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Originally published In Press as doi:10.1074/jbc.M313709200 on March 16, 2004

J. Biol. Chem., Vol. 279, Issue 22, 23477-23485, May 28, 2004
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Inhibition of NF{kappa}B Increases the Efficacy of Cisplatin in in Vitro and in Vivo Ovarian Cancer Models*

Seiji Mabuchi{ddagger}, Masahide Ohmichi{ddagger}§, Yukihiro Nishio{ddagger}, Tadashi Hayasaka§, Akiko Kimura{ddagger}, Tsuyoshi Ohta§, Maki Saito§, Jun Kawagoe§, Kazuhiro Takahashi§, Namiko Yada-Hashimoto{ddagger}, Masahiro Sakata{ddagger}, Teiichi Motoyama||, Hirohisa Kurachi§, Keiichi Tasaka{ddagger}, and Yuji Murata{ddagger}

From the {ddagger}Department of Obstetrics and Gynecology, Osaka University Medical School, 2-2, Yamadaoka, Suita, Osaka 565-0871, Japan and the Departments of §Obstetrics and Gynecology and ||Pathology, Yamagata University, School of Medicine, 2-2-2 Iidanishi, Yamagata 990-9585, Japan

Received for publication, December 15, 2003 , and in revised form, March 12, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Whether or not inhibition of NF{kappa}B increases the efficacy of cisplatin in in vitro and in vivo ovarian cancer models was investigated. We compared the basal levels of phosphorylation of I{kappa}B{alpha} and activity of NF{kappa}B between cisplatin-sensitive A2780 cells and cisplatin-resistant Caov-3 cells. The basal levels of phosphorylation of I{kappa}B{alpha} and activity of NF{kappa}B in Caov-3 cells were significantly higher than those in A2780 cells. Cisplatin caused a more marked decrease in the phosphorylation of I{kappa}B{alpha} and activity of NF{kappa}B in A2780 cells than in Caov-3 cells. Thus, high basal levels of phosphorylation of I{kappa}B{alpha} and activation of NF{kappa}B and less marked inhibition of the phosphorylation of I{kappa}B{alpha} and activation of NF{kappa}B by cisplatin seem to reduce the sensitivity of cells to cisplatin. Inhibition of NF{kappa}B activity either by treatment with the I{kappa}B{alpha} phosphorylation inhibitor (BAY 11-7085) or a specific NF{kappa}B nuclear translocation inhibitor (SN-50) or by transfection of p50{Delta}NLS (which lacks the nuclear localization signal domain) increased the efficacy of both the cisplatin-induced attenuation of I{kappa}B{alpha} phosphorylation and NF{kappa}B activity and the cisplatin-induced apoptosis. In addition, treatment with BAY 11-7085 increased the efficacy of the cisplatin-induced attenuation of both the expression of X-linked inhibitor of apoptosis protein (XIAP) and cell invasion through Matrigel. Moreover, treatment with BAY 11-7085 increased the efficacy of the cisplatin-induced inhibition of the intra-abdominal dissemination and production of ascites using athymic nude mice inoculated intraperitoneally with Caov-3 cells. These results suggest that combination therapy of cisplatin with the NF{kappa}B inhibitor should increase the therapeutic efficacy of cisplatin.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The sensitivity of cells to chemotherapeutic drug-induced apoptosis appears to depend on the balance between proapoptotic and antiapoptotic signals. Therefore, it is possible that antiapoptotic signals such as the PI3K1-Akt survival cascade are involved in sensitivity to chemotherapeutic drugs. We reported that Akt inactivation sensitizes human ovarian cancer cells to cisplatin (1) and paclitaxel (2), suggesting that Akt inactivation could be a hallmark for examining the sensitivity of cells to some chemotherapeutic drugs. Possible mechanisms by which Akt promotes cell survival include phosphorylation and inactivation of the proapoptotic proteins BAD and caspase-9 (3, 4). Akt also phosphorylates and inactivates the Forkhead transcription factors, resulting in reduced expression of the cell cycle inhibitor p27Kip1 and the Fas ligand (5-7). Via the phosphorylation of I{kappa}B kinase, Akt also activates NF{kappa}B, a transcription factor that has been implicated in cell survival (8, 9).

NF{kappa}B is activated in certain cancers and in response to chemotherapy and radiation. NF{kappa}B normally resides in the cytoplasm as an inactivated form in a complex with I{kappa}B{alpha}. Phosphorylation of I{kappa}B{alpha} by upstream kinases promotes its degradation, allowing NF{kappa}B to translocate to the nucleus and induce target genes (6, 7). The transcriptional activation of genes associated with cell proliferation (10), angiogenesis (11, 12), metastasis (13, 14), and suppression of apoptosis (15) appears to lie at the heart of the ability of NF{kappa}B to promote oncogenesis (16) and cancer therapy resistance (17, 18). While activation of NF{kappa}B may induce apoptosis in certain situations (19-22), most reports suggest that NF{kappa}B mediates survival signals that counteract apoptosis (23-28). It has been reported that intrinsically or constitutively activated NF{kappa}B may be critical in the development of drug resistance in cancer cells (29-31). Therefore, several agents that are able to inhibit NF{kappa}B function might be considered as an adjuvant approach in combination with chemotherapy for a variety of cancers.

