Proton beam irradiation inhibits the migration of melanoma cells

In recent years experimental data have indicated that low-energy proton beam radiation might induce a difference in cellular migration in comparison to photons. We therefore set out to compare the effect of proton beam irradiation and X-rays on the survival and long-term migratory properties of two cell lines: uveal melanoma Mel270 and skin melanoma BLM.Cells treated with either proton beam or X-rays were analyzed for their survival using clonogenic assay and MTT test. Long-term migratory properties were assessed with time-lapse monitoring of individual cell movements, wound test and transpore migration, while the expression of the related proteins was measured with western blot.Exposure to proton beam and X-rays led to similar survival but the quality of the cell colonies was markedly different. More paraclones with a low proliferative activity and fewer highly-proliferative holoclones were found after proton beam irradiation in comparison to X-rays. At 20 or 40 days post-irradiation, migratory capacity was decreased more by proton beam than by X-rays. The beta-1-integrin level was decreased in Mel270 cells after both types of radiation, while vimentin, a marker of EMT, was increased in BLM cells only.We conclude that proton beam irradiation induced long-term inhibition of cellular motility, as well as changes in the level of beta-1 integrin and vimentin. If confirmed, the change in the quality, but not in the number of colonies after proton beam irradiation might favor tumor growth inhibition after fractionated proton therapy

Estimation of the response of host to photodynamic therapy of murine lung carcinoma tumors

Terapia fotodynamiczna (PDT) wymaga do działania fotosensybilizatora (PS) efektywnie gromadzącego się w tkance nowotworowej oraz obecności tlenu. Po podaniu fotosensybilizatora do układu w określonym czasie przeprowadzane jest naświetlanie miejsca, w którym znajduje się nowotwór. Można przez to wyróżnić protokół skierowany preferencyjnie przeciwko unaczynieniu guza (VTP) lub przeciwko jego komórkom (CTP). W wyniku terapii indukowane jest powstawanie reaktywnych form tlenu (ROS) oraz wysoce reaktywnego tlenu singletowego, powodujących niszczenie komórek na drodze nekrozy i/lub apoptozy. Na skutek indukcji stresu oksydacyjnego i niszczenia błon cytoplazmatycznych następuje uwalnianie czynników prozapalnych, a przez to indukcja reakcji zapalnej i napływ komórek układu immunologicznego. Celem pracy była obserwacja odpowiedzi nieswoistej w postaci napływu neutrofili i makrofagów do guza LLC i przylegającej tkanki u myszy oraz zbadanie poziomu czynników IL-6, IL-10, TNF-α i VEGF po 24 h od terapii. Przeprowadzono również analizę unaczynienia guza po terapii i analizę narządów gospodarza. W tych celach porównywano protokoły terapeutyczne: PDT15, skierowanego przeciwko unaczynieniu i PDT72, skierowanego przeciwko komórkom guza, gdzie podawano fotosensybilizator w dawce 2 mg/kg i 4 mg/kg. W pracy wykorzystano jako fotosensybilizator halogenową pochodną bakteriochloryny.PDT indukuje napływ komórek odpowiedzi nieswoistej, neutrofili i makrofagów. Neutrofile silnie infiltrują tkankę tuż po terapii, szczególnie po PDT15, oraz napływają do tkanki falami. Z kolei napływ makrofagów po PDT72 2 mg/kg jest wolniejszy. Ponadto zauważono różnice po PDT72 z zastosowaniem dawki 2 mg/kg i 4 mg/kg. W przypadku większej dawki infiltracja makrofagów jest silniejsza. Jednakże w obu tych przypadkach następuje większy napływ makrofagów po kilku dniach od terapii. Wszystkie protokoły powodowały spadek gęstości naczyń, który był największy po PDT15. Poznanie dokładnych mechanizmów działających w następstwie terapii fotodynamicznej może pozwolić na modyfikację metody i zwiększenie jej skuteczności.Photodynamic therapy (PDT) requires a photosensitizer (PS), which accumulates in tumor tissue, and the presence of oxygen. In specified time after application of photosensitizer to the system, irradiation is performed at the site of tumor. Therefore we can distinguish either protocol directed against tumor vasculature (VTP), or against tumor cells (CTP). On account of therapy reactive oxygen species (ROS) and singlet oxygen are produced, which entail destroying tumor cells via apoptosis and/or necrosis. Because of oxidative stress and cell membranes deterioration, a release of proinflammatory proteins takes place, so development of inflammatory reaction and infiltration by immunological system cells is expected.Purpose of this thesis was to observe and evaluate an innate response manifested by neutrophils and macrophages infiltration to LLC tumor and adjacent tissue, and also to verify levels of IL-6, IL-10, TNF-α and VEGF during 24 h after therapy. In this case two therapeutic protocols were compared: PDT15 directed against tumor vasculature with PS dose of 2 mg/kg, and PDT72 directed against tumor cells with two different doses: 2 mg/kg or 4 mg/kg. Bacteriochlorine derivative was used as photosensitizer.PDT induced neutrophil and macrophage inflow. Neutrophils strongly infiltrated tumor tissue within minutes after therapy, specially after PDT15 and flowed to the tissue in waves. On the other hand, influx of macrophages was slower. Furthermore a difference was observed in PDT72 between usage of dose 2 mg/kg and 4 mg/kg. Reaction was stronger with grater dosage. But yet in both cases there was a higher inflow of macrophages in a few days after therapy. All protocols entailed decrease in vascular density, which was the largest after PDT15. Knowledge about mechanisms occurring in photodynamic therapy may let us modify this method and increase its effectiveness

