Isoflurane increases adenosine generation in cultured endothelial cells.

<p>Adenosine in media from human umbilical vein endothelial cells (EA.hy926, A) or mouse glomerular endothelial cells (GENC, B) measured with high pressure liquid chromatography. Isoflurane treatment (0–2.5%) for 6 hr increased adenosine concentrations when compared to carrier gas-treated controls (N = 5). Data are presented as means ± SEM. *P<0.05 vs. carrier gas-treated controls. Error bars represent 1 SEM.</p

Isoflurane releases CD73 containing microparticles in cultured endothelial cells.

<p>A. Human umbilical vein endothelial (EA.hy926) cells were treated with 0–2.5% isoflurane for 1 hr and endothelial cell culture media microparticles (MP) were isolated and assayed for CD73 activity. Isoflurane caused a significant increase in human endothelial cell microparticle CD73 activity (N = 5–8). B. Microparticles isolated from mouse glomerular endothelial cells (GENC) treated with 2.5% isoflurane for 1 hr also had higher CD73 activity compared to carrier gas-treated cells (N = 4–6). C and D. Representative CD73 immunoblotting images (C) and band intensity quantifications (D) from microparticles isolated from EA.hy926 cells. Beta-actin protein expression was also quantified to normalize lane loading. Isoflurane treatment (2.5% for 1 hr) significantly increased CD73 protein expression in EA.hy926 cell microparticles compared to carrier gas-treated cells. *P<0.05 vs. carrier gas group. Error bars represent 1 SEM.</p

Isoflurane transiently increases CD73 activity without changing CD73 synthesis in cultured endothelial cells.

<p>A. Human umbilical vein endothelial (EA.hy926) cells treated with 2.5% isoflurane showed a significant but transient induction of CD73 activity. CD73 activity peaked at 3 hr and then decreased to near baseline at 6–16 hr after isoflurane treatment (N = 6–8). B. Isoflurane treatment for 3 hr caused dose-dependent increase in CD73 activity in EA.hy926 cells compared to carrier gas-treated cells (N = 4–5). Data are presented as means ± SEM. *P<0.05 vs. CD73 activity measured at baseline (A) or in cells treated with 0% isoflurane (B). C and D. Representative images for CD73 mRNA (RT-PCR) and protein (immunoblotting) expression in EA.hy926 cells. EA.hy926 cells were treated with carrier gas or with 2.5% isoflurane for 6 hr (C) or for 16 hr (D). Isoflurane treatment did not increase CD73 mRNA or protein expression in EA.hy926 cells. Representative of 3–4 experiments.</p

Flow cytometric analyses of endothelial cell culture media microparticles.

<p>A. Representative flow cytometric analyses of microparticles isolated from EA.hy926 endothelial cell culture media. EA.hy926 cells were treated with 2.5% isoflurane or with carrier gas for 1 hr and isolated microparticles were incubated with CD73 antibody and Annexin V. B. EA.hy926 cells treated with 2.5% isoflurane for 1 hr had significantly higher CD73+ Annexin V+ microparticles compared to microparticles isolated from carrier gas-treated endothelial cells (N = 5). *P<0.05 vs. carrier gas group. Error bars represent 1 SEM.</p

Isoflurane stimulates Rho kinase activity in human endothelial cells.

<p>A. Rho kinase activity in human endothelial (EA.hy9262) cells was measured by detecting myosin phosphatase target protein-1 phosphorylation after treatment with 2.5% isoflurane or with carrier gas for 30 min. Isoflurane significantly increased Rho kinase activity in EA.hy9262 cells compared to carrier gas-treated cells (N = 5). *P<0.05 vs. carrier gas group. Error bars represent 1 SEM. B. EA.hy9262 endothelial Rho kinase activity was also assessed by detecting myosin light chain (MLC) phosphorylation in EA.hy9262 cells with immunoblotting. MLC phosphorylation increased in EA.hy9262 cells treated with 2.5% isoflurane for 30 min compared to carrier gas-treated cells. Total MLC immunoreactivity did not change with isoflurane treatment. Representative of 2 experiments performed in triplicate.</p

Primers used to amplify cDNAs based on published GenBank sequences for human.

<p>bp, base pairs; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ICAM-1, intercellular adhesion molecule-1; TNF-α, tumor necrosis factor-alpha; VCAM-1, vascular cell adhesion molecule-1; CD73, Ecto-5′-nucleotidase. Respective anticipated PCR product size (bp, base pairs), PCR cycle number for linear amplification, and annealing temperatures used for each primer are also provided.</p

GFRA1 promotes cisplatin-induced chemoresistance in osteosarcoma by inducing autophagy

<p>Recent progress in chemotherapy has significantly increased its efficacy, yet the development of chemoresistance remains a major drawback. In this study, we show that GFRA1/GFRα1 (GDNF family receptor α 1), contributes to cisplatin-induced chemoresistance by regulating autophagy in osteosarcoma. We demonstrate that cisplatin treatment induced GFRA1 expression in human osteosarcoma cells. Induction of GFRA1 expression reduced cisplatin-induced apoptotic cell death and it significantly increased osteosarcoma cell survival via autophagy. GFRA1 regulates AMPK-dependent autophagy by promoting SRC phosphorylation independent of proto-oncogene <i>RET</i> kinase. Cisplatin-resistant osteosarcoma cells showed NFKB1/NFκB-mediated GFRA1 expression. GFRA1 expression promoted tumor formation and growth in mouse xenograft models and inhibition of autophagy in a GFRA1-expressing xenograft mouse model during cisplatin treatment effectively reduced tumor growth and increased survival. In cisplatin-treated patients, treatment period and metastatic status were associated with GFRA1-mediated autophagy. These findings suggest that GFRA1-mediated autophagy is a promising novel target for overcoming cisplatin resistance in osteosarcoma.</p