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Fluopsin C is an antibiotic compound derived from secondary metabolism of different microorganisms, which possesses antitumor, antibacterial, and antifungal activity. Related to fluopsin C antiproliferative activity, the aim of this study was to examine the following parameters: cytotoxicity, genotoxicity, cell cycle arrest, cell death induction (apoptosis), mitochondrial membrane potential (MMP), colony formation, and mRNA expression of genes involved in adaptive stress responses and cellular death utilizing a monolayer. In addition, a three-dimensional cell culture was used to evaluate the effects on growth of tumor spheroids. Fluopsin C was cytotoxic (1) producing cell division arrest in the G1 phase, (2) elevating expression of mRNA of the CDKN1A gene and (3) decrease in expression of mRNA H2AFX gene. Further, fluopsin C enhanced DNA damage as evidenced by increased expression of mRNA of GADD45A and GPX1 genes, indicating that reactive oxygen species (ROS) may be involved in the observed genotoxic response. Reticulum stress was also detected as noted from activation of the ribonuclease inositol-requiring protein 1 (IRE1) pathway, since a rise in mRNA expression of the ERN1 and TRAF2 genes was observed. During the cell death process, an increase in mRNA expression of the BBC3 gene was noted, indicating participation of this antibiotic in oncotic (ischemic) cell death. Data thus demonstrated for the first time that fluopsin C interferes with the volume of tumor spheroids, in order to attenuate their growth. Our findings show that fluopsin C modulates essential molecular processes in response to stress and cell death.

Zerumbone (ZER) is a phytochemical with antioxidant and antiproliferative properties. This study evaluated the cytoxicity of ZER combined with chemotherapeutic agents and the expression of mRNA genes related to cell cycle, cell death, xenobiotic metabolism, DNA damage, and endoplasmic reticulum (ER) stress in HepG2/C3A cells. ZER was cytotoxic (IC

We consider random changepoint segmented regression models to analyse data from a study conducted to verify whether treatment with stem cells may delay the onset of a symptom of amyotrophic lateral sclerosis in genetically modified mice. The proposed models capture the biological aspects of the data, accommodating a smooth transition between the periods with and without symptoms. An additional changepoint is considered to avoid negative predicted responses. Given the nonlinear nature of the model, we propose an algorithm to estimate the fixed parameters and to predict the random effects by fitting linear mixed models iteratively via standard software. We compare the variances obtained in the final step with bootstrapped and robust ones.

Background Cell culture (spheroid and 2D monolayer cultures) is an essential tool in drug discovery. Piperlongumine (PLN), a naturally occurring alkaloid present in the long pepper (Piper longum), has been implicated in the regulation of GSTP1 activity. In vitro treatment of cancer cells with PLN increases ROS (reactive oxygen species) levels and induces cell death, but its molecular mode of action has not been entirely elucidated. Methods In this study, we correlated the antiproliferative effects (2D and 3D cultures) of PLN (CAS 20069–09-4, Sigma-Aldrich) with morphological and molecular analyses in HepG2/C3A cell line. We performed assays for cytotoxicity (MTT), comet assays for genotoxicity, induction of apoptosis, analysis of the cell cycle phase, and analysis of the membrane integrity by flow cytometry. Relative expression of mRNA of genes related to proliferation, apoptosis, cell cycle control, metabolism of xenobiotics, and reticulum endoplasmic stress. Results PLN reduced the cell proliferation by the cell cycle arrest in G2/M. Changes in the mRNA expression for CDKN1A (4.9x) and CCNA2 (0.5x) of cell cycle control genes were observed. Cell death occurred due to apoptosis, which may have been induced by increased expression of proapoptotic mRNAs (BAK1, 3.1x; BBC3, 2.4x), and by an increase in 9 and 3/7 active caspases. PLN induced cellular injury by ROS generation and DNA damage. DNA damage induced MDM2 signaling (3.0x) associated with the appearance of the monastral spindle in mitosis. Genes associated with ROS degradation also showed increased mRNA expression (GSR, 2.0x; SOD1, 2.1x). PLN induce endoplasmic reticulum stress with the increase in the mRNA expression of ERN1 (4.5x) and HSPA14 (2.2x). The xenobiotic metabolism showed increased mRNA expression for CYP1A2 (2.2x) and CYP3A4 (3.4x). In addition to 2D culture, PLN treatment also inhibited the growth of 3D culture (spheroids). Conclusion Thus, the findings of our study show that several gene expression biomarkers (mRNAs) and monastral spindle formation indicated the many pathways of damage induced by PLN treatment that contributes to its antiproliferative effects. Graphical abstract

