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AZD2014 Radiosensitizes Oral Squamous Cell Carcinoma by Inhibiting AKT/mTOR Axis and Inducing G1/G2/M Cell Cycle Arrest.

Yu CC, Huang HB, Hung SK, Liao HF, Lee CC, Lin HY, Li SC, Ho HC, Hung CL, Su YC - PLoS ONE (2016)

Bottom Line: To address this question, we investigated the effect of AZD2014, a novel small molecular ATP-competitive inhibitor of mTORC1 and mTORC2 kinase, as a radiosensitizer in primary OSCC and OSCC-derived cell line models.The radiosensitizing effect of AZD2014 were then assessed using cell viability assays, clonogenic survival assays, and cell cycle analyses.Combination treatment with AZD2014 and irradiation resulted in significant reduction in OSCC cell line and primary OSCC cell colony formation due to the enhanced inhibition of AKT and both mTORC1 and mTORC2 activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science and Institute of Molecular Biology, National Chung Cheng University, Chia-Yi, Taiwan, R.O.C.

ABSTRACT

Background: Oral squamous cell carcinoma (OSCC) is one of the most common malignant neoplasms in Taiwan. Activation of the mTOR signaling pathway has been linked to decreased radiation responsiveness in human oral cancer, thus it limits efficacy of radiotherapy. To address this question, we investigated the effect of AZD2014, a novel small molecular ATP-competitive inhibitor of mTORC1 and mTORC2 kinase, as a radiosensitizer in primary OSCC and OSCC-derived cell line models.

Methods: We isolated primary tumor cells from OSCC tissues and cell lines. AZD2014 was administered with and without ionizing radiation. The radiosensitizing effect of AZD2014 were then assessed using cell viability assays, clonogenic survival assays, and cell cycle analyses. Western blotting was used to detect protein expression.

Results: Combination treatment with AZD2014 and irradiation resulted in significant reduction in OSCC cell line and primary OSCC cell colony formation due to the enhanced inhibition of AKT and both mTORC1 and mTORC2 activity. Pre-treatment with AZD2014 in irradiated oral cancer cells induced tumor cell cycle arrest at the G1 and G2/M phases, which led to disruption of cyclin D1-CDK4 and cyclin B1-CDC2 complexes. Moreover, AZD2014 synergized with radiation to promote both apoptosis and autophagy by increasing caspase-3 and LC3 in primary OSCC cells.

Conclusions: These findings suggest that in irradiated OSCC cells, co-treatment with AZD2014, which targets mTORC1 and mTORC2 blockade, is an effective radiosensitizing strategy for oral squamous cell carcinoma.

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Related in: MedlinePlus

Expression of AKT and mTOR signaling pathway in OSCC cell lines.Cell lines were homogenized in lysate buffer as described in Materials and Methods. A 50 μM sample of protein was analyzed by Western blotting using specific antibodies against components of the AKT/mTOR pathway.
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pone.0151942.g001: Expression of AKT and mTOR signaling pathway in OSCC cell lines.Cell lines were homogenized in lysate buffer as described in Materials and Methods. A 50 μM sample of protein was analyzed by Western blotting using specific antibodies against components of the AKT/mTOR pathway.

Mentions: We first investigated AKT/mTOR signaling in OSCC-derived cell lines SCC4 and SCC25. Activation of mTOR can be reliably determined by phosphorylation of Ser2448, a site is that is phosphorylated by AKT. Activation of mTOR results in phosphorylation of two main downstream targets the ribosomal protein p70S6K and subsequent phosphorylation of the S6 ribosomal protein (p-S6). Another, eukaryotic translation initiation factor 4E binding protein1 (4EBP1) phosphorylated at Ser-65 corresponding to the hyperphosphorylated form, is regulated by mTORC1. Compared with the SCC4 cell line, activation of phospho-AKT at Ser473 was observed in SCC25 cells. High levels of phospho-mTOR at Ser2448 and phospho-4EBP1 at Ser65 were detected. Also phospho-S6 at Ser235/236 was not elevated in SCC25 cells. The mTORC2 adaptor protein, rictor displayed a significantly higher expression in the SCC25 cell line (Fig 1). The above results suggested the presence of highly activate mTOR signaling in OSCC cell lines.


