Enhancement of Annexin V in response to combination of epigallocatechin gallate and quercetin as a potent arrest the cell cycle of colorectal cancer / Aumento da anexina V em resposta à combinação de galato de epigalocatequina e quercetina como uma potente parada do ciclo celular do câncer colorretal

Abstract Colorectal cancer (CRC) is one of the most common cancers leading to comorbidities and mortalities globally. The rational of current study was to evaluate the combined epigallocatechin gallate and quercetin as a potent antitumor agent as commentary agent for therapeutic protocol. The present study investigated the effect of epigallocatechin Gallate (EGCG) (150mg) and quercetin (200mg) at different proportions on proliferation and induction of apoptosis in human colon cancer cells (HCT-116). Cell growth, colonogenic, Annexin V in addition cell cycle were detected in response to phytomolecules. Data obtained showed that, the colony formation was inhibited significantly in CRC starting from the lowest concentration tested of 10 µg/mL resulting in no colonies as visualized by a phase-contrast microscope. Data showed a significant elevation in the annexin V at 100 µg/mL EGCG(25.85%) and 150 µg/mL quercetin (48.35%). Moreover, cell cycle analysis showed that this combination caused cell cycle arrest at the G1 phase at concentration of 100 µg/mL (72.7%) and 150 µg/mL (75.25%). The combined effect of epigallocatechin Gallate and quercetin exert antiproliferative activity against CRC, it is promising in alternative conventional chemotherapeutic agent.

Resumo O câncer colorretal (CCR) é um dos cânceres mais comuns, levando a comorbidades e mortalidade em todo o mundo. O racional do presente estudo foi avaliar a combinação de galato de epigalocatequina e quercetina como um agente antitumoral potente como agente de comentário para protocolo terapêutico. O presente estudo investigou o efeito de galato de epigalocatequina (EGCG) (150 mg) e quercetina (200 mg) em diferentes proporções na proliferação e indução de apoptose em células de câncer de cólon humano (HCT-116). O crescimento celular, colonogênico, anexina V, além do ciclo celular foram detectados em resposta a fitomoléculas. Os dados obtidos mostraram que a formação de colônias foi inibida significativamente no CRC a partir da concentração mais baixa testada de 10 µg/mL, resultando em nenhuma colônia conforme visualizado por um microscópio de contraste de fase. Os dados mostraram uma elevação significativa na anexina V a 100 µg/mL de EGCG (25,85%) e 150 µg/mL de quercetina (48,35%). Além disso, a análise do ciclo celular mostrou que essa combinação causou parada do ciclo celular na fase G1 na concentração de 100 µg/mL (72,7%) e 150 µg/mL (75,25%). O efeito combinado da epigalocatequina galato e quercetina exerce atividade antiproliferativa contra o CCR, é promissor como agente quimioterápico alternativo convencional.

Cell cycle related long non-coding RNAs as the critical regulators of breast cancer progression and metastasis

Cell cycle is one of the main cellular mechanisms involved in tumor progression. Almost all of the active molecular pathways in tumor cells directly or indirectly target the cell cycle progression. Therefore, it is necessary to assess the molecular mechanisms involved in cell cycle regulation in tumor cells. Since, early diagnosis has pivotal role in better cancer management and treatment, it is required to introduce the non-invasive diagnostic markers. Long non-coding RNAs (LncRNAs) have higher stability in body fluids in comparison with mRNAs. Therefore, they can be used as efficient non-invasive markers for the early detection of breast cancer (BCa). In the present review we have summarized all of the reported lncRNAs involved in cell cycle regulation in BCa. It has been reported that lncRNAs mainly affect the cell cycle in G1/S transition through the CCND1/CDK4-6 complex. Present review paves the way of introducing the cell cycle related lncRNAs as efficient markers for the early detection of BCa.

Alterações sistêmicas na assinatura do ciclo celular de pacientes com COVID-19 / Systemic changes in the cell cycle signature of patients with COVID-19

Com base nas perturbações fosfoproteômicas de moléculas associadas ao ciclo celular em células infectadas pelo coronavírus causador da síndrome respiratória aguda grave (SARSCoV)-2, a hipótese de inibidores do ciclo celular como uma terapia potencial para a doença de coronavírus 2019 (COVID-19) foi proposta. No entanto, o cenário das alterações do ciclo celular em COVID-19 permanece inexplorado. Aqui, realizamos uma análise integrativa de sistemas imunológicos de proteoma publicamente disponível (espectrometria de massa) e dados de transcriptoma (sequenciamento de RNA em massa e de célula única [scRNAseq]), com o objetivo de caracterizar mudanças globais na assinatura do ciclo celular de pacientes com COVID-19. Além de módulos de co-expressão de genes significativos enriquecidos associados ao ciclo celular, encontramos uma rede interconectada de proteínas diferencialmente expressas associadas ao ciclo celular (DEPs) e genes (DEGs) integrando dados moleculares de 1.480 indivíduos (974 pacientes infectados por SARS-CoV-2 e 506 controles [controles saudáveis ou indivíduos com outras doenças respiratórias]). Entre esses DEPs e DEGs estão várias ciclinas (CCNs), ciclo de divisão celular (CDCs), quinases dependentes de ciclinas (CDKs) e proteínas de manutenção de minicromossomos (MCMs). Embora os pacientes com COVID-19 compartilhem parcialmente o padrão de expressão de algumas moléculas associadas ao ciclo celular com outras doenças respiratórias, eles exibiram uma expressão significativamente maior de moléculas associadas ao ciclo celular relacionadas à gravidade da doença. Notavelmente, a assinatura do ciclo celular predominou nos leucócitos do sangue dos pacientes, mas não nas vias aéreas superiores. Os dados de scRNAseq de 229 indivíduos (159 pacientes com COVID- 19 e 70 controles) revelaram que as alterações das assinaturas do ciclo celular predominam nas células B, T e NK. Esses resultados fornecem uma compreensão global única das alterações nas moléculas associadas ao ciclo celular em pacientes com COVID-19, sugerindo novas vias putativas para intervenção terapêutica

