CSE1L participates in regulating cell mitosis in human seminoma.

OBJECTIVES: CSE1L has been reported to be highly expressed in various tumours. Testicular germ cell tumours are common among young males, and seminoma is the major type. However, whether CSE1L has functions in the seminoma is unclear. MATERIALS AND METHODS: The expression of CSE1L was detected by immunohistochemistry in seminoma tissues and non-tumour normal testis tissues from patients. CSE1L distribution during cell mitosis was determined by immunofluorescent staining with CSE1L, α-tubulin and γ-tubulin antibodies. The effects of Cse1L knockdown on cell proliferation and cell cycle progression were determined by Cell Counting Kit-8 assay, flow cytometry, PH3 staining and bromodeoxyuridine incorporation assay. RESULTS: CSE1L was significantly enriched in the seminoma tissue compared with the non-tumour normal testis tissue. CSE1L also co-localized with α-tubulin in the cells with a potential to divide. In the seminoma cell line TCam-2, CSE1L was associated with the spindles and the centrosomes during cell division. The knockdown of CSE1L in TCam-2 cells attenuated the cells' proliferative capacity. Cell cycle assay revealed that the CSE1L-deficient cells were mainly arrested in the G0/G1 phase and moderately delayed in the G2/M phase. The proportion of cells with multipolar spindle and abnormal spindle geometry was obviously increased by CSE1L expression silencing in the TCam-2 cells. CONCLUSIONS: Overall, these findings showed that CSE1L plays a pivotal role in maintaining cell proliferation and cell division in seminomas.

High expression of long non-coding RNA ATB indicates a poor prognosis and regulates cell proliferation and metastasis in non-small cell lung cancer

Background and aim. Long non-coding RNAs (lncRNAs) have been demonstrated to act as a critical regulator in the processes of tumor biology. In this study, whether lncRNA-ATB is a potential indicator for non-small cell lung cancer (NSCLC) was investigated and its biological function in NSCLC was also determined. Methods. The expression levels of lncRNA-ATB in NSCLC tissues and cell lines were measured. A549 cell line was explored to investigate the functions of lncRNA-ATB in NSCLC. Results. Real-time PCR results showed that lncRNA-ATB expression was up-regulated in both in NSCLC tissues and cell lines. High lncRNA-ATB expression in tumor tissue was associated with larger tumor size, lymph node metastasis, and distant metastasis in patients with NSCLC, respectively. In addition, the patients with high expression of lncRNA-ATB presented a lower survival probability. In vitro experiments showed that down-regulation of lncRNA-ATB promoted the cell apoptosis, whereas inhibited the cell viability, cell migration, and cell invasion. Conclusion. High expression of lncRNA-ATB indicated a poor prognosis and led to the cell proliferation and metastasis in NSCLC (AU)

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The relationship between CSE1L expression and axillary lymph node metastasis in breast cancer.

AIM: CSE1L is the human homologue to the yeast gene CSE1 and CSE1L is a gene related to cancer progression. Thus, CSE1L may regulate the invasion and metastasis of breast cancer. The aim of this study is to show the relationship between CSE1L and axillary lymph node metastasis. METHODS: Sixty-six breast cancer patients were evaluated according to patient and tumor characteristics. Immunohistochemistry was carried out on formalin-fixed, paraffin-embedded archival breast tumor tissues. The results of CSE1L staining were analyzed according to the percentage of immunoreactive cells. RESULTS: There were 29 patients without axillary lymph node metastasis and 37 patients with nodal metastasis. The mean age of the patients was 50.6 ± 11.3 years. Age, tumor size, nuclear grade and hormone receptor status were similar in the axillary lymph node positive and negative groups. There was a statistically significant relationship between cytoplasmic CSE1L expression and axillary lymph node metastasis. However, nuclear CSE1L expression did not have any effect on axillary lymph node metastasis. CONCLUSIONS: Cytoplasmic CSE1L overexpression may be a valuable tool for prognosis of breast cancer in future.