Oxidative stress (OS) is involved in the onset of various pathological processes, and sodium selenite (Na2SeO3) is known to have antioxidant activity. This study evaluated the cellular response of human HepG2/C3A cells supplemented with Na2SeO3 when exposed to hydrogen peroxide (H2O2)-induced OS. We analyzed cytotoxicity, cell proliferation, and genotoxicity in comparison with molecular data of mRNA and protein expression. The MTT and comet assays revealed that Na2SeO3 conferred cytoprotective and anti-genotoxic effects. In contrast, RTCA (Real-Time Cell Analysis) and flow cytometry analysis revealed that Na2SeO3 did not inhibit H2O2-induced anti-proliferative effects or cell cycle arrest (G2/M). Cells exposed simultaneously to Na2SeO3 and H2O2 showed overexpression of GPX1 mRNA, indicating that Na2SeO3 influenced the cellular antioxidant system. Furthermore, downregulation of CAT mRNA and SOD1 and PRX2 proteins induced by H2O2, was minimal after the Na2SeO3+H2O2 treatment. Although normalization of CCN2B mRNA expression by Na2SeO3 was observed after the Na2SeO3+H2O2 treatment, this was not observed for other genes such as CDKN1A, CDKN1C, and CDKN2B, which are related to cell cycle control, nor for GADD45A, which is involved in the cellular response to DNA damage. Furthermore, both CDKN1B and CDKN1C expression were downregulated in HepG2/C3A cells treated with Na2SeO3 only. Our results indicate that cellular response to Na2SeO3 involved the modulation of the antioxidant system. Na2SeO3 was unable completely recover HepG2/C3A cells from H2O2-induced oxidative damage, as evidenced by analysis of cell proliferation kinetics, cell cycle assay, and expression of key genes involved in cell cycle progression and response to DNA damage.

Background: Genistein (5,7-Dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) is the most abundant isoflavone in soybean, which has been associated with a lower risk of development of cancer and cardiovascular diseases. Of particular interest regarding cancer preventive properties of flavo-noids is their interaction with cytochrome P450 enzymes (CYPs). However, contradictory data report the effect of genistein on expression of СYPs enzymes. Objective: The aim of this study was to investigate the effects of genistein on cytochrome P450 (CYP) gene expression levels in human hepatocellular carcinoma (HepG2/C3A) and colon adenocarcinoma (HT29) cells. Methods: Real-time RT–PCR was used to examine the expression of genes families involved in xenobi-otic metabolism, such as CYP1 (CYP1A1, CYP1B1), CYP2 (CYP2E1, CYP2D6), CYP3 (CYP3A4); and of a family involved in the catabolism of the all-trans-retinoic acid (ATRA), CYP26 (CYP26A1, CYP26B1). Results: RT-qPCR data analysis showed that after 12 h of exposure of HepG2/C3A cells to genistein (5 and 50 µM) there was an upregulation of CYP1A1 and CYP1B1 and downregulation of CYP2D6, CYP26A1 and CYP26B1 mRNA levels. There was no change in the mRNA levels of CYP P450 genes in HT29 cells. Conclusion: Our results suggest that treatment with genistein in non-toxic concentrations may impact the expression level of CYPs involved in the biotransformation of xenobiotics and drug metabolizing en-zymes. Moreover, the downregulation of ATRA metabolism-related genes opens a new research path for the study of genistein as retinoic acid metabolism blocking agent for treating cancer and other patholo-gies.

Duchenne muscular dystrophy is the most common and severe form of progressive muscular dystrophy. Previous results showed an increased survival in double knockout mice (dko) when treated with adipose-derived CD146+ cells. In this study, we analyzed the effect of CD146+ cells compared to mesenchymal stem/stromal cells (MSCs) derived from the same human adipose sample when injected in the dko mouse model without immunosuppression. Both CD146+ cells and MSCs increased the survival of treated mice when compared to vehicle-injected mice, with a more prominent effect of CD146+ cells than MSCs. Both CD146+ cells and MSCs suppressed peripheral blood mononuclear cell proliferation, indicating immunomodulatory properties. Co-culture experiments showed that MSCs have a more inflammatory profile expression, and angiogenesis assay showed that CD146+ cells can improve blood vessel formation. CD146+ cells can extend survival of muscular dystrophy mice more efficiently than MSCs, possibly due to immunomodulatory and angiogenic properties. Further investigations focusing on exogenous CD146+ cell role in vivo will improve cell therapy understanding and effectiveness.