These considerations led us to examine whether the status of NF{kappa}B activity is involved in sensitivity to cisplatin. In the present study, we show that the basal levels of phosphorylation of I{kappa}B{alpha} and activity of NF{kappa}B in cisplatin-resistant Caov-3 cells are significantly higher than those in cisplatin-sensitive A2780 cells. BAY 11-7085, a known pharmacological inhibitor of I{kappa}B{alpha} phosphorylation (32), enhanced the cisplatin-induced inhibition of I{kappa}B{alpha} phosphorylation and NF{kappa}B activity and increased the efficacy of cisplatin in in vitro and in vivo ovarian cancer models.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—The anti-I{kappa}B{alpha} and phospho-I{kappa}B{alpha} antibodies were obtained from Cell Signaling Technology (Beverly, MA). The anti-NF{kappa}B p65 was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The I{kappa}B{alpha} phosphorylation inhibitor BAY 11-7085 was purchased from Alexis Biochemicals (San Diego, CA). The specific NF{kappa}B nuclear translocation inhibitor SN-50 was purchased from Biomol (Plymouth Meeting, PA). Anti-PARP, cleaved PARP, and the X-linked inhibitor of apoptosis protein (XIAP) antibodies were obtained from Cell Signaling Technology. Anti-{beta}-actin antibody was purchased from Sigma. ECL Western blotting detection reagents were obtained from Amersham Biosciences. The cell titer 96-well proliferation assay was obtained from Promega (Madison, WI). The terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) kit (ApopTag®) was obtained from Chemicon (Temecula, CA).

Cell Cultures—Human ovarian papillary adenocarcinoma cell line Caov-3 was obtained from the American Type Culture Collection. The human ovarian cancer A2780 cell line derived from a patient prior to treatment was kindly provided by Dr. T. Tsuruo (Institute of Molecular and Cellular Biosciences, Tokyo, Japan) and Drs. R. F. Ozols and T. C. Hamilton (NCI, National Institutes of Health, Bethesda, MD) (33). The cells were cultured at 37 °C in Dulbecco's modified Eagle's medium with 10% fetal bovine serum in a water-saturated atmosphere of 95% air and 5% CO2.

Constructs—The NF{kappa}B reporter plasmid (pElam-luc) was a kind gift from Dr. J. Cheng (University of South Florida College of Medicine) (34). pCR-FLAG-p50 (encoding full-length human p50 with the FLAG epitope) and pCR-FLAG-p50{Delta}NLS (encoding nuclear localization signal (NLS) polypeptide-deficient p50 with the FLAG epitope) constructs were kind gifts from Dr. Gourisankar Ghosh (University of California, San Diego) Chen et al. (39).

Proliferation Assay—Cell proliferation (35) was assessed by the addition of cisplatin at the indicated concentrations for 48 h, 1 day after seeding test cells into 96-well plates. The number of surviving cells was determined 24 h later by determination of A590 of the dissolved formazan product after the addition of MTS for 1 h as described by the manufacturer (Promega). All experiments were carried out in quadruplicate, and the viability was expressed as the ratio of the number of viable cells with cisplatin treatment to that without treatment.

Western Blotting—Cells were incubated without serum for 16 h and then treated with various agents. They were then washed twice with phosphate-buffered saline and lysed in ice-cold HNTG buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA, 10 mM sodium pyrophosphate, 100 µM sodium orthovanadate, 100 mM NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) (2). The lysates were centrifuged at 12,000 x g at 4 °C for 15 min, and the protein concentrations of the supernatants were determined using the Bio-Rad protein assay reagent. Equal amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Blocking was done in 10% bovine serum albumin in 1x Tris-buffered saline. Western blot analyses were performed with various specific primary antibodies.

NF{kappa}B Transcriptional Activation Analysis—Cells were seeded in 60-mm dishes and transfected with 2 µg of NF{kappa}B reporter plasmid (pElam-luc) for 24 h using LipofectAMINE Plus (Invitrogen) according to the manufacturer's protocol. Cells were treated with various agents and then harvested and subjected to luciferase assays using the luciferase assay system (Promega) as described previously (36). A plasmid expressing the bacterial {beta}-galactosidase gene was also cotransfected in each experiment to serve as an internal control for transfection efficiency.

Assay of Invasion through Matrigel—Polyvinylpyrrolidone-free polycarbonate filters (8-µm pore size; Chemotaxicell, Kurabo, Japan) were coated with a mixture of basement membrane components (Matrigel, 25 µg/filter) and placed in modified Boyden chambers (37). The cells (5 x 104) were released from their culture dishes by brief exposure to EDTA (1 mmol/liter) and centrifuged, resuspended in 0.1% bovine serum albumin-Dulbecco's modified Eagle's medium, and placed in the upper compartment of the Boyden chamber. Fibroblast-conditioned medium in the lower compartment served as a chemoattractant. After incubation for 24 h at 37 °C, the cells on the lower surface of the filter were fixed, stained with Mayer's hematoxylin solution, and enumerated using an ocular micrometer, and at least 10 fields/filter were counted. All of the experiments were independently performed in triplicate.

