The present studies defined the biological effects of a GST fusion protein of melanoma differentiation-associated gene-7 (mda-7), GST-MDA-7 (1 and 30 nmol/L), on cell survival and cell signaling in primary human glioma cells in vitro. GST-MDA-7, in a dose- and time-dependent fashion killed glioma cells with diverse genetic characteristics; 1 nmol/L caused arrest without death, whereas 30 nmol/L caused arrest and killing after exposure. Combined inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT function was required to enhance 1 nmol/L GST-MDA-7 lethality in all cell types, whereas combined activation of MEK1 and AKT was required to suppress 30 nmol/L GST-MDA-7 lethality; both effects are mediated in part by modulating c-Jun NH2-terminal kinase (JNK) 1-3 activity. The geldanamycin 17AAG inhibited AKT and ERK1/2 in GBM cells and enhanced GST-MDA-7 lethality. JNK1-3 signaling promoted BAX activation and mitochondrial dysfunction. In GBM6 cells, GST-MDA-7 (30 nmol/L) transiently activated p38 mitogen-activated protein kinase, which was modestly protective against JNK1-3-induced toxicity, whereas GST-MDA-7 (300 nmol/L) caused prolonged intense p38 mitogen-activated protein kinase activation, which promoted cell death. In GBM12 cells that express full-length mutant activated ERBB1, inhibition of ERBB1 did not modify GST-MDA-7 lethality; however, in U118 established glioma cells, stable overexpression of wild-type ERBB1 and/or truncated active ERBB1vIII suppressed GST-MDA-7 lethality. Our data argue that combined inhibition of ERK1/2 and AKT function, regardless of genetic background, promotes MDA-7 lethality in human primary human glioma cells via JNK1-3 signaling and is likely to represent a more ubiquitous approach to enhancing MDA-7 toxicity in this cell type than inhibition of ERBB1 function. [Mol Cancer Ther 2008;7(2):314–29]

Glioblastoma multiforme (GBM) is diagnosed in ∼20,000 patients per annum in the United States, with anaplastic astrocytoma and GBM accounting for the majority of astrocytic tumors (1, 2). Despite surgical removal of the tumor and intensive use of radiation and chemotherapy, the mean survival of this disease is only in general extended by these interventions from 2 to 3 months to 1 year (15).

The gene for mda-7 [interleukin (IL)-24], an IL-10 family member (612), was isolated from human melanoma cells induced to undergo terminal differentiation by treatment with IFN-β and mezerein (6). In agreement with MDA-7/IL-24 acting as a tumor suppressor, MDA-7/IL-24 protein expression is decreased in advanced melanomas with nearly undetectable levels in metastatic disease (6, 13, 14). Enforced expression of MDA-7/IL-24 inhibits the growth of transformed cells and ultimately causes cell death, without apparently exerting deleterious effects in normal human epithelial cells, fibroblast cells, melanocytes, and astrocytes (6, 1426). The mechanisms by which Ad.mda-7 induces tumor cell death are not fully understood; however, several studies suggest the involvement of proteins important for the onset of growth inhibition and apoptosis, including BCL-2 family members (1626). For example, in melanoma, malignant glioma, and prostate and breast cancer cell lines but not in normal melanocytes, astrocytes, or prostate or breast epithelial cells, respectively, infected with Ad.mda-7, the ratio of proapoptotic to antiapoptotic proteins in cancer cells changes in favor of cell death, thereby facilitating induction of apoptosis (12, 1620, 25, 26). In prostate cancer lines, overexpression of either BCL-2 or BCL-xL in a cell type–dependent fashion can protect cells from Ad.mda-7 toxicity (24, 26). Thus, MDA-7/IL-24 lethality can occur by different pathways in different cell types. More recently, MDA-7 toxicity has been linked to alterations in homeostatic endoplasmic reticulum stress signaling as judged by its binding to BiP/GRP78 (27).

The ability of MDA-7/IL-24 to modulate cell signaling processes in transformed cells has been investigated by several groups, with the majority of studies presenting data from adenoviral transduction and expression of the MDA-7/IL-24 transgene in the infected cell population. Our laboratories have presented evidence that Ad.mda-7 kills established glioma and melanoma cells in part by promoting p38 mitogen-activated protein kinase (MAPK)–dependent expression of GADD153 (25, 28). Other groups have argued that inhibition of phosphatidylinositol-3-kinase (PI3K) signaling, but not extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, modestly promotes Ad.mda-7 lethality in breast and lung cancer cells (29, 30). Prior studies using purified MDA-7/IL-24 proteins have tended to treat tumor cells with high nanomolar to micromolar concentrations of the cytokine in studies to modulate tumor cell biology, which are unlikely to be therapeutically achievable in vivo. Prior work by our groups has shown, using bacterially synthesized GST-MDA-7 protein, that in the 0.25 to 2.0 nmol/L concentration range GST-MDA-7 primarily causes growth arrest with little cell killing, whereas at ∼20-fold greater concentrations the cytokine causes profound growth arrest and a delayed induction of tumor cell death, 48 to 96 h after exposure (3133). The toxicity of low nanomolar GST-MDA-7 concentrations were elevated by multiple agents that generate reactive oxygen species, which correlated with prolonged activation of the c-Jun NH2-terminal kinase (JNK) 1/2 pathway that was linked to tumor cell killing. However, the dose-dependent effects of GST-MDA-7 on tumor cell signaling processes have not been studied in any cell line in a detailed manner, and the relative abilities of low (∼1 nmol/L) and higher (∼30 nmol/L) GST-MDA-7 concentrations to alter signaling pathway activities in tumor cells, and the relevance of any changes in cell signaling processes to cell survival, are presently unknown.

Using primary human GBM isolates, maintained in vivo and cultured in vitro, and established human glioma cell lines, we examined the effect of GST-MDA-7 on cell viability and cell signaling and linked changes in cell signaling to some of the molecular mechanisms by which GST-MDA-7 enhances glioma cell death.

Materials

Antibody reagents, kinase inhibitors, caspase inhibitors cell culture reagents, primary human GBM cells, and noncommercial recombinant adenoviruses, which were kindly provided by Dr. K. Valerie (Virginia Commonwealth University) and Dr. J. Moltken (University of Cincinnati), have been previously described in refs. 1820, 2428, 3135.

Methods

Generation of Ad.mda-7 and Synthesis of GST-MDA-7. Recombinant type V adenovirus to express MDA-7 (Ad.mda-7), control (CMV vector), or control (β-galactosidase) were generated using recombination in HEK293 cells as described in refs. 1820, 2428, 32.

Culture and In vitro Exposure of Cells to GST-MDA-7 and Drugs. All established cell lines (U251 and U118) were cultured at 37°C [5% (v/v) CO2] in vitro using RPMI supplemented with 5% (v/v) FCS and 10% (v/v) nonessential amino acids. Primary human glioma cells were initially subcultured following isolation from the animal in 2% (v/v) FCS to prevent growth of contaminating rodent fibroblasts for 1 week before in vitro analyses, after which cells were cultured in 5% (v/v) FCS. For short-term cell killing assays and immunoblotting, cells were plated at a density of 3 × 103 per cm2 and 36 h after plating treated with GST-MDA-7 and/or various drugs as indicated. Cells were not cultured in reduced/no serum medium during any study in this research.

Cell Treatments, SDS-PAGE, and Western Blot Analysis. Cells were treated with various GST-MDA-7 concentrations as indicated in the figure legend. For SDS-PAGE and immunoblotting, cells were lysed in a nondenaturing lysis buffer and prepared for immunoprecipitation as described in refs. 2433.

Recombinant Adenoviral Vectors: Infection In vitro. We generated and purchased previously noted recombinant adenoviruses to express constitutively activated and dominant-negative AKT (dnAKT) and MEK1 (dnMEK1) proteins, dominant-negative caspase-9 (dncasp.9), XIAP, c-FLIP-s, CRM A, and BCL-xL (Vector Biolabs). Cells were infected with these adenoviruses at an approximate multiplicities of infection (MOI) of 50. As noted above, cells were further incubated for 24 h to ensure adequate expression of transduced gene products before drug exposures.

Detection of Cell Death by Trypan Blue, Hoechst, Terminal Deoxynucleotidyl Transferase–Mediated dUTP Nick End Labeling, and Flow Cytometric Assays. Cells were harvested by trypsinization with trypsin/EDTA for ∼10 min at 37°C. As some apoptotic cells detached from the culture substratum into the medium, these cells were also collected by centrifugation of the medium at 1,500 rpm for 5 min. The pooled cell pellets were resuspended and mixed with trypan blue dye. Trypan blue stain, in which blue dye incorporating cells were scored as being dead, was done by counting of cells using a light microscope and a hemacytometer. Five hundred cells from randomly chosen fields were counted and the number of dead cells was counted and expressed as a percentage of the total number of cells counted. For confirmatory purposes, the extent of apoptosis was evaluated by assessing Hoechst-stained and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling–stained cytospin slides under fluorescent light microscopy and scoring the number of cells exhibiting the “classic” morphologic features of apoptosis and necrosis. For each condition, 10 randomly selected fields per slide were evaluated, encompassing at least 1,500 cells. Alternatively, the Annexin V/propidium iodide assay was carried to determine cell viability out as per the manufacturer’s instructions (BD PharMingen) using a Becton-Dickinson FACScan flow cytometer (18, 32, 34).

Cell Survival Analyses. Cells were assayed for the effect(s) of GST-MDA-7 cell survival. Cells were plated (250-2,000 per 60-mm dish) and 12 h after plating treated with GST or GST-MDA-7. Ninety-six hours after GST-MDA-7 treatment, cells were washed with fresh medium and permitted to continue growing for 20 to 28 days in the absence of GST or GST-MDA-7 (32).

