Abstract

Materials and Methods

Results

Initial experiments focused on defining the dose-dependent impact of GST-MDA-7 on OCC growth and viability. GST-MDA-7 in a dose-dependent manner suppressed the proliferation of SKOVIII and OVCAR cells (Fig. 1, A and B) with a reduced effect in SKOVIII. These findings correlated with dose-dependent cell killing by GST-MDA-7 in these cells; it is noteworthy that GST-MDA-7 suppressed tumor cell proliferation without causing a large degree of cell death in both OCCs (compare Fig. 1, B with A). In a similar manner as observed with GST-MDA-7 protein, increasing multiplicities of infection of a type 5 recombinant adenovirus expressing MDA-7/IL-24, Ad.5-mda-7, resulted in dose-dependent tumor cell killing (Fig. 1C). In contrast to several prior studies in other tumor cell types, we noted in OCC that infection with a recombinant adenovirus to express MDA-7/IL-24 was at least as efficacious at killing cells as was treatment with GST-MDA-7. As a result of this observation, we focused our studies using Ad.5-mda-7.

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Fig. 1.

MDA-7/IL-24 causes a dose-dependent induction of growth arrest and OCC death. A and B, OVCAR cells and SKOVIII cells were treated 24 h after plating with GST-MDA-7 (0–200 nM). At the indicated time point after GST-MDA-7 treatment (96 h), cell viability was determined by trypan blue exclusion assay, and cell numbers were determined using hemocytometer (± S.E.M., n = 3). C, OVCAR and SKOVIII cells were infected with type 5 recombinant adenoviruses empty vector (Ad.5-cmv) or to express MDA-7/IL-24 (Ad.5-mda-7) at an m.o.i. of 20 or 80. Cells were isolated 48 h after exposure, and the loss of cell viability above empty vector Ad.5-cmv infection was determined by using Annexin V propidium iodide staining assays in triplicate using a flow cytometer (± S.E.M., n = 3).

Infection of OCC (OVCAR) with Ad.5-mda-7 promoted cleavage of caspase 3 and PARP1 processing of LC3 within 48 h (Fig. 2A). Similar data were obtained in SKOVIII cells (Supplemental Fig. S1). Expression of MDA-7/IL-24 induced phosphorylation of PERK and eIF2α. In a PERK-dependent fashion, MDA-7/IL-24 also reduced ERK1/2 and AKT phosphorylation and activated JNK1/2 and p38 MAPK (Fig. 2B). MDA-7/IL-24 lowered expression of MCL-1 and BCL-XL and increased BAX levels via PERK signaling. Inhibition of both p38 MAPK and JNK1/2 signaling blocked Ad.5-mda-7 lethality that correlated with reduced activation of BAX and BAK (Fig. 2, C and D). Sustained activation of AKT or of MEK1 using molecular tools suppressed Ad.5-mda-7 toxicity (Fig. 2E). Additional suppression of AKT or MEK1 function using dominant-negative constructs further enhanced MDA-7/IL-24 lethality. In SKOVIII cells that were more resistant to MDA-7/IL-24 lethality than OVCAR cells, we noted that inhibition of the extrinsic pathway using c-FLIP-s or CRM A expression blunted MDA-7/IL-24-induced killing, whereas in OVCAR cells, neither c-FLIP-s nor CRM A had an impact on MDA-7/IL-24 toxicity (Fig. 2E).

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Fig. 2.

