Abstract
Despite recent advances in tumour cell biology, the prognosis for patients suffering
from malignant glioma remains poor. Although primary glioma rarely metastasises
outside the central nervous system (primary being defined as the mass of tumour
cells at the original site of the neoplastic event) median survival of adults is less than
1 year after diagnosis.The efficacy of existing therapeutic interventions is limited by
poor penetration of chemotherapeutic drugs across the blood brain barrier, the
inherent radioresistance of glioma tissue and the infiltrating nature of the tumour.
Further progress is likely to be achieved through analysis of the complex biology of
these tumours and the development of novel therapeutic strategies. The purpose of
this study was to investigate the therapeutic potential of the n-6 essential fatty acids
arachidonic acid and gamma-linolenic acid, which may inhibit tumour proliferation
by acting as substrates for the production of potentially cytotoxic reactive oxygen
intermediates and stimulating apoptotic cell death, both alone and in conjunction
with radiation.
Experiments were undertaken to investigate the effects of exogenous arachidonic
acid and gamma-linolenic acid on cellular peroxidation, proliferation, viability and
apoptosis. These investigations were carried out on single cell suspensions of
morphologically heterogeneous fresh human glioma tissue and associated normal
brain, human phagocytes and the rat C6 glioma cell line. It was shown that oxidative
activity was impaired in human glioma tissue. Addition of 4-40μM arachidonic acid
and gamma-linolenic acid induced a concentration dependant increase in tumour
reactive oxygen intermediate production and apoptotic activity. Although the
kinetics of reactive oxygen intermediate formation in the presence of arachidonic
acid and gamma-linolenic acid followed an exponential function in both normal and
tumour cell preparations, tumour cells showed a significantly higher sensitivity to
exogenous essential fatty acid stimulus. The kinetics of this stimulation were grade
dependent, with high grade tumours responding in a more rapid and sustained
manner in comparison with lower grade tumours. The morphological heterogeneity
of the human glioma preparations was confirmed with immunohistochemical
analysis and flow cytometry using monoclonal and polyclonal anti-Glial Acidic
Fibrillary Protein (GFAP). GFAP positive cells responded to exogenous arachidonic
acid and gamma-linolenic acid with increased reactive oxygen intermediate
production, indicating a high sensitivity of glioma cells to essential fatty acid
stimulus. Reactive oxygen intermediate production was also investigated in
phagocyte preparations of patients undergoing pulmonary resection for lung cancer.
It was found that reactive oxygen intermediate generation was stimulated in patient
and control phagocytes by exogenous 1 -40μM arachidonic acid and gamma-linolenic
acid both pre and post-operatively. Increased reactive oxygen intermediate formation
was detected in the cell population identified as leukocytes in preparations of human
primary glioma, although this response was less than that of associated tumour. It
was also found that surgery was associated with an increase in phagocyte reactive
oxygen intermediate at 2 and 7 days post-operatively in lung cancer patients. The
interactive effects of arachidonic acid, gamma-linolenic acid and therapeutic
radiation were demonstrated in the rat C6 glioma cell line. The rate ofreactive
XVI
oxygen intermediate production in response to exogenous arachidonic acid and
gamma-linolenic acid increased within the first hour, and elevated oxidative activity
was detected for up to three hours. However, a different pattern ofreactive oxygen
intermediate generation was observed in response to radiation alone. Similarly, an
early apoptotic response was observed following exogenous arachidonic acid and
gamma-linolenic acid stimulation. In comparison, radiation induced stimulation of
apoptosis occurred over the 12 hour period of incubation and was maximal between
6 and 8 hours post-irradiation. An enhanced radiation response was observed when
the stimulation of apoptosis induced by essential fatty acid stimulus alone was low,
suggesting that essential fatty acids and radiation may interact to potentiate reactive
oxygen intermediate generation and apoptosis.
In conclusion, this study has provided evidence that glioma tissue has low basal
oxidative activity in comparison with associated normal brain, and that addition of
exogenous arachidonic acid and gamma-linolenic acid stimulates peroxidative and
apoptotic activity in glioma tissue a grade dependant manner. Studies on the cellular
heterogeneity of human glioma samples indicate that the stimulation ofreactive
oxygen intermediate production by exogenous arachidonic acid and gamma-linolenic
acid occurs in GFAP positive cells. This indicates high sensitivity of human glioma
to exogenous essential fatty acid stimulus. Phagocyte populations from lung cancer
and malignant glioma patients also respond with increased reactive oxygen
intermediate production to exogenous arachidonic acid and gamma-linolenic acid,
although the magnitude of this increase is less than that observed for tumour cells. In
addition, there is evidence ofpotentiation ofthe oxidative and apoptotic response of
the rat C6 cell line to exogenous arachidonic acid and gamma-linolenic acid in the
presence of therapeutically relevant doses ofradiation. These results are consistent
with a clinical role for arachidonic acid and gamma-linolenic acid in the treatment of
malignant glioma.