Based on phosphoproteomics perturbations of cell cycle-associated molecules in severe acute respiratory syndrome coronavirus (SARS-CoV)-2-infected cells, the hypothesis of cell cycle inhibitors as a potential therapy for Coronavirus disease 2019 (COVID-19) has been proposed. However, the landscape of cell cycle alterations in COVID-19 remains mostly unexplored. Here, we performed an integrative systems immunology analysis of publicly available proteome (mass spectrometry) and transcriptome data (bulk and single-cell RNA sequencing [scRNAseq]), aiming to characterize global changes in the cell cycle signature of COVID-19 patients. Beyond significant enriched cell cycle-associated gene co-expression modules, we found an interconnected network of cell cycle-associated differentially expressed proteins (DEPs) and genes (DEGs) by integrating molecular data of 1,480 individuals (974 SARS-CoV- 2 infected patients and 506 controls [either healthy controls or individuals with other respiratory illness]). Among these DEPs and DEGs are several cyclins (CCNs), cell division cycle (CDCs), cyclin-dependent kinases (CDKs), and mini-chromosome maintenance proteins (MCMs). Although COVID-19 patients partially shared the expression pattern of some cell cycleassociated molecules with other respiratory illnesses, they exhibited a significantly higher expression of cell cycle-associated molecules associated with disease severity. Notably, the cell cycle signature predominated in the patients blood leukocytes but not in the upper airways. The scRNAseq data from 229 individuals (159 COVID-19 patients and 70 controls) revealed that the alterations of cell cycle signatures predominate in B, T, and NK cells. These results provide a unique global comprehension of the alterations in cell cycle-associated molecules in COVID-19 patients, suggesting new putative pathways for therapeutic intervention

Speckle type POZ adaptor protein (SPOP) and its role in cancer / La proteína adaptadora Speckle-type POZ (SPOP) y su papel en cáncer

Abstract Proteasomal degradation is an essential regulatory mechanism for cellular homeostasis maintenance. The speckle-type POZ adaptor protein (SPOP) is part of the ubiquitin ligase E3 cullin-3 RING-box1 complex, responsible for the ubiquitination and proteasomal degradation of biomolecules involved in cell cycle control, proliferation, response to DNA damage, epigenetic control, and hormone signaling, among others. Changes in SPOP have been associated with the development of different types of cancer, since it can act as a tumor suppressor mainly in prostate, breast, colorectal, lung cancer and liver cancer, due to point mutations and/or reduced expression, or as an oncogene in kidney cancer by protein overexpression. In endometrial cancer it has a dual role, since it can act as a tumor suppressor or as an oncogene. SPOP is a potential prognostic biomarker and a promising therapeutic target.

Resumen La degradación proteosómica es un mecanismo de regulación esencial para el mantenimiento de la homeostasis celular. La proteína adaptadora Speckle-type POZ (SPOP) hace parte del complejo ubiquitin ligasa E3 cullin-3 RING-box1, encargado de la ubiquitinación y degradación proteosomal de biomoléculas involucradas en el control del ciclo celular, proliferación, respuesta al daño de ADN, control epigenético, señalización hormonal, entre otros. Las alteraciones en SPOP han sido asociadas al desarrollo de diferentes tipos de cáncer, ya que puede actuar como supresor tumoral principalmente en cáncer de próstata, mama, colorrectal y pulmón, debido a mutaciones puntuales y/o expresión reducida o como oncogén en cáncer riñón por sobreexpresión de la proteína. En cáncer endometrial tiene un rol dual, ya que puede actuar como supresor tumoral o como oncogén. SPOP es considerado como un potencial biomarcador pronóstico y un objetivo terapéutico prometedor.

Effect of Huangkui on mouse podocytes under high glucose environment / Efecto de Huangkui en podocitos de ratón en un entorno de glucosa alta

To explore a new underlying molecular mechanism of Huangkui Extract Powder (HKEP) in the alleviation of diabetic nephropathy (DN). Murine immortalized podocytes were divided into (i) normal glucose (NG, 5.6 mM), (ii) NG + HKEP (0.45 g/L), (iii) HG, and (iv) HG + HKEP (0.45 g/L) groups. MTT assay and flow cytometry were used to detect the podocyte proliferation, apoptosis and cell cycle. Cell viability was inhibited, and apoptosis increased in(iii) HG group compared with (i) NG group (p<0.05). mRNA and protein expression of nephrin and podocin significantly decreased in (iii) HG group compared with (i) NG group (p<0.05). When compared with (iii) HG group, (iv) HG + HKEP group had higher cell viability, lower apoptotic rate and higher mRNA and protein expression of nephrin and podocin (p<0.05). HKEP can attenuate HG-induced podocyte damage, which may be one of the mechanisms of HKEP for attenuating DN.

Explorar un nuevo mecanismo molecular subyacente del extracto del polvo de Huangkui (HKEP) en el alivio de la nefropatía diabética (ND). Los podocitos murinos inmortalizados se dividieron en (i) grupos de glucosa normal (NG, 5,6 mM), (ii) NG + HKEP (0,45 g/L), (iii) HG y (iv) HG + HKEP (0,45 g/L). Se utilizaron el ensayo MTT y la citometría de flujo para detectar la proliferación de podocitos, la apoptosis y el ciclo celular. La viabilidad celular se inhibió y la apoptosis aumentó en el grupo (iii) HG en comparación con el grupo (i) NG (p<0,05). La expresión de ARNm y proteínas de nefrina y podocina disminuyó significativamente en el grupo (iii) HG en comparación con el grupo (i) NG (p<0,05). En comparación con el grupo (iii) HG, el grupo (iv) HG + HKEP tuvo una mayor viabilidad celular, una tasa de apoptosis más baja y una expresión de ARNm y proteínas más altas de nefrina y podocina (p<0,05). HKEP puede atenuar el daño de los podocitos inducido por HG, que puede ser uno de los mecanismos de HKEP para atenuar la DN.

