EBM Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

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 Google Scholar
Google Scholar
Right arrow Articles by Izevbigie, E. B.
Right arrow Articles by Walker, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Izevbigie, E. B.
Right arrow Articles by Walker, A.
Experimental Biology and Medicine 229:163-169 (2004)
© 2004 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

A Novel Natural Inhibitor of Extracellular Signal-Regulated Kinases and Human Breast Cancer Cell Growth

Ernest B. Izevbigie*,1, Joseph L. Bryant{dagger} and Alice Walker*

* The Molecular Genetics and Molecular & Cellular Signaling Laboratory, Department of Biology, and National Institutes of Health Center for Environmental Health, Jackson State University, Jackson, Mississippi 39217; and {dagger} Institute of Human Virology, Animal Model Division, University of Maryland Biotechnology Institute, Baltimore, Maryland 21201

1 To whom requests for reprints should be addressed at Molecular and Cellular Signaling Laboratory, Center for Environmental Health, Jackson State University, Department of Biology, 1400 John R. Lynch Street, P.O. Box 18540, Jackson, MS 39217. E-mail: rnest.b.izevbigie{at}jsums.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Water-soluble extracts of edible Vernonia amygdalina leaves were recently reported as potent inhibitors of cultured MCF-7 cells. The mechanism by which V. amygdalina inhibits MCF-7 cell growth has not been previously studied. The objective of this study was to evaluate the effects of V. amygdalina on the activities, DNA synthesis, and subsequent cell growth of extracellular signal-regulated protein kinases 1 and 2 (ERKs 1/2;). Treatment of cells with various concentrations (3–100 mg/ml) of water-soluble V. amygdalina extract potently inhibited ERK activities, DNA synthesis (P < 0.005), and cell growth (P < 0.01) in a concentration-dependent fashion, both in the absence and presence of serum. The growth rate of cells pretreated with 10 mg/ml V. amygdalina for 48 hrs before transfer to V. amygdalina-free medium was not significantly different (P > 0.05) from untreated cells. These results suggest that V. amygdalina, at least at concentrations up to 10 mg/ml, exhibits cytostatic action to retard the growth of human breast cancer cells. In addition, the ERK signaling pathways may be one or more of the intracellular targets for V. amygdalina antineoplastic actions.

Key Words: human breast cancer • DNA synthesis • ERKs activities • V. amygdalina extract


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Breast cancer is the most commonly diagnosed cancer in women, representing approximately 30% of all types of cancer in women (1). One out of every eight women will be diagnosed with breast cancer in her lifetime. It is estimated that in the United States in 2002, breast cancer will have accounted for more than 15% of all new cancer cases and 7% of cancer-related deaths (1). Overall, cancer mortality rates in several locations have improved in the past decade, but the incidence for some types of cancer among certain ethnic groups continues to rise. Conventional methods of treatment have yielded some benefits, but novel chemotherapeutic agents are needed to improve cancer incidence and survival rates. Plant-derived products are considered excellent sources for the discovery and development of such novel cancer chemoprotective and chemotherapeutic agents (2). During the past decade, the number of people using herbs and related products in the United States has increased from 34% in 1990 to 42% in 1995, with related out-of-pocket costs of about $27 billion (3).

Several factors have contributed to the increase in the use of herbal products. First, results from cell culture and animal model experiments suggest that some herbal products have potential for use as chemotherapeutic or chemopreventive agents for cancer (4–29). Second, the deregulating effect of the 1994 Dietary Supplement Health and Education Act, which weakens the Food and Drug Administration’s ability to regulate ‘‘dietary supplements,’’ also contributed to the increase in herbal product use. Third, the belief that herbs can provide some benefits over and above allopathic medicine also allows users to believe that they have some control in their choice of medications (30). As a result, it is estimated that 50% of patients with breast cancer use herbal products (3). A survey conducted by the World Health Organization revealed that about 80% of the world’s population, in nearly all countries, still depend on herbal medicine as their main source for therapy (31).