MDA-7/IL-24 promotes transformed cell killing through ER stress signaling and activation of the JNK/p38 MAPK pathways. A, OVCAR cells were infected with Ad.5-cmv or Ad.5-mda-7 at an m.o.i. of 50. Cells were isolated 12–48 h after infection and processed for SDS-PAGE and immunoblotting against the indicated proteins (n = 3). B, OVCAR cells were transfected with empty vector plasmid (CMV) or with a plasmid to express dominant-negative PERK. Twelve hours after transfection, cells were infected with Ad.5.cmv or Ad.5.mda-7 at an m.o.i. of 50. Cells were isolated 48 h after infection and processed for SDS-PAGE and immunoblotting against the indicated proteins (n = 3). C, OVCAR and SKOV3 cells were infected at an m.o.i. of 50 and 100, respectively, with Ad.5-cmv or Ad.5-mda-7. In parallel as indicated, cells were infected with a virus to express dominant-negative p38 MAPK. Twenty-four hours after infection, cells were treated with vehicle or with JNK-IP (10 μM). At the indicated time points in the graphs, cells were isolated, and the loss of cell viability was determined by trypan blue exclusion assays in triplicate (±S.E.M., n = 3). D, OVCAR cells were infected with viruses and treated with JNK-IP as in C. Forty-eight hours after infection, cells were isolated, and portions of the lysates were subjected to immunoprecipitation for isolation of activated forms of BAX and BAK. The precipitates were subjected to SDS-PAGE alongside an equal portion of the total cell lysates, and blotting was performed to determine the levels of BAX, BAK, and GAPDH (n = 2). E, OVCAR and SKOVIII cells were infected at an m.o.i. of 50 and 100, respectively, with Ad.5-cmv or Ad.5-mda-7. In parallel as indicated, cells were infected with recombinant adenoviruses to express activated and dominant-negative forms of AKT and MEK1 or with viruses to express apoptosis inhibitory proteins (c-FLIP-s, BCL-XL, dominant-negative caspase 9). Treatment with LY294002 (10 μM) occurred 24 h after virus infection. At the indicated time points in the graphs, cells were isolated, and the loss of cell viability was determined by trypan blue exclusion assays in triplicate (± S.E.M., n = 3). F, OVCAR cells were transfected as indicated with expression plasmids (empty vector CMV, dominant-negative PERK, BiP/GRP78, MCL-1) or with siRNA (siSCR, siATG5) and 12 h after transfection, cells were infected with Ad.5-cmv or Ad.5-mda-7 at an m.o.i. of 50. Cells were isolated 72 h after infection, and the loss of cell viability was determined by trypan blue exclusion assays in triplicate (± S.E.M., n = 3).

Prior studies by our laboratories have demonstrated that MDA-7/IL-24-induced activation of PERK was a toxic signal that promoted activation of the JNK pathway as well as a toxic form of autophagy (Yacoub et al., 2008a). In OVCAR cells, Ad.5-mda-7 infection promoted a PERK-dependent vesicularization of a transfected LC3-GFP construct; vesicularization was blocked by knockdown of ATG5 expression (data not shown). Expression of dominant-negative PERK, BiP/GRP78, or knockdown of ATG5 suppressed Ad.mda-7 lethality (Fig. 2F).

Prior studies from our laboratories, including those in Figs. 1 and ​and2,2, have used recombinant type 5 adenoviruses to deliver MDA-7/IL24 to brain cancer cells (Ad.5-mda-7) (Yacoub et al., 2008b). Some OCC tumor cell lines and other tumor cell types such as renal carcinoma and malignant GBM cells are in general not readily amenable to gene therapy approaches using low particle levels of type 5 adenovirus because of their reduced expression of Coxsackie and adenovirus receptor (Park et al., 2009). Because of this, we generated a recombinant adenovirus to express MDA-7/IL24, which contained a modified serotype viral knob from types 5 and 3 adenoviruses (Ad.5/3-mda-7) (Dash et al., 2010). OCCs were poorly killed after exposure to an Ad.5-mda-7 infection at a multiplicity of infection of 20 particles per cell (Fig. 3A). Infection of OCC using an Ad.5/3-mda-7 virus caused significant amounts of cell killing at an m.o.i. of either 20 or 80 (Fig. 3A). These data argue that modified serotype type 5/serotype 3 recombinant viruses may represent useful gene delivery system in ovarian carcinoma.

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Fig. 3.

