Tumor-Stromal Interactions in a Co-Culture Model of Human Pancreatic Adenocarcinoma Cells and Fibroblasts and Their Connection with Tumor Spread.

One key feature of pancreatic ductal adenocarcinoma (PDAC) is a dense desmoplastic reaction that has been recognized as playing important roles in metastasis and therapeutic resistance. We aim to study tumor-stromal interactions in an in vitro coculture model between human PDAC cells (Capan-1 or PL-45) and fibroblasts (LC5). Confocal immunofluorescence, Enzyme-Linked Immunosorbent Assay (ELISA), and Western blotting were used to evaluate the expressions of activation markers; cytokines arrays were performed to identify secretome profiles associated with migratory and invasive properties of tumor cells; extracellular vesicle production was examined by ELISA and transmission electron microscopy. Coculture conditions increased FGF-7 secretion and α-SMA expression, characterized by fibroblast activation and decreased epithelial marker E-cadherin in tumor cells. Interestingly, tumor cells and fibroblasts migrate together, with tumor cells in forming a center surrounded by fibroblasts, maximizing the contact between cells. We show a different mechanism for tumor spread through a cooperative migration between tumor cells and activated fibroblasts. Furthermore, IL-6 levels change significantly in coculture conditions, and this could affect the invasive and migratory capacities of cells. Targeting the interaction between tumor cells and the tumor microenvironment might represent a novel therapeutic approach to advanced PDAC.

Mechanistic added value of a trans-Sulfonamide-Platinum-Complex in human melanoma cell lines and synergism with cis-Platin.

BACKGROUND: Cisplatin is a potent antitumor agent. However, toxicity and primary and secondary resistance are major limitations of cisplatin-based chemotherapy, leading to therapeutic failure. We have previously reported that mono-sulfonamide platinum complexes have good antitumor activity against different tumoral cell lines and with a different and better cytotoxic profile than cisplatin. Besides, N-sulfonamides have been used extensively in medicinal chemistry as bactericides, anticonvulsant, inhibitors of the carbonic anhydrase, inhibitors of histone deacetylases, and inhibitors of microtubule polymerization, among others. METHODS: We aimed to compare the cytotoxic effects of cisplatin and a trans-sulfonamide-platinum-complex (TSPC), in two human melanoma cell lines that differ in their TP53 status: SK-MEL-5, TP53 wild type, and SK-MEL-28, TP53 mutated. We performed cytotoxicity assays with both drugs, alone and in combination, cell cycle analyses, western blotting and immunoprecipitation, and fluorescence immunocytochemistry. RESULTS: TSPC had similar antiproliferative activity than cisplatin against SK-MEL-5 (3.24 ± 1.08 vs 2.89 ± 1.12 µM) and higher against SK-MEL-28 cells (5.83 ± 1.06 vs 10.17 ± 1.29 µM). Combination of both drugs inhibited proliferation in both cell lines, being especially important in SK-MEL-28, and showing a synergistic effect. In contrast to cisplatin, TSPC caused G1 instead G2/M arrest in both cell lines. Our present findings indicate that the G1 arrest is associated with the induction of CDKN1A and CDKN1B proteins, and that this response is also present in melanoma cells containing TP53 mutated. Also, strong accumulation of CDKN1A and CDKN1B in cells nuclei was seen upon TSPC treatment in both cell lines. CONCLUSIONS: Overall, these findings provide a new promising TSPC compound with in vitro antitumor activity against melanoma cell lines, and with a different mechanism of action from that of cisplatin. Besides, TSPC synergism with cisplatin facilitates its potential use for co-treatment to reduce toxicity and resistance against cisplatin. TSPC remains a promising lead compound for the generation of novel antineoplastic agent and to explore its synergism with other DNA damaging agents.

Blood-based biomarkers for monitoring antiangiogenic therapy in non-small cell lung cancer.

Tumor angiogenesis pathways have been identified as important therapeutic targets in non-small cell lung cancer. However, no biomarkers have been described as predictors of response to antiangiogenic therapy in these patients. In this study, plasma levels of VEGF, bFGF, E-selectin, and S-ICAM and gene expression profiles of peripheral blood mononuclear cells from non-small cell lung cancer patients treated with chemotherapy plus bevacizumab were analyzed before and after treatment. Values were correlated with clinicopathological characteristics and treatment response. Plasma factor levels were measured using commercially available ELISA kits. The TaqMan(®) human angiogenesis array was used to investigate the effect of treatment on gene expression profiles. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment analysis was performed for differentially expressed genes using WEB-based GEne SeT AnaLysis Toolkit. Our results suggest a benefit for patients with increased plasma levels of VEGF, E-selectin, and S-ICAM in the course of bevacizumab treatment. Also, we identified differentially expressed genes between paired blood samples from patients before and after treatment, and significantly perturbed pathways were predicted. These changes in gene expression and levels of plasma factors could be used to assess the effectiveness of antiangiogenic therapy, in addition to standard clinical and radiological evaluations.