Herbal medicinal use is popular in Nigeria, where there are anecdotal reports on the efficacies of many herbal products against many illnesses. One such herb, Vernonia amygdalina, is purported to have several health benefits. More than 30 years ago, Kupchan and colleagues (32) first demonstrated that purified subfractions of organic solvent extracts of V. amygdalina were cytotoxic to human carcinoma cells of the nasopharynx. Twenty-four years later, Jisaka et al. (33) reported similar anticancer activities of purified organic solvent fractions of V. amygdalina against other cancerous cell lines. In the same year, Obaseiki-Ebor et al. (34) reported that organic solvent extracts of V. amygdalina inhibited ethyl methanesulfonate-induced mutations in Salmonella typhimurium, thus suggesting that antimutagenic activity represents one mechanism used by the organic solvent extracts of V. amygdalina to elicit their anticancer benefits. By the end of 2002, only two studies (32, 33) showed anticancer activities of organic solvent extracts of V. amygdalina or its fractions, and only one study (34) suggested a possible mechanism to explain the cancer protective effects.

The effects of water-soluble extracts of V. amygdalina were unknown before Izevbigie (35) reported that V. amygdalina inhibited the growth of human MCF-7 breast cancer cells in vitro. The latter findings may have more relevant clinical implications because water-soluble extract preparations represent the traditional method by which V. amygdalina is administered to patients by herbalists. In addition, mechanisms to explain the antiproliferative actions of V. amygdalina remain largely uninvestigated. Because many anticancer agents, including tamoxifen, share the attenuation of extracellular signal-regulated protein kinases 1 and 2 (ERKs 1/2; Refs. 36–39) activities, we sought to investigate the effects of V. amygdalina on ERK activities.

In this study we demonstrate that concentrations (3–100 µg/ml) of V. amygdalina potently inhibited DNA synthesis in a concentration-dependent fashion in the absence or presence of serum. The inhibitory effects of 10 µg/ml V. amygdalina were reversed by switching cells to V. amygdalina-free medium. We also observed that ERK activities were markedly decreased in cells exposed to V. amygdalina compared to those of unexposed cells, thus suggesting ERK-dependent cytostatic actions of V. amygdalina. These data further suggest that V. amygdalina shows promise for use as an agent to prevent or delay the onset of breast cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human breast tumor cell line MCF-7 (passage #169) was a generous gift from Dr. Adrian Senderowicz, National Institute of Dental and Craniofacial Research, who purchased them from American Type Culture Collection lot 2431315 starting at the 149th passage with a 90%–100% viability. Fetal bovine serum (FBS), RPMI 1640 medium, and phosphate-buffered saline (PBS) were purchased from Gibco BRL (Grand Island, NY). BCA protein assay kits were obtained from Pierce (Rockford, IL). [3H]Thymidine (1 mCi/ml) was purchased from ICN Pharmaceuticals (Irving, CA). All other chemicals were obtained from Sigma Chemical Company (St. Louis, MO).

V. amygdalina Preparation.
Pesticide-free fresh V. amygdalina leaves, collected in Benin City, Nigeria, were rinsed with cold, distilled water. The leaves were soaked in cold water (1:1 w/v) overnight at 4°C before being crushed by a gentle means to a mixture. The mixture was then filtered through clean white gauze to remove particulate matter before filtration through a 0.45-lm filtration unit for sterilization. The resulting solution was lyophilized (5 g) and stored at -20°C.

Cell Culture.
MCF-7 cells were seeded at a density of 4 x 104 cells in 35-mm-diameter tissue culture plates and propagated in RPMI 1640 medium containing 10% FBS and 1% pen/strep/fungisome mixture. They were then grown in a humidified incubator under an atmosphere of 95% air and 5% CO2 at 37°C to subconfluence. Fresh medium was supplied every 48 hrs.

DNA Synthesis Assay.
DNA synthesis was determined by [3H]thymidine incorporation assays as we previously described (35), in the absence and presence of 10% FBS (serum). For DNA synthesis determination in the absence of serum, cells were grown to subconfluence in medium supplemented with serum and 1% pen/strep/ fungisome mixture before overnight serum starvation for synchronization. Fresh serum-free medium was provided, and then cells were treated with different concentrations (3–100 µg/ml) of V. amygdalina for 18 hrs before 1 µCi/ml [3H]-thymidine was added to each 35-mm-diameter dish, and the cells were grown for an additional 6-hr period. In contrast, for the DNA synthesis determination experiments in the presence of serum, log-phase proliferating cells were treated with different concentrations of V. amygdalina (3–100 µg/ml) for 18 hrs, and incubated with 1 µCi/ml [3H]-thymidine/35-mm dish for an additional 6-hr period. All incubations were terminated by aspirating the culture medium, doing three sequential washes with cold PBS, followed by the addition of 2 ml/35-mm dish of ice-cold 10% TCA for 20 mins at 4°C. After washing the cells three times with ice-cold water, cells were solubilized with 1 ml of 0.5 M NaOH/35-mm dish at 37°C for 30 mins. Upon solubilization, contents were transferred to scintillation vials, 5 ml scintillation cocktail was added to each vial, and radioactivities were determined with a scintillation counter.