A tropism-modified type 5/type 3 virus infects ovarian cancer cells more readily than a type 5 virus and enhances cisplatin toxicity. A, OVCAR and SKOVIII cells were infected with Ad.5-cmv or Ad.5-mda-7 or with tropism-modified viruses Ad.5/3-cmv or Ad.5/3-mda-7 at an m.o.i. of 20 or 80, as indicated. Cells were isolated 48 h after infection, and the loss of cell viability was determined by trypan blue exclusion assays in triplicate (± S.E.M., n = 3). Inset, cells were infected with the indicated viruses, and 24 h after infection, cells were isolated to determine the expression of MDA-7/IL-24 and the cleavage status of PARP1. B, OVCAR and SKOVIII cells were infected with Ad.5-cmv or Ad.5-mda-7 or with tropism-modified viruses Ad.5/3-cmv or Ad.5/3-mda-7 at an m.o.i. of 80. Twenty-four h after infection, cells were treated with vehicle or cisplatin (CDDP, 3 μM). Seventy-two hours after infection, cells were isolated, and the loss of cell viability was determined by trypan blue exclusion assays in triplicate (± S.E.M., n = 3). Inset, identical portions of OVCAR cells, infected as indicated, were isolated 72 h after exposure, and the loss of cell viability was determined by using Annexin V/propidium iodide staining assays in triplicate using a flow cytometer (± S.E.M., n = 3). C, OVCAR and SKOVIII cells were infected with Ad.5-cmv or Ad.5-mda-7 at an m.o.i. of 80, and 24 h after infection, cells were treated with vehicle or BBR3464 (BBR3464, 100–300 nM). Forty-eight hours after infection, cells were isolated, and the loss of cell viability was determined by trypan blue exclusion assays in triplicate (± S.E.M., n = 3). D, OVCAR cells were plated in sextuplicate as single cells were infected with Ad.5-cmv or Ad.5-mda-7 at an m.o.i. of 80, and 24 h after infection, cells were treated with vehicle or cisplatin (CDDP, 3 μM). The media were changed to drug-free media 48 h after CDDP treatment, and colonies were permitted to form for 10 to 14 days (± S.E.M., n = 2).

Platinum-containing chemotherapeutic drugs are a well established modality for the treatment of ovarian cancer. Infection of tumor cells with Ad.5-mda-7 enhanced the toxicity of cisplatin (Fig. 3B). Data similar to those in OCCs using cisplatin were also observed in primary human GBM cells (Supplemental Fig. S2). Enhanced toxicity of cisplatin, when combined with an oncolytic adenovirus expressing MDA-7/IL-24, has also been observed in hepatocellular carcinoma cells (Wu et al., 2009). BBR3464 is a novel platinum-based drug that has undergone phase II evaluation. In a manner similar to cisplatin, infection of tumor cells with Ad.5-mda-7 enhanced the toxicity of BBR3464 (Fig. 3C). Data similar to those in OCCs using BBR3464 were also observed in primary human GBM cells (Supplemental Fig. S3). In colony-formation assays, Ad.5-mda-7 and cisplatin interacted in a greater than additive fashion to suppress clonogenic survival (Fig. 3D).

We next determined the impact of Ad.5-mda-7 infection and cisplatin treatment on the activities of survival-signaling kinases and apoptosis-regulatory caspases. Expression of MDA-7/IL-24 promoted the cleavage of caspase 3 and the activation of JNK1/2, p38 MAPK, and PERK (Fig. 4A). Treatment of cells expressing MDA-7/IL-24 with cisplatin caused additional cleavage of caspase 3 and activation of JNK1/2 but not additional activation of PERK. Expression of activated MEK1 EE increased the protein levels of BiP/GRP78 and blocked MDA-7/IL-24-induced activation of PERK. These effects correlated with sustained activation of ERK1/2 and reduced activation of JNK1/2 and of caspase 3. Expression of dominant-negative MEK1 suppressed ERK1/2 activity and BiP/GRP78 levels and enhanced MDA-7/IL-24-induced JNK1/2 and PERK activation and cleavage of caspase 3. Coexpression of dominant-negative MEK1 and dominant-negative AKT significantly enhanced the toxicities of MDA-7/IL-24, cisplatin, and MDA-7/IL-24 + cisplatin treatments (Fig. 4B). Expression of constitutively activated forms of either MEK1 or AKT suppressed MDA-7/IL-24 + cisplatin toxicity. Inhibition of caspase-9 or JNK pathway signaling blocked MDA-7/IL-24 + cisplatin-induced killing (Fig. 4C). Overexpression of BiP/GRP78 also suppressed MDA-7/IL-24 and MDA-7/IL-24 + cisplatin-induced killing as measured in trypan blue dye exclusion assays, which correlated with reduced activation of PERK and sustained expression of MCL-1 and c-FLIP-s (Fig. 4C, inset). Data very similar to those using trypan blue were obtained using Annexin V/PI flow cytometry analyses (Fig. 4D).

