Tumor necrosis factor α (TNFα) has been used to treat patients with certain tumor types. However, its antitumor activity has been undermined by the activation of IκBα kinase (IKK), which in turn activates nuclear factor-κB (NF-κB) to help cancer cells survive. Therefore, inhibition of TNFα-induced IKK activity with specific IKK inhibitor represents an attractive strategy to treat cancer patients. This study reveals IKI-1 as a potent small molecule inhibitor of IKKα and IKKβ, which effectively blocked TNFα-mediated IKK activation and subsequent NF-κB activity. Using gene profiling analysis, we show that IKI-1 blocked most of the TNFα-mediated mRNA expression, including many genes that play important roles in cell survival. We further show that in vitro and in vivo combination of TNFα with IKI-1 had superior potency than either agent alone. This increased potency was due primarily to the increased apoptosis in the presence of both TNFα and IKI-1. Additionally, IKKβ small interfering RNA transfected cells were more sensitive to the treatment of TNFα. The study suggests that the limited efficacy of TNFα in cancer treatment was due in part to the activation of NF-κB, allowing tumor cells to escape apoptosis. Therefore, the combination of IKI-1 with TNFα may improve the efficacy of TNFα for certain tumor types. [Cancer Res 2008;68(22):9519–24]

Tumor necrosis factor α (TNFα) is a pleiotropic protein that initially isolated from the serum of mice treated with bacterial endotoxin (13). It is involved in many diseases, such as autoimmune diseases and cancer. Interestingly, TNFα has been shown to have antitumor activity in various tumors (4, 5). Multiple clinical trials have been conducted to treat cancer patients with various tumor types with mixed success (4, 5). However, many tumors have been found to be resistant to TNFα (6, 7). Additionally, TNFα has been shown to cause significant systemic toxicity, which prevents it from becoming an effective anticancer agent (6, 8). Currently, its usages are limited to limb perfusion and isolated hepatic perfusion for the treatment of locally advanced solid tumors (4, 5).

TNFα mediates its biological effect through its receptor TNF-R1 and TNF-R2 (9, 10), which recruit many cellular proteins and consequently regulate multiple signaling pathways. One of the major pathways TNFα activates is the IκBα kinase (IKK)/nuclear factor-κB (NF-κB) pathway. NF-κB is regulated by its upstream kinase, IKK complex, which comprises at least IKKα and IKKβ and a noncatalytic regulatory protein, NEMO (1113). In cytoplasm, NF-κB forms a tight complex with IκBα, which blocks the nuclear translocation signal of NF-κB (1113). When IKK is activated, it phosphorylates IκBα, which in turn triggers ubiquitination and proteasome-dependent degradation of IκBα (1113). NF-κB is thus released and translocated to the nucleus, where it regulates an array of genes responsible for cell survival and growth, which paradoxically blocked the apoptotic potential exerted by TNFα. Using genetic or pharmacologic approaches, several groups showed clearly that the potency of TNFα can be dramatically increased in certain tumors when NF-κB activity is inhibited (1416). Because NF-κB is controlled primarily by IKK, it is conceivable that blocking IKK will result in dramatic cell death, thus improving the anticancer activity of TNFα. This notion has been strongly supported by many studies using nonselective IKK inhibitors (1722). Recently, Frelin and colleagues (23) showed that AS602868, a specific small molecule IKKβ inhibitor, potentiated the proapoptotic effect of TNFα in Jukat leukemia cells.

We show here that IKI-1 is a potent IKK inhibitor,1

1

S. Sum, L. Chen, D. Powell, T. Mansour, Y. Zhang, M. Gavriil, T. Saddler. N-[3-(dimethylamino)propyl]-4-{[4-(3-fluoro-4-methoxyphenyl)pyrimidin-2-yl]amino}benzenesulfonamide: an orally available IKK inhibitor with antitumor activity, manuscript in preparation.

which blocked TNFα-mediated activation of IKK/NF-κB pathway and downstream target genes in pancreatic tumor cells. Additionally, IKI-1 greatly potentiated the effect of TNFα on the growth of tumor cells through activation of apoptosis. Furthermore, the combination resulted in shrinkage of tumors grown in nude mice, suggesting that combination of an IKK inhibitor with TNFα is a potential treatment for irresectable pancreatic cancer.

