Neuronal differentiation by indomethacin and IBMX inhibits proliferation of small cell lung cancer cells in vitro.

BACKGROUND: Small cell lung cancer (SCLC) is one of the most aggressive malignancies implying a very poor prognosis for patients even under therapy. Since it is known that SCLC cells exhibit neurone-like characteristics, we investigated whether a neuronal induction medium (NID) consisting of indomethacin (200 µM), 3-isobutyl-1-methylxanthine (IBMX, 500 µM) and insulin (5 µg/ml) induces neuronal differentiation and by this reduces malignancy of SCLC in vitro. METHODS: Anti-proliferative effects were tested by incubating five SCLC cell lines (OH1, OH3, SW2, H69 and H82) with NID for 72 h (XTT-assay). Afterwards, anti-proliferative as well as cytotoxic effects (lactate dehydrogenase [LDH] assay, electron microscopy) of a range of drug concentrations (indomethacin 6.25-800 µM, IBMX 15.625-2000 µM and combinations of both) regarding H82 and SW2 were analysed. We further investigated the presence of cyclooxygenase- (COX-) 1 and 2 (IHC, Western blot) as well as levels of COX-2 before and after treatment. Neuronal differentiation was evaluated by morphological analyses (electron microscopy), detection of CD 56 and CD 171 (FACS) and recording Na(+) and K(+) currents (patch clamp). RESULTS: Proliferation of all cell lines was inhibited significantly in a dose dependent manner (linear regression), whereas SW2 and H82 were most sensitive. Treatment with insulin alone had no effect at all. Cytotoxic effects were only observed after incubation with high concentrations of indomethacin (H82) and combined treatment (SW2). COX-1 and 2 were detectable in H82 and SW2, whereas the level of COX-2 remained unaffected under treatment. By electron microscopy, we could not observe distinct neurone-like morphological changes after 72 h of treatment. However, the majority of H82 and SW2 cells expressed both CD 56 (NCAM) and CD 171 (L1), showing an increase of NCAM and L1 intensity at the cell surface after 7 and 14 days of treatment. We further demonstrated an up-regulation of neurone-specific Na(+) currents as well as a significant down-regulation of herg K(+) currents after NID treatment. CONCLUSION: Our findings demonstrate significant anti-proliferative, non-toxic effects of indomethacin and IBMX on SCLC cells in vitro. Treated SCLC cells further possess increased neuronal characteristics in vitro, possibly leading to a reduced malignant potential.

Increased numbers of spontaneous SCLC metastasis in absence of NK cells after subcutaneous inoculation of different SCLC cell lines into pfp/rag2 double knock out mice.

Spontaneous metastases in small cell lung cancer (SCLC) occur regularly in patients but seldom if any in conventional xenograft mouse models. To overcome this problem, SCLC cells were grafted subcutaneously onto pore forming protein and recombination activating gene 2 double knock out (pfp/rag2) mice and in severe combined immunodeficient (scid) mice. Primary tumours grew well in both mouse strains, while metastases occurred frequently in the pfp/rag2 mice and infrequently in scid mice. Hence NK cells, which are inactive in pfp/rag2 mice, play an important role in SCLC metastasis formation in xenograft models. This observation is in agreement with clinical studies, where a high NK cell number in the blood is correlated with a better prognosis of the patient.

[18F]FDG and [18F]FLT PET-CT and MR imaging of human neuroblastomas in a SCID mouse xenograft model.

BACKGROUND: Finding new therapeutic agents is of great clinical interest in neuroblastoma research because prognosis of children with disseminated stages of disease is still poor. As xenograft mouse models are frequently used for studying anticancer drugs in vivo, small animal imaging is an important method of monitoring in anticancer research. MATERIALS AND METHODS: SCID mice inoculated with human neuroblastoma SK-N-SH cells were examined with positron-emission tomography-computed tomography (PET-CT) using [18F]fluorodeoxyglucose (FDG) or [18F]fluoro-L-thymidine (FLT) and with magnetic resonance imaging (MRI). RESULTS: All neuroblastomas were detected by MRI. In PET-CT imaging, no tumour was visualized with [18F]FDG, but 13 out of 14 (93%) were found with [18F]FLT. Uptake of [18F]FLT was significantly associated with tumour weight. Necrotic areas could not be identified either by MR imaging or on PET-CT scans. CONCLUSION: Both MR and PET-CT imaging with [18F]FLT are highly qualified for the detection of neuroblastomas grown in SCID mice. However, [18F]FDG, which is the standard tracer in clinical PET-CT imaging, is not suited for PET-CT imaging in the neuroblastoma model.

Visualisation of neuroblastoma growth in a Scid mouse model using [18F]FDG and [18F]FLT-PET.

BACKGROUND: Tumor therapy has been monitored using the metabolic indicator [18F]fluorodeoxyglucose ([18F]FDG). However, the nucleotide precursor [18F]fluoro-thymidine ([18F]FLT) is in principle more specific as it is incorporated into DNA. Thus, the [18F]FDG and [18F]FLT uptake by human neuroblastomas grown in Scid mice are compared in this study. MATERIALS AND METHODS: Scid mice were inoculated with human neuroblastoma cells. Tumor imaging was performed with a human whole-body full-ring PET scanner. Furthermore, the tumor weight and the cell proliferation rate were determined. RESULTS: Neuroblastomas could be visualised using [18F]FDG in 40% and with [18F]FLT in 70% of the cases. [18F]FDG or [18F]FLT uptake could not be visualised in neuroblastomas less than 1.0 g in weight. No correlation between the cell proliferation rate and tracer uptake could be detected. CONCLUSION: [18F]FLT showed a higher uptake than [18F]FDG and, therefore, might be more suitable for monitoring anticancer therapy, at least in this tumor model.

An optimized fixation and extraction technique for high resolution of inositol phosphate signals in rodent brain.

Members of lower and higher inositol phosphates distinctly participate in signal transduction (1). Relatively little is known regarding possible biological functions of inositol phosphates in functionally different areas of the intact brain. A detailed study on the regional distribution of biologically important inositol phosphates may help elucidate their physiological functions in different brain regions in the regional tissue context. We now show a novel technique which allows fixation and subsequent dissection of whole rat brains into small volume elements for mapping of the whole range of inositol phosphates from Ins(1,4,5)P3 to InsP6. The method has been successfully applied to investigate regional differences of a broader spectrum of inositol phosphates in microdissected brain tissue and to construct 3D-maps of these signaling compounds. The technique can be particularly well employed to investigate regional changes in the spectrum of higher inositol phosphates and phosphoinositides upon neuronal stimulation induced by motor activity or drug treatment.