Advertisement
Epidemiology and Prevention

N-(4-Hydroxyphenyl)Retinamide Inhibits Invasion, Suppresses Osteoclastogenesis, and Potentiates Apoptosis through Down-regulation of IκBα Kinase and Nuclear Factor-κB–Regulated Gene Products

Shishir Shishodia, Angelica M. Gutierrez, Reuben Lotan and Bharat B. Aggarwal
DOI: 10.1158/0008-5472.CAN-05-1585 Published October 2005

Abstract

N-(4-hydroxyphenyl) retinamide [4-HPR], a synthetic retinoid, has been shown to inhibit tumor cell growth, invasion, and metastasis by a mechanism that is not fully understood. Because the nuclear factor-κB (NF-κB) has also been shown to regulate proliferation, invasion, and metastasis of tumor cells, we postulated that 4-HPR modulates the activity of NF-κB. To test this postulate, we examined the effect of this retinoid on NF-κB and NF-κB–regulated gene products. We found that 4-HPR potentiated the apoptosis induced by tumor necrosis factor (TNF) and chemotherapeutic agents, suppressed TNF-induced invasion, and inhibited RANKL-induced osteoclastogenesis, all of which are known to require NF-κB activation. We found that 4-HPR suppressed both inducible and constitutive NF-κB activation without interfering with the direct DNA binding of NF-κB. 4-HPR was found to be synergistic with Velcade, a proteasome inhibitor. Further studies showed that 4-HPR blocked the phosphorylation and degradation of IκBα through the inhibition of activation of IκBα kinase (IKK), and this led to suppression of the phosphorylation and nuclear translocation of p65. 4-HPR also inhibited TNF-induced Akt activation linked with IKK activation. NF-κB–dependent reporter gene expression was also suppressed by 4-HPR, as was NF-κB reporter activity induced by TNFR1, TRADD, TRAF2, NIK, and IKK but not that induced by p65 transfection. The expression of NF-κB–regulated gene products involved in antiapoptosis (IAP1, Bfl-1/A1, Bcl-2, cFLIP, and TRAF1), proliferation (cyclin D1 and c-Myc), and angiogenesis (vascular endothelial growth factor, cyclooxygenase-2, and matrix metalloproteinase-9) were also down-regulated by 4-HPR. This correlated with potentiation of apoptosis induced by TNF and chemotherapeutic agents.

  • Growth
  • Apoptosis
  • TNF
  • AKT
  • NF-κB
  • IKK
  • N-(4-hydroxyphenyl)retinamide

Introduction

A synthetic retinoid, N-(4-hydroxyphenyl)retinamide (4-HPR), also called fenretinide, was first reported by Sporn et al. 25 years ago. It was found to be less toxic than retinyl acetate, inhibited N-nitroso-N-methylurea–induced breast cancer in rats, had superior pharmacology to retinyl acetate, accumulated selectively in breast tissue, and exhibited antiproliferative effects against mammary epithelium ( 1). This retinamide has since been shown to suppress the proliferation of a wide variety of tumor cells, including hematopoietic tumor cells ( 2– 6), breast cancer cells ( 7), glioma ( 8), neuroblastoma ( 9, 10), lung cancer ( 11, 12), squamous cell carcinoma ( 12– 14), melanoma ( 15), cervical carcinoma ( 16), uterine leiomyoma ( 17), epidermoid carcinoma ( 18), colon adenocarcinoma ( 19), prostate cancer ( 20), hepatoma ( 21), and ovarian carcinoma ( 22). 4-HPR is also effective in organotypic cultures of ovarian carcinoma ( 23). Several studies in animals have also shown that 4-HPR has activity against neuroblastoma ( 24), prostate cancer ( 25, 26), and Kaposi's sarcoma ( 27). These preclinical studies have led to clinical testing of the effectiveness of 4-HPR against breast cancer ( 28– 30) and prostate cancer ( 31). 4-HPR is also quite effective against actinic keratoses, a precursor to squamous cell carcinoma of the skin ( 32).

Exactly how 4-HPR mediates its anticancer effects is poorly understood. It has been found to induce apoptosis of tumor cells independently of the retinoid receptor pathways retinoic acid receptor (RAR) and retinoid X receptor (RXR). Modulation of the antiapoptotic proteins Bcl-2 ( 33, 34), cyclooxygenase-2 (COX-2; refs. 35, 36), cyclin D1 ( 36), and c-Jun NH2-terminal kinase ( 37) has been implicated in 4-HPR-induced apoptosis. Other reports suggest that the mitochondrial pathway plays a special role in apoptosis induced by this retinamide ( 18, 34, 37). 4-HPR induces G1-S arrest in the cells ( 4), which is most likely mediated through its effects on cyclin D1 and pRb-cyclin-dependent kinase (CDK) regulation ( 38). In vivo, 4-HPR has been shown to exhibit anti-invasive ( 39), antiangiogenic ( 40), and antimetastatic ( 25) activities. Tumor cell proliferation, invasion, angiogenesis, and metastasis have been shown to be regulated by the nuclear transcription factor nuclear factor-κB (NF-κB; ref. 41).

NF-κB is a family of Rel domain–containing proteins present in the cytoplasm of all cells, where they are kept in an inactive state by a family of anchorin domain–containing proteins, which includes IκBα, IκBβ, IκBγ, IκBε, Bcl-3, p105, and p100. Under resting conditions, NF-κB consists of a heterotrimer of p50, p65, and IκBα in the cytoplasm; only when activated and translocated to the nucleus is the sequence of events leading to activation initiated. Most carcinogens, inflammatory agents, and tumor promoters, including cigarette smoke, phorbol ester, okadaic acid, H2O2, and tumor necrosis factor (TNF), have been shown to activate NF-κB. The activation of NF-κB involves the phosphorylation, ubiquitination, and degradation of IκBα and phosphorylation of p65, which in turn leads to the translocation of NF-κB to the nucleus where it binds to specific response elements in the DNA. The phosphorylation of IκBα is catalyzed by IκBα kinase (IKK), which is essential for NF-κB activation by most agents. NF-κB has been shown to regulate the expression of several genes whose products are involved in tumorigenesis ( 41). These include antiapoptotic genes (e.g., cIAP, survivin, TRAF, cFLIP, Bfl-1, Bcl-2, and Bcl-xL); COX-2, matrix metalloproteinase-9 (MMP-9); vascular endothelial growth factor (VEGF); genes encoding adhesion molecules, chemokines, and inflammatory cytokines; and cell cycle regulatory genes (e.g., cyclin D1 and c-myc).

Because 4-HPR regulates the expression of various gene products that are regulated by NF-κB and suppresses tumor cell proliferation, invasion, angiogenesis, and metastasis, which are also regulated by NF-κB, we postulated that this retinamide must mediate its effects through modulation of NF-κB activation pathway. To test this postulate, we investigated the effect of 4-HPR on NF-κB activation induced by a variety of inflammatory agents and carcinogens in various cell types, including those where NF-κB is constitutively active. We found that 4-HPR inhibited the activation of NF-κB through inhibition of IκB kinase, IκBα phosphorylation and degradation, p65 nuclear translocation, DNA binding, and NF-κB–dependent reporter gene expression. The suppression of NF-κB by 4-HPR inhibited TNF-induced cell invasion and abrogated RANKL-induced osteoclastogenesis. 4-HPR also led to the down-regulation of gene products that prevent apoptosis and promote inflammation and tumor metastasis.

Materials and Methods

Materials. 4-HPR (Sigma-Aldrich, St. Louis, MO) was dissolved in DMSO as a 10 mmol/L stock solution and stored at −20°C. Bacteria-derived human TNF, purified to homogeneity with a specific activity of 5 × 107 units/mg, was kindly provided by Genentech, Inc. (South San Francisco, CA). Penicillin, streptomycin, RPMI 1640, fetal bovine serum (FBS), and LipofectAMINE 2000 were obtained from Invitrogen (Grand Island, NY). The following polyclonal antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA): anti-p65, against the epitope corresponding to amino acids mapping within the NH2-terminal domain of human NF-κB p65; anti-p50, against a peptide 15 amino acids long mapping at the nuclear localization sequence region of NF-κB p50; anti-IκBα, against amino acids 297 to 317 mapping at the COOH terminus of IκBα/MAD-3; anti-c-Rel; anti–cyclin D1 against amino acids 1 to 295, which represents full-length cyclin D1 of human origin; anti-MMP-9; anti–poly(ADP-ribose) polymerase (PARP); anti-IAP1; anti-Bcl-2; anti-Bfl-1/A1; and anti-TRAF1. Anti-COX-2 and anti-MMP-9 antibodies were obtained from BD Biosciences (San Diego, CA), and phosphospecific anti-IκBα (Ser32), phosphorylated Akt, and phosphorylated p65 (Ser536) antibody from Cell Signaling (Beverly, MA). Anti-IKKα and anti-IKKβ antibodies were kindly provided by Imgenex (San Diego, CA). Velcade (PS-341) was obtained from Millennium (Cambridge, MA).

