1-Benzyl-indole-3-carbinol is a novel indole-3-carbinol derivative with significantly enhanced potency of anti-proliferative and anti-estrogenic properties in human breast cancer cells

Abstract

Indole-3-carbinol (I3C), a natural autolysis product of a gluccosinolate present in Brassica vegetables such as broccoli and cabbage, has anti-proliferative and anti-estrogenic activities in human breast cancer cells. A new and significantly more potent I3C analogue, 1-benzyl-I3C was synthesized, and in comparison to I3C, this novel derivative displayed an approximate 1000-fold enhanced potency in suppressing the growth of both estrogen responsive (MCF-7) and estrogen-independent (MDA-MB-231) human breast cancer cells (I3C IC(50) of 52 microM, and 1-benzyl-I3C IC(50) of 0.05 microM). At significantly lower concentrations, 1-benzyl-I3C induced a robust G1 cell cycle arrest and elicited the key I3C-specific effects on expression and activity of G1-acting cell cycle genes including the disruption of endogenous interactions of the Sp1 transcription factor with the CDK6 promoter. Furthermore, in estrogen responsive MCF-7 cells, with enhanced potency 1-benzyl-I3C down-regulated production of estrogen receptor-alpha protein, acts with tamoxifen to arrest breast cancer cell growth more effectively than either compound alone, and inhibited the in vivo growth of human breast cancer cell-derived tumor xenografts in athymic mice. Our results implicate 1-benzyl-I3C as a novel, potent inhibitor of human breast cancer proliferation and estrogen responsiveness that could potentially be developed into a promising therapeutic agent for the treatment of indole-sensitive cancers.

Fig. 1

Synthesis and structure of 1-Benzyl-indole-3-carbinol. The upper diagram shows the structure of indole-3-carbinol. The synthesis of 1-Benzyl-indole-3-carbinol was accomplished by the benzylation of indole, then formylation to the aldehyde, and finally reduction to the carbinol. The structure of 1-Benzyl-indole-3-carbinol is shown in the lower diagram.

Fig. 2

Dose-dependent effects of 1-benzyl-I3C and I3C on the DNA synthesis of human MCF-7 breast cancer cells. (A) Triplicate samples of asynchronously growing human MCF-7 breast cancer cells were treated with increasing concentrations of 1-benzyl-I3C, I3C or the vehicle control (DMSO) for 72 hours. During the last three hours, cells were pulsed with 3 μCi [³H]thymidine (84 Ci/mmol), and the incorporation into DNA was determined by acid precipitation as described in the Materials and Methods section. Triplicates were averaged and expressed as counts per minute per well. (B) To determine the IC₅₀ values of the I3C and 1-benzyl-I3C inhibition of DNA synthesis of MCF-7 cells, the results of the [³H]thymidine incorporation data are plotted as the percentage of vehicle control treated cells for each indole. Data are the mean of three experiments; bars, +/-SE.

Fig. 3

1-benzyl-I3C inhibits DNA synthesis and induces a G1 cell cycle arrest of estrogen-responsive and estrogen-independent unresponsive human breast cancer cell lines. (A) Estrogen responsive MCF-7 cells and estrogen independent MDA-MB-231 cells were treated for 72 hours with the indicated concentrations of 1-benzyl-I3C and during the last three hours, cells were pulsed labeled with 3 μCi [³H]thymidine and the incorporation into DNA and percent incorporation calculated as described in the figure 2 legend. Data represent mean of three independent experiments. (B) MCF-7 cells and MBA-MD-231 cells were treated for 72 hours with either 0.2 μM 1-benzyl-I3C or the DMSO vehicle control, and effects on the cell cycle monitored by flow cytometry for DNA content of propidium iodide stained nuclei. (C) MCF-7 and MDA-MB-231 cells were treated with or without 0.2 μM 1-benzyl-I3C for 72 hours, and western blots of electrophoretically fractionated total cell extracts analyzed for total phosphorylated-Rb (pRb) and hyperphosphorylated-Rb (ppRB) using the corresponding Rb-specific antibodies.

Fig. 4

Time course of effects of 1-benzyl-I3C on expression of G1 cell cycle components. Upper panel: Human MCF-7 breast cancer cells were treated with 0.2 μM 1-benzyl-I3C or the DMSO vehicle control for 0, 24, 48 or 72 hours followed by western blot analysis of the indicated G1 cell cycle components. Actin protein levels were used a gel loading control. Lower panel: The level of observed cell cycle protein in 1-benzyl-I3C treated cells was compared to control cells and quantified at each time point by densitometric analysis of the western blot.

