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Inhibition of NFκB Increases the Efficacy of Cisplatin in in Vitro and in Vivo Ovarian Cancer Models

Inhibition of NFκB Increases the Efficacy of Cisplatin in in Vitro and in Vivo Ovarian Cancer Models THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 22, Issue of May 28, pp. 23477–23485, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Inhibition of NFB Increases the Efficacy of Cisplatin in in Vitro and in Vivo Ovarian Cancer Models* Received for publication, December 15, 2003, and in revised form, March 12, 2004 Published, JBC Papers in Press, March 16, 2004, DOI 10.1074/jbc.M313709200 Seiji Mabuchi‡, Masahide Ohmichi‡§ , Yukihiro Nishio‡, Tadashi Hayasaka§, Akiko Kimura‡, Tsuyoshi Ohta§, Maki Saito§, Jun Kawagoe§, Kazuhiro Takahashi§, Namiko Yada-Hashimoto‡, Masahiro Sakata‡, Teiichi Motoyama , Hirohisa Kurachi§, Keiichi Tasaka‡, and Yuji Murata‡ From the ‡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 are involved in sensitivity to chemotherapeutic drugs. We re- Whether or not inhibition of NFB increases the effi- cacy of cisplatin in in vitro and in vivo ovarian cancer ported that Akt inactivation sensitizes human ovarian cancer models was investigated. We compared the basal levels cells to cisplatin (1) and paclitaxel (2), suggesting that Akt of phosphorylation of IB and activity of NFB be- inactivation could be a hallmark for examining the sensitivity tween cisplatin-sensitive A2780 cells and cisplatin-re- of cells to some chemotherapeutic drugs. Possible mechanisms sistant Caov-3 cells. The basal levels of phosphorylation by which Akt promotes cell survival include phosphorylation of IB and activity of NFB in Caov-3 cells were signif- and inactivation of the proapoptotic proteins BAD and icantly higher than those in A2780 cells. Cisplatin caspase-9 (3, 4). Akt also phosphorylates and inactivates the caused a more marked decrease in the phosphorylation Forkhead transcription factors, resulting in reduced expression of IB and activity of NFB in A2780 cells than in Kip1 of the cell cycle inhibitor p27 and the Fas ligand (5–7). Via Caov-3 cells. Thus, high basal levels of phosphorylation the phosphorylation of IB kinase, Akt also activates NFB, a of IB and activation of NFB and less marked inhibi- transcription factor that has been implicated in cell survival tion of the phosphorylation of IB and activation of (8, 9). NFB by cisplatin seem to reduce the sensitivity of cells NFB is activated in certain cancers and in response to to cisplatin. Inhibition of NFB activity either by treat- chemotherapy and radiation. NFB normally resides in the ment with the IB phosphorylation inhibitor (BAY 11- cytoplasm as an inactivated form in a complex with IB. 7085) or a specific NFB nuclear translocation inhibitor Phosphorylation of IB by upstream kinases promotes its (SN-50) or by transfection of p50NLS (which lacks the degradation, allowing NFB to translocate to the nucleus and nuclear localization signal domain) increased the effi- induce target genes (6, 7). The transcriptional activation of cacy of both the cisplatin-induced attenuation of IB genes associated with cell proliferation (10), angiogenesis (11, phosphorylation and NFB activity and the cisplatin- 12), metastasis (13, 14), and suppression of apoptosis (15) ap- induced apoptosis. In addition, treatment with BAY 11- 7085 increased the efficacy of the cisplatin-induced at- pears to lie at the heart of the ability of NFB to promote tenuation of both the expression of X-linked inhibitor of oncogenesis (16) and cancer therapy resistance (17, 18). While apoptosis protein (XIAP) and cell invasion through Ma- activation of NFB may induce apoptosis in certain situations trigel. Moreover, treatment with BAY 11-7085 increased (19 –22), most reports suggest that NFB mediates survival the efficacy of the cisplatin-induced inhibition of the signals that counteract apoptosis (23–28). It has been reported intra-abdominal dissemination and production of asci- that intrinsically or constitutively activated NFB may be crit- tes using athymic nude mice inoculated intraperitone- ical in the development of drug resistance in cancer cells (29 – ally with Caov-3 cells. These results suggest that combi- 31). Therefore, several agents that are able to inhibit NFB nation therapy of cisplatin with the NFB inhibitor function might be considered as an adjuvant approach in com- should increase the therapeutic efficacy of cisplatin. bination with chemotherapy for a variety of cancers. These considerations led us to examine whether the status of NFB activity is involved in sensitivity to cisplatin. In the The sensitivity of cells to chemotherapeutic drug-induced present study, we show that the basal levels of phosphoryla- apoptosis appears to depend on the balance between proapop- tion of IB and activity of NFB in cisplatin-resistant totic and antiapoptotic signals. Therefore, it is possible that Caov-3 cells are significantly higher than those in cisplatin- antiapoptotic signals such as the PI3K -Akt survival cascade sensitive A2780 cells. BAY 11-7085, a known pharmacologi- cal inhibitor of IB phosphorylation (32), enhanced the cis- platin-induced inhibition of IB phosphorylation and NFB * The costs of publication of this article were defrayed in part by the activity and increased the efficacy of cisplatin in in vitro and payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to in vivo ovarian cancer models. indicate this fact. To whom correspondence should be addressed: Osaka University EXPERIMENTAL PROCEDURES Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: Materials—The anti-IB and phospho-IB antibodies were ob- 81-6-6879-3354; Fax: 81-6-6879-3359; E-mail: [email protected]. tained from Cell Signaling Technology (Beverly, MA). The anti-NFB ac.jp. The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; NFB, nuclear factor-B; IB, inhibitor of NFB; PBS, phosphate- buffered saline; MTS, 3-[4,5,dimethylthiazol-2-yl]-5-[3-carboxyme- TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick thoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt; PARP, poly- end-labeling; IAP, inhibitor of apoptosis protein; XIAP, X-linked IAP; (ADP-ribose) polymerase; BAD, Bcl-2-associated death protein; i.p., intraperitoneally; NLS, nuclear localization signal. This is an Open Access article under the CC BY license. This paper is available on line at http://www.jbc.org 23477 23478 Role of NFB in Cisplatin-induced Apoptosis p65 was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, nu/nu athymic mice (n  30). Two weeks after inoculation, one group of CA). The IB phosphorylation inhibitor BAY 11-7085 was purchased mice (n  6) was treated with BAY 11-7085 (5 mg/kg) three times from Alexis Biochemicals (San Diego, CA). The specific NFB nuclear weekly plus cisplatin (5 mg/kg) once a week for 4 weeks. A second group translocation inhibitor SN-50 was purchased from Biomol (Plymouth of mice (n  6) was treated with BAY 11-7085 alone (5 mg/kg) three Meeting, PA). Anti-PARP, cleaved PARP, and the X-linked inhibitor of times weekly for 4 weeks. A third group (n  6) was treated with apoptosis protein (XIAP) antibodies were obtained from Cell Signaling cisplatin alone (5 mg/kg) once a week for 4 weeks. The remainder of the Technology. Anti--actin antibody was purchased from Sigma. ECL mice (n  6) received vehicle (PBS) alone. Abdominal circumference and Western blotting detection reagents were obtained from Amersham body weight were measured twice weekly. At the end of the experiment, Biosciences. The cell titer 96-well proliferation assay was obtained from mice underwent euthanasia with CO . The volume of ascites was meas- Promega (Madison, WI). The terminal deoxynucleotidyltransferase-me- ured, and tumor tissue was excised and fixed in 4% paraformaldehyde diated dUTP nick end-labeling (TUNEL) kit (ApopTag®) was obtained and embedded in paraffin. Paraffin sections (5 m) were used for from Chemicon (Temecula, CA). histochemical analysis. Cell Cultures—Human ovarian papillary adenocarcinoma cell line Immunohistochemistry—Formalin-fixed, paraffin-embedded tissues Caov-3 was obtained from the American Type Culture Collection. The were used. Apoptosis was assessed by TUNEL staining using an human ovarian cancer A2780 cell line derived from a patient prior to ApopTag® Plus peroxidase in situ apoptosis kit according to the man- treatment was kindly provided by Dr. T. Tsuruo (Institute of Molecular ufacturer’s protocol. For analysis of expression of phospho-IB and and Cellular Biosciences, Tokyo, Japan) and Drs. R. F. Ozols and T. C. localization of NFB p65, slides were incubated in methanol with 