Telomere-homologous G-rich oligonucleotides sensitize human ovarian cancer cells to TRAIL-induced growth inhibition and apoptosis

Abstract

G-rich T-oligos (GT-oligos; oligonucleotides with homology to telomeres) elicit a DNA damage response in cells and induce cytotoxic effects in certain tumor cell lines. We have previously shown that GT-oligo inhibits growth, arrests cell cycle, and induces apoptosis in ovarian, pancreatic, and prostate cancer cells. However, not all ovarian cancer cell lines are susceptible to GT-oligo exposure. GT-oligo was found to induce transcript expression of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors DR-4 and DR-5, which are generally silenced in ovarian cancer cells, rendering them insensitive to TRAIL. Exposure of TRAIL- and GT-oligo-resistant cell lines to GT-oligo rendered them sensitive to the cytotoxic effects of TRAIL, producing more than additive inhibition of growth. An intracellular inhibitor of the extrinsic apoptotic pathway, FLICE-like Inhibitory Protein-Short (FLIPs), was down-regulated and Jun kinase (JNK) was activated by exposure to GT-oligo. JNK inhibition partially reversed the growth inhibition caused by the combination of GT-oligo and TRAIL indicating partial involvement of the Jun kinase pathway in the resulting cytotoxic effect. Both capase-8 and caspases 3/7 were activated by exposure to GT-oligo plus TRAIL, consistent with activation of the extrinsic apoptotic pathway. These results demonstrate a novel way of sensitizing resistant ovarian cancer cells to TRAIL-mediated cytotoxicity.

FIG. 1.

Effect of GT-oligo exposure on death receptor expression: SKOV-3 ovarian cancer cells (A, B, C) and CAOV-3 ovarian cancer cells (D, E) were exposed to GT-oligo at 10 μM or 20 μM concentrations, for 48 hours. Real-time quantitative polymerase chain reaction was performed and the results are expressed as relative transcript expression compared with the untreated control cells. The results were normalized to β-actin transcript expression levels in each sample. (A) Death receptor (DR)-6 transcripts in SKOV-3 cells; (B) DR-4 transcripts in SKOV-3 cells; (C) DR-5 transcripts in SKOV-3 cells; (D) DR-4 transcripts in CAOV-3 cells; (E): DR-5 transcripts in CAOV-3 cells. Panel A: *P=0.008 compared with control. Panel B: *P=0.0648 compared with control; **P=0.003 compared with control. Panel C: *P=0.104 compared with control; **P=0.0104 compared with control. Panel D: *P=0.001 compared with control; **P=0.001 compared with control. Panel E: *P=0.001 compared with control; **P=0.0001 compared with control.

FIG. 2.

Ovarian cancer cells were exposed GT-oligo at 10 μM for 72 hours, harvested, fixed, and stained with FITC-conjugated DR-4 or DR-5 antibodies. After flow cytometric analysis, the histograms of DR-4 and DR-5 from GT-oligo-treated cells and vehicle-treated cells were superimposed. (A) Results from SKOV-3. (B) Results from CAOV-3 cells.

FIG. 3.

Effects of GT-oligo and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the growth of SKOV-3 and CAOV-3 ovarian cancer cells: SKOV-3 (A) and CAOV-3 (B) cells were treated for 96 hours with G-rich T-oligo (GT-oligo) at 10 μM, TRAIL at 20 ng/mL, or with the combination of GT-oligo (10 μM) and TRAIL (20 ng/mL). Live cells were enumerated. The number of % live cells in vehicle-treated cultures was normalized to 100%. (C) A dose-response experiment with SKOV-3 ovarian cancer cells was performed by exposing the cells to TRAIL at 20 or 40 ng/mL for 96 hours. Results were expressed as percentage of live cells at that time compared with control, as described in A and B. Panel A: *P=0.0001 compared with control; **P=0.006 compared with GT-oligo alone. Panel B: *P=0.0001 compared with control; **P=0.0039 compared with GT-oligo alone.

FIG. 4.

Effects of GT-oligo and TRAIL on caspase-8 and caspases 3/7 and expression levels of FLIP_S, and mitigation of effects on growth by inhibition of JNK. SKOV-3 cells were exposed to vehicle, GT-oligo (10 μM), TRAIL (20ng/mL), or to the combination of GT-oligo (10 μM) and TRAIL (20ng/mL) for 96 hours. Cells were harvested and lysed and caspase 8 (A) and caspase 3/7 (B) assays were performed on the lysates. The induction of caspase 8 and caspase 3/7 activity induced by the combination of TRAIL plus GT-oligo compared with the control or the single agents alone was significant (panel A: *P=0.004; panel B: *P=0.006). (C) SKOV-3 ovarian cancer cells were exposed to GT-oligo at 10 μM for 48 hours. Results are expressed as relative transcript expression levels compared to the untreated control cells. The results were normalized to β-actin transcript expression levels in each sample. The decrease in FLIP_S transcript level compared to control was highly significant (*P=0.0001). (D) SKOV-3 cells were exposed for 96 hours to either vehicle or GT-oligo (10 μM), protein lysates were prepared and equal amount of proteins were resolved in SDS-PAGE, transferred to membranes, and blotted against anti-phospho JNK antibody, stripped, and blotted against anti-JNK antibody. The position of bands corresponding to phospho-JNK (pJNK) and total JNK are indicated. (E) SKOV-3 cells were exposed for 96 hours to vehicle, GT-oligo (10 μM), TRAIL (20 ng/mL), SP60025, the combination of GT-oligo+TRAIL, or the combination of GT-oligo+TRAIL+SP60025. Cells were harvested and enumerated using trypan blue dye exclusion as marker of live cells. Y-axis indicates the percentage of live cells in each treatment group after normalizing to the vehicle-treated control cells, which were assigned a value of 100%. The effect of the combination of GT-oligo plus TRAIL compared with the control (*P=0.0019) and compared with the addition of SP60025 (**P=0.026) were significant.