Ion Channels as Promising Therapeutic Targets for Melanoma

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Tumour-derived and host-derived nitric oxide differentially regulate breast carcinoma metastasis to the lungs

To study the role of nitric oxide (NO) in lung metastasis of breast carcinoma, we isolated two cell clones (H and J) from the parental EMT-6 murine breast carcinoma cell line, based on their differential NO production. In vitro, EMT-6 J cells, but not EMT-6H cells, constitutively expressed inducible NO synthase (NOS II) and secreted high levels of NO. IL-1β increased NO production in both clones, and TNF-α had a synergistic effect on IL-1β-induced NO production, but NO production by EMT-6 J cells was always higher than by EMT-6H cells. Proliferation, survival and adhesion to lung-derived endothelial cells of both clones were similar and were not affected by NO. In vivo, both clones similarly located in the lungs of syngeneic mice 48 h after injection. However, EMT-6H cells were significantly more tumorigenic than EMT-6 J cells as assessed at later time points. Injection of EMT-6 J cells and simultaneous treatment of mice with aminoguanidine (AG), a NOS II inhibitor, significantly increased tumour formation. Injection of EMT-6H and EMT-6 J cells into NOS II-deficient mice resulted in a significant survival increase as compared with wild-type animals. Simultaneous administration of AG increased the death rate of NOS II-deficient mice injected with EMT-6 J cells. These results demonstrate that: (i) NO does not influence the early stages of tumour metastasis to the lungs and (ii) NOS II expression in tumour cells reduces, while NOS II expression in host cells enhances, tumour nodule development. In conclusion, the cellular origin and the local NO production are critical in the metastatic proces

Automatic Nuclei Cell Counting in Low-Resolution Fluorescence Images

International audienceThis paper deals with the problem of counting nuclei in low-resolution fluorescence images. The low resolution is a consequence of the usage of a large field of view, this to reduce the number of experiments, implying time saving and money saving. But the small size of nuclei increases the risk of error in counting. In this work we used some image processing basic operators in order to extract potential shapes. These shape are then characterized (size, shape, edges) to decide if they are noise or not. Two approaches are presented for this: the first one correspond to a translation of some basic knowledge used by practitioners; the second one uses a supervised classification technique where it is the computer who discovers the knowledge by the analysis of a database of cases. This leads to a fast technique which gives very good results, validated by cell biology experts

Two Approaches for Automatic Nuclei Cell Counting in Low Resolution Fluorescence Images

This paper deals with the problem of counting nuclei in low-resolution fluorescence images. The low resolution is a consequence of the usage of a large field of view, this to reduce the number of experiments, implying time saving and money saving. But the small size of nuclei increases the risk of error in counting. In this work we used some image processing basic operators in order to extract potential shapes. These shape are then characterized (size, shape, edges) to decide if they are noise or not. Two approaches are presented for this: the first one correspond to a translation of some basic knowledge used by practitioners; the second one uses a supervised classification technique where it is the computer who discovers the knowledge by the analysis of a database of cases. This leads to a fast technique which gives very good results, validated by cell biology experts

The Calcium-Sensing Receptor is A Marker and Potential Driver of Neuroendocrine Differentiation in Prostate Cancer

The mechanisms underlying neuroendocrine (NE) differentiation in prostate cancer (PCa) remain mostly uncharacterized. Since a deregulated calcium homeostasis has been reported in neuroendocrine prostate cancer (NEPC), we explored herein the link between NE differentiation and the calcium-sensing receptor (CaSR). CaSR expression was evaluated by immunohistochemistry—together with NE markers—on tissue microarrays containing samples of normal prostate, localized PCa, metastatic castration resistant PCa (MCRPC) and NEPC. In prostate tissues, we observed a strong association between CaSR and chromogranin expression. Both markers were strongly expressed in all cases of NEPC and co-expression was confirmed by double immunostaining. In MCRPC, the expression of CaSR was significantly associated with shorter overall survival. The involvement of CaSR in NE differentiation was evaluated in PCa cell lines. Inhibition of CaSR led to decrease the expression of neuronal (NSE, βtubulinIII) and NE (chromogranin, synaptophysin) markers in the NE PCa cell line NCI-H660. A decrease of neuronal and NE markers was also observed in siCaSR-transfected PC3 and 22RV1 cells, respectively, whereas CaSR activation increased both NSE and synaptophysin expression in PC3 cells. These results strongly suggest that CaSR is a marker and a driver of NE differentiation in PCa and emphasize the potential of CaSR directed therapy for NEPC patients

Glyco-Phospho-Glycero Ether Lipids (GPGEL): synthesis and evaluation as small conductance Ca2+-activated K+ channel (SK3) inhibitors

