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Clinical Cancer Research 13, 6217, October 15, 2007. doi: 10.1158/1078-0432.CCR-07-1421
© 2007 American Association for Cancer Research
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Cancer Therapy: Preclinical

Adenovirally Delivered Tumor Necrosis Factor-{alpha} Improves the Antiglioma Efficacy of Concomitant Radiation and Temozolomide Therapy

Bakhtiar Yamini1, Xiaohong Yu1, Peter Pytel2, Nicholas Galanopoulos1, Vinay Rawlani1, Jula Veerapong3, Kai Bickenbach3 and Ralph R. Weichselbaum3

Authors' Affiliations: 1 Section of Neurosurgery, Department of Surgery, 2 Department of Pathology, and 3 Department of Radiation and Cellular Oncology, Pritzker School of Medicine, The University of Chicago, Chicago, Illinois

Requests for reprints: Bakhtiar Yamini, MC 4066, Section of Neurosurgery, University of Chicago Hospitals, 5841 S. Maryland Avenue, Chicago, IL 60637. Phone: 773-702-2475; Fax: 773-702-5234; E-mail: byamini{at}surgery.bsd.uchicago.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Treatment of malignant glioma involves concomitant temozolomide and ionizing radiation (IR). Nevertheless, overall patient survival remains poor. This study was designed to evaluate if addition of Ad.Egr–tumor necrosis factor (TNF), a replication defective adenovector encoding a cDNA for TNF-{alpha}, to temozolomide and IR can improve overall antiglioma effect.

Experimental Design: The efficacy of combination treatment with Ad.Egr-TNF, IR, and temozolomide was assessed in two glioma xenograft models. Animal toxicity and brain histopathology after treatment were also examined. In addition, in an attempt to explain the antitumor interaction between these treatments, the activation status of the transcription factor nuclear factor-{kappa}B was examined.

Results: Triple therapy (Ad.Egr-TNF, IR, and temozolomide) leads to significantly increased survival in mice bearing glioma xenografts compared with dual treatment. Fifty percent of animals treated with the triple regimen survive for >130 days. Pathologic examination shows that triple therapy leads to a complete response with formation of a collagenous scar. No significant change in myelination pattern is noted after triple therapy, compared with any double treatment. Treatment of intracranial glioma bearing mice with Ad.Egr-TNF and IR leads to cachexia and poor feeding that does not improve, whereas triple therapy results in less toxicity, which improves over 21 days. Both Ad.Egr-TNF and IR activate nuclear factor-{kappa}B, and temozolomide inhibits this activity in an inhibitor of {kappa}B{alpha} (I{kappa}B{alpha})–independent manner.

Conclusion: This work shows that the addition of adenoviral TNF-{alpha} gene delivery to temozolomide and IR significantly improves antiglioma efficacy and illustrates a potential new treatment regimen for use in patients with malignant glioma.

For over 30 years, standard postsurgical management of malignant glioma has involved the combination of radiation and chemotherapy, yet overall patient prognosis has only slightly improved in this time (1, 2). One strategy that can potentially enhance antiglioma treatment efficacy involves combining conventional radio/chemotherapy with death ligands, such as tumor necrosis factor {alpha} (TNF-{alpha}). Such a therapeutic regimen can improve glioma cell killing by either targeting nonoverlapping cytotoxic pathways, thus bypassing resistance to a single agent, or by sensitizing cells with concurrently given therapies. The primary advantage of incorporating TNF-{alpha} into a combination treatment protocol involves its direct (and indirect) antitumor action (3, 4). Although systemic administration of TNF-{alpha} may cause dose-limiting side effects, such as shock, fatigue, and vascular collapse (5), high regional and intratumoral concentrations of TNF-{alpha} can be maintained safely (6, 7). In this regard, the adenoviral construct Ad.Egr-TNF, which carries a cDNA for TNF-{alpha} under the control of chemoinducible and radio-inducible elements of the Egr-1 promoter, enables in vivo delivery of TNF-{alpha} in combination with ionizing radiation (IR) or chemotherapy (8, 9).

