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Clinical Cancer Research Vol. 10, 7645-7654, November 15, 2004
© 2004 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Inhibition of Inhibitor of Nuclear Factor-{kappa}B Phosphorylation Increases the Efficacy of Paclitaxel in in Vitro and in Vivo Ovarian Cancer Models

Seiji Mabuchi1,4, Masahide Ohmichi1,2, Yukihiro Nishio1, Tadashi Hayasaka2, Akiko Kimura1, Tsuyoshi Ohta2, Jun Kawagoe2, Kazuhiro Takahashi2, Namiko Yada-Hashimoto1, Hozumi Seino-Noda1, Masahiro Sakata1, Teiichi Motoyama3, Hirohisa Kurachi2, Joseph R. Testa4, Keiichi Tasaka1 and Yuji Murata1

1 Department of Obstetrics and Gynecology, Osaka University Medical School, Osaka, Japan; Departments of 2 Obstetrics and Gynecology and 3 Pathology, Yamagata University, School of Medicine, Yamagata, Japan; and 4 Human Genetics Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We investigated whether inhibition of nuclear factor-{kappa}B (NF{kappa}B) increases the efficacy of paclitaxel in in vitro and in vivo ovarian cancer models. Treatment of paclitaxel-sensitive Caov-3 cells with paclitaxel transiently activated the phosphorylation of Akt, the phosphorylation of I{kappa}B kinase (IKK), and the phosphorylation of inhibitor of NF{kappa}B (I{kappa}B{alpha}). Paclitaxel also caused a transient increase in NF{kappa}B activity, followed by a decrease in NF{kappa}B activity. We show an association between Akt and IKK and show that the phosphorylation of IKK induced by paclitaxel is blocked by treatment with a phosphatidylinositol 3-kinase inhibitor (wortmannin or LY294002). Furthermore, interference of the Akt signaling cascade inhibits the transient induction of I{kappa}B{alpha} phosphorylation and NF{kappa}B activity by paclitaxel. Inhibition of NF{kappa}B activity by treatment with an I{kappa}B{alpha} phosphorylation inhibitor (BAY 11-7085) attenuated both basal and transient induction of I{kappa}B{alpha} phosphorylation by paclitaxel. Treatment with BAY 11-7085 also enhanced the inhibition of NF{kappa}B activity by paclitaxel for up to 24 hours. In addition, treatment with BAY 11-7085 decreased the viability of cells treated with paclitaxel. Moreover, treatment with BAY 11-7085 increased the efficacy of paclitaxel-induced inhibition of intraabdominal dissemination and production of ascites in athymic nude mice inoculated intraperitoneally with Caov-3 cells. These results suggest that paclitaxel transiently induces NF{kappa}B activity via the phosphatidylinositol 3-kinase/Akt cascade and that combination therapy with paclitaxel and an NF{kappa}B inhibitor would increase the therapeutic efficacy of paclitaxel.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Paclitaxel, a natural product originally isolated from the bark of Taxus brevifolia, has significant antitumor activity against several human cancers, particularly advanced ovarian and breast carcinomas (1 , 2) . Unlike other antimicrotubule agents that interfere with tubulin polymerization, paclitaxel increases tubulin polymerization, stabilizes microtubules, and prevents tubulin depolymerization, ultimately causing tubulin bundling (3, 4, 5) . These effects of the drug are associated with cell cycle arrest in G2-M phase of the cell cycle, as well as cellular toxicity (5, 6, 7) . The inclusion of paclitaxel in the treatment of patients with newly diagnosed ovarian cancer has led to improved response rates and prolonged median survival compared with the results of prior therapeutic regimens (8) . Nevertheless, most of patients with advanced ovarian cancer are destined to relapse and develop resistance to initially active drugs such as paclitaxel (9) .

The sensitivity of cells to chemotherapeutic drug-induced apoptosis seems to depend on the balance between proapoptotic and antiapoptotic signals. Therefore, it is possible that antiapoptotic signals such as the phosphatidylinositol 3-kinase (PI3K)/Akt survival cascade are involved in the sensitivity to chemotherapeutic drugs. We reported that Akt inactivation sensitizes human ovarian cancer cells to cisplatin (10) and paclitaxel (11) , suggesting that Akt inactivation could be a hallmark of 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 (12 , 13) . Akt also phosphorylates and inactivates the Forkhead transcription factors, resulting in reduced expression of the cell cycle inhibitor p27Kip1 and the Fas ligand (14, 15, 16) . Via the phosphorylation of I{kappa}B kinase (IKK), Akt also activates nuclear factor-{kappa}B (NF{kappa}B), a transcription factor that has been implicated in cell survival (17 , 18) .