Studies that evaluated the mechanisms of action of Plumbagin (PLB) and its toxicity may contribute to future therapeutic applications of this compound. We investigate biomarker important in the mechanisms of action correlate the expression of mRNA with the cytotoxic and genotoxic effects of PLB on HepG2/C3A. In the analysis of cytotoxicity, PLB decreased cell viability and membrane integrity at concentrations ≥ 15μM. Xenobiotic-metabolizing system showed strong mRNA induction of CYP1A1, CYP1A2, and CYP3A4, suggesting extensive metabolization. PLB induced apoptosis and an increase in the mRNA expression of genes BBC3, CASP3, and CASP8. At a concentration of 15μM, there was a reduction in the expression of PARP1 mRNA and an increase in the expression of BECN1 mRNA, suggesting that PLB may also induce cell death by autophagy. PLB induced an arrest at the G2/M phase due to DNA damage, as observed in the comet assay. This damage is associated with the increased mRNA expression of genes p21, GADD45A, and H2AFX and with changes in the expression of proteins H2AX, p21, p53, Chk1, and Chk2. These results allow a better understanding of the cellular action of PLB and of its toxicity, thereby contributing to the development of PLB-based drugs, with markers of mRNA expression possibly playing a role as indicators for monitoring toxicity in human cells.

Plumbagin (PLB) is a phytochemical being used for centuries in traditional medicines. Recently, its capacity to inhibit the development of human tumors has been observed, through the induction of apoptosis, cell cycle arrest, and inhibition of angiogenesis and metastasis. Here we evaluated the mechanism of action of PLB in the kidney adenocarcinoma 786-O cell line, which are metabolizing cells important for toxicology studies. After the treatment with PLB, we observed increased apoptosis and cell cycle arrest in S and G2/M phases, starting at 5 µM. In addition, PLB was cytotoxic, genotoxic and induced loss of cell membrane integrity. Regarding gene expression, treatment with 7.5 µM PLB reduced the amount of MTOR, BCL2 and ATM transcripts, and increased CDKN1A (p21) transcripts. Phosphorylation levels of yH2AX was increased and MDM2 protein level was reduced following the treatment with PLB, demonstrating its genotoxic effect. Our results suggest that PLB acts in molecular pathways related to the control of proliferation and cell death in 786-O cells.

In amyotrophic lateral sclerosis (ALS), motor neurons die selectively. Therefore, initial symptoms that include fasciculation, spasticity, muscle atrophy, and weakness emerge following axons retraction and consequent muscles' denervation. Patients lose the ability to talk and swallow and rely on parenteral nutrition and assisted ventilation to survive. The degeneration caused by ALS is progressive and irreversible. In addition to the autonomous mechanism of neuronal cell death, non-autonomous mechanisms have been proved to be toxic for motor neurons, such as the activation of astrocytes and microglia. Among the cells being studied to unveil these toxic mechanisms are pericytes, cells that help keep the integrity of the blood-brain barrier and blood-spinal cord barrier. In this chapter, we aim to discuss the role of pericytes in ALS.

In amyotrophic lateral sclerosis (ALS), motor neurons die selectively. Therefore, initial symptoms that include fasciculation, spasticity, muscle atrophy, and weakness emerge following axons retraction and consequent muscles’ denervation. Patients lose the ability to talk and swallow and rely on parenteral nutrition and assisted ventilation to survive. The degeneration caused by ALS is progressive and irreversible. In addition to the autonomous mechanism of neuronal cell death, non-autonomous mechanisms have been proved to be toxic for motor neurons, such as the activation of astrocytes and microglia. Among the cells being studied to unveil these toxic mechanisms are pericytes, cells that help keep the integrity of the blood–brain barrier and blood–spinal cord barrier. In this chapter, we aim to discuss the role of pericytes in ALS.

Curcumin (Cur), is a pigment with antiproliferative activity but has some pharmacokinetic limitations, which led researchers to look for more effective structure analogs. This work investigated the effects of Cur and compared them with the two analogs, demethoxycurcumin (DeMC) and dimethoxycurcumin (DiMC), to elucidate their mechanisms of action. The cytotoxic, antiproliferative, and genotoxic effects these compounds were correlated based on gene expression analysis in the human renal adenocarcinoma cells (786-O). Cur decreased CYP2D6 expression and exhibited cytotoxic effects, such as inducing monopolar spindle formation and mitotic arrest mediated by the increase in CDKN1A (p21) mRNA. This dysregulation induced cell death through a caspase-independent pathway but was mediated by decrease in MTOR and BCL2 mRNA expression, suggesting that apoptosis occurred by autophagy. DeMC and DiMC had similar effects in that they induced monopolar spindle and mitotic arrest, were genotoxic, and activated GADD45A, an important molecule in repair mechanisms, and CDKN1A. However, the induction of apoptosis by DeMC was delayed and regulated by the decrease of antiapoptotic mRNA BCL.XL and subsequent activation of caspase 9 and caspase 3/7. DiMC treatment increased the expression of CYP1A2, CYP2C19, and CYP3A4 and exhibited higher cytotoxicity compared with other compounds. It induced apoptosis by increasing mRNA expression of BBC3, MYC, and CASP7 and activation of caspase 9 and caspase 3/7. These data revealed that different gene regulation processes are involved in cell death induced by Cur, DeMC, and DiMC. All three can be considered as promising chemotherapy candidates, with DiMC showing the greatest potency.

