Efficacy of phosphocreatine pre-administration on XIAP and Smac in ischemic penumbra of rats with focal cerebral ischemia reperfusion injury

Abstract Purpose: To observe the efficacy of phosphocreatine pre-administration (PCr-PA) on X-linked inhibitor of apoptosis protein (XIAP), the second mitochondia-derived activator of caspase (Smac) and apoptosis in the ischemic penumbra of rats with focal cerebral ischemia-reperfusion injury (CIRI). Methods: A total of 60 healthy male Sprague Dawley (SD) rats were randomly divided into three groups (n=20): group A (the sham operation group), group B <intraperitoneally injected with 20 mg/kg (10 mg/ml) of saline before preparing the ischemia-reperfusion (IR) model>, and group C <intraperitoneally injected with 20 mg/kg (10 mg/ml) of PCr immediately before preparing the IR model>. After 24 h for reperfusion, the neurological function was evaluated and the tissue was sampled to detect expression of XIAP, Smac and caspase-3 positive cells in the ischemic penumbra so as to observe the apoptosis. Results: Compared with group B, neurological deficit scores, numbers of apoptotic cells, expression of Smac,caspase-9 and the numbers of Caspase-3 positive cells were decreased while expression of XIAP were increased in the ischemic penumbra of group C. Conclusions: Phosphocreatine pre-administration may elicit neuroprotective effects in the brain by increasing expression of X-linked inhibitor of apoptosis protein, reducing expression of second mitochondia-derived activator of caspase, and inhibiting the apoptosis in the ischemic penumbra.

High expression of XIAP and Bcl-2 may inhibit programmed cell death in glioblastomas / Alta expressão de XIAP e Bcl-2 pode inibir morte celular programada em glioblastomas

ABSTRACT Glioblastoma (GBM) is the most malignant glioma and represents 29% of all brain tumors. Tumorigenesis is intimately connected with characteristics acquired in the physiologic pathway of cellular death. Objective: In the present study, the expression of anti-apoptotic (XIAP and Bcl-2) and apoptotic (cytochrome C, caspase 9, APAF-1), caspase 3 and the Smac/DIABLO genes related to the apoptosis pathway were evaluated in 30 samples of glioblastoma. Methods: The gene expression was evaluated in 30 glioblastomas (WHO grade IV) and compared to 10 white matter control samples with real-time PCR. Results and Conclusion: There were higher expressions of XIAP (p = 0.0032) and Bcl-2 (p = 0.0351) in the glioblastoma samples compared to the control samples of normal brain. These results raise the question of whether Bcl-2 and XIAP genes can be responsible for the inhibition of programmed cell death in glioblastomas. Moreover, they provide additional information capable of allowing the development of new target therapy strategies.

RESUMO O glioblastoma (GBM) é o glioma mais maligno e representa 29% de todos os tumores cerebrais. A tumorigênese está intimamente ligada à características adquiridas na via fisiológica de morte celular. Objetivo: Avaliar a expressão de genes anti-apoptóticos (XIAP e Bcl-2) e apoptóticos (citocromo C, a caspase 9, APAF-1), caspase 3 e SMAC/DIABLO, relacionados à apoptose, em 30 amostras de tecido de pacientes com glioblastoma. Métodos: A expressão gênica foi avaliada em trinta glioblastomas e comparada a dez amostras controles de substância branca por PCR em tempo real. Resultados e Conclusão: Houve maior nível de expressão de XIAP (p = 0,0032) e Bcl-2 (p = 0,0351) em comparação com as amostras controle, de cérebro normal. Estes resultados levantam a questão de que os genes Bcl-2 e XIAP podem ser responsáveis pela inibição da morte celular programada em glioblastomas, além disso, proporcionam informação adicional capaz de permitir o desenvolvimento de novas estratégias de terapia alvo.

Efecto mutagénico y genotóxico, y expresión de los genes Rad51C , Xiap , P53 y Nrf2 inducidos por extractos antipalúdicos de plantas recolectadas en el Vaupés medio, Colombia / Mutagenicity, genotoxicity and gene expression of Rad51C, Xiap, P53 and Nrf2 induced by antimalarial extracts of plants collected from the middle Vaupés region, Colombia

Resumen Introducción. Dada la resistencia de Plasmodium a los medicamentos antipalúdicos, es necesario encontrar nuevas alternativas terapéuticas para su tratamiento y control. Con base en el saber indígena colombiano, se recopilaron extractos de plantas del Vaupés medio con potencial efecto antipalúdico. Objetivo. Evaluar el efecto mutagénico y genotóxico, y la expresión de los genes Rad51C, Xiap, P53 yNrf2, inducidos por cuatro extractos etanólicos con actividad anti-Plasmodium(R001, T002, T015 y T028). Materiales y métodos. Se evaluó el potencial mutagénico de cuatro extractos etanólicos con efecto antiplasmódico utilizando el test de Ames y el efecto genotóxico, con un ensayo del cometa; asimismo, se analizó la expresión de los genes Rad51C, Xiap, P53 y Nrf2 en células HepG2. Resultados. Los extractos no fueron mutágenos en la cepa TA98 de Salmonella typhimurium en presencia y ausencia de actividad metabólica de la fracción S9. En la cepa TA100, los extractos R001, T015 y T028 se comportaron como mutágenos débiles en presencia de S9, con índices mutagénicos de 1,58; 1,38; 1,53 y 1,61, respectivamente; T015 tuvo el mismo comportamiento en ausencia de S9, con un índice mutagénico de 1,36. En el ensayo del cometa, todos los extractos provocaron daño de categorías 1 o 2, con colas de cometas entre 36,7 y 51,48 µm de longitud; sin embargo, el índice dedaño genético sugirió que los tratamientos afectaron la mayoría de las células. En los genes en estudio, los extractos R001 y T028 indujeron una sobreexpresiónde 1,84 a 3,99 frente a las células sin tratar de los genes Xiap y P53. Conclusiones. Los resultados evidenciaron que el extracto T002 fue el más seguro, ya que presentó actividad anti-Plasmodium, no fue citotóxico en las células HepG2, no fue mutágeno, causó daño de categoría 1 en el ADN y no modificó la expresión de los genes evaluados.