Defensin c-thionin from Capsicum chinense improves butyrate cytotoxicity on human colon adenocarcinoma cell line Caco-2

BACKGROUND: Butyrate is a histone deacetylase inhibitor that induces apoptosis and inhibits cell proliferation of colorectal cancer cells. To improve its anticancer activity, butyrate has been evaluated mixed with drugs and different molecules. Plant antimicrobial peptides are attractive anticancer alternative molecules because they show selective cytotoxic activity against different cancer cell lines. In this work, we explore if the plant defensin c-thionin (Capsicum chinense) can improve butyrate activity on Caco-2 cell line and we also determined the mechanism of death activated. RESULTS: The combined treatment of c-thionin (3.5 mM) and butyrate (50 mM) showed higher cytotoxicity on Caco-2 cells with respect to single treatments. Also, the combined treatment reduced cell proliferation and exhibited a higher rate of apoptosis than single treatments. Combined treatment induced caspases 8 and 9 activation to an extent comparable with that of butyrate while c-thionin did not activate caspases. Additionally, reactive oxygen species generation preceded the onset of apoptosis, and superoxide anion production was higher in cells treated with the combined treatment. CONCLUSIONS: The c-thionin from Habanero chili pepper improved the butyrate cytotoxicity on Caco-2 cells. This effect occurred through apoptosis induction associated with reactive oxygen species production. Therefore, the combination of butyrate with cytotoxic antimicrobial peptides could be an attractive strategy for cancer therapy.

Juniperus communis extract induces cell cycle arrest and apoptosis of colorectal adenocarcinoma in vitro and in vivo

Juniperus communis (JCo) is a well-known traditional Chinese medicinal plant that has been used to treat wounds, fever, swelling, and rheumatism. However, the mechanism underlying the anticancer effect of JCo extract on colorectal cancer (CRC) has not yet been elucidated. This study investigated the anticancer effects of JCo extract in vitro and in vivo as well as the precise molecular mechanisms. Cell viability was evaluated using the MTT assay. Cell cycle distribution was examined by flow cytometry analysis, and cell apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Protein expression was analyzed using western blotting. The in vivo activity of the JCo extract was evaluated using a xenograft BALB/c mouse model. The tumors and organs were examined through hematoxylin-eosin (HE) staining and immunohistochemistry. The results showed that JCo extract exhibited higher cytotoxicity against CRC cells than against normal cells and showed synergistic effects when combined with 5-fluorouracil. JCo extract induced cell cycle arrest at the G0/G1 phase via regulation of p53/p21 and CDK4/cyclin D1 and induced cell apoptosis via the extrinsic (FasL/Fas/caspase-8) and intrinsic (Bax/Bcl-2/caspase-9) apoptotic pathways. In vivo studies revealed that JCo extract suppressed tumor growth through the inhibition of proliferation and induction of apoptosis. In addition, there was no obvious change in body weight or histological morphology of normal organs after treatment. JCo extract suppressed CRC progression by inducing cell cycle arrest and apoptosis in vitro and in vivo, suggesting the potential application of JCo extract in the treatment of CRC.

Regorafenib Suppresses Migration of and Induces Cell Cycle Arrest and Apoptosis in MCF-7 Cells

This study investigated the mechanism underlying the suppression of estrogen receptor-positive MCF-7 cell growth by regorafenib. MCF-7 cells were treated with regorafenib, and the effect of regorafenib on multiple cancer-associated pathways was evaluated. Although regorafenib effectively inhibited the proliferation of MCF-7 cells, it had no effect on the proliferation of the normal breast epithelial cell line MCF10A. Regorafenib suppressed MCF-7 cell migration, probably by regulating the homeostatic expression of matrix metalloproteinases and the tissue inhibitor of MMPs. Furthermore, it upregulated p21 expression, downregulated cyclin B1 and cyclin D1 expresssions, and caused cell cycle arrest. In addition, regorafenib induced apoptosis in MCF-7 cells by reducing Mcl-1 expression and activating caspase signaling. These results demonstrate that regorafenib has the potential to be an effective drug for treating breast cancer

Dysregulation of KIF14 regulates the cell cycle and predicts poor prognosis in cervical cancer: a study based on integrated approaches

Cervical cancer (CC) is the most common malignant tumor in females. Although persistent high-risk human papillomavirus (HPV) infection is a leading factor that causes CC, few women with HPV infection develop CC. Therefore, many mechanisms remain to be explored, such as aberrant expression of oncogenes and tumor suppressor genes. To identify promising prognostic factors and interpret the relevant mechanisms of CC, the RNA sequencing profile of CC was downloaded from the Cancer Genome Atlas and the Gene Expression Omnibus databases. The GSE63514 dataset was analyzed, and differentially expressed genes (DEGs) were obtained by weighted coexpression network analysis and the edgeR package in R. Fifty-three shared genes were mainly enriched in nuclear chromosome segregation and DNA replication signaling pathways. Through a protein-protein interaction network and prognosis analysis, the kinesin family member 14 (KIF14) hub gene was extracted from the set of 53 shared genes, which was overexpressed and associated with poor overall survival (OS) and disease-free survival (DFS) of CC patients. Mechanistically, gene set enrichment analysis showed that KIF14 was mainly enriched in the glycolysis/gluconeogenesis signaling pathway and DNA replication signaling pathway, especially in the cell cycle signaling pathway. RT-PCR and the Human Protein Atlas database confirmed that these genes were significantly increased in CC samples. Therefore, our findings indicated the biological function of KIF14 in cervical cancer and provided new ideas for CC diagnosis and therapies.

