Response Profiling Using Shotgun Proteomics Enables Global Metallodrug Mechanisms of Action To Be Established

Abstract

Response profiling using shotgun proteomics for establishing global metallodrug mechanisms of action in two colon carcinoma cell lines, HCT116 and SW480, has been applied and evaluated with the clinically approved arsenic trioxide. Surprisingly, the complete established mechanism of action of arsenic trioxide was observed by protein regulations in SW480, but not HCT116 cells. Comparing the basal protein expression in the two cell lines revealed an 80 % convergence of protein identification, but with significant expression differences, which in turn seem to affect the extent of protein regulation. A clear-cut redox response was observed in SW480 cells upon treatment with arsenic, but hardly in HCT116 cells. Response profiling was then used to investigate four anti-cancer metallodrugs (KP46, KP772, KP1339 and KP1537). Proteome alterations were mapped to selected functional groups, including DNA repair, endocytosis, protection from oxidative stress, protection from endoplasmatic reticulum (ER) stress, cell adhesion and mitochondrial function. The present data suggest that knowledge of the mechanism of action of anti-cancer metallodrugs and improved patient stratification strategies are imperative for the design of clinical studies.

Time-dependent shotgun proteomics revealed distinct effects of an organoruthenium prodrug and its activation product on colon carcinoma cells

“…It was found that response profiling is applicable across diverse classes of metallodrugs, whose representatives induced specific protein regulation patterns, thus suggesting specific modes of action. 6 This method confirmed known and suggested unrecognized drug effects. Besides elucidating the modes of action of metallodrugs on a cellular level, target identification approaches emerged that frequently make use of MS-based techniques.…”

“…The cancer cells were expanded in T25 flasks for response profiling experiments and treated with half the IC 50 -concentrations of plecstatin-2 (15 mM) or arsenic trioxide (5 mM) for 3 h. The cells were fractionated into cytoplasmic (CYT) and nuclear (NE) fractions before digestion. 6 For the PCR experiments, the cells were analogously treated with plecstatin-2 or arsenic trioxide for 24 h. Three biological replicates were used for each condition.…”

“…1 RomeroCanelón and Sadler suggested the use of systems pharmacology approaches to assess metallodrug effects in 2015 2 and proteomics [3][4][5][6] or transcriptomics 7,8 approaches are being reported with increasing frequency. Such cell-level investigations are still challenging because metallodrugs are considered to be rather unspecific and the detailed modes of action are indeed elusive in most cases.…”

“…12 The former is preferentially employed to quantify metal isotopes in complex biological matrices, while the latter was initially used to study model systems and provides molecular information on the interaction products between metallodrugs and biomolecules. Recently, considerable efforts were undertaken to extend these techniques to the study of metallodrugs in the cellular context by applying MS-based shotgun proteomics, termed response profiling, 6 besides other techniques based on gel electrophoresis. [14][15][16][17] The main advantage of the MS-based shotgun approach relates to the online combination of peptide separation and analysis by LC-MS/MS allowing for the routine identification of five peptides per second and a total of 45000 proteins.…”

“…[14][15][16][17] The main advantage of the MS-based shotgun approach relates to the online combination of peptide separation and analysis by LC-MS/MS allowing for the routine identification of five peptides per second and a total of 45000 proteins. 6 Response profiling yields an indirect, but global assessment of effects on the cellular proteome upon drug treatment in living cells. It was found that response profiling is applicable across diverse classes of metallodrugs, whose representatives induced specific protein regulation patterns, thus suggesting specific modes of action.…”