Materials. All cell culture reagents were purchased from Invitrogen, except for charcoal-stripped fetal bovine serum (FBS), which was obtained from Hyclone. Steady-Glo Lucifierase Assay System and CellTiter-Glo were purchased from Promega. Apoptotic DNA-Ladder kit was purchased from Roche Applied Science. CaspaseOne kit was purchased from Promega. Small interfering RNAs (siRNA) ON-TARGETplus SMARTpool for IKKα, IKKβ and nontarget control were purchased from Dharmacon. Inhibitor IV and ZVAD-FMK were purchased from Calbiochem. The synthesis of IKI-1 will be described in a future publication.1

Cell culture and Western blot analysis. BxPC3 cells were obtained from American Type Culture Collection and were maintained in DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Cells were harvested in M-PER reagent (Pierce). Thirty micrograms of protein were electrophoresed on an 8% to 16% SDS-PAGE gel and then transferred to polyvinylidene difluoride using a Bio-Rad liquid transfer apparatus. The Western blot analysis was performed with enhanced chemiluminescence (SuperSignal, Pierce) using the anti-IκBα antibody (Santa Cruz) at 1 μg/mL and the horseradish peroxidase–conjugated antirabbit secondary antibody (Bio-Rad) diluted at 1:4,000. Phosphorylation of IκBα was detected using anti–phosphorylated IκBα antibodies (Cell Signaling Technology). IKKβ was detected using the anti-IKKβ antibody (Cell Signaling Technology).

Transfection of siRNA. Two thousand BxPC3 cells were plated in each well of 96-well plates. siRNA of IKKβ or nontarget was prepared in OptiMem (Invitrogen) medium containing Optifect (Invitrogen) and added to each well. Twenty-four hours later, siRNA was removed and replaced with fresh medium. After 24 h, cells were treated with or without TNFα (final concentration, 5 ng/mL). Cell growth was measured after 96 h after TNFα treatment with the addition of 100 μL of CellTiter-Glo reagent, and signals were read with a Vector reader (PerkinElmer).

Apo-One homogeneous caspase-3/caspase-7 assay (Promega). Ten thousand BxPC3 cells were plated in each well of 96-well plates. Cells were treated with various concentration of IKI-1, as indicated for 1 h before the addition of TNFα (5 ng/mL). Twenty four hours later, 100 μL of Apo-One reagent was added to each well containing 100 μL of culture medium. After 1 h of incubation, fluorescence of the samples was determined with a fluorescence plate reader (GeminiXS, Molecular Devices).

Cell death detection ELISA. Ten thousand BxPC3 cells were plated in each well of 96-well plates. Cells were treated with IKI-1 for 1 h before the addition of TNFα. After 24 h, cell death was evaluated according to manufacturer's instruction (Roche Applied Science).

NF-κB nuclear translocation assay. Six thousand BxPC3 cells were plated in each well of clear-bottomed, 96-well plates (PerkinElmer) and incubated overnight at 37°C. On the next day, cells were treated with IKI-1 for 1 h before the addition of TNFα (final concentration, 5 ng/mL). NF-κB nuclear translocation assay was performed using HitKit HCS reagent (Thermo Fisher Scientific), according to manufacturer's protocol. NF-κB signals were detected using an ArrayScan VTI HCS Reader (Thermo Fisher Scientific).

For the standard immunofluorescence staining procedure, BxPC3 cells were incubated on Biocoat chamber slides (BD Biosciences) overnight. Cells were treated with IKI-1 for 1 h and then with 5 ng/mL TNFα for 20 min. The medium was aspirated, and cells were fixed with 3.7% formaldehyde in PBS for 10 min at room temperature, followed by permeabilization with 0.15% Triton X-100 for 10 min. Cell were rehydrated in PBS and then incubated in Cellomics NF-κB p65 antibody for 1 h at 37°C. Cells were washed in PBS and stained with Alexa Fluor 488 secondary antibody (Cellomics) for 1 h at room temperature. The slide was washed, and the coverslip was mounted using Prolong Gold Antifade with 4′,6-diamidino-2-phenylindole (Invitrogen).

Gene profiling experiments. RNA extraction and preparation, chip hybridization and data reduction, and data filtering and statistics were similar to those detailed in ref. 24, with the exception that U95v2 chips (Affymetrix GeneChips) were used in this study.

