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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Mol Cancer Ther. Author manuscript; available in PMC Dec 1, 2009.
Published in final edited form as:
PMCID: PMC2653264
NIHMSID: NIHMS84623

IL-24 overcomes TMZ-resistance and enhances cell death by downregulation of MGMT in human melanoma cells

Abstract

Melanoma is the most malignant of skin cancers, highly resistant to chemotherapy and radiotherapy. Temozolomide (TMZ), a promising new derivative of dacarbazine is currently being tested for treatment of metastatic melanoma. Resistance to alkylating agents such as TMZ correlates with increased expression of DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). Interleukin 24 (IL-24, mda-7), is a tumor suppressor cytokine that selectively inhibits tumor cell growth by inducing apoptosis and cell-cycle arrest in melanoma cell lines and solid tumors. This tumor-selective activity has been observed in multiple pre-clinical animal models and in clinical trials. In this study, we analyzed the ability of Ad-IL-24 and its protein product, IL-24, to overcome TMZ-resistance in human melanoma cells.

We have demonstrated that Ad-IL-24 via exogenous IL-24 protein induces combinatorial synergy of TMZ-induced cell killing in TMZ-resistant melanoma cells by inhibition of MGMT. Neutralizing antibodies against IL-24 or its receptors significantly blocked the apoptotic activity of IL-24 + MGMT treatment. We show that accumulation of functional p53 is essential for IL-24-induced downregulation of MGMT. Using either MGMT siRNA, p53 siRNA, or a p53 dominant negative mutant to block MGMT protein expression resulted in increased sensitization to TMZ. However, MGMT blockade in combination with IL-24 + TMZ resulted in loss of combinatorial synergy, indicating that MGMT expression is required for the reversal of TMZ-resistance in melanoma cells. This study demonstrates that IL-24 can play a significant role in overcoming TMZ-resistance and that the clinical efficacy of TMZ may be improved by using a biochemotherapy combination with IL-24.

Keywords: IL-24, adenovirus, O6-methylguanine-DNA methyltransferase, Temozolomide, melanoma, chemoresistance

Introduction

Melanoma is a common skin cancer resulting in high morbidity and mortality. Current standard therapy for metastatic melanoma is adjuvant therapy with IFN-α, or IL-2; however, response rates have not exceeded 15-20%. Clinical studies using chemotherapeutic agents as monotherapy or in combination with biotherapies have not improved response rates (1). At the present time dacarbazine (DTIC) is the only FDA-approved chemotherapeutic for melanoma, and is being tested in combination with other chemotherapies; however, the toxicity of these regimens is high. Temozolomide (TMZ; Temodar®) is also being tested as a promising chemotherapeutic treatment (2). Given the limitations of the current therapies for metastatic melanoma there is an urgent need for developing novel treatments that can overcome acquired drug resistance.

TMZ is an oral methylating agent derivative of DTIC. At physiologic pH, TMZ degrades into 5-(3-methyltriazen-1-yl) imidazole-4-carboxamide (MTIC), the active metabolite of DTIC. TMZ has been approved for treatment of resistant anaplastic astrocytomas, and is being tested for treatment of metastatic melanoma (3-5). A phase III study comparing it to DTIC in advanced metastatic melanoma reports significantly longer progression-free survival, improved quality of life, and higher systemic exposure to MTIC for the TMZ arm. TMZ has the advantage of crossing the blood brain barrier, and thus may prevent or treat CNS metastases (4, 6). Regional therapy in an animal model of advanced melanoma comparing intra-arterial delivery of alkylating agents showed that TMZ was more effective than melphalan (7).

The mechanism of action of TMZ is primarily mediated by DNA alkylation, although it can also inhibit other enzymes (esterase and glyoxalase) (8, 9). Three repair mechanisms counteract the action of TMZ: the primary mechanism involves O6-methylguanine-DNA methyltransferase (MGMT), although DNA mismatch repair and poly (ADP-ribose) polymerase also contribute. High levels of MGMT are reported in brain and other tumors, and correlate with resistance to TMZ (10-12). Methylation of the MGMT promoter inhibits its repair activity, and correlates with reduced MGMT protein expression in gliomas and B-cell lymphomas (13, 14). Additionally, low or absent MGMT expression correlates with improved overall and disease free survival in B-cell lymphoma and glioma patients (14, 15). Confirmation for MGMT’s regulation of TMZ activity comes from reports that demonstrate TMZ can decrease the activity of MGMT (16, 17), and inhibition of MGMT enhances TMZ cytotoxicity (18, 19).

