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. 2007 Mar;30(3):641-50.

The low-toxicity 9-cis UAB30 novel retinoid down-regulates the DNA methyltransferases and has anti-telomerase activity in human breast cancer cells

Nathan J Hansen  1 , Rebecca C Wylie, Sharla M O Phipps, William K Love, Lucy G Andrews, Trygve O Tollefsbol
Affiliations

The low-toxicity 9-cis UAB30 novel retinoid down-regulates the DNA methyltransferases and has anti-telomerase activity in human breast cancer cells

Nathan J Hansen et al. Int J Oncol. 2007 Mar.

Abstract

Retinoic acids and their derivatives potentiate anti-cancer effects in breast cancer cells. The aberrant expression of telomerase is critical to the continued proliferation of most cancer cells. Thus, telomerase is an attractive target for chemoprevention and treatment of breast cancer. 9cUAB30 is a novel synthetic retinoid X receptor-selective retinoic acid (RA) that effectively reduces the tumorigenic phenotype in mouse breast carcinoma with lower toxic effects than natural retinoid treatments. We have assessed 9cUAB30 retinoic acid treatment of human breast cancer cells to determine the potential of this drug as an effective telomerase inhibitor and its application to cancer therapy. 9cUAB30 was found to decrease DNA methyltransferase and telomerase expression in MDA-MB-361, T-47D, and MCF-7 human breast cancer cells and to inhibit the proliferation of these cells. This low-toxicity retinoid also reduced colony formation in soft agar assays in each of these cell types. Combination treatments of 9cUAB30 and all-trans RA proved to be synergistically more effective than either RA alone, further suggesting a possible general epigenetic mechanism that contributes to the anti-telomerase activity of the retinoids. Therefore, the novel retinoid, 9cUAB30, is effective in inhibiting the growth of human breast cancer cells, its anti-cancer effects appear to be related to telomerase inhibition and the mechanism for this process could be mediated through epigenetic modifications.

