Knockdown of MADD and c-FLIP overcomes resistance to TRAIL-induced apoptosis in ovarian cancer cells

Abstract

Objective: The clinical utility of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in the treatment of established human malignancies is limited by the development of resistance to TRAIL. We hypothesized that knockdown of map-kinase activating death domain containing protein (MADD), a TRAIL-resistance factor, may overcome TRAIL resistance in ovarian cancer cells.

Study design: MADD expression in resected ovarian cancer specimens and cell lines was quantified with the use of polymerase chain reaction. Sensitivity of ovarian cancer cell lines to TRAIL, with or without MADD knockdown, was assessed.

Results: MADD is expressed at relatively higher levels in human malignant ovarian cancer tissues and cell lines, compared with normal ovarian tissues. The cell lines OVCA429 and OVCAR3 were susceptible, and cell lines CAOV-3 and SKOV-3 were resistant to TRAIL. MADD knockdown in CAOV-3 cells, but not in SKOV-3 cells, conferred TRAIL sensitivity. Knockdown of cellular Fas-associated death domain-like interleukin-1 beta-converting enzyme-inhibitory protein (c-FLIP) in SKOV-3 cells increased spontaneous and TRAIL-induced apoptosis, which was further increased on MADD knockdown.

Conclusion: MADD/c-FLIP(L) knockdown can render TRAIL-resistant ovarian cancer cells susceptible to TRAIL.

Figure 1. Ovarian cancer tissues and cell lines

A. Detection of IG20 gene expression in ovarian cancer tissues (supplementary Table-1) and cell lines by qRT-PCR. One μg of total RNA was extracted from ovarian cancer tissues or cell lines, and subjected to qRT-PCR by using TaqMan one-step RT-PCR Master Mix Kit (Applied Biosystems, CA, USA). Primer set and probe # 1 listed in supplementary Table-3 was used, and 18srRNA primer was used as internal control. B. Expression of IG20 SVs in different ovarian cancer cell lines. One μg of total RNA was used for the RT-PCR using the Super-Script III One-Step RT-PCR system (Invitrogen Life Technologies, Carlsbad, CA, USA). F1/B1 primer set was used to amplify a region spanning exon 13L and 16 of the IG20 gene, and the GAPDH served as a loading control. C. Immunofluorescence staining shows the endogenous MADD expression in different ovarian cancer cells.

Figure 2. Susceptibility of different ovarian cancer cell lines to TRAIL- induced apoptosis

Approximately, 4 × 10⁵ of OVCA-429, OVCAR-3, CAOV-3 and SKOV-3 ovarian cancer cells were cultured in 6-well plates, and treated with different doses of TRAIL for 4 h. Subsequently, cells were fixed, permeabilized and stained with PE-conjugated anti-caspase-3 antibody for 30 min at room temperature and subjected to FACS analysis. ** P< 0.01 in OVCA-429 vs control; + P< 0.05 in OVCAR-3 vs control; ## P<0.01 in SKOV-3 vs control. Representative data are shown from three independent experiments.

Figure 3. IG20 knockdown sensitizes resistant ovarian cancer cells to TRAIL- induced apoptosis

A. Shows specific knockdown of IG20 in OVCA429, OVCAR-3, CAOV-3 and SKOV-3 ovarian cancer cells transduced with lentivirus expressing by Mid-shRNA and not the control Scr-shRNA (shRNA sequences are shown in supplementary Table-2). The GAPDH served as a loading control. Previously reported F1/B1 primer set was used. B. Western Blot shows loss of protein expression upon IG20 gene knockdown using Mid-shRNA in ovarian cancer cell lines. Representative data are shown from three independent experiments. C. Ovarian cancer cells were transduced with SCR or MID shRNA carrying lentivirus for 72 hrs. These cells were incubated with different doses of TRAIL for an additional 4 h and stained with PE-conjugated anti-caspase-3 antibody and subjected to FACS analysis. Summary of data from three independent experiments is shown.

Figure 4. Expression of death, decoy receptors and anti-apoptotic proteins in ovarian cancer cell lines

A. Shows expression levels of death receptors (DRs) and decoy receptors (DcRs) in different ovarian cancer cell lines. Representative data are shown from two independent experiments. B. Expression levels of anti-apoptotic proteins in ovarian tissues and cancer cell lines. The experiment was carried out as described under materials and methods and the primer sequences are shown in supplementary Table-2. Data shown are mean normalized delta CT from threshold cycles for the target and control samples vs 18s rRNA.

Figure 5. Down modulation of IG20 and c-FLIP expression sensitizes SKOV-3 cells to TRAIL-induced apoptosis

A. Shows efficiency of down modulation of c-FLIP (siRNA sequence used to knockdown c-FLIP is shown in supplementary Table-3). Representative data are shown from three independent experiments. B. Effect of c-FLIP and IG20 knockdown on TRAIL-induced apoptosis in TRAIL resistant SKOV-3 cell lines. Data Summarized from three independent experiments are shown.