Human lung-derived mesenchymal stem cell-conditioned medium exerts in vitro antitumor effects in malignant pleural mesothelioma cell lines.

BACKGROUND The soluble factors secreted by mesenchymal stem cells are thought to either support or inhibit tumor growth. Herein, we investigated whether the human lung-derived mesenchymal stem cell-conditioned medium (hlMSC-CM) exerts antitumor activity in malignant pleural mesothelioma cell lines H28, H2052 and Meso4. METHODS hlMSC-CM was collected from the human lung-derived mesenchymal stem cells. Inhibition of tumor cell growth was based on the reduction of cell viability and inhibition of cell proliferation using the XTT and BrdU assays, respectively. Elimination of tumor spheroids was assessed by the anchorage-independent sphere formation assay. The cytokine profile of hlMSC-CM was determined by a chemiluminescence-based cytokine array. RESULTS Our data showed that hlMSC-CM contains a broad range of soluble factors which include: cytokines, chemokines, hormones, growth and angiogenic factors, matrix metalloproteinases, metalloproteinase inhibitors and cell-cell mediator proteins. The 48- and 72-hour hlMSC-CM treatments of H28, H2052 and Meso4 cell lines elicited significant decreases in cell viability and inhibited cell proliferation. The 72-hour hlMSC-CM incubation of H28 cells completely eliminated the drug-resistant sphere-forming cells, which is more potent than twice the half maximal inhibitory concentration of cisplatin. CONCLUSIONS Our findings indicate that the cell-free hlMSC-CM confers in vitro antitumor activities via soluble factors in the tested mesothelioma cells and, hence, may serve as a therapeutic tool to augment the current treatment strategies in malignant pleural mesothelioma.

Comparative Cytotoxicity Study of Silver Nanoparticles (AgNPs) in a Variety of Rainbow Trout Cell Lines (RTL-W1, RTH-149, RTG-2) and Primary Hepatocytes.

Among all classes of nanomaterials, silver nanoparticles (AgNPs) have potentially an important ecotoxicological impact, especially in freshwater environments. Fish are particularly susceptible to the toxic effects of silver ions and, with knowledge gaps regarding the contribution of dissolution and unique particle effects to AgNP toxicity, they represent a group of vulnerable organisms. Using cell lines (RTL-W1, RTH-149, RTG-2) and primary hepatocytes of rainbow trout (Oncorhynchus mykiss) as in vitro test systems, we assessed the cytotoxicity of the representative AgNP, NM-300K, and AgNO3 as an Ag+ ion source. Lack of AgNP interference with the cytotoxicity assays (AlamarBlue, CFDA-AM, NRU assay) and their simultaneous application point to the compatibility and usefulness of such a battery of assays. The RTH-149 and RTL-W1 liver cell lines exhibited similar sensitivity as primary hepatocytes towards AgNP toxicity. Leibovitz's L-15 culture medium composition (high amino acid content) had an important influence on the behaviour and toxicity of AgNPs towards the RTL-W1 cell line. The obtained results demonstrate that, with careful consideration, such an in vitro approach can provide valuable toxicological data to be used in an integrated testing strategy for NM-300K risk assessment.

The stem cell gene "inhibitor of differentiation 1" (ID1) is frequently expressed in non-small cell lung cancer

Inhibitor of differentiation 1 (ID1) plays a role in cellular differentiation, proliferation, angiogenesis and tumor invasion. As shown recently, ID1 is positively regulated by the tyrosine kinase SRC in lung carcinoma cell lines and with that appears as a potential new therapeutic target in non-small cell carcinoma (NSCLC). To substantiate this hypothesis we examined ID1, SRC and matrix metalloproteinase-9 (MMP-9) immunohistochemically in human NSCLC specimens.

Synergistic induction of cell death in liver tumor cells by TRAIL and chemotherapeutic drugs via the BH3-only proteins Bim and Bid

Although death receptors and chemotherapeutic drugs activate distinct apoptosis signaling cascades, crosstalk between the extrinsic and intrinsic apoptosis pathway has been recognized as an important amplification mechanism. Best known in this regard is the amplification of the Fas (CD95) signal in hepatocytes via caspase 8-mediated cleavage of Bid and activation of the mitochondrial apoptosis pathway. Recent evidence, however, indicates that activation of other BH3-only proteins may also be critical for the crosstalk between death receptors and mitochondrial triggers. In this study, we show that TNF-related apoptosis-inducing ligand (TRAIL) and chemotherapeutic drugs synergistically induce apoptosis in various transformed and untransformed liver-derived cell lines, as well as in primary human hepatocytes. Both, preincubation with TRAIL as well as chemotherapeutic drugs could sensitize cells for apoptosis induction by the other respective trigger. TRAIL induced a strong and long lasting activation of Jun kinase, and activation of the BH3-only protein Bim. Consequently, synergistic induction of apoptosis by TRAIL and chemotherapeutic drugs was dependent on Jun kinase activity, and expression of Bim and Bid. These findings confirm a previously defined role of TRAIL and Bim in the regulation of hepatocyte apoptosis, and demonstrate that the TRAIL-Jun kinase-Bim axis is a major and important apoptosis amplification pathway in primary hepatocytes and liver tumor cells.

