Modulation of pancreatic tumor potential by overexpression of protein kinase C ß1.

OBJECTIVE: This study aimed to investigate whether the overexpression of protein kinase C ß1 (PKCß1) is able to modulate the malignant phenotype displayed by the human ductal pancreatic carcinoma cell line PANC1. METHODS: PKCß1 overexpression was achieved using a stable transfection approach. PANC1-PKCß1 and control cells were analyzed both in vitro and in vivo. RESULTS: PANC1-PKCß1 cells displayed a lower growth capacity associated with the down-regulation of the MEK/ERK pathway and cyclin expression. Furthermore, PKCß1 overexpression was associated with an enhancement of cell adhesion to fibronectin and with reduced migratory and invasive phenotypes. In agreement with these results, PANC1-PKCß1 cells showed an impaired ability to secrete proteolytic enzymes. We also found that PKCß1 overexpressing cells were more resistant to cell death induced by serum deprivation, an event associated with G0/G1 arrest and the modulation of PI3K/Akt and NF-κB pathways. Most notably, the overexpression of PKCß1 completely abolished the ability of PANC1 cells to induce tumors in nude mice. CONCLUSIONS: Our results established an important role for PKCß1 in PANC1 cells suggesting it would act as a suppressor of tumorigenic behavior in pancreatic cancer.

Determination of robustness and optimal work conditions for a purification process of a therapeutic recombinant protein using response surface methodology.

A typical chromatographic purification step has numerous operating parameters that can impact its performance. As it is not feasible to evaluate the influence of each one, the current practice in biopharmaceutical industry is to apply risk analysis approach to identify process parameters that should be examined during process characterization. Once these parameters are identified, a response surface study can be run to help understand the relationship between critical inputs and outputs. We performed a study comprising optimization and robustness determination for a Blue-Sepharose purification step of rhEPO, a well-known therapeutic glycoprotein. Initially, risk analysis was fulfilled to identify key parameters. A small-scale model was created and qualified before its use in experimental studies, given by a Box-Behnken design with three factors. This method proved to be a very useful tool in bioprocess validation studies in which many input variables can affect product quality and safety.

PKC Delta (PKCdelta) promotes tumoral progression of human ductal pancreatic cancer.

OBJECTIVE: Our objective was to study the role of protein kinase C delta (PKCdelta) in the progression of human pancreatic carcinoma. METHODS: Protein kinase C delta expression in human ductal carcinoma (n = 22) was studied by immunohistochemistry. We analyzed the effect of PKCdelta overexpression on in vivo and in vitro properties of human ductal carcinoma cell line PANC1. RESULTS: Human ductal carcinomas showed PKCdelta overexpression compared with normal counterparts. In addition, in vitro PKCdelta-PANC1 cells showed increased anchorage-independent growth and higher resistance to serum starvation and to treatment with cytotoxic drugs. Using pharmacological inhibitors, we determined that phosphatidylinositol-3-kinase and extracellular receptor kinase pathways were involved in the proliferation of PKCdelta-PANC1. Interestingly, PKCdelta-PANC1 cells showed a less in vitro invasive ability and an impairment in their ability to migrate and to secrete the proteolytic enzyme matrix metalloproteinase-2. In vivo experiments indicated that PKCdelta-PANC1 cells were more tumorigenic, as they developed tumors with a significantly lower latency and a higher growth rate with respect to the tumors generated with control cells. Besides, only PKCdelta-PANC1 cells developed lung metastasis. CONCLUSION: Our results showed that the overexpression of PKCdelta in PANC1 cells induced a more malignant phenotype in vivo, probably through the modulation of cell proliferation and survival, involving phosphatidylinositol-3-kinase and extracellular receptor kinase signaling pathways.

[Retinoid expression (RARbeta and CRBP1) in non-small-cell lung carcinoma]. / Retinoides (RARbeta y CRBP1) en carcinoma de pulmón a celulas no pequeñas.

Although early-stage non-small-cell lung carcinoma (NSCLC) patients have a relative by favorable prognosis, the risk of a bad outcome remains substantial. Identification of reliable prognostic markers for disease recurrence and death has meaningful clinical application. Retinoids are involved in cell growth and differentiation and may antagonize cancer progression. Their effects are mediated through nuclear receptors called Retinoic Acid Receptor (RAR) and regulated by molecules such as Cellular Retinol-Binding Protein 1 (CRBP1) that function in retinol storage. The aim of this work was to analyze by immunohistochemistry the expression patterns of RARbeta and CRBP1, involved in retinoid-mediated signaling, in the tumoral tissue of a cohort of stage I/II NSCLC patients (n = 49) who underwent a successful surgical resection. Prognostic evaluation was performed with the multivariate Cox proportional hazard model: 44.9% of tumors were positive for RARbeta staining at cytoplasmic level, while 34.7% showed nuclear staining. CRBP1 staining was observed in 61.2% of the lung tumors. No relationship was found between the number of cells expressing the studied molecules and clinical pathological features, including sex, T and N (stage), histopathology and p53 expression. Univariate analysis showed a significant association between positive cytoplasmatic expression of RARbeta with shorter overall survival (Log-rank test 4.17, p = 0.0412). Multivariate studies indicated that RARbeta expression was not influenced by other clinical pathological parameters. In conclusion, in this cohort of stage I and II NSCLC, only the expression of RARbeta at cytoplasmatic level is a significant independent unfavorable prognostic factor.

