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Discovery Research Laboratories, Nippon Shinyaku Co. Ltd., Kyoto 601-8550 [H. T., K. K., F. K.]; Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya 464-8681 [N. O.]; Division of Demyelinating Disease and Aging, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo 187-8502 [M. I.]; and D. Western Therapeutics Institute, Nagoya 466-0825 [H. H.], Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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50%. To explore the molecular mechanism of this effect, we assessed the expression of the multidrug resistance gene (MDR1), which is constitutive in K2/ARS cells, at both the protein and the mRNA level. Western and reverse transcription-PCR analysis revealed that the expression of MDR1 was significantly suppressed by treatment with HMN-176. Furthermore, when administered p.o., HMN-214 suppressed the expression of MDR1 mRNA in a mouse xenograft model implanted with KB-A.1, an Adriamycin-resistant cell line. Luciferase reporter fusion gene analysis demonstrated that HMN-176 inhibited the Y-box-dependent promoter activity of the MDR1 gene in a dose-dependent manner. Moreover, we show by electrophoretic mobility shift assay that HMN-176 inhibits the binding of NF-Y, which is thought to be an essential factor for the basal expression of MDR1, to its target Y-box consensus sequence in the MDR-1 promoter. Inhibition of MDR-1 expression was achieved with pharmacological concentrations of HMN-176, suggesting that HMN-176 may act by two different mechanisms—cytotoxicity and MDR1 down-regulation—simultaneously. The data presented strongly suggest that the antitumor mechanism of HMN-176 (or its prodrug HMN-214 in vivo) is quite different from those of known antitumor agents. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Amplification of the MDR1 gene associated with the increased expression of P-gp is frequently observed in cell lines selected in vitro by resistance to antitumor agents, although studies in humans using tumor samples and normal tissue have demonstrated increased P-gp expression without gene amplification (2, 3, 4) . The MDR phenotype may also be induced by other mechanisms (5) . However, transfection of the MDR1 gene into mammalian cells results in the full MDR phenotype (6) , demonstrating the possibility that MDR1 alone could play a major role in MDR. Several agents capable of modulating and decreasing MDR1 in vitro (7, 8, 9, 10, 11, 12, 13, 14) have been used clinically. Of these, however, PSC388 yielded no apparent benefit in randomized Phase III trials of patients with ovarian cancer, whereas ET-743 is being developed as an antitumor agent rather than a chemosensitizer.
MDR1 promoters contain putative Sp1 binding sequences and CCAAT elements (Y-boxes). The Y-box especially is absolutely required for the basal expression of human MDR1 (15 , 16) . This sequence is recognized by the nuclear protein NF-Y complex, which consists of A, B, and C subunits (17 , 18) . Tax, a viral transactivator encoded by human T-cell leukemia virus type I, has also been shown to activate the transcription of MDR1 through direct interaction with NF-YB, the B subunit of NF-Y (19) . This observation may have important implications for the chemotherapy of refractory virus-associated malignancies. Thus, NF-Y plays a pivotal role in the regulation of MDR1 gene expression, and it is thought to be a good molecular target for manipulating the MDR phenotype.
HMN-176 ((E)-4-{[2-N-[4-methoxybenzenesulfonyl]amino]-stilbazole}1-oxide) is an active metabolite of the synthetic antitumor compound HMN-214 ((E)-4-{2-[2-(N-acetyl-N-[4-methoxybenzenesulfonyl]amino)stilbazole]}1-oxide). HMN-176 shows potent cytotoxicity toward various human tumor cell lines, and in mitotic cells, it causes cell cycle arrest at M phase through the destruction of spindle polar bodies, followed by the induction of DNA fragmentation. However, no direct interactions of HMN-176 with tubulin are observed (2) . Moreover, in animal models, we have observed that oral administration of the prodrug HMN-214 caused no significant nerve toxicity, a severe side effect often associated with microtubule binding agents such as Taxol and VCR.3 In Phase I clinical trials, HMN-214 has caused sensory neuropathy and ileus in some patients. However, the grade and frequency of these adverse effects were much lower than those of typical microtubule binding agents. As expected from the mechanism of action of HMN-214 (induction of G2-M arrest in dividing cells), the main adverse effect was neutropenia.4
Although the exact molecular mechanism of HMN-176 remains to be elucidated, our recent in vitro observations have suggested the involvement of polo-like kinase in HMN-176-induced cell cycle arrest. HMN-176 does not inhibit polo-like kinase; instead, it alters its spatial distribution. This change may keep the maturation promoting factor complex inactive, resulting in cell cycle arrest at G2-M (unpublished data).
