Introduction

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Paclitaxel (Taxol), a naturally occurring antineoplastic agent, has shown great promise in the therapeutic treatment of certain human solid tumors, particularly in drug-refractory ovarian cancer and metastatic breast cancer (Wani et al., 1971; Holmes et al., 1991; Tishler et al., 1992). However, the exact mechanism by which paclitaxel exerts its cytotoxic action remains unclear. Previous studies demonstrated that paclitaxel is a unique antimicrotubule agent that acts by inhibiting microtubule depolymerization and promoting the formation of unusually stable microtubules, thereby disrupting the normal dynamic reorganization of the microtubule network required for mitosis and cell proliferation (Schiff et al., 1979; Rowinsky et al., 1990; Williams and Smith, 1993; Willingham and Bhalla, 1994). Thus, it was generally believed that the antitumor effects of paclitaxel resulted mainly from interference with the normal function of microtubules and blockage of cell cycle progression in the late G₂-M phase via prevention of mitotic spindle formation (Fuchs and Johnson, 1978).

In recent years, several laboratories demonstrated that paclitaxel, at clinically relevant concentrations, was able to induce typical internucleosomal DNA fragmentation and other morphological features of apoptosis in a number of solid tumor cell lines (Bhalla et al., 1993; Fan et al., 1994; Cheng et al., 1995). These results clearly indicated that, in addition to its classical activity against microtubules and cell cycle arrest, paclitaxel also possesses cell-killing activity by induction of apoptosis. It is currently unclear whether this finding suggests a novel mechanism of action for paclitaxel against tumor cells or just represents an end product of the well known action of paclitaxel on microtubules and cell cycle arrest. Recent studies in this laboratory have revealed that glucocorticoids selectively inhibit paclitaxel-induced apoptotic cell death in a number of solid tumor cells but do not affect the ability of paclitaxel to induce microtubule bundling and mitotic arrest (Fan et al., 1994, 1996a,b). This selective inhibition of glucocorticoids on paclitaxel's cell-killing activity implies that paclitaxel-induced apoptosis may take place via a signaling pathway independent of cell cycle arrest. In other words, paclitaxel may cause cell death through a gene-directed process; i.e., paclitaxel may directly induce or activate apoptosis-associated genes or regulatory proteins, which in turn triggers the apoptotic process.

Although there is no solid evidence that paclitaxel-induced apoptosis occurs via a pathway independent of mitotic arrest, the possible existence of such a pathway has been proposed by many investigators (Jordan et al., 1996; Torres et al., 1997; Miller et al., 1999). In addition to the features of apoptotic cell death induced by low concentrations of paclitaxel and the selective inhibition by glucocorticoids, a number of apoptosis-associated genes or proteins have been reported to be activated or regulated by paclitaxel (Haldar et al., 1995; Strober et al., 1996; Moos and Fitzpatrick, 1998; Fan, 1999). One of these factors, NF-B, a member of the Rel transcription factor family, and its specific intracellular inhibitor IB, participate in the regulation of many biological processes, including inflammation and immune response, cell proliferation, and apoptotic cell death (Brown et al., 1993; Baldwin, 1996). NF-B normally resides in the cytoplasm as an inactivated form in a complex with IB. IB modulates the function or activity of NF-B through its proteolytic degradation in response to different extracellular stimuli (Baeuerle, 1991; Sun et al., 1995). A key player in this cascade of events is IB kinase complex (IKK and ) that is responsible for the phosphorylation and degradation of IB (Zandi et al., 1997; Delhase et al., 1999).

In recent years, increasing evidence indicates that activation of NF-B plays an important role in coordinating the control of apoptotic cell death, which either promotes or inhibits apoptosis, depending on different apoptotic stimuli and cell types (Beg and Baltimore, 1996; Grimm et al., 1996; Wang et al., 1996; Qin et al., 1998; Ryan et al., 2000). By using the unique inhibitory action of glucocorticoids on paclitaxel-induced apoptosis, we recently discovered that paclitaxel significantly down-regulated IB, which in turn promoted the nuclear translocation of NF-B and its DNA-binding activity. In contrast, we found that glucocorticoids could antagonize paclitaxel-mediated NF-B nuclear translocation and activation through induction of IB protein synthesis (Huang et al., 2000). Further investigation demonstrated that tumor cells stably transfected with antisense IB expression vectors exhibited a marked increase in sensitivity to paclitaxel-induced apoptosis (Huang et al., 2000). These results suggest that the NF-B/IB signaling pathway may contribute to the mediation of paclitaxel-induced cell death in solid tumor cells.