Cell Proliferation Studies.
Log-phase proliferating cells were treated with different concentrations (12.5–100 µg/ml) of extracts with the appropriate controls. Twenty-four hours following treatment, triplicate 100-mm wells/ treatment were randomly selected for cell number determination. The medium was aspirated from cell monolayers and washed with PBS pH 7.4 to more easily detach the cells from the substratum of the culture. The resulting cell monolayers were treated with 1 ml trypsin/100-mm well and incubated briefly at 37°C. Cells were viewed microscopically to ensure a complete cell detachment. Cells were then resuspended in RPMI 1640 medium and counted with a hemocytometer. For the studies on cytostatic inhibition of cell growth, following cell exposure to V. amygdalina for 48 hrs with unexposed cells as controls, medium removal, PBS washing, trypsinization, and cell number determination, equal number of cells (300 x 103)/100 mm plate of either exposed or unexposed cells were plated. At 24 and 48 hrs, triplicate plates per treatment were harvested and counted.

Determination of IC50 Concentration of V. amygdalina.
The absorbance values obtained per treatment were converted to percentage cell viability. Regression analysis was performed on the cell viability data and the resulting equation was used to compute the inhibition concentration required to produce a 50% reduction (IC50) in cell viability.

Mitogen-Activated Protein Kinase (MAPK) Assays.
Cells at approximately 80% confluence were serum-starved overnight, stimulated with different concentrations (12.5–100) µg/ml of V. amygdalina, and incubated at 37°C for 10 mins. After incubation, the culture medium was aspirated, cells were washed with cold PBS, and lysed in a buffer containing 20 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM ethyleneglycotetraacetic acid, 1 mM ß-glycerophosphate, 1% Triton X-100, 2.5 mM MgCl2, 1 mM dithiothreitol (DTT), 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 2.5 mM sodium pyrophosphate, and 10 µg/ml leupeptin. Cells were scraped, transferred to Eppendorf tubes, and centrifuged. Two-hundred microliters containing 200 µg of total protein were immunoprecipitated with immobilized phospho-p44/42 kinase (Thr202/Tyr204) monoclonal antibody with gentle rocking overnight at 4°C. Pellets were recovered and washed twice with lysis buffer and twice more with kinase buffer containing 25 mM Tris pH 7.5, 5 mM ß-glycerophosphate, 2 mM DTT, 0.1 mM sodium vanadate, and 10 mM MgCl2. The pellets were suspended in 50 µl of kinase buffer supplemented with 200 µM ATP and 2 µl ELK-1 fusion protein (substrate for MAPK) and incubated for 30 mins at 37°C. The reactions were terminated by the addition of 15 µl 5x Laemmli buffer. Samples were then boiled and electrophoresed in 12% polyacrylamide gel electrophoresis. The resulting gels were transferred onto a nitrocellulose membrane in buffer containing 25 mM Tris base, 0.2 M glycine, 25% methanol pH 8.5 at 70 mA overnight.

Western Immunoblotting.
After transfer, the membrane was washed with Tris-buffered saline (TBS) for 5 mins at room temperature, followed by incubation in blocking buffer for 2 hrs at room temperature. The membrane was then incubated with primary antibody (ELK-1 at 1:1000 dilution) in antibody dilution buffer containing TBS, 0.1% Tween-20, and 5% bovine serum albumin, with gentle agitation overnight at 4°C. The membrane was washed three times for 5 mins each with TBS-Tween (TBST), followed by incubation with a secondary antibody conjugated to horseradish peroxidase at 1:2000 dilution in blocking buffer containing TBS, 0.1% Tween, and 5% w/v nonfat dry milk, with gentle agitation for 1 hr at room temperature. Finally, each membrane was washed with TBST three times for 5 mins at room temperature.