Treatments in Vivo—All of the procedures involving animals and their care in this study were approved by the animal care committee of Osaka University in accordance with institutional and Japanese government guidelines for animal experiments. Caov-3 cells were harvested in 0.25% trypsin/PBS/EDTA, washed once each with medium and PBS, and resuspended in PBS at 106 cells/200 µl. One million Caov-3 cells were injected intraperitoneally (i.p.) into 5-week-old female nu/nu athymic mice (n = 30). Two weeks after inoculation, one group of mice (n = 6) was treated with BAY 11-7085 (5 mg/kg) three times weekly plus cisplatin (5 mg/kg) once a week for 4 weeks. A second group of mice (n = 6) was treated with BAY 11-7085 alone (5 mg/kg) three times weekly for 4 weeks. A third group (n = 6) was treated with cisplatin alone (5 mg/kg) once a week for 4 weeks. The remainder of the mice (n = 6) received vehicle (PBS) alone. Abdominal circumference and body weight were measured twice weekly. At the end of the experiment, mice underwent euthanasia with CO2. The volume of ascites was measured, and tumor tissue was excised and fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin sections (5 µm) were used for histochemical analysis.

Immunohistochemistry—Formalin-fixed, paraffin-embedded tissues were used. Apoptosis was assessed by TUNEL staining using an ApopTag® Plus peroxidase in situ apoptosis kit according to the manufacturer's protocol. For analysis of expression of phospho-I{kappa}B{alpha} and localization of NF{kappa}B p65, slides were incubated in methanol with 0.3% hydrogen peroxidase to eliminate endogenous peroxidase activity. Sections were stained by the immunoperoxidase method with the streptavidin-biotin (SAB) complex system (Nichirei, Tokyo, Japan). The anti-phospho-I{kappa}B{alpha} and anti-NF{kappa}B p65 antibodies were used at a 1:50 dilution. Background reactivity for TUNEL was determined by processing slides in the absence of terminal deoxynucleotidyltransferase (negative control). For determination of TUNEL expression in tissue sections, we counted the number of apoptotic events in five random fields at x400 magnification and divided that number by the total number of cells per field. Control samples for phospho-I{kappa}B{alpha} and NF{kappa}B p65 staining exposed to secondary antibody alone showed no nonspecific staining.

Statistics—Statistical analysis was performed using one-way analysis of variance followed by Fisher's least significant difference test, and p < 0.05 was considered significant. Data are expressed as the mean ± S.E.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Differences of I{kappa}B{alpha} Phosphorylation and NF{kappa}B Activity Depending on the Sensitivity to Cisplatin—The sensitivity to cisplatin of A2780 and Caov-3 cells was examined using the MTS assay (Fig. 1A). It was first confirmed thereby that A2780 cells are sensitive and Caov-3 cells are resistant to cisplatin, as reported previously (1, 35). Since constitutive phosphorylation of I{kappa}B{alpha} together with increased NF{kappa}B activity has been reported to reduce sensitivity to chemotherapeutic drugs and is associated with invasive behavior of cancer cells (38), we next compared the I{kappa}B{alpha} phosphorylation (Fig. 1B) and NF{kappa}B activity (Fig. 1C) between cisplatin-sensitive A2780 cells and cisplatin-resistant Caov-3 cells. Cells were treated with 200 µM cisplatin for 30 min and used to prepare lysates that were analyzed by Western blotting with anti-phospho-I{kappa}B{alpha} (Fig. 1B, panel ii), anti-I{kappa}B{alpha} (Fig. 1B, panel iii), or anti-{beta}-actin antibody (Fig. 1B, panel iv). Although the basal expression of {beta}-actin did not differ between A2780 and Caov-3 cells (Fig. 1B, panel iv), the basal level of phosphorylation of I{kappa}B{alpha} in Caov-3 cells was significantly higher than that in A2780 cells (Fig. 1B, panels i and ii). Although cisplatin caused a decrease in the level of phosphorylated I{kappa}B{alpha} in both Caov-3 and A2780 cells, the degree of the decrement of phosphorylated I{kappa}B{alpha} by cisplatin in A2780 cells was more marked than that in Caov-3 cells (Fig. 1B, panels i and ii). Total levels of I{kappa}B{alpha} did not differ among the lanes (Fig. 1B, panel iii), suggesting that cisplatin had no effect on the degradation of I{kappa}B{alpha} in either Caov-3 or A2780 cells. To assess the NF{kappa}B activity, cells were transfected with a NF{kappa}B-luciferase reporter plasmid. The basal NF{kappa}B activity in Caov-3 cells was significantly higher than that in A2780 cells (Fig. 1C). Although cisplatin caused a decrease in NF{kappa}B activity in both Caov-3 and A2780 cells, the degree of the decrement of NF{kappa}B activity caused by cisplatin in A2780 cells was more marked than that in Caov-3 cells (Fig. 1C). These results suggest that both high basal levels of phosphorylation of I{kappa}B{alpha} and activation of NF{kappa}B and weaker decrements of the phosphorylation of I{kappa}B{alpha} and activation of NF{kappa}B by cisplatin seem to reduce the sensitivity of cells to cisplatin.