0.3% Hamilton (NCI, National Institutes of Health, Bethesda, MD) (33). The hydrogen peroxidase to eliminate endogenous peroxidase activity. Sec- cells were cultured at 37 °C in Dulbecco’s modified Eagle’s medium with tions were stained by the immunoperoxidase method with the strepta- 10% fetal bovine serum in a water-saturated atmosphere of 95% air and vidin-biotin (SAB) complex system (Nichirei, Tokyo, Japan). The anti- 5% CO . phospho-IB and anti-NFB p65 antibodies were used at a 1:50 Constructs—The NFB reporter plasmid (pElam-luc) was a kind gift dilution. Background reactivity for TUNEL was determined by process- from Dr. J. Cheng (University of South Florida College of Medicine) ing slides in the absence of terminal deoxynucleotidyltransferase (neg- (34). pCR-FLAG-p50 (encoding full-length human p50 with the FLAG ative control). For determination of TUNEL expression in tissue sec- epitope) and pCR-FLAG-p50NLS (encoding nuclear localization signal tions, we counted the number of apoptotic events in five random fields (NLS) polypeptide-deficient p50 with the FLAG epitope) constructs at 400 magnification and divided that number by the total number of were kind gifts from Dr. Gourisankar Ghosh (University of California, cells per field. Control samples for phospho-IB and NFB p65 stain- San Diego) Chen et al. (39). ing exposed to secondary antibody alone showed no nonspecific Proliferation Assay—Cell proliferation (35) was assessed by the ad- staining. dition of cisplatin at the indicated concentrations for 48 h, 1 day after Statistics—Statistical analysis was performed using one-way analy- seeding test cells into 96-well plates. The number of surviving cells was sis of variance followed by Fisher’s least significant difference test, determined 24 h later by determination of A of the dissolved forma- and p  0.05 was considered significant. Data are expressed as the zan product after the addition of MTS for1has described by the mean  S.E. manufacturer (Promega). All experiments were carried out in quadru- RESULTS plicate, and the viability was expressed as the ratio of the number of viable cells with cisplatin treatment to that without treatment. Differences of IB Phosphorylation and NFB Activity De- Western Blotting—Cells were incubated without serum for 16 h and pending on the Sensitivity to Cisplatin—The sensitivity to cis- then treated with various agents. They were then washed twice with platin of A2780 and Caov-3 cells was examined using the MTS phosphate-buffered saline and lysed in ice-cold HNTG buffer (50 mM assay (Fig. 1A). It was first confirmed thereby that A2780 cells HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM are sensitive and Caov-3 cells are resistant to cisplatin, as MgCl ,1mM EDTA, 10 mM sodium pyrophosphate, 100 M sodium reported previously (1, 35). Since constitutive phosphorylation orthovanadate, 100 mM NaF, 10 g/ml aprotinin, 10 g/ml leupeptin, and1mM phenylmethylsulfonyl fluoride) (2). The lysates were centri- of IB together with increased NFB activity has been re- fuged at 12,000  g at 4 °C for 15 min, and the protein concentrations ported to reduce sensitivity to chemotherapeutic drugs and is of the supernatants were determined using the Bio-Rad protein assay associated with invasive behavior of cancer cells (38), we next reagent. Equal amounts of proteins were separated by SDS-polyacryl- compared the IB phosphorylation (Fig. 1B) and NFB activ- amide gel electrophoresis and transferred to nitrocellulose membranes. ity (Fig. 1C) between cisplatin-sensitive A2780 cells and cis- Blocking was done in 10% bovine serum albumin in 1 Tris-buffered platin-resistant Caov-3 cells. Cells were treated with 200 M saline. Western blot analyses were performed with various specific primary antibodies. cisplatin for 30 min and used to prepare lysates that were NFB Transcriptional Activation Analysis—Cells were seeded in analyzed by Western blotting with anti-phospho-IB (Fig. 1B, 60-mm dishes and transfected with 2 gofNFB reporter plasmid panel ii), anti-IB (Fig. 1B, panel iii), or anti--actin antibody (pElam-luc) for 24 h using LipofectAMINE Plus (Invitrogen) according to (Fig. 1B, panel iv). Although the basal expression of -actin did the manufacturer’s protocol. Cells were treated with various agents and not differ between A2780 and Caov-3 cells (Fig. 1B, panel iv), then harvested and subjected to luciferase assays using the luciferase the basal level of phosphorylation of IB in Caov-3 cells was assay system (Promega) as described previously (36). A plasmid express- ing the bacterial -galactosidase gene was also cotransfected in each significantly higher than that in A2780 cells (Fig. 1B, panels i experiment to serve as an internal control for transfection efficiency. and ii). Although cisplatin caused a decrease in the level of Assay of Invasion through Matrigel—Polyvinylpyrrolidone-free poly- phosphorylated IB in both Caov-3 and A2780 cells, the de- carbonate filters (8-m pore size; Chemotaxicell, Kurabo, Japan) were gree of the decrement of phosphorylated IB by cisplatin in coated with a mixture of basement membrane components (Matrigel, 25 A2780 cells was more marked than that in Caov-3 cells (Fig. g/filter) and placed in modified Boyden chambers (37). The cells (5 1B, panels i and ii). Total levels of IB did not differ among 10 ) were released from their culture dishes by brief exposure to EDTA (1 mmol/liter) and centrifuged, resuspended in 0.1% bovine serum al- the lanes (Fig. 1B, panel iii), suggesting that cisplatin had no bumin-Dulbecco’s modified Eagle’s medium, and placed in the upper effect on the degradation of IB in either Caov-3 or A2780 compartment of the Boyden chamber. Fibroblast-conditioned medium cells. To assess the NFB activity, cells were transfected with in the lower compartment served as a chemoattractant. After incuba- aNFB-luciferase reporter plasmid. The basal NFB activity tion for 24 h at 37 °C, the cells on the lower surface of the filter were in Caov-3 cells was significantly higher than that in A2780 cells fixed, stained with Mayer’s hematoxylin solution, and enumerated us- (Fig. 1C). Although cisplatin caused a decrease in NFB activ- ing an ocular micrometer, and at least 10 fields/filter were counted. All of the experiments were independently performed in triplicate. ity in both Caov-3 and A2780 cells, the degree of the decrement Treatments in Vivo—All of the procedures involving animals and of NFB activity caused by cisplatin in A2780 cells was more their care in this study were approved by the animal care committee of marked than that in Caov-3 cells (Fig. 1C). These results sug- Osaka University in accordance with institutional and Japanese gov- gest that both high basal levels of phosphorylation of IB and ernment guidelines for animal experiments. Caov-3 cells were har- activation of NFB and weaker decrements of the phosphoryl- vested in 0.25% trypsin/PBS/EDTA, washed once each with medium ation of IB and activation of NFB by cisplatin seem to and PBS, and resuspended in PBS at 10 cells/200 l. One million Caov-3 cells were injected intraperitoneally (i.p.) into 5-week-old female reduce the sensitivity of cells to cisplatin. Role of NFB in Cisplatin-induced Apoptosis 23479 FIG.1. Differences of IB phosphorylation and NFB 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 M cisplatin for 30 min. Cell lysates were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with anti-phospho-IB (panel ii), anti-IB (panel iii), or anti--actin (panel iv) antibody. The positions of molecular mass markers are noted on the left. Relative densitometric units of the phospho-IB 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 M 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 NFB Activity on Cisplatin-induced that the NLS polypeptide (p50) is required for the translocation Apoptosis—We next examined whether inhibition of NFB ac- of the dimer to the nucleus (40) and that p50NLS lacking the tivity increased the efficacy with which cisplatin induced the NLS domain inhibits the nucleocytoplasmic shuttling of NFB attenuation of IB phosphorylation and NFB activity and dimers. Therefore, we examined the effect of p50NLS on the also induced apoptosis. We confirmed that treatment with an cisplatin-induced attenuation of NFB activity (Fig. 2B, panel IB phosphorylation inhibitor (BAY 11-7085) (32) attenuated iv). Transfection of p50NLS significantly increased the effi- both the phosphorylation of IB (Fig. 2A, ii, panels a and b) cacy with which cisplatin induced the attenuation of NFB and NFB activity (Fig. 2B, panel i), and a specific NFB activity compared with the effects in cells expressing wild-type nuclear translocation