Preparation of S-100 Fractions and Assessment of Cytochrome c Release. Cells were harvested after GST-MDA-7 treatment as described previously by centrifugation at 600 rpm for 10 min at 4°C and washed in PBS. Cells (∼1 × 106) were lysed by incubation for 3 min in 100 μL lysis buffer containing 75 mmol/L NaCl, 8 mmol/L Na2HPO4, 1 mmol/L NaH2PO4, 1 mmol/L EDTA, and 350 μg/mL digitonin. The lysates were centrifuged at 12,000 rpm for 5 min, and the supernatant was collected and added to an equal volume of 2× Laemmli buffer. The protein samples were quantified and separated by 15% SDS-PAGE.

Data Analysis. Comparison of the effects of various treatments was done using one-way ANOVA and a two-tailed Student’s t test. Differences with P < 0.05 were considered statistically significant. Experiments shown are the individual experimental mean ± SE (of multiple individual data points) from multiple separate experiments.

The effect of low (∼1 nmol/L), intermediate (∼5 nmol/L), and high (∼30 nmol/L) GST-MDA-7 concentrations on cell signaling and viability in primary human glioma cells expressing activated ERBB1vIII (GBM6) and mutant active wild-type ERBB1 (GBM12) was determined (Fig. 1A-C). Low concentrations of GST-MDA-7 had a modest effect on cell viability 0 to 96 h after exposure as judged by caspase-3 cleavage, poly(ADP-ribose) polymerase cleavage, trypan blue staining, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling staining, and Hoechst staining; however, low concentrations of GST-MDA-7 significantly suppressed cell growth as judged by cell counting and by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays (Fig. 1B and C; data not shown). Higher concentrations of GST-MDA-7 promoted significant higher amounts of cell killing that were manifested 48 to 96 h after treatment, which correlated with cleavage of caspase-3 and poly(ADP-ribose) polymerase, and a significantly greater suppression of cell numbers than were observed with lower cytokine doses (Fig. 1A-C). Near identical data to that in GBM6 and GBM12 cells were obtained in GBM5 cells that express a constitutively active PI3K protein (Supplementary Fig. S1).7

7

Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

Figure 1.

GST-MDA-7 causes a dose- and time-dependent induction of primary human glioma cell death. A, primary human glioma cells (GBM6) were treated with 1, 5, or 30 nmol/L GST-MDA-7 or GST 36 h after plating. Cell viability was determined 48, 72, and 96 h after GST-MDA-7 treatment by trypan blue exclusion assays in triplicate using a hemacytometer (±SE; n = 5; *, P < 0.05, greater cell killing than a 30 nmol/L GST exposure). Inset, cells 96 h after GST-MDA-7 treatment were immunoblotted for the appearance of the cleaved form of caspase-3. B, cells were treated with 1, 5, or 30 nmol/L GST-MDA-7 or GST 36 h after plating. Total cell numbers were determined 96 h after GST-MDA-7 treatment in triplicate by hemacytometer (±SE; n = 5; #, P < 0.05, less cell numbers than GST control). Inset, right, top, cells 96 h after GST-MDA-7 treatment were immunoblotted for the appearance of the cleaved form of poly(ADP-ribose) polymerase. Bottom, cells 96 h after GST-MDA-7 treatment were fixed to glass slides and stained (terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling for DNA double-strand breaks; Hoechst for nuclear condensation). Total apoptotic/dead cell numbers were determined 96 h after GST-MDA-7 treatment in triplicate by fluorescent light microscopy (±SE; n = 2). C, primary human glioma cells (GBM12) were treated with 1, 5, or 30 nmol/L GST-MDA-7 or GST 36 h after plating. Cell viability was determined 48, 72, and 96 h after GST-MDA-7 treatment by trypan blue exclusion assays in triplicate using a hemacytometer (±SE; n = 5; *, P < 0.05, greater cell killing than a 30 nmol/L GST exposure). Inset, top left, cells, 96 h after GST-MDA-7 treatment, were immunoblotted for the appearance of the cleaved form of caspase-3. Inset, right, cells were treated with 1, 5, or 30 nmol/L GST-MDA-7 or GST, 36 h after plating. Total cell numbers were determined 96 h after GST-MDA-7 treatment in triplicate by hemacytometer (±SE; n = 5; #, P < 0.05, less cell numbers than GST control). Immunoblotting, cells 96 h after GST-MDA-7 treatment were immunoblotted for the appearance of the cleaved form of poly(ADP-ribose) polymerase.

Figure 1.

GST-MDA-7 causes a dose- and time-dependent induction of primary human glioma cell death. A, primary human glioma cells (GBM6) were treated with 1, 5, or 30 nmol/L GST-MDA-7 or GST 36 h after plating. Cell viability was determined 48, 72, and 96 h after GST-MDA-7 treatment by trypan blue exclusion assays in triplicate using a hemacytometer (±SE; n = 5; *, P < 0.05, greater cell killing than a 30 nmol/L GST exposure). Inset, cells 96 h after GST-MDA-7 treatment were immunoblotted for the appearance of the cleaved form of caspase-3. B, cells were treated with 1, 5, or 30 nmol/L GST-MDA-7 or GST 36 h after plating. Total cell numbers were determined 96 h after GST-MDA-7 treatment in triplicate by hemacytometer (±SE; n = 5; #, P < 0.05, less cell numbers than GST control). Inset, right, top, cells 96 h after GST-MDA-7 treatment were immunoblotted for the appearance of the cleaved form of poly(ADP-ribose) polymerase. Bottom, cells 96 h after GST-MDA-7 treatment were fixed to glass slides and stained (terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling for DNA double-strand breaks; Hoechst for nuclear condensation). Total apoptotic/dead cell numbers were determined 96 h after GST-MDA-7 treatment in triplicate by fluorescent light microscopy (±SE; n = 2). C, primary human glioma cells (GBM12) were treated with 1, 5, or 30 nmol/L GST-MDA-7 or GST 36 h after plating. Cell viability was determined 48, 72, and 96 h after GST-MDA-7 treatment by trypan blue exclusion assays in triplicate using a hemacytometer (±SE; n = 5; *, P < 0.05, greater cell killing than a 30 nmol/L GST exposure). Inset, top left, cells, 96 h after GST-MDA-7 treatment, were immunoblotted for the appearance of the cleaved form of caspase-3. Inset, right, cells were treated with 1, 5, or 30 nmol/L GST-MDA-7 or GST, 36 h after plating. Total cell numbers were determined 96 h after GST-MDA-7 treatment in triplicate by hemacytometer (±SE; n = 5; #, P < 0.05, less cell numbers than GST control). Immunoblotting, cells 96 h after GST-MDA-7 treatment were immunoblotted for the appearance of the cleaved form of poly(ADP-ribose) polymerase.

Close modal

Based on these observations, we next explored the effect 1 to 30 nmol/L GST-MDA-7 had on the activities of multiple signal transduction pathways in primary human GBM cells over a 72-h time course as assessed by the phosphorylation of individual protein kinases. Treatment of GBM6 and GBM12 cells with 1 nmol/L GST-MDA-7 in a cell type–dependent fashion modestly suppressed or enhanced ERK1/2 and AKT activity and modestly enhanced p38 MAPK and JNK1-3 signaling (Fig. 2A and B; cf. data in ref. 32). Treatment of these cells with 30 nmol/L GST-MDA-7 abolished ERK1/2 (GBM6) or AKT (GBM12) activity and more strongly promoted phosphorylation of JNK1-3 and p38 MAPK. JNK1-3 activity, as measured using immunoprecipitation of the enzyme and a 96-well colorimetric plate assay, was also noted to be increased in GBM6 cells in a dose-dependent fashion (data not shown). GBM5 cells responded to GST-MDA-7 exposure in a similar manner to GBM6 cells (Supplementary Fig. S1, inset).7 An intermediate GST-MDA-7 concentration of 5 nmol/L, which caused a similar level of toxicity to that observed in cells treated with 1 nmol/L GST-MDA-7, enhanced JNK1-3 and p38 MAPK activity to the same extent as that observed in cells treated with 30 nmol/L GST-MDA-7, but in contrast to a 30 nmol/L GST-MDA-7 exposure, treatment with 5 nmol/L GST-MDA-7 did not profoundly suppress ERK1/2 or AKT signaling (Fig. 2; cf. data in Fig. 1).

Figure 2.

In primary human GBM cells, GST-MDA-7 causes a dose- and time-dependent inactivation of ERK1/2 and/or AKT and a dose-dependent activation of JNK1-3 and p38 MAPK. A, primary human glioma cells (GBM6) were treated with 1, 5, and 30 nmol/L GST-MDA-7 or with 5 and 30 nmol/L GST 36 h after plating. Cells were isolated at the indicated time points and 100 μg protein at each time point was subjected to SDS-PAGE on 12% gels followed by immunoblotting to determine the phosphorylation status of ERK1/2, AKT (S473), ERK5, p38α/β MAPK, JNK1-3, and total expression of ERK2 (n = 2 for the full-time course and n = 4 for selected 48- and 72-h time points). B, primary human glioma cells (GBM12) were treated with 1, 5, and 30 nmol/L GST-MDA-7 or with 5 and 30 nmol/L GST 36 h after plating. Cells were isolated at the indicated time points and 100 μg protein at each time point was subjected to SDS-PAGE on 12% gels followed by immunoblotting to determine the phosphorylation status of ERK1/2, AKT (S473), ERK5, p38α/β MAPK, JNK1-3, and total expression of ERK2 (n = 2 for the full-time course and n = 4 for selected 48- and 72-h time points). C, GBM6 cells were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing (CMV), dominant-negative Cdc42 N17, dominant-negative MEKK1, dominant-negative RhoA N19, dominant-negative Ras N17, dominant-negative Rac1 N17, dominant-negative MKK4, dominant-negative MKK7, and dominant-negative MKK3 as indicated. Cells were cultured for 24 h after infection and then treated with GST or GST-MDA-7 (30 nmol/L). Cells were isolated 48 h after GST-MDA-7 treatment and total cell lysates were subjected to SDS-PAGE and immunoblotting to determine the phosphorylation of JNK1-3 and the total expression of ERK2 and β-actin (n = 2). Bottom, a possible JNK signaling pathway induced by GST-MDA-7 in glioma cells.

