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Experimental Therapeutics |
B Inhibitor in Nude Mice
Department of Urology, Keio University, School of Medicine, Tokyo 160-8582 [E. K., Y. H., J. N., K. K., M. O., T. O., M. M.], and Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama 223-0061 [N. T., Y. S., K. U.] Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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B inhibitor, a dehydroxymethylepoxyquinomicin designated as DHMEQ, against prostate cancer. The activity of NFB, evaluated by transient transfection of a luciferase reporter DNA containing a specific binding sequence for NFB, was inhibited by DHMEQ in three human hormone-refractory prostate cancer cell lines, DU145, JCA-1, and PC-3. Statistically significant growth inhibition was achieved by 20 µg/ml of DHMEQ, and marked levels of apoptosis were induced 48 h after DHMEQ administration in vitro. Electrophoretic mobility shift assay showed that DHMEQ completely inhibited NFB DNA binding activity in JCA-1 cells. Furthermore, i.p. administrations of DHMEQ significantly inhibited pre-established JCA-1 s.c. tumor growth in nude mice without any side effects. Our result indicates the possibility of using a novel NFB activation inhibitor, DHMEQ, as a new treatment strategy against hormone-refractory prostate cancer. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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,2
IL-6, and granulocyte colony-stimulating factor, which are thought to play a crucial role in tumor growth and progression (1)
. Previously, it has been demonstrated that both JCA-1 and PC-3 hormone-refractory prostate cancer cells secreted large amounts of IL-6, which was enhanced by exogenous TNF- (2)
. Moreover, we have reported recently that the serum level of IL-6 was a significant prognostic factor for prostate cancer patients, especially with disseminated diseases (3)
. These cytokines that might enhance the malignant potential of hormone-refractory prostate cancer are regulated by several transcription factors through various signal transduction pathways. Among such transcription factors, NFB is one of the most critical regulators of cytokine-inducible gene expression (4)
. It is well known that NFB is often constitutively activated in hormone-refractory prostate cancer cells, which may increase expression of antiapoptosis proteins, thereby decreasing the effectiveness of anticancer therapy and contributing to the development of the chemoresistant phenotypes (5
, 6)
. Palayoor et al. (5)
have reported that NFB is constitutively activated in the hormone-refractory prostate cancer cell lines PC-3 and DU145, but not in the hormone-dependent LNCaP prostate cancer cell line. It is suggested that TNF- did not induce cytotoxic effects on hormone-refractory prostate cancer cells, because the NFB protective pathway prevents the cells from undergoing apoptosis (7)
. These findings support the hypothesis that NFB plays an important role in maintaining the chemoresistant nature of hormone-refractory prostate cancers. Therefore, approaches that inhibit NFB function may be beneficial in the treatment of chemoresistant prostate cancers. On the basis of this understanding, we have investigated the effectiveness of a newly synthesized unique NFB inhibitor as a potent treatment modality for hormone-refractory prostate cancers.
Epoxyquinomicin C was originally isolated from Amycolatopsis as a weak antibiotic and anti-inflammatory agent, having a 4-hydroxy-5, 6-epoxycyclohexenone structure like panepoxydone (8)
. Panepoxydone isolated from the basidiomycete Lentinus crinitus was found to inhibit TNF--induced activation of NFB (9)
. Therefore, we have newly designed and synthesized DHMEQ as a 5-dehydroxymethyl derivative of epoxyquinomicin C, which showed anti-NFB activity in cultured human leukemia Jurkat cells and inhibited type II collagen-induced rheumatoid arthritis in mice (10)
. Recently, DHMEQ was found to inhibit activation of NFB at the level of nuclear translocation (11)
. Thus far, in the field of anticancer research, no investigations have been reported to regulate NFB activation using a single agent synthesized from a natural product. In the present study, we evaluated the inhibitory effects of a novel NFB activation inhibitor, DHMEQ, against hormone-refractory prostate cancer cells on: (a) constitutive activation of NFB; (b) the levels of antiapoptosis; and (c) tumor growth in a xenograft animal model to establish an innovative and effective treatment strategy for hormone-refractory prostate cancers.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Chemicals.