Abstracts Introduction: Due to Plasmodium resistance to antimalarial drugs, it is important to find new therapeutic alternatives for malaria treatment and control. Based on the knowledge of Colombian indigenous communities, we collected extracts of plants with potential antimalarial effects from the middle Vaupés region. Objective: To evaluate the mutagenic and genotoxic effects, as well as the gene expression of Rad51C, Xiap, P53 and Nrf2 induced by four ethanolic extracts with antimalarial activity (R001, T002, T015 and T028). Materials and methods: We evaluated four ethanolic extracts with antimalarial activity using the Ames test to assess mutagenicity, and the comet assay on HepG2 cells to determine the genotoxicicity. We also evaluated the expression of Rad51C, Xiap, P53 and Nrf2 from HepG2 cells stimulated with the four extracts. Results: None of the four extracts was mutagenic in Salmonella typhimurium TA98 strain in the presence and absence of S9 metabolic activity. Extracts R001, T015 and T028 were weakly mutagenic on the TA100 strain in the presence of S9, with mutagenic indexes (MI) of 1.58, 1.53 and 1.61, respectively. The T015 strain showed the same behavior without S9 with an MI of 1.36. The results of the comet assay showed that the four extracts produced category 1 or 2 damage, with comets between 36.7 and 51.48 µm in length. However, the genetic damage index suggested that most of the cells were affected by the treatments. Regarding gene expression, extracts R001 and T028 induced an overexpression of genes Xiap and P53 with an 1.84 to 3.99 fold-change compared with untreated cells. Conclusions: These results revealed that the T002 extract was the safest as it had antimalarial activity and was not cytotoxic on HepG2 cells. Moreover, it was not mutagenic and it only produced category 1 damage on the DNA. Also, the extract did not induce a change in the expression of the tested genes.

Localização subcelular de XIAP: implicações na proliferação celular, resistência às drogas e prognóstico no câncer de mama / [Subcellular localization of XIAP: implications for cell proliferation, drug resistance and prognosis in breast cancer]

O câncer de mama é o tipo tumoral que mais acomete as mulheres no Brasil e no mundo, além de causar elevadas taxas de morbidade e mortalidade. Essa neoplasia tem como importante característica o desbalanço entre proliferação e morte celular. Nesse contexto, se insere a XIAP, uma proteína inibidora da apoptose (IAP), que exerce sua função antiapoptótica através da ligação e inibição de caspases, bem como ubiquitinação de proteínas-alvo. A XIAP é encontrada principalmente na porção citoplasmática tanto em células tumorais quanto em não neoplásicas, porém estudos mostram que também é possível detectar a sua expressão no núcleo. Dados prévios do nosso grupo mostram que a XIAP pode estar localizada tanto no citoplasma quanto no núcleo em pacientes com câncer de mama, porém não há muitos relatos acerca dos papéis exercidos pela XIAP em diferentes compartimentos celulares. O objetivo do presente estudo é elucidar o impacto da XIAP e sua localização subcelular na proliferação celular, resistência às drogas e no prognóstico no câncer de mama. Nossos dados mostram que todas as linhagens celulares investigadas apresentaram XIAP citoplasmática, exceto as células MCF-7 DoxR , resistentes à doxorrubicina (dox), que também apresentaram XIAP na fração nuclear, como avaliado por fracionamento subcelular e Western blotting. Pelos ensaios de MTT e clonogênico, observamos que o tratamento com a dox diminuiu a viabilidade celular e a capacidade de formação de colônias nas células MDA-MB-231 e MCF-7, porém o mesmo resultado não foi observado nas células MCF-7 DoxR , sugerindo que a localização nuclear de XIAP esteja associada ao fenótipo de resistência à dox. Corroborando esses achados, as células MCF-7 TaxR , resistentes ao paclitaxel, apresentaram expressão nuclear de XIAP, confirmando uma possível correlação da presença de XIAP nuclear com o perfil de resistência aos quimioterápicos utilizados no tratamento do câncer de mama, independentemente do mecanismo de ação. Além disso, o tratamento com as drogas não alterou a localização subcelular de XIAP em nenhuma das células testadas. Adicionalmente, a indução da superexpressão dos mutantes XIAP∆RING e XIAPNLS C-term por transfecção transiente, onde é possível detectar expressão de XIAP nuclear, resultou no aumento da capacidade proliferativa das células MCF-7, como examinado pela contagem de células, ensaio clonogênico e de viabilidade celular. De maneira consistente, a indução de XIAP no núcleo promoveu a resistência ao tratamento com dox, confirmando os nossos achados prévios referentes à presença da XIAP no núcleo de células quimiorresistentes. Por fim, a análise de curvas de Kaplan-Meyer revelou que a localização nuclear da XIAP conferiu um prognóstico adverso nas pacientes negativas para receptores hormonais, enquanto que a presença de XIAP citoplasmática conferiu uma tendência ao melhor prognóstico nas pacientes desse subgrupo. De acordo, a expressão de XIAP citoplasmática foi associada à idade igual ou superior a 50 anos e ao tamanho de tumor T1, fatores de prognóstico favorável no câncer de mama. Em conjunto, nossos dados mostram que a expressão de XIAP pode ser encontrada em diferentes compartimentos subcelulares em linhagens celulares e amostras de pacientes com câncer de mama, estando a presença de XIAP no núcleo associada a um fenótipo de maior proliferação e resistência às drogas in vitro, além de um prognóstico desfavorável em pacientes com câncer de mama negativas para receptores hormonais.

