Cell death and lumen formation in spheroids of MCF-7 cells

3D (three-dimensional) cell culture permits a more integrated analysis of the relationship between cells, inserting them into a structure more closely resembling the cellular microenvironment in vivo. The development of in vitro parameters to approximate in vivo 3D cellular environments makes a less reductionist interpretation of cell biology possible. For breast cells, in vitro 3D culture has proven to be an important tool for the analysis of luminal morphogenesis. A greater understanding of this process is necessary because alterations in the lumen arrangement are associated with carcinogenesis. Following lumen formation in 3D cell culture using laser scanning confocal microscopy, we observed alterations in the arrangement of cytoskeletal components (F-actin and microtubules) and increasing levels of cell death associated with lumen formation. The formation of a polarized monolayer facing the lumen was characterized through 3D reconstructions and the use of TEM (transmission electron microscopy), and this process was found to occur through the gradual clearing of cells from the medullary region of the spheroids. This process was associated with different types of cell death, such as apoptosis, autophagy and entosis. The present study showed that changes in the extracellular matrix associated with long periods of time in 3D cell culture lead to the formation of a lumen in MCF-7 cell spheroids and that features of differentiation such as lumen and budding formation occur after long periods in 3D culture, even in the absence of exogenous extracellular compounds.

Insulin-like growth factor-1 and 17 beta-estradiol down-regulate prostate apoptosis response-4 expression in MCF-7 breast cancer cells

The PKC apoptosis WTI regulator gene, also named prostate apoptosis response-4 (PAR-4), encodes a pro-apoptotic protein that sensitizes cells to numerous apoptotic stimuli. Insulin-like growth factor-1 (IGF-1) and 17 beta-estradiol (E2), two important factors for breast cancer development and progression, have been shown to down-regulate PAR-4 expression and inhibit apoptosis induced by PAR-4 in neuronal cells. In this study, we sought to investigate the mechanisms of regulation of PAR-4 gene expression in MCF-7 cells treated with E2 or IGF-1. E2 (10 nM) and IGF-1 (12.5 nM) each down-regulated PAR-4 expression in MCF-7 cells after 24 h of treatment. The effect of E2 was dependent on ER activation, as demonstrated by an increase in PAR-4 expression when cells were pretreated for 1 h with 1 mu M ICI-182,780 (ICI) before receiving E2 plus ICI. The effect of IGF-1 was abolished by pre-treatment for 1 h with 30 mu M LY294002 (a specific PI3-K inhibitor), and significantly inhibited by 30 mu M SB202190 (a specific p38MAPK inhibitor). We also demonstrated that E2 acts synergistically with IGF-1, resulting in greater down-regulation of PAR-4 mRNA expression compared with E2 or IGF-1 alone. Our results show for the first time that E2 and IGF-1 inhibit PAR-4 gene expression in MCF-7 cells, suggesting that this down-regulation may provide a selective advantage for breast cancer cell survival.

Cytotoxic effects of butanolic extract from Pfaffia paniculata (Brazilian Ginseng) on cultured human breast cancer cell line MCF-7

Roots of Pfaffia paniculata have been well documented for multifarious therapeutic values and have also been used for cancer therapy in folk medicine. This study has been performed in a human breast tumor cell line, the MCF-7 cells. These are the most commonly used model of estrogen-positive breast cancer, and it has been originally established in 1973 at the Michigan Cancer Foundation from a pleural effusion taken from a woman with metastatic breast cancer. Butanolic extract of the roots of P. paniculata showed cytotoxic effect MCF-7 cell line. as determined with crystal violet assay, cellular death with acridine orange/ethidium bromide staining, and cell proliferation with immunocytochemistry of bromodeoxyuridine (BrdU). Subcellular alterations were evaluated by electron microscopy. Cells treated With butanolic extract showed degeneration of cytoplasmic components and profound morphological and nuclear alterations. The results show that this butanolic extract indeed presents cytotoxic substances, and its fractions merit further investigations. (C) 2008 Elsevier GmbH. All rights reserved.