In vivo studies. Animal care, injection of cancer cells, and tumor measurement were described in detail in our previously published paper (25). IKI-1 (suspended in methocel/Tween 20) was given daily for 5 d/wk for a total of 2 wk, and TNFα (prepared in PBS) was given twice a week for 2 wk. The control vehicles were given on the same regimen as the drugs. Control groups were 10 mice per vehicle, whereas the drug groups were 10 mice per drug.

IKI-1 inhibited TNFα-mediated phosphorylation and degradation of IκBα in BxPC3 cells. IKI-1 is a potent IKK inhibitor, which inhibited equally the activity of both IKKα (IC50 = 80 nmol/L) and IKKβ (IC50 = 70 nmol/L)1 TNFα treatment resulted in IκBα phosphorylation and degradation in BxPC3 pancreatic cells, which occurred within 5 min (Fig. 1). IKI-1 effectively blocked the phosphorylation and degradation of IκBα in a dose-dependent manner.

Figure 1.

IKI-1 inhibited TNFα-mediated IκBα phosphorylation and degradation. BxPC3 pancreatic cells were treated with indicated concentration of IKI-1 for 30 min before the addition of 10 ng/mL of TNFα (final concentration). Cells were harvested after 15 min of TNFα treatment. Phosphorylated IκBα, IκBα, and actin protein levels were analyzed by Western blot analysis.

Figure 1.

IKI-1 inhibited TNFα-mediated IκBα phosphorylation and degradation. BxPC3 pancreatic cells were treated with indicated concentration of IKI-1 for 30 min before the addition of 10 ng/mL of TNFα (final concentration). Cells were harvested after 15 min of TNFα treatment. Phosphorylated IκBα, IκBα, and actin protein levels were analyzed by Western blot analysis.

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IKI-1 inhibited TNFα-induced p65 translocation. Activation of IKK triggers the translocation of NF-κB to the nucleus. The effect of IKI-1 on p65 nuclear translocation was examined with the use of ArrayScan VTI HCS Reader (Thermo Fisher Scientific). In the absence of TNFα, p65 was primarily located in the cytoplasm (Fig. 2A). Upon TNFα treatment, nearly all p65 signals were detected in the nucleus (Fig. 2A). IKI-1 inhibited p65 nuclear translocation in a dose-dependent manner with an IC50 of ∼200 nmol/L (200 ± 15, n = 8). To confirm the data generated by ArrayScan, we performed standard immunofluorescence staining of p65. The results were similar and supportive of the conclusion based on ArrayScan analysis (Fig. 2B). To rule out the possibility that IKI-1 is a nonselective inhibitor of transcription factors, we tested its effect on Stat3 translocation induced by TNFα. We found no significant inhibition of Stat3 translocation using up to 10 μmol/L of IKI-1 (data not shown).

Figure 2.

Effect of IKI-1 on the nuclear p65 level induced by TNFα. A, high content analysis of p65 localization. BxPC3 pancreatic cells were plated (6,000 cells per well, 96-well plate) and treated with indicated concentration of IKI-1 for 30 min before the addition of 5 ng/mL of TNFα (final concentration). At 15 min after TNFα treatment, p65 localization was stained using HitKit (Cellomics) according to manufacturer's instruction. Quantitation of p65 was analyzed by ArrayScan VTI HCS Reader according to manufacturer's instruction. IC50 is the average of eight replicates. B, standard immunofluorescence staining of p65 localization. Standard immunofluorescence was carried, as described in Materials and Methods.

Figure 2.

Effect of IKI-1 on the nuclear p65 level induced by TNFα. A, high content analysis of p65 localization. BxPC3 pancreatic cells were plated (6,000 cells per well, 96-well plate) and treated with indicated concentration of IKI-1 for 30 min before the addition of 5 ng/mL of TNFα (final concentration). At 15 min after TNFα treatment, p65 localization was stained using HitKit (Cellomics) according to manufacturer's instruction. Quantitation of p65 was analyzed by ArrayScan VTI HCS Reader according to manufacturer's instruction. IC50 is the average of eight replicates. B, standard immunofluorescence staining of p65 localization. Standard immunofluorescence was carried, as described in Materials and Methods.