inhibitor (SN-50) attenuated the NFB p50 (Fig. 2B, panel iv). Thus, these data indicated that NFB activity (Fig. 2B, panel ii). In addition, treatment with BAY inhibitors increased the efficacy with which cisplatin induced 11-7085 increased the efficacy with which cisplatin induced the the attenuation of NFB activity. attenuation of IB phosphorylation (Fig. 2A, iii, panels a and In the presence of BAY 11-7085 or SN-50, the ability of b) and NFB activity (Fig. 2B, panel iii), whereas treatment cisplatin to induce growth inhibition was significantly en- with SN-50 increased the efficacy with which cisplatin induced hanced (Fig. 2C). Moreover, we examined the effects of BAY the attenuation of NFB activity (Fig. 2B, panel iii). Total 11-7085 (Fig. 2D, panel i) and SN-50 (Fig. 2D, panel ii)onthe levels of IB did not differ among the lanes (Fig. 2A, panel c), cisplatin-induced cleavage of PARP by Western blotting with like the basal expression of -actin (Fig. 2A, panel d), suggest- anti-PARP antibody (Fig. 2D, panel b), anti-cleaved PARP an- ing that cisplatin and/or BAY 11-7085 had no effect on the tibody (Fig. 2D, panel c), or anti--actin antibody (Fig. 2D, degradation of IB. Furthermore, we examined the effect of panel d). Cisplatin induced the cleavage of PARP (Fig. 2D, another NFB inhibitor on the cisplatin-induced attenuation of panels a, b, and c). BAY 11-7085 (Fig. 2D, i, panels a, b, and c) NFB activity. The best characterized species of NFB dimer is and SN-50 (Fig. 2D, ii, panels a, b, and c) significantly en- the p50/p65 heterodimer (39). A previous report demonstrated hanced the ability of cisplatin to induce the cleavage of PARP, 23480 Role of NFB in Cisplatin-induced Apoptosis FIG.2. Effect of inhibitors of NFB activity on cisplatin-induced apoptosis. A, Caov-3 cells were treated with 200 M cisplatin (panel i), 5 M BAY 11-7085 (panel ii), or 200 M cisplatin 5 M 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-IB antibody (panel b), anti-IB antibody (panel c), or anti--actin (panel d) antibody. The positions of molecular mass markers are noted on the left. Relative densitometric units of the phospho-IB 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 M BAY 11-7085 or 10 M SN50 (panel iii) for 6 h. Caov-3 cells were cotransfected with pElam-luc and pCR-FLAG-p50 or pCR-FLAG-p50NLS. 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 M BAY 11-7085 or 10 M 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 M cisplatin  5 M BAY 11-7085 (panel i)or200 M cisplatin  10 M SN50 (panel ii) for 24 h. Lysates (250 g of protein) were subjected to Western blotting using anti-PARP (panel b), anti-cleaved PARP (panel c), or anti--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. whereas the expression of -actin was not changed by any of lanes, both cisplatin and BAY 11-7085 partially attenuated the these treatments (Fig. 2D, panel d). expression of XIAP, and co-treatment with cisplatin plus BAY Effect of Inhibition of NFB Activity on the Cisplatin-induced 11-7085 completely inhibited the expression of XIAP (Fig. 3). Attenuation of the Expression of Survival Genes—NFB regu- Effect of Inhibition of NFB Activity on the Cisplatin-induced lates the expression of a number of antiapoptotic genes (41– Attenuation of Invasion of Caov-3 Cells through Matrigel— 43). Among them are the family of inhibitor of apoptosis pro- Because it was reported that NFB is involved in invasiveness teins (IAPs), which play a central role in repressing caspase- and metastatic properties (46), we examined the effects of mediated cell death. It was reported that cisplatin inhibits the cisplatin and BAY 11-7085 alone and in combination on the expression of XIAP (44) and that down-regulation of XIAP invasion of Caov-3 cells through Matrigel. Under laboratory induces apoptosis and increases cisplatin sensitivity (45), sug- conditions, Caov-3 cells migrated through Matrigel. Whereas gesting that XIAP is a determinant of cisplatin sensitivity in either cisplatin or BAY 11-7085 partially inhibited cell invasion ovarian cancer. Therefore, we examined the effect of cisplatin through Matrigel, co-treatment with cisplatin plus BAY 11- and BAY 11-7085 alone and in combination on the expression of 7085 completely inhibited cell invasion through Matrigel XIAP. Although the expression of -actin did not vary among the (Fig. 4). Role of NFB in Cisplatin-induced Apoptosis 23481 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 M BAY 11-7085  the indicated concentrations of cisplatin (lanes 8 and 9) for 24 h. Lysates (250 g of protein) were subjected to Western blotting using anti-XIAP (middle panel) or anti--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. FIG.4. BAY 11-7085 enhances the cisplatin-induced attenua- tion of invasion. Caov-3 cells were treated with 200 M cisplatin, 5 M BAY 11-7085, or 200 M cisplatin 5 M BAY 11-7085 for 24 h. The cells (5  10 ) 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 FIG.5. Appearance of and ascites formation in mice after main process of progression in ovarian cancer and the amount treatment with cisplatin, BAY 11-7085 alone, or the combination of ascitic fluid and the size of the disseminated tumor are thereof. Athymic nude mice were inoculated (i.p.) with Caov-3 cells or correlated with the patient’s prognosis (48), controlling ascitic growth medium (Control). Two weeks after inoculation, athymic nude mice inoculated (i.p.) with Caov-3 cells were randomized into four fluid and peritoneal dissemination are crucial in ovarian cancer groups and treated as follows for 4 weeks: panel a, vehicle alone (PBS); therapy. We therefore examined the effect of cisplatin and BAY panel b, cisplatin (5 mg/kg) once a week; panel c, BAY 11-7085 (5 mg/kg) 11-7085 alone and in combination on the control of intra-ab- three times a week; and panel d, cisplatin (5 mg/kg) once a week  BAY dominal dissemination of ovarian cancer and ascites formation 11-7085 (5 mg/kg) three times a week. Representative mice are shown to assess whether combination therapy would increase the in A. At autopsy, the volume of ascites was measured (B). therapeutic efficacy of each agent. Athymic nude mice were inoculated (i.p.) with Caov-3 cells or growth medium. Two (5 mg/kg) three times a week. The appearance of the mice is weeks after inoculation, athymic nude mice inoculated (i.p.) shown in Fig. 5A. The volume of ascites was measured at with Caov-3 cells were randomized into four groups treated autopsy (Fig. 5B). The volume of ascites was significantly with the following for 4 weeks: (a) vehicle (PBS), (b) cisplatin (5 higher in athymic nude mice inoculated (i.p.) with Caov-3 cells mg/kg) once a week, (c) BAY 11-7085 (5 mg/kg) three times a than in athymic nude mice inoculated (i.p.) with growth me- week, and (d) cisplatin (5 mg/kg) once a week  BAY 11-7085 dium. Pathological examination was performed to determine 23482 Role of NFB in Cisplatin-induced Apoptosis 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 (200 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). the extent of intra-abdominal dissemination at autopsy. Intra- Intra-abdominally disseminated tumors harvested from the abdominal dissemination was clearly detected in athymic nude different groups were processed for immunohistochemical anal- mice inoculated (i.p.) with Caov-3 cells followed by treatment yses. Apoptosis was analyzed by the TUNEL method (Fig. 7). with vehicle (Fig. 6, A, panel a, and B) and the intra-abdominal The fraction of TUNEL-positive cells was higher in tumors dissemination was confirmed by the histological findings to from mice treated with cisplatin  BAY 11-7085 than in tu- consist of mucinous cystoadenocarcinoma (Fig. 6C), which is mors from mice treated with vehicle alone (Fig. 7A). Apoptosis consistent with Caov-3 cells. Cisplatin alone (Fig. 5B, panel b) was quantitated in tumors of mice treated with vehicle alone or BAY 11-7085 alone (Fig. 5B, panel c) significantly dimin- (panel a), cisplatin alone (panel b), BAY 11-7085 alone (panel ished the volume of ascites compared with vehicle control. The c), and cisplatin  BAY 11-7085 (panel d) (Fig. 7B) as described combination of cisplatin  BAY 11-7085 (Fig. 5B, panel d) for the experiment shown in Fig. 5. Cisplatin alone (Fig. 7B, further enhanced the inhibitory effects on the production of panel b) or BAY 11-7085 alone (Fig. 7B, panel c) significantly ascites. Cisplatin alone (Fig. 6A, panel b) or BAY 11-7085 alone increased the proportion of apoptotic cells in tumors compared (Fig. 6A, panel c) also apparently diminished the extent of with vehicle alone (Fig. 7B, panel a). The combination of cis- intra-abdominal dissemination. The