Differential Cell Death Effects in Glioblastoma after Drug- Induced DNA Damage
Object: Glioblastomas are difficult tumors to eradicate because
of resistance to apoptosis and other mechanisms of programmed
cell death such as autophagy. We hypothesized that DNA damage,
regardless of etiology, would cause autophagy. To test our
hypothesis, we examined the ability of two DNA damaging agents,
1, 3-Bis (2-chloroethyl)-1-nitrosourea (BCNU) and cisplatin, to
Materials and methods: DNA damage was assessed by western
blot for γ-H2AX and immunofluorescence for phospho-53BP1.
Autophagy was measured by microtubule-associated protein 1 light
chain (LC3) and beclin 1 western blots, acridine orange staining,
response to the 3-MA inhibitor, and autophagosome detection
using electron microscopy. To study apoptosis, we examined levels
of BAX and BAK, TUNEL staining, inhibition with ZVAD.fmk and
caspase 3/7 activation.
Results: The levels of the DNA damage indicators γ-H2AX and
53BP1 increased with both BCNU and cisplatin. While LC3-II
autophagy proteins were highly expressed in BCNU samples,
LC3-II levels were below the limits of detection in cells treated
with cisplatin. Caspase 3/7 activation only slightly increased
with BCNU, but markedly increased with cisplatin. Surprisingly,
BAX and BAK levels did not change in response to either
chemotherapeutic compound. Significant TUNEL staining was
evident in cisplatin, but not BCNU-treated cells, and the pancaspase
inhibitor, ZVAD.fmk, did not diminish cell death after
Conclusion: Although both drugs caused DNA damage, we
concluded not all DNA damage results in a specific type of cell
death, as BCNU-related cell death in glioblastomas occurs
through autophagy and cisplatin predominantly induces apoptosis.
The specific molecular mechanisms underlying the activation of
autophagy remain obscure.