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Experimental Therapeutics |
Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218 [J. D. H., J. K., R. C. C. H.]; Department of Pathology, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205 [T. C. W.]; and Cancer Immunology Program, Cardinal Bernardin Cancer Center, Loyola University, Chicago, Illinois 60626 [W. M. K.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells pass through many checkpoints as they proceed through the cell cycle, and certain criteria must be met to pass each of these checkpoints. In the G2-M transition, the most essential regulator is the cyclin-dependent kinase CDC2. This kinase binds to the regulatory protein cyclin B, and this complex, also called the MPF, is responsible for stimulating a myriad of events that lead to the entry of the cell into early prophase (1) . Not surprisingly, the loss or deactivation of either component of the MPF will block cellular progression out of G2.
Transcription of the genes coding for the two protein components of the MPF is regulated by a number of promoter elements. The most essential of these have been determined by Cogswell et al. (2) for cyclin B and Tommasi and Pfeifer (3) for CDC2. The cyclin B promoter contains an upstream stimulating factor element, an E-box, an AP-2 site, an NF-Y site, and two Sp1 sites. The CDC2 promoter has binding sites for the CCAAT box factor Cp1, Y-box proteins, ets-2, E2F-4, and two Sp1 sites. Deletion of the Sp1 sites in the cyclin B promoter has little effect, but the binding of Sp1 to the CDC2 promoter is essential for the production of CDC2 (3 , 4) .
Our laboratory has studied extensively the use of derivatives of the plant lignan NDGA for blocking viral replication through the inhibition of viral transcription. We have shown that NDGA derivatives, originally isolated from Larrea tridentata and now synthesized chemically, can inhibit the production of HIV (5
, 6)
, herpes simplex virus (7)
, and HPV transcripts (8)
by the deactivation of their Sp1-dependent promoters. Unexpectedly, one of these derivatives, M4N (Fig. 1)
, appears to also induce cell cycle arrest in mammalian cell lines. The research presented in this study indicates that M4N is capable of inducing G2 arrest in transformed mammalian cells and suggests that this arrest is attributable to inhibition of the production of the cyclin-dependent kinase CDC2. Additionally, intratumoral injection of M4N in a mouse system caused a substantial decrease in tumor size and viability without detected host toxicity. Necrosis of the tumor cells correlated with reduction of CDC2 protein levels. This work demonstrates that M4N may be a potential chemotherapeutic agent for the control of cancer growth.
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MATERIALS AND METHODS |
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Cell Culture.
The C3 cell line was generated by transfection of EJras-transformed C57Bl/6 (B6) mouse embryo cells with full length HPV16 (10)
. The cells were grown and maintained in Iscove’s modified Dulbecco’s medium (Life Technologies, Inc., Bethesda Research Laboratories, Bethesda, MD) supplemented with 5% FCS, 10 µM ß-mercaptoethanol, and penicillin/streptomycin. The TC-1 cell line (11)
was grown and maintained in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% FCS, nonessential amino acids, L-glutamine, sodium pyruvate, and penicillin/streptomycin. The C33a cell line (ATCC accession no. HTB31) was grown and maintained in MEM (Life Technologies, Inc.) supplemented with 10% FCS, nonessential amino acids, sodium pyruvate, and penicillin/streptomycin. The CEMT4 cell line was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, NIH, CEM-T4 was obtained (12)
from Dr. J. P. Jacobs, and they were grown and maintained in MEM (Life Technologies, Inc.) and supplemented with 10% FCS and penicillin/streptomycin.
M4N Treatment and Cell Growth Analysis.
Three days after seeding, cultured cells from two representative dishes were harvested and counted with a hemocytometer. Growth media from the remaining dishes was then removed and replaced with media containing 1% DMSO and a range of concentrations of M4N. After 3 days in the presence of M4N, the cells were counted with a hemocytometer, and cell viability was assessed by trypan blue exclusion.
Flow Cytometry.