Figure 2.

In primary human GBM cells, GST-MDA-7 causes a dose- and time-dependent inactivation of ERK1/2 and/or AKT and a dose-dependent activation of JNK1-3 and p38 MAPK. A, primary human glioma cells (GBM6) were treated with 1, 5, and 30 nmol/L GST-MDA-7 or with 5 and 30 nmol/L GST 36 h after plating. Cells were isolated at the indicated time points and 100 μg protein at each time point was subjected to SDS-PAGE on 12% gels followed by immunoblotting to determine the phosphorylation status of ERK1/2, AKT (S473), ERK5, p38α/β MAPK, JNK1-3, and total expression of ERK2 (n = 2 for the full-time course and n = 4 for selected 48- and 72-h time points). B, primary human glioma cells (GBM12) were treated with 1, 5, and 30 nmol/L GST-MDA-7 or with 5 and 30 nmol/L GST 36 h after plating. Cells were isolated at the indicated time points and 100 μg protein at each time point was subjected to SDS-PAGE on 12% gels followed by immunoblotting to determine the phosphorylation status of ERK1/2, AKT (S473), ERK5, p38α/β MAPK, JNK1-3, and total expression of ERK2 (n = 2 for the full-time course and n = 4 for selected 48- and 72-h time points). C, GBM6 cells were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing (CMV), dominant-negative Cdc42 N17, dominant-negative MEKK1, dominant-negative RhoA N19, dominant-negative Ras N17, dominant-negative Rac1 N17, dominant-negative MKK4, dominant-negative MKK7, and dominant-negative MKK3 as indicated. Cells were cultured for 24 h after infection and then treated with GST or GST-MDA-7 (30 nmol/L). Cells were isolated 48 h after GST-MDA-7 treatment and total cell lysates were subjected to SDS-PAGE and immunoblotting to determine the phosphorylation of JNK1-3 and the total expression of ERK2 and β-actin (n = 2). Bottom, a possible JNK signaling pathway induced by GST-MDA-7 in glioma cells.

Close modal

Additional studies then attempted to further delineate the pathway by which JNK1-3 became activated. Expression of dominant-negative MEKK1 abolished GST-MDA-7-induced activation of JNK1-3 in GBM6 and GBM12 cells (Fig. 2C). Expression of dominant-negative RhoA N19 also abolished JNK1-3 activation by GST-MDA-7, whereas expression of dominant-negative Rac1 N17/Cdc42 N17/Ras N17 had no effect on JNK1-3 activation (Fig. 2C). Expression of dominant-negative MKK4 or MKK7 weakly suppressed JNK1-3 activation, whereas combined expression of dominant-negative MKK4 and MKK7 abolished GST-MDA-7-induced activation of JNK1-3 in GBM6 cells (Fig. 2C).

Further analyses then determined using molecular approaches whether any of the alterations in GBM cell viability were causally linked to altered signaling pathway function(s) in cells treated with 30 nmol/L GST-MDA-7. In GBM6 and GBM12 cells, combined inhibition of MEK1 and AKT function promoted the lethality of GST-MDA-7 (Fig. 3A and B). In GBM6 and GBM12 cells, activation of AKT or MEK1 variably and modestly suppressed GST-MDA-7 lethality in the primary GBM cells, whereas activation of both AKT and MEK1 was required to promote a potent antiapoptotic effect regardless of the cell type under investigation (Fig. 3). In all GBM cells, inhibition of JNK1-3 signaling suppressed GST-MDA-7 toxicity; surprisingly, based on prior work in established tumor cell lines, wherein p38 MAPK signaling was toxic, expression of dominant-negative p38α MAPK (dnp38α) enhanced basal levels of GBM cell morbidity as well as GST-MDA-7 lethality: the enhanced level of killing observed in cells expressing dnp38α was abolished by inhibition of JNK1-3 function. In agreement with the hypothesis that the majority of cell killing was being mediated via the intrinsic apoptosis pathway, expression of dncasp.9 and overexpression of BCL-xL suppressed GST-MDA-7 lethality (Fig. 3A-C). Identical data to that observed in GBM6 and GBM12 cells were obtained in GBM5 cells (Supplementary Fig. S2).7

Figure 3.

Modulation of GST-MDA-7 (30 nmol/L) toxicity by manipulation of AKT, MEK1, JNK1-3, and p38 MAPK activities. A, primary human glioma cells (GBM6) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing, dnAKT, constitutively active AKT (caAKT), dnMEK1, constitutively active MEK1 (caMEK1), dnp38α, dncasp.9, and the antiapoptotic protein BCL-xL. Cells were cultured for 24 h after infection and then treated with the JNK inhibitory peptide based on sequence from JIP-1 (10 μmol/L; JNK-IP) 30 min before addition of GST or GST-MDA-7 (30 nmol/L). Cells were isolated 72 h after GST-MDA-7 treatment and viability was assessed in triplicate by trypan blue assay (±SE; n = 3-4; *, P < 0.05, greater than vector control-infected GST-MDA-7 exposure; #, P < 0.05, lower than vector control-infected GST-MDA-7 exposure; ##, P < 0.05, lower than either caAKT- or caMEK1-infected cell GST-MDA-7 exposure). Inset, 24 h after infection, the activities of AKT (S473) and ERK1/2 were assessed by immunoblotting in infected GBM6 cells (n = 2). B, primary human glioma cells (GBM12) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing, dnAKT, caAKT, dnMEK1, caMEK1, dnp38α, dncasp.9, and the antiapoptotic protein BCL-xL. Cells were cultured for 24 h after infection and then treated with the JNK inhibitory peptide based on sequence from JIP-1 (10 μmol/L) 30 min before addition of GST or GST-MDA-7 (30 nmol/L). Cells were isolated 72 h after GST-MDA-7 treatment and viability was assessed in triplicate by trypan blue assay (±SE; n = 3; *, P < 0.05, greater than vector control-infected GST-MDA-7 exposure; #, P < 0.05, lower than vector control-infected GST-MDA-7 exposure; ##, P < 0.05, lower than either caAKT- or caMEK1-infected cell GST-MDA-7 exposure). Inset, 24 h after infection, the activities of AKT (S473) and ERK1/2 were assessed by immunoblotting in infected GBM12 cells (n = 2). C, primary human glioma cells (GBM6) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing or dncasp.9. Cells were cultured for 24 h after infection and then treated with the JNK inhibitory peptide based on sequence from JIP-1 (10 μmol/L) 30 min before addition of GST or GST-MDA-7 (30 nmol/L). Cells were isolated 72 h after GST-MDA-7 treatment and viability was assessed in triplicate by Annexin V/propidium iodide staining and flow cytometry. Data are expressed as the true percentage of cell death above that observed in Ad.cmv-infected GST-treated cells (±SE; n = 3).

Figure 3.

Modulation of GST-MDA-7 (30 nmol/L) toxicity by manipulation of AKT, MEK1, JNK1-3, and p38 MAPK activities. A, primary human glioma cells (GBM6) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing, dnAKT, constitutively active AKT (caAKT), dnMEK1, constitutively active MEK1 (caMEK1), dnp38α, dncasp.9, and the antiapoptotic protein BCL-xL. Cells were cultured for 24 h after infection and then treated with the JNK inhibitory peptide based on sequence from JIP-1 (10 μmol/L; JNK-IP) 30 min before addition of GST or GST-MDA-7 (30 nmol/L). Cells were isolated 72 h after GST-MDA-7 treatment and viability was assessed in triplicate by trypan blue assay (±SE; n = 3-4; *, P < 0.05, greater than vector control-infected GST-MDA-7 exposure; #, P < 0.05, lower than vector control-infected GST-MDA-7 exposure; ##, P < 0.05, lower than either caAKT- or caMEK1-infected cell GST-MDA-7 exposure). Inset, 24 h after infection, the activities of AKT (S473) and ERK1/2 were assessed by immunoblotting in infected GBM6 cells (n = 2). B, primary human glioma cells (GBM12) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing, dnAKT, caAKT, dnMEK1, caMEK1, dnp38α, dncasp.9, and the antiapoptotic protein BCL-xL. Cells were cultured for 24 h after infection and then treated with the JNK inhibitory peptide based on sequence from JIP-1 (10 μmol/L) 30 min before addition of GST or GST-MDA-7 (30 nmol/L). Cells were isolated 72 h after GST-MDA-7 treatment and viability was assessed in triplicate by trypan blue assay (±SE; n = 3; *, P < 0.05, greater than vector control-infected GST-MDA-7 exposure; #, P < 0.05, lower than vector control-infected GST-MDA-7 exposure; ##, P < 0.05, lower than either caAKT- or caMEK1-infected cell GST-MDA-7 exposure). Inset, 24 h after infection, the activities of AKT (S473) and ERK1/2 were assessed by immunoblotting in infected GBM12 cells (n = 2). C, primary human glioma cells (GBM6) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing or dncasp.9. Cells were cultured for 24 h after infection and then treated with the JNK inhibitory peptide based on sequence from JIP-1 (10 μmol/L) 30 min before addition of GST or GST-MDA-7 (30 nmol/L). Cells were isolated 72 h after GST-MDA-7 treatment and viability was assessed in triplicate by Annexin V/propidium iodide staining and flow cytometry. Data are expressed as the true percentage of cell death above that observed in Ad.cmv-infected GST-treated cells (±SE; n = 3).