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Figures

Figure 1
Figure 1
Novel retinoid treatment decreases the proliferation rates of human breast cancer cells. (A) Human T47D breast carcinoma cells were grown in untreated control media (◇) or either 2 μM ATRA (□) or 10 μM 9cUAB30 (◆) for 12 days of concurrent culture. (B) Human MDA-MB-361 cells remained untreated (◇) or were treated with 10 μM 9cUAB30 (■), 2 μM ATRA (□) or a combination of 5 μM UAB-30 and 1 μM ATRA (▲). (C) Human MCF-7 cells were untreated (◇), treated with 10 μM 9cUAB-30 (◆), or treated with 2 μM ATRA (□). Proliferation is expressed as the percentage of the ratio of live cells harvested at days 3, 6, 9, and 12 of treatment to the number of live cells originally plated. Average cell numbers obtained by trypan blue exclusionary staining performed with a standard hemacytometer are presented with SEMs from triplicate culturing of each treatment group.
Figure 2
Figure 2
hTERT mRNA expression decreases with retinoic acid treatment. (A) RT-PCR amplification of a 219-bp segment of hTERT mRNA in T47D human breast carcinoma cells treated with either 10 μM RA or 9cUAB30 over 12 days of concurrent culture. (B) hTERT expression in MDA-MB-361 cells. +, samples treated with retinoic acid; -, untreated samples; d, days. (C) hTERT cDNA in MCF-7 cells with and without retinoid treatment. β-actin and GAPDH served as controls to normalize the loading and quality of RNA. The data shown are representative of triplicate gels from three individual experiments.
Figure 3
Figure 3
DNMT mRNA expression is altered with retinoid treatment. (A) DNA methyltransferase (DNMT) expression in T47D cells exposed to 10 μM ATRA over 12 days of concurrent culture. Product bands are shown for DNMT1, DNMT3a, and DNMT3b and the ubiquitous housekeeping enzyme GAPDH. (B) RT-PCR amplification of DNMT mRNAs in T47D breast cancer cells treated with 10 μM 9cUAB30 over 12 days of concurrent culture. Product bands are shown for DNMT1, DNMT3a, and DNMT3b and GAPDH. d, days.
Figure 4
Figure 4
Analysis of telomerase activity in breast cancer cells in response to retinoids. (A1) Dose-responsive inhibition of telomerase activity with ATRA and 9cUAB30. DNA products from the TRAP assay are presented for samples treated with 0.1, 1, and 10 μM ATRA or 9cUAB30 for 6 days of concurrent treatment. (A2) Inhibition of telomerase over 12-day treatment with 10 μM ATRA. TRAP assay results are presented for T47D breast cancer cells exposed to 10 μM ATRA for 12 days of concurrent treatment. (A3) Inhibition of telomerase over 12 days of treatment with 9cUAB30. C, untreated control cell samples. (B) Retinoid-induced down-regulation of telomerase activity in MDA-MB-361 cells. Cells were treated with 10 μM 9cUAB30, 2 μM ATRA, or a combination of 5 μM 9cUAB30 and 1 μM ATRA. (C) Telomerase activity in MCF-7 cells following treatment with retinoids. Treatments were 10 μM 9cUAB30 or 2 μM ATRA. +, treated samples; and -, untreated samples. In all gels shown a 61-bp internal control (IC) included in the reaction mixture for TRAP assays was used to standardize loading. The ladder formed above the IC represents 6-bp increments of TTAGGG telomeric repeats. The height of the ladder formed and the intensity of fluorescence are proportional to telomerase activity in the sample.
Figure 5
Figure 5
Soft agar analysis of retinoid-treated T47D and MDA-MB-361 cells. (A) Representative photograph of an MDA-MB-361 colony of greater than 25 cells that were counted at each time point taken at x400 magnification. (B) Examination of contact inhibition growth requirements was performed using soft agar analysis in untreated control T47D cells and cells exposed to 0.1, 1.0, or 10.0 μM ATRA or 9cUAB30 for 28 days. Colony counting results for both groups are summarized as a percent of untreated control cell colony counts. (C) Levels of tumorigenicity of MDA-MB-361 cells following 10 μM 9cUAB30 (green horizontal stripes), 2 μM ATRA (red diagonal stripes), and 5 μM 9cUAB30 and 1 μM ATRA combination (yellow dots) treatments compared to untreated MDA-MB-361 cells (blue vertical stripes). Level of tumorigenicity is expressed as a ratio of the percentage of treated colonies to the number of untreated colonies.
Figure 6
Figure 6
Treatment with retinoids significantly affected apoptosis in breast cells. (A-E) TUNEL-based labeling of apoptotic DNA fragments. Terminal deoxynucleotidyl transferase (TdT)-anti-peroxidase conjugation was performed using the ApopTag analysis (Serologicals) in untreated control cells and 10 μM ATRA- and 9cUAB30-treated T47D cells through 6 days of culture. (A) Control, (B) day 3 ATRA-treated, and (C) day 3 9cUAB30-treated breast cancer cells stained with peroxidase substrate. (D and E) TUNEL-staining of retinoid-exposed cells from 6 days of culture. Arrows indicate the presence of brown peroxidase-stained cells while green background is indicative of normal nuclei labeled with methyl green counterstain. (F and G) Fluorescence-activated cell sorting (FACS) analysis of apoptosis and death of MDA-MB-361 and MCF-7 cells in response to 12 days of retinoid exposure. Early apoptotic cells (lower right quadrants) were detected by FACS analysis as cells that incorporated Alexa®Fluor 488 (Alexa-488)-labeled Annexin V into the plasma membrane. Late apoptosis/necrosis (upper right quadrant) was detected as those cells that stained with both Alexa-488 and propidium iodide (PI). The lower left quadrant includes cells that did not incorporate either Alexa-488 or PI and represent live cells not undergoing apoptosis. The upper left quadrant displays cells that stained with PI and represent dead cells. Cell sorting was performed using FACScalibur, and analysis was achieved utilizing CellQuest software. (H) Comparative analysis of apoptosis induction in breast cancer cells. MDA-MB-361 cells (purple) and MCF-7 cells (blue dots) were treated for 12 days with 10 μM 9cUAB30, 2 μM ATRA, and a combination of 5 μM 9cUAB30 and 1 μM ATRA (orange).
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