Pilot screening of potential matrikines resulting from collagen breakages through ionizing radiation.

Little is known regarding radiation-induced matrikines and the possible degradation of extracellular matrix following therapeutic irradiation. The goal of this study was to determine if irradiation can cut collagen proteins at specific sites, inducing potentially biologically active peptides against cartilage cells. Chondrocytes cultured as 3D models were evaluated for extracellular matrix production. Bystander molecules were analyzed in vitro in the conditioned medium of X-irradiated chondrocytes. Preferential breakage sites were analyzed in collagen polypeptide by mass spectrometry and resulting peptides were tested against chondrocytes. 3D models of chondrocytes displayed a light extracellular matrix able to maintain the structure. Irradiated and bystander chondrocytes showed a surprising radiation sensitivity at low doses, characteristic of the presence of bystander factors, particularly following 0.1 Gy. The glycine-proline peptidic bond was observed as a preferential cleavage site and a possible weakness of the collagen polypeptide after irradiation. From the 46 collagen peptides analyzed against chondrocytes culture, 20 peptides induced a reduction of viability and 5 peptides induced an increase of viability at the highest concentration between 0.1 and 1 µg/ml. We conclude that irradiation promoted a site-specific degradation of collagen. The potentially resulting peptides induce negative or positive regulations of chondrocyte growth. Taken together, these results suggest that ionizing radiation causes a degradation of cartilage proteins, leading to a functional unbalance of cartilage homeostasis after exposure, contributing to cartilage dysfunction.

A Proteomic Study Suggests Stress Granules as New Potential Actors in Radiation-Induced Bystander Effects.

Besides the direct effects of radiations, indirect effects are observed within the surrounding non-irradiated area; irradiated cells relay stress signals in this close proximity, inducing the so-called radiation-induced bystander effect. These signals received by neighboring unirradiated cells induce specific responses similar with those of direct irradiated cells. To understand the cellular response of bystander cells, we performed a 2D gel-based proteomic study of the chondrocytes receiving the conditioned medium of low-dose irradiated chondrosarcoma cells. The conditioned medium was directly analyzed by mass spectrometry in order to identify candidate bystander factors involved in the signal transmission. The proteomic analysis of the bystander chondrocytes highlighted 20 proteins spots that were significantly modified at low dose, implicating several cellular mechanisms, such as oxidative stress responses, cellular motility, and exosomes pathways. In addition, the secretomic analysis revealed that the abundance of 40 proteins in the conditioned medium of 0.1 Gy irradiated chondrosarcoma cells was significantly modified, as compared with the conditioned medium of non-irradiated cells. A large cluster of proteins involved in stress granules and several proteins involved in the cellular response to DNA damage stimuli were increased in the 0.1 Gy condition. Several of these candidates and cellular mechanisms were confirmed by functional analysis, such as 8-oxodG quantification, western blot, and wound-healing migration tests. Taken together, these results shed new lights on the complexity of the radiation-induced bystander effects and the large variety of the cellular and molecular mechanisms involved, including the identification of a new potential actor, namely the stress granules.

High LET Radiation Overcomes In Vitro Resistance to X-Rays of Chondrosarcoma Cell Lines.

Chondrosarcomas are malignant tumors of the cartilage that are chemoresistant and radioresistant to X-rays. This restricts the treatment options essential to surgery. In this study, we investigated the sensitivity of chondrosarcoma to X-rays and C-ions in vitro. The sensitivity of 4 chondrosarcoma cell lines (SW1353, CH2879, OUMS27, and L835) was determined by clonogenic survival assays and cell cycle progression. In addition, biomarkers of DNA damage responses were analyzed in the SW1353 cell line. Chondrosarcoma cells showed a heterogeneous sensitivity toward irradiation. Chondrosarcoma cell lines were more sensitive to C-ions exposure compared to X-rays. Using D10 values, the relative biological effectiveness of C-ions was higher (relative biological effectiveness = 5.5) with cells resistant to X-rays (CH2879) and lower (relative biological effectiveness = 3.7) with sensitive cells (L835). C-ions induced more G2 phase blockage and micronuclei in SW1353 cells as compared to X-rays with the same doses. Persistent unrepaired DNA damage was also higher following C-ions irradiation. These results indicate that chondrosarcoma cell lines displayed a heterogeneous response to conventional radiation treatment; however, treatment with C-ions irradiation was more efficient in killing chondrosarcoma cells, compared to X-rays.

Bystander effectors of chondrosarcoma cells irradiated at different LET impair proliferation of chondrocytes.

While the dose-response relationship of radiation-induced bystander effect (RIBE) is controversial at low and high linear energy transfer (LET), mechanisms and effectors of cell-to-cell communication stay unclear and highly dependent of cell type. In the present study, we investigated the capacity of chondrocytes in responding to bystander factors released by chondrosarcoma cells irradiated at different doses (0.05 to 8 Gy) with X-rays and C-ions. Following a medium transfer protocol, cell survival, proliferation and DNA damages were quantified in bystander chondrocytes. The bystander factors secreted by chondrosarcoma cells were characterized. A significant and major RIBE response was observed in chondrocyte cells (T/C-28a2) receiving conditioned medium from chondrosarcoma cells (SW1353) irradiated with 0.1 Gy of X-rays and 0.05 Gy of C-ions, resulting in cell survivals of 36% and 62%, respectively. Micronuclei induction in bystander cells was observed from the same low doses. The cell survival results obtained by clonogenic assays were confirmed using impedancemetry. The bystander activity was vanished after a heat treatment or a dilution of the conditioned media. The cytokines which are well known as bystander factors, TNF-α and IL-6, were increased as a function of doses and LET according to an ELISA multiplex analysis. Together, the results demonstrate that irradiated chondrosarcoma cells can communicate stress factors to non-irradiated chondrocytes, inducing a wide and specific bystander response related to both doses and LET.

siPGK1 sensitizes chemoresistant human ovarian cancer cell lines to cisplatin.

Enhanced glycolysis provides essential intermediates for cancer cell proliferation. Its inhibition could be a promising approach for destroying tumors, especially those developing in hypoxic conditions, which are presumably the most chemoresistant. In hypoxic cells, glycolysis provides the main part of ATP. Phosphoglycerate kinase-1 (PGK1) catalyzes a crucial reaction of glycolysis that reconstitutes the two molecules of ATP previously consumed. PGK1 inhibition could arrest growth or kill hypoxic and/or chemoresistant cells. We tested siPGK1 transfection in two human ovarian cancer cells lines of increasing chemoresistance, and showed that: Expression of PGK1 was significantly reduced and associated with blockade of cell growth in the G(1) phase; siPGK1 associated with cisplatin was more effective than cisplatin-alone at inhibiting proliferation of chemoresistant cells; siPGK1 -alone and -associated with cisplatin strongly increased expression of the BH3-only pro-apoptotic protein BCL-2 Interacting Mediator of cell death (BIM). PGK1 might be a key target for sensitizing chemoresistant cells to cisplatin.