Close modal

Based on the above findings, we did studies to determine how or whether signaling pathway cross-talk occurred in the control of cell viability in cells treated with GST-MDA-7. In GBM6 and GBM12 cells, treatment with 30 nmol/L GST-MDA-7 suppressed ERK1/2 and AKT activity, respectively (Fig. 4A; cf. data in Fig. 2). Although inhibition of JNK1-3 signaling protected GBM6 and GBM12 cells against the toxic effects of GST-MDA-7, it did not restore ERK1/2 and AKT activity to basal levels. These findings are in contrast to our prior work in which we combined 1 nmol/L GST-MDA-7 and ionizing radiation exposure, where inactivation of ERK1/2 was JNK1-3 dependent and caspase-3 dependent (30). Expression of dominant-negative p38 MAPK enhanced basal levels of cell death as well as 30 nmol/L GST-MDA-7 toxicity, which in the case of basal viability levels correlated with modest inactivation of AKT, and in the case of GST-MDA-7 toxicity correlated with a weak (GBM12) and stronger (GBM6) promotion of JNK1-3 activation, which was causal in cell killing (Fig. 4A; cf. data in Fig. 3). Activation of JNK1-3 has been linked by many groups to the induction of mitochondrial dysfunction, and we examined the regulation of BAX activity in GST-MDA-7-treated glioma cells. GST-MDA-7 (30 nmol/L) activated BAX in a JNK1-3-dependent fashion and promoted cell killing: constitutive activation of MEK1 and AKT suppressed MDA-7/IL-24-induced activation of JNK1-3 and of BAX and suppressed cell killing (Fig. 4B). To confirm a role for JNK1-3 signaling in long-term cell survival, we did colony formation assays; a 96-h treatment of GBM12 or U251 glioma cells with GST-MDA-7 (30 nmol/L) suppressed colony formation 14 to 21 days after exposure, an effect that was blocked by inhibition of JNK1-3 signaling and by inhibition of caspase-9 function (Fig. 4C).

Figure 4.

Modulation of GST-MDA-7 (30 and 300 nmol/L) toxicity by manipulation of AKT, MEK1, JNK1-3, and p38 MAPK activities. A, primary human glioma cells (GBM6 and GBM12) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing or dnp38α. Cells were cultured for 24 h after infection and then treated with the JNK inhibitory peptide based on sequence from JIP-1 (10 μmol/L) 30 min before addition of GST or GST-MDA-7 (30 nmol/L). Cells were isolated 48 h after GST-MDA-7 treatment and the activities of JNK1-3, p38 MAPK, AKT (S473), and ERK1/2 were assessed by immunoblotting in infected GBM6 and GBM12 cells (a representative study from n = 3). B,bottom, primary human glioma cells (GBM6) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing, caAKT, and caMEK1 as indicated. Cells were cultured for 24 h after infection and then treated with the JNK inhibitory peptide based on sequence from JIP-1 (10 μmol/L) 30 min before addition of GST or GST-MDA-7 (30 nmol/L). Cells were isolated 24 h after GST-MDA-7 treatment and cell lysates were split into two portions. In one portion, equal amounts of protein lysate were subjected to immunoprecipitation using the anti-active BAX 6A7 antibody. Immunoprecipitates were subjected to SDS-PAGE and immunoblotted for BAX. In the second portion, equal amounts of protein lysate were subjected to SDS-PAGE and immunoblotted for phosphorylated JNK1-3 (P-JNK1-3), BCL-xL, cleaved caspase-3, BAX, and ERK2. Data are from a representative experiment (n = 3). Top, primary human glioma cells (GBM6) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing or caAKT and caMEK1. Cells were cultured for 24 h after infection and then treated with GST or GST-MDA-7 (30 nmol/L). Cells were isolated 72 h after GST-MDA-7 treatment and viability was assessed in triplicate by Annexin V/propidium iodide staining and flow cytometry. Data are expressed as the true percentage of cell death above that observed in Ad.cmv-infected GST-treated cells (±SE; n = 3). C, primary human glioma cells (GBM12) and established human glioma cells (U251) were infected with either a vector control adenovirus (Ad.cmv; CMV) or an adenovirus to express dncasp.9. Twenty-four hours after infection, cells were plated in sextuplicate as single cells and 12 h after plating treated with either vehicle (DMSO) or the JNK inhibitory peptide (10 μmol/L). Thirty minutes later, cells were treated with GST-MDA-7 or GST (30 nmol/L). Cells were incubated for 96 h after which time the medium was removed and replaced with medium lacking GST-MDA-7 but retaining JNK inhibitory peptide. Fourteen to 21 days after treatment, the medium was removed and colonies were fixed, stained, and counted (±SE; n = 2 independent experiments; *, P < 0.05, greater survival than Ad.cmv only). D, primary human GBM cells (GBM6 and GBM12) were plated and 24 h after plating treated with either the p38α/β MAPK inhibitor SB203580 or the inactive analogue of the inhibitor drug SB202474 (both 1 μmol/L) followed 30 min later by addition of GST or GST-MDA-7 (30 and 300 nmol/L). Cells were isolated 48 h after GST-MDA-7 treatment and viability was assessed in triplicate by trypan blue assay (±SE; n = 3; *, P < 0.05, greater than SB203580-treated cells; #, P < 0.05, less than SB203580-treated cells). Top, inset, left, cells were isolated 48 h after SB203580/GST-MDA-7 treatment and subjected to SDS-PAGE and immunoblotting was done to determine phosphorylation of p38α/β MAPK and HSP27 and expression of GADD153, GADD45α, GADD34, and ERK2.

Figure 4.

Modulation of GST-MDA-7 (30 and 300 nmol/L) toxicity by manipulation of AKT, MEK1, JNK1-3, and p38 MAPK activities. A, primary human glioma cells (GBM6 and GBM12) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing or dnp38α. Cells were cultured for 24 h after infection and then treated with the JNK inhibitory peptide based on sequence from JIP-1 (10 μmol/L) 30 min before addition of GST or GST-MDA-7 (30 nmol/L). Cells were isolated 48 h after GST-MDA-7 treatment and the activities of JNK1-3, p38 MAPK, AKT (S473), and ERK1/2 were assessed by immunoblotting in infected GBM6 and GBM12 cells (a representative study from n = 3). B,bottom, primary human glioma cells (GBM6) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing, caAKT, and caMEK1 as indicated. Cells were cultured for 24 h after infection and then treated with the JNK inhibitory peptide based on sequence from JIP-1 (10 μmol/L) 30 min before addition of GST or GST-MDA-7 (30 nmol/L). Cells were isolated 24 h after GST-MDA-7 treatment and cell lysates were split into two portions. In one portion, equal amounts of protein lysate were subjected to immunoprecipitation using the anti-active BAX 6A7 antibody. Immunoprecipitates were subjected to SDS-PAGE and immunoblotted for BAX. In the second portion, equal amounts of protein lysate were subjected to SDS-PAGE and immunoblotted for phosphorylated JNK1-3 (P-JNK1-3), BCL-xL, cleaved caspase-3, BAX, and ERK2. Data are from a representative experiment (n = 3). Top, primary human glioma cells (GBM6) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing or caAKT and caMEK1. Cells were cultured for 24 h after infection and then treated with GST or GST-MDA-7 (30 nmol/L). Cells were isolated 72 h after GST-MDA-7 treatment and viability was assessed in triplicate by Annexin V/propidium iodide staining and flow cytometry. Data are expressed as the true percentage of cell death above that observed in Ad.cmv-infected GST-treated cells (±SE; n = 3). C, primary human glioma cells (GBM12) and established human glioma cells (U251) were infected with either a vector control adenovirus (Ad.cmv; CMV) or an adenovirus to express dncasp.9. Twenty-four hours after infection, cells were plated in sextuplicate as single cells and 12 h after plating treated with either vehicle (DMSO) or the JNK inhibitory peptide (10 μmol/L). Thirty minutes later, cells were treated with GST-MDA-7 or GST (30 nmol/L). Cells were incubated for 96 h after which time the medium was removed and replaced with medium lacking GST-MDA-7 but retaining JNK inhibitory peptide. Fourteen to 21 days after treatment, the medium was removed and colonies were fixed, stained, and counted (±SE; n = 2 independent experiments; *, P < 0.05, greater survival than Ad.cmv only). D, primary human GBM cells (GBM6 and GBM12) were plated and 24 h after plating treated with either the p38α/β MAPK inhibitor SB203580 or the inactive analogue of the inhibitor drug SB202474 (both 1 μmol/L) followed 30 min later by addition of GST or GST-MDA-7 (30 and 300 nmol/L). Cells were isolated 48 h after GST-MDA-7 treatment and viability was assessed in triplicate by trypan blue assay (±SE; n = 3; *, P < 0.05, greater than SB203580-treated cells; #, P < 0.05, less than SB203580-treated cells). Top, inset, left, cells were isolated 48 h after SB203580/GST-MDA-7 treatment and subjected to SDS-PAGE and immunoblotting was done to determine phosphorylation of p38α/β MAPK and HSP27 and expression of GADD153, GADD45α, GADD34, and ERK2.

Close modal

In prior studies, Ad.mda-7 and GST-MDA-7 have been shown to promote cell killing via activation of the p38 MAPK pathway and increased expression of GADD transcription factors (25, 28, 33). Hence, we determined whether the role of p38 MAPK signaling in GST-MDA-7 toxicity in GBM6 and GBM12 cells was dose dependent and different from established tumor cell types examined previously. Treatment of GBM6 cells with GST-MDA-7 (1-300 nmol/L) enhanced in a dose-dependent fashion phosphorylation of p38 MAPK and its substrate heat shock protein (HSP) 27 (Fig. 4D, inset; cf. data in Fig. 2A). In contrast to GBM6 cells, treatment of GBM12 cells with GST-MDA-7 (1-300 nmol/L) resulted in a peak p38 MAPK activation, which occurred at ∼30 nmol/L GST-MDA-7 and that was not significantly further elevated by exposure to 300 nmol/L GST-MDA-7 (Fig. 4D, inset; cf. data in Fig. 2B). In agreement with our data expressing dominant-negative p38 MAPK, treatment of GBM6 and GBM12 cells with a low specific concentration of the p38α/β MAPK inhibitor SB203580 (1 μmol/L), but not the inactive analogue of this drug SB202474, enhanced basal levels of cell morbidity and modestly enhanced GST-MDA-7 (30 nmol/L) lethality within 48 h (Fig. 4D, bottom). Treatment of GBM6 cells with GST-MDA-7 (300 nmol/L) caused ∼4-fold more cell killing than treatment with GST-MDA-7 (30 nmol/L), and inhibition of p38 MAPK suppressed the lethality of GST-MDA-7 (300 nmol/L). In GBM12 cells, however, inhibition of p38 MAPK signaling enhanced GST-MDA-7 lethality regardless of GST-MDA-7 concentration (30-300 nmol/L).

