Growth inhibition and apoptosis induced by daunomycin-conjugated triplex-forming oligonucleotides targeting the c-myc gene in prostate cancer cells

Abstract

Covalent attachment of intercalating agents to triplex-forming oligonucleotides (TFOs) is a promising strategy to enhance triplex stability and biological activity. We have explored the possibility to use the anticancer drug daunomycin as triplex stabilizing agent. Daunomycin-conjugated TFOs (dauno-TFOs) bind with high affinity and maintain the sequence-specificity required for targeting individual genes in the human genome. Here, we examined the effects of two dauno-TFOs targeting the c-myc gene on gene expression, cell proliferation and survival. The dauno-TFOs were directed to sequences immediately upstream (dauno-GT11A) and downstream (dauno-GT11B) the major transcriptional start site in the c-myc gene. Both dauno-TFOs were able to down-regulate promoter activity and transcription of the endogenous gene. Myc-targeted dauno-TFOs inhibited growth and induced apoptosis of prostate cancer cells constitutively expressing the gene. Daunomycin-conjugated control oligonucleotides with similar sequences had only minimal effects, confirming that the activity of dauno-TFOs was sequence-specific and triplex-mediated. To test the selectivity of dauno-TFOs, we examined their effects on growth of normal human fibroblasts, which express low levels of c-myc. Despite their ability to inhibit c-myc transcription, both dauno-TFOs failed to inhibit growth of normal fibroblasts at concentrations that inhibited growth of prostate cancer cells. In contrast, daunomycin inhibited equally fibroblasts and prostate cancer cells. Thus, daunomycin per se did not contribute to the antiproliferative activity of dauno-TFOs, although it greatly enhanced their ability to form stable triplexes at the target sites and down-regulate c-myc. Our data indicate that dauno-TFOs are attractive gene-targeting agents for development of new cancer therapeutics.

Figure 1

Triplex DNA formation by daunomycin-conjugated TFOs in the c-myc gene promoter. (A) Target sites in the c-myc promoter. Target A is an 11 bp sequence located 40 bp upstream of the P2 promoter. The 11 bp target B sequence is about 100 bp downstream the P2 promoter. (B) Sequence of dauno-TFOs and control oligonucleotides. Both dauno-GT11A and dauno-GT11B were designed to bind in antiparallel orientation to the purine-rich strand of the respective targets. Dauno-CO11 and dauno-GT11C are control oligonucleotides unable to form triplex DNA with sequences in the c-myc promoter. (C) EMSA. Oligonucleotides corresponding to the pyrimidine-rich strands of target A (lower panel) and target B (upper panel) were 5′ end labeled with [γ-³²P]ATP and annealed to the complementary strand. Duplex DNA (1 nM) was incubated for 18 h at 37°C with the indicated concentrations of either dauno-GT11A or dauno-GT11B. Gel electrophoresis was carried out under non-denaturating conditions. Positions of duplex and triplex DNA are indicated.

Figure 2

Inhibition of c-myc transcription by daunomycin-conjugated TFOs. (A) Luciferase reporter assay. Normal fibroblasts were transfected for 4 h with the p262-Myc reporter, pRL-SV40 and 1 µM of dauno-CO11, dauno-GT11A or dauno-GT11B. Luciferase activity was measured after 24 h. Data are presented as percent of luciferase activity compared to cells transfected with control oligonucleotide. ^*P < 0.05 compared to control trasfected cells. (B) RT–PCR. DU145 cells were left untreated (lane 1) or transfected with 1 µM of dauno-GT11C (lane 2), dauno-GT11A (lane 3) and dauno-GT11B (lane 4). Total RNA was extracted after 24 h. c-myc and β-actin RNA were determined by RT–PCR. (C) DU145 cells were left untreated (Control) or transfected with 1 µM of dauno-CO11 or dauno-GT11B. Cells were harvested after 24 h and c-myc protein level was examined by FACS. (D) DU145 cells were transfected with 1 µM of dauno-CO11 (lane 1) or dauno-GT11B (lane 2). Cell lysates were prepared after 24 h and c-myc protein level examined by western blot. (E) DU145 cells were transfected with 1 µM of the oligonucleotides along with either PMT-2T-Myc or PMT-2T. c-myc protein level was determined 24 h later by western blot. Lane 1, PMT-2T-Myc and dauno-CO11; lane 2, PMT-2T and dauno-CO11; lane 3, PMT-2T-Myc and dauno-GT11A; lane 4, PMT-2T and dauno-GT11A.