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  		FIG. 1.
Differences of I{kappa}B{alpha} phosphorylation and NF{kappa}B activity depending on the sensitivity to cisplatin. A, Caov-3 or A2780 cells were treated with the indicated concentrations of cisplatin. Twenty-four hours later, cell viability was assessed by the MTS assay as described under "Experimental Procedures." Significant differences from Caov-3 cells are indicated by asterisks. **, p < 0.01. B, Caov-3 or A2780 cells were treated with 200 µM cisplatin for 30 min. Cell lysates were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with anti-phospho-I{kappa}B{alpha} (panel ii), anti-I{kappa}B{alpha} (panel iii), or anti-{beta}-actin (panel iv) antibody. The positions of molecular mass markers are noted on the left. Relative densitometric units of the phospho-I{kappa}B{alpha} bands are shown in panel i with the density of the control bands in Caov-3 cells set arbitrarily at 1.0. Values shown represent the mean ± S.E. from at least three separate experiments. Significant differences are indicated by asterisks. **, p < 0.01. C, Caov-3 or A2780 cells were transfected with pElam-luc. After transfection, the cells were incubated with 100 µM cisplatin. Six hours later, cell pellets were collected and used to prepare lysates that were subjected to luciferase assays. The transcriptional activity of each plasmid was normalized with respect to that of the vehicle control of Caov-3 cells taken as 1.0. Values shown represent the mean ± S.E. from at least three separate experiments. Significant differences are indicated by asterisks. **, p < 0.01.

 
Effect of Inhibition of NF{kappa}B Activity on Cisplatin-induced Apoptosis—We next examined whether inhibition of NF{kappa}B activity increased the efficacy with which cisplatin induced the attenuation of I{kappa}B{alpha} phosphorylation and NF{kappa}B activity and also induced apoptosis. We confirmed that treatment with an I{kappa}B{alpha} phosphorylation inhibitor (BAY 11-7085) (32) attenuated both the phosphorylation of I{kappa}B{alpha} (Fig. 2A, ii, panels a and b) and NF{kappa}B activity (Fig. 2B, panel i), and a specific NF{kappa}B nuclear translocation inhibitor (SN-50) attenuated the NF{kappa}B activity (Fig. 2B, panel ii). In addition, treatment with BAY 11-7085 increased the efficacy with which cisplatin induced the attenuation of I{kappa}B{alpha} phosphorylation (Fig. 2A, iii, panels a and b) and NF{kappa}B activity (Fig. 2B, panel iii), whereas treatment with SN-50 increased the efficacy with which cisplatin induced the attenuation of NF{kappa}B activity (Fig. 2B, panel iii). Total levels of I{kappa}B{alpha} did not differ among the lanes (Fig. 2A, panel c), like the basal expression of {beta}-actin (Fig. 2A, panel d), suggesting that cisplatin and/or BAY 11-7085 had no effect on the degradation of I{kappa}B{alpha}. Furthermore, we examined the effect of another NF{kappa}B inhibitor on the cisplatin-induced attenuation of NF{kappa}B activity. The best characterized species of NF{kappa}B dimer is the p50/p65 heterodimer (39). A previous report demonstrated that the NLS polypeptide (p50) is required for the translocation of the dimer to the nucleus (40) and that p50{Delta}NLS lacking the NLS domain inhibits the nucleocytoplasmic shuttling of NF{kappa}B dimers. Therefore, we examined the effect of p50{Delta}NLS on the cisplatin-induced attenuation of NF{kappa}B activity (Fig. 2B, panel iv). Transfection of p50{Delta}NLS significantly increased the efficacy with which cisplatin induced the attenuation of NF{kappa}B activity compared with the effects in cells expressing wild-type p50 (Fig. 2B, panel iv). Thus, these data indicated that NF{kappa}B inhibitors increased the efficacy with which cisplatin induced the attenuation of NF{kappa}B activity.



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  		FIG. 2.
Effect of inhibitors of NF{kappa}B activity on cisplatin-induced apoptosis. A, Caov-3 cells were treated with 200 µM cisplatin (panel i), 5 µM BAY 11-7085 (panel ii), or 200 µM cisplatin+ 5 µM BAY 11-7085 (panel iii) for the indicated times. Cell lysates were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with anti-phospho-I{kappa}B{alpha} antibody (panel b), anti-I{kappa}B{alpha} antibody (panel c), or anti-{beta}-actin (panel d) antibody. The positions of molecular mass markers are noted on the left. Relative densitometric units of the phospho-I{kappa}B{alpha} bands are shown in panel a, with the density of the control bands set arbitrarily at 1.0. Values shown represent the mean ± S.E. from at least three separate experiments. Significant differences are indicated by asterisks. **, p < 0.01. B, Caov-3 cells were transfected with pElam-luc. After transfection, the cells were incubated with the indicated concentrations of BAY-11-7085 (panel i), SN50 (panel ii), or the indicated concentrations of cisplatin ± 5 µM BAY 11-7085 or 10 µM SN50 (panel iii) for 6 h. Caov-3 cells were cotransfected with pElam-luc and pCR-FLAG-p50 or pCR-FLAG-p50{Delta}NLS. After transfection, the cells were incubated with the indicated concentrations of cisplatin (panel iv). Six hours later, cell pellets were collected and used to prepare lysates that were subjected to luciferase assays. The transcriptional activity of each plasmid was normalized with respect to that of vehicle-treated cells taken as 1.0. Values shown represent the mean ± S.E. from at least three separate experiments. Significant differences are indicated by asterisks. **, p < 0.01. C, Caov-3 cells were treated with the indicated concentrations of cisplatin ± 5 µM BAY 11-7085 or 10 µM SN50. Twenty-four hours later, cell viability was assessed by the MTS assay as described under "Experimental Procedures." Significant differences from the values in cells treated with cisplatin alone are indicated by asterisks. **, p < 0.01. D, Caov-3 cells were treated with 200 µM cisplatin ± 5 µM BAY 11-7085 (panel i) or 200 µM cisplatin ± 10 µM SN50 (panel ii) for 24 h. Lysates (250 µg of protein) were subjected to Western blotting using anti-PARP (panel b), anti-cleaved PARP (panel c), or anti-{beta}-actin (panel d) antibody. The positions of molecular mass markers are noted on the left. Relative densitometric units of the cleaved PARP bands (panels b and c) are shown in panel a, with the density of the vehicle bands set arbitrarily at 1.0. Values shown represent the mean ± S.E. from at least three separate experiments. Significant differences are indicated by asterisks. **, p < 0.01.