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 inhibitor of NF{kappa}B (I{kappa}B{alpha}). Phosphorylation of I{kappa}B{alpha} by IKK promotes its degradation, allowing NF{kappa}B to translocate to the nucleus and induce target genes (15 , 16) . The transcriptional activation of genes associated with cell proliferation (19) , angiogenesis (20 , 21) , metastasis (22 , 23) , and suppression of apoptosis (24) seems to lie at the heart of the ability of NF{kappa}B to promote oncogenesis (25) and cancer therapy resistance (26 , 27) . Although it was proposed that NF{kappa}B might be required for paclitaxel-induced cell death (28) , most reports suggest that paclitaxel-induced NF{kappa}B activity mediates survival signals that counteract apoptosis (29, 30, 31) . 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) . Therefore, several agents that are able to inhibit NF{kappa}B function might be considered as an adjuvant approach in combination with paclitaxel for lung cancer (30) , prostate cancer (31) , pancreatic cancer (32) , and breast cancer (33) .

These considerations led us to examine whether the status of NF{kappa}B activity is involved in the sensitivity to paclitaxel in human ovarian cancer cells and whether agents that are able to inhibit NF{kappa}B function might be considered as an adjuvant approach in combination with paclitaxel for ovarian cancer. In the present study, we show that BAY 11-7085, a known pharmacologic inhibitor of I{kappa}B{alpha} phosphorylation (34) , inhibits both basal and transient induction of I{kappa}B{alpha} phosphorylation and NF{kappa}B activity by paclitaxel for 3 hours. In addition, BAY 11-7085 enhances inhibition of NF{kappa}B activity by paclitaxel for up to 24 hours and increases the efficacy of paclitaxel in in vitro and in vivo ovarian cancer models.


   EXPERIMENTAL PROCEDURES
Top
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
Antiphospho-IKK{alpha} (Ser-180)/IKKß (Ser-181), anti-IKK{alpha}, Antiphospho-I{kappa}B{alpha}, anti-I{kappa}B{alpha}, anticleaved poly(ADP-ribose) polymerase (PARP) and anti-X-linked inhibitor of apoptosis protein (anit-XIAP) antibodies were obtained from Cell Signaling Technology (Beverly, MA). Anti-matrix metalloproteinase-9 (anti-MMP-9) antibody was purchased from Chemicon (Temecula, CA). Anti-ß-actin antibody was purchased from Sigma. (St. Louis, MO). The I{kappa}B{alpha} phosphorylation inhibitor BAY 11-7085 was purchased from Alexis Biochemicals (San Diego, CA). Enhanced chemiluminescence Western blotting detection reagents were obtained from Amersham (Arlington Heights, IL). The Cell Titer 96-well proliferation assay was obtained from Promega (Madison, WI).

Cell Cultures.
The human ovarian papillary adenocarcinoma cell line Caov-3 was obtained from American Type Culture Collection (Rockville, MD). The cells were cultured at 37°C in DMEM 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; ref. 35 ).

Proliferation Assay.
Cell proliferation (36) was assessed by the addition of paclitaxel at the indicated concentrations for 48 hours 1 day after seeding test cells into 96-well plates. The number of surviving cells was determined 24 hours later by determination of A490 of the dissolved formazan product after the addition of MTS {3-[4,5,dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt}for 1 hour 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 paclitaxel treatment to that without treatment.

Western Blotting.
Cells were incubated without serum for 16 hours and then treated with various agents. They were then washed twice with PBS and lysed in ice-cold HNTG buffer [50 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 10% glycerol, 1% Triton X-100, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, 10 mmol/L sodium PPI, 100 µmol/L sodium orthovanadate, 100 mmol/L NaF, 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride; ref. 11 ). The lysates were centrifuged at 12,000 x g at 4°C for 15 minutes, and the protein concentrations of the supernatants were determined with Bio-Rad protein assay reagent. Equal amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blocking was carried out with 10% bovine serum albumin in 1x Tris-buffered saline. Western blotting analyses were doned 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 hours with LipofectAMINE Plus (Life Technologies, Inc. Gaithersburg, MD) according to the manufacturer’s protocol. Cells were treated with various agents, and then harvested and subjected to luciferase assays with the Luciferase Assay System (Promega) as described previously (37) . A plasmid expressing the bacterial ß-galactosidase gene was also cotransfected in each experiment to serve as an internal control for transfection efficiency.

