An In Vitro Analysis of TKI-Based Sequence Therapy in Renal Cell Carcinoma Cell Lines

The tyrosine kinase inhibitor (TKI) cabozantinib might impede the growth of the sunitinibresistant cell lines by targeting MET and AXL overexpression in metastatic renal cell carcinoma (mRCC). We studied the role of MET and AXL in the response to cabozantinib, particularly following long-term administration with sunitinib. Two sunitinib-resistant cell lines, 786-O/S and Caki-2/S, and the matching 786-O/WT and Caki-2/WT cells were exposed to cabozantinib. The drug response was cell-line-specific. The 786-O/S cells were less growth-inhibited by cabozantinib than 786-O/WT cells (p-value = 0.02). In 786-O/S cells, the high level of phosphorylation of MET and AXL was not affected by cabozantinib. Despite cabozantinib hampering the high constitutive phosphorylation of MET, the Caki-2 cells showed low sensitivity to cabozantinib, and this was independent of sunitinib pretreatment. In both sunitinib-resistant cell lines, cabozantinib increased Src-FAK activation and impeded mTOR expression. The modulation of ERK and AKT was cell-line-specific, mirroring the heterogeneity among the patients. Overall, the MET- and AXL-driven status did not affect cell responsiveness to cabozantinib in the second-line treatment. The activation of Src-FAK might counteract cabozantinib activity and contribute to tumor survival and may be considered an early indicator of therapy response

Ion channels in control of pancreatic stellate cell migration

Pancreatic stellate cells (PSCs) play a critical role in the progression of pancreatic ductal adenocarcinoma (PDAC). Once activated, PSCs support proliferation and metastasis of carcinoma cells. PSCs even co-metastasise with carcinoma cells. This requires the ability of PSCs to migrate. In recent years, it has been established that almost all “hallmarks of cancer” such as proliferation or migration/invasion also rely on the expression and function of ion channels. So far, there is only very limited information about the function of ion channels in PSCs. Yet, there is growing evidence that ion channels in stromal cells also contribute to tumor progression. Here we investigated the function of K(Ca)3.1 channels in PSCs. K(Ca)3.1 channels are also found in many tumor cells of different origin. We revealed the functional expression of K(Ca)3.1 channels by means of Western blot, immunofluorescence and patch clamp analysis. The impact of K(Ca)3.1 channel activity on PSC function was determined with live-cell imaging and by measuring the intracellular Ca2(+) concentration ([Ca(2+)](i)). K(Ca)3.1 channel blockade or knockout prevents the stimulation of PSC migration and chemotaxis by reducing the [Ca(2+)](i) and calpain activity. K(Ca)3.1 channels functionally cooperate with TRPC3 channels that are upregulated in PDAC stroma. Knockdown of TRPC3 channels largely abolishes the impact of K(Ca)3.1 channels on PSC migration. In summary, our results clearly show that ion channels are crucial players in PSC physiology and pathophysiology

A Novel NHE1-Centered Signaling Cassette Drives Epidermal Growth Factor Receptor–Dependent Pancreatic Tumor Metastasis and Is a Target for Combination Therapy

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers principally because of early invasion and metastasis. The epidermal growth factor receptor (EGFR) is essential for PDAC development even in the presence of Kras, but its inhibition with erlotinib gives only a modest clinical response, making the discovery of novel EGFR targets of critical interest. Here, we revealed by mining a human pancreatic gene expression database that the metastasis promoter Na+/H+ exchanger (NHE1) associates with the EGFR in PDAC. In human PDAC cell lines, we confirmed that NHE1 drives both basal and EGF-stimulated three-dimensional growth and early invasion via invadopodial extracellular matrix digestion. EGF promoted the complexing of EGFR with NHE1 via the scaffolding protein Na +/H + exchanger regulatory factor 1, engaging EGFR in a negative transregulatory loop that controls the extent and duration of EGFR oncogenic signaling and stimulates NHE1. The specificity of NHE1 for growth or invasion depends on the segregation of the transient EGFR/Na +/H + exchanger regulatory factor 1/NHE1 signaling complex into dimeric subcomplexes in different lipid raftlike membrane domains. This signaling complex was also found in tumors developed in orthotopic mice. Importantly, the specific NHE1 inhibitor cariporide reduced both three-dimensional growth and invasion independently of PDAC subtype and synergistically sensitized these behaviors to low doses of erlotinib

