Docetaxel, a member of the taxane family, has been shown to induce apoptosis in a variety of cancer cells. However, toxicity at therapeutic doses has precluded the use of docetaxel alone for the management of cancer patients. PSK, a protein-bound polysaccharide, is widely used in Japan as an immunopotentiating biological response modifier for cancer patients. Our previous study showed that PSK induced downregulation of several invasion-related factors, suggesting an interaction of PSK with transcriptional factors. In this study, we showed that PSK dose dependently enhanced apoptosis induced by 1 nM of docetaxel in a human pancreatic cancer cell line NOR-P1. Furthermore, PSK inhibited docetaxel-induced nuclear factor kappa B (NF-κB) activation. Moreover, the expression of cellular inhibitor of apoptosis protein (cIAP-1), which is transcriptionally regulated by NF-κB and functions as an antiapoptotic molecule through interrupting the caspase pathway, was also inhibited by treatment with PSK plus docetaxel. As a result, PSK enhanced the docetaxel-induced caspase-3 activation. In addition, treatment by transfection of NF-κB decoy oligodeoxynucleotides (ODNs), but not scramble ones, inhibited the expression of cIAP-1 in NOR-P1 cells and induced a significant increase in docetaxel-induced apoptosis. Our data indicate that PSK suppresses the docetaxel-induced NF-κB activation pathway. Combination of PSK with a low dose of docetaxel may be a new therapeutic strategy to treat patients with pancreatic cancer.
Docetaxel, a chemotherapeutic agent belonging to the taxane family, has been shown to possess significant cell-killing activity in a variety of tumor cells through induction of apoptosis (Haldar et al., 1997; Kolfschoten et al., 2002). As an agent of second-line chemotherapy, docetaxel is one of the most important new and active chemotherapeutic agents developed in recent years, and has potential activity against human solid tumors including pancreatic cancer that are refractory to conventional anticancer agents (Bissery et al., 1995; Kaye, 1995). Recently, several phase II trials of advanced pancreatic cancer have reported high response rates (20%) and relatively long survival after treatment with docetaxel administered at 100 mg/m2 over a 1-h period (Rougier et al., 2000; Lenzi et al., 2002). However, the use of high-dose docetaxel always induced different levels of toxic reactions, and significant toxicity has precluded the use of docetaxel as a monotherapy for cancer (Cortes and Pazdur, 1995). Since low or moderate doses of docetaxel have no significant antitumor activity in patients with pancreatic cancer (Okada et al., 1999), ways to reduce the dose of docetaxel without affecting its antitumor activity should be examined.
PSK, a protein-bound polysaccharide, has been used as a nonspecific immunostimulant for treating cancer patients in Japan for more than 20 years. The antitumor activity for PSK has been documented in experimental animal models and beneficial therapeutic effects have been shown in clinical studies of several types of tumor (Tsukagoshi et al., 1984; Fukushima, 1996; Fisher and Yang, 2002). Despite these observations, the effect of PSK on the modulation of chemotherapy-induced apoptosis has not been investigated.
The nuclear factor kappa B (NF-κB) is a transcription factor involved in regulating a variety of cellular behavior. Constitutive NF-κB activity is required for cellular proliferation and protection of tumor cells from apoptosis (Bargou et al., 1997; Wang et al., 1999; Arlt et al., 2001). Although the molecular mechanism involved in this antiapoptotic function is not fully understood, recent studies suggest that cellular inhibitor of apoptosis protein (cIAP)-1 and cIAP-2 transcriptionally regulated by NF-κB function cooperatively to suppress apoptosis through inhibition of caspase-3 activity (Aota et al., 2000). Therefore, downregulation of NF-κB activity would be a therapeutic target for inducing apoptosis in cancer cells. We have recently shown that PSK downregulates plasminogen activator (uPA) and matrix metalloproteinases-9 (MMP-9) in a human pancreatic cancer cell line (NOR-P1) (Zhang et al., 2000), implying that PSK may have some action on NF-κB activity.
In the present study, we examined the effects of combined PSK and docetaxel treatment on apoptosis in NOR-P1 cells. Our results indicated that PSK rendered tumor cells more susceptible to cytotoxicity induced by a low dose of docetaxel, resulting in an efficient execution of apoptosis. Furthermore, the enhanced apoptosis was because of the action of PSK in suppressing NF-κB activation, and was followed by inactivation of cIAP-1 and augmentation of caspase-3 activity. Thus, PSK is potentially useful when used in combination with cancer chemotherapy such as taxane-including regimens to improve outcome.
