Table of Contents  

Pallichankandy, Rahman, Thayyullathil, and Galadari: Sphingosine analogue FTY720 sensitizes arsenic trioxide-induced autophagic cell death in human malignant glioma cells

Introduction

Malignant glioma is the most common primary tumour of the central nervous system and the most lethal among all cancers. The current standard treatments for the management of malignant gliomas include surgical resection, radiation therapy and chemotherapy. Gliomas are notoriously resistant to therapies that induce apoptosis (type I programmed cell death). Interestingly, several lines of evidence indicate that gliomas are less resistant to therapies that induce autophagy (type II programmed cell death).1 Indeed, the autophagic cell death pathway is being explored in multiple apoptosis-resistant cancers, including gliomas, and is a promising avenue for further therapeutic development.1

Autophagic cell death is characterized by the extensive degradation of intracellular organelles and cytoplasmic proteins in double-membraned vesicles known as autophagosomes.2 Recently, interest in autophagy has been renewed among cancer biologists because different types of cancer cells were shown to undergo autophagy in response to anticancer therapies.3,4 Temozolomide, one of the most efficacious cytotoxic drugs employed in glioma therapy, exerts its activity by inducing autophagic cell death and has demonstrated a real therapeutic benefit in apoptosis-resistant glioblastoma patients.5

Arsenic trioxide (ATO) has been successfully employed in the treatment of patients with acute promyelocytic leukaemia.6,7 Recent preclinical studies have demonstrated that ATO can induce apoptotic cell death in prostate and ovarian cancers.8 It has been reported that ATO induces autophagic cell death, but not apoptotic cell death, in malignant gliomas.9 A combination treatment of glioma cells with ATO and ionizing radiation induced the synergistic augmentation of autophagic cell death and thereby increased the therapeutic efficacy of ionizing radiation.10

FTY720 is an immunosuppressant and is the first US Food and Drug Administration-approved oral drug for the treatment of multiple sclerosis.11 In addition to immunosuppression, FTY720 has also been shown to induce apoptotic cell death in a wide variety of cancers including T-cell leukaemia, multiple myeloma12 and cancers of the liver,13 breast,14 prostate,15 kidney16 and bladder.17 Moreover, FTY720 has been shown to induce autophagic cell death in ovarian cancer.18

Acquired resistance to apoptosis-inducing drugs is a roadblock in glioma therapy. Because glioma cells are less resistant to autophagy, therapeutic agents that can induce autophagic cell death and overcome tumour resistance are urgently needed. In our study we investigated the mechanism of FTY720-induced autophagic cell death in a human malignant glioma. Simultaneously, we studied the mechanisms underlying synergistic enhancement of ATO-induced autophagic cell death by FTY720 in human malignant glioma cells. We demonstrate that FTY720 potentiates the efficacy of ATO through its ability to increase the extent of ATO-induced autophagic cell death.

Materials and methods

Glioma cell lines, cell culture conditions and drug treatment

The U87MG and U118MG cells (ATCC, Rockville, MD, USA) were grown in Dulbecco’s modified essential medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum. All cell lines were grown in an incubator containing a humidified atmosphere of 95% air and 5% CO2 at 37°C. ATO (Sigma Chemical Co., St. Louis, MO, USA) at a concentration of 1 mM fresh stock was prepared before every experiment. FTY720 (Enzo Life Sciences, San Diego, CA, USA) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM and was stored in a dark-coloured bottle at –20°C. Acridine orange (AO), benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (z-VAD-fmk) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. MegaTran 1.0 transfection reagent was purchased from OriGene (Rockville, MD, USA).

Cell viability assay

Cells were grown in 96-well microtitre plates (5000 cells per well) and were incubated for 24 hours with or without drug. At the required time point, 25 μl of MTT (5 mg/ml) was added to each well. The plates were then incubated for a further 3 hours at 37°C and the formazan crystals formed were solubilized in 200 µl of DMSO. The coloured solution was quantified at 570 nm by using a 96-well plate reader (Victor X3 PerkinElmer spectrofluorometer, Waltham, MA, USA). The viability was expressed as percentage over control.

Protein lysate preparation and Western blot analysis

The protein lysate preparation, sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis were carried out as described in ‘Cell viability assay’.19 The following antibodies were used: anti-actin, anti-p62/SQSTM1 and donkey anti-goat immunoglobulin G (IgG) from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); anti-PARP, anti-light chain 3 (LC3), anti-phospho AktSer473, anti-Akt, anti-phospho mTORSer2448 and anti-mTOR from Cell Signalling Technology (Beverly, MA, USA); and anti-rabbit IgG and anti-mouse IgG were purchased from Sigma Chemical Co.

