Polyana Lopes Benfica

Investigation of Ehrlich ascites tumor cell death mechanisms induced by Synadenium umbellatum Pax.

ETHNOPHARMACOLOGICAL RELEVANCE Synadenium umbellatum Pax. is widely found in South America and empirically used in Brazil for the treatment of several diseases, mainly cancer. The aim of the study was to investigate cell death mechanisms induced by Synadenium umbellatum Pax. using Ehrlich ascites tumor (EAT) cells, as well as the myelotoxicity potential of this plant. MATERIALS AND METHODS S. umbellatum cytotoxicity was evaluated in EAT cells by trypan blue exclusion and MTT reduction test and the mechanisms involved in EAT cell death were investigated by light and fluorescence microscopy, flow cytometry and immunocytochemistry. Investigation of S. umbellatum myelotoxicity was performed by clonogenic assay of colony forming unit- granulocyte macrophage (CFU-GM). RESULTS AND CONCLUSION Our results demonstrated that S. umbellatum decreased the viability of EAT cells using both methods. Morphological analyses revealed that S. umbellatum-treatment induced EAT cell death by apoptotic pathway. We demonstrated the occurrence of reactive oxygen species (ROS) overgeneration, increased intracellular Ca(2+) concentration, alteration in mitochondrial membrane potential, phosphatydylserine externalization, and activation of caspases 3, 8, and 9. However, S. umbellatum produced myelotoxicity in bone marrow cells in a concentration-dependent manner. In comparison to EAT cells, the effects of S. umbellatum in bone marrow cells were 8-fold lower. Taken together, our results showed that S. umbellatum induced apoptosis in EAT cells at several levels and seems more toxic to tumor cells than to normal bone marrow cells.

Antitumoral and antiangiogenic activity of Synadenium umbellatum Pax

AIM OF THE STUDY Synadenium umbellatum Pax (SU), a plant used in the Midwestern region of Brazil, was tested for its antitumor and antiangiogenic activities in vitro, using K-562 and Ehrlich ascites tumor (EAT) cells, and in vivo, using the EAT-bearing model. MATERIALS AND METHODS The viability of tumor cells was evaluated by MTT and trypan blue exclusion assays, after incubation with the ethanolic extract of SU (EESU) (0.15-20mg/mL) or equivalent concentrations of its partitioned fractions (chloroformic, hexanic, and methanolic). In vivo studies were performed in EAT-bearing mice treated intraperitoneally with 5, 10, and 25mg/kg of the EESU or equivalent doses of the fractions for 10 days. The methotrexate (1.5mg/kg), for 10 days, was used as control. RESULTS SU and fractions, except the methanolic, decreased the viability of the cells in a concentration-dependent manner. In vivo results showed a significant dose-dependent antitumoral efficacy of SU against EAT growth. The best results in prolonging life span were produced by 25mg/kg of EESU. In these animals, the levels of vascular endothelial growth factor were markedly decreased after the treatment. CONCLUSIONS The data presented herein could open interesting perspectives for further research of SU as a candidate anticancer agent.

Assessment of mutagenic and antimutagenic effects of Punica granatum in mice

In the present study, the ability of Punica granatum ethanolic leaf extract (PGL) and Punica granatum ethanolic fruit extract (PGF) to induce mutagenicity or to modulate the genotoxic effects induced by the alkylating agent cyclophosphamide (CP) was evaluated. Swiss male mice were treated by gavage for 10 days with PGL or PGF (12.5, 25, 50, and 75 mg/kg/day) prior to exposure to CP (i.p. 200 mg/kg), 24 h after the end of the treatment. Initial observations revealed that normal mice treated with both extracts (12.5, 25, 50, and 75 mg/kg/day) showed a similar micronucleated polychromatic erythrocyte (MNPCE) frequency to that of the control group. Investigation of the protective effect of PGL and PGF based on data analysis revealed that, irrespective of dose or extract, oral administration of PGL or PGF for 10 days prior to exposure had reduced, in a dose-dependent manner, the frequency of MNPCE induced by CP in all groups studied. Higher reductions were observed at PGF doses of 50 and 75 mg/kg. Taken together, these results demonstrate that mice treated with P. granatum showed an absence of mutagenic effects and dose-dependent protective effects against CP-induced oxidative DNA damage.

