Profiling of apoptotic changes in human breast cancer cells using SELDI-TOF mass spectrometry.
Apoptosis is a key process in the response of tumours to chemotherapeutic agents. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many tumor cells, while sparing most normal cells. Several chemotherapeutic drugs synergize with TRAIL in reducing tumor growth and inducing apoptosis. Because some tumour cells respond poorly to these treatments, biomarkers that predict clinical responsiveness are needed. This study used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to identify novel apoptotic markers in TRAIL and etoposide (T+E)-treated MDA-MB-231 and ZR-75-1 breast cancer cells and MCF-10A non-transformed breast cells. T+E induced apoptosis, increasing caspase-3 activity at 4-8h, in all cell lines. Protein profiles revealed two prominent peaks, m/z 10090 and 8560, which decreased significantly during apoptosis. Mass spectrometry sequencing of tryptic peptides identified these proteins as S100A6 (confirmed immunologically) and ubiquitin (confirmed against a purified standard), respectively. Caspase inhibition prevented the decrease in both proteins during T+E-induced apoptosis whereas proteasome inhibition combined with T+E further decreased ubiquitin, possibly by preventing its recycling. Using SELDI-TOF MS we have identified S100A6 and ubiquitin as potential protein markers of apoptosis. Further validation using patient samples is required to confirm their potential utility in monitoring the effectiveness of anti-cancer drugs in inducing tumour cell apoptosis.
Leong
Sharon
S
Kolling Institute of Medical Research, The University of Sydney, Royal North Shore Hospital, Sydney, Australia. sleong@med.usyd.edu.au
Christopherson
Richard I
RI
Baxter
Robert C
RC
D017209:Apoptosis / Q000187:drug effects; D015415:Biomarkers; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology*; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism; D045744:Cell Line, Tumor; D003720:Densitometry; D005047:Etoposide / Q000494:pharmacology; D006801:Humans; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D011485:Protein Binding; D040901:Proteomics; D000075383:S100 Calcium Binding Protein A6; D009418:S100 Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000494:pharmacology; D013997:Time Factors
D015415:Biomarkers; D061945:Caspase Inhibitors; D018797:Cell Cycle Proteins; D011480:Protease Inhibitors; D061988:Proteasome Inhibitors; D000075383:S100 Calcium Binding Protein A6; D009418:S100 Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C055351:S100A6 protein, human; D005047:Etoposide; D020169:Caspases; D046988:Proteasome Endopeptidase Complex