Comparison of immortalized bEnd5 and primary mouse brain microvascular endothelial cells as in vitro blood-brain barrier models for the study of T cell extravasation

Important insights into the molecular mechanism of T cell extravasation across the blood-brain barrier (BBB) have already been obtained using immortalized mouse brain endothelioma cell lines (bEnd). However, compared with bEnd, primary brain endothelial cells have been shown to establish better barrier characteristics, including complex tight junctions and low permeability. In this study, we asked whether bEnd5 and primary mouse brain microvascular endothelial cells (pMBMECs) were equally suited as in vitro models with which to study the cellular and molecular mechanisms of T cell extravasation across the BBB. We found that both in vitro BBB models equally supported both T cell adhesion under static and physiologic flow conditions, and T cell crawling on the endothelial surface against the direction of flow. In contrast, distances of T cell crawling on pMBMECs were strikingly longer than on bEnd5, whereas diapedesis of T cells across pMBMECs was dramatically reduced compared with bEnd5. Thus, both in vitro BBB models are suited to study T cell adhesion. However, because pMBMECs better reflect endothelial BBB specialization in vivo, we propose that more reliable information about the cellular and molecular mechanisms of T cell diapedesis across the BBB can be attained using pMBMECs.

In vivo analysis of uropod function during physiological T cell trafficking

Migrating lymphocytes acquire a polarized phenotype with a leading and a trailing edge, or uropod. Although in vitro experiments in cell lines or activated primary cell cultures have established that Rho-p160 coiled-coil kinase (ROCK)-myosin II-mediated uropod contractility is required for integrin de-adhesion on two-dimensional surfaces and nuclear propulsion through narrow pores in three-dimensional matrices, less is known about the role of these two events during the recirculation of primary, nonactivated lymphocytes. Using pharmacological antagonists of ROCK and myosin II, we report that inhibition of uropod contractility blocked integrin-independent mouse T cell migration through narrow, but not large, pores in vitro. T cell crawling on chemokine-coated endothelial cells under shear was severely impaired by ROCK inhibition, whereas transendothelial migration was only reduced through endothelial cells with high, but not low, barrier properties. Using three-dimensional thick-tissue imaging and dynamic two-photon microscopy of T cell motility in lymphoid tissue, we demonstrated a significant role for uropod contractility in intraluminal crawling and transendothelial migration through lymph node, but not bone marrow, endothelial cells. Finally, we demonstrated that ICAM-1, but not anatomical constraints or integrin-independent interactions, reduced parenchymal motility of inhibitor-treated T cells within the dense lymphoid microenvironment, thus assigning context-dependent roles for uropod contraction during lymphocyte recirculation.

MicroRNA-21 suppression impedes medulloblastoma cell migration

Medulloblastoma (MB), the most common malignant brain tumour in children, is characterised by a high risk of leptomeningeal dissemination. But little is known about the molecular mechanisms that promote cancer cell migration in MB. Aberrant expression of miR-21 is recognised to be causatively linked to metastasis in a variety of human neoplasms including brain tumours; however its function in MB is still unknown. In this study we investigated the expression level and the role of miR-21 in MB cell migration. miR-21 was found to be up-regulated, compared to normal cerebellum, in 29/29 MB primary samples and 6/6 MB-derived cell lines. Inverse correlation was observed between miR-21 expression and the metastasis suppressor PDCD4, while miR-21 repression increased the release of PDCD4 protein, suggesting negative regulation of PDCD4 by miR-21 in MB cells. Anti-miR-21 decreased protein expression of the tumour cell invasion mediators MAP4K1 and JNK, which are also known to be negatively regulated by PDCD4, and down-regulated integrin protein that is essential for MB leptomeningeal dissemination. Moreover miR-21 knockdown in MB cells increased the expression of two eminent negative modulators of cancer cell migration, E-Cadherin and TIMP2 proteins that are known to be positively regulated by PDCD4. Finally and importantly, suppression of miR-21 decreased the motility of MB cells and reduced their migration across basement membranes in vitro. Together, these compelling data propose miR-21 pathway as a novel mechanism impacting MB cell dissemination and raises the possibility that curability of selected MB may be improved by pharmaceutical strategies directed towards microRNA-21.

