Clinical Cancer Research AACR Conference on Cancer Prevention
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Park, R.
Right arrow Articles by Huang, R. C. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Park, R.
Right arrow Articles by Huang, R. C. C.
Clinical Cancer Research Vol. 11, 4601-4609, June 15, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

Systemic Treatment with Tetra-O-Methyl Nordihydroguaiaretic Acid Suppresses the Growth of Human Xenograft Tumors

Richard Park, Chih-Chuan Chang, Yu-Chuan Liang, Yousun Chung, Ryan A. Henry, Elaine Lin, David E. Mold and Ru Chih C. Huang

Authors' Affiliation: Department of Biology, Johns Hopkins University, Baltimore, Maryland

Requests for reprints: Ru Chih C. Huang, Biology Department, Johns Hopkins University, 249 Mudd Hall, 3400 North Charles Street, Baltimore, MD 21218-2685. Phone: 410-516-5181; Fax: 410-516-5213; E-mail: rhuang{at}jhu.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: We have previously shown that the transcriptional inhibitor tetra-O-methyl nordihydroguaiaretic acid (M4N) induces growth arrest in tumor cells and exhibits tumoricidal activity when injected intratumorally into tumor cell explants in mice. The experiments reported here were designed to determine whether M4N can be given systemically and inhibit the growth of five different human xenograft tumors.

Experimental Design: Nude (nu/nu) mice bearing xenografts of each of five human tumor types (i.e., hepatocellular carcinoma, Hep 3B; prostate carcinoma, LNCaP; colorectal carcinoma, HT-29; breast carcinoma, MCF7; and erythroleukemia, K-562) were treated with M4N given i.v. or i.p. in a Cremophor EL–based solvent system or orally in a corn oil based diet. Tumors from the treated animals were measured weekly and analyzed for the expression of the Cdc2 and survivin genes, both previously shown to be down-regulated by M4N.

Results: Systemic M4N treatment suppressed the in vivo growth of xenografts in each of the five human tumor types. Four of the five tumor models were particularly sensitive to M4N with tumor growth inhibitions (T/C values) of ≤42%, whereas the fifth, HT-29, responded to a lesser extent (48.3%). Growth arrest and apoptosis in both the xenograft tumors and in the tumor cells grown in culture were accompanied by reductions in both Cdc2 and tumor-specific survivin gene expression. Pharmacokinetic analysis following oral and i.v. administration to ICR mice indicated an absolute bioavailability for oral M4N of ~88%. Minimal drug-related toxicity was observed.

Conclusion: These preclinical studies establish that when given systemically, M4N can safely and effectively inhibit the growth of human tumors in nude mice.

Key Words: M4N • CDC2 • survivin • Cremophor EL

Cancers of all types are characterized by defects in the control or regulation of the cell cycle which result in unrestrained and aberrant cell proliferation. The CDC2-cyclin B1 complex, or MPF, is well established as the universal and key regulator of the G2-M phase transition of the cell cycle (1), and recent clinical studies showing elevated CDC2 expression in breast and colorectal carcinomas and the independent and prognostic association of CDC2 expression with tumor metastases, underscore the importance of this key cell cycle regulator in tumor progression (2, 3). Consequently, Cdc2 gene expression and activity represent an attractive target for antitumor drug development whose strategy is to selectively attenuate or prevent uncontrolled cell proliferation. We were first to report that the transcription inhibitor, tetra-O-methyl nordihydroguaiaretic acid (M4N), is capable of arresting the proliferation of several transformed and tumor cell lines in culture and exhibits tumoricidal activity in a C3 cell-induced mouse tumor model system (4). M4N-mediated arrest occurred at the G2 stage of the cell cycle which correlated with a reduction in Cdc2 RNA levels, protein levels, and enzymatic activity. Our report was immediately followed by another which showed that M4N inhibits the growth of both murine and human melanomas and human colon cancer in vivo (5). Subsequently, we have shown that after M4N treatment and cell cycle arrest, expression of the survivin gene, a member of the inhibitor of apoptosis family, is also suppressed and the mitochondrial apoptotic pathway is activated (6).

M4N is a hydrophobic molecule with limited water solubility. In our previous published experiments, M4N was dissolved in DMSO and either added to cell cultures or injected intratumorally (4, 6). Intratumoral injection is a highly efficient method of delivering M4N to localized nonmetastatic inoperable tumors without causing tissue toxicity. However, the clinical usefulness of nonsystemic intratumoral chemotherapy is limited and the conventional standard of care in clinical oncology remains surgery followed by systemic chemotherapy and/or radiation as deemed appropriate to the clinical situation. In this study, we considerably expand the cancer therapeutic potential of M4N by investigating its ability to be given systemically, either parenterally when formulated in a Cremophor EL–based solvent system or orally when included in a corn oil–based diet, and its efficacy as an antitumor agent in vivo against five human cancer xenograft models. The tissue distribution and bioavailability of systemically delivered M4N and its effect on Cdc2 and survivin gene expression both in humor tumor cell culture and xenograft tumors are also examined.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and culture conditions. Human tumor cell lines were obtained from American Type Culture Collection (Mannassas, VA). The human hepatocellular carcinoma cell line, Hep 3B, was maintained in Eagle's MEM supplemented with 10% fetal bovine serum (FBS); the human breast cancer cell line, MCF7, was grown in DMEM containing 10% FBS; the human colorectal carcinoma cell line, HT-29, was cultured in McCoy's 5a medium with 10% FBS; the human prostate carcinoma cell line, LNCaP, was maintained in RPMI 1640 with 10% FBS and the human erythroleukemia cell line, K-562, was propagated in Iscove's modified Dulbecco's medium containing 10% FBS. The C3 cell line was generated by transfection of the EJras-transformed C57Bl/6 (B6) mouse embryo cells with full-length HPV16 (7). The cells were grown and maintained in Iscove's modified Dulbecco's medium supplemented with 5% FBS and 10 µmol/L ß-mercaptoethanol. All of the cultures contained the antibiotics penicillin and streptomycin.

