Investigation of the effect of nocodazole on the PI3K/Akt signaling pathway in COS7 cells

Shi,Huaiping; Wu,Wujun; Luo,Jun

doi:10.3892/mmr.2014.2632

Investigation of the effect of nocodazole on the PI3K/Akt signaling pathway in COS7 cells

Authors:
- Huaiping Shi
- Wujun Wu
- Jun Luo
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Affiliations: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, P.R. China, Department of Hepatobiliary Surgery, Shaanxi Provincial People's Hospital, Xi'an City, Shaanxi 710068, P.R. China
Published online on: October 14, 2014 https://doi.org/10.3892/mmr.2014.2632
Pages: 3233-3240

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Abstract

A number of studies have demonstrated that nocodazole suppresses Akt phosphorylation; however, the underlying molecular mechanism remains unclear. In the current study, the mechanism of nocodazole‑induced suppression of Akt phosphorylation was investigated. The cell cycle was analyzed using flow cytometry and EGF receptor dimerization was evaluated through a cross-linking assay. Immunoprecipitation experiments were performed to investigate the interaction between P85 and EGFR and cell migration was evaluated through a wound healing assay. COS7 cells were observed to be rounded following a 24‑h treatment with nocodazole, and the results revealed that ~45% of COS7 cells were arrested at the G2/M phase and that the cyclin B1 expression level was greatly increased. EGF‑mediated Akt phosphorylation was markedly inhibited in nocodazole‑treated cells. In addition, the levels of internalized EGF‑EGFR complexes in nocodazole‑treated cells were reduced, and EGF‑EGFR dimerization was found to be affected by nocodazole. Akt phosphorylation in COS7 cells was demonstrated to be overridden by AG1478 and wortmannin. The results also showed that p85 did not bind to activated EGFR in nocodazole‑treated cells, and that nocodazole and protein inhibitors reduced cell migration. In summary, these results indicate that nocodazole inhibits the PI3K/Akt pathway by interfering with the binding of p85 binding to activated EGFR and further affects the growth of cells.

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