em A /em , HUVEC had been starved overnight and were treated with 50 nmol/L BEZ235 for 1 h before irradiation (4 Gy)
em A /em , HUVEC had been starved overnight and were treated with 50 nmol/L BEZ235 for 1 h before irradiation (4 Gy). left for 17 h upon and the medium was replaced. Cells were transferred to normoxia at 1 h post-irradiation. em B /em , clonogenic survival of cells after irradiation with 6 Gy and treatment with 50 nmol/L BEZ235 in oxic and hypoxic (0.5% O2) conditions, as described above (A) and in Figure ?Figure1.1. *, em P /em 0.05; **, em P /em 0.01; *, em P /em 0.001 over DMSO-treated control. 1748-717X-7-48-S2.PPT (341K) GUID:?6414A5B1-BCC2-48EB-827B-528F50FCAC7D Additional file 3 Figure S3 Time-course of H2AX foci in irradiated tumor cells treatedwith BEZ235. FaDu and SQ20B cells were exposed to 50 nmol/L BEZ235 for 1 h followed Docosapentaenoic acid 22n-3 by irradiation with 4 Gy. Drugs were left up to a maximum of 24 h. Residual H2AX foci were counted at the indicated time points. *, em P /em 0.05; **, em P /em 0.01; ***, em P /em 0.001 over DMSO-treated control. 1748-717X-7-48-S3.PPTX (112K) GUID:?56F5A4B4-7EE2-469B-9F53-4F7D34BE3C31 Abstract Background The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is activated in tumor cells and promotes tumor cell survival after radiation-induced DNA damage. Because the pathway may not be completely inhibited after blockade of PI3K itself, due to feedback through mammalian target of rapamycin (mTOR), more effective inhibition might be expected by targeting both PI3K and mTOR inhibition. Materials and methods We investigated the effect of two dual PI3K/mTOR (both mTORC1 and mTORC2) inhibitors, NVP-BEZ235 and NVP-BGT226, on SQ20B laryngeal and FaDu hypopharyngeal cancer cells characterised by EGFR overexpression, on T24 bladder tumor cell lines with H-Ras mutation and on endothelial cells. Analysis of target protein phosphorylation, clonogenic survival, number of residual H2AX foci, cell cycle and apoptosis after radiation was performed in both tumor and endothelial cells. In vitro angiogenesis assays were conducted as well. Results Both compounds effectively inhibited phosphorylation of Akt, mTOR and S6 target proteins and reduced clonogenic survival in irradiated tumor cells. Persistence of DNA damage, as evidenced by increased number of H2AX foci, was detected after irradiation in the presence of PI3K/mTOR inhibition, together with enhanced G2 cell cycle delay. Docosapentaenoic acid 22n-3 Treatment with one of the inhibitors, NVP-BEZ235, also resulted in decreased clonogenicity after irradiation of tumor cells under hypoxic conditions. In addition, NVP-BEZ235 blocked VEGF- and IR-induced Akt phosphorylation and increased radiation killing in human umbilical venous endothelial cells (HUVEC) and human dermal microvascular dermal cells (HDMVC). NVP-BEZ235 inhibited VEGF-induced cell migration and capillary tube formation in vitro and enhanced the antivascular effect of irradiation. Treatment with NVP-BEZ235 moderately increased apoptosis in SQ20B and HUVEC cells but not in FaDu cells, and increased necrosis in both tumor and endothelial all cells tumor. Conclusions The results of this study demonstrate that PI3K/mTOR inhibitors can enhance radiation-induced killing in tumor and endothelial cells and may be of benefit when combined with radiotherapy. strong class=”kwd-title” Keywords: PI3K, mTOR, Radiosensitization, Endothelial cells, VEGF Background Radiotherapy is one of the most important modalities for the management of cancer. However, despite progress in radiation technology and significant gains achieved with the use of combined radio-chemotherapy, there is a substantial proportion of patients that fail to achieve long-term control [1]. The latter provides a strong rationale for combining molecular targets with radiation to improve patient outcome. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway controls tumor cell proliferation, growth, and survival after DNA damage [2]. Activation of this pathway is frequent in many cancers and can occur through diverse mechanisms such as amplification of the epidermal growth factor receptor (EGFR) gene, mutations of the Ras oncogene, PI3K mutations and loss of phosphatase and tensin homologue deleted in chromosome 10 (PTEN) [1-3]. This pathway consists of EGFR/Ras/PI3K/Akt and is a prime target for inhibition in the context of radiotherapy [4-6]. We and others have previously shown that inhibition of the EGFR/Ras/PI3K/Akt pathway can increase susceptibility to radiation-induced tumor killing [3,7-11]. Inhibition of Ras, PI3 kinase and Akt reduce tumor clonogenic survival after radiation at clinically Rabbit Polyclonal to BAX relevant doses [3-5,7,10,12]. A phase III randomized clinical trial evaluated the addition of cetuximab, an EGFR inhibitor, to radiotherapy and