ABSTRACT
LOVTRAP is an optogenetic approach for reversible light-induced protein dissociation using protein A fragments that bind to the LOV domain only in the dark, with tunable kinetics and a >150-fold change in the dissociation constant (Kd). By reversibly sequestering proteins at mitochondria, we precisely modulated the proteins' access to the cell edge, demonstrating a naturally occurring 3-mHz cell-edge oscillation driven by interactions of Vav2, Rac1, and PI3K proteins.
Subject(s)
Light , Optogenetics/methods , Phosphatidylinositol 3-Kinase/chemistry , Photoreceptors, Plant , Proto-Oncogene Proteins c-vav/chemistry , rac1 GTP-Binding Protein/chemistry , Avena/metabolism , HeLa Cells , Humans , Kinetics , Phosphatidylinositol 3-Kinase/genetics , Phosphatidylinositol 3-Kinase/radiation effects , Photoreceptors, Plant/chemistry , Photoreceptors, Plant/genetics , Photoreceptors, Plant/radiation effects , Protein Interaction Mapping , Protein Structure, Tertiary , Proto-Oncogene Proteins c-vav/genetics , Proto-Oncogene Proteins c-vav/radiation effects , Recombinant Fusion Proteins , rac1 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/radiation effectsABSTRACT
BACKGROUND: Mutations in key tumor suppressor genes such as tumor protein 53 (TP53) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) are the main genetic alterations in cancers. TP53 mutations have been found in most patients with non-small cell lung cancer (NSCLC), whereas PTEN mutations are rarely found in lung cancer, though most NSCLCs lack PTEN protein synthesis. However, the signaling involved in radio- and chemotherapy of NSCLC with wild-type PTEN and nonfunctional p53 is not clearly understood. METHODS: In this study, we established a xenograft tumor model with H358 NSCLC cells expressing wild-type PTEN, but nonfunctional p53. Protein expression and phosphorylation of PTEN and its downstream signal molecules in NSCLC tissues were detected by Western blot. RESULTS: We demonstrated that radiation and paclitaxel alone inhibited tumor growth, but a combined therapy of radiation and paclitaxel was more effective in inhibiting NSCLC tumor growth. Interestingly, both radiation and paclitaxel significantly increased PTEN protein expression and phosphorylation. Further identification of the affected PTEN downstream molecules showed that Akt phosphorylation at Ser(473) and Thr(308) residues was significantly decreased, whereas Bax and cleaved caspase-3 levels were significantly increased in tumor tissues treated with both radiation and paclitaxel. The combined treatment was more effective than either treatment alone in regulating the studied molecules. We also found that paclitaxel, but not radiation, inhibited phosphoinositide 3-kinase (PI3K) activity. CONCLUSIONS: Our study suggested that a PTEN-PI3K-Akt-Bax signaling cascade is involved in the therapeutic effect of combined radiation/paclitaxel treatment in NSCLC without p53 expression. Our study also suggested that PTEN is an ideal target in tumors with wild-type PTEN and a lack of functional p53.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms/genetics , PTEN Phosphohydrolase/genetics , Paclitaxel/pharmacology , Signal Transduction/genetics , Tumor Suppressor Protein p53/genetics , Animals , Antineoplastic Agents, Phytogenic/therapeutic use , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/therapy , Caspase 3/drug effects , Caspase 3/metabolism , Caspase 3/radiation effects , Chemoradiotherapy , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/therapy , Mice , Mice, Inbred BALB C , Mutation , Neoplasm Transplantation , Oncogene Protein v-akt/drug effects , Oncogene Protein v-akt/metabolism , Oncogene Protein v-akt/radiation effects , PTEN Phosphohydrolase/metabolism , Paclitaxel/therapeutic use , Phosphatidylinositol 3-Kinase/drug effects , Phosphatidylinositol 3-Kinase/metabolism , Phosphatidylinositol 3-Kinase/radiation effects , Phosphorylation/drug effects , Phosphorylation/radiation effects , bcl-2-Associated X Protein/drug effects , bcl-2-Associated X Protein/metabolism , bcl-2-Associated X Protein/radiation effectsABSTRACT
PURPOSE: To test the radiosensitising effects of a tumour-suppressor gene, phosphatase and tensin homologue deleted from chromosome 10 (PTEN), in hepatocellular carcinoma cells (HCC). MATERIALS AND METHODS: Radiation-induced wild-type PTEN or mutant PTEN was transfected into the SMMC-7721 human hepatocellular carcinoma cells. The expressions of PTEN and serine/threonine protein kinase (Akt) were detected by Western blot analysis. 3-(4,5)-dimethylthiahiazo-(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) absorbance and clonogenic survival assays were used to determine cell viability, proliferation and radiosensitivity. By performing cell-cycle analysis, terminal deoxynucleotidyltransferase (TdT)-mediated dUTP biotin nick end labelling (TUNEL) assays and gamma-histone H2A (γ-H2AX) formation assays, we were able to explore the mechanism of PTEN enhancement of radiosensitivity. RESULTS: Restoration of wild-type PTEN induced growth suppression and sensitised the cells to ionising radiation specifically by its lipid phosphatase activity through the PTEN-phosphatidylinositol 3-kinase (PI3K)-Akt signalling pathway. Restoring PTEN function correlated with G2/M arrest, apoptosis and the retardation of repair of radiation-induced double strand breaks (DSB). CONCLUSIONS: Our study suggests that strategies designed to restore the expression of PTEN may represent promising new therapies for sensitising HCC cells to ionising radiation.