a chemopreventive agent free of adverse effects, could effectively inhibit lung tumorigenesis without affecting body weight gain. We tested the hypothesis by assessing macroscopic and microscopic lung tumors, cell proliferation- and apoptosis-related proteins in the lung tissues of mice pretreated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and given in the diet I3C, silibinin or I3C plus silibinin. This study showed that combinatorial treatment with I3C and silibinin could be a promising approach for the prevention of lung cancer in current and former smokers.
Chemicals, reagents and diets
I3C and silibinin were from Sigma (St Louis, MO). NNK was synthesized as described (21). Anti-phospho-Akt, anti-total Akt, anti-phospho-extracellular signal-regulated kinase (ERK), anti-total ERK, anti-cyclin D1, anti-poly (ADP-ribose) polymerase (PARP), anti-β-actin and goat anti-rabbit IgG secondary antibody were from Cell Signaling Technology (Beverly, MA). Mouse diets (AIN-93G and AIN-93M) were purchased from Harlan Teklad (Madison, WI). The AIN-93G diet, high in protein and fat, was used to support rapid growth of the mice until the animals became mature adults (16 weeks old); thereafter, AIN-93G diet was replaced by AIN-93M diet, a low-protein and low-fat diet, which is recommended for adult maintenance (22). AIN-93 diets are standard diets for lung tumorigenesis studies in A/J mice.
Cells and cell culture
Human lung adenocarcinoma A549 cell line and human large-cell lung cancer cell line H460 were obtained from American Type Culture Collection (Rockville, MD). The cells were maintained in exponential growth as monolayers in RPMI 1640 medium supplemented with 1% penicillin–streptomycin and 10% fetal calf serum at 37°C, in a humidified atmosphere containing 5% CO2/95% air.
Cell proliferation assay
Cell growth was determined using the methylthiazoletetrazolium (MTT; Biotium, Hayward, CA) assay as follows. Ten thousand A549 or H460 cells were grown in a 96-well plate for 24 h and then exposed to I3C and silibinin, alone or in combination, for 24 h or 72 h followed by MTT treatment (100 μl per well) for 3 h. Subsequently, culture media were aspirated, 100 μl of dimethyl sulfoxide (DMSO) was added to each well and absorbance at 570 nm was read with a plate reader. Each treatment with the chemopreventive agents and the corresponding solvent control (DMSO) was carried out in triplicate. The MTT assays were repeated three times on different days.
Annexin V/propidium iodide assays for apoptosis
For Annexin V/propidium iodide (PI) assays, cells were stained with Annexin V-fluorescein isothiocyanate and PI and then evaluated for apoptosis by flow cytometry according to the manufacturer’s protocol (BD Pharmingen, San Diego, CA). Briefly, after treatment of A549 or H460 cells with I3C (50 μM) and silibinin (50 μM), individually or in combination, for 72 h, 1 × 106 cells were washed twice with cold phosphate-buffered saline and stained with 5 μl Annexin V-fluorescein isothiocyanate and 10 μl PI (5 μg/ml) in ×1 binding buffer [10 mmol/l N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (pH 7.4), 140 mmol/l NaOH, 2.5 mmol/l CaCl2] for 15 min at room temperature in the dark. The apoptotic cells were determined using a Becton Dickinson FACScan cytofluorometer. Both early apoptotic (Annexin V-positive, PI-negative) and late apoptotic (Annexin V-positive and PI-positive) cells were included in cell death determinations.
