Breast cancer (BC) patients use alternative and natural remedies more than patients with other malignancies. Specifically, 63%–83% use at least one type of alternative medicine and 25%–63% use herbals and vitamins. Propolis is a naturopathic honeybee product, and CAPE (caffeic acid phenethyl ester), is a major medicinal component of propolis. CAPE, in a concentration dependent fashion, inhibits MCF-7 (hormone receptor positive, HR+) and MDA-231 (a model of triple-negative BC (TNBC) tumor growth, both in vitro and in vivo without much effect on normal mammary cells and strongly influences gene and protein expression. It induces cell cycle arrest, apoptosis and reduces expression of growth and transcription factors, including NF-κB. Notably, CAPE down-regulates mdr-1 gene, considered responsible for the resistance of cancer cells to chemotherapeutic agents. Further, CAPE dose-dependently suppresses VEGF formation by MDA-231 cells and formation of capillary-like tubes by endothelial cells, implicating inhibitory effects on angiogenesis. In conclusion, our results strongly suggest that CAPE inhibits MDA-231 and MCF-7 human breast cancer growth via its apoptotic effects, and modulation of NF-κB, the cell cycle, and angiogenesis.
Estrogen alone cannot explain the differences in breast cancer (BC) recurrence and incidence rates in pre-and postmenopausal women. In the present study, we have tested a hypothesis that, in addition to estrogen, both iron deficiency due to menstruation and iron accumulation as a result of menstrual stop play important roles in menopause-related BC outcomes. We first tested this hypothesis in cell culture models mimicking the high estrogen and low iron premenopausal condition and the low estrogen and high iron postmenopausal condition, respectively. Subsequently, we examined this hypothesis in mice that were fed iron deficient and iron overload diets. We have shown that estrogen only slightly up-regulates vascular endothelial growth factor (VEGF), an angiogenic factor known to be important in BC recurrence. It is, rather, iron deficiency that significantly promotes VEGF by stabilizing hypoxia inducible factor-1α (HIF-1α). Conversely, high iron levels increase oxidative stress and sustain mitogen-activated protein kinase (MAPK) activation, which are mechanisms of known significance in BC development. Taken together, our results suggest, for the first time, that an iron deficiency-mediated pro-angiogenic environment could contribute to the high recurrence of BC in young patients, and iron accumulation-associated pro-oxidant conditions could lead to the high incidence of BC in older women.
BackgroundYoung women diagnosed with breast cancer are known to have a higher mortality rate from the disease than older patients. Specific risk factors leading to this poorer outcome have not been identified. In the present study, we hypothesized that iron deficiency, a common ailment in young women, contributes to the poor outcome by promoting the hypoxia inducible factor-1α (HIF-1α and vascular endothelial growth factor (VEGF) formation. This hypothesis was tested in an in vitro cell culture model system.ResultsHuman breast cancer MDA-MB-231 cells were transfected with transferrin receptor-1 (TfR1) shRNA to constitutively impair iron uptake. Cellular iron status was determined by a set of iron proteins and angiogenesis was evaluated by levels of VEGF in cells as well as by a mouse xenograft model. Significant decreases in ferritin with concomitant increases in VEGF were observed in TfR1 knockdown MDA-MB-231 cells when compared to the parental cells. TfR1 shRNA transfectants also evoked a stronger angiogenic response after the cells were injected subcutaneously into nude mice. The molecular mechanism appears that cellular iron deficiency elevates VEGF formation by stabilizing HIF-1α. This mechanism is also true in human breast cancer MCF-7 and liver cancer HepG2 cells.ConclusionsCellular iron deficiency increased HIF-1α, VEGF, and angiogenesis, suggesting that systemic iron deficiency might play an important part in the tumor angiogenesis and recurrence in this young age group of breast cancer patients.
Chronic exposure to low doses of arsenite causes transformation of human osteogenic sarcoma (HOS) cells. Although oxidative stress is considered important in arsenite-induced cell transformation, the molecular and cellular mechanisms by which arsenite transforms human cells are still unknown. In the present study, we investigated whether altered iron homeostasis, known to affect cellular oxidative stress, can contribute to the arsenite-mediated cell transformation. Using arsenite-induced HOS cell transformation as a model, it was found that total iron levels are significantly higher in transformed HOS cells in comparison to parental control HOS cells. Under normal iron metabolism conditions, iron homeostasis is tightly controlled by inverse regulation of ferritin and transferrin receptor (TfR) through iron regulatory proteins (IRP). Increased iron levels in arsenite transformed cells should theoretically lead to higher ferritin and lower TfR in these cells than in controls. However, the results showed that both ferritin and TfR are decreased, apparently through two different mechanisms. A lower ferritin level in cytoplasm was due to the decreased mRNA in the arsenitetransformed HOS cells, while the decline in TfR was due to a lowered IRP-binding activity. By challenging cells with iron, it was further established that arsenite-transformed HOS cells are less responsive to iron treatment than control HOS cells, which allows accumulation of iron in the transformed cells, as exemplified by significantly lower ferritin induction. On the other hand, caffeic acid phenethyl ester (CAPE), an antioxidant previously shown to suppress As-mediated cell transformation, prevents As-mediated ferritin depletion. In conclusion, our results suggest that altered iron homeostasis contributes to arsenite-induced oxidative stress and, thus, may be involved in arsenite-mediated cell transformation.
