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SNAB AS BIOLOGY UNIT 3 (2015)

TREATING GLIOBLASTOMAS WITH CHEMICALS FOUND IN CANNABIS

1.1 The Problem


The problem I have decided to explore is that of brain tumours.
According to Cancer Research UK[1] , in 2011, 9,365 people in the UK
were diagnosed with brain or other CNS and intracranial tumours
and currently, only 14% of those are predicted to live more than 10
years after diagnosis. These cancerous brain tumours (including
aggressive brain tumours called glioblastomas) cause 3% of deaths
due to cancer and if we can inhibit the growth of these cells, then
the patient will live longer and/or have a better quality of life.
Some symptoms of brain tumours include seizures, fits,
feeling nauseous or drowsy and experiencing severe/frequent
headaches. However, depending on the position of the tumour in
the brain, the individual may experience symptoms anywhere from
changes in personality (becoming more aggressive/irritable) and
forgetfulness, to problems with sight, and difficulty comprehending
seemingly simple words/ phrases; even after going through
chemotherapy, the patient may still have side effects such as loss of
hair, tiredness, and lower resistance to infection.

1.2 Biological Processes To Produce Data


A study conducted by Jahan P. Marcu et al. (and published by the
American Association for Cancer Research[2]) investigates whether
cannabidiol would modulate the ability of 9-THC to inhibit
glioblastomas cell proliferation and survival.
The cell lines that were used were that of SF126, U251 and
U87 (glioblastoma cells) and were maintained at a temperature of
37c and 5% CO2 and in all experiments, the cells were first cultured
in RPMI media which contained 10% fetal bovine serum. The
glioblastomas cells were then seeded into 96-well plates and on the
first day of the treatment, the media were replaced with the vehicle
control or the drug (and 0.1% FBS) in a 2x2 factorial design; the
media were replaced every 24 hours.
In order to obtain the data, a range of analysis methods were
carried out; one of which was apoptosis analysis. The cells, which
were grown in 6-well culture dishes and treated every 24 hours for 3
days, were collected pelleted, washed once with a buffer solution
and then briefly, the cell pellet was resuspended in 300 l of the
supplied reaction buffer along with 3 l of both PI and FITC-tagged
annexin. After a 15-min incubation period at room temperature, the
labelled cells were analysed by flow cytometry. By using flow
cytometry, annexin staining and a PI emission signal detector, they
could quantify the percentage of cells going through apoptosis (in

both control and treatment groups) as well as ignore the necrotic


cells.
As well as an apoptosis analysis, a cell cycle analysis was also
carried out. The cells that were grown in petri dishes and had been
receiving drug treatments for two days were, on the third day,
harvested and centrifuged. After being washed again, the pellet was
centrifuged once more, and resuspended in 0.5 ml of 2%
paraformaldehyde before fixed overnight at room temperature. The
next day the cells were pelleted, resuspended in 0.5 ml 0.3% Titron
in a buffer solution and incubated for 5 minutes at room
temperature. The cells were then washed once again, and then
suspended in PBS (0.1% BSA) with 10 g/ml Propidium Iodide and
100 g/ml RNAse. After this, the cells were incubated for 30 mins
and stored at 4C; Cell cycle was measured using a FACS Calibur
using Cell Quest Pro and Modfit software.

1.3 Appropriateness of Method/Solutions


The methods used during the experiment are appropriate for
measuring to what extent cannabidiol is effective at modulating the
ability of 9-THC to inhibit glioblastomas cell proliferation and
survival. By measuring the rate at which the cells are growing and
then dying as well as the number of cells that are in G2-GM state, you
can quantify the amount by which the cells have (in this case)
decreased. This allows you to compare the effects of the different
conditions; below are the findings from this experiment I will be
focussing on.
9-THC And Cannabidiol Both Have Inhibitory Effects On The
Glioblastoma Cell Lines Studied
When the SF126, U251 and U87 cells were treated with a range of
concentrations of either 9-THC or cannabidiol, they inhibited the
growth of the cells. The average increase in size of the tumours
treated with 9-THC for the 3 cell lines was 3.03 m however the
average increase in size of the glioblastomas for cannabidiol was 0.8
m; this clearly shows that cannabidiol has a much stronger
inhibitory effect than 9-THC in the three cell lines studied.
Cannabidiol Improves The Inhibitory Effects Of 9-THC On
Glioblastoma Cell Growth

Shown in the graph above are the results of treating the cells with
different combinations of the chemicals (and different
concentrations of the chemicals). This way, one is able to compare
the inhibitory effect of the different concentrations and chemicals.
You can see the best combination is that in graph D: 0.4 mol/L of
CBD alongside 1.7 mol/L of 9-THC. As well as this, you can also
see that in every case where cannabidiol and 9-THC are used in
conjunction with each other, the inhibitory effect of 9-THC is
amplified.
This means if we were to use both 9-THC as well as cannabidiol as
a treatment to inhibit the growth of glioblastomas, we would be
much more likely to prolong the life of the patient as well as reduce
the likelihood of the cells spreading to other parts of the body as the
combination approach inhibits the growth of the cells and induces
apoptosis.

Cannabinoids have been shown to be successful in the past for


inhibiting the growth of tumours. In order to solidify these findings,
scientists from the department of oncology at the university of
London have conducted a study investigating whether the
combination of cannabidiol and 9-THC enhances the anticancer
effects of radiation therapy. Cannabinoids were used in two forms,
pure (P) and as a botanical drug substance (BDS). Results
demonstrated a duration- and dose-dependent reduction in cell
viability with each cannabinoid and suggested that THC-BDS was
more efficacious than THC-P, whereas, conversely, CBD-P was more
efficacious than CBD-BDS. Analysis showed any/all of the
combinations of cannabinoids to be hyperadditive. However, the
combination that proved to be most effective at increasing the radio
sensitivity of the glioma cells was THC-P + CBD-P in conjunction with
each other. The treatment was administered 4 hours before
irradiation began and when comparing the results of treating the
glioma cells with either of the cannabinoids individually, there was
no competition. The increase in radio sensitivity was also
accompanied by an increase in markers of autophagy (self
degrading of the cells) and apoptosis (the death of cells which
occurs as a normal and controlled part of an organism's growth and
development). These results (in vivo) show dramatic reductions in
tumour volumes when both cannabinoids are used alongside
radiation.
Bibliography
1. www.cancerresearchuk.org
2. www.ncbi.nlm.nih.gov/pmc/articles/PMC2806496/
3. http://science.howstuffworks.com/life/cellularmicroscopic/apoptosis.htm