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IKI-1 inhibited TNFα-induced gene expression in BxPC3. Because NF-κB controls many genes, we used oligonucleotide array to study the effect of IKI-1 on genes regulated by TNFα. Genes, whose expression was induced by TNFα treatment from 1.5-fold to 21-fold (P ≤ 0.01), were selected for further analysis. TNFα alone induced ∼236 nonredundant genes (Supplementary Table S2). Cotreatment with IKI-1 (1 μmol/L) inhibited most of the TNFα-induced genes (Fig. 3A). Among all the genes, 102 genes were inhibited by >70%, 80 genes were inhibited from 30% to 70%, and the remaining 54 genes were inhibited by <30% (Fig. 3B). Examples of NF-κB–regulated genes are illustrated in Fig. 3C, confirming that increase in cell death by TNFα and IKI-1 was due to the inhibition of key molecules involved in cell survival. A complete list of TNFα-induced genes with and without IKI-1 is submitted as Supplementary Data.

Figure 3.

Inhibition of NF-κB target genes by IKI-1. BxPC3 cells were treated with vehicle, IKI-1 (1 μmol/L), TNFα 5 ng/mL, or combination of IKI-1 and TNFα. Cells were harvested, and genes were profiled according to what was described in Materials and Methods. Genes that were induced by TNFα with signals of >30-fold and 1.5-fold over the vehicle control (P < 0.01) were selected. A, effect of IKI-1, TNFα, and combination of both on the expression of genes. B, effect of IKI-1 on TNFα-induced genes. C, representative NF-κB target genes inhibited by IKI-1. Columns, means of three experiments carried out in triplicate; bars, SE.

Figure 3.

Inhibition of NF-κB target genes by IKI-1. BxPC3 cells were treated with vehicle, IKI-1 (1 μmol/L), TNFα 5 ng/mL, or combination of IKI-1 and TNFα. Cells were harvested, and genes were profiled according to what was described in Materials and Methods. Genes that were induced by TNFα with signals of >30-fold and 1.5-fold over the vehicle control (P < 0.01) were selected. A, effect of IKI-1, TNFα, and combination of both on the expression of genes. B, effect of IKI-1 on TNFα-induced genes. C, representative NF-κB target genes inhibited by IKI-1. Columns, means of three experiments carried out in triplicate; bars, SE.

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IKI-1 enhanced TNFα-mediated growth inhibition of BxPC3 cells. Inhibition of NF-κB pathway was shown to enhance TNFα apoptotic effect in multiple cell lines. The combination effect on the growth of BxPC3 pancreatic cancer cells was thus tested. Figure 4A shows that IKI-1 itself had moderate effect on the growth of BxPC3 cells. However, in the presence of a non–growth inhibitory dose of TNFα (5 ng/mL), the IC50 of IKI-1 decreased ∼5-fold. To confirm that this combination effect was mediated through the inhibition of IKKβ in BxPC3 cells, we treated cells with siRNA of IKKα and IKKβ in the presence or absence of TNFα. Synergistic effect was seen from cells treated with siRNA of IKKβ, but not IKKα, or nontarget siRNA control (Fig. 4B and C).

Figure 4.

Potentiation of TNFα-mediated growth inhibition by IKI-1. A, cells (2,000 per well, 96-well plate) were treated with increasing amount of IKI-1 for 30 min, after which cells were treated with 10 ng/mL of TNFα. Relative cell growth was assayed using CellTiteGlo after 96 h. B, effect of siRNA IKKβ on the growth of BxPC3 cells. C, lack of effect of siRNA IKKα on the growth of BxPC3 cells. IKKα and IKKβ siRNA-transfected cells were treated with or without TNFα (final concentration, 5 ng/mL). Cells in multiple wells were trypsinized and subjected to Western blot analysis for IKKα and IKKβ protein level (top). Growth assay was performed on cells in other wells after 96 h using CellTiterGlo according to manufacturer's instruction (bottom). Columns, means of three experiments carried out in triplicate; bars, SE.

Figure 4.

