Ernest B. Izevbigie

Discovery of Water-Soluble Anticancer Agents (Edotides) from a Vegetable Found in Benin City, Nigeria

Cancer claims the lives of more than six million people each year in the world. About 1,268,000 new cancer cases, and 553,400 deaths were reported in the United States in 2001. Current treatment approaches have yielded significant progress in the fight against cancer, but the incidence of developing certain types of cancer continues to rise. This is especially true in the African-American communities. African Americans are about 33% more likely to die of cancer than are whites and more than twice likely to die of cancer as are Asian-Islander, American-Indians, and Hispanics. This increase coupled with the harsh side effects of some of the cancer chemotherapies have led to the search for more natural biological products, especially those derived from plant products, currently known as herbal medicine. There is a need for a continued search for novel natural products that may be used as cancer chemopreventive and/or chemotherapeutic agents. The objective of this study was to evaluate the effect(s) of a novel water-soluble leaf extract of Vernonia amygdalina (VA) on human breast cancer cell DNA synthesis. MCF-7 cell line, considered a suitable model, was used in this study. Treatment of cells with physiologically relevant concentrations of water-soluble VA extract potently inhibited DNA synthesis in a concentration-dependent fashion both in the absence and presence of serum. Fractions of VA extract separated using preparative reverse-phase chromatography also inhibited DNA synthesis (P < 0.005). These results suggest that VA vegetable, if incorporated in the diet, may prevent or delay the on-set of breast cancer.

A novel natural inhibitor of extracellular signal-regulated kinases and human breast cancer cell growth

Water-soluble extracts of edible Vernonia amygdalitis 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 μg/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 μg/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 μg/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.

Ethanol modulates the growth of human breast cancer cells in vitro

The role of ethanol or its metabolites on breast neoplasm has not been characterized. We hypothesized that ethanol may alter the growth rate of human breast tumor epithelial cells by modulating putative growth-promoting signaling pathways such as p44/42 mitogen-activated protein kinases (MAPKs). The MCF-7 cell line, considered a suitable model, was used in these studies to investigate the effects of ethanol on [3H]thymidine incorporation, cell number, and p44/42 MAPK activities in the presence or absence of a MAPK or extracellular signal-regulated kinase ERK-1, and (MEK1) inhibitor (PD098059). Treatment of MCF-7 cells with a physiologically relevant concentration of ethanol (0.3% or 65 mM) increased p44/42 activities by an average of 400% (P < 0.02), and subsequent cell growth by 200% (P < 0.05) in a MEK1 inhibitor (PD098059)-sensitive fashion, thus suggesting that the Ras/MEK/MAPK signaling pathways are crucial for ethanol-induced MCF-7 cell growth.

Isolation and structure determination of a sesquiterpene lactone (vernodalinol) from Vernonia amygdalina extracts

Context: Vernonia amygdalina Del. (VA; Asteraceae or Compositae) is a small tree growing throughout tropical Africa. It is widely used for food and medicinal purposes by local people. It was reported that it had several qualities, including anticancer activity. Objective: A sesquiterpene lactone, vernodalinol, was isolated from VA leaves. The first reported source of vernodalinol was in 2009 from a different plant, only 1H NMR spectrum and no detailed structural analysis were carried out. No whole spectroscopic data were provided. Materials and methods: VA dried leaves were extracted with 85% ethanol followed by further separation into four fractions by liquid–liquid extraction technique using various solvents: hexane, chloroform, and n-butanol. Vernodalinol was separated from the n-butanol fraction by column chromatography. The biological activity of vernodalinol was evaluated in estrogen receptor-positive (ER+) human breast carcinoma cells (MCF-7) in vitro. Results: Results indicated that vernodalinol (25 and 50 μg/mL) inhibited breast cancerous cell growth (DNA synthesis) by 34% (Pu2009<u20090.025) and 40% (Pu2009<u20090.025), respectively. It is reasonable to expect an LC50 of 70–75 μg/mL for vernodalinol in MCF-7 cells. Discussion and conclusion: Vernodalinol structure was confirmed using a battery of spectroscopic methods, 1D and 2D NMR, high-resolution mass spectrometry (HR-MS), UV, IR, and X-ray. These results suggest that vernodalinol, although it has some biological activity, is likely to work in concert with other ingredients responsible for the anticancer activity exhibited of VA.