In established glioma cells, GADD transcription factors have been linked to the toxic effects of MDA-7/IL-24. We noted that GADD153 expression was enhanced in a p38 MAPK-dependent fashion in GBM6 cells treated with GST-MDA-7 (30 and 300 nmol/L), but in contrast to the dose-dependent increases in p38 MAPK phosphorylation that correlated with changes in cell viability, there was no marked dose-dependent increase in GADD153 levels with increasing GST-MDA-7 doses (Fig. 4D, inset, left). Similar findings for GADD153 expression were noted in GBM12 cells. GST-MDA-7 very weakly modulated GADD34 and GADD45 protein levels in GBM6 and GBM12 cells. Collectively, these findings argue that the role of p38 MAPK signaling and GADD transcription factors in modulating GST-MDA-7 toxicity in primary human glioma cells is a more complicated process than has been observed in other established tumor cell types. Collectively, the findings in Figs. 2 to 4 show that GST-MDA-7-induced activation of the JNK pathway coupled to inactivation of the MEK1/2 pathway and/or the AKT pathway plays a central role in the lethality of GST-MDA-7 in primary human glioma cells. Our data also argue that, in a dose-dependent manner, activation of p38 MAPK by GST-MDA-7 has the potential to either promote cell survival or cell killing.

The probability that physicians will be able to achieve systemic prolonged high toxic concentrations of MDA-7/IL-24 (>30 nmol/L) in patients is low. Hence, further analyses determined whether the toxicity of lower GST-MDA-7 concentrations (∼1 nmol/L) could be enhanced by the manipulation of AKT and MEK1 signaling. In GBM6 and GBM12 cells, the promotion of 1 nmol/L GST-MDA-7 toxicity to a level approximating the lethality of a 30 nmol/L GST-MDA-7 treatment required inhibition of both the PI3K-AKT pathways and the MEK-ERK1/2 pathways (Fig. 5A and B). Similar data were obtained in GBM5 cells (Supplementary Fig. S3).7 In GBM6 and GBM12 cells, expression of dnMEK1 enhanced GST-MDA-7-induced activation of JNK1-3 that was further enhanced by inhibition of AKT (Fig. 5A and B, inset). In GBM6 and GBM12 cells, suppression of JNK1-3 signaling abolished the promotion of GST-MDA-7 toxicity caused by inhibition of MEK1 and AKT (Fig. 5C, inset, left and bottom; data not shown). Inhibition of MEK1 and AKT enhanced the activation of BAX by low 1 nmol/L concentrations of GST-MDA-7; the activation of BAX was also JNK1-3 dependent (Fig. 5C, inset, right). Collectively, our data show that simultaneous inhibition of both PI3K-AKT and MEK-ERK1/2 pathways enhances the toxicity of low 1 nmol/L GST-MDA-7 concentrations via a JNK1-3-dependent and BAX-dependent mechanism.

Figure 5.

Expression of dnAKT and dnMEK1 is required to enhance 1 nmol/L GST-MDA-7 toxicity to the approximate level of toxicity induced by 30 nmol/L GST-MDA-7: suppression of AKT and ERK1/2 function correlates with GST-MDA-7-induced activation of proapoptotic JNK1-3 signaling. A, primary human glioma cells (GBM6) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing, dnAKT, or dnMEK1. Cells were cultured for 24 h after infection and then treated with GST or GST-MDA-7 (1 and 30 nmol/L as indicated). Cells were isolated 72 h after GST-MDA-7 treatment and viability was assessed in triplicate by trypan blue assay (±SE; n = 3; *, P < 0.05, greater than vector control-infected GST-MDA-7 exposure; **, P < 0.05, greater than either dnAKT- or dnMEK1-infected cell GST-MDA-7 exposure). Inset, primary human glioma cells (GBM6) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing, dnAKT, or dnMEK1. Cells were cultured for 24 h after infection and then treated with GST or GST-MDA-7 (30 nmol/L). Cells were isolated 48 h after GST-MDA-7 treatment (1 nmol/L) and the activities of JNK1-3, p38 MAPK, AKT (S473), and ERK1/2 were assessed by immunoblotting in infected GBM6 cells (n = 3). B, primary human glioma cells (GBM12) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing, dnAKT, or dnMEK1. Cells were cultured for 24 h after infection and then treated with GST or GST-MDA-7 (1 and 30 nmol/L as indicated). Cells were isolated 72 h after GST-MDA-7 treatment and viability was assessed in triplicate by trypan blue assay (±SE; n = 3; *, P < 0.05, greater than vector control-infected GST-MDA-7 exposure; **, P < 0.05, greater than either dnAKT- or dnMEK1-infected cell GST-MDA-7 exposure). Inset, primary human glioma cells (GBM12) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing, dnAKT, or dnMEK1. Cells were cultured for 24 h after infection and then treated with GST or GST-MDA-7 (30 nmol/L). Cells were isolated 48 h after GST-MDA-7 treatment (1 nmol/L) and the activities of JNK1-3, p38 MAPK, AKT (S473), and ERK1/2 were assessed by immunoblotting in infected GBM6 cells (a representative study; n = 3). C, primary human glioma cells (GBM6) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing, dnAKT, or dnMEK1. Cells were cultured for 24 h after infection and then treated with the JNK inhibitory peptide based on sequence from JIP-1 (10 μmol/L) 30 min before addition of GST or GST-MDA-7 (1 nmol/L). Cells were isolated 72 h after GST-MDA-7 treatment and viability was assessed in triplicate by trypan blue assay (±SE; n = 3; #, P < 0.05, lower than dnAKT and dnMEK1 infected with GST-MDA-7 exposure in the absence of JNK inhibitory peptide). Inset, top left, primary human glioma cells (GBM6) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing or dnAKT (caAKT) and dnMEK1 (caMEK1). Cells were cultured for 24 h after infection and then treated with GST or GST-MDA-7 (30 nmol/L). Cells were isolated 72 h after GST-MDA-7 treatment and viability was assessed in triplicate by Annexin V/propidium iodide staining and flow cytometry. Data are expressed as the true percentage of cell death above that observed in Ad.cmv-infected GST-treated cells (±SE; n = 3). Inset, top right, primary human glioma cells (GBM6) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing, dnAKT (caAKT), and dnMEK1 (caMEK1) as indicated. Cells were cultured for 24 h after infection and then treated with the JNK inhibitory peptide based on sequence from JIP-1 (10 μmol/L) 30 min before addition of GST or GST-MDA-7 (1 nmol/L). Cells were isolated 24 h after GST-MDA-7 treatment and cell lysates were split into two portions. In one portion, equal amounts of protein lysate were subjected to immunoprecipitation using the anti-active BAX 6A7 antibody. Immunoprecipitates were subjected to SDS-PAGE and immunoblotted for BAX. In the second portion, equal amounts of protein lysate were subjected to SDS-PAGE and immunoblotted for phosphorylated JNK1-3, BCL-xL, cleaved caspase-3, BIM, BAX, and ERK2. Data are from a representative experiment (n = 3).

Figure 5.

Expression of dnAKT and dnMEK1 is required to enhance 1 nmol/L GST-MDA-7 toxicity to the approximate level of toxicity induced by 30 nmol/L GST-MDA-7: suppression of AKT and ERK1/2 function correlates with GST-MDA-7-induced activation of proapoptotic JNK1-3 signaling. A, primary human glioma cells (GBM6) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing, dnAKT, or dnMEK1. Cells were cultured for 24 h after infection and then treated with GST or GST-MDA-7 (1 and 30 nmol/L as indicated). Cells were isolated 72 h after GST-MDA-7 treatment and viability was assessed in triplicate by trypan blue assay (±SE; n = 3; *, P < 0.05, greater than vector control-infected GST-MDA-7 exposure; **, P < 0.05, greater than either dnAKT- or dnMEK1-infected cell GST-MDA-7 exposure). Inset, primary human glioma cells (GBM6) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing, dnAKT, or dnMEK1. Cells were cultured for 24 h after infection and then treated with GST or GST-MDA-7 (30 nmol/L). Cells were isolated 48 h after GST-MDA-7 treatment (1 nmol/L) and the activities of JNK1-3, p38 MAPK, AKT (S473), and ERK1/2 were assessed by immunoblotting in infected GBM6 cells (n = 3). B, primary human glioma cells (GBM12) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing, dnAKT, or dnMEK1. Cells were cultured for 24 h after infection and then treated with GST or GST-MDA-7 (1 and 30 nmol/L as indicated). Cells were isolated 72 h after GST-MDA-7 treatment and viability was assessed in triplicate by trypan blue assay (±SE; n = 3; *, P < 0.05, greater than vector control-infected GST-MDA-7 exposure; **, P < 0.05, greater than either dnAKT- or dnMEK1-infected cell GST-MDA-7 exposure). Inset, primary human glioma cells (GBM12) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing, dnAKT, or dnMEK1. Cells were cultured for 24 h after infection and then treated with GST or GST-MDA-7 (30 nmol/L). Cells were isolated 48 h after GST-MDA-7 treatment (1 nmol/L) and the activities of JNK1-3, p38 MAPK, AKT (S473), and ERK1/2 were assessed by immunoblotting in infected GBM6 cells (a representative study; n = 3). C, primary human glioma cells (GBM6) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing, dnAKT, or dnMEK1. Cells were cultured for 24 h after infection and then treated with the JNK inhibitory peptide based on sequence from JIP-1 (10 μmol/L) 30 min before addition of GST or GST-MDA-7 (1 nmol/L). Cells were isolated 72 h after GST-MDA-7 treatment and viability was assessed in triplicate by trypan blue assay (±SE; n = 3; #, P < 0.05, lower than dnAKT and dnMEK1 infected with GST-MDA-7 exposure in the absence of JNK inhibitory peptide). Inset, top left, primary human glioma cells (GBM6) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing or dnAKT (caAKT) and dnMEK1 (caMEK1). Cells were cultured for 24 h after infection and then treated with GST or GST-MDA-7 (30 nmol/L). Cells were isolated 72 h after GST-MDA-7 treatment and viability was assessed in triplicate by Annexin V/propidium iodide staining and flow cytometry. Data are expressed as the true percentage of cell death above that observed in Ad.cmv-infected GST-treated cells (±SE; n = 3). Inset, top right, primary human glioma cells (GBM6) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing, dnAKT (caAKT), and dnMEK1 (caMEK1) as indicated. Cells were cultured for 24 h after infection and then treated with the JNK inhibitory peptide based on sequence from JIP-1 (10 μmol/L) 30 min before addition of GST or GST-MDA-7 (1 nmol/L). Cells were isolated 24 h after GST-MDA-7 treatment and cell lysates were split into two portions. In one portion, equal amounts of protein lysate were subjected to immunoprecipitation using the anti-active BAX 6A7 antibody. Immunoprecipitates were subjected to SDS-PAGE and immunoblotted for BAX. In the second portion, equal amounts of protein lysate were subjected to SDS-PAGE and immunoblotted for phosphorylated JNK1-3, BCL-xL, cleaved caspase-3, BIM, BAX, and ERK2. Data are from a representative experiment (n = 3).