Actividad citotóxica de la fracción clorofórmica de Piper aduncum y su efecto en el ciclo celular en líneas celulares de cáncer gástrico / Cytotoxic activity of the chloroform fraction of Piper aduncum and its effect on the cell cycle in gastric cancer cell lines

RESUMEN Objetivos: Evaluar la actividad citotóxica de la fracción clorofórmica del extracto metanólico de Piper aduncum (PAMoCl) y su efecto en el ciclo celular en dos líneas celulares de cáncer gástrico: AGS y KATO III. Materiales y métodos: El efecto citotóxico de PAMoCl se evaluó en las líneas celulares: AGS y KATO III. Se probaron concentraciones de PAMoCl: 1,25; 2,5; 5; 10; 20; 40; 80 y 160 µg/mL. Para evaluar la viabilidad celular se usó el reactivo resazurina. En el ensayo de ciclo celular las células fueron tratadas con 19,62 µg/mL y 39,23 µg/mL de PAMoCl para AGS, así como 87,49 µg/mL y 160 µg/mL para KATO III. Después de 24 horas ambas líneas celulares fueron analizadas por citometría de flujo. Resultados: PAMoCl mostró actividad citotóxica con una inhibición del crecimiento celular en un 50% (IC50) de 39,23 µg/mL y 87,49 µg/mL a las 24 horas y un IC50 de 49,47 µg/mL y 64,68 µg/mL a las 48 horas frente a las líneas celulares AGS y KATO III, respectivamente. Además, se observó que PAMoCl tiene efecto a nivel del ciclo celular: provoca una acumulación de células en la fase G2/M. Conclusiones: PAMoCl contiene metabolitos secundarios con actividad citotóxica que tienen efecto en la fase G2/M del ciclo celular, en dos líneas celulares de cáncer gástrico tanto primario como metastásico. Los resultados de este estudio permitirán profundizar en la búsqueda de principios activos presentes en PAMoCl que tengan mayor eficacia en la eliminación de células de cáncer gástrico, pero con menor toxicidad en células sanas.

ABSTRACT Objectives: To evaluate the cytotoxic activity of the chloroform fraction of the Piper aduncum methanolic extract (PAMoCl) and its effect on the cell cycle in two gastric cancer cell lines: AGS and KATO III. Materials and methods: The cytotoxic effect of PAMoCl was evaluated in cell lines AGS and KATO III. The following PAMoCl concentrations were tested, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 μg/mL. Resazurine was used to evaluate cell viability. In the cell cycle assay, the cells were treated with 19.62 μg/mL and 39.23 μg/mL of PAMoCl for AGS as well as 87.49 μg/mL and 160 μg/mL for KATO III. After 24 hours both cell lines were analyzed by flow cytometry. Results: PAMoCl showed cytotoxic activity, inhibiting cell growth by 50%. It presented a (IC50) of 39.23 μg/mL and 87.49 μg/mL at 24 hours and a (IC50) of 49.47 μg/mL and 64.68 μg/mL at 48 hours against AGS and KATO III cell lines, respectively. In addition, it was observed that PAMoCl has an effect on the cell cycle, it causes an accumulation of cells in the G2/M phase. Conclusions: PAMoCl contains secondary metabolites with cytotoxic activity that have an effect on the G2/M phase of the cell cycle, in two gastric cancer cell lines, both primary and metastatic. The results of this study will allow us to deepen the search for more effective active ingredients found in PAMoCl for eliminating gastric cancer cells, but with less toxicity for healthy cells.

Long non-coding RNA LINC01268 promotes cell growth and inhibits cell apoptosis by modulating miR-217/SOS1 axis in acute myeloid leukemia

The aim of this study was to evaluate the pathogenic role of newly identified long non-coding (lnc)-RNA LINCO1268 in acute myeloid leukemia (AML), and investigate its therapeutic potential. The expression level of LINC01268 in AML was measured by quantitative PCR (qPCR). The viability, cell cycle progression, and apoptosis of AML cells were measured by CCK-8 assay and flow cytometry, respectively. The interaction between LINC01268 and miR-217 were predicted by the miRDB website, and then verified by luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The relationship between miR-217 and SOS1 was predicted by TargetScan website, and verified by luciferase reporter assay. LINC01268 was significantly upregulated by 1.6 fold in bone marrow samples of AML patients, which was associated with poor prognosis. LINC01268 was also significantly upregulated in AML cells. LINC01268 knockdown inhibited viability and cell cycle progression but promoted apoptosis of AML cells. Furthermore, LINC01268 functioned as a ceRNA via competitively binding to miR-217, and SOS1 was identified as a target of miR-217. Moreover, LINC01268 positively regulated SOS1 expression to promote AML cell viability and cell cycle progression but inhibited apoptosis via sponging miR-217. LINC01268 promoted cell growth and inhibited cell apoptosis through modulating miR-217/SOS1 axis in AML. This study offers a novel molecular mechanism for a better understanding of the pathology of AML. LINC01268 could be considered as a potential biomarker for the therapy and diagnosis of AML.