Two death pathways induced by sorafenib in myeloma cells: puma-mediated apoptosis and necroptosis

Purpose. Sorafenib is a multikinase inhibitor that targets the MAPK pathway and is currently used for the treatment of hepatocellular and renal carcinoma. Recently, it has been shown that sorafenib is also cytotoxic to multiple myeloma (MM) cells. Here, we have further analyzed the mechanism of sorafenib-induced death in MM cells. Methods. Cell death induced by sorafenib in MM cell lines and in plasma cells from MM patients was evaluated by analysis of gene expression by RT-MLPA and quantitative PCR, protein levels and functionality by Western blot and flow cytometry and gene silencing with siRNA. Results. Cell death was characterized by phosphatidylserine exposure, ΔΨm loss, cytochrome c release and caspase activation, hallmarks of apoptosis. DL50 at 24 h ranged from 6 to 10 µM. Ex vivo treatment with 20 µM sorafenib induced apoptosis in around 80 % myeloma cells from six multiple myeloma patients. Sorafenib induced caspase-dependent degradation of Bcl-xL and Mcl-1 proteins, destabilizing the mitochondria and speeding up the development of apoptosis. Sorafenib treatment increased levels of Puma at mRNA and protein level and gene silencing with siRNA confirmed a relevant role for Puma in the induction of apoptosis. Co-treatment with the pan-caspase inhibitor Z-VAD-fmk prevented cell death to a variable degree depending on the cell line. In RPMI 8226 cells, Z-VAD-fmk prevented most of sorafenib-induced death. However, death in MM.1S was only prevented by co-incubation with both Z-VAD-fmk and the RIP1K inhibitor necrostatin-1, indicating that under conditions of inefficient caspase activation, sorafenib induces death by necroptosis. Conclusion. Our results demonstrate a key role for Puma in the triggering of sorafenib-induced apoptosis and that this drug can also induce death by necroptosis in multiple myeloma cells (AU)

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La elevada glucosa altera la respuesta anti-apoptótica del estímulo mecánico en osteocitos de ratón MLO-Y4 / High glucose alters the antiapoptotic response to mechanical stimulation in MLO-Y4 osteocytic cells

El estrés oxidativo es clave en el envejecimiento y en los estados diabéticos. La carga mecánica es decisiva para mantener la masa ósea. La respuesta del hueso a los estímulos mecánicos parece reducirse con el envejecimiento y probablemente en la enfermedad ósea provocada por la diabetes. Entender los mecanismos mediante los que el estrés oxidativo afecta a la función de las células óseas, y en concreto a la mecanotransducción en el osteocito, podría proporcionar nuevas dianas moleculares para mejorar los tratamientos actuales y el diseño de otros nuevos para prevenir la pérdida de masa ósea. Nuestros resultados indican que un medio de alta glucosa («diabético») ejerce un efecto negativo sobre la capacidad de los osteocitos para responder a estímulos mecánicos, a través de la interacción con la β-catenina. Además, estos hallazgos sugieren que el estímulo mecánico promueve la viabilidad osteocítica, al menos en parte, a través de la producción de la proteína relacionada con la parathormona (PTHrP) (AU)

Oxidative stress is a key factor in aging and diabetes. Mechanical loading is critical to maintain bone mass. The response of bone to mechanical stimuli appears to be reduced with aging and probably in bone disease caused by diabetes. Understanding the mechanisms by which oxidative stress affects function of bone cells, and specifically to osteocyte mechanotransduction, may provide new molecular targets to improve current treatments and design new treatments to prevent bone loss. Our results indicate that high glucose medium («diabetic ») has a negative effect on the ability of osteocytes to respond to mechanical stimuli affecting b-catenin and apoptosis. Moreover, these findings suggest that the mechanical stimulus promotes viability osteocytic, at least in part, through production of parathyroid hormone related protein (PTHrP) (AU)

Las proteínas de choque térmico (heat shock proteins) como potenciales dianas terapéuticas en aterosclerosis / Heat shock proteins as potential therapeutic targets in atherosclerosis