Detection of Phospho ELK-1 (Serine 383).
According to the manufacturer’s instructions, the membrane was incubated with 10 ml of LumiGLO (chemiluminescent reagent) with gentle agitation for 1 min at room temperature. The membrane was drained of excess developing solution, wrapped in Saran wrap, and exposed to Kodak X-OMAT AR film. Phosphorylated ELK-1 fusion protein was visualized by autoradiography and quantitated by densitometry.

Statistical Analysis.
Results are expressed as the mean ± SD of values obtained in triplicate from at least three different experiments. Differences between groups were compared by Student’s t test; P values < 0.05 were considered significant. When more than two means were compared, significance was determined by one-way analysis of variance followed by multiple comparisons using the Student-Neuman-Keul’s test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
V. amygdalina Inhibits DNA Synthesis in the Absence or Presence of Serum.
V. amygdalina exposure markedly inhibited DNA synthesis by MCF-7 cells in a concentration-dependent fashion (3–100 µg/ml) in the absence or presence of serum. The highest concentration used (100 µg/ml) caused a 7-fold growth inhibition in the absence of serum (P < 0.01) and 20-fold growth inhibition (P < 0.005) in the presence of serum compared to and expressed as percentages of controls (Fig. 1Go). Two different controls were used in these experiments; one received serum (serum positive) and the other did not (serum negative). The average DNA synthesis for the serum-positive controls was approximately 5-fold that of serum-negative controls. Therefore, DNA synthesis inhibition by V. amygdalina, expressed as percentages of controls, were higher in the presence of serum. To confirm the DNA synthesis data presented in Figure 1Go, cell count studies, using a hemocytometer to determine cell number, were carried out.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 1. V. amygdalina inhibits DNA synthesis in the absence or presence of serum. Either subconfluent cells were serum-starved overnight for DNA synthesis in the absence of serum experiments or log-phase proliferating cells in the presence of serum were treated with various concentrations of V. amygdalina as described in ‘‘Materials and Methods.’’ Each data point represents the mean ± SD of three independent experiments.

 
V. amygdalina Inhibits Cell Growth.
Consistent with the DNA synthesis data shown in Figure 1Go, treatment of cells with V. amygdalina inhibited cell proliferation in a concentration-dependent fashion. Twelve-and-a-half microgram/milliliter concentration produced a 40% decrease in cell proliferation (P < 0.05) compared to that of untreated cells. The highest concentration of 100 µg/ml resulted in an approximately 15-fold decrease in cell proliferation (P < 0.005; Fig. 2Go).



View larger version (12K):
[in this window]
[in a new window]
  		Figure 2. V. amygdalina inhibits mitosis. Log-phase proliferating cells were treated for DNA synthesis in the presence of serum with various concentrations of V. amygdalina as described in ‘‘Materials and Methods.’’ Each data point represents the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01.

 
Effects of V. amygdalina Pretreatment on MCF-7 Cell Growth.
Although the DNA synthesis (Fig. 1Go) and cell count studies (Fig. 2Go) have shown cell growth inhibitory (anticancer) effects of V. amygdalina, we wanted to know whether such inhibition was reversible or irreversible upon drug withdrawal. Because Izevbigie (35) had previously reported the IC50 value of V. amygdalina in these cells to be 5.68 ± 0.2 µg/ml, in the present studies we almost doubled the IC50 value (10 µg/ml) to determine the effects of V. amygdalina pretreatment and withdrawal on cell growth.

Table 1Go shows that pretreatment of cells with 10 µg/ml V. amygdalina for up to 48 hrs before returning cells to V. amygdalina-free medium did not affect subsequent cell growth (P < 0.05) for either 24 or 48 hrs of growth compared to that of untreated cells, thus suggesting cytostatic actions of V. amygdalina, at least up to 10 µg/ ml. These findings are corroborated by the cytotoxicity studies previously reported by Izevbigie (35).


View this table:
[in this window]
[in a new window]
  		Table 1. Effect of V. amygdalina Pretreatment on MCF-7 Growthab
 
So far, in our estimation, V. amygdalina has yielded some encouraging data on the inhibition of DNA synthesis and subsequent cell growth, cytotoxicity, and mechanisms of inhibition, and thus further investigation was warranted. Next, we sought to determine the intracellular targets mediating V. amygdalina actions. Because the inhibition activities of ERKs 1/2 is a common characteristic shared by many anticancer agents including tamoxifen (36), paclitaxel, and estramustine (37), we chose to investigate the effects of V. amygdalina on ERK activities.

