Transcript Chloroquine-mediated blockade of the cyclical mTOR

Garreth D’Souza, Anita Kumar,
Durga Chougule, Dr. Yasmin Khan
Department of Life Sciences,
Sophia College, Mumbai
Abstract
A large amount of evidence indicates that autophagy may be one of the most common
drug resistance mechanisms in cancers. Consequently, pharmacological agents capable of
blocking/inhibiting autophagy could make a huge impact on cancer therapeutics. The
historically used and well-tolerated anti-malarial drug chloroquine has been shown to have
exactly such autophagy blocking potential. Consequently numerous studies have
demonstrated that adding chloroquine to autophagy-inducing anti-cancer regimens greatly
enhances their therapeutic efficacy not just in cell culture and animal models but in patients as
well.
Recently, a landmark pharmacological screen conducted by Williams et al identified
pharmacological agents capable of inducing autophagy via a novel mTOR-independent
autophagy pathway. Here we report, for the first time the synergistic effects of chloroquine
alongside such mTOR-independent autophagy-inducers using the C6 Glioma cell line as our
model system. The efficacy of this combination is being assessed using cell morphology and
the MTT and Trypan Blue assays for estimating cell viability.
Introduction
The
most well-characterized means of autophagy induction is nutrient
deprivation, which centers around the inhibition of a key signaling molecule called
mTOR (mammalian target of rapamycin)1. A large number of anti-cancer drugs also
cause mTOR inhibition in cancer cells which consequently leads to autophagy
induction.2 The use of Chloroquine alongside such mTOR inhibitors has had
hugely positive results in cell culture and xenograft studies in animal models3 and
clinical studies are now underway for testing the potential anti-cancer efficacies of
such combinations in patients.4
Recently a novel mTOR-independent pathway for autophagy induction was
elucidated as a result of a landmark pharmacological screen conducted by Williams
et al.5 In our study we make use of the clinically-approved pharmacological agents
capable of inducing autophagy via this novel pathway, with a focus on L-type
calcium channel (LTCC) antagonists.
The use of chloroquine in combination with the aforementioned pharmacological
agents has to the authors knowledge not been previously attempted and
consequently the study may have many important implications for the
development of potential anti-cancer therapeutic strategies.
Methodologies:
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MTT Assay:6
MTT - (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
In metabolically active cells, mitochondrial dehydrogenases convert:
MTT (yellow tetrazole) → Purple formazan crystals.
The purple crystals are solubilized using 10% SDS solution in 0.01 N HCl and then
estimated spectrophotometrically at 540/690nm.
Mitochondrial activity is thus used as a measure of cell viability and proliferation of
cultured cells.
Trypan Blue Assay:
Trypan blue staining is a popular vital staining technique used for differentiating
between live and dead cells. Only dead cells take up the stain.
Treated cells are trypsinized and suspended in Whole medium (DMEM with 10%
FBS) for neutralization.
The cell suspension is centrifuged at 5000rpm for 10 minutes.
The supernatant is discarded and the pellet resuspended in 100µL of 1:1 :: Whole
Medium : 4% Trypan Blue solution.
After a two minute interval, the total number of live and dead cells are counted using
a haemocytometer and the percentage viability is then calculated.
Effect of Chloroquine and/or Nimodipine on
C6 Glioma cells after 72hrs of drug exposure:
Control
+100X
+100X
30µL Nimodipine
+100X
10µL Chloroquine
30µL Nimodipine + 10µL Chloroquine
+100X
Cells treated with both Nimodipine and Chloroquine (but not either drug on its own)
showed a dramatic decrease in number and prominent rounding up indicative of cell death.
Images captured using Nikon Inverted Microscope with Green Interference Filter (GIF).
Effect of Chloroquine and/or Verapamil on
C6 Glioma cells after 72hrs of drug exposure:
10µL Chloroquine
Control
100X
+100X
+100X
30µL Verapamil
+100X
30µL Verapamil + 10µL Chloroquine
+100X
Similar synergistic cell-death promoting effects observed with the use of Verapamil in
combination with Chloroquine but not when either drug was used on its own.
Images captured using Nikon Inverted Microscope with Green Interference Filter (GIF).