Assay of Invasion through Matrigel.
Polyinylprolidone-free polycarbonate filters (8-µm pore size; Chemotaxicell; KURABO, Osaka, Japan) were coated with a mixture of basement membrane components (Matrigel; 25 µg/filter) and placed in modified Boyden chambers (38) . The cells (5 x 104) were released from their culture dishes by brief exposure to EDTA (1 mmol/L), centrifuged, resuspended in 0.1% BSA-DMEM, 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 hours at 37°C, the cells on the lower surface of the filter were fixed, stained with Mayer’s hematoxylin solution, and enumerated with an ocular micrometer and counting at least 10 fields/filter. All of the experiments were independently done 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 into 5-week-old female nu/nu athymic mice (n = 24). Two weeks after inoculation, one group of mice (n = 6) was treated with BAY 11-7085 (5 mg/kg) 3 times weekly plus paclitaxel (20 mg/kg) 3 times weekly for 4 weeks. A second group of mice (n = 6) was treated with BAY 11-7085 alone (5 mg/kg) 3 times weekly for 4 weeks. A third group (n = 6) was treated with paclitaxel alone (20 mg/kg) 3 times weekly for 4 weeks. A fourth group of the mice (n = 6) received vehicle (PBS) alone. An additional six mice inoculated with growth medium only served as a control group. Abdominal circumference and body weight were measured twice weekly. At the end of the experiment, mice underwent euthanasia with CO2. Final abdominal circumference and volume of ascites were measured, tumor tissue was excised, fixed in 4% paraformaldehyde, and embedded in paraffin. Paraffin sections (5 µm) were used for histochemical analysis.

Statistics.
Statistical analysis was done with one-way ANOVA followed by Fisher’s least significant difference test, and P < 0.05 was considered significant. Data are expressed as the mean ± SEM.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Paclitaxel on IKK Phosphorylation, I{kappa}B{alpha} Phosphorylation, and NF{kappa}B Activity.
We first examined the effect of paclitaxel on the NF{kappa}B signaling cascade. A putative Akt phosphorylation site (39) at amino acids 18–23 in IKK{alpha} has been reported (15) . To examine whether phosphorylated Akt and IKK{alpha} were physically associated, cells were treated with (Fig. 1ACitation , right) or without (Fig. 1ACitation , left) paclitaxel for 3 hours, after which endogenous IKK{alpha} and phosphorylated Akt were immunoprecipitated (Fig. 1A)Citation . Endogenous phosphorylated Akt and endogenous IKK{alpha} were coimmunoprecipitated from cells treated with paclitaxel, as in the case of cells stimulated with platelet-derived growth factor (16) . Next, we examined the effect of paclitaxel on the phosphorylation of Akt and the phosphorylation of IKK (Fig. 1B and C)Citation . Cells were treated with the indicated concentrations of paclitaxel for 30 minutes (Fig. 1B)Citation or 200 nmol/L paclitaxel for the indicated times (Fig. 1C)Citation and used to prepare lysates that were analyzed by Western blotting with antiphospho-IKK{alpha} (Ser-180)/IKKß (Ser-181), anti-IKK{alpha}, antiphospho-Akt, or anti-Akt antibody. Although each expression of IKK{alpha} and Akt was not changed (Fig. 1B and CCitation , iii and v), Caov-3 cells treated with paclitaxel showed a dose-dependent (Fig. 1B)Citation and a transient (Fig. 1C)Citation increase in the phosphorylation of Akt (i and iv) and IKK (i and ii). Pretreatment with either of the PI3K inhibitors LY294002 (Fig. 1DCitation , Lane 3) or wortmannin (Fig. 1DCitation , Lane 4) inhibited the paclitaxel-induced phosphorylation of both IKK (Fig. 1D, iCitation and ii) and Akt (Fig. 1D, i and ivCitation ).



