Effects of leptin on proliferation and differentiation of hypoxic rat retinal progenitor cells in vitro / 南方医科大学学报

OBJECTIVE@#To investigate the the effects of leptin on the proliferation, differentiation and PTEN expression of rat retinal progenitor cells (RPCs) cultured under hypoxic condition.@*METHODS@#SD rat RPCs were cultured in normoxic conditions or exposed to hypoxia in the presence of 0, 0.3, 1.0, 3.0, 10, and 30 nmol/L leptin for 12, 48 and 72 h, and the cell viability was assessed using cell counting kit 8 (CCK 8) assay. The RPCs in primary culture were divided into control group, hypoxia group, and hypoxia+leptin group, and after 48 h of culture, the cell medium was replaced with differentiation medium and the cells were further cultured for 6 days. Immunofluorescence staining was employed to detect the cells positive for β-tubulin III and GFAP, and Western blotting was used to examine the expression of PTEN at 48 h of cell culture.@*RESULTS@#The first generation of RPCs showed suspended growth in the medium with abundant and bright cellular plasma and formed mulberry like cell spheres after 2 days of culture. Treatment with low-dose leptin (below 3.0 nmol/L) for 48 h obviously improved the viability of RPCs cultured in hypoxia, while at high concentrations (above 10 nmol/L), leptin significantly suppressed the cell viability (P < 0.05). The cells treated with 3.0 nmol/L leptin for 48 h showed the highest viability (P < 0.05). After treatment with 3.0 nmol/L leptin for 48 h, the cells with hypoxic exposure showed similar GFAP and β-tubulin Ⅲ positivity with the control cells (P>0.05), but exhibited an obvious down-regulation of PTEN protein expression compared with the control cells (P < 0.05).@*CONCLUSION@#In rat RPCs with hypoxic exposure, treatment with low dose leptin can promote the cell proliferation and suppress cellular PTEN protein expression without causing significant effects on cell differentiation.

Chemerin promotes proliferation and migration of ovarian cancer cells by upregulating expression of PD-L1 / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Ovarian cancer is the third-most-common malignant reproductive tumor in women. According to the American Cancer Society, it has the highest mortality rate of gynecological tumors. The five-year survival rate was only 29% during the period from 1975 to 2008 (Reid et al., 2017). In recent decades, the five-year survival rate of ovarian cancer has remained around 30% despite continuous improvements in surgery, chemotherapy, radiotherapy, and other therapeutic methods. However, because of the particularity of the volume and location of ovarian tissue, the early symptoms of ovarian cancer are hidden, and there is a lack of highly sensitive and specific screening methods. Most patients have advanced metastasis, including abdominal metastasis, when they are diagnosed (Reid et al., 2017). Therefore, exploring the mechanism of ovarian cancer metastasis and finding early preventive measures are key to improving the survival rate and reducing mortality caused by ovarian cancer.

Puerarin Inhibits the Proliferation,Invasion,and Migration of Non-small Cell Lung Cancer Cells through Regulating miR-490/Denticleless E3 Ubiquitin Protein Ligase / 中国医学科学院学报