Las heat shock proteins (HSP) son una familia de proteínas que se producen en grandes cantidades por la mayoría de las células como respuesta al estrés. Las HSP se localizan tanto en arterias sanas, como en placas ateroscleróticas, aunque su papel en la aterosclerosis está aún por definir. En el presente estudio, nuestro objetivo fue analizar el efecto de la modulación de las HSP en procesos involucrados en la formación de la placa de ateroma, como el estrés oxidativo, la inflamación y la apoptosis. Para ello, hemos usado un inhibidor de la HSP90 (17-AAG), el cual es capaz de inducir un aumento en los valores de expresión de la HSP70/HSP27. Inicialmente, hemos observado que el tratamiento con 17-AAG es capaz de reducir la actividad de la NAD(P)H oxidasa inducida por factor de necrosis tumoral alfa. Asimismo, el tratamiento moduló distintas vías de señalización proliferativas (p. ej., MAPK), así como la activación del factor nuclear kappa B (NF-κB) inducidas por un cócktail de citocinas (interferón gamma/interleucina 6 [IL-6]). En estas condiciones, se observó una disminución en los niveles de citocinas proinflamatorias (p. ej., IL-6). Por último, mientras el tratamiento con 17-AAG provocó una disminución en la apoptosis después de la incubación con diversos estímulos proapoptóticos, la inhibición de la HSP27 mediante transfección con un siARN específico aumentó la apoptosis celular inducida por elastasa. Estos resultados indican que la modulación de los valores de ciertas HSP puede representar una nueva aproximación terapéutica en el tratamiento de enfermedades inflamatorio-proliferativas como la aterosclerosis (AU)

Heat shock proteins (HSP) are a family of proteins present in the majority of cells and are produced as a cell protection mechanism against adverse conditions. HSP are present in both atherosclerotic plaques and healthy arteries, although their role in atherosclerosis remain to be defined. Our objective was to investigate the effect of HSP90 inhibitors in the modulation of the different processes involved in atherogenesis. We have used an HSP90 inhibitor (17-AAG), which is able to increase the heat shock response. Treatment of monocytes and vascular smooth muscle cells with 17-AAG increased HSP70 expression. Under these conditions, 17-AAG was able to decrease NADPH oxidase activity induced by TNF-α. Stimulation of cells with cytokines induced the activation of different signalling pathways (e.g. MAPKs and NF-κB) and increased the levels of cytokines such as IL-6, which was diminished by HSP90 inhibitors. Finally, while treatment with 17-AAG prevented the apoptosis of cells induced proapoptotic stimuli, HSP27 silencing increased apoptosis induced by elastase. HSP90 inhibitors decrease pro-atherogenic processes, suggesting that these drugs may have beneficial effects in the treatment of atherosclerosis (AU)

Cellular apoptosis susceptibility protein and proliferation in human hepatocellular carcinoma.

The cellular apoptosis susceptibility protein (CAS) is the human homologue of the product of the essential yeast chromosome segregation gene, CSE1, and has important roles in tumor necrosis factor (TNF)-induced apoptosis and cell proliferation. In this study, we used immunoblotting and immunohistochemistry to look at CAS expression in human hepatocellular carcinoma (HCC) cells. We also studied the correlation between CAS expression and cell proliferation. To do this, we studied the expression of proliferating cell nuclear antigen (PCNA) by immunostaining and at apoptosis by in situ nick end-labeling (TUNEL), followed by calculation of the PCNA labeling index (PCNA LI) and TUNEL labeling index (TUNEL LI). CAS was constitutively expressed in human HCC cell lines and was primarily confined to the cytoplasm of the cells. PCNA LI and TUNEL LI were significantly higher in HCC than in non-tumor tissue (p<0.01); however, the ratio of TUNEL LI/PCNA LI in HCC was significantly lower than that of non-tumor tissue. Immunohistochemistry revealed that the staining intensity score of CAS in HCC was significantly higher than that of non-tumor tissue (p<0.05). These results indicate that there is an augmentation of pro-liferative activity and apoptosis in HCC tissue, as compared to non-tumor tissue. There was a significant positive correlation between CAS and PCNA LI (p<0.05). In addition, we observed an inverse relationship between CAS expression and TUNEL LI, although the correlation did not reach statistical significance. These results suggest that CAS is expressed at higher levels in human HCC tissue than in non-tumor tissue. CAS may be associated with cell proliferation rather than apoptosis, and further, CAS might play an important role in the development of human HCCs.