Activity Markers of the Anti-Breast Carcinoma Cell Growth Fractions of Vernonia amygdalina Extracts

Vernonia amygdalina (VA) is an edible plant of the Asteraceae family used in many herbal formulations prescribed by herbalists for many diseases. We have previously reported that aqueous VA extracts inhibit the growth of estrogen receptor-positive human breast cancerous cells in vitro. Activity markers of the VA extracts have not been previously identified or characterized. Hence, the objective of this study was to identify activity markers of the VA extracts associated with cell growth inhibition. Extraction of VA with multiple solvents of various polarity indexes yielded three fractions (A-1–2, B-1–3) that significantly inhibited cell growth (P < 0.05) at 0.1 mg/ml concentration. At a higher concentration of 1 mg/ml, six fractions of hexane, chloroform, butanol, and ethyl acetate (A-1–3, B-1–4) inhibited DNA synthesis by 76%, 98%, 94%, 98%, 98%, and 96%, respectively. These fractions were UV-detected from 250–730 nm; and all showed three distinct peaks around 410, 431, and 664 nm. Furthermore, HPLC analysis of the fractions revealed similar retention times of 2.213, 2.167, and 2.151 min, respectively. Bioactivity assays showed that HPLC retention of approximately 2 min is required for cell growth-inhibitory activity of VA fractions. Interestingly, all active fractions exhibited HPLC peaks at approximately 2 min. Therefore, the UV and HPLC peaks may be used as predictive tools to determine VA extracts activities.

The Fem1c genes: conserved members of the Fem1 gene family in vertebrates.

The fem-1 gene of Caenorhabditis elegans functions in a signaling pathway that controls sex determination. Homologs of fem-1 in mammals have been characterized, consisting of two family members, Fem1a and Fem1b. We report here on Fem1c, a third member of the Fem1 gene family, in three vertebrate species: human, mouse, and zebrafish. The proteins encoded by these Fem1c genes share >99% amino acid identity between human and mouse, 79% amino acid identity between mouse and zebrafish, and end with a C-terminal Arginine residue, which distinguishes them from other FEM-1 proteins reported thus far. The human and mouse Fem1c coding regions show conservation of intron-exon structure and expression pattern in adult tissues. Human FEM1C maps to 5q22, mouse Fem1c maps to chromosome 18, and zebrafish fem1c maps to Linkage Group 8. The Fem1c genes in vertebrates may play a conserved role in the development and/or physiologic function of these organisms.

Vernonia amygdalina—Induced Growth Arrest and Apoptosis of Breast Cancer (MCF-7) Cells

Breast cancer is the second leading cause of cancer-related deaths of women in the United States. Fortunately, the mortality rate from breast cancer has decreased in recent years due to an increased emphasis on early detection and more effective treatments. Although great advancements have been made in the treatment and control of cancer progression, significant deficiencies and room for improvement remain. The central objective of this research was to further determine the in vitro mechanisms of Vernonia amygdalina (VA) leaf extracts as an anticancer candidate for the treatment of breast cancer. To achieve our objective, MCF-7 cells were treated with different concentrations of VA for 24 hand 48 h. Cell viability, live and dead cells were determined by the means of trypan blue exclusion test. Live and dead cells were further evaluated by propidium iodine (PI) assay using the Cellometer Vision. Cell apoptosis was measured by flow cytometry assessment using annexin V/PI kit. Data obtained from the trypan blue test demonstrated that VA treatment reduces cell viability in a concentration- and time-dependent manner. Result of the PI assay showed a gradual increase in the population of necrotic cells (fluorescence positive cells) in VA-treated cells compared to the control cells (fluorescence negative cells). Treatment of these cancer cells (MCF-7) for 48 h at concentrations ranging from 250 μg/mL to 1000 μg/mL caused early signs of apoptosis resulting from phosphatidylserine externalization as judged by annexin V assay. We observed a strong concentration-response relationship with regard to VA exposure and annexin V/PI positive cells. In summary, our finding demonstrates that VA-induced cytotoxicity and apoptosis in MCF-7 cells involve phosphatidylserine externalization accompanied by secondary necrotic cell death. With previous findings in our laboratory, the data generated in the present study confirms that VA is a valuable botanical therapeutic agent for the treatment of breast cancer.

Sensitivity and mechanisms of taxol-resistant prostate adenocarcinoma cells to Vernonia amygdalina extract.

