Recent perspectives on the anticancer properties of aqueous extracts of Nigerian Vernonia amygdalina.

Innovative developments are necessary for treating and defeating cancer, an oftentimes deadly group of diseases characterized by the uncontrolled growth and spread of abnormal cells. Breast cancer (BC) is the second leading cause of cancer-related deaths of women in the USA, and prostate cancer (PC) is the second leading cause of cancer-related deaths of American men. Although some efficacious BC drugs are pharmaceutically marketed, they affect the quality of life for some patients because they are toxic in that their usages have been accompanied by side effects such as stroke, thrombosis, slow heart rate, seizure, increased blood pressure, nausea, emesis, and more. Therefore, there is an urgent need for the discovery of molecular markers for early detection of this disease and discovery of targets for the development of novel, less toxic therapeutics. A botanical plant Vernonia amygdalina has been widely used in Nigerian and other Central and West African cultures for centuries as an herbal medicine. Mounting evidence suggests that treatment with low concentrations of aqueous leaf extracts of the edible Nigerian V. amygdalina plant (Niger-VA) arrests the proliferative activities and induces apoptosis in estrogen receptor-positive, estrogen receptor-negative, and triple-negative human breast cancerous cells and in androgen-independent human PC-3. Also, in athymic mice, Niger-VA potentiates increased efficacies and optimizes treatment outcomes when given as a cotreatment with conventional chemotherapy drugs. Evidence of its noticeable cytostatic activities ranging from changes in DNA synthesis to growth inhibition, mechanisms of inducing apoptosis in different cancer cell lines, and in vivo antitumorigenic activities and chemopreventive efficacy reinforce the idea that Niger-VA deserves increased attention for further development as a phytoceutical, anticancer drug entity. Hence, the present review article highlights impactful published literature on the anticancer effects of Niger-VA in multiple cancerous cell lines and in a nude mouse model, supporting its potential usefulness as a natural product, chemotherapeutic medicine for treatment of both BC and PC.

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, VA's induction was more intense, but transient, compared to TAX's 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 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 (1)H 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% (P < 0.025) and 40% (P < 0.025), respectively. It is reasonable to expect an LC(50) 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.

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.

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.

Vernonia amygdalina: anticancer activity, authentication, and adulteration detection.

Evidence suggests that most chemotherapeutic agents are less effective as treatment in patients with estrogen receptor-negative (ER-) breast carcinomas compared to those with estrogen receptor-positive (ER+) breast carcinomas. Moreover, African American Women (AAW) is disproportionately diagnosed with ER- breast cancer compared to their white counterparts. Novel therapies effective against ER- breast carcinomas are urgently needed to ameliorate the health disparity. Previous reports show that low concentrations (microgram/ml) of water-soluble leaf extracts of a Nigerian edible plant, V. amygdalina (VA), potently retards the proliferative activities of ER+ human breast cancerous cells (MCF-7) in vitro in a concentration-dependent fashion. However, the anti-proliferative activities of VA in either ductal or ER- carcinoma cells have not been characterized. The exposure of BT-549 to increasing concentrations of VA (10, 100, and 1000 microg/mL) inhibited cell growth by approximately 14 % (P<0.05), 22 % (p<0.05), and 50 % (p<0.005) respectively. The cell count studies were corroborated by DNA synthesis studies. Treatments of BT-549 with 10, 100, and 1000 microg/mL VA inhibited DNA synthesis in a concentration dependent fashion by 22 %, 76 % (P<0.05), and 86 % (p<0.01) respectively. BT-549 cells were insensitive to 10 and 100 nM paclitaxel (TAX) treatments. Isolation of DNA from dried VA leaves yielded approximately 12.2 and 1 kbp genomic DNA that were Eco RI-insensitive but Hind III and Bam HI-sensitive. These pieces of information may be used to enhance the safety of medicinal botanical VA through authentication, and adulteration detection.

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.

Aqueous Vernomia amygdalina extracts alter MCF-7 cell membrane permeability and efflux.

Breast cancer is the second leading cause of cancer related deaths of women in the United States. Several treatment strategies have been developed over the past decade to reduce cancer morbidity and mortality rates. While mortality rates have declined in some ethnic populations, the overall cancer incidence continues to grow. Hence, chemotherapeutic agents are needed to improve cancer treatment outcome. Previous studies show that low concentrations (microgram/ml) of water-soluble leaf extracts of a Nigerian edible plant, V. amygdalina (VA), potently retard the proliferative activities of estrogen receptor positive (ER+) human breast cancerous cells (MCF-7) cells in vitro in a concentration-dependent fashion. The anti-proliferative activities of VA are extracellular signal-regulated kinases (1/2) (ERKs (1/2))-dependent. Cell culture and animal model studies, conducted by other investigators using other plant extracts, have also revealed that plant extract components called thionins may be responsible for their anticancer activities. These thionins are believed to interact with the cells in ways that compromise membrane potential/permeability resulting in the alteration of efflux, cytosolic activities, and subsequent cell death. Therefore, we hypothesized that VA exposure may compromise cell membrane as another mode of action to elicit its anticancer activities in MCF-7 cells. The exposure of cells to VA decreased [3H]thymidine uptake in a concentration-dependent (0, 30, and 100 mug/ml VA) manner (p < 0.05) but increased [3H]thymidine release, expressed as percent of [3H]thymidine incorporated, into the medium (p < 0.05). The amount of [3H]thymidine released into the medium was 1.7, 7.4, and 11.0 % for 0, 30, and 100 mug/ml VA respectively. Thus suggesting the membranes in VA-treated cells were compromised in a concentration-dependent fashion.

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

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.

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.