Artemisia vulgaris Induces Tumor-Selective Ferroptosis and Necroptosis via Lysosomal Ca2+ Signaling.

OBJECTIVE: To evaluate the chemical composition and effects of Artemisia vulgaris (AV) hydroalcoholic extract (HEAV) on breast cancer cells (MCF-7 and SKBR-3), chronic myeloid leukemia (K562) and NIH/3T3 fibroblasts. METHODS: Phytochemical analysis of HEAV was done by high-performance liquid chromatography-mass (HPLC) spectrometry. Viability and cell death studies were performed using trypan blue and Annexin/FITC-7AAD, respectively. Ferrostatin-1 (Fer-1) and necrostatin-1 (Nec-1) were used to assess the mode of HEAV-induced cell death and acetoxymethylester (BAPTA-AM) was used to verify the involvement of cytosolic calcium in this event. Cytosolic calcium measurements were made using Fura-2-AM. RESULTS: HEAV decreased the viability of MCF-7, SKBR-3 and K562 cells (P<0.05). The viability of HEAV-treated K562 cells was reduced compared to HEAV-exposed fibroblasts (P<0.05). Treatment of K562 cells with HEAV induced cell death primarily by late apoptosis and necrosis in assays using annexin V-FITC/7-AAD (P<0.05). The use of Nec-1 and Fer-1 increased the viability of K562 cells treated with HEAV relative to cells exposed to HEAV alone (P<0.01). HEAV-induced Ca2+ release mainly from lysosomes in K562 cells (P<0.01). Furthermore, BAPTA-AM, an intracellular Ca2+ chelator, decreased the number of non-viable cells treated with HEAV (P<0.05). CONCLUSIONS: HEAV is cytotoxic and activates several modalities of cell death, which are partially dependent on lysosomal release of Ca2+. These effects may be related to artemisinin and caffeoylquinic acids, the main compounds identified in HEAV.

Effective Synergy of Sorafenib and Nutrient Shortage in Inducing Melanoma Cell Death through Energy Stress.

Skin melanoma is one of the most aggressive and difficult-to-treat human malignancies, characterized by poor survival rates, thus requiring urgent novel therapeutic approaches. Although metabolic reprogramming has represented so far, a cancer hallmark, accumulating data indicate a high plasticity of cancer cells in modulating cellular metabolism to adapt to a heterogeneous and continuously changing microenvironment, suggesting a novel therapeutic approach for dietary manipulation in cancer therapy. To this aim, we exposed melanoma cells to combined nutrient-restriction/sorafenib. Results indicate that cell death was efficiently induced, with apoptosis representing the prominent feature. In contrast, autophagy was blocked in the final stage by this treatment, similarly to chloroquine, which also enhanced melanoma cell sensitization to combined treatment. Energy stress was evidenced by associated treatment with mitochondrial dysfunction and glycolysis impairment, suggesting metabolic stress determining melanoma cell death. A reduction of tumor growth after cycles of intermittent fasting together with sorafenib treatment was also observed in vivo, reinforcing that the nutrient shortage can potentiate anti-melanoma therapy. Our findings showed that the restriction of nutrients by intermittent fasting potentiates the effects of sorafenib due to the modulation of cellular metabolism, suggesting that it is possible to harness the energy of cancer cells for the treatment of melanoma.

Medicinal properties of Angelica archangelica root extract: Cytotoxicity in breast cancer cells and its protective effects against in vivo tumor development.

OBJECTIVE: Although Angelica archangelica is a medicinal and aromatic plant with a long history of use for both medicinal and food purposes, there are no studies regarding the antineoplastic activity of its root. This study aimed to evaluate the cytotoxicity and antitumor effects of the crude extract of A. archangelica root (CEAA) on breast cancer. METHODS: The cytotoxicity of CEAA against breast adenocarcinoma cells (4T1 and MCF-7) was evaluated by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Morphological and biochemical changes were detected by Hoechst 33342/propidium iodide (PI) and annexin V/PI staining. Cytosolic calcium mobilization was evaluated in cells staining with FURA-4NW. Immunoblotting was used to determine the effect of CEAA on anti- and pro-apoptotic proteins (Bcl-2 and Bax, respectively). The 4T1 cell-challenged mice were used for in vivo assay. RESULTS: Using ultra-high-performance liquid chromatography-mass spectrometry analysis, angelicin, a constituent of the roots and leaves of A. archangelica, was found to be the major constituent of the CEAA evaluated in this study (73 µg/mL). The CEAA was cytotoxic for both breast cancer cell lines studied but not for human fibroblasts. Treatment of 4T1 cells with the CEAA increased Bax protein levels accompanied by decreased Bcl-2 expression, in the presence of cleaved caspase-3 and cytosolic calcium mobilization, suggesting mitochondrial involvement in breast cancer cell death induced by the CEAA in this cell line. No changes on the Bcl-2/Bax ratio were observed in CEAA-treated MCF7 cells. Gavage administration of the CEAA (500 mg/kg) to 4T1 cell-challenged mice significantly decreased tumor growth when compared with untreated animals. CONCLUSION: Altogether, our data show the antitumor potential of the CEAA against breast cancer cells in vitro and in vivo. Further research is necessary to better elucidate the pharmacological application of the CEAA in breast cancer therapy.