PSK enhanced docetaxel-induced growth inhibition in NOR-P1 cells
First, the sensitivity of NOR-P1 cells to docetaxel was determined after 48 h incubation at concentrations from 100 pM to 1 μ M. Growth of NOR-P1 cells was inhibited by docetaxel in a dose-dependent manner (Figure 1a). The concentration corresponding to 10% growth inhibition (1 nM) was used in the subsequent combined treatment experiments.
Second, to investigate whether PSK alters the susceptibility of cells to docetaxel, NOR-P1 cells were cultured in docetaxel (1 nM) with or without various concentrations of PSK. PSK dose dependently enhanced the cytotoxic effect of docetaxel in NOR-P1 cells (Figure 1b), although PSK alone had no significant effect on cell proliferation (data not shown). Therefore, these results indicate that PSK enhances the growth inhibitory effect of docetaxel in NOR-P1 cells.
PSK enhanced docetaxel-induced apoptosis in NOR-P1 cells
Docetaxel has been known to induce apoptosis in many tumor cell types (Haldar et al., 1997; Rowinsky, 1997). To determine whether PSK-enhanced growth inhibition in NOR-P1 cells is a result of induced apoptosis, electrophoretic analysis of DNA fragments was performed. An increase in DNA laddering was observed after docetaxel treatment (Figure 2a), indicating that apoptosis was induced by docetaxel in NOR-P1 cells. However, PSK alone did not produce this apoptotic effect. Strikingly, DNA laddering was significantly increased following treatment with docetaxel plus PSK compared to docetaxel alone. This DNA ladder result was further confirmed by FACS analysis. As shown in Figure 2b, the proportion of pre-G1 cells, presumably apoptotic cells, were found to be 3.26, 3.88, and 14.08% in control, PSK, and docetaxel treatment, respectively. Cells treated with docetaxel plus PSK reached an apoptotic population (pre-G1) of 33.46%.
For further quantification of apoptotic cells induced by docetaxel, PSK or both, Hoechst 33342 staining was performed. Observation of the stained cells after treatment revealed that cells showing small, condensed, and fragmented nuclei (indicative of apoptosis) were present in wells treated with docetaxel and docetaxel plus PSK, but not PSK alone (Figure 2c). The percentage of apoptotic cells was calculated in NOR-P1 cells treated with docetaxel and docetaxel plus PSK. Combined treatment with docetaxel and PSK substantially increased apoptosis compared to docetaxel alone (Figure 2d), showing 68.4, 113.5, and 178.3% induction of apoptotic cells in response to 50, 100, and 500 μg/ml, respectively, of PSK. These data show that PSK increases the sensitivity of NOR-P1 cells to apoptosis induced by docetaxel.
PSK inhibited docetaxel-induced NF-κB activation in NOR-P1 cells
Since NF-κB plays an essential role in the upregulation of antiapoptotic activity in various cell types (Bargou et al., 1997; Wang et al., 1999; Arlt et al., 2001), we investigated the involvement of NF-κB activity in the PSK-mediated increase in susceptibility to apoptosis induced by docetaxel in NOR-P1 cells. In electrophoretic mobility shift assay (EMSA), nuclear protein extracts were incubated with a 32P-labeled oligonucleotide containing a conserved NF-κB binding sequence. As shown in Figure 3a, NOR-P1 cells constitutivelly expressed NF-κB activity, and docetaxel alone enhanced NF-κB nuclear translocation. Furthermore, the docetaxel-induced NF-κB activation was inhibited by adding PSK to the treatment. However, this inhibition was not observed when the cells were exposed to PSK alone. To confirm the specificity of binding, an excess of unlabeled NF-κB binding oligonucleotide abolished binding of NF-κB (Figure 3a: 10-fold in lane 5 and 100-fold in lane 6), while oligonucleotide containing AP-1 binding sequence (Figure 3a: lane 7) did not change NF-κB binding activity, demonstrating that binding of the NF-κB probe was specific. Densitometric analysis of autoradiographic bands showed that combined treatment with docetaxel and 100 μg of PSK decreased docetaxel-induced NF-κB activation by 43% (Figure 3b). Thus, PSK probably interferes with the signaling cascade that leads to inhibition of NF-κB activation.
