Chloroquine-mediated Blockade of mTOR-independent Autophagy in Cancer

1. Chloroquine-mediated Blockade of mTOR-independent Autophagy in Cancer Garreth D’Souza, Anita Kumar, Durga Chougule, Dr. Yasmin Khan Department of Life Sciences, Sophia College, Mumbai

2. 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.

3. 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.

4. Methodologies: 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.

5. Effect of Chloroquine and/or Nimodipine on C6 Glioma cells after 72hrs of drug exposure: Control 10µL Chloroquine +100X +100X 30µL Nimodipine + 10µL Chloroquine 30µL Nimodipine +100X +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).

6. Effect of Chloroquine and/or Verapamil on C6 Glioma cells after 72hrs of drug exposure: 10µL Chloroquine Control 100X +100X +100X 30µL Verapamil 30µL Verapamil + 10µL Chloroquine +100X +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).

7. 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.

8. PRELIMINARY RESULTS Alive Dead Synergistic effects of Nimodipine and Chloroquine on induction of cell death in C6 Glioma cells in vitro. n = 6; Sets performed in duplicates.

9. CONTROL - 48 HRS 30µM NIMODIPINE + 10µM CQ – 48 HRS +400X +400X Cells treated with L-type Calcium channel blockers and/or with Chloroquine display an intensely vacuolated cytoplasm probably indicative of autophagosomes. Untreated cells lack a vacuolated cytoplasm.

10. 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 autophagy-specific construct to measure cellular levels of autophagy.

11. 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 well-tolerated 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.

12. References • Autophagy and the Integrated Stress Response. Kroemer et al. (2010) • Phosphatidylinositol 3-kinase inhibitors: promising drug candidates for cancer therapy. Kong et al. (2008) • Akt inhibition promotes autophagy and sensitizes PTEN-null tumors to lysosomotropic agents. Degtyarev et al. (2008) • Chloroquine and its analogs - A new promise of an old drug for effective and safe cancer therapies. Solomon and Lee (2009) • Novel targets for Huntington’s disease in an mTOR-independent autophagy pathway. Williams et al. (2008) • MTT ASSAY - Rapid colorimetric assay for cellular growth and survival - application to proliferation and cytotoxicity assays. Mosmann. (1983) • Calcium and cancer: targeting Ca2+ transport. Monteith et al. (2007) • Control of basal autophagy by calpain1 mediated cleavage of ATG5. Xia et al. (2010) • Reversal of chloroquine resistance in Plasmodium falciparum by verapamil. Martin et al. (1987) • Rapamycin and mTOR-independent autophagy inducers ameliorate toxicity of polyglutamine expanded huntingtin and related proteinopathies. Sarkar et al. (2009) • Identification of the Multidrug Resistance-related Membrane Glycoprotein as an Acceptor for Calcium Channel Blockers. Safa et al .(1987) • Is Resistance Useless? Multidrug Resistance and Collateral Sensitivity. Gottesman et al. (2009) Acknowledgements Dr. Neelam Shirsat from ACTREC for providing our laboratory with the C6 Glioma cell line.