Systemic chemotherapy-induced microsatellite instability in the mononuclear cell fraction of women with breast cancer can be reproduced in vitro and abrogated by amifostine

Objectives Microsatellite instability (MSI) induction by alkylating agent-based chemotherapy (ACHT) may underlie both tumor resistance to chemotherapy and secondary leukaemias in cancer patients. We investigated if ACHT could induce MSI in tumor-derived plasma-circulating DNA (pfDNA) and in normal peripheral blood mononuclear (PBMN) cells. We also evaluated if amifostine could interfere with this process in an in-vitro model. Methods MSI was determined in pfDNA, PBMN cells and urine cell-free DNA (ufDNA) of 33 breast cancer patients before and after ACHT. MCF-7 cells and PBMN from normal donors were exposed in vitro to melphalan, with or without amifostine. Results We observed at least one MSI event in PBMN cells, pfDNA or ufDNA of 87, 80 and 80% of patients, respectively. In vitro, melphalan induced MSI in both MCF-7 and normal PBMN cells. In PBMN cells, ACHT-induced MSI occurred together with a significant decrease in the expression of the DNA mismatch repair gene hMSH2. Amifostine decreased hMSH2 expression and also prevented MSI induction only in normal PBMN cells. Conclusions ACHT induced MSI in PBMN cells and in tumour-derived pfDNA. Because of its protective effect against ACHT induction of MSI in normal PBMN cells in vitro, amifostine may be a potential agent for preventing secondary leukaemias in patients exposed to ACHT.

MicroRNA expression is differentially altered by xenobiotic drugs in different human cell lines

Several noncoding microRNAs (miR or miRNA) have been shown to regulate the expression of drug-metabolizing enzymes and transporters. Xenobiotic drug-induced changes in enzyme and transporter expression may be associated with the alteration of miRNA expression. Therefore, this study investigated the impact of 19 xenobiotic drugs (e. g. dexamethasone, vinblastine, bilobalide and cocaine) on the expression of ten miRNAs (miR-18a, -27a, -27b, -124a, -148a, -324-3p, -328, -451, -519c and -1291) in MCF-7, Caco-2, SH-SY5Y and BE(2)-M17 cell systems. The data revealed that miRNAs were differentially expressed in human cell lines and the change in miRNA expression was dependent on the drug, as well as the type of cells investigated. Notably, treatment with bilobalide led to a 10-fold increase of miR-27a and a 2-fold decrease of miR-148a in Caco-2 cells, but no change of miR-27a and a 2-fold increase of miR-148a in MCF-7 cells. Neuronal miR-124a was generally down-regulated by psychoactive drugs (e. g. cocaine, methadone and fluoxetine) in BE(2)-M17 and SH-SY5Y cells. Dexamethasone and vinblastine, inducers of drug-metabolizing enzymes and transporters, suppressed the expression of miR-27b, -148a and -451 that down-regulate the enzymes and transporters. These findings should provide increased understanding of the altered gene expression underlying drug disposition, multidrug resistance, drug-drug interactions and neuroplasticity. Copyright (C) 2011 John Wiley & Sons, Ltd.

Expression of a dendritic cell maturation marker CD83 on tumor cells from lung cancer patients and several human tumor cell lines: is there a biological meaning behind it?

The present paper shows, for the first time, the membrane expression of the dendritic cell maturation marker CD83 on tumor cells from lung cancer patients. CD83 was also detected on freshly cultured fibroblast-like cells from these tissues and on several adherent human tumor cell lines (lung adenocarcinomas P9, A459 and A549, melanomas A375 and C81-61, breast adenocarcinomas SKBR-3 and MCF-7 and colon carcinoma AR42-J), but not in the non-adherent MOT leukemia cell line. CD83 may have immunosuppressive properties and its expression by cancer cells could have a role in facilitating tumor growth.