We have previously reported that HMN-154, which is closely related to HMN176, interacts with NF-YB and thereby interrupts the binding of the NF-Y heterotrimer to DNA (20) . These results strongly suggest that HMN-176 can inhibit the expression of MDR1 at the transcriptional level by inhibiting the activity of NF-Y. The main purpose of this study was to examine the inhibitory effect of HMN-176 on the expression of MDR1 in multidrug-resistant cells and the resultant restoration of sensitivity to Adriamycin.
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MATERIALS AND METHODS |
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. HMN-176 (A) was dissolved in DMSO to a concentration of 10 mM and stored at 4°C in the dark. Before each experiment, the stock solution was diluted with PBS to the appropriate concentration. Suspensions of HMN-214 (B), a prodrug of HMN-176 for oral administration, was prepared as follows: HMN-214 was placed in an agate mortar and finely ground with an agate pestle. The ground HMN-214 was suspended with an agate pestle by gradually adding 0.5% methylcellulose 4000 solution to make a 3 mg/ml suspension. This was additionally diluted with methylcellulose 4000 solution to obtain suspensions of the appropriate concentration. Adriamycin was purchased from Kyowa Hakko Kogyo Co., Ltd. (Tokyo, Japan) and dissolved in saline for injection.
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Tumor Transplantation and Drug Administration.
Tumor tissue was grown in advance by s.c. transplantation into nude mice. The resulting tumors were removed, cut into cubic fragments of 8 mm3, and transplanted s.c. into the right axillary region of nude mice with a trocar. When the theoretical volume of the tumor had reached about 145 mm3, oral administration of HMN-214 was started (day 1).
Western Analysis.
Whole-cell extract (5 µg) was electrophoresed on a 10% polyacrylamide gel and electroblotted onto a nitrocellulose membrane, after which immunodetection was performed with a 1:1000 dilution of anti-MDR1 antibody (SC-1517; Santa Cruz Biotechnology, Santa Cruz, CA).
RT-PCR Analysis.
Total RNA from cell culture and the tumor sample were extracted by the acid guanidinium thiocyanate-phenol-chloroform method (21)
. Reverse transcription of total RNA was performed using SuperScript (Life Technologies, Inc., Rockville, MD) according to the supplier’s instructions. A set of primers used for MDR1-cDNA-specific amplification are derived from different exons of the MDR1 gene to prevent amplification of genomic DNA. The sequences of the primers were 5'-TCACAGGGAAGAGATCGTGAG-3' (sense) and 5'-CAAGGCAGTCAGTTACAGTC-3' (antisense). The reverse transcription mixture was subjected directly to the PCR. After PCR, samples were electrophoresed on a 2% agarose gel and stained with ethidium bromide.
EMSA.
The purified 27-mer 5'-GTGGTGAGGCTGATTGGCTGGGCAGGA-3', which contains the Y-box element of the human MDR1 gene (16)
, was end labeled by T4 polynucleotide kinase in the presence of [
-32P]dATP (>3000 Ci/mol) and made double stranded by annealing it to the corresponding antisense oligonucleotide. Binding reactions were performed as follows: 5 µg of HeLa nuclear extract (Promega, Madison, WI) were incubated with 18 µl of 10 mM Tris-HCl (pH 7.3), containing 0.05 mM EDTA, 40 mM NaCl, 3 mM MgCl2, 10 mM 2-mercaptoethanol, 0.25 mM DTT, 2 µg poly(dI·dC) and various concentrations of HMN-176 for 30 min. Antibody supershift of the NF-Y complex was performed by including 2 µg of anti-NF-YA or anti-NF-YB antibody (anti-YA, PharMingen, San Diego, CA; anti-YB, kindly provided by Dr. Roberto Mantovani, University of Milan, Milan, Italy) in the reaction mixture. 32P-labeled probe (10,000 cpm) was added and the reaction mixture additionally incubated for 30 min. All reactions were performed at room temperature. Samples were run on 5% nondenaturing polyacrylamide gels in 0.5x TBE (100 mM Tris, 83 mM boric acid, and 1 mM EDTA) at room temperature and 10 V/cm. The gels were dried and exposed to a BAS 2000 Bio-Image Analyzer (Fujifilm, Tokyo, Japan).