Mice. Female ICR mice, 6 to 8 weeks of age, were purchased from Harlan Sprague-Dawley (Indianapolis, IN). C57Bl/6 mice and T cell–deficient nude (nu/nu) mice, males and females 5 to 6 weeks of age, were purchased from Charles River Laboratories (Wilmington, MA). C57Bl/6 mice bearing C3 cell tumors were prepared as described previously (4). The nude mice were housed in a pathogen-free room and all experiments involving the mice were carried out in accordance with the Johns Hopkins University Animal Care and Use Committee guidelines.

Tissue distribution of M4N. Two ICR mice and one C57Bl/6 mouse bearing a C3 cell-induced tumor were injected via tail vein and i.p., respectively, with 100 µL of Cremophor EL/ethanol–based solvent containing 100 µCi of tritiated M4N (specific activity, 4.68 µCi/µg) and 60 mmol/L of unlabeled M4N (2.14 mg/mouse). After 3 hours for the ICR mice and 4 hours for the tumor-bearing mouse, the animals were euthanized and various organs were harvested, weighed, and dissolved overnight in 4 mol/L guanidine isothiocyanate. Insoluble material was further extracted with ethanol and the tritium content of both the guanidine isothiocyanate extract and the ethanol extract was measured on a Packard scintillation counter. The concentration (µg/g) of M4N in each organ was calculated based on the specific activity of the inoculum.

For long-term oral administration experiments, mice were individually fed pellets consisting of M4N dissolved in corn oil and basal mix (Harlan Teklad, Indianapolis, IN) for 14 weeks. The food pellets weighed 9 g and contained ~300 mg M4N each. Organs from two orally M4N fed mice were cut into small pieces, dried under vacuum, and pulverized into a rough powder using a mortar and pestle. The samples were then extracted overnight in absolute ethanol with vigorous shaking. The extracts were cleared by centrifugation and the insoluble residue was extracted two more times with absolute ethanol. The ethanol extracts were pooled, evaporated, extracted with ethyl acetate, and dried completely under vacuum. The samples were then sent to KP Pharmaceuticals (Bloomington, IN) for quantitative high-performance liquid chromatography analysis using a reverse-phase C-18 column with a mobile phase of 35%: 0.1% TFA in H2O, 65%: CAN.

Pharmacokinetics of M4N in mice. Healthy female ICR mice were treated i.v. or orally with 44 mg/kg of M4N. Orally given M4N was dissolved at a concentration of 2.8 mmol/L (1 mg/mL) in corn oil and delivered by gavage with a 25-mm, 22-guage feeding needle with a 1.25-mm-diameter tip (Fine Science Tools, Foster City, CA). At predetermined times (5 and 40 minutes, 2, 4, 8, and 16 hours) two to three mice were euthanized by cervical dislocation and blood was collected from the heart and pericardial cavity into heparinized tubes. The blood samples were pooled and dried under vacuum before acetonitrile extraction (thrice) and high-performance liquid chromatography analysis. The area under the plasma concentration-time curve (AUC0-inf) was calculated by the trapezoidal rule up to the last measured concentration time point (AUC0-t) using KaleidaGraph software (Synergy Software, Reading, PA). Beyond the last measured time point (Ct), the area was estimated using the equation:

where {lambda}z is the slope of the extrapolated portion of the concentration-time curve (8).

Tumor models. T cell–deficient nude (nu/nu) mice, males and females 5 to 6 weeks of age, were purchased from Charles River Laboratories and housed and handled in a pathogen-free environment in accordance with the Johns Hopkins University Animal Care and Use Committee guidelines. The mice were implanted s.c. in their flanks with 2.5 x 106 Hep3B cells, 1 x 107 HT-29 cells, 2 x 106 MCF7 cells or 1 x 106 K-562 cells suspended in HBSS, or 2 x 106 LNCaP cells suspended in HBSS/Matrigel (50:50, v/v). Female mice receiving MCF7 cells were implanted s.c. with a pellet of 17ß-estradiol (0.72 mg/pellet, 60-day release; Innovative Research of America, Sarasota, FL) several days before injection. When the tumors exhibited a mean diameter of 7 to 8 mm, the mice were assigned to a treatment group that received M4N and a control group that received the vehicle only. To ensure that the M4N-treated groups and the control groups started with approximately equal distributions of tumor sizes, the mice were grouped into one of three categories: those bearing small tumors (<4 mm in length), medium tumors (4-8 mm), and large tumors (>8 mm). Both control and M4N-treated groups received roughly equal numbers of mice from each of the three categories.

