Dual targeting of HER3 and MEK may overcome HER3-dependent drug-resistance of colon cancers

Although the medical treatment of colorectal cancer has evolved greatly in the last years, a significant portion of early-stage patients develops recurrence after therapies. The current clinical trials are directed to evaluate new drug combinations and treatment schedules. By the use of patient-derived or established colon cancer cell lines, we found that the tyrosine kinase receptor HER3 is involved in the mechanisms of resistance to therapies. In agreement, the immunohistochemical analysis of total and phospho-HER3 expression in 185 colorectal cancer specimens revealed a significant correlation with lower disease-free survival. Targeting HER3 by the use of the monoclonal antibody patritumab we found induction of growth arrest in all cell lines. Despite the high efficiency of patritumab in abrogating the HER3-dependent activation of PI3K pathway, the HER2 and EGFR-dependent MAPK pathway is activated as a compensatory mechanism. Interestingly, we found that the MEK-inhibitor trametinib inhibits, as expected, the MAPK pathway but induces the HER3-dependent activation of PI3K pathway. The combined treatment results in the abrogation of both PI3K and MAPK pathways and in a significant reduction of cell proliferation and survival. These data suggest a new strategy of therapy for HER3-overexpressing colon cancers

A protein kinase a-independent pathway controlling aquaporin 2 trafficking as a possible cause for the syndrome of inappropriate antidiuresis associated with polycystic kidney disease 1 haploinsufficiency.

Renal water reabsorption is controlled by vasopressin (AVP) which binds to V2 receptors resulting in PKA activation, phosphorylation of AQP2 at serine 256 (pS256) and translocation to the plasma membrane. Besides S256, AVP causes dephosphorylation of S261. Recent studies showed that cyclin-dependent kinases can phosphorylate S261 AQP2 peptides in vitro. In an attempt to investigate the possible role of cdks on AQP2 phosphorylation, we identified a new PKA-independent pathway regulating AQP2 trafficking. In ex-vivo kidney slices and MDCK-AQP2 cells, R-roscovitine, a specific cdks inhibitor, increased pS256 and decreased pS261. The changes in AQP2 phosphorylation were paralleled by an increase in cell surface AQP2 expression and osmotic water permeability in the absence of forskolin stimulation. Of note, R-roscovitine didn’t alter cAMP-dependent PKA activity. Because phosphorylation results from the balance between kinase and phosphatase activity, we evaluated the possible contribution of protein phosphatases PP1, PP2A and PP2B. Of these, R-roscovitine treatment specifically reduced PP2A protein expression and activity in MDCK cells. Interestingly, in PKD1+/- mice displaying a syndrome of inappropriate antidiuresis with high level of pS256 despite unchanged AVP and cAMP, we found a reduced PP2A expression and activity and reduced pS261. Similarly to what previously found in PKD1+/- mice, R-roscovitine treatment caused a significant decrease in intracellular calcium in MDCK cells. Our data indicate that a reduced activity of PP2A, secondary to reduced intracellular Ca2+ levels, promotes AQP2 trafficking independently of the AVP-PKA axis. This pathway may be relevant for explaining pathological states characterized by inappropriate AVP secretion and positive water balance

Haemocompatible polymer surfaces obtained by bonding of thrombin inhibitors

The covalent bonding of thrombin inhibitors to surfaces of vinyl alcohol copolymers is described. As inhibitors p-amino benzamidine and dansyl arginine were used. The haemocompatibility of these surfaces was evaluated by in vitro tests (PTT and APTT)

Differential modulation of intracellular Ca2+ responses associated with calcium-sensing receptor activation in renal collecting duct cells

In this work, we studied G protein-coupled Extracellular Calcium Sensing Receptor (CaR) signaling in mouse cortical collecting duct cells (MCD4) expressing endogenous CaR. Intracellular [Ca2+] measurements performed with real time video imaging revealed that CaR stimulation with 5mM Ca2+, 300 mu M Gd3+ and with 10 mu M of specific allosteric modulator NPS-R 568, all resulted in an increase in [Ca2+](i) although displaying different features. Specifically, Ca2+ as well as stimulation with NPS-R 568 induced a rapid peak of [Ca2+](i) while stimulation with Gd3+ induced transient intracellular Ca2+ oscillations. PLC inhibition completely abolished any [Ca2+](i) increase after stimulation with CaR agonists. Inhibition of Rho or Rho kinase (ROK) abolished [Ca2+](i) oscillations induced by Gd3+, while the peak induced by high Ca2+ was similar to control. Conversely, emptying the intracellular calcium stores abolished the response to Gd3+. On the other hand, the inhibition of calcium influx did not alter calcium changes. We conclude that in our cell model, CaR stimulation with distinct agonists activates two distinct transduction pathways, both PLC-dependent. The transient cytosolic Ca2+ oscillations produced by Gd3+ are modulated by Rho-Rho kinase signaling, whereas the rapid peak of intracellular Ca2+ in response to 5mM [Ca2+](o) is mainly due to PLC/IP3 pathway activation

Proinflammatory cytokine production in HaCaT cells treated by eosin: implications for the topical treatment of psoriasis

Psoriasis is a multifactorial skin dermatosis characterized in its classical form by erythematous and hyperkeratotic plaques on extensor surfaces of the body, that in most cases can be managed therapeutically by topical agents. Hyperproliferation and a marked inflammation in both epidermis and dermis are thought to be driven by interaction of activated type-1 T lymphocytes and antigen-presenting cells and keratinocytes that release several proinflammatory and immunomodulating molecules. The aim of this study is to investigate whether tetrabromofluorecin, commonly know as eosin, a classical compound traditionally topically used in psoriasis for its presumed anti-inflammatory activities, is able to modulate the production of TNF-alpha, IL-6 and IL-8 that are recognized as the most active and characterized cytokines in the pathogenesis of this skin disorder. HaCaT cell line was used to verify the effects on epidermal inflammation by eosin at scalar doses after testing the viability of cells. Two different population of cells, one stimulated by IFNgamma and one non-stimulated, were cultivated in presence of tolerable concentrations. The expression and release of IL-6, IL-8, IL-10, and TNF-alpha were analysed by RT-PCR and ELISA, respectively. Our results show that tolerable concentrations of eosin were 0.05%, 0.02%, and 0.01%. The expression and production of TNFalpha, IL-8 and IL-6 were dramatically reduced in presence of eosin 0.05% and 0.02% and the action of eosin was more pronounced on TNF-alpha. In agreement with clinical data, our results show that in presence of tolerable concentrations, eosin seems to influence remarkably the production of three important cytokines involved in the hyperproliferation and inflammatory process, giving a specific explanation of its efficacy and supporting its topical use in the clinical setting