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Fig. 4.

Ad.5-mda-7 + cisplatin toxicity in OCCs is dependent on PERK, JNK and CD95 signaling. A, OVCAR cells were infected with Ad.5-cmv or Ad.5-mda-7 in parallel with Ad.cmv, Ad.MEK1 EE, or Ad.dnMEK1. Twenty-four hours after infection, cells were treated with vehicle or CDDP (3 μM). Forty-eight hours after infection, cells were isolated and processed for SDS-PAGE and immunoblotting against the indicated proteins (n = 3). B, OVCAR and SKOVIII cells were infected with the indicated viruses at the stated m.o.i., and 24 h after infection, cells were treated with vehicle or cisplatin (CDDP, 3 μM). Cells were isolated 72 h after infection, and the loss of cell viability was determined by trypan blue exclusion assays in triplicate (± S.E.M., n = 3). C, OVCAR cells were infected with Ad.5-cmv or Ad.5-mda-7 at an m.o.i. of 80 in parallel with viruses to express dominant-negative caspase 9, BCL-XL, or dominant-negative p38 MAPK. In parallel, cells were transfected with empty vector plasmid or plasmid to express Grp78/BiP. Twenty-four hours after infection as indicated, cells were treated with JNK-IP (10 μM) followed by cisplatin treatment (CDDP, 3 μM). Cells were isolated 72 h after infection, and the loss of cell viability was determined by trypan blue exclusion assays in triplicate (± S.E.M., n = 3). Inset, cells infected to express MDA-7/IL-24 and/or BiP/GRP78 were isolated 48 h after infection and processed for SDS-PAGE and blotting to determine the expression of MCL-1, c-FLIP-s, and GAPDH and the phosphorylation of PERK. D, OVCAR cells were infected and treated with cisplatin in a fashion identical with that described in C. Cells were isolated 72 h after infection, and the loss of cell viability was determined by using Annexin V/propidium iodide staining assays in triplicate using a flow cytometer (± S.E.M., n = 3). E, OVCAR cells were infected with caspase 8 inhibitors (c-FLIP-s, CRM A) or dominant-negative p38 MAPK or were transfected to express dominant-negative PERK or to knockdown ceramide synthase 6 (LASS6). Twenty-four hours after infection, cells where indicated were treated with JNK-IP (10 μM). Cells were isolated 72 h after infection, and the loss of cell viability was determined by trypan blue exclusion assays in triplicate (± S.E.M., n = 3). Inset, OVCAR cells growing in glass-chambered slides in triplicate were infected to express MDA-7/IL-24 and were treated 24 h after infection with CDDP (3 μM) for 6 h. Cells were fixed but not permeabilized, and the levels of cell surface CD95 was determined by immunohistochemistry (± S.E.M., n = 2). dn, dominant negative.

In contrast to the minimal effects on altering MDA-7/IL-24 lethality as a single agent, inhibition of caspase-8 function expressing either c-FLIP-s or CRM A prevented cisplatin-enhancing MDA-7/IL-24-induced killing (Fig. 4E). Prior studies in renal cancer cells have shown that ceramide synthase 6 plays a key role in MDA-7/IL-24 lethality, implicating the de novo ceramide synthesis pathway as a mediator of MDA-7/IL-24 effects (Park et al., 2009). Knockdown of ceramide synthase 6 expression suppressed MDA-7/IL-24 and MDA-7/IL-24 + cisplatin lethality (Fig. 4E). Knockdown of ATG5 also suppressed MDA-7/IL-24 + cisplatin-induced killing (Supplemental Fig. S4). Cisplatin treatment activated CD95, and this effect was enhanced by the expression of MDA-7/IL-24 (Fig. 4E, inset). Our data demonstrate that MDA-7/IL-24-induced activation of PERK plays a key role in regulating mitochondrial stability through MCL-1 and that cisplatin enhances MDA-7/IL-24 toxicity via activation of the extrinsic pathway and de novo ceramide synthesis.