Interleukin 24 (IL-24, MDA-7) was originally identified as melanoma differentiation-associated gene-7 (mda-7), a cytokine-tumor suppressor located within a cluster on chromosome 1q32 that encodes IL-10; IL-19 and IL-20 (20). IL-24 protein expression is lost during melanoma tumor progression and virtually all metastatic melanomas lack IL-24 expression (21). IL-24 functions as a pro-Th1 cytokine in human peripheral blood mononuclear cells and induces secretion of IL-6, IFN-γ and TNF-α (22). Adenovirus-mediated IL-24 gene delivery (Ad-IL-24) induces apoptosis selectively in cancer cells while sparing normal cells (23, 24). This tumor-cell-specific growth-inhibitory effect has also been demonstrated using multiple in vivo animal models and has been observed in human clinical trials (25-27). The tumor suppressor activity of IL-24 is independent of the status of other tumor suppressor genes such as p53, Rb, p16 or Ras (24, 28). IL-24 regulates many proliferative control mechanisms in tumor cells and can down-regulate anti-apoptotic proteins (Bcl-2/BCL-xL) and upregulate pro-apoptotic proteins (Bax, Bak); this effect is not seen in normal cells (29, 30). IL-24 also regulates p38 MAPK signaling in melanoma and glioma cells (31). Many studies have established IL-24 as a promising therapeutic candidate with potent antitumor, antiangiogenic, and cytokine activities. In this study, we test the effect of IL-24 on overcoming resistance to TMZ in melanoma and have identified the role of MGMT in overcoming chemoresistance by IL-24. We also explored the mechanism by which IL-24 can reverse resistance to TMZ.

Materials and methods

Cell culture and reagents

All melanoma cell lines were obtained from the America Type Culture Collection (ATCC, Manassas, VA) and were maintained in DMEM (Hyclone, Inc., Logan, Utah) supplemented with 10% fetal bovine serum (Life Technologies, Inc.), 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and HEPES buffer (Life Technologies, Inc., Grand Island, NY). The cells were screened routinely to verify lack of mycoplasma contamination and were used in the log phase of growth. 3,4-dihydro-3-methyl-4-oxoimidazo [5,1-d]-as-tetrazine-8-carboxamide, (TMZ, Schering-Plough Corporation, Kenilworth, N.J., USA) was dissolved in phosphate-buffered saline (PBS) and used at concentrations ranging from 0-6400 μM, although most studies employed 200 μM TMZ. Monoclonal anti-IL-24 antibody was prepared as described previously (32). Rabbit Serine-15 phosphorylated-p53 (p-p53) antibody and SignalSilence® p53 siRNA Kit were purchased from Cell Signaling Technology Inc. (Beverly, MA), β-Actin monoclonal antibody and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) kits were purchased from Oncogene Research Products (San Diego, CA). Antibodies for IL-20R1, IL-22R1 was purchased from R&D Systems, Inc. (Minneapolis, MN). MGMT siRNA, p53 siRNA and control siRNA and transfected medium, Antibodies for MGMT, p53, p21, p-Akt, p-ERK, p-MAPK and all other primary and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cell viability was analyzed by trypan blue exclusion assay. Cells were trypsinized and an aliquot suspended 1:1 volume with 0.4% trypan blue. Total cell numbers and cell viability counts were assessed using a hemocytometer by light microscopy.

Gene transfer

Replication-deficient human type 5 adenovirus (Ad5) carrying the IL-24 gene was previously described (24). The IL-24 gene was linked to an internal CMV-IE promoter, followed by an SV40 polyadenylation sequence. The same adenoviral vector containing the sequence for expression of luciferase (Ad-luc) was used as control virus. Cells were plated 1 day before infection. Target cells were infected with adenoviral vectors (Ad-IL-24 or Ad-luc) using 1,000-3,000 viral particles per cell (50-150 pfu/cell). Experimental conditions were optimized to achieve IL-24 protein expression in >70% of cells, based on results of immunohistochemical staining. The transfections of MGMT and p53 siRNA were executed according to Santa Cruz provided siRNA transfection protocol. MGMT plasmid (ORF Clone that contains full-length of homo sapiens MGMT cDNA) was purchased from OriGene Technologies, Inc. (Catalog: RC201612, Rockville, MD), amplified, and transfected into A375, a MGMT negative cell line, using Lipofectamine2000™ according to standard procedure described above. Transfected cells were incubated at 37°C for 18-48 hours prior to testing for transgene expression. The cells were then passaged at a 1:10 dilution into fresh growth medium 24 hours after transfection and maintained in selective medium (containing 400 μM G418) for stable clone selection. This MGMT expressing A375 subclone was named “A375M”. To establish a MGMT-knock-down cell line from a MGMT highly-expressing melanoma cell line, MGMT-targeted short hairpin (shRNA) and control vectors encoding a neomycin selectable marker (purchased from SuperArray Bioscience Corporation, Frederick, MD) were used to transfect MeWo melanoma cell line according to the manufacturer’s protocol. Western blot analysis was used to evaluate MGMT expression. The pEGFP-N1 plasmid was provided by Dr. Roger Bryan Sutton, at Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston. The pP53wt and pP53mut (p53 codons 22-23 were mutated from Leu-Trp to Gln-Ser) vector constructs were provided by Drs. Kishor K. Bhakat and Sankar Mitra, at the Sealy Center for Molecular Sciences, University of Texas Medical Branch at Galveston (33). MeWo cells were seeded in 6-well plates with antibiotics free medium to 80% confluence. MeWo cells were transfected with 2.5 μg/well pEGFP-N1, pP53wt or pP53mut by Lipofectmine™ 2000 reagent using the methods recommended by the manufacturer (Invitrogen, Carlsbad, CA, USA).