AZD2014 Radiosensitizes Oral Squamous Cell Carcinoma by Inhibiting AKT/mTOR Axis and Inducing G1/G2/M Cell Cycle Arrest.

Yu CC, Huang HB, Hung SK, Liao HF, Lee CC, Lin HY, Li SC, Ho HC, Hung CL, Su YC - PLoS ONE (2016)

Expression of AKT and mTOR signaling pathway in OSCC cell lines.Cell lines were homogenized in lysate buffer as described in Materials and Methods. A 50 μM sample of protein was analyzed by Western blotting using specific antibodies against components of the AKT/mTOR pathway.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4816280&req=5

pone.0151942.g001: Expression of AKT and mTOR signaling pathway in OSCC cell lines.Cell lines were homogenized in lysate buffer as described in Materials and Methods. A 50 μM sample of protein was analyzed by Western blotting using specific antibodies against components of the AKT/mTOR pathway.
Mentions: We first investigated AKT/mTOR signaling in OSCC-derived cell lines SCC4 and SCC25. Activation of mTOR can be reliably determined by phosphorylation of Ser2448, a site is that is phosphorylated by AKT. Activation of mTOR results in phosphorylation of two main downstream targets the ribosomal protein p70S6K and subsequent phosphorylation of the S6 ribosomal protein (p-S6). Another, eukaryotic translation initiation factor 4E binding protein1 (4EBP1) phosphorylated at Ser-65 corresponding to the hyperphosphorylated form, is regulated by mTORC1. Compared with the SCC4 cell line, activation of phospho-AKT at Ser473 was observed in SCC25 cells. High levels of phospho-mTOR at Ser2448 and phospho-4EBP1 at Ser65 were detected. Also phospho-S6 at Ser235/236 was not elevated in SCC25 cells. The mTORC2 adaptor protein, rictor displayed a significantly higher expression in the SCC25 cell line (Fig 1). The above results suggested the presence of highly activate mTOR signaling in OSCC cell lines.

Bottom Line: To address this question, we investigated the effect of AZD2014, a novel small molecular ATP-competitive inhibitor of mTORC1 and mTORC2 kinase, as a radiosensitizer in primary OSCC and OSCC-derived cell line models.The radiosensitizing effect of AZD2014 were then assessed using cell viability assays, clonogenic survival assays, and cell cycle analyses.Combination treatment with AZD2014 and irradiation resulted in significant reduction in OSCC cell line and primary OSCC cell colony formation due to the enhanced inhibition of AKT and both mTORC1 and mTORC2 activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science and Institute of Molecular Biology, National Chung Cheng University, Chia-Yi, Taiwan, R.O.C.

ABSTRACT

Background: Oral squamous cell carcinoma (OSCC) is one of the most common malignant neoplasms in Taiwan. Activation of the mTOR signaling pathway has been linked to decreased radiation responsiveness in human oral cancer, thus it limits efficacy of radiotherapy. To address this question, we investigated the effect of AZD2014, a novel small molecular ATP-competitive inhibitor of mTORC1 and mTORC2 kinase, as a radiosensitizer in primary OSCC and OSCC-derived cell line models.

Methods: We isolated primary tumor cells from OSCC tissues and cell lines. AZD2014 was administered with and without ionizing radiation. The radiosensitizing effect of AZD2014 were then assessed using cell viability assays, clonogenic survival assays, and cell cycle analyses. Western blotting was used to detect protein expression.

Results: Combination treatment with AZD2014 and irradiation resulted in significant reduction in OSCC cell line and primary OSCC cell colony formation due to the enhanced inhibition of AKT and both mTORC1 and mTORC2 activity. Pre-treatment with AZD2014 in irradiated oral cancer cells induced tumor cell cycle arrest at the G1 and G2/M phases, which led to disruption of cyclin D1-CDK4 and cyclin B1-CDC2 complexes. Moreover, AZD2014 synergized with radiation to promote both apoptosis and autophagy by increasing caspase-3 and LC3 in primary OSCC cells.

Conclusions: These findings suggest that in irradiated OSCC cells, co-treatment with AZD2014, which targets mTORC1 and mTORC2 blockade, is an effective radiosensitizing strategy for oral squamous cell carcinoma.

Show MeSH
Related in: MedlinePlus