Cytotoxicity and antiangiogenic activity of grandisin.

Objectives The antitumoural properties of grandisin, a tetrahydrofuran neolignan from Piper solmsianum, were investigated by in‐vitro and in‐vivo assays using the Ehrlich ascites tumoural (EAT) model.

The compound cis-(dichloro)tetrammineruthenium(III) chloride induces caspase-mediated apoptosis in K562 cells

Ruthenium(III) complexes are increasingly attracting the interest of researchers due to their promising pharmacological properties. In the present study, we investigated the ability of cis-(dichloro)tetrammineruthenium(III) chloride to produce lethal effects in human chronic myelogenous leukemia K562 cells. The MTT tetrazolium reduction test and the trypan blue exclusion assay revealed that the IC(50) for the compound after 48 h of incubation with K562 cells was approximately 10.74 and 73.45 microM, respectively. Interestingly, it was observed that this compound exhibits mild cytotoxicity towards MRC-5 human fibroblast cells (IC(50)>383 microM). Flow cytometric analysis revealed that cis-(dichloro)tetrammineruthenium(III) chloride was capable of change cell cycle distribution since the percentage of cells in the G1, S and G2 phases decreased. In addition, treatment with this compound induced apoptotic cell death in K562 cells, demonstrated by increased DNA content in the sub-G1-peak and a significant increase in caspase-3 activity. Assay using cyclosporin A, an inhibitor of the mitochondrial permeability transition pore (MPT) showed that the preincubation of K562 cells with this inhibitor had not effect on cis-(dichloro)tetrammineruthenium(III) chloride induced caspase-3 activation. In summary, cis-(dichloro)tetrammineruthenium(III) chloride displayed a significant cytotoxic effect through cell cycle arrest and apoptotic induction in K562 cells, which suggests that cis-(dichloro)tetrammineruthenium(III) chloride might have therapeutic potential against leukemia.

Synadenium umbellatum Pax. promotes cell cycle arrest and induces apoptosis in K-562 leukemia cells

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disease that shows apoptosis resistance. The introduction of imatinib mesylate has revolutionized the treatment of CML, but imatinib resistance may develop at any time and inevitably leads to disease progression. Synadenium umbellatum Pax. belongs to the Euphorbiaceae family and is popularly used in Brazil for the treatment of cancer. The cytotoxicity of Euphorbiaceae is associated with the ability of these plants and their bioactive compounds to induce apoptotic tumor cell death. Therefore, we aimed to investigate the cytotoxicity and the mechanisms of death induced by S. umbellatum extract in leukemic cells. S. umbellatum cytotoxicity was evaluated by trypan blue exclusion assay and flow cytometric analysis of the cell cycle; the mechanisms involved in K-562 cell death were investigated by light microscopy and flow cytometry. The results demonstrate that S. umbellatum is cytotoxic to leukemic cells in a concentration-dependent manner. Morphological analysis revealed that S. umbellatum treatment induced K-562 cell death by an apoptotic pathway. Furthermore, data indicate ROS overproduction, alterations in mitochondrial membrane potential, phosphatidylserine externalization and activation of caspase 9. Taken together, the results demonstrate that S. umbellatum extract arrested the cell cycle and triggered apoptosis at several levels in K-562 cells.

Induction of apoptosis in Ehrlich ascites tumour cells via p53 activation by a novel small-molecule MDM2 inhibitor – LQFM030

The activation of the p53 pathway through the inhibition of MDM2 has been proposed as a novel therapeutic strategy against tumours. A series of cis‐imidazoline analogues, termed nutlins, were reported to displace the recombinant p53 protein from its complex with MDM2 by binding to MDM2 in the p53 pocket, and exhibited an antitumour activity both in vitro and in vivo. Thus, the purpose of this study was to evaluate the antitumour properties of LQFM030 (2), a nutlin analogue created by employing the strategy of molecular simplification.