Finally, we determined whether Ad.5-mda-7 interacted with another clinically relevant therapeutic agent used in OCC therapy: paclitaxel. In both OVCAR and SKOVIII cells, paclitaxel interacted in an additive fashion to promote Ad.5-mda-7 lethality (Fig. 5A). Cisplatin and paclitaxel interacted to kill OVCAR cells in at least an additive fashion and the drug combination facilitated Ad.5-mda-7 lethality significantly greater than that caused by the cisplatin + Ad.5-mda-7 combination (Fig. 5B). In true percentage terms, Ad.5-mda-7 and cisplatin caused 6.2 ± 0.1% more killing than either agent alone, whereas Ad.5-mda-7 and cisplatin and paclitaxel treatment caused 22.2 ± 2.3% (p < 0.05) more killing than each agent alone. These findings correlated with greater levels of procaspase 3 and PARP1 cleavage (Fig. 5B, top inset). The results also correlated with further increases in BAX levels without any significant further decrease in BCL-XL levels; this suggests that the apoptotic rheostat is further altered toward prodeath signaling by the treatment with paclitaxel. Cisplatin and paclitaxel facilitated Ad.5-mda-7 lethality to kill OVCAR cells in at least a greater than additive fashion (Supplemental Fig. S6). In agreement with the concept that enhanced levels of BAX were causal in the elevated levels of tumor cell killing, knockdown of BAX expression significantly reduced the lethal interaction between Ad.5-mda-7, cisplatin, and paclitaxel (Fig. 5C). Collectively, the data in Figs. 3 to ​to55 demonstrate that expression of MDA-7/IL-24, via Ad.5-mda-7 or Ad.5/3-mda-7, enhances the toxic effects of established OCC therapeutic drugs in OCCs.

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Fig. 5.

Paclitaxel enhances the toxicity of Ad.5-mda-7 + cisplatin in a greater than additive fashion. A, OVCAR and SKOVIII cells were infected with Ad.5-cmv or Ad.5-mda-7 at an m.o.i. of 80. Twenty-four hours after infection, as indicated, cells were treated with paclitaxel (paclitx., 0–100 nM). Cells were isolated 48 h after infection, and the loss of cell viability was determined by trypan blue exclusion assays in triplicate (± S.E.M., n = 3). B, OVCAR cells were infected with Ad.5-cmv or Ad.5-mda-7 at an m.o.i. of 80. Twenty-four hours after infection, as indicated, cells were treated with paclitaxel (paclitx., 10 nM) and/or cisplatin (CDDP, 3 μM). Cells were isolated 48 h after infection, and the loss of cell viability was determined by trypan blue exclusion assays in triplicate (± S.E.M., n = 3). Top inset, cells were isolated 24 h after virus infection and processed for SDS-PAGE and immunoblotting against the indicated proteins to determine expression/phosphorylation (n = 2). C, OVCAR cells were transfected with scrambled siRNA (siSCR) or an siRNA to knock down BAX expression and 24 h later were infected with Ad.5-cmv or Ad.5-mda-7 at an m.o.i. of 80. Twenty-four hours after infection, as indicated, cells were treated with paclitaxel (paclitx., 10 nM) and cisplatin (CDDP, 3 μM). Cells were isolated 48 h after infection, and the loss of cell viability was determined by trypan blue exclusion assays in triplicate (± S.E.M., n = 3).

Discussion

Previous studies have noted that GST-MDA-7 (or Ad.5-mda-7) reduces proliferation and causes tumor cell- and transformed cell-specific killing, as well as radiosensitization in malignant glioma, prostate, and ovarian cancer cells (Su et al., 2006; Yacoub et al., 2004, 2008b; Emdad et al., 2006). However, the precise signaling pathways modified by GST-MDA-7 (or Ad.5-mda-7) as a single agent and causally related to its cancer-specific cell killing effects in human ovarian carcinoma cells are still not well understood.

Infection of OCCs with a dose of Ad.5-mda-7 virus particles that caused profound toxicity after ~72 h correlated with strong activation of the JNK1/2 and p38 MAPK pathways as well as of PERK and eIF2α. This treatment, in parallel, nearly abolished ERK1/2 and AKT signaling. Multiple studies using a variety of cytokine and toxic stimuli document that prolonged JNK1–3 and/or p38 MAPK activation in a wide variety of cell types can trigger cell death (Xia et al., 1995; Matsuzawa et al., 2002; Park et al., 2009). The balance between the readouts of ERK1/2 and JNK1–3 signaling may also represent a common key homeostatic mechanism that regulates cell survival versus cell death processes. Inhibition of JNK1/2 or p38 MAPK reduced MDA-7/IL-24 toxicity, and inhibition of both pathways abolished killing; this was associated with reduced activation of BAX and BAK.