Relevance of insulin-like growth factor 1 receptor gene expression as a prognostic factor in non-small-cell lung cancer.

PURPOSE: Signalling through the insulin-like growth factor 1 receptor (IGF-1R) is implicated in carcinogenesis, metastasis, and resistance to cytotoxic cancer therapies. The purpose of this study was to investigate the prognostic role of IGF-1R expression in surgically resected non-small-cell lung cancer (NSCLC), and responses to IGF-1R tyrosine kinase inhibitor NVP-ADW742 in a panel of lung cancer cell lines. METHODS: Insulin-like growth factor 1 receptor (IGF-1R) expression was evaluated by quantitative RT-PCR in 115 NSCLC samples and in a panel of 6 NSCLC cell lines. Cytotoxicity experiments with IGF-1R inhibitor and conventional systemic drugs such as paclitaxel in cell lines were realised. RESULTS: Insulin-like growth factor 1 receptor (IGF-1R) was differentially expressed across histologic subtypes, with the lowest levels observed in squamous cell tumours. Median survival was longer in patients with squamous tumour histology expressing low IGF-1R levels. In multivariable analysis, ageing and high tumour stage were significant predictors of worse overall survival. The hazard of death was lower in patients with squamous histology and low IGF-1R gene expression. There was no correlation between IGF-1R expression and response to tyrosine kinase inhibitor in cell lines tested. However, combination drug treatment resulted in synergistically enhanced antiproliferative effects on several cell lines. CONCLUSIONS: These findings suggest that IGF-1R is a potential target for therapy in NSCLC patients. Combination therapies will have an important role in treatment.

Prognostic value of dual-specificity phosphatase 6 expression in non-small cell lung cancer.

Dual-specificity phosphatase 6 (DUSP6/MKP-3) is a mitogen-activated protein kinase phosphatase that regulates extracellular signal-regulated kinases (ERKs) activity via feedback mechanisms, with an increasingly recognized role in tumour biology. The aim of this study was to explore the role of DUSP6 expression in the prognosis of human non-small cell lung cancer (NSCLC). DUSP6 expression levels were evaluated by real-time quantitative reverse transcription polymerase chain reaction (PCR) in 60 NSCLC samples from patients who underwent pulmonary resection at 12 de Octubre University Hospital. We performed a statistical analysis to investigate the correlation of DUSP6 expression and the clinical outcomes. We found that 66.7% of the tumour samples show the downregulation of DUSP6 at the messenger RNA (mRNA) levels compared to benign epithelial lung tissues and 55% of them show at least twofold downregulation of DUSP6 gene expression. Patients were classified into three groups according to their DUSP6 expression levels and those with very low levels (at least twofold downregulation) had the worst outcomes. Using the value of twice below the mean value in benign epithelial lung tissue as a cutoff, the overall survival of patients with very low DUSP6 levels was significantly lower than that in the rest of patients (31.9 ± 18.8 months vs. not reached, P = 0.049). This was most pronounced in adenocarcinoma histology and high-stage tumour samples. Our results suggest that DUSP6 gene expression in tumour samples may be a prognostic marker in NSCLC.

Evaluation of novel trans-sulfonamide platinum complexes against tumor cell lines.

Platinum-based drugs, mainly cisplatin, are employed for the treatment of solid malignancies. However, cisplatin treatment often results in the development of chemoresistance, leading to therapeutic failure. Here, the antitumor activity of different trans-sulfonamide platinum complexes in a panel of human cell lines is presented. The cytotoxicity profiles and cell cycle analyses of these platinum sulfonamide complexes were different from those of cisplatin. These studies showed that complex 2b with cyclohexyldiamine and dansyl moieties had the best antitumoral activities.

DICER1, DROSHA and miRNAs in patients with non-small cell lung cancer: implications for outcomes and histologic classification.