Administration of M4N to tumor-explanted nude mice. For i.p. administration, M4N was dissolved in 6% Cremophor EL, 6% ethanol, and 88% saline (v/v/v) as described for paclitaxel in Loganzo et al. (9). Mice received either a single daily 100-µL injection containing 2 mg of M4N for 3 weeks or 100 µL of placebo for 3 weeks (control). This dosage of M4N was determined in initial experiments with mice bearing hepatocellular carcinoma tumor HT-29 to be close to the maximum amount that could be injected without solvent toxicity. In oral administration experiments, mice were fed pellets consisting of M4N dissolved in corn oil and basal mix (Harlan Teklad) for 3 weeks. Each food pellet weighed 9 g and contained 300 mg of M4N. The mice were caged separately and allowed to eat freely. The amount of M4N consumed by each mouse was determined by counting the number of pellets eaten and weighing any residual pellets.

Evaluation of antitumor effect. Tumors were measured in two perpendicular dimensions once every 7 days, and the tumor volumes were calculated according to the following formula:

where a is the width of the tumor (smaller diameter) and b is the length (larger diameter; ref, 10). The relative tumor volume (RTV) of each tumor was defined as the ratio of the volume at a given time and the volume at the start of treatment (11). The mean RTV and SE was calculated for each treatment group. Antitumor activity was determined by calculating the tumor growth inhibition (TGI) value using the following equation:

where T is the mean RTV of the treated tumors at the experiment end point (3 weeks) and C is the mean RTV of the control group (10, 11). The National Cancer Institute standard for the minimal level of antitumor activity (T/C ≤ 42%) was adopted. At the termination of the experiment, the tumors were excised and fixed in formaldehyde. Tissue samples were then sent to Paragon Bioservices (Baltimore, MD) for histology and immunohistochemistry using antibodies against human CDC2 and survivin.

Cell proliferation and viability assay. One day after seeding, cultured cells from three representative dishes were counted with a hemocytometer. Growth medium was then removed from the remaining dishes and replaced with medium containing 1% DMSO and a range of concentrations of M4N in triplicate. After 3 days in the presence of M4N, the cells were counted with a hemocytometer, and cell viability was assessed by trypan blue exclusion.

Northern blots. After 24 and 72 hours of M4N treatment, cells were washed twice with calcium and magnesium-free phosphate buffered saline (PBS) and total RNA was isolated using the Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Equal amounts (24 µg) of RNA were separated on a 1.5% formaldehyde-agarose gel and transferred to a Hybond Nytran Supercharge nylon membrane filter (Schleicher and Schuell, Keene, NH) by capillary blotting. The RNA was hybridized to random-primed 32P-labeled cDNA probes specific for the human CDC2, survivin, and ß-actin genes. Hybridization was carried out in 50% deionized formamide, 5x SSPE [1x = 0.18 mol/L NaCl, 10 mmol/L sodium phosphate (pH 7.7), and 1 mmol/L EDTA], 5x Denhardt's solution, 0.5% SDS, and 20 µg/mL salmon sperm DNA at 42°C for 17 hours. The blots were washed four times for 15 minutes in decreasing concentrations of salt (with a final concentration of 0.1x SSPE, 0.1% SDS at 45°C) and exposed to X-ray film for 72 hours. For repeated hybridizations, the blots were stripped in 0.1% SDS at 100°C for 10 minutes and washed in 5x SSPE for 10 minutes.

Western blots. After 24 and 72 hours of M4N treatment, cell monolayers were washed with PBS and harvested with 10 mmol/L EDTA and 10 mmol/L EGTA in PBS. The washed cells were pelleted and resuspended in lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1% NP40, 1% sodium deoxycholate, and 1 mmol/L EDTA] containing a protease inhibitor cocktail. The lysate was cleared by centrifugation and protein concentrations were determined with the Bio-Rad Laboratories (Hercules, CA) protein concentration assay solution. Twenty-five micrograms of protein were separated on a 14% SDS-PAGE gel and electroblotted to a Hybond enhanced chemiluminescence nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ) using a semidry electroblot apparatus. Primary rabbit polyclonal antibodies against CDC2 (Oncogene Research Products, Boston, MA) and cyclin B (Santa Cruz Biotechnology, Santa Cruz, CA) and primary mouse polyclonal antibody against actin (Santa Cruz Biotechnology) were used at a final concentration of 0.2 µg/mL. The secondary antibodies were anti-rabbit or anti-mouse immunoglobulin G conjugated to horseradish peroxidase. The filters were developed with the enhanced chemiluminescence Western Blot Detection Kit (Amersham Biosciences). The chemiluminescence filters were placed against X-ray film for detection of protein bands.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Tissue distribution and pharmacokinetics of M4N after systemic administration. The hydrophobic nature and poor water solubility of M4N have until now limited its use as a chemotherapeutic agent to tumor ablation via intratumoral injection. Other hydrophobic, water-insoluble pharmaceuticals, such as tenipocide, cyclosporin A, and paclitaxel, have been successfully solubilized for systemic administration using Cremophor EL (12), a polyethoxylated castor oil. We therefore investigated whether a Cremophor EL–based formulation could be developed for M4N. In addition, M4N dissolved in corn oil and formulated into mouse chow pellets with a basal nutrient mix was used in tests of oral M4N administration.