ERK1/2-mediated Cytoplasmic Accumulation of hnRNPK Antagonizes TRAIL-induced Apoptosis through Upregulation of XIAP in H1299 Cells / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance greatly limits the clinical therapeutic efficacy of TRAIL. Elucidating the molecular mechanism underlying TRAIL resistance will be fundamental to resolving this problem.</p><p><b>METHODS</b>Nuclear and cytoplasmic protein extraction and immuno？uorescence (IF) assay were used to detect changes in heterogeneous nuclear ribonucleoprotein K (hnRNPK) localization in H1299 cells. The evaluation of cell apoptosis in cells transfected with GFP-hnRNPK, GFP-hnRNPK S284/353A, or GFP-hnRNPK S284/353D mutant was performed using cleaved caspase-3 antibody. The gene expression of XIAP was tested by quantitative RT-PCR.</p><p><b>RESULTS</b>Previously, we reported that hnRNPK antagonized TRAIL-induced apoptosis through inhibition of PKC-mediated GSK3β phosphorylation. In this study, we further demonstrate that TRAIL treatment induces cytoplasmic accumulation of hnRNPK in H1299 cells. The hnRNPK localized in the cytoplasm has a higher capacity to antagonize TRAIL-induced apoptosis. Both ERK1/2 signaling inhibitor U0126 and ERK-phosphoacceptor-site mutant (GFP-hnRNPK S284/353A) diminish cytoplasmic accumulation of hnRNPK induced by TRAIL. Moreover, we show that XIAP is involved in hnRNPK-mediated TRAIL resistance in H1299 cells.</p><p><b>CONCLUSION</b>Taken together, these results give new insights into the understanding of the molecular mechanism associated with TRAIL resistance in lung adenocarcinoma.</p>

Morphological and immunohistochemical analysis of apoptosis in the cerebellum of rats subjected to focal cerebral ischemia with or without alcoholism model

ABSTRACT PURPOSE: To evaluated histopathological changes, morphometric and expression of proteins CASPASE-3, BCL-2 and XIAP related to apoptosis in the cerebellum after induction of temporary focal cerebral ischemia followed by reperfusion, with or without a model of chronic alcoholism. METHODS: Fifty Wistar rats were used and divided into: control group (C), sham group (S), ischemic group (I), alcoholic group (A), and ischemic and alcoholic group (IA). The cerebellum samples collected were stained for histopathological and morphometric analysis and immunohistochemistry study. RESULTS: Histopathological changes were observed a greater degree in animals in groups A and IA. The morphometric study showed no difference in the amount of cells in the granular layer of the cerebellum between the groups. The expression of CASPASE-3 was higher than BCL-2 and XIAP in the groups A and IA. CONCLUSION: We observed correlation between histopathological changes and the occurrence of apoptosis in cerebellar cortex.

Effects of LCL161, a Smac mimetic on the proliferation and apoptosis in hepatocellular carcinoma cells / 中南大学学报(医学版)

OBJECTIVE@#To investigate the effects of LCL161, a Smac mimetic, on the proliferation and apoptosis in hepatocellular carcinoma cells and the underlying mechanisms. 
@*METHODS@#The effect of LCL161 on the cell viability of HepG2 and SMMC7721 cells was measured by MTT assay. The effect of LCL161 at lower concentrations on the proliferation in hepatocellular carcinoma (HCC) cells was detected by colony formation assay. Apoptosis was assessed by flow cytometry with PI staining. The mitochondrial membrane potential was measured by JC-1 staining. The expression of PARP, p-Akt, cIAP1 and XIAP protein was analyzed by Western blot.
@*RESULTS@#LCL161 displayed notable antiproliferative activity on HCC cells at the concentrations of 1-16 μmol/L (P<0.01), with IC50 values of 4.3 and 4.9 μmol/L for HepG2 and SMMC7721 cells, respectively, after treatment for 48 h. LCL161 at lower concentrations obviously inhibited the colony formation of HCC cells. LCL161 induced significant apoptosis in HCC cells (P<0.01), and resulted in the apoptotic rate at (1.5±0.8)% or (1.8±0.6)% , (15.2±2.8)% or (12.2±2.4)%, (28.7±3.0)% or (22.4±2.7)%, (34.6±2.3)% or (30.2±2.4)% for HepG2 cells or SMMC7721 cells at the concentration of 0, 2, 4 or 8 μmol/L, respectively. The result of JC-1 staining indicated that the mitochondrial membrane potential of HCC cells was reduced by LCL161. In addition, LCL161 promoted the cleavage of PARP, down-regulated the protein expression of p-Akt, and degraded cIAP1.
@*CONCLUSION@#LCL161 possesses significant anti-proliferative activity and pro-apoptotic action in HepG2 and SMMC7721 cells, which might be correlated with reduction in mitochondrial membrane potential, down-regulation of p-Akt and degradation of cIAP1.