demonstrated improved overall survival in the combined modality arm over radiation alone [13]. The kinase mTOR consists of TORC1 and TORC2, two functionally distinct multiprotein complexes [14]. TORC1 includes mTOR and raptor (regulatory-associated protein of mTOR). TORC2 is composed of mTOR and rictor (rapamycin-insensitive companion of TOR) and regulates the activity of Akt [14]. mTOR inhibitors have radiosensitising potential in tumor and vascular cells [15,16]. Inhibition of TORC1 activity alone can result in TORC2-mediated.Different drug-radiation schedules were tested (see “Results”). H2AX foci in irradiated tumor cells treatedwith BEZ235. FaDu and SQ20B cells were exposed to 50 nmol/L BEZ235 for 1 h followed by irradiation with 4 Gy. Drugs were left up to a maximum of 24 h. Residual H2AX foci were counted at the indicated time points. *, em P /em 0.05; **, em P /em 0.01; ***, em P /em 0.001 over DMSO-treated control. 1748-717X-7-48-S3.PPTX (112K) GUID:?56F5A4B4-7EE2-469B-9F53-4F7D34BE3C31 Abstract Background The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is activated in tumor cells and promotes tumor cell survival after radiation-induced DNA damage. Because the pathway may not be completely inhibited after blockade of Docosapentaenoic acid 22n-3 PI3K itself, due to feedback through mammalian target of rapamycin (mTOR), more effective inhibition might be expected by targeting both PI3K and mTOR inhibition. Materials and methods We investigated the effect of two dual PI3K/mTOR (both mTORC1 and mTORC2) inhibitors, NVP-BEZ235 and NVP-BGT226, on SQ20B laryngeal and FaDu hypopharyngeal cancer cells characterised by EGFR overexpression, on T24 bladder tumor cell lines with H-Ras mutation and on endothelial cells. Analysis of target protein phosphorylation, clonogenic survival, number of residual H2AX foci, cell cycle and apoptosis after radiation was performed in both tumor and endothelial cells. In vitro angiogenesis assays were conducted as well. Results Both compounds effectively inhibited phosphorylation of Akt, mTOR and S6 target proteins and reduced clonogenic survival in irradiated tumor cells. Persistence of DNA damage, as evidenced by increased number of H2AX foci, was detected after irradiation in the presence of PI3K/mTOR inhibition, together with enhanced G2 cell cycle delay. Treatment with one of the inhibitors, NVP-BEZ235, also resulted in decreased clonogenicity after irradiation of tumor cells under hypoxic conditions. In addition, NVP-BEZ235 blocked VEGF- and IR-induced Akt phosphorylation and increased radiation killing in human umbilical venous endothelial cells (HUVEC) and human dermal microvascular dermal cells (HDMVC). NVP-BEZ235 inhibited VEGF-induced cell migration and capillary tube formation in vitro and enhanced the antivascular effect of irradiation. Treatment with NVP-BEZ235 moderately increased apoptosis in SQ20B and HUVEC cells but not in FaDu cells, and increased necrosis in both tumor and endothelial all cells tumor. Conclusions The results of this study demonstrate that PI3K/mTOR inhibitors can enhance radiation-induced killing in tumor and endothelial cells and may be of benefit when combined with radiotherapy. strong class=”kwd-title” Keywords: PI3K, mTOR, Radiosensitization, Endothelial cells, VEGF Background Radiotherapy is one of the most important modalities for the management of cancer. However, despite progress in radiation technology and significant gains achieved with the use of combined radio-chemotherapy, there is a substantial proportion of patients that fail to achieve long-term control [1]. The latter provides a strong rationale for combining molecular targets with radiation to improve patient outcome. The phosphatidylinositol Docosapentaenoic acid 22n-3 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway controls tumor cell proliferation, growth, and survival after DNA damage [2]. Activation of this pathway is frequent in many cancers and can occur through diverse mechanisms such as amplification of the epidermal growth factor receptor (EGFR) gene, mutations of the Ras oncogene, PI3K mutations and loss of phosphatase and tensin homologue deleted in chromosome 10 (PTEN) [1-3]. This pathway consists of EGFR/Ras/PI3K/Akt and is a prime target for inhibition in the context of radiotherapy [4-6]. We and others have previously shown that inhibition of the EGFR/Ras/PI3K/Akt pathway can increase susceptibility to radiation-induced tumor killing [3,7-11]. Inhibition of Ras, PI3 kinase and Akt reduce tumor clonogenic survival after radiation at clinically relevant doses [3-5,7,10,12]. A phase III randomized clinical trial evaluated the addition of cetuximab, an EGFR inhibitor, to radiotherapy and demonstrated improved overall survival in the combined modality arm over radiation alone [13]. The kinase mTOR consists of TORC1 and TORC2,.