Retinoides (RARβ y CRBP1) en carcinoma de pulmón a células no pequeñas / Retinoid expression (RARβ and CRBP1) in non-small-cell lung carcinoma

Aunque los pacientes con cáncer de pulmón a células no pequeñas en estadios tempranos (NSCLC) tienen buen pronóstico, el 20-30% recae, siendo relevante la identificación de biomarcadores pronósticos. Los retinoides regulan crecimiento y diferenciación, y pueden antagonizar la progresión tumoral. Su efecto depende del transporte citosólico mediado por moléculas como CRBP1, y de la unión a receptores específicos (RARβ). Alteraciones en esta vía se asociaron con cáncer. Nuestro objetivo fue estudiar la expresión, mediante inmunohistoquímica, de RARβ y CRBP1 en el tejido tumoral de 49 pacientes NSCLC Estadio I/II, obtenido durante la cirugía. La supervivencia se analizó mediante los test Log Rank y multivariado de Cox. El 44.9% de los tumores fueron positivos para RARβ con expresión a nivel citoplasmático, mientras que el 34.7% lo expresó a nivel nuclear. La tinción para CRBP1 se observó en el 61.2% de los tumores. No se encontró asociación entre la expresión de ambas moléculas y las características clinicopatológicas (sexo, tamaño tumoral, nódulos línfáticos comprometidos, histopatología y p53). Tampoco se encontró asociación con el hábito de fu-mar. La presencia de células tumorales en el lavado pleural se asoció significativamente con la expresión de CRBP1. Por otro lado, se demostró asociación entre la expresión elevada de RARβ citoplasmático y menor supervivencia global (LR 4.17, p=0.0412). El análisis multivariado no mostró asociación con otras variables de pronóstico en NSCLC. En conclusión, en este grupo de pacientes NSCLC Estadio I/II, RARβ pareciera predecir la supervivencia global en forma independiente.

Although early-stage non-small-cell lung carcinoma (NSCLC) patients have a relative by favorable prognosis, the risk of a bad outcome remains substantial. Identification of reliable prognostic markers for disease recurrence and death has meaningful clinical application. Retinoids are involved in cell growth and differentiation and may antagonize cancer progression. Their effects are mediated through nuclear receptors called Retinoic Acid Receptor (RAR) and regulated by molecules such as Cellular Retinol-Binding Protein 1 (CRBP1) that function in retinol storage. The aim of this work was to analyze by immunohistochemistry the expression patterns of RARβ and CRBP1, involved in retinoid-mediated signaling, in the tumoral tissue of a cohort of stage I/II NSCLC patients (n=49) who underwent a successful surgical resection. Prognostic evaluation was performed with the multivariate Cox proportional hazard model: 44.9% of tumors were positive for RARβ staining at cytoplasmic level, while 34.7% showed nuclear staining. CRBP1 staining was observed in 61.2% of the lung tumors. No relationship was found between the number of cells expressing the studied molecules and clinical pathological features, including sex, T and N (stage), histopathology and p53 expression. Univariate analysis showed a significant association between positive cytoplasmatic expression of RARβ with shorter overall survival (Log-rank test 4.17, p=0.0412). Multivariate studies indicated that RARβ expression was not influenced by other clinical pathological parameters. In conclusion, in this cohort of stage I and II NSCLC, only the expression of RARβ at cytoplasmatic level is a significant independent unfavorable prognostic factor.

Association between mast cells of different phenotypes and angiogenesis in colorectal cancer.