Cell lines. The cell lines used in our studies included human non–small cell lung carcinoma cells (H1299), human lung epithelial cell carcinoma cells (1170-I), human leukemia (Jurkat), human myelogenous leukemia (KBM-5), human multiple myeloma cell lines U266 and RPMI 8226, and human head and neck squamous cell carcinoma (HNSCC) cell lines MDA 1986, HN5, and FADU. HNSCC cell lines were obtained from Dr. Gary Clayman (University of Texas, M.D. Anderson Cancer Center, Houston, TX). 1170-I cell lines were obtained from Dr. Klein-Szanto (Fox Chase Cancer Center, Philadelphia, PA). All the other cell lines were obtained from the American Type Culture Collection (Manassas, VA). Jurkat, H1299, and multiple myeloma cells were cultured in RPMI 1640 with 10% FBS, and KBM-5 cells were cultured in Iscove's modified DMEM with 15% FBS. Raw 264.7 cells were cultured in DMEM/F-12. For 1170-I cells, keratinocyte serum-free medium was supplemented with 3% FBS. All HNSCC cell lines were cultured in DMEM containing 10% FBS, nonessential amino acids, pyruvate, glutamine (1%), and vitamins (2%). All media were supplemented with 100 units/mL penicillin and 100 μg/mL streptomycin.

Cytotoxicity assay. Cytotoxicity was assayed by the modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously ( 42).

Poly(ADP-ribose) polymerase cleavage assay. For detection of cleavage products of PARP, whole-cell extracts were prepared by subjecting 4-HPR-treated cells to lysis in lysis buffer [20 mmol/L Tris (pH 7.4), 250 mmol/L NaCl, 2 mmol/L EDTA (pH 8.0), 0.1% Triton X-100, 0.01 μg/mL aprotinin, 0.005 μg/mL leupeptin, 0.4 mmol/L phenylmethylsulfonyl fluoride, 4 mmol/L NaVO4]. Lysates were spun at 14,000 rpm for 10 minutes to remove insoluble material, resolved by 10% SDS-PAGE, and probed with PARP antibodies.

Live and dead assay. To measure apoptosis, we used the Live and Dead assay (Molecular Probes, Eugene, OR), which determines intracellular esterase activity and plasma membrane integrity. This assay employs calcein, a polyanionic dye, which is retained within the live cells and provides green fluorescence ( 42). It also employs the ethidium monomer dye (red fluorescence), which can enter the cells only through damaged membranes and bind to nucleic acids but is excluded by the intact plasma membrane of live cells. Briefly, 1 × 105 cells were incubated with 10 μmol/L 4-HPR for 24 hours and then treated with 1 nmol/L TNF for 16 hours at 37°C. Cells are stained with the Live and Dead reagent (5 μmol/L ethidium homodimer, 5 μmol/L calcein-AM) and then incubated at 37°C for 30 minutes. Cells were analyzed under a fluorescence microscope (Labophot-2, Nikon, Tokyo, Japan).

Invasion assay. The membrane invasion culture system was used to assess cell invasion because invasion through the extracellular matrix is a crucial step in tumor metastasis. The BD BioCoat Tumor Invasion System is a chamber that has a light-tight polyethylene terephthalate membrane with 8-μm-diameter pores and is coated with a reconstituted basement membrane gel (BD Biosciences). A total of 2.5 × 104 H1299 cells were suspended in serum-free medium and seeded into the upper wells. After incubation overnight, cells were treated with 10 μmol/L 4-HPR for 12 hours and then stimulated with 1 nmol/L TNF for a further 24 hours in the presence of 1% FBS and the 4-HPR. The cells that invaded through the Matrigel (i.e., those that migrated to the lower chamber during incubation) were stained with 4 μg/mL calcein-AM (Molecular Probes) in PBS for 30 minutes at 37°C and scanned for fluorescence with a Victor 3 multiplate reader (Perkin-Elmer Life and Analytical Sciences, Boston, MA); fluorescent cells were counted.

Osteoclast differentiation assay. To determine the effect of 4-HPR on RANKL-induced osteoclastogenesis, we cultured RAW 264.7 cells, which can differentiate into osteoclasts by RANKL in vitro ( 42). RAW 264.7 cells were cultured in 24-well dishes at a density of 1 × 104 per well and allowed to adhere overnight. The medium was then replaced, and the cells were pretreated with 5 μmol/L 4-HPR for 12 hours and then treated with 5 nmol/L RANKL. At days 4 and 5, the cells were stained for tartrate-resistant acid phosphatase (TRAP) expression as described previously using an acid phosphatase kit (Sigma-Aldrich), and the TRAP-positive multinucleated osteoclasts (>3 nuclei) per well were counted.

Nuclear factor-κB activation. To determine NF-κB activation by TNF (which has a well-established role in inflammation, tumor proliferation, promotion, invasion, and metastasis; ref. 43), we carried out electrophoretic mobility shift assay (EMSA) essentially as described previously ( 44). Briefly, nuclear extracts prepared from TNF-treated cells (1 × 106/mL) were incubated with 32P-end-labeled 45-mer double-stranded NF-κB oligonucleotide (15 μg protein with 16 fmol DNA) from the HIV long terminal repeat, 5′-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3′ (boldface indicates NF-κB-binding sites), for 30 minutes at 37°C, and the DNA-protein complex formed was separated from free oligonucleotide on 6.6% native polyacrylamide gels. A double-stranded mutated oligonucleotide, 5′-TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3′, was used to examine the specificity of binding of NF-κB to the DNA. The specificity of binding was also examined by competition with the unlabeled oligonucleotide. For supershift assays, nuclear extracts prepared from TNF-treated cells were incubated with antibodies against either the p50 or the p65 subunit of NF-κB for 30 minutes at 37°C before the complex was analyzed by EMSA. Preimmune serum was included as a negative control. The dried gels were visualized with a Storm820 and radioactive bands were quantitated using ImageQuant software (Amersham Pharmacia Biotechnology, Piscataway, NJ).

Western blot analysis. To determine the effect of 4-HPR on TNF-dependent IκBα phosphorylation, IκBα degradation, p65 translocation, and p65 phosphorylation, cytoplasmic extracts were prepared as described previously ( 44) from H1299 cells (2 × 106/mL) that had been pretreated with 25 μmol/L 4-HPR for 24 hours and then exposed to 0.1 nmol/L TNF for various times. Cytoplasmic protein (30 μg) was resolved on 10% SDS-PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% nonfat milk, and probed with specific antibody against IκBα, phosphorylated IκBα, p65, and phosphorylated p65. To determine the expression of cyclin D1, COX-2, MMP-9, cIAP1, TRAF1, Bcl-2, Bfl-1, and cFLIP in whole-cell extracts of treated cells (2 × 106 cells in 2 mL medium), protein (30-50 μg) was resolved on SDS-PAGE and probed by Western blot with specific antibodies as per manufacturer's recommended protocol. The blots were washed, exposed to horseradish peroxidase–conjugated secondary antibodies for 1 hour, and finally detected by enhanced chemiluminescence reagent (Amersham Pharmacia Biotechnology). The bands were quantitated using a Personal Densitometer Scan version 1.30 using ImageQuant software version 3.3 (Molecular Dynamics).

IκBα kinase assay. To determine the effect of 4-HPR on TNF-induced IKK activation, we analyzed IKK by a method essentially as described previously ( 44). Briefly, the IKK complex from whole-cell extracts was precipitated with antibody against IKKα and IKKβ and then treated with protein A/G-Sepharose beads (Pierce, Rockford, IL). After 2 hours, the beads were washed with lysis buffer and then resuspended in a kinase assay mixture containing 50 mmol/L HEPES (pH 7.4), 20 mmol/L MgCl2, 2 mmol/L DTT, 20 μCi [γ-32P]ATP, 10 μmol/L unlabeled ATP, and 2 μg substrate glutathione S-transferase-IκBα (amino acids 1-54). After incubation at 30°C for 30 minutes, the reaction was terminated by boiling with SDS sample buffer for 5 minutes. Finally, the protein was resolved on 10% SDS-PAGE, the gel was dried, and the radioactive bands were visualized with a Storm820. To determine the total amounts of IKKα and IKKβ in each sample, whole-cell proteins (50 μg) were resolved on 7.5% SDS-PAGE, electrotransferred to a nitrocellulose membrane, and then blotted with either anti-IKK-α or anti-IKK-β antibody.

