Glargine and degludec: solution behaviour of higher dose synthetic insulins

Single, double and triple doses of the synthetic insulins glargine and degludec currently used in patient therapy are characterised using macromolecular hydrodynamic techniques (dynamic light scattering and analytical ultracentrifugation) in an attempt to provide the basis for improved personalised insulin profiling in patients with diabetes. Using dynamic light scattering and sedimentation velocity in the analytical ultracentrifuge glargine was shown to be primarily dimeric under solvent conditions used in current formulations whereas degludec behaved as a dihexamer with evidence of further association of the hexamers (“multi-hexamerisation”). Further analysis by sedimentation equilibrium showed that degludec exhibited reversible interaction between mono- and-di-hexamer forms. Unlike glargine, degludec showed strong thermodynamic non-ideality, but this was suppressed by the addition of salt. With such large injectable doses of synthetic insulins remaining in the physiological system for extended periods of time, in some case 24–40 hours, double and triple dose insulins may impact adversely on personalised insulin profiling in patients with diabetes

Receptor Tyrosine Kinases in Osteosarcoma: 2019 Update

The primary conclusions of our 2014 contribution to this series were as follows: Multiple receptor tyrosine kinases (RTKs) likely contribute to aggressive phenotypes in osteosarcoma and, therefore, inhibition of multiple RTKs is likely necessary for successful clinical outcomes. Inhibition of multiple RTKs may also be useful to overcome resistance to inhibitors of individual RTKs as well as resistance to conventional chemotherapies. Different combinations of RTKs are likely important in individual patients. AXL, EPHB2, FGFR2, IGF1R, and RET were identified as promising therapeutic targets by our in vitro phosphoproteomic/siRNA screen of 42 RTKs in the highly metastatic LM7 and 143B human osteosarcoma cell lines. This chapter is intended to provide an update on these topics as well as the large number of osteosarcoma clinical studies of inhibitors of multiple tyrosine kinases (multi-TKIs) that were recently published

Nuclear insulin-like growth factor-1 receptor (IGF1R) displays proliferative and regulatory activities in non-malignant cells.

The insulin-like growth factor-1 receptor (IGF1R) mediates the biological actions of IGF1 and IGF2. The IGF1R is involved in both physiological and pathological activities and is usually overexpressed in most types of cancer. In addition to its classical mechanism of action, recent evidence has shown a nuclear presence of IGF1R, associated with novel genomic/transcriptional types of activities. The present study was aimed at evaluating the hypothesis that nuclear IGF1R localization is not restricted to cancer cells and might constitute a novel physiologically relevant regulatory mechanism. Our data shows that nuclear translocation takes place in a wide array of cells, including normal diploid fibroblasts. In addition, we provide evidence for a synergistic effect of a nuclear translocation blocker along with selective IGF1R inhibitors in terms of decreasing cell proliferation. Given the important role of the IGF1R in mitogenesis, the present results may be of translational relevance in cancer research. In conclusion, results are consistent with the concept that nuclear IGF1R fulfills important physiological and pathological roles

Effects of dansylcadaverine on cell proliferation.

<p>(A) MCF7, MCF10A, M12 and P69 cells were treated with dansylcadaverine, with or without IGF1, for 24 hr, after which absorbance was measured. (B) Stable MCF7/IGF1R KO and empty vector-transfected (control) cells were treated with dansylcadaverine for 24 hr, after which absorbance was measured. (*) significantly different versus control. Results of a representative experiment repeated three times with similar results are shown. The inset depicts the basal IGF1R levels in IGF1R-silenced and control (empty vector-transfected) cells. Heat shock protein-70 (hsp70) was used as a loading control).</p

Confocal microscopy analysis of IGF1R nuclear localization in primary human fibroblasts.

<p>Fluorescence confocal microscope imaging of IGF1R in primary human fibroblasts. Cells were seeded in 24-well plates and after 24 hr were fixed and stained for IGF1R with a fluorescent donkey anti-rabbit antibody (green- 488) and DAPI (blue). Results of a representative experiment repeated two times with similar results are shown.</p

Western blot analysis of cytoplasmic and nuclear IGF1R in MCF10A cells.

<p>MCF10A cells were transfected with IGF1R siRNA (10 nM) or non-targeting (NT) siRNA and harvested 48 hr post transfection. (A) Western blot analysis of total IGF1R, Sumo-1, tubulin [a marker for the cytoplasmic <b>(C)</b> fraction] and SP1 [a marker for the nuclear <b>(N)</b> fraction]. (B) Densitometric analysis of Western blots. The results of a representative experiment repeated three times with similar results are shown. Analyses were done using the “ImageJ software”. Blots of the cytoplasmic fraction were exposed for short periods (2–5 min) whereas blots of the nuclear fraction were exposed for longer periods (10–15 min). Thus, it is not possible to compare between relative abundance of proteins between fractions.</p

Effects of dansylcadaverine on IGF1R nuclear translocation in MCF7, MCF10A, M12 and P69 cells.

<p>Cells were treated with dansylcadaverine for 24 hr, after which cell fractionation was performed as described in Materials and Methods. <b>(A)</b> Western blot analysis of total IGF1R, total IR and tubulin (as a control for the cytoplasmic fraction) and lamin B1 (as a control for the nuclear fraction) in MCF7 and MCF10A cells. Given that different exposure times were carried out for the cytoplasmic and nuclear fractions, it is not possible to compare expression levels between both fractions. <b>(B)</b> Quantitative analysis of the results. Analyses were done using “ImageJ” software. <b>(C)</b> Western blot analysis of total IGF1R, tubulin and lamin B1 in M12 and P69 cells. <b>(D)</b> Quantitative analysis of the results. Results of a representative experiment repeated three times with similar results are shown. The “-“and “+” symbols represent control and dansylcadaverine-treated cells, respectively.</p

Effects of dansylcadaverine and IGF1R inhibitors on cell proliferation of M12 cells.

<p>M12 cells were treated with dansylcadaverine, or with the selective IGF1R inhibitors AEW541 or AG1024, or both dansylcadaverine and IGF1R inhibitors for 24 hr, after which absorbance was measured. (*) significantly different versus control. Results of a representative experiment repeated three times with similar results are shown.</p

Confocal microscopy analysis of IGF1R nuclear localization.

<p>Confocal immunofluorescence microscopic imaging of IGF1R-expressing MCF10A <b>(A, B)</b> and MCF7 <b>(D, E)</b> cells. Cells were transfected with an IGF1R siRNA or NT siRNA for 48 h. <b>Effect of IGF1 treatment on IGF1R expression</b>. Serum-starved MCF10A <b>(C)</b> and MCF7 <b>(F)</b> cells were treated with IGF1 (50 ng/ml) for 7 hr, and IGF1R localization was evaluated by fluorescence imaging. Control experiment using only secondary antibody <b>(G).</b> Fixed cells were stained for IGF1R with a fluorescent donkey anti-rabbit antibody (green- 488) and DAPI (blue). Results of a representative experiment repeated three times with similar results are shown. Images <b>A-G</b> were photographed using x63NA1.4 amplification.</p