combination of cisplatin  platin  BAY 11-7085 (Fig. 7B, panel d) further enhanced the BAY 11-7085 (Fig. 6A, panel d) further enhanced each inhibi- increase of the proportion of apoptotic cells in tumors. We tory effect on intra-abdominal dissemination. These results further confirmed whether BAY 11-7085 blocked the NFB suggest that combination therapy of cisplatin with BAY 11- cascade in vivo.IB phosphorylation status and subcellular 7085 would increase the therapeutic efficacy of cisplatin. localization of NFB p65 were assessed by immunohistochem- Role of NFB in Cisplatin-induced Apoptosis 23483 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 (400 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 400 magnification. Significant differences are indicated by asterisks. **, p  0.01. istry with anti-phospho-IB antibody (Fig. 8A) and anti-NFB pression of apoptosis. Therefore, NFB inhibitors might increase p65 antibody (Fig. 8B), respectively. Immunoreactivity for the efficacy of chemotherapies in both primary and metastatic phosphorylated IB was found to be prominent in the cyto- lesions. It was reported that NFB inhibitors induce adhesion- plasm in tumors treated with vehicle alone and was apparently dependent colon cancer apoptosis (52). We showed in this study reduced in tumors treated with BAY 11-7085 (Fig. 8A), sug- that treatment of athymic mice with BAY 11-7085 enhanced the gesting that BAY 11-7085 inhibited the IB phosphorylation ability of cisplatin to inhibit tumor implantation into the liver in vivo. Whereas immunoreactivity for NFB p65 in tumors and peritoneum (Fig. 6A). In addition, BAY 11-7085 increased treated with vehicle alone was observed in both the cytoplasm the ability of cisplatin to inhibit both cell proliferation in an MTS and nuclei, immunoreactivity for NFB p65 in tumors treated assay (Fig. 2C) and cellular invasion in an in vitro invasion assay with BAY 11-7085 was only observed in the cytoplasm (Fig. (Fig. 4). Thus, NFB inhibitors might increase the efficiency with 8B), suggesting that BAY 11-7085 inhibited the partial nuclear which cisplatin inhibits both primary and metastatic lesions. translocation of NFB p65 in vivo. These results confirmed the Glycogen synthase kinase-3 (53) and endothelial nitric-oxide appropriate drug targeting in vivo. synthase (54, 55) are also other Akt substrates, and Akt is thus also involved in metabolic processes and vessel dilatation, respec- DISCUSSION tively. Therefore, it is possible that inhibition of PI3K/Akt acti- The form of chemotherapy used might determine whether vation is not a safe strategy for preventing chemoresistance. NFB has a pro- or antiapoptotic effect (10). For example, the Accordingly, NFB inhibitors might be more useful for sensitiza- majority of chemotherapies induce some form of DNA damage, tion to chemotherapeutic drugs than agents that are able to and inhibition of NFB activation promotes cell death. How- inhibit PI3K/Akt activity. ever, it was proposed recently that NFB might be required for Constitutive activation of NFB has been described in a Taxol-induced cell death (49). This is consistent with studies great number of solid tumors, and this activation appears to that suggest that NFB can function proapoptotically depend- support cancer cell survival and to reduce the sensitivity to ing on the type of stimulus to which cells are exposed. For chemotherapeutic drugs. We showed in this study that whereas example, it has been reported that the induction of cell death by cisplatin-sensitive A2780 cells do not have constitutive activa- hydrogen peroxide requires NFB and that NFB is required tion of NFB, cisplatin-resistant Caov-3 cells do have constitu- for p53-dependent cell death (50). More studies will be needed tive activation of NFB. Thus, it appears that constitutive to determine the chemotherapy specificity and potential tumor activation of NFB mediates cisplatin resistance in ovarian specificity of NFB in controlling cancer therapy efficacy. cancer cells and inhibition of NFB activation sensitizes the We reported that Akt inactivation and inhibition of BAD phos- ovarian cancer cells to cisplatin. phorylation sensitize human ovarian cancer cells to cisplatin (1) How do NFB inhibitors cause the inhibition of growth of and paclitaxel (2). In addition, we recently demonstrated that human ovarian cancer cells? It was reported that NFB inhib- inhibition of Forkhead phosphorylation sensitizes human ovar- ian cancer cells to cisplatin. Although NFB is a substrate of Akt itors diminished the expression of survival genes regulated by NFB, such as c-IAP-2, TRAF-1, TRAF-2, XIAP, or IEX-1L (42, (like BAD and Forkhead), NFB activation is involved in angio- genesis (11, 12) and metastasis (13, 14) in addition to the sup- 43). We also showed that NFB inhibitors caused the inhibition 23484 Role of NFB in Cisplatin-induced Apoptosis FIG.8. BAY 11-7085 blocks the NFB 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 IB phosphorylation status and subcellular localization of NFB p65 were assessed by immunohistochemistry with anti-phospho-IB antibody (A) and anti-NFB p65 antibody (B), respectively. Scale bars,15 m. of the expression of survival genes in human ovarian cancer of NFB. The advantage of soluble inhibitors is that their cells (Fig. 3). The fact that NFB mediates the expression of delivery would be easier and more efficient than gene transfer multiple survival genes makes it an important and rational in vivo. We did not detect significant renal, hepatic, or pulmo- target for cancer chemotherapy. nary tissue toxicity in this study. A previous study also showed BAY 11-7082 is also a known pharmacological inhibitor of that using up to 20 mg/kg/day of these agents in rats for 21 IB phosphorylation (32), like BAY 11-7085. In the presence days did not cause obvious toxicity (32). of BAY 11-7082, the cisplatin-induced attenuation of IB Activation of NFB via phosphorylation of an inhibitor pro- phosphorylation was significantly enhanced (data not tein (IB) leads to degradation of IB through the ubiquitin- shown). BAY 11-7082 and BAY 11-7085 also activate the proteasome pathway. Inhibition of IB degradation by protea- c-Jun N-terminal protein kinase and p38 (32), both of which some inhibitors keeps NFB in the cytoplasm, thereby are known to be involved in the induction of apoptosis (56). preventing it from acting on nuclear DNA (57, 58). PS-341, Thus, the effects of BAY 11-7082 and BAY 11-7085 do not which is a potent, boronic acid dipeptide that is highly selective exclude the role of cellular proteins other than NFB. Five for proteasome inhibition, can be systemically administered homologous polypeptides, p50, p65, c-Rel, RelB, and p52, clinically (59). PS-341 has been shown to enhance the apoptotic comprise the mammalian Rel/NFB transcription factor fam- response to chemotherapy in a variety of in vitro and in vivo ily. The subunits associate in a combinatorial fashion to form models (18, 60 – 62). A Phase I trial of PS-341 and carboplatin transcriptionally active homo- and heterodimers. The best in recurrent ovarian cancer is currently ongoing (51). A Phase characterized species of NFB dimer is the p50/p65 het- II trial of PS-341 for the treatment of recurrent platinum- erodimer (39). A previous report demonstrated that the NLS sensitive ovarian or primary or peritoneal cancer (GOG 146-N) polypeptide of p50 is required for its translocation to the is also being conducted. We now await the results of these nucleus (40) and that p50NLS lacking the NLS domain currently ongoing clinical trials. inhibits the nucleocytoplasmic shuttling of NFB dimers. Acknowledgments—We thank Dr. J. Cheng (University of South Therefore, we examined the effect of p50NLS on the atten- Florida College of Medicine) for providing the NFB reporter plasmid uation of IB phosphorylation by cisplatin. Transfection of (pElam-luc), and Dr. Gourisankar Ghosh (Department of Chemistry p50NLS significantly inhibited the increased the efficacy of and Biochemistry, University of California, San Diego) for providing pCR-FLAG-p50 and pCR-FLAG-p50NLS constructs. We are also the cisplatin-induced attenuation of NFB activity compared grateful to Dr. Junko Takeda and Dr. Kazue Onuma for technical with the effect in cells expressing wild-type p50 (Fig. 2B, assistance and Ayako Okamura and Tomoko Iwaki for secretarial panel iv). Thus, the similarity of the effects caused by treat- assistance. ment with BAY 11-7082 or BAY 11-7085 and by transfection with p50NLS suggests that inhibition of NFB activity has REFERENCES strong potential as a novel adjuvant chemotherapy. 1. 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M., Pien, C. S., Worland, P., Adams, J., and Kauffman, M. G. (2002) 277, 29973–29982 Proc. Am. Assoc. Cancer Res. 43, 158 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Inhibition of NFκB Increases the Efficacy of Cisplatin in in Vitro and in Vivo Ovarian Cancer Models