Estudos funcionais e biológicos associados à interação de DUSP3 com a proteína HNRNPC1/C2 / Functional and biological studies associated to DUSP3 and HNRNPC1/C2 protein inte ractions

A regulação da fosforilação/desfosforilação das proteínas é o eixo central de muitas cascatas de sinalização. A fosfatase DUSP3, constituída apenas por um único domínio catalítico, desempenha papéis fundamentais na proliferação e senescência celular. Nas células HeLa, submetidas ao estresse genotóxico, o DUSP3 interage fisicamente com as proteínas HNRNPC, mas o efeito dessa função molecular ainda é desconhecido. Aqui demostramos que a ausência de DUPS3 mantem a proteína HNRNPC1/C2 num estado hiperfosforilado. Para entender melhor o envolvimento da interação DUSP3-HNRNPC nas funções biológicas da HNRNPC1/C2, foram estudadas células de fibroblasto deficientes de DUSP3. Foi analisado o efeito da deficiência de DUSP3 na biogênese dos ribossomos através do ensaio de perfil de polirribossomos e quantificação dos rRNAs com RT-qPCR. Os resultados mostraram que a deficiência de DUSP3 não afeta a maturação das subunidades ribossômicas, mas teria um impacto na transcrição dos pré-rRNAs e no acumulo das espécies 47S/45S. A expressado de genes contendo sequencias IRES foi analisado através do RT-qPCR e sua tradução ao longo do ciclo e em condições de estresse. Da expressão, não existe nenhuma diferença nos níveis de transcrição dos genes c-myc e xiap nas células normais e deficientes de DUSP3 em condições basais. Embora a síntese destas proteínas é maior nas células deficientes, mantendo um nível maior de tradução ao longo de todo o ciclo. Sob condições de estresse, esta duas proteínas sempre mantem uma maior expressão nas células Knockdown para DUSP3. Neste trabalho também foi estabelecido a presença de DUSP3 nos complexos da subunidade 40S, através do analise das frações obtidas do ensaio de polirribossomos e interação in vitro (Co-IP). A presença de DUSP3 nas subunidades 40S, os monossomas 80S e polissomos poderia ser através da interação direta com proteínas que possuem um domínio RRM e seria dependente dos complexos formados pelas proteínas e seus RNAs alvos. Aqui mostramos a interação in vitro de DUSP3 com a proteína PABP (com quatro domínios RRM), proteína que tem um papel importante na manutenção da taxa global de tradução, esta interação é enfraquecida na ausência de RNAs. A deficiência de DUSP3 também teria um impacto na interação das proteínas HNRNPC1/C2 e P53 in vitro. A ausência de DUSP3 diminui a interação HNRNPC-P53 através da hiperfosforilação da proteina HNRNPC1/C2. A perda desta interação, aumentaria os níveis da proteína P53 na célula deficiente de DUSP3 e poderia gerar parada no ciclo celular. Através de ensaios de imunofluorescência, se observo uma maior taxa de transcrição global na célula deficiente de DUSP3. Por fim, aqui demostramos que a interação direta de DUSP3 e HNRNPC1/C2 vai permitir a regulação das funções biológicas desta proteína, e a ausência de DUSP3 vai ter efeitos pleiotrópicos na homeostase da célula

inglêsProtein phosphorylation/dephosphorylation regulation is a central axis of many signaling cascades. DUSP3 phosphatase, consisting only of a single catalytic domain, plays key roles in cell proliferation and senescence. In HeLa cells subjected to genotoxic stress, DUSP3 physically interacts with HNRNPC proteins, but the effect of this molecular function is still unknown. Here we demonstrate that the absence of DUPS3 keeps the HNRNPC1/C2 proteins in a hyperphosphorylated state. To better understand the involvement of DUSP3- HNRNPC interaction on the biological functions of HNRNPC1/C2, DUSP3 deficient fibroblast cells were studied. The effect of DUSP3 deficiency on ribosome biogenesis was analyzed by polyribosome profile assay and RT-qPCR for rRNA quantification. The results showed that DUSP3 deficiency does not affect ribosomal subunit maturation, but would have an impact on transcription of pre-rRNAs and accumulation of 47S / 45S species. The expression of genes containing IRES sequences was analyzed by RT-qPCR and their translation throughout the cycle and under stress conditions. From expression, there is no difference in transcriptional levels of c-myc and xiap genes in normal and DUSP3 deficient cells under basal conditions. Although, the synthesis of these proteins is higher in deficient cells and these maintain a higher level of translation throughout the cell cycle. Under stress conditions, these two proteins always maintain higher expression in Knockdown cells for DUSP3. In this work, the presence of DUSP3 in the 40S ribosomal subunit complexes was also established by analyzing the fractions obtained from the polyribosome assay and in vitro interaction (CoIP). The presence of DUSP3 in the 40S subunits, 80S monosomes and polysomes could be through direct interaction with proteins that have an RRM domain and would be dependent on the complexes formed by the proteins and their target RNAs. Here we show the in vitro interaction of DUSP3 with PABP protein (with four RRM domains), a protein that plays an important role in maintaining the overall translation rate, this interaction is weakened in the absence of RNAs. DUSP3 deficiency would also have an impact on the interaction of HNRNPC1/C2 and P53 proteins in vitro. The absence of DUSP3 decreases HNRNPC-P53 interaction through hyperphosphorylation of the HNRNPC1/C2 proteins. Loss of this interaction would increase P53 protein levels in the DUSP3 deficient cell and could lead to cell cycle arrest. Through immunofluorescence assays, a higher overall transcription rate is observed in the DUSP3 deficient cell. Finally, we demonstrate that the direct interaction of DUSP3 and HNRNPC1/C2 will allow the regulation of the biological functions of this protein, and the absence of DUSP3 will have pleiotropic effects on cell homeostasis

Acute toxicity of Jatropha curcas oil on plant cell cycle / Toxicidade aguda do óleo de jatropha curcas no ciclo celular de plantas

Today, a great interest in Jatropha-based products exists worldwide, mainly for the production of biofuel.However, the oil obtained from this plant is known to be toxic due to contained curcins andphorbol esters. Bioassays, including plant cytogenetic assays based on cell cycle observation, are useful for determining the toxicity of J. curcas oil. Hence, the aim of this study was to describe the mechanism of action of J. curcas oil by cell cycle analysis using Lactuca sativa as plant testing model. A decrease in root growth was observed, closely related to the reduction in mitotic index, along with an increase in condensed nuclei. J. curcas chemicals act both as aneugenic agents, leading to the formation of lagged, sticky chromosomes and c-metaphase cells, as well as clastogenic agents, inducing the formation of chromosome bridges and fragments. The cytotoxicity and genotoxicity of phorbol esters and other chemical components of J. curcas oil was determined and discussed.