NF-kB decoy augmented docetaxel-induced apoptosis in NOR-P1 cells
To determine whether the activation of NF-κB by docetaxel is directly associated with docetaxel-induced apoptosis in NOR-P1 cells, we examined the effect of NF-κB decoy ODN transfection on the apoptotic response to docetaxel. After introduction of NF-κB decoy ODN into cells by transient transfection, we stimulated cells by docetaxel for 24 h, and then stained the nuclei of cells using Hoechst 33342 method for analysis of apoptotic cells. As shown in Figure 4, the transfection of NF-κB decoy ODN increased the percentage of apoptotic cells on docetaxel-treated NOR-P1 cells, but scrambled ODN did not have any effect on docetaxel-induced apoptosis of NOR-P1 cells. This result may suggest that NF-κB activation regulates negatively docetaxel-induced apoptosis in NOR-P1 cells.
PSK decreased docetaxel-induced expression of cIAP-1 and increased docetaxel- induced caspase-3 activation in NOR-P1 cells
NF-κB positively regulates the expression of anti-apoptotic molecules such as cIAP-1 and cIAP-2, and consequently blocks the apoptotic signals directly at the step of caspase activation (Aota et al., 2000). We therefore examined whether PSK-mediated NF-κB inhibition affects cIAP-1, cIAP-2, and caspase-3. Activation of caspase-3 was determined by a decrease in the expression of procaspase-3 and an increase in the expression of cleaved caspase-3 fragments. As shown in Figure 5a, caspase-3 activation and slight cIAP-1 induction were observed after exposure to docetaxel alone, whereas cIAP-2 was not changed. When cells were treated with docetaxel plus PSK, a significant reduction in cIAP-1 expression and further casepase-3 activation were observed, although PSK alone had no effect on these activities. However, no remarkable change in cIAP-2 expression was observed in the treated cells. Furthermore, the mRNA expression of cIAP-1 was also examined by RT–PCR, and this RT–PCR pattern was similar to the result of Western blot (Figure 5b), indicating that the expression of cIAP-1 was also transcriptionally regulated by docetaxel plus PSK treatment.
NF-κB decoy inhibited the expression of cIAP-1 in NOR-P1 cells
To further elucidate that there is a link between NF-κB and cIAP-1 in the treated NOR-P1 cells, we evaluated the expression of cIAP-1 in NF-κB decoy and scrambled ODN-treated cells at 48 h after transfection. NOR-P1 cells treated with NF-κB decoy ODN showed a significantly reduced cIAP-1 expression at both mRNA and protein levels compared with control and scrambled ODN-treated cells (Figure 6). However, no remarkable change in cIAP-2 and caspase-3 expression was observed in the treated cells (data not shown). These results indicated that the expression of cIAP-1 could be regulated by NF-κB in NOR-P1 cells, and the reduction in cIAP-1 by PSK plus treatment is likely because of the inhibition of NF-κB.
In the present study, combined treatment with PSK resulted in a significant enhancement of apoptosis induced by a low dose of docetaxel in a human pancreatic cancer cell line NOR-P1. Furthermore, we speculate a signal transduction mechanism responsible for PSK-enhanced apoptosis as follows: PSK inhibits the activation of NF-κB, then downregulates the antiapoptotic molecules cIAP-1 and leads to the activation of caspase-3, resulting in apoptosis of cancer cells. Our findings provide a possible mechanism for the synergistic induction of apoptosis by a low dose of docetaxel in combination with PSK.
Recently, docetaxel is commonly used to treat patients with pancreatic cancer. However, the use of high-dose docetaxel in these patients always induced various levels of toxic reaction (Cortes and Pazdur, 1995). Several investigators have studied the efficacy of docetaxel combined with other agents such as gemcitabine in the treatment of patients with pancreatic cancer, but the addition of these agents to docetaxel did not appear to be useful in patients with advanced pancreatic cancer since single agent produced similar results (Cascinu et al., 1999; Stathopoulos et al., 2000). Our results indicate that combined treatment with PSK may allow dose reduction of docetaxel without affecting its antitumor activity in NOR-P1 cells, which may circumvent the problem of toxic reactions induced by high doses of docetaxel. Thus, PSK is potentially useful when combined with docetaxel in chemotherapy for clinical treatment of pancreatic cancer.