In the present study, we further investigated the molecular mechanism of paclitaxel-induced apoptosis via activation of NF-B signaling pathway. Using an IB phosphorylation inhibitor and stable transfection of a mutant IB, we demonstrated that the prevention of IB phosphorylation and degradation could significantly inhibit NF-B activation and apoptotic cell death induced by paclitaxel. Furthermore, we found that paclitaxel could activate IKK activity and up-regulate its upstream regulator, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1). Our results suggest that IKK might play a crucial role in the mediation or regulation of paclitaxel-induced apoptosis in solid tumor cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Drugs and Cell Culture. Paclitaxel was purchased from Calbiochem (La Jolla, CA) and dissolved in 100% dimethyl sulfoxide to make a 1.0 mM stock solution, which was then diluted in culture medium to obtain the desired concentrations. Glucocorticoids (triamcinolone acetonide) were dissolved in 100% ethanol as 10² to 10⁵ M stock solutions. The IB phosphorylation inhibitor compound Bay 117821 was purchased from Alexis Co. (San Diego, CA) and dissolved in 100% dimethyl sulfoxide to make a 10 mM stock solution. Human wild-type breast tumor BCap37 cells, BCap37 cell lines stably transfected with sense or antisense IB cDNA (Huang et al., 2000), and human ovary tumor OV2008 cells were cultured in RPMI 1640 medium (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Hyclone Laboratories, Logan, UT).

Plasmids and Recombinant Proteins. pGEX-4T-IB fusion protein expression vectors were constructed by subcloning IB cDNA restriction enzyme fragments from pCR2.1-IB vectors. The constructs were confirmed by DNA sequencing. Glutathione S-transferase (GST)-IB fusion proteins were purified from Escherichia coli. cells transformed with pGEX-IB expression vectors by using glutathione-agarose affinity chromatography (Amersham Biosciences, Piscataway, NJ) and confirmed by Western blot. Mutated human IB gene (deletion of NH₂-terminal 36 amino acids, including Ser³² and Ser³⁶) was obtained by using polymerase chain reaction with wild-type IB cDNA as template and the pair of primers MUTIB-5' (5'-ATGAAAGACGAGGAGTACGAG-3') and MUTIB-3' (5'-CTTTGCACTCATAACGTCAGA-3'). The polymerase chain reaction products were inserted into pCR 2.1 vectors (Invitrogen, Carlsbad, CA) and sequenced. Subsequently, mutant IB expression vectors were constructed from unique restriction sites available within the pCR2.1 vector. Mutant IB cDNAs were excised from pCR2.1 vectors and inserted into the high-level pcDNA3 mammalian expression vector system (Invitrogen). All constructs were confirmed by DNA sequencing.

Stable Transfection and Selection of Mutant IB cDNA-Transfected Cells. Transfections were performed by Lipofectin (Invitrogen) as recommended by the manufacturer. Briefly, BCap37 cells were washed twice with Opti-MEM reduced-serum medium, and 3 ml of the same medium was added to the cells. Plasmid DNA (2 µg/6-cm plate) containing mutant IB- inserts was mixed with Lipofectin before addition to the tumor cells. Stable transfectants were selected by incubating the cells in the medium containing 500 µg/ml Geneticin (G418). Surviving colonies were picked approximately 2 weeks later. Single colonies were amplified and continually grew in medium containing G418. Cells from each individual colony were examined for mutant IB expression by Western blot assays. Positive colonies were maintained in culture medium with G418 for further experiments. All transfectants were routinely cultured in RPMI 1640 medium containing 10% fetal calf serum and 1% penicillin/streptomycin.

Western Blotting. Cells treated with different agents were harvested by trypsinization and washed with phosphate-buffered saline (PBS). Cellular protein was isolated using the protein extraction buffer containing 150 mM NaCl, 10 mM Tris, pH 7.2, 5 mM EDTA, 0.1% Triton X-100, 5% glycerol, 2% SDS. Protein concentrations were determined using Bio-Rad DC Protein Assay (Bio-Rad, Hercules, CA). Equal amounts of proteins (50 µg/lane) were fractionated on a 12.5% SDS-polyacrylamide gel electrophoresis (PAGE) gel and transferred to PVDF membranes. The membranes were incubated with anti-IB, IKK, MEKK1 primary antibodies, respectively (1:3000; Santa Cruz Biotechnology, Santa Cruz, CA). After washing with PBS, the membranes were incubated with peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibody (1:4000; Jackson Immunoresearch Laboratories, Inc., West Grove, PA) followed by enhanced chemiluminescent staining using the enhanced chemiluminescence system (Amersham Biosciences). -Actin was used to normalize for protein loading.