 
In the presence of BAY 11-7085 or SN-50, the ability of cisplatin to induce growth inhibition was significantly enhanced (Fig. 2C). Moreover, we examined the effects of BAY 11-7085 (Fig. 2D, panel i) and SN-50 (Fig. 2D, panel ii) on the cisplatin-induced cleavage of PARP by Western blotting with anti-PARP antibody (Fig. 2D, panel b), anti-cleaved PARP antibody (Fig. 2D, panel c), or anti-{beta}-actin antibody (Fig. 2D, panel d). Cisplatin induced the cleavage of PARP (Fig. 2D, panels a, b, and c). BAY 11-7085 (Fig. 2D, i, panels a, b, and c) and SN-50 (Fig. 2D, ii, panels a, b, and c) significantly enhanced the ability of cisplatin to induce the cleavage of PARP, whereas the expression of {beta}-actin was not changed by any of these treatments (Fig. 2D, panel d).

Effect of Inhibition of NF{kappa}B Activity on the Cisplatin-induced Attenuation of the Expression of Survival Genes—NF{kappa}B regulates the expression of a number of antiapoptotic genes (41-43). Among them are the family of inhibitor of apoptosis proteins (IAPs), which play a central role in repressing caspase-mediated cell death. It was reported that cisplatin inhibits the expression of XIAP (44) and that down-regulation of XIAP induces apoptosis and increases cisplatin sensitivity (45), suggesting that XIAP is a determinant of cisplatin sensitivity in ovarian cancer. Therefore, we examined the effect of cisplatin and BAY 11-7085 alone and in combination on the expression of XIAP. Although the expression of {beta}-actin did not vary among the lanes, both cisplatin and BAY 11-7085 partially attenuated the expression of XIAP, and co-treatment with cisplatin plus BAY 11-7085 completely inhibited the expression of XIAP (Fig. 3).



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  		FIG. 3.
BAY 11-7085 enhances the cisplatin-induced attenuation of the expression of survival genes. Caov-3 cells were treated with the indicated concentrations of cisplatin (lanes 2 and 3), BAY 11-7085 (lanes 5 and 6), or 5 µM BAY 11-7085 + the indicated concentrations of cisplatin (lanes 8 and 9) for 24 h. Lysates (250 µg of protein) were subjected to Western blotting using anti-XIAP (middle panel) or anti-{beta}-actin (lower panel) antibody. The positions of the molecular mass markers are noted on the left. Relative densitometric units of the XIAP bands are shown in the top panel, with the density of the vehicle bands set arbitrarily at 1.0. Values shown represent the mean ± S.E. from at least three separate experiments. Significant differences are indicated by asterisks. **, p < 0.01.

 
Effect of Inhibition of NF{kappa}B Activity on the Cisplatin-induced Attenuation of Invasion of Caov-3 Cells through Matrigel—Because it was reported that NF{kappa}B is involved in invasiveness and metastatic properties (46), we examined the effects of cisplatin and BAY 11-7085 alone and in combination on the invasion of Caov-3 cells through Matrigel. Under laboratory conditions, Caov-3 cells migrated through Matrigel. Whereas either cisplatin or BAY 11-7085 partially inhibited cell invasion through Matrigel, co-treatment with cisplatin plus BAY 11-7085 completely inhibited cell invasion through Matrigel (Fig. 4).



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  		FIG. 4.
BAY 11-7085 enhances the cisplatin-induced attenuation of invasion. Caov-3 cells were treated with 200 µM cisplatin, 5 µM BAY 11-7085, or 200 µM cisplatin+ 5 µM BAY 11-7085 for 24 h. The cells (5 x 104) were released from their culture dishes and placed on Matrigel as described under "Experimental Procedures." The relative number of cells that penetrated through Matrigel is shown, with the number of penetrating cells in the vehicle control set arbitrarily at 1.0 (100%). Values shown represent the mean ± S.E. from at least three separate experiments. Significant differences are indicated by asterisks. **, p < 0.01.

