Insulin-like growth factor-I receptor inhibition by specific tyrosine kinase inhibitor NVP-AEW541 in endometroid and serous papillary endometrial cancer cell lines

Endometrial cancer is the most widespread gynecological cancer in Western countries and constitutes a major public health issue. The role of the insulin-like growth factor (IGF) system in endometrial biology as well as in endometrial cancer has been well established. The IGF-I receptor (IGF-IR) emerged in recent years as a promising therapeutic target in a number of cancers. NVP-AEW541 (Novartis Pharma) is a pyrrolo(2,3-d)pyrimidine derivative with specific IGF-IR tyrosine kinase inhibitory activity. NVP-AEW541 has been shown to specifically abrogate IGF-I-mediated IGF-IR autophosphorylation and to reduce activation of the IGF-IR downstream signaling pathways. The aim of the present study was to investigate the potential anti-proliferative activities of NVP-AEW541 in Type I (endometrioid) and Type II (uterine serous papillary endometrial carcinoma, USPC) endometrial cancer cell lines. Results obtained showed that NVP-AEW541 abolished the IGF-I stimulated IGF-IR phosphorylation in all of the cell lines investigated (ECC-1, Ishikawa, USPC-1, USPC-2), whereas it abolished AKT and ERK phosphorylation in ECC-1 and USPC-1 cells. Furthermore, the inhibitor prevented from IGF-I from exerting its antiapoptotic effect in ECC-1, USPC-1 and USPC-2 cells. In addition, proliferation assays showed that NVP-AEW541 caused a significant decrease in proliferation rate in all of the cell lines. NVP-AEW541 had no major effect on the insulin receptor. In summary, our results suggest that specific IGF-IR inhibition by NVP-AEW541 is a promising therapeutic tool in endometrial cancer

The health and economic impact of switching vaccines in universal varicella vaccination programs using a dynamic transmission model: An Israel case study

Currently available health economic models for varicella infection are designed to inform the cost-effectiveness of universal varicella vaccination (UVV) compared with no vaccination. However, in countries with an existing UVV program, these models cannot be used to evaluate whether to continue with the current varicella vaccine or to switch to an alternative vaccine. We developed a dynamic transmission model that incorporates the historical vaccination program to project the health and economic impact of changing vaccination strategies. We applied the model to Israel, which initiated UVV in 2008 with a quadrivalent vaccine, MMRV-GSK, and switched to MMRV-MSD in 2016. The model was calibrated to pre-vaccination incidence data before projecting the impact of the historical and future alternative vaccination strategies on the clinical burden of varicella. Total costs and QALYs lost due to varicella infections were projected to compare continuing with MMRV-MSD versus switching to MMRV-GSK in 2022. Over a 50-year time horizon, continuing with MMRV-MSD reduced varicella incidence further by 64%, reaching 35 cases per 100,000 population by 2072, versus a 136% increase in incidence with MMRV-GSK. Continuing with MMRV-MSD reduced cumulative hospitalization and outpatient cases by 48% and 58% (vs. increase of 137% and 91% with MMRV-GSK), respectively. Continuing with MMRV-MSD resulted in 139 fewer QALYs lost with total cost savings of 3% compared with switching to MMRV-GSK, from the societal perspective. In Israel, maintaining the UVV strategy with MMRV-MSD versus switching to MMRV-GSK is projected to further reduce the burden of varicella and cost less from the societal perspective

Effect of metformin on the cell cycle in USC.

<p>USPC-2 and USPC-1 cells were seeded in quadruplicate dishes, serum-starved for 24 h, and treated with metformin (or left untreated, controls) for 72 h. Cell cycle distribution was assessed by FACS analysis. The values in the table denote mean ± SEM;</p>*<p>p<0.05 versus control cells.</p

Scanning densitometry analysis of the effect of metformin on pTen, p53 and specific cell cycle regulatory proteins.