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  		Fig. 1. Effect of paclitaxel on phosphorylation of IKK. A, association between Akt and IKK{alpha}. Serum-deprived cells were treated with or without 200 nmol/L paclitaxel for 3 hours and then harvested and lysed. The lysates were subjected to immunoprecipitation with antirabbit IgG antibody (Lane 1), anti-IKK{alpha} antibody (Lane 2), or antiphospho-Akt antibody (Lane 3). The immunoprecipitates were subjected to Western blotting with antiphospho-Akt antibody (top) or anti-IKK{alpha} (bottom) antibody. The positions of molecular weight markers are noted on the left. Caov-3 cells were treated with the indicated concentrations of paclitaxel (B), 200 nmol/L paclitaxel for the indicated times (C), or pretreated with 50 µmol/L LY294002 for 20 minutes (Lane 3) or 100 nmol/L wortmannin for 15 minutes (Lane 4), and then treated with 200 nmol/L paclitaxel for 30 minutes (D). Cell lysates were subjected to Western blotting by antiphospho-IKK{alpha} (Ser-180)/IKKß (Ser-181; ii), anti-IKK{alpha} (iii), antiphospho-Akt (iv), or anti-Akt (v) antibody. The positions of molecular weight markers are noted on the left. Relative densitometric units of the phospho-IKK bands (open bars) or the phospho-Akt bands (closed bars) are shown (in i) with the density of the control bands in Caov-3 cells set arbitrarily at 1. Values shown represent the mean ± SEM from at least three separate experiments. Significant differences are indicated by asterisks. **, P < 0.01.

 
We next examined whether paclitaxel induces the phosphorylation and degradation of I{kappa}B{alpha} (Fig. 2A)Citation . Cells were treated with 200 nmol/L paclitaxel for the indicated times and used to prepare lysates that were analyzed by Western blotting with antiphospho-I{kappa}B{alpha}, anti-I{kappa}B{alpha}, or anti-ß-actin antibody. Although the expression of ß-actin was not changed (Fig. 2A, ivCitation ), paclitaxel-stimulated Caov-3 cells showed a transient increase in the phosphorylation of I{kappa}B{alpha} (Fig. 2A, i and iiCitation ) and in its subsequent degradation (Fig. 2A, i and iiiCitation ). Pretreatment with either LY294002 (Fig. 2BCitation , Lane 3) or wortmannin (Fig. 2BCitation , Lane 4) inhibited both the paclitaxel-induced phosphorylation of I{kappa}B{alpha} (Fig. 2B, i and iiCitation ) and the degradation of I{kappa}B{alpha} (Fig. 2B, i and iiiCitation ).



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  		Fig. 2. Effect of paclitaxel on phosphorylation and degradation of I{kappa}B{alpha}, and on NF{kappa}B activity. Caov-3 cells were treated with 200 nmol/L paclitaxel for the indicated times (A) or were pretreated with 50 µmol/L LY294002 for 20 minutes (Lane 3) or 100 nmol/L wortmannin for 15 minutes (Lane 4), followed by treatment with 200 nmol/L paclitaxel for 30 minutes (B). Cell lysates were subjected to Western blotting by antiphospho-I{kappa}B{alpha} (ii), anti-I{kappa}B{alpha} (iii), or anti-ß-actin (iv) antibody. The positions of molecular weight markers are noted on the left. Relative densitometric units of the phospho-I{kappa}B{alpha} bands (open bars) or the I{kappa}B bands (closed bars) are shown (in i) with the density of the control bands in Caov-3 cells set arbitrarily at 1. Values shown represent the mean ± SEM from at least three separate experiments. Significant differences are indicated by asterisks. **, P < 0.01. Caov-3 cells were transfected with pElam-luc. After transfection, the cells were incubated with 200 nmol/L paclitaxel for the indicated times (C) or pretreated with 50 µmol/L LY294002 for 20 minutes (Lanes 3 and 4), and then treated with 200 nmol/L paclitaxel for 3 hours (D). 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. Values shown represent the mean ± SEM from at least three separate experiments. Significant differences are indicated by asterisks. **, P < 0.01.