Objective To explore the mechanism of puerarin inhibiting the proliferation,invasion,and migration of non-small cell lung cancer cells. Methods A549 cells were cultured and treated with different concentrations of puerarin.The inhibition rate (IR) on cell proliferation was detected by CCK-8,and qRT-PCR was performed to detect the mRNA levels of miR-490 and denticleless E3 ubiquitin protein ligase(DTL).Double luciferase reporter assay was employed to identify the targets of miR-490 and DTL based on the establishment of NC mimic group,miR-490 mimic group,NC inhibitor group,and miR-490 inhibitor group.The cells treated by 20 μmol/L puerarin were classified into six groups:DMSO,puerarin,puerarin+NC inhibitor,puerarin+miR-490 inhibitor,puerarin+miR-490 inhibitor+Si-NC,and puerarin+miR-490 inhibitor+Si-DTL.Transwell was used to detect cell migration and invasion.Western blotting was performed to detect the protein levels of epithelial-mesenchymal transition-related markers E-cadherin,N-cadherin,and Vimentin. Results With the increase in puerarin concentration,the IR gradually elevated (F=105.375,P<0.001),miR-490 expression gradually increased (F=32.919,P<0.001),and DTL expression gradually decreased (F=116.120,P<0.001).Compared with NC mimic group,miR-490 mimic group had decreased luciferase activity (t=7.762,P=0.016),raised miR-490 mRNA level (t=13.319,P<0.001),and declined DTL mRNA level (t=7.415,P=0.002).Compared with those in NC inhibitor group,miR-490 demonstrated decreased mRNA level (t=9.523,P=0.001) and DTL presented increased mRNA level (t=11.305,P<0.001) in miR-490 inhibitor group.Western blotting showed that the protein level of DTL was higher in NC mimic group (t=7.953,P=0.001) than in miR-490 mimic group and higher in miR-490 inhibitor group than in NC inhibitor group (t=10.552,P<0.001).Compared with DMSO group,puerarin group showed up-regulated mRNA level of miR-490 (t=10.255,P=0.001) while down-regulated mRNA level of DTL (t=6.682,P=0.003).Compared with those in puerarin+NC inhibitor group,the mRNA level of miR-490 declined (t=10.995,P<0.001) while that of DTL raised (t=12.478,P<0.001) in puerarin+miR-490 inhibitor group.The mRNA level of miR-490 had no significant difference between puerarin+miR-490 inhibitor+Si-NC group and puerarin+miR-490 inhibitor+Si-DTL group (t=1.081,P=0.341),and that of DTL was lower in the latter group (t=14.321,P<0.001).The protein level of DTL was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=11.423,P<0.001),and lower in puerarin+miR-490 inhibitor+Si-DTL group than in puerarin+miR-490 inhibitor+Si-NC group (t=12.080,P<0.001).Compared with DMSO group,puerarin group showed inhibited cell proliferation (F=129.27,P<0.001).The activity of cell proliferation was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (F=75.12,P<0.001),and higher in puerarin+miR-490 inhibitor+Si-NC group than in puerarin+miR-490 inhibitor+Si-DTL group (F=52.59,P<0.001).Compared with DMSO group,puerarin group had suppressed cell migration (t=8.963,P=0.001).The cell migration ability was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=12.117,P<0.001) and higher in puerarin+miR-490 inhibitor+Si-NC group than in puerarin+miR-490 inhibitor+Si-DTL group (t=12.934,P<0.001).Puerarin group showed weakened cell invasion ability compared with DMSO group (t=4.710,P=0.009).The cell invasion ability was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=13.264,P<0.001) and lower in puerarin+miR-490 inhibitor+Si-DTL group than in puerarin+miR-490 inhibitor+Si-NC group (t=13.476,P<0.001).Compared with DMSO group,puerarin group showed up-regulated protein level of E-cadherin (t=7.137,P=0.002) while down-regulated protein levels of N-cadherin (t=8.828,P=0.001) and vimentin (t=6.594,P=0.003).Compared with those in puerarin+NC inhibitor group,the protein level of E-cadherin (t=12.376,P<0.001) decreased while those of N-cadherin (t=13.436,P<0.001) and vimentin (t=11.467,P<0.001) increased in puerarin+miR-490 inhibitor group.Compared with puerarin+miR-490 inhibitor+Si-NC group,puerarin+miR-490 inhibitor+Si-DTL group up-regulated the protein level of E-cadherin (t=13.081,P<0.001) while down-regulated the protein levels of N-cadherin (t=10.835,P<0.001) and vimentin (t=11.862,P<0.001). Conclusion Puerarin could inhibit the proliferation,invasion,and migration of non-small cell lung cancer cells by up-regulating miR-490 and down-regulating DTL.

Sideritis perfoliata inhibits cell proliferation, induces apoptosis and exhibits cellular antioxidant activity in cervical cancer cells / Sideritis perfoliata inhibe la proliferación celular, induce apoptosis y exhibe actividad antioxidante celular en células de cáncer de cuello uterino

In this study, it was aimed to determine the antioxidant and anticancer activities of Sideritis perfoliata methanolic extract (SPE) on cervical cancer cells (HeLa). Different doses (25, 50,100 and 200 µg/mL) of SPE were used to determine proliferation of HeLa cells by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) staining method. Induction of apoptosis was determined by Annexine-V and propidium iodide staining method. Interleukin (IL) 6-8 levels were measured by ELISA method. Antioxidant activities of SPE were determined by DPPH, DNA (plasmid pBR322) protecting and cellular antioxidant activity tests. Some phytochemicals of SPE were also screened by LC-MS-MS. It was determined that SPE reduced the proliferation of HeLa cells and also induced apoptosis. IL6-8 levels importantly decreased at 200 µg/mL. SPE exhibited moderately antioxidant activities in tests used. Among the phenolics identified, vanillic acid had the highest amount. As a result, it was determined to have the anticancer activity of SPE by decreasing cell proliferation, inducing apoptosis and decreasing IL6-8 in HeLa cells.

En este estudio, se tuvo como objetivo determinar las actividades antioxidantes y anticancerígenas del extracto metanólico de Sideritis perfoliata (SPE) en las células de cáncer de cuello uterino (HeLa). Se utilizaron diferentes dosis (25, 50, 100 y 200 µg/mL) de SPE para determinar la proliferación de células HeLa mediante el método de tinción con bromuro de 3-[4,5-dimetiltiazol-2-il] -2,5-difenil-tetrazolio (MTT). La inducción de apoptosis se determinó mediante el método de tinción con anexina-V y yoduro de propidio. Los niveles de interleucina (IL) 6-8 se midieron mediante el método ELISA. Las actividades antioxidantes de SPE se determinaron mediante pruebas de DPPH, protección de ADN (plásmido pBR322) y actividad antioxidante celular. Algunos fitoquímicos de SPE también se analizaron mediante LC-MS-MS. Se determinó que SPE redujo la proliferación de células HeLa y también indujo apoptosis. Los niveles de IL6-8 disminuyeron de manera importante a 200 µg/mL. SPE mostró actividades moderadamente antioxidantes en las pruebas utilizadas. Entre los fenólicos identificados, el ácido vainílico tuvo la mayor cantidad. Como resultado, se determinó que tenía la actividad anticancerígena de SPE al disminuir la proliferación celular, inducir apoptosis y disminuir la IL6-8 en las células HeLa.