Aspidospermine is an indole alkaloid with biological properties associated with combating parasites included in the genera Plasmodium, Leishmania and Trypanossoma. The present study evaluated the cytotoxicity (resazurin test), genotoxicity (comet assay) and mechanism of action (gene expression analysis via qRT-PCR) of this alkaloid in human HepG2 cells. The results demonstrated that treatment with aspidospermine was both cytotoxic (starting at 75 μM) and genotoxic (starting at 50 μM). There was no significant modulation of the expression of the following genes: GSTP1 and GPX1 (xenobiotic metabolism); CAT (oxidative stress); TP53 and CCNA2 (cell cycle); HSPA5, ERN1, EIF2AK3 and TRAF2 (endoplasmic reticulum stress); CASP8, CASP9, CASP3, CASP7, BCL-2, BCL-XL BAX and BAX (apoptosis); and PCBP4, ERCC4, OGG1, RAD21 and MLH1 (DNA repair). At a concentration of 50 μM (non-cytotoxic, but genotoxic), there was a significant increase in the expression of CYP1A1 (xenobiotic metabolism) and APC (cell cycle), and at a concentration of 100 μM, a significant increase in the expression of CYP1A1 (xenobiotic metabolism), GADD153 (endoplasmic reticulum stress) and SOD (oxidative stress) was detected, with repression of the expression of GR (xenobiotic metabolism and oxidative stress). The results of treatment with aspidospermine at a 100 μM concentration (the dose indicated in the literature to achieve 89 % reduction of the growth of L. amazonensis) suggest that increased oxidative stress and an unfolded protein response (UPR) occurred in HepG2 cells. For the therapeutic use of aspidospermine (antiparasitic), chemical alteration of the molecule to achieve a lower cytotoxicity/genotoxicity in host cells is recommended.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease whose pathophysiology is poorly understood. Aiming to better understand the cause of motor neuron death, the use of experimental cell-based models increased significantly over the past years. In this scenario, much knowledge has been generated from the study of motor neurons derived from embryonic stem cells and induced pluripotent stem cells. These methods, however, have advantages and disadvantages, which must be balanced on experimental design. Preclinical studies provide valuable information, making it possible to combine diverse methods to build an expanded knowledge of ALS pathophysiology. In addition to using stem cells as experimental models for understanding disease mechanism, these cells had been quoted for therapy in ALS. Despite ethical issues involved in its use, cell therapy with neural stem cells stands out. A phase I clinical trial was recently completed and a phase II is on its way, attesting the method's safety. In another approach, mesenchymal stromal cells capable of releasing neuroregulatory and anti-inflammatory factors have also been listed as candidates for cell therapy for ALS, and have been admitted as safe in a phase I trial. Despite recent advances, application of stem cells as an actual therapy for ALS patients is still in debate. Here, we discuss how stem cells have been useful in modeling ALS and address critical topics concerning their therapeutic use, such as administration protocols, injection site, cell type to be administered, type of transplantation (autologous vs. allogeneic) among other issues with particular implications for ALS therapy.

In cancer, mesenchymal stem/stromal cells (MSCs) have been considered as vehicles for targeted delivery of drugs due to their inherent tropism toward primary and metastatic tumors. However, it is still unclear whether MSCs could be therapeutically explored without significant harm, since a great amound of evidence indicates that MSCs are able to exert both tumor-suppressive and pro-oncogenic effects. Here, we discuss how MSCs might adopt a pro- or anti-inflammatory profile in response to changes within the tumor microenvironment and how these features may lead to opposite outcomes in tumor development. Additionally, we address how differences in experimental design might impact interpretation and consistency of the current literature in this specific field. Finally, we point-out critical issues to be addressed at a pre-clinical stage, regarding safety and therapeutic effectiveness of MSCs application in cancer treatment.