 
Effect of BAY 11-7085 on the Cisplatin-induced Inhibition of Intra-abdominal Dissemination of Ovarian Cancer—Ovarian cancer is clinically silent and half of the patients are first detected at an advanced stage with ascites and peritoneal dissemination (47). Because peritoneal dissemination is the main process of progression in ovarian cancer and the amount of ascitic fluid and the size of the disseminated tumor are correlated with the patient's prognosis (48), controlling ascitic fluid and peritoneal dissemination are crucial in ovarian cancer therapy. We therefore examined the effect of cisplatin and BAY 11-7085 alone and in combination on the control of intra-abdominal dissemination of ovarian cancer and ascites formation to assess whether combination therapy would increase the therapeutic efficacy of each agent. Athymic nude mice were inoculated (i.p.) with Caov-3 cells or growth medium. Two weeks after inoculation, athymic nude mice inoculated (i.p.) with Caov-3 cells were randomized into four groups treated with the following for 4 weeks: (a) vehicle (PBS), (b) cisplatin (5 mg/kg) once a week, (c) BAY 11-7085 (5 mg/kg) three times a week, and (d) cisplatin (5 mg/kg) once a week + BAY 11-7085 (5 mg/kg) three times a week. The appearance of the mice is shown in Fig. 5A. The volume of ascites was measured at autopsy (Fig. 5B). The volume of ascites was significantly higher in athymic nude mice inoculated (i.p.) with Caov-3 cells than in athymic nude mice inoculated (i.p.) with growth medium. Pathological examination was performed to determine the extent of intra-abdominal dissemination at autopsy. Intra-abdominal dissemination was clearly detected in athymic nude mice inoculated (i.p.) with Caov-3 cells followed by treatment with vehicle (Fig. 6, A, panel a, and B) and the intra-abdominal dissemination was confirmed by the histological findings to consist of mucinous cystoadenocarcinoma (Fig. 6C), which is consistent with Caov-3 cells. Cisplatin alone (Fig. 5B, panel b) or BAY 11-7085 alone (Fig. 5B, panel c) significantly diminished the volume of ascites compared with vehicle control. The combination of cisplatin + BAY 11-7085 (Fig. 5B, panel d) further enhanced the inhibitory effects on the production of ascites. Cisplatin alone (Fig. 6A, panel b) or BAY 11-7085 alone (Fig. 6A, panel c) also apparently diminished the extent of intra-abdominal dissemination. The combination of cisplatin + BAY 11-7085 (Fig. 6A, panel d) further enhanced each inhibitory effect on intra-abdominal dissemination. These results suggest that combination therapy of cisplatin with BAY 11-7085 would increase the therapeutic efficacy of cisplatin.



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  		FIG. 5.
Appearance of and ascites formation in mice after treatment with cisplatin, BAY 11-7085 alone, or the combination thereof. Athymic nude mice were inoculated (i.p.) with Caov-3 cells or growth medium (Control). Two weeks after inoculation, athymic nude mice inoculated (i.p.) with Caov-3 cells were randomized into four groups and treated as follows for 4 weeks: panel a, vehicle alone (PBS); panel b, cisplatin (5 mg/kg) once a week; panel c, BAY 11-7085 (5 mg/kg) three times a week; and panel d, cisplatin (5 mg/kg) once a week + BAY 11-7085 (5 mg/kg) three times a week. Representative mice are shown in A. At autopsy, the volume of ascites was measured (B).

 



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  		FIG. 6.
BAY 11-7085 enhances the cisplatin-induced inhibition of intra-abdominal dissemination. A, athymic nude mice were inoculated (i.p.) with Caov-3 cells. Two weeks after inoculation, athymic nude mice inoculated (i.p.) with Caov-3 cells were randomized into four groups and treated as follows for 4 weeks: panel a, vehicle alone (PBS); panel b, cisplatin (5 mg/kg) once a week; panel c, BAY 11-7085 (5 mg/kg) three times a week; and panel d, cisplatin (5 mg/kg) once a week + BAY 11-7085 (5 mg/kg) three times a week. At autopsy, pathological examination was performed to determine the extent of intra-abdominal dissemination. B, magnified view of intra-abdominal dissemination of athymic nude mice inoculated (i.p.) with Caov-3 cells followed by treatment with vehicle (PBS) alone. Panel i, metastasis to the liver; panel ii, metastasis to the kidney; panels iii and iv, visceral peritoneal dissemination; panel v, parietal peritoneal dissemination. C, histological findings (x200 magnification) of hematoxylin and eosin staining of parietal peritoneal dissemination of athymic nude mice inoculated (i.p.) with Caov-3 cells followed by treatment with vehicle alone (PBS).

