Chemosensitization by diverging modulation by short-term and long-term TNF-α action on ABCB1 expression and NF-κB signaling in colon cancer

  • Authors:
    • Wolfgang Walther
    • Dennis Kobelt
    • Lisa Bauer
    • Jutta Aumann
    • Ulrike Stein
  • View Affiliations

  • Published online on: October 5, 2015     https://doi.org/10.3892/ijo.2015.3189
  • Pages: 2276-2285
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Multidrug resistance (MDR) is a major cause for cancer chemotherapy failure. Among the numerous strategies to overcome persistent action of proinflammatory cytokines, such as tumor necrosis factor α (TNF-α) permits downregulation of MDR-associated genes, including ATP-binding cassette, subfamily B 1 gene (ABCB1). A key regulator of ABCB1 expression is the transcription factor nuclear factor κ light chain enhancer (NF-κB)/p65. We analyzed diverging short- and long-term effects of TNF-α regarding modulation of NF-κB/p65 signaling and ABCB1 expression in colon cancer cells. Highly resistant ABCB1 overexpressing human HCT15 colorectal carcinoma cells were subjected to short- (30-120 min) or long-term (24-96 h) TNF-α treatment. TNF-α mediated modulation of ABCB1 expression was analyzed by real-time RT-PCR and western blot analysis. The TNF-mediated chemosensitization was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay. The involvement of TNF receptors and of NF-κB/p65 signaling was analyzed by western blot analysis, ABCB1 promoter analysis and electrophoretic mobility shift assay (EMSA). The study revealed, that long-term, but not short-term TNF-α treatment leads to TNF-receptor 1 (TNFR1) mediated downregulation of ABCB1 resulting in sensitization towards drug treatment. It dampens NF-κB/p65 activation and nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor α (IκBα) resynthesis, associated with reduced nuclear accumulation of NF-κB/p65 and reduced binding to its consensus sequence in the ABCB1 promoter. The study reveals the diverging effects of short- or long-term TNF-α action and provides novel insights on downregulation of ABCB1 expression by TNF-mediated repression of NF-κB signaling.

Introduction

The phenomenon of multidrug resistance (MDR) still represents a major obstacle for successful chemotherapy of cancer (14). The emergence of the MDR phenotype is mainly caused by the increase in expression of MDR-associated genes, such as ATP-binding cassette, subfamily B 1 gene (ABCB1) (MDR1), ABCC1 (MRP1) and ABCG2 (BCRP1). Numerous efforts have been made to develop strategies for modulation of MDR-associated genes. The classical type of MDR is mediated by overexpression of the P-glycoprotein (PgP), the gene product of the ATP-binding cassette (ABC) transporter ABCB1. Therefore reversal approaches target the expression and function of ABCB1/PgP (4). Among these approaches the use of cytokines has shown promise for downregulation of ABCB1 in association with increased drug uptake and chemosensitization of tumor cells (5). Several studies have shown, that cytokines, such as tumor necrosis factor α (TNF-α), interferon γ (IFN-γ), leukoregulin and interleukin-2 (IL-2) are capable of reducing ABCB1 gene expression and increasing chemosensitivity of different cancer cell lines in vitro, and more recently also for endothelial cells in the blood-brain barrier (612). It has been shown, that particularly persistent treatment with these cytokines is of importance to achieve significant reduction in ABCB1 gene expression (6,7,9,13). Consistent with the pre-clinical findings of cytokine-mediated chemosensitization, clinical trials have demonstrated synergism of combinations of cytostatic drugs with TNF-α or interferones in cancer patients (14,15). Among all cytokines used for ABCB1 expression modulation, TNF-α has shown the highest efficiency in vitro, in vivo and in clinical approaches (9,11,13,16). Although the TNF-α mediated effects on ABCB1 expression were repeatedly shown at functional level, it is still not fully understood, what mechanism in cell signalling is reponsible for these effects.

TNF-α exerts its intracellular activities via TNF-receptor 1 (TNFR1, p55) and TNF-receptor 2 (TNFR2, p75). Among other factors, the receptor-mediated intracellular signalling cascade requires nuclear factor κ light chain enhancer (NF-κB) as one key mediator and TNF-α is one of the most potent activators of NF-κB signaling (17).

Activation of NF-κB, which is sequestered in the cytoplasm as an inactive factor, is mediated via the nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor (IκB) kinase (IKK) complex, which is triggered by proinflammatory cytokines including TNF-α or cytostatic drugs (18). IKK phosphorylates the NF-κB inhibitor IκBα, which is then degraded by the 26S proteasome complex. NF-κB is released, unmasking its nuclear localization signal and enters the nucleus to activate transcription of specific target genes, including of its own inhibitor IκBα. Within this signaling cascade IκBα mediates both, rapid activation of NF-κB and strong negative feedback (19).

The link between NF-κB and ABCB1 gene expression regulation has been analyzed in several studies (20). These studies demonstrate that transient induction of NF-κB is associated with increase in ABCB1 gene expression and inversely, inhibition of NF-κB leads to downregulation of ABCB1 expression (2023). Promoter analyses revealed that the human ABCB1 promoter harbors NF-κB responsive elements, which bind NF-κB to mediate regulation of ABCB1 gene expression (24). However, other studies revealed a more complex picture of TNF-α triggered NF-κB activation and its target gene regulation. They provide detailed insights into the diverging nature of TNF-α/NF-κB signaling. This is strongly dependent on duration of pathway activation, showing rather contrary effects of early transient and of late persistent phase effects, mediated either by IκBα or IκBβ (25). Interestingly, such differential action can lead to negative feedback mechanisms of TNF-α/NF-κB signaling which dampen the initial early transient phase effects, in which IκBα and IκBβ are important regulators (2628).