Recombinant rabies virus glycoprotein synthesis in bioreactor by transfected Drosophila melanogaster S2 cells carrying a constitutive or an inducible promoter

S2 cell populations (S2AcRVGP2K and S2MtRVGP-Hy) were selected after transfection of gene expression vectors carrying the cDNA encoding the rabies virus glycoprotein (RVGP) gene under the control of the constitutive (actin) or inductive (metallothionein) promoters. These cell populations were cultivated in a 1 L bioreactor mimicking a large scale bioprocess. Cell cultures were carried out at 90 rpm and monitored/controlled for temperature (28 degrees C) and dissolved oxygen (10 or 50% air saturation). Cell growth attained similar to 1.5-3 x 10(7) cells/mL after 3-4 clays of cultivation. The constitutive synthesis of RVGP in S2AcRVGP2K cells led to values of 0.76 mu g/10(7) cells at day 4 of culture. The RVGP synthesis in S2MtRVGP-Hy cell fraction increased upon CuSO(4) induction attaining specific productivities of 1.5-2 mu g/10(7) cells at clays 4-5. RVGP values in supernatant as a result of cell lysis were always very low (<0.2 mu g/mL) indicating good integrity of cells in culture. Overall the RVGP productivity was of 1.5-3 mg/L. Our data showed an important influence of dissolved oxygen on RVGP synthesis allowing a higher and sustained productivity by S2MtRVGP-Hy cells when cultivated with a DO of 10% air saturation. The RVGP productivity in bioreactors shown here mirrors those previously observed for T-flasks and shaker bottles and allow the preparation of the large RVGP quantities required for studies of structure and function. (C) 2010 Elsevier B.V. All rights reserved.

Antiproliferative Effect of Benzophenones and their Influence on Cathepsin Activity

The antiproliferative activity of two prenylated benzophenones isolated from Rheedia brasiliensis. the tri-prenylated garciniaphenone and the tetraprenylated benzophenone 7-epiclusianone, was investigated against human cancer cell lines. The antiproliferative activity on melanoma (UACC-62), breast (MCF-7), drug-resistant breast (NCI-ADR), lung/non-small cells (NCI460), ovarian (OVCAR 03), prostate (PC03), kidney (786-0), lung (NCI-460) and tongue (CRL-1624 and CRL-1623) cancer cells was determined using spectrophotometric quantification of the cellular protein content. The effect of these benzophenones on the activity of cathepsins B and G was also investigated. Garciniaphenone displayed cytostatic activity in all cell lines, whereas 7-epiclusianone showed a dose-dependent cytotoxic effect. The IC(50) values for cell proliferation revealed that 7-epiclusianone is more active than garciniaphenone against most of the cell lines. Furthermore, the antiproliferative effects demonstrated by garciniaphenone and 7-epiclusianone were related to their cathepsin inhibiting properties. In conclusion, 7-epiclusianone is a promising naturally occurring agent which displays multiple inhibitory effects which may be working in concert to inhibit cancer cell proliferation in vitro. The putative pathway by which 7-epiclusianone affects cancer cell development may involve cathepsin inhibition. Copyright (C) 2009 John Wiley & Sons, Ltd.

New antitumoral agents I: In vitro anticancer activity and in vivo acute toxicity of synthetic 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one and derivatives

This paper describes a new method for the preparation of 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one 1 and its derivatives 2-5. This set of synthetic compounds exhibited high antitumoral activities regarding in vitro screening against several human tumor cell lines as lung carcinoma NCI-460, melanoma UACC-62, breast MCF-7, colon HT-29, renal 786-O, ovarian OVCAR-03 and ovarian expressing the resistance phenotype for adriamycin NCI-ADR/ RES, prostate PC-3, and leukemia K-562. Compounds were also tested against murine tumor cell line B16F10 melanoma and lymphocytic leukemia L1210 as well as to their effect toward normal macrophages. Specific activity against colon cancer cells HT-29 was observed for all tested compounds and suggests further studies with models of colon cancer. Compounds 1, 2, and 4 showed significant cytotoxic activity with IC(50) values <= 2.3 mu M for all human cancer cell lines. Intraperitoneal acute administration of compound 1 and 2 showed very low toxicity rate. (C) 2010 Elsevier Ltd. All rights reserved.

Silencing of LRRC49 and THAP10 genes by bidirectional promoter hypermethylation is a frequent event in breast cancer

Previously we found that levels of LRRC49 (leucine rich repeat containing 49; FLJ20156) transcripts were elevated in ER-positive breast tumors compared with ER-negative breast tumors. The LRRC49 gene is located on chromosome 15q23 in close proximity to the THAP10 (THAP domain containing 10) gene. These two genes have a bidirectional organization being arranged head-to-head on opposite strands, possibly sharing the same promoter region. Analysis of the promoter region of this gene pair revealed the presence of potential estrogen response elements (EREs), suggesting the potential of this promoter to be under the control of estrogen. We used quantitative real-time PCR (qPCR) to evaluate the expression of LRRC49 and THAP10 in a series of 72 primary breast tumors, and found reduced LRRC49 and THAP10 expression in 61 and 46% of the primary breast tumors analyzed, respectively. In addition, the occurrence of LRRC49/THAP10 promoter hypermethylation was examined by methylation specific PCR (MSP) in a sub-group of the breast tumors. Hypermethylation was observed in 57.5% of the breast tumors analyzed, and the levels of mRNA expression of both genes were inversely correlated with promoter hypermethylation. We investigated the effects of 17 beta-estradiol on LRRC49 and THAP10 expression in MCF-7 breast cancer cells and found both transcripts to be up-regulated 2- to 3-fold upon 17 beta-estradiol treatment. Our results show that the transcripts of LRRC49/THAP10 bidirectional gene pair are co-regulated by estrogen and that hypermethylation of the bidirectional promoter region simultaneously silences both genes. Further studies will be necessary to elucidate the role of LRRC49/THAP10 down-regulation in breast cancer.

In vitro effect of Aloe vera, Coriandrum sativum and Ricinus communis fractions on Leishmania infantum and on murine monocytic cells

In South America, visceral leishmaniasis is a zoonosis caused by the protozoan species Leishmania infantum (syn. L. chagasi) and is primarily transmitted through the bite of the female Lutzomyia longipalpis. Its main reservoir in urban areas is the dog. The application of control measures recommended by health agencies have not achieved significant results in reducing the incidence of human cases, and the lack of effective drugs to treat dogs resulted in the prohibition of this course of action in Brazil. Therefore, it is necessary to search new alternatives for the treatment of canine and human visceral leishmaniasis. The objectives of this study were to evaluate the in vitro effect of fractions from Aloe vera (aloe), Coriandrum sativum (coriander), and Ricinus communis (castor) on promastigotes and amastigotes of L. infantum and to analyze the toxicity against the murine monocytic cells RAW 264.7. To determine the viability of these substances on 50% parasites (IC50), we used a tetrazolium dye (MU) colorimetric assay (bromide 3-4.5-dimethylthiazol-2-yl-2,5-dephenyltetrazolium), and on amastigotes we performed an in situ ELISA. All fractions were effective against L. infantum promastigotes and did not differ from the positive control pentamidine (p > 0.05). However, the R. communis ethyl acetate and chloroform fractions, as well as the C. sativum methanol fraction, were the most effective against amastigotes and did not differ from the positive control amphotericin B (p > 0.05). The R. communis ethyl acetate fraction was the least toxic, presenting 83.5% viability of RAW 264.7 cells, which was similar to the results obtained with amphotericin B (p > 0.05). Based on these results, we intend to undertake in vivo studies with R. communis ethyl acetate fractions due the high effectiveness against amastigotes and promastigotes of L. infantum and the low cytotoxicity towards murine monocytic cells. (C) 2011 Elsevier B.V. All rights reserved.

Fast and Simple Nuclear Magnetic Resonance Method To Measure Conjugated Linoleic Acid in Beef

Conjugated linoleic acids (CLAs) are a group of linoleic acid isomers that are naturally found in food products originating from ruminants (meat and dairy). These acids have received special attention in recent years due to their potential human health benefits. Research efforts have been proposed to increase the CLA content in beef to improve public health. However, because there are more than 30 million beef cattle used each year by the American food industry, it will be necessary to ensure their content in a large number of samples. Therefore, it is important to have an inexpensive and rapid analytical method to measure CLA content in food products. Because gas chromatography (GC), a current popular method for measuring CLAs, is slow, this paper describes a nuclear magnetic resonance spectroscopy ((1)H NMR) method that is potentially >10 times faster than the GC method. Analyses show a correlation coefficient of 0.97, indicating the capacity of NMR to quantify the CLA content in beef samples. Furthermore, the method proposed herein is simple and does not require sophisticated sample preparation.

Constituents and antiproliferative activity of extracts from leaves of Croton macrobothrys

Croton macrobothrys Baill, Euphorbiaceae, is a tree from the Atlantic Forest in Southern Brazil, used in traditional medicine and popularly known as "dragon's blood" and "pau-sangue". Leaf n-hexane, dichloromethane and methanol extracts were analyzed by GC/MS and evaluated for their in vitro antiproliferative activity on cell lines 786-0 (kidney), HT-29 (colon), K562 (leukemia), NCI-ADR/RES (drug resistant ovary), NCI-H460 (lung), MCF-7 (mammary), PC-3 (prostate), OVCAR-3 (ovary), U251 (glioma) and UACC-62 (melanoma). The dicloromethane extract exhibited activity against all cell lines at the concentration 25 µg/mL, in particular on cell lines NCI-H460 (GI50 0.33 μg/mL) and K5662 (GI50 0.91 μg/mL). Relevant constituents in dichloromethane extract are the alkaloids corydine and salutaridine, as well as the diterpenes geranylgeraniol and crotonin-derived clerodanes.

PAMM: A Redox Regulatory Protein That Modulates Osteoclast Differentiation

The central role of reactive oxygen species (ROS) in osteoclast differentiation and in bone homeostasis prompted us to characterize the redox regulatory system of osteoclasts. In this report, we describe the expression and functional characterization of PAMM, a CXXC motif-containing peroxiredoxin 2-like protein expressed in bone marrow monocytes on stimulation with M-CSF and RANKL. Expression of wild-type (but not C to G mutants of the CXXC domain) PAMM in HEK293 cells results in an increased GSH/GSSG ratio, indicating a shift toward a more reduced environment. Expression of PAMM in RAW264.7 monocytes protected cells from hydrogen peroxide-induced oxidative stress, indicating that PAMM regulates cellular redox status. RANKL stimulation of RAW 264.7 cells caused a decrease in the GSH/GSSG ratio (reflecting a complementary increase in ROS). In addition, RANKL-induced osteoclast formation requires phosphorylation and translocation of NF-kappa B and c-Jun. In stably transfected RAW 264.7 cells, PAMM overexpression prevented the reduction of GSH/GSSG induced by RANKL. Concurrently, PAMM expression completely abolished RANKL-induced p100 NF-kappa B and c-Jun activation, as well as osteoclast formation. We conclude that PAMM is a redox regulatory protein that modulates osteoclast differentiation in vitro. PAMM expression may affect bone resorption in vivo and help to maintain bone mass. Antioxid. Redox Signal. 13, 27-37.

Synthesis, characterization, and bactericidal properties of composites based on crosslinked resins containing silver

Two different commercial crosslinked resins (Amberlite GT73 and Amberlite IRC748) were employed for anchoring silver. The -SH and -N(CH2COOH)2 groups, respectively, present on these resins were used for Ag+ chelation from an aqueous solution. The Ag+ ions were reduced with three different reductants: hydrazine, hydroxylamine, and formaldehyde (under an alkaline pH). The produced composites were characterized with thermogravimetry/differential thermogravimetry and scanning electron microscopy combined with a backscattered scanning electron detector. Energy-dispersive X-ray spectroscopy coupled to scanning electron microscopy allowed the observation of submicrometer particles of silver, and chemical microanalysis of emitted X-rays revealed the presence of metal on the internal and external surfaces of the composite microspheres. The amount of incorporated silver was determined by titration. The antibacterial activity of the silver/resin composites was determined toward 10(3)-10(7) cells/mL dilutions of the auxotrophic AB1157 Escherichia coli strain; the networks containing anchored submicrometer silver particles were completely bactericidal within a few minutes because of the combined action of silver and functional groups of the resins. (c) 2007 Wiley Periodicals, Inc.