Melanoma Differentiation Associated Gene-7, mda-7/Interleukin-24, Induces Apoptosis in Prostate Cancer Cells by Promoting Mitochondrial Dysfunction and Inducing Reactive Oxygen Species

Introduction

Subtraction hybridization and microarray analyses are defining the spectrum of gene expression changes occurring as a consequence of induction of irreversible growth arrest and terminal differentiation in human melanoma cells (1 , 2) . Application of the subtraction hybridization approach to terminal differentiation in human melanoma cells resulted in the cloning of mda-7 7 (3) . Expression of mda-7 inversely correlates with tumorigenic progression from melanocyte to malignant melanoma suggesting a putative function as an inhibitor of melanoma progression (3 , 4) . On the basis of its structure, cytokine-like properties, and proposed mode of action, mda-7 has now been classified as IL-24 (5 , 6) . Initial studies focused on the growth inhibitory properties of this molecule, resulting in the provocative observation that mda-7/IL-24 selectively suppresses the growth of a broad spectrum of tumor cells, without affecting growth of normal cells (3 , 7) . When introduced into tumor cells by adenovirus transduction, Ad.mda-7 induces apoptosis in multiple cancer cell types but not in normal cells (8 , 9) . On the basis of the apparent selectivity of mda-7/IL-24 toward cancer cells in vitro and animal studies confirming antitumor properties in vivo, Ad.mda-7 was evaluated recently in a Phase I/II clinical trial in patients with advanced carcinomas (10) . These studies document that Ad.mda-7 (INGN 241) is safe, and a single intratumoral injection can result in >70% tumor apoptosis with a concomitant inhibition in disease progression.

On the basis of the potential utility of mda-7/IL-24 for the gene-based therapy of multiple cancers, understanding the mechanism by which this gene selectively induces apoptosis in cancer cells will be of immense value. Studies initially performed in the context of human breast carcinoma cells documented that Ad.mda-7 up-regulates expression of the proapoptotic protein Bax and decreases expression of the antiapoptotic protein Bcl-2, thereby shifting the balance from survival to death (8) . Moreover, both overexpression of Bcl-2 or the adenovirus type 5 E1B proteins were able to protect breast cancer cells from loss of survival after infection with Ad.mda-7 suggesting that changes in the levels of Bcl-2-family proteins and mitochondrial function are relevant to mda-7/IL-24 action (8) . Studies in additional model systems, including lung, pancreatic and prostate carcinomas, melanomas, and malignant gliomas provide additional support for a functional relationship between changes in the ratio of proapoptotic proteins, Bax and/or Bak, to antiapoptotic proteins, such as Bcl-2 and/or Bcl-x_L, in Ad.mda-7-induced apoptosis (5 , 11) . In addition, recent studies demonstrate that overexpression of Bcl-2 and Bcl-x_L can differentially protect specific prostate cancer cell lines from Ad.mda-7-induced apoptosis (12) . Although these studies provide support for a role of mitochondria in Ad.mda-7-induced apoptosis, definitive evidence for such an involvement is lacking. On the basis of these considerations, we have presently investigated the relationship between mitochondrial function and cellular redox status, and resistance or sensitivity to Ad.mda-7-induced apoptosis in normal and tumor-derived human prostate cells. These studies document the importance of dysregulation of mitochondrial functions and redox changes as cellular pathways specifically perturbed by Ad.mda-7, which culminate in the selective induction of apoptosis in prostate cancer cells.

Materials and Methods

Cell Lines and Virus Infection Protocol.

Human DU-145, PC-3, and LNCaP prostate carcinoma cells were obtained from the American Type Culture Collection and cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 1% MEM sodium pyruvate, and nonessential amino acids. P69, an SV40-immortalized human prostate epithelial cell line was provided by Dr. Joy Ware (Virginia Commonwealth University, Richmond, VA) and grown under serum-free conditions as described previously (13) . Bcl-2 and Bcl-x_L stable overexpressing clones of each prostate cancer cell line were generated and cultured as described (12) . The recombinant replication-defective Ad.mda-7 virus was created in two steps as described previously (8) and plaque purified by standard procedures. Cells were infected with 100 plaque forming units/cell of Ad.mda-7 or Ad.vec(30 plaque forming units/cell for LNCaP cells) and analyzed as described.

MTT Viability Assays.

Cell viability was assessed by MTT assays as described (12) . Briefly, cells were seeded in 96-well tissue culture plates (1.5 × 10³ cells/well) and treated with various agents. At the indicated times, medium was removed, and fresh medium containing 0.5 mg/ml MTT was added to each well. The cells were incubated at 37°C for 4 h and then an equal volume of solubilization solution (0.01 N HCl in 10% SDS) was added to each well and mixed thoroughly. The absorbance from the plates was read on a Bio-Rad Microplate Reader Model 550 at 595 nm.