DNA Transfection and Analysis of Transient Transfections.
Transient transfection of vector DNA into HeLa cells was accomplished by the lipofectin method according to the manufacture’s recommendations (Life Technologies, Inc.). The MDR-Luc construction contains 150 bp of genomic sequence from MDR1, upstream of the transcription initiation site (positions -99 to +50), in the reporter plasmid vector pGL-3 (Promega). This vector (2 µg) was cotransfected with 40 ng of pRL-SV40, an internal control vector. The cells were allowed to recover for 24 h before being treated for 48 h with various concentrations of HMN-176. Cell extracts were prepared according to the manufacturer’s directions, and the reaction was initiated by the addition of 100 µl of luciferase assay reagent. The luciferase activity was determined for each sample by integration of the luminescence signals over a 20-s period with a Berthold Lumat LB 9507 luminometer.
Enhanced Cytotoxicity of Adriamycin in the Presence of HMN-176.
K2/ARS cells were seeded into a 96-well microplate at a density of 1.5 x 103 cells/well. After incubation at 37°C and 5% CO2 for 24 h, HMN-176 was added at the indicated concentrations and incubation continued for an additional 48 h. The HMN-176 was then washed out by replacing the medium three times and Adriamycin added at the indicated concentrations. The cells were additionally incubated for 72 h at 37°C, and inhibition of growth was measured by MTT assay (22)
. The absorbance at 595 nm after 72-h treatment was calculated by setting the absorbance at zero time to 100%. GI50 was calculated by the Scansoft 96 software program (Dainippon Pharmaceutical Co., Osaka, Japan).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. A 790-fold increase in the Adriamycin concentration was required to cause a 50% reduction in the growth of resistant cells compared with the sensitive parent K2 cells. Much higher degrees of cross-resistance were observed for Taxol and VCR (1650- and 1800-fold, respectively). A much lower degree of cross-resistance of K2/ARS cells to HMN-176 was observed (14.3-fold).
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. Furthermore, Western blotting revealed the presence of MDR1 on K2/ARS cells but not on the parent K2 cells (Fig. 3)
. Thus, the multidrug-resistant phenotype of K2/ARS cells was associated with the overexpression of MDR1.
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56% (Fig. 2)
and also reduced the expression of MDR1 protein as detected by Western blotting (Fig. 3)
. Treatment with 1 µM HMN-176 had no significant effect on the expression of MDR1 mRNA or protein.
Because K2 and K2/ARS cells failed to grow after transplantation into nude mice, we used KB and its multidrug-resistant subline KB-A.1 (23)
as the transplants. For in vivo experiments, we used HMN-214, an acetylated derivative HMN-176 (Fig. 1)
. HMN-214 is easily absorbed when p.o. administered and is readily hydrolyzed to HMN-176 in serum (data not shown). HMN-214 was p.o. administered at various doses from days 1 to 28. On day 29, tumors were isolated from animals bearing KB and KB-A.1 cells in the HMN-214-treated and untreated groups. Total RNA was extracted from the tumor samples, and the amount of MDR1 mRNA was determined by RT-PCR. The density of the bands was assessed, and the MDR1/GAPDH ratios were calculated. The expression of MDR1 mRNA was below the detection limit in the KB strain (Fig. 4)
; in contrast, high levels of MDR1 mRNA were detected in KB-A.1 cells, and these levels significantly decreased upon treatment with HMN-214.