Production and treatment with human IL-24

Ad-IL-24 was transfected (1000 vp/cell, 96 h) into 10 liter wave bioreactor containing 1,700,000/ml HeLa cells grown in serum-free media and supernatant was concentrated 10 times by tangential flow filtration (100K Pellicon II membranes were purchased from Millipore Corporate, Billerica, MA, USA, Feeding pressure ~8 PSI) followed by diafiltration (Feeding pressure ~8 PSI, with four volumes PBS) to approximately 35 μg/ml IL-24. Cells were treated with purified IL-24 protein at 0-39 ng/ml.

Other treatments

For combination temozolomide (TMZ) treatments, 2×105 cells cells were plated and allowed to attach overnight, the next day, cells were treated with either Ad-IL-24, Ad-Luc (both at 0-2000 vp/cell, as indicated in each figure), Temozolomide (200 μM), or a combination of these. 3 or 4 days after treatment, cells were harvested and processed to determine percent cell death, changes in protein expression, or apoptosis, as described in each subsection.

Immunoblotting assay

Immunoblotting was performed using standard procedures as described elsewhere (34). Briefly, 1 × 105 cells/well were plated in 6-well tissue culture plates (Corning Incorporated, Corning, NY) and treated. 4 days later, cells were rinsed in PBS, scraped and lyzed (lysis buffer and protease inhibitor cocktail was purchased from BD Biosciences, San Jose, CA). Protein concentration was determined using a modified Bradford assay (Protein concentration assay reagent was purchased from Bio-Rad Laboratories, Inc., Hercules, CA), and proteins separated by SDS-PAGE in 4-20% Tris Glycine gels (Invitrogen Corporation, Carlsbad, CA). Proteins were then transferred to a nitrocellulose membrane (Invitrogen Corporation, Carlsbad, CA) using standard procedures (70 volts, 1.5 hours, 4 C) and visualized using enhanced chemiluminescence (GE Healthcare Bio-Sciences Corpation, Piscataway, NJ, USA) after incubation with primary or secondary antibodies.

FACS analysis

Cell surface receptor subunits IL-20R1 and IL-22R1 were examined by flow cytometry following protocols described by Sant Cruz Biotechnology, Inc. Briefly, 1 × 105 cells/well were plated in 6-well tissue culture plates (Corning Incorporated, Corning, NY); 4 days later, a monolayer of cells was detached by adding 0.2% Trypsin:EDTA/PBS, washed once with ice-cold PBS, pelleted and resuspended into 0.1 ml 1% FBS in PBS and incubated with either anti-IL-20R1, anti-IL-22R1 (R&D Systems, Inc. Minneapolis, MN) or normal IgG control antibody (Santa Cruz Biotechlogy, Inc., Santa Cruz, CA) for 60 min at room temperature. Cells were then washed and incubated in FITC-conjugated secondary antibody (Santa Cruz Biotechlogy, Inc., Santa Cruz, CA) in 1% FBS in PBS for 30 min on ice. The cells were washed 3 times with 0.1% Tween 20 in PBS, pelleted and resuspended in 500 μl of 1% paraformaldehyde and data acquired and analyzed.

Apoptosis was determined via FACS analysis and FragEL™ DNA Fragmentation Detection Kit (EMD Chemicals, Inc., San Diego, CA) performed according to the manufacturer’s protocol. The cells were analyzed by flow cytometric analysis on a FACScalibur flow cytometer (BD Biosciences, San Jose CA). A minimum sample population of 10,000 cells was used for each analysis.

