Microtubule S-glutathionylation as a potential approach for antimitotic agents.
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.
Zhejiang Cancer Research Institute, Zhejiang Cancer Hospital, Hangzhou, Zhejiang 310022, China.
Wei
W
Chen
Teresa
T
Seefeldt
Alan
A
Young
Xiaoying
X
Zhang
Yong
Y
Zhao
John
J
Ruffolo
Radhey S
RS
Kaushik
Xiangming
X
Guan
D050256:Antimitotic Agents; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D005434:Flow Cytometry; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000737:chemistry* / Q000378:metabolism; D011422:Propionates / Q000494:pharmacology
C526052:2-amino-3-(4-aminophenyl)propionic acid; D050256:Antimitotic Agents; D011422:Propionates; D005978:Glutathione