Annexin V Binding Assays.

Cells were trypsinized, washed once with complete medium, and stained with FITC-labeled Annexin-V (kit from Oncogene Research Products, Boston, MA) according to the manufacturer’s instructions. Flow cytometry was performed immediately after staining.

Assessment of Mitochondrial Δψ_m and ROS Production.

Changes in mitochondrial transmembrane potential Δψ_m were determined by staining cells in 20 nm of DiOC₆ (3) in PBS for 30 min at 37°C in the dark. The dye accumulates in actively respiring mitochondria depending on Δψ_m (14) . Controls were performed in the presence of 50 μm mitochondrial uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (Sigma). To determine ROS production, cells were stained with 2.5 μm HE or 5 μm DCFH-DA in PBS for 30 min at 37°C in the dark (15) . Immediately after staining, cells were analyzed by flow cytometry (FACSscan; Becton-Dickinson, Mountain View, CA), and data were analyzed using CellQuest software, version 3.1 (Becton Dickinson). For inhibition experiments, NAC, CsA, BA (all from Sigma) or z-VAD.fmk (Calbiochem, La Jolla, CA) were added 2 h before infection with Ad.mda-7. In all of the cases, cells were gated to exclude cell debris.

Statistical Analysis.

All of the experiments were performed at least three times. Results are expressed as mean ± SE Statistical comparisons were made using an unpaired two-tailed Student’s t test. A P < 0.05 was considered significant.

Results and Discussion

Ad.mda-7 Induces ROS and Apoptosis Selectively in Prostate Cancer Cells.

Ad.mda-7 infection inhibits proliferation and induces apoptosis in diverse prostate cancer cell lines but not in normal human prostate epithelial cells (12) . Moreover, overexpression of antiapoptotic members of the Bcl-2-family differentially protects prostate carcinoma cells from Ad.mda-7-induced apoptosis. In the present study, we used these model systems to determine whether Ad.mda-7 regulates the levels of intracellular ROS and whether a rise in ROS is necessary for Ad.mda-7-mediated apoptosis.

ROS (including singlet oxygen and hydrogen peroxide, as well as free radicals such as superoxide anion and hydroxyl radicals) regulate apoptosis and proliferation in response to a variety of stimuli, including tumor necrosis factor-α, UV and γ-irradiation, and anthracyclines (16) . To determine whether ROS production contributes to apoptosis induction by Ad.mda-7 in prostate cancer cells, normal immortal prostate epithelial cells (P69; Ref. 13 ) and prostate carcinoma cells were infected with Ad.vec or Ad.mda-7, and the effect of antioxidants (NAC and Tiron) on Ad.mda-7-induced cell death was evaluated by MTT assays. In the presence of noncytotoxic doses of a general antioxidant, NAC (5 mm), or a free radical and hydrogen peroxide scavenger, Tiron (1 mm), the cell death promoting activity of Ad.mda-7 was abrogated in all three of the prostate carcinoma cell lines (Fig. 1A) ⇓ . This inhibitory effect of antioxidant treatment was not associated with altered MDA-7 cellular or secreted protein levels (data not shown). To explore the relationship between ROS and Ad.mda-7-induced apoptosis additional experiments were performed with As₂O₃ and NSC656240 (a dithiophene), two agents that can promote ROS production and apoptosis in cancer cells (Refs. 17 , 18 ; Fig. 1B ⇓ ). 8 Cotreatment of Ad.mda-7-infected prostate cancer cells with noncytotoxic doses of As₂O₃ (0.5 μm) or NSC656240 (400 nm) potentiated cell death in all three of the prostate carcinoma cell lines but not in normal immortal P69 prostate epithelial cells (Fig. 1B) ⇓ . These observations suggest that free radicals may contribute to Ad.mda-7 induction of apoptosis in prostate carcinoma cells.

To confirm selective induction of ROS in prostate carcinoma cells after infection with Ad.mda-7, we measured the levels of intracellular free radicals in P69 and prostate cancer cells before and after Ad.mda-7 infection using two dyes: DCFH-DA and HE. Nonfluorescent DCFH-DA diffuses into cells, where it is deacetylated to DCF, which fluoresces on reaction with hydrogen peroxide or nitrous oxide. HE enters the cell and can be oxidized by superoxide or free hydroxyl radicals to yield fluorescent ethidium (15) . Comparing these two dyes in prostate carcinoma cells indicated that Ad.mda-7 induced DCF fluorescence (indicating hydrogen peroxide and nitrous oxide production) to a somewhat greater extent than HE fluorescence (indicating free hydroxyl radical formation; data not shown). On the basis of this consideration and because both values (DCF and HE fluorescence) were found to change coordinately, we used DCF fluorescence as our readout for ROS production.