It is known that mast cells proliferate in solid tumours and increase tumour angiogenesis. Nevertheless, there is no consensus regarding their role in colorectal cancer (CRC). In this study, we aimed to clarify the relationship of mast cells positive for tryptase (MCts) and tryptase-chymase (MCtcs) with microvessel density (MVD) in the intratumoral zone and the invasive edge of 80 CRC patient tumours. We evaluated these parameters and associated their expression with clinicopathological parameters, including survival rate. Tumour sections from each patient were immunostained for tryptase to evaluate MCts, chymase to evaluate MCtcs, and CD34 to evaluate microvessel counts under x100 microscopy. The number of MCs of both phenotypes and the MVD counts were higher in the invasive edge than in the intratumoral zone (p<0.001). MCt numbers were higher than those of MCtcs in all Astler-Coller stages in both regions. A positive correlation between MVD and MCts or MCtcs was observed (Pearson's test p<0.001). Neither the number of MCs nor MVD was associated with overall survival (log rank test). However, only 8.3% of patients with low numbers of MCtcs in the invasive edge succumbed to the disease, compared to 32% with high numbers of MCtcs. Our results indicate that angiogenesis and MC hyperplasia are events which appear early during CRC development. The correlation of MC phenotypes with MVD is in agreement with the role attributed to MCs, that of angiogenesis enhancement. Collectively, these findings suggest that screening during the early malignization of CRC can provide valuable clinical information.

Protein kinase C delta inhibits the production of proteolytic enzymes in murine mammary cells.

In previous studies we have determined that protein kinase C (PKC) delta, a widely expressed member of the novel PKC serine-threonine kinases, induces in vitro changes associated with the acquisition of a malignant phenotype in NMuMG murine mammary cells. In this study we show that PKCdelta overexpression significantly decreases urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) production, two proteases associated with migratory and invasive capacities. This effect is markedly enhanced by treatment with phorbol 12-myristate 13-acetate (PMA). On the other hand, depletion of PKCdelta using RNAi led to a marked increase in both uPA and MMP-9 secretion, suggesting a physiological role for PKCdelta in controlling protease secretion. The MEK-1 inhibitor PD98059 reverted the characteristic pattern of proteases secretion and phospho-ERK1/2 up-regulation observed in PKCdelta overexpressors, suggesting that the PKCdelta effect is mediated by the MEK/ERK pathway. Our results suggest a dual role for PKCdelta in murine mammary cell cancer progression. While this kinase clearly promotes mitogenesis and favors malignant transformation, it also down-modulates the secretion of proteases probably limiting metastatic dissemination.

Prognostic value of E-cadherin, beta-catenin, MMPs (7 and 9), and TIMPs (1 and 2) in patients with colorectal carcinoma.

BACKGROUND AND OBJECTIVES: Therapy of colorectal tumors (CRC) based on histology and clinical factors is insufficient to predict the evolution of each patient. The finding of molecular abnormalities able to differentiate subgroups of patients with bad prognosis will improve our ability to treat them successfully. Our purpose was to analyze retrospectively the prognostic input of E-cadherin, beta-catenin, metalloproteinases (MMPs) (7 and 9), and tissue inhibitors of metalloproteinases (TIMPs) (1 and 2) in patients with a follow-up period of 5 years. METHODS: Antigen expression was analyzed by immunohistochemistry. Prognostic evaluation was performed with the multivariate proportional hazards model. RESULTS: We demonstrated a concomitant loss of E-cadherin and beta-catenin at membranous level and an abnormal accumulation of nuclear beta-catenin. Besides, we found that all MMPs and TIMPs studied were overexpressed in CRC tissue. There was no association between the expression of any of these molecules and the known clinical-pathological parameters employed in CRC pathology. A multivariate analysis demonstrated that the overall survival could be independently predicted by the loss of E-cadherin and the overexpression of TIMP-2. CONCLUSIONS: The expression of E-cadherin and TIMP-2 could be relevant in determining the prognosis of CRC patients and providing a more accurate mechanism for their classification.

Phylogeny of triatomine vectors of Trypanosoma cruzi suggested by mitochondrial DNA sequences.

The subfamily Triatominae (Hemiptera: Reduviidae) comprises hematophagous insects, most of which are actual or potential vectors of Trypanosoma cruzi, the protozoan agent of Chagas' disease (American trypanosomiasis). DNA sequence comparisons of mitochondrial DNA (mtDNA) genes were used to infer phylogenetic relationships among 32 species of the subfamily Triatominae, 26 belonging to the genus Triatoma and six species of different genera. We analyzed mtDNA fragments of the 12S and 16S ribosomal RNA genes (totaling 848-851 bp) from each of the 32 species, as well as of the cytochrome oxidase I (COI, 1447 bp) gene from nine. The phylogenetic analyses unambiguously supported several clusters within the genus Triatoma. In the morphological classification, T. costalimai was placed tentatively within the infestans complex while T. guazu was not included in any Triatoma complex. The placement of these species in the molecular phylogeny indicated that both belong to the infestans complex. We confirmed with a strong support the inclusion of T. circummaculata, a member of a different complex based on morphology, within the infestans complex. On the other hand, the present phylogenetics analysis did not support the monophyly of the infestans complex species as it was suggested in our previous studies. While no strong inference of polyphyly of the genus Triatoma was provided by the bootstrap analyses, the other species belonging to Triatomini analyzed could not be distinguished from the species of Triatoma.