 
Intra-abdominally disseminated tumors harvested from the different groups were processed for immunohistochemical analyses. Apoptosis was analyzed by the TUNEL method (Fig. 7). The fraction of TUNEL-positive cells was higher in tumors from mice treated with cisplatin + BAY 11-7085 than in tumors from mice treated with vehicle alone (Fig. 7A). Apoptosis was quantitated in tumors of mice treated with vehicle alone (panel a), cisplatin alone (panel b), BAY 11-7085 alone (panel c), and cisplatin + BAY 11-7085 (panel d) (Fig. 7B) as described for the experiment shown in Fig. 5. Cisplatin alone (Fig. 7B, panel b) or BAY 11-7085 alone (Fig. 7B, panel c) significantly increased the proportion of apoptotic cells in tumors compared with vehicle alone (Fig. 7B, panel a). The combination of cisplatin + BAY 11-7085 (Fig. 7B, panel d) further enhanced the increase of the proportion of apoptotic cells in tumors. We further confirmed whether BAY 11-7085 blocked the NF{kappa}B cascade in vivo. I{kappa}B{alpha} phosphorylation status and subcellular localization of NF{kappa}B p65 were assessed by immunohistochemistry with anti-phospho-I{kappa}B{alpha} antibody (Fig. 8A) and anti-NF{kappa}B p65 antibody (Fig. 8B), respectively. Immunoreactivity for phosphorylated I{kappa}B{alpha} was found to be prominent in the cytoplasm in tumors treated with vehicle alone and was apparently reduced in tumors treated with BAY 11-7085 (Fig. 8A), suggesting that BAY 11-7085 inhibited the I{kappa}B{alpha} phosphorylation in vivo. Whereas immunoreactivity for NF{kappa}B p65 in tumors treated with vehicle alone was observed in both the cytoplasm and nuclei, immunoreactivity for NF{kappa}B p65 in tumors treated with BAY 11-7085 was only observed in the cytoplasm (Fig. 8B), suggesting that BAY 11-7085 inhibited the partial nuclear translocation of NF{kappa}B p65 in vivo. These results confirmed the appropriate drug targeting in vivo.



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  		FIG. 7.
Analysis of apoptosis in tumors growing intra-abdominally in athymic nude mice. Athymic nude mice were inoculated (i.p.) with Caov-3 cells. Two weeks after inoculation, athymic nude mice inoculated (i.p.) with Caov-3 cells were randomized into four groups and treated as follows for 4 weeks: panel a, vehicle alone (PBS); panel b, cisplatin (5 mg/kg) once a week; panel c, BAY 11-7085 (5 mg/kg) three times a week; and panel d, cisplatin (5 mg/kg) once a week + BAY 11-7085 (5 mg/kg) three times a week as described for the experiment shown in Fig. 5. A, at autopsy, tumors growing intra-abdominally were excised and stained by immunohistochemistry for TUNEL. Representative areas are shown (x400 magnification). B, TUNEL-positive cells were expressed as a percentage of total cells. Values shown represent the mean ± S.E. from evaluation of five random areas at x400 magnification. Significant differences are indicated by asterisks. **, p < 0.01.

 



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  		FIG. 8.
BAY 11-7085 blocks the NF{kappa}B cascade in tumors growing intra-abdominally in athymic nude mice. Athymic nude mice were inoculated (i.p.) with Caov-3 cells. Two weeks after this inoculation, mice were treated with vehicle alone (PBS) (left panel) or BAY 11-7085 (5 mg/kg) three times a week (right panel). At autopsy, tumors growing intra-abdominally were excised, and I{kappa}B{alpha} phosphorylation status and subcellular localization of NF{kappa}B p65 were assessed by immunohistochemistry with anti-phospho-I{kappa}B{alpha} antibody (A) and anti-NF{kappa}B p65 antibody (B), respectively. Scale bars, 15 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The form of chemotherapy used might determine whether NF{kappa}B has a pro- or antiapoptotic effect (10). For example, the majority of chemotherapies induce some form of DNA damage, and inhibition of NF{kappa}B activation promotes cell death. However, it was proposed recently that NF{kappa}B might be required for Taxol-induced cell death (49). This is consistent with studies that suggest that NF{kappa}B can function proapoptotically depending on the type of stimulus to which cells are exposed. For example, it has been reported that the induction of cell death by hydrogen peroxide requires NF{kappa}B and that NF{kappa}B is required for p53-dependent cell death (50). More studies will be needed to determine the chemotherapy specificity and potential tumor specificity of NF{kappa}B in controlling cancer therapy efficacy.

We reported that Akt inactivation and inhibition of BAD phosphorylation sensitize human ovarian cancer cells to cisplatin (1) and paclitaxel (2). In addition, we recently demonstrated that inhibition of Forkhead phosphorylation sensitizes human ovarian cancer cells to cisplatin. Although NF{kappa}B is a substrate of Akt (like BAD and Forkhead), NF{kappa}B activation is involved in angiogenesis (11, 12) and metastasis (13, 14) in addition to the suppression of apoptosis. Therefore, NF{kappa}B inhibitors might increase the efficacy of chemotherapies in both primary and metastatic lesions. It was reported that NF{kappa}B inhibitors induce adhesion-dependent colon cancer apoptosis (52). We showed in this study that treatment of athymic mice with BAY 11-7085 enhanced the ability of cisplatin to inhibit tumor implantation into the liver and peritoneum (Fig. 6A). In addition, BAY 11-7085 increased the ability of cisplatin to inhibit both cell proliferation in an MTS assay (Fig. 2C) and cellular invasion in an in vitro invasion assay (Fig. 4). Thus, NF{kappa}B inhibitors might increase the efficiency with which cisplatin inhibits both primary and metastatic lesions. Glycogen synthase kinase-3{alpha} (53) and endothelial nitric-oxide synthase (54, 55) are also other Akt substrates, and Akt is thus also involved in metabolic processes and vessel dilatation, respectively. Therefore, it is possible that inhibition of PI3K/Akt activation is not a safe strategy for preventing chemoresistance. Accordingly, NF{kappa}B inhibitors might be more useful for sensitization to chemotherapeutic drugs than agents that are able to inhibit PI3K/Akt activity.