 
To assess NF{kappa}B activity, cells were transfected with an NF{kappa}B-luciferase reporter plasmid and treated with 200 nmol/L paclitaxel for the indicated times. Paclitaxel caused a transient increase in NF{kappa}B activity lasting 2 to 4 hours, followed by a decrease in NF{kappa}B activity thereafter (Fig. 2C)Citation . Pretreatment with LY294002 inhibited the transient up-regulation of NF{kappa}B activity by paclitaxel for 3 hours (Fig. 2D)Citation . Collectively, these results indicate that paclitaxel transiently induces phosphorylation of IKK and I{kappa}B{alpha} as well as activation of NF{kappa}B via a PI3K/Akt cascade, followed by the decrease in NF{kappa}B activity thereafter. As we reported previously (11) , similar findings were also detected in other ovarian cancer cell lines (data not shown).

Inhibition of NF{kappa}B Activity Sensitizes Caov-3 Cells to Paclitaxel.
The involvement of the NF{kappa}B signaling cascade in the paclitaxel-induced inhibition of cell viability was examined with an I{kappa}B{alpha} phosphorylation inhibitor (BAY 11-7085; ref. 34 ). We first confirmed that treatment with BAY 11-7085 attenuated both basal and transient induction of I{kappa}B{alpha} phosphorylation by paclitaxel (Fig. 3A)Citation . Treatment with BAY 11-7085 inhibited the transient induction of NF{kappa}B activity by paclitaxel for 3 hours (Fig. 3B, i)Citation and enhanced the inhibition of NF{kappa}B activity by paclitaxel for 24 hours (Fig. 3B, ii)Citation . Whereas either treatment with paclitaxel for 24 hours (Fig. 3CCitation , right) or treatment with BAY 11-7085 for 24 hours (Fig. 3CCitation , left) inhibited cell viability, cotreatment with paclitaxel plus BAY 11-7085 for 24 hours further enhanced the inhibitory effects on cell viability (Fig. 3CCitation , right).



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  		Fig. 3. Effect of BAY 11-7085 on paclitaxel-induced phosphorylation and degradation of I{kappa}B{alpha}, and on NF{kappa}B activity. A, Caov-3 cells were treated with 200 nmol/L paclitaxel (left), 5 µmol/L BAY 11-7085 (center), or 200 nmol/L paclitaxel plus 5 µmol/L BAY 11-7085 (right) for the indicated times. Cell lysates were subjected to Western blotting by antiphospho-I{kappa}B{alpha} (ii), anti-I{kappa}B{alpha} (iii), or anti-ß-actin (iv) antibody. The positions of molecular weight markers are noted on the left. Relative densitometric units of the phospho-I{kappa}B{alpha} bands (open bars) or the I{kappa}B{alpha} bands (closed bars) are shown (in i) with the density of the control bands set arbitrarily at 1. Values shown represent the mean ± SEM 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 treated with 200 nmol/L paclitaxel and/or 5 µmol/L BAY 11-7085 for 3 hours (i) or 24 hours (ii). 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. Values shown represent the mean ± SEM from at least three separate experiments. Significant differences are indicated by asterisks. **, P < 0.01. C, Caov-3 cells were treated with 5 µmol/L BAY 11-7085 (left) or the indicated concentrations of paclitaxel ± 5 µmol/L BAY 11-7085 (right). Twenty-four hours later, cell viability was assessed by the MTS assay as described in "Materials and Methods." Significant differences from the values in cells treated with paclitaxel alone are indicated by asterisks. **, P < 0.01. D, Caov-3 cells were treated with 200 nmol/L paclitaxel and/or 5 µmol/L BAY 11-7085 for 24 hours. Lysates (50 µg of protein) were subjected to Western blotting by anticleaved PARP (top) or anti-ß-actin (bottom) antibody. The positions of molecular weight markers are noted on the left.

 
We further examined the effect of treatment with paclitaxel for 24 hours and treatment with BAY 11-7085 for 24 hours alone and in combination on apoptosis by Western blotting with anticleaved PARP antibody (Fig. 3D)Citation . Paclitaxel induced cleavage of PARP. BAY 11-7085 seemed to enhance the ability of paclitaxel to induce cleavage of PARP, whereas the expression of ß-actin was not changed by any of these treatments.