Nuclear Factor-κB Signaling Mediates Antimony-induced Astrocyte Activation / 生物医学与环境科学（英文）

Objective@#Antimony (Sb) has recently been identified as a novel nerve poison, although the cellular and molecular mechanisms underlying its neurotoxicity remain unclear. This study aimed to assess the effects of the nuclear factor kappa B (NF-κB) signaling pathway on antimony-induced astrocyte activation.@*Methods@#Protein expression levels were detected by Western blotting. Immunofluorescence, cytoplasmic and nuclear fractions separation were used to assess the distribution of p65. The expression of protein in brain tissue sections was detected by immunohistochemistry. The levels of mRNAs were detected by Quantitative real-time polymerase chain reaction (qRT-PCR) and reverse transcription-polymerase chain reaction (RT-PCR).@*Results@#Antimony exposure triggered astrocyte proliferation and increased the expression of two critical protein markers of reactive astrogliosis, inducible nitric oxide synthase (iNOS) and glial fibrillary acidic protein (GFAP), indicating that antimony induced astrocyte activation @*Conclusion@#Antimony activated astrocytes by activating the NF-κB signaling pathway.

mRNAs biomarker related to the control of proliferation and cell death in HepG2/C3A spheroid and monolayer cultures treated with piperlongumine

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 20069­09-4, 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

Lidocaine in oncological surgery: the role of blocking in voltage-gated sodium channels. A narrative review / Lidocaína em cirurgia oncológica: o papel do bloqueio dos canais de sódio dependentes de voltagem. Revisão narrativa

Abstract Background: The current evidence suggests that oncological surgery, which is a therapy used in the treatment of solid tumors, increases the risk of metastasis. In this regard, a wide range of tumor cells express Voltage-Gated Sodium Channels (VGSC), whose biological roles are not related to the generation of action potentials. In epithelial tumor cells, VGSC are part of cellular structures named invadopodia, involved in cell proliferation, migration, and metastasis. Recent studies showed that lidocaine could decrease cancer recurrence through its direct effects on tumor cells and immunomodulatory properties on the stress response. Objective: The aim of this narrative review is to highlight the role of VGSC in tumor cells, and to describe the potential antiproliferative effect of lidocaine during the pathogenesis of metastasis. Contents: A critical review of literature from April 2017 to April 2019 was performed. Articles found on PubMed (2000-2019) were considered. A free text and MeSH-lidocaine; voltage-gated sodium channels; tumor cells; invadopodia; surgical stress; cell proliferation; metastasis; cancer recurrence - for articles in English, Spanish and Portuguese language - was used. A total of 62 were selected. Conclusion: In animal studies, lidocaine acts by blocking VGSC and other receptors, decreasing migration, invasion, and metastasis. These studies need to be replicated in humans in the context of oncological surgery.

Resumo Justificativa: As evidências atuais sugerem que a cirurgia oncológica, usada no tratamento de tumores sólidos, aumenta o risco de metástase. Nesse sentido, uma ampla gama de células tumorais expressa Canais de Sódio Dependentes de Voltagem (CSDV), cujos papéis biológicos não estão relacionados à produção de potencial de ação. Nas células epiteliais tumorais, o CSDV é parte integrante de estruturas celulares denominadas invadópodes, que participam da proliferação, migração e metástase celular. Estudos recentes mostraram que a lidocaína pode diminuir a recorrência do câncer através de efeitos diretos nas células tumorais e de propriedades imunomoduladoras na resposta ao estresse. Objetivo: O objetivo desta revisão narrativa é analisar o papel do CSDV nas células tumorais e descrever o possível efeito antiproliferativo da lidocaína na patogênese das metástases. Conteúdo: Foi realizada uma revisão crítica da literatura de Abril de 2017 a Abril de 2019. Os artigos encontrados no PubMed (2000 − 2019) foram analisados. Pesquisamos textos de linguagem livre e descritores MeSH-lidocaína; canais de sódio dependentes de voltagem; células tumorais; invadópodes; estresse cirúrgico; proliferação celular; metástase; recorrência do câncer − em artigos publicados em inglês, espanhol e português. Foram selecionadas 62 publicações. Conclusão: Em estudos empregando animais, a lidocaína atua bloqueando o CSDV e outros receptores, diminuindo a migração, invasão e metástase. Esses estudos precisam ser replicados em humanos submetidos a cirurgia oncológica.

Cichoric acid from extracted Echinacea purpurea induces the proliferation and apoptosis of peripheral blood mononuclear cells from yaks