Statistical Analysis

All studies were repeated 2-3 times as indicated, using triplicate samples for cell counting analyses. Data are shown as mean + SD and were considered significant when p<0.05. The statistical significance of the experimental results was evaluated using the Students t-test. Co-effects of two drugs were analyzed by Isobologram method: fitting two-way ANOVA model with the percentage of dead cell as the response. Fixed effect of IL-24 protein, TMZ, and the interaction was categorized to combinatorial synergy or non-synergistic interaction.

Results

TMZ sensitivity inversely correlates to endogenous MGMT levels in melanoma

Previous studies have indicated that endogenous MGMT levels are related to TMZ resistance (10, 11, 18, 35). In order to explore TMZ resistance mechanisms, we examined the endogenous MGMT levels of five human melanoma cell lines (A375, MeWo, WM35, SK-MEL-28 and WM1341) by Western blot assay. WM35 cells have no detectable MGMT protein, A375 cells express low levels of MGMT protein, and MeWo, SK-MEL-28 and WM1341 express relatively high levels of MGMT (Fig 1 upper panel). We examined the growth inhibitory effect of TMZ on the five melanoma cell lines using increasing concentrations up to 800 μM. There was a clear distinction between TMZ-sensitive and TMZ-resistant cell lines (Figure 1 lower panel). TMZ, at the concentration of 200 μM induced cell death in TMZ-sensitive melanoma cells, A375 and WM35 (29% and 23% cell death respectively), while the resistant cells showed minimal death: MeWo (4%), SK-MEL-28 (4%) and WM1341 (5% cell death). TMZ at 800 μM killed about 90% of A375 and WM35 cells, which express low MGMT levels. In contrast, cells expressing high endogenous MGMT (MeWo, SK-MEL-28 and WM1341) did not show more than 20% killing at this TMZ dose (Fig. 1 lower panel). Therefore, cells with high levels of endogenous MGMT (WeMo, SK-MEL-28 and WM1341) were classified as TMZ-resistant, but cells with low levels of endogenous MGMT (A375 and WM35) were TMZ-sensitive. In subsequent studies we use the TMZ-resistant cells, MeWo, SK-MEL-28 and WM1341 which displayed IC50 values of 1563 μM, 1111 μM and 1508 μM, respectively and compared them to the sensitive cell lines WM35 and A375, which had IC50 values of 281 μM and 313 μM respectively.

Fig. 1
Comparison of endogenous MGMT levels and TMZ sensitivity among human melanoma cells. Upper panel: Melanoma cells (A375, WM35, MeWo, SK-MEL-28 and WM1341) were harvested and cellular lysates were examined for MGMT expression using rabbit anti-MGMT polyclonal ...

Ad-IL-24 overcomes resistance to TMZ and enhances cell death by downregulation of MGMT via p53 induction

To examine whether IL-24 expression modulates TMZ-induced cell killing, we treated melanoma cells with Ad-IL-24 and TMZ. TMZ—resistant (MeWo, SK-MEL-28, WM1341) and TMZ-sensitive (A375, WM35) melanoma cells were exposed to PBS control, Ad-luc control virus, Ad-IL-24, TMZ, Adluc + TMZ or Ad-IL-24 + TMZ. The combination of Ad-IL-24 and TMZ produced combinatorial synergy in cell killing in all three TMZ-resistant cells but not in TMZ-sensitive cells; for example, in SK-MEL-28 cells, Ad-IL-24 treatment induced 17.2%±1.6% killing and TMZ caused 5.1%±0.5% killing when used as single agents, however, the combination of Ad-IL-24 + TMZ killed 30.9%±2.0% cells (Fig. 2A). This synergistic effect was not observed with the Ad-luc + TMZ combination. Treatment with Ad-luc killed 7.8%±1.6% of SK-MEL-28 cells, and the Ad-luc + TMZ combination generated only 9.6%±0.2% killing, indicating IL-24 and not adenovirus was responsible for the enhanced TMZ-induced killing. Combinatorial synergy was induced by Ad-IL-24 + TMZ in all three TMZ-resistant melanoma cells (MeWo, SK-MEL-28, WM1341), indicating that Ad-IL-24 could partially overcome TMZ-resistance. Ad-IL-24 also enhanced the killing activity of TMZ in TMZ-sensitive (A375 and WM35) cells. In A375, Ad-IL-24 alone or TMZ alone killed 18.1%±1.3% and 12.3%±1.5% of the treated cells respectively; however, the combination of Ad-IL-24 and TMZ induced 27.2%±1.8% cell death. The killing manner of Ad-IL-24 and TMZ in WM35 was similar to A375.

Fig. 2Fig. 2Fig. 2
Reversal of TMZ resistance by Ad-IL-24 occurs via downregulation of MGMT. A. Ad-IL-24 enhanced TMZ-induced killing and overcame TMZ-resistance in melanoma cells. TMZ-resistant cell lines (MeWo, SK-MEL-28 and WM1341) and TMZ-sensitive cell line (A375 and ...