4-Nerolidylcatechol: apoptosis by mitochondrial mechanisms with reduction in cyclin D1 at G0/G1 stage of the chronic myelogenous K562 cell line

Abstract Context: 4-Nerolidylcatechol (4-NRC) has showed antitumor potential through apoptosis. However, its apoptotic mechanisms are still unclear, especially in leukemic cells. Objectives: To evaluate the cytotoxic potential of 4-NRC and its cell death pathways in p53-null K562 leukemic cells. Materials and methods: Cytotoxicity of 4-NRC (4.17–534.5 μM) over 24 h of exposure was evaluated by MTT assay. 4-NRC-induced apoptosis in K562 cells was investigated by phosphatidylserine (PS) externalization, cell cycle, sub-G1, mitochondrial evaluation, cytochrome c, cyclin D1 and intracellular reactive oxygen species (ROS) levels, and caspase activity analysis. Results: IC50 values obtained were 11.40, 27.31, 15.93 and 15.70 μM for lymphocytes, K562, HL-60 and Jurkat cells, respectively. In K562 cells, 4-NRC (27 μM) promoted apoptosis as verified by cellular morphological changes, a significant increase in PS externalization and sub-G1 cells. Moreover, it significantly arrested the cells at the G0/G1 phase due to a reduction in cyclin D1 expression. These effects of 4-NRC also significantly promoted a reduction in mitochondrial activity and membrane depolarization, accumulation of cytosolic cytochrome c and ROS overproduction. Additionally, it triggered an increase in caspases -3/7, -8 and -9 activities. When the cells were pretreated with N-acetyl-l-cysteine ROS scavenger, 4-NRC-induced apoptosis was partially blocked, which suggests that it exerts cytotoxicity though not exclusively through ROS-mediated mechanisms. Discussion and conclusion: 4-NRC has antileukemic properties, inducing apoptosis mediated by mitochondrial-dependent mechanisms with cyclin D1 inhibition. Given that emerging treatment concepts include novel combinations of well-known agents, 4-NRC could offer a promising alternative for chemotherapeutic combinations to maximize tumour suppression.

4-Fluorobenzaldehyde limonene-based thiosemicarbazone induces apoptosis in PC-3 human prostate cancer cells

Aims: This study evaluated parameters of toxicity and antiproliferative effects of (+)‐N(1)‐4‐fluorobenzaldehyde‐N(4)‐{1‐methyl‐1‐[(1R)‐4‐methylcyclohexene‐3‐il]‐ethyl}‐thiossemicarbazone (4‐FTSC) in PC‐3 adenocarcinoma prostate cells. Main methods: Cytotoxicity of 4‐FTSC in PC‐3 cells was evaluated using MTT assay. Morphology examination of PC‐3 cells treated with 4‐FTSC was also performed as well as the cell death mechanisms induced were investigated using flow cytometry. Parameters of toxicity of 4‐FTSC was conducted by the investigation of its potential myelotoxicity and lymphotoxicity, hemolytic activity and acute oral toxicity profile. Key findings: 4‐FTSC showed promising cytotoxic effects against PC‐3 cells (IC50 = 18.46 &mgr;M). It also triggered apoptotic morphological changes, phosphatidylserine externalization and a significant increase of DNA fragmentation in PC‐3 cells. Moreover, 4‐FTSC did not show changes in the PC‐3 cell cycle with levels of p21, p27, NF&kgr;B and cyclin D1 similar to those found in both control and treated cells. 4‐FTSC also promoted an increase of p53 levels associated with mitochondrial impairment through loss of &dgr;&PSgr;m and ROS overproduction. 4‐FTSC‐induced cell death mechanism in PC‐3 cells involved activation of caspase‐3/‐7 through apoptosis intrinsic pathway via caspase‐9. Regarding toxicological profile, 4‐FTSC showed in vitro lymphotoxicity, although with low cytotoxicity for bone marrow progenitors and no hemolytic potential. Moreover, it was classified as GHS category 5 (LD50 > 2000–5000 mg/Kg), suggesting it has low acute oral systemic toxicity. Significance: 4‐FTSC seems to be a promising candidate to be used as a clinical tool in prostate cancer treatment. Further studies are required to better clarify its toxicopharmacological effects found in this compound.