Green fluorescent protein-light chain 3 dot assay

The green fluorescent protein (GFP)-tagged microtubule-associated protein LC3 expression vector pEGFP-LC3 was obtained from Addgene 21073 (Dr Tamotsu Yoshimori’s laboratory, Osaka University, Osaka, Japan). LC3 is recruited to the autophagosomal membrane during autophagy. Therefore, GFP-tagged LC3-expressing cells have been used to demonstrate the induction of autophagy.20 During the induction of autophagy, the diffuse distribution of GFP-LC3 changes to a punctate pattern (GFP-LC3 dots). The assay was performed in both U87MG and U118MG cells stably transfected with pEGFP-LC3, using MegaTran 1.0 transfection reagent in accordance with the manufacturer’s instructions. To quantify autophagic cells after drug treatment, GFP-LC3 punctate spots were counted in 100 GFP-LC3 cells that had more than 10 bright GFP-LC3 punctate spots.

Detection and quantification of acidic vesicular organelles with acridine orange staining

Autophagy is the process of sequestering cytoplasmic proteins into lytic components and is characterized by the formation and promotion of acidic vesicular organelles (AVOs). To assess the occurrence of AVOs, after drug treatment, glioma cells were stained with AO, as described in ‘Glioma cell lines, cell culture conditions and drug treatment’, above.5 Briefly, cells were incubated with AO (1 µg/ml) for 20 minutes then examined under an Olympus DP71 fluorescent microscope (Tokyo, Japan). To quantify the development of AVOs, drug-treated cells were incubated with media containing AO (1 µg/ml) for 20 minutes and harvested using trypsin-ethylenediaminetetraacetic acid. The cells were then resuspended in phenol red-free DMEM and analysed by BD FACSCanto II using BD FACSDiva software (version 6, BD Biosciences, San Jose, CA, USA). A minimum of 10 000 cells within the gated region were analysed.

Results

FTY720 induces autophagy in human malignant glioma cells

To examine the antitumour effect of FTY720 on malignant glioma cells, two malignant glioma cell lines (U87MG and U118MG) were treated with FTY270 at concentrations ranging from 0 to 20 µM for 24 hours. As shown in Figure 1, FTY720 inhibited the viability of both cell lines in a dose-dependent manner. An increasing number of studies have shown that cancer cells, including malignant glioma cells, undergo autophagic cell death in response to various anticancer drugs.3,5 Thus, we examined whether or not FTY720 induces autophagy in these malignant glioma cells. First we determined the effect of FTY720 on the levels of AVOs by staining the cells with AO, as described previously.5 As shown in Figure 2, FTY720-treated glioma cells demonstrated AVOs by displaying bright red fluorescence. Quantitatively, fluorescence intensity was dramatically increased in U87MG cells from 3.6% to 18%, and in U118MG cells from 4.5% to 33% following FTY720 treatment (Figure 3). In order to further confirm the formation of autophagic vesicles, glioma cells stably transfected with GFP-LC3 were treated with different concentrations of FTY720. As shown in Figure 4, diffuse expression of GFP-LC3 in the untreated control cells was turned into high-intensity punctate expression in FTY720-treated cells. Quantitative analysis revealed a significant increase in GFP-LC3 punctate expression in FTY720-treated cells compared with control cells (Figure 5). Furthermore, Western blot analysis showed a drastic conversion of non-autophagic soluble LC3 (LC3-I) to autophagic LC3 (LC3-II) in response to FTY720 treatment (Figure 6). Altogether, our data establish the possible involvement of the autophagic cell death pathway in the tumour-suppressive action of FTY720 against malignant glioma cells.

FIGURE 1

Cells were treated with the indicated concentration of FTY720 for 24 hours. Following the treatment, cell viability was measured by using the MTT assay; data shown are mean ± standard deviation (SD) (n = 3).

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FIGURE 2

Cells were stained with 1 µg/ml AO at 37°C for 20 minutes. A representative slide under a fluorescent microscope shows FTY720-induced AVO formation displaying bright red fluorescence.

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FIGURE 3

Fluorescence intensity was analysed quantitatively by flow cytometry.

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FIGURE 4

The effect of FTY720 on intracellular localization of GFP-LC3 protein – glioma cells stably transfected with GFP-LC3 were treated with the indicated concentration of FTY720 for 24 hours. After treatment, GFP-LC3 dots were assessed by fluorescence microscopy.

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FIGURE 5

Puncta were counted, as described in Materials and methods. Data shown are means ± SD (n = 3).

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FIGURE 6

Effect of FTY720 on the expression of LC3-II protein – cells were treated with the indicated concentration of FTY720 for 24 hours. After the treatment, whole-cell lysates were prepared and a Western blot analysis of LC3-I and LC3-II was carried out. Actin was used as a loading control.

