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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

AZD2014 sensitized primary OSCC cells to radiation, which led to reduced clonogenic survival by inhibiting AKT/mTOR signaling and inducing cell death.Primary cells were established from three individual patients with OSCC, and were treated with AZD2014 alone, IR alone, or the two treatments in combination. (A) Typical image of colony growth for the different treatments are shown. (B)AKT/mTOR pathway apoptosis, autophagy, and DNA damage pathway-related proteins were analyzed by Western blotting. SD = standard deviation.
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pone.0151942.g006: AZD2014 sensitized primary OSCC cells to radiation, which led to reduced clonogenic survival by inhibiting AKT/mTOR signaling and inducing cell death.Primary cells were established from three individual patients with OSCC, and were treated with AZD2014 alone, IR alone, or the two treatments in combination. (A) Typical image of colony growth for the different treatments are shown. (B)AKT/mTOR pathway apoptosis, autophagy, and DNA damage pathway-related proteins were analyzed by Western blotting. SD = standard deviation.

Mentions: To confirm the biological effects of AZD2014, we isolated and expanded three primary tumor cells from OSCC patients and then treated the cells with AZD2014 with and without ionizing radiation. Our results indicated that combination treatment with AZD2014 and IR significantly reduced colony formation in all primary OSCC cells compared with RAD001 or RT alone (Fig 6A). To further explore the mechanisms of AKT/mTOR signaling and cell death we also found that AZD2014 treatment effectively inhibited phospho-AKT, phospho-mTOR, mTORC1 targets (p70S6K1 and 4E-BP1), and the regulatory associated protein mTORC2 (rictor). Combination treatment with AZD2014 and RT slightly increased the expression of the active caspase-3, although no significant differences in the expressions of BAX and BAK were observed. Moreover, the level of LC3 was found to be increased in combination of AZD2014 and IR, but no significant differences in γ-H2AX expression were found after AZD2014 treatment (Fig 6B). Thus, the induction of cell death and inhibition of the AKT/mTOR pathway contributed to the augmented growth-inhibitory effect induced by the combination treatment.


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)

AZD2014 sensitized primary OSCC cells to radiation, which led to reduced clonogenic survival by inhibiting AKT/mTOR signaling and inducing cell death.Primary cells were established from three individual patients with OSCC, and were treated with AZD2014 alone, IR alone, or the two treatments in combination. (A) Typical image of colony growth for the different treatments are shown. (B)AKT/mTOR pathway apoptosis, autophagy, and DNA damage pathway-related proteins were analyzed by Western blotting. SD = standard deviation.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0151942.g006: AZD2014 sensitized primary OSCC cells to radiation, which led to reduced clonogenic survival by inhibiting AKT/mTOR signaling and inducing cell death.Primary cells were established from three individual patients with OSCC, and were treated with AZD2014 alone, IR alone, or the two treatments in combination. (A) Typical image of colony growth for the different treatments are shown. (B)AKT/mTOR pathway apoptosis, autophagy, and DNA damage pathway-related proteins were analyzed by Western blotting. SD = standard deviation.
Mentions: To confirm the biological effects of AZD2014, we isolated and expanded three primary tumor cells from OSCC patients and then treated the cells with AZD2014 with and without ionizing radiation. Our results indicated that combination treatment with AZD2014 and IR significantly reduced colony formation in all primary OSCC cells compared with RAD001 or RT alone (Fig 6A). To further explore the mechanisms of AKT/mTOR signaling and cell death we also found that AZD2014 treatment effectively inhibited phospho-AKT, phospho-mTOR, mTORC1 targets (p70S6K1 and 4E-BP1), and the regulatory associated protein mTORC2 (rictor). Combination treatment with AZD2014 and RT slightly increased the expression of the active caspase-3, although no significant differences in the expressions of BAX and BAK were observed. Moreover, the level of LC3 was found to be increased in combination of AZD2014 and IR, but no significant differences in γ-H2AX expression were found after AZD2014 treatment (Fig 6B). Thus, the induction of cell death and inhibition of the AKT/mTOR pathway contributed to the augmented growth-inhibitory effect induced by the combination treatment.

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