Caffeic acid phenethyl ester (CAPE), derived from a honeybee product propolis, exhibits a diversity of anti-tumor effects in pre-clinical models of human breast cancer.

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.

Effects of iron deficiency and iron overload on angiogenesis and oxidative stress-a potential dual role for iron in breast cancer.

Estrogen alone cannot explain the differences in breast cancer (BC) recurrence and incidence rates in pre- and postmenopausal women. In this 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 or the low-estrogen and high-iron postmenopausal condition. Subsequently, we examined this hypothesis in mice that were fed iron-deficient and iron-overloaded diets. We show 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. Conversely, high iron levels increase oxidative stress and sustain mitogen-activated protein kinase activation, which are mechanisms of known significance in BC development. Taken together, our results suggest, for the first time, that an iron-deficiency-mediated proangiogenic 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.

Effects of cellular iron deficiency on the formation of vascular endothelial growth factor and angiogenesis. Iron deficiency and angiogenesis.

BACKGROUND: Young 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. RESULTS: Human 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. CONCLUSIONS: Cellular 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.

Altered iron homeostasis involvement in arsenite-mediated cell transformation.

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 arsenite-transformed 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.

Caffeic acid phenethyl ester (CAPE) prevents transformation of human cells by arsenite (As) and suppresses growth of As-transformed cells.

Recent evidence suggests that inflammatory cytokines and growth factors contribute to arsenite (As)-induced human carcinogenesis. We investigated the expression of inflammatory cytokine mRNAs during the transformation process induced by chronic As exposure in non-tumorigenic human osteogenic sarcoma (N-HOS) cells using gene arrays, and results were confirmed by RT-PCR and protein arrays. Caffeic acid phenethyl ester (CAPE), a naturally occurring immunomodulating agent, was used to evaluate the role of inflammatory factors in the process of As-mediated N-HOS cell transformation and in As-transformed HOS (AsT-HOS) cells. We found that an 8-week continuous exposure of N-HOS to 0.3 microM arsenite resulted in HOS cell transformation. That exposure also caused substantial decreases in inflammatory cytokine mRNAs, such as interleukin (IL) IL-1alpha, IL-2, IL-8, IL-18, MCP-1, TGF-beta2, and TNF-alpha, while it increased c-jun mRNA in a time-dependent manner. Co-incubation of N-HOS with As and CAPE (0.5-2.5 microM) prevented As-mediated declines in cytokine mRNAs in the co-treated cells, as well as their transformation to anchorage independence, while it caused decreases in c-jun mRNA. CAPE (up to 10 microM) had no effect on growth of N-HOS cells. However, CAPE (1-10 microM) treatment of AsT-HOS cells inhibited cell growth, induced cell cycle G2/M arrest, and triggered apoptosis, accompanied by changes in cytokine gene expression, as well as decreases in cyclin B1 and cdc2 abundance. Resveratrol (RV) and (-)(.) epigallocatechin gallate (EGCG), preventive agents present in grapes and green tea, respectively, induced similar changes in AsT-HOS cell growth but required much higher doses than CAPE to cause 50% growth arrest (<2.5 microM CAPE versus 25 microM RV or 50 microM EGCG). Overall, our findings suggest that inflammatory cytokines play an important role in the suppressive effects of CAPE on As-induced cell transformation and in the selective cytotoxicity of CAPE to As-transformed HOS cells.

Interleukin-1alpha up-regulation in vivo by a potent carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) and control of DMBA-induced inflammatory responses.

Tumor-initiating properties of complete carcinogens such as 7,12-dimethylbenz(a)anthracene (DMBA) are well known but not the mechanism of DMBA-mediated tumor promotion. Our hypothesis is that interleukin (IL)-1alpha, an early proinflammatory cytokine that initiates a cascade of other cytokines and growth factors, is up-regulated by DMBA and contributes to inflammation and carcinogenesis. We found that topical exposure of SENCAR mice to a carcinogenic DMBA dose indeed triggers significant increases in mouse skin IL-1alpha and IL-1alpha mRNA. Five DMBA applications (200 nmol each) caused a statistically significant (P = 0.02) increase in serum IL-1alpha, comparable with that induced by 12-O-tetradecanoylphorbol-13-acetate, a potent tumor promoter. IL-1alpha increase in serum was evident 24 h after the first DMBA application, whereas that in skin required five DMBA doses and became statistically significant (P < 0.0003) 48 h later. Skin IL-1alpha enhancement was preceded by a 6-fold up-regulation of IL-1alpha mRNA. A pretreatment with antimurine IL-1alpha antibody (Ab) nearly abolished DMBA-induced IL-1alpha mRNA (P = 0.0001) in skin and substantially decreased IL-1alpha in serum. Infiltration of polymorphonuclear leukocytes into skin was elevated 6-fold (P = 0.002) and >10-fold (P = 0.001) 24 h and 48 h after the fifth DMBA exposure, respectively. A pretreatment with anti-IL-1alpha Ab decreased polymorphonuclear leukocyte infiltration by >65% (P < 0.02), which suggests that this process is at least 65% under IL-1alpha control. Anti-IL-1alpha antibodies had no effect on edema, thus dissociating the two inflammation markers. Injecting anti-IL-1alpha Ab before DMBA applications significantly (P < 0.04) decreased the volume of carcinomas (CAs) in comparison with CAs that arose in mouse skin injected with a nonspecific serum. These results prove that IL-1alpha is induced by a carcinogenic DMBA dose and contributes to DMBA-induced inflammation and volume of CAs, hallmarks of tumor promotion and progression.