Figure 3

Inhibition of prostate cancer cell growth by daunomycin-conjugated TFOs. Prostate cancer cells DU145 (A and C) and PC3 (B) were transfected for 4 h with the indicated oligonucleotides using DOTAP. Viable cell number was determined after 96 h using MTT assays. Data are presented as percentage of viable cells compared to untreated control cells and are mean ± SD of triplicate samples from representative experiments. ^*P < 0.01 compared with untreated and control-transfected cells.

Figure 4

Reduced colony forming ability of prostate cancer cells treated with daunomycin-conjugated TFOs. DU145 cells were transfected with DOTAP alone or 1 µM dauno-CO11, dauno-GT11A or dauno-GT11B. Cells were counted and plated to determine colony forming ability in anchorage-dependent conditions. Colonies were stained with crystal violet after 8–10 days and counted. (A) Percentage of colonies relative to mock-transfected cells. Data are mean ± SD of triplicate samples from a representative experiment. ^*P < 0.05 compared to untreated and control-transfected cells. (B) Colonies formed by control and dauno-TFO-treated cells from a representative experiment.

Figure 5

Induction of apoptotic cell death by daunomycin-conjugated TFOs in prostate cancer cells. DU145 cells were left untransfected (control) or transfected with 1 µM of dauno-CO11 and dauno-GT11B. After 24 h cells were harvested, stained with FITC-Annexin-V and analyzed by FACS to detect FITC and daunomycin positive cells.

Figure 6

Cellular uptake and apoptosis in daunomycin-conjugated TFO-treated prostate cancer cells. DU145 cells were transfected with increasing concentrations of dauno-GT11A. FITC-Annexin-V staining and daunomycin uptake were measured by FACS. (A) Scatter plots of Annexin-V and daunomycin staining distribution. (B) Fluorescence intensity distribution in control and dauno-TFO-treated cells. (C) Plot of mean cell fluorescent intensity as function of dauno-TFO concentration. (D) Percentages of Annexin-V positive cells at increasing doses of dauno-TFO.

Figure 7

Uptake and intracellular distribution of daunomycin-conjugated TFOs in normal fibroblasts and prostate cancer cells. Fibroblasts (A and B) and DU145 cells (C and D) were treated with DOTAP alone (upper panels) or transfected with 1 µM of dauno-TFO using DOTAP (lower panels). After staining with DAPI, cells were examined on fluorescence microscope and images collected using DAPI and Texas red filter sets. Merged images of control and TFO-treated cells are shown.

Figure 8

Activity of daunomycin-conjugated TFOs in normal human fibroblasts. (A) Fibroblasts were mock-transfected (Control) or transfected with 1 µM of dauno-TFO (Dauno-TFO) using DOTAP and cellular uptake determined by FACS. (B) Cells were transfected with dauno-GT11A, dauno-GT11B or dauno-CO11 and viable cell numbers determined by MTT assays after 96 h. (C) Fibroblasts were left untreated (lane 1) or transfected with 1 µM of dauno-GT11A (lane 2) and dauno-GT11B (lane 3). After 24 h, c-myc protein level was determined by immunoblotting. (D) Fibroblasts, DU145 and PC3 prostate cancer cells were incubated with daunomycin. Viable cell numbers were determined after 96 h by MTT assays.