Silencing of Pin1 suppresses hyperoxia-induced apoptosis of A549 cells / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To explore the effect of silence of Pin1 expression on hyperoxia-induced apoptosis in alveolar epithelial cells A549.</p><p><b>METHODS</b>A549 cells were divided into four groups: control, hyperoxia, negative lentivirus and Pin1-shRNA hyperoxia. The hyperoxia group was exposed to a mixture of 95%O2 and 5%CO2 for 10 minutes. Then cells were cultured in a closed environment. After 24 hours, the changes of morphology were observed under an inverted microscope. Cell apoptosis was detected by flow cytometry (FCM). The expression of X-linked inhibitor of apoptosis protein (XIAP) and Caspase-9 were detected by immunohistochemistry. The production of reactive oxygen species (ROS) and cellular mitochondria membrane potential (△Ψm) were determined by fluorescence microscopy.</p><p><b>RESULTS</b>Under the inverted microscope, the A549 cells grew slowly and the changes in morphology of the cells were most obvious in the hyperoxia and negative lentivirus groups. The changes in morphology of A549 cells were obviously improved in the Pin1-shRNA hyperoxia group. The FCM results showed that the apoptosis rate of A549 cells increased, Caspase-9 expression increased, XIAP expression decreased, mitochondrial ROS production increased and mitochondrial membrane potential decreased in the hyperoxia and negative lentivirus groups compared with the control group (P<0.05). Compared with the hyperoxia and negative lentivirus groups, the apoptosis rate of A549 cells decreased, Caspase-9 expression decreased, XIAP expression increased, mitochondrial ROS production decreased and mitochondrial membrane potential increased in the Pin1-shRNA hyperoxia group (P<0.05), although the levels of the indexes did not reach to those of the control group.</p><p><b>CONCLUSIONS</b>Silencing of Pin1 could suppress hyperoxia-induced apoptosis of A549 cells.</p>

Anthocyanins From the Fruit of Vitis coignetiae Pulliat Potentiate the Cisplatin Activity by Inhibiting PI3K/Akt Signaling Pathways in Human Gastric Cancer Cells

BACKGROUND: Cisplatin (cis-diaminedichloroplatinum, CDDP) is a widely used chemotherapeutic agent for the treatment of many cancers. However, initial resistance to CDDP is a serious problem in treating these cancers. Vitis coignetiae Pulliat (Meoru in Korea) have shown anti-nuclear factor kappa B and anti-epidermal growth factor receptor activities in cancer cells. METHODS: In this study, in order to seeking an approach to increase the anti-cancer effects of CDDP with natural products. Here, we investigated anthocyanins isolated from Vitis coignetiae Pulliat (anthocyanidins isolated from meoru, AIMs) can enhance anti-cancer effects of cisplatin (CDDP) in stomach cancer cells. The cell viability of SNU-1 and SNU-16 cells after treated with AIMs and CDDP were analyzed by MTT assay. The expressions of Akt and X-linked inhibitor of apoptosis protein (XIAP) proteins were examined by western blot in AIMs- and CDDP-treated cells. RESULTS: We found that AIMs enhanced anticancer effects of CDDP, which activity was additive but not synergistic. AIMs suppressed Akt activity of the cancer cells activated by CDDP. AIMs also suppressed in XIAP an anti-apoptotic protein. CONCLUSIONS: This study suggests that the anthocyanins isolated from fruits of Vitis coignetiae Pulliat enhanced anti-cancer effects of CDDP by inhibiting Akt activity activated by CDDP.

Is Whole Exome Sequencing Clinically Practical in the Management of Pediatric Crohn's Disease?

BACKGROUND/AIMS: The aim of this study was to identify the profile of rare variants associated with Crohn's disease (CD) using whole exome sequencing (WES) analysis of Korean children with CD and to evaluate whether genetic profiles could provide information during medical decision making. METHODS: DNA samples from 18 control individuals and 22 patients with infantile, very-early and early onset CD of severe phenotype were used for WES. Genes were filtered using panels of inflammatory bowel disease (IBD)-associated genes and genes of primary immunodeficiency (PID) and monogenic IBD. RESULTS: Eighty-one IBD-associated variants and 35 variants in PID genes were revealed by WES. The most frequently occurring variants were carried by nine (41%) and four (18.2%) CD probands and were ATG16L2 (rs11235604) and IL17REL (rs142430606), respectively. Twenty-four IBD-associated variants and 10 PID variants were predicted to be deleterious and were identified in the heterozygous state. However, their functions were unknown with the exception of a novel p.Q111X variant in XIAP (X chromosome) of a male proband. CONCLUSIONS: The presence of many rare variants of unknown significance limits the clinical applicability of WES for individual CD patients. However, WES in children may be beneficial for distinguishing CD secondary to PID.