Um grande interesse mundial existe em produtos à base de pinhão manso, principalmente para a produção de biocombustíveis. No entanto, o óleo obtido a partir desta planta é conhecidamente tóxico por conter curcina e ésteres de forbol. Bioensaios, incluindo ensaios citogenéticos em plantas-modelo com base na observação do ciclo celular, são úteis para determinar a toxicidade do óleo de J. curcas. Assim, o objetivo deste estudo foi descrever o mecanismo de ação do óleo de J. curcas por análise do ciclo celular usando Lactuca sativa como modelo de teste em plantas. Foi observada uma redução no crescimento das raízes, intimamente relacionada com a redução do índice mitótico e com um aumento de núcleos condensados. Os constituintes químicos de J. curcas atuam simultaneamente como agentes aneugênicos, levando à formação de cromossomos perdidos e pegajosos e células em c-metáfase, bem como agentes clastogênicos, induzindo a formação de pontes e fragmentos cromossômicos. A citotoxicidade e genotoxicidade do éster de forbol e outros componentes químicos do óleo de J. curcas foram determinados e discutidos.

Skp2 inhibitor SKPin C1 decreased viability and proliferation of multiple myeloma cells and induced apoptosis

Multiple myeloma (MM) is a malignant neoplasm of plasma, and exhibits several harmful effects including osteolytic injuries, hypercalcemia, and immune dysfunction. Many patients with MM succumb to the underlying malignancy. An S-phase kinase-related protein 2 (Skp2) inhibitor, designated SKPin C1, has been developed and confirmed to have an inhibitory effect on metastatic melanoma cells. This study aimed to determine the effect of SKPin C1 on MM. Normal B lymphocytes, THP-1 cells, and MM U266 and RPMI 8226 cells were exposed to various dosages of SKPin C1 for 48 h. Cell proliferation was determined by MTT, EdU staining, and cell cycle assays. Western blot assays were performed to assess intracellular protein levels of Skp2, p27, and cleaved caspase-3. The amount of ubiquitin attached to p27 was determined using an immunoprecipitation assay. The viability of U266 and RPMI 8226 cells was significantly inhibited by 10 μM SKPin C1 and the inhibitory effect was enhanced with increasing doses of SKPin C1. In contrast, 50 μM SKPin C1 only marginally decreased viability of normal B lymphocytes in 12 h. Skp2 and p27 expression in U266 and RPMI 8226 cells was higher and lower, respectively, than that in the normal B lymphocytes. Treatment with SKPin C1 or Skp2 knockdown increased p27 protein levels in U266 and RPMI 8226 cells by preventing p27 from being ubiquitinated, which slowed the cell cycle, inhibited cell proliferation, and triggered apoptosis. Therefore, this study suggested SKPin C1 as a potent inhibitor against aberrant proliferation and immortalization of MM.

Antineoplastic Effect of Procyanidin-rich Extract of Lafoensia Pacari in Lung Carcinoma Cells

Abstract Lafoensia pacari A. St. Hill has been used in traditional medicine as an anti-ulcerogenic and anti-inflammatory. Although there is an ethnopharmacological indication for cancer treatment, only a few studies have demonstrated its possible anticancer activity. Thus, the aims of this study were: (1) to evaluate the antineoplastic effect of L. pacari ethanolic extract (LPE) in lung carcinoma cells, (2) to determine the mode of action of LPE and (3) to identify the substances present in LPE. Human and murine lung cancer cell lines were grown in vitro and treated with different concentrations of LPE. Cell cycle and caspase-3 activity assays were performed in order to verify the mode of action. LC-ESI-MS screening was performed to detect the compounds present in LPE. LPE showed a dose-dependent cytotoxic effect, where neoplastic cells were more sensitive than non-neoplastic. The LPE induced sub-G1 cell cycle arrest in cancer cells suggesting cell death, which was confirmed as apoptosis by the activation of caspase-3. The LC-ESI-MS analysis indicated a high level of procyanidins, which could be responsible for the antineoplastic effect of LPE. Thus, we concluded that a Lafoensia pacari extract, rich in procyanidins, is cytotoxic against lung cancer cells through activation of caspase-3-dependent apoptosis.

Gramicidin inhibits human gastric cancer cell proliferation, cell cycle and induced apoptosis

BACKGROUND: Gastric cancer is a common malignant tumor with high morbidity and mortality worldwide, which seriously affects human health. Gramicidin is a short peptide antibiotic which could be used for treating infection induced by bacteria or fungi. However, the anti-cancer effect of gramicidin on gastric cancer cells and its underlying mechanism remains largely unknown. RESULTS: Gastric cancer cells SGC-7901, BGC-823 and normal gastric mucosal cells GES-1 were treated with different concentrations of gramicidin respectively. The results of CCK-8 experiment revealed cellular toxicity of gramicidin to cancer cells while cell colony formation assay showed that gramicidin significantly inhibited the proliferation of gastric cancer cells, but had little effect on normal gastric mucosal cells. In addition, the wound healing assay showed that gramicidin inhibited the migration of SGC-7901 cell. Meanwhile, apoptosis and cell cycle analysis revealed that gramicidin induced cell apoptosis with G2/M cell cycle inhibition. Furthermore, western blot analysis demonstrated that gramicidin down-regulated the expression of cyclinD1 and Bcl-2 as well as the FoxO1 phosphorylation. CONCLUSIONS: The current study illustrated the anti-tumor activity of gramicidin on gastric cancer cells, providing a possibility for gramicidin to be applied in clinical practice for the treatment of gastric cancer.