To prepare the cells for flow cytometry evaluation, cells were seeded at a density of 105 to 106 cells/dish. Twenty-four h later, the growth media was removed, and half of the cells were given fresh media containing M4N in 1% DMSO. The control cells received 1% DMSO in the growth media. Cell samples were incubated for 3 days and then counted as described above. Approximately 2 x 106 cells/condition were aliquoted, washed twice with PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4), and pelleted in a clinical centrifuge. Samples were fixed by dropwise addition of -20°C 70% ethanol, followed by a 45-min incubation at 4°C. Then the cells were washed in PBS twice, and a solution of 10 µg/ml propidium iodide, 100 µg/ml RNase A, and 38 mM sodium citrate in PBS was added. Cells were stored overnight at 4°C and subsequently analyzed by flow cytometry in a EPICS 752 flow cytometer (Coulter Instruments). Each sample analyzed comprised a minimum of 3 x 104 cells. The indicated cell cycle positions are estimates based on the DNA content of control cultures.
Immunofluorescence and DAPI Staining.
C3 cells were grown on coverslips under the conditions described for flow cytometry. Immunofluorescence evaluation of 
- and 
-tubulin was done as described by Quintyne et al. (13)
. Briefly, coverslips were washed twice with PBS and subsequently fixed with methanol. Blocking was carried out with 2% BSA, 0.05% Tween 20 in PBS. Primary antibodies (rabbit anti--tubulin and mouse anti--tubulin DM1A; Sigma Chemical Co.) were diluted 1:500 and 1:100, respectively, in TTBS [25 mM Tris (pH 7.4), 137 mM NaCl, 2.7 mM KCl, and 0.1% Tween 20] and incubated with the cells for 30 min at room temperature. After three washes in TTBS, cells were exposed to FITC (1:500 in TTBS) and Texas red-conjugated (1:200 in TTBS) horse antimouse and antirabbit, respectively (Vector Laboratories, Inc) and incubated for 30 min at room temperature. After three washes in TTBS and a final wash in water, coverslips were mounted to slides in 3:1 Mowiol 4-88 (Calbiochem Corp.):N-propyl gallate (Sigma Chemical Co.) in PBS plus 50% glycerol and viewed by fluorescence microscopy.
For DAPI (Sigma Chemical Co.) staining, cells were prepared as for immunofluorescence but were treated with DAPI stain instead of antibody at a concentration of 1 µg/ml in PBS before being mounted and analyzed by fluorescence microscopy.
Western Blots and Kinase Assays.
C3 cells were prepared as described above for flow cytometry. After the desired incubation times, samples were washed with PBS and harvested with 10 mM EDTA and 10 mM EGTA in PBS. The cells were pelleted and lysed in a 250-µl solution of 50 mM HEPES (pH 7.0), 250 mM NaCl, 0.1% NP40, 10% glycerol, 1x protease inhibitor cocktail (Sigma Chemical Co.), and 1 mM DTT. Protein extracts were then quantified with the Bio-Rad protein concentration assay solution and stored at -80°C.
For the Western blots, 40 µg of protein was separated on a 12% SDS PAGE gel and electroblotted to a Hybond enhanced chemiluminescence nitrocellulose membrane (Amersham). The Western Light Immudetection Kit (Tropix) was used to detect CDC2 and cyclin B. Primary antibodies were rabbit polyclonals against CDC2 (Oncogene Research Products, Cambridge, MA; catalogue DO4431-1) and cyclin B (Santa Cruz Biotechnology, Santa Cruz, CA; catalogue SCBT H-433). Both primary antibodies were used at a final concentration of 2 µg/ml. Chemiluminescence filters were placed against X-ray film for detection of protein bands.
The kinase assays were performed after immunoprecipitation by combining 100 µg of total protein extract with 3 µg of mouse monoclonal anticyclin B1 (PharMingen; catalogue number 14541A) and incubating at 4°C for 30 min with mild agitation. Protein A/G agarose pellets (Santa Cruz Biotechnology; catalogue SC-2003) were equilibrated with lysis buffer [50 mM HEPES (pH 7.0), 250 mM NaCl, 0.1% NP40, 10% glycerol, protease inhibitor cocktail (Sigma Chemical Co.), and 1 mM DTT] and added to protein extract solutions. Samples were then incubated at 4°C overnight, followed by four 5-min washes with lysis buffer. A solution consisting of 20 mM HEPES (pH 7.9), 5 mM MgCl2, 2 mM DTT, 20 µM ATP, 50 µCi of [-32P]ATP, and 10 µg of histone H1 was added to the protein A/G pellets and incubated at 37°C for 30 min. Reactions were stopped by the addition of 25 µl of 2x SDS running buffer. Proteins were separated by boiling this mixture and running the supernatants on 12% SDS PAGE gels. The gels were stained with Coomassie Brilliant Blue, dried, and exposed to X-ray film for 17 h.