PSK is a biological response modifier used in adjuvant immunotherapy of malignant diseases. Combination therapies with PSK and antitumor agents such as mitomycin C, 5-fluorouracil, cyclophosphamide, bleomycin, CPT-11, and cisplatin have been reported: in these reports, advantageous biological effects of PSK have been documented in terms of preventing damage to the host defense mechanism and allowing more efficient antitumor treatment (Mori et al., 1987; Iwaguchi et al., 1989; Anai et al., 1991; Ebina and Murata, 1992; Iino et al., 1992; Takahashi and Mai, 2001; Shibata et al., 2002). Our studies showed another advantage of PSK when combined with chemotherapeutic agents, which is augmentation of apoptosis induced by a low dose of docetaxel.
In an attempt to elucidate the mechanism of the effect of PSK on docetaxel-induced apoptosis, we used EMSA to investigate the activity of NF-κB, which is a key mediator in the inhibition of an apoptosis response. We focused on this antiapoptosis factor because we have previously shown that PSK inhibits uPA and MMP-9 expression, which have been identified as targets of NF-κB transcriptional activity in this cell line (Zhang et al., 2000). In the present study, we showed that docetaxel alone activated NF-κB binding activity and this activation was dose dependent (data not shown). Recently, two different effects of paclitaxel, another chemotherapeutic agent of the taxane family, on NF-κB activity have been reported. Spencer et al. reported that paclitaxel selectively inhibited phorbol ester-induced NF-κB activation via the inhibition of IκB-α phosphorylation and subsequent degradation (Spencer et al., 1999), whereas Hung et al. showed that paclitaxel induced NF-κB activation by upregulating IκB kinase (Huang et al., 2000). Therefore, in the situation of treatment by taxane agents, the role of NF-κB in the regulation of cell death has not yet been established. Our results first showed that docetaxel induced NF-κB activation in tumor cell lines, indicating that docetaxel-induced apoptosis was not caused primarily by the inhibition of NF-κB activation. More importantly, when tumor cells were treated with docetaxel plus PSK, the docetaxel-induced NF-κB activation was suppressed although PSK alone had no effect on NF-κB activity, implying that PSK selectively inhibited the docetaxel-induced NF-κB activity rather than the endogenous cellular NF-κB activity. These changes may explain the increased susceptibility of cells to undergo apoptosis. Furthermore, NF-κB decoy ODN treatment confirmed the role of NF-κB in docetaxel-induced apoptosis, the NF-κB activation that regulates negatively docetaxel-induced apoptosis in NOR-P1 cells. This result further supports the conclusion that PSK-mediated inhibition of NF-κB activation promotes docetaxel-induced apoptosis.
NF-κB is generally presumed to control the expression of the antiapoptotic protein cIAP-1 and cIAP-2 (Chu et al., 1997; Wang et al., 1998), and that these antiapoptotic molecules block the proapoptotic signals at the step where external stimuli activate caspase (Roy et al., 1997; Wright et al., 2000). Thus, we examined the expression of these two proteins after treatment with docetaxel plus PSK, and found that cIAP-1 was inhibited by the combination of docetaxel and PSK compared to docetaxel alone, but cIAP-2 was not affected by PSK combined treatment. These results indicated that NF-κB regulated the expression of cIAP-1 but not cIAP-2 in NOR-P1 cells. The relation between NF-κB and cIAP-1 in NOR-P1 cells was further determined by NF-κB decoy ODN treatment, which showed that NF-κB decoy ODN treatment inhibited the expression of cIAP-1. This result indicated that PSK-mediated cIAP-1 reduction might result from PSK-mediated NF-κB inhibition. Finally, we estimated the activity of caspase-3, which is the terminal downstream caspase of the apoptotic pathway and functions as an executioner in apoptosis. Docetaxel alone activated caspase-3, indicating that caspase-3 actually participates in docetaxel-induced apoptosis in NOR-P1 cells. Furthermore, the combination of PSK with docetaxel accelerated the activation of caspase-3, although PSK alone failed to activate it. These data suggest that NF-κB-mediated cIAP-1 inhibition by PSK further activates docetaxel-induced caspase activation.
In summary, we have demonstrated that PSK may augment apoptosis induced by low-dose docetaxel in NOR-P1 cells through inhibition of the NF-κB signaling pathway. Exposure of tumor cells to docetaxel and PSK leads to inhibition of the NF-κB activation induced by docetaxel, then causes the decreased cIAP-1 expression, and leads to the activation of caspase-3. Our data suggest that PSK may have a potential use in the chemotherapy of pancreatic cancer.