Constitutive activation of NF{kappa}B has been described in a great number of solid tumors, and this activation appears to support cancer cell survival and to reduce the sensitivity to chemotherapeutic drugs. We showed in this study that whereas cisplatin-sensitive A2780 cells do not have constitutive activation of NF{kappa}B, cisplatin-resistant Caov-3 cells do have constitutive activation of NF{kappa}B. Thus, it appears that constitutive activation of NF{kappa}B mediates cisplatin resistance in ovarian cancer cells and inhibition of NF{kappa}B activation sensitizes the ovarian cancer cells to cisplatin.

How do NF{kappa}B inhibitors cause the inhibition of growth of human ovarian cancer cells? It was reported that NF{kappa}B inhibitors diminished the expression of survival genes regulated by NF{kappa}B, such as c-IAP-2, TRAF-1, TRAF-2, XIAP, or IEX-1L (42, 43). We also showed that NF{kappa}B inhibitors caused the inhibition of the expression of survival genes in human ovarian cancer cells (Fig. 3). The fact that NF{kappa}B mediates the expression of multiple survival genes makes it an important and rational target for cancer chemotherapy.

BAY 11-7082 is also a known pharmacological inhibitor of I{kappa}B{alpha} phosphorylation (32), like BAY 11-7085. In the presence of BAY 11-7082, the cisplatin-induced attenuation of I{kappa}B{alpha} phosphorylation was significantly enhanced (data not shown). BAY 11-7082 and BAY 11-7085 also activate the c-Jun N-terminal protein kinase and p38 (32), both of which are known to be involved in the induction of apoptosis (56). Thus, the effects of BAY 11-7082 and BAY 11-7085 do not exclude the role of cellular proteins other than NF{kappa}B. Five homologous polypeptides, p50, p65, c-Rel, RelB, and p52, comprise the mammalian Rel/NF{kappa}B transcription factor family. The subunits associate in a combinatorial fashion to form transcriptionally active homo- and heterodimers. The best characterized species of NF{kappa}B dimer is the p50/p65 heterodimer (39). A previous report demonstrated that the NLS polypeptide of p50 is required for its translocation to the nucleus (40) and that p50{Delta}NLS lacking the NLS domain inhibits the nucleocytoplasmic shuttling of NF{kappa}B dimers. Therefore, we examined the effect of p50{Delta}NLS on the attenuation of I{kappa}B{alpha} phosphorylation by cisplatin. Transfection of p50{Delta}NLS significantly inhibited the increased the efficacy of the cisplatin-induced attenuation of NF{kappa}B activity compared with the effect in cells expressing wild-type p50 (Fig. 2B, panel iv). Thus, the similarity of the effects caused by treatment with BAY 11-7082 or BAY 11-7085 and by transfection with p50{Delta}NLS suggests that inhibition of NF{kappa}B activity has strong potential as a novel adjuvant chemotherapy.

Most studies of the inhibition of NF{kappa}B activity in vivo have used a gene therapy approach through the introduction of a nondegradable I{kappa}B mutant that prevents nuclear translocation of NF{kappa}B. The advantage of soluble inhibitors is that their delivery would be easier and more efficient than gene transfer in vivo. We did not detect significant renal, hepatic, or pulmonary tissue toxicity in this study. A previous study also showed that using up to 20 mg/kg/day of these agents in rats for 21 days did not cause obvious toxicity (32).

Activation of NF{kappa}B via phosphorylation of an inhibitor protein (I{kappa}B) leads to degradation of I{kappa}B through the ubiquitin-proteasome pathway. Inhibition of I{kappa}B degradation by proteasome inhibitors keeps NF{kappa}B in the cytoplasm, thereby preventing it from acting on nuclear DNA (57, 58). PS-341, which is a potent, boronic acid dipeptide that is highly selective for proteasome inhibition, can be systemically administered clinically (59). PS-341 has been shown to enhance the apoptotic response to chemotherapy in a variety of in vitro and in vivo models (18, 60-62). A Phase I trial of PS-341 and carboplatin in recurrent ovarian cancer is currently ongoing (51). A Phase II trial of PS-341 for the treatment of recurrent platinum-sensitive ovarian or primary or peritoneal cancer (GOG 146-N) is also being conducted. We now await the results of these currently ongoing clinical trials.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-3354; Fax: 81-6-6879-3359; E-mail: masa{at}med.id.yamagata-u.ac.jp.

1 The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; NF{kappa}B, nuclear factor-{kappa}B; I{kappa}B{alpha}, inhibitor of NF{kappa}B; PBS, phosphate-buffered saline; MTS, 3-[4,5,dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt; PARP, poly-(ADP-ribose) polymerase; BAD, Bcl-2-associated death protein; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; IAP, inhibitor of apoptosis protein; XIAP, X-linked IAP; i.p., intraperitoneally; NLS, nuclear localization signal. Back


   ACKNOWLEDGMENTS
 
We thank Dr. J. Cheng (University of South Florida College of Medicine) for providing the NF{kappa}B reporter plasmid (pElam-luc), and Dr. Gourisankar Ghosh (Department of Chemistry and Biochemistry, University of California, San Diego) for providing pCR-FLAG-p50 and pCR-FLAG-p50{Delta}NLS constructs. We are also grateful to Dr. Junko Takeda and Dr. Kazue Onuma for technical assistance and Ayako Okamura and Tomoko Iwaki for secretarial assistance.



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