Activation of the PI3K-Akt pathway is known to induce tumor radioresistance. In the current study, we examined the ability of 17AAG, which decreases the levels of Hsp90 client proteins including components of the PI3K-Akt pathway, to sensitize radioresistant human squamous cell carcinoma cells to Xirradiation. Human squamous cell carcinoma cell lines (SQ20B, SCC61 and SCC13) were incubated for 16 h at 37°°°°C in medium containing 17AAG. Radiation sensitivity was determined by clonogenic assays, and protein levels were examined by western blotting. Apoptosis was determined in monolayer cells by AO/EB double staining and in spheroids using the TdT-mediated dUTP nick end labeling assay. 17AAG (0.2 µ µ µ µM) enhanced the radiosensitivity more effectively in radioresistant SQ20B and SCC13 cells than in radiosensitive SCC61 cells. However, in all three cell lines, 17AAG increased radiation-induced apoptosis by reducing the expression of EGFR and ErbB-2 and inhibiting the phosphorylation of Akt. Furthermore, 17AAG (1 µ µ µ µM) sensitized SQ20B spheroids to radiation, and inhibition of Akt activation by 17AAG increased radiation-induced apoptosis in spheroids. The findings suggest that 17AAG effectively sensitizes radioresistant cells to radiation by inhibiting the PI3K-Akt pathway. Targeting the PI3K-Akt pathway with 17AAG could be a useful strategy for radiosensitization of carcinomas. (Cancer Sci 2005; 96: 911-917) R adiation therapy is commonly used for squamous cell carcinoma of the head and neck because of its minimal cosmetic effects and its ability to preserve voice in laryngeal cancer. However, treatment failure correlates with a poorer prognosis and decreased patient survival.The EGFR family contributes to resistance to radiation and chemotherapy in many human tumors, including head and neck cancer.(1-14) ErbB-2, a member of the EGFR family, is also overexpressed in 30% of breast tumors as well as in a significant number of other cancers. (15,16) Blockade of the EGFRmediated signaling pathway enhances the sensitivity of tumor cells to radiation and several chemotherapeutic agents.The PI3K-Akt pathway is a major downstream pathway initiated by activation of the EGFR family members. (17)(18)(19)(20)(21) This pathway plays an important role in cell growth and survival. (22,23) The serine/threonine kinase Akt (also known as protein kinase B) is thought to be a downstream target of PI3K. Akt activation is responsible for desensitizing cells to apoptotic stimuli. This occurs by regulation of the transcriptional activity of both Forkhead family members (24,25) and NF-κB, (26,27) and through phosphorylation and inactivation of the apoptotic machinery including Bcl-2 homolog, Bad (28,29) and caspase-9.(30) Furthermore, Akt is amplified or overexpressed in a wide variety of human tumors.(31-33) PTEN, a tumor suppressor gene, is thought to negatively regulate the PI3K-Akt pathway.(34-36) Mutations causing PTEN to be functionally inactive are frequently detected in many human cancers and enhance Akt activity.(37) Akt acti...
Choline is essential for the synthesis of the major membrane phospholipid phosphatidylcholine and the neurotransmitter acetylcholine (ACh). Elevated levels of choline and up-regulated choline kinase activity have been detected in cancer cells. Thus, the intracellular accumulation of choline through choline transporters is the rate-limiting step in phospholipid metabolism and a prerequisite for cancer cell proliferation. However, the uptake system for choline and the functional expression of choline transporters in lung cancer cells are poorly understood. We examined the molecular and functional characterization of choline uptake in the small cell lung carcinoma cell line NCI-H69. Choline uptake was saturable and mediated by a single transport system. Interestingly, removal of Na(+) from the uptake buffer strongly enhanced choline uptake. This increase in choline uptake under the Na(+)-free conditions was inhibited by dimethylamiloride (DMA), a Na(+)/H(+) exchanger (NHE) inhibitor. Various organic cations and the choline analog hemicholinium-3 (HC-3) inhibited the choline uptake and cell viability. A correlation analysis of the potencies of organic cations for the inhibition of choline uptake and cell viability showed a strong correlation (R=0.8077). RT-PCR revealed that choline transporter-like protein 1 (CTL1) mRNA and NHE1 are mainly expressed. HC-3 and CTL1 siRNA inhibited choline uptake and cell viability, and increased caspase-3/7 activity. The conversion of choline to ACh was confirmed, and this conversion was enhanced under Na(+)-free conditions, which in turn was sensitive to HC-3. These results indicate that choline uptake through CTL1 is used for ACh synthesis. Both an acetylcholinesterase inhibitor (eserine) and a butyrylcholinesterase inhibitor (ethopropazine) increased cell proliferation, and these effects were inhibited by 4-DAMP, a mAChR3 antagonist. We conclude that NCI-H69 cells express the choline transporter CTL1 which uses a directed H(+) gradient as a driving force, and its transport functions in co-operation with NHE1. This system primarily supplies choline for the synthesis of ACh and secretes ACh to act as an autocrine/paracrine growth factor, and the functional inhibition of CTL1 could promote apoptotic cell death. Identification of this new CTL1-mediated choline transport system provides a potential new target for therapeutic intervention.
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