Prostate cancer (PC) patients once Paclitaxel (TAX) treatment responsive later develop hormone refractory PC, thus becoming TAX-insensitive. This underscores the urgent need to develop novel anti-PC therapies. Vernonia amygdalina (VA) could be one such candidate agent. We have shown that androgen-independent PC-3 cells are sensitive to VA treatment in vitro. VA extract (0.01, 0.1 and 1 mg/ml) inhibited DNA synthesis by 12%, 45% (p<0.05), and 73% (p<0.01) respectively. In contrast, TAX (0.01, 0.1, and 1 μM) failed to significantly affect cell growth, suggesting TAX resistance. We tested molecular mechanisms which may lend to the observed PC-3 cell VA sensitivity/TAX resistance. Though both VA and TAX stimulated MAPK activity, VAs induction was more intense, but transient, compared to TAXs sustained action. NF-κB activation was inhibited on average by 50% by either 1 mg/ml VA or 1 μM TAX. VA extract caused 35% and 45% increases in c-Myc activity at 10 and 60 min intervals respectively, with the highest stimulation attained 1h after treatment. In contrast, similar levels were attained by TAX rapidly (within 5 min) and were sustained compared to the slow/multi-phasic action of VA. VA extract treatments had no effect on AKT gene expression, while TAX treatments yielded a four-fold (P<0.01) increase; and P-glycoprotein (P-gp) activity was inhibited by VA and stimulated by TAX, compared to control (basal ATPase activity). This study shows that TAX-resistant PC-3 cells are sensitive to VA, perhaps explained by differential regulatory patterns of MAPK, c-Myc, AKT, and Pgp activities/expressions.

Isolation and characterization of the antibreast carcinoma cell growth components of Vernonia amygdalina extracts

Vernonia amygdalina (VA) is widely used for medicinal and food purposes in tropical Africa. Many health benefits (antioxidant, antimicrobial, anticancer activities and more) of VA extracts have been reported. The mechanisms of actions have also been described. We have previously reported that VA extracts elicited growth inhibitory activities in human estrogen receptor-positive (ER+) cells (MCF-7 cells) and ductal carcinoma cells (BT-549) in vitro. The active components in the organic solvent (chloroform)-extracted VA have been previously determined. However, the active components in the ethanolic extracts of VA have not been previously studied. Hence, the objectives of this study are to isolate and characterize the active components of the ethanolic extracts of VA using liquid–liquid extraction, thin layer chromatography and column techniques. Fractionation of the ethanolic extracts of VA yielded three fractions named A1, A2 and A3, and A2 retained the DNA synthesis-inhibitory activity of the extracts. Subsequent fractionation of A2 yielded fraction A2B whose activity was 16 and three times more potent than the ethanolic fraction and fraction A2, respectively. The treatment of cells with 100 μg/mL of either the ethanolic VA extracts, fraction A2 or fraction A2B resulted in a 23% (P < 0.01), 86% (P < 0.0001) and 97% (P < 0.0001) inhibition of DNA synthesis compared with vehicle-treated controls, respectively. Further purification of A2B by high-speed countercurrent chromatography and confirmed by spectroscopic analysis revealed that the major active components of A2B (65% by weight) were steroid glucosides.

V. Amygdalina: Folk Medicine, Analysis, and Potential Application for Cancer Treatment

Folk medicine (FM) is practiced by people without access to conventional medical services; it usually involves the use of natural remedies such as herbs or vegetable substances. Before the use of pharmaceutical drugs, and surgical procedures, these healing methods were used, and are still in use today. It is estimated that twenty five percent of all therapeutic drugs trace their origins to plants, and almost two-thirds of the people of the world rely on their healing powers. One hundred years ago, health care in the U.S. was provided by a highly competitive medical sect, and quite infrequently, folk medicine practitioners were patronized. However, FM usage in the U.S. has increased drastically during the past decade. National surveys of adults (18 years of age or older) show that one in three adults use unconventional therapies or Complementary and Alternative Medicine (CAM) in the U.S. The rate of CAM usage is more than eighty percent among cancer patients. Vernonia amygdalina (VA) is well known for its medicinal importance. Fractionation of the VA extracts with solvents of varying polarities, by silica gels analyses, UV Spectrophotometer, HPLC, TLC and NMR techniques have yielded some biologically-active fractions.