3-bromopyruvate enhances cisplatin sensitivity of hepatocellular carcinoma cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of 3-bromopyruvate (3-BP) in sensitizing hepatocellular carcinoma cells to cisplatin-induced apoptosis and its possible mechanism.</p><p><b>METHODS</b>The growth inhibition of HepG2 and SMMC7721 cells following exposures to different concentrations of 3-BP and cisplatin was measured by MTT assay. The apoptosis of cells treated with 100 µmol/L 3-BP with or without 8 µmol/L cisplatin was assessed using flow cytometry with PI staining, and the activity of caspase-3 and intracellular ATP level were detected using commercial detection kits; the expression of XIAP and PARP was analyzed using Western blotting.</p><p><b>RESULTS</b>3-BP produced obvious inhibitory effects on HepG2 and SMMC7721 cells at the concentrations of 50-400 µmol/L with IC50 values of 238.9∓13.9 µmol/L and 278.7∓11.7 µmol/L for a 48-h treatment, respectively. Cisplatin also inhibited the growth of HepG2 and SMMC7721 cells at the concentrations of 2-32 µmol/L, with IC50 values of 16.4∓0.9 µmol/L and 20.9∓1.8 µmol/L after a 48-h treatment, respectively. Treatment with 100 µmol/L 3-BP combined with 8 µmol/L cisplatin for 48 h resulted in a growth inhibition rate of (60.6∓2.2)% in HepG2 cells and (56.8∓2.3)% in SMMC7721 cells, which were significantly higher than those in cells treated with 3-BP or cisplatin alone. The combined treatment for 48 h induced an apoptotic rate of (51.1∓4.3)% in HepG2 cells and (46.5∓3.9)% in SMMC7721 cells, which were also markedly higher than those in cells with 3-BP or cisplatin treatment alone.</p><p><b>CONCLUSION</b>3-BP can sensitize HepG2 and SMMC7721 cells to cisplatin-induced apoptosis possibly by causing intracellular ATP deficiency, down-regulating XIAP, and increasing caspase-3 activity.</p>

XIAP gene mutation screening in children with hemophagocytic lymphohistiocytosis / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the prevalence of mutations and sequence variations in X-linked inhibitor of apoptosis (XIAP) gene among Chinese pediatric patients with hemophagocytic lymphohistiocytosis (HLH).</p><p><b>METHODS</b>Sixty-five children who were diagnosed with HLH between January 2009 and December 2012 (case group), as well as 70 healthy children (control group), were enrolled in the study. The exons of XIAP gene (1-1, 1-2, 2-6) were amplified by PCR and directly sequenced. The genotypic and allelic frequencies of single nucleotide polymorphism (SNP) were analyzed.</p><p><b>RESULTS</b>None of the HLH patients showed mutations in these exons of XIAP gene. Only one nonsynonymous SNP, rs5956583 located in exon 5, was observed, but there were no significant differences in the genotypic and allelic frequencies of this SNP between the case and control groups (P>0.05).</p><p><b>CONCLUSIONS</b>HLH caused by XIAP mutations may be rare in children. SNP rs5956583 of XIAP gene may have little contribution to the development of childhood HLH.</p>

Avaliação de survivina e XIAP e dos fatores de transcrição foxo3a e foxM1 como potenciais biomarcadores de quimiorresistência no câncer de mama / [Evaluation of survivin and XIAP and transcription factors foxo3a and foxM1 as potential chemoresistance biomarkers in breast cancer]

