Laura M. Bystrom

Distinct roles for hepcidin and interleukin-6 in the recovery from anemia in mice injected with heat-killed Brucella abortus

Anemia of inflammation (AI) is commonly observed in chronic inflammatory states and may hinder patient recovery and survival. Induction of hepcidin, mediated by interleukin 6, leads to iron-restricted erythropoiesis and anemia. Several translational studies have been directed at neutralizing hepcidin overexpression as a therapeutic strategy against AI. However, additional hepcidin-independent mechanisms contribute to AI, which are likely mediated by a direct effect of inflammatory cytokines on erythropoiesis. In this study, we used wild-type, hepcidin knockout (Hamp-KO) and interleukin 6 knockout (IL-6-KO) mice as models of AI. AI was induced with heat-killed Brucella abortus (BA). The distinct roles of iron metabolism and inflammation triggered by interleukin 6 and hepcidin were investigated. BA-treated wild-type mice showed increased expression of hepcidin and inflammatory cytokines, as well as transitory suppression of erythropoiesis and shortened red blood cell lifespan, all of which contributed to the severe anemia of these mice. In contrast, BA-treated Hamp-KO or IL-6-KO mice showed milder anemia and faster recovery compared with normal mice. Moreover, they exhibited different patterns in the development and resolution of anemia, supporting the notion that interleukin 6 and hepcidin play distinct roles in modulating erythropoiesis in AI.

Iron and Reactive Oxygen Species: Friends or Foes of Cancer Cells?

SIGNIFICANCE In this review, the dual nature of both iron and reactive oxygen species (ROS) will be explored in normal and cancer cell metabolism. Although iron and ROS play important roles in cellular homeostasis, they may also contribute to carcinogenesis. On the other hand, many studies have indicated that abrogation of iron metabolism, elevation of ROS, or modification of redox regulatory mechanisms in cancer cells, should be considered as therapeutic approaches for cancer. RECENT ADVANCES Drugs that target different aspects of iron metabolism may be promising therapeutics for cancer. The ability of iron chelators to cause iron depletion and/or elevate ROS levels indicates that these types of compounds have more potential as antitumor medicines than originally expected. Other natural and synthetic compounds that target pathways involved in ROS homeostasis also have potential value alone or in combination with current chemotherapeutics. CRITICAL ISSUES Although ROS induction and iron depletion may be targets for cancer therapies, the optimal therapeutic strategies have yet to be identified. This review highlights some of the research that strives to identify such therapeutics. FUTURE DIRECTIONS More studies are needed to better understand the role of iron and ROS in carcinogenesis not only as cancer promoters, but also as cytotoxic agents to cancer cells and cancer stem cells (CSCs). Moreover, the structure-activity effects of iron chelators and other compounds that increase ROS and/or disrupt iron metabolism need to be further evaluated to assess the effectiveness and selectivity of these compounds against both cancer and CSCs.

Cancer Cells with Irons in the Fire

Iron is essential for the growth and proliferation of cells, as well as for many biological processes that are important for the maintenance and survival of the human body. However, excess iron is associated with the development of cancer and other pathological conditions, due in part to the pro-oxidative nature of iron and its damaging effects on DNA. Current studies suggest that iron depletion may be beneficial for patients that have diseases associated with iron overload or other iron metabolism disorders that may increase the risk for cancer. On the other hand, studies suggest that cancer cells are more vulnerable to the effects of iron depletion and oxidative stress in comparison to normal cells. Therefore, cancer patients might benefit from treatments that alter both iron metabolism and oxidative stress. This review highlights the pro-oxidant effects of iron, the relationship between iron and cancer development, the vulnerabilities of the iron-dependent cancer phenotype, and how these characteristics may be exploited to prevent or treat cancer.

Phenolics, Sugars, Antimicrobial and Free-Radical-Scavenging Activities of Melicoccus bijugatus Jacq. Fruits from the Dominican Republic and Florida