Cafestol, a diterpene molecule found in coffee, induces leukemia cell death.

To evaluate the antitumor properties of Cafestol four leukemia cell lines were used (NB4, K562, HL60 and KG1). Cafestol exhibited the highest cytotoxicity against HL60 and KG1 cells, as evidenced by the accumulation of cells in the sub-G1 fraction, mitochondrial membrane potential reduction, accumulation of cleaved caspase-3 and phosphatidylserine externalization. An increase in CD11b and CD15 differentiation markers with attenuated ROS generation was also observed in Cafestol-treated HL60 cells. These results were similar to those obtained following exposure of the same cell line to cytarabine (Ara-C), an antileukemic drug. Cafestol and Ara-C reduced the clonogenic potential of HL60 cells by 100%, but Cafestol spared murine colony forming unit- granulocyte/macrophage (CFU-GM), which retained their clonogenicity. The co-treatment of Cafestol and Ara-C reduced HL60 cell viability compared with both drugs administered alone. In conclusion, despite the distinct molecular mechanisms involved in the activity of Cafestol and Ara-C, a similar cytotoxicity towards leukemia cells was observed, which suggests a need for prophylactic-therapeutic pre-clinical studies regarding the anticancer properties of Cafestol.

Chlorella vulgaris restores bone marrow cellularity and cytokine production in lead-exposed mice.

Chlorella vulgaris (CV) was examined for its modulating effects on the reduction induced by lead (Pb) on the numbers of marrow hematopoietic stem cells (HSCs) (c-Kit(+)Lin(-)), granulocyte-macrophage progenitors (Gr1(+)Mac1(+)) and total bone marrow cellularity. In mice gavage-treated daily with 50mg/kg dose of CV for 10 days, concomitant to a continuous offering of 1300 ppm lead acetate in drinking water, the treatment with the algae recovered the significantly reduced numbers of these cell populations to control values. As CV may have a myelostimulating effect through the induction of cytokines, we evaluated its modulating effects on the production of IL-1α, TNF-α, IFN-γ, IL-10 and IL-6. Our results demonstrated that lead significantly impairs the production of IFN-γ, IL-1α and TNF-α and increases the production of IL-10 and IL-6 and that these effects are successfully modulated by the CV treatment. The activity of NK cells, reduced in Pb-exposed animals, was raised to levels higher than those of controls in the exposed group treated with CV. Treatment with the algae also stimulated the production of IFN-γ, IL-1α, TNF-α and NK cells activity in normal mice. In addition, zinc bone concentrations, reduced in lead-exposed mice, were partially, but significantly, reversed by the treatment with CV.

Bone marrow progenitor cells from chemically exposed workers display an intrinsic ability for autonomous proliferation.

In this study, the autonomous proliferation of bone marrow progenitor cells (CFU-C), a pathological phenomenon observed in many hematological abnormalities, was investigated in 31 individuals who had been diagnosed as having neutropenia. Of these subjects, 18 had been chronically exposed (range of exposure 5-30 years) to a variety of petroleum distillates. We observed that the group of exposed individuals presented higher numbers of autonomous CFU-C when compared with those unexposed subjects. In addition, follow-up data demonstrated that 20% of the exposed population (4 of the 18) developed malignant hematological diseases. The autonomous CFU-C obtained from all individuals studied was composed predominantly of macrophages. This suggests an involvement of these cells in the development of hematological abnormalities, probably as a result of increased production of chemical myelotoxic metabolites.

Effects produced by Royal Jelly on haematopoiesis: relation with host resistance against Ehrlich ascites tumour challenge.

Royal jelly (RJ) was shown to exhibit immunomodulatory properties, although its biological activity is still unclear. In order to elucidate the mechanism whereby RJ activates the immunological system, we examined the role of this substance on the haematopoietic response of Ehrlich ascites tumour (EAT)-bearing mice. Our results demonstrated that RJ prevented the myelosupression induced by the temporal evolution of the tumour and abrogated the splenic haematopoiesis observed in EAT-bearing mice. The stimulating effect of RJ was also observed in vitro on the multipotent bone marrow stem cells, evaluated by the long-term bone marrow cultures (LTBMCs). The study of survival clearly showed the antitumour activity of RJ. Treatment was given prophylactically for 20 days and therapeutically for 3, 8 and 13 days. Except for the treatment with the lower dose of 500 mg/kg, given for 23 days, all the other dose schedules were able to prolong survival. A more effective antitumoural response was observed with the more prolonged treatment regimen. In this regard, the administration of RJ for 33 days produced the highest protection reaching an extension of survival at about 38%, 71% and 85% for the doses of 500, 1000 and 1500 mg/kg, respectively, whereas with the 23 and 28 days treatment schedules, survival increased at a rate of 19% and 23%, respectively, and comparable results were found among the effective doses of RJ. Increased survival rate might be related to the decreased Prostaglandin E2 (PGE2) levels observed in EAT-bearing mice after RJ treatment. These results point to RJ as a promising modifier of biological response leading to myeloprotection and antitumour activity.