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 22, Issue of May 28, pp. 23477–23485, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Inhibition of NFB Increases the Efficacy of Cisplatin in in Vitro and in Vivo Ovarian Cancer Models* Received for publication, December 15, 2003, and in revised form, March 12, 2004 Published, JBC Papers in Press, March 16, 2004, DOI 10.1074/jbc.M313709200 Seiji Mabuchi‡, Masahide Ohmichi‡§ , Yukihiro Nishio‡, Tadashi Hayasaka§, Akiko Kimura‡, Tsuyoshi Ohta§, Maki Saito§, Jun Kawagoe§, Kazuhiro Takahashi§, Namiko Yada-Hashimoto‡, Masahiro Sakata‡, Teiichi Motoyama , Hirohisa Kurachi§, Keiichi Tasaka‡, and Yuji Murata‡ From the ‡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 are involved in sensitivity to chemotherapeutic drugs. We re- Whether or not inhibition of NFB increases the effi- cacy of cisplatin in in vitro and in vivo ovarian cancer ported that Akt inactivation sensitizes human ovarian cancer models was investigated. We compared the basal levels cells to cisplatin (1) and paclitaxel (2), suggesting that Akt of phosphorylation of IB and activity of NFB be- inactivation could be a hallmark for examining the sensitivity tween cisplatin-sensitive A2780 cells and cisplatin-re- of cells to some chemotherapeutic drugs. Possible mechanisms sistant Caov-3 cells. The basal levels of phosphorylation by which Akt promotes cell survival include phosphorylation of IB and activity of NFB in Caov-3 cells were signif- and inactivation of the proapoptotic proteins BAD and icantly higher than those in A2780 cells. Cisplatin caspase-9 (3, 4). Akt also phosphorylates and inactivates the caused a more marked decrease in the phosphorylation Forkhead transcription factors, resulting in reduced expression of IB and activity of NFB in A2780 cells than in Kip1 of the cell cycle inhibitor p27 and the Fas ligand (5–7). Via Caov-3 cells. Thus, high basal levels of phosphorylation the phosphorylation of IB kinase, Akt also activates NFB, a of IB and activation of NFB and less marked inhibi- transcription factor that has been implicated in cell survival tion of the phosphorylation of IB and activation of (8, 9). NFB by cisplatin seem to reduce the sensitivity of cells NFB is activated in certain cancers and in response to to cisplatin. Inhibition of NFB activity either by treat- chemotherapy and radiation. NFB normally resides in the ment with the IB phosphorylation inhibitor (BAY 11- cytoplasm as an inactivated form in a complex with IB. 7085) or a specific NFB nuclear translocation inhibitor Phosphorylation of IB by upstream kinases promotes its (SN-50) or by transfection of p50NLS (which lacks the degradation, allowing NFB to translocate to the nucleus and nuclear localization signal domain) increased the effi- induce target genes (6, 7). The transcriptional activation of cacy of both the cisplatin-induced attenuation of IB genes associated with cell proliferation (10), angiogenesis (11, phosphorylation and NFB activity and the cisplatin- 12), metastasis (13, 14), and suppression of apoptosis (15) ap- induced apoptosis. In addition, treatment with BAY 11- 7085 increased the efficacy of the cisplatin-induced at- pears to lie at the heart of the ability of NFB to promote tenuation of both the expression of X-linked inhibitor of oncogenesis (16) and cancer therapy resistance (17, 18). While apoptosis protein (XIAP) and cell invasion through Ma- activation of NFB may induce apoptosis in certain situations trigel. Moreover, treatment with BAY 11-7085 increased (19 –22), most reports suggest that NFB mediates survival the efficacy of the cisplatin-induced inhibition of the signals that counteract apoptosis (23–28). It has been reported intra-abdominal dissemination and production of asci- that intrinsically or constitutively activated NFB may be crit- tes using athymic nude mice inoculated intraperitone- ical in the development of drug resistance in cancer cells (29 – ally with Caov-3 cells. These results suggest that combi- 31). Therefore, several agents that are able to inhibit NFB nation therapy of cisplatin with the NFB inhibitor function might be considered as an adjuvant approach in com- should increase the therapeutic efficacy of cisplatin. bination with chemotherapy for a variety of cancers. These considerations led us to examine whether the status of NFB activity is involved in sensitivity to cisplatin. In the The sensitivity of cells to chemotherapeutic drug-induced present study, we show that the basal levels of phosphoryla- apoptosis appears to depend on the balance between proapop- tion of IB and activity of NFB in cisplatin-resistant totic and antiapoptotic signals. Therefore, it is possible that Caov-3 cells are significantly higher than those in cisplatin- antiapoptotic signals such as the PI3K -Akt survival cascade sensitive A2780 cells. BAY 11-7085, a known pharmacologi- cal inhibitor of IB phosphorylation (32), enhanced the cis- platin-induced inhibition of IB phosphorylation and NFB * The costs of publication of this article were defrayed in part by the activity and increased the efficacy of cisplatin in in vitro and payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to in vivo ovarian cancer models. indicate this fact. To whom correspondence should be addressed: Osaka University EXPERIMENTAL PROCEDURES Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: Materials—The anti-IB and phospho-IB antibodies were ob- 81-6-6879-3354; Fax: 81-6-6879-3359; E-mail: [email protected]. tained from Cell Signaling Technology (Beverly, MA). The anti-NFB ac.jp. The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; NFB, nuclear factor-B; IB, inhibitor of NFB; PBS, phosphate- buffered saline; MTS, 3-[4,5,dimethylthiazol-2-yl]-5-[3-carboxyme- TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick thoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt; PARP, poly- end-labeling; IAP, inhibitor of apoptosis protein; XIAP, X-linked IAP; (ADP-ribose) polymerase; BAD, Bcl-2-associated death protein; i.p., intraperitoneally; NLS, nuclear localization signal. This is an Open Access article under the CC BY license. This paper is available on line at http://www.jbc.org 23477 23478 Role of NFB in Cisplatin-induced Apoptosis p65 was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, nu/nu athymic mice (n  30). Two weeks after inoculation, one group of CA). The IB phosphorylation inhibitor BAY 11-7085 was purchased mice (n  6) was treated with BAY 11-7085 (5 mg/kg) three times from Alexis Biochemicals (San Diego, CA). The specific NFB nuclear weekly plus cisplatin (5 mg/kg) once a week for 4 weeks. A second group translocation inhibitor SN-50 was purchased from Biomol (Plymouth of mice (n  6) was treated with BAY 11-7085 alone (5 mg/kg) three Meeting, PA). Anti-PARP, cleaved PARP, and the X-linked inhibitor of times weekly for 4 weeks. A third group (n  6) was treated with apoptosis protein (XIAP) antibodies were obtained from Cell Signaling cisplatin alone (5 mg/kg) once a week for 4 weeks. The remainder of the Technology. Anti--actin antibody was purchased from Sigma. ECL mice (n  6) received vehicle (PBS) alone. Abdominal circumference and Western blotting detection reagents were obtained from Amersham body weight were measured twice weekly. At the end of the experiment, Biosciences. The cell titer 96-well proliferation assay was obtained from mice underwent euthanasia with CO . The volume of ascites was meas- Promega (Madison, WI). The terminal deoxynucleotidyltransferase-me- ured, and tumor tissue was excised and fixed in 4% paraformaldehyde diated dUTP nick end-labeling (TUNEL) kit (ApopTag®) was obtained and embedded in paraffin. Paraffin sections (5 m) were used for from Chemicon (Temecula, CA). histochemical analysis. Cell Cultures—Human ovarian papillary adenocarcinoma cell line Immunohistochemistry—Formalin-fixed, paraffin-embedded tissues Caov-3 was obtained from the American Type Culture Collection. The were used. Apoptosis was assessed by TUNEL staining using an human ovarian cancer A2780 cell line derived from a patient prior to ApopTag® Plus peroxidase in situ apoptosis kit according to the man- treatment was kindly provided by Dr. T. Tsuruo (Institute of Molecular ufacturer’s protocol. For analysis of expression of phospho-IB and and Cellular Biosciences, Tokyo, Japan) and Drs. R. F. Ozols and T. C. localization of NFB p65, slides were incubated in