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Open Access Research article

Microtubule S-glutathionylation as a potential approach for antimitotic agents

Wei Chen1, Teresa Seefeldt2, Alan Young3, Xiaoying Zhang4, Yong Zhao5, John Ruffolo6, Radhey S Kaushik36 and Xiangming Guan2*

Author Affiliations

1 Zhejiang Cancer Research Institute, Zhejiang Cancer Hospital, Hangzhou, Zhejiang, 310022, China

2 Department of Pharmaceutical Sciences, South Dakota State University, Brookings, SD 57007, USA

3 Department of Veterinary and Biomedical Sciences, South Dakota State University, Brookings, SD, 57007, USA

4 ACEA Bio CO., Ltd., Hangzhou, Zhejiang, 310030, China

5 Department of Physiology, Michigan State University, East Lansing, MI, 48824, USA

6 Department of Biology and Microbiology, South Dakota State University, Brookings, SD, 57007, USA

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BMC Cancer 2012, 12:245  doi:10.1186/1471-2407-12-245

Published: 15 June 2012

Abstract

Background

Microtubules have been one of the most effective targets for the development of anticancer agents. Cancer cells treated by these agents are characterized by cell arrest at G2/M phase. Microtubule-targeting drugs are, therefore, referred to as antimitotic agents. However, the clinical application of the current antimitotic drugs is hampered by emerging drug resistance which is the major cause of cancer treatment failure. The clinical success of antimitotic drugs and emerging drug resistance has prompted a search for new antimitotic agents, especially those with novel mechanisms of action. The aim of this study was to determine whether microtubules can be S-glutathionylated in cancer cells and whether the glutathionylation will lead to microtubule dysfunction and cell growth inhibition. The study will determine whether microtubule S-glutathionylation can be a novel approach for antimitotic agents.

Methods

2-Acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino)phenyl carbamoylsulfanyl]propionic acid (2-AAPA) was used as a tool to induce microtubule S-glutathionylation. UACC-62 cells, a human melanoma cell line, were used as a cancer cell model. A pull-down assay with glutathione S-transferase (GST)-agarose beads followed by Western blot analysis was employed to confirm microtubule S-glutathionylation. Immunofluorescence microscopy using a mouse monoclonal anti-α-tubulin-FITC was used to study the effect of the S-glutathionylation on microtubule function; mainly polymerization and depolymerization. Flow cytometry was employed to examine the effect of the S-glutathionylation on cell cycle distribution and apoptosis. Cell morphological change was followed through the use of a Zeiss AXIO Observer A1 microscope. Cancer cell growth inhibition by 2-AAPA was investigated with ten human cancer cell lines.

Results

Our investigation demonstrated that cell morphology was changed and microtubules were S-glutathionylated in the presence of 2-AAPA in UACC-62 cells. Accordingly, microtubules were found depolymerized and cells were arrested at G2/M phase. The affected cells were found to undergo apoptosis. Cancer growth inhibition experiments demonstrated that the concentrations of 2-AAPA required to produce the effects on microtubules were compatible to the concentrations producing cancer cell growth inhibition.

Conclusions

The data from this investigation confirms that microtubule S-glutathionylation leads to microtubule dysfunction and cell growth inhibition and can be a novel approach for developing antimitotic agents.