Edible fruits of the native South American tree Melicoccus bijugatus Jacq. are consumed fresh or in traditional food, drink and medicinal preparations. Some therapeutic effects of these fruits may be due to phenolics and sugars. Aqueous acetone, methanol or ethanol tissue extracts of different cultivars or collections of M. bijugatus fruits from the Dominican Republic and Florida were analyzed for total phenolics and free radical scavenging activity by UV-vis spectroscopy, sugars by gas chromatography, and antimicrobial activity by the disc diffusion assay. Total phenolics and free radical scavenging activities ranked: seed coat > embryo > pulp extracts. Montgomery cultivar fruits had the highest total phenolics. For sugars: pulp > embryo and highest in Punta Cana fruit pulp. In all extracts: sucrose > glucose and fructose. Glucose:fructose ratios were 1:1 (pulp) and 0.2:1 (embryo). Pulp extracts had dose-response antibacterial activity and pulp and embryo extracts had antifungal activity against one yeast species. Phenolics and sugars were confirmed with thin-layer chromatography and nuclear magnetic resonance. Sugar-free pulp fractions containing phenolics had slightly more antimicrobial activity than H2O-soluble pulp fractions with sugars. Results indicate M. bijugatus fruits contain phenolics, sugars and other H2O-soluble compounds consistent with therapeutic uses.

Dehydroleucodine, a Sesquiterpene Lactone from Gynoxys verrucosa, Demonstrates Cytotoxic Activity against Human Leukemia Cells

The sesquiterpene lactones dehydroleucodine (1) and leucodine (2) were isolated from Gynoxys verrucosa, a species used in traditional medicine in southern Ecuador. The activity of these compounds was determined against eight acute myeloid leukemia (AML) cell lines and compared with their activity against normal peripheral blood mononuclear cells. Compound 1 showed cytotoxic activity against the tested cell lines, with LD50 values between 5.0 and 18.9 μM. Compound 2 was inactive against all of the tested cell lines, demonstrating that the exocyclic methylene in the lactone ring is required for cytotoxic activity. Importantly, compound 1 induced less toxicity to normal blood cells than to AML cell lines and was active against human AML cell samples from five patients, with an average LD50 of 9.4 μM. Mechanistic assays suggest that compound 1 has a similar mechanism of action to parthenolide (3). Although these compounds have significant structural differences, their lipophilic surface signatures show striking similarities.

The potential health effects of Melicoccus bijugatus Jacq. fruits: phytochemical, chemotaxonomic and ethnobotanical investigations.

Most natural product research is market-driven and thus many plant species are overlooked for their health value due to lack of financial incentives. This may explain the limited information available about the health effects of the edible fruit species Melicoccus bijugatus, a member of the Sapindaceae family that grows mostly in the Caribbean and in parts of South America. However, recent phytochemical studies of these fruits have shed some light on their biological effects. In this review the health effects of M. bijugatus fruit pulp and seeds are assessed in relation to phytochemical and ethnobotanical studies, as well as chemotaxonomic information and medicinal uses of other Sapindaceae species. The chemistry of M. bijugatus fruits was found to be different than the other Sapindaceae fruits, although some of the medicinal uses were similar. Specific phenolics or sugars in M. bijugatus fruits may contribute to their therapeutic uses, especially for gastrointestinal problems, and to some extent toxicological effects. This review focuses our understanding about the specific biological effects of M. bijugatus fruits, which may be useful for predicting other medicinal uses, potential drug or food interactions and may benefit people where the fruits are prevalent and healthcare resources are scarce.

Abstract 3992: Allogeneic TCRαβ-deficient CAR T-cells targeting CD123 effectively eliminate myeloid leukemia cells in vitro and in vivo PDX mice