DHMEQ was synthesized in our laboratory (10)
. It was dissolved in DMSO to prepare a 10 µg/ml solution and subsequently diluted in culture medium to a final DMSO concentration of <0.1%.
Cell Growth Assay.
The growth-inhibitory effects of DHMEQ were determined as reported previously (13)
. Briefly, 2 x 104 cells were seeded in each well of 96-well plates and allowed to grow overnight. Then, cells were treated with various concentrations of DHMEQ. Cells treated with the same concentrations of DMSO were served as controls. After 48 h of incubation, cytotoxicity was determined by staining the plates with 0.2% crystal violet. The absorbance value of each well was determined at 550 nm with a 405 nm reference beam by a microplate reader (Bio-Rad, Tokyo, Japan).
Measurement of Activity of NFB.
The activity of NFB was monitored using a luciferase plasmid DNA that contains a specific binding sequence for NFB (10)
. Transfection was carried out using cationic liposome, Gene PORTER 2 (Gene Therapy Systems, San Diego, CA). Briefly, 2 x 104 cells per each well were plated in 96-well plates. When cultured cells reached 
80% confluence, cells were overlaid with 0.2 µg DNA/1 µl Gene PORTER 2 complexes in a total volume of 50 µl of serum-free RPMI 1640 for 14 h. Subsequently, various concentrations of DHMEQ in 50 µl of RPMI 1640 containing 10% fetal bovine serum were added. After additional incubation for 8 h, cells were washed with PBS and harvested in 50 µl of Reporter Lysis Buffer provided in Luciferase Assay kit (Promega, Madison, WI). Luciferase activity in 10 µl of lysate was measured by a luminometer (Lumat 9501; Berthold). Obtained raw luciferase activities were normalized by dividing with percent cytotoxicity at each concentration.
EMSA.
JCA-1 cells treated with 20 µg/ml of DHMEQ were incubated for different time periods. The cells were harvested, washed with PBS, suspended in 400 µl of buffer A (10 mM HEPES, 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, and 0.1 mM phenylmethylsulfonyl fluoride), and incubated on ice for 15 min. Nuclei were pelleted by centrifugation for 5 min at 14,000 rpm, resuspended in 40 µl of buffer C [50 mM HEPES, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, and 25% glycerol (v/v)], incubated on ice for 20 min, and centrifuged for 5 min at 14,000 rpm at 4°C. The supernatant was used as a nuclear extract. The binding reaction mixture containing 5 µg of protein of nuclear extract, 2 µg of poly(deoxyinosinic-deoxycytidylic acid), and 32P-labeled probe was incubated for 20 min at room temperature. The complexes were separated from free DNA on 4% native polyacrylamide gel. The DNA probe used for NFB binding was the double-stranded oligonucleotide containing the B site from the mouse light chain enhancer (5'-ATGTGAGGGGACTTTCCCAGGC-3').
Western Blot Analysis.
The expression levels of IB, Bcl-2, and Bcl-xL protein in JCA-1 cells were determined by Western blot analysis. Samples containing equal amounts of protein (30 µg) were subjected to electrophoresis on a SDS polyacrylamide gel and transferred to a nitrocellulose filter. The filters were blocked with Tris-buffered saline containing 5% nonfat milk at 4°C overnight and then incubated for 1 h with anti-IB rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Bcl-2 mouse monoclonal antibody, anti-Bcl-xL mouse monoclonal antibody (Transduction Laboratories, Lexington, KY), or anti-ß-actin mouse antibody (Sigma Chemical Co., St. Louis, MO). The filters were then incubated for 1 h with antirabbit or antimouse secondary antibody (Dako A/S, Glostrup, Denmark), and reactivity was detected by enhanced chemiluminescence system (Amersham Life Science, Inc., Little Chalfont, United Kingdom).
Detection of Apoptosis.