BACKGROUND: Cichoric acid (CA) is extracted from Echinacea purpurea. It is well known and widely used for its immunological function. However, the effect of CA on peripheral blood mononuclear cells (PBMCs) from yaks is still unclear. This study investigated the potential influences of CA on the proliferation, cytokine induction, and apoptosis of PBMCs from Datong yak in vivo, and aimed to provide a basis for exploring the pharmacological activities of CA on yaks. RESULTS: In this study, CA promoted PBMCs proliferation by combining concanavalin A (Con A) and exhibited a dose-dependent effect as demonstrated by a Cell Counting Kit-8. The concentration of 60 µg/ml CA was the best and promoted the transformation from the G0/G1 phase to the S and G2/M phases with Con A. Furthermore, 60 µg/ml CA significantly increased IL-2, IL-6, and IFN-γ levels and PCNA, CDK4 and Bcl-2 expression levels, but it significantly inhibited the TP53, Bax, and Caspase-3 expression levels. Transcriptome analysis revealed a total of 6807 differentially expressed genes (DEGs) between the CA treatment and control groups. Of these genes, 3788 were significantly upregulated and 3019 were downregulated. Gene Ontology and pathway analysis revealed that DEGs were enriched in cell proliferation and immune function signaling pathways. The expression level of some transcription factors (BTB, Ras, RRM_1, and zf-C2H2) and genes (CCNF, CCND1, and CDK4) related to PBMCs proliferation in yaks were significantly promoted after CA treatment. By contrast, anti-proliferation-associated genes (TP53 and CDKN1A) were inhibited. CONCLUSIONS: In summary, CA could regulate the immune function of yaks by promoting proliferation and inhibiting inflammation and apoptosis of PBMCs.

Toxicity evaluation of water extract of tissue-cultured Taraxacum formosanum by acute, subacute administration, and Ames test

BACKGROUND: Taraxacum species (commonly known as dandelion) used as herbal medicine have been reported to exhibit an antiproliferative effect on hepatoma cells and antitumor activity in non-small-cell lung cancer cells. Although several investigations have demonstrated the safety of Taraxacum officinale, the safety of tissue-cultured plants of T. formosanum has not been assessed so far. Therefore, the present study examines the safety of the water extract of the entire plant of tissue cultured T. formosanum based on acute and subacute toxicity tests in rats, as well as the Ames tests. RESULTS: No death or toxicity symptoms were observed in the acute and subacute tests. The results of the acute test revealed that the LD50 (50% of lethal dose) value of the T. formosanum water extract for rats exceeded 5 g/kg bw. No abnormal changes in the body weight, weekly food consumption, organ weight, or hematological, biochemical, and morphological parameters were observed in the subacute toxicity test. Thus, the no observed adverse effect level (NOAEL) of T. formosanum water extract was estimated to be higher than 2.0 g/kg. Finally, the results of the Ames test revealed that T. formosanum water extract was not genotoxic at any tested concentration to any of five Salmonella strains. CONCLUSIONS: The water extract of tissue-cultured T. formosanum was non-toxic to rats in acute and subacute tests and exhibited no genotoxicity to five Salmonella strains.

Ethnobotany of Plectranthus neochilus Schltr (Meprobamate) in Cuba / Estudio etnobotánico de Plectranthus neochilus Schltr (Meprobamato) en Cuba

Chloroform extract (CE) and fractions obtained from Aldama arenaria roots were evaluated for their in vitro antiproliferative activity against 10 human tumor cell lines [leukemia (K-562), breast (MCF-7), ovary expressing a multidrug-resistant phenotype (NCI/ADR-RES), melanoma (UACC-62), lung (NCI-H460), prostate (PC-3), colon (HT29), ovary (OVCAR-3), glioma (U251), and kidney (786-0)]. CE presented weak to moderate antiproliferative activity (mean log GI50 1.07), whereas fractions 3 and 4, enriched with pimaranetype diterpenes [ent-pimara-8(14),15-dien-19-oic acid and ent-8(14),15-pimaradien-3ß-ol], presented moderate to potent activity for most cell lines, with mean log GI50 of 0.62 and 0.59, respectively. The results showed promising in vitro antiproliferative action of the samples obtained from A. arenaria, with the best results for NCI/ADR-RES, HT29, and OVCAR-3, and TGI values ranging from 5.95 to 28.71 µg.ml -1, demonstrating that compounds of this class may be potential prototypes for the discovery of new therapeutic agents.

El extracto de cloroformo (CE) y las fracciones obtenidas de las raíces de Aldama arenaria fueron evaluadas por su actividad antiproliferativa in vitro contra 10 líneas celulares tumorales humanas [leucemia (K-562), mama (MCF-7), ovario que expresa un fenotipo resistente a múltiples fármacos (NCI/ADR-RES), melanoma (UACC-62), pulmón (NCI-H460), próstata (PC-3), colon (HT29), ovario (OVCAR-3), glioma (U251) y riñón (786-0)]. CE presentó actividad antiproliferativa débil a moderada (log GI50 promedio de 1.07), mientras que las fracciones 3 y 4, enriquecidas con diterpenos de tipo pimarane [ent-pimara-8 (14), ácido 15-dien-19-oico y ent-8 (14), 15-pimaradien-3ß-ol], presentaron actividad moderada a potente para la mayoría de las líneas celulares, con un log GI50 promedio de 0.62 y 0.59, respectivamente. Los resultados mostraron una prometedora acción antiproliferativa in vitro de las muestras obtenidas de A. arenaria, con los mejores resultados para NCI/ADR-RES, HT29 y OVCAR-3, y valores de TGI que van desde 5.95 a 28.71 µg.ml -1, lo que demuestra que los compuestos de esta clase pueden ser prototipos potenciales para el descubrimiento de nuevos agentes terapéuticos.