Flow cytometry analysis of cellular fluorescence revealed that Ad.mda-7 infection produced a 3–5-fold increase in ROS production in prostate carcinoma cells but not in normal P69 cells (Fig. 1C) ⇓ . The largest ROS induction effect was apparent in LNCaP cells followed by DU-145 cells with the smallest changes occurring in PC-3 cells. Similarly, when treated with NSC656240 or As₂O₃, ROS levels increased in all of the prostate carcinoma cells with the greatest level of induction being apparent in LNCaP cells (Fig. 1C) ⇓ . Treatment with a noncytotoxic dose of NAC suppressed Ad.mda-7-induced ROS generation, and also inhibited As₂O₃ and NSC656240 stimulated ROS generation in prostate cancer cells (Fig. 1 ⇓ C; data not shown). The increase in ROS production coincided with apoptosis induction in the prostate cancer cell lines, as confirmed by Annexin V binding (Fig. 1D) ⇓ . As observed for ROS induction, pretreatment with nontoxic doses of NAC prevented Ad.mda-7-induced apoptosis. Moreover, a combination treatment with Ad.mda-7 plus As₂O₃ or NSC656240 increased apoptosis to variable extents in the three prostate cancer cell lines, without inducing apoptosis in normal P69 cells (Fig. 1D) ⇓ .

Ad.mda-7 Temporally and Selectively Induces ROS Production and Δψ_m Reduction in Prostate Carcinoma Cells.

Because ROS may play a dual role in apoptosis, either being a modulator of mitochondrial membrane potential loss or a consequence of this change, depending on the death stimuli (14 , 19) , we determined the time course of mitochondrial changes (ROS, Δψ_m, and membrane apoptotic changes (Annexin V binding) after Ad.vecor Ad.mda-7 infection (Fig. 2) ⇓ . Cells were infected with Ad.vecor Ad.mda-7 at the indicated multiplicity of infection, collected at different times up to 60 h, and stained for ROS production with DCFH-DA and with the cationic mitochondrial dye DiOC₆ (3) , which accumulates in active mitochondria, to determine changes in Δψ_m (14) . In parallel, annexin V binding assays were performed as described in “Materials and Methods.”

A detailed analysis of the time course of ROS production and Δψ_m changes confirmed that in all three of the prostate cancer cell lines, initial decreases in Δψ_m occurred before ROS induction in Ad.mda-7-treated cells (Fig. 2) ⇓ . The initial small decrease in Δψ_m at early times (6–8 h) was followed by increased ROS production (10–20 h). The decline in Δψ_m continued up to 12 h in LNCaP and up to 30 h in DU-145 and PC-3. At 45–50 h, a secondary burst of ROS production and a concurrent final steep drop in Δψ_m occurred, documenting complete mitochondrial dysfunction. As shown in Fig. 2 ⇓ , the decline in Δψ_m and the increase in annexin V binding (an early indicator of cytoplasmic apoptosis) occurred concomitantly, consistent with prior reports of apoptosis-associated events in other cell types (14) . Thus, our present studies suggest that Ad.mda-7-induced apoptosis may follow a similar chronology. Time-lapse video microscopy is one approach that can be used to confirm these temporal changes in ROS production and Δψ_m in prostate carcinoma cells after infection with Ad.mda-7.

It is worth noting that in all of the prostate cancer cell lines, a correlation was observed between mitochondrial changes and MDA-7 protein expression. Mitochondrial changes in Ad.mda-7-infected prostate cancer cells first became apparent when MDA-7 protein was initially detected by immunoblotting (data not shown). In a previous study, we demonstrated that MDA-7 protein first appeared by 6–9 h after Ad.mda-7 infection of prostate cancer cells (12) . However, despite similar kinetic changes in MDA-7 protein expression in Ad.mda-7-infected normal prostate epithelial cells, no decline in survival, mitochondrial functions, or induction of apoptosis were evident (Fig. 2 ⇓ ; Ref. 12 ).

Ad.mda-7 Infection Modulates MPT in Prostate Cancer Cells.