In this context our study analyzed both, the transient and more importantly persistent TNF-α mediated effects on ABCB1 expression in human colorectal cancer cells as diverging action of this cytokine. We show that NF-κB/p65 exerts signal transduction via TNFR1, which is strictly time-dependent and tightly associated with the modulation of ABCB1 expression. These findings extend the picture of previously described diverging nature of TNF-α action. They provide a potential link to chronic inflammation, persistent TNF-α release and drug sensitivity in TNF-α exposed cells or tissues, as is clinically observed in e.g. inflammatory intestine diseases (2932). More importantly, this study might open new insights for targeted interventions of MDR reversal in the treatment of colon cancer.

Materials and methods

Cell culture

The human colorectal carcinoma cell line HCT15 was cultured at 37ºC, 5% CO2 in RPMI-1640 medium (Gibco BRL, Gaithersburg, MD, USA), containing 10% FCS. This cell line endogenously expresses high-level ABCB1 and possesses a strong MDR phenotype (33). Authentification of the cell line was performed by STR DNA typing (DSMZ, Braunschweig, Germany).

Treatment with TNF-α

Briefly, 5×104 cells were seeded into 24-well plates and grown for 24 h. Then, cells were treated with 30 ng TNF-α/ml (Invitrogen, Carlsbad, CA, USA) for 5–120 min in short-term incubations and 24–72 h in long-term incubations. The cells were harvested for further analyses at indicated time points (5, 10, 15, 30, 60 and 120 min; 24, 48 and 72 h). In the long-term incubations in addition cells were harvested shortly after TNF-α application at the respective days, indicated as 24, 48 and 72 h+.

Blocking of TNF-receptors

The blocking of TNF-receptors was performed in cells treated for 72 h with TNF-α. For this, 5×104 cells were seeded into 24-well plates and grown for 24 h. One hour before each treatment with 30 ng TNF-α/ml (Invitrogen) cells were incubated with 8 or 20 μg mouse anti-TNFR1 monoclonal antibody (R&D Systems, Inc., Minneapolis, MN, USA) and 4 or 12 μg mouse anti-TNFR2 monoclonal antibody (R&D Systems, Inc.) respectively, to specifically block the receptor (as recommended by manufacturer). The control cells were treated with 30 ng TNF-α/ml only, or remained untreated.

Real-time quantitative RT-PCR (qRT-PCR)

Total RNA from cells was isolated using the TRIzol™ method (Invitrogen). Reverse transcriptase (RT) reaction was performed with 50 ng of total RNA (MuLV reverse transcriptase; Perkin-Elmer, Weiterstadt, Germany). Each quantitative real-time PCR (95ºC for 10 sec, 45 cycles of 95ºC 10 sec, 60ºC 30 sec and 72ºC 1 sec) was done using the LightCycler (LightCycler Fast Start DNA master hybridization probes kit; Roche Diagnostics GmbH, Mannheim, Germany). Expression of human ABCB1, NF-κB/p65, IκBα, IκBβ and of the housekeeping gene glucose-6-phosphate dehydrogenase (G6PDH) was determined in parallel from the same RT-reaction, each done in duplicate per sample. For ABCB1 a 167 bp amplicon (forward, 5′-CCCATCATTGCAATAGCAGG-3′; FITC-labeled probe forward, 5′-CACTGAAAGATAAGAAAGAACTAGAAGGTGCT-3′; LCRed640-labeled probe forward, 5′-GGAAGATCGCTACTGAAGCAATAGAAAACT-3′ and reverse, 5′-GTTCAAACTTCTGCTCCTGA-3′); for NF-κB/p65 a 365 bp amplicon (forward, 5′-AGATCAATGGCTACACAGGA-3′ and reverse, 5′-GATGGGATGAGAAAGGACA-3′); for IκBα a 354 bp amlicon (forward, 5′-CCGAGACTTTCGAGGAAAT-3′ and reverse, 5′-GTGAGCTGGTAGGGAGAATA-3′); for IκBβ a 417 bp amplicon (forward, 5′-AGTACATGGACCTGCAGAAT-3′ and reverse, 5′-GGACCATCTCCACATCTTTg-3′), and for G6PDH a 123 bp amplicon was produced, which were detected by gene-specific fluorescein- and LCRed640-labeled hybridization probes [(primers for ABCB1, NF-κB, IκBα, IκBβ; BioTeZ, Berlin, Germany); (probes for ABCB1; TIB Molbiol, Berlin, Germany); (primers and probes for G6PDH; Roche Diagnostics GmbH)]. The calibrator cDNA, derived from the human ABCB1 expressing cell line HCT15, was employed in serial dilutions (in duplicate) simultaneously in each run.

Western blotting

Cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1X complete protease inhibitor cocktail (Roche Diagnostics GmbH). Cytoplasmic and nuclear extracts were prepared from cells by using the NE-PER extraction kit according to manufacturer's instructions (Pierce Biotechnology, Inc., Rockford, IL, USA). The protein content was quantified by using the Coomassie Plus protein assay according to manufacturer's instructions (Pierce Biotechnology, Inc.). Precast NuPAGE 4–12% gradient polyacrylamide gels (Invitrogen) were loaded with 50 μg protein of either total cell lysates, cytoplasmic extracts, or 10 μg of nuclear extracts and electrophorezed at 200 V, 60 min. The gels were blotted onto nitrocellulose filter (Hybond-C Extra; Amersham, Freiburg, Germany). The filters were blocked 1 h at room temperature in TBS blocking buffer (50 mM Tris, 150 mM NaCl, pH 7.5, 5% fat-free dry milk) and washed in TBST (0.05% Tween-20 in TBS buffer) at RT.