Translocation of chromatin proteins to nucleoli—The influence of protein dynamics on post‐fixation localization

International audienceIt is expected that the subnuclear localization of a protein in a fixed cell, detected by microscopy, reflects its position in the living cell. We demonstrate, however, that some dynamic nuclear proteins can change their localization upon fixation by either crosslinking or non-crosslinking methods. We examined the subnuclear localization of the chromatin architectural protein HMGB1, linker histone H1, and core histone H2B in cells fixed by formaldehyde, glutaraldehyde, glyoxal, ethanol, or zinc salts. We demonstrate that some dynamic, weakly binding nuclear proteins, like HMGB1 and H1, may not only be unexpectedly lost from their original binding sites during the fixation process, but they can also diffuse through the nucleus and eventually bind in nucleoli. Such translocation to nucleoli does not occur in the case of core histone H2B, which is more stably bound to DNA and other histones. We suggest that the diminished binding of some dynamic proteins to DNA during fixation, and their subsequent translocation to nucleoli, is induced by changes of DNA structure, arising from interaction with a fixative. Detachment of dynamic proteins from chromatin can also be induced in cells already fixed by non-crosslinking methods when DNA structure is distorted by intercalating molecules. The proteins translocated during fixation from chromatin to nucleoli bind there to RNA-containing structures

Cellular migration properties of BLM cells treated with proton beam radiation or X rays.

<p>Individual cell movements were evaluated at 20 days after irradiation (A, B, C) and at 40 days after irradiation (D, E, F) and were evaluated in terms of ‘Speed’, i.e. average speed of cell movement; ‘Displacement’, i.e. the total linear length of the cell displacement from the starting point (μm) and CME (coefficient of movement efficiency), i.e. the ratio of cell displacement to the cell trajectory length. Mean values presented as percent of control; *p<0.05, **p<0.01, ***p<0.001.</p

Proton beam irradiation inhibits the migration of melanoma cells - Fig 1

<p><b>Clonogenic assay of cell survival of Mel270 (A) and BLM (B) cells, treated with proton beam (■) or X rays (●).</b> Representative images of colonies are presented at <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186002#pone.0186002.s001" target="_blank">S1 Fig</a>. Cell were seeded immediately after radiation. Mean values with SEM, *p<0.05; **p<0.001. RBE values were determined from a linear-quadratic model and were 1.10 for Mel270, and 1.13 for BLM cells. (C–E) Three types of colonies formed by Mel270 (C) and BLM (D) cells in two weeks after irradiation with 1–5 Gy of proton beam or X-rays, determined as the percentage of the total number of colonies. Mel270 and BLM cells form three types of colonies described as holo-, mero- and paraclones (E). Holoclones are large, packed colonies displaying heterogeneity, which are believed to be derived from cancer initiating cells; meroclones are putatively derived from transit-amplifying cells and paraclones are loosely packed cells, derived from differentiated cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186002#pone.0186002.ref017" target="_blank">17</a>].</p

Cellular migration properties of Mel270 cells treated with proton beam radiation or X rays.

<p>Individual cell movements were evaluated at 20 days after irradiation (A, B, C) and at 40 days after irradiation (D, E, F) and three parameters were calculated: ‘Speed’, i.e. average speed of cell movement; ‘Displacement’, i.e. the total linear length of the cell displacement from the starting point (μm) and CME (coefficient of movement efficiency), i.e. the ratio of cell displacement to the cell trajectory length. Mean values presented as percent of control; *p<0.05, **p<0.01, ***p<0.001.</p