Immunolocalization of nuclear factor-κB p65. The effect of 4-HPR on TNF-induced nuclear translocation of p65 was examined by an immunocytochemical method using an epifluorescence microscope (Labophot-2; Nikon, Tokyo, Japan) and a Photometrics Coolsnap CF color camera (Nikon, Lewisville, TX) as described previously ( 44).

Nuclear factor-κB–dependent reporter gene transcription. The effect of 4-HPR on TNF-induced NF-κB-dependent reporter gene transcription in H1299 cells was measured as described previously ( 44).

Cyclooxygenase-2 promoter-dependent reporter luciferase gene expression. COX-2 promoter activity was examined as described elsewhere ( 44). To further determine the effect of 4-HPR on COX-2 promoter, A293 cells were seeded at a concentration of 1.5 × 105 cells per well in six-well plates. After overnight culture, the cells in each well were transfected with 2 μg DNA consisting of COX-2 promoter-luciferase reporter plasmid along with 6 μL LipofectAMINE 2000 according to the manufacturer's protocol. The COX-2 promoter (−375 to +59), which was amplified from human genomic DNA by using the primers 5′-GAGTCTCTTATTTATTTTT-3′ (sense) and 5′-GCTGCTGAGGAGTTCCTGGACGTGC-3′ (antisense), was kindly provided by Dr. Xiao-Chun Xu (M.D. Anderson Cancer Center). After a 6-hour exposure to the transfection mixture, the cells were incubated in medium containing 4-HPR for 12 hours. The cells were exposed to TNF (0.1 nmol/L) for 24 hours and then harvested. Luciferase activity was measured by using the Luclite (Perkin-Elmer) luciferase assay system according to the manufacturer's protocol and detected by luminometer (Victor 3, Perkin-Elmer). All experiments were done in triplicate and repeated at least twice to prove their reproducibility.

Results

The goal of this study was to investigate the effect of a synthetic retinoid, 4-HPR, on the transcription factor NF-κB signaling pathway, on NF-κB–regulated gene products, and on NF-κB-mediated cellular responses. The structure of this retinoid is shown in Fig. 1A . The concentration of 4-HPR used and the duration of exposure had minimal effect on the viability of H1299 cells as determined by trypan blue dye exclusion test. For most studies, human small cell lung carcinoma H1299 cells were used because these cells have been shown to be sensitive to 4-HPR ( 11, 12). To examine the effect of 4-HPR on the NF-κB activation pathway, most studies employed TNF because the pathway activated by this agent is well understood.

Figure 1.
Figure 1.

A, structure of 4-HPR. B, 4-HPR enhances apoptosis induced by TNF and chemotherapeutic agents. KBM-5 cells (5,000/0.1 mL) were incubated at 37°C with TNF, Taxol, or doxorubicin (Doxo) in the presence and absence of 5 μmol/L 4-HPR, as indicated, for 72-hour duration, and the viable cells were assayed using MTT reagent. Columns, mean cytotoxicity from triplicate cultures; bars, SD. C, 4-HPR enhances TNF-induced PARP cleavage. KBM-5 cells (2 × 106/1 mL) were serum starved for 24 hours and then incubated with TNF (1 nmol/L) alone or in combination with 4-HPR (25 μmol/L) for the indicated times, and PARP cleavage was determined by Western blot analysis as described in Materials and Methods. Values (bottom) indicate the densitometry analysis of the 87,000 Da band. D, 4-HPR enhances TNF-induced cell death. KBM-5 cells (2 × 106/mL) were serum starved for 24 hours and then incubated with TNF (1 nmol/L) alone or in combination with 4-HPR (10 μmol/L) as indicated for 24 hours. Cell death was determined by calcein-AM–based Live and Dead assay as described in Materials and Methods. Representative experiment of the three independent ones showing similar results.

N-(4-hydroxyphenyl) retinamide potentiates the apoptotic effects of tumor necrosis factor and chemotherapeutic drugs. Because NF-κB activation has been shown to suppress the apoptosis induced by various agents ( 45), whether 4-HPR will modulate the apoptosis induced by TNF and chemotherapeutic agents was investigated. The effect of 4-HPR on TNF and chemotherapeutic agent–induced apoptosis was examined by the MTT assay. We found that 4-HPR enhances the cytotoxic effects of TNF, paclitaxel, and doxorubicin ( Fig. 1B). By using caspase-activated PARP cleavage, we show that the enhanced cytotoxicity was due to apoptosis. TNF-induced PARP-cleavage was enhanced in the 4-HPR-treated cells ( Fig. 1C). The Live and Dead assay, which measures intracellular esterase activity and plasma membrane integrity, also indicated that 4-HPR up-regulated TNF-induced apoptosis from 18% to 58% ( Fig. 1D). The results of all the assays together suggest that 4-HPR enhances the apoptotic effects of TNF and chemotherapeutic agents.

N-(4-hydroxyphenyl) retinamide suppresses tumor necrosis factor–induced tumor cell invasion activity. It is known that NF-κB regulates the expression of gene products (e.g., MMP-9, COX-2, and VEGF) that mediate tumor cell invasion ( 46). Whether 4-HPR can modulate TNF-induced tumor cell invasion activity was investigated in vitro. To determine this, tumor cells were seeded to the top chamber of the Matrigel invasion chamber with TNF in the presence or absence of 4-HPR and then examined for invasion. As shown in Fig. 2A , TNF induced tumor cell invasion by ∼15-fold and 4-HPR suppressed this activity. 4-HPR alone had no invasion activity.

Figure 2.
Figure 2.

4-HPR suppresses TNF-induced invasive activity and RANKL-induced osteoclastogenesis. A, H1299 cells (2.5 × 104) were seeded into the upper wells of a Matrigel invasion chamber overnight in the absence of serum, pretreated with 10 μmol/L 4-HPR for 12 hours, treated with 1 nmol/L TNF for 24 hours in the presence of 1% serum, and then subjected to invasion assay. The value for no 4-HPR and no TNF was set to 1.0. Columns, mean of three experiments; bars, SD. *, P < 0.05; **, P < 0.001. B, RAW 264.7 cells (1 × 104) were plated overnight, pretreated with 5 μmol/L 4-HPR for 12 hours, and then treated with 5 nmol/L RANKL. Four and 5 days later, cells were stained for TRAP and evaluated for osteoclastogenesis. Photographs were taken after 5 days of incubation with RANKL. The numbers of TRAP-positive multinucleated osteoclasts (>3 nuclei) per well were counted (C).

N-(4-hydroxyphenyl) retinamide suppresses RANKL-induced osteoclastogenesis. Because RANKL, a member of the TNF superfamily, induces osteoclastogenesis through the activation of NF-κB ( 47), we determined whether 4-HPR can suppress RANKL-induced osteoclastogenesis. We found that RANKL induced osteoclast differentiation, as indicated by the expression of TRAP, and that 4-HPR suppressed it ( Fig. 2B and C).

N-(4-hydroxyphenyl) retinamide inhibits tumor necrosis factor–induced cyclooxygenase-2, matrix metalloproteinase-9, and vascular endothelial growth factor expression. Above results indicate that 4-HPR inhibits TNF-induced tumor cell invasion. Whether these effects of 4-HPR are mediated through the suppression of COX-2, MMP-9, and VEGF gene products was investigated. As shown in Fig. 3A , TNF treatment induced the expression of VEGF, COX-2, and MMP-9 gene products and 4-HPR abolished the expression ( Fig. 3A).

Figure 3.
Figure 3.

4-HPR inhibits TNF-induced NF-κB–regulated gene products. A, 4-HPR inhibits MMP-9, COX-2, and VEGF expression induced by TNF. H1299 cells (2 × 106/mL) were left untreated or incubated with 25 μmol/L 4-HPR for 24 hours and then treated with 0.1 nmol/L TNF for different times. Whole-cell extracts were prepared, and whole-cell lysate (80 μg) was analyzed by Western blotting using antibodies against VEGF, MMP-9, and COX-2. B, 4-HPR inhibits cyclin D1 and c-myc expression induced by TNF. H1299 cells (2 × 106/mL) were left untreated or incubated with 25 μmol/L 4-HPR for 24 hours and then treated with 0.1 nmol/L TNF for different times. Whole-cell extracts were prepared, and whole-cell lysate (80 μg) was analyzed by Western blotting using antibodies against cyclin D1 and c-myc. Representative experiment of the three independent ones showing similar results. C, 4-HPR inhibits the expression of antiapoptotic gene products cIAP1, Bfl-1/A1, Bcl-2, TRAF1, and cFLIP: H1299 cells (2 × 106/mL) were left untreated or incubated with 25 μmol/L 4-HPR for 24 hours and then treated with 0.1 nmol/L TNF for different times. Whole-cell extracts were prepared and whole-cell lysate (50 μg) was analyzed by Western blotting using antibodies against IAP1, Bfl-1/A1, Bcl-2, TRAF1, and cFLIP as indicated. Representative experiment of the three independent ones showing similar results.