Northern Blots.
C3 cells were prepared as described above for flow cytometry. After the desired incubation times, cells were washed with PBS, harvested with trypsin, and pelleted in a clinical centrifuge. Total RNA was prepared from the cells with guanidine isothiocyanate and quantified by absorbance at 260 nm. Equal amounts of RNA were separated on a 4-morpholinepropanesulfonic acid-EDTA-sodium acetate gel and transferred to a nylon filter as described by Sambrook et al. (14)
. Hybridization was carried out in 50% deionized formamide, 5 x SSPE, 5 x Denhardt’s solution, 0.5% SDS, and 20 µg/ml salmon sperm DNA at 42°C for 1 h using random primed 32P-labeled DNA probes for CDC2 (nucleotides 369–390 of CDC2 cDNA; Ref. 15
) and GAPDH (nucleotides 438–459 of GAPDH cDNA; Ref. 16
). The blots were washed four times for 15 min in decreasing concentrations of salt (with a final concentration of 0.1 x SSPE, 0.1% SDS at 42°C) and exposed to film for 72 h. For repeated testing, blots were stripped with 100°C 0.1% SDS for 15 min and washed in 5 x SSPE for 15 min.
M4N Treatment of C3 Cell-induced Tumors in Mice.
C3 cells were grown, harvested, and counted as described above for flow cytometry. The cells were given two additional washes with HBSS (Life Technologies, Inc.), and 5 x 105 cells were injected s.c. between the shoulders of fourteen C57bl/6 mice. Tumors were allowed to develop, with 11 mice developing single tumor masses and 3 mice developing two separate tumor masses. After approximately 20 days, four mice received daily intratumoral injections of 0.1 ml of DMSO, and seven mice received daily intratumoral injections containing 0.1 ml of preheated drug solution (20 mg of M4N in DMSO at 55°C). The mice that developed two tumors received daily intratumoral injections of 0.1 ml of DMSO containing 20 mg of M4N into one of their tumors. After 2 weeks of treatment, the mice were euthanized and photographed, and the tumors were weighed. Excised tumors were immediately fixed and then stored in 4% formaldehyde in PBS. The fixed tissue was then dehydrated through a series of graded alcohols and xylene and embedded in paraffin. The paraffin tissue blocks were thin sectioned and stained for microscopy with H&E (dehydration, embedding, sectioning, and H&E staining done by AML Laboratory, Baltimore, MD) or analyzed by immunocytochemical methods for CDC2 (embedded tissue blocks sectioned and tested for CDC2 by BD PharMingen, San Diego, CA).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We tested increasing amounts of the NDGA derivative M4N on cultures of the HPV-16/ras-transformed C3 cell line (10)
, the non-Sp1 regulated HPV-16 E6/E7/ras-transformed TC-1 cell line (11)
, the HPV-negative human cervical cancer cell line C33a, and the human leukemia cell line CEM-T4 (Ref. 12
; Fig. 2
). A modest reduction in cell growth was observed at low concentrations of the drug, and some cell death was seen at high amounts. Interestingly, all of the samples demonstrated arrest without cell death when exposed to moderate concentrations of M4N. After 3 days at these concentrations, the number of cells remained equal to the count at the initiation of treatment (Arrest; Fig. 2
). Surprisingly, arrested cells maintained >95% viability after exposure to the drug for 8 days (data not shown).
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. The most extreme responses were seen with the C3 and CEMT4 cell lines, in which >90% of the cells show G2-M DNA content.