<p>Optical density was expressed as pTen, p53, cyclin D1, p21, Ras, Rb and E2F1 values normalized to the corresponding actin levels. A value of 100% was given to the optical density of IGF-I treated cells. The table shows the result of a typical experiment, repeated three times with similar results.</p><p>−, untreated cells; I, IGF-I-treated cells; M, metformin-treated cells; M+I, metformin and IGF-I-treated cells.</p

Effect of metformin on cell migration.

<p>Wounds were made on monolayers of USPC-2 (A) and USPC-1 (B) cells grown to 100% confluence. Cells were then incubated in serum-free media containing IGF-I (50 ng/ml), metformin (10 mM), or both, for 48, 72 and 96 h (USPC-2) and for 48 and 72 h (USPC-1). Treated or untreated (control) cells were photographed just after scratch (time 0), and after 48, 72 and 96 h. Results presented here are representative of triplicate independent samples of each cell line. The rate of migration was measured by quantifying the total distance that the cells (as indicated by rulers) moved from the edge of the scratch toward the centre of the scratch. A value of 100% was given to the wound area at time 0. The migration of IGF-I and/or metformin treated samples was compared to wound area at time 0.</p

Scanning densitometry analysis of the effect of metformin on IGF-I-stimulated IGF-IR, AKT and ERK1/2 phosphorylation.

<p>Optical density was expressed as pIGF-IR, pAKT and pERK values normalized to the corresponding total proteins. A value of 100% was given to the optical density of IGF-I treated cells. The table shows the result of a typical experiment, repeated three times with similar results.</p><p>−, untreated cells; I, IGF-I-treated cells; M, metformin-treated cells; M+I, metformin and IGF-I-treated cells.</p

Effect of metformin on GSK3ß and Foxo1 expression.

<p>A, Western blot of pGSK3ß and GSK3ß in USPC-2 and USPC-1 cells treated with metformin for 24 h and/or IGF-I. The figure shows the results of a typical experiment repeated three times. B, Western blot analysis of Foxo1 on USPC-2 and USPC-1 cells treated for 24 h with metformin and/or IGF-I. The figure shows the results of a characteristic experiment, repeated three times with similar results.</p

Effect of metformin on IGF-I-mediated signal transduction and mTOR and Ampk signalling pathway in endometrial cancer cells.

<p>A, Ishikawa, ECC-1, USPC-2 and USPC-1 cells were treated with metformin (10 mM) for 24 h (or left untreated) in the presence or absence of IGF-I (50 ng/ml) during the last 10 min of the incubation period. Whole cell lysates (100 µg) were resolved by SDS-PAGE and immunoblotted with antibodies against pIGF-IR, TIGF-IR, IR, pAKT, TAKT, pERK1/2, TERK1/2 and actin, followed by incubation with an HRP-conjugated secondary antibody. The figure shows the results of a typical experiment, repeated three times with similar results. B, USPC-2 and USPC-1 cell lines were treated with metformin for 24 h (or left untreated) and/or IGF-I during the last 10 min of the incubation. Whole cell lysates (100 µg) were resolved by SDS-PAGE and immunoblotted with antibodies against pmTOR, TmTOR, pAmpk, TAmpk, and p85. The figure shows the results of a typical experiment, repeated three times with similar results.</p

Effect of metformin on proliferation and cell cycle regulatory proteins in USC cells.

<p>Cells were plated in 24-well plates at a density of 5×10⁴ cells/well for USPC-2 (A) and 3.6×10⁴ cells/well for USPC-1 (B). Cells were incubated in the absence (open bars) or presence (solid bars) of metformin, and proliferation was evaluated at 24, 48 and 72 h by MTT measurements. A value of 100% was given to the cell number at time 0. The bars represent the mean ± S.E.M. of three independent experiments, performed each in triplicate samples; *p<0.05 versus untreated cells. C, Western blot analysis of cyclin D1, p21, Ras, Rb and E2F1 in USPC-2 and USPC-1 cells treated with metformin for 24 h in the absence or presence of IGF-I. Whole-cell lysates (100 µg) were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Results are representative of three independent experiments.</p