Direct cell killing by TNF-{alpha} occurs through the extrinsic apoptotic cascade (10). In many cells, however, TNF-{alpha}–induced activation of the transcription factor, nuclear factor-{kappa}B (NF-{kappa}B), results in the expression of many downstream genes that promote cell survival and inflammation (11, 12). The mammalian NF-{kappa}B family consists of five structurally related proteins, the most abundant form of which consists of the heterodimer of p50 (NF-{kappa}B1) and p65 (RelA; ref. 13). In unstimulated cells, NF-{kappa}B is sequestered in the cytosol bound to inhibitor of {kappa}B (I{kappa}B) proteins. After TNF-{alpha} stimulation, I{kappa}B{alpha} is phosphorylated and degraded, releasing the NF-{kappa}B subunits which translocate into the nucleus and bind {kappa}B elements in DNA. In addition to mediating inflammation and resistance to TNF-{alpha}–induced cytotoxicity, NF-{kappa}B–regulated genes also promote resistance to killing by IR (14, 15).

Temozolomide is an oral monofunctional alkylating agent with a favorable toxicity profile that is commonly used in the management of malignant glioma (16). The cytotoxicity of temozolomide is thought to be due to the formation of O6-methylguanine adducts, which mispair with thymine and result in stimulation of the mismatch repair system. It has been proposed that subsequent futile cycles of mismatch repair cause DNA strand breaks and cell death (17). The concomitant and adjuvant use of temozolomide with IR has been shown to modestly increase the survival of patients with newly diagnosed glioblastoma (18). Despite this success, the mechanism by which these two treatment modalities interact is not well understood (19). We have recently shown that SN1-type alkylators, like temozolomide, block NF-{kappa}B activity by inhibiting {kappa}B-element binding (20).

It has previously been reported that combination treatment of hind-limb glioma xenografts with Ad.Egr-TNF and IR results in 70% tumor cure (21). In the present study, we initially investigated the effect of Ad.Egr-TNF and IR in an intracranial glioma. Subsequently, in both hind-limb and intracranial xenografts, we evaluated whether intratumoral Ad.Egr-TNF improves the efficacy of temozolomide and IR. Triple therapy with Ad.Egr-TNF, temozolomide, and IR was found to significantly increase antiglioma efficacy compared with standard temozolomide and IR without causing toxicity greater than any of the two treatments together. Temozolomide was also noted to inhibit adenovirus-induced and IR-induced NF-{kappa}B, suggesting an additional mechanism, in conjunction with spatio-temporal synergy, for the strong antiglioma interaction of these treatments.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cells, reagents and vector
Temozolomide was obtained from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program (National Cancer Institute, NIH) and was dissolved in DMSO (final concentration, <0.1% v/v). U87MG human glioblastoma cells were purchased from American Type Culture Collection and cultured in DMEM supplemented with 10% fetal bovine serum, penicillin (100 IU/mL), and streptomycin (100 µg/mL) at 37°C and 5% CO2. The replication incompetent adenoviral vector Ad.Egr-TNF has been previously described (22).

Luciferase assay
U87 cells (5 x 103) were plated overnight and cotransfected with the NF-{kappa}B luciferase reporter Ig-{kappa}B-Luc (containing three repeats of the immunoglobulin {kappa}-light chain enhancer B site; ref. 23) and the Renilla reniformis pRL-TK expression vectors (ratio of Ig-{kappa}B-Luc/pRL-TK, 10:1) using the SuperFectin transfection kit (Qiagen). After 24 h, cells were pretreated with temozolomide (or 0.1% DMSO, vehicle) and then with 10 ng/mL human TNF-{alpha} or Ad.Egr-TNF (MOI, 100) for 8 h and 5 Gy IR for 1 h. NF-{kappa}B and Renilla luciferase activities were measured with the Dual-Luciferase reporter assay system (Promega Corp.) 5 h after TNF-{alpha} stimulation. Relative luminescence was calculated as the ratio of firefly luminescence to Renilla luminescence.

