The induction of autophagy in gliomas treated by temozolomide (pp.99-110)
Authors: (Manabu Natsumeda, Hiroshi Aoki, and Yukihiko Fujii)
Abstract: Autophagy, rather than apoptosis, is induced in gliomas by various treatments, such as radiation and temozolomide (TMZ). The mechanisms of autophagy are gradually being clarified, however, the role of autophagy in cancer therapy remains controversial. Our goal is to elucidate the role of autophagy for application to clinical treatment. We set out to establish methods of monitoring autophagy in in vitro studies and in glioma samples. There are only a few previous reports discussing the detection of autophagy in tissue samples.
In vitro studies employing electron microscopy, analysis of LC3I/II conversion by western blotting, and GFP-LC3B immunofluorescence assays were undertaken to detect the induction of autophagy. None of these methods alone are sufficient for monitoring autophagy. Therefore, multiple assays to verify an autophagic response were incorporated.
The development of a reliable method to monitor autophagy in surgically obtained tissues is vital in elucidating the role of autophagy in the clinical setting. To achieve this goal, immunohistochemistry was utilized in surgical tissue. Macroautophagy was monitored by immunohistochemistry employing anti-LC3B and anti-lysosome associated membrane protein 1 (LAMP1) antibodies. Likewise, chaperone-mediated autophagy was monitored using anti-LAMP2A antibodies. An increase of autophagy after TMZ treatment was noted. LC3B expression by western blotting in frozen tissue samples showed increases in both LC3-I and LC3-II expression after TMZ treatment.
By employing immunohistochemical approaches mentioned, it is possible to monitor autophagy in gliomas, opening the door for monitoring therapeutic effects and/or resistance to treatments in gliomas in the clinical setting.