The clinical and functional significance of RNA-interference machinery in lung cancer is poorly understood. Besides, microRNAs (miRNA) have the potential to serve both as biomarkers and therapeutic agents, by personalizing diagnosis and therapy. In this study, we investigated whether the expression levels of DICER1 and DROSHA, components of the RNA-interference machinery, can predict survival, and whether the miRNA expression profiles can differentiate histologic subtypes in non-small cell lung cancer (NSCLC). Levels of DICER1, DROSHA and five different miRNAs were measured in NSCLC specimens (N = 115) by qRT-PCR assay and correlated with clinical outcomes. Low expression of DROSHA was associated with an increased median survival (154.2 versus 39.8 months, P = 0.016). Also, high DROSHA expression was associated with decreased median survival in the following subgroups: adenocarcinoma (P = 0.011), grade III tumors (P = 0.038) and low-stage patients (P = 0.014). In multivariate analyses, we found two independent predictors of reduced disease-specific survival: high DROSHA expression [hazards ratio = 2.24; P = 0.04] and advanced tumor stage (hazards ratio = 1.29, P = 0.02). In general, the overall tumor miRNA expression was downregulated in our cohort compared with normal tissues. Expression levels of hsa-let-7a (P = 0.005) and miR-16 (P = 0.003) miRNA were significantly higher in squamous cell carcinoma than in adenocarcinoma samples. This study supports the value of the expression profiling of the components of the miRNA-processing machinery in the prognosis of NSCLC patients, especially DROSHA expression levels. In addition, differential expression of miRNAs, such as hsa-let-7a and miR-16 may be helpful tools in the histologic subclassification of NSCLC.

Role of protein kinase C and mitochondrial permeability transition pore in the neuroprotective effect of ceramide in ischemia-induced cell death.

In this study, we investigated the role of protein kinase C (PKC) and mitochondrial permeability transition pore (mPTP) on the effect of ceramide in an in vitro model of ischemia in SH-SY5Y neuroblastoma cells. In ischemic cell viability studies, a dual effect of ceramide was observed, depending on ceramide concentration. PKC isoforms are involved in the protective effect of low concentrations of ceramide. During ischemia, ceramide treatment leads to an increase in the formation of reactive oxygen species (ROS), which induces a controlled opening of mPTP. This fact prevents mitochondrial Ca(2+) overload, which is clearly protective.

Involvement of mitochondria on neuroprotective effect of sphingosine-1-phosphate in cell death in an in vitro model of brain ischemia.

Sphingosine-1-phosphate (S1P) has been demonstrated to be an important regulator of cell death and survival. Although it has been suggested that the sphingolipid may act as a neuroprotector in the cell apoptosis induced by traumatic brain injury, the mechanisms involved in this action are unknown. In this study, the relationship between S1P and neuroprotective effect was studied in an in vitro model of ischemia, maintaining SH-SY5Y human neuroblastoma cells under oxygen-glucose deprivation (OGD). When cells were treated with 1 microM S1P simultaneously with OGD and recovery, cell viability increases in a dose-response manner. S1P treatment reduces significantly both necrosis and apoptosis cell death. On the other hand, the treatment with specific PKC epsilon (V1-2), prevents S1P protective effect of OGD/recovery-induced necrosis. Moreover, S1P treatment provokes the translocation of PKC epsilon to the mitochondria. From these results, it is reasonable to assume that S1P protection from necrosis is mediated by PKC epsilon. We also studied the action of S1P on mitochondrial inner membrane potential and mitochondrial Ca(2+) levels during ischemia. In this regard, we must point out that S1P treatment reduces the OGD-induced membrane depolarization and also reduces the increase of Ca(2+) in mitochondria during OGD. Results also indicate that mitochondria from OGD treated cells have significantly less ability to resist swelling on Ca(2+) loading than those obtained in presence of oxygen and glucose. Nevertheless, when S1P was added, this resistance increases considerably. These findings suggest that S1P may have a potential role as a neuroprotective agent in brain injury.

Sphingosylphosphorylcholine induces mitochondria-mediated apoptosis in neuro 2a cells: involvement of protein kinase C.

We demonstrate that sphingosylphosphorylcholine-mediated cell death involves the activation of different protein kinase C isozymes in different manners. Treating cells with sphingosylphosphorylcholine resulted in activation of protein kinase C delta, which is necessary, together with elevation of Ca2+ for sphingosylphosphorylcholine-induced apoptosis. A rapid translocation from cytosol to membrane, and a proteolytic protein kinase C delta cleavage was found, probably due to activation of caspase-3, to give a catalytically active fragment involved in cellular apoptosis. Moreover, sphingosylphosphorylcholine also induced translocation of protein kinase C zeta, resulting in an anti-apoptotic effect. To explore whether a mitochondrial pathway is involved in sphingosylphosphorylcholine-induced apoptosis, we analyzed the effect of sphingosylphosphorylcholine on cytochrome c release and caspase-3 activity. We must point out that the sphingolipid caused an increase of cytochrome c release from mitochondria to cytosol concomitantly with an increase of caspase-3 activity. Furthermore, a translocation of Bax was found, after sphingosylphosphorylcholine treatment.