Tumor bioassay
Female A/J mice, 5–6 weeks of age, were obtained from The Jackson Laboratory (Bar Harbor, ME). Female mice are more docile and easier to work with than their male counterparts. The experimental design for the tumor bioassay is shown in Figure 2A. Upon arrival (Week 0), the mice were housed in the specific-pathogen-free animal quarters of Research Animal Resources, University of Minnesota Academic Health Center, randomized into different groups and maintained on AIN-93G-pelleted diet. One week after arrival, the mice were switched to AIN-93G-powdered diet and treated intraperitoneally with NNK (50 mg/kg, in 0.1 ml physiological saline solution, (groups 1–4) or the vehicle (0.1 ml of physiological saline solution, group 5) twice weekly for a total of four treatments. I3C (10 μmol/g diet) and silibinin (7 μmol/g diet), individually or in combination, were added to the diet beginning 1 week after the last dose of NNK (Groups 2, 3 and 4, Table I). The doses of the chemopreventive agents were chosen on the basis of previous studies (17,19). Mice in group 1 (carcinogen control) and group 5 (untreated control) were maintained on non-supplemented diet. At week 10 of the study, the diet was changed from AIN-93G to AIN-93M. Diet consumption was measured twice weekly, and body weights were determined weekly until termination. The experiment was terminated at week 27 by euthanizing the mice with an overdose of carbon dioxide. The lungs were harvested and tumors on the surface of the lung counted and their sizes determined under a dissecting microscope. All left lung lobes from carcinogen-treated mice and controls were preserved in 10% buffered formalin for subsequent histopathological analyses. Tumors on the remaining lobes of carcinogen-treated mice were microdissected and stored, together with the right lung lobes from control mice, at −80°C, for western immunoblotting studies.
Fig. 2.
Fig. 2.
Effect of I3C and silibinin on the body weight of A/J mice. (A) Experimental protocol to assess inhibition of NNK-induced lung tumorigenesis by I3C and silibinin in A/J mice. (B) Growth curves of mice given I3C and silibinin in the diet for 27 weeks.
Table I.
Table I.
Effects of I3C and silibinin, alone or in combination, on NNK-induced lung tumors in A/J micea
Histopathological analysis
Formalin-fixed left lung lobes from all mice were routinely processed, embedded in paraffin and three 4 μm thick step sections (each 200 μm apart) were cut and stained with hematoxylin and eosin. Proliferative lesions were counted in each step section, and the total number of each lesion type per mouse was expressed as an average number of each lesion per section (sum of each lesion in three step sections divided by three).
Proliferative lesions in the lungs were classified as hyperplasia, adenoma, adenoma with dysplasia or adenocarcinoma based on our previous reports (20,23) and the recommendations published by the Mouse Models of Human Cancers Consortium (24). The category adenoma with dysplasia is an adenoma in which 10 cells are pleomorphic, characterized by large cell and/or nuclear size; increased cytoplasmic-to-nuclear ratio; prominent nucleoli; nuclear crowding and increased numbers of mitotic figures with no evidence of parenchymal invasion by pleomorphic cells (20,23).
Western immunoblot analyses
For the preparation of cell lysates, A549 or H460 cells (1 × 106) were treated with DMSO, I3C, silibinin or a combination of I3C and silibinin, for 24 or 72 h, harvested and suspended for 1 h in a lysis buffer composed of the following constituents. Fifty mmol/l Tris–HCl, 150 mmol/l NaCl, 1 mmol/l ethyleneglycol-bis(aminoethylether)-tetraacetic acid, 1 mmol/l ethylenediaminetetraacetic acid, 20 mmol/l, 1% Triton X-100, pH 7.4 and protease inhibitors [aprotinin (1 μg/ml), leupeptin (1 μg/ml), pepstatin (1 μmol/l) and phenylmethylsulfonyl fluoride (0.1 mmol/l)] and phosphatase inhibitors Na3VO4 (1 mmol/l) and NaF (1 mmol/l).The preparations were centrifuged (14 000g for 25 min at 4°C), the supernatants collected, aliquoted and stored at −80°C. For the analysis of mouse lung tissue lysates, aliquots of normal lungs (vehicle control mice, 30 mg/mouse) or microdissected tumors (carcinogen-treated mice, 30 mg/mouse) from six mice were pooled, ground with a mortar and pestle on liquid nitrogen, and the powder suspended in ice-cold lysis buffer for 1 h and processed similar to the cell lysates.