Fig. 5

Effects of I3C and 1-benzyl-I3C on CDK6 transcript expression and on endogenous interactions of Sp1 with the CDK6 promoter. (A) MCF-7 cells were treated with 1 μM 1-benzyl-I3C (1-B-I3C), 200 μM I3C or the DMSO vehicle control for 48 hours, total RNA isolated and the levels of CDK6 transcripts determined by RT-PCR analysis using CDK6-specific oligonucleotide primers. The level of the constitutively expressed GAPDH was used as a positive control for each condition. (B) Chromatin immunoprecipitation was used to evaluate the effects of 1-benzyl-I3C and I3C on endogenous interactions of the Sp1 transcription factor with the Ets-Sp1 composite element in the CDK6 promoter. Chromatin was isolated from MCF-7 cells treated with or without either 200 μM I3C or 1 μM 1-benzyl-I3C for 48 hours. Sp1 was immunoprecipitated from total cell extracts using Sepharose G bound to anti-Sp1 antibody and DNA released from Sp1 was amplified using the oligonucleotide primers described in the methods and materials section. Input samples represent total genomic DNA from each treatment (loading control).

Fig. 6

Effects of 1-benzyl-I3C on the specific enzymatic activities of the G1 acting Cyclin Dependent Kinases. (A) MCF-7 cells were treated with either 0.2 μM 1-benzyl-I3C or the DMSO vehicle control for 72 hours followed by immunoprecipitations of CDK2, CDK4, or CDK6 as described in Materials and Methods section. One aliquot of each immunoprecipitation was assayed in vitro for protein kinase activity using GST-Rb as a substrate (left panels), and the other aliquot was used to assess total CDK protein levels in each immunoprecipitation by western blot analysis (right panels). The No IP lane represents an immunoprecipitation with a non-specific IgG. (B) MCF-7 were treated with 0.2 μM 1-benzyl-I3C for indicated times, and at each time point, the enzymatic activities and protein levels of immunoprecipitated CDKs were performed as described above. The amount of immunoprecipitated CDK protein was quantified by densitometry, and the level of [³²P]GST-Rb generated from the corresponding kinase assays was determined by phosphoimager analysis as described in Materials and Methods. The values are expressed as percentage of growing control cells not with the vehicle control DMSO at each time point, which was calculated by dividing the values of 1-benzyl-I3C treated cells with the values of vehicle control treated cells. Error bars represent standard error of the mean three independent experiments.

Fig. 7

Cooperative effects of 1-benzyl-I3C and I3C with tamoxifen on the inhibition of DNA synthesis and stimulation in G1 phase arrested cells in MCF-7 cells. MCF-7 cells were treated with the indicated combinations of 100 μM I3C, 0.5 μM 1-benzyl-I3C (1-B-I3C) and/or 1 μM tamoxifen for 72 hours. The effects on DNA synthesis was monitored by pulsed labeling with 3 μCi [³H]thymidine during the last three hours of incubation (upper panel). The effects on the number of cells arrested in the G1 phase of the cell cycle was monitored by flow cytometry for DNA content of propidium iodide stained nuclei (lower panel). The reported data represent the mean of three independent experiments.

Fig. 8

Dose-dependent effects of 1-benzyl-I3C and I3C on expression of estrogen receptor-alpha protein. MCF-7 cells were treated with indicated concentrations of 1-benzyl-I3C or I3C for 24 hours, and the level of estrogen receptor-alpha (ERα) determined by western blot analysis of electrophoretically fractionated total cell extracts. Western blot analysis of actin levels was used as a gel loading control.

Fig. 9

In vivo effects of 1-benzyl-I3C and I3C on the growth of human MCF-7 breast cancer cells derived tumors from xenografts in athymic mice. MCF-7 cell xenografts were grown to a tumor size of 35 mm³ in athymic mice, and then the animals were injected with 30 mg/kg body mass 1-benzyl-I3C, 300 mg/kg body mass I3C or with the DMSO vehicle control as described in the Methods and Materials section. Tumor size was monitored throughout a three-week time course, and the residual tumors were excised and photographically visualized (lower panel).