Irradiation is one of the cornerstones used in the treatment of
malignant glioma. However, the effect is modest and
glioma cells generally display a pronounced radio-resistance. In this study, the effect of irradiation, alone and in combination with the antimicrotubule
drug estramustine (EaM), was investigated in vitro using the BT4C rat
glioma cell line, and in vivo the BT4C rat intracerebral
glioma model was used. Apoptosis was detected by analysing
DNA laddering, in situ end labelling (ISEL) and
Annexin V reactivity. In addition, phosphorylation status of MAPK, JNK, p38, and AKT,
proteins involved in pro- and anti-apoptotic signalling pathways was analysed by Western blotting. Irradiation did not induce apoptosis, neither in vitro nor in vivo. EaM, however, induced apoptosis in vivo and in vitro, regardless of whether EaM was given alone, before or after irradiation. When BT4C cells were treated with the
caspase-3 inhibitor
Ac-DEVD-CHO prior to EaM, the number of apoptotic cells was decreased, indicating an involvement of
caspase-3. The signalling pathways regulating apoptosis are complex and involve
kinases such as MAPK, JNK, p38 and AKT. Irradiation did not induce any changes in the expression levels or phosphorylation status of these
proteins. On the other hand, the phosphorylation level of AKT was reduced after EaM treatment, which might, in part, propose how EaM induces apoptosis in
glioma cells.