For western immunoblotting, 60 μg of protein from the cell or tissue lysates were loaded onto a 4–12% Novex Tris–glycine gel (Invitrogen, Carlsbad, CA) and run for 60 min at 200 V. The proteins were then transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA) for 1 h at 30 vol. Protein transfer was confirmed by staining membranes with BLOT-FastStain (Chemicon, Temecula, CA). Subsequently, membranes were blocked in 5% Blotto non-fat dry milk in Tris buffer containing 1% Tween 20 for 1 h and probed overnight with the following primary antibodies obtained from Cell Signaling Technology: anti-phospho-Akt, anti-total-Akt, anti-phospho- ERK, anti-total ERK and anti-cyclin D1 and anti-PARP. All primary antibodies were used at a dilution of 1:1000. After incubating the membranes with a secondary antibody (goat anti-rabbit IgG; 1:5000) for 1 h, chemiluminescent immunodetection was used. Signal was visualized by exposing membranes to HyBolt CL autoradiography film. All membranes were stripped and reprobed with anti-β-actin (1:1000) to check for differences in the amount of protein loaded in each lane. For each protein, at least three western assays were carried out.
Statistical analysis
Wilcoxon rank sum test was used for pairwise comparisons of the number of tumors on the surface of the lung (groups treated with NNK and received chemopreventive agents versus the group treated with NNK only). The same test was also used to compare the effect of individual chemopreventive agents versus combinatorial treatment. Since the frequency of the different histopathological lesions (hyperplasia, adenoma, adenoma with progression and adenocarcinoma) was approximately normally distributed, two-sample-t-test was employed for the pairwise comparison of this variable between groups treated with carcinogen and given chemopreventive agents versus the group treated with NNK only. Descriptive statistics including means and standard deviations were reported for all variables. Two-sided P-values ≤ 0.05 were considered statistically significant. All analyses were conducted in SAS 9.1.3. For the analyses of results from the MTT assay, the two-sided Student’s t-test was used. Data are reported as mean ± standard deviation of triplicate determinations. P values of 0.05 were considered statistically significant.
Combinatorial treatment with I3C and silibinin inhibited cell proliferation, reduced activation of Akt and ERK and caused apoptosis in A549 and H460 lung cancer cells
To determine if I3C and silibinin, alone or in combination, cause growth inhibition in A549 and H460 cells, the cells were exposed to different concentrations of the compounds for 24 or 72 h. As shown in Figure 1A, treatment of the cells with I3C for 24 did not affect cell proliferation at any of the concentrations tested, whereas silibinin significantly reduced the growth of both cell lines by 20–30% at concentrations >75 μM. Combination of I3C and silibinin caused stronger antiproliferative effects than the individual agents. Exposure of A549 cells to 100 μM of I3C plus 75 μM of silibinin or 200 μM of I3C plus 75 μM for 24 h reduced the proliferation of the cells by 40 and 62%, respectively. The corresponding effects in H460 cells were 31 and 69%. Next, we examined if the proliferation of A549 and H460 cells could be further decreased upon treatment with I3C and silibinin, alone or in combination, for a longer period of time, 72 h. As depicted in Figure 1B, I3C did not affect the growth of the cells except at the highest concentration (400 μM), in which the proliferation of A549 and H460 cells was reduced by 75 and 87%, respectively. Silibinin reduced the growth of A549 and H460 by 58 and 30% at a concentration of 100 μM and 47 and 22% at a concentration of 75 μM, respectively. Interestingly, combinations of I3C and silibinin at lower concentrations caused stronger effects than that of the single agents used at higher concentrations. Treatment of A549 cells and H460 cells with 25 μM I3C plus 25 μM silibinin, 50 μM I3C plus 25 μM silibinin, 100 μM I3C plus 25 μM silibinin or 50 μM I3C plus 50 μM silibinin reduced the growth of the cells by ~25, 35, 59, and 64%, respectively. The corresponding reductions in H460 cells were 27, 31, 46 and 58%.
Fig. 1.
Fig. 1.