Three hours after a single i.v. injection (Fig. 1A) and 4 hours after an i.p. injection of M4N formulated in 6% Cremophor EL/6% ethanol/88% saline (Fig. 1B), M4N was quickly distributed to the organs via the bloodstream. In each case, the highest concentration of M4N was found in the duodenum (i.v., 18.6 µg/g; i.p., 6.1 µg/g) followed by the colon (14.3 µg/g) and liver (7.5 µg/g) for i.v. administration and adipose tissue (2.5 µg/g) and liver (1.1 µg/g) for i.p. injection. Measurable levels of M4N (0.2 µg/g) were also found in the brain. Of particular relevance to this study was the presence of M4N in C3 cell tumor explants (0.24 µg/g) following i.p. administration (Fig. 1B). Assuming a tissue density near 1 g/mL, the concentration of M4N found in the C3 tumors after a single 2 mg dose is ~0.67 µmol/L. This is significant because the growth inhibitory effects of M4N on cells in culture are seen at the micromolar level. M4N was still detectable in most of the organs 18 hours after administration; however, nearly all of the drug was eliminated within 6 days (data not shown).



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 1. Tissue distribution of M4N following systemic administration. The amount of M4N per gram of tissue for 10 mouse organs following i.v., i.p., and oral administration. A, 3 hours after a single i.v. injection; B, 3 hours after a single i.p. injection; and C, 14 weeks after continuous oral administration. Organs where M4N concentrations were not determined (ND). Groups A and C were normal mice while Group B mice were carrying C3 tumors of ~2.5 cm3 in volume.

 
Oral administration of chemotherapeutic agents is better tolerated than parental administration for long-term post-surgical adjuvant treatment of cancer. Thus, the ability to systemically distribute M4N orally was also investigated. Mice were fed a diet of food pellets containing M4N dissolved in corn oil daily for 14 weeks to determine what steady-state tissue concentrations of M4N could be achieved. Each mouse consumed ~90 mg of M4N daily. Steady-state levels of between 350 and 900 µg M4N/g of dry tissue was detected in the gastrointestinal tract organs and the spleen; 17 to 23 µg/g in the lungs and adipose tissue; and 5 to 13 µg/g M4N in the heart, liver, and kidney (Fig. 1C). Assuming that mouse tissues are about 65% water and their density is not too different than 1 g/mL, then it is estimated that the concentrations of M4N in the tissues after daily oral administration range from 4.9 µmol/L to 0.9 mmol/L. The steady-state plasma concentration was found to be 23 µg/g or 22.4 µmol/L. These levels are within the range of concentrations found to effect the growth of tumor cells in vitro.

Pharmacokinetic analysis of M4N (44 mg/kg) was carried out following oral and i.v. administration to healthy ICR mice. The time course of plasma levels of M4N is shown in Fig. 2 and the pharmacokinetic variables for each route are given in Table 1. After oral gavage, M4N plasma levels rose gradually, reaching the Cmax 8 hours after treatment. Integration of the concentration-time curves revealed an absolute bioavailability for oral M4N of ~88%.



View larger version (14K):
[in this window]
[in a new window]
  		Fig. 2. Plasma concentration-time curves of M4N (44 mg/kg) after i.v. (-{bullet}-) or oral (-{circ}-) administration in ICR mice. Plasma from two to three mice was pooled for each time point.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Pharmacokinetic variables of M4N after oral and i.v. administration (44 mg/kg)

 
Systemic M4N treatment inhibits the in vivo growth of human tumor xenografts. To determine the effect of systemic administration of M4N on the in vivo growth of human tumors, nude mice were inoculated s.c. in each flank with HT-29 colorectal carcinoma cells, LNCaP prostate carcinoma cells, MCF7 breast adenocarcinoma cells, K-562 erythroleukemia cells, and Hep 3B hepatocellular carcinoma cells. When the tumors attained the appropriate size, the mice were treated for 3 weeks with single daily i.p. injections containing 2 mg of M4N formulated in the Cremophor EL–based solvent. For control mice receiving the vehicle only, the mean RTV increased rapidly over the 3-week treatment period (Fig. 3). In contrast, the rate of increase of the mean RTV was significantly reduced in the M4N-treated mice and in the case of the K-562 tumors the mean RTV decreased over the treatment period. As summarized in Table 2, M4N treatment resulted in significant differences (P < 0.05) in the end point mean RTV of the control and treated mice. The tumor growth inhibition (T/C) values for four of the five tumor models were ≤42%, the minimum level for antitumor activity according to National Cancer Institute standards (10). The T/C value for HT-29 derived tumors was 48.3%.



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 3. Effect of systemic M4N treatment on the growth of human xenograft tumors in nude mice. Nude mice with human tumor xenografts of human tumor cell lines HT-29, LNCaP, Hep 3B, K-562, and MCF7 were treated for 3 weeks with daily i.p. injections of M4N. Weekly measurements of tumor size were made and the mean relative tumor volume (RTV) was plotted as a function of time after the start of treatment. (—) control; (– –) i.p. M4N treatment; (···) oral M4N treatment.