Effect of Inhibition of NF{kappa}B Activity on Paclitaxel-Induced Attenuation of the Expression of Survival Genes.
NF{kappa}B regulates the expression of a number of antiapoptotic genes (40, 41, 42) . Among them are the family of inhibitor of apoptosis proteins, which play a central role in repressing caspase-mediated cell death. It was reported that cisplatin inhibits the expression of XIAP (43 , 44) and down-regulation of XIAP induces apoptosis and increases paclitaxel sensitivity (45, 46, 47) , suggesting that XIAP is a determinant of paclitaxel sensitivity in ovarian cancer. Therefore, we examined the effect of paclitaxel and BAY 11-7085 alone and in combination on the expression of XIAP. Although paclitaxel treatment alone had no effect on XIAP expression under these conditions, BAY 11-7085 partially attenuated the expression of XIAP, and cotreatment with paclitaxel plus BAY 11-7085 almost completely abolished the expression of XIAP (Fig. 4)Citation .



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  		Fig. 4. Effect of BAY 11-7085 on the expression of survival genes. Caov-3 cells were treated with the indicated concentrations of 200 nmol/L paclitaxel (Lane 2), 5 µmol/L BAY 11-7085 (Lane 4), or 5 µmol/L BAY 11-7085 + 200 nmol/L paclitaxel (Lane 6) for 24 hours. Lysates (50 µg of protein) were subjected to Western blotting by anti-XIAP (middle) or anti-ß-actin (bottom) antibody. The positions of molecular weight markers are noted on the left. Relative densitometric units of the XIAP bands are shown in the top, with the density of the vehicle bands set arbitrarily at 1.0. Values shown represent the mean ± SEM from at least three separate experiments. Significant differences are indicated by asterisks. **, P < 0.01.

 
Effect of Inhibition of NF{kappa}B Activity on Paclitaxel-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 (48) , we examined the effects of paclitaxel and BAY 11-7085 alone and in combination on the invasion of Caov-3 cells through Matrigel. Whereas either paclitaxel or BAY 11-7085 partially inhibited cell invasion through Matrigel, cotreatment with paclitaxel plus BAY 11-7085 almost completely inhibited cell invasion through Matrigel (Fig. 5A)Citation .



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  		Fig. 5. BAY 11-7085 enhances paclitaxel-induced attenuation of invasion. A, Caov-3 cells were treated with 200 nmol/L paclitaxel, 5 µmol/L BAY 11-7085, or 200 nmol/L paclitaxel + 5 µmol/L BAY 11-7085 for 24 hours. The cells (5 x 104) were plated on Matrigel as described in "Materials and Methods." The fraction of cells that penetrated through Matrigel is shown, with the fraction of penetrating cells in the vehicle control set arbitrarily at 100%. Values shown represent the mean ± SEM from at least three separate experiments. Significant differences are indicated by asterisks. **, P < 0.01. B, Caov-3 cells were treated with 200 nmol/L paclitaxel and/or 5 µmol/L BAY 11-7085 for 24 hours. Lysates (50 µg of protein) were subjected to Western blotting by anti-MMP-9 (ii) or anti-ß-actin (iii) antibody. The positions of molecular weight markers are noted on the left. Relative densitometric units of the MMP-9 bands are shown (in i), with the density of the vehicle bands set arbitrarily at 1. Values shown represent the mean ± SEM from at least three separate experiments. Significant differences are indicated by asterisks. **, P < 0.01.

 
To confirm that inhibitory effects on invasion are not secondary to the cells simply being nonviable, the expression of MMP-9 was examined. Although either paclitaxel or BAY 11-7085 partially inhibited MMP-9 expression, cotreatment with paclitaxel plus BAY 11-7085 almost completely inhibited MMP-9 expression (Fig. 5B, i and ii)Citation . The expression of ß-actin was not changed by any of these treatments (Fig. 5B, iii)Citation .