N-(4-hydroxyphenyl) retinamide inhibits tumor necrosis factor–induced cyclin D1 and c-myc expression. Both cyclin D1 and c-myc regulate cellular proliferation and are regulated by NF-κB ( 41). Whether 4-HPR controls the expression of these gene products was also examined. Our results show that 4-HPR abolished, in a time-dependent fashion, the TNF-induced expression of cyclin D1 and c-myc ( Fig. 3B).

N-(4-hydroxyphenyl) retinamide inhibits tumor necrosis factor–induced activation of antiapoptotic gene products. Above results indicate that 4-HPR potentiates the apoptosis induced by TNF. Whether this effect of 4-HPR is through suppression of antiapoptotic gene products was investigated. NF-κB up-regulates the expression of several genes implicated in facilitating tumor cell survival, including cIAP1, Bfl-1, Bcl-2, TRAF1, and cFLIP ( 41). We found that 4-HPR inhibited the TNF-induced as well as the basal expression of all of these proteins ( Fig. 3C).

N-(4-hydroxyphenyl) retinamide blocks nuclear factor-κB activation induced by various agents. We found that the expression of various gene products that are involved in antiapoptosis (IAP1, Bfl-1/A1, Bcl-2, cFLIP, and TRAF1), proliferation (cyclin D1, and c-myc), and invasion (VEGF, COX-2, and MMP-9) were down-regulated by 4-HPR. All of these gene products are regulated by NF-κB. Whether 4-HPR modulates NF-κB activation was investigated. We first examined the effect of 4-HPR on the activation of NF-κB induced by various agents, including TNF, pervanadate, phorbol 12-myristate 13-acetate (PMA), okadaic acid, cigarette smoke condensate, and H2O2. DNA-binding assay (EMSA) showed that 4-HPR suppressed the NF-κB activation induced by all these agents ( Fig. 4A ). These results suggest that 4-HPR acted at a step in the NF-κB activation pathway that is common to all these agents.

Figure 4.

4-HPR inhibits NF-κB. A, 4-HPR blocks NF-κB activation induced by TNF, sodium pervanadate, PMA, okadaic acid, and H2O2. H1299 cells (2 × 106/mL) were preincubated for 24 hours at 37°C with 25 μmol/L 4-HPR and then treated with TNF (0.1 nmol/L), sodium pervanadate (250 μmol/L, 2 hours), PMA (100 ng/mL, 1 hours), okadaic acid (500 nmol/L, 4 hours), cigarette smoke condensate (10 μg/mL, 30 minutes), or H2O2 (250 μmol/L, 1 hour). Nuclear extracts were prepared and tested for NF-κB activation as described in Materials and Methods. Representative experiment of the three independent ones showing similar results. B, 4-HPR inhibits TNF-dependent NF-κB activation in a dose-dependent manner. H1299 cells (2 × 106/mL) were preincubated with the indicated concentrations of 4-HPR for 24 hours at 37°C and then treated with 0.1 nmol/L TNF for 30 minutes. Nuclear extracts were prepared and tested for NF-κB activation as described in Materials and Methods. C, 4-HPR inhibits TNF-dependent NF-κB activation in a time-dependent manner. H1299 cells (2 × 106/mL) were preincubated with 25 μmol/L 4-HPR for the indicated times at 37°C and then treated with 0.1 nmol/L TNF for 30 minutes at 37°C. Nuclear extracts were prepared and then tested for NF-κB activation. Cell viability (%) is indicated. D, 4-HPR does not modulate the ability of NF-κB to bind to the DNA. Nuclear extracts from H1299 cells (2 × 106/mL) treated or not treated with 0.1 nmol/L TNF for 30 minutes were treated with the indicated concentrations of 4-HPR for 2 hours at room temperature and then assayed for DNA binding by EMSA. Representative experiment of the three independent ones showing similar results. E, Velcade synergizes with 4-HPR in inhibiting TNF-induced NF-κB activation. H1299 cells (2 × 106/mL) were pretreated with 10 μmol/L 4-HPR and/or 0.05 μmol/L Velcade as indicated for 24 hours and then treated with 0.1 nmol/L TNF for 30 minutes. Nuclear extracts were prepared and then assayed for DNA binding by EMSA. Representative experiment of the three independent ones showing similar results.

N-(4-hydroxyphenyl) retinamide suppresses nuclear factor-κB activation in a dose- and time-dependent manner. We next determined the dose and time of exposure to 4-HPR required to suppress NF-κB activation. EMSA results showed that 4-HPR alone had no effect on NF-κB activation. However, it inhibited TNF-mediated NF-κB activation in a dose-dependent manner ( Fig. 4B). The suppression of NF-κB activation by 4-HPR was also found to be time dependent ( Fig. 4C). When nuclear extracts from TNF-activated cells were incubated with antibodies to the p50 (NF-κB1) and the p65 (RelA) subunit of NF-κB, the resulting bands were shifted to higher molecular masses (data not shown), suggesting that the TNF-activated complex consisted of p50 and p65. Preimmune serum had no effect on DNA binding. Addition of excess unlabeled NF-κB (cold oligonucleotide; 100-fold) caused complete disappearance of the band, whereas mutated oligonucleotide had no effect on the DNA binding.

N-(4-hydroxyphenyl) retinamide does not directly affect binding of nuclear factor-κB to the DNA. Some NF-κB inhibitors, including N-tosyl-l-phenylalanine chloromethyl ketone (TPCK; the serine protease inhibitor), herbimycin A (protein tyrosine kinase inhibitor), and caffeic acid phenethyl ester (CAPE), directly modify NF-κB to suppress its DNA binding ( 48– 50). Whether 4-HPR mediates its effect through similar mechanism was examined. EMSA showed that 4-HPR did not modify the DNA-binding ability of NF-κB proteins prepared from TNF-treated cells ( Fig. 4D). These results suggest that 4-HPR inhibits NF-κB activation by a mechanism different from that of TPCK, herbimycin A, or CAPE.

Inhibition of nuclear factor-κB by N-(4-hydroxyphenyl) retinamide is potentiated by Velcade. Our results until now show that 25 μmol/L 4-HPR is required to inhibit TNF-induced NF-κB activation in vitro. This dose, however, is not physiologically achievable ( 51, 52). We, therefore, examined whether Velcade, a proteasome inhibitor, synergize with 4-HPR to inhibit NF-κB activation. We found that 0.05 μmol/L Velcade or 10 μmol/L 4-HPR alone had no effect on TNF-induced NF-κB activation ( Fig. 4E). However, when 10 μmol/L 4-HPR was combined with 0.05 μmol/L Velcade, it completely suppressed TNF-induced NF-κB activation ( Fig. 4E). These results suggest that physiologically achievable level of 4-HPR when combined with Velcade can abrogate NF-κB activation.

Inhibition of nuclear factor-κB activation by N-(4-hydroxyphenyl) retinamide is not cell type specific. It has been reported that NF-κB induction pathway in epithelial cells may differ from lymphoid cells ( 53). We therefore determined whether 4-HPR inhibited NF-κB activation in three different cell types. 4-HPR completely inhibited TNF-induced NF-κB activation in lung epithelial cell carcinoma (1170-I), T-cell leukemia (Jurkat), and myeloid leukemia (KBM-5) cells ( Fig. 4F), indicating a lack of cell type specificity.

N-(4-hydroxyphenyl) retinamide inhibits constitutive nuclear factor-κB activation. Various tumor cells are known to express constitutively active NF-κB ( 41). To determine whether 4-HPR can inhibit constitutive NF-κB activation, we treated multiple myeloma (U266) cells with varying doses of 4-HPR. We found that 4-HPR inhibited the constitutive NF-κB activation in a dose-dependent manner with optimum inhibition occurring at 25 μmol/L ( Fig. 4G). Whether constitutive NF-κB in other tumor cells is also affected by 4-HPR was also examined. As shown in Fig. 4H, 4-HPR inhibited constitutively active NF-κB in other multiple myeloma (RPMI 8226) and HNSCC (MDA 1986, HN5, and FADU) tumor cells.