 

View this table:
[in this window]
[in a new window]
  		Table 2. Effect of systemic M4N on human xenograft tumors

 
An additional set of mice bearing LNCaP xenografts were fed food pellets containing M4N to investigate the effect of oral M4N treatment on tumor growth. The mice received an average of 78 mg of M4N/d for 3 weeks. Over the course of the treatment, the mean RTV did not increase significantly (Fig. 3). At the end of treatment period, the mean RTV was 1.1, significantly (P < 0.05) lower than the 3.40 value of the control group (Table 2). For oral M4N treatment, the T/C value was 36.0%, below the minimum level for antitumor activity.

Mice treated systemically with M4N exhibit minimal toxicity. The health and well-being of the mice were assessed by recording of their body weights at the beginning and end of treatment. For each of the tumor models, the mean change in body weight for M4N-treated mice was small (–0.1 to –2.7 g/mouse) and not significantly different from that for the control mice (Table 2). The greatest weight loss associated with M4N treatment was observed in the nude mice with LNCaP xenografts (–2.7 g/mouse). This morbidity was likely tumor related rather than drug related because the control mice exhibited a similarly large mean body weight change (–2.8 g/mouse). Mortality rates for the treatment and control groups did not differ significantly except in the HT-29 tumor–bearing mice (Table 2). The higher mortality rate observed for the M4N-treated mice in this group (61.5% versus 7.7%) was attributable to the fact that M4N treatment in these mice was initiated at a dose of 3 mg/d. After significant body weight change and four deaths were recorded in the M4N-treated mice in the first week of exposure, the dose was lowered to 1 mg/d until the toxicity was reversed (5 days) and a dose of 2 mg/d was maintained for the remainder of the experiment. When corrected for the HT-29-explanted mice lost during the first week overdose, the overall mortality rates were 22.2% for the M4N-treated mice and 15.8% for the control mice.

M4N inhibits the growth of human tumor cells in culture and their expression of CDC2 and survivin. We have previously shown in mouse C3 tumor cells that M4N arrests cells at the G2-M phase of the cell cycle by reducing the levels of CDC2 protein and kinase activity (4) and induces apoptosis by suppressing survivin gene expression and stability and activating the mitochondrial apoptosis pathway (6). To investigate the mechanism of M4N's inhibition of the in vivo growth of the five human tumor xenografts, in vitro experiments exploring the effect of M4N on each of the cell lines in culture were done. Cells in culture were treated for 3 days with a range of concentrations of M4N after which the number of viable cells was determined and compared with the starting cell number. M4N inhibited the growth of all of the cell lines tested with IC50 values of 5 to 10 µmol/L (Fig. 4). Cell cycle arrest is evident when over a wide range of drug concentrations the number of viable cells remains nearly the same as the pretreatment number. Two of the cell lines, HT-29 and LNCaP, exhibited growth arrest between 20 and 60 µmol/L M4N, whereas the other three cell lines, Hep 3B, K-562, and MCF7, were more sensitive to M4N and exhibited growth inhibition curves without a plateau and viable cell numbers below the pretreatment values at M4N concentration as low as 10 µmol/L. From these data, a single M4N concentration that effectively inhibited or arrested cell growth was chosen for each cell line to analyze the effect of the drug on CDC2 and survivin gene expression.



View larger version (26K):
[in this window]
[in a new window]
  		Fig. 4. Effect of M4N on the proliferation of human tumor cell lines. The number of viable cells after 72 hours of treatment with different concentrations of M4N was determined for cell lines HT-29, LNCaP, Hep 3B, K-562, and MCF 7. The horizontal line in each graph represents the number of viable at the initiation of M4N exposure.

 
Northern blot analysis of total RNA isolated from each of the five human tumor cell lines was done to examine the effect of M4N on CDC2 and survivin mRNA levels. Cell cultures were exposed to a growth inhibitory dose of M4N and RNA was extracted after 24 and 72 hours of treatment. Hybridization of the RNA to cDNA probes specific for human CDC2 and survivin yielded the predicted transcripts of 1.4 and 2.0 kb for CDC2 (13) and 1.9 kb for survivin (14). By 72 hours, the relative amounts of CDC2 and survivin mRNA in the M4N-treated cells was greatly reduced when compared with RNA from untreated cells (Fig. 5A). For cell line HT-29, the inhibitory effect was evident by 24 hours. The amount of ß-actin mRNA was unaffected by the M4N treatment and it was used to standardize the results. The relative amounts of CDC2 and survivin protein were examined by Western blot analysis with similar results. Protein extracts from the M4N-treated cells contained decreased amounts of CDC2 and survivin when compared with the extracts from untreated cells (Fig. 5B). The results were most dramatic for the HT-29, LNCaP, Hep 3B, and MCF7 cell lines whereas less so for K-562. ß-Actin protein levels did not change with the drug treatment.



View larger version (59K):
[in this window]
[in a new window]
  		Fig. 5. Effect of M4N on CDC2 and survivin mRNA and protein levels in human tumor cell lines. Total RNA and protein extracts were prepared from control cells (–) or cells exposed to M4N (+) for 24 and 72 hours. Levels of CDC2, survivin and ß-actin mRNA and protein were determined by Northern (A) and Western blot (B) analysis.