A sensitized RNA interference screen identifies a novel role for the PI3K p110γ isoform in medulloblastoma cell proliferation and chemoresistance

Medulloblastoma is the most common malignant brain tumor in children and is associated with a poor outcome. We were interested in gaining further insight into the potential of targeting the human kinome as a novel approach to sensitize medulloblastoma to chemotherapeutic agents. A library of small interfering RNA (siRNA) was used to downregulate the known human protein and lipid kinases in medulloblastoma cell lines. The analysis of cell proliferation, in the presence or absence of a low dose of cisplatin after siRNA transfection, identified new protein and lipid kinases involved in medulloblastoma chemoresistance. PLK1 (polo-like kinase 1) was identified as a kinase involved in proliferation in medulloblastoma cell lines. Moreover, a set of 6 genes comprising ATR, LYK5, MPP2, PIK3CG, PIK4CA, and WNK4 were identified as contributing to both cell proliferation and resistance to cisplatin treatment in medulloblastoma cells. An analysis of the expression of the 6 target genes in primary medulloblastoma tumor samples and cell lines revealed overexpression of LYK5 and PIK3CG. The results of the siRNA screen were validated by target inhibition with specific pharmacological inhibitors. A pharmacological inhibitor of p110γ (encoded by PIK3CG) impaired cell proliferation in medulloblastoma cell lines and sensitized the cells to cisplatin treatment. Together, our data show that the p110γ phosphoinositide 3-kinase isoform is a novel target for combinatorial therapies in medulloblastoma.

Novel agents targeting the IGF-1R/PI3K pathway impair cell proliferation and survival in subsets of medulloblastoma and neuroblastoma

The receptor tyrosine kinase (RTK)/phosphoinositide 3-kinase (PI3K) pathway is fundamental for cancer cell proliferation and is known to be frequently altered and activated in neoplasia, including embryonal tumors. Based on the high frequency of alterations, targeting components of the PI3K signaling pathway is considered to be a promising therapeutic approach for cancer treatment. Here, we have investigated the potential of targeting the axis of the insulin-like growth factor-1 receptor (IGF-1R) and PI3K signaling in two common cancers of childhood: neuroblastoma, the most common extracranial tumor in children and medulloblastoma, the most frequent malignant childhood brain tumor. By treating neuroblastoma and medulloblastoma cells with R1507, a specific humanized monoclonal antibody against the IGF-1R, we could observe cell line-specific responses and in some cases a strong decrease in cell proliferation. In contrast, targeting the PI3K p110α with the specific inhibitor PIK75 resulted in broad anti-proliferative effects in a panel of neuro- and medulloblastoma cell lines. Additionally, sensitization to commonly used chemotherapeutic agents occurred in neuroblastoma cells upon treatment with R1507 or PIK75. Furthermore, by studying the expression and phosphorylation state of IGF-1R/PI3K downstream signaling targets we found down-regulated signaling pathway activation. In addition, apoptosis occurred in embryonal tumor cells after treatment with PIK75 or R1507. Together, our studies demonstrate the potential of targeting the IGF-1R/PI3K signaling axis in embryonal tumors. Hopefully, this knowledge will contribute to the development of urgently required new targeted therapies for embryonal tumors.

Defining new criteria for selection of cell-based intestinal models using publicly available databases

The criteria for choosing relevant cell lines among a vast panel of available intestinal-derived lines exhibiting a wide range of functional properties are still ill-defined. The objective of this study was, therefore, to establish objective criteria for choosing relevant cell lines to assess their appropriateness as tumor models as well as for drug absorption studies.

High NRBP1 expression in prostate cancer is linked with poor clinical outcomes and increased cancer cell growth

We recently established the rationale that NRBP1 (nuclear receptor binding protein 1) has a potential growth-promoting role in cell biology. NRBP1 interacts directly with TSC-22, a potential tumor suppressor gene that is differently expressed in prostate cancer. Consequently, we analyzed the role of NRBP1 expression in prostate cancer cell lines and its expression on prostate cancer tissue microarrays (TMA).