The influence of phytoestrogens or estrogens on the proliferation of the rat endocervical mucosa

SUMMARY INTRODUCTION Although estrogen therapy is widely used against post-menopausal symptoms, it can present adverse effects, including endometrial cancer. Soy isoflavones are considered a possible alternative to estrogen therapy. However, there are still concerns whether isoflavones exert trophic effects on the uterine cervix. OBJECTIVES To evaluate the histomorphometric and immunohistochemical alterations in the uterine cervix of ovariectomized rats treated with soy isoflavones (Iso). METHODS Fifteen adult Wistar rats were ovariectomized (Ovx) and divided into three groups: Group I (Ovx), administered with vehicle solution; Group II (OVX-Iso), administered with concentrated extract of Iso (150 mg/kg) by gavage; and Group III (OVX-E2), treated with 17β-estradiol (10 µg/kg), subcutaneously. After 30 days of treatments, the uterine cervix was fixed in 10% formaldehyde and processed for paraffin-embedding. Sections were stained with Hematoxylin and eosin for morphological and morphometric studies or subjected to immunohistochemistry for detections of Ki-67 and vascular endothelial growth factor-A (Vegf-A). The data obtained were subjected to statistical analysis (p ≤ 0.05). RESULTS We noted an atrophic uterine cervix in GI, whereas it was more voluminous in GII and even more voluminous in GIII. The thickness of the cervical mucosa was significantly higher in GIII, as compared to GI and GII. The cell proliferation (Ki-67) was significantly elevated in the estradiol and isoflavones treated groups, whereas Vegf-A immunoexpression was significantly higher in GIII, as compared to groups GII and GI. CONCLUSIONS Soy isoflavones cause less trophic and proliferative effects in the uterine cervix of rats as compared to estrogen.

RESUMO INTRODUÇÃO Embora a terapia estrogênica seja amplamente utilizada contra sintomas pós-menopausais, ela pode apresentar efeitos adversos, incluindo câncer de mama e endometrial. Assim, as isoflavonas da soja são consideradas uma alternativa possível à terapia estrogênica. No entanto, ainda há controvérsias se estes compostos exercem efeitos tróficos significativos no colo do útero. OBJETIVOS Avaliar as alterações histomorfométricas e imuno-histoquímicas no colo do útero de ratas ovariectomizadas tratadas com isoflavonas da soja (iso). MÉTODOS Quinze ratas Wistar adultas foram ovariectomizadas bilateralmente (Ovx) e separadas em três grupos: Grupo I (Ovx) - veículo (propilenoglicol); Grupo II (Ovx-Iso) - receberam extrato concentrado de Iso (150 mg/kg) e Grupo III (Ovx-E2) - tratado com 17β-estradiol (10 µg/kg); as soluções foram administradas via gavagem por 30 dias consecutivos. Posteriormente, os colos uterinos foram retirados, fixados em formaldeído a 10% tamponado e processados para inclusão em parafina. Cortes (4 µm) foram coradas com hematoxilina e eosina para estudo morfológico e morfométricos, enquanto outros foram submetidos à imuno-histoquímica para detecção de Ki-67 e do fator de crescimento endotelial vascular-A (Vegf-A). Os dados obtidos foram submetidos à análise estatística (p≤0,05). RESULTADOS Observamos a presença de colo uterino atrófico no GI (Ovx), sendo este mais volumoso no GII (Ovx+Iso) e ainda mais volumoso no GIII (Ovx+E2). A espessura da mucosa cervical foi significativamente maior no GIII (Ovx-E2), em comparação ao GI (Ovx) e ao GII (Ovx-Iso). A proliferação celular (Ki-67) foi significativamente mais elevada nos grupos tratados com estradiol e isoflavonas, enquanto a imunoexpressão de Vegf-A foi significativamente maior no GIII (Ovx-E2), em comparação ao GII (Ovx-Iso) e ao GI (Ovx-E2). CONCLUSÕES As isoflavonas da soja causam menos efeitos tróficos e proliferativos no colo do útero de ratas em comparação ao estrogênio.

Enhancement of cellular activity in hyperglycemic mice dermal wounds dressed with chitosan-alginate membranes

The use of specially designed wound dressings could be an important alternative to facilitate the healing process of wounds in the hyperglycemic state. Biocompatible dressings combining chitosan and alginate can speed up wound healing by modulating the inflammatory phase, stimulating fibroblast proliferation, and aiding in remodeling phases. However, this biomaterial has not yet been explored in chronic and acute lesions of diabetic patients. The aim of this study was to evaluate the effect of topical treatment with a chitosan-alginate membrane on acute skin wounds of hyperglycemic mice. Diabetes mellitus was induced by streptozotocin (60 mg · kg-1 · day-1 for 5 days, intraperitoneally) and the cutaneous wound was performed by removing the epidermis using a surgical punch. The results showed that after 10 days of treatment the chitosan and alginate membrane (CAM) group exhibited better organization of collagen fibers. High concentrations of interleukin (IL)-1α, IL-1β, granulocyte colony-stimulating factor (G-CSF), and tumor necrosis factor-alpha (TNF-α) were detected in the first and second days of treatment. G-CSF and TNF-α level decreased after 5 days, as well as the concentrations of TNF-α and IL-10 compared with the control group (CG). In this study, the inflammatory phase of cutaneous lesions of hyperglycemic mice was modulated by the use of CAM, mostly regarding the cytokines IL-1α, IL-1β, TNF-α, G-CSF, and IL-10, resulting in better collagen III deposition. However, further studies are needed to better understand the healing stages associated with CAM use.