 
CDC2 and survivin expression is suppressed in M4N-treated human xenograft tumors. In cell culture, M4N-induced growth inhibition correlated with reduced CDC2 and survivin gene expression in all five human tumor types tested. To determine if a similar reduction of CDC2 and survivin gene expression was associated with the in vivo growth inhibition of human tumor xenografts by systemic administration of M4N, tissue sections of xenograft tumor biopsies from M4N-treated and control mice were analyzed histologically and immunochemically for CDC2 and survivin protein levels. In the case of all five tumor types, antibody staining for both CDC2 and survivin consistently and clearly showed reduced expression levels of both proteins in M4N-treated samples compared with the control samples (Fig. 6). In addition, H&E staining showed clearly elevated levels of necrosis and apoptosis in all of the M4N-treated tumors compared with the controls.



View larger version (74K):
[in this window]
[in a new window]
  		Fig. 6. Effect of systemic M4N treatment on CDC2 and survivin protein levels of human xenograft tumors in nude mice. Formaldehyde-fixed tumors from mice treated daily for 3 weeks with i.p. injections of M4N or placebo (Control) were sectioned and analyzed by H&E staining and immunochemical analysis using antibodies specific for human CDC2 and survivin.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
We have shown that the water-insoluble anticancer agent, M4N, can be given systemically to mice either i.p. or i.v. when dissolved in a Cremophor EL/ethanol–based solvent or orally when dissolved in corn oil. Tissue levels of M4N achieved after a single dose parenterally or after continuous feeding are in the range required to inhibit tumor cells in culture and when tested against five different tumor cell types in nude mice xenografts, M4N, given daily as an i.p. injection, was able to inhibit the growth of all five with minimal toxicity. The inhibition in tumor growth, both in vitro and in vivo correlated with reduced levels of CDC2 and survivin mRNA and protein, corroborating our earlier findings in the mouse C3 tumor cell system (4, 6).

CDC2 (CDK1) is the catalytic component of the M-phase promoting factor that ushers the eukaryotic cell into mitosis. It is just one of many cyclin-dependent kinases that regulate the cell cycle and whose deregulation can lead to various diseases including cancer (15). Because of their ability to arrest cellular proliferation, cyclin-dependent kinase inhibitors are being pursued as potential cancer chemotherapeutics. Of the >50 cyclin-dependent kinase inhibitors that have been identified, all act by competitive inhibition at the ATP-binding pocket and whereas there is some selectivity, the search for inhibitors that target individual cyclin-dependent kinases has thus far been unsuccessful (16). M4N uses an entirely different mechanism to inhibit CDC2 kinase activity. It suppresses the production of CDC2 mRNA and thereby reduces the level of CDC protein and kinase activity. Data from our previous studies on the inhibition of HIV replication by M4N (17), suggest that it interferes with binding of Sp1 to its cognate promoter sites and therefore may have some selectivity for the SP1-dependent CDC2 promoter. In our previous studies of cells in culture, M4N exposure has always caused cell cycle arrest. The in vitro cell growth inhibition curves presented here suggest that some tumor cells may respond to M4N differently than others. For example, cells lines MCF7, Hep 3B, and K-562 are more sensitive to M4N than HT-29 and LNCaP. In cells like these, the progression from arrest to apoptosis may be accelerated. Whether this is due to cell to cell variation in the kinetics of M4N inhibition of survivin versus CDC2 gene expression has yet to be determined.

Sp1 is also involved in regulating expression of the survivin gene; therefore, it is not surprising that M4N inhibits the production of survivin mRNA in the human tumor cell lines. Survivin is an inhibitor of apoptosis protein that is overexpressed in most common cancers and other chemotherapeutic agents that inhibit survivin based on antisense or small interfering RNA technology are being developed (18, 19). Survivin is also a target of CDC2 kinase activity in mitotic cells. Phosphorylation of survivin by CDC2 kinase leads to increased stability of survivin in its complex with caspase 9 and XIAP (20, 21). M4N, by its ability to inhibit CDC2 levels and therefore CDC2 kinase activity, also destabilizes any survivin not eliminated by M4N inhibition of its expression, thereby enhancing its effectiveness. Recently, it has been shown that hedamycin, a member of the pluramycin class of antitumor antibiotics, interacts with GC-rich DNA sequences in the survivin promoter abrogating the binding of Sp1 and resulting in the down regulation of survivin gene expression and the modulation of cancer cell viability (22). Whether hedamycin effects CDC2 expression like M4N is not yet known.

Survivin expression is not limited to cancer cells. It is also plays essential roles in normal hematopoietic cell proliferation (23); T-cell development, maturation, and homeostasis (24); and terminally differentiated neutrophils (25). Drugs that target survivin may affect these normal processes as well. There is recent evidence to suggest that survivin overexpression in tumor cells is the result of transcriptional deregulation, possibly mediated by mutations in the CDE/CHR repressor binding region in the survivin promoter (26). If survivin gene expression is regulated by different mechanisms in normal and malignant cells, it is possible that the transcription inhibitor, M4N, may only affect tumor cell expression of survivin.

M4N is a small molecule derived from a plant lignan. It is generally assumed that small inhibitors of transcription cannot be particularly selective. However, it has recently been shown that the bisanthracycline, WP631, inhibits Sp1-mediated transcription by specifically recognizing GC-rich tracts present in Sp1 binding sites and bisintercalating the DNA with a binding constant close to that of the transcription factor itself (27). WP631 induces a G2-M arrest, polyploidy, and nonapoptotic cell death in culture cells (28, 29). Imperatorin, a dietary furanocoumarin, is another Sp-1 inhibitor that has been described (30). It inhibits HIV-1 replication through an Sp1-dependent pathway like M4N; however, its effect on tumor cell proliferation and mechanism of action have not been well studied. The number of genes affected by M4N has not been determined; however, it is important to note that we have been able to significantly reverse M4N-induced cell arrest and apoptosis simply by resupplying the cells with CDC2 or survivin (6). It was also recently shown that the survivin promoter is highly active in both established tumor cell lines and the primary melanoma cells, whereas survivin promoter activities were extremely low in major mouse organs (31).