We have published studies arguing that signaling via PERK can promote tumor cell death or alternatively cell survival based on the stimulus (Yacoub et al., 2008a,b; Park et al., 2008). There are three primary unfolded protein response sensors: PERK, activating transcription factor 6, and IRE1. As unfolded proteins accumulate, BiP (GRP78), the 70-kDa heat shock protein ER resident chaperone, dissociates from PERK, activating transcription factor 6, or IRE1. BiP/GRP78 dissociation from PERK allows this protein to dimerize, autophosphorylate, and then phosphorylate eukaryotic translation initiation factor α, the protein required for bringing the initiator methionyl-transfer RNA to the 40S ribosome (Park et al., 2008; Zhang et al., 2008). Phosphorylated eIF2α thus leads to the repression of global translation, helping to allow cells to recover from the accumulation of unfolded proteins. Reduced translation, however, can also lower the expression of some prosurvival proteins such as MCL-1 and c-FLIP-s, as noted in OCCs after the expression of MDA-7/IL-24, leading to increased cell death. MDA-7/IL24 binds to BiP/GRP78, and in OCCs, we hypothesized that this binding would play a central role in regulating PERK activity/activation and regulating MDA-7/IL24-induced cell killing (Gupta et al., 2006a). Indeed, a constitutively active form of MEK1 maintained ERK1/2 phosphorylation in cells expressing MDA-7/IL-24, and increased BiP/GRP78 expression and blocked PERK activation. Overexpression of BiP/GRP78 blocked MDA-7/IL-24–induced PERK activation, and expression of dominant-negative PERK blocked MDA-7/IL-24-induced activation of JNK1/2 as well as reduced expression of MCL-1. Thus, in OCCs, MDA-7/IL-24, by binding to BiP/GRP78, results in the disruption of the BiP/GRP78-PERK chaperone interaction, resulting in enhanced PERK signaling into the eIF2α and JNK downstream pathways that act to promote mitochondrial dysfunction via decreased protective BCL-2 family protein expression and increased activity of toxic BH3 domain proteins, respectively.

In one ovarian cancer cell line, MDA-7/IL-24 lethality has been shown previously to be mediated by CD95-caspase-8 signaling, indicating that the extrinsic pathway to apoptosis could be activated by this cytokine (Gopalan et al., 2005). However, in many of our prior analyses with MDA-7/IL24 in breast, prostate, pancreatic, and brain tumor cells, as well as from the work of others, it was noted that MDA-7/IL-24-induced cell death was mediated by the disruption of mitochondrial function with little or no involvement of death receptors or caspase-8 in the killing process. Our present studies suggest that in SKOVIII cells, but not OVCAR cells, extrinsic pathway signaling also plays a role in MDA-7/IL-24 lethality. In renal carcinoma cells, we have noted recently that overexpression of c-FLIP-s or the caspase-8 inhibitory protein CRM A blocked GST-MDA-7 lethality, and knockdown of CD95 or FADD expression also reduced GST-MDA-7 toxicity (Park et al., 2009).

In addition to JNK pathway-mediated activation of BAX and BAK, our findings in OVCAR cells also argued that MDA-7/IL-24 caused altered ratios in the total expression of proapoptotic BH3 domain-containing proteins, particularly BAX, and antiapoptotic proteins, such as MCL-1 and BCL-XL, with the subsequent activation of caspases-9 and -3; this finding is similar to data obtained by expressing MDA-7/IL-24 in other tumor cell types (e.g., LNCaP prostate cancer cells) (Fisher, 2005). In other prostate cancer cell types, such as DU-145, which lack the expression of BAX, Ad.5-mda-7 is an even more potent inducer of tumor cell death than is observed in LNCaP cells. In GBM cells, our prior data argued that at least five BH3 domain-containing proteins could potentially mediate GST-MDA-7 toxicity downstream of GST-MDA-7-stimulated activation of PERK and JNK1–3 (Yacoub et al., 2008a,c). Together, based on these findings, it is probable that the reason why multiple transformed cell types exhibit MDA-7/IL-24 toxicity regardless of their genetic background is because of the pleiotropic range of proapoptotic proteins and pathways that can be recruited by this cytokine to initiate mitochondrial cell death processes.