Inhibition of hepatic tumor cell proliferation in vitro and tumor growth in vivo by taltobulin, a synthetic analogue of the tripeptide hemiasterlin

AIM: To investigate the inhibitory effects of taltobulin (HTI-286), a synthetic analogue of natural hemiasterlin derived from marine sponges, on hepatic tumor growth in vitro and in vivo. METHODS: The potential anti-proliferative effects of HTI-286 on different hepatic tumor cell lines in vitro and in vivo were examined. RESULTS: HTI-286 significantly inhibited proliferation of all three hepatic tumor cell lines (mean IC50 = 2 nmol/L +/- 1 nmol/L) in vitro. Interestingly, no decrease in viable primary human hepatocytes (PHH) was detected under HTI-286 exposure. Moreover, intravenous administration of HTI-286 significantly inhibited tumor growth in vivo (rat allograft model). CONCLUSION: HTI-286 might be considered a potent promising drug in treatment of liver malignancies. HTI-286 is currently undergoing clinical evaluation in cancer patients.

A pedigree with autosomal dominant thrombocytopenia, red cell macrocytosis, and an occurrence of t(12:21) positive pre-B acute lymphoblastic leukemia

Sampling and analyzing new families with inherited blood disorders are major steps contributing to the identification of gene(s) responsible for normal and pathologic hematopoiesis. Familial occurrences of hematological disorders alone, or as part of a syndromic disease, have been reported, and for some the underlying genetic mutation has been identified. Here we describe a new autosomal dominant inherited phenotype of thrombocytopenia and red cell macrocytosis in a four-generation pedigree. Interestingly, in the youngest generation, a 2-year-old boy presenting with these familial features has developed acute lymphoblastic leukemia characterized by a t(12;21) translocation. Tri-lineage involvement of platelets, red cells and white cells may suggest a genetic defect in an early multiliear progenitor or a stem cell. Functional assays in EBV-transformed cell lines revealed a defect in cell proliferation and tubulin dynamics. Two candidate genes, RUNX1 and FOG1, were sequenced but no pathogenic mutation was found. Identification of the underlying genetic defect(s) in this family may help in understanding the complex process of hematopoiesis.

Cell replacement therapy for intracerebral hemorrhage

Intracerebral hemorrhage (ICH), for which no effective treatment strategy is currently available, constitutes one of the most devastating forms of stroke. As a result, developing therapeutic options for ICH is of great interest to the medical community. The 3 potential therapies that have the most promise are cell replacement therapy, enhancing endogenous repair mechanisms, and utilizing various neuroprotective drugs. Replacement of damaged cells and restoration of function can be accomplished by transplantation of cells derived from different sources, such as embryonic or somatic stem cells, umbilical cord blood, and genetically modified cell lines. Early experimental data showing the benefits of cell transplantation on functional recovery after ICH have been promising. Nevertheless, several studies have focused on another therapeutic avenue, investigating novel ways to activate and direct endogenous repair mechanisms in the central nervous system, through exposure to specific neuronal growth factors or by inactivating inhibitory molecules. Lastly, neuroprotective drugs may offer an additional tool for improving neuronal survival in the perihematomal area. However, a number of scientific issues must be addressed before these experimental techniques can be translated into clinical therapy. In this review, the authors outline the recent advances in the basic science of treatment strategies for ICH.

miR-15a and miR-16 are implicated in cell cycle regulation in a Rb-dependent manner and are frequently deleted or down-regulated in non-small cell lung cancer

MicroRNAs (miRNA) are negative regulators of gene expression at the posttranscriptional level, which are involved in tumorigenesis. Two miRNAs, miR-15a and miR-16, which are located at chromosome 13q14, have been implicated in cell cycle control and apoptosis, but little information is available about their role in solid tumors. To address this question, we established a protocol to quantify miRNAs from laser capture microdissected tissues. Here, we show that miR-15a/miR-16 are frequently deleted or down-regulated in squamous cell carcinomas and adenocarcinomas of the lung. In these tumors, expression of miR-15a/miR-16 inversely correlates with the expression of cyclin D1. In non-small cell lung cancer (NSCLC) cell lines, cyclins D1, D2, and E1 are directly regulated by physiologic concentrations of miR-15a/miR-16. Consistent with these results, overexpression of these miRNAs induces cell cycle arrest in G(1)-G(0). Interestingly, H2009 cells lacking Rb are resistant to miR-15a/miR-16-induced cell cycle arrest, whereas reintroduction of functional Rb resensitizes these cells to miRNA activity. In contrast, down-regulation of Rb in A549 cells by RNA interference confers resistance to these miRNAs. Thus, cell cycle arrest induced by these miRNAs depends on the expression of Rb, confirming that G(1) cyclins are major targets of miR-15a/miR-16 in NSCLC. Our results indicate that miR-15a/miR-16 are implicated in cell cycle control and likely contribute to the tumorigenesis of NSCLC.