Determination of Internucleosomal DNA Cleavage. After tumor cells were treated with various drug regimes as indicated, cells were harvested, counted, and washed with PBS at 4°C. Then cells were suspended in lysis solution [5 mM Tris-HCl, 20 mM EDTA, and 0.5% (v/v) Triton X-100] for 20 min on ice. Detection of DNA fragmentation was performed as described previously (Cheng et al., 1995). DNA samples were analyzed by electrophoresis in a 1.2% agarose slab gel containing 0.2 µg/ml ethidium bromide, and visualized under UV illumination.

MTT Assays. Cells were harvested with trypsin and resuspended to a final concentration of 4 × 10⁴ cells/ml in fresh medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. Aliquots of the cell suspension were evenly distributed into 96-well tissue culture plates (100 µl/well) with lids (Falcon, Oxnard, CA). Designated columns were treated with the various drug regimes. One column from each plate contained medium alone and another column contained cells in drug-free media. At the end of each time points, the 96-well plates were centrifuged to collect all the detached cells and the media were carefully removed. Then 100 µl of a 1 mg/ml MTT solution, diluted in culture media, was added to each well. The plates were incubated at 37°C in 5% CO₂ atmosphere for 3 h, allowing viable cells to reduce the yellow tetrazolium salt (MTT) into dark blue formazan crystals. At the end of the 3-h incubation, the MTT solution was removed and 100 µl of dimethyl sulfoxide (Sigma, St. Louis, MO) was added to each well to dissolve the formazan crystals. To ensure complete dissolution of the formazan crystals, the plates were vortexed gently at low speed for 10 min. The absorbance in individual wells was determined at 560 nm by a microplate reader (Molecular Devices, Sunnyvale, CA).

Immunoprecipitation and Kinase Assays. Cells treated with various drug regimes were harvested by trypsinization and washed with PBS buffer and the pellet was resuspended in 60 to 90 µl of immunoprecipitation lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 5 mM EDTA, 1 mM dithiothreitol, 100 mM NaF, 2 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg each of aprotinin and leupeptin per milliliter) and stored on ice for 20 min before centrifugation (14,000g, 20 min, 4°C). IB kinase complex was immunoprecipitated by incubation for 1 h at 4°C with IKK rabbit polyclonal antibodies (Santa Cruz Biotechnology) bound to protein-A Sepharose (Amersham Biosciences). The immunoprecipitates were washed twice with immunoprecipitation buffer and twice with kinase buffer (20 mM HEPES, pH 7.4, 20 mM -glycerophosphate, 20 mM MgCl₂, 2 mM dithiothreitol, 0.1 mM sodium orthovanadate). The kinase assays were initiated by the addition of 1 mg of GST-IB fusion protein as substrate and 10 Ci/mmol [-³²P]ATP. Reaction mixtures were incubated for 30 min at 30°C and stopped by the addition of 2× SDS-PAGE sample buffer. The phosphorylation of the IB proteins was examined by SDS-polyacrylamide gel electrophoresis followed by autoradiography. The portion containing IKK was analyzed by Western blotting for IKK protein as control.

Flow Cytometry Analysis. Cell sample preparation and propidium iodide (PI) staining for flow cytometry analysis were performed according to the method described by Nicoletti et al. (1991). BCap37 cells transfected with empty expression vector pcDNA3 (Vector), IB sense cDNA (WT IB), and mutant IB cDNA (MUT IB) were treated with paclitaxel in different concentrations (10, 100, and 500 nM) for 48 h. Cells were then harvested by trypsinization and washed twice with PBS followed by fixation in 1% formaldehyde and dehydration in 70% ethanol diluted in PBS. Cells were then incubated in PBS containing 100 µg/ml RNase and 40 µg/ml PI at 37°C for 1 h before flow cytometry analysis. Cell cycle distribution was determined using a Coulter Epics V instrument (Beckman Coulter, Inc., Fullerton, CA) with an argon laser set to excite at 488 nm. The results were analyzed using Elite 4.0 software (Phoenix Flow System, San Diego, CA). The percentage of cells at the sub-G₁ was taken as measure of the apoptotic rate of the cell population.

