Background:The objectives of this phase I study were to assess the safety and tolerability of E7080 in patients with advanced, refractory solid tumours; to determine the maximum tolerated dose (MTD) and pharmacokinetics profile of E7080; and to explore preliminary evidence of its anti-tumour efficacy.Methods:E7080 was administered orally in escalating doses on a once-daily continuous schedule in 28-day cycles to eligible patients. Samples for pharmacokinetic analyses were collected on days 1, 8, 15 and 22 of cycle 1 and day 1 of cycle 2. Anti-tumour efficacy was assessed every two cycles.Results:Eighty-two patients received E7080 in dose cohorts from 0.2 to 32 mg. Dose-limiting toxicities were grade 3 proteinuria (two patients) at 32 mg, and the MTD was defined as 25 mg. The most frequently observed cumulative toxicities (all grades) were hypertension (40% of patients), diarrhoea (45%), nausea (37%), stomatitis (32%) and vomiting (23%). Seven patients (9%) had a partial response and 38 patients (46%) had stable disease as best response. E7080 has dose-linear kinetics with no drug accumulation after 4 weeks' administration.Conclusion:E7080 is well tolerated at doses up to 25 mg per day. Encouraging anti-tumour efficacy was observed in patients with melanoma and renal cell carcinoma.

SummaryFormins are actin polymerization factors that are known to nucleate and elongate actin filaments at the barbed end. In the present study we show that human FHOD1 lacks actin nucleation and elongation capacity, but acts as an actin bundling factor with capping activity toward the filament barbed end. Constitutively active FHOD1 associates with actin filaments in filopodia and lamellipodia at the leading edge, where it moves with the actin retrograde flow. At the base of lamellipodia, FHOD1 is enriched in nascent, bundled actin arcs as well as in more mature stress fibers. This function requires actin-binding domains located N-terminally to the canonical FH1-FH2 element. The bundling phenotype is maintained in the presence of tropomyosin, confirmed by electron microscopy showing assembly of 5 to 10 actin filaments into parallel, closely spaced filament bundles. Taken together, our data suggest a model in which FHOD1 stabilizes actin filaments by protecting barbed ends from depolymerization with its dimeric FH2 domain, whereas the region N-terminal to the FH1 domain mediates F-actin bundling by simultaneously binding to the sides of adjacent F-actin filaments.

Inherited cardiomyopathies are caused by point mutations in sarcomeric gene products, including α-cardiac muscle actin (ACTC1). We examined the biochemical and cell biological properties of the α-cardiac actin mutations Y166C and M305L identified in hypertrophic cardiomyopathy (HCM). Untagged wild-type (WT) cardiac actin, and the Y166C and M305L mutants were expressed by the baculovirus/Sf9-cell system and affinity purified by immobilized gelsolin G4-6. Their correct folding was verified by a number of assays. The mutant actins also displayed a disturbed intrinsic ATPase activity and an altered polymerization behavior in the presence of tropomyosin, gelsolin, and Arp2/3 complex. Both mutants stimulated the cardiac β-myosin ATPase to only 50 % of WT cardiac F-actin. Copolymers of WT and increasing amounts of the mutant actins led to a reduced stimulation of the myosin ATPase. Transfection of established cell lines revealed incorporation of EGFP- and hemagglutinin (HA)-tagged WT and both mutant actins into cytoplasmic stress fibers. Adenoviral vectors of HA-tagged WT and Y166C actin were successfully used to infect adult and neonatal rat cardiomyocytes (NRCs). The expressed HA-tagged actins were incorporated into the minus-ends of NRC thin filaments, demonstrating the ability to form hybrid thin filaments with endogenous actin. In NRCs, the Y166C mutant led after 72 h to a shortening of the sarcomere length when compared to NRCs infected with WT actin. Thus our data demonstrate that a mutant actin can be integrated into cardiomyocyte thin filaments and by its reduced mode of myosin interaction might be the basis for the initiation of HCM.

Opioid use disorder is classified as a chronic recurrent disease of the central nervous system (CNS) which leads to personality disorders, co-morbidities and premature death. It develops as a result of long-term administration of various abused substances, along with morphine. The pharmacological action of morphine is associated with its stimulation of opioid receptors. Opioid receptors are a group of G protein-coupled receptors and activation of these receptors by ligands induces significant molecular changes inside the cell, such as an inhibition of adenylate cyclase activity, activation of potassium channels and reductions of calcium conductance. Recent data indicate that other signalling pathways also may be involved in morphine activity. Among these are phospholipase C, mitogen-activated kinases (MAP kinases) or β-arrestin. The present review focuses on major mechanisms which currently are considered as essential in morphine activity and dependence and may be important for further studies.