N-(4-hydroxyphenyl) retinamide inhibits tumor necrosis factor–dependent IκBα degradation. Because IκBα degradation is required for activation of NF-κB ( 41), we determined whether 4-HPR's inhibition of TNF-induced NF-κB activation was due to inhibition of IκBα degradation. We found that TNF induced IκBα degradation in control cells as early as 10 minutes, but in 4-HPR-pretreated cells TNF had no effect on IκBα degradation ( Fig. 5A, top ).

Figure 5.
Figure 5.

A, 4-HPR inhibits TNF-induced phosphorylation and degradation of IκBα. H1299 cells (2 × 106/mL) were incubated with 25 μmol/L 4-HPR for 24 hours at 37°C, treated with 0.1 nmol/L TNF for the indicated times at 37°C, and then tested for IκBα (top) and phosphorylated IκBα (middle) in cytosolic fractions by Western blot analysis. Equal protein loading was evaluated by β-actin (bottom). B, 4-HPR inhibits TNF-induced IKK activity. H1299 cells (2 × 106/mL) were treated with 25 μmol/L 4-HPR for 24 hours and then treated with 0.1 nmol/L TNF for the indicated time intervals. Whole-cell extracts were prepared and extract (200 μg) was immunoprecipitated with antibodies against IKKα and IKKβ. Thereafter, immune complex kinase assay was done as described in Materials and Methods. To examine the effect of 4-HPR on the level of expression of IKK proteins, whole-cell extract (30 μg) was run on 10% SDS-PAGE, electrotransferred, and immunoblotted with indicated antibodies as described in Materials and Methods. C, 4-HPR has no direct effect on IKK activity. Whole-cell extracts were prepared from untreated and TNF (0.1 nmol/L)–treated H1299 cells (2 × 106/mL); protein/sample whole-cell extract (200 μg) was immunoprecipitated with antibodies against IKKα and IKKβ. The immune complex was treated with the indicated concentrations of 4-HPR for 30 minutes at 30°C; then, a kinase assay was done as described in Materials and Methods. Equal protein loading was evaluated by IKKβ. D, 4-HPR inhibits Akt phosphorylation. H1299 cells (2 × 106/mL) were incubated with 25 μmol/L 4-HPR for 24 hours and then treated with 0.1 nmol/L TNF for the indicated times. The whole cell extracts were analyzed by Western blotting using antibodies against the phosphorylated Akt. Equal protein loading was evaluated by Akt. Representative experiment of the three independent ones showing similar results. E, 4-HPR inhibits TNF-induced phosphorylation of p65. H1299 cells (2 × 106/mL) were incubated with 25 μmol/L 4-HPR for 24 hours and then treated with 0.1 nmol/L TNF for the indicated times. The cytoplasmic extracts were analyzed by Western blotting using antibodies against the phosphorylated form of p65. F, 4-HPR inhibits TNF-induced nuclear translocation of p65. H1299 cells (1 × 106/mL) were either untreated or pretreated with 25 μmol/L 4-HPR for 24 hours at 37°C and then treated with 0.1 nmol/L TNF for the indicated times. Nuclear extracts were prepared and analyzed by Western blotting using antibodies against p65. G, 4-HPR inhibits TNF-induced nuclear translocation of p65. H1299 cells (1 × 106/mL) were first treated with 25 μmol/L 4-HPR for 24 hours at 37°C and then exposed to 0.1 nmol/L TNF. After cytospin, immunocytochemical analysis was done as described in Materials and Methods. Representative experiment of the three independent ones showing similar results.

N-(4-hydroxyphenyl) retinamide inhibits tumor necrosis factor–dependent IκBα phosphorylation. We next determined whether 4-HPR affected TNF-induced IκBα phosphorylation needed for IκBα degradation. Western blot analysis using antibody that detects only the serine-phosphorylated form of IκBα indicated that TNF induced IκBα phosphorylation as early as 5 minutes, and 4-HPR completely suppressed it ( Fig. 5A, middle). Thus, 4-HPR inhibited TNF-induced NF-κB activation by inhibiting phosphorylation and degradation of IκBα.

N-(4-hydroxyphenyl) retinamide inhibits tumor necrosis factor–induced IκBα kinase activation. Because 4-HPR inhibits the phosphorylation and degradation of IκBα, we tested the effect of 4-HPR on TNF-induced IKK activation, which is required for TNF-induced phosphorylation of IκBα. As shown in Fig. 5B (top), 4-HPR completely suppressed TNF-induced activation of IKK. TNF or 4-HPR had no direct effect on the expression of IKK protein (bottom).

Next, we examined the effect of 4-HPR on IKK activity in vitro. We found that 4-HPR did not directly interfere with the IKK activity ( Fig. 5C). Because treatment of cells inhibits TNF-induced IKK activity, 4-HPR must suppress the activation of IKK.

N-(4-hydroxyphenyl) retinamide inhibits tumor necrosis factor–induced Akt activation. TNF has been shown to activate IKK through activation of Akt ( 54). Therefore, we also tested the effect of 4-HPR on TNF-induced activation of Akt. As shown in Fig. 5D, TNF induced the activation of Akt in a time-dependent manner, and 4-HPR treatment suppressed the activation.

N-(4-hydroxyphenyl) retinamide inhibits tumor necrosis factor–induced phosphorylation and nuclear translocation of p65. We also tested the effect of 4-HPR on TNF-induced phosphorylation of p65, because phosphorylation is also required for transcriptional activity of p65 ( 41). As shown in Fig. 5E, 4-HPR suppressed p65 phosphorylation almost completely. Likewise, Western blot analysis ( Fig. 5F) and immunocytochemistry ( Fig. 5G) indicated that 4-HPR abolished TNF-induced nuclear translocation of p65.

N-(4-hydroxyphenyl) retinamide represses tumor necrosis factor–induced nuclear factor-κB–dependent reporter gene expression. Our results up to this point show that 4-HPR inhibited the translocation of p65 inside the nucleus. To further determine that 4-HPR inhibited NF-κB–dependent gene transcription, we transiently transfected cells with the NF-κB–regulated secretory alkaline phosphatase (SEAP) reporter construct and then stimulated them with TNF. We found that TNF produced an ∼20-fold increase in SEAP activity over vector control ( Fig. 6A ), which was inhibited by dominant-negative IκBα, indicating specificity. When the cells were pretreated with 4-HPR, TNF-induced NF-κB–dependent SEAP expression was inhibited in a dose-dependent manner. These results show that 4-HPR inhibits NF-κB–dependent reporter gene expression induced by TNF.

Figure 6.
Figure 6.

A, 4-HPR inhibits TNF-induced NF-κB–dependent reporter gene (SEAP) expression. H1299 cells were transiently transfected with a NF-κB-containing plasmid linked to the SEAP gene and then treated with the indicated concentrations of 4-HPR. After 24 hours in culture with 0.1 nmol/L TNF, cell supernatants were collected and assayed for SEAP activity as described in Materials and Methods. Results are expressed as fold activity over the activity of the vector control. B, 4-HPR inhibits NF-κB–dependent reporter gene expression induced by TNFR, TRADD, TRAF, NIK, and IKKβ. H1299 cells were transiently transfected with the indicated plasmids along with a NF-κB-containing plasmid linked to the SEAP gene and then left either untreated or treated with 25 μmol/L 4-HPR for 24 hours. Cell supernatants were assayed for SEAP activity as described in Materials and Methods. Results are expressed as fold activity over the activity of the vector control. Bars, SD. C, 4-HPR inhibits TNF-induced COX-2 promoter activity. H1299 cells were transiently transfected with a COX-2 promoter plasmid linked to the luciferase gene and then treated with the indicated concentrations of 4-HPR. After 24 hours in culture with 0.1 nmol/L TNF, cell supernatants were collected and assayed for luciferase activity as described in Materials and Methods. Results are expressed as fold activity over the activity of the vector control. Columns, mean of triplicate cultures of a representative experiment of the three independent ones showing similar results; bars, SD. D, schematic representation of the effect of 4-HPR on TNF-induced NF-κB activation and apoptosis.

We next determined where 4-HPR acts in the sequence of TNFR1, TRADD, TRAF2, NIK, and IKK recruitment that characterizes TNF-induced NF-κB activation ( 41). In cells transfected with TNFR1, TRADD, TRAF2, NIK, IKKβ, and p65 plasmids, NF-κB–dependent reporter gene expression was induced; 4-HPR suppressed SEAP expression in all cells, except those transfected with p65 ( Fig. 6B).

N-(4-hydroxyphenyl) retinamide represses tumor necrosis factor–induced cyclooxygenase-2 promoter activity. We next determined whether 4-HPR affected COX-2 promoter activity, which is regulated by NF-κB ( 41). As shown in Fig. 6C, 4-HPR inhibited the TNF-induced COX-2 promoter activity in a dose-dependent manner.