methanol with 0.3% Hamilton (NCI, National Institutes of Health, Bethesda, MD) (33). The hydrogen peroxidase to eliminate endogenous peroxidase activity. Sec- cells were cultured at 37 °C in Dulbecco’s modified Eagle’s medium with tions were stained by the immunoperoxidase method with the strepta- 10% fetal bovine serum in a water-saturated atmosphere of 95% air and vidin-biotin (SAB) complex system (Nichirei, Tokyo, Japan). The anti- 5% CO . phospho-IB and anti-NFB p65 antibodies were used at a 1:50 Constructs—The NFB reporter plasmid (pElam-luc) was a kind gift dilution. Background reactivity for TUNEL was determined by process- from Dr. J. Cheng (University of South Florida College of Medicine) ing slides in the absence of terminal deoxynucleotidyltransferase (neg- (34). pCR-FLAG-p50 (encoding full-length human p50 with the FLAG ative control). For determination of TUNEL expression in tissue sec- epitope) and pCR-FLAG-p50NLS (encoding nuclear localization signal tions, we counted the number of apoptotic events in five random fields (NLS) polypeptide-deficient p50 with the FLAG epitope) constructs at 400 magnification and divided that number by the total number of were kind gifts from Dr. Gourisankar Ghosh (University of California, cells per field. Control samples for phospho-IB and NFB p65 stain- San Diego) Chen et al. (39). ing exposed to secondary antibody alone showed no nonspecific Proliferation Assay—Cell proliferation (35) was assessed by the ad- staining. dition of cisplatin at the indicated concentrations for 48 h, 1 day after Statistics—Statistical analysis was performed using one-way analy- seeding test cells into 96-well plates. The number of surviving cells was sis of variance followed by Fisher’s least significant difference test, determined 24 h later by determination of A of the dissolved forma- and p  0.05 was considered significant. Data are expressed as the zan product after the addition of MTS for1has described by the mean  S.E. manufacturer (Promega). All experiments were carried out in quadru- RESULTS plicate, and the viability was expressed as the ratio of the number of viable cells with cisplatin treatment to that without treatment. Differences of IB Phosphorylation and NFB Activity De- Western Blotting—Cells were incubated without serum for 16 h and pending on the Sensitivity to Cisplatin—The sensitivity to cis- then treated with various agents. They were then washed twice with platin of A2780 and Caov-3 cells was examined using the MTS phosphate-buffered saline and lysed in ice-cold HNTG buffer (50 mM assay (Fig. 1A). It was first confirmed thereby that A2780 cells HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM are sensitive and Caov-3 cells are resistant to cisplatin, as MgCl ,1mM EDTA, 10 mM sodium pyrophosphate, 100 M sodium reported previously (1, 35). Since constitutive phosphorylation orthovanadate, 100 mM NaF, 10 g/ml aprotinin, 10 g/ml leupeptin, and1mM phenylmethylsulfonyl fluoride) (2). The lysates were centri- of IB together with increased NFB activity has been re- fuged at 12,000  g at 4 °C for 15 min, and the protein concentrations ported to reduce sensitivity to chemotherapeutic drugs and is of the supernatants were determined using the Bio-Rad protein assay associated with invasive behavior of cancer cells (38), we next reagent. Equal amounts of proteins were separated by SDS-polyacryl- compared the IB phosphorylation (Fig. 1B) and NFB activ- amide gel electrophoresis and transferred to nitrocellulose membranes. ity (Fig. 1C) between cisplatin-sensitive A2780 cells and cis- Blocking was done in 10% bovine serum albumin in 1 Tris-buffered platin-resistant Caov-3 cells. Cells were treated with 200 M saline. Western blot analyses were performed with various specific primary antibodies. cisplatin for 30 min and used to prepare lysates that were NFB Transcriptional Activation Analysis—Cells were seeded in analyzed by Western blotting with anti-phospho-IB (Fig. 1B, 60-mm dishes and transfected with 2 gofNFB reporter plasmid panel ii), anti-IB (Fig. 1B, panel iii), or anti--actin antibody (pElam-luc) for 24 h using LipofectAMINE Plus (Invitrogen) according to (Fig. 1B, panel iv). Although the basal expression of -actin did the manufacturer’s protocol. Cells were treated with various agents and not differ between A2780 and Caov-3 cells (Fig. 1B, panel iv), then harvested and subjected to luciferase assays using the luciferase the basal level of phosphorylation of IB in Caov-3 cells was assay system (Promega) as described previously (36). A plasmid express- ing the bacterial -galactosidase gene was also cotransfected in each significantly higher than that in A2780 cells (Fig. 1B, panels i experiment to serve as an internal control for transfection efficiency. and ii). Although cisplatin caused a decrease in the level of Assay of Invasion through Matrigel—Polyvinylpyrrolidone-free poly- phosphorylated IB in both Caov-3 and A2780 cells, the de- carbonate filters (8-m pore size; Chemotaxicell, Kurabo, Japan) were gree of the decrement of phosphorylated IB by cisplatin in coated with a mixture of basement membrane components (Matrigel, 25 A2780 cells was more marked than that in Caov-3 cells (Fig. g/filter) and placed in modified Boyden chambers (37). The cells (5 1B, panels i and ii). Total levels of IB did not differ among 10 ) were released from their culture dishes by brief exposure to EDTA (1 mmol/liter) and centrifuged, resuspended in 0.1% bovine serum al- the lanes (Fig. 1B, panel iii), suggesting that cisplatin had no bumin-Dulbecco’s modified Eagle’s medium, and placed in the upper effect on the degradation of IB in either Caov-3 or A2780 compartment of the Boyden chamber. Fibroblast-conditioned medium cells. To assess the NFB activity, cells were transfected with in the lower compartment served as a chemoattractant. After incuba- aNFB-luciferase reporter plasmid. The basal NFB activity tion for 24 h at 37 °C, the cells on the lower surface of the filter were in Caov-3 cells was significantly higher than that in A2780 cells fixed, stained with Mayer’s hematoxylin solution, and enumerated us- (Fig. 1C). Although cisplatin caused a decrease in NFB activ- ing an ocular micrometer, and at least 10 fields/filter were counted. All of the experiments were independently performed in triplicate. ity in both Caov-3 and A2780 cells, the degree of the decrement Treatments in Vivo—All of the procedures involving animals and of NFB activity caused by cisplatin in A2780 cells was more their care in this study were approved by the animal care committee of marked than that in Caov-3 cells (Fig. 1C). These results sug- Osaka University in accordance with institutional and Japanese gov- gest that both high basal levels of phosphorylation of IB and ernment guidelines for animal experiments. Caov-3 cells were har- activation of NFB and weaker decrements of the phosphoryl- vested in 0.25% trypsin/PBS/EDTA, washed once each with medium ation of IB and activation of NFB by cisplatin seem to and PBS, and resuspended in PBS at 10 cells/200 l. One million Caov-3 cells were injected intraperitoneally (i.p.) into 5-week-old female reduce the sensitivity of cells to cisplatin. Role of NFB in Cisplatin-induced Apoptosis 23479 FIG.1. Differences of IB phosphorylation and NFB 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 M cisplatin for 30 min. Cell lysates were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with anti-phospho-IB (panel ii), anti-IB (panel iii), or anti--actin (panel iv) antibody. The positions of molecular mass markers are noted on the left. Relative densitometric units of the phospho-IB 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 M 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 NFB Activity on Cisplatin-induced that the NLS polypeptide (p50) is required for the translocation Apoptosis—We next examined whether inhibition of NFB ac- of the dimer to the nucleus (40) and that p50NLS lacking the tivity increased the efficacy with which cisplatin induced the NLS domain inhibits the nucleocytoplasmic shuttling of NFB attenuation of IB phosphorylation and NFB activity and dimers. Therefore, we examined the effect of p50NLS on the also induced apoptosis. We confirmed that treatment with an cisplatin-induced attenuation of NFB activity (Fig. 2B, panel IB phosphorylation inhibitor (BAY 11-7085) (32) attenuated iv). Transfection of p50NLS significantly increased the effi- both the phosphorylation of IB (Fig. 2A, ii, panels a and b) cacy with which cisplatin induced the attenuation of NFB and NFB activity (Fig. 2B, panel i), and a specific NFB activity compared with the effects in cells expressing wild-type nuclear