Effects of ICI 182, 780, an ERα and ERß antagonist, and G-1, a GPER agonist, on autophagy in breast cancer cells / Efeitos do antagonista seletivo do REα e do REß ICI 182, 780 e do agonista de GPER G-1 no processo de autofagia em células de câncer de mama

ABSTRACT Objective To investigate if ICI 182,780 (fulvestrant), a selective estrogen receptor alpha/beta (ERα/ERβ) antagonist, and G-1, a selective G-protein-coupled receptor (GPER) agonist, can potentially induce autophagy in breast cancer cell lines MCF-7 and SKBr3, and how G-1 affects cell viability. Methods Cell viability in MCF-7 and SKBr3 cells was assessed by the MTT assay. To investigate the autophagy flux, MCF-7 cells were transfected with GFP-LC3, a marker of autophagosomes, and analyzed by real-time fluorescence microscopy. MCF-7 and SKBr3 cells were incubated with acridine orange for staining of acidic vesicular organelles and analyzed by flow cytometry as an indicator of autophagy. Results Regarding cell viability in MCF-7 cells, ICI 182,780 and rapamycin, after 48 hours, led to decreased cell proliferation whereas G-1 did not change viability over the same period. The data showed that neither ICI 182,780 nor G-1 led to increased GFP-LC3 puncta in MCF-7 cells over the 4-hour observation period. The cytometry assay showed that ICI 182,780 led to a higher number of acidic vesicular organelles in MCF-7 cells. G-1, in turn, did not have this effect in any of the cell lines. In contrast, ICI 182,780 and G-1 did not decrease cell viability of SKBr3 cells or induce formation of acidic vesicular organelles, which corresponds to the final step of the autophagy process in this cell line. Conclusion The effect of ICI 182,780 on increasing acidic vesicular organelles in estrogen receptor-positive breast cancer cells appears to be associated with its inhibitory effect on estrogen receptors, and GPER does notseem to be involved. Understanding these mechanisms may guide further investigations of these receptors' involvement in cellular processes of breast cancer resistance.

RESUMO Objetivo Avaliar o efeito dos compostos ICI 182,780 (fulvestranto), um antagonista seletivo dos receptores de estrógeno alfa/beta (REα/REβ), e do G-1, um agonista seletivo de receptores de estrógeno acoplados a proteínas-G (GPER), na possível indução de autofagia em linhagens de câncer de mama MCF-7 e SKBr3, bem como o efeito de G-1 na viabilidade celular. Métodos A viabilidade celular de células MCF-7 e SKBr3 foi avaliada pelo ensaio com MTT. Para investigar a indução da autofagia, células MCF-7 foram transfectadas com GFP-LC3, um marcador de autofagossomos, e analisadas por microscopia de fluorescência em tempo real. As células MCF-7 e SKBr3 foram incubadas com o indicador de compartimentos ácidos laranja de acridina e analisadas por citometria de fluxo como indicativo para autofagia. Resultados Em células MCF-7, o ICI 182,780 e rapamicina após 48 horas levaram à diminuição da viabilidade celular, enquanto o G-1 não alterou a viabilidade no mesmo período de tratamento. Nem o ICI 182,780 e nem o G-1 induziram aumento na pontuação de GFP-LC3 em células MCF-7 até 4 horas. Já os ensaios de citometria de fluxo demonstraram que ICI 182,780 levou ao aumento de compartimentos ácidos em células MCF-7. O G-1 não aumentou estes parâmetros em ambas as linhagens. Por outro lado, ICI 182,780 e G-1 não induziram à redução da viabilidade em células SKBr3 e nem à formação de compartimentos ácidos, como etapa final do processo autofágico. Conclusão O aumento de compartimentos ácidos pelo ICI 182,780 em células de câncer de mama positivas para receptores de estrógeno parece estar associado com seu efeito inibidor de receptores de estrógeno, mas sem o envolvimento de GPER. A compreensão desses mecanismos pode direcionar estudos sobre o envolvimento dos receptores nos processos celulares de resistência do câncer de mama.

Enhanced anti-tumor efficacy of 5-aminolevulinic acid-gold nanoparticles-mediated photodynamic therapy in cutaneous squamous cell carcinoma cells

The objective of this study was to investigate whether the conjugation of gold nanoparticles (GNPs) to 5-aminolevulinic acid (5-ALA) could enhance the anti-tumor efficiency of photodynamic therapy (PDT) in epidermoid carcinoma cells. The mRNA and protein expression levels were determined by quantitative real-time PCR and western blot, respectively. Cell viability, apoptosis, invasion, and migration were determined by MTT assay, flow cytometry, transwell invasion assay, and migration assay, respectively. Singlet oxygen generation was detected by the singlet oxygen sensor green reagent assay. Our results showed that PDT with 5-ALA and GNPs-conjugated 5-ALA (5-ALA-GNPs) significantly suppressed cell viability, increased cell apoptosis and singlet oxygen generation in both HaCat and A431 cells, and PDT with 5-ALA and 5-ALA-GNPs had more profound effects in A431 cells than that in HaCat cells. More importantly, 5-ALA-GNPs treatment potentiated the effects of PDT on cell viability, cell apoptosis, and singlet oxygen generation in A431 cells compared to 5-ALA treatment. Further in vitro assays showed that PDT with 5-ALA-GNPs significantly decreased expression of STAT3 and Bcl-2 and increased expression of Bax in A431 cells compared with PDT with 5-ALA. In addition, 5-ALA-GNPs treatment enhanced the inhibitory effects of PDT on cell invasion and migration and Wnt/β-catenin signaling activities in A431 cells compared to 5-ALA treatment. In conclusion, our results suggested that GNPs conjugated to 5-ALA significantly enhanced the anti-tumor efficacy of PDT in A431 cells, which may represent a better strategy to improve the outcomes of patients with cutaneous squamous cell carcinoma.