Nuclear Extraction Preparation and Electrophoretic Mobility Shift Assays. Nuclear extracts were prepared via procedures described previously (Huang et al., 2000). In brief, after BCap37 cells transfected with empty pCDNA3 vectors or mutant IB were treated with paclitaxel for different concentrations for 24 h, cells were harvested and resuspended in 800 µl of hypotonic lysis buffer (10 mM HEPES, pH 7.8, 10 mM KCl, 2 mM MgCl₂. 1 mM dithiothreitol, 0.1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride). Then cells were incubated on ice for 15 min. After that, 50 µl of 10% Nonidet P-40 was added, and cells were vigorously mixed and centrifuged. The nuclear pellets were suspended in 50 µl of buffer containing 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and 10% glycerol (v/v) and mixed for 20 min and centrifuged to produce supernatant containing nuclear proteins. Protein concentrations were determined using the Bio-Rad DC Protein Assay (Bio-Rad).

Northern Blotting. BCap37 cells were treated with different concentrations of paclitaxel for 24 h. Total RNA was isolated and 20 µg was fractionated in 1% agarose-formaldehyde gel, transferred to nitrocellulose membrane, and UV cross-linked. The membrane was probed with [³²P]UTP-labeled antisense MEKK1 RNA probes generated from the subcloned MEKK1 cDNA fragments in pCDNA3 vectors. The membrane was then washed and autoradiographed. The same membrane was stripped and reprobed with human antisense -actin RNA probes to normalize RNA loading.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

IB Phosphorylation Inhibitor Prevents Paclitaxel-Induced IB Degradation and Inhibits Paclitaxel-Induced Apoptosis. Our previous studies revealed that paclitaxel induced IB protein degradation in BCap37, OV2008, and other solid tumor cells (Huang et al., 2000). Because IB degradation was mainly caused by its phosphorylation and ubiquitination (Baeuerle, 1991), we used a novel IB phosphorylation inhibitor, Bay 117821, that was recently identified to selectively inhibit cytokine-induced IB phosphorylation and degradation in human endothelial cells (Pierce et al., 1997), to examine whether the inhibition of IB degradation could affect paclitaxel-induced apoptotic cell death. By Western blot, we determined that cotreatment with Bay 117821 (10 µM) significantly blocked the degradation of IB induced by paclitaxel in both BCap37 and OV2008 cells (Fig. 1). Subsequently, we performed DNA fragmentation and MTT assays to evaluate the influence of the IB phosphorylation inhibitor on paclitaxel-induced apoptotic cell death and overall cytotoxicity. The results shown in Fig. 2A indicate that paclitaxel alone was able to induce characteristic DNA fragmentation at 10 nM or greater concentrations within 48 h. However, concurrent treatment of Bay 117821 (10 µM) with paclitaxel significantly inhibited paclitaxel-induced apoptosis. Furthermore, MTT assays also showed that the specific inhibitor of IB phosphorylation interfered with the cytotoxicity of paclitaxel in both BCap37 cells and OV2008 cells (Fig. 2B). These results suggest that the phosphorylation and degradation of IB might be a critical step for the activation of NF-B and the mediation of paclitaxel-induced apoptosis.

View larger version (45K): [in this window] [in a new window]   Fig. 1.   IB phosphorylation inhibitor prevents paclitaxel-induced IB degradation. BCap37 and OV2008 cells were exposed to the indicated concentrations of paclitaxel (PTX) for 24 h with or without the simultaneous treatment of compound Bay 117821 (10 µM). Equal amounts (50 µg/lane) of cellular protein were fractionated on 12.5% SDS-PAGE gel and transferred to PVDF membranes followed by immunoblotting with an anti-IB polyclonal antibody.

View larger version (70K): [in this window] [in a new window]   Fig. 2.   IB phosphorylation inhibitor represses paclitaxel-induced apoptosis. BCap37 and OV2008 cells were treated with different concentrations of paclitaxel (PTX) with or without the cotreatment of 10 µM compound Bay 117821 for 48 h. Cells were then harvested for DNA fragmentation assays (A) or MTT assays (B). The results of the MTT assay were presented as means ± S.D. based on three independent experiments.

