Targeting sphingosine kinase 1 in carcinoma cells decreases proliferation and survival by compromising PKC activity and cytokinesis

Sphingosine kinases (SK) catalyze the phosphorylation of proapoptotic sphingosine to the prosurvival factor sphingosine 1-phosphate (S1P), thereby promoting oncogenic processes. Breast (MDA-MB-231), lung (NCI-H358), and colon (HCT 116) carcinoma cells were transduced with shRNA to downregulate SK-1 expression or treated with a pharmacologic SK-1 inhibitor. The effects of SK-1 targeting were investigated by measuring the level of intracellular sphingosine, the activity of protein kinase C (PKC) and cell cycle regulators, and the mitotic index. Functional assays included measurement of cell proliferation, colony formation, apoptosis, and cell cycle analysis. Downregulation of SK-1 or its pharmacologic inhibition increased intracellular sphingosine and decreased PKC activity as shown by reduced phosphorylation of PKC substrates. In MDA-MB-231 cells this effect was most pronounced and reduced cell proliferation and colony formation, which could be mimicked using exogenous sphingosine or the PKC inhibitor RO 31-8220. SK-1 downregulation in MDA-MB-231 cells increased the number of cells with 4N and 8N DNA content, and similar effects were observed upon treatment with sphingosine or inhibitors of SK-1 or PKC. Examination of cell cycle regulators unveiled decreased cdc2 activity and expression of Chk1, which may compromise spindle checkpoint function and cytokinesis. Indeed, SK-1 kd cells entered mitosis but failed to divide, and in the presence of taxol also failed to sustain mitotic arrest, resulting in further increased endoreduplication and apoptosis. Our findings delineate an intriguing link between SK-1, PKC and components of the cell cycle machinery, which underlines the significance of SK-1 as a target for cancer therapy.

Radiolabeled somatostatin receptor antagonists are preferable to agonists for in vivo peptide receptor targeting of tumors

Targeting neuroendocrine tumors expressing somatostatin receptor subtypes (sst) with radiolabeled somatostatin agonists is an established diagnostic and therapeutic approach in oncology. While agonists readily internalize into tumor cells, permitting accumulation of radioactivity, radiolabeled antagonists do not, and they have not been considered for tumor targeting. The macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was coupled to two potent somatostatin receptor-selective peptide antagonists [NH(2)-CO-c(DCys-Phe-Tyr-DAgl(8)(Me,2-naphthoyl)-Lys-Thr-Phe-Cys)-OH (sst(3)-ODN-8) and a sst(2)-selective antagonist (sst(2)-ANT)], for labeling with (111/nat)In. (111/nat)In-DOTA-sst(3)-ODN-8 and (111/nat)In-DOTA-[4-NO(2)-Phe-c(DCys-Tyr-DTrp-Lys-Thr-Cys)-DTyr-NH(2)] ((111/nat)In-DOTA-sst(2)-ANT) showed high sst(3)- and sst(2)-binding affinity, respectively. They did not trigger sst(3) or sst(2) internalization but prevented agonist-stimulated internalization. (111)In-DOTA-sst(3)-ODN-8 and (111)In-DOTA-sst(2)-ANT were injected intravenously into mice bearing sst(3)- and sst(2)-expressing tumors, and their biodistribution was monitored. In the sst(3)-expressing tumors, strong accumulation of (111)In-DOTA-sst(3)-ODN-8 was observed, peaking at 1 h with 60% injected radioactivity per gram of tissue and remaining at a high level for >72 h. Excess of sst(3)-ODN-8 blocked uptake. As a control, the potent agonist (111)In-DOTA-[1-Nal(3)]-octreotide, with strong sst(3)-binding and internalization properties showed a much lower and shorter-lasting uptake in sst(3)-expressing tumors. Similarly, (111)In-DOTA-sst(2)-ANT was injected into mice bearing sst(2)-expressing tumors. Tumor uptake was considerably higher than with the highly potent sst(2)-selective agonist (111)In-diethylenetriaminepentaacetic acid-[Tyr(3),Thr(8)]-octreotide ((111)In-DTPA-TATE). Scatchard plots showed that antagonists labeled many more sites than agonists. Somatostatin antagonist radiotracers therefore are preferable over agonists for the in vivo targeting of sst(3)- or sst(2)-expressing tumors. Antagonist radioligands for other peptide receptors need to be evaluated in nuclear oncology as a result of this paradigm shift.