O câncer de mama é a neoplasia que apresenta maior mortalidade entre as mulheres ao redor do mundo, apesar dos avanços na descoberta de novas modalidades terapêuticas e marcadores prognósticos. A resistência ao tratamento quimioterápico é uma das principais causas de falha terapêutica, apontando para a necessidade da identificação de biomarcadores preditivos de resposta. Evidências científicas mostram que a superexpressão de FoxM1 e das proteínas antiapoptóticas Survivina e XIAP, bem como a inativação do fator de transcrição Foxo3a, estão associados a quimioresistência e a um prognóstico desfavorável no câncer de mama. Dessa forma, o objetivo desse estudo é avaliar o papel das proteínas Survivina, XIAP, Foxo3a e FoxM1 como potenciais fatores de resistência à doxorrubicina (doxo), quimioterápico amplamente empregado no tratamento no câncer de mama. Nossos dados mostram que a doxo foi capaz de inibir a viabilidade celular nas linhagens celulares derivadas de carcinoma de mama MCF7 (não-invasiva, positiva para receptores de estrogênio e Her2) e MDA-MB-231 (invasiva, triplo-negativa), como avaliado pelo ensaio de MTT. A droga induziu a perda da adesão celular e fragmentação do DNA, como observado pela análise morfológica com quantificação das células não-aderidas e avaliação do conteúdo de DNA por citometria de fluxo. A análise da ativação das caspases-3, -7 e -9 por Western blotting revelou que a doxo induziu apoptose em células com diferentes status de p53. Em paralelo, o tratamento com a doxo resultou na redução dos níveis proteicos e de RNAm de XIAP e Survivina, como avaliado por Western blotting e PCR em tempo real, respectivamente. Entretanto, a indução da superexpressão da Survivina, por transfecção plasmidial, não foi capaz de conferir resistência ao quimioterápico. Corroborando esses resultados, observamos que o silenciamento gênico por siRNA de XIAP e Survivina, isoladamente ou em combinação, não sensibilizou as células à morte celular induzida pela doxo, indicando que tais proteínas não desempenham papel na resistência à droga. Contrariando dados da literatura, a doxo foi capaz de induzir a fosforilação de Foxo3a e Akt e reduzir a expressão de seu alvo transcricional Bim e dos níveis de RNAm de FOXO3A. De maneira consistente, a droga promoveu a translocação de Foxo3a do núcleo para o citoplasma, como examinado por fracionamento subcelular, apontando para a inativação da sua função. Além disso, a expressão do fator de transcrição FoxM1 foi reduzida, mediante o estímulo apoptótico induzido pela doxo. A indução da superexpressão de FoxM1 foi capaz de reverter a sensibilidade das células MDA-MB-231 à doxo, processo que envolveu a indução dos níveis de Survivina e XIAP. O mesmo efeito não foi observado nas células MCF7 superexpressando FoxM1, uma vez que se mantiveram sensíveis ao quimioterápico e apresentaram inalterados níveis de Survivina e XIAP. O conjunto dos nossos dados indica que a via de sinalização oncogênica mediada pelo fator de transcrição FoxM1 é capaz de promover a resistência à doxo e sugere que a combinação clínica de inibidores de FoxM1 com a doxo tem o potencial de sobrepujar a quimiorresistência no câncer de mama, principalmente em tumores triplo-negativos.

Breast cancer is the leading cause of deaths in women around the world, despite recent advances regarding novel therapeutic options and identification of prognostic factors. Resistance to therapy is the main cause of treatment failure and still there is no predictive biomarker for response to systemic therapy available. Increasing evidence shows that Survivin and XIAP antiapoptotic proteins and FoxM1 overexpression, as well as the inactivation of Foxo3a transcription factor, are closely associated with chemoresistance and poor prognosis in breast cancer. Thus, this study aimed to investigate Survivin, XIAP, Foxo3a and FoxM1 potential role on resistance to doxorubicin (dox), a chemotherapeutic agent widely used to treat breast cancer. Our data demonstrates that dox inhibited cell viability in the breast cancer derived cell lines MCF7 (non-invasive, Her2 and estrogen receptor positive) and MDA-MB- 231 (invasive, triple-negative), as evaluated through the MTT assay. The drug induced loss of cell adhesion and DNA fragmentation, as examined by morphological analysis followed by quantification of non-adherent cells and flow cytometry DNA content analysis. Western blotting evaluation of caspases-3, -7 and -9 activation revealed that dox induced apoptosis in cells with different p53 activation status. In parallel, exposure to dox resulted in reduction in Survivin and XIAP protein and mRNA levels, as evaluated by Western blotting and real time PCR, respectively. However, when we transfected cells with a Survivin-encoding plasmid, we did not observe a cell death-resistant phenotype. Accordingly, XIAP and Survivin silencing through siRNA, individually or in combination, had little effect on breast cancer cells sensitivity towards dox, suggesting that the drug can induce apoptosis independently of their expression. Contrasting data in the literature, dox treatment induced Foxo3a and Akt phosphorylation and reduced the expression of its transcriptional target Bim and FOXO3A mRNA levels. In agreement, dox-exposed cells displayed Foxo3a expression in cytoplasm, differently from predominantly nuclear Foxo3a observed in untreated cells, as examined through subcellular localization. These data point to dox-induced Foxo3a inactivation in breast cancer cells. In addition, we observed that FoxM1 transcription factor expression was inhibited upon dox-mediated apoptotic stimuli. Importantly, FoxM1 overexpression could counteract apoptosis in MDA-MB-231 cells, along with induction of Survivin and XIAP expression. This effect was not observed in MCF7 cells, which remained similarly sensitive to dox and displayed Survivin and XIAP levels unaltered. Altogether, our results demonstrate that FoxM1 signaling pathway can promote dox resistance and suggest that combining FoxM1 inhibitors with dox has the potential to circumvent chemoresistance in breast cancer, specially in triple negative tumors.

TcpC induces apoptosis of human vascular endothelial cells and its mechanisms / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effects of TcpC on human umbilical vascular endothelial cells (HUVECs) and its mechanisms.</p><p><b>METHODS</b>HUVECs were co-cultured with TcpC secreting wild-type E. coli strain CFT073 (TcpC(wt)) or tcpc gene-deleted CFT073 mutant strain (TcpC(mut)) in transwell system,respectively. Apoptosis of HUVECs was analyzed by Annexin-V/PI double staining. Mitochondrial membrane depolarization was detected by JC-1 staining. Expression of apoptosis-related proteins in HUVECs was determined by Western blot.</p><p><b>RESULTS</b>HUVECs showed morphological changes after co-cultured with TcpC(wt) for 24 h: the cells became detached and cell debris increased,and cell number was also decreased when compared to HUVECs co-cultured with TcpC(mut). The apoptosis of HUVEC cells co-cultured with TcpC(wt) for 24 h significantly increased,compared to that of control group and TcpC(mut) group (60.1% 9.7% compared with 9.0% 1.3% and 16.9% 0.4%,respectively, P<0.05); meanwhile the mitochondrial depolarization of HUVECs co-cultured with TcpC(wt) was significantly increased,compared to that in control and TcpC(mut) groups (64.5% 0.9% compared with 14.5% 2.1% and 15.6% 3.3%, respectively,P<0.05). Cleavage of PARP and inhibition of Mcl-1 and XIAP expression were seen in HUVECs co-cultured with TcpC(wt),but not in groups of control and TcpC(mut).</p><p><b>CONCLUSION</b>TcpC secreted from CFT073 can induce apoptosis of HUVECs through mitochondrial pathway, in which PARP is cleaved and Mcl-1 and XIAP expressions are inhibited.</p>