Paclitaxel Activates IB Kinase Activity. To study the intrinsic mechanisms by which paclitaxel down-regulates IB protein and leads to the activation of NF-B, we next examined the effect of paclitaxel on endogenous IKK activity. As described under Materials and Methods, GST-IB fusion proteins were purified from isopropyl -D-thiogalactoside-induced E. coli cells that were transformed with pGEX-IB fusion vectors and used as subtracts for kinase assays, whereas the IKK complexes were prepared by immunoprecipitation of cell extracts harvested from BCap37 and OV2008 cells treated with a variety of concentrations or different time points of paclitaxel. The results of kinase assays depicted in Fig. 3 indicate that the phosphorylation of substrate GST-IB protein (P-GST-IB) was remarkably stimulated by IKK complexes obtained from BCap37 or OV2008 cells treated with 10 nM or greater concentrations of paclitaxel for 24 h (Fig. 3A). When tumor cells were treated with 100 nM paclitaxel, the IKK activation was observed as early as 3 h (Fig. 3B). These results indicate that paclitaxel activated IB kinase activity, which in turn led to IB phosphorylation and degradation. In addition, we also examined the possible effect of glucocorticoids on IKK activity. The result showed that glucocorticoids did not interfere with IKK activity induced by paclitaxel in both BCap37 and OV2008 cells (Fig. 3C). These data provide another piece of evidence that glucocorticoids antagonize paclitaxel-induced NF-B activation through induction of IB protein expression rather than inhibition of IB degradation.

View larger version (70K): [in this window] [in a new window]   Fig. 3.   Paclitaxel activates IB kinase activity. BCap37 cells and OV2008 cells were treated with different concentrations of paclitaxel (PTX) for 24 h (A); or 100 nM paclitaxel in a time course as indicated (B); or 100 nM paclitaxel for 24 h with/without pretreatment of glucocorticoids (107 M) (C). IKK complex was immunoprecipitated with an anti-IKK antibody and then was subjected to the in vitro kinase assay (KA) by using GST-IB as the substrate. After SDS-PAGE, the gel containing the substract was dried and processed for autoradiography. The portion containing IKK was analyzed by Western blotting for IKK protein.

Mutant IB Lacking Ser³² and Ser³⁶ Suppresses Paclitaxel-Induced NF-B Activation. Proteolytic degradation of IB is essential for activation of NF-B (Baeuerle, 1991; Sun et al., 1995). Previous studies have revealed that the degradation of IB protein is mainly due to the inducible phosphorylation of IB at Ser³² and Ser³⁶ by IB kinase complex (Brown et al., 1995; Traenckner et al., 1995; DiDonato et al., 1997). To further confirm that paclitaxel down-regulates IB through induction of IB phosphorylation and degradation, we constructed a mutant IB expression vector by deleting 36 amino acids, including Ser³² and Ser³⁶ from the NH₂ terminus of IB gene. Such a mutant IB protein cannot be degraded by the IB kinase complex but still possesses the ability to bind to NF-B through the interior ankyrin motif domain and functions as a super suppressor of NF-B molecules (Brown et al., 1995; Shinohara et al., 2001). As shown in Fig. 4, BCap37 cells with stable transfection of this mutant IB expressed a smaller size of IB protein, which was not degraded in the presence of paclitaxel. Furthermore, we examined the effect of mutant IB on paclitaxel-induced DNA-binding activity of NF-B. By EMSAs, an increased level of DNA-binding activity was clearly detected in empty vector-transfected BCap37 cells exposed to paclitaxel, but this elevated DNA-binding activity of NF-B by paclitaxel was markedly inhibited in the cells transfected with the mutant IB (Fig. 5). These findings demonstrated that the mutant IB could interfere with paclitaxel-induced NF-B activation.

View larger version (40K): [in this window] [in a new window]   Fig. 4.   Paclitaxel does not degrade mutant IB. Equal amounts (50 µg/lane) of cellular proteins from wild-type BCap37 cells (lane 1), pcDNA 3 vector transfectants (lane 2), and mutant IB transfectants treated with different concentrations of paclitaxel for 24 h (lanes 3-7) were fractionated on 12.5% SDS-PAGE gel and transferred to PVDF membranes followed by immunoblot with anti-IB polyclonal antibody. -Actin protein was used as a control.