Close modal

ERBB1 is a growth factor receptor whose actions have been linked via the ERK1/2 and PI3K-AKT pathways in many gliomas to an aggressive phenotype and to rapid patient morbidity (3, 5, 32). As ERBB1 signaling can modulate ERK1/2 and AKT activity, and inhibition of these pathways enhances GST-MDA-7 toxicity (Figs. 35), we determined whether inhibition of ERBB1 function enhanced GST-MDA-7 toxicity and could replicate our observations for inhibition of ERK1/2 and AKT. In GBM12 cells that express a full-length mutated active ERBB1 protein, expression of dominant-negative ERBB1 (COOH-terminal deletion of 533 amino acids, CD533) or treatment with an ERBB1 inhibitor AG1478 caused a similar amount of cell death to that induced by GST-MDA-7 (30 nmol/L) exposure. Combined treatment of GBM12 cells with GST-MDA-7 and inhibition of ERBB1 resulted in essentially a null effect on cell killing (Fig. 6A). To further explore whether ERBB1 signaling alters the survival of glioma cells exposed to GST-MDA-7, we treated established U118 human glioma cells transfected to express wild-type ERBB1 or ERBB1vIII with GST-MDA-7. Expression of either wild-type ERBB1 or ERBB1vIII suppressed the toxicity of GST-MDA-7 in U118 cells compared with cells transfected with vector control plasmid (Fig. 6B). Collectively, these findings argue in human glioma cells that downstream inhibition of PI3K and MEK1/2 may represent a better approach to enhance GST-MDA-7 lethality that upstream inhibition of ERBB1.

Figure 6.

ERBB1 function affects on GST-MDA-7 toxicity in primary and established human glioma cells. A, primary human glioma cells (GBM12) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing or dominant-negative ERBB1 (ERBB1-CD533). Cells were cultured for 24 h after infection and then treated with vehicle (DMSO) or the ERBB1 inhibitor AG1478 (1 μmol/L) followed 30 min later by treatment with GST or GST-MDA-7 (30 nmol/L). Cells were isolated 72 h after GST-MDA-7 treatment and viability was assessed in triplicate by trypan blue assay (±SE; n = 3). B, U118 established glioma cells transfected with vector plasmid to express truncated active ERBB1vIII or to express wild-type full-length ERBB1 (ERBB1 WT) were plated and 24 h after plating treated with GST or GST-MDA-7 (0-100 nmol/L). Cells were isolated 72 h after GST-MDA-7 treatment and viability was assessed in triplicate by trypan blue assay (±SE; n = 3). C, primary human glioma cells (GBM6 and GBM12) were plated and 36 h after plating treated with the near simultaneous addition of vehicle (DMSO) or 17AAG (200 nmol/L) and GST or GST-MDA-7 (1-10 nmol/L). Cells were isolated for viability analyses 72 h after GST-MDA-7 treatment as judged in triplicate by trypan blue dye exclusion assay (±SE; n = 3; *, P < 0.05, greater than 17AAG exposure). Inset, cells were treated in an identical fashion to those for trypan blue assays, except that cells were isolated 24 h after 17AAG treatment for immunoblotting. Immunoblotting was done to determine the expression of ERK2 and the phosphorylation of AKT (S473), ERK1/2, and JNK1-3. Data are from a representative experiment (n = 3). D, primary human glioma cells (GBM6) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing, caAKT, and caMEK1 as indicated. Cells were cultured for 24 h after infection and then treated with the JNK inhibitory peptide based on sequence from JIP-1 (10 μmol/L) 30 min before the near simultaneous addition of vehicle (DMSO) or 17AAG (200 nmol/L) and GST or GST-MDA-7 (10 nmol/L). Cells were isolated for viability analyses 72 h after GST-MDA-7 treatment as judged in triplicate by trypan blue dye exclusion assay (±SE; n = 3; #, P < 0.05, lower than CMV-infected 17AAG and GST-MDA-7-treated cells; ##, P < 0.05, lower than that observed in 17AAG and GST-MDA-7-treated cells infected with either caAKT or ca MEK1). Inset, cells were treated in an identical fashion to those for trypan blue assays, except that cells were isolated 24 h after 17AAG treatment for immunoblotting. Immunoblotting was done to determine the expression of HSP90 and ERK2 and the phosphorylation of AKT (S473), ERK1/2, and JNK1-3. Data are from a representative experiment (n = 2).

Figure 6.

ERBB1 function affects on GST-MDA-7 toxicity in primary and established human glioma cells. A, primary human glioma cells (GBM12) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing or dominant-negative ERBB1 (ERBB1-CD533). Cells were cultured for 24 h after infection and then treated with vehicle (DMSO) or the ERBB1 inhibitor AG1478 (1 μmol/L) followed 30 min later by treatment with GST or GST-MDA-7 (30 nmol/L). Cells were isolated 72 h after GST-MDA-7 treatment and viability was assessed in triplicate by trypan blue assay (±SE; n = 3). B, U118 established glioma cells transfected with vector plasmid to express truncated active ERBB1vIII or to express wild-type full-length ERBB1 (ERBB1 WT) were plated and 24 h after plating treated with GST or GST-MDA-7 (0-100 nmol/L). Cells were isolated 72 h after GST-MDA-7 treatment and viability was assessed in triplicate by trypan blue assay (±SE; n = 3). C, primary human glioma cells (GBM6 and GBM12) were plated and 36 h after plating treated with the near simultaneous addition of vehicle (DMSO) or 17AAG (200 nmol/L) and GST or GST-MDA-7 (1-10 nmol/L). Cells were isolated for viability analyses 72 h after GST-MDA-7 treatment as judged in triplicate by trypan blue dye exclusion assay (±SE; n = 3; *, P < 0.05, greater than 17AAG exposure). Inset, cells were treated in an identical fashion to those for trypan blue assays, except that cells were isolated 24 h after 17AAG treatment for immunoblotting. Immunoblotting was done to determine the expression of ERK2 and the phosphorylation of AKT (S473), ERK1/2, and JNK1-3. Data are from a representative experiment (n = 3). D, primary human glioma cells (GBM6) were plated and 12 h after plating infected with adenoviruses at a final MOI of 50 to express nothing, caAKT, and caMEK1 as indicated. Cells were cultured for 24 h after infection and then treated with the JNK inhibitory peptide based on sequence from JIP-1 (10 μmol/L) 30 min before the near simultaneous addition of vehicle (DMSO) or 17AAG (200 nmol/L) and GST or GST-MDA-7 (10 nmol/L). Cells were isolated for viability analyses 72 h after GST-MDA-7 treatment as judged in triplicate by trypan blue dye exclusion assay (±SE; n = 3; #, P < 0.05, lower than CMV-infected 17AAG and GST-MDA-7-treated cells; ##, P < 0.05, lower than that observed in 17AAG and GST-MDA-7-treated cells infected with either caAKT or ca MEK1). Inset, cells were treated in an identical fashion to those for trypan blue assays, except that cells were isolated 24 h after 17AAG treatment for immunoblotting. Immunoblotting was done to determine the expression of HSP90 and ERK2 and the phosphorylation of AKT (S473), ERK1/2, and JNK1-3. Data are from a representative experiment (n = 2).

Close modal

Geldanamycins are therapeutic agents that act by inhibiting the function of HSP90 and lower expression, and reduce the activity, of the PI3K-AKT and the MEK-ERK1/2 pathways (35, 36). This suggests that geldanamycins could be a useful family of clinically relevant compounds that may interact with MDA-7. In GBM6 and GBM12 cells, 17AAG modestly suppressed the activity of ERK1/2 and AKT, and combined with GST-MDA-7 to further suppress ERK1/2 and AKT activity, as well as activating JNK1-3 (Fig. 6C). As 17AAG suppressed both AKT and ERK1/2 activity and activated JNK1-3, we determined whether constitutive activation AKT and MEK1 prevented the toxic interaction between GST-MDA-7 and 17AAG. In GBM6 cells, the toxicity of 17AAG was suppressed by combined, but not individual, activation of AKT and MEK1 (Fig. 6D). Individual activation of either AKT or MEK1 suppressed the toxic interaction between 17AAG and GST-MDA-7. Tumor cell viability in cells treated with 17AAG and GST-MDA-7 was maintained by inhibition of JNK1-3 signaling.

