STAT3 differential scanning fluorimetry and differential scanning light scattering assays: Addressing a missing link in the characterization of STAT3 inhibitor interactions

STAT3 protein is an established target for the development of new cancer therapeutic agents. Despite lacking a traditional binding site for small molecule inhibitors, many STAT3 inhibitors have been identified and explored for their anti-cancer activity. Because STAT3 signaling is mediated by protein-protein interactions, indirect methods are often employed to determine if proposed STAT3 inhibitors bind to STAT3 protein. While established STAT3 inhibition assays (such as the fluorescence polarization assay, electrophoretic mobility shift assay and ELISAs) have been used to identify novel inhibitors of STAT3 signaling, methods that directly assess STAT3 protein-inhibitor interactions could facilitate the development of novel inhibitors. In this context, we herein report new STAT3 binding assays based on differential scanning fluorimetry (DSF) and differential scanning light scattering (DSLS) to characterize interactions between STAT3 protein and inhibitors. Several peptide and small molecule STAT3 inhibitors have been evaluated, and new insight into how these compounds may interact with STAT3 is provided

Microstructural and mechanical characterisation of laser-welded high-carbon and stainless steel

The final publication is available at Springer via http://dx.doi.org/10.1007/s00170-015-7111-5Laser welding is becoming an important joining technique for welding of stainless steel to carbon steel and is extensively used across various sectors, including aerospace, transportation, power plants, electronics and other industries. However, welding of stainless steel to high-carbon steel is still at its early stage, predominantly due to the formation of hard brittle phases, which undermine the mechanical strength of the joint. This study reports a scientific investigation on controlling the brittle phase formation during laser dissimilar welding of high-carbon steel to stainless steel. Attempts have been made to tailor the microstructure and phase composition of the fusion zone through influencing the alloying composition and the cooling rate. Results show that the heat-affected zone (HAZ) within the high-carbon steel has significantly higher hardness than the weld area, which severely undermines the weld quality. To reduce the hardness of the HAZ, a new heat treatment strategy was proposed and evaluated using a finite element analysis-based numerical simulation model. A series of experiments has been performed to verify the developed thermo-metallurgical finite element analysis (FEA) model, and a qualitative agreement of predicted martensitic phase distribution is shown to exist

PKN1 modulates TGFß and EGF signaling in HEC-1-A endometrial cancer cell line

Sanaz Attarha,1,2 Ravi Kanth Rao Saini,3 Sonia Andersson,2 Miriam Mints,2 Serhiy Souchelnytskyi1,4,5 1Department of Oncology–Pathology, 2Department of Women's and Children's Health, Karolinska Institutet, Stockholm, 3Department of Biomedicine, Sahlgrenska Cancer Center, University of Gothenburg, Gothenburg, Sweden; 4OCD-AB, Uppsala, Sweden; 5Neurocentrum, Karolinska University Hospital, Solna, Sweden Background: The response of cells to TGFβ and EGF is mediated by a network of various intracellular regulators. The signaling crosstalk between different regulators is of key importance for tumorigenesis. The crosstalk may explain the modulation of cellular responses to the same regulator by another signaling molecule. As PKN1 – a serine/threonine kinase implicated in tumorigenesis – was identified as potential crosstalk node for TGFβ and EGF signaling, the cellular functions that may be affected by PKN1 in a crosstalk of TGFβ and EGF were explored. Methods: To investigate the contribution of PKN1 to TGFβ and EGF signaling, transiently PKN1-transfected HEC-1-A endometrial cancer cells were generated and subjected to treatment with TGFβ1, EGF, and their combination. Proliferation, apoptosis, invasion, wound healing, and migration assays were performed. The impact of PKN1 on the expression and phosphorylation of intracellular proteins was monitored by immunoblotting. Results: It was demonstrated that PKN1 modulated the responses of HEC-A-1 endometrial cancer cells to TGFβ1 and EGF. PKN1 had an inhibitory effect on the stimulation of cell migration, and PKN1 kinase activity was required for the inhibitory effect of TGFβ and EGF on cell proliferation and invasiveness. It was observed that phosphorylation of Smad2, FAK, and Erk1/2 correlated with responses of the cells to TGFβ1 and EGF. Conclusion: PKN1 modulates TGFβ- and EGF-dependent regulation of cell proliferation, migration, and invasiveness, and therefore is a component of the network signaling downstream of TGFβ and EGF. Keywords: PKN1 kinase, TGFβ, EGF, cell migration, proliferation, invasivenes

Irreversible TrxR1 inhibitors block STAT3 activity and induce cancer cell death

Because of its key role in cancer development and progression, STAT3 has become an attractive target for developing new cancer therapeutics. While several STAT3 inhibitors have progressed to advanced stages of development, their underlying biology and mechanisms of action are often more complex than would be expected from specific binding to STAT3. Here, we have identified and optimized a series of compounds that block STAT3-dependent luciferase expression with nanomolar potency. Unexpectedly, our lead compounds did not bind to cellular STAT3 but to another prominent anticancer drug target, TrxR1. We further identified that TrxR1 inhibition induced Prx2 and STAT3 oxidation, which subsequently blocked STAT3-dependent transcription. Moreover, previously identified inhibitors of STAT3 were also found to inhibit TrxR1, and likewise, established TrxR1 inhibitors block STAT3-dependent transcriptional activity. These results provide new insights into the complexities of STAT3 redox regulation while highlighting a novel mechanism to block aberrant STAT3 signaling in cancer cells