Quantification of apoptosis was determined using flow cytometric analysis of cells labeled with TUNEL assay. Cells (1 x 105) were plated in T-25 flask, incubated overnight, and treated with 20 µg/ml of DHMEQ for 48 h. TUNEL assay was performed using ApopTag kit (Intergen Co., Purchase, NY), and apoptosis was detected by flow cytometer (Beckman Coulter, Inc., Fullerton, CA). The staining by annexin V/PI, using Mebcyto Apoptosis kit (Medical & Biological Laboratories, Nagoya, Japan), was also used to monitor apoptosis in JCA-1 cells. Forty-eight h after the treatment with various concentrations of DHMEQ, cells were harvested, washed, and then incubated with annexin V-FITC and PI at room temperature for 15 min in the dark. The fluorescence was analyzed by flow cytometer, and subsequent analysis was carried out according to the manufacturer’s instructions.
Treatments in Vivo.
All of the procedures involving animals and their care in this study were approved by the animal care committee of Keio University in accordance with institutional and Japanese government guidelines for animal experiments. Male BALB/c-nu/nu mice were obtained from Sankyo Lab Service Co. (Tokyo, Japan). JCA-1 cells (5 x 106) were implanted s.c. in the flank of each nude mouse. When animals developed palpable tumors, mice were randomly assigned into two groups. Then 8 µg/kg DHMEQ was administered i.p. once daily for consecutive 14 days. Control animals only received vehicle medium injection. Each experimental group consisted of 8 mice. Animals were carefully monitored, and tumor size as well as body weight was measured weekly for 5 weeks. Tumor volume was calculated according to the formula a2 x b x 0.52, where a and b are the smallest and largest diameters, respectively.
Statistical Analysis.
The statistical analysis was performed using Student’s t test. Unless otherwise indicated, average values were expressed as mean values with SD. Ps < 0.01 were interpreted as statistically significant.
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RESULTS AND DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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). DHMEQ at a concentration of 20 µg/ml inhibited cell growth of all of the prostate cancer cells in a time-dependent manner (Fig. 1B)
. DMSO, which was used as a solvent for DHMEQ, was not toxic even at the highest concentration used in this study. In hormone-dependent LNCaP prostate cancer cells, DHMEQ did not induce significant growth inhibition at concentrations of 20 µg/ml or less (data not shown), suggesting that DHMEQ is more effective in hormone-refractory prostate cancer cells in which NFB is constitutively activated. We performed a transient transfection assay using a NFB luciferase reporter gene to evaluate the effect of DHMEQ on NFB activity (Fig. 2, A–C)
. The significant decrease of NFB activity was observed in DU145, JCA-1, and PC-3 cells treated with DHMEQ at concentrations of 10 µg/ml or higher, 2.5 µg/ml or higher, and 10 µg/ml or higher, respectively (P < 0.01). These results confirmed that DHMEQ inhibited constitutively activated NFB in these hormone-refractory prostate cancer cells.
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B binding activity to the B DNA site in JCA-1 cells was examined by EMSA (Fig. 3)
. The DNA binding activity was completely inhibited after 2, 4, and 6 h of exposure to 20 µg/ml of DHMEQ. Then, 8 h after the DHMEQ treatment, DNA binding activity had recovered slightly. This result suggested that DHMEQ completely inhibited NFB DNA binding activity in JCA-1 cells, in which NFB was constitutively activated. Because the activation of NFB normally proceeds through the ubiquitination and degradation of the IB inhibitory protein, Western blotting was carried out to evaluate the effects of DHMEQ on the expression levels of the IB protein in JCA-1 cells. IB was not modulated even by the highest dose of DHMEQ (Fig. 4)
. This result indicated that inhibition of NFB activity in JCA-1 cells by DHMEQ was not associated with phosphorylation and degradation of IB. The molecular target of DHMEQ on NFB activity is still under investigation, however, we have already shown that DHMEQ inhibited TNF--induced activation of NFB in human T-cell leukemia Jurkat cells possibly by inhibiting the nuclear translocation of NFB from the cytoplasm (11)
.
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B plays an important role in antiapoptotic mechanism. Previous studies have shown that the absence or inhibition of NFB subunits in culture cells such as fibroblasts and macrophages resulted in potentiation of apoptosis in response to TNF- (14
, 15)
. In our current study, significant apoptosis was induced by DHMEQ (20 µg/ml) treatment in all of the three prostate cancer cell lines (Fig. 5)
. With staining by annexin V/PI, in JCA-1 cells, apoptotic indexes induced by 20 or 40 µg/ml of DHMEQ and DMSO were 44.1%, 23.8%, and 2.9%, respectively. Taken together, DHMEQ has the potent inhibitory effects on NFB activation and produces a significant decrease in cell viabilities through apoptotic mechanism. We also investigated whether Bcl-2 and Bcl-xL, which were important regulators of apoptosis, could be affected by DHMEQ treatment (Fig. 4)
. DHMEQ did not lower the levels of both Bcl-2 and Bcl-xL proteins, which suggested that DHMEQ might induce apoptosis through a different pathway from the Bcl-2 family.