We also evaluated cell proliferation. In SK-MEL-28 cells, 200 μM TMZ alone inhibited 8.4%±1.6% proliferation as compared to controls, and Ad-IL-24 induced 38.8%±3.6% growth inhibition; however, treatment with the combination of Ad-IL-24 and TMZ inhibited 53.1%±2.5% proliferation (data not shown).

Previous studies have shown that IFNβ is able to inhibit MGMT mRNA expression and also to stimulate IL-24 mRNA in human melanoma (23, 36). To test whether Ad-IL-24 can regulate MGMT protein expression, we treated MeWo and SK-MEL-28 cells with Ad-luc control vector or Ad-IL-24. Ad-luc did not inhibit MGMT; in contrast, Ad-IL-24 suppressed MGMT in a dose-dependent manner (Fig 2B).

Since Ad-IL-24 enhanced cell death, after treatment with TMZ, in TMZ—resistant melanoma cells (Figure 2A) and since sensitivity to TMZ is related to MGMT, mismatch repair and p53 status (6, 10, 37), we next examined whether Ad-IL-24 (a MGMT inhibitor) also regulates p53. We treated MeWo (has a p53 point mutation in the 72th amino acid mutaed from Glu to Lys yet it has normal p53 function) with Ad-IL-24 p53 expression increased, as compared to cells treated with Ad-luc. To assess function of the induced p53, we evaluated expression of p21, phospho-p53 (serine-15) and MGMT. Ad-IL-24 induced p53 phosphorylation with concomitant increases in p21 in both TMZ-resistant cell lines. P27 was also induced by Ad-IL-24 in both lines (data not shown). Treatment with the combination of Ad-IL-24 + TMZ increased phosphorylated-p53 (pp53) and p21 as compared to cells treated with Ad-luc + TMZ. Treatment with Ad-IL-24 + TMZ potently inhibited MGMT in MeWo, a TMZ-resistant cell line (Fig 2C). P53 activation, p21 increase and MGMT inhibition were also observed in another TMZ-resistant cell, SK-MEL-28, a 245G/S point mutation of p53 and overexpresses p53 protein (data not shown).

IL-24 specifically down-regulates MGMT via IL-20R/IL-22R receptors in human melanoma cells

Previous studies showed that Ad-IL-24 induces both intracellular and secreted IL-24 protein and that both forms can induce tumor cell death (24, 34, 38). To investigate whether exogenous IL-24 is involved in the downregulation of MGMT, we treated SK-MEL-28 cells with IL-24; Western blot data showed that MGMT was inhibited by IL-24 in a dose-response manner (Fig. 3A, top panel). When treated MeWo with IL-24, MGMT also was inhibited in the same manner (data not shown). Supporting the specificity of the effect is the cell death analysis results shown in Fig 3A (bottom panel); cell death after treatment with a combination of TMZ + IL-24 + anti-IL-24 antibodies was significantly decreased (35.2% and 20.9% in MeWo and SK-MEL-28 respectively), in a dose-dependent manner, as compared to cells treated with TMZ + IL-24 + IgG (44.9% and 36.9% in MeWo and SK-MEL-28 respectively). Since Ad-IL-24 inhibits MGMT and induces p53 (Fig. 2C), we asked whether IL-24 protein has the same function. We treated MeWo and SK-MEL-28 cells with IL-24, TMZ (T) or both IL-24 + TMZ (IL-24 + T) to evaluate MGMT, p21 and p53 protein expression. Western blot results show that IL-24 strongly induced p53 and p21 expression, whereas MGMT expression was completely inhibited (Fig. 3B). In contrast, TMZ was a weak inducer of p53 and p21 and did not reduce MGMT expression. The combination of IL-24 + TMZ, like IL-24 treatment, resulted in complete inhibition of MGMT in both cell lines.

Fig. 3Fig. 3Fig. 3
Inhibition of MGMT by IL-24 occurs via IL-24 receptors. A. IL-24 Enhanced TMZ-mediated cell death by inhibition of MGMT. SK-MEL-28 was treated with PBS (C) or varying concentrations of IL-24 as indicated for 72 h and MGMT examined by Western blotting. ...

Our previous studies show that exogenous IL-24 binds specific receptors to mediate its biological effects (34, 38). To evaluate whether IL-24-mediated downregulation of MGMT involves IL-20R/IL-22R receptors, we treated two melanoma lines expressing high levels of endogenous MGMT (MeWo and SK-MEL-28) with IL-24 in combination with or without IL-24 neutralizing antibodies. Western blot analyses showed that when the cells were treated with IL-24, MGMT protein levels were suppressed by 40% in SK-MEL-28 and 70% in MeWo (Fig. 3C); whereas either anti-IL24 antibody or antibodies against the IL-24 receptors were able to block the inhibitory effect on MGMT of IL-24. Treatment with single anti-IL-20R1 or anti-IL22R1 antibodies also reversed IL-24 inhibition of MGMT, although to a lesser extent than the combination of the two (data not shown).