translocation inhibitor (SN-50) attenuated the NFB p50 (Fig. 2B, panel iv). Thus, these data indicated that NFB activity (Fig. 2B, panel ii). In addition, treatment with BAY inhibitors increased the efficacy with which cisplatin induced 11-7085 increased the efficacy with which cisplatin induced the the attenuation of NFB activity. attenuation of IB phosphorylation (Fig. 2A, iii, panels a and In the presence of BAY 11-7085 or SN-50, the ability of b) and NFB activity (Fig. 2B, panel iii), whereas treatment cisplatin to induce growth inhibition was significantly en- with SN-50 increased the efficacy with which cisplatin induced hanced (Fig. 2C). Moreover, we examined the effects of BAY the attenuation of NFB activity (Fig. 2B, panel iii). Total 11-7085 (Fig. 2D, panel i) and SN-50 (Fig. 2D, panel ii)onthe levels of IB did not differ among the lanes (Fig. 2A, panel c), cisplatin-induced cleavage of PARP by Western blotting with like the basal expression of -actin (Fig. 2A, panel d), suggest- anti-PARP antibody (Fig. 2D, panel b), anti-cleaved PARP an- ing that cisplatin and/or BAY 11-7085 had no effect on the tibody (Fig. 2D, panel c), or anti--actin antibody (Fig. 2D, degradation of IB. Furthermore, we examined the effect of panel d). Cisplatin induced the cleavage of PARP (Fig. 2D, another NFB inhibitor on the cisplatin-induced attenuation of panels a, b, and c). BAY 11-7085 (Fig. 2D, i, panels a, b, and c) NFB activity. The best characterized species of NFB dimer is and SN-50 (Fig. 2D, ii, panels a, b, and c) significantly en- the p50/p65 heterodimer (39). A previous report demonstrated hanced the ability of cisplatin to induce the cleavage of PARP, 23480 Role of NFB in Cisplatin-induced Apoptosis FIG.2. Effect of inhibitors of NFB activity on cisplatin-induced apoptosis. A, Caov-3 cells were treated with 200 M cisplatin (panel i), 5 M BAY 11-7085 (panel ii), or 200 M cisplatin 5 M 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-IB antibody (panel b), anti-IB antibody (panel c), or anti--actin (panel d) antibody. The positions of molecular mass markers are noted on the left. Relative densitometric units of the phospho-IB 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 M BAY 11-7085 or 10 M SN50 (panel iii) for 6 h. Caov-3 cells were cotransfected with pElam-luc and pCR-FLAG-p50 or pCR-FLAG-p50NLS. 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 M BAY 11-7085 or 10 M 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 M cisplatin  5 M BAY 11-7085 (panel i)or200 M cisplatin  10 M SN50 (panel ii) for 24 h. Lysates (250 g of protein) were subjected to Western blotting using anti-PARP (panel b), anti-cleaved PARP (panel c), or anti--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. whereas the expression of -actin was not changed by any of lanes, both cisplatin and BAY 11-7085 partially attenuated the these treatments (Fig. 2D, panel d). expression of XIAP, and co-treatment with cisplatin plus BAY Effect of Inhibition of NFB Activity on the Cisplatin-induced 11-7085 completely inhibited the expression of XIAP (Fig. 3). Attenuation of the Expression of Survival Genes—NFB regu- Effect of Inhibition of NFB Activity on the Cisplatin-induced lates the expression of a number of antiapoptotic genes (41– Attenuation of Invasion of Caov-3 Cells through Matrigel— 43). Among them are the family of inhibitor of apoptosis pro- Because it was reported that NFB is involved in invasiveness teins (IAPs), which play a central role in repressing caspase- and metastatic properties (46), we examined the effects of mediated cell death. It was reported that cisplatin inhibits the cisplatin and BAY 11-7085 alone and in combination on the expression of XIAP (44) and that down-regulation of XIAP invasion of Caov-3 cells through Matrigel. Under laboratory induces apoptosis and increases cisplatin sensitivity (45), sug- conditions, Caov-3 cells migrated through Matrigel. Whereas gesting that XIAP is a determinant of cisplatin sensitivity in either cisplatin or BAY 11-7085 partially inhibited cell invasion ovarian cancer. Therefore, we examined the effect of cisplatin through Matrigel, co-treatment with cisplatin plus BAY 11- and BAY 11-7085 alone and in combination on the expression of 7085 completely inhibited cell invasion through Matrigel XIAP. Although the expression of -actin did not vary among the (Fig. 4). Role of NFB in Cisplatin-induced Apoptosis 23481 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 M BAY 11-7085  the indicated concentrations of cisplatin (lanes 8 and 9) for 24 h. Lysates (250 g of protein) were subjected to Western blotting using anti-XIAP (middle panel) or anti--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. FIG.4. BAY 11-7085 enhances the cisplatin-induced attenua- tion of invasion. Caov-3 cells were treated with 200 M cisplatin, 5 M BAY 11-7085, or 200 M cisplatin 5 M BAY 11-7085 for 24 h. The cells (5  10 ) 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 FIG.5. Appearance of and ascites formation in mice after main process of progression in ovarian cancer and the amount treatment with cisplatin, BAY 11-7085 alone, or the combination of ascitic fluid and the size of the disseminated tumor are thereof. Athymic nude mice were inoculated (i.p.) with Caov-3 cells or correlated with the patient’s prognosis (48), controlling ascitic growth medium (Control). Two weeks after inoculation, athymic nude mice inoculated (i.p.) with Caov-3 cells were randomized into four fluid and peritoneal dissemination are crucial in ovarian cancer groups and treated as follows for 4 weeks: panel a, vehicle alone (PBS); therapy. We therefore examined the effect of cisplatin and BAY panel b, cisplatin (5 mg/kg) once a week; panel c, BAY 11-7085 (5 mg/kg) 11-7085 alone and in combination on the control of intra-ab- three times a week; and panel d, cisplatin (5 mg/kg) once a week  BAY dominal dissemination of ovarian cancer and ascites formation 11-7085 (5 mg/kg) three times a week. Representative mice are shown to assess whether combination therapy would increase the in A. At autopsy, the volume of ascites was measured (B). therapeutic efficacy of each agent. Athymic nude mice were inoculated (i.p.) with Caov-3 cells or growth medium. Two (5 mg/kg) three times a week. The appearance of the mice is weeks after inoculation, athymic nude mice inoculated (i.p.) shown in Fig. 5A. The volume of ascites was measured at with Caov-3 cells were randomized into four groups treated autopsy (Fig. 5B). The volume of ascites was significantly with the following for 4 weeks: (a) vehicle (PBS), (b) cisplatin (5 higher in athymic nude mice inoculated (i.p.) with Caov-3 cells mg/kg) once a week, (c) BAY 11-7085 (5 mg/kg) three times a than in athymic nude mice inoculated (i.p.) with growth me- week, and (d) cisplatin (5 mg/kg) once a week  BAY 11-7085 dium. Pathological examination was performed to determine 23482 Role of NFB in Cisplatin-induced Apoptosis 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 (200 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). the extent of intra-abdominal dissemination at autopsy. Intra- Intra-abdominally disseminated tumors harvested from the abdominal dissemination was clearly detected in athymic nude different groups were processed for immunohistochemical anal- mice inoculated (i.p.) with Caov-3 cells followed by treatment yses. Apoptosis was analyzed by the TUNEL method (Fig. 7). with vehicle (Fig. 6, A, panel a, and B) and the intra-abdominal The fraction of TUNEL-positive cells was higher in tumors dissemination was confirmed by the histological findings to from mice treated with cisplatin  BAY 11-7085 than in tu- consist of mucinous cystoadenocarcinoma (Fig. 6C), which is mors from mice treated with vehicle alone (Fig. 7A). Apoptosis consistent with Caov-3 cells. Cisplatin alone (Fig. 5B, panel b) was quantitated in tumors of mice treated with vehicle alone or BAY 11-7085 alone (Fig. 5B, panel c) significantly dimin- (panel a), cisplatin alone (panel b), BAY 11-7085 alone (panel ished the volume of ascites compared with vehicle control. The c), and cisplatin  BAY 11-7085 (panel d) (Fig. 7B) as described combination of cisplatin  BAY 11-7085 (Fig. 5B, panel d) for the experiment shown in Fig. 5. Cisplatin alone (Fig. 7B, further enhanced the inhibitory effects on the production of panel b) or BAY 11-7085 alone (Fig. 7B, panel c) significantly ascites. Cisplatin alone (Fig. 6A, panel b) or BAY 11-7085 alone increased the proportion of apoptotic cells in tumors compared (Fig. 6A, panel c) also apparently diminished the extent of with vehicle alone (Fig. 7B, panel a). The combination of cis- intra-abdominal dissemination. The