Cell fractionation and electrophoretic mobility shift assay
Cells were grown and treated as indicated. They were then pelleted by centrifugation at 1,000 rpm for 5 min at 4°C and resuspended in ice-cold buffer A [10 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 0.1 mmol/L EDTA, 1 mmol/L DTT, 0.5 mmol/L phenylmethylsulfonyl fluoride, 1 µg/mL leupeptin, 5 µg/mL aprotinin]. After the addition of 25 µL 10% NP40, the suspension was vortexed and centrifuged at 14,500 rpm for 1 min at 4°C; the supernatant was designated as the cytoplasmic fraction. Nuclei were resuspended in 50 µL of ice-cold buffer B [20 mmol/L HEPES (pH 7.9), 0.4 mol/L NaCl, 1 mmol/L EDTA, 1 mmol/L DTT, 1 mmol/L phenylmethylsulfonyl fluoride, 25% glycerol, 1 µg/mL leupeptin, 5 µg/mL aprotinin] and centrifuged at 14,500 rpm for 5 min. The supernatant was used as the nuclear fraction, and protein concentration was determined by the Bradford method. Electrophoretic mobility shift assay was done with 10 µg nuclear protein using the Promega gel shift assay system and 32P-labeled NF-{kappa}B consensus oligonucleotide. Nuclear extract was also preincubated with 100-fold excess unlabeled NF-{kappa}B (specific competitor) or activator protein consensus sequence (nonspecific competitor).

Western blotting
Cellular lysate (20 µg) was subjected to SDS-PAGE. After electrotransfer, Immobilon-P membranes (Millipore Corp.) were probed with primary rabbit polyclonal antibody against I{kappa}B{alpha} (Cell Signaling Technology, Inc.) diluted 1:1,000 overnight at 4°C. Antirabbit IgG horseradish peroxidase–linked secondary antibody (Cell Signaling Technology) was used at 1:1,000. Immunoreactive bands were detected by SuperSignal enhanced chemiluminescence (Pierce) and exposed to Kodak X-Omat film.

Animals and xenograft studies
Six-week-old to seven-week-old female athymic nude mice (Frederick Cancer Research Institute) were used, and surgery was done in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of Chicago.

Hind limb studies. U87 cells (5 x 106) were injected into the right hind limb, and the animals randomized into six treatment groups (as noted in the figure legend) when tumors reached an average of 200 mm3 (length x width x thickness / 2; day 0). Ad.Egr-TNF [2 x 108 particle units (pu)] in 10 µL serum-free medium was then given i.t. twice a week for 2 weeks. Temozolomide was given i.p. at a dose of 5 mg/kg 3 h after vector (total, 20 mg/kg). IR was delivered to the tumor area at a dose of 5 Gy every 2 to 3 days (on the days of temozolomide and vector injection) to a total of 30 Gy; animals were restrained in lucite chambers and protected with lead shields during irridation. Control animals were injected i.t. or i.p. with serum-free medium and placed in lucite chambers without IR administration. Xenograft volume was measured twice a week, and fractional tumor volumes were calculated (V/V0, wherein V0 is volume on day 0).

Intracranial studies. U87 cells (5 x 105) were inoculated into the right caudate nucleus on day 0 using a screw guide technique as described (24). On day 5, mice were randomized into treatment groups, and 5 x 108 pu Ad.Egr-TNF in 5 µL serum-free medium were i.t. injected once. In some animals, 5 mg/kg temozolomide were given i.p. 3 h after vector every 2 days for three doses (total, 15 mg/kg). IR was delivered to the tumor at a dose of 5 Gy and repeated every 2 to 3 days (total, 30 Gy). Control animals received serum-free medium i.p. and i.t. Daily assessment of animal skin turgor, feeding behavior, and appearance was made; also, animals were weighed before treatment and at 7 and 21 days after initiation of therapy. Mice were sacrificed when moribund.

Pathologic analysis
Moribund animals (and long-term survivors) were sacrificed, and brains were harvested after intracardiac perfusion with paraformaldehyde and fixed with 10% neutral buffered formalin. Paraffin-embedded specimens were sectioned (8 µm), stained with H&E, and analyzed for the presence of viable tumor and necrosis. Luxol fast blue–stained sections were also examined to assess white matter damage.

Statistical analysis
Results are expressed as mean ± SD. Statistical significance was taken as P < 0.05, using a two-tailed Student's t test. Kaplan-Meier survival curves were plotted for the intracranial experiment and analyzed by the log-rank method.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Ad.Egr-TNF and IR treatment of intracranial glioma xenografts. As the intracranial environment is quite different from the periphery (25), we initially examined whether the peripheral antiglioma effect of Ad.Egr-TNF and IR (21) is replicated in an intracranial tumor model. Treatment of nude mice bearing intracranial U87 glioma xenografts with intratumoral Ad.Egr-TNF and fractionated local IR results in a significant increase in animal survival when compared with treatment with IR alone (P = 0.05; Fig. 1 ). However, the mice treated with combination Ad.Egr-TNF and IR feed poorly have decreased skin turgor and seem cachectic. Furthermore, 30% of these combination-treated animals require early euthanasia (i.e., prior to that of untreated animals) due to their moribund state. On pathologic examination, animals requiring early sacrifice have small tumors that are of insufficient size to cause death on their own. These results show that combination treatment of animals bearing intracranial tumors with Ad.Egr-TNF and IR results in significant increase in survival with some increase in animal toxicity.


Figure 1
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  		Fig. 1. Kaplan-Meier survival curves of nude mice bearing U87 intracranial xenografts. Animals were treated on day 5 after tumor inoculation with 5 x 108 pu Ad.Egr-TNF injected i.t. and 5 Gy IR delivered to the tumor daily to a total of 30 Gy (n = 6 animals per treatment group). Experiment was repeated with similar results. Log rank, P = 0.05, IR versus Ad.Egr-TNF + IR.

 
Ad.Egr-TNF-, temozolomide, and IR treatment of glioma xenografts. In patients with malignant glioma, a standard postsurgical treatment regimen involves the concomitant use of temozolomide and IR. We examined if triple therapy with Ad.Egr-TNF, temozolomide, and IR can improve the overall antitumor effect. Treatment efficacy was initially assessed in hind-limb xenografts by comparing the volume of tumor after treatment (V) to the initial volume (V0). Nude mice (10 of 10) bearing U87 xenografts treated with temozolomide, IR, and Ad.Egr-TNF had complete disappearance of tumor by 30 days compared with 0 of 10 animals in the temozolomide alone, IR alone, IR/temozolomide, and temozolomide/Ad.Egr-TNF groups (P < 0.00001 temozolomide + IR + Ad.Egr-TNF versus Ad.Egr-TNF + temozolomide; Fig. 2A ).


Figure 2
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  		Fig. 2. A, U87 hind-limb xenografts were allowed to grow to an average of 200 mm3 (V0) and animals randomized into the groups indicated (n = 10 per group). Treatment was then initiated with 5 x 108 pu Ad.Egr-TNF given i.t. every 2 d for four doses. Temozolomide (TMZ) was delivered i.p. at a dose of 5 mg/kg for four doses, and IR (5 Gy) was given to the tumor daily to a total of 30 Gy. Tumor volume (V) was measured twice a week, and fractional tumor volume (V/V0) was calculated. Points, value; bars, SD. P < 0.00001, temozolomide + IR + Ad.Egr-TNF versus temozolomide + Ad.Egr-TNF. B, Kaplan-Meier survival curves of nude mouse bearing intracranial U87 tumors. Mice were randomized into eight groups as indicated (n = 8 per group) on day 5 after tumor inoculation. Ad.Egr-TNF (5 x 108 pu) was injected i.t. once; temozolomide (5 mg/kg) was given i.p. 3 h after vector inoculation and repeated every 2 d for three total doses; IR (5 Gy) was delivered to the area of tumor daily to a total of 30 Gy. P < 0.01. Log rank, Ad.Egr-TNF + temozolomide + IR versus Ad.Egr-TNF + temozolomide. The experiment was repeated with similar results.

 
To further investigate the efficacy and toxicity of triple therapy, an intracranial xenograft model was incorporated. U87 cells (5 x 105) were implanted into the right caudate nucleus of nude mice and allowed to grow. On day 5, animals were treated with intratumoral Ad.Egr-TNF, followed by temozolomide and IR. Animals treated with triple therapy had a significant increase in median survival compared with those in all other treatment groups and specifically compared with the standard antiglioma regimen of temozolomide and IR (Fig. 2B). Fifty percent of the animals treated with temozolomide, IR, and Ad.Egr-TNF were still alive and healthy 130 days posttumor inoculation compared with 0% of animals in the other groups. Animals treated with the double regimen of Ad.Egr-TNF and IR (Fig. 2B, solid black line) appeared cachectic and were found to have a 12% (±5%) decrease in body weight 7 days after the initiation of treatment. In none of these animals did the decrease in body weight improve over the observation period, and in the repeat experiment, early animal death (prior to untreated controls) was seen in two of nine mice (data not shown). Although the animals receiving triple therapy showed a 10% (±4%) decrease in body weight 7 days after initiation of treatment, they all remained active and their body weights returned to pretreatment levels by 21 days. Importantly, none of these triple therapy animals died early in any experiment. Body weight change in the animals in all the other treatment groups was noted to be <5% 7 days after initiation of treatment.

Pathologic examination of the brains was then done. All animals that required early euthanasia were found to have tumors in the cerebral hemispheres. Representative brain specimens in untreated animals and in those receiving Ad.Egr-TNF alone are shown (Fig. 3A and B ). The tumors in nonresponders that received triple therapy and those treated with Ad.Egr-TNF and IR alone did not show any histomorphologic alterations in the form of necrosis, increased inflammatory response, or vascular thrombosis when compared with untreated control animals. In the long-term survivors (all treated with the triple regimen of Ad.Egr-TNF, temozolomide, and IR), no residual viable tumor cells were found. In three of four of these surviving animals, regressive changes in the form of a collagenous scar and organizing necrosis with cholesterol clefts were noted in the region of tumor inoculation (Fig. 3C and D). None of the brains of other animals showed similar regressive changes or collagenous scar formation. In addition, myelin staining of the long-term survivors did not show any additional changes compared with the animals in the other treatment groups, suggesting that triple therapy does not lead to additional myelin breakdown.


Figure 3
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  		Fig. 3. Pathologic analysis of mouse brains. A–D, low (2x, left), intermediate (4x, center), and high (10x, right) power images of the tumor area in four different animals. A, untreated control. B, Ad.Egr-TNF alone. C and D, long-term surviving animals treated with Ad.Egr-TNF, temozolomide, and IR. Arrowhead, collagenous scar; arrow, injection site.

 
To investigate how triple therapy affects tumor growth early on, animals bearing intracranial xenografts were treated with Ad.Egr-TNF, temozolomide, and IR alone and in combination and sacrificed 10 days after initiation of therapy. In one of three mice treated with triple therapy, regressive changes with collagenous scar formation were found in the region of tumor inoculation. The tumors in the other animals all showed similar histomorphology with no evidence of necrosis, inflammation, or vascular thrombosis.

Temozolomide inhibits NF-{kappa}B induced by IR and Ad.Egr-TNF. In an attempt to explain the potent antiglioma interaction seen with combination Ad.Egr-TNF, temozolomide, and IR, we examined the effect of therapy on NF-{kappa}B activity. Activation of NF-{kappa}B, by TNF-{alpha}, IR, or adenovirus, can potentially attenuate the antitumor effect (14, 26, 27). We initially showed that 5 Gy IR increase NF-{kappa}B–dependent luciferase in glioma cells, and the combination of IR and TNF-{alpha} leads to an even greater increase in NF-{kappa}B activity (Fig. 4A ). Pretreatment of cells with 100 µmol/L temozolomide (peak patient plasma temozolomide level in clinical trials is ~100 µmol/L; ref. 28) reduces TNF-{alpha}–induced and IR-induced NF-{kappa}B both on luciferase reporter analysis (Fig. 4A) and on gel shift assay (Fig. 4B). Combination treatment with TNF-{alpha}, temozolomide, and IR results in <10% decrease in cell viability at the time point tested in the luciferase studies (data not shown). The effect of temozolomide on IR-induced NF-{kappa}B is independent of I{kappa}B{alpha} degradation (Fig. 4C). We then evaluated how temozolomide affects Ad.Egr-TNF–induced NF-{kappa}B. Pretreatment with temozolomide blocks NF-{kappa}B activated by Ad.Egr-TNF alone and NF-{kappa}B activated by combination Ad.Egr-TNF and IR (Fig. 4D). These data show that temozolomide inhibits NF-{kappa}B activated by Ad.Egr-TNF and IR and suggest a mechanism by which temozolomide may interact with IR, Ad.Egr-TNF, and induced TNF-{alpha} to result in a potent antiglioma effect.


Figure 4
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  		Fig. 4. Temozolomide inhibits IR-induced and Ad.Egr-TNF–induced NF-{kappa}B. A and D, NF-{kappa}B–dependent luciferase assay. Columns, mean fold change of relative luminescence of triplicate samples; bars, SD. Value of untreated sample = 1. A, U87 cells were cotransfected with Ig-{kappa}B-Luc and R. reniformis and were then treated with 100 µmol/L temozolomide (16 h pretreatment), 10 ng/mL TNF-{alpha}, and 5 Gy IR alone or in combination as indicated. IR was delivered 1 h after TNF-{alpha}. *, P < 0.005. B, nuclear fractions were isolated from U87 cells treated with 100 µmol/L temozolomide (16 h pretreatment), 10 ng/mL TNF-{alpha}, and/or 5Gy IR. Electrophoretic mobility shift assay was done with NF-{kappa}B consensus oligonucleotide (oligo). A hundred-fold excess cold NF-{kappa}B or Ap-1 oligonucleotide was given where indicated. C, U87 cells were untreated or treated with 10 ng/mL TNF-{alpha} for 15 min, 100 µmol/L temozolomide (16 h pretreatment), and 5 Gy IR (1 h) alone and in combination, as indicated. Western blot with anti-I{kappa}B{alpha} was done, and ß-actin was used as loading control. D, cells were treated with 100 µmol/L temozolomide (16 h pretreatment), Ad.Egr-TNF (MOI 100, 8 h), and 5 Gy IR (16 h). *, P < 0.02. Representative of three separate experiments.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The heterogeneous make up of malignant gliomas enables them to resist treatments that target a single cytotoxic pathway, and it is likely that a combinatorial treatment regimen is necessary for their successful management (29). Radiochemotherapy-activated gene delivery is an attractive strategy for effectively combining diverse treatment modalities (30). The antiglioma efficacy of combination Ad.Egr-TNF, temozolomide, and IR is clearly shown in the current study using two separate mouse models. In the intracranial experiment, three of four animals that survived long term (>130 days) were noted to have regressive changes with formation of a collagenous scar in the area that would have been occupied by tumor. Although the regressive changes implicate tumor necrosis as the antitumor mechanism, no necrosis was noted in the animal brains from either the late or early treatment period. This may well be because the time point of our evaluation of the brains, even in the early period, was after the period of peak necrosis. Interestingly, regressive changes have not been previously reported in glioma-bearing animals treated with TNF-{alpha} and IR (22, 31–33), although a small fibrous scar was noted in the tumor region when TNF-related apoptosis-inducing ligand was used in combination with temozolomide (34).

In addition to efficacy, we also looked at treatment-associated toxicity, specifically as TNF-{alpha} in the central nervous system has been linked with neuronal demyelination (35). Examination of mouse brains with Luxol fast blue staining did not reveal major differences in myelination pattern between the animals in the various treatment groups. Such a lack of overall difference in myelination may be explained by the fact that the expression of TNF-{alpha} is localized to the injection area and not distributed throughout the entire brain.

Although toxicity in the form of major myelination change was not seen, animals receiving dual intracranial therapy with Ad.Egr-TNF and IR had significant systemic toxicity with 30% requiring early euthanasia. The combination of Ad.Egr-TNF and IR does not produce such toxicity when given intracranially to animals that do not bear tumors (data not shown). It is therefore unlikely that the toxic effects are due to systemic leakage of TNF-{alpha}, a finding supported by the observation that no systemic toxicity was noted in animals with hind-limb tumors that were given similar treatment. In addition, it has been previously noted that even with doses of Ad-Egr.TNF as high as 4 x 1011 pu, systemic TNF-{alpha} levels, after IR stimulation, remain <15.6 ng/L, a value several hundred-fold lower than the level at which dose-limiting TNF-{alpha} toxicity has been reported (30). It is more likely that an intracranial phenomenon, such as uncontrolled cerebral edema, underlies the systemic toxicity. High intracranial TNF-{alpha} expression, in combination with IR in animals with tumors, may result in enough edema to cause intracranial hypertension. In this regard, cerebral edema has previously been reported in mice bearing intracranial gliomas that produce TNF-{alpha} (33). In these animals, toxicity was reversed by inhibition of edema after expression of transforming growth factor ß1 (36). Despite the toxicity of combination Ad.Egr-TNF and IR, animals that receive the triple regimen of Ad.Egr-TNF, temozolomide, and IR do not display such severe negative signs. Although it is possible that rapid tumor lysis, secondary to the use of triple therapy, may account for these effects, decreased intracranial edema due to inhibition of NF-{kappa}B by temozolomide (Fig. 4D) may also contribute to the improved animal health. Such a contention is supported by a previous report demonstrating that inhibition of NF-{kappa}B in vivo results in diminished edema formation (37).

Ad.Egr-TNF is a replication defective adenovirus that can efficiently transfect cells and produce long-term TNF-{alpha} expres sion. Using an adenoviral vector bearing the ß-galactosidase gene, we and others have noted that there is >95% uptake efficiency of replication-defective Ad5 vectors by glioma cells when the MOI is 100 (ref. 38; data not shown). Although adenoviruses in general have several advantages as gene delivery agents, these vectors have been shown to induce long-lasting inflammation that can be quite detrimental (39). Activation of NF-{kappa}B has been reported to mediate the inflammatory response after adenoviral infection (26), and furthermore, this NF-{kappa}B activity can attenuate the cytotoxicity of the genes transcribed (27). NF-{kappa}B activated by Ad.Egr-TNF may be especially detrimental as it will not only reduce TNF-{alpha} gene expression but also attenuate TNF-{alpha}–induced cytotoxicity. Thus, inhibition of Ad.Egr-TNF–induced NF-{kappa}B by temozolomide can further enhance the antitumor effect.

Activation of NF-{kappa}B also plays a significant role in mediating radio resistance (14), and inhibition of I{kappa}B{alpha} phosphorylation increases glioma radio sensitivity, possibly by decreasing potentially lethal damage repair (40). Although IR is the primary therapeutic modality in the management of malignant glioma (2), the addition of temozolomide enhances overall patient survival. Specifically, the clinical success seen when IR and temozolomide are used together is noted when these modalities are given concomitantly as opposed to consecutively (18). The mechanism for this interaction is not clear. Although, temozolomide-induced G2-M cell cycle arrest (a radio-sensitive phase of the cell cycle; ref. 41) has been cited as a possible explanation, inhibition of IR-induced NF-{kappa}B by temozolomide (Fig. 4A) shows an additional mechanism by which these two treatment modalities interact.

Malignant gliomas represent a diverse group of lesions that rarely metastasize outside of the brain. As these tumors almost always recur within 2 cm of the border of the original lesion (42), a locoregional treatment approach, like the one described in this report, has significant therapeutic value. The success of concomitant temozolomide and IR in glioblastoma patients (18) means that new treatment strategies will be compared with the efficacy of this protocol. In this regard, the increase is animal survival seen when Ad.Egr-TNF is added to temozolomide and IR illustrates the potential of using such a triple treatment regimen in a clinical setting.


   Footnotes
 
Grant support: American Cancer Society, Illinois Division grant 05-15 and Cancer Research Foundation Young Investigator grant (B. Yamini), R01 CA111423-01A1 (R.R. Weichselbaum), and National Cancer Institute grant P50 CA 097247 (B. Yamini and R.R. Weichselbaum).

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.

Received 6/ 8/07; revised 7/31/07; accepted 8/ 9/07.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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S. OSHIRO, H. TSUGU, F. KOMATSU, T. OHMURA, M. OHTA, S. SAKAMOTO, T. FUKUSHIMA, and T. INOUE
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