IL-24 enhanced TMZ-induced cell killing and apoptosis via IL-20R/IL-22R receptors in human melanoma cells

Since IL-24 reduced MGMT protein expression via IL-20R1 and IL-22R1 receptor binding (Fig. 3C), we wanted to evaluate IL-24-induced cell death in melanoma cells. Thus, MeWo and SK-MEL-28 cells were treated with IL-24, TMZ, or combinations of IL-24 + TMZ as well as specific neutralizing antibodies for IL-20R and IL22R. IL-24 killed both melanoma cells and co-treatment with anti-IL-20R1, anti-IL22R1, or a combination of the two, significantly inhibited (p<0.01) IL-24 mediated cell killing (Fig 4A). TMZ (200 μM) monotherapy induced similar activity against MeWo and SK-MEL-28 cells (5.3%±1.1% and 7.3%±0.8% cell death, respectively). IL-24 monotherapy induced cell death in both MeWo and SK-MEL-28 (16.7±1.1%% and 18.7%±1.1%, respectively) cells. The TMZ + IL-24 combination showed combinatorial synergy in cell killing against both MeWo cells (37.8%±2.8%) and SM-MEL-28 cells (34.0%±0.5%). Therefore, IL-24 enhances TMZ-induced killing to MeWo and SK-MEL-28 cell and shows combinatorial synergy to both MeWo and SK-MEL-28 cells (Fig. 4A).

Fig. 4Fig. 4
Reversal of TMZ-resistance by IL-24 occurs via IL-24 receptors. A. MeWo and SK-MEL-28 cells in triplicate were treated with IL-24 (39 ng/ml), 200 μM TMZ, normal mouse IgG, anti-IL-20R1 or anti-IL-22R monoclonal antibody as indicated. After 96 ...

Anti-IL-22R1 and anti-IL20R1 treatments induced a modest reduction in cell death triggered by TMZ + IL-24 treatment in both MeWo and SK-MEL-28 cells, while nonspecific normal mouse IgG had no effect. Co-treatments with anti-IL-20R1 + IL-24 + TMZ induce (12.4%±2.2% in MeWo and 16.8%±0.5% in SK-MEL-28, respectively); Co-treatments with anti-IL-22R1 + IL-24 + TMZ induced 16.1%±0.4% and 21.4%±0.9% cell death in MeWo and SK-MEL-28 respectively. Combining neutralizing antibodies against both receptors further reduced killing to levels comparable to single-agent treated controls.

Induction of cell-cycle arrest and apoptosis by IL-24 and TMZ, used as single agents, has been were well documented (22, 39, 40). To evaluate the role of IL-24 in the activation of programmed cell death pathways when TMZ-resistant melanoma cell lines (MeWo, SK-MEL-28) are treated with a combination of IL-24 and TMZ, we performed TUNEL assays (FACS assays after DNA fragmentation labeling). Treatment with IL-24 caused 10.7%±1.4% and 8.1%±0.4% apoptosis in MeWo and SK-MEL-28 cells, treatment with TMZ induced 7.5%±2.1% and 2.6%±0.2% apoptosis respectively. Addition of TMZ to IL-24 resulted in combinatorial synergy of melanoma cell killing, and IL-24 relieved the resistance to TMZ in both cell lines (p<0.01) inducing a higher apoptotic index, as compared to that of IL-24 or TMZ single-agent treatment (Fig. 4B).

P53 plays a critical role in IL-24 reversal of resistance to TMZ by inhibition of MGMT

In order to examine if blockade of MGMT modifies killing by the combination of TMZ and IL-24, we next transfected MGMT siRNA or control siRNA into MeWo cells. Analysis of lysates from the transfected cells showed that MGMT expression was significantly reduced, whereas p53 and p21 expression were not altered, by MGMT siRNA (Fig. 5A left panel). A375 is a wild type p53 cell line. We confirmed inhibition of MGMT expression using A375M cells transfected with MGMT-targeted shRNA (Fig. 5A middle panel). Treatment with TMZ induced increased cell death in MGMT shRNA transfected (36.9%±2.7%) MeWo and (42.9%±2.9%) A375M cells as compared to control-shRNA transfected cells (6.7%±1.0% and 6.4%±2.2% for MeWo and A375M respectively), indicating MGMT shRNA transfected cells were more sensitive to TMZ (P<0.01, Fig. 5A right panel). The combination of TMZ + IL-24 induced combinatorial synergy in control shRNA transfected cells (45.3%±2.1% in MeWo and 39.3%±1.7% in A375M). However, the synergistic cell death effect in IL-24 + TMZ treated cells disappeared upon transfection with MGMT shRNA (38.4%±2.9% in MeWo and 43.1%±4.5% in A375M), indicating that the reversal of TMZ-resistance by IL-24 was abrogated when MGMT was inhibited (Fig. 5A right panel).