Introduction: D/L-alpha-metyrosine (SM-88, racemetyrosine) is a clinical stage chemically modified racemic amino acid therapy with broad anticancer activity in patients across 15 different indications. While amino acid metabolism has been leveraged in oncology imaging for decades, SM-88 offers a novel therapeutic approach to selectively disrupt cancer cells. This study explored phenotypic and mechanistic effects of exposing cancer cells to D/L-alpha-metyrosine as well as potential immunomodulation of the in vivo tumor microenvironment related to tyrosine hydroxylase inhibition and catecholamine depletion. Methods: In vitro experiments were conducted in a range of human and mouse cancer cell lines, including MCF-7, 4T1, HCT116, CAPAN2, PAN02, PANC01. The effect of SM-88 methyl-ester (SM-88 ME) alone on viability, apoptosis, migration/invasion, protein synthesis inhibition, autophagy, cell cycle arrest, and ROS generation was assessed. In vivo experiments included xenograph studies of HCT116 with monotherapy consisting of either IP injections up to 200 mg/kg/day of SM-88 ME, or orally administered SM-88 up to 324 mg/kg/day. Orthotopic xenographs of 4T1 and PAN02 models using monotherapy doses up to 200 mg/kg/day are ongoing. Ongoing immunohistochemistry and flow cytometry is focused on correlation with LAT1 expression and efficacy, alteration in macrophage polarization (M1/M2 ratio), Tregs, and various T cell populations in the tumor microenvironment. Results: Exposure to SM-88 ME exhibited a dose dependent increase in ROS, as well as decreases in caspase 3/7 activation. An increase in ROS following exposure to SM-88 ME was also observed in PAN02 cells. Similar decreases in caspase 3/7 activation were observed in CAPAN2, HCT116, and MCF7 cells. Interestingly, across a variety of cell lines including PAN02, PANC1, and 4T1 cell morphology changes suggested the formation of autophagic vacuoles. Viability across all cell lines was reduced in a dose and time dependent manner. In a mouse HCT116 SC xenograft model, mice receiving 324 mg/kg/day SM-88 ME showed significantly reduced tumor size compared to control mice. Additional in vitro and in vivo experiments are ongoing. Conclusions: The importance of amino acid metabolism in cancer has gained greater awareness over the past decade, however potential therapeutic approaches in this area remain limited. SM-88 has been dosed in over 180 cancer patients with encouraging efficacy and safety findings to date. Early data suggest increases in ROS generation and decreases in apoptosis may occur in certain cancer types (PAN02 pancreatic cancer), suggesting a shift away from apoptosis and towards autophagy. Potential immune modulatory effects of SM-88, including alterations in macrophage polarization related to tyrosine hydroxylase inhibition and subsequent reductions in catecholamine production are being examined. Citation Format: Alexander Vandell, Jonathan Eckard, Steve Hoffman, Giuseppe Del Priore, Martin Fernandez-Zapico. In vitro and in vivo anticancer effects of D/L-alpha-metyrosine (SM-88), a novel metabolism-based therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5998.
Background: A wide range of cancers, including pancreatic cancer, display altered expression of amino acid transporter LAT-1. LAT-1 overexpression has been shown to be an important prognostic factor and predicts chemo resistance in pancreatic cancer (Kaira K, 2012; Altan B, 2018). Radiolabeled amino acids for PET imaging, including 3-18F-l-α-methyl-tyrosine ([18F] FAMT), have successfully leveraged cancer’s aberrant LAT1 expression and amino acid uptake for decades. SM-88 is a denatured D/L racemic form of α-methyl-tyrosine designed for selective uptake and disruption of protein synthesis in cancer cells, as well as disruption of catecholamine production via inhibition of tyrosine hydroxylase. Catecholamines have been reported to potentially have a role in driving pancreatic cancer progression and changes in the tumor immune status (Renz B, Cancer Cell 2018; Calvani M, Oncotarget 2014). Initial clinical and preclinical results with SM-88 indicate that this agent could pose a novel approach in the treatment of pancreatic cancer. Methods and Results: Initial xenograft models of human gastrointestinal cancers using monotherapy SM-88 showed a >50% decrease in tumor growth at three weeks of treatment (p<0.05). Ongoing preclinical experiments are testing the full pharmacodynamic effects of SM-88 on pancreatic cancer cells, as well as potential effects on the tumor microenvironment due to catecholamine interruption. In results from the ongoing phase 2 trial of SM-88 in heavily pretreated metastatic pancreatic cancer patients, SM-88 has shown monotherapy antitumor activity based on RECIST 1.1 criteria. Additionally, 16/24 patients demonstrated a reduction in circulating tumor cells, with the median nadir decline of 63%. LAT1 has been reported to be important in EMT transition and mesenchymal cell survival (Holldorsson S, Cancer letters 2017); examination of the impact of SM-88 on this important emerging biomarker is ongoing. Conclusion: Altered amino acid (LAT1) metabolism has been shown to be a negative prognosticator in pancreatic cancer outcomes and progression. Targeting LAT1 and amino acid pathways has been identified as a therapeutic approach in multiple cancers (Fuchs BC, Semin Ca Biol 2005). Initial and ongoing studies of SM-88 may clarify the potential utility of this approach in the treatment of pancreatic cancer. Citation Format: Martin Fernandez-Zapico, Dae Won Kim, Philip Philip, Alexander Vandell, Jonathan Eckard, Ron Korn, Giuseppe Del Priore, Diane Simeone. Therapeutic potential of targeting amino acid metabolism in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B15.
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