Platinum-based chemotherapeutic drugs are a mainstay of ovarian cancer therapy (Coleman and Sood, 2006). MDA-7/IL-24 and cisplatin, as well as the novel platinum-containing drug BBR3464, interacted in a greater than additive fashion to kill OCCs in vitro via CD95. In prior work, treating primary hepatocytes with low doses of bile acids, which cause ligand-independent activation of CD95, we discovered that JNK1/2 activation was CD95-dependent, and our recent studies in renal carcinoma cells also demonstrated that JNK1/2 and, to a lesser extent, p38 MAPK activation after GST-MDA-7 treatment required CD95 signaling. In OCCs, cisplatin further enhanced MDA-7/IL-24-induced JNK1/2 activation, and this enhanced JNK1/2 activation was required for the toxic interaction between the two agents. MDA-7/IL-24-induced PERK activation was required for MDA-7/IL-24 and MDA-7/IL-24 + cisplatin-induced JNK1/2 and p38 MAPK activation. Matsuzawa et al., (2002) have implicated previously a TRAF2-ASK1-JNK cascade downstream of IRE1 in ER stress responses in multiple cell types, and based on our data, PERK-dependent signaling could also feed into this cell survival regulatory process. Further studies are necessary to determine whether the enhanced activity of MDA-7/IL24, when administered by an oncolytic adenovirus, with cisplatin in the context of hepatocellular carcinoma (Wu et al., 2009) involve similar signaling pathway alterations as observed in OCCs.

In our recent studies treating primary hepatocytes with bile acids; in RCC/HCC/pancreatic tumor cells with the drugs sorafenib and vorinostat; and in renal carcinoma cells treated with GST-MDA-7, we discovered that ligand-independent activation of CD95 was dependent in part on the actions of ASMase and the de novo ceramide synthesis pathway (Park et al., 2008; Zhang et al., 2008). Recent studies have suggested that the de novo and ASMase pathways of ceramide generation can cooperate to regulate lipid raft function (Zhang et al., 2009); cisplatin was shown to promote CD95 activation and that ceramide generation may play a role in this process (Min et al., 2007). Determining the molecular mechanisms by which MDA-7/IL-24 enhances cisplatin-induced activation of CD95 and whether cisplatin-induced DNA damage increases expression of ceramide synthase genes require studies beyond the scope of the present article.

The studies in this article were also engaged to determine whether we could enhance the infectivity of adenoviral gene transduction in OCCs, using a chimeric adenovirus containing the knob protein expressing portions of the types 5 and 3 adenoviruses. A type 5/3 recombinant adenovirus was more efficacious at delivering mda-7/IL-24 to tumor cells than a type 5 virus, resulting in greater expression of MDA-7/IL-24. The greater expression of MDA-7/IL-24 using the type 5/3 virus resulted in a greater amount of tumor cell killing, as observed using this virus in low Coxsackie and adenovirus receptor prostate cancer cells (Dash et al., 2010). It is noteworthy that whereas infection using Ad.5/3-mda-7 generated at least 10-fold more MDA-7/IL-24 protein, it only enhanced apoptosis ~3-fold more than Ad.5-mda-7 infection. This argues that there is a certain threshold at which MDA-7/IL-24 becomes toxic to a tumor cell, and producing more MDA-7/IL-24 may not per se increase killing. We have observed a similar dose-dependent effect of GST-MDA-7 on GBM and renal carcinoma cells in vitro (Yacoub et al., 2008a; Park et al., 2009). However, based on the pleiotropic antitumor effects of MDA-7/IL-24 in vivo, including the inhibition of tumor angiogenesis, potent “bystander” anticancer activity and immune modulating activity, having enhanced the expression of MDA-7/IL-24, may directly contribute to and enhance the anticancer properties of this cytokine in vivo (Fisher, 2005; Sarkar et al., 2005, 2008; Sauane et al., 2008; Emdad et al., 2009; Greco et al., 2009).

Supplementary Material

References