Expression of pGSK-3α/β Tyr279/216 and XIAP proteins in cholangiocarcinoma and their clinical significance / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the expressions of the active form of glycogen synthase kinase-3(GSK-3)-pGSK-3α/β (Tyr279/216) and its downstream moleculor X-linked inhibitor of apoptosis protein (XIAP) in cholangiocarcinoma and to analyze their correlation with clinicopathological and survival significance.</p><p><b>METHODS</b>Immunohistoehemistry was used to detect the expressions of the active form of GSK-3- pGSK-3α/β (Tyr279/216) and its downstream moleculor XIAP proteins in 50 cholangiocarcinoma tissues and 20 normal bile duct tissues.</p><p><b>RESULTS</b>The positive rates of pGSK-3α/β (Tyr279/216) and XIAP were 62.0% and 68.0% in cholangiocarcinoma, and 10.0% and 25.0% in normal bile duct tissues, respectively. The intensity of pGSK-3α/β (Tyr279/216) and XIAP expressions in cholangiocarcinoma were significantly higher than that in the normal bile duct tissues (P < 0.001), and there was a significant correlation between pGSK-3α/β (Tyr279/216) and XIAP expressions (r = 0.544, P < 0.001). The expression of pGSK-3α/β(Tyr279/216) protein in cholangiocarcinoma was associated with TNM stage (P = 0.042), histological grade (P = 0.031), whereas the expression of XIAP protein in cholangiocarcinoma was correlated with CEA level (P = 0.006). Patients with positive expression of pGSK-3α/β (Tyr279/216) and XIAP demonstrate a significantly worse prognosis than that of patients with negative expression of pGSK-3α/β (Tyr279/216) and XIAP for overall survival (P = 0.002, P = 0.018). Multivariate survival analysis revealed that positive pGSK-3α/β (Tyr279/216) expression provided significant independent prognostic value for overall survival (P = 0.002).</p><p><b>CONCLUSIONS</b>The expressions of pGSK-3α/β(Tyr279/216) and XIAP proteins were significantly associated with the development and progression of cholangiocarcinoma. pGSK-3α/β(Tyr279/216) may be an important prognostic factor for survival of patients with cholangiocarcinoma.</p>

Effect of bortezomib on reverse multidrug resistance and XIAP expression in imatinib-resistant primary cells of chronic myeloid leukemia in blastic crisis / 中国实验血液学杂志

This study was aimed to explore the effects of proteasome inhibitor bortezomib on the drug sensitivity of imatinib-resistant primary cells in blastic phase of chronic myeloid leukemia (CML) and the expression of XIAP. MTT method was used to detect the inhibitory effect on cell growth, flow cytometry was used to assay the apoptosis and P170 expression, and RT-PCR was used to monitor the expression of XIAP mRNA. The results showed that the effect of imatinib or bortezomib alone showed an inhibitory effect on MNC in time-and dose-dependent manner; 5 and 10 nmoL/L bortezomib combined with imatinib could significantly enhance the sensitivity of mononuclear cells to imatinib. The increase of apoptosis rate, and the decrease of P170 expression could be observed by flow cytometry during treatment with bortezomib. The over-expression of XIAP could be down regulated by bortezomib. It is concluded that the bortezomib could inhibit primary cells of leukemia and enhance sensitivity of CML primary cells to imatinib.The bortezomib may increase the cell apoptosis by inhibition of XIAP expression, so as to provide the experiment evidence to spread bortezomib for the clinical treatment of chronic myeloid leukemia.

Characterization of H460R, a Radioresistant Human Lung Cancer Cell Line, and Involvement of Syntrophin Beta 2 (SNTB2) in Radioresistance

A radioresistant cell line was established by fractionated ionizing radiation (IR) and assessed by a clonogenic assay, flow cytometry, and Western blot analysis, as well as zymography and a wound healing assay. Microarray was performed to profile global expression and to search for differentially expressed genes (DEGs) in response to IR. H460R cells demonstrated increased cell scattering and acidic vesicular organelles compared with parental cells. Concomitantly, H460R cells showed characteristics of increased migration and matrix metalloproteinase activity. In addition, H460R cells were resistant to IR, exhibiting reduced expression levels of ionizing responsive proteins (p-p53 and gamma-H2AX); apoptosis-related molecules, such as cleaved poly(ADP ribose) polymerase; and endoplasmic reticulum stress-related molecules, such as glucose-regulated protein (GRP78) and C/EBP-homologous protein compared with parental cells, whereas the expression of anti-apoptotic X-linked inhibitor of apoptosis protein was increased. Among DEGs, syntrophin beta 2 (SNTB2) significantly increased in H460R cells in response to IR. Knockdown of SNTB2 by siRNA was more sensitive than the control after IR exposure in H460, H460R, and H1299 cells. Our study suggests that H460R cells have differential properties, including cell morphology, potential for metastasis, and resistance to IR, compared with parental cells. In addition, SNTB2 may play an important role in radioresistance. H460R cells could be helpful in in vitro systems for elucidating the molecular mechanisms of and discovering drugs to overcome radioresistance in lung cancer therapy.