The experiments reported here represent an important step in the development of M4N as a viable chemotherapeutic agent. Human tumor xenografts implanted in immunosuppressed mice have been widely used in preclinical anticancer drug development. A retrospective study of compounds that have been tested with xenografts and in phase II trials revealed that 45% with activity against more than one third of the xenografts tested also showed clinical activity (32). M4N was effective against all five of the human tumor xenografts we tested and therefore is predicted successful in clinical trials. A small phase I clinical trial of M4N has already been conducted with human head and neck tumor patients using an intratumoral injection dose of 20 mg M4N/cm3 tumor volume. The unpublished results of that trial showed that at this dosage, M4N was tolerable to patients and produced tumor necrosis in the majority of the treated patients. Furthermore, the high absolute bioavailibility of M4N reported here make it a candidate for development as an oral chemotheurapeutic, which is clinically desirable.


   Acknowledgments
 
We thank Dr. Jonathan Heller of Erimos Pharmaceuticals for his invaluable comments and suggestions, Ming-Hua Hsu and Stacy Cheng for their generous assistance with the nude mice experiments, and Liane Lee and Dr. Ming Chuan Shao for lending their expertise to the pharmacokinetic experiments.


   Footnotes
 
Grant support: Erimos Pharmaceuticals LLC grant P690-C25-2407 (R.C.C. Huang) and National Science Council, Taiwan, ROC (Y-C. Liang).

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.

Note: R. Park is currently at the Department of Microbiology and Immunology, Cornell University, Ithaca, NY.

E. Lin is currently at the New York University School of Medicine, New York, NY.

Y-C. Liang is a visiting predoctoral fellow from Tunghai University, Taiwan, ROC.