For detection anti-human-TNFR1 mouse IgG monoclonal antibody (1:100), anti-human-TNFR2 mouse IgG monoclonal antibody (1:250) (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-human PgP monoclonal mouse IgG-antibody C219 (1:100; Calbiochem, San Diego, CA, USA), and anti-human NF-κB/p65 mouse monoclonal IgG-antibody (1:500), anti-human IκBα mouse monoclonal IgG-antibody (1:500), anti-human IκBβ mouse monoclonal IgG-antibody (1:100) (all from Santa Cruz Biotechnology, Inc.), anti-human β-tubulin mouse monoclonal IgM antibody (1:500; BD Pharmingen, Heidelberg, Germany), anti-human nuclear matrix protein (NMP) p84 mouse monoclonal IgG antibody (1:2,000; Abcam, Cambridge, UK) and anti-human GAPDH goat polyclonal antibody (1:1,000; Santa Cruz Biotechnology, Inc.) were used. As secondary antibodies HRP-labeled goat anti-mouse IgG-antibody (1:6,000; Pierce Biotechnology, Inc.), goat anti-mouse IgM-antibody (1:5,000; Sigma-Aldrich, St. Louis, MO, USA) or mouse anti-goat antibody (1:5,000; Santa Cruz Biotechnology, Inc.) were used. All antibodies were diluted in TBST containing 5% BSA. After incubation with the respective primary and secondary antibodies, the filters were washed in TBST. After washing, the respective protein was detected using ECL-solution (Amersham) and exposed to Kodak X-Omat AR film.

Electrophoretic mobility shift assay (EMSA)

The nuclear extracts were prepared from cells by using the NE-PER extraction kit according to manufacturer's instructions (Pierce Biotechnology, Inc.). The EMSA was performed with 3 μg of nuclear extract using biotin end-labeled double-stranded oligonucleotides harboring the NF-κB/p65-binding site of the wild-type human ABCB1 promoter (sense, 5′-GCTGCTCTGGCCGCGATGGGCACTGCAGGGGCTTTCCTGTGCGCGGGGTCTCCAGCATCT-3′ and antisense, 5′-AGATGCTGGAGACCCCGCGCACAGGAAAGCCCCTGCAGTGCCCATCGCGGCCAGAGCAGC-3′); or mutant (sense, 5′-GCTGCTCTGGCCGCGATGGGCACTGCACTCGCTTTCCTGTGCGCGGGGTCTCCAGCATCT-3′ and antisense, 5′-AGATGCTGGAGACCCCGCGCACAGGAAAGCGAGTGCAGTGCCCATCGCGGCCAGAGCAGC-3′). In competition experiments unlabeled double-stranded oligonucleotides were used to control binding specificity of NF-κB/p65. For the supershift experiments, the NF-κB/p65 mouse monoclonal IgG-antibody (0.5 or 1.0 μg; Santa Cruz Biotechnology, Inc.) was used. For the EMSA 6% TBS pre-cast gels (Invitrogen) were used. Gels were blotted onto nitrocellulose filters (Amersham). After UV cross-linking shifted and supershifted bands were detected by the LightShift kit (Pierce Biotechnology, Inc.) according to manufacturer's instructions and exposed to Kodak X-Omat AR film.

Drug uptake assay

After the pretreatment with 30 ng TNF-α/ml (Invitrogen) for 2 or 72 h, respectively, cells were incubated with doxorubicin (50 μM; Sigma, Taufkirchen, Germany) in phenol red-free RPMI-1640 medium for 3 h at 37ºC, and were washed with phenol red-free medium and kept on ice. Fluorescence intensity of 1×104 cells was measured in duplicate per sample by using the FACSCalibur (Cell Quest program; Becton-Dickinson, San Diego, CA, USA). The drug uptake is expressed as fold-increase compared to untreated cells, which did not receive TNF-α.

Cytotoxicity assay 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)

For the cytotoxicity assay, 5×103 cells were seeded into 96-well plates. The short- or long-term TNF-α effects on drug sensitivity of cells was determined by 2 or 72 h pretreatment with 30 ng TNF-α/ml. Thereafter cells were washed and treated with 50, 100, 200, 400, 1,000, 1,250 and 1,500 ng doxorubicin/ml (Sigma) for 72 h at 37ºC. Then, MTT (5 mg/ml; Sigma) was added and absorbance was measured in triplicates at 560 nm in a microplate reader (Tecan Group Ltd., Männedorf, Switzerland). Values are expressed as percent of untreated controls. Cells treated 2 or 72 h with TNF-α only served as additional control.

Cell signalling reporter assay

To determine TNF-α mediated short- and long-term induction via NF-κB/p65, we used the Cignal NF-κB/p65 Renilla/firefly dual luciferase reporter assay kit (SABiosciences, Frederick, MD, USA). For each well of a 96-well plate 100 ng Cignal NF-κB reporter or Cignal negative control plasmid-DNA, respectively, were reverse transfected with FuGENE HD (Roche Diagnostics GmbH) into 1×104 cells and seeded in serum-free conditions, as recommended by the manufacturer. Then, 24 h after transfection medium was replaced with RPMI-1640 medium +10% FBS and treatment with 30 ng TNF-α/ml (Invitrogen) was started. For short-term TNF-α affected reporter expression cells were incubated 2, 4, 6, 8, 10 and 12 h, washed with PBS and lysed in passive lysis buffer (Promega). For evaluation of TNF-α affected reporter expression in long-term treatment, transfected cells were incubated for 8, 24, 48 and 72 h with 30 ng TNF-α/ml. In addition, time points of 8 h after the respective TNF-α treatment were included, indicated as 24, 48 and 72 h+. Cells were washed with PBS and lysed in passive lysis buffer. The luciferase assay was performed in triplicates with Dual-Luciferase Reporter Assay system (Promega) using 3 μl cell lysate in triplicates and using the Centro LB 960 luminometer (Berthold Technologies GmbH & Co., Bad Wildbad, Germany).

ABCB1 promoter driven luciferase reporter assay

To analyze the short- and long-term effects of TNF-α on the human ABCB1 promoter activity, the NF-κB/p65 consensus harboring 990 bp promoter sequence was PCR-cloned (24). This sequence spans the first exon and intron, and was used to drive the pGL3 plasmid luciferase reporter expression (Promega).