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). DHMEQ treatment at the dosage used was well tolerated, leading to no body weight loss in animals when compared with controls (data not shown).
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B activation including IB gene delivery have been investigated (16)
. A recombinant adenovirus vector expressing the stable form of IB clearly induced apoptosis, when cancer cells showed a constitutive NFB activity in vitro (17)
. However, the clinical feasibility of gene therapy approaches for inhibiting NFB are quite limited by requisite intratumoral delivery of a vector that expresses the NFB inhibitor, and few studies reported the usefulness of this strategy in an in vivo model.
Nonsteroidal anti-inflammatory drugs could also inhibit the activation of NFB. Dexamethasone activated intracellular IB synthesis (18)
. Ibuprofen inhibited an upstream regulator of IB kinase (19)
. Although these agents have been shown to specifically inhibit NFB and have antitumor effect in vitro, again, in vivo study was not fully investigated. In reality, these agents may not be used as a single agent, rather they can be used in combination with other cytotoxic agents for clinical application.
Natural products reported to inhibit activation of NFB are panepoxydone (9)
, cycloepoxydon (20)
, and gliotoxin (21)
. Panepoxydone and cycloepoxydon from the deuteromycete strain were suggested to inhibit TNF--induced phosphorylation and degradation of IB, and activation of NFB. Gliotoxin produced by fungi was also reported to inhibit NFB by preventing the degradation of IB. Few investigations were reported thus far in the literature to use such natural products that had inhibitory effects on NFB activation in the field of cancer research as we clearly demonstrated in this study using DHMEQ.
Recently, it has been reported that there is some possibility that JCA-1 cells are cross-contaminant (22)
. However, it has also been suggested that a subline may have acquired certain phenotypic characteristics. At least NFB is constitutively activated as clearly shown in our EMSA results in which untreated JCA-1 cells displayed elevated levels of NFB DNA binding activity, as shown previously in PC-3 and DU145 cells (5)
. Furthermore, DHMEQ apparently inhibited the NFB activities in vitro and the tumor growth in vivo.
In conclusion, our results indicate that DHMEQ inhibits NFB activity resulting in promoting apoptotic mechanisms in hormone-refractory prostate cancer cell lines in vitro and has a potent inhibitory effect on JCA-1 tumor growth in animal models. This is the first report to certify the evidence that a single agent synthesized from a natural product has significant cytotoxic effects against hormone-refractory prostate cancers. These results demonstrated that a novel and unique NFB inhibitor, DHMEQ, is a promising candidate as an antitumor agent in hormone-refractory prostate cancer. Additional studies on safety and efficacy of DHMEQ would provide a rationale for eventual clinical trails in hormone-refractory prostate cancer patients.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Department of Urology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Phone: 81-3-5363-3825; Fax: 81-3-3225-1985; E-mail: horigchi{at}aol.com 
2 The abbreviations used are: IL, interleukin; TNF, tumor necrosis factor; NFB, nuclear factor B; DHMEQ, dehydroxymethylepoxyquinomicin; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling; PI, propidium iodide; EMSA, electrophoretic mobility shift assay.
Received 2/20/02. Accepted 10/29/02.
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), JCA-1 (
), and PC-3 (
)] were treated with various concentrations of DHMEQ (A). Cytotoxic effects of DHMEQ on various time points (B). The cells [DU145 (
), JCA-1 (
)] were treated with 20 µg/ml of DHMEQ and incubated for various time periods. Control cells were treated with the same concentration of DMSO as used in DHMEQ treatment. The cell viabilities were measured by quantitative crystal violet assay. Each value represents the mean derived from at least three individual experiments; bars, ±SD. *, more statistically significant than controls (P < 0.01).
