Identification of novel proteins with changed expression in resistant cancer cells could be helpful in elucidation mechanisms involved in the development of acquired resistance to paclitaxel. In this study, we carried out a 2D-PAGE using the mitochondrial-enriched fraction from paclitaxel-resistant MCF7/PacR cells compared to original paclitaxel-sensitive MCF7 breast cancer cells. Differentially expressed proteins were identified employing mass spectrometry. We found that lysosomal cathepsin D and mitochondrial abhydrolase-domain containing protein 11 (ABHD11) had decreased expression in MCF7/PacR cells. On the other hand, mitochondrial carbamoyl-phosphate synthetase 1 (CPS1) and ATPase family AAA-domain containing protein 3A and 3B (ATAD3A, ATAD3B) were overexpressed in MCF7/PacR cells. Further, we showed that there was no difference in localization of CPS1 in MCF7 and MCF7/PacR cells. We demonstrated a significant increase in the number of CPS1 positive MCF7/PacR cells, using FACS analysis, compared to the number of CPS1 positive MCF7 cells. Silencing of CPS1 expression by specific siRNA had no significant effect on the resistance of MCF7/PacR cells to paclitaxel. To summarize, we identified several novel proteins of a mitochondrial fraction whose role in acquired resistance to paclitaxel in breast cancer cells should be further assessed.

• Keywords
• ATPase family AAA-domain containing protein 3A and 3B (ATAD3A, 3B), abhydrolase-domain containing protein 11 (ABHD11), breast cancer cells, carbamoyl-phosphate synthetase 1 (CPS1), cathepsin D, mitochondria, paclitaxel resistance, two-dimensional electrophoresis,
• MeSH
• Drug Resistance, Neoplasm * drug effects   MeSH
• Cell Fractionation MeSH
• Carbamoyl-Phosphate Synthase (Ammonia) genetics   metabolism   MeSH
• Humans MeSH
• MCF-7 Cells MeSH
• Mitochondrial Proteins genetics   metabolism   MeSH
• Mitochondria genetics   metabolism   MeSH
• Breast Neoplasms drug therapy   genetics   metabolism   MeSH
• Paclitaxel pharmacology   MeSH
• Proteome MeSH
• Proteomics methods   MeSH
• Gene Expression Regulation, Neoplastic MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
• Tandem Mass Spectrometry MeSH
• Gene Silencing MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• CPS1 protein, human MeSH Browser
• Carbamoyl-Phosphate Synthase (Ammonia) MeSH
• Mitochondrial Proteins MeSH
• Paclitaxel MeSH
• Proteome MeSH

Wedelolactone is a multi-target natural plant coumestan exhibiting cytotoxicity towards cancer cells. Although several molecular targets of wedelolactone have been recognized, the molecular mechanism of its cytotoxicity has not yet been elucidated. In this study, we show that wedelolactone acts as an inhibitor of chymotrypsin-like, trypsin-like, and caspase-like activities of proteasome in breast cancer cells. The proteasome inhibitory effect of wedelolactone was documented by (i) reduced cleavage of fluorogenic proteasome substrates; (ii) accumulation of polyubiquitinated proteins and proteins with rapid turnover in tumor cells; and (iii) molecular docking of wedelolactone into the active sites of proteasome catalytic subunits. Inhibition of proteasome by wedelolactone was independent on its ability to induce reactive oxygen species production by redox cycling with copper ions, suggesting that wedelolactone acts as copper-independent proteasome inhibitor. We conclude that the cytotoxicity of wedelolactone to breast cancer cells is partially mediated by targeting proteasomal protein degradation pathway. Understanding the structural basis for inhibitory mode of wedelolactone might help to open up new avenues for design of novel compounds efficiently inhibiting cancer cells.

• Keywords
• breast cancer, copper, proteasome, reactive oxygen species, wedelolactone,
• MeSH
• Proteasome Inhibitors chemistry   pharmacology   toxicity   MeSH
• Coumarins chemistry   pharmacology   toxicity   MeSH
• Humans MeSH
• Copper metabolism   MeSH
• Cell Line, Tumor MeSH
• Breast Neoplasms metabolism   MeSH
• Proteasome Endopeptidase Complex chemistry   metabolism   MeSH
• Proteolysis MeSH
• Reactive Oxygen Species metabolism   MeSH
• Molecular Docking Simulation MeSH
• Ubiquitination MeSH
• Protein Binding MeSH
• Cell Survival drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Proteasome Inhibitors MeSH
• Coumarins MeSH
• Copper MeSH
• Proteasome Endopeptidase Complex MeSH
• Reactive Oxygen Species MeSH
• wedelolactone MeSH Browser