px-478 and Carcinoma--Squamous-Cell

Purpose. To validate the function of autophagy with the regulation of hypoxia inhibitor-induced glycosylation in oral squamous cell carcinoma cell. Methods. Human Tca8113 cell line was used to detect autophagy and glycosylation related protein expression by western blotting and immunofluorescence with HIF-1α inhibitor. Short interfering RNA (siRNA) transfection blocked human ATG12 and ATG1. Results. HIF-1α inhibitor PX-478 reduced the amount of LC3-II and LC3-I in Tca8113 cells. PX-478 decreased the expression of O-GlcNAc and OGT and increased OGA expression. The tendency of O-GlcNAc showed a similar pattern to OGT. PX-478 gradually decreased OGT expression in Tca8113 cells. Protein level of O-GlcNAc and OGT increased in ATG12 and ATG1 depletion. The expression of OGT decreased at first and then rose slowly with the treatment of Atg12 and Atg1 siRNA and PX-478 fluctuant. Autophagy affected the stability of OGT when HIF-1α signaling was blocked. Conclusions. Autophagy reduced by hypoxic stress inhibited. HIF-1α inhibitor decreased glycosylation. OGT became unstable in the absence of autophagy when HIF-1α signaling was blocked.

Topics: Autophagy; Autophagy-Related Protein 12; Autophagy-Related Protein-1 Homolog; Carcinoma, Squamous Cell; Cell Line, Tumor; Enzyme Inhibitors; Glycosylation; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Intracellular Signaling Peptides and Proteins; Microtubule-Associated Proteins; Mouth Neoplasms; Mustard Compounds; N-Acetylglucosaminyltransferases; Phenylpropionates; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Small Ubiquitin-Related Modifier Proteins

Hypoxia inducible factor-1 (HIF-1) promotes tumor cell adaptation to microenvironmental stress. HIF-1 is up-regulated in irradiated tumors and serves as a promising target for radiosensitization. We initially confirmed that the orally bioavailable HIF-1 inhibitor PX-478 reduces HIF-1 protein levels and signaling in vitro in a dose-dependent manner and provides direct radiosensitization of hypoxic cancer cells in clonogenic survival assays using C6 glioma, HN5 and UMSCCa10 squamous cells, and Panc-1 pancreatic adenocarcinoma cell lines. However, PX-478 yields striking in vivo tumor sensitization to single-dose irradiation, which cannot be explained by incremental improvement in direct tumor cell killing. We show that PX-478 prevents postradiation HIF-1 signaling and abrogates downstream stromal adaptation in C6 and HN5 reporter xenografts as measured by serial ultrasound, vascular magnetic resonance imaging, and hypoxia response element-specific micro-positron emission tomography imaging. The primacy of indirect PX-478 in vivo effects was corroborated by our findings that (a) either concurrent or early postradiation sequencing of PX-478 provides roughly equivalent sensitization and (b) constitutive vascular endothelial growth factor expression maintains refractory tumor vessel function and progression following combined radiation and PX-478. These results confirm that disruption of postradiation adaptive HIF-1 signaling by PX-478 imparts increased therapeutic efficacy through blockade of HIF-1-dependent reconstitution of tumor stromal function. Successful translation of targeted HIF-1 radiosensitization to the clinical setting will require specific consideration of tumor microenvironmental effects and mechanisms.

Topics: Adenocarcinoma; Animals; Blotting, Western; Carcinoma, Squamous Cell; Cell Hypoxia; Enzyme-Linked Immunosorbent Assay; Glioma; Head and Neck Neoplasms; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoenzyme Techniques; Magnetic Resonance Imaging; Mice; Mice, Nude; Mustard Compounds; Pancreatic Neoplasms; Phenylpropionates; Positron-Emission Tomography; Stromal Cells; Tumor Cells, Cultured; Tumor Stem Cell Assay; Vascular Endothelial Growth Factor A; Whole-Body Irradiation