V. amygdalina Vitiates ERK Activities.
As shown in Figure 3Go, exposure of cells to different concentrations (12.5–100 µg/ml) of V. amygdalina vitiated ERK activities in a concentration-dependent fashion. The concentration of 12.5 µg/ml elicited about 40% inhibition of ERK activities. The inhibition remained steady at 25 µg/ml but became stronger at 50 µg/ml (6-fold inhibition) and again remained steady at 100 µg/ml.



View larger version (95K):
[in this window]
[in a new window]
  		Figure 3. V. amygdalina vitiates ERK activities. Cells propagated to subconfluence were serum-starved overnight and treated with various concentrations of V. amygdalina (12.5–100 µg/ml) and incubated at 37°C for 10 mins. After incubation, MAPK assays were performed as described in ‘‘Materials and Methods.’’ The result shown here is representative of at least three independent experiments.

 
Comparison of V. amygdalina to Some Previously Reported Anticancer Plant Extracts Against MCF-7 Cells.
An exhaustive review of the literature revealed that about 14 crude plant extracts (excluding the purified fractions) have been studied in these cells (human breast cancerous cells or MCF-7). We thought that it would be of interest to present, in a tabular form, the efficacies (IC50) of the 14 anticancer plant extracts previously reported by others (40) in comparison to our extract (V. amygdalina; Ref. 35).

Table 2Go shows that the IC50 values for 14 previously reported plant crude extracts with human breast cancerous cell growth inhibitory properties ranged from the least potent (> 1.0 mg/ml; Ligustrum lucidum, Taraxacum mongolici, Sarcandra glabra) to the most potent of 0.1 mg/ml (Anemarrhena asphodeloides, Artemisia argyi, Rubia cordiforlia). In comparison, V. amygdalina is more than 17-fold more potent than the most potent and more than 170-fold more potent than the least potent of the plant extracts previously reported by others (40).


View this table:
[in this window]
[in a new window]
  		Table 2. Comparison of V. amygdalina Anticancer Activity (35) to Some Previously Reported Plant Extracts Against MCF-7 Cells (40)
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There are anecdotal reports of the biological activities of V. amygdalina against a wide range of human diseases. But evidence to support these health benefit claims is still scanty. The anticancer activity of organic extracts of V. amygdalina was first reported for human carcinoma cells of the nasopharynx 34 years ago by Kupchan and colleagues (32). Twenty-four years later, Jisaka and others (33) showed that components of organic extracts of V. amygdalina, vernodaline and vernolide, retarded the growth of P-388 and L1210 leukemia cells with IC50 values of 0.11 and 0.17 µg/ ml for vernodaline and 0.13 and 0.11 for vernolide, respectively. Obaseiki-Ebor et al. (34) reported an anti-mutagenic activity of organic solvent extracts of V. amygdalina. These investigators observed that V. amygdalina prevented the development of ethyl methansulfonate–induced mutations in Salmonella typhimurium. Most recently, Izevbigie (35) reported anticancer activities of V. amygdalina with an IC50 value of 0.0056 ± 0.0002 mg/ml. These extracts represent the first crude extracts, with this magnitude of potency against human breast cancerous cells, reported to date.

We have now advanced our understanding of how V. amygdalina extracts inhibit cell growth: by actions that were reversed by the replacement of V. amygdalina-containing medium with V. amygdalina-free medium. Hence, cells exposed to 10 µg/ml of this extract for up to 48 hrs grew at the same rate after withdrawal of extracts compared to the rate of unexposed cells. This suggests that V. amygdalina, at least up to a concentration of 10 µg/ml, uses cytostatic actions to inhibit cell growth.