Comparação entre os perfis de expressão de microRNAs em Linfomas de Hodgkin Clássico / Comparison between microRNA expression profiles in Classic Hodgkin Lymphoma

Introdução: Linfoma de Hodgkin (LH) é um dos linfomas mais frequentes no mundo ocidental, com cerca de 3 a 4 novos casos a cada 100 mil pessoas. Os microRNAs são pequenas moléculas que regulam a transcrição gênica e estão associadas ao desenvolvimento e progressão das neoplasias. Em LH clássico (LHC), alguns microRNAs já foram identificados a partir de células infectadas pelo EBV. Além destes, outros microRNAs já foram descritos em LHC, tanto em estudos onde foram realizadas esta avaliação em culturas celulares como utilizando a massa tumoral total de linfonodos, mas com pouca correlação com o comportamento biológico. Objetivo: O presente estudo tem como objetivo principal avaliar o perfil de expressão de microRNA em LHC, comparando casos livres de doença após a terapia com casos que exibiram recidivas. Material e métodos: Foram avaliados 106 pacientes com o diagnóstico de LHC, sendo coletados dados clínicos e avaliados os marcadores imuno-histoquímicos CD30, CD15, CD20 e CD15 destes casos, além da presença de EBV por CISH. Vinte casos foram selecionados para a avaliação da expressão de 377 microRNAs, 10 casos com recidiva e 10 casos sem evidência de doença. Como grupos comparadores, foram selecionadas 8 amostras de linfonodos reativos, 4 com hiperplasia folicular e 4 com hiperplasia paracortical. Os resultados do perfil de expressão foram submetidos ao método de agrupamento hierárquico não supervisionado. Resultados e discussão: Os parâmetros clínico-patológicos desta coorte não diferiram daqueles encontrados na literatura. A sobrevida global e a sobrevida livre de progressão foram de 92,1% e 83,6% em 5 anos, respectivamente. Os fatores associados ao prognóstico em análise multivariada foram idade, níveis séricos de albumina e DHL e estadiamento. O perfil de expressão dos microRNAs de todos os casos de LHC possibilitaram a separação completa dos mesmos em relação aos linfonodos reativos, independentemente do tipo de hiperplasia. Na comparação entre os casos de LHC, foram identificados 3 microRNAs diferencialmente expressos: miR-502-3p e miR-363, ambos hipoexpressos no grupo que continha todos os casos com recidivas e miR-886-5p, hiperexpresso neste grupo. Os dois primeiros microRNAs estão associados a supressão tumoral, através da inibição da proliferação e migração celular. O miR-886-5p é associado ao aumento da proliferação celular e inibição da apoptose. Em linfomas, este já foi descrito como estando hiperexpresso em linfomas T, incluindo linfoma anaplásico de grandes células. Conclusão: Os LHC possuem um perfil distinto de expressão de microRNAs, com muitos deles envolvendo mecanismos fundamentais na oncogênese, como ciclo celular e apoptose. Os microRNAs encontrados diferencialmente expressos nos casos recidivados no presente estudo podem estar associados ao comportamento menos indolente destas neoplasias, podendo ser alvo de futuras investigações, incluindo seu uso como potenciais alvos de terapia

Introduction: Hodgkin lymphoma (HL) is one of the most common lymphomas in the western world, with about 3 to 4 new cases per 100,000 people. MicroRNAs are small molecules that regulate gene transcription and are associated with the development and progression of neoplasms. In classic HL (CHL), some microRNAs have already been identified from EBV-infected cells. In addition, other microRNAs have already been described in CHL, both in studies where this evaluation was performed in cell cultures and using whole lymph node tumor tissue, but with little correlation with biological behavior. Objective: The present study aims to evaluate the microRNA expression profile in CHL, comparing disease-free cases after therapy with cases that showed recurrences. A total of 106 patients diagnosed with CHL were evaluated. Clinical data were collected and the immunohistochemical markers CD30, CD15, CD20 and CD15 of these cases were evaluated, as well as the presence of EBV by CISH. Twenty cases were selected for the analysis of the expression of 377 microRNAs, 10 cases with recurrences and 10 cases without evidence of disease. For comparison, 8 samples of reactive lymph nodes were selected, 4 with follicular hyperplasia and 4 with paracortical hyperplasia. The expression profile results were submitted to the unsupervised hierarchical clustering method. Results and discussion: The clinico-pathological parameters of this cohort did not differ from those found in the literature. Overall survival and progression-free survival were 92.1% and 83.6% at 5 years, respectively. Factors associated with prognosis in multivariate analysis were age, serum albumin and DHL levels, and staging. The microRNA expression profile of all CHL cases allowed their complete separation from reactive lymph nodes, regardless of the type of hyperplasia. When comparing the cases of CHL, 3 differentially expressed microRNAs were identified: miR-502-3p and miR-363, both hypoexpressed in the group containing all relapsed cases and miR-886-5p, hyperexpressed in this group. The first two microRNAs are associated with tumor suppression through inhibition of cell proliferation and migration. The miR-886-5p is associated with increased cell proliferation and inhibition of apoptosis. In lymphomas, it has been described as being overexpressed in T-cell lymphomas, including anaplastic large cell lymphoma. Conclusion: CHLs have a distinct microRNA expression profile, with many involving key mechanisms in oncogenesis, such as cell cycle and apoptosis. The differentially expressed microRNAs found in the relapsed cases in the present study may be associated with the less indolent behavior of these neoplasms and may be the subject of future investigations, including their use as potential therapeutic targets

Gliomas malignos: biología molecular y detalles oncogenéticos / Wicked gliomas: molecular biology and oncogenetics detail

RESUMEN La biología de los gliomas malignos se asocia con el balance de la expresión de las proteínas que controlan de manera positiva o negativa el ciclo celular, la proliferación, la motilidad, la neoformación vascular y el reconocimiento del sistema inmune. La frecuencia de las alteraciones genéticas que están presentes en GBM2 y GBM1 son diferentes así como la edad de los pacientes en la que se presentan. Mientras que los GBM1 suelen aparecer en edades más tardías, alrededor de los 60-70 años, los GBM2 suelen presentarse en edades más tempranas, 40-50 años. En la génesis del glioblastoma existen alteraciones moleculares a nivel de genes supresores de tumores, oncogenes y genes reparadores de ADN (AU).