Because ROS production and the decline in Δψ_m were directly associated with apoptosis or reduced cell survival in prostate carcinoma cells infected with Ad.mda-7 (Fig. 2) ⇓ , the role of MPT in Ad.mda-7-induced apoptosis was investigated. MPT is characterized by the opening of mitochondrial megachannels to allow solutes and water to enter the mitochondria (20) . MPT can be triggered by ROS or other agents resulting in a decrease in Δψ_m, followed by depletion of ATP or activation of caspases/endonucleases (reviewed in Ref. 19 ). This process is controlled by a multiprotein complex found in the inner and outer membranes of the mitochondria known as the PTP (20) . The PTP consists of voltage-dependent anion channel/porin, adenosine nucleotide translocator, cyclophilin D, the complex forming the PBzRs, and other proteins (20) . Upon PTP opening, the mitochondria lose their Δψ_m across the inner membrane culminating in apoptosis accompanied by an immediate shutdown of mitochondrial biogenesis. Another consequence of Δψ_m disruption is the uncoupling of oxidative phosphorylation (21) and the generation of superoxide anion on the uncoupled respiratory chain resulting in additional damage to proteins and membranes (14) .

CsA and BA specifically bind to different components of the PTP complex (cyclophilin D and adenosine nucleotide translocator, respectively), thereby preventing mitochondrial membrane permeabilization and apoptosis in a wide variety of cell types (14 , 22, 23, 24) . On the other hand, the PBzR agonist (PK11195) can potentiate the induction of MPT (25) . On the basis of these considerations, prostate cancer cells and normal P69 cells were pretreated with nontoxic doses of CsA (200 nm), BA (50 μm), or PK11195 (50 μm) for 2 h postinfection with Ad.vec or Ad.mda-7, and cellular viability and early (cytoplasmic) apoptosis were accessed 18 h (LNCaP cells) and 24 h (DU-145, PC-3, and P69 cells) after infection. Pretreatment with CsA or BA prevented cell death (Fig. 3A) ⇓ and annexin V exposure in Ad.mda-7-infected prostate cancer cells (Fig. 3C) ⇓ . Analysis of mitochondrial changes [DiOC₆ (3) retention] and ROS production (DCFH-DA staining) confirmed that CsA and BA abrogated the decline in Δψ_m (Fig. 3B) ⇓ . In a recent study using two lung cancer cell lines, H1299 and A549, Ad.mda-7 induced changes in Δψ_m only in A549 cells and CsA did not prevent Ad.mda-7-induced cell death in either of these cell types or alter Δψ_m in A549 cells (26) . The reason for these differences among prostate carcinoma and lung carcinoma cell lines is not currently known but could reflect inherent differences in the mode of action of mda-7/IL-24 in these two tumor cell types or simply differences in experimental protocols, i.e., the dose of CsA used, the temporal kinetics of changes investigated, and/or a different protocol to measure Δψ_m (15) . PK11195 increased mitochondrial depolarization, annexin V staining, and enhanced Ad.mda-7-induced killing in DU-145, PC-3, and LNCaP, without inducing any of these changes in P69 cells (Fig. 3, A–C) ⇓ . The ability of CsA and BA to inhibit Δψ_m decline, ROS production, and apoptosis, and the ability of a PBzR agonist to promote these changes highlights the importance of mitochondria and MPT as arbiters of Ad.mda-7-induced death in prostate carcinoma cells (Fig. 4C) ⇓ .

NAC only minimally inhibited Ad.mda-7-induced Δψ_m in DU-145 and LNCaP cells without affecting this parameter in PC-3 cells (Fig. 3B) ⇓ , in contrast with its ability to prevent Ad.mda-7-induced decreases in cell viability up to 48 h (Fig. 1A) ⇓ and induction of ROS (Fig. 1C) ⇓ in all three of the prostate cancer cell lines, when evaluated at 18 h (LNCaP) and 24 h (DU-145, PC-3, and P69). Similarly, whereas the general caspase inhibitor z-VAD.fmk inhibited cell death in all three of the prostate cancer cells (Fig. 3A) ⇓ , it only partially blocked Ad.mda-7-induced Δψ_m in DU-145 cells without affecting Δψ_m in PC-3 or LNCaP cells (Fig. 3C) ⇓ , suggesting that Ad.mda-7 may initially facilitate mitochondrial changes via a caspase-independent mechanism (27 , 28) .

Bcl-2 and Bcl-x_L Differentially Protect Prostate Cancer Cells from ROS Induction and Decreased Δψ_m After Infection with Ad.mda-7.