To gain insights on the intracellular targets of V. amygdalina extracts, we chose to study their effects on ERK activities because many breast cancer chemotherapeutic drugs modulate their activities to elicit their antineoplastic actions (36–39). ERKs are serine/threonine kinases that are rapidly activated upon stimulation of a variety of cell surface receptors (41, 42). They function to convert extracellular stimuli to intracellular signals regulating the expression of genes that are important for many cellular processes, including cell growth and differentiation (43). These kinases play a central role in mitogenic signaling, as impediment of their functions may prevent agonist-induced cell proliferation. Evidence shows that elevated ERK expression or activities are associated with breast tumori-genesis (39). In tissue samples from three groups of patients: (i) human breast tumors and their matched adjacent normal breast tissue, (ii) breast tumors and their matched lymph node metastases, and (iii) breast tumors from patients who later proved to be tamoxifen-sensitive or resistant, Adeyinka et al. (39) reported positive associations between ERK expression, breast tumorigenicity, and metastases compared to those of control normal cells. These finding were corroborated by reports by Wang et al. (44) based on studies using tumor specimens from 48 patients. Taken together, these findings suggest that cancer chemotherapeutic drugs may elicit their antineoplastic actions by attenuating ERK expression or activities, and this characteristic is shared by the V. amygdalina extracts.

Other investigators have also reported MAPK inhibitory activities of other natural products such as catechins, one of the active ingredients in green tea (45) and resveratrol (18). Thus, MAPKs appear to be a critical component of growth-promoting pathways.

Hitherto, a commercially available drug (PD098059), to the best of our knowledge, was the only MEK1-specific inhibitor used to block the ERK pathway for research purposes only. We now have preliminary data (not shown) to show that on an equal concentration basis, V. amygdalina extracts are more potent inhibitors of ERK activities than PD098059. Even more interesting, V. amygdalina is edible (34, 46), which makes the use of this extract as a cancer chemopreventive or a chemotherapeutic agent (or both) very likely in the very near future. Work is currently ongoing in our laboratory to examine the effects of V. amygdalina on other crucial cell cycle regulators such as the cyclin-dependent kinases and their inhibitors.


   Acknowledgments
 
We thank Drs. Abdul K. Mohamed and Felix A. Okojie for their assistance on this project.


   Footnotes
 
This work was supported by Research Centers in Minority Institutions/the National Institutes of Health Grant G122RR13459.