ABSTRACT The glioblastoma it is the primary wicked tumor of the central nervous system more common in adults and it invariably associates to a bad presage. The biology of the wicked gliomas associates with the balance of the expression of the proteins that they control of positive way or negative the cellular cycle, the proliferation, the motility, the vascular neoformation and the recognition of the immune system. The frequency of the genetic alterations that they are present in GBM2 and GBM1 is different. While the GBM1 usually appears in later ages, around the 60-70 years, the GBM2 usually presents in earlier ages, 40-50 years. In the genesis of the glioblastoma exist molecular alterations at level of suppressive genes of tumors (GST), oncogenes and reparative genes of DNA (AU).

Enhanced apoptosis and inhibition of gastric cancer cell invasion following treatment with LDH@Au loaded Doxorubicin

Background: The suppression of cancer cell growth and invasion has become a challenging clinical issue. In this study, we used nanotechnology to create a new drug delivery system to enhance the efficacy of existing drugs. We developed layered double hydroxide by combing Au nanosol (LDH@Au) and characterized the compound to prove its function as a drug delivery agent. The anti-cancer drug Doxorubicin was loaded into the new drug carrier to assess its quality. We used a combination of apoptosis assays, cell cycle assays, tissue distribution studies, cell endocytosis, transwell invasion assays, and immunoblotting to evaluate the characteristics of LDH@Au as a drug delivery system. Results: Our results show that the LDH@Au-Dox treatment significantly increased cancer cell apoptosis and inhibited cell invasion compared to the control Dox group. Additionally, our data indicate that LDH@Au-Dox has a better target efficiency at the tumor site and improved the following: cellular uptake, anti-angiogenesis action, changes in the cell cycle, and increased caspase pathway activation. Conclusions: Our findings suggest the nano drug is a promising anti-cancer agent and has potential clinical applications.

Expression of cell cycle inhibitors in canine prostate with proliferative inflammatory atrophy and carcinoma / Expressão de inibidores do ciclo celular na próstata canina com atrofia inflamatória proliferativa e carcinoma

Gene expression of CDKN1A, CDKN1B, and TP53, and immunostaining of p21, p27 and p53 were evaluated to verify the role of these cell cycle inhibitors in canine prostates with proliferative inflammatory atrophy-PIA and prostatic carcinoma-PC. Seventy samples, 15 normal, 30PIA and 25PC. Regarding number of p27 and p53 labeled cells, difference between normal and PIA and PC was observed, as well as between PIA and PC for p53. Immunostaining intensities of p21, p27 and p53 were different when comparing normal tissues to PIA and PC. Sixteen cDNA of canine prostatic FFPE tissue were subjected to RT-PCR and RT-qPCR, four normal, three PIA, and nine PC. CDKN1A mRNA was detected in four PC by RT-PCR, and it was overexpressed when compared to normal by RT-qPCR, in one PIA and six PC. CDKN1B mRNA was detected in three PC by RT-PCR and it was overexpressed in three PC and decreased in one PC. TP53 mRNA was overexpressed in one PIA and three PC. In conclusion, when overexpressed in canine prostate with premalignant and malignant, p21 and p27 play a role controlling cell proliferation, working as a protective factor in the evolution of PIA to PC, and in the PC development, even in the presence of altered p53.(AU)

A expressão gênica de CDKN1A, CDKN1B e TP53, assim como imunomarcação de p21, p27 e p53 foram realizadas a fim de verificar o papel desses inibidores do ciclo celular na próstata canina com atrofia inflamatória proliferativa (PIA) e carcinoma prostático (PC). Foram obtidas70 amostras de próstata canina, sendo 15 de tecido normal, 30 de PIA e 25 de PC. Quanto ao número de células imunomarcadas foi observada diferença entre amostras normais, com PIA e PC para p27 e p53, assim como entre PIA e PC para p53. Para a intensidade de imunomarcação houve diferença entre os tecidos normais e com PIA e PC para p21, p27 e p53. Foram obtidas dezesseis amostras de cDNA a partir de amostras de próstatas caninas embebidas em parafina para a realização da RT-PCR e RT-qPCR, sendo quatro normais, três com PIA, e nove com o PC. O gene CDKN1A foi detectado em quatro das amostras com PC por RT-PCR, e pela RT-qPCR este estava superexpresso em uma PIA e em seis PC quando da comparação com o tecido prostático normal. O CDKN1B foi detectado em três PC por RT-PCR e pela RT-qPCR estava superexpresso em três PC e reduzido em um PC. O TP53 foi detectado em todas as próstatas caninas com PIA e PC por RT-PCR, sendo também superexpresso em uma glândula com PIA e em três com PC. Concluiu-se que p21 e p27 quando superexpressas na próstata canina com lesões pré-malignas (PIA) e malignas (PC) desempenham ação no controle da proliferação celular, possivelmente atuando como fator de proteção na evolução da PIA para PC, e no desenvolvimento do PC, mesmo na presença de p53 alterada. Assim, o próximo passo é avaliar essas proteínas do ciclo celular em casos de PC canino com metástase.(AU)