International audienceThe SK3 channel, a member of the small conductance calcium-dependent potassium channels (SKCa), plays a central role with respect to the motility of highly metastatic cancerous cells (e.g. MDA-MB435s – breast cancer cell). Edelfosine, an ether lipid derivative, is able to partially inhibit this channel activity and thus reduce the SK3-dependent cell motility, but, due to its toxicity, analogues of this compound were highly desired. Ohmline, an edelfosine analogue that possesses a lactose unit in its polar domain, was the first efficient and non-toxic SK3 inhibitor that exhibits an amphiphilic structure. In the present work, we have modified the polar head of Ohmline by placing a disubstituted phosphate group between a disaccharide unit (lactose, maltose, and melibiose) and the glycerol ether-lipid moiety. It was first observed that this modification increases the water solubility of these compounds. All these novel compounds are efficient SK3 channel inhibitors with an activity comparable to Ohmline (patch-clamp measurements). These compounds are also able to reduce the SK3-dependent cell motility with similar efficacies to Ohmline. In a broader perspective it is shown that the presence of one anionic charge (coming from the presence of a phosphate group) in the polar head group does not alter the SK3 channel inhibition and provides insights into the future development of a class of migration-targeted anticancer agents

KCa2.3 channel-dependent hyperpolarization increases melanoma cell motility.

International audienceCell migration and invasion are required for tumour cells to spread from the primary tumour bed so as to form secondary tumours at distant sites. We report evidence of an unusual expression of KCa2.3 (SK3) protein in melanoma cell lines but not in normal melanocytes. Knockdown of the KCa2.3 channel led to plasma membrane depolarization, decreased 2D and 3D cell motility. Conversely, enforced production of KCa2.3 protein in KCa2.3 non-expressing cells led to the plasma membrane becoming hyperpolarized, and enhanced cell motility. In contrast, KCa3.1 channels had no effect on cell motility despite an active role in regulating membrane potential. Our data also suggest that membrane hyperpolarization increases melanoma cell motility and that this occurs through the KCa2.3 channel. Our findings reveal a previously unknown function of the KCa2.3 channel, and suggest that the KCa2.3 channel might be the only member of the Ca(2+)-activated K(+) channel family involved in melanoma cell motility pathways

Altered SK3/KCa2.3-mediated migration in adenomatous polyposis coli (Apc) mutated mouse colon epithelial cells.

International audienceLost of adenomatous polyposis coli gene (Apc) disturbs the migration of intestinal epithelial cells but the mechanisms have not been fully characterized. Since we have demonstrated that SK3/KCa2.3 channel promotes cancer cell migration, we hypothesized that Apc mutation may affect SK3/KCa2.3 channel-mediated colon epithelial cell motility. We report evidence that SK3/KCa2.3 channel promotes colon epithelial cells motility. Following Apc mutation SK3/KCa2.3 expression is largely reduced leading to a suppression of the SK3/KCa2.3 channel mediated-cell migration. Our findings reveal a previously unknown function of the SK3/KCa2.3 channel in epithelial colonic cells, and suggest that Apc is a powerful regulator SK3/KCa2.3 channel

Development of pyrene-based fluorescent ether lipid as inhibitor of SK3 ion channels

International audienceWe report the synthesis of three bioactive pyrene-based fluorescent analogues of Ohmline which is the most efficient and selective inhibitor of SK3 ion channel. The interaction of these Ohmline-pyrene (OP1-3) with liposomes of different composition reveals that only OP2 and OP3 are readily integrated into liposomes. Fluorescence measurements indicate that, depending on their concentration, OP2 and OP3 exist either as monomer or as a mixture of monomer and excimers within the liposome bilayer. Among the three Ohmline Pyrene compounds (OP1-3) only OP2 is able to reduce SK3 currents and is the first efficient fluorescent modulator of SK3 channel as revealed by patch clamp measurements (- 71.3 ± 13.3% at 10 mM) and by its inhibition of SK3-dependent cancer cell migration at (32.5% ± 4.8% at 1 mM). We also report the first fluorescence study on living breast cancer cells (MDA-MB-231) showing that OP2 is rapidly integrated in bio-membranes followed by cell internalization.Nous décrivons la synthèse d’analogues de l’Ohmline comportant au niveau de la partie lipidique une sonde fluorescente de type pyrène. Nous montrons que l’un d’entre eux (le composé OP2) est capable de réduire les courants potassiques SK3 de 71% et de réduire la migration cellulaire SK3-dépendante de 32%. Il s’agit, à notre connaissance, du premier inhibiteur des canaux ioniques SK3 de nature amphiphile et fluorescent