View larger version (76K): [in this window] [in a new window]   Fig. 5.   Expression of mutant IB blocks paclitaxel-induced NF-B activation. BCap37 cells transfected with mutant IB (MUT IB) or pCDNA3 vector only (Vector) were treated with different concentrations of paclitaxel for 24 h. Equal amounts of nuclear cell extracts were subjected to EMSAs with a -32P-labeled oligonucleotide encompassing the NF-B binding site.

Transfection of Mutant IB Reduces Sensitivity of Tumor Cells to Paclitaxel-Induced Apoptosis. Next, the tumor cells with stable transfection of the mutant IB were compared with their parental cells to determine whether the expression of the mutant IB altered the sensitivity of tumor cells to paclitaxel-induced apoptosis. As depicted in Fig. 6, BCap37 cells transfected with empty pcDNA3 expression vectors (Vector), wild-type IB cDNA (WTIB) (Huang et al., 2000), and mutant IB cDNA (MUTIB) were treated with a series of increasing concentrations of paclitaxel (10-500 nM) for 48 h followed by the DNA fragmentation assay. We observed that the cells transfected with the mutant IB exhibited more resistance to paclitaxel-induced apoptosis. By flow cytometric analyses, we also observed that the percentage of cells at sub-G₁ DNA, which is believed to represent apoptotic cell populations, was dramatically decreased in mutant IB transfectants in comparison with those transfected with the empty vector or wild-type IB (Fig. 7, A and B). MTT assays showed that mutant IB significantly increased the cell viability in presence of paclitaxel (Fig. 7C). These results indicate that the introduction of the mutant IB resulted in the decreased sensitivity of tumor cells to paclitaxel-induced apoptosis.

View larger version (101K): [in this window] [in a new window]   Fig. 6.   Transfection of mutant IB inhibits paclitaxel-induced DNA fragmentation. BCap37 cells transfected with empty vectors (lanes 2-5), wild-type sense IB cDNA (lanes 6-9), and mutant IB cDNA (lanes 10-13) were treated with 10, 100, and 500 nM paclitaxel for 48 h. Fragmented DNA was analyzed by electrophoresis in 1.2% agarose gel containing 0.1% ethidium bromide. Lane 1 was 1-kilobase DNA marker.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Transfection of mutant IB suppresses paclitaxel-induced apoptosis. BCap37 cells transfected with empty vectors (Vectors), wild-type sense IB cDNA (WT IB), and mutant IB cDNA (MUT IB) were treated with 10, 100, and 500 nM paclitaxel for 48 h. Then cells were harvested and stained for DNA with propidium iodide (PI) for flow cytometric analysis. The peaks corresponding to G1 and G2/M phases of the cell cycle are indicated. The sub-G1 peaks labeled as AP represent apoptotic cells (A). The percentage of cells with a sub-G1 DNA content was taken as a measure of apoptotic rate of the cell population (B). The cell viability was measured by MTT assays (C). Data in the bar graph are means ± S.D. of three independent experiments.

Paclitaxel Up-Regulates MEKK1 Expressions. Latest studies revealed that MEKK1 phosphorylates the IKK subunit, preferentially IKK, resulting in the activation of NF-B in response to some cytokine stimuli [such as tumor necrosis factor- (TNF-), interleukin-1] (Karin and Delhase, 1998; May and Ghosh, 1999). To investigate whether paclitaxel activates IKK through regulation of MEKK1 activity, we examined the possible alteration of MEKK1 protein in the tumor cells treated with different concentrations of paclitaxel. By Western blot, we found that paclitaxel enhanced the protein levels of MEKK1 in both BCap37 and OV2008 cells (Fig. 8A). The increase of MEKK1 protein level was observed as early as 3 h after paclitaxel treatment (Fig. 8B). Furthermore, Northern blot analysis showed that the mRNA expressions of MEKK1 were stimulated by paclitaxel treatment (Fig. 8C), suggesting that MEKK1 might be the primary target of paclitaxel in the NF-B/IB signaling pathway. In addition, we examined whether glucocorticoids exposure affects paclitaxel-induced MEKK1 activity. As expected, glucocorticoids did not change paclitaxel-enhanced MEKK1 protein expressions (Fig. 8D), implying that glucocorticoids do not interfere with paclitaxel-mediated activities of upstream regulators of NF-B/IB signaling pathway.