Previously, we have noted that treatment of cells with GST-MDA-7 reduced proliferation and caused tumor-specific cell killing and radiosensitization (25, 31, 32). The studies in this research were designed to determine how primary human glioma cells respond to GST-MDA-7 exposure over a range of cytokine concentrations with respect to alterations in cell signaling and how, mechanistically, alterations in signaling affect viability.

Low concentrations of GST-MDA-7 (∼1 nmol/L) suppressed glioma cell growth with minimal toxicity, which correlated with transient weak enhancement of p38 MAPK and JNK1-3 activity and with a modest reduction in ERK1/2 and AKT signaling. An intermediate 5-fold higher concentration of GST-MDA-7 also suppressed glioma cell growth with modest toxicity, which correlated with a stronger transient activation of p38 MAPK and JNK1-3 but with only a modest reduction in ERK1/2 and AKT signaling. A 30-fold higher GST-MDA-7 concentration caused profound levels of toxicity that correlated with stronger activation of JNK1-3 and with a stronger transient activation of p38 MAPK and that in a cell type–dependent fashion nearly abolished either ERK1/2 and/or AKT signaling.

Multiple studies using a variety of cytokine and toxic stimuli have shown that JNK1-3 activation in astrocytes, neurons, and transformed versions of these cells can cause cell death (37, 38). The balance between the outputs of ERK1/2 and AKT signaling and that of JNK1-3 signaling has been argued to represent a key homeostatic mechanism, which regulates cell survival versus cell death processes (39, 40). GST-MDA-7-induced JNK1-3 signaling was causal in BAX activation that will act to promote caspase-9-dependent cell killing. Maintenance of ERK1/2 and/or AKT signaling in cells exposed to high toxic concentrations of GST-MDA-7 suppressed JNK1-3 phosphorylation as well as abolishing subsequent BAX activation and cell killing. Inhibition of AKT and ERK1/2 promoted the toxicity of GST-MDA-7, which was associated with increased JNK1-3 signaling, and that was causal in BAX activation and tumor cell death. Thus, GST-MDA-7, at low apparently nontoxic concentrations, activates the JNK1-3 pathway in glioma cells but does not suppress ERK1/2 and/or AKT signaling, and for GST-MDA-7 to become a toxic agent, even in glioma cells exhibiting relatively high levels of JNK1-3 activity requires the suppression of ERK1/2 and/or AKT function. Thus, the loss of ERK1/2 and AKT signaling appears to be a primary response associated with cell killing, and the gain of JNK1-3 signaling a secondary response, in GST-MDA-7-induced lethality in primary human glioma cells.

In prior studies, we and others have argued that Ad.mda-7 and GST-MDA-7-induced activation of the p38 MAPK pathway is causal in the lethal effects of MDA-7 expression (27, 28). The present studies in primary human glioma cells, using molecular and small-molecule inhibitor approaches, argue that basal levels of p38 MAPK activity and GST-MDA-7-induced p38 MAPK activation in the 1 to 30 nmol/L concentration range are protective signals. We noted that inhibition of p38 MAPK function could suppress basal levels of AKT phosphorylation in GBM6 cells, in general agreement with data reported by Park et al. (41). Sainz-Perez et al. argued that loss of MDA-7 expression in leukemia cells reduced p38 MAPK activity, which was causal in reduced viability (42). Inhibition of p38 MAPK function also modestly enhanced GST-MDA-7-induced JNK1-3 phosphorylation, and enhanced JNK1-3 signaling was causal in BAX activation and elevated levels of cell death.

The reasons why virus-mediated intracellular overexpression of MDA-7 or high dose ∼400 to 600 nmol/L GST-MDA-7 treatment may cause cell killing via p38 MAPK signaling whereas we observed lower dose 1 to 30 nmol/L MDA-7 acting in a protective manner in glioma cells were unclear. In other works, we and others have shown that the biological effect of transient intense or prolonged low-level activation of the ERK1/2 or JNK1/2 pathways can promote cell growth and cell survival, whereas prolonged intense activation of these pathways inhibits growth and causes cell death (43). Thus, it is possible that based on the dosage of MDA-7 the relative amplitude and duration of p38 MAPK activation could be a marker for whether the “readout” signals from this pathway are protective or toxic. In agreement with this hypothesis, we noted in GBM6 cells that an order of magnitude higher GST-MDA-7 concentration (300 nmol/L) promoted intense activation of p38 MAPK compared with a 30 nmol/L GST-MDA-7 exposure; 300 nmol/L GST-MDA-7-induced p38 MAPK activation was a cytotoxic signal. However, in GBM12 cells, regardless of the GST-MDA-7 concentration, p38 MAPK signaling appeared to act in a protective manner. Hence, our findings suggest that p38 MAPK signaling may promote or diminish GST-MDA-7 toxicity, which is dependent on the cell type and the relative cytokine concentration.

ERBB1 expression in glioblastoma, either as a full-length receptor or as a truncated active mutant receptor (variant III), is known to be an indicator for a more malignant cellular phenotype and for poorer patient survival (3, 5). Recently, inhibition of ERBB1 function was shown to enhance the lethality of Ad.mda-7 in lung cancer cells, in part by derepressing inhibitory signaling from the ERBB1 receptor, which acts to suppress MDA-7/IL-24 protein synthesis (44). To our surprise, we found that inhibition of ERBB1 using small-molecule or molecular approaches did not significantly alter GST-MDA-7 toxicity in GBM12 cells that express a full-length mutant active ERBB1 protein. In contrast to these findings, expression of ERBB1 or ERBB1vIII suppressed GST-MDA-7 lethality in stably transfected U118 established human glioma cells. Collectively, these findings in glioma cells argue against a simplistic interpretation that ERBB1 inhibition obligatorily promoting GST-MDA-7 toxicity and suggest that combined inhibition of PI3K-AKT signaling and MEK1/2-ERK1/2 signaling may represent a more ubiquitous approach to promoting cytokine toxicity.

Treatment with GST-MDA-7 in a dose-dependent fashion caused a JNK-dependent activation of BAX. BAX is a BH3 domain protein that promotes pore formation in the mitochondrial outer membrane leading to loss of mitochondrial integrity, including the release into the cytosol of cytochrome c (16, 17, 45, 46). In agreement with data showing BAX activation, expression of dncasp.9 or of BCL-xL suppressed GST-MDA-7 lethality, showing that activation of the intrinsic pathway was a central response to GST-MDA-7 exposure. Prior studies, in which low concentrations of GST-MDA-7 were shown to radiosensitize primary human GBM cells by activation of JNK1-3 and the intrinsic pathway, together with the present findings using higher individual concentrations of GST-MDA-7, suggest that in human glioma cells agents that promote activation of other apoptosis pathways could have potential for combinatorial therapeutic approaches.

Based on our finding that the toxicity of GST-MDA-7 correlated with reduced ERK1/2 and AKT activity, we hypothesized that geldanamycin-induced inhibition of AKT and ERK1/2, by suppressing HSP90 function, would enhance cytokine toxicity. The toxic interaction between the clinically relevant geldanamycin 17AAG and GST-MDA-7 was suppressed by constitutive activation of either AKT or MEK1, whereas the toxicity of 17AAG as a single agent was only suppressed by expression of both activated AKT and MEK1. A recent contemporaneous study by Pataer et al. noted that viability of established lung cancer cells was reduced in a greater than additive manner in cells infected with Ad.mda-7 and treated with HSP90 antagonist geldanamycins without suppression of HSP90 expression (47). In this study, the relative role of AKT versus MEK1 signaling was not investigated. Several groups have shown that geldanamycins, alone or in combination with other agents, can alter the viability of brain cancer cells in vitro. Additional studies will be required to determine whether the lethality of MDA-7 towards glioma tumors in vivo can be enhanced by agents that simultaneously suppress both AKT and ERK1/2 activity.

Grant support: Public Health Service grants P01-CA104177, R01-CA108325, and R01-DK52825, Jim Valvano “V” Foundation, and Department of Defense award DAMD17-03-1-0262 (P. Dent); Public Health Service grants R01-CA63753 and R01-CA77141 and a Leukemia Society of America grant 6405-97 (S. Grant); Public Health Service grant P01-CA104177 (D.T. Curiel); and Public Health Service grants P01-CA104177, R01-CA97318, R01-CA98172, and P01-NS31492, Samuel Waxman Cancer Research Foundation, and Michael and Stella Chernow Endowment (P.B. Fisher).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: P. Dent is The Universal, Inc. Professor in Signal Transduction Research and P.B. Fisher is The Michael and Stella Chernow Urological Cancer Research Scientist.