Materials and methods
Cell line and reagents
Human pancreatic cancer cell line NOR-P1 (Sato et al., 2000) was maintained in RPMI-1640 medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal calf serum (Gibco BRL) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) at 37°C. Docetaxel was purchased from Rhone-Poulenc Rorer (Antony, France) and PSK was from Kureha Chemical Industry (Tokyo, Japan).
Cell proliferation assay
The sensitivity of NOR-P1 to docetaxel, PSK, or both was determined by the WST-1 proliferation assay kit (Takara, Kyoto, Japan) as previously described (Ishiyama et al., 1996). Cells were seeded in 96-well culture plates at a density of 1 × 104 cells/well, and incubated at 37°C for 24 h. Then the cells were further incubated with docetaxel, PSK, or both at the indicated concentration for 48 h. The cells were then incubated with the WST-1 reagents, and absorbance of formazan products at 450 nm was measured with a CS-9300 microplate reader (Shimadzu, Tokyo, Japan).
Detection of apoptosis
For analysis of DNA laddering characteristic of apoptotic cell death, DNA was isolated from cells treated with docetaxel, PSK, or both after 48 h. Cells were resuspended in a lysis buffer containing 10 mM EDTA, 10 mM Tris (pH 8.0), and 0.5% Triton X-100 at 4°C for 10 min and centrifuged at 16 000 r.p.m. for 20 min. The supernatant was treated with RNase A at 37°C for 1 h and proteinase K at 50°C for 30 min, then precipitated with isopropanol. The DNA was resuspended, and electrophoresed in a 2% agarose gel at 50 V for 3 h. DNA was visualized by ethidium bromide staining and photographed under UV light. FACS analysis of propidium iodide (PI)-stained nuclei was performed as previously described (Nicoletti et al., 1991). Briefly, cells were plated at a density of 2 × 105 cells/well in six-well dishes, and incubated at 37°C for 24 h. Then the cells were further incubated with docetaxel, PSK, or both for 24 h. The cells were then harvested by trypsinization, collected by centrifugation, and suspended in hypotonic lysis buffer (0.1% Triton X-100 in PBS), 0.1 mg/ml RNase, and 40 μg/ml PI. After 30 min at 37°C, the cells were analysed with a FACSCalibur cytofluoremeter (Becton Dickinson, San Jose, CA, USA), and the percentage of hypodiploid cells was determined. Morphologic changes in the nuclear chromatin of cells undergoing apoptosis were detected by staining with Hoechst 33342 (Wako, Osaka, Japan). Briefly, cells were plated in 96-well plates (2 × 105 cells/well); treated with docetaxel, PSK, or both for 24 h; stained with Hoechst 33342, and then observed under fluorescence microscopy. A total of 1000 cells were observed in five randomly chosen fields. Cells with condensed or fragmented nuclei were considered to be apoptotic. The data are expressed as the percentage of apoptotic cells.
NF-κB activity in nuclei isolated from NOR-P1 cells was determined by EMSA. Extraction of nuclear proteins and EMSA were performed as described previously (Kojima et al., 2000). Briefly, 5 μg of nuclear protein was incubated for 30 min at room temperature with a binding buffer (20 mM HEPES-NaOH, pH 7.9, 2 mM EDTA, 100 mM NaCl, 10% glycerol, 0.2% NP-40), poly (dI-dC), and 32P-labeled double-stranded oligonucleotide containing the NF-κB binding motif (Promega Corp., Madison, WI, USA). The sequence of the double-stranded oligomer used for EMSA was as follows: 5′-IndexTermAGTTGAGGGGACTTTCCCAGGC-3′ (only sense strand is shown). The reaction mixtures were loaded on a 4% polyacrylamide gel and electrophoresed with a running buffer of 0.25 × TBE. After the gel was dried, the DNA–protein complexes were visualized by autoradiography. We used activation protein-1 (AP-1) probe (Promega Corp.) in competitive studies. Sequence of AP-1 probe was as follows: 5′-IndexTermCGCTTGATGAGTCAGCCGGAA-3′.