For the assay 150 ng ABCB1 promoter luciferase or promotorless pGL3 plasmid-DNA as negative control respectively were reverse transfected in 1×104 cells in each well of a 96-well plate with LTX-RG (Invitrogen) in serum-free conditions. Medium was replaced with RPMI-1640 medium and 10% FBS 24 h after transfection and TNF-α treatment was started. For determination of short-term TNF-α affected reporter expression cells were incubated with 30 ng TNF-α/ml (Biosource) for 2, 4, 6, 8, 10 and 12 h, washed with PBS and lysed passive lysis buffer (Promega). For evaluation of TNF-α-affected reporter expression in long-term treatment, transfected cells were incubated for 8, 24, 48 and 72 h with TNF-α, washed with PBS and lysed in passive lysis buffer. In addition, cells were harvested 8 h after the respective TNF-α application in the long-term treatment, indicated as 24, 48 and 72 h+. The luciferase activity was quantified by luciferase reporter assay in triplicates by the Centro LB 960 luminometer.

Statistical analysis

Analyses for statistical significance were performed with GraphPad Prism version 5 (GraphPad Software Inc., La Jolla, CA, USA). Comparison of several groups was done by one-way analysis of variance (ANOVA) and Bonferroni post hoc multiple comparison. Statistical significance was set at P-values <0.05.

Results

TNF-α mediated regulation of ABCB1 expression, drug uptake and cytotoxicity

The TNF-α mediated effects on ABCB1 expression were analyzed in HCT15 human colon carcinoma cells, which is an intrinsically high expressor of ABCB1. First, we evaluated the effects of human TNF-α on ABCB1 expression and ABCB1-mediated resistance. The treatment with 30 ng TNF-α/ml significantly reduced ABCB1 expression at mRNA level starting from 24–72 h (Fig. 1A). This long-term treatment caused 1.9-fold reduction in mRNA expression after 24 h, 3.8-fold reduction after 48 h and 6-fold reduction after 72 h, whereas short-term treatment (0.5–2 h) did not reduce ABCB1 expression. Similarly, western blot analysis revealed reduced PgP-expression at 24 h up to 96 h of TNF-α treatment (Fig. 1B). The short-term treatment (5 min to 2 h) however, did not alter PgP-expression. In a next step we determined possible functional effects of TNF-α mediated ABCB1 downregulation. For this, the uptake of the fluorescent drug doxorubicin as PgP substrate was determined (Fig. 1C). TNF-α treatment for 72 h leads to significant up to 3.8-fold increase in doxorubicin uptake, whereas 2 h TNF-α treatment caused only 1.4-fold increase in drug uptake. We next asked if the increased drug uptake after long-term TNF-α treatment has impact on doxorubicin cytotoxicity. Long-term treatment did increase cytotoxicity leading to 64% growth inhibition at highest dose of 1,500 ng/ml (Fig. 1E). By comparison, 2 h TNF-α treatment did not exert any effect on doxorubicin cytotoxicity even at highest drug doses (Fig. 1D). Taken together, we demonstrated in our model that long-term TNF-α treatment downregulated ABCB1 expression at mRNA and protein levels in colorectal cancer cells. This leads to high drug accumulation and sensitization towards doxorubicin, reflected by increased cytotoxicity.

TNFR signaling and PgP expression

Next we determined, whether TNFR1/p55 or TNFR2/p75 is essential in mediating the observed TNF-α effects on ABCB1 expression. Western blot analysis revealed, that both receptors are present in the colorectal cancer cells (Fig. 2A). For blocking the TNF-α binding to either TNFR1 or TNFR2 we pre-treated the cells with anti-TNFR1 or anti-TNFR2 antibody (Fig. 2B). As shown, addition of anti-TNFR1 antibody prevented PgP downregulation of the long-term 72 h TNF-α treatment, whereas addition of the anti-TNFR2 antibody could not prevent downregulation of PgP. In control experiments sole addition of the antibodies, however, did not alter PgP expression. This excluded any antibody mediated unspecific effect on PgP expression (Fig. 2C). Thus, binding of TNF-α to TNFR1 is essential for TNF-α mediated downregulation of ABCB1 expression after long-term TNF-α treatment.

Effects of short- and long-term TNF-α mediated NF-κB/p65 signaling on ABCB1

TNFR1 mediates signaling via the NF-κB pathway. Therefore, we determined the effect of short- and long-term TNF-α on NF-κB/p65 and on IκBα, IκBβ as regulators of NF-κB/p65 (Fig. 3A and B). The short-term (5–120 min) TNF-α mediated effects show strong cytoplasmatic reduction in IκBα levels after 10 min of TNF-α treatment, whereas, no detectable alteration for cytoplasmic NF-κB level was observed (Fig. 3A). As early as 10 min of TNF-α treatment the NF-κB/p65 protein starts to accumulate in the cell nuclei. This differs from what is observed for long-term (24–72 h) TNF-α mediated effects. Here IκBα levels remain at very low level and NF-κB shuttling to the nucleus is reduced. This is paralleled by reduction in PgP levels in the same experiment (Fig. 3B). Therefore, long-term TNF-α treatment reduces shuttling of NF-κB/p65 to the nucleus and prevents re-appearance of IκBα to levels of control cells in the cytoplasm. The analysis of IκBα mRNA expression revealed that this reduction in IκBα for long-term TNF-α treatment is the result of significant up to 3-fold reduced transcription (Fig. 3C). The cytoplasmic NF-κB level, however, seems to be unaffected by long-term TNF-α action. Even if analyzing the time point of 30 min after re-application of TNF-α (indicated as 24, 48 and 72 h+), no increase in nuclear accumulation of NF-κB/p65 is seen. For NF-κB/p65 shows only slight and insignificant reduction in mRNA expression by the long-term TNF-α treatment as seen in Fig. 3C.