Acute myeloid leukemia (AML) is incurable in the majority of patients. While allogeneic stem cell transplantation remains the most effective therapy for AML to date, other types of cellular therapy have not yet been successful in this disease. The success of autologous T-cells expressing chimeric antigen receptors (CARs) in patients with advanced B cell leukemia and lymphomas has encouraged the investigation of CAR technology for the treatment of AML by targeting distinct tumor-specific antigens. We have developed an allogeneic CAR-T cell platform using T-cells from third-party healthy donors to generate T-cells targeting CD123, the transmembrane alpha chain of the interleukin-3 receptor, which is expressed on blasts, leukemic progenitor and leukemic stem cells from the majority of patients with acute myeloid leukemia (AML). Transcription Activator-Like Effector Nuclease (TALEN) gene-editing technology was used to inactivate the TCRα constant (TRAC) gene, eliminating the potential for engineered T-cells to cause graft versus host disease (GvHD). Using leukemia cell lines in both in vitro and in vivo models, we confirmed that TCR-deficient T-cells expressing an anti-CD123 CAR display significant antitumor activity. We next evaluated the in vitro cytotoxic activity of TCR KO CD123-CAR T-cells (UCART123) in primary AML (n = 6) samples and normal cells (normal bone and umbilical cord blood; n = 3) using T-cell:AML cell ratios of 5:1, 2:1 and 0.5:1; and T-cell:normal cell ratio of 10:1 and 1:1. Degranulation and IFNγ release assays revealed potent activation of UCART123 cells when exposed to CD123 leukemia cells but not to normal hematopoietic cells. Cytotoxicity of UCART123 cells was observed as early as 4 hours upon initiation of the co-cultures. However, at 24 hours, more than 80% of leukemic cells (blasts, progenitors and stem cells) were eliminated at all ratios tested, whereas only approximately 20% CAR-independent cell death was observed with the TCR-deficient T-cells. Normal hematopoietic cells showed an average of 30% cell death when exposed to either UCART123 or TCR-deficient T-cells, demonstrating the selectivity of CD123 CAR-T cells toward leukemic cells. Finally, we evaluated the in vivo activity of the CAR-T cells against established patient derived xenografts (PDX) using AML and normal CB CD34+ cells. We found no significant difference between PBS, TCR-deficient (10e6/mouse) and CAR T-cell treatments (10 e6/mouse) in PDX mice transplanted with normal CD34+ CB. Strikingly, 14 days of treatment eliminated most of the leukemic cells from the AML-PDX mice. T-cells were still detected at day 14 after treatment with UCART123 cells (mean 52% CD123 CAR and mean 1.5% TCR-), without evidence of GVHD. Efforts are underway to develop allogeneic CD123 CAR-T cells for clinical trials in AML. Citation Format: Monica L. Guzman, Hongliang Zong, Mayumi Sugita, Luis A. Lara-Martinez, Laura M. Bystrom, Nicole M. Cruz, Roman Galetto, Agnes Gouble, Celine Lebuhotel, Alexander Bank, Julianne Smith, Gail J. Roboz. Allogeneic TCRαβ-deficient CAR T-cells targeting CD123 effectively eliminate myeloid leukemia cells in vitro and in vivo PDX mice. [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 3992.

Abstract 1232: A-type proanthocyanidins selectively target acute myeloid leukemia cells in vitro and in vivo

Acute myelogenous leukemia (AML) is often a fatal disease where after strong induction therapy most patients relapse and die. A-type proanthocyanidins (A-PACs) are a unique class of compounds found in cranberries (Vaccinium macrocarpon Ait.) that we have found to be effective against several leukemia cell lines and primary AML samples in vitro. Moreover, A-PACs possess a unique ether bond and have ortho-hydroxyl phenolic groups that have the potential to bind to iron, alter redox status, and other biological effects. We found that pre-treatment with antioxidants or holo-transferrin (iron-saturated transferrin) partially protected AML cells from A-PAC induced cell death (p AML-PDX mice (n = 15), were treated for 2.5 weeks via intraperitoneal injections of A-PACs (25 or 50 mg/kg dose every 3 days) or a vehicle control (PBS every 3 days). Mice were sacrificed and leukemia engraftment was evaluated using anti-human CD45 and CD33. Moreover, primary cells treated with A-PACs were assessed for effects on iron metabolism, oxidative stress, cytokine response, and survival pathways by gene expression analysis or flow cytometry. Administration of A-PACs to AML-PDX tumors reduced tumor burden. Mice that were treated with the vehicle control had engraftment of AML primary cells equivalent to 12.51% (95% CI: 4.9, 20.11; n = 5), whereas the mice treated with the 50 mg/kg and 25 mg/kg A-PACs showed a level of engraftment of 5.2% (95% CI: 1.5, 8.9; n = 5) and 5.4% (95% CI: 2.3, 8.5; n = 5), respectively. These results indicated more than a 50% reduction in engraftment, which was better or equal to the effects we observed in mice treated with high-dose cytarabine, a standard care drug. Moreover, no toxic effects were observed in the mice. It was also found that both cells and mice treated with A-PACs lead to the production of specific subset of cytokines. Global gene expression data showed consistent upregulation of some of these cytokines, and also upregulation of NF-κB and enzymes indicative of oxidative stress. The results indicate that A-PACs not only target primary AML cells in vitro, but are also effective in vivo by a potentially novel mechanism. Further elucidation of this mechanism may uncover new vulnerabilities of this disease. Citation Format: Laura M. Bystrom, Luis Andres Lara-Martinez, Bernardo Gomel, Burak Isal, Hongliang Zong, Sabrina Martinez, Catherine Neto, Stefano Rivella, Monica L. Guzman. A-type proanthocyanidins selectively target acute myeloid leukemia cells in vitro and in vivo. [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 1232.