Molecular radiobiology
Autophagy contributes to resistance of tumor cells to ionizing radiation

https://doi.org/10.1016/j.radonc.2011.06.002Get rights and content

Abstract

Background and purpose

Autophagy signaling is a novel important target to improve anticancer therapy. To study the role of autophagy on resistance of tumor cells to ionizing radiation (IR), breast cancer cell lines differing in their intrinsic radiosensitivity were used.

Materials and methods

Breast cancer cell lines MDA-MB-231 and HBL-100 were examined with respect to clonogenic cell survival and induction of autophagy after radiation exposure and pharmacological interference of the autophagic process. As marker for autophagy the appearance of LC3-I and LC3-II proteins was analyzed by SDS–PAGE and Western blotting. Formation of autophagic vacuoles was monitored by immunofluorescence staining of LC3.

Results

LC3-I and LC3-II formation differs markedly in radioresistant MDA-MB-231 versus radiosensitive HBL-100 cells. Western blot analyses of LC3-II/LC3-I ratio indicated marked induction of autophagy by IR in radioresistant MDA-MB-231 cells, but not in radiosensitive HBL-100 cells. Indirect immunofluorescence analysis of LC3-II positive vacuoles confirmed this differential effect. Pre-treatment with 3-methyladenine (3-MA) antagonized IR-induced autophagy. Likewise, pretreatment of radioresistant MDA-231 cells with autophagy inhibitors 3-MA or chloroquine (CQ) significantly reduced clonogenic survival of irradiated cells.

Conclusion

Our data clearly indicate that radioresistant breast tumor cells show a strong post-irradiation induction of autophagy, which thus serves as a protective and pro-survival mechanism in radioresistance.

Section snippets

Materials and antibodies

Cell culture medium was from Gibco-Invitrogen (Karlsruhe-Germany). 3-Methyladenine (3-MA), chloroquine diphosphate (CQ), rapamycin, and anti-actin antibody were purchased from Sigma–Aldrich (Taufkirchen-Germany). Mouse monoclonal anti-LC3 antibody was purchased from nanoTools (Teningen, Germany).

Cell culture

Breast cancer cell lines MDA-MB-231 (MDA-231) and HBL-100 were used. Cells were cultures in RPMI Medium containing 10% fetal calf serum (FCS) and 1% penicillin–streptomycin and incubated in a humidified

Induction of autophagy by IR

To assess the effect of different radiation doses on the autophagic pathway the breast cancer cell lines MDA-231 and HBL-100, presenting significant different intrinsic IR sensitivities as previously reported from our laboratory [34] and shown in Fig. 1A, were analyzed at different time points after IR exposure (3, 7, and 24 h). As shown in Fig. 1B untreated radioresistant MDA-231 cells presented a low amount of LC3-II. However, exposure to IR at different doses (1, 2, and 4 Gy) markedly

Discussion

Autophagy is a catabolic mechanism used by cells to overcome different stress situations. On the one hand autophagy eliminates toxic and damaged cellular components. On the other hand this process delivers new precursors for synthesis of macromolecules. The interest in the recently discovered mechanism autophagy has increased in the last decade. Many studies have shown its importance by unraveling autophagy-dependent signaling in the development of various diseases, such as neurodegenerative

Acknowledgments

This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft (DFG, SFB-824/1), BMBF (MOBITUM, 01EZ0826; Kompetenzverbund Strahlenforschung, 03NUK007E; Spitzencluster m4, 01EX102C) awarded to GM. Further financial support was received from the Deutsche Forschungsgemeinschaft (SFB-773, TP-B02) awarded to H.P.R.

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