Received 10/27/04; revised 3/25/05; accepted 3/31/05.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Pines J. Four-dimensional control of the cell cycle. Nat Cell Biol 1999;1:EE73–9.
  2. Kourea HP, Koutras AK, Scopa CD, et al. Expression of the cell cycle regulatory proteins p34(cdc2), p21(waf1), and p53 in node negative invasive ductal breast carcinoma. Mol Pathol 2003;56:328–35.[Abstract/Free Full Text]
  3. Nozoe T, Honda S, Inutsuka S, Korenaga D. p34cdc2 expression is an independent indicator for lymph node metastasis in colorectal carcinoma. J Cancer Res Clin Oncol 2003;129:498–502.[CrossRef][Medline]
  4. Heller JD, Kuo J, Wu TC, Kast M, Huang RCC. Tetra-O-methyl nordihydroguaiaretic acid induces G2 arrest in mammalian cells and exhibits tumoricidal activity in vivo. Cancer Res 2001;61:5499–504.[Abstract/Free Full Text]
  5. Lambert JD, Meyers RO, Timmermann BN, Dorr RT. Tetra-O-methylnordihydroguaiaretic acid inhibits melanoma in vivo. Cancer Lett 2001;171:47–56.[CrossRef][Medline]
  6. Chang CC, Heller JD, Kuo J, Huang RCC. Tetra-O-methyl nordihydroguaiaretic acid induces growth arrest and cellular apoptosis by inhibiting Cdc2 and survivin expression. Proc Natl Acad Sci U S A 2004;101:13239–44.[Abstract/Free Full Text]
  7. Feltkamp MCW, Smits HL, Vierboom MPM, et al. Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells. Eur J Immunol 1993;23:2242–9.[Medline]
  8. Toutain PL, Bousquet-Melou A. Bioavailability and its assessment. J Vet Pharmacol Ther 2004;27:455–66.[CrossRef][Medline]
  9. Loganzo F, Discafani CM, Annable T, et al. HTI-286, an synthetic analogue of the tripeptide hemiasterlin, is a potent antimicrotubule agent that circumvents p-glycoprotein-mediated resistance in vitro and in vivo. Cancer Res 2003;63:1838–45.[Abstract/Free Full Text]
  10. Bissery MC, Guenard D, Gueritte-Voegelein F, Lavelle F. Experimental antitumor activity of Taxotere (RP 56976, NSC 628503), a Taxol analogue. Cancer Res 1991;51:4845–52.[Abstract/Free Full Text]
  11. Sanceau J, Poupon MF, Delattre O, Sastre-Garau X, Wietzerbin J. Strong inhibition of Ewing tumor xenograft growth by combination of human interferon-{alpha} or interferon-ß with ifosfamide. Oncogene 2002;21:7700–9.[CrossRef][Medline]
  12. Strickley RG. Solubilizing excipients in oral and injectable formulations. Pharm Res 2004;21:201–30.[CrossRef][Medline]
  13. Tommasi S, Pfeifer GP. In vivo structure of the human cdc2 promoter: release of a p130-E2F-4 complex from sequences immediately upstream of the transcription initiation site coincides with induction of cdc2 expression. Mol Cell Biol 1995;15:6901–13.[Abstract]
  14. Ambrosini G, Adida C, Altieri DC. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med 1997;3:917–21.[CrossRef][Medline]
  15. Malumbres M, Barbacid M. To cycle or not to cycle: a critical decision in cancer. Nat Rev 2001;1:222–31.
  16. Knockaert M, Greengard P, Meijer L. Pharmacological inhibitors of cyclin-dependent kinases. Trends Pharmacol Sci 2002;23:417–25.[CrossRef][Medline]
  17. Gnabre JN, Brady JN, Clanton DJ, et al. Inhibition of human immunodeficiency virus type 1 transcription and replication by DNA sequence-selective plant lignans. Proc Natl Acad Sci U S A 1995;92:11239–43.[Abstract/Free Full Text]
  18. Ansell SM, Arendt BK, Grote DM, et al. Inhibition of survivin expression suppresses the growth of aggressive non-Hodgkin's lymphoma. Leukemia 2004;18:616–23.[CrossRef][Medline]
  19. Uchida H, Tanaka T, Sasaki K, et al. Adenovirus-mediated transfer of siRNA against survivin induced apoptosis and attenuated tumor cell growth in vitro and in vivo. Mol Ther 2004;10:162–71.[CrossRef][Medline]
  20. O'Connor DS, Grossman D, Plescia J, et al. Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin. Proc Natl Acad Sci U S A 2000;97:13103–7.[Abstract/Free Full Text]
  21. Dohi T, Okada K, Xia F, et al. An IAP-IAP complex inhibits apoptosis. J Biol Chem 2004;279:34087–9.[Abstract/Free Full Text]
  22. Wu J, Ling X, Pan D, et al. Molecular mechanism of inhibition of survivin transcription by the GC-rich sequence selective DNA-binding antitumor agent, hedamycin: evidence of survivin downregulation associated with drug sensitivity. J Biol Chem. In press 2005.
  23. Fukuda S, Mantel CR, Pelus LM. Survivin regulates hematopoietic progenitor cell proliferation through p21WAF1/Cip1-dependent and -independent pathways. Blood 2004;103:120–7.[Abstract/Free Full Text]
  24. Xing Z, Conway EM, Kang C, Winoto A. Essential role of survivin, an inhibitor of apoptosis protein, in T cell development, maturation, and homeostasis. J Exp Med 2004;199:69–80.[Abstract/Free Full Text]
  25. Altznauer F, Martinelli S, Yousefi S, et al. Inflammation-associated cell cycle-independent block of apoptosis by survivin in terminally differentiated neutrophils. J Exp Med 2004;199:1343–54.[Abstract/Free Full Text]
  26. Yong X, Fang F, Ludewig G, Jones G, Jones D. A mutation found in the promoter region of the human survivin gene is correlated to overexpression of survivin in cancer cells. DNA Cell Biol 2004;23:419–29.[CrossRef][Medline]
  27. Mansilla S, Priebe W, Portugal J. Sp1-targeted inhibition of gene transcription by WP631 in transfected lymphocytes. Biochemistry 2004;43:7584–92.[CrossRef][Medline]
  28. Villamarin S, Ferrer-Miralles N, Mansilla S, Priebe W, Portugal J. Induction of G2/M arrest and inhibition of c-myc and p53 transcription by WP631 in Jurkat T lymphocytes. Biochem Pharmacol 2002;63:1251–8.[CrossRef][Medline]
  29. Villamarin S, Mansilla S, Ferrer-Miralles N, Priebe W, Portugal J. A comparative analysis of the time-dependent antiproliferative effects of daunorubicin and WP631. Eur J Biochem 2003;270:764–70.[Medline]
  30. Sancho R, Marquez N, Gomez-Gonzalo M, et al. Imperatorin inhibits HIV-1 replication through an Sp1-dependent pathway. J Biol Chem 2004;279:37349–59.[Abstract/Free Full Text]
  31. Zhu ZB, Makhija SK, Lu B, et al. Transcriptional targeting of tumors with a novel tumor-specific survivin promoter. Cancer Gene Ther 2004;11:256–62.[CrossRef][Medline]
  32. Johnson JI, Decker S, Zaharevitz D, et al. Relationships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials. Br J Cancer 2001;84:1424–31.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Molecular Cancer TherapeuticsHome page
D. L. Rowe, T. Ozbay, L. M. Bender, and R. Nahta
Nordihydroguaiaretic acid, a cytotoxic insulin-like growth factor-I receptor/HER2 inhibitor in trastuzumab-resistant breast cancer
Mol. Cancer Ther., July 1, 2008; 7(7): 1900 - 1908.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
M. Pennati, M. Folini, and N. Zaffaroni
Targeting survivin in cancer therapy: fulfilled promises and open questions
Carcinogenesis, June 1, 2007; 28(6): 1133 - 1139.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
H.-C. Yeh, C. M. Puleo, T. C. Lim, Y.-P. Ho, P. E. Giza, R. C. C. Huang, and T.-H. Wang
A microfluidic-FCS platform for investigation on the dissociation of Sp1-DNA complex by doxorubicin
Nucleic Acids Res., December 4, 2006; 34(21): e144 - e144.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Park, R.
Right arrow Articles by Huang, R. C. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Park, R.
Right arrow Articles by Huang, R. C. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online