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-tubulin (green, cytoskeleton) and -tubulin (red, centrosomes) were used to determine the status of the centrosomes in the C3 cell line after 72 h of M4N treatment. As shown in Fig. 4a
, the centrosomes of M4N-treated cells are duplicated but are still located next to each other in the nucleus of the cell. Because centrosomes separate during early prophase, it can be concluded that these cells have not begun mitosis. In contrast, the -tubulin staining of the control cells has the diffuse pattern characteristic of G1 or S-phase (19)
. A lack of chromatin condensation in the M4N-treated cells was also observed with DAPI staining (Fig. 4b)
, additional evidence that the cells have not moved forward out of G2 phase (20)
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. These experiments demonstrate that the drug may act by inhibiting the production of the CDC2 protein, resulting in a loss in the activity of the MPF.
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, the amount of CDC2 mRNA is reduced after only 24-h treatment with M4N and nearly eliminated after 72 h. Production of the non-Sp1-regulated housekeeping gene GAPDH was used as an RNA loading control, and its levels were not affected by 40 µM M4N. The kinase assays, Western blots, and Northern blots were repeated three to five times each with the same results.
Intratumoral Injections of M4N Cause Necrosis of C3 Cell-induced Tumors in Mice.
Upon discovering the potent antiproliferative effects of M4N on transformed cells in culture, we sought to investigate whether the drug could have a significant impact on the growth of tumors in mammals. C3 cell-induced tumors were treated with intratumoral injections of either M4N dissolved in DMSO or DMSO alone. Drug treatments were performed daily for 2 weeks, at which point the mice were euthanized and examined. As seen in Table 1
, those mice that received intratumoral injections of M4N showed a marked reduction in tumor mass as compared to mice treated with the drug vehicle alone. In mice with two tumors, tumor regression was only observed at the M4N-treated site. There was no toxicity observed in any of the mice, as determined by daily evaluation of activity and overall body weight change during the course of treatment. The general weight increase observed in both control and drug-treated mice can be attributed to the fact that the mice were not fully grown when treatment began.
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; Fig. 6
). The treated lesion is condensed and necrotic, whereas the untreated tumor site appears viable. Notably, no toxic effects were seen in the surrounding tissues and organs, and it appeared that M4N was still present around the treated tumor when the animal was evaluated.
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. In the sections from the control tumors (Fig. 7, a, c, and e)
, the cells have a high nuclear/cytoplasmic ratio, pleomorphic nuclear changes, high mitotic figures, spindle-like sarcoma shape, and infiltration into the surrounding tissue, features that are characteristic of cancer cells. In contrast, sections from M4N-treated tumors show apoptotic structures in focal areas and significant necrosis throughout. Multiple stages of tumor regression were evident in the tumor biopsies taken from different mice. In mouse number 14 (Fig. 7f)
, focal areas show apoptotic nuclear fragmentation, whereas in mouse number 13 (Fig. 7d)
, the tumor had become fully necrotic and large holes are visible where dead tissue had been cleared. The tumor in mouse number 12 (Fig. 7b)
had almost completely regressed by the time of the biopsy. In all of the tumor biopsies there was a small amount of drug precipitate visible, and in focal areas there was chronic inflammation and fibrosis replacing the dead cells at the tumor site. Normal tissue surrounding the tumors appeared to be unaffected by M4N treatment. Data given for mice 12, 13, and 14 are representative of all of the mice used in the present study, and the same conclusions were obtained in two separate experimental repeats under similar conditions.
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, using antibodies specific to CDC2, revealed that tumor tissues exposed to the drug show substantially less staining than the untreated control. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In contrast to the observed cell cycle arrest seen in M4N-treated cell lines, cells incubated with the parent compound of M4N, NDGA, undergo apoptosis without exhibiting a cell cycle specific block, even at low concentrations. This apoptosis is widely believed to be attributable to the inhibition of lipoxygenases (21 , 22) . Recent evidence (17) has shown that glutathione oxidation and mitochondrial depolarization may also be possible mechanisms for the observed effects of NDGA on cellular systems. Our observations suggest that the primary effects of M4N on cellular systems are unique, because cells treated with a moderate amount of the drug exhibit clear G2 arrest. However, it is possible that these other pathways may play a role in the necrosis of tumor tissue in the mouse model system.
The Tumoricidal Actions of M4N in the Mouse Model System.