Amyotrophic Lateral Sclerosis (ALS) is one of the most common adult-onset motor neuron disease causing a progressive, rapid and irreversible degeneration of motor neurons in the cortex, brain stem and spinal cord. No effective treatment is available and cell therapy clinical trials are currently being tested in ALS affected patients. It is well known that in ALS patients, approximately 50% of pericytes from the spinal cord barrier are lost. In the central nervous system, pericytes act in the formation and maintenance of the blood-brain barrier, a natural defense that slows the progression of symptoms in neurodegenerative diseases. Here we evaluated, for the first time, the therapeutic effect of human pericytes in vivo in SOD1 mice and in vitro in motor neurons and other neuronal cells derived from one ALS patient. Pericytes and mesenchymal stromal cells (MSCs) were derived from the same adipose tissue sample and were administered to SOD1 mice intraperitoneally. The effect of the two treatments was compared. Treatment with pericytes extended significantly animals survival in SOD1 males, but not in females that usually have a milder phenotype with higher survival rates. No significant differences were observed in the survival of mice treated with MSCs. Gene expression analysis in brain and spinal cord of end-stage animals showed that treatment with pericytes can stimulate the host antioxidant system. Additionally, pericytes induced the expression of SOD1 and CAT in motor neurons and other neuronal cells derived from one ALS patient carrying a mutation in FUS. Overall, treatment with pericytes was more effective than treatment with MSCs. Our results encourage further investigations and suggest that pericytes may be a good option for ALS treatment in the future. Graphical Abstract ᅟ.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder, caused by mutations in the dystrophin gene, affecting 1:3500–5000 boys worldwide. The lack of dystrophin induces degeneration of muscle cells and elicits an immune response characterized by an intensive secretion of pro-inflammatory cytokines. Immunoglobulins modulate the inflammatory response through several mechanisms and have been widely used as an adjuvant therapy for autoimmune diseases. Here we evaluated the effect of immunoglobulin G (IG) injected intraperitoneally in a severely affected double knockout (dko) mouse model for Duchenne muscular dystrophy. The IG dko treated mice were compared regarding activity rates, survival and histopathology with a control untreated group. Additionally, dendritic cells and naive lymphocytes from these two groups and WT mice were obtained to study in vitro the role of the immune system associated to DMD pathophysiology. We show that IG therapy significantly enhances activity rate and lifespan of dko mice. It diminishes muscle tissue inflammation by decreasing the expression of costimulatory molecules MHC, CD86 and CD40 and reducing Th1-related cytokines IFN-γ, IL-1β and TNF-α release. IG therapy dampens the effector immune responses supporting the hypothesis according to which the immune response accelerates DMD progression. As IG therapy is already approved by FDA for treating autoimmune disorders, with less side-effects than currently used glucocorticoids, our results may open a new therapeutic option aiming to improve life quality and lifespan of DMD patients.

Dimethoxycurcumin (DiMC), a synthetic analog of curcumin, was shown to have antiproliferative activity in human tumor cell lines. Therefore, we investigated its cytotoxic, antiproliferative, genotoxic, and apoptotic effect and correlated these evaluations with the expression of transcripts and proteins in the human hepatocellular carcinoma cell line (HepG2/C3A). Treatment with DiMC resulted in increased CYP2E1, CYP2C19 and CYP1A2 transcripts levels and was cytotoxic (≥10 μM). DiMC caused mitotic arrest by inducing monopolar spindle formation and was genotoxic increasing expression of the CDKN1A, GADD45A and PARP1 gene, key effectors in the cell cycle arrest and DNA repair pathways, respectively. This genotoxicity was caused by generation of reactive oxygen species and reduction of antioxidant proteins levels. Furthermore, we observed a decrease in important proteins involved in DNA repair. In addition to the observed apoptotic morphology and the presence of annexin labeling, we observed increased expression of BAK1 and CASP7 genes and caspase 3/7 protein activity, showing that these effects caused apoptosis through the intrinsic pathway in HepG2/C3A cells. Our results indicate that DiMC modulates important molecular targets leading to cell death even in metabolic competent cells models has considerable potential in anticancer therapy.

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder caused by null mutations in the dystrophin gene. Although the primary defect is the deficiency of muscle dystrophin, secondary events, including chronic inflammation, fibrosis, and muscle regeneration failure are thought to actively contribute to disease progression. Despite several advances, there is still no effective therapy for DMD. Therefore, the potential regenerative capacities, and immune-privileged properties of mesenchymal stromal cells (MSCs), have been the focus of intense investigation in different animal models aiming the treatment of these disorders. However, these studies have shown different outcomes according to the sources from which MSCs were obtained, which raise the question whether stem cells from distinct sources have comparable clinical effects. Here, we analyzed the protein content of the secretome of MSCs, isolated from three different sources (adipose tissue, skeletal muscle, and uterine tubes), obtained from five donors and evaluated their in vitro properties when cocultured with DMD myoblasts. All MSC lineages showed pathways enrichment related to protein metabolic process, oxidation-reduction process, cell proliferation, and regulation of apoptosis. We found that MSCs secretome proteins and their effect in vitro vary significantly according to the tissue and donors, including opposite effects in apoptosis assay, indicating the importance of characterizing MSC secretome profile before its use in animal and clinical trials. Despite the individual differences a pool of conditioned media from all MSCs lineages was able to delay apoptosis and enhance migration when in contact with DMD myoblasts.