Fig. 5Fig. 5Fig. 5
Requirement of p53 accumulation for MGMT expression. A. Blocking MGMT abolishes synergistic cell killing by IL-24 + TMZ combination. MeWo cells were transfected with control siRNA or MGMT siRNA for 72 h. Left panel: Cell lysates were extracted and western ...

To confirm that MGMT expression is dependent on p53 status, we used p53 siRNA from different sources to block p53 expression and examined both MGMT expression and killing mediated by TMZ + IL-24. Western blot analysis showed partial inhibition of p53 expression using p53 siRNA, and also inhibition of p21WAF1/CIP1 expression (Fig. 5B left panel). We confirmed p53 knock down in p53 siRNA transfected A375M cells (Fig. 5B middle panel). Evaluation of cell death revealed that TMZ-induced cell death increased in p53 siRNA transfected (29.9%±0.9%) MeWo and (44.5%±2.1%) A375M cells as compared to that in control siRNA transfected cells (7.0%±1.6% in MeWo and 8.4%±1.8% in A375M) (p<0.01). The combination of TMZ + IL-24 induced similar percent cell death in control siRNA transfected MeWo (44.8%±2.6%) and A375M (43.2%±5.0%) to in p53 siRNA transfected cells MeWo (39.1%±3.5%) and A375M(34.2%±1.2%), indicating that the TMZ-sensitization induced by IL-24 was mediated by p53-dependent activation of MGMT (Fig. 5B right panel).

A previous study has shown that the transcriptional activity of p53 is inhibited when a p53 plasmid with mutated residues at positions 22 and 23 (Leu22Gln and Trp23Ser, pP53mut) is transfected into SAOS2 cells. These two residues are required for p53’s trans-activation activity, and for p53 binding of human mdm-2 (hdm-2) and in vitro binding to the adenovirus 5 (Ad5) E1B 55kD (33). In order to investigate if abnormal p53 protein is capable to induce MGMT expression, we transfected plasmids encoding for GFP (pEGFP-N1), wild type p53 (pP53wt) or a dominant-negative mutant p53 (pP53mut) into MeWo cells. Both pP53wt and pP53mut transfected MeWo cells expressed higher levels of p53 than pEGFP-N1 transfected cells. P21 level was reduced in pP53mut transfected cells as compared to controls, indicating that mutant p53 repressed p21WAF1/CIP1 expression (Fig. 5C left panel). TMZ induced increased cell death in pP53mut-transfected cells (11.6%±2.0%) compared to pEGFP-N1transfected MeWo cells (7.1%±1.1%, P<0.01). The combination of TMZ + IL-24 induced increased cell death in pEGFP-N1-transfected cells (43.6%±2.4%) compared to pP53mut-transfected MeWo cells (26.4%±2.5% P<0.01), indicating that the reversal of TMZ-resistance induced by IL-24 can be inhibited by a dominant-negative p53 (Fig. 5C right panel). Collectively, these data indicate that functional p53 protein accumulation is essential for expression of MGMT and inhibition of MGMT abolishes IL-24-induced sensitization of melanoma cells to TMZ.

Discussion

Although TMZ is a promising chemotherapeutic agent for patients with advanced melanoma, resistance develops quickly and with high frequency. In melanoma cells, a single cycle of TMZ is sufficient to up-regulate MGMT expression causing increased drug resistance (35). The literature demonstrates that endogenous MGMT correlates with resistance to TMZ and other alkylating agents such as 1,3-bis(2-chloroethyl)-1-nitrosourea, and cisplatin (35, 41, 42). Thus, due to their potential for application in the clinical setting, agents that effectively decrease the levels of MGMT have been evaluated in combination regimens with TMZ; two main agents in this group are O6-benzylguanine and IFNβ. O6-benzylguanine (O6-BG) is a potent inhibitor of MGMT and acts as its substrate. Treatment with O6-BG potentiates TMZ-induced cytotoxicity in melanoma cells in vitro and increases sensitivity to other alkylating agents Treatment of human melanoma xenografts with a combination of O6-BG and TMZ, results in greater antitumor effect (5-fold inhibition of tumor growth, P<0.005) than an equivalent dose of TMZ (43). Thus far, clinical trials testing the combination of TMZ and O6-BG have failed to demonstrate efficacy and show significant hematological toxicity (43, 44). Another agent investigated for use in combination with TMZ is the anti-inflammatory cytokine, Interferon beta (IFNβ). IFNβ sensitizes glioma cells that harbor an unmethylated MGMT promoter and are resistant to TMZ (36). In a nude mouse xenograft model, adenoviral delivery of IFNβ or p53 suppressed MGMT promoter activity while treatment of gliomas with IFNβ + TMZ was reported to induce synergistic inhibition of tumor growth (45).