Received for publication June 10, 2003. Accepted for publication October 1, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Jemal A, Thomas A, Murray T, Thun M. Cancer statistics. CA Cancer J Clin 52:23–47, 2002.[Abstract/Free Full Text]
  2. Abdulaev FI. Plant-derived agents against cancer. In: Gupta SK, Ed. Pharmacology and Therapeutics in the New Millennium. New Delhi: Narosa Publishing House, pp345–354, 1993.
  3. Richardson MA. Biopharmacologic and herbal therapies for cancer: research update from NCCAM. J Nutr 131(11S):3037S–3040S, 2001.[Abstract/Free Full Text]
  4. Pezzuto JM. Plant-derived anticancer agents. Biochem Pharmacol 53:121–133, 1997.[Medline]
  5. Zeegers MP, Goldbohm RA, Van den Brandt PA. Consumption of vegetables and fruits and urothelial cancer incidence: a prospective study. Cancer Epidemiol Biomarkers Prev 10:1121–1128, 2001.[Abstract/Free Full Text]
  6. Kobayashi T, Nakata T, Kuzumaki T. Effect of flavonoids on cell cycle progression in prostate cancer cells. Cancer Lett 176:17–23, 2000.
  7. Block G, Patterson B, Subar A. Fruits, vegetables, and cancer prevention: a review of the epidemiological evidence. Nutr Cancer 18:1–29, 1992.[Medline]
  8. Fahey JW, Zhang Y, Talalay P. Broccoli sprouts: an exceptionally rich source of inducers of enzymes that protect against chemical carcinogens. Proc Natl Acad Sci. U S A 94:10367–10372, 1997.[Abstract/Free Full Text]
  9. Singletary K. Natural products and cancer chemoprevention. J Nutr 130:465–466, 2000.[Abstract/Free Full Text]
  10. Dragsted L, Srube M, Larsen J. Cancer-preventive factors in fruits and vegetables: biochemical and biological background. Pharmacol Toxicol 72(suppl.1):116–135, 1993.
  11. Maoka T, Mochida K, Kozuka M, Ito Y, Fujiwara Y, Hashimoto K, Enjo F, Ogata M, Nobukuni Y, Tokuda H, Nishino H. Cancer chemopreventive activity of carotenoids in the fruits of red paprika capsicum annuum L. Cancer Lett 172:103–109, 2001.[Medline]
  12. Littman AJ, Beresford SA, White E. The association of dietary fat and plant foods with endometrial cancer (United States). Cancer Causes Control 12:691–702, 2001.[Medline]
  13. Terry P, Lagergren J, Hansen H, Wolk A, Nyren O. Fruits and vegetables consumption in the prevention of esophageal and cardiac cancers. Eur J Cancer Prev 10:365–369, 2001.[Medline]
  14. Rock CL, Moskowitz A, Huizar B, Saenz CC, Clark JT, Daly TL, Chin H, Behling C, Ruffin MT 4th. High vegetable and fruit diet intervention in premenopausal women with cervical intraepithelial neoplasia. J Am Diet Assoc 101:1167–1174, 2001.[Medline]
  15. Simopoulos AP. The Mediterranean diets: what is so special about the diet of Greece? The scientific evidence. J Nutr 131(11 Suppl):3065S–3073S, 2001.[Abstract/Free Full Text]
  16. Heber D, Bowerman S. Applying science to changing dietary patterns. J Nutr 131(11 Suppl):3078S–3081S, 2001.[Abstract/Free Full Text]
  17. Tatman D, Mo H. Volatile isoprenoid constituents of fruits, vegetables and herbs cumulatively suppress the proliferation of murine B16 melanoma and human HL-60 leukemia cells. Cancer Lett 175:129–139, 2002.[Medline]
  18. Yu R, Hebbar V, Kim DW, Mandlekar S, Pezzuto JM, Kong AN. Resveratrol inhibits phorbol ester and UV-induced activator protein 1 activation by interfering with mitogen-activated protein kinase pathways. Mol Pharmacol 60:217–224, 2001.[Abstract/Free Full Text]
  19. Meta-Greenwood E, Ito A, Westenburg H, Cui B, Mehta RG, Kinghorn AD, Pezzuto JM. Discovery of novel inducers of cellular differentiation using HL-60 promyelocyte cells. Anticancer Res 21:1763–1770, 2001.[Medline]
  20. Bhat KP, Lantvit D, Christov K, Mehta RG, Moon RC, Pezzuto JM. Estrogenic and antiestrogenic properties of resveratrol in mammary tumor models. Cancer Res 61:7456–7463, 2001.[Abstract/Free Full Text]
  21. Jang M, Pezzuto JM. Effects of resveratrol on 12-o-tetradecanolyl-phorbol-13- acetate-induced oxidative events and gene expression in mouse skin. Cancer Lett 134:81–89, 1998.[Medline]
  22. Jang M, Pezzuto JM. Cancer chemopreventive activity of resveratrol. Drugs Exp Clin Res 25:65–77, 1999.[Medline]
  23. Song LL, Kosmeder JW, Lee SK, Gerhauser C, Lantvit D, Moon RC, Moriarty RM, Pezzuto JM. Cancer chemopreventive activity mediated by 4'-bromoflavone, a potent inducer of phase II detoxification enzyme. Cancer Res 59:578–585, 1999.[Abstract/Free Full Text]
  24. Tan GT, Lee S, Lee IS, Chen J, Leitner P, Besterman JM, Kingkong AD, Pezzuto JM. Natural-product inhibitors of human DNA ligase 1. Biochem J 313(pt 3):993–1000, 1996.