Potentiation of TNFα-mediated growth inhibition by IKI-1. A, cells (2,000 per well, 96-well plate) were treated with increasing amount of IKI-1 for 30 min, after which cells were treated with 10 ng/mL of TNFα. Relative cell growth was assayed using CellTiteGlo after 96 h. B, effect of siRNA IKKβ on the growth of BxPC3 cells. C, lack of effect of siRNA IKKα on the growth of BxPC3 cells. IKKα and IKKβ siRNA-transfected cells were treated with or without TNFα (final concentration, 5 ng/mL). Cells in multiple wells were trypsinized and subjected to Western blot analysis for IKKα and IKKβ protein level (top). Growth assay was performed on cells in other wells after 96 h using CellTiterGlo according to manufacturer's instruction (bottom). Columns, means of three experiments carried out in triplicate; bars, SE.

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IKI-1 enhanced TNFα-mediated apoptosis. To understand the mechanisms by which IKI-1 enhanced TNFα-mediated growth inhibition, we sought to study the combination effect on caspase activity. Whereas either TNFα or IKI-1 alone had little effect on the activity of caspase-3 and caspase-7, together, they dramatically increased the activity of caspase-3 and caspase-7. Additionally, IKI-1 dramatically increased TNFα-mediated apoptosis measured by DNA fragmentation (Fig. 5B). This effect is primarily due to the activation of caspases, because cells treated with a pan caspase inhibitor (ZVAD-FMK) showed much reduced apoptosis induced by the combination (Fig. 5B).

Figure 5.

Effect of IKI-1 on TNFα-mediated apoptosis. A, BxPC3 pancreatic cancer cells were treated with various concentrations of IKI-1 for 30 min followed by TNFα treatment. At 24 h later, caspase-3/caspase-7 activity was measured, as described in detail under Materials and Methods. B, BxPC3 pancreatic cancer cells were treated with 0.5 μmol/L of IKI-1 for 30 min followed by TNFα treatment (final concentration, 10 ng/mL). At 24 h later, DNA fragmentation was determined with an ELISA kit (Roche Applied Science). Columns, means of eight replicates; bars, SE.

Figure 5.

Effect of IKI-1 on TNFα-mediated apoptosis. A, BxPC3 pancreatic cancer cells were treated with various concentrations of IKI-1 for 30 min followed by TNFα treatment. At 24 h later, caspase-3/caspase-7 activity was measured, as described in detail under Materials and Methods. B, BxPC3 pancreatic cancer cells were treated with 0.5 μmol/L of IKI-1 for 30 min followed by TNFα treatment (final concentration, 10 ng/mL). At 24 h later, DNA fragmentation was determined with an ELISA kit (Roche Applied Science). Columns, means of eight replicates; bars, SE.

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IKI-1 potentiated TNFα-induced apoptosis in BxPC3 xenograft model. The combination effect observed in vitro prompted us to study the efficacy of this combination in BxPC3 mouse xenograft model. IKI-1 was given 100 mg/kg p.o. twice daily for 5 days a week for a total of 2 weeks. TNFα (1 μg/tumor) was injected intratumorally twice a week. Whereas neither IKI-1 nor TNFα had any effect on tumor growth, the combination of both resulted in significant inhibition of tumor growth compared with the control (Fig. 6).

Figure 6.

Effect of combination of IKI-1 and TNFα on the growth of BxPC3 pancreatic tumor. Tumor-bearing mice were given IKI-1 twice daily for 5 d/wk for a total of 2 wk. TNFα was injected intratumorally twice a week (arrow). Each group contained 10 mice. Handling of mice, treatment, and tumor measurement were described in detail under Materials and Methods. Points, means of 10 animals; bars, SE.

Figure 6.

Effect of combination of IKI-1 and TNFα on the growth of BxPC3 pancreatic tumor. Tumor-bearing mice were given IKI-1 twice daily for 5 d/wk for a total of 2 wk. TNFα was injected intratumorally twice a week (arrow). Each group contained 10 mice. Handling of mice, treatment, and tumor measurement were described in detail under Materials and Methods. Points, means of 10 animals; bars, SE.