Adipose tissue is considered an important source of mesenchymal stromal cells since it is an abundant tissue with great distribution throughout the body and human adipose-derived stem cells (hASCs) have high proliferation rates in vitro. These adult stromal cells have the ability to differentiate into tissues including bone, cartilage and adipose in vitro, and also display some biological functions such as trophic, paracrine and immunomodulatory effects that may have the greatest therapeutic impact in vivo for diverse pathologies. The considerable therapeutic potential of hASCs turned them of substantial interest in many areas of research. hASCs transplantation is currently being tested in more than 70 clinical trials for many diseases, including bone fractures, graft versus host disease, multiple sclerosis, brain injury, acute respiratory distress syndrome, idiopathic pulmonary fibrosis and diabetes. In this chapter, we will describe the protocols for hASCs isolation and characterization, besides summarizing current reported studies about their potential use.

A Esclerose Lateral Amiotrófica (ELA), também conhecida como Doença de Lou Gehrig, é a forma mais comum de doença do neurônio motor. Tem início geralmente tardio (4ª/5ª década de vida), afetando tanto os neurônios motores superiores quanto os inferiores. A degeneração provocada pela ELA é progressiva e irreversível. Em geral, a evolução da doença é rápida, levando os pacientes ao óbito entre 3 e 5 anos após o início dos sintomas, devido principalmente à falência respiratória. Atualmente, o único medicamento liberado pelo FDA (Food and Drug Administration) para o uso em ELA é o Riluzol, que tem um efeito mínimo na expectativa de vida dos pacientes. Neste cenário, a terapia celular vem sendo avaliada como uma possível alternativa. Estudos pré-clínicos indicam efeitos benéficos do tratamento de camundongos SOD1 (modelo animal para ELA) com células estromais mesenquimais ou simplesmente células mesenquimais (MSCs), atribuída principalmente à ação de fatores solúveis. Aqui propusemos o uso de pericitos, uma linhagem celular ainda não testada para tratamento pré-clinico em modelo murinho de ELA. Pericitos são células perivasculares que circundam células endoteliais e que desempenham importantes funções celulares como por exemplo participação da formação e manutenção da barreira hematoencefálica, essencial para proteger o sistema nervoso central de danos em doenças neurodegenerativas. Dessa forma, este trabalho pretendeu comparar o potencial terapêutico de células mesenquimais e pericitos obtidos do tecido adiposo humano de um mesmo doador, em camundongos SOD1. Para tal, testes físicos (peso, PaGE, motor score, rotarod) foram aplicados semanalmente e a sobrevida dos animais foi avaliada. Os resultados demonstram que, com exceção dos benefícios observados nos testes do PaGE e do motor score em uma fase mais inicial da doença, o tratamento com MSCs ou pericitos não resulta em efeitos significativos no quadro clínico de camundongos SOD1 do sexo feminino. Para os machos, o tratamento com pericitos se destaca em relação aos tratamentos com MSCs ou HBSS (veículo), resultando em efeitos benéficos na sobrevida e em determinadas funções motoras dos animais, com destaque para os testes do motor score e do rotarod, onde há uma melhora na fase inicial da doença. A análise da expressão gênica no cérebro e na medula de animais em fase final da doença sugere que o tratamento de machos com pericitos é capaz de estimular as defesas antioxidantes do animal. Ainda nestes órgãos, não foram encontrados vestígios das células humanas injetadas, indicando um possível efeito sistêmico das mesmas Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig\'s disease, is the most common form of motor neuron disease. Most cases are characterized by an adult onset of symptoms, usually in the fourth or fifth decade of life, affecting both upper and lower motor neurons. The degeneration caused by ALS is progressive and irreversible. On average, the survival ranges from 3 to 5 years after onset, mainly due to respiratory failure. Currently, the only Food and Drug Administration (FDA)-approved medication for this disorder is Riluzole, but its effects on survival are minimal. In this scenario, cell therapy is being evaluated as a possible alternative. Preclinical studies indicate beneficial effects of treatment of SOD1 mice (animal model for ALS) with mesenchymal stromal cells or simply mesenchymal cells (MSCs), mainly attributed to the action of soluble factors. Here we propose the use of pericytes, a cell line not yet tested for preclinical treatment in of ALS. Pericytes are perivascular cells surrounding endothelial cells and play important cellular roles such as assistance of formation and maintenance of the blood-brain barrier, which is essential to protect the central nervous system from damage in neurodegenerative diseases. Thus, this study sought to compare the therapeutic potential of mesenchymal cells and pericytes, both obtained from the same human adipose tissue, in SOD1 mice. For this purpose, survival and physical performance (weight, PaGE, motor score and rotarod) were evaluated. Except for the benefits observed in PaGE and the motor score tests in an early stage of the disease, treatment with MSCs and pericytes does not result in significant effects on disease progression of SOD1 female mice. For males, treatment with pericytes stands out compared to treatment with MSCs or HBSS (vehicle), resulting in beneficial effects on survival and in certain physical functions of the animals, particularly for the motor score and rotarod tests, where improvement was observed in the initial stage of the disease. The analysis of gene expression in the brain and spinal cord in end-stage animals suggests that treatment of males with pericytes can stimulate the animals\' antioxidant defense. No traces of injected human cells were observed in brain or spinal cord of mice, indicating a possible systemic effect of the transplant

