Carolyn B. Howard

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.

Basic Apoptotic Mechanisms of Lead Toxicity in Human Leukemia (Hl-60) Cells

Lead exposure represents a medical and public health emergency, especially in children consuming high amounts of lead-contaminated flake paints. It may also cause hematological effects to people of all ages. Recent studies in our laboratory have indicated that apoptosis may be associated with the lead-induced oxidative stress and DNA damage. However, the mechanisms underlying its effect on lymphocytes are still largely unknown. Therefore, the aim of the present study was to investigate the apoptotic mechanisms of lead nitrate [Pb(NO3)2] using HL-60 cells as a test model. HL-60 cells were treated with different concentrations of Pb(NO3)2 for 24 h prior to cell viability assay and flow cytometry assessment. The results obtained from the trypan blue exclusion test indicated that at very low concentration, Pb(NO3)2 has no effect on the viability of HL-60 cells. A significant (p < 0.05) decrease in cell viability was observed when exposed to high level of Pb(NO3)2. Data generated from the flow cytometric assessment indicated that Pb(NO3)2 exposure significantly (p < 0.05) increased the proportion of annexin V positive cells (apoptotic cells) compared to the control. Pb(NO3)2 induced apoptosis of HL-60 cells was associated with the activation of caspase-3. In summary, these studies demonstrated that Pb(NO3)2 represents an apoptosis-inducing agent in HL-60 promyelocytic leukemia cells and its apoptotic mechanism functions, at least in part via, induction of phosphatidylserine externalization and caspase-3 activation.

Ascorbic Acid Potentiation of Arsenic Trioxide Anticancer Activity Against Acute Promyelocytic Leukemia

Introduction Acute promyelocytic leukemia (APL) is a malignant disorder of the white blood cells. Arsenic trioxide (As2O3) has been used as a therapeutic agent to treat APL and other tumors. Studies suggest that ascorbic acid (AA) supplementation may improve the clinical outcome of As2O3 for APL patients. Our aim was to use human leukemia (HL-60) APL-cells as an in vitro test model to evaluate the effect of physiologic doses of AA on As2O3-induced toxicity and apoptosis of HL-60 cells. Methods HL-60 cells were treated either with a pharmacologic dose of As2O3 alone and with several physiologic doses of AA. Cell survival was determined by trypan blue exclusion test. The extent of oxidative cell/tissue damage was determined by measuring lipid hydroperoxide concentration by spectrophotometry. Cell apoptosis was measured by flow cytometry using Annexin-V and propidium iodide (PI) staining. Results AA treatment potentiates the cytotoxicity of As2O3 in HL-60 cells. Viability decreased from (58 ± 3)% in cells with As2O3 alone to (47 ± 2)% in cells treated with 100 µM AA and 6 µg/mL As2O3 with P < 0.05. There was a significant (P < 0.05) increase in lipid hydroperoxide concentrations in HL-60 cells co-treated with AA compared to As2O3 alone. Flow cytometry assessment (Annexin V FITC/PI) suggested that AA co-treatment induces more apoptosis of HL-60 cells than did As2O3 alone, but this was not statistically significant. Taken together, our experiment indicates that As2O3 induced in vitro cell death and apoptosis of HL-60 cells. Administration of physiologic doses of AA enhanced As2O3-induced cytotoxicity, oxidative cell/tissue damage, and apoptosis of HL-60 cells through externalization of phosphatidylserine. Conclusions These suggest that AA may enhance the cytotoxicity of As2O3, suggesting a possible future role of AA/As2O3 combination therapy in patients with APL.

Increased expression of beige/brown adipose markers from host and breast cancer cells influence xenograft formation in mice.