The dynamic reorganization of the actin cytoskeleton is regulated by a number of actin binding proteins (ABPs). Four human colon adenocarcinoma cell lines – parental and three selected sublines, which differ in motility and metastatic potential, were used to investigate the expression level and subcellular localization of selected ABPs. Our interest was focused on cofilin and ezrin. These proteins are essential for cell migration and adhesion. The data received for the three more motile adenocarcinoma sublines (EB3, 3LNLN, 5W) were compared with those obtained for the parental LS180 adenocarcinoma cells and fibroblastic NRK cells. Quantitative densitometric analysis and confocal fluorescence microscopy were used to examine the expression levels and subcellular distribution of the selected ABPs. Our data show distinct increase in the level of cofilin in adenocarcinoma cells accompanied by the reduction of inactive phosphorylated form of cofilin. In more motile cells, cofilin was accumulated at cellular periphery in co-localization with actin filaments. Furthemore, we indicated translocation of ezrin towards the cell periphery within more motile cells in comparison with NRK and parental adenocarcinoma cells.In summary, our data indicate the correlation between migration ability of selected human colon adenocarcinoma sublines and subcellular distribution as well as the level of cofilin and ezrin. Therefore these proteins might be essential for the higher migratory activity of invasive tumor cells.

Non-muscle actins have been studied for many decades; however, the reason for the existence of both isoforms is still unclear. Here we show, for the first time, a successful inactivation of the ACTB (CRISPR clones with inactivated ACTB, CR-ACTB) and ACTG1 (CRISPR clones with inactivated ACTG1, CR-ACTG1) genes in human melanoma cells (A375) via the RNA-guided D10A mutated Cas9 nuclease gene editing [CRISPR/Cas9(D10A)] technique. This approach allowed us to evaluate how melanoma cell motility was impacted by the lack of either β actin coded by ACTB or γ actin coded by ACTG1. First, we observed different distributions of β and γ actin in the cells, and the absence of one actin isoform was compensated for via increased expression of the other isoform. Moreover, we noted that γ actin knockout had more severe consequences on cell migration and invasion than β actin knockout. Next, we observed that the formation rate of bundled stress fibers in CR-ACTG1 cells was increased, but lamellipodial activity in these cells was impaired, compared to controls. Finally, we discovered that the formation rate of focal adhesions (FAs) and, subsequently, FA-dependent signaling were altered in both the CR-ACTB and CR-ACTG1 clones; however, a more detrimental effect was observed for γ actin-deficient cells. Our research shows that both non-muscle actins play distinctive roles in melanoma cells’ FA formation and motility.

Thymosin β4 (Tβ4), a multifunctional 44-amino acid polypeptide and a member of actin-binding proteins (ABPs), plays an important role in developmental processes and wound healing. In recent years an increasing number of data has been published suggesting Tβ4's involvement in tumorigenesis. However, Tβ4's role in melanoma tumor development still remains to be elucidated. In our study we demonstrate that Tβ4 is crucial for melanoma adhesion and invasion. For the purpose of our research we tested melanoma cell lines differing in invasive potential. Moreover, we applied shRNAs to silence TMSB4X (gene encoding Tβ4) expression in a cell line with high TMSB4X expression. We found out that Tβ4 is not only a component of focal adhesions (FAs) and interacts with several FAs components but also regulates FAs formation. We demonstrate that Tβ4 level has an impact on FAs' number and morphology. Moreover, manipulation with TMSB4X expression resulted in changes in cells' motility on non-coated and Matrigel TM (resembling basement membrane composition)-coated surfaces and drastically decreased invasion abilities of the cells. Additionally, a correlation between Tβ4 expression level and exhibition of mesenchymal-like [epithelialmesenchymal transition (EMT)] features was discovered. Cells with lowered TMSB4X expression were less EMT-progressed than control cells. Summarizing, obtained results show that Tβ4 by regulating melanoma cells' adhesion has an impact on motility features and EMT. Our study not only contributes to a better understanding of the processes underlying melanoma cells' capacity to create metastases but also highlights Tβ4 as a potential target for melanoma management therapy.

The understanding of melanoma malignancy mechanisms is essential for patient survival, because melanoma is responsible for ca. 75% of deaths related to skin cancers. Enhanced formation of invadopodia and extracellular matrix (ECM) degradation are two important drivers of cell invasion, and actin dynamics facilitate protrusive activity by providing a driving force to push through the ECM. We focused on the influence of epidermal growth factor (EGF), hepatocyte growth factor (HGF) and transforming growth factor β (TGFβ) on melanoma cell invasiveness, since they are observed in the melanoma microenvironment. All three factors stimulated invasion of A375 and WM1341D cells derived from primary tumor sites. In contrast, only EGF and HGF stimulated invasion of WM9 and Hs294T cells isolated from lymph node metastasis. Enhanced formation of invadopodia and ECM degradation underlie the increased amount of invasive cells after stimulation with the tested agents. Generally, a rise in invasive potential was accompanied by a decrease in actin polymerization state (F:G ratio). The F:G ratio remained unchanged or was even increased in cell lines from a metastasis treated with TGFβ. Our findings indicate that the effects of stimulation with EGF, HGF and TGFβ on melanoma cell invasiveness could depend on melanoma cell progression stage.