SPOAN syndrome is a neurodegenerative disorder mainly characterized by spastic paraplegia, optic atrophy and neuropathy (SPOAN). Affected patients are wheelchair bound after 15 years old, with progressive joint contractures and spine deformities. SPOAN patients also have sub normal vision secondary to apparently non-progressive congenital optic atrophy. A potential causative gene was mapped at 11q13 ten years ago. Here we performed next-generation sequencing in SPOAN-derived samples. While whole-exome sequencing failed to identify the causative mutation, whole-genome sequencing allowed to detect a homozygous 216-bp deletion (chr11.hg19:g.66,024,557_66,024,773del) located at the non-coding upstream region of the KLC2 gene. Expression assays performed with patient’s fibroblasts and motor neurons derived from SPOAN patients showed KLC2 overexpression. Luciferase assay in constructs with 216-bp deletion confirmed the overexpression of gene reporter, varying from 48 to 74%, as compared with wild-type. Knockdown and overexpression of klc2 in Danio rerio revealed mild to severe curly-tail phenotype, which is suggestive of a neuromuscular disorder. Overexpression of a gene caused by a small deletion in the non-coding region is a novel mechanism, which to the best of our knowledge, was never reported before in a recessive condition. Although the molecular mechanism of KLC2 up-regulation still remains to be uncovered, such example adds to the importance of non-coding regions in human pathology Fil: Melo, Uira S.. Universidade de Sao Paulo; Brasil Fil: Macedo Souza, Lucia I.. Universidade de Sao Paulo; Brasil Fil: Figueiredo, Thalita. Federal University of Paraiba; Brasil. Paraiba State University; Brasil Fil: Muotri, Alysson R. University of California at San Diego; Estados Unidos Fil: Gleeson, Joseph G.. The Rockefeller University; Estados Unidos Fil: Coux, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Armas, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Calcaterra, Nora Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Kitajima, João P.. Mendelics Genomic Analysis; Brasil Fil: Amorim, Simone. Universidade de Sao Paulo; Brasil Fil: Olávio, Thiago R.. Universidade de Sao Paulo; Brasil Fil: Griesi Oliveira, Karina. Universidade de Sao Paulo; Brasil Fil: Coatti, Giuliana C.. Universidade de Sao Paulo; Brasil Fil: Rocha, Clarissa R.R. Universidade de Sao Paulo; Brasil Fil: Martins Pinheiro, Marinalva. Universidade de Sao Paulo; Brasil Fil: Menck, Carlos F.M.. Universidade de Sao Paulo; Brasil Fil: Zaki, Maha S.. National Research Center. EL Cairo; Egipto Fil: Kok, Fernando. Universidade de Sao Paulo; Brasil Fil: Zatz, Mayana. Universidade de Sao Paulo; Brasil Fil: Santos, Silvana. Federal University of Paraiba; Brasil. Paraiba State University; Brasil

A aspidospermina é um alcalóide com propriedades biológicas no combate aos parasitos inclusos nos gêneros Plasmodium, Leishmania e Trypanossoma. Testes toxicológicos em células humanas devem ser realizados para a avaliação de riscos aos seres humanos, visando o uso futuro do composto como medicamento contra tais parasitos. Assim, o objetivo do presente estudo foi avaliar a citotoxicidade (ensaio da resazurina) a genotoxicidade (ensaio do cometa) e o mecanismo de ação (expressão gênica por qRT-PCR) deste alcalóide em sistema de cultura de células metabolizadoras HepG2. Os resultados demonstram que o tratamento com aspidospermina foi citotóxico a partir da concentração de 75.M e apresentou efeito genotóxico a partir da concentração de 50.M. Para esta mesma concentração de 50.M (não citotóxica e genotóxica), houve aumento significativo da expressão de CYP1A1 e APC, em relação ao controle não tratado. Para a concentração de 100M, houve aumento significativo da expressão de CYP1A1, GADD153 e SOD, e repressão da expressão de GR. Os resultados indicam que o tratamento com aspidospermina a 100μM, dose que comprovadamente provoca redução de 89% do crescimento de L. amazonenses, pode desencadear o aumento do estresse oxidativo, aumento da resposta UPR e direcionamento à apoptose em células HepG2. Em virtude dos efeitos relatados, para que a aspidospermina seja utilizada com segurança, recomenda-se alteração química da molécula inicial visando uma menor citotoxicidade/genotoxicidade em células hospedeiras. Abstract.