View larger version (48K): [in this window] [in a new window]   Fig. 8.   Paclitaxel induces MEKK1 expression. BCap37 cells or OV2008 cells treated with increasing concentrations of paclitaxel (PTX) as indicated for 24 h (A) or 100 nM paclitaxel in a time course as indicated (B) were immunoblotted with anti-MEKK1 polyclonal antibody; BCap37 cells were treated with different concentrations of paclitaxel (PTX) for 24 h (C). RNA (20 µg/lane) was size fractionated by formaldehyde/agarose gel electrophoresis. After transfer to the nitrocellulose membrane, RNA was hybridized with [32P]UTP-labeled antisense riboprobes synthesized from MEKK1 pCDNA3 vectors; BCap37 cells were exposed 100 nM paclitaxel with or without the preincubation of glucocorticoids (107 M) for 24 h followed by immunoblotting with anti-MEKK1 polyclonal antibody (D).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

This study investigates the molecular mechanisms of paclitaxel-induced apoptosis via the activation of the NF-B/IB signaling pathway. In previous studies, we have demonstrated that paclitaxel could degrade IB protein and promote the nuclear translocation and DNA-binding activity of transcription factor NF-B (Huang et al., 2000). Activation of NF-B has been believed to play an important role in coordinating the control of apoptotic cell death, either as a promoter or, perhaps more commonly, as a blocker of apoptosis (Beg and Baltimore, 1996; Grimm et al., 1996; Qin et al., 1998; Ryan et al., 2000). The purpose of this study was to determine how activation of NF-B regulates paclitaxel-induced apoptotic cell death and whether IB phosphorylation and degradation are essential for paclitaxel-induced NF-B activation. BCap37 and OV2008 cells were first tested by cotreatment with paclitaxel and a recently identified IB phosphorylation inhibitor, Bay 117821, which has been shown to specifically inhibit the phosphorylation of IB induced by some cytokines such as TNF- (Pierce et al., 1997). The results indicate that Bay 117821 can prevent paclitaxel-induced IB degradation in both BCap37 and OV2008 cells (Fig. 1). Meanwhile, we determined that the tumor cells cotreated with Bay 117821 exhibited reduced sensitivity to paclitaxel-induced apoptosis (Fig. 2). These results suggest that the proteolytic degradation of IB might be an important step for the activation of NF-B, which in turn mediates paclitaxel-induced apoptosis in these solid tumor cells. The precise molecular target for IB phosphorylation inhibitor Bay 117821 is not yet clear. Although Bay 117821 was shown to inhibit IB phosphorylation and degradation, this may be the result of direct inhibition of a paclitaxel-inducible IB kinase or due to inhibition of a signaling event upstream of the IB kinase. The exact mechanism of the inhibitory action of Bay 117821 on paclitaxel-induced IB degradation needs to be investigated further.

Recent studies have identified a high molecular mass complex of IB kinases (IKK and IKK) that plays a key role in IB protein phosphorylation and degradation (DiDonato et al., 1997; Delhase et al., 1999; May and Ghosh, 1999). We therefore examined the potential effect of paclitaxel on IKK activity. By in vitro IB kinase assay, we demonstrated that IKK activities were significantly stimulated by paclitaxel in both BCap37 and OV2008 cells (Fig. 3). Next, we constructed a mutant IB expression vector in which an N-terminal fragment containing Ser³² and Ser³⁶ was deleted. Based on current knowledge, the degradation of IB is mainly due to the inducible phosphorylation of Ser³² and Ser³⁶. Deletion or substitution of these two amino acids with other residues has been reported to prevent IB from signal-induced phosphorylation (Brown et al., 1995; Shinohara et al., 2001). Through stable transfection of this mutant IB into wild-type BCap37 cells, we demonstrated that the mutant IB protein was insensitive to IKK-mediated phosphorylation and degradation but still possessed the ability to interact with cytoplasmic NF-B and inhibit paclitaxel-induced NF-B activation. (Figs. 4 and 5). Meanwhile, the results from DNA fragmentation and flow cytometric assays revealed that the expression of the mutant IB significantly inhibited paclitaxel-induced apoptotic cell death (Figs. 6 and 7). These findings further indicate that paclitaxel-stimulated IKK is critical for IB degradation and consequent activation of NF-B. Blockage of NF-B activation by the mutant IB disrupts the signaling pathway leading to paclitaxel-induced apoptotic cell death.

