Accumulation of cyclophilin A isoforms in conditioned medium of irradiated breast cancer cells

Abstract

Secreted proteins play a key role in cell signaling and communication. We recently showed that ionizing radiations induced a delayed cell death of breast cancer cells, mediated by the death receptor pathways through the expression of soluble forms of "death ligands." Using the same cell model, the objective of our work was the identification of diffusible factors, secreted following cell irradiation, potentially involved in cell death signaling. Differential proteomic analysis of conditioned media using 2DE resulted in detection of numerous spots that were significantly modulated following cell irradiation. The corresponding proteins were identified using MALDI-TOF MS and LC-MS/MS approaches. Interestingly, five isoforms of cyclophilin A were observed as increased in conditioned medium of irradiated cells. These isoforms differed in isoelectric points and in accumulation levels. An increase of cyclophilin A secretion was confirmed by Western blotting of conditioned media of irradiated or radiosentive mammary cells. These isoforms displayed an interesting pattern of protein maturation and post-translational modifications, including an alternating removal of N-terminal methionine, associated with a combination of acetylations and methylations. The role of the protein is discussed in relation with its potential involvement in the mechanisms of intercells relationships and radiosensitivity.

Secreted cyclophilin A mediates G1/S phase transition of cholangiocarcinoma cells via CD147/ERK1/2 pathway

“…Secondly, the levels of sCypA were positively correlated with inCypA levels; and thirdly, suppression of CypA expression lowered the level of inCypA and consequently reduced the sCypA level. Concomitantly, CypA has also been identified as a protein secreted by human cancer cells, e.g., head and neck cancer [37], irradiated breast cancer [38], and prostate cancer [39].…”

Proteomic overview and perspectives of the radiation-induced bystander effects

“…An increased secretion of Cyclophilin A (CyPA) was observed in 5 modulated spots, and confirmed by a 1D and 2D Western blotting analysis. As observed subsequently by a deeper MS analysis, these CyPA isoforms were modified by several posttranslational modifications such as methylations and/or acetylations [61]. Moreover, the secreted form of CyPA was increased in radio-sensitive cell lines and potentially linked to a RIBE of that type of cells.…”

“…7). Gel-based analysis of such samples allows a global analysis of IR consequences on the selected biological system and presents the advantages of limiting the selection of false-positive BF markers and the identification of direct effects and BE within the same examination [61]. The proteomic comparison of irradiated cells and bystander cells, using un-irradiated cells as control, will display protein biomarkers of direct and indirect effects of IR, with common or distinct cellular mechanisms.…”

“…In addition, the extraction process of BF from conditioned medium should be compatible with the subsequent proteomic technology. Several protocols were developed by proteomic and biochemistry researchers to improve the extraction capacity and allow a correct separation of proteins by 2DE [61,72]. The main objective of such protein extraction is to concentrate proteins in a small amount of buffer, or to precipitate them as a pellet using centrifugation (Fig.…”

“…Using a 2DE gel-based proteomic strategy, the secretion of bystander factors from several human breast cell lines following irradiation at 10 Gy was analyzed in vitro [61]. To limit the identification of false-positive secreted proteins, total and cytosolic cell extracts were performed in parallel to secretory protein extracts with un-irradiated controls and irradiated samples.…”