Discussion

The present study was designed to investigate the effect of 4-HPR on the NF-κB activation pathway and on the NF-κB–regulated gene products that control tumor cell survival, proliferation, invasion, angiogenesis, and metastasis (see Fig. 6D). We found that 4-HPR potentiated the apoptosis induced by TNF and chemotherapeutic agents and inhibited TNF-induced invasion and RANKL-induced osteoclastogenesis. 4-HPR suppressed NF-κB activated by carcinogens, tumor promoters, and inflammatory stimuli in a variety of cell lines. This inhibition was mediated through inhibition of IKK by 4-HPR, which led to suppression of phosphorylation and degradation of IκBα. 4-HPR also inhibited the TNF-induced phosphorylation of p65, nuclear p65 translocation, and NF-κB–dependent reporter gene activity. The expression of gene products involved in antiapoptosis (IAP1, Bfl-1/A1, Bcl-2, TRAF1, and cFLIP), proliferation (cyclin D1 and c-Myc), and metastasis (MMP-9, COX-2, and VEGF) were also down-regulated by 4-HPR.

Our results indicate that 4-HPR suppresses NF-κB activated by a wide variety of agents. This is the first report to examine the effect of 4-HPR on NF-κB activated by various stimuli. These results suggest that 4-HPR must act at a step common to all these agents. We found that 4-HPR blocked the activation of NF-κB without directly interfering with the DNA binding of NF-κB. Further analysis of the pathway indicated that 4-HPR targets at the level of IKK. It seems that our results differ from that of Bayon et al. where they did not find any IKK inhibition with 4-HPR. The difference in the results is actually due to the dose and time used. They used 6 μmol/L 4-HPR for 4 hours, and this dose and time had no effect in our system either. We found that treatment of cells with 25 μmol/L 4-HPR for 24 hours is needed to inhibit IKK activation. However, our in vitro kinase assay results show that 4-HPR is not a direct inhibitor of IKK. Thus, it seems that 4-HPR blocks the activation of IKK by interfering with some upstream regulatory kinases. Akt, NIK, mitogen-activated protein kinase kinase kinase 1, and atypical protein kinase C are candidates because they are upstream kinases that regulate IKK ( 41). Indeed, our study shows that 4-HPR inhibits TNF-induced Akt activation. Suppression of TNF-induced Akt activation may lead to inhibition of IKK activation. The effect of 4-HPR on Akt activation was specific as TNF-induced extracellular signal-regulated kinase activation was unaffected (data not shown).

We found that 4-HPR inhibited not only inducible NF-κB activation but also constitutively activated NF-κB in multiple myeloma and HNSCC cells. These results agree with a previous report that showed suppression of constitutive NF-κB by 4-HPR in human prostate DU145 and PC3 cells ( 55). Constitutive active NF-κB activation has been found to be critical for the survival and proliferation of various tumor cell types ( 41); however, the mechanism of constitutive NF-κB activation is not well understood. Some of the potential mechanisms are overexpression of IκBα without inhibition of NF-κB activity, mutations in the IκBα gene, enhanced IκBα degradation, and constitutive expression of TNF and interleukin-1 ( 41).

The genes that are involved in the proliferation and metastasis of cancer have been shown to be regulated by NF-κB ( 41). We show in this report that 4-HPR inhibits the expression of cyclin D1 and c-myc, which are both regulated by NF-κB. Our results are consistent with recent reports where 4-HPR has been shown to block the G1-S transition by inhibiting the phosphorylation of Rb, CDK4, and cyclin D protein complex ( 4, 38, 56). 4-HPR has also been shown to inhibit the expression of c-Myc in several tumor cell lines ( 33). Our results also show that the expression of COX-2, MMP-9, and VEGF, also regulated by NF-κB, are down-regulated by 4-HPR. Indeed, 4-HPR has been shown to down-regulate COX-2 gene expression in human colon adenocarcinoma cell lines ( 19). Activation of NF-κB is known to promote angiogenesis and invasion, and 4-HPR is anti-invasive ( 39) and antiangiogenic ( 27). These results imply that 4-HPR exercises its anticancer properties through the inhibition of NF-κB.

NF-κB is known to regulate the expression of IAP1, xIAP, Bfl-1/A1, TRAF1, Bcl-2, cFLIP, and survivin, and their overexpression in numerous tumors has been linked to survival, chemoresistance, and radioresistance. Our results indicate that 4-HPR treatment down-regulates most of these gene products. Earlier studies have shown that 4-HPR induces apoptosis through a mitochondrial pathway regulated by proteins from the Bcl-2 family. The levels of Bcl-2 mRNA were markedly diminished by 4-HPR treatment in several tumor cell types ( 33, 34). Our studies also show that 4-HPR potentiated the apoptotic effects of TNF, Taxol, and doxorubicin. These effects are similar to that reported with a specific inhibitor of NF-κB ( 57). These results are also consistent with a recent report that 4-HPR potentiates the apoptotic effects of TRAIL ( 58), another member of the TNF superfamily.

Our results indicate that exposure of cells to 25 μmol/L 4-HPR is needed to suppress NF-κB activation. Although it is difficult to correlate in vitro concentrations to that in vivo, a plasma concentration of 12.9 μmol/L in the patients and 7.9 μmol/L in the tissue have been reported ( 51, 52). We found that when combined with 50 nmol/L Velcade, 10 μmol/L 4-HPR is sufficient to inhibit NF-κB activation. We found that 24-hour treatment was needed to suppress NF-κB activation. This suggests slow cellular accumulation of 4-HPR. Similar observations have been made by others ( 33– 35, 55). Because 4-HPR was also effective in cells where NF-κB is constitutively active (see Fig. 4G and H), it suggests that the chemopreventive effects of 4-HPR could be mediated through NF-κB suppression.

Most retinoids are known to act through binding to the RARs and RXRs. Evidence exists for and against participation of retinoid receptors in 4-HPR–elicited cellular events. Transactivation studies have shown that 4-HPR can activate certain RAR and RXR ( 59), but studies in human breast carcinomas have shown that 4-HPR–mediated biological actions involve retinoid receptor–independent pathways ( 60). Overall, our results indicate that antiproliferative, proapoptotic, anti-invasive, antiosteoclastogenic, antiangiogenic, and antimetastatic effects assigned to 4-HPR may be mediated through suppression of NF-κB–regulated gene products. Considering the lack of effect on normal cells ( 61), 4-HPR has a great potential for prevention and treatment of cancer.

Acknowledgments

Grant support: Clayton Foundation for Research (B.B. Aggarwal), Department of Defense, U.S. Army Breast Cancer Research Program grant BC010610 (B.B. Aggarwal), NIH Lung Chemoprevention PO1 grant CA91844 (B.B. Aggarwal), and NIH P50 Head and Neck Specialized Programs of Research Excellence grant (B.B. Aggarwal).

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.

We thank Walter Pagel for a careful review of the article. Dr. Aggarwal is a Ransom Horne, Jr., Distinguished Professor of Cancer Research. This work was supported in part by the Odyssey Program and the Theodore N. Law Award for Scientific Achievement at The University of Texas M.D. Anderson Cancer Center (to S. Shishodia).

Footnotes

    • Received May 9, 2005.
    • Revision received July 20, 2005.
    • Accepted August 9, 2005.

