Ionizing radiation enhances adenoviral vector expressing mda-7/IL-24-mediated apoptosis in human ovarian cancer

Abstract

Ovarian cancer is the fifth most common cause of cancer-related death in women. Current interventional approaches, including debulking surgery, chemotherapy, and/or radiation have proven minimally effective in preventing the recurrence and/or mortality associated with this malignancy. Subtraction hybridization applied to terminally differentiating human melanoma cells identified melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), whose unique properties include the ability to selectively induce growth suppression, apoptosis, and radiosensitization in diverse cancer cells, without causing any harmful effects in normal cells. Previously, it has been shown that adenovirus-mediated mda-7/IL-24 therapy (Ad.mda-7) induces apoptosis in ovarian cancer cells, however, the apoptosis induction was relatively low. We now document that apoptosis can be enhanced by treating ovarian cancer cells with ionizing radiation (IR) in combination with Ad.mda-7. Additionally, we demonstrate that mda-7/IL-24 gene delivery, under the control of a minimal promoter region of progression elevated gene-3 (PEG-3), which functions selectively in diverse cancer cells with minimal activity in normal cells, displays a selective radiosensitization effect in ovarian cancer cells. The present studies support the use of IR in combination with mda-7/IL-24 as a means of augmenting the therapeutic benefit of this gene in ovarian cancer, particularly in the context of tumors displaying resistance to radiation therapy.

Fig. 1

GFP, Luc, and MDA-7/IL-24 expression following infection of normal mesothelial and ovarian carcinoma cells with various adenoviruses and de novo levels of Jun, PEA-3, and EF-1α protein in mesothelial and ovarian carcinoma cells. A: Variable infectivity of ovarian cancer cells by adenovirus as determined by GFP expression. The indicated cells were infected with either Ad.CMV-GFP or Ad.PEG-GFP at a moi of 100 pfu/cell, and GFP expression was analyzed by fluorescence microscopy at 2d post-infection. B: Upper part: PEG-promoter drives high luciferase expression in ovarian cancer cells, but not in normal human mesothelial cells. The indicated cells were infected with Ad.PEG-luc at a moi of 100 pfu/cell, and luciferase activity was measured at 48 h post-infection and expressed as RLU/mg protein. The data represent the mean ±SD of three independent experiments, each performed in triplicate. Lower part: The expressions of JUN, PEA-3, and EF-1α protein were analyzed by Western blot analysis in ovarian cancer cells and normal human mesothelial cells. C: Infection of ovarian cancer cells and normal human mesothelial cells with Ad.CMV-mda-7 or Ad.PEG-mda-7 results in variable amounts of intracellular MDA-7/IL-24 protein. Cells were infected with the indicated viruses at a moi of 100 pfu/cell and total cell lysates were prepared at d 1, d 2, and d 3 post-infection. The cell lysate used for Ad.vec was the lysate prepared at d 3 post-infection. The expression of MDA-7/IL-24 and EF-1α proteins were evaluated by Western blot analysis.

Fig. 2

Effect of infection of normal mesothelial and ovarian carcinoma cells with Ad.CMV-mda-7 or Ad.PEG-mda-7 on cell viability and apoptosis induction. A: Infection with Ad.CMV-mda-7 or Ad.PEG-mda-7 results in a decrease in cell viability in ovarian cancer cells. Cells were infected with the indicated viruses and cell viability was assessed by MTT assay at 3 d and 6 d post-infection. A significant loss in cell viability was evident in the SKOV3 and OV-4 cell lines. Results are the average from at least three experiments ±SD. B: Infection with Ad.CMV-mda-7 or Ad.PEG-mda-7 variably induces apoptosis in different ovarian cancer cells. The indicated cell type was infected with the designated viruses for 72 h, and Annexin-V binding assay was performed as described in Materials and Methods. Results are the average from at least three experiments ±SD.

Fig. 3

Infection with Ad.CMV-mda-7 or Ad.PEG-mda-7 induces p38 MAPK phosphorylation and treatment with SB203580 protects ovarian cancer cells from mda-7-mediated apoptosis. A: Cells were infected with the indicated viruses as described in Figure 1C. Total cellular extracts were prepared at 48 h post-infection and the levels of phospho-p38 and total p38 MAPK were analyzed by Western blot analysis. B: Cells were infected with the indicated viruses and treated with 1 μM SB203580. Cell viability was assessed by MTT assay at 4 d post-infection. Results are the average from at least three experiments ±SD.

Fig. 4

Combination of Ad.CMV-mda-7 or Ad.PEG-mda-7 plus IR induces enhanced killing and apoptosis in ovarian cancer cells. A: Effect of single and combination treatment with Ad. CMV-mda-7 or Ad.PEG-mda-7 plus IR on apoptosis induction in ovarian cancer cells. The indicated cell type was either uninfected or infected with Ad.vec, Ad.CMV-mda-7, or Ad.PEG-mda-7 and the next day was exposed to 2 Gy of IR. An Annexin-V binding assay was performed at 48 h post-irradiation as described in Materials and Methods. Results are the average from at least three experiments ±SD. B: Effect of combination treatment with Ad.CMV-mda-7 or Ad.PEG-mda-7 plus IR on growth of ovarian cancer cells. Cells were either uninfected or infected and untreated or treated with IR as described in Figure 4A and colony forming assays were performed as described in the Materials and Methods section. Data are presented as a percentage of colony formation to that of the uninfected and untreated group. Results shown are averages ±SD of triplicate samples.

Fig. 5

Combination treatment with mda-7/IL-24 and IR reverses the translational block in ovarian cancer cells. A: Infection of Ad.CMV-mda-7 induces an abundant amount of MDA-7/IL-24 RNA. Total RNA was isolated at d 1, d 2, and d 3 after infection as described in Materials and Methods, and analyzed by Northern blotting. The blots were probed with a α-³²P[dCTP]-labeled, full-length human mda-7/IL-24 cDNA probe and exposed for autoradiography. B: Combination treatment of IR plus MDA-7/IL-24 enhances mda-7/IL-24 protein. Cells were infected with Ad.vec, Ad.CMV-mda-7, or Ad.PEG-mda-7 and untreated or treated with IR the next day. Protein lysates were prepared 24 h after IR and Western blotting was performed as described in Materials and Methods to determine the levels of MDA-7/IL-24 protein. Equal protein loading was confirmed by Western blotting with an EF-1α antibody.