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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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AZD2014 or IR alone and in combination treatment induced cell cycle arrest by altering the cell cycle checkpoints in OSCC cell lines.SCC4 and SCC25 cells were treated with AZD2014 alone, IR alone, and the two treatments in combination for 48 hours. Cell cycle profiles were determined using the NucleoCounter NC-3000. (A) Shows the percentage of cells in G1, S, and G2/M phases were quantified and results expressed at the mean ± SD of three independent experiments performed in triplicate. *p < 0.05 compared to the IR alone. (B) The cells were treated with AZD2014, IR alone, and in combination for 48 hours. Cell lysates were prepared and immunoblotted for cyclin D1, CDK4, cyclinB1 and CDC2 and actin as a loading control. SD = standard deviation.
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pone.0151942.g005: AZD2014 or IR alone and in combination treatment induced cell cycle arrest by altering the cell cycle checkpoints in OSCC cell lines.SCC4 and SCC25 cells were treated with AZD2014 alone, IR alone, and the two treatments in combination for 48 hours. Cell cycle profiles were determined using the NucleoCounter NC-3000. (A) Shows the percentage of cells in G1, S, and G2/M phases were quantified and results expressed at the mean ± SD of three independent experiments performed in triplicate. *p < 0.05 compared to the IR alone. (B) The cells were treated with AZD2014, IR alone, and in combination for 48 hours. Cell lysates were prepared and immunoblotted for cyclin D1, CDK4, cyclinB1 and CDC2 and actin as a loading control. SD = standard deviation.

Mentions: The mTOR pathway positively controls cell cycle progression and cell proliferation by regulating S6K1 and the eukaryotic translation initiation factor, 4EBP1[14]. As radiosensitization by inhibition of mTOR signaling has been shown to cause cell cycle arrest [11,12], we assessed AZD2014-induced radiosensitization in SCC4 and SCC25 cells. A higher proportion of the cell population was detected in the G1 phase after treatment with AZD2014 as compared with radiation alone. Otherwise, we found that the proportion of G2/M phase cells increased in IR-treated cells. SCC4 and SCC25 cells pre-treated with AZD2014 and then irradiated showed significantly increased proportions at the G1 stage and a G2/M delay (Fig 5A). To understand the mechanisms by which AZD2014 combined with radiation induces G1 and G2/M phase arrest to inhibit cell growth, we tested the likelihood of changes in expression levels of diverse regulators of the cell cycle particularly associated with G1 checkpoints such as cyclin D1, and CDK4 [15]. In the presence of AZD2014, cyclin D1 and CDK4 were significantly decreased, but there were no obvious changes in the control and IR alone. In contrast, levels of cyclin B1 and CDC2, which are proteins also associated with the G2/M transition [16], treatment with IR reduced cyclin B1 and CDC2 expression, but did not alter cyclin B1 levels altered by treatment with AZD2014 (Fig 5B). Taken together, we concluded that the cytostatic effect of AZD2014 combined with ionizing radiation exhibits an inhibitory additive effect on the progression of cells through inhibits cyclin D1-CDK4 and cyclin B1-CDC2 kinase activation, leading to G1 and G2/M arrest.


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 or IR alone and in combination treatment induced cell cycle arrest by altering the cell cycle checkpoints in OSCC cell lines.SCC4 and SCC25 cells were treated with AZD2014 alone, IR alone, and the two treatments in combination for 48 hours. Cell cycle profiles were determined using the NucleoCounter NC-3000. (A) Shows the percentage of cells in G1, S, and G2/M phases were quantified and results expressed at the mean ± SD of three independent experiments performed in triplicate. *p < 0.05 compared to the IR alone. (B) The cells were treated with AZD2014, IR alone, and in combination for 48 hours. Cell lysates were prepared and immunoblotted for cyclin D1, CDK4, cyclinB1 and CDC2 and actin as a loading control. SD = standard deviation.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0151942.g005: AZD2014 or IR alone and in combination treatment induced cell cycle arrest by altering the cell cycle checkpoints in OSCC cell lines.SCC4 and SCC25 cells were treated with AZD2014 alone, IR alone, and the two treatments in combination for 48 hours. Cell cycle profiles were determined using the NucleoCounter NC-3000. (A) Shows the percentage of cells in G1, S, and G2/M phases were quantified and results expressed at the mean ± SD of three independent experiments performed in triplicate. *p < 0.05 compared to the IR alone. (B) The cells were treated with AZD2014, IR alone, and in combination for 48 hours. Cell lysates were prepared and immunoblotted for cyclin D1, CDK4, cyclinB1 and CDC2 and actin as a loading control. SD = standard deviation.
Mentions: The mTOR pathway positively controls cell cycle progression and cell proliferation by regulating S6K1 and the eukaryotic translation initiation factor, 4EBP1[14]. As radiosensitization by inhibition of mTOR signaling has been shown to cause cell cycle arrest [11,12], we assessed AZD2014-induced radiosensitization in SCC4 and SCC25 cells. A higher proportion of the cell population was detected in the G1 phase after treatment with AZD2014 as compared with radiation alone. Otherwise, we found that the proportion of G2/M phase cells increased in IR-treated cells. SCC4 and SCC25 cells pre-treated with AZD2014 and then irradiated showed significantly increased proportions at the G1 stage and a G2/M delay (Fig 5A). To understand the mechanisms by which AZD2014 combined with radiation induces G1 and G2/M phase arrest to inhibit cell growth, we tested the likelihood of changes in expression levels of diverse regulators of the cell cycle particularly associated with G1 checkpoints such as cyclin D1, and CDK4 [15]. In the presence of AZD2014, cyclin D1 and CDK4 were significantly decreased, but there were no obvious changes in the control and IR alone. In contrast, levels of cyclin B1 and CDC2, which are proteins also associated with the G2/M transition [16], treatment with IR reduced cyclin B1 and CDC2 expression, but did not alter cyclin B1 levels altered by treatment with AZD2014 (Fig 5B). Taken together, we concluded that the cytostatic effect of AZD2014 combined with ionizing radiation exhibits an inhibitory additive effect on the progression of cells through inhibits cyclin D1-CDK4 and cyclin B1-CDC2 kinase activation, leading to G1 and G2/M arrest.

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