    References

    1. ↵
      Moon RC, Thompson HJ, Becci PJ, et al. N-(4-hydroxyphenyl)retinamide, a new retinoid for prevention of breast cancer in the rat. Cancer Res 1979; 39: 1339–46.
      OpenUrlAbstract/FREE Full Text
    2. ↵
      Zou CP, Kurie JM, Lotan D, Zou CC, Hong WK, Lotan R. Higher potency of N-(4-hydroxyphenyl)retinamide than all-trans-retinoic acid in induction of apoptosis in non-small cell lung cancer cell lines. Clin Cancer Res 1998; 4: 1345–55.
      OpenUrlAbstract
    3. Delia D, Aiello A, Lombardi L, et al. N-(4-hydroxyphenyl)retinamide induces apoptosis of malignant hemopoietic cell lines including those unresponsive to retinoic acid. Cancer Res 1993; 53: 6036–41.
      OpenUrlAbstract/FREE Full Text
    4. ↵
      DiPietrantonio AM, Hsieh TC, Olson SC, Wu JM. Regulation of G1-S transition and induction of apoptosis in HL-60 leukemia cells by fenretinide (4HPR). Int J Cancer 1998; 78: 53–61.
      OpenUrlCrossRefPubMed
    5. O'Donnell PH, Guo WX, Reynolds CP, Maurer BJ. N-(4-hydroxyphenyl)retinamide increases ceramide and is cytotoxic to acute lymphoblastic leukemia cell lines, but not to non-malignant lymphocytes. Leukemia 2002; 16: 902–10.
      OpenUrlCrossRefPubMed
    6. ↵
      Darwiche N, Hatoum A, Dbaibo G, et al. N-(4-hydroxyphenyl)retinamide induces growth arrest and apoptosis in HTLV-I-transformed cells. Leukemia 2004; 18: 607–15.
      OpenUrlCrossRefPubMed
    7. ↵
      Marth C, Bock G, Daxenbichler G. Effect of 4-hydroxyphenylretinamide and retinoic acid on proliferation and cell cycle of cultured human breast cancer cells. J Natl Cancer Inst 1985; 75: 871–5.
    8. ↵
      Puduvalli VK, Saito Y, Xu R, Kouraklis GP, Levin VA, Kyritsis AP. Fenretinide activates caspases and induces apoptosis in gliomas. Clin Cancer Res 1999; 5: 2230–5.
      OpenUrlAbstract/FREE Full Text
    9. ↵
      Mariotti A, Marcora E, Bunone G, et al. N-(4-hydroxyphenyl)retinamide: a potent inducer of apoptosis in human neuroblastoma cells. J Natl Cancer Inst 1994; 86: 1245–7.
      OpenUrlFREE Full Text
    10. ↵
      Lovat PE, Oliverio S, Ranalli M, et al. GADD153 and 12-lipoxygenase mediate fenretinide-induced apoptosis of neuroblastoma. Cancer Res 2002; 62: 5158–67.
      OpenUrlAbstract/FREE Full Text
    11. ↵
      Kalemkerian GP, Slusher R, Ramalingam S, Gadgeel S, Mabry M. Growth inhibition and induction of apoptosis by fenretinide in small-cell lung cancer cell lines. J Natl Cancer Inst 1995; 87: 1674–80.
      OpenUrlAbstract/FREE Full Text
    12. ↵
      Sun SY, Li W, Yue P, Lippman SM, Hong WK, Lotan R. Mediation of N-(4-hydoxyphenyl)retinamide-induced apoptosis in human cancer cells by different mechanisms. Cancer Res 1999; 59: 2493–8.
      OpenUrlAbstract/FREE Full Text
    13. Oridate N, Suzuki S, Higuchi M, Mitchell MF, Hong WK, Lotan R. Involvement of reactive oxygen species in N-(4-hydroxyphenyl)retinamide-induced apoptosis in cervical carcinoma cells. J Natl Cancer Inst 1997; 89: 1191–8.
      OpenUrlAbstract/FREE Full Text
    14. ↵
      Kabbout M, Hatoum A, Abou-Lteif G, Chakroun I, Homaidan FR, Darwiche N. Stage-specific effect of N-(4-hydroxyphenyl)retinamide on cell growth in squamous cell carcinogenesis. Mol Carcinog 2004; 40: 12–23.
      OpenUrlCrossRefPubMed
    15. ↵
      Montaldo PG, Pagnan G, Pastorino F, et al. N-(4-hydroxyphenyl) retinamide is cytotoxic to melanoma cells in vitro through induction of programmed cell death. Int J Cancer 1999; 81: 262–7.
      OpenUrlCrossRefPubMed
    16. ↵
      Suzuki S, Higuchi M, Proske RJ, Oridate N, Hong WK, Lotan R. Implication of mitochondria-derived reactive oxygen species, cytochrome c and caspase-3 in N-(4-hydroxyphenyl)retinamide-induced apoptosis in cervical carcinoma cells. Oncogene 1999; 18: 6380–7.
      OpenUrlCrossRefPubMed
    17. ↵
      Broaddus RR, Xie S, Hsu CJ, Wang J, Zhang S, Zou C. The chemopreventive agents 4-HPR and DFMO inhibit growth and induce apoptosis in uterine leiomyomas. Am J Obstet Gynecol 2004; 190: 686–92.
      OpenUrlCrossRefPubMed
    18. ↵
      Ulukaya E, Pirianov G, Kurt MA, Wood EJ, Mehmet H. Fenretinide induces cytochrome c release, caspase 9 activation and apoptosis in the absence of mitochondrial membrane depolarisation. Cell Death Differ 2003; 10: 856–9.
      OpenUrlCrossRefPubMed
    19. ↵
      Merritt G, Aliprandis ET, Prada F, Rigas B, Kashfi K. The retinoid fenretinide inhibits proliferation and downregulates cyclooxygenase-2 gene expression in human colon adenocarcinoma cell lines. Cancer Lett 2001; 164: 15–23.
      OpenUrlCrossRefPubMed
    20. ↵
      Igawa M, Tanabe T, Chodak GW, Rukstalis DB. N-(4-hydroxyphenyl) retinamide induces cell cycle specific growth inhibition in PC3 cells. Prostate 1994; 24: 299–305.
      OpenUrlPubMed
    21. ↵
      Kim DG, You KR, Liu MJ, Choi YK, Won YS. GADD153-mediated anticancer effects of N-(4-hydroxyphenyl)retinamide on human hepatoma cells. J Biol Chem 2002; 277: 38930–8.
      OpenUrlAbstract/FREE Full Text
    22. ↵
      Supino R, Crosti M, Clerici M, et al. Induction of apoptosis by fenretinide (4HPR) in human ovarian carcinoma cells and its association with retinoic acid receptor expression. Int J Cancer 1996; 65: 491–7.
      OpenUrlCrossRefPubMed
    23. ↵
      Guruswamy S, Lightfoot S, Gold MA, et al. Effects of retinoids on cancerous phenotype and apoptosis in organotypic cultures of ovarian carcinoma. J Natl Cancer Inst 2001; 93: 516–25.
      OpenUrlAbstract/FREE Full Text
    24. ↵
      Ponthan F, Lindskog M, Karnehed N, Castro J, Kogner P. Evaluation of anti-tumour effects of oral fenretinide (4-HPR) in rats with human neuroblastoma xenografts. Oncol Rep 2003; 10: 1587–92.
      OpenUrlPubMed
    25. ↵
      Pollard M, Luckert PH. The inhibitory effect of 4-hydroxyphenyl retinamide (4-HPR) on metastasis of prostate adenocarcinoma-III cells in Lobund-Wistar rats. Cancer Lett 1991; 59: 159–63.
      OpenUrlCrossRefPubMed
    26. ↵
      Pollard M, Luckert PH, Sporn MB. Prevention of primary prostate cancer in Lobund-Wistar rats by N-(4-hydroxyphenyl)retinamide. Cancer Res 1991; 51: 3610–1.
      OpenUrlAbstract/FREE Full Text
    27. ↵
      Ferrari N, Morini M, Pfeffer U, Minghelli S, Noonan DM, Albini A. Inhibition of Kaposi's sarcoma in vivo by fenretinide. Clin Cancer Res 2003; 9: 6020–9.
      OpenUrlAbstract/FREE Full Text
    28. ↵
      Sabichi AL, Modiano MR, Lee JJ, et al. Breast tissue accumulation of retinamides in a randomized short-term study of fenretinide. Clin Cancer Res 2003; 9: 2400–5.
      OpenUrlAbstract/FREE Full Text
    29. Decensi A, Johansson H, Miceli R, et al. Long-term effects of fenretinide, a retinoic acid derivative, on the insulin-like growth factor system in women with early breast cancer. Cancer Epidemiol Biomarkers Prev 2001; 10: 1047–53.
      OpenUrlAbstract/FREE Full Text
    30. ↵
      Formelli F, Carsana R, Costa A, et al. Plasma retinol level reduction by the synthetic retinoid fenretinide: a one year follow-up study of breast cancer patients. Cancer Res 1989; 49: 6149–52.
      OpenUrlAbstract/FREE Full Text
    31. ↵
      Pienta KJ, Esper PS, Zwas F, Krzeminski R, Flaherty LE. Phase II chemoprevention trial of oral fenretinide in patients at risk for adenocarcinoma of the prostate. Am J Clin Oncol 1997; 20: 36–9.
      OpenUrlCrossRefPubMed
    32. ↵
      Moglia D, Formelli F, Baliva G, et al. Effects of topical treatment with fenretinide (4-HPR) and plasma vitamin A levels in patients with actinic keratoses. Cancer Lett 1996; 110: 87–91.