Furthermore, we analyzed the fate of IκBβ in long-term TNF-α treatment where IκBβ protein persists in the cytoplasm, however, for 24–72 h TNF-α treatment at slightly lower level (Fig. 3C). The expression analysis also revealed an up to 2.5-fold reduction of IκBβ mRNA for the long-term TNF-α treatment, which correlates with the reduction at protein level (Fig. 3B and C).

NF-κB/p65 binding to its consensus sequence in the ABCB1 promoter after long-term TNF-α treatment

Since long-term TNF-α reduced nuclear NF-κB/p65 we determined if this is associated with reduced binding of NF-κB/p65 to its consensus sequence in the human ABCB1 promoter. In the EMSA we first analyzed, if binding to the NF-κB consensus sequence of the ABCB1 promoter is specific (Fig. 4A). Under non-stimulated conditions there is binding of the nuclear extract, which by addition of excess nonlabeled oligo disappers due to competition. The mutated oligo however shows no binding. After treatment with TNF-α for 30 min the mutated oligo shows binding of nuclear extract. However, addition of anti-NF-κB/p65 antibody does not lead to supershift. By contrast, use of the wild-type oligo shows binding of the nuclear extract, and supershift bands after addition of the anti-p65 antibody. This indicates, that the short-term TNF-α treatment induces NF-κB binding to the consensus sequence. Next we analyzed the impact of long-term TNF-α action on NF-κB binding to its consensus sequence within the human ABCB1 promoter by supershift experiment using the anti-p65 antibody. As positive control, the 30 min TNF-α stimulation again induced NF-κB binding, verified by appearace of a supershift band (Fig. 4B). This supershift disappeared when TNF-α was added for 24, 48 and 72 h, respectively. Interestingly, this is also seen for the samples, collected 30 min after each re-application of TNF-α, indicated as 24, 48 and 72 h+. This suggests that long-term TNF-α treatment desensitizes the colorectal cancer cells to TNF-α mediated NF-κB signaling and prevents NF-κB from binding to its consensus sequence within the ABCB1 promoter. This in turn leads to downregulation of ABCB1 expression.

Diverging effect of TNF-α mediated regulation on ABCB1 promoter activity

Since long-term TNF-α action prevents nuclear translocation and binding to consensus sequence of NF-κB, determination of functional consequences of this is important. First, we analyzed the effects of short- and long-term TNF-α treatment using the Cignal NF-κB/p65 Renilla/firefly dual luciferase reporter assay, which harbors tandem repeats of NF-κB transcriptional response element (Fig. 5). In this assay short-term application of TNF-α induces reporter expression peaking at 8 h with a 1.7-fold increase compared to the control (Fig. 5A). The long-term effects of TNF-α dramatically differ from this. Here, persistent TNF-α application leads to significant downregulation of reporter expression compared to the short-term TNF-α effects (Fig. 5B). This is reflected by the 3- to 5-fold decreases in promoter activity at 24–72 h of TNF-α application. Even at the time points of 8 h after each respective re-application of TNF-α (indicated as 24, 48 and 72 h+) the reporter system remains unresponsive towards TNF-α mediated induction. To further validate this observation, we used the NF-κB consensus sequence containing human ABCB1 promoter for luciferase expression. These assays revealed that short-term TNF-α application slightly reduced basal promoter activity in the colorectal cancer cells (Fig. 5C). The picture again drastically changes for long-term TNF-α treatment. The results show significant 2- to 9-fold decreases in ABCB1 promoter activity (Fig. 5D). At 8 h after respective TNF-α re-application (indicated as 24, 48 and 72 h+) this reporter system remains also unresponsive for TNF-α mediated induction and the reporter expression is even further reduced. Taken together, these analyses support that long-term TNF-α inhibits ABCB1 promoter activity. This results in reduced ABCB1 transcription and PgP expression (Fig. 1) leading to MDR reversal in the resistant cells.

Discussion

MDR still represents a leading obstacle for successful chemotherapy of cancer. Many different approaches are used to overcome MDR including the use of MDR reversing drugs or cytokines (1,5,34). One long known molecule, which mediates MDR in cancer is the ATP binding cassette ABCB1/PgP transporter. This membrane protein is responsible for the drug extrusion from cancer cells causing drug resistance. Therefore great efforts are aimed at downregulation of ABCB1 expression to sensitize tumor cells towards chemotherapy. One such approach is the use of pro-inflammatory cytokines, such as TNF-α or INF-γ for chemosensitization of resistant tumor cells (7,35). From numerous studies it is well accepted that particularly TNF-α is able to modulate the ABCB1 expression. In this regard several studies report, that treatment with TNF-α mediates ABCB1 downregulation, which in turn leads to improved drug accumulation in association with increased cytotoxicity. This has been found in different tumor models, including glioblastoma, colon, breast and hepatocellular cancer, and more recently also for endothelial cells in the blood-brain barrier (5,812). By contrast there are reports with the opposite observations, stating that treatment with TNF-α or other factors, which lead to activation of the NF-κB pathway rather act as inducers of ABCB1 expression (20,3638), thus, interference in NF-κB signaling by inhibitors has been shown to downregulate ABCB1 expression, which then sensitizes tumor cells towards chemotherapy (20).

Due to the complex and partially contradictory picture on TNF-α mediated effects we were interested to determine the interplay between short- and long-term TNF-α treatment, NF-κB signaling and the resulting modulation in this signaling, which leads to ABCB1 repression in colon cancer cells.

In this study we determined in more detail the impact of short-term and of long-term action of TNF-α on the expression regulation of ABCB1 in intrinsically resistant human HCT15 colon carcinoma cells. We have shown, that persistent treatment with TNF-α for 24–72 h leads to significant downregulation of ABCB1 in association with sensitization of these cells towards drug treatment. This is in line with previous publications, in which TNF-α leads to MDR reversal (5,9).

