Indole-3-carbinol downregulation of telomerase gene expression requires the inhibition of estrogen receptor-alpha and Sp1 transcription factor interactions within the hTERT promoter and mediates the G1 cell cycle arrest of human breast cancer cells

Abstract

Indole-3-carbinol (I3C), a naturally occurring hydrolysis product of glucobrassicin from cruciferous vegetables such as broccoli, cabbage and Brussels sprouts, is an anticancer phytochemical that triggers complementary sets of antiproliferative pathways to induce a cell cycle arrest of estrogen-responsive MCF7 breast cancer cells. I3C strongly downregulated transcript expression of the catalytic subunit of the human telomerase (hTERT) gene, which correlated with the dose-dependent indole-mediated G(1) cell cycle arrest without altering the transcript levels of the RNA template (hTR) for telomerase elongation. Exogenous expression of hTERT driven by a constitutive promoter prevented the I3C-induced cell cycle arrest and rescued the I3C inhibition of telomerase enzymatic activity and activation of cellular senescence. Time course studies showed that I3C downregulated expression of estrogen receptor-alpha (ERα) and cyclin-dependent kinase-6 transcripts levels (which is regulated through the Sp1 transcription factor) prior to the downregulation of hTERT suggesting a mechanistic link. Chromatin immunoprecipitation assays demonstrated that I3C disrupted endogenous interactions of both ERα and Sp1 with an estrogen response element-Sp1 composite element within the hTERT promoter. I3C inhibited 17β-estradiol stimulated hTERT expression and stimulated the production of threonine-phosphorylated Sp1, which inhibits Sp1-DNA interactions. Exogenous expression of both ERα and Sp1, but not either alone, in MCF7 cells blocked the I3C-mediated downregulation of hTERT expression. These results demonstrate that I3C disrupts the combined ERα- and Sp1-driven transcription of hTERT gene expression, which plays a significant role in the I3C-induced cell cycle arrest of human breast cancer cells.

Fig. 1.

I3C inhibited expression of hTERT correlates with the indole-mediated cell cycle arrest of human breast cancer cells. (A) Hormone-sensitive MCF7 human breast cancer cells were treated with the indicated doses of I3C or the DMSO vehicle control for 48 h. Expression of hTERT, hTR, CDK6, CDK4, ERα and progesterone receptor (PR) transcripts was determined by RT–PCR analysis of total isolated RNA (left panel). The PCR products were visualized on a 1% agarose gel stained with ethidium bromide. GAPDH provided a gel-loading control for the RT–PCR (left panel). Expression of the indicated gene transcripts was also quantified by qPCR (right panel). qPCR data are expressed as fold change after normalization to GAPDH for each treatment and to DMSO for comparative purposes. In a parallel experiment over the same indole concentrations, the I3C-induced G₁ cell cycle arrest was analyzed by flow cytometry of propidium stained nuclei. At each I3C concentration, the number of cells with a G₁ phase DNA content was plotted in comparison with the densitometric analysis of hTERT and hTR transcript levels. (B) MCF7 cells were treated with DMSO vehicle control, 200 μM I3C, 50 μM of the I3C dimer DIM, 200 μM of the inactive indole tryptophol, 1 μM tamoxifen or 1 nM fulvestrant for 48 h and the levels of hTERT and hTR transcripts were determined by RT–PCR analysis of total isolated RNA. The cell cycle arrest in each treatment condition was monitored by flow cytometry as described above. The G₁ arrest of at least 70% of the cells compared with the 55% of cells with a G₁ DNA content in control growing cells was considered positive for the cell cycle arrest.

Fig. 2.

Exogenous expression of hTERT overcomes I3C-dependent G₁ cell cycle arrest of MCF7 cells. MCF7 cells were stably transfected with a hTERT expression vector (forming MCF7-hTERT cells or with the neomycin empty expression vector (forming MCF7-neo cells). Western blots of total cell extracts were probed with hTERT antibodies or with hsp90 antibodies as a gel-loading control (top panel). MCF7-neo and MCF7-hTERT cells were treated with 200 μM I3C or with the DMSO vehicle control for 48 h. Cells were hypotonically lysed and stained with propidium iodide prior to analysis by coulter cell counter. Cell count versus PI staining is displayed (n = 10 000) per treatment. Cell cycle phase analyzed using Win-MultiCycle software. All analysis was performed in triplicate.