We demonstrated previously that overexpression of Bcl-2 and Bcl-x_L can differentially protect prostate carcinoma cells from apoptosis induced by Ad.mda-7 (12) . These bcl-2-family members have also been found to protect diverse cell types from ROS-dependent (29 , 30) and ROS-independent (31) apoptosis. Extensive research on the mechanism of inhibition of apoptosis by Bcl-2 has focused on its interaction with and regulation of mitochondrial function, particularly the mitochondrial permeability pore (32 , 33) . However, the localization of Bcl-2 to intracellular sites of oxygen-free radical generation, including the cytoplasmic face of the mitochondrial outer membrane, endoplasmic reticulum, and nuclear membranes predict that Bcl-2 may exhibit antioxidant properties (30) . Furthermore, Bcl-2 knockout mice express a phenotype consistent with that of mice exposed to chronic oxidative stress (polycystic kidney disease and follicular hypopigmentation; Ref. 34 ).

In the present study, we examined the effect of overexpression of the antiapoptotic Bcl-2-family members on generation of ROS and changes in Δψ_m during Ad.mda-7-induced apoptosis. To examine the relationship between Bcl-2-family members, and changes in mitochondrial function and ROS we used a series of well-characterized prostate cancer cell clones displaying stable overexpression of Bcl-2 or Bcl-x_L (12) . In DU-145 and PC-3 cells overexpression of Bcl-x_L and not Bcl-2 prevented Ad.mda-7-induced apoptosis, whereas in LNCaP cells overexpression of Bcl-2 and not Bcl-x_L was protective (12) . As shown on Fig. 4 ⇓ A, overexpression of Bcl-x_L completely abrogated the decline in ΔΨ_m in DU-145 and PC-3 cells after Ad.mda-7 infection, without preventing this change in LNCaP cells. In contrast, Bcl-2 overexpression, but not Bcl-x_L overexpression, prevented the reduction in Δψ_m in LNCaP cells on Ad.mda-7 infection. Similarly, Bcl-x_L, but not Bcl-2, protected DU-145 and PC-3 cells from Ad.mda-7-induced ROS induction, whereas Bcl-2, but not Bcl-x_L, protected LNCaP cells from this biochemical change (Fig. 4B) ⇓ . These studies provide additional evidence that induction of apoptosis in prostate cancer cells by Ad.mda-7 is linked to changes in mitochondrial function (reduction in Δψ_m) and ROS production.

Concluding Remarks.

It is now firmly established that mda-7/IL-24 can induce apoptosis in a selective manner in a broad spectrum of cancer cells (7 , 8 , 11 , 12 , 35 , 36) . Although progress has been made in defining the signaling and biochemical events involved in mda-7/IL-24-induced apoptosis in cancer cells (5 , 36) , the precise mechanism underlying the cancer specificity of mda-7/IL-24 and the cellular targets of mda-7/IL-24 action remain essentially unknown. In the case of prostate, Ad.mda-7 induces apoptosis in carcinomas but not in normal prostate epithelial cells (12) . In contrast, overexpression of specific antiapoptotic members of the Bcl-2-family, Bcl-2 and Bcl-x_L, differentially protects prostate cancer cells from apoptosis (12) . On the basis of our present studies, a hypothetical model of Ad.mda-7-induced apoptosis in prostate cancer cells can be constructed (Fig. 4C) ⇓ . In this model, Ad.mda-7 infection of prostate carcinoma cells, but not normal prostate epithelial cells, results in biochemical changes specifically in these tumor cells that result in mitochondrial dysfunction and ROS induction. Ad.mda-7 infection leads to the loss of Δψ_m and a burst of ROS, before the visualization of the apoptotic events such as phosphatidylserine exposure (as monitored by annexin V staining). In the apoptotic process, Ad.mda-7 induces mitochondrial depolarization, which is associated with MPT followed by ROS production. This apoptotic process is inhibited by simultaneous treatment with ROS inhibitors, NAC and Tiron, and potentiated by simultaneous treatment with ROS inducers, As₂O₃, NSC656240, and the PBzR agonist PK11195. The ability of Ad.mda-7 to induce a loss in Δψ_m, enhance ROS production, and trigger apoptosis is inhibited in specific prostate cancer cells by forced overexpression of antiapoptotic members of the Bcl-2 gene family, Bcl-2 or Bcl-x_L. These studies document a relationship between mitochondrial dysfunction and ROS induction and sensitivity to apoptosis induction by mda-7/IL-24 in prostate cancer cells. Our results also suggest that the ability of Ad.mda-7 to selectively kill prostate cancer cells can be augmented by agents that enhance mitochondrial dysfunction and induce ROS production. This finding may suggest strategies for enhancing the cancer-specific, apoptosis-inducing properties of this novel cytokine for translational applications in cancer therapy (36) .