Protein expression of cIAP-1, cIAP-2, and caspase-3 was investigated by Western blotting. Cells (2 × 106) cultured as described above were collected and lysed with an extraction buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 0.1% SDS, 5 mM EDTA (pH 8.0), 1 mM PMSF, 10 μg/ml trypsin inhibitor, and 50 mM iodoacetamide. After 30 min at 4°C, cell debris was eliminated by centrifugation at 15 000 r.p.m. for 20 min, and the supernatant was collected. After measurement of protein concentration using a protein assay kit (Bio-Rad, Hercules, CA, USA), 40 μg of protein was mixed with SDS sample buffer, separated by SDS–PAGE, transferred to a polyvinylidene difluoride membrane (Bio-Rad), and blocked with 0.1% Tween and 5% skim milk overnight. The membranes were incubated with anti-cIAP-1, cIAP-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and caspase-3 (R&D Systems, Minneapolis, MN, USA) antibodies in PBS with 1% BSA for 1 h. Anti-human β-actin antibody (Biomedical Technologies, Stoughton, MA, USA) was used as a control. The membranes were washed three times with 0.1% Tween 20 in PBS and stained with horseradish peroxidase-conjugated secondary antibody. All immunoblots were detected by the enhanced chemiluminescence system (Amersham, Buckinghamshire, England) according to the manufacturer's instructions. For quantitation, the bands were scanned with a Scan Jet 4C/T (Hewlett Packard, San Diego, CA, USA), and densitometry was performed using the NIH Image Program (Version 1.60; NIH Division of Computer Research and Technology, Bethesda, MD, USA) on a Macintosh personal computer (Apple Computer, Inc., Cupertino, CA, USA). The ratios of cIAP-1, cIAP-2, and caspase-3 to the corresponding β-actin were calculated.
RNA isolation and semiquantitative RT–PCR
Total RNA was extracted from cultured NOR-P1 cells using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. The concentration of RNA was determined spectrophotometrically, and the integrity of all samples was confirmed by visualizing 28S and 18S ribosomal RNA bands under UV light after gel electrophoresis. Semiquantitative RT–PCR was performed as described previously (Zhang et al., 2000). Briefly, 1 μg of total RNA was reverse transcribed with reverse transcriptase (Takara, Kyoto, Japan) using random primers. Subsequently, each RT reaction mixture was subjected to PCR amplification using Taq Gold polymerase (Perkin-Elmer, Branchburg, NJ, USA) with cycle numbers varying from 15 to 40. Cycles consisted of heat denaturation (94°C for 1 min), annealing (55°C for 1 min), and extension (72°C for 2 min). The PCR products were size fractionated on a 2% agarose gel, and visualized under UV light. Sequences of oligonucleotide primers used for RT–PCR to determine expression of the target gene are listed, preceded by accession number for Gene Bank or references, and followed by expected transcript sizes: cIAP1 (Cui et al., 2000): sense 5′-IndexTermCCTGTGGTTAAATCTGCCTTG-3′, antisense 5′ IndexTermCAATTCGGCACCATAACTCTG-3′, 106 bp. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (NM002046): sense 5′-IndexTermCCACCCATGGCAAATTCCATGGCA-3′, antisense 5′-IndexTermTCTAGACGGCAGGTCAGGTCCACC-3′, 593 bp.
NF-κB decoy oligodeoxynucleotide (ODN) treatment
Phosphorothioated and double-stranded NF-κB decoy ODNs (single-stranded sequence: 5′-IndexTermAGTTGAGGGGACTTTCCCAGGC-3′) and scrambled ODNs (single-stranded sequence: 5′-IndexTermTTGCCGTACCTGACTTAGCCGT-3′) were synthesized according to the previous report (Vos et al., 2000). To study the effect of the NF-κB decoy, NOR-P1 cells were transiently transfected with 10 μ M NF-κB decoy or scrambled ODNs using lipofectamin (GIBCO-BRL, Gaithersburg, MD, USA) according to the manufacturer's instruction. Cells were incubated for 48 h and the expression of cIAP-1 was examined by RT–PCR and Western blotting. For apoptosis analysis, cells are treated with docetaxel 24 h after transfection, and then further incubated for 24 h. Thereafter, cells were stained with Hoechst 33342, and observed under fluorescence microscopy as described above.
Differences were analysed using Student's t-test, and P<0.05 was considered significant. All experiments were performed at least in triplicate. Data are shown as the mean±standard deviation (s.d.).
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Zhang, H., Morisaki, T., Nakahara, C. et al. PSK-mediated NF-κB inhibition augments docetaxel-induced apoptosis in human pancreatic cancer cells NOR-P1. Oncogene 22, 2088–2096 (2003). https://doi.org/10.1038/sj.onc.1206310
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