Combinatorial treatment with I3C and silibinin suppresses growth and induces apoptosis in A549 and H460 cells. (A) Effect of I3C and silibinin, alone or in combination, on the growth of A549 cells and H460 cells treated with the compounds for 24 h. ( (more ...)
To determine if the strong antiproliferative activities of combinations of I3C and silibinin are paralleled by modulation of cell proliferation-related proteins, we examined the effect of the compounds on activation of Akt and ERK. In line with the cell proliferation assay, exposure of A549 or H460 cells to I3C alone (100–400 μM) or silibinin alone (75 μM) for 24 h did not affect activation of Akt or ERK (Figure 1C). On the other hand, combinatorial treatment with the agents caused marked reduction in Akt activation and moderate effects on ERK; as the concentration of I3C in the mixture increased, the effect on Akt and ERK activation also was enhanced, in particular in H460 cells. Extended treatment (72 h) of the cells with 50 μM I3C plus 50 μM silibinin completely inhibited Akt activation in both cell lines; ERK activation was completely inhibited in H460 cells but only partially inactivated in A549 cells (Figure 1D).
Analysis of apoptosis induction in I3C plus silibinin(50 μM of each)-treated A549 and H460 cells, by annexin V/PI assay, showed 32 and 57% apoptotic cells, respectively (Figure 1E). On the other hand, the frequency of apoptosis rate in cells treated with the individual agents was similar to that measured in cells treated with DMSO, the vehicle control.
Effects of I3C and silibinin, alone or in combination, on NNK-induced lung tumors
Treatment with I3C, silibinin or their combination did not affect food consumption (data not shown) or body weight gain (Figure 2B). The final body weight of mice treated with NNK and given I3C or I3C plus silibinin was reduced by 1.8 and 2%, respectively, relative to the weight of mice in the NNK only group, whereas the body weight of mice maintained on silibinin-supplemented diet was the same as that of the group treated with NNK alone.
Upon termination of the tumor bioassay, lungs were harvested and tumors on the surface of the lung counted and their size determined. The results are shown in Table I. Mice in group 1 which were treated with NNK and maintained on control diet had 35.1 ± 7.6 tumors per mouse. Mice in groups 2, 3 and 4 which were treated with NNK and given I3C, silibinin or I3C plus silibinin in the diet had 20.1 ± 3.8, 22.6 ± 4.6 and 14.2 ± 2.0 tumors per mouse, corresponding to a significant reduction by 43, 36 and 60%, respectively (Table I). Comparison of the efficacy of I3C plus silibinin with that of I3C alone or silibinin alone showed a significantly stronger efficacy for the combinatorial treatment. Tumor incidence was 100% in all carcinogen-treated mice.
Upon counting the lung tumors, the size of the tumors was categorized into four classes: <0.5 mm, 0.5–1 mm, >1 mm but <2 mm and ≥2 mm. Multiplicities of the lung tumors in the various treatment groups, subdivided according to the tumor size, are depicted in Figure 3. Generally, regardless of the treatment group, the majority of the tumors had a diameter of 0.5–2 mm (Figure 3A). In mice treated with NNK and maintained on a control diet, the frequency of tumors with the size of <0.5 mm, 0.5–1 mm, >1 mm but <2 mm and ≥2 mm was 3.9 ± 2.6, 14.0 ± 5.5, 15.3 ± 4.1 and 1.8 ± 1.6, respectively. Supplementation of the diet with I3C plus silibinin significantly reduced the frequency of tumors with a diameter of <0.5 mm, 0.5–1 mm, >1 mm but <2 mm and ≥2 mm to 1.5 ± 1.3, 8.6 ± 2.1, 3.9 ± 1.2 and 0.1 ± 0.4, respectively. Similarly, in mice given the individual chemopreventive agents, the frequency of the various classes of tumors was significantly reduced (Figure 3). Comparisons of the effects of combinatorial treatment with single agent treatment showed that I3C plus silibinin was significantly more effective toward larger tumors compared with I3C alone or silibinin alone.
Fig. 3.
