Microtubule S-glutathionylation as a potential approach for antimitotic agents

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.

Figure 1

Chemical structure of 2-AAPA.

Scheme 1

Protein S-glutathionylation.

Figure 2

2-AAPA induces protein S-glutationylation in UACC-62 cells. UACC-62 cells were treated with 50 μM 2-AAPA for different time periods and protein S-glutathionylation was determined by the HPLC method described by Chen et al. [16]. The results were presented as the mean ± SD of triplet determination of one set of samples. No protein S-glutathionylation was detected in untreated cells. Control 1 and control 2 were conducted at 5 min and 4 h respectively. No S-glutathionylation was detected in both control 1 and control 2.

Figure 3

2-AAPA induces tubulin S-glutathionylation. UACC-62 cells were treated with 100 μM 2-AAPA for 20 min and S-glutathionylated proteins were captured by a pull-down assay using GST-agarose beads followed by Western blot analysis. α- and β-Tubulins were detected as described in the method.

Figure 4

2-AAPA induces microtubule depolymerization in UACC-62 cells. UACC-62 cells were treated with the indicated concentrations of 2-AAPA for 12 h (a), or with 50 μM 2-AAPA for indicated time periods (b), followed by fixation, permeabilization and indirect immunofluorescent analysis with an anti-α-tubulin-FITC. Nuclei were stained with DAPI. Paclitaxel and vinblastine were employed as positive controls for microtubule stabilization and microtubule depolymerization respectively. The data are derived from one of the three independent experiments.

Figure 5

Effect of 2-AAPA on cell morphological changes in UACC-62 cells. UACC-62 cells were treated with 50 μM 2-AAPA. Some of the cells undergoing morphological changes are marked by red arrows.

Figure 6

Effect of 2-AAPA on cell cycle distribution in UACC-62 cells. The histograms of UACC-62 cells treated with different concentrations of 2-AAPA for 24 h and 48 h are presented (a). The bar presentations reflects the quantification of cell cycle distribution at 24 h (b) and 48 h (c) respectively. Results are presented as the mean ± SD of three independent experiments. *, P < 0.05 indicates statistical significance in 2-AAPA treated groups as compared to the control.

Figure 7

Effect of 2-AAPA on the induction of apoptosis in UACC-62 cells. UACC-62 cells were treated with different concentrations of 2-AAPA for 12 h and 24 h and stained with Annexin V and PI. Apoptotic cells were analyzed by flow cytometry (a). The percentages of apoptotic cells are presented for 12 h (b) and 24 h (c) treatment. Results are presented as the mean ± SD of three independent experiments. *, P < 0.05 indicates statistical significance in 2-AAPA treated groups as compared to the control.