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In this study, we show that IKI-1, a potent IKK inhibitor, effectively inhibited the TNFα-induced phosphorylation of IκBα and nuclear translocation of p65 in BxPC3 pancreatic cancer cells. We further investigated the effect of IKI-1 on TNFα-mediated biological responses, such as NF-κB target genes regulation, apoptosis, growth inhibition, and tumor growth. Our results show that IKI-1 dramatically potentiated the effect of TNFα on the growth of pancreatic cancer cells through the activation of apoptotic pathway. Similar effect was also observed in another pancreatic cancer line, Panc-1 cells (Supplementary Fig. S1) and multiple nonpancreatic lines (Supplementary Table S1), suggesting that the potentiation in growth inhibition is mechanism-based rather than a cell line phenomena. Using RNA interference to knockdown IKKβ and gene profiling approaches, we determined that the potentiation correlated with the inhibition of IKKβ and, consequently, NF-κB–mediated target genes. Our study shows that combination of TNFα and IKK inhibitor may have a potential to treat pancreatic cancer patients whose tumors are irresectable.

Earlier studies using cells from either NF-κB knockout mice or cells transfected with an IκBα superrepressor revealed that NF-κB plays an important role in the resistance to TNFα-mediated cell death (1416). This is because NF-κB regulates multiple survival genes to protect cells from cell death. Therefore, when NF-κB activity is blocked, the TNFα prodeath effect is potentiated tremendously. Multiple studies using NF-κB inhibitors have resulted in similar results (1722). However, these NF-κB inhibitors usually have multiple activities, which obscure the interpretation of the results. Recently, Frelin and colleagues showed that an IKKβ inhibitor AS602868 potentiated the effect of TNFα in Jurkat leukemic cells (23). It remains unclear if this potentiation is specific to AS602868 or the mechanism of inhibiting IKK/NF-κB pathway. To address this question, we used siRNA of IKKα and IKKβ to show that, indeed, the effect of TNFα can be potentiated in the IKKβ, but not IKKα, knockdown pancreatic cells. Similar augmentation was obtained with our small molecule inhibitor IKI-1 (Fig. 4A). The knockdown study results seemed to suggest that IKKβ is responsible for the survival of cells under TNFα treatment. Studies with a selective IKKβ inhibitor IV and our own selective inhibitors also supported this notion (data not shown).

Using caspase and DNA fragmentation assays, we showed that the synergistic inhibition of cellular growth with the combination of TNFα and IKI-1 was largely due to increased apoptosis (Fig. 5A and B). In the latter assay, a pan caspase inhibitor abrogated majority of the caspase activity. These results are in agreement with Frelin's finding in Jukat cells (23). The dramatic increase in apoptosis also explains the initial shrinkage of tumor in a pancreatic tumor xenograft model when both TNFα and IKI-1 were given (Fig. 6).

Pancreatic cancer is presently the fourth leading cause of cancer death in Western countries, with the poorest survival rate among all cancers (2008 Cancer Facts). The high fatality rate is due to undetectable progression of the disease until late stages, the lack of specific and sensitive markers for early detection and the largely refractory response to available surgical, chemotherapeutic, and radiotherapeutic treatment modalities (2630). In ∼45% of patients with pancreatic cancer, the primary tumor has already metastasized by the time of diagnosis to distal sites, including the liver, lungs, duodenum, and peritoneum, whereas an additional 35% to 45% of patients suffer from irresectable or locally advanced disease.

The current treatment is inadequate, given the prevalence of the disease. TNFα is not a feasible anticancer agent for pancreatic cancer due to its systemic toxicities. To overcome systemic toxicities, many innovative approaches have been tried. Targeted delivery using vasculature-specific binding peptide (31), receptor-selective TNFα mutant to reduce toxic effect (32), liposomal delivery, and viral delivery of TNFα have been explored but fall short of being effective enough to treat patients. A promising intratumoral delivery of radiation-induced activation of viral vector expressing TNFα gene is currently in clinical trials for several cancer types, including pancreatic cancer (33, 34). Initial results showed reduced systemic toxicities and safety. Recently, colloidal gold nanoparticles coupled with TNFα also showed promising results by enhancing the level of TNFα in tumors from 7-fold to 10-fold without causing significant systemic toxicities (35). Our data, along with those of others', strongly suggest that the combination of an IKK inhibitor with TNFα provides an effective treatment option for pancreatic cancer patients through the use of reduced dose of TNFα.

All of the authors have an ownership interest in Wyeth.

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Current address for Y. Zhang: Enzon Pharmaceutical, 20 Kingsbridge Road, Piscataway, NJ 08854.

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.

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