Fig. 3.

I3C-dependent inhibition of telomerase activity is rescued by exogenous expression of hTERT. (A) MCF7-neo and MCF7-hTERT cells were treated with 200 μM I3C or with the DMSO vehicle control for 48 h and then subjected to cellular lysis using CHAPS lysis buffer. Telomeric repeat DNA extention from template DNA was measured using fluorescent probes (telomere extension = increased fluorescence) as indicated in Millipore TRAP assay kit. Internal sulforophane probe to nonspecific DNA sequence served as loading control. Analysis was performed in triplicate. (B) MCF7-neo and MCF7-hTERT cells were treated with 200 μM I3C or the DMSO vehicle control for 48 h. Cells were incubated with senescence detection reagent (X-gal solution) to measure internal β-galactosidase expression. Analysis was performed in triplicate. Positive staining cells were visualized using light microscope. Images captured with 10 megapixel digital camera. Prism software was used to perform a paired t-test to determine significance of difference between treated and untreated cells (P = 0.0225).

Fig. 4.

I3C inhibited constitutive and estrogen regulated hTERT expression, I3C disruption of endogenous interactions of ERα and Sp1 with the hTERT promoter and indole stimulated Sp1 phosphorylation. (A) MCF7 cells were treated with 200 μM I3C or DMSO vehicle control for a 72 h time course. hTERT, hTR, CDK6 and ERα transcript expression was determined at the indicated time points by RT–PCR analysis of total isolated RNA. The PCR products were visualized on a 1% agarose gel stained with ethidium bromide (left panel). GAPDH provided a gel-loading control for the RT–PCR. Expression of the indicated genes over the 72 h time course was also quantified by qPCR (right panel). qPCR data are expressed as fold change after normalization to GAPDH for each treatment and to T = 0 h for comparative purposes. (B) ChIP was employed to characterize endogenous ERα and Sp1 interactions with the ERE–Sp1 composite element within the hTERT promoter. Chromatin was isolated from MCF7 cells treated with or without 200 μM I3C for 48 h. ERα or Sp1 was immunoprecipitated from total cell extracts using Sepharose G bound to either anti-ERα antibody or anti-Sp1 antibody and DNA was amplified using the oligonucleotide primers defined in the Materials and Methods (top panel). Input samples represent total genomic DNA from each treatment (loading control). This result was repeated twice. Isolated chromatin from an independent experiment using the same antibodies and conditions was quantified using qPCR at the same loci (bottom panel). qPCR data were normalized to input levels and is expressed as fold change from DMSO. (C) T47D cells were treated with 200 μM I3C or DMSO control for 48 h in full media. Western blots of total cell extracts were probed with hTERT antibody and with ACTIN antibody as a gel-loading control (top panel). MCF7 cells were grown in steroid-deficient media for 24 h and then treated with the indicated combinations of 200 μM I3C and 10 nM β-2-estradiol (E₂). hTERT transcript expression was determined by RT–PCR analysis and GAPDH provided a gel loading control for the RT–PCR (lower panel). (D) MCF7 cells were treated with the 200 μM I3C or with the DMSO vehicle control for 48 h. Total cell extracts were immunoprecipitated with mouse anti-Sp1 antibody and electrophoretically fractionated samples blotted with either rabbit anti-Sp1 or rabbit anti-phospho-threonine antibodies (Sp1-THR^P).

Fig. 5.

I3C disrupts endogenous binding of ERα and Sp1 at a critical regulatory site within the hTERT promoter. MCF7 cells were transfected with CMV-ERα, CMV-Sp1, a combination of CMV-ERα and CMV-Sp1, or with the CMV-Neo vector control and treated with or without 200 μM I3C for 48 h. Total RNA was collected and the level of ERα, Sp1 and hTERT transcripts determined by RT–PCR analysis. GAPDH was used as total RNA loading control. PCR products were visualized on a 1% agarose gel stained with ethidium bromide. Result was repeated twice, representative gel shown.