Effect of focal adhesion kinase inhibition on osteoblastic cells grown on titanium with different topographies

Abstract Objective The present study aimed to investigate the participation of focal adhesion kinases (FAK) in interactions between osteoblastic cells and titanium (Ti) surfaces with three different topographies, namely, untreated (US), microstructured (MS), and nanostructured (NS). Methodology Osteoblasts harvested from the calvarial bones of 3-day-old rats were cultured on US, MS and NS discs in the presence of PF-573228 (FAK inhibitor) to evaluate osteoblastic differentiation. After 24 h, we evaluated osteoblast morphology and vinculin expression, and on day 10, the following parameters: gene expression of osteoblastic markers and integrin signaling components, FAK protein expression and alkaline phosphatase (ALP) activity. A smooth surface, porosities at the microscale level, and nanocavities were observed in US, MS, and NS, respectively. Results FAK inhibition decreased the number of filopodia in cells grown on US and MS compared with that in NS. FAK inhibition decreased the gene expression of Alp, bone sialoprotein, osteocalcin, and ALP activity in cells grown on all evaluated surfaces. FAK inhibition did not affect the gene expression of Fak, integrin alpha 1 ( Itga1 ) and integrin beta 1 ( Itgb1 ) in cells grown on MS, increased the gene expression of Fak in cells grown on NS, and increased the gene expression of Itga1 and Itgb1 in cells grown on US and NS. Moreover, FAK protein expression decreased in cells cultured on US but increased in cells cultured on MS and NS after FAK inhibition; no difference in the expression of vinculin was observed among cells grown on all surfaces. Conclusions Our data demonstrate the relevance of FAK in the interactions between osteoblastic cells and Ti surfaces regardless of surface topography. Nanotopography positively regulated FAK expression and integrin signaling pathway components during osteoblast differentiation. In this context, the development of Ti surfaces with the ability to upregulate FAK activity could positively impact the process of implant osseointegration.

Perillyl alcohol has antibacterial effects and reduces ROS production in macrophages

Abstract Natural products have emerged as a rich source of bioactive compounds for adjunctive treatments of many infectious and inflammatory conditions, including periodontitis. Among the monoterpenes with significant biological properties, there is the perillyl alcohol (POH), which can be found in several essential oils and has shown immunomodulatory properties in recent studies, which may be interesting in the treatment of non-neoplastic inflammatory disorders. Objective To determine the antibacterial and immune modulatory activities of the POH. Methodology The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the POH for two significant Gram-negative periodontal pathogens were determined by macrodilution and subculture, respectively. Cell proliferation and cytotoxicity in RAW 264.7 macrophages were determined by Trypan Blue and mitochondrial enzymatic activity assay. The modulation of reactive oxygen species (ROS) was analyzed by flow cytometry and expression of TNF and arginase-1 by real-time PCR. Results The POH was effective against P. gingivalis (ATCC 33277) and F. nucleatum (ATCC 25586) with MIC= MBC=1600 μM. No cytotoxicity up to 100 µM was observed on macrophages. The cell proliferation was inhibited from 48 hours at 100 μM (p<0.05) and 250 μM (p<0.01). The POH increased ROS production at both 10 μM and 100 μM (p<0.05) in unstimulated cells. The PMA-induced ROS production was not affected by POH, whereas 100 μM significantly reduced lipopolysaccharide-induced (LPS-induced) ROS. The expression of TNF was not affected by POH in unstimulated cells or in cells polarized to M1 phenotype, whereas both concentrations of POH reduced (p<0.05) the expression of arginase-1 in M2-polarized macrophages. Conclusion The POH has antibacterial activity against periodontal pathogens and reduced proliferation of murine macrophages without significant cytotoxicity at concentrations up to 100 μM. In addition, the POH reduced the LPS-induced ROS and the expression of arginase-1 in M2-polarized macrophages.

Antiproliferative and photoprotective activities of the extracts and compounds from Calea fruticosa

In this paper, we complement our previous study on the antiproliferative activity of Calea fruticosa (Asteraceae) by isolating the compounds apigenin-4',7-dimethyl ether (1), budlein A (2), quercetin (3), and cichoriin (4) from the plant's aerial parts. The antiproliferative activity of these compounds was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method against human tumor cell lines. Compound 3 displayed moderate antiproliferative activity in three cell lines (HCT-116, PC-3, and SF-295, with cell growth inhibition values of 72.97, 74.55, and 68.94%) and high antiproliferative activity (90.86%) in the HL-60 cell line. The in vitro sun protection factor (SPF) of the extracts and compound 4, with and without sunscreen, was determined by a spectrophotometric method. The ethanol extract exhibited the highest SPF (9.67) at a concentration of 0.100 mg/mL, while compound 4, isolated from this extract, showed a SPF of 13.79 at the same concentration. A relative increased efficacy of SPF was observed for the extracts and compound 4 when sunscreen was also used. Compound 4 has not been reported previously from any species within the genus Calea. Compounds 1-4 were obtained from this species for the first time.