Anandamide inhibits oxidative phosphorylation in isolated liver mitochondria

A study on the effect of anandamide (AEA) in energy coupling of rat liver mitochondria is presented. Micromolar concentrations of AEA, while almost ineffective on substrate supported oxygen consumption rate and on uncoupler stimulated respiration, strongly inhibited the respiratory state III. AEA did not change the rate and the extent of substrate generated membrane potential, but markedly delayed rebuilding by respiration of the potential collapsed by ADP addition. Overall, these data suggest that anandamide inhibits the oxidative phosphorylation process. Direct measurement of the F"oF"1 ATP synthase activity showed that the oligomycin sensitive ATP synthesis was inhibited by AEA, (IC"5"0, 2.5@mM), while the ATP hydrolase activity was unaffected. Consistently, AEA did not change the membrane potential generated by ATP hydrolysis

New excavations in the urban area of Caere

Excavations conducted by Quenn's University in the central area of the Etruscan city of Caere, near the so-called hypogaeum of Clepsina, have brought to light a sequence of phases indicative of a longer and more complex history of urban occupation at the site than previously thought. Excavation and geophysical prospection have revealed a regular urban plan in the area between the centre of the city plateau and the sanctuary of Manganello. Research within the hypogaeum has allowed for a better reading of wall paintings, drawings and inscriptions, including two previously unknown texts.Les fouilles conduites par l’Université Queen’s au centre de la cité étrusque de Caeré, près du monument connu comme l’hypogée de Clepsina, ont mis en lumière une série de phases d’occupation du site qui révèle une présence urbaine plus longue et plus complexe qu’on ne le pensait jusqu’à présent. Des fouilles et des prospections géophysiques ont révélé un plan urbain régulier dans la zone située entre le centre du plateau de la cité et le sanctuaire de Manganello. Des recherches menées à l’intérieur de l’hypogée, particulièrement à l’aide de l’imagerie à infrarouge, ont permis une meilleure lecture des peintures murales, des dessins et des inscriptions, révélant entre autres deux textes inconnus jusqu’à présent

Mitochondria isolated in nearly isotonic KCl buffer: focus on cardiolipin and organelle morphology

Rat liver mitochondria were isolated in parallel in two different isolation buffers: a standard buffer containing mannitol/sucrose and a nearly physiological KCl based solution. The two different organelle preparations were comparatively characterized by respiratory activity, heme content, microsomal and Golgi contamination, electron microscopy and lipid analyses. The substitution of saccharides with KCl in the isolation buffer does not induce the formation of mitoplasts or disruption of mitochondria. Mitochondria isolated in KCl buffer are coupled and able to maintain a stable transmembrane charge separation. A number of biochemical and functional differences between the two organelle preparations are described; in particular KCl mitochondria exhibit lower cardiolipin content and smaller intracristal compartments in comparison with the standard mitochondrial preparation

Tumor-reducing effect of the clinically used drug clofazimine in a SCID mouse model of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) represents the most common form of pancreatic cancer with rising incidence in developing countries. Unfortunately, the overall 5-year survival rate is still less than 5%. The most frequent oncogenic mutations in PDAC are loss-of function mutations in p53 and gain-of-function mutations in KRAS. Here we show that clofazimine (Lamprene), a drug already used in the clinic for autoimmune diseases and leprosy, is able to efficiently kill in vitro five different PDAC cell lines harboring p53 mutations. We provide evidence that clofazimine induces apoptosis in PDAC cells with an EC50 in the \ub5M range via its specific inhibitory action on the potassium channel Kv1.3. Intraperitoneal injection of clofazimine resulted in its accumulation in the pancreas of mice 8 hours after administration. Using an orthotopic PDAC xenotransplantation model in SCID beige mouse, we show that clofazimine significantly and strongly reduced the primary tumor weight. Thus, our work identifies clofazimine as a promising therapeutic agent against PDAC and further highlights ion channels as possible oncological targets