Effects of Epstein-Barr virus on the development of dendritic cells derived from cord blood monocytes: an essential role for apoptosis

OBJECTIVE: Epstein-Barr virus (EBV) is a ubiquitous human γ-herpes virus, which can adapt and evade host immune defense. Dendritic cells (DCs) play a pivotal role in the initiation and maintenance of immune responses. This study investigated the effects of EBV on cord blood monocytes derived DCs (CBDC). METHODS: Monocytes were isolated from cord blood and cultured in medium containing recombinant IL-4 and GM-CSF to induce DCs development. B95-8 supernatant was added in monocytes culture medium for EBV infection at day 0. Phenotypic characterization of DCs, apoptotic cells, and mitochondrial membrane potential (MMP) were detected by flow cytometry. The morphology was observed by Hoechst 33258 staining and TUNEL staining, the expression of X-linked inhibitor of apoptosis protein (XIAP) was detected by Western blotting assay and caspase 3, 8 and 9 activity was measured. RESULTS: Phenotypic characterization of DCs was changed in EBV-treated group. Chromatin condensation and DNA fragmentation were observed in EBV induced CBDC apoptosis. In addition, caspase 3, caspase 8, and caspase 9 activation were enhanced in the EBV-treated group. This was accompanied by the loss of MMP. Furthermore, XIAP expression was down-regulated in the EBV-treated group and compared to mock-infected group. CONCLUSION: These results suggested that EBV could inhibit CBDC phenotypic differentiation, and induce CBDC apoptosis in caspase-dependent manner with involvement of the mitochondrial pathway. This might help EBV to evade host immune responses to establish persistent infection.

Simultaneous inhibition of XIAP and survivin expression on EMT and invasion of human pancreatic cancer cells / 中南大学学报(医学版)

OBJECTIVE@#To investigate the simultaneous inhibition of X-linked inhibitor of apoptosis protein (XIAP) and survivin expression on epithelial-mesenchymal transition (EMT) and invasiion of pancreatic cancer cells Panc-1, and its mechanism.@*METHODS@#On the established human pancreatic cancer cells Panc-1-XS, the expression of XIAP and survivin was inhibited simultaneously. Cell invasion and migration were detected by Transwell chamber experiments and scratch test, and the expression of epithelial marker E-cadherin, mesenchymal markers Slug, phosphatase and tensin homolog deleted on chromosome ten (PTEN) and P-Akt protein was determined by Western blot.@*RESULTS@#Cell invasion and migration of Panc-1-XS cells decreased significantly, accompanied by significantly upregulated protein expression of E-cadherin, and significantly declined protein expression of the Slug, indicating increased mesenchymal-epithelial conversion (MET); and increased protein expression of PTEN, and declined protein expression of P-Akt.@*CONCLUSION@#Simultaneously inhibiting the expression of XIAP and survivin can partially reverse EMT phenotype of pancreatic cancer Panc-1 cells, which then significantly reduces the cell invasion and migration of Panc-1 cell lines. This process may be regulated by PTEN/PI3K/Akt signaling pathway.

The expression and significance of smac, XIAP, caspase-3 in nonnasal inverted papilloma / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To explore the expression and significance of second mitochondria derived activator of caspase (Smac), X-linked inhibitor of apoptosis protein (XIAP)and cysteine containing aspartate specific protease 3 (caspase-3) in the growth, development and carcinogenesis of the nonnasal inverted papilloma (NIP).@*METHOD@#Immunohistochemical method was used to detect the expression of Smac, XIAP, caspase-3 in 10 cases of nasal cavity mucosae (NM) and 45 cases of NIP, the group of NIP including 25 cases of NIP without dysplasia, 11 cases of NIP with dysplasia, and 9 cases of NIP with malignant transformation to squamous cell carcinoma (SCC).@*RESULT@#The intensity of the positive expression of Smac, Caspase-3 in NIP were lower than NM, the intensity of the positive expression decreased with the decreasing degree of histological differentiation. There was a significant difference between NIP without dysplasia and SCC. It was presented with a progressive tendency for the expression of XIAP in the group of NM and NIP. The lower degree of histological differentiation, the higher intensity of the positive expression. The expression between NIP without dysplasia and SCC had a significant difference. Smac negatively correlated with XIAP (r(s) = -0.323, P < 0.05), XIAP negatively correlated with caspase-3 (r(s) = -0.408, P < 0.01), Smac positively correlated with caspase 3 (r(s) = 0.424, P < 0.01).@*CONCLUSION@#Smac, XIAP, caspase 3 might be associated with the growth and carcinogenesis of NIP.