Drug targeting using OX7-immunoliposomes: correlation between Thy1.1 antigen expression and tissue distribution in the rat

OX7 monoclonal antibody F((ab')2) fragments directed against Thy1.1 antigen can be used for drug targeting by coupling to the surface of drug-loaded liposomes. Such OX7-conjugated immunoliposomes (OX7-IL) were used recently for drug delivery to rat glomerular mesangial cells, which are characterized by a high level of Thy1.1 antigen expression. In the present study, the relationship between OX7-IL tissue distribution and target Thy1.1 antigen localization in different organs in rat was investigated. Western blot and immunohistofluorescence analysis revealed a very high Thy1.1 expression in brain cortex and striatum, thymus and renal glomeruli. Moderate Thy1.1 levels were observed in the collecting ducts of kidney, lung tissue and spleen. Thy1.1 was not detected in liver and heart. There was a poor correlation between Thy1.1 expression levels and organ distribution of fluorescence- or (14)C-labeled OX7-IL. The highest overall organ density of OX7-IL was observed in the spleen, followed by lung, liver and kidney. Heart and brain remained negative. With respect to intra-organ distribution, a localized and distinct signal was observed in renal glomerular mesangial cells only. As a consequence, acute pharmacological (i.e. toxic) effects of doxorubicin-loaded OX7-IL were limited to renal glomeruli. The competition with unbound OX7 monoclonal antibody F((ab')2) fragments demonstrated that the observed tissue distribution and acute pharmacological effects of OX7-IL were mediated specifically by the conjugated OX7 antibody. It is concluded that both the high target antigen density and the absence of endothelial barriers are needed to allow for tissue-specific accumulation and pharmacological effects of OX7-IL. The liposomal drug delivery strategy used is therefore specific toward renal glomeruli and can be expected to reduce the risk of unwanted side effects in other tissues.

Linking livelihood strategies with a regional level landscape mosaic to explore trade-offs between forest conservation and human wellbeing in a Madagascar biodiversity hotspot

The north-eastern escarpment of Madagascar contains the island’s last remaining large-scale humid forest massifs surrounded by diverse small-scale agricultural mosaics. There is high deforestation mainly caused by shifting cultivation practiced by local land users to produce upland rice for subsistence. Today, large protected areas restrict land users’ access to forests to collect wood and other forest products. Moreover, they are no more able to expand their cultivated land, which leads to shorter shifting cultivation cycles and decreasing plot sizes for irrigated rice and cash crop cultivation. Cash crop production of clove and vanilla is exposed to risks such as extreme inter-annual price fluctuations, pests and cyclones. In the absence of work opportunities, agricultural extension services and micro-finance schemes people are stuck in a poverty trap. New development strategies are needed to mitigate the trade-offs between forest conservation and human well-being. As landscape composition and livelihood strategies vary across the region, these strategies need to be spatially differentiated to avoid implementing generic solutions, which do not fit the local context. However, up to date, little is known about the spatial patterns of shifting cultivation and other land use systems at the regional level. This is mainly due to the high spatial and temporal dynamics inherent to shifting cultivation, which makes it difficult to monitor the dynamics of this land use system with remote sensing methods. Furthermore, knowledge about land users’ livelihood strategies and the risks and opportunities they face stems from very few local case studies. To overcome this challenge, firstly, we used remote sensing data and a landscape mosaic approach to delineate the main landscape types at the regional level. Secondly, we developed a land user typology based on socio-ecological data from household surveys in 45 villages spread throughout the region. Combining the land user typology with the landscape mosaic map allowed us to reveal spatial patterns of the interaction between landscapes and people and to better understand the trade-offs between forest conservation and local wellbeing. While shifting cultivation systems are being transformed into more intensive permanent agricultural systems in many countries around the globe, Madagascar seems to be an exception to this trend. Linking land cover information to human-environmental interactions over large areas is crucial to designing policies and to inform decision making for a more sustainable development of this resource-rich but poverty-prone context.

Targeting the PI3K/AKT/mTOR signaling pathway in medulloblastoma

Medulloblastoma is the most common malignant childhood brain tumor and is associated with a poor outcome. There is an urgent need to develop novel targeted therapeutic approaches for medulloblastoma, which will arise from an enhanced understanding of the disease at the molecular level. Medulloblastoma has been recognized to be a heterogeneous disease, and no recurrent cancer gene mutations have been found, although many of the mutations described so far affect key intracellular signaling pathways, such as sonic hedgehog (SHH) and Wnt/β-catenin. The PI3K/AKT/mTOR (PAM) signaling pathway controls key cellular responses, such as cell growth and proliferation, survival, migration and metabolism. Over the last decades, it has been recognized that this intracellular signaling pathway is frequently activated by genetic and epigenetic alterations in malignant brain tumors, including medulloblastoma. Clinical trials have started to evaluate the safety and efficacy of agents targeting this pathway in malignant brain tumors. Due to the complexity of the PAM signaling pathway, there remain significant difficulties in the development of novel therapeutic approaches. The future challenges in developing effective treatments for cancer patients include the development of predictive biomarkers and combinatorial approaches to effectively target multiple signal transduction pathways. In this review article, we will summarize the current knowledge about the role of PAM signaling in medulloblastoma and discuss the strategies that are currently being evaluated with targeted agents against this pathway.