In light of these experimental results and our previous studies, the activation of NF-B seems to act as a promoter in paclitaxel-induced apoptosis. However, it is currently unclear how the activated NF-B triggers the downstream apoptotic machinery. NF-B is a nuclear transcriptional factor. Theoretically, it is assumed to mediate paclitaxel-induced apoptosis through the regulation of gene expressions, particularly for those genes whose expressions are associated with apoptotic cell death. To date, NF-B has been reported to participate in the transcription of more than 150 target genes (Pahl, 1999). Many of these NF-B target genes are considered as proapoptotic genes, such as FAS/APO-1 ligand (FasL), c-myc, ICE, and p53 (Suda et al., 1993; Wu and Lozano, 1994; Brown et al., 1995). Moreover, some of these genes, including p53, c-myc, and FasL were even found to respond to paclitaxel in certain normal and tumor cells (La Rosa et al., 1994; Blagosklonny et al., 1995; el Khyari et al., 1997; Srivastava et al., 1999). Therefore, although the effecter gene(s) that potentially contributes to paclitaxel-induced apoptosis remains unidentified, it should be reasonable to hypothesize that activated NF-B might stimulate the expression of a specific proapoptotic gene that eventually triggers the downstream signaling pathway, leading to the paclitaxel-induced apoptotic cell death.

MEKK1 is a 196-kDa enzyme that is involved in the regulation of the c-Jun NH₂-terminal kinase pathway and apoptosis (Lange-Carter et al., 1993). Latest evidence shows that MEKK1 can phosphorylate and activate IKK (preferentially IKK) in response to a variety of cytokine stimuli (May and Ghosh, 1999). It was also reported that under different circumstances overexpression of MEKK1 was found to stimulate NF-B activities (Hirano et al., 1996; Meyer et al., 1996), and MEKK1-induced NF-B activation can be inhibited by the dominant negative IKK and IKK mutation (Lee et al., 1998). These findings suggest that the IKK complex may be the major substrate of MEKK1 and that IKK activation depends on MEKK1 activity. In this study, we analyzed the expression of MEKK1 in the cells treated with paclitaxel and found that paclitaxel was able to up-regulate both protein and mRNA levels of MEKK1 in BCap37 and OV2008 cells (Fig. 8). Based on this finding, we suspect that MEKK1 may be the primary target of paclitaxel. The up-regulated MEKK1 then, in turn, activates the IKK activity and the NF-B signaling pathway.

Glucocorticoids are routinely used in the clinical application of paclitaxel to prevent hypersensitivity reactions (McEvoy, 1995). Glucocorticoids have been previously demonstrated to inhibit paclitaxel-induced apoptosis and NF-B activation through induction of IB synthesis (Fan, et, 1996a,b; Huang et al., 2000). To exclude the possibility that glucocorticoids may directly affect IKK and MEKK1, we also examined IKK activity and MEKK1 expression in the cells exposed to glucocorticoids. Our results indicate that glucocorticoids do not interfere with either IKK activity or MEKK1 expression in the presence or absence of paclitaxel (Figs. 3D and 8D). These expected results support our previous hypothesis that glucocorticoids antagonize paclitaxel-induced IB degradation by stimulating IB synthesis rather than by interfering with the IB degradation or its upstream events.

On the basis of these observations and our previous data on the opposite regulation of NF-B activation by paclitaxel and glucocorticoids, we would hypothesize the following pathway to explain paclitaxel-induced apoptosis and the inhibitory action of glucocorticoids (Fig. 9). Briefly, exposure of tumor cells to paclitaxel leads to the increased expression of MEKK1, which in turn activates IKK. The activated IKK then causes the degradation of IB and the disassociation of the NF-B/IB- complex. Subsequently, the released cytoplasmic NF-B translocates into the nucleus, where it functions as a transcription factor to regulate apoptosis-associated gene expression. Conversely, glucocorticoids inhibit paclitaxel-induced apoptosis through induction of IB- protein synthesis, which antagonizes paclitaxel-mediated NF-B nuclear translocation and activation. Given this hypothesized pathway, MEKK1 might be the primary target of paclitaxel, whereas the activation of IKK plays a critical role in the subsequent activation of NF-B and the regulation of paclitaxel-induced apoptotic cell death in solid tumor cells.

View larger version (15K): [in this window] [in a new window]   Fig. 9.   Hypothesized mechanism of paclitaxel-induced apoptosis mediated by the IB-F-B signaling pathway and the inhibitory action of glucocorticoids. GC, glucocorticoids; GCR, glucocorticoids receptor.