Tissue array analysis of expression microarray candidates identifies markers associated with tumor grade and outcome in serous epithelial ovarian cancer.

Molecular profiling is a powerful approach to identify potential clinical markers for diagnosis and prognosis as well as providing a better understanding of the biology of epithelial ovarian cancer. On the basis of the analysis of HuFL expression data, we have previously identified genes that distinguish low malignant potential and invasive serous epithelial ovarian tumors. In this study, we used immunohistochemistry to monitor a subset of differently expressed candidates (Ahr, Paep, Madh3, Ran, Met, Mek1, Ccne1, Ccd20, Cks1 and Cas). A tissue array composed of 244 serous tumors of different grades (0-3) and stages (I-IV) was used in this analysis. All markers assayed presented differential protein expression between serous tumors of low and high grade. Significant differences in Ccne1 and Ran expression were observed in a comparison of low malignant potential and grade 1 tumor samples (p<0.01). In addition, irrespective of the grade, Ccne1, Ran, Cdc20 and Cks1 showed significant differences of expression in association with the clinical stage of disease. While high level of Ccne1 have previously been associated with poor outcomes, here we found that high level of either Ran or Cdc20 appear to be more tightly associated with a poor prognosis (p<0.001, 0.03, respectively). The application of these biomarkers in both the initial diagnosis and prognostic attributes of patients with epithelial ovarian tumors should prove to be useful in patient management.

Intracellular signal cascade in CD4+ T-lymphocyte migration stimulated by interferon-gamma-inducible protein-10.

The intracellular signal cascades involved in chemokine-stimulated migration of in vitro activated human peripheral blood CD4+ T-lymphocytes were investigated. IP-10-mediated chemotactic response of lymphocytes was decreased in the presence of selective inhibitors of Src-kinases (by 40-45%), PI3-kinases (35-40%), and MAP-kinases ERK1/2 (35-40%) and p38 (20%). Combined addition of specific inhibitors of Src-kinases and PI3-kinases and inhibitors of Src-kinases and ERK1/2 MAP-kinases did not result in the further increase of the inhibitory effect, while the combined addition of specific inhibitors of PI3-kinases and ERK1/2 MAP-kinases decreased migration of CD4+ T-lymphocytes more effectively (by 55-60%) than any individual inhibitor. Immunoblotting analysis of activation of MAP-kinases ERK1/2 and p38 revealed increased level of phosphorylation of ERK1/2 and p38 MAP-kinases in the presence IP-10. Selective inhibitors of Src-kinases and PI3-kinases significantly inhibited phosphorylation of p38 but did not influence phosphorylation of ERK1/2 MAP-kinases. Our results suggest that Src-kinases, PI3-kinases, and ERK1/2 MAP-kinases are involved in intracellular signal cascade activated during IP-10-stimulated migration of T-lymphocytes, whereas p38 MAP-kinases do not participate in the migration process, although its activation induced by IP-10 depends on Src-kinases and PI3-kinases.

Valoración de la molécula FAS en el testículo contralateral tras torsión testicular unilateral. Estudio experimental en ratas / Valoration of the fas in the contralateral testis after unilateral testicular torsion. Experimental study in rat

Investigamos la implicación de la molécula FAS y BCL2en la apoptosis de células testiculares que ocurre en el testículo contralateral tras torsión testicular unilateral. Se compara con un grupo control. El estudio se realiza en ratas macho Wistar en edad prepuberal. La determinación de FAS y BCL-2 se realiza en co-cultivos celulares obtenidos del testículo contralateral y de los testículos controles. La valoración de las moléculas se realiza por técnica de inmunofluorescencia indirecta (IFI) con la aplicación de anticuerpos monoclonales específicos y su expresión se determina en citometría de flujo según protocolo. Observamos un incremento de la expresión de FAS y disminución de BCL-2 en el testículo contralateral con respecto al grupo control. Este estudio indica que la expresión de FAS y BCL-2 puede estar implicada en la apoptosis celular que ocurre en el testículo contralateral en la torsión testicular unilatera (AU)