Apart from these studies, the question remained, what mechanism is responsible for the TNF-α mediated downregulation of ABCB1, which is mostly observed for persistent treatment. We therefore evaluated by which TNFR-signaling such modulation of ABCB1 expression is permitted. This analysis revealed, that TNFR1 mediated signaling is important for this process. Since TNF-α triggers NF-κB activation and nuclear translocation via TNFR1, we focused on this pathway under conditions of short- and of long-term TNF-α activation. The human ABCB1 promoter harbors NF-κB binding sites linking NF-κB signaling and ABCB1 expression regulation (24). Our study shows short-term TNF-α treatment triggers NF-κB activation. This is demonstrated by nuclear accumulation of NF-κB, accompanied by disappearance of IκBα from the cytoplasm, and binding of the transcription factor to its consensus sequence in the EMSA. By contrast, long-term TNF-α treatment does the opposite: NF-κB is not shuttled to the nucleus, even at time points shortly after TNF-α re-stimulation, although NF-κB is still present in the cytoplasm. This is associated with lack of IκBα re-appearance in the cytoplasm. This re-appearance would be expected after TNF-α treatment to rescue NF-κB from the nucleus and to reduce its DNA-binding ability leading to the known termination of NF-κB signaling (39). Interestingly, similar observation regarding lack of IκBα re-appearance was made in FS-4 fibroblasts treated for up to 15 h with TNF-α. In this study long-term TNF-α prevents re-appearance of IκBα due to persistent proteasome-mediated degradation of the protein (28,40). It is known that IκBα is not only important for the termination of NF-κB signaling but is also essential for re-initiation of the signaling after its resynthesis (27). The absence of IκBα leads to delayed and reduced cytokine-induced NF-κB activation (27). Here we show, that the persistent TNF-α action prevents re-appearance of IκBα due to reduced IκBα transcription, which is then unavailable for maintenance of ABCB1 expression. In addition, we did also observe significant reduction in IκBβ transcription and reduced protein level in the long-term TNF-α treated HCT15 cells. This reduction however, does not reach the extent observed for IκBα. It is suggested that the remaining IκBβ level might be sufficient for dumping the long-term oscillations in NF-κB signaling, as described by others (26). In this context almost unaltered presence and action of IκBβ might overrule the remaining small IκBα activity and hinder NF-κB activation, which finally results in ABCB1 downregulation.

As we observed, persistent TNF-α treatment not only reduces nuclear translocation of NF-κB, but also DNA binding to its consensus sequence in the ABCB1 promoter, explaining why ABCB1 transcription is reduced. This is further supported by the reporter assays. They clearly show, that long-term TNF-α treatment has inhibitory effects, whereas for short-term TNF-α treatment induction in reporter gene expression was seen. Interestingly, even re-application of TNF-α in the long-term treatment was unable to restimulate reporter gene expression, from either the Cignal NF-κB/p65 Renilla/firefly dual luciferase reporter system or the authentic ABCB1 promoter construct.

In conclusion, our study provides new perspectives on the mechanism how long-term treatment of TNF-α is reducing NF-κB signaling, which results in the downregulation of ABCB1. This strongly supports the MDR reversing potential of the pro-inflammatory cytokine, which in turn mediates effective chemosensitization of colon cancer cells. Furthermore, this mechanism might also apply to the phenomena observed in situations of chronic inflammation of the gut associated with persistent presence of TNF-α, such as ulcerative colitis. These conditions are reported to be associated with reduced expression of ABC transporters, including ABCB1 (29,30). Interestingly, apart from colon cancer such observation has recently also been made in an inflammation model for microglia parenchymal cells, where ABCB1 expression is reduced pointing to a potential more general property of TNF-α on regulation of ABCB1 (41).

Acknowledgements

We thank Liselotte Malcherek for excellent technical assistance.

Abbreviations:

ABCB1

ATP-binding cassette, subfamily B 1 gene

EMSA

electromobility shift assay

MDR

multidrug resistance

NF-κB

nuclear factor κ light chain enhancer

IκBα/IκBβ

nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α/β

PgP

P-glycoprotein

TNFR

TNF-receptor

TNF-α

tumor necrosis factor α

References

1 

Binkhathlan Z and Lavasanifar A: P-glycoprotein inhibition as a therapeutic approach for overcoming multidrug resistance in cancer: Current status and future perspectives. Curr Cancer Drug Targets. 13:326–346. 2013. View Article : Google Scholar : PubMed/NCBI

2 

Breier A, Gibalova L, Seres M, Barancik M and Sulova Z: New insight into P-glycoprotein as a drug target. Anticancer Agents Med Chem. 13:159–170. 2013. View Article : Google Scholar

3 

Nooter K and Stoter G: Molecular mechanisms of multidrug resistance in cancer chemotherapy. Pathol Res Pract. 192:768–780. 1996. View Article : Google Scholar : PubMed/NCBI

4 

Teodori E, Dei S, Martelli C, Scapecchi S and Gualtieri F: The functions and structure of ABC transporters: Implications for the design of new inhibitors of Pgp and MRP1 to control multidrug resistance (MDR). Curr Drug Targets. 7:893–909. 2006. View Article : Google Scholar : PubMed/NCBI

5 

Stein U and Walther W: Cytokine-mediated reversal of multidrug resistance. Cytotechnology. 27:271–282. 1998. View Article : Google Scholar

6 

Walther W and Stein U: Influence of cytokines on mdr1 expression in human colon carcinoma cell lines: Increased cytotoxicity of MDR relevant drugs. J Cancer Res Clin Oncol. 120:471–478. 1994. View Article : Google Scholar : PubMed/NCBI

7 

Stein U, Walther W and Shoemaker RH: Modulation of mdr1 expression by cytokines in human colon carcinoma cells: An approach for reversal of multidrug resistance. Br J Cancer. 74:1384–1391. 1996. View Article : Google Scholar : PubMed/NCBI