  25. Udeani GO, Gerhauser C, Thomas CF, Moon RC, Kosmeder JW, Kingkong AD, Moriarty RM, Pezzuto JM. Cancer chemopreventive activity mediated by deguelin, a naturally occurring rotenoid. Cancer Res 57:3424–3428, 1997.[Abstract/Free Full Text]
  26. Talalay P, Fahey JW, Holtzclaw WD, Prestera T, Zhang Y. Chemo-protection against cancer by phase 2 enzyme induction. Toxicol Lett 82–83:173–179, 1995.
  27. Talalay P. Chemoprotection against cancer by induction of phase 2 enzymes. Biofactors 12:5–11, 2000.[Medline]
  28. Talalay P, Talalay P. The importance of using scientific principles in the development of medicinal agents from plants. Acad Med 76:238–247, 2001.[Medline]
  29. Talalay P, Fahey JW. Phytochemicals from cruciferous plants protect against cancer by modulating carcinogen metabolism. J Nutr 131(11 Suppl):3027S–3033S, 2001.[Abstract/Free Full Text]
  30. Wargovich MJ, Wood C, Hollis DM, Zander ME. Herbals, cancer prevention and health. J Nutr 131:3034S–3036S, 2001.[Abstract/Free Full Text]
  31. Olayiwole A. WHO guidelines for the assessment of herbal medicines. ESCOP Symposium: the Scientific Assessment of Phytomedicines, March 13, 1992. Milan, Italy: Fitoterapia 1:XIII(2), 1992.
  32. Kupchan SM, Hemingway RJ, Karim A, Werner D. Tumor inhibitors. XLVII. Vernodalin and vernomygdin. Two new cytotoxic sesquiterpene lactones from Vernonia amygdalina Del. J Org Chem 34:3908–3911, 1969.[Medline]
  33. Jisaka M, Ohigashi H, Takegawa K, Huffman MA, Koshimizu K. Antitumoral and antimicrobial activities of bitter sesquiterpene lactones of Vernonia amygdalina, a possible medical used by wild chimpanzees. Biosci Biochem 57:833–834, 1993.
  34. Obaseiki-Ebor EE, Odukoya K, Telikepalli H, Mitscher LA, Shankel D. Antimutagenic activity of extracts of leaves of four common edible vegetable plants in Nigeria (West Africa). Mutat Res 302:109–117, 1993.[Medline]
  35. Izevbigie EB. Discovery of water-soluble anticancer agents (edotides) from a vegetable found in Benin City, Nigeria. Exp Biol Med 228:293–298, 2003.[Abstract/Free Full Text]
  36. Atanaskova N, Keshamouni VG, Krueger JS, Schwartz JA, Miller F, Reddy KB. MAP Kinase/estrogen receptor cross-talk enhances estrogen-mediated signaling and tumor growth but does not confer tamoxifen resistance. Oncogene 21:4000–4008, 2002.[Medline]
  37. Liu XM, Wang LG, Kreis W, Budman DR, Adams LM. Differential effect of vinorelbine versus paclitaxel on ERK2 kinase activity during apoptosis in MCF-7 cells. Br J Cancer 85:1403–1411, 2001.[Medline]
  38. Mandlekar S, Kong AN. Mechanisms of tamoxifen apoptosis. Apoptosis 6:469–477, 2001.[Medline]
  39. Adeyinka A, Nui Y, Cherlet T, Snell L, Watson PH, Murphy LC. Activated mitogen-activated protein kinase expression during human breast tumorigenesis and progression. Clin Cancer Res 8:1747–1753, 2002.[Abstract/Free Full Text]
  40. Campbell MJ, Hamilton B, Shoemaker M, Tagliaferri M, Cohen I, Tripathy D. Antiproliferative activity of Chinese medicinal herbs on breast cancer cells in vitro. Anticancer Res 22:3843–3852, 2002.[Medline]
  41. Cobb MH, Boulton TG, Robbins DJ. Extracellular signal-regulated kinases: ERKs in progress. Cell Regul 2:965–978, 1991.[Medline]
  42. Ray LB, Sturgill TW. Rapid stimulation by insulin of a serine/threonine kinase in 3T3-L1 adipocytes that phosphorylate microtubule-associated protein 2 in vitro. Proc Natl Acad Sci U S A 84:1502–1506, 1987.[Abstract/Free Full Text]
  43. Davis RJ. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem 268:14553–14556, 1993.[Free Full Text]
  44. Wang Z, Wang S, Zhu F, Ye Y, Yu Y, Qiao X. Expression of extracellular signal-regulated kinase and its relationship with clinico-pathological characteristics of breast cancer. Zhonghua Zhong Liu Za Zhi 24:360–363, 2002.[Medline]
  45. Deguchi H, Fuji T, Nakagawa S, Koga T, Shirouzu K. Analysis of cell growth inhibitory effects of catechin through MAPK in human breast cancer cell line T47D. Int J Oncol 21:1301–1305, 2002.[Medline]
  46. Uhegbu FO. Dietary secondary amines and liver hepatoma in Port Harcourt, Nigeria. Plant Foods Hum Nutr 51:257–263, 1997.[Medline]




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 Google Scholar
Google Scholar
Right arrow Articles by Izevbigie, E. B.
Right arrow Articles by Walker, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Izevbigie, E. B.
Right arrow Articles by Walker, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS