Traditional and Advanced Cell Cultures in Hematopoietic Stem Cell Studies.

Hematopoiesis is the main function of bone marrow. Human hematopoietic stem and progenitor cells reside in the bone marrow microenvironment, making it a hotspot for the development of hematopoietic diseases. Numerous alterations that correspond to disease progression have been identified in the bone marrow stem cell niche. Complex interactions between the bone marrow microenvironment and hematopoietic stem cells determine the balance between the proliferation, differentiation and homeostasis of the stem cell compartment. Changes in this tightly regulated network can provoke malignant transformation. However, our understanding of human hematopoiesis and the associated niche biology remains limited due to accessibility to human material and the limits of in vitro culture models. Traditional culture systems for human hematopoietic studies lack microenvironment niches, spatial marrow gradients, and dense cellularity, rendering them incapable of effectively translating marrow physiology ex vivo. This review will discuss the importance of 2D and 3D culture as a physiologically relevant system for understanding normal and abnormal hematopoiesis.

Redox signaling in ischemic postconditioning protection involves PKCε and Erk1/2 pathways and converges indirectly in Nrf2 activation.

Ischemic-postconditioning (iPostC) exerts cardioprotection by preserving redox homeostasis in the reperfused heart. This protective effect has been associated with the activation of endogenous antioxidant response driven by transcription factor Nrf2 and with the activation of 'reperfusion injury salvage kinases' (RISK) as PI3K, PKC and Erk1/2. Redox homeostasis is essential for normal cell physiology since reactive oxygen species (ROS) are crucial for processes that involve protein signaling. Thus, it has become clear that not only the perturbation of redox balance to oxidative state is deleterious but also towards a reductive state contributing to pathogenesis of diseases. However, there is still a scarce knowledge about the role of ROS in the cardioprotective signals mediated by RISK in postconditioned hearts. Therefore, we studied the role of ROS as initiator of RISK signaling molecules in iPostC-conferred cardioprotection. With the aim to study the relationship between redox-dependent RISK activation and the downstream activation of the transcription factor Nrf2, we evaluated the effect of redox signaling disruption by the effect of ascorbic acid in iPostC hearts. Our results showed that PKCε and Erk1/2 activation is redox-dependent and that concurs downstream with Nrf2 deficient activation. Besides, using inhibitors we found that neither PI3K nor Erk1/2 are directly related with Nrf2 activation, indicating that these kinases have other targets. We conclude that redox signaling participates in cardioprotection triggered by iPostC through the action of kinase-dependent and -independent mechanisms and concurred with the downstream regulation of Nrf2-mediated antioxidant response to prolonged redox balance during long reperfusion.

Oxysterols in adipose tissue-derived mesenchymal stem cell proliferation and death.

Mesenchymal stem cells (MSCs) are multipotent cells characterized by self-renewal and cellular differentiation capabilities. Oxysterols comprise a very heterogeneous group derived from cholesterol through enzymatic and non-enzymatic oxidation. Potent effects in cell death processes, including cytoxicity and apoptosis induction, were described in several cell lines. Very little is known about the effects of oxysterols in MSCs. 7-ketocholesterol (7-KC), one of the most important oxysterols, was shown to be cytotoxic to human adipose tissue-derived MSCs. Here, we describe the short-term (24h) cytotoxic effects of cholestan-3α-5ß-6α-triol, 3,5 cholestan-7-one, (3α-5ß-6α)- cholestane-3,6-diol, 7-oxocholest-5-en-3ß-yl acetate, and 5ß-6ß epoxy-cholesterol, on MSCs derived from human adipose tissue. MSCs were isolated from adipose tissue obtained from three young, healthy women. Oxysterols, with the exception of 3,5 cholestan-7-one and 7-oxocholest-5-en-3ß-yl acetate, led to a complex mode of cell death that include apoptosis, necrosis and autophagy, depending on the type of oxysterol and concentration, being cholestan-3α-5ß-6α-triol the most effective. Inhibition of proliferation was also promoted by these oxysterols, but no changes in cell cycle were observed.

Tamoxifen inhibits mitochondrial oxidative stress damage induced by copper orthophenanthroline.

In this work, we studied the effect of tamoxifen and cyclosporin A on mitochondrial permeability transition caused by addition of the thiol-oxidizing pair Cu2+ -orthophenanthroline. The findings indicate that tamoxifen and cyclosporin A circumvent the oxidative membrane damage manifested by matrix Ca2+ release, mitochondrial swelling, and transmembrane electrical gradient collapse. Furthermore, it was found that tamoxifen and cyclosporin A prevent the generation of TBARs promoted by Cu2+ -orthophenanthroline, as well as the inactivation of the mitochondrial enzyme aconitase and disruption of mDNA. Electrophoretic analysis was unable to demonstrate a cross-linking reaction between membrane proteins. Yet, it was found that Cu2+ -orthophenanthroline induced the generation of reactive oxygen species. It is thus plausible that membrane leakiness is due to an oxidative stress injury.

Apoptotic effect of eugenol envolves G2/M phase abrogation accompanied by mitochondrial damage and clastogenic effect on cancer cell in vitro.

BACKGROUND: Eugenol (EUG) is a major phenolic compound present in clove whose anti-cancer properties have been demonstrated previously. These anti-cancer properties may involves the modulation of different mechanisms, including α-estrogen receptor (αER) in luminal breast cancer cells, COX-2 inhibition in melanoma cells or p53 and caspase-3 activation in colon cancer cells. HYPOTHESIS: EUG promotes a burst in ROS production causing cell-cycle perturbations, mitochondria toxicity and clastogenesis triggering apoptosis in melanoma breast- and cervix-cancer cells in vitro. METHODS: Morphological changes were evaluated through the light- and electronic- microscopy. Cell-cycle, ROS, PCNA and Apoptosis was detected by flow cytometry and clastogenicity was evaluated by Comet-assay. RESULTS: The results obtained herein pointed out that EUG promotes, increasing ROS production leading to abrogation of G2/M of phase of cell-cycle, and consecutively, clastogenesis in vitro. In addition, EUG induces Proliferation Cell Nuclear Antigen (PCNA) downregulation and decreasing in mitochondria potential (ΔΨm). Of note, a Bax up-regulation was also observed on cells treated with EUG. All of these findings cooperate in order to induce apoptosis in cancer cells. CONCLUSION: These promising results presented herein shed new light on the mechanisms of action of EUG suggesting a possible applicability of this phenylpropanoid as adjuvant in anti-cancer therapy.

ABCB1, ABCC1, and LRP gene expressions are altered by LDL, HDL, and serum deprivation in a human doxorubicin-resistant uterine sarcoma cell line.

Multidrug resistance (MDR) is the major cause of cancer treatment failure. The ATP-binding cassette-B1 (ABCB1) transporter, also known as MDR1 or P-glycoprotein, is thought to promote the efflux of drugs from cells. MDR is also associated with the multidrug resistance-associated protein 1 (ABCC1) and the lung resistance-related protein (LRP), a human major vault protein. Moreover, MDR has a complex relationship with lipids. The ABCB1 has been reported to modulate cellular cholesterol homeostasis. Conversely, cholesterol has been reported to modulate multidrug transporters. However, results reported to date are contradictory and confusing. The aim of this study was to investigate whether LDL, HDL, and serum deprivation could influence ABCB1, ABCC1, and LRP expression in a human doxorubicin-resistant uterine sarcoma cell line. ABCB1 and ABCC1 expression increased after 24 h of serum deprivation, and expression returned to basal levels after 72 h. LDL, depending on concentration, increased ABCB1, ABCC1, and LRP expression. ABCB1 expression increased at low HDL, and decreased at high HDL concentrations. We demonstrated that serum deprivation and lipoproteins, particularly LDL, modulated ABCB1 expression and, to a lesser extent, ABCC1 expression. This finding may link the phenomena of drug transport, cholesterol metabolism and cancer.

Short-term effects of 7-ketocholesterol on human adipose tissue mesenchymal stem cells in vitro.

Oxysterols comprise a very heterogeneous group derived from cholesterol through enzymatic and non-enzymatic oxidation. Among them, 7-ketocholesterol (7-KC) is one of the most important. It has potent effects in cell death processes, including cytoxicity and apoptosis induction. Mesenchymal stem cells (MSCs) are multipotent cells characterized by self-renewal and cellular differentiation capabilities. Very little is known about the effects of oxysterols in MSCs. Here, we describe the short-term cytotoxic effect of 7-ketocholesterol on MSCs derived from human adipose tissue. MSCs were isolated from adipose tissue obtained from two young, healthy women. After 24 h incubation with 7-KC, mitochondrial hyperpolarization was observed, followed by a slight increase in the level of apoptosis and changes in actin organization. Finally, the IC50 of 7-KC was higher in these cells than has been observed or described in other normal or cancer cell lines.

Organochlorine pesticide residues in bovine milk from organic farms in Chiapas, Mexico.

Thirty six samples of bovine milk were collected from Chiapas State, Mexico between January 2011 and December 2011 with the intention of identifying and quantifying organochlorine pesticide residues in organic farms. The analyses were done using gas chromatography with an electron capture detector (Ni(63)). In general the values found in raw milk were lower than the permissible limit proposed by FAO/WHO/Codex Alimentarius 2006. Average concentrations for alpha + beta HCH were 3.62 ng/g, gamma HCH 0.34 ng/g, heptachlor + epoxide 0.67 ng/g, DDT and isomers 1.53 ng/g, aldrin + dieldrin 0.77 ng/g, and endrin 0.66 ng/g (only present in samples from farm 2). The organic milk from Chiapas has shown low concentrations of pesticide residues in recent years and satisfies international and national regulations for commercialization.

Caracterização de uma nanopartícula lipídica semelhante à LDL (LDE) como vetor para RNA de interferência / Characterization of an lipid nanoparticle resembles LDL (LDE) with vector for interference RNA

As nanopartículas são consideradas promissores vetores para a liberação eficaz e segura de ácidos nucléicos para tipos específicos de célula ou tecido, proporcionando uma alternativa aos vetores virais para terapia gênica. No entanto, com a maioria destes sistemas não torna possível a entrega de oligonucleotídeos nas células in vivo de forma especifica. O uso de uma nanoemulsão funcionalmente semelhante à lipoproteina de baixa densidade poderia resolver esse problema, pois esta particula é capaz de direcionar o transporte das moléculas para a internalização celular através de receptores de LDL. Aqui, descreve-se um sistema lipidico semelhante à lipoproteína de baixa densidade, LDE, capaz de direcionar e liberar RNA de interferência (RNAi) para as células tumorais in vitro e in vivo em um modelo celular que expressa resistência a múltiplas drogas (células de sarcoma uterino; MESSA/ Dx5). Estudou-se também as caracteristicas de captação do complexo LDE-RNAi e a regulação especifica do gene mdr-1. Os resultados sugerem que a LDE é estavel e liga-se com alta afinidade aos RNAis permitindo que eles entrem nas células tumorais, com localização citoplasmática. Em conclusão, a LDE, por direcionar o RNAi a receptores de LDL favorece o silenciamento do gene mdr-1 por RNAi nas células MES-SA/Dx5 aumentando sua sensibilidade a quimioterápicos.

Nanoparticles are considered promising vectors for efficient and safe delivery of nucleic acids to specific types of cell or tissue, providing an alternative to viral vectors for gene therapy. However, most of these systems cannot target and deliver oligonucleotides to specific cells in vivo. The use of nanoemulsion functionally resemble low density emulsion could solve this problem, once particle is capable of direct the molecules transport to the cell through internalization in LDL receptors. Here, we describe a lipid system similar to low density lipoprotein, LDE, capable of targeting and release small interfering RNA (siRNA) to tumor cells in vitro and in vivo in a cell model that expresses multidrug resistance (sarcoma uterine cell line; MES-SA/Dx5). Were also studied the characteristics of uptake of the complex LDE-siRNA, as well as the downregulation of mdr-1 gene. The results suggest that LDE is stable and bind with high affinity to siRNAs allowing them to enter tumor cells, with cytoplasmic localization enhanced. In conclusion, LDE, binds to siRNA through LDL receptors, and promotes mdr-1 silenciament by RNAi in MES-sa/Dx5 cells, increasing their sensitivity to chemotherapeutics agents.