8 

Stein U, Walther W and Shoemaker RH: Reversal of multidrug resistance by transduction of cytokine genes into human colon carcinoma cells. J Natl Cancer Inst. 88:1383–1392. 1996. View Article : Google Scholar : PubMed/NCBI

9 

Ding L, Chen XP, Zhang ZW, Guan J, Zhang WG, Wang HP, Wang ZH and Li CL: Synergistic effect of bromocriptine and tumor necrosis factor-alpha on reversing hepatocellular carcinoma multidrug resistance in nude mouse MDR1 model of liver neoplasm. World J Gastroenterol. 11:5621–5626. 2005. View Article : Google Scholar : PubMed/NCBI

10 

Lee NY and Kang YS: The decrease of paclitaxel efflux by pretreatment of interferon-γ and tumor necrosis factor-α after intracerebral microinjection. Brain Res. 1499:158–162. 2013. View Article : Google Scholar : PubMed/NCBI

11 

Lee G and Piquette-Miller M: Cytokines alter the expression and activity of the multidrug resistance transporters in human hepatoma cell lines; analysis using RT-PCR and cDNA micro-arrays. J Pharm Sci. 92:2152–2163. 2003. View Article : Google Scholar : PubMed/NCBI

12 

Iqbal M, Ho HL, Petropoulos S, Moisiadis VG, Gibb W and Matthews SG: Pro-inflammatory cytokine regulation of P-glycoprotein in the developing blood-brain barrier. PLoS One. 7:e430222012. View Article : Google Scholar : PubMed/NCBI

13 

Belliard AM, Lacour B, Farinotti R and Leroy C: Effect of tumor necrosis factor-alpha and interferon-gamma on intestinal P-glycoprotein expression, activity, and localization in Caco-2 cells. J Pharm Sci. 93:1524–1536. 2004. View Article : Google Scholar : PubMed/NCBI

14 

Kreuser ED, Wadler S and Thiel E: Biochemical modulation of cytotoxic drugs by cytokines: Molecular mechanisms in experimental oncology. Recent Results Cancer Res. 139:371–382. 1995. View Article : Google Scholar : PubMed/NCBI

15 

Vacchelli E, Galluzzi L, Eggermont A, Galon J, Tartour E, Zitvogel L and Kroemer G: Trial Watch: Immunostimulatory cytokines. OncoImmunology. 1:493–506. 2012. View Article : Google Scholar : PubMed/NCBI

16 

Lejeune FJ and Rüegg C: Recombinant human tumor necrosis factor: An efficient agent for cancer treatment. Bull Cancer. 93:E90–E100. 2006.PubMed/NCBI

17 

Baud V and Karin M: Signal transduction by tumor necrosis factor and its relatives. Trends Cell Biol. 11:372–377. 2001. View Article : Google Scholar : PubMed/NCBI

18 

Karin M: How NF-kappaB is activated: The role of the IkappaB kinase (IKK) complex. Oncogene. 18:6867–6874. 1999. View Article : Google Scholar : PubMed/NCBI

19 

Huang TT, Kudo N, Yoshida M and Miyamoto S: A nuclear export signal in the N-terminal regulatory domain of IkappaBalpha controls cytoplasmic localization of inactive NF-kappaB/IkappaBalpha complexes. Proc Natl Acad Sci USA. 97:1014–1019. 2000. View Article : Google Scholar : PubMed/NCBI

20 

Bentires-Alj M, Barbu V, Fillet M, Chariot A, Relic B, Jacobs N, Gielen J, Merville MP and Bours V: NF-kappaB transcription factor induces drug resistance through MDR1 expression in cancer cells. Oncogene. 22:90–97. 2003. View Article : Google Scholar : PubMed/NCBI

21 

Takada Y, Kobayashi Y and Aggarwal BB: Evodiamine abolishes constitutive and inducible NF-kappaB activation by inhibiting IkappaBalpha kinase activation, thereby suppressing NF-kappaB-regulated antiapoptotic and metastatic gene expression, up-regulating apoptosis, and inhibiting invasion. J Biol Chem. 280:17203–17212. 2005. View Article : Google Scholar : PubMed/NCBI

22 

Wang W, McLeod HL and Cassidy J: Disulfiram-mediated inhibition of NF-kappaB activity enhances cytotoxicity of 5-fluorouracil in human colorectal cancer cell lines. Int J Cancer. 104:504–511. 2003. View Article : Google Scholar : PubMed/NCBI

23 

Nakanishi C and Toi M: Nuclear factor-kappaB inhibitors as sensitizers to anticancer drugs. Nat Rev Cancer. 5:297–309. 2005. View Article : Google Scholar : PubMed/NCBI

24 

Ogretmen B and Safa AR: Negative regulation of MDR1 promoter activity in MCF-7, but not in multidrug resistant MCF-7/Adr, cells by cross-coupled NF-κ B/p65 and c-Fos transcription factors and their interaction with the CAAT region. Biochemistry. 38:2189–2199. 1999. View Article : Google Scholar : PubMed/NCBI

25 

Thompson JE, Phillips RJ, Erdjument-Bromage H, Tempst P and Ghosh S: I kappa B-beta regulates the persistent response in a biphasic activation of NF-kappa B. Cell. 80:573–582. 1995. View Article : Google Scholar : PubMed/NCBI

26 

Hoffmann A, Levchenko A, Scott ML and Baltimore D: The IkappaB-NF-kappaB signaling module: Temporal control and selective gene activation. Science. 298:1241–1245. 2002. View Article : Google Scholar : PubMed/NCBI

27 

Schmidt C, Peng B, Li Z, Sclabas GM, Fujioka S, Niu J, Schmidt-Supprian M, Evans DB, Abbruzzese JL and Chiao PJ: Mechanisms of proinflammatory cytokine-induced biphasic NF-kappaB activation. Mol Cell. 12:1287–1300. 2003. View Article : Google Scholar : PubMed/NCBI

