title: Dominant negative Rac1 attenuates paclitaxel-induced apoptosis in human melanoma cells through upregulation of heat shock protein 27: a functional proteomic analysis.
abstract: GTPase ras-related C3 botulinum toxin substrate 1 (Rac1) plays a role in various cellular processes pertinent to cancer development. In the present study, we investigated the molecular mechanisms underlying apoptosis regulation by Rac1 through functional proteomic analysis of three human melanoma M14 cell lines stably transfected with constitutively active Rac1V12, dominant negative Rac1N17, and empty vector (pIRES), respectively. We found that paclitaxel evoked apoptosis in the melanoma cell lines through the intrinsic (mitochondria) pathway in a caspsae-3-dependent manner. Compared to the Rac1pIRES and Rac1V12 cells, Rac1N17 cells were more resistant to paclitaxel-triggered caspase-3 activation and apoptosis. Protein composition comparisons amongst the three cell lines identified two peptide spots of interest. One was Hsp27, which was upregulated in Rac1N17 cells as assessed in our gel image interpretation, PMF and Western blot analysis. The other was identified as SR-25 protein (also known as the ADP-ribosylation factor-like factor 6-interacting protein 4; ARL6IP4) using PMF, which was separated only from the Rac1N17 cells under the experimental conditions. Moreover, knockdown of the protein level of Hsp27 using small interfering RNA in Rac1N17 cells significantly increased the paclitaxel-elicited caspase-3 activation and apoptosis. In conclusion, our results implicate that Hsp27 and SR-25 are mediators in Rac1 signaling pathway(s). It appears that the dominant negative Rac1N17 reduces the apoptosis sensitivity toward paclitaxel in the melanoma cells through upregulation of Hsp27, which inhibits its down stream drug-elicited caspase-3 activation.
authors.affiliation: National University Medical Institute, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. lee_shao@hotmail.com
authors.forename: Shao Chin
authors.initials: SC
authors.lastname: Lee
authors.forename: Nigel
authors.initials: N
authors.lastname: Sim
authors.forename: Marie-Veronique
authors.initials: MV
authors.lastname: Clement
authors.forename: Sanjiv Kumar
authors.initials: SK
authors.lastname: Yadav
authors.forename: Shazib
authors.initials: S
authors.lastname: Pervaiz
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000187:drug effects / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D005799:Genes, Dominant; D006360:Heat-Shock Proteins / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008545:Melanoma / Q000737:chemistry / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D017239:Paclitaxel / Q000494:pharmacology*; D010455:Peptides / Q000032:analysis; D040901:Proteomics; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000187:drug effects; D020830:rac1 GTP-Binding Protein / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*
chemical_list: D006360:Heat-Shock Proteins; D010455:Peptides; D053148:Caspase 3; D020830:rac1 GTP-Binding Protein; D017239:Paclitaxel