Fig. 3.
Effect of I3C and silibinin, alone or in combination, on the multiplicity and growth of NNK-induced lung tumors and the expression of growth- and apoptosis-related proteins in the tumor tissues. (A) Differential effects of I3C and silibinin on the growth (more ...)
Effect of I3C, silibinin or I3C plus silibinin on the incidence and multiplicity of NNK-induced histopathological lesions
The microscopic lesions observed in lung tissues of mice were classified as hyperplastic foci, adenoma, adenoma with dysplasia and adenocarcinoma based on established criteria (19). Images of representative lung tissue sections showing the frequencies of the various histopathological lesions in the different treatment groups are depicted in Figure 3B.
As shown in Table II, in mice treated with NNK and maintained on control diet, multiplicities of hyperplasic foci, adenoma, adenoma with dysplasia and adenocarcinoma were 2.6 ± 0.6, 3.6 ± 1.4, 2.5 ± 1.0 and 0.6 ± 0.4, respectively. None of the chemoprevention regimens significantly reduced the multiplicities of hyperplastic foci or adenoma, with the exception of the effect of silibinin on hyperplastic foci. On the other hand, multiplicities of adenoma with cellular pleomorphism were significantly decreased to 0.5 ± 0.5, 0.8 ± 0.9 and 0.2 ± 0.2, corresponding to reductions by 84, 68 and 92% in mice given I3C, silibinin or I3C plus silibinin, respectively. Similarly, the multiplicities of adenocarcinoma were significantly reduced to 0.1 ± 0.3, 0.3 ± 0.4 and 0.03 ± 0.09, corresponding to reductions by 83, 50 and 95% in mice given I3C, silibinin or I3C plus silibinin, respectively.
Table II.
Table II.
Effects of I3C and silibinin, alone or in combination, on NNK-induced pulmonary lesions in A/J micea
The overall inhibitory activities of I3C plus silibinin against adenoma with dysplasia and adenocarcinoma were clearly stronger than that of I3C alone or silibinin alone. However, the differences were not statistically significantly different due to the low incidence of the lesions and the wide intragroup variations in the number of the lesions.
Effects of I3C, silibinin or their combination on cell proliferation-, survival- and apoptosis-related proteins in mouse lung tissues
Our studies in A549 cells and H460 cells showed that combinatorial treatment with I3C and silibinin is effective in inhibiting activation of proteins related to cell proliferation and survival. Therefore, we asked if dietary feeding of a mixture of I3C and silibinin induces similar effects in mouse lung tumor tissues. Western blot analysis of normal lung tissues from vehicle-treated mice and lung tumor tissues from mice treated with NNK alone or with the chemopreventive agents showed a higher level of p-Akt, p-ERK and cyclin D1 in lung tumors tissues from mice treated with NNK alone as compared with the level in normal tissues from the vehicle control group (Figure 3C). In mice treated with NNK and given the chemopreventive agents, the level of p-Akt, p-ERK, cyclin D1 was reduced, compared with the level in the NNK only group, with the exception of p-ERK in NNK plus I3C-treated mice; the greatest reduction in the level of the proteins was seen in the group given the combination of I3C plus silibinin. Moreover, in the groups given the preventive agents, a cleaved fragment of PARP, an indicator of apoptosis, was observed.
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. (25) 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 (13,30). 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 (32,33). 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. (34) 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 (12,35), 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.
Funding
National Institutes of Health (CA-128801 to F.K.).
Acknowledgments
We thank Bob Carlson for help in the preparation of the Figures and Josh Parker and Paula Overn, from Comparative Pathology Shared Resource, for help with the mouse lung tumor microdissection and for technical histology assistance, respectively.
Conflict of Interest Statement: None declared.
Glossary
Abbreviations
DMSOdimethyl sulfoxide
ERKextracellular signal-regulated kinase
I3Cindole-3-carbinol
MTTmethylthiazoletetrazolium
NNK4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
PARPpoly (ADP-ribose) polymerase
PIpropidium iodide

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