28 

Ladner KJ, Caligiuri MA and Guttridge DC: Tumor necrosis factor-regulated biphasic activation of NF-kappa B is required for cytokine-induced loss of skeletal muscle gene products. J Biol Chem. 278:2294–2303. 2003. View Article : Google Scholar

29 

Gibson PR: Increased gut permeability in Crohn's disease: Is TNF the link? Gut. 53:1724–1725. 2004. View Article : Google Scholar : PubMed/NCBI

30 

Englund G, Jacobson A, Rorsman F, Artursson P, Kindmark A and Rönnblom A: Efflux transporters in ulcerative colitis: Decreased expression of BCRP (ABCG2) and Pgp (ABCB1). Inflamm Bowel Dis. 13:291–297. 2007. View Article : Google Scholar : PubMed/NCBI

31 

Petrovic V, Teng S and Piquette-Miller M: Regulation of drug transporters during infection and inflammation. Mol Interv. 7:99–111. 2007. View Article : Google Scholar : PubMed/NCBI

32 

Blokzijl H, Vander Borght S, Bok LI, Libbrecht L, Geuken M, van den Heuvel FA, Dijkstra G, Roskams TA, Moshage H, Jansen PL, et al: Decreased P-glycoprotein (P-gp/MDR1) expression in inflamed human intestinal epithelium is independent of PXR protein levels. Inflamm Bowel Dis. 13:710–720. 2007. View Article : Google Scholar : PubMed/NCBI

33 

Wu L, Smythe AM, Stinson SF, Mullendore LA, Monks A, Scudiero DA, Paull KD, Koutsoukos AD, Rubinstein LV, Boyd MR, et al: Multidrug-resistant phenotype of disease-oriented panels of human tumor cell lines used for anticancer drug screening. Cancer Res. 52:3029–3034. 1992.PubMed/NCBI

34 

Wu CP, Calcagno AM and Ambudkar SV: Reversal of ABC drug transporter-mediated multidrug resistance in cancer cells: Evaluation of current strategies. Curr Mol Pharmacol. 1:93–105. 2008. View Article : Google Scholar : PubMed/NCBI

35 

Ho EA and Piquette-Miller M: Regulation of multidrug resistance by pro-inflammatory cytokines. Curr Cancer Drug Targets. 6:295–311. 2006. View Article : Google Scholar : PubMed/NCBI

36 

Ros JE, Schuetz JD, Geuken M, Streetz K, Moshage H, Kuipers F, Manns MP, Jansen PL, Trautwein C and Müller M: Induction of Mdr1b expression by tumor necrosis factor-alpha in rat liver cells is independent of p53 but requires NF-kappaB signaling. Hepatology. 33:1425–1431. 2001. View Article : Google Scholar : PubMed/NCBI

37 

Um JH, Kang CD, Lee BG, Kim DW, Chung BS and Kim SH: Increased and correlated nuclear factor-kappa B and Ku auto-antigen activities are associated with development of multidrug resistance. Oncogene. 20:6048–6056. 2001. View Article : Google Scholar : PubMed/NCBI

38 

Kuo MT, Liu Z, Wei Y, Lin-Lee YC, Tatebe S, Mills GB and Unate H: Induction of human MDR1 gene expression by 2-acetyl aminofluorene is mediated by effectors of the phosphoinositide 3-kinase pathway that activate NF-kappaB signaling. Oncogene. 21:1945–1954. 2002. View Article : Google Scholar : PubMed/NCBI

39 

Arenzana-Seisdedos F, Thompson J, Rodriguez MS, Bachelerie F, Thomas D and Hay RT: Inducible nuclear expression of newly synthesized I kappa B alpha negatively regulates DNA-binding and transcriptional activities of NF-kappa B. Mol Cell Biol. 15:2689–2696. 1995. View Article : Google Scholar : PubMed/NCBI

40 

Poppers DM, Schwenger P and Vilcek J: Persistent tumor necrosis factor signaling in normal human fibroblasts prevents the complete resynthesis of I kappa B-alpha. J Biol Chem. 275:29587–29593. 2000. View Article : Google Scholar : PubMed/NCBI

41 

Gibson CJ, Hossain MM, Richardson JR and Aleksunes LM: Inflammatory regulation of ATP binding cassette efflux transporter expression and function in microglia. J Pharmacol Exp Ther. 343:650–660. 2012. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

December-2015
Volume 47 Issue 6

Print ISSN: 1019-6439
Online ISSN:1791-2423

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Walther W, Kobelt D, Bauer L, Aumann J and Stein U: Chemosensitization by diverging modulation by short-term and long-term TNF-α action on ABCB1 expression and NF-κB signaling in colon cancer. Int J Oncol 47: 2276-2285, 2015
APA
Walther, W., Kobelt, D., Bauer, L., Aumann, J., & Stein, U. (2015). Chemosensitization by diverging modulation by short-term and long-term TNF-α action on ABCB1 expression and NF-κB signaling in colon cancer. International Journal of Oncology, 47, 2276-2285. https://doi.org/10.3892/ijo.2015.3189
MLA
Walther, W., Kobelt, D., Bauer, L., Aumann, J., Stein, U."Chemosensitization by diverging modulation by short-term and long-term TNF-α action on ABCB1 expression and NF-κB signaling in colon cancer". International Journal of Oncology 47.6 (2015): 2276-2285.
Chicago
Walther, W., Kobelt, D., Bauer, L., Aumann, J., Stein, U."Chemosensitization by diverging modulation by short-term and long-term TNF-α action on ABCB1 expression and NF-κB signaling in colon cancer". International Journal of Oncology 47, no. 6 (2015): 2276-2285. https://doi.org/10.3892/ijo.2015.3189