Effect of BAY 11-7085 on Paclitaxel-Induced Inhibition of Intraabdominal Dissemination of Ovarian Cancer.
Ovarian cancer is initially clinically silent, and half of patients are first detected at an advanced stage with ascites and peritoneal dissemination (28) . 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 (49) , controlling ascitic fluid and peritoneal dissemination are crucial in the therapy of ovarian cancer. We therefore examined the effect of paclitaxel and BAY 11-7085 alone and in combination on the control of intraabdominal 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 intraperitoneally with Caov-3 cells or growth medium. Two weeks after inoculation, athymic nude mice inoculated intraperitoneally with Caov-3 cells were randomized into four groups treated with the following for 4 weeks: (a) vehicle (PBS); (b) paclitaxel (20 mg/kg) 3 times weekly; (c) BAY 11-7085 (5 mg/kg) 3 times weekly; and (d) paclitaxel (20 mg/kg) 3 times weekly plus BAY 11-7085 (5 mg/kg) 3 times weekly. The appearance of the mice is shown in Fig. 6ACitation . The abdominal circumference was measured (Fig. 6B)Citation , and the volume of ascites was also measured at autopsy (Fig. 6C)Citation . Both abdominal circumference and volume of ascites were significantly higher in athymic nude mice inoculated intraperitoneally with Caov-3 cells than in athymic nude mice inoculated intraperitoneally with growth medium. Pathologic examination was done to determine the extent of intraabdominal dissemination at autopsy. Intra-abdominal dissemination was clearly detected in athymic nude mice inoculated intraperitoneally with Caov-3 cells followed by treatment with vehicle (Fig. 7A)Citation , and the intraabdominal dissemination was shown by histologic assessment to consist of mucinous cystoadenocarcinoma (Fig. 7B)Citation , which is consistent with Caov-3 cells. Paclitaxel alone (Fig. 6B and C, b)Citation or BAY 11-7085 alone (Fig. 6B and C, c)Citation significantly diminished both the abdominal circumference and the volume of ascites compared with those in the vehicle control. The combination of paclitaxel + BAY 11-7085 (Fig. 6B and CCitation , d) further enhanced the inhibitory effects on the abdominal circumference and the production of ascites. Paclitaxel alone or BAY 11-7085 alone also diminished the extent of intraabdominal dissemination; combination of paclitaxel + BAY 11-7085 further enhanced the inhibitory effect on intraabdominal dissemination (data not shown). These results suggest that combination therapy of paclitaxel with BAY 11-7085 would increase the therapeutic efficacy of paclitaxel.



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  		Fig. 6. Appearance and ascites formation of mice after treatment with paclitaxel, BAY 11-7085 alone, or the combination thereof. Athymic nude mice were inoculated intraperitoneally with Caov-3 cells or growth medium (Control). Two weeks after inoculation, athymic nude mice inoculated intraperitoneally with Caov-3 cells were randomized into four groups treated with the following for 4 weeks: (a) vehicle alone (PBS); (b) paclitaxel (20 mg/kg) 3 times weekly; (c) BAY 11-7085 (5 mg/kg) 3 times weekly; and (d) paclitaxel (20 mg/kg) 3 times weekly + BAY 11-7085 (5 mg/kg) 3 times a week. Representative mice are shown in A. The abdominal circumference (AC) was measured (B). At autopsy, the volume of ascites was measured (C).

 


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  		Fig. 7. Pathological examination of in vivo ovarian cancer model. A, athymic nude mice were inoculated intraperitoneally with Caov-3 cells. Two weeks after inoculation, athymic nude mice inoculated intraperitoneally with Caov-3 cells were treated with vehicle alone (PBS) for 4 weeks. At autopsy, pathological examination was done to determine the extent of intraabdominal dissemination. B, histologic findings (x200) of parietal peritoneal dissemination of athymic nude mice inoculated intraperitoneally with Caov-3 cells followed by treatment with vehicle (PBS) alone.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The novel finding in this study is that an NF{kappa}B inhibitor, which enhanced paclitaxel-induced inhibition of I{kappa}B{alpha} phosphorylation and NF{kappa}B activity, increased the efficacy of paclitaxel in both in vitro and in vivo ovarian cancer models. Although adjuvant approaches in combination with paclitaxel have been reported for lung cancer (30) , prostate cancer (31) , pancreatic cancer (32) , and breast cancer (33) , there have been no reports about an adjuvant approach in combination with paclitaxel for ovarian cancer. In addition, in vitro models were used in previous reports, and there have been no reports using in vivo models.

It was previously proposed that NF{kappa}B might be required for paclitaxel-induced cell death (28) . Thus, although NF{kappa}B is a transcription factor that has been implicated in cell survival (17 , 18) , there is a possibility that the function of NF{kappa}B depends on tumor type. However, most reports suggest that paclitaxel-induced NF{kappa}B activity mediates survival signals that counteract apoptosis (29, 30, 31) .