The initiation and progression of breast cancer is a complex process that is influenced by heterogeneous cell populations within the tumor microenvironment. Although adipocytes have been shown to promote breast cancer development, adipocyte characteristics involved in this process remain poorly understood. In this study, we demonstrate enrichment of beige/brown adipose markers, contributed from the host as well as tumor cells, in the xenografts from breast cancer cell lines. In addition to uncoupling protein-1 (UCP1) that is exclusively expressed in beige/brown adipocytes, gene expression for classical brown (MYF5, EVA1, and OPLAH) as well as beige (CD137/TNFRSF9 and TBX1) adipocyte markers was also elevated in the xenografts. Enrichment of beige/brown characteristics in the xenografts was independent of the site of implantation of the breast tumor cells. Early stages of xenografts showed an expansion of a subset of mammary cancer stem cells that expressed PRDM16, a master regulator of brown adipocyte differentiation. Depletion of UCP1+ or Myf5+ cells significantly reduced tumor development. There was increased COX2 (MT-CO2) expression, which is known to stimulate formation of beige adipocytes in early xenografts and treatment with a COX2 inhibitor (SC236) reduced tumor growth. In contrast, treatment with factors that induce brown adipocyte differentiation in vitro led to larger tumors in vivo. A panel of xenografts derived from established breast tumor cells as well as patient tumor tissues were generated that expressed key brown adipose tissue–related markers and contained cells that morphologically resembled brown adipocytes. Implications: This is the first report demonstrating that beige/brown adipocyte characteristics could play an important role in breast tumor development and suggest a potential target for therapeutic drug design. Mol Cancer Res; 14(1); 78–92. ©2015 AACR.

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.

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.

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.

Abstract 2606: Chemo-preventive efficacy of a natural product anti-cancer agent which surpasses Taxol

Breast cancer (BC), a major public health problem, is one of the leading causes of cancer-related deaths among women. In particular, triple-negative breast cancer (TNBC), the most aggressive form of BC poses significant therapeutic challenges. TNBCs are most common in women with BRCA1 gene mutations, younger women, and in pre-menopausal African American (AA) women compared to White women, significantly contributing to health disparities. Consisting of many heterogeneous, undifferentiated cell types including very high mammary cancer stem cell (MCSCs) content, TNBCs are diseases with high recurrence rates. Combining therapeutics to target a variety of growth pathways has inhibited growth and shrunk aggressive TN tumors. Yet, a major drawback of such therapy is the severe side effects commonly used chemotherapeutic agents have on normal cells. Therefore there is a critical need to develop new combinations of therapeutics that target MCSCs and improve the anticancer effects, while reducing harmful side effects. We present compelling preliminary findings comparing the anticancer effects of aqueous extracts of a plant, Vernonia amygdalina (VA), alone or in combination with Paclitaxel (TAX) in vitro and against TNBC cells injected into athymic mice intra-peritoneally. The effects of TAX alone or VA extracts alone or with TAX, were evaluated in Hsd:Athymic Nude-Foxn1nu, age 5-6 weeks, female mice inoculated subcutaneously with Hras MCSCs. Animals were grouped as follows: 1-Control, inoculated with Hras cells only; 2-VA treatment (10 mg/kg) simultaneously with injection of Hras cells, followed by 16-day VA treatment; 3-replicate of 2; 4-pretreatment with VA 10 days before injection of Hras cells, followed by 16-day VA treatment; 5-IP inoculation of TAX (10 mg/kg) simultaneously with injection of Hras cells; and 6-subcutaneous inoculation of VA (5 mg/kg) plus IP injection of TAX (5 mg/kg) simultaneously with injection of Hras cells. Studies revealed that though there was a lag in tumor growth for all treatment groups, the most significant reduction in tumor size was observed in group 4 animals, those which had been pretreated with VA each day for 10 days prior to inoculation with Hras cells. In the second round of experiments, we evaluated the efficacy of VA extracts (10-day pretreatment, high dose, 20 mg/kg) administered simultaneously with injections of both Hras cells and AA woman-derived MDA-MB-468 cells, at opposing sites, versus combined treatment with very low doses of VA and TAX (5 mg/kg of each agent, subcutaneously) toward inhibition of growth of tumors in our nude mice model. Based upon our findings, VA extract exhibits chemopreventative efficacy, addressing a major health problem which is the dominant biological cause of population-based disparities in BC mortality in the U.S. Our expectation is that this work could direct further in vivo studies and eventually translate to human clinical trials. Citation Format: Carolyn B. Howard. Chemo-preventive efficacy of a natural product anti-cancer agent which surpasses Taxol. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2606.