EUROPEAN RADIATION RESEARCH 2012

Sensitivity of Melanoma B16 Side Population Cells to Ionising Radiation and Methotrexate

18 Oct 2012, 16:55

1m

Poster Hall (Vietri sul Mare)

poster preferred Stem Cells Radiobiology Poster Session 3

Speaker

Ms Olga Matchuk (Medical Radiological Research Center of the Ministry of Health of the Russian Federation)

Description

Cancer stem cells (CSC), detected in variety of tumor types and cancer cell lines, were shown to be more resistant to the low-LET radiation in comparison to the other cancer cells. CSC are supposed to determine long-term efficiency of cancer therapy. Therefore, exploration of regularities and mechanisms of CSC radio- and chemosensitivity is of great interest at present. The aim of this investigation is to compare CSC and the other cancer cells sensitivity to the low (60Со gamma-rays) radiation and methotrexate. To identify putative CSC we used flow cytometry-based side population (SP) technique, founded on high capacity of CSC to efflux the vital dye Hoechst 33342. SP and non SP cells were sorted and exposed to radiation at doses of 1-10 Gy. Colony-formation test was applied to evaluate SP and non SP survival rate after the irradiation. To study cytotoxic effect of methotrexate 40% confluent melanoma B-16 culture was treated with the drug at final concentrations 1µg/ml or 5µg/ml during 2 days. Cell number and percent of SP were assessed at the end of the incubation. Proportion of SP was 0.9±0.2% in control 70% confluent cultures. Radiosensitivity of SP cells was below in comparison to that of the other cells: D0 average values (±SE) were 2.3±0.3 Gy and 1.4±0.2 Gy, accordingly (р=0.047). Total number of cells incubated with methotrexate was approximately 20 times less than that in control probes. However percent of SP was significantly increased up to 5.7±2.1 and 2.8±0.7 after methotrexate treatment (1µg/ml or 5µg/ml, accordingly) (p<0.01 as compared with control). The results indicate that cytotoxic effect of methotrexate extends more to non SP than to SP cells. In unexposed samples only 14.5 % of SP cells were in S+G2+M phases in comparison with 61.6 % in non SP (p<0.01), as it was shown by flow cytometry of sorted cells after standard staining with propidium iodide. Therefore one of the possible reasons for different sensitivity of examined cell populations to low-LET radiation and methotrexate is distinction in proliferative activity. According to multiple world-wide investigations, quiescent state appears to be necessary for preserving the self-renewal of stem cells and is a critical factor in the resistance of CSC to radio-, chemotherapy and targeted therapies. Our results confirm this thesis for SP of melanoma B16 cells. This work was supported by the Foundation for Assistance to Small Innovation Enterprises (FASIE), Russia.