Knocking down LSD1 inhibits the stemness features of colorectal cancer stem cells

As a top leading cause of cancer death in many countries, colorectal cancer (CRC) has drawn increasing attention to the study of the pathological mechanism. According to the "cancer stem cell hypothesis", malignancies originate from a small fraction of cancer cells that show self-renewal properties to initiate and sustain tumor growth and tumor metastasis. Therefore, these cancer stem cells (CSC) probably play important roles in tumor recurrence, metastasis, and drug resistance. Previous research reported that lysine-specific histone demethylase 1 (LSD1) maintains cancer stemness through up-regulating stemness markers SOX2 and OCT4. CD133 is believed to be the most robust surface marker for CRC stem cells, however the regulatory effect of LSD1 on stemness of CD133+ CRC has never been reported. In this study, our objectives included: 1) to isolate pure CD133+ and CD133− cells from SW620 cell line; 2) to investigate the effect of LSD1 on the characteristics of CD133+ stem cancer cells by knocking down the target gene. Results suggested that the SW620 cell line had both CD133+ and CD133− subsets. The CD133+ subset exhibited more CSC-like characteristics compared with the CD133− subset with higher viability, colony formation rate, migration and invasion rate, resistance to anti-cancer drugs, and apoptosis in vitro. The CD133+ also induced faster tumor formation and larger tumors in vivo. In the LSD1-knockdown CD133+ cells, the CSC-like characteristics had been all weakened. We conclude that LSD1 was important for CSCs to maintain their "stemness" features, which could be a potential therapeutic target of CRC.

Effect of ethanol extracts of Antrodia cinnamomea on head and neck squamous cell carcinoma cell line

Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignant tumors. Ethanol extract of Antrodia cinnamomea (EEA) has been widely studied for its health benefits including anticancer effects. The purpose of this study was to assess the effects of EEA on HNSCC. Cell proliferation, transwell, and wound healing assays were performed. The impact of EEA on tumor growth was investigated using a xenograft model. Expressions of migration-related proteins (MMP-2, MMP-9, TIMP-1, and TIMP-2) and apoptosis-related proteins (cleaved caspase-9 and cleaved PARP) were determined using western blot analysis. The results indicated that EEA significantly inhibited the capacities of proliferation, invasion, and migration of HNSCC cells in a dose-dependent manner. Cleaved caspase-9 and cleaved PARP expressions were increased in cells treated with an increasing concentration of EEA, which suggested that EEA induced apoptosis of HNSCC. MMP-2 and MMP-9 were downregulated when cells were administered EEA, while TIMP-1 and TIMP-2 were not affected, which uncovered the mechanisms mediating the EEA-induced inhibition on cell invasion and migration. The animal experiment also suggested that EEA inhibited tumor growth. Our study confirmed the inhibitive effects of EEA on cell proliferation, invasion, and migration of HNSCC in vitro and in vivo, providing the basis for further study of the application of EEA as an effective candidate for cancer treatment.

Treatment with ß-elemene combined with paclitaxel inhibits growth, migration, and invasion and induces apoptosis of ovarian cancer cells by activation of STAT-NF-κB pathway

In this study, we aimed to analyze the anti-cancer effects of β-elemene combined with paclitaxel for ovarian cancer. RT-qPCR, MTT assay, western blot, flow cytometry, and immunohistochemistry were used to analyze in vitro and in vivo anti-cancer effects of combined treatment of β-elemene and paclitaxel. The in vitro results showed that β-elemene+paclitaxel treatment markedly inhibited ovarian cancer cell growth, migration, and invasion compared to either paclitaxel or β-elemene treatment alone. Results demonstrated that β-elemene+paclitaxel induced apoptosis of SKOV3 cells, down-regulated anti-apoptotic Bcl-2 and Bcl-xl gene expression and up-regulated pro-apoptotic P53 and Apaf1 gene expression in SKOV3 cells. Administration of β-elemene+paclitaxel arrested SKOV3 cell cycle at S phase and down-regulated CDK1, cyclin-B1, and P27 gene expression and apoptotic-related resistant gene expression of MDR1, LRP, and TS in SKOV3 cells. In vivo experiments showed that treatment with β-elemene+paclitaxel significantly inhibited ovarian tumor growth and prolonged the overall survival of SKOV3-bearing mice. In addition, the treatment inhibited phosphorylated STAT3 and NF-κB expression in vitro and in vivo. Furthermore, it inhibited migration and invasion through down-regulation of the STAT-NF-κB signaling pathway in SKOV3 cells. In conclusion, the data suggested that β-elemene+paclitaxel can inhibit ovarian cancer growth via down-regulation of the STAT3-NF-κB signaling pathway, which may be a potential therapeutic strategy for ovarian cancer therapy.