We reported that Akt inactivation and inhibition of BAD phosphorylation sensitize human ovarian cancer cells to cisplatin (10) and paclitaxel (11) . In addition, we showed recently that inhibition of Forkhead phosphorylation sensitizes human ovarian cancer cells to cisplatin (50) . Although NF{kappa}B is a substrate of Akt, like BAD and Forkhead, NF{kappa}B activation is involved in angiogenesis (20 , 21) and metastasis (22 , 23) as well as in suppression of apoptosis. Therefore, NF{kappa}B inhibitors might increase the efficacy of chemotherapy against both primary and metastatic lesions. It was reported that NF{kappa}B inhibitors induce adhesion-dependent colon cancer apoptosis (51) . We showed in this study that treatment of athymic mice with BAY 11-7085 enhances the ability of paclitaxel to inhibit tumor implantation into the liver and peritoneum (Fig. 7)Citation . In addition, BAY 11-7085 increased the ability of paclitaxel to inhibit both cell proliferation in an MTS assay (Fig. 3C)Citation and cellular invasion in an in vitro invasion assay (Fig. 5A)Citation . Thus, NF{kappa}B inhibitors might increase the efficiency with which paclitaxel inhibits both primary and metastatic lesions. Glycogen synthase kinase 3{alpha} (52) and endothelial nitric oxide synthase (53 , 54) are also Akt substrates, and Akt is thus also involved in metabolic processes and vessel dilation, 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. Moreover, we also showed that the signaling cascade of NF{kappa}B is involved in the mechanism of maintaining the cell viability after cisplatin treatment of ovarian cancer cells (55) , as in the case of paclitaxel. Thus, because NF{kappa}B inhibitors might augment the effects of both cisplatin and paclitaxel, which together constitute the first line regimen of treatment for ovarian cancer, the combination of NF{kappa}B inhibitors with cisplatin and paclitaxel might be useful for molecularly targeted therapy.

How do NF{kappa}B inhibitors cause the inhibition of growth of human ovarian cancer cells? It was reported that NF{kappa}B inhibitors reduced the expression of survival genes regulated by NF{kappa}B, such as c-IAP-2, TRAF-1, TRAF-2, XIAP, or IEX-1L (41 , 42) . We also showed that NF{kappa}B inhibitors inhibited the expression of survival genes in human ovarian cancer cells (Fig. 4)Citation . The fact that NF{kappa}B mediates the expression of multiple survival genes makes it an important and rational target for cancer chemotherapy.

Activation of NF{kappa}B via phosphorylation of an inhibitor protein (I{kappa}B{alpha}) leads to degradation of I{kappa}B{alpha} through the ubiquitin-proteasome pathway. Inhibition of I{kappa}B{alpha} degradation by proteasome inhibitors keeps NF{kappa}B in the cytoplasm, thereby preventing it from acting on nuclear DNA (56 , 57) . PS-341, which is a potent boronic acid dipeptide that is highly selective for proteasome inhibition, can be systemically administered clinically (58) . PS-341 has been shown to enhance apoptotic response to chemotherapy in a variety of in vitro and in vivo models (59, 60, 61, 62) . A phase I trial of PS-341 and carboplatin in recurrent ovarian cancer is currently ongoing (63) . A phase II trial of PS-341 for the treatment of recurrent platinum-sensitive ovarian or primary peritoneal cancer (GOG 146-N) is also being conducted. It was also reported that proteasome inhibitor 1 enhances paclitaxel-induced apoptosis in a human lung adenocarcinoma cell line (30) . This is the first report that an NF{kappa}B inhibitor sensitizes human ovarian cancer cells to the effect of paclitaxel and the first to suggest that future clinical trials designed to examine the effect of NF{kappa}B inhibitors on the sensitivity to paclitaxel are warranted.


   ACKNOWLEDGMENTS
 
We are grateful to Ayako Okamura and Tomoko Iwaki for technical and secretarial assistance.


   FOOTNOTES
 
Grant support: National Cancer Institute grant CA83638 (SPORE in Ovarian Cancer) (S. Mabuchi and J. Testa).

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.

Requests for reprints: Masahide Ohmichi, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 011-81-6-6879-3354; Fax: 011-81-6-6879-3359; E-mail: masa{at}med.id yamagata-u.ac.jp.

Received 5/14/04; revised 8/ 4/04; accepted 8/ 6/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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