The results of the intratumoral injection study suggest that M4N may have effects on tumor cells that go beyond the down-regulation of CDC2 and subsequent cell cycle arrest. Most notable of these are the focal apoptosis and widespread necrosis that are observed on histological examination of biopsies of the M4N-treated tumors. These effects are also observed in cultured cells at high concentrations of M4N and, therefore, may simply be the result of the increased dose of drug used to inject the tumors. However, closer histological examination of the tumor biopsies seems to indicate that there are more factors involved. Although distant organs in the animal are unaffected by the drug because of its low solubility and resultant lack of systemic circulation, the tissue surrounding the treated tumors is also undamaged except for the signs of chronic inflammation characteristic of a healing response. There is no evidence that the growth, viability, or protein expression of the normal cells is inhibited by M4N; therefore, its cytotoxic capacity is likely specific to dividing cells and not a direct result of its concentration. Additionally, the low solubility of M4N in water suggests that the concentration of the drug inside effected cells is considerably lower than the injected dose. We believe that the tumoricidal effects of M4N seen in the mouse model are likely attributable to interactions in several pathways, and it is the sum of these events that eventually leads to cell death. Further investigation will be required to elucidate the precise mechanism by which M4N causes tumor-specific necrosis.
Prospects of Using M4N as an Anticancer Agent by Intratumoral Injection.
The selection of M4N as an anticancer agent administered directly into tumors provides several distinctive advantages: (a) M4N is a hydrophobic compound and is exceedingly soluble in DMSO (200 mg/ml; 55°C). Therefore, only a small volume of the drug solution is needed for injection to achieve effective dosage of the drug. In our mouse study, daily injections of 100 µl for several days were sufficient to completely stop the tumor growth in mice; (b) systemic toxicity is avoided because M4N, which is relatively insoluble in bodily fluids, remains concentrated in the area of the tumor when injected intratumorally. Retention of M4N in the tumor region has been verified by injecting tritium-labeled M4N intratumorally into mice, followed by liquid scintillation analysis of labeled drug recovered from the tumor, blood, urine, feces, and several major organs. In addition, because the drug is slow to be cleared from the area, continued injection of M4N should become unnecessary after only a few treatments. Thus, when the drug is directly targeted to the tumor, size becomes the determining factor for the required amount of drug that is necessary. There is no reason to believe that the dosage would need to be increased for the treatment of human tumors of comparable size. This should reduce the risk considerably in human trials; (c) because M4N is injected into the tumor site, inoperable tumors, such as those of the brain, may be treated effectively without high-risk surgery; (d) the drug should have unlimited antitumor applications as demonstrated by its effectiveness against a wide range of transformed cell lines; and (e) M4N can be chemically synthesized with high yield and low cost from the parent compound NDGA, and the parent compound can be obtained from the creosote bush in large amounts. For these reasons, clinical trials that use M4N to remove tumor tissue are likely to be effective and should reduce the need for extensive surgery. Such treatment can proceed easily and cheaply, without much delay should the trials be successful.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from NIH (1 RO1 DE12165) and Biocure Medical, LLC (to R. C. H.) along with a grant from NIH (1 RO1 ICA/AI78399; to W. M. K.) J. D. H. is a predoctoral fellow at the Johns Hopkins University, Department of Biology, Baltimore, Maryland. 
2 To whom requests for reprints should be addressed, at Department of Biology, The Johns Hopkins University, Baltimore, MD 21218. Phone: (410) 516-5181; Fax: (410) 516-5213; E-mail: huang{at}jhunix.hcf.jhu.edu
3 The abbreviations used are: M4N, tetra-O-methyl nordihydroguaiaretic acid; MPF, maturation-promoting factor; NDGA, nordihydroguaiaretic acid; HPV, human papillomavirus; DAPI, 4',6-diamidino-2-phenylindole; SSPE, saline-sodium phosphate-EDTA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TTBS, Tris-Tween-buffered saline.
Received 2/ 5/01. Accepted 5/ 9/01.
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-tubulin complexes. Binding to the centrosome. Regulation and microtubule nucleation. Curr. Opin. Cell Biol., 12: 113-118, 2000.[Medline]
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