      OpenUrlCrossRefPubMed
    33. ↵
      Delia D, Aiello A, Formelli F, et al. Regulation of apoptosis induced by the retinoid N-(4-hydroxyphenyl) retinamide and effect of deregulated bcl-2. Blood 1995; 85: 359–67.
      OpenUrlAbstract/FREE Full Text
    34. ↵
      Boya P, Morales MC, Gonzalez-Polo RA, et al. The chemopreventive agent N-(4-hydroxyphenyl)retinamide induces apoptosis through a mitochondrial pathway regulated by proteins from the Bcl-2 family. Oncogene 2003; 22: 6220–30.
      OpenUrlCrossRefPubMed
    35. ↵
      Simeone AM, Li YJ, Broemeling LD, Johnson MM, Tuna M, Tari AM. Cyclooxygenase-2 is essential for HER2/neu to suppress N-(4-hydroxyphenyl)retinamide apoptotic effects in breast cancer cells. Cancer Res 2004; 64: 1224–8.
      OpenUrlAbstract/FREE Full Text
    36. ↵
      Levine L. N-(4-hydroxyphenyl)retinamide: a synthetic analog of vitamin A that is a potent inhibitor of prostaglandin biosynthesis. Prostaglandins Med 1980; 4: 285–96.
      OpenUrlCrossRefPubMed
    37. ↵
      Hail N, Jr., Lotan R. Mitochondrial respiration is uniquely associated with the prooxidant and apoptotic effects of N-(4-hydroxyphenyl)retinamide. J Biol Chem 2001; 276: 45614–21.
      OpenUrlAbstract/FREE Full Text
    38. ↵
      Panigone S, Debernardi S, Taya Y, Fontanella E, Airoldi R, Delia D. pRb and Cdk regulation by N-(4-hydroxyphenyl)retinamide. Oncogene 2000; 19: 4035–41.
      OpenUrlCrossRefPubMed
    39. ↵
      Kim JH, Tanabe T, Chodak GW, Rukstalis DB. In vitro anti-invasive effects of N-(4-hydroxyphenyl)-retinamide on human prostatic adenocarcinoma. Anticancer Res 1995; 15: 1429–34.
      OpenUrlPubMed
    40. ↵
      Ribatti D, Alessandri G, Baronio M, et al. Inhibition of neuroblastoma-induced angiogenesis by fenretinide. Int J Cancer 2001; 94: 314–21.
      OpenUrlCrossRefPubMed
    41. ↵
      Aggarwal BB. Nuclear factor-κB: the enemy within. Cancer Cell 2004; 6: 203–8.
      OpenUrlCrossRefPubMed
    42. ↵
      Bharti AC, Takada Y, Shishodia S, Aggarwal BB. Evidence that receptor activator of nuclear factor (NF)-κB ligand can suppress cell proliferation and induce apoptosis through activation of a NF-κB-independent and TRAF6-dependent mechanism. J Biol Chem 2004; 279: 6065–76.
      OpenUrlAbstract/FREE Full Text
    43. ↵
      Aggarwal BB. Signalling pathways of the TNF superfamily: a double-edged sword. Nat Rev Immunol 2003; 3: 745–56.
      OpenUrlCrossRefPubMed
    44. ↵
      Shishodia S, Majumdar S, Banerjee S, Aggarwal BB. Ursolic acid inhibits nuclear factor-κB activation induced by carcinogenic agents through suppression of IκBα kinase and p65 phosphorylation: correlation with down-regulation of cyclooxygenase 2, matrix metalloproteinase 9, and cyclin D1. Cancer Res 2003; 63: 4375–83.
      OpenUrlAbstract/FREE Full Text
    45. ↵
      Wang CY, Mayo MW, Baldwin AS, Jr. TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-κB. Science 1996; 274: 784–7.
      OpenUrlAbstract/FREE Full Text
    46. ↵
      Liotta LA, Thorgeirsson UP, Garbisa S. Role of collagenases in tumor cell invasion. Cancer Metastasis Rev 1982; 1: 277–88.
      OpenUrlCrossRefPubMed
    47. ↵
      Abu-Amer Y, Tondravi MM. NF-κB and bone: the breaking point. Nat Med 1997; 3: 1189–90.
      OpenUrlCrossRefPubMed
    48. ↵
      Finco TS, Beg AA, Baldwin AS, Jr. Inducible phosphorylation of IκBα is not sufficient for its dissociation from NF-κB and is inhibited by protease inhibitors. Proc Natl Acad Sci U S A 1994; 91: 11884–8.
      OpenUrlAbstract/FREE Full Text
    49. Mahon TM, O'Neill LA. Studies into the effect of the tyrosine kinase inhibitor herbimycin A on NF-κB activation in T lymphocytes. Evidence for covalent modification of the p50 subunit. J Biol Chem 1995; 270: 28557–64.
      OpenUrlAbstract/FREE Full Text
    50. ↵
      Natarajan K, Singh S, Burke TR, Jr., Grunberger D, Aggarwal BB. Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-κB. Proc Natl Acad Sci U S A 1996; 93: 9090–5.
      OpenUrlAbstract/FREE Full Text
    51. ↵
      Camerini T, Mariani L, De Palo G, et al. Safety of the synthetic retinoid fenretinide: long-term results from a controlled clinical trial for the prevention of contralateral breast cancer. J Clin Oncol 2001; 19: 1664–70.
      OpenUrlAbstract/FREE Full Text
    52. ↵
      Vaishampayan U, Heilbrun LK, Parchment RE, et al. Phase II trial of fenretinide in advanced renal carcinoma. Invest New Drugs 2005; 23: 179–85.
      OpenUrlCrossRefPubMed
    53. ↵
      Bonizzi G, Piette J, Merville MP, Bours V. Distinct signal transduction pathways mediate nuclear factor-κB induction by IL-1β in epithelial and lymphoid cells. J Immunol 1997; 159: 5264–72.
      OpenUrlAbstract
    54. ↵
      Ozes ON, Mayo LD, Gustin JA, Pfeffer SR, Pfeffer LM, Donner DB. NF-κB activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 1999; 401: 82–5.
      OpenUrlCrossRefPubMed
    55. ↵
      Shimada K, Nakamura M, Ishida E, Kishi M, Yonehara S, Konishi N. Contributions of mitogen-activated protein kinase and nuclear factor κB to N-(4-hydroxyphenyl)retinamide-induced apoptosis in prostate cancer cells. Mol Carcinog 2002; 35: 127–37.
      OpenUrlCrossRefPubMed
    56. ↵
      Zhou Q, Stetler-Stevenson M, Steeg PS. Inhibition of cyclin D expression in human breast carcinoma cells by retinoids in vitro. Oncogene 1997; 15: 107–15.
      OpenUrlCrossRefPubMed
    57. ↵
      Takada Y, Singh S, Aggarwal BB. Identification of a p65 peptide that selectively inhibits NF-κB activation induced by various inflammatory stimuli and its role in down-regulation of NF-κB-mediated gene expression and up-regulation of apoptosis. J Biol Chem 2004; 279: 15096–104.
      OpenUrlAbstract/FREE Full Text
    58. ↵
      Cuello M, Coats AO, Darko I, et al. N-(4-hydroxyphenyl) retinamide (4HPR) enhances TRAIL-mediated apoptosis through enhancement of a mitochondrial-dependent amplification loop in ovarian cancer cell lines. Cell Death Differ 2004; 11: 527–41.
      OpenUrlCrossRefPubMed
    59. ↵
      Fanjul AN, Delia D, Pierotti MA, et al. 4-Hydroxyphenyl retinamide is a highly selective activator of retinoid receptors. J Biol Chem 1996; 271: 22441–6.
      OpenUrlAbstract/FREE Full Text
    60. ↵
      Sheikh MS, Shao ZM, Li XS, et al. N-(4-hydroxyphenyl)retinamide (4-HPR)-mediated biological actions involve retinoid receptor-independent pathways in human breast carcinoma. Carcinogenesis 1995; 16: 2477–86.
      OpenUrlAbstract/FREE Full Text
    61. ↵
      Ulukaya E, Kurt A, Wood EJ. 4-(N-hydroxyphenyl)retinamide can selectively induce apoptosis in human epidermoid carcinoma cells but not in normal dermal fibroblasts. Cancer Invest 2001; 19: 145–54.
      OpenUrlPubMed
    View Abstract
    Back to top
    Cancer Research: 65 (20)
    October 2005
    Volume 65, Issue 20

    Sign up for alerts

    View this article with LENS

    Open full page PDF
    Article Alerts
    Email Article
    Citation Tools
    N-(4-Hydroxyphenyl)Retinamide Inhibits Invasion, Suppresses Osteoclastogenesis, and Potentiates Apoptosis through Down-regulation of IκBα Kinase and Nuclear Factor-κB–Regulated Gene Products
    Shishir Shishodia, Angelica M. Gutierrez, Reuben Lotan and Bharat B. Aggarwal
    Cancer Res October 15 2005 (65) (20) 9555-9565; DOI: 10.1158/0008-5472.CAN-05-1585
    del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

    Jump to section

    Advertisement