1
Levin VA, Gutin PH, Leibel S. Neoplasms of the central nervous system, cancer: principles and practice of oncology. 4th ed. Philadelphia: JB Lippincott; 1993. p. 1679–737.
2
Greenlee RT, Murray T, Bolden S. Cancer statistics 2000.
Cancer J Clin
2000
;
50
:
7
–33.
3
Jelsma R, Bucy PC. The treatment of glioblastoma multiforme of the brain.
J Neurosurg
1967
;
27
:
388
–400.
4
Eley K, Benedict S, Chung T, et al. The effects of pentoxifylline on the survival of human glioma cells with continuous and intermittent stereotactic radiosurgery irradiation.
Int J Radiat Oncol Biol Phys
2002
;
54
:
542
–50.
5
Nieder C, Grosu AL, Molls M. A comparison of treatment results for recurrent malignant gliomas.
Cancer Treat Rev
2000
;
26
:
397
–409.
6
Jiang H, Lin JJ, Su ZZ, Goldstein NI, Fisher PB. Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma differentiation, growth and progression.
Oncogene
1995
;
11
:
2477
–6.
7
Huang EY, Madireddi MT, Gopalkrishnan RV, et al. Genomic structure, chromosomal localization and expression profile of a novel melanoma differentiation associated (mda-7) gene with cancer specific growth suppressing and apoptosis inducing properties.
Oncogene
2001
;
20
:
7051
–63.
8
Goris A, Marrosu MG, Vandenbroeck K. Novel polymorphisms in the IL-10 related AK155 gene (chromosome 12q15).
Genes Immun
2001
;
2
:
284
–6.
9
Parrish-Novak J, Xu W, Brender T, et al. Interleukins 19, 20, and 24 signal through two distinct receptor complexes. Differences in receptor-ligand interactions mediate unique biological functions.
J Biol Chem
2002
;
277
:
47517
–23.
10
Caudell EG, Mumm JB, Poindexter N, et al. The protein product of the tumor suppressor gene, melanoma differentiation-associated gene 7, exhibits immunostimulatory activity and is designated IL-24.
J Immunol
2002
;
168
:
6041
–6.
11
Wang M, Tan Z, Zhang R, Kotenko SV, Liang P. Interleukin 24 (MDA-7/MOB-5) signals through two heterodimeric receptors, IL-22R1/IL-20R2 and IL-20R1/IL-20R2.
J Biol Chem
2002
;
277
:
7341
–47.
12
Pestka S, Kraus CD, Sarkar D, Walter MR, Shi Y, Fisher PB. Interleukin-10 and related cytokines and receptors.
Annu Rev Immunol
2004
;
22
:
929
–79.
13
Ekmekcioglu S, Ellerhorst J, Mhashilkar AM, et al. Down-regulated melanoma differentiation associated gene (mda-7) expression in human melanomas.
Int J Cancer
2001
;
94
:
54
–9.
14
Ellerhorst JA, Prieto VG, Ekmekcioglu S. Loss of MDA-7 expression with progression of melanoma.
J Clin Oncol
2002
;
20
:
1069
–74.
15
Jiang H, Su ZZ, Lin JJ, Goldstein NI, Young CSH, Fisher PB. The melanoma differentiation associated gene mda-7 suppresses cancer cell growth.
Proc Natl Acad Sci U S A
1996
;
93
:
9160
–5.
16
Sauane M, Gopalkrishnan RV, Sarkar D, et al. MDA-7/IL-24: novel cancer growth suppressing and apoptosis inducing cytokine.
Cytokine Growth Factor Rev
2003
;
14
:
35
–51.
17
Sarkar D, Su ZZ, Lebedeva IV, et al. mda-7 (IL-24): signaling and functional roles.
Biotechniques
2002
;
10
:
30
–9.
18
Su ZZ, Lebedeva IV, Gopalkrishnan RV, et al. A combinatorial approach for selectively inducing programmed cell death in human pancreatic cancer cells.
Proc Natl Acad Sci U S A
2001
;
98
:
10332
–7.
19
Su ZZ, Madireddi MT, Lin JJ, et al. The cancer growth suppressor gene mda-7 selectively induces apoptosis in human breast cancer cells and inhibits tumor growth in nude mice.
Proc Natl Acad Sci U S A
1998
;
95
:
14400
–5.
20
Lebedeva IV, Su ZZ, Chang Y, Kitada S, Reed JC, Fisher PB. The cancer growth suppressing gene mda-7 induces apoptosis selectively in human melanoma cells.
Oncogene
2002
;
21
:
708
–8.
21
Saeki T, Mhashilkar A, Chada S, Branch C, Roth JA, Ramesh R. Tumor-suppressive effects by adenovirus-mediated mda-7 gene transfer in non-small cell lung cancer cell in vitro.
Gene Ther
2000
;
7
:
2051
–7.
22
Saeki T, Mhashilkar A, Swanson X, et al. Inhibition of human lung cancer growth following adenovirus-mediated mda-7 gene expression in vivo.
Oncogene
2002
;
21
:
4558
–6.
23
Madireddi MT, Su ZZ, Young CS, Goldstein NI, Fisher PB. mda-7, a novel melanoma differentiation associated gene with promise for cancer gene therapy.
Adv Exp Med Biol
2000
;
465
:
239
–61.
24
Su ZZ, Lebedeva IV, Sarkar D, et al. Ionizing radiation enhances therapeutic activity of mda-7/IL-24: overcoming radiation- and mda-7/IL-24-resistance in prostate cancer cells overexpressing the antiapoptotic proteins bcl-xL or bcl-2.
Oncogene
2006
;
25
:
2339
–48.
25
Su ZZ, Lebedeva IV, Sarkar D, et al. Melanoma differentiation associated gene-7, mda-7/IL-24, selectively induces growth suppression, apoptosis and radiosensitization in malignant gliomas in a p53-independent manner.
Oncogene
2003
;
22
:
1164
–80.
26
Lebedeva IV, Sarkar D, Su ZZ, et al. Bcl-2 and Bcl-xL differentially protect human prostate cancer cells from induction of apoptosis by melanoma differentiation associated gene-7, mda-7/IL-24.
Oncogene
2003
;
22
:
8758
–73.
27
Gupta P, Walter MR, Su ZZ, et al. BiP/GRP78 is an intracellular target for MDA-7/IL-24 induction of cancer-specific apoptosis.
Cancer Res
2006
;
66
:
8182
–91.
28
Sarkar D, Su ZZ, Lebedeva IV, et al. mda-7 (IL-24) mediates selective apoptosis in human melanoma cells by inducing the coordinated overexpression of the GADD family of genes by means of p38 MAPK.
Proc Natl Acad Sci U S A
2002
;
99
:
10054
–9.
29
Mhashilkar AM, Stewart AL, Sieger K, et al. MDA-7 negatively regulates the β-catenin and PI3K signaling pathways in breast and lung tumor cells.
Mol Ther
2003
;
8
:
207
–19.
30
Chada S, Bocangel D, Ramesh R, et al. mda-7/IL24 kills pancreatic cancer cells by inhibition of the Wnt/PI3K signaling pathways: identification of IL-20 receptor-mediated bystander activity against pancreatic cancer.
Mol Ther
2005
;
11
:
724
–33.
31
Yacoub A, Mitchell C, Brannon J, et al. MDA-7 (interleukin-24) inhibits the proliferation of renal carcinoma cells and interacts with free radicals to promote cell death and loss of reproductive capacity.
Mol Cancer Ther
2003
;
2
:
623
–32.
32
Yacoub A, Mitchell C, Hong Y, et al. MDA-7 regulates cell growth and radiosensitivity in vitro of primary (non-established) human glioma cells.
Cancer Biol Ther
2004
;
3
:
739
–51.
33
Sauane M, Gopalkrishnan RV, Choo HT, et al. Mechanistic aspects of mda-7/IL-24 cancer cell selectivity analysed via a bacterial fusion protein.
Oncogene
2004
;
23
:
7679
–90.
34
Yacoub A, Park MA, Hanna D, et al. OSU-03012 promotes caspase-independent but PERK-, cathepsin B, BID-, and AIF-dependent killing of transformed cells.
Mol Pharmacol
2006
;
70
:
589
–603.
35
Mitchell C, Park MA, Zhang G, et al. 17AAG enhances the lethality of deoxycholic acid in primary rodent hepatocytes and established cell lines via a PERK-, ceramide-, and Ca2+-dependent increase in mitochondrial ROS production.
Mol Cancer Ther
2007
;
6
:
618
–32.
36
Sharp S, Workman P. Inhibitors of the HSP90 molecular chaperone: current status.
Adv Cancer Res
2006
;
95
:
323
–48.
37
Kanzawa T, Iwado E, Aoki H, et al. Ionizing radiation induces apoptosis and inhibits neuronal differentiation in rat neural stem cells via the c-Jun NH2-terminal kinase (JNK) pathway.
Oncogene
2006
;
25
:
3638
–48.
38
Yoon S, Choi J, Yoon J, Huh JW, Kim D. Okadaic acid induces JNK activation, bim overexpression and mitochondrial dysfunction in cultured rat cortical neurons.
Neurosci Lett
2006
;
394
:
190
–5.
39
Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis.
Science
1995
;
270
:
1326
–31.
40
Valerie K, Yacoub A, Hagan MP, et al. Radiation-induced signaling: inside out and outside in.
Mol Cancer Ther
2007
;
6
:
789
–801.
41
Park CM, Park MJ, Kwak HJ, et al. Ionizing radiation enhances matrix metalloproteinase-2 secretion and invasion of glioma cells through src/epidermal growth factor receptor-mediated p38/Akt and phosphatidylinositol 3-kinase/Akt signaling pathways.
Cancer Res
2006
;
66
:
8511
–9.
42
Sainz-Perez A, Gary-Gouy H, Portier A, et al. High Mda-7 expression promotes malignant cell survival and p38 MAP kinase activation in chronic lymphocytic leukemia.
Leukemia
2006
;
20
:
498
–504.
43
Auer KL, Park JS, Seth P, et al. Prolonged activation of the mitogen-activated protein kinase pathway promotes DNA synthesis in primary hepatocytes from p21Cip-1/WAF1-null mice, but not in hepatocytes from p16INK4a-null mice.
Biochem J
1998
;
336
:
551
–60.
44
Emdad L, Lebedeva IV, Su ZZ, et al. Combinatorial treatment of non-small-cell lung cancers with gefitinib and Ad.mda-7 enhances apoptosis-induction and reverses resistance to a single therapy.
J Cell Physiol
2007
;
210
:
549
–59.
45
Fisher PB. Is mda-7/IL-24 a “magic bullet” for cancer?
Cancer Res
2005
;
65
:
10128
–38.
46
Lebedeva IV, Emdad L, Su ZZ, et al. mda-7/IL-24, a novel anticancer cytokine: focus on bystander antitumor, radiosensitization and antiangiogenic properties and overview of the phase I clinical experience.
Int J Oncol
2007
;
31
:
985
–1007.
47
Pataer A, Bocangel D, Chada S, Roth JA, Hunt KK, Swisher SG. Enhancement of adenoviral MDA-7-mediated cell killing in human lung cancer cells by geldanamycin and its 17-allyl-amino-17-demethoxy analogue.
Cancer Gene Ther
2007
;
14
:
12
–8.