Apoptosis en el testículo criptorquídico. Implicación de la molécula FAS en su modulación / Apoptosis in the cryptorchidism. Role of the fas protein in it's modulation

La degeneración celular que ocurre en la criptorquidia con alteración de la espermatogénesis normal se produce por un mecanismo de «apoptosis» o muerte celular programada. Son varias las vías que activan o modulan dicha apoptosis y entre ellas la vía mediada inmunológicamente por reacción antígeno-anticuerpo. Cuando la muerte por apoptosis se produce por vía inmnológica, una molécula «FAS» es la implicada en la modulación de dicha apoptosis. En este trabajo investigamos el papel que la molécula FAS tiene en el incremento de apoptosis en el artículo criptorquídico. El estudio se realiza en biopsias testiculares de pacientes criptorquídicos tomadas durante la orquidopexia, y la determinación de FAS se realiza por técnicas de inmunohistoquímica. Los resultados obtenidos indican que la molécula FAS no parece tener una clara implicación en la modulación de la apoptosis en el testículo criptorquídico (AU)

Cellular apoptosis susceptibility gene expression in endometrial carcinoma: correlation with Bcl-2, Bax, and caspase-3 expression and outcome.

Deregulation of proliferation and apoptosis is known to contribute to neoplastic transformation and growth. Using specific antibodies for the cellular apoptosis susceptibility (CAS) gene, caspase-3, Bcl-2, and Bax, we examined the protein expression in 89 endometrial carcinomas and in 56 samples of nonneoplastic adjacent endometrium for comparison. Immunostaining results were scored with regard to approximate percentage of positive tumor cells (< 10%, 10% to 50%, > 50%) and relative immunostaining intensity (1+, 2+, 3+). In nonneoplastic endometrium, CAS protein was expressed in 70.6%, Bax in 64%, caspase-3 in 52%, and Bcl-2 in 87%. In neoplastic tissue, CAS was present in 93% of the tumors, Bax in 88%, caspase-3 in 77%, and Bcl-2 in 51%. Bcl-2:Bax ratio was > 1 in 9 cases (10%). In cases of atrophy (n = 24) and simple (n = 10) and complex (n = 22) hyperplasia in the adjacent endometrium, lower levels of expression compared with carcinoma were observed for CAS (p = 0.003), caspase-3 (p = 0.034), and Bax (p = 0.04) and higher levels for Bcl-2, although for this protein the results were not statistically significant (p = 0.32). There was no association between immunoscores and FIGO stage. High caspase-3 levels were seen in endometrioid tumor type (p = 0.017). CAS expression was higher in grade 3 tumors (p = 0.002) and older patients (p = 0.013). All tumors of younger patients (< 50 years) were Bcl-2 negative (p = 0.037). Caspase-3 correlated positively with CAS (p = 0.008), Bax (p = 0.04), and low Bcl-2:Bax ratio (p = 0.043), and inversely (as a trend) with Bcl-2 (p = 0.056). Survival analysis (Kaplan-Meier and Cox regression) established a strong association between prognosis and stage, grade, and histologic type (all p < or = 0.0036). In addition, shorter survival was observed for patients whose tumors contained > 50% of positive cells for caspase-3 (p = 0.024) or for CAS (p = 0.04). Age, Bcl-2, Bax, and Bcl-2:Bax ratio did not provide prognostic information. Our results suggest a role of CAS, Bcl-2, Bax, and caspase-3, which are apparently involved in the progressive deregulation of proliferation and apoptosis leading from simple and complex hyperplasia to carcinoma. In addition, CAS and caspase-3 protein levels may be useful markers in predicting the outcome of the patients.