A novel candidate in combination regimens for melanoma is IL-24, which was first identified in melanoma cells treated with a combination of IFNβ and mezerein to induce terminal differentiation (46). Because of its tumor specific anti-angiogenic, pro-apoptotic, and growth inhibitory activities IL-24 is a good candidate for sensitization of tumor cells to TMZ without exacerbation of this agent’s toxicity. Consistent with its tumor specific selectivity, IL-24 has been safely and efficiently used to treat various types of tumors in cancer patients (22, 26, 27). Importantly, evaluation of matched primary and metastatic clinical melanoma samples has revealed that expression of the IL-24 protein is lost during the pathologic progression of melanomas into more aggressive phenotypes (21). Thus, we hypothesized that restoring IL-24 in melanoma cells could reduce their metastatic capability and reverse TMZ-resistance. In this study, we demonstrate that IL-24 synergistically enhances TMZ-induced cytotoxicity in melanoma cells resistant to this agent (Figure 2A). Further, we show that IL-24 induces this effect via inhibition of MGMT protein expression (Figures (Figures2B,2B, ,3C3C and and4A4A).

Because of its function as a sensor of DNA damage and promoter of DNA repair, several studies have investigated a role for p53 as a regulator of MGMT; however, these have yielded conflicting results. While it is well established that p53 activates expression of target genes that contain a p53 binding sequence and TATA box, p53 can also regulate promoter activity of genes that do not possess a p53-binding site; the MGMT promoter belongs to this category. Various mechanisms have been proposed to explain this phenomenon but no clear consensus has been reached. Importantly, p53 regulation of MGMT expression has important implications in the development of TMZ-resistance, and in tumor cell responses against TMZ and other alkylating agents (14, 16, 18, 19, 37, 47). Our present study shows that abrogation of p53 in TMZ-resistant melanoma cells, via transfection with p53 siRNA or a dominant-negative p53 mutant plasmid, results in loss of IL-24-induced synergy and decreased sensitivity to TMZ (Fig. 5B), thus supporting that p53 signaling mediates the effect of IL-24 on MGMT. In our study, direct inhibition of MGMT expression -via MGMT siRNA- did not alter expression of p53 or its direct downstream target, p21 (Fig. 5A); this is consistent with MGMT being a downstream target of p53. Interestingly siRNA blockade of MGMT expression abolished the synergistic effect of IL-24 on TMZ; implicating the need to have a minimum threshold of MGMT for the combinatorial synergy of cell death.

Taken together, our results confirm that decreased expression of MGMT is the mechanism underlying the synergistic effect of IL-24+TMZ and reversal of TMZ-resistance, and that IL-24 down-regulation of MGMT requires p53 function. In our study, we also demonstrate that IL-24 can inhibit MGMT and overcome melanoma resistance to TMZ via type 1 and type 2 IL-24 receptors, resulting in activation of p53 and subsequent downregulation of MGMT. These results support a promising role for IL-24 as part of a novel biochemotherapy approach to treat advanced melanoma, and overcome TMZ-resistance.

Acknowledgments

We thank Dr. Roger Bryan Sutton at Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston for pEGFP-N1 plasmid, Dr. Kishor K. Bhakat and Dr. Sankar Mitra at the Sealy Center for Molecular Sciences, University of Texas Medical Branch, Galveston for plasmids of p53 wild type and p53 mutant (codons 22-23 mutant), Mr. Hai Pham and Dr. Shuyuan Zhang at Introgen Therapeutics, Inc. for production and purification of IL-24 protein.

Grant support: This work was supported by NCI grants CA89778, CA88421 and CA097598 (SC), the Texas Higher Education Coordinating Board ATP/ARP grant 003657-0078-2001 (RR), by Institutional Research Grant (RR), and by W. M. Keck Gene Therapy grant (RR) and R41-CA 89778 and R42-CA 89778 (EAG and SC); P50 CA093459 (EAG).

Footnotes

Conflicts of Interest: MZ, DB and SC report being employees of Introgen. No other potential conflict to this article was reported.

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