Combined EGFR and ROCK inhibition in TNBC leads to cell death via impaired autophagic flux

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with very limited therapeutic options. We have recently shown that the combined inhibition of EGFR and ROCK in TNBC cells results in cell death, however, the underlying mechanisms remain unclear. To investigate this, here we applied a mass spectrometry-based proteomic approach to identify proteins altered upon single and combination treatments. Our proteomic data revealed autophagy as the major molecular mechanism implicated in the cells' response to combinatorial treatment. In particular, we here show that EGFR inhibition by gefitinib treatment alone induces autophagy, a cellular recycling process that acts as a cytoprotective response for TNBC cells. However, combined inhibition of EGFR and ROCK leads to autophagy blockade and accumulation of autophagic vacuoles. Our data show impaired autophagosome clearance as a likely cause of antitumor activity. We propose that the inhibition of the autophagic flux upon combinatorial treatment is attributed to the major cytoskeletal changes induced upon ROCK inhibition, given the essential role the cytoskeleton plays throughout the various steps of the autophagy process

Determination and validation of aprepitant in rat plasma using LC−MS/MS

Aim: The assessment of efficacy should be paralleled with extensive pharmacokinetic parameters, and a valid bioanalytical method is a pre-condition for accurate plasma concentration. Materials &amp; methods: A simple, specific, rapid and sensitive LC−MS/MS method has been developed for quantitative analysis of aprepitant in rat plasma. A C18 column was used as stationary phase and the mobile phase consisted of a mixture of formic acid in water and formic acid in acetonitrile. Quantification was performed using multiple reaction monitoring mode. Results: The selectivity, linearity, accuracy, precision, robustness and ruggedness of the method were evaluated in accordance with bioanalytical method validation guideline of ICH and all results were within the acceptable range. Conclusion: The validated LC−MS/MS method was found to be useful for the quantitative analysis of aprepitant in rat plasma samples. </jats:p

Combined EGFR and ROCK inhibition in TNBC leads to cell death via impaired autophagic flux

AbstractTriple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with very limited therapeutic options. We have recently shown that the combined inhibition of EGFR and ROCK in TNBC cells results in cell death, however, the underlying mechanisms remain unclear. To investigate this, here we applied a mass spectrometry-based proteomic approach to identify proteins altered upon single and combination treatments. Our proteomic data revealed autophagy as the major molecular mechanism implicated in the cells’ response to combinatorial treatment. In particular, we here show that EGFR inhibition by gefitinib treatment alone induces autophagy, a cellular recycling process that acts as a cytoprotective response for TNBC cells. However, combined inhibition of EGFR and ROCK leads to autophagy blockade and accumulation of autophagic vacuoles. Our data show impaired autophagosome clearance as a cause of antitumor activity. We propose that the inhibition of the autophagic flux upon combinatorial treatment is attributed to the major cytoskeletal changes induced upon ROCK inhibition, given the essential role the cytoskeleton plays throughout the various steps of the autophagy process.</jats:p

Determination and validation of aprepitant in rat plasma using LC-MS/MS.

Aim: The assessment of efficacy should be paralleled with extensive pharmacokinetic parameters, and a valid bioanalytical method is a pre-condition for accurate plasma concentration. Materials & methods: A simple, specific, rapid and sensitive LC-MS/MS method has been developed for quantitative analysis of aprepitant in rat plasma. A C18 column was used as stationary phase and the mobile phase consisted of a mixture of formic acid in water and formic acid in acetonitrile. Quantification was performed using multiple reaction monitoring mode. Results: The selectivity, linearity, accuracy, precision, robustness and ruggedness of the method were evaluated in accordance with bioanalytical method validation guideline of ICH and all results were within the acceptable range. Conclusion: The validated LC-MS/MS method was found to be useful for the quantitative analysis of aprepitant in rat plasma samples