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. Antibodies against both NF-YA and NF-YB produced supershifted bands, suggesting that this DNA-protein complex was NF-Y. To verify the inhibition of MDR1 promoter activity, we performed a reporter assay in the presence of HMN-176. We chose to evaluate human MDR1 promoter function in human tumor cells (HeLa), rather than drug-resistant cell lines selected in vitro because these cells conform more closely to cells from clinical isolates. Upon treatment with HMN-176, luciferase expression driven by the MDR1 promoter was suppressed in a dose-dependent manner (Fig. 5B)
. At a concentration of 300 nM HMN-176, 40% inhibition of promoter activity was achieved in this system. We could not examine the effect of HMN-176 at higher concentrations because of its cytotoxicity against HeLa cells. Deletion of the Y-box (nucleotides -82 to -73) resulted in the loss of >85% of the promoter activity, and HMN-176 had no significant effect on the residual promoter activity (data not shown).
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. A 40% reduction in the GI50 for Adriamycin was achieved by pretreatment with 3 µM HMN-176.
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DISCUSSION |
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. Because Taxol is known to be involved in the typical multidrug phenotype associated with MDR1 overexpression (24)
, K2/ARS cells appear to have a classical MDR phenotype. We detected >50% reduction in MDR1 mRNA expression in K2/ARS cells after treatment with 3 µM HMN-176 for 48 h. Although a longer exposure may have a greater effect, we could not test this possibility in our experimental system because of the exposure-time-dependent cytotoxicity of HMN-176. The doses used in this study were within the range achieved in the plasma of patients undergoing Phase I clinical trials, which we know to be well tolerated. Cytotoxicity and MDR1 down-regulation are, therefore, concomitant effects at pharmacological concentrations, but the molecular targets mediating these effects seem to be different. We have also shown that down-regulation of MDR1 can be attributed to the inhibition of NF-Y activity by HMN-176. Mutations and/or deletions that abolish NF-Y binding to the Y-box have been shown to result in a significant loss of MDR1 promoter activity (16) , which indicates that NF-Y regulates the basal and inducible expression of MDR1. The Y-box, on which NF-Y acts, binds NF-Y even before induction (13 , 14) . In our experiments, the inhibition of DNA binding was detected when HMN-176 was preincubated with HeLa nuclear extract, which suggests that the affinity between HMN-176 and NF-Y is not so high as to displace the preexisting NF-Y complex from the high-affinity Y-boxes. Therefore we consider that in our experimental systems, NF-Y newly synthesized during physiological turnover might be a target of HMN-176. Accordingly, we could not detect a significant decrease in MDR1 mRNA or protein expression when K2/ARS cells were exposed to 3 µM HMN-176 for periods shorter than 48 h (data not shown).
Scotto et al. (25) and Abolhoda et al. (26) reported that in some metastatic sarcomas, tumor MDR1 mRNA levels increase after exposure to the DNA-damaging agent Adriamycin. They have additionally shown that the transcriptional activation of MDR-1 is mediated by NF-Y.5 The response of cancer cells to continuous or repetitive multiple stress is thought to be a crucial step in the acquisition of the MDR phenotype. However, as shown in our studies, HMN-176 treatment induced no significant up-regulation of MDR1 in either parent or drug-resistant K2 cells. Additional work is necessary to find out whether HMN-176, as well as ET-743, interferes with the acute activation of MDR1 expression.
We tested the cross-resistance of other K2 sublines selected for by cisplatin, Adriamycin, camptothecin, and Taxol. In all cases, much lower degrees of cross-resistance were observed for HMN-176 than for the other agents tested. Because it seems unlikely that resistance to these very different agents is mediated solely by MDR1, we speculate that NF-Y is a common factor involved in drug resistance mediated by multiple upstream factors, including MDR-related protein and the lung resistance protein. However, to our knowledge, the involvement of NF-Y in the regulation of the multidrug resistance protein expression has not yet been clearly established. Effects of HMN-176 on the expression of P-gp species other than MDR1 remain to be assessed. Furthermore, because MDR1 is expressed in normal tissues such as brain and kidney, the effect of HMN-176 in these tissues needs to be estimated in vivo. Our efforts to establish a cell line resistant to HMN-176 have not succeeded. Although it is not yet clear whether HMN-176 can be a substrate for MDR1, these results suggest that enhanced cytotoxicity caused by the down-regulation of MDR1 might make it difficult to establish a cell line resistant to HMN-176 itself. Therefore, we can expect HMN-176 to provide benefit by inhibiting the emergence of multidrug-resistant cells through the down-regulation of MDR1 within a tolerable dose-range. All in all, NF-Y seems to be a promising target for manipulating the MDR phenotype.