MTT Assay For Cell Viability:
Combined Effects of Verapamil and Chloroquine
140
100
71.8
54.2
40.0
20
53.0
85.34
40
69.2
60
77.5
122.3
80
100
PERCENTILE VIABILITY
120
0
CONTROL
5 µM CQ
10µM CQ
30 µM VERA 30 µM VERA + 30 µM VERA + 60 µM VERA 60 µM VERA + 60 µM VERA +
5 µM CQ
10µM CQ
5 µM CQ
10µM CQ
MTT data further corroborate the ability of Chloroquine to enhance the cell-death induced by
Verapamil. Notably even a non-toxic dose of Chloroquine (5μM) was able to enhance cell death
of Verapamil.
n = 6; Sets performed in duplicates.
PRELIMINARY
RESULTS
Effect of Chloroquine on
Nimodipine-treated Cell
Viability (By Trypan Blue
Assay)
MTT Assay For Cell Viability:
Combined Effects of Nimodipine and
Chloroquine
120
120
7.2
7.17
80
8.0
100
69.0
CONTROL
10µM CQ
92.8
92.7
40
91.9
20
60
51.6
54.7
60.1
40
66.4
104.7
60
% OF CELLS
80
100
20
0
30.9
PERCENTILE VIABILITY
100
0
CONTROL
10µM CQ
10µM NIMO 10µM NIMO 40µM NIMO 40µM NIMO
+ 10µM CQ
+ 10µM CQ
Synergistic effects of Nimodipine and Chloroquine
on induction of cell death in C6 Glioma cells in
vitro.
n = 6; Sets performed in duplicates.
30µM NIMO 30µM NIMO
+ 10µM CQ
Alive
Dead
CONTROL - 48 HRS
+400X
+400X
Untreated cells
cytoplasm.
30µM NIMODIPINE + 10µM CQ – 48 HRS
lack
a vacuolated
Cells treated with L-type Calcium
channel
blockers
and/or
with
Chloroquine display an intensely
vacuolated
cytoplasm
probably
indicative of autophagosomes.
Discussion
L-type Calcium channels are present in excitable as well as certain non-excitable
tissues and are upregulated in certain cancers7 where they mediate increased calcium
influx into cells. Since calcium plays a key role in proliferation and cell cycle
progression, the increase in cytosolic calcium levels has been shown to correlate with
increased proliferation rates.
The use of LTCC antagonists decreases cytosolic calcium levels leading to cellular
stress and the concomitant induction of autophagy as a cell survival mechanism.5 The
induction of autophagy has been shown to occur via an mTOR-independent
mechanism involving inhibition of calpain activity5,8 as a result of the decreased
cytosolic calcium levels. While this has been shown to be true for neuronal cells, it
remains to be seen if this holds true for Gliomas. Nonetheless, when chloroquine was
used in combination with the LTCC antagonists it substantially potentiated the death
of cancer cells in vitro consistent with the LTCC antagonists’ autophagy-inducing
abilities.
Our future direction involves determining if the use of the LTCC antagonists leads to
autophagy induction in gliomas as has been theoretically proposed. This shall involve
the use of Monodansyl cadaverine and Acridine Orange as well as an autophagyspecific construct to measure cellular levels of autophagy.
Conclusion
Our study is important on several counts:
• Firstly, we have demonstrated for the first time the potential anti-cancer
efficacy of the use of Chloroquine in combination with mTOR-independent
autophagy inducers. The LTCC antagonists used for the study are clinically welltolerated drugs used for the successful treatment of hypertension. The use of a
verapamil-chloroquine combination has been previously conducted for the
treatment of drug resistant malaria. 9
• Secondly, mTOR-dependent and -independent pathways have been previously
shown to have additive effects on autophagy induction.10 Hence, the
simultaneous use of agents targeting both pathways could theoretically lead to a
greater potentiation of cancer cell death when combined with chloroquine. This
is an interesting hypothesis that merits further study.
• Lastly, certain LTCC antagonists like verapamil are well known to have
multidrug-resistance reversal capabilities.11,12 Consequently, this could be
another reason why patients could potentially benefit from the addition of such
drugs to their anti-cancer regimens.
References
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Akt inhibition promotes autophagy and sensitizes PTEN-null tumors to lysosomotropic agents.
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Chloroquine and its analogs - A new promise of an old drug for effective and safe cancer therapies.
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Acknowledgements
Dr. Neelam Shirsat from ACTREC for providing our laboratory with the C6 Glioma cell line.