combination of cisplatin  platin  BAY 11-7085 (Fig. 7B, panel d) further enhanced the BAY 11-7085 (Fig. 6A, panel d) further enhanced each inhibi- increase of the proportion of apoptotic cells in tumors. We tory effect on intra-abdominal dissemination. These results further confirmed whether BAY 11-7085 blocked the NFB suggest that combination therapy of cisplatin with BAY 11- cascade in vivo.IB phosphorylation status and subcellular 7085 would increase the therapeutic efficacy of cisplatin. localization of NFB p65 were assessed by immunohistochem- Role of NFB in Cisplatin-induced Apoptosis 23483 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 (400 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 400 magnification. Significant differences are indicated by asterisks. **, p  0.01. istry with anti-phospho-IB antibody (Fig. 8A) and anti-NFB pression of apoptosis. Therefore, NFB inhibitors might increase p65 antibody (Fig. 8B), respectively. Immunoreactivity for the efficacy of chemotherapies in both primary and metastatic phosphorylated IB was found to be prominent in the cyto- lesions. It was reported that NFB inhibitors induce adhesion- plasm in tumors treated with vehicle alone and was apparently dependent colon cancer apoptosis (52). We showed in this study reduced in tumors treated with BAY 11-7085 (Fig. 8A), sug- that treatment of athymic mice with BAY 11-7085 enhanced the gesting that BAY 11-7085 inhibited the IB phosphorylation ability of cisplatin to inhibit tumor implantation into the liver in vivo. Whereas immunoreactivity for NFB p65 in tumors and peritoneum (Fig. 6A). In addition, BAY 11-7085 increased treated with vehicle alone was observed in both the cytoplasm the ability of cisplatin to inhibit both cell proliferation in an MTS and nuclei, immunoreactivity for NFB p65 in tumors treated assay (Fig. 2C) and cellular invasion in an in vitro invasion assay with BAY 11-7085 was only observed in the cytoplasm (Fig. (Fig. 4). Thus, NFB inhibitors might increase the efficiency with 8B), suggesting that BAY 11-7085 inhibited the partial nuclear which cisplatin inhibits both primary and metastatic lesions. translocation of NFB p65 in vivo. These results confirmed the Glycogen synthase kinase-3 (53) and endothelial nitric-oxide appropriate drug targeting in vivo. synthase (54, 55) are also other Akt substrates, and Akt is thus also involved in metabolic processes and vessel dilatation, respec- DISCUSSION tively. Therefore, it is possible that inhibition of PI3K/Akt acti- The form of chemotherapy used might determine whether vation is not a safe strategy for preventing chemoresistance. NFB has a pro- or antiapoptotic effect (10). For example, the Accordingly, NFB inhibitors might be more useful for sensitiza- majority of chemotherapies induce some form of DNA damage, tion to chemotherapeutic drugs than agents that are able to and inhibition of NFB activation promotes cell death. How- inhibit PI3K/Akt activity. ever, it was proposed recently that NFB might be required for Constitutive activation of NFB has been described in a Taxol-induced cell death (49). This is consistent with studies great number of solid tumors, and this activation appears to that suggest that NFB can function proapoptotically depend- support cancer cell survival and to reduce the sensitivity to ing on the type of stimulus to which cells are exposed. For chemotherapeutic drugs. We showed in this study that whereas example, it has been reported that the induction of cell death by cisplatin-sensitive A2780 cells do not have constitutive activa- hydrogen peroxide requires NFB and that NFB is required tion of NFB, cisplatin-resistant Caov-3 cells do have constitu- for p53-dependent cell death (50). More studies will be needed tive activation of NFB. Thus, it appears that constitutive to determine the chemotherapy specificity and potential tumor activation of NFB mediates cisplatin resistance in ovarian specificity of NFB in controlling cancer therapy efficacy. cancer cells and inhibition of NFB activation sensitizes the We reported that Akt inactivation and inhibition of BAD phos- ovarian cancer cells to cisplatin. phorylation sensitize human ovarian cancer cells to cisplatin (1) How do NFB inhibitors cause the inhibition of growth of and paclitaxel (2). In addition, we recently demonstrated that human ovarian cancer cells? It was reported that NFB inhib- inhibition of Forkhead phosphorylation sensitizes human ovar- ian cancer cells to cisplatin. Although NFB is a substrate of Akt itors diminished the expression of survival genes regulated by NFB, such as c-IAP-2, TRAF-1, TRAF-2, XIAP, or IEX-1L (42, (like BAD and Forkhead), NFB activation is involved in angio- genesis (11, 12) and metastasis (13, 14) in addition to the sup- 43). We also showed that NFB inhibitors caused the inhibition 23484 Role of NFB in Cisplatin-induced Apoptosis FIG.8. BAY 11-7085 blocks the NFB 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 IB phosphorylation status and subcellular localization of NFB p65 were assessed by immunohistochemistry with anti-phospho-IB antibody (A) and anti-NFB p65 antibody (B), respectively. Scale bars,15 m. of the expression of survival genes in human ovarian cancer of NFB. The advantage of soluble inhibitors is that their cells (Fig. 3). The fact that NFB mediates the expression of delivery would be easier and more efficient than gene transfer multiple survival genes makes it an important and rational in vivo. We did not detect significant renal, hepatic, or pulmo- target for cancer chemotherapy. nary tissue toxicity in this study. A previous study also showed BAY 11-7082 is also a known pharmacological inhibitor of that using up to 20 mg/kg/day of these agents in rats for 21 IB phosphorylation (32), like BAY 11-7085. In the presence days did not cause obvious toxicity (32). of BAY 11-7082, the cisplatin-induced attenuation of IB Activation of NFB via phosphorylation of an inhibitor pro- phosphorylation was significantly enhanced (data not tein (IB) leads to degradation of IB through the ubiquitin- shown). BAY 11-7082 and BAY 11-7085 also activate the proteasome pathway. Inhibition of IB degradation by protea- c-Jun N-terminal protein kinase and p38 (32), both of which some inhibitors keeps NFB in the cytoplasm, thereby are known to be involved in the induction of apoptosis (56). preventing it from acting on nuclear DNA (57, 58). PS-341, Thus, the effects of BAY 11-7082 and BAY 11-7085 do not which is a potent, boronic acid dipeptide that is highly selective exclude the role of cellular proteins other than NFB. Five for proteasome inhibition, can be systemically administered homologous polypeptides, p50, p65, c-Rel, RelB, and p52, clinically (59). PS-341 has been shown to enhance the apoptotic comprise the mammalian Rel/NFB transcription factor fam- response to chemotherapy in a variety of in vitro and in vivo ily. The subunits associate in a combinatorial fashion to form models (18, 60 – 62). A Phase I trial of PS-341 and carboplatin transcriptionally active homo- and heterodimers. The best in recurrent ovarian cancer is currently ongoing (51). A Phase characterized species of NFB dimer is the p50/p65 het- II trial of PS-341 for the treatment of recurrent platinum- erodimer (39). A previous report demonstrated that the NLS sensitive ovarian or primary or peritoneal cancer (GOG 146-N) polypeptide of p50 is required for its translocation to the is also being conducted. We now await the results of these nucleus (40) and that p50NLS lacking the NLS domain currently ongoing clinical trials. inhibits the nucleocytoplasmic shuttling of NFB dimers. Acknowledgments—We thank Dr. J. Cheng (University of South Therefore, we examined the effect of p50NLS on the atten- Florida College of Medicine) for providing the NFB reporter plasmid uation of IB phosphorylation by cisplatin. Transfection of (pElam-luc), and Dr. Gourisankar Ghosh (Department of Chemistry p50NLS significantly inhibited the increased the efficacy of and Biochemistry, University of California, San Diego) for providing pCR-FLAG-p50 and pCR-FLAG-p50NLS constructs. We are also the cisplatin-induced attenuation of NFB activity compared grateful to Dr. Junko Takeda and Dr. Kazue Onuma for technical with the effect in cells expressing wild-type p50 (Fig. 2B, assistance and Ayako Okamura and Tomoko Iwaki for secretarial panel iv). Thus, the similarity of the effects caused by treat- assistance. ment with BAY 11-7082 or BAY 11-7085 and by transfection with p50NLS suggests that inhibition of NFB activity has REFERENCES strong potential as a novel adjuvant chemotherapy. 1. 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