Is the inhibition of NF-Y associated with the cytotoxicity of HMN-176? After incubation of K2/ARS cells at 3 µM for 48 h, cell growth was significantly retarded because of a prolonged G2-M phase, but DNA fragmentation was hardly detected by the terminal deoxynucleotidyltransferase-mediated nick end labeling method at this time point (data not shown). In addition, we have demonstrated by RT-PCR that the mRNA levels of some housekeeping genes such as ß-tubulin (data not shown) and GAPDH (Fig. 2)
in treated cells are not altered so that these experimental conditions do not induce cell death, although they do retard cell growth. Therefore, it seems unlikely that the inhibitory effect of HMN-176 on NF-Y correlates with its cytotoxicity. Although HMN-176, as well as HMN-154, targets NF-Y, there may well be other targets that more directly determine the fate of the cell. We have recently obtained evidence that BRAP2, initially identified as a molecule associated with the suppressor oncogene product BRCA1 (27)
, also binds HMN-176.6
It has been suggested that BRAP2 plays a crucial role in the nuclear-cytoplasmic shuttling of BRCA1. A defect in a molecule involved in nuclear-cytoplasmic shuttling can lead to cell death after cell cycle arrest at G2-M phase (28)
. In this context, BRAP2 is a possible target for HMN-176 that mediates its cytotoxic effect.
In conclusion, we have shown that the antitumor agent HMN-176 can suppress MDR1 expression by inhibiting NF-Y activity in MDR1- overexpressing multidrug-resistant tumor cells. All resistant cell lines used in our experiments were artificially established by constitutive exposure to drugs. However, in clinical settings, the emergence of such highly resistant cells seems unlikely. Therefore, we believe that the half reduction of MDR1 expression by HMN-176 may be effective in the much-less-resistant cells usually seen in clinical settings. Furthermore, a preliminary in vitro study in our laboratory revealed that the use of HMN-176 in combination with various common antitumor agents such as Adriamycin, Taxol, VCR, and etoposide showed an additive effect, suggesting that the combination of these agents is a promising approach.
On the basis of these findings, we have initiated in vitro studies with HMN-176 and in vivo studies with HMN-214 (as a prodrug for HMN-176) to test whether it enhances the antitumor activity of other antitumor agents.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Discovery Research Laboratories, Nippon Shinyaku Co. Ltd., Nishinosho-Monguchi-cho, Kisshoin, Minami-ku, Kyoto 601-8550, Japan. Phone: 81-75-321-9163; Fax: 81-75-321-9038; E-mail: hi.tanaka{at}po.nippon-shinyaku.co.jp 
2 The abbreviations used are: MDR, multidrug resistance; RT-PCR, reverse transcription-PCR; EMSA, electrophoretic mobility shift assay; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; P-gp, P-glycoprotein; glyceraldehyde-3-phosphate dehydrogenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; VCR, vincristine.
3 F. Katoh, T. Honmura, R. Yamaguchi, M. Matsuda, K. Kimura, and H. Hidaka, unpublished results.
4 A. Patnaik, L. Forero, A. Goetz, A. W. Tolcher, L. A. Hammond, C. Takimoto, L. Wood, G. R. Weiss, J. Bono, L. Smith, and E. K. Rowinsky, unpublished results.
5 Z. Hu, S. Jin, and K. W. Scotto, unpublished results.
6 H. Tanaka and N. Ohshima, BRAP2-mediated tumor cell death induced by the synthetic antitumor agent, HMN-176, manuscript in preparation.
Received 4/17/03. Revised 7/18/03. Accepted 8/ 5/03.
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