Diversos estudos indicam o selenito de sódio (uma forma inorgânica do micronutriente selênio) como um composto com potencial para o tratamento do câncer. Análises em linhagens de células tumorais têm demonstrado a ação apoptótica, anti-carcinogênica e quimiopreventiva deste composto. No entanto, o seu mecanismo de ação em linhagens de células não-tumorais ainda é pouco compreendido, sendo este, um importante requisito para o desenvolvimento de novas drogas. As células de linhagem não-tumoral de queratinócitos –HaCat, têm se mostrado um excelente modelo para estudos toxicológicos. Assim, este estudo teve como objetivo avaliar o potencial citotóxico (ensaio do MTT), a genotoxicidade (ensaio do cometa) e o mecanismo de ação (expressão gênica por qRT-PCR) do selenito de sódio em células HaCat, e analisar os seus efeitos na membrana celular, interferência no ciclo celular e indução de apoptose por citometria de fluxo. Os resultados demonstram que o tratamento com selenito de sódio por 24h de exposição apresentou citotóxicidade a partir da concentração de 10µM, apresentando alterações no padrão morfológico das células com o aparecimento de grânulos citoplasmáticos, porém não apresentou efeito genotóxico. Os dados obtidos por meio da citometria de fluxo revelaram que o selenito de sódio foi indutor de danos na membrana celular, de apoptose, e interfere no ciclo celular. Em 12h de tratamento com 10 µM do selenito de sódio, notou-se redução significativa na expressão dos genes mTOR e ATR e um aumento na expressão do gene PUMA em relação ao controle. Entretanto, não foi encontrado alteração significativa na expressão das caspases (CASP 3, CASP 7, CASP 8 e CASP 9) bem como dos genes PARP 1, BIRC 5, BECN 1 e c-MYC, o que indica que o selenito de sódio induz a apoptose por mecanismo independente de caspases em células HaCat. Além disso, os genes analisados relacionados ao estresse oxidativo (CATB, GPX 1 e SOD 1), e os demais envolvidos no dano e reparo do DNA (ATM, GADD 45A, H2AX e MDM 2) e no ciclo celular (CCNA 1, CCNB 1, CCNB 2, CCND 1, CHK 1, CHK 2, p53 e p21) não apresentaram alteração significativa quando comparados com o controle. Através da análise de cinética celular em tempo real notou-se que o selenito de sódio interfere na proliferação de queratinócitos por, pelo menos, 72h após o tratamento. Com base nos efeitos relatados, pode-se inferir que o selenito de sódio apresenta um efeito tóxico para as células não-tumorais em concentrações que são semelhantes a testes em linhagens células tumorais, alterando o padrão morfológico e a curva de proliferação celular, modulando o padrão de expressão gênica induzindo a célula à apoptose mediado pelo PUMA. Several studies indicate sodium selenite (an inorganic form of the selenium micronutrient) as a compound with potential for the treatment of cancer. Analyzes in tumor cell lines there are demonstrated the apoptotic, anti-carcinogenic and chemopreventive action of this compound. However, its mechanism of action in non-tumor cell lines is still poorly understood, and this is an important requirement for the development of new drugs. Non-tumor cell lines of keratinocytes-HaCat, have been shown to be an excellent model for toxicological studies. This study evaluated the cytotoxic potential (MTT assay), genotoxicity (comet assay) and mechanism of action (qRT-PCR gene expression) of sodium selenite in HaCat cells, and to analyze its effects in the cell membrane, interference in the cell cycle and induction of apoptosis by flow cytometry. The results show that treatment with sodium selenite for 24h was cytotoxic from the concentration of 10 μM, presenting changes in cell morphology with the appearance of cytoplasmic granules, but did not resulted in genotoxic effect. The flow cytometry data showed the sodium selenite was inducer of cell membrane damage, apoptosis, and interferes with the cell cycle. In 12h treatment sodium selenite 10 μM, significant reduction in mTOR and ATR gene expression and an increase in PUMA gene expression was observed in relation to the control. However, there was no significant change in the expression of caspases (CASP 3, CASP 7, CASP 8 and CASP 9) as well as PARP 1, BIRC 5, BECN 1 and c-MYC genes, indicating that sodium selenite induces Apoptosis by caspase-independent mechanism in HaCat cells. In addition, the analyzed genes related to oxidative stress (CAT B, GPX 1 and SOD 1), and the others involved in DNA damage and repair (ATM, GADD 45A, H2AX and MDM 2) and in the cell cycle (CCNA 1, CCNB 1, CCNB 2, CCND 1, CHK 1, CHK 2, p53 and p21) showed no significant change when compared to the control. Through the real-time cell kinetics analysis we noticed that sodium selenite interferes in the proliferation of HaCat for at least 72h after treatment. Our results suggest that sodium selenite has a toxic effect to non-tumor cells at such like concentration in tumor cell lines tests, altering the morphological arrangement and the cell proliferation curve, modulating the pattern of gene expression , and inducing the cell to PUMA-mediated apoptosis.