HMJ-607-fig6.jpg

FTY720 induces caspase-independent cell death in human malignant glioma cells

FTY720 has been shown to induce apoptosis in some cancer cell lines.12,14,15 In order to determine if FTY720 induces apoptosis in malignant glioma cells, one of the prime hallmarks of apoptotic cell death, PARP cleavage, was examined. FTY720 did not induce PARP cleavage even at higher concentrations in both glioma cell lines (Figure 7). Moreover, pre-treatment of U87MG cells with the pancaspase inhibitor z-VAD-fmk failed to inhibit FTY720-induced cell death (Figure 8), suggesting that the caspase-mediated apoptotic pathway is not involved in FTY720-induced cell death in malignant glioma.

FIGURE 7

Effect of FTY720 on PARP cleavage: cells were treated with the indicated concentration of FTY720 for 24 hours. After the treatment, whole-cell lysates were prepared and a Western blot analysis of PARP was carried out.

HMJ-607-fig7.jpg
FIGURE 8

U87MG cells were pretreated with 50 µM z-VAD-fmk followed by 15 µM FTY720 for 24 hours. After the treatment, cell viability was assessed by a MTT assay. Data shown are means ± SD (n = 3).

HMJ-607-fig8.jpg

Effects of bafilomycin A1 on FTY720-induced autophagic cell death in U87MG cells

In order to validate the contribution of autophagy in FTY720-induced cell death, we analysed the effect of bafilomycin A1 (Baf A1), a well-established inhibitor of autophagy that prevents the maturation of autophagic vacuoles by inhibiting the fusion between autophagosomes and lysosomes.21 Pretreatment of U87MG cells with Baf A1 for 1 hour followed by FTY720 treatment and AO staining resulted in a significant decrease in FTY720-induced AVOs displaying bright red fluorescence compared with FTY720-only treated cells (Figure 9). Quantitatively, fluorescence intensity was measured using fluorescence-activated cell sorting analysis (Figure 10). Next we examined the autophagic flux by using a LC3 turnover assay, which measures the autolysosomal degradation of LC3-II by comparing the amount of LC3-II in the presence and absence of Baf A1. Pretreatment of U87MG cells with Baf A1 resulted in an increase in FTY720-induced LC3-II accumulation, suggesting an increase in the autophagic flux (Figure 11). In addition, pretreatment with Baf A1 significantly protected U87MG cells from FTY720-induced loss of viability (Figure 12).

FIGURE 9

A representative slide under a fluorescent microscope shows AVOs displaying bright red fluorescence. Cells were pretreated with 250 nM Baf A1 followed by 15 µM FTY720 treatment for 24 hours. Following the treatment, cells were stained with 1 µg/ml AO at 37°C for 20 minutes.

HMJ-607-fig9.jpg
FIGURE 10

Acridine orange-stained cells were quantitatively analysed by flow cytometry.

HMJ-607-fig10.jpg
FIGURE 11

Western blot analysis of LC3-I and -II. Actin was used as a loading control.

HMJ-607-fig11.jpg
FIGURE 12

Cell viability was assessed by MTT assay. Data shown are means ± SD (n = 3).

HMJ-607-fig12.jpg

FTY720 augments arsenic trioxide-induced autophagic cell death in malignant glioma cells

Next we examined whether or not FTY720 treatment in combination with ATO has any sensitizing effect on cell death in human malignant glioma. As shown in Figure 13, treatment with either ATO (2 µM) or FTY720 (5 µM) resulted in very little cell death. However, the combination treatment synergistically enhanced cell death in a significant manner (see Figure 13).

FIGURE 13

Cells were treated with or without FTY720 (5 µM) for 24 hours in the presence or absence of ATO (2 µM). Following the treatment, cell viability was assessed by MTT assay. Data shown are means ± SD (n = 3).

HMJ-607-fig13.jpg

Arsenic trioxide alone has been reported to induce autophagy in malignant glioma cells.9 In order to determine if autophagy is the mode of cell death that is induced by FTY720–ATO combination, U87MG cells were treated with FTY720, ATO or FTY720–ATO in combination for 24 hours. Following the treatment, the cells were stained with AO and the formation of AVOs was examined by fluorescence microscopy and flow cytometry. The fluorescence intensity was significantly enhanced in the cells treated with FTY720–ATO combination when compared with either FTY720 or ATO treatment (Figures 14 and 15). Furthermore, compared with either FTY720 or ATO alone, FTY720–ATO combination treatment significantly increased GFP-LC3 puncta formation (Figures 16 and 17) and LC3-II conversion (Figure 18, top panel). In order to conclusively establish the induction of autophagy, the effect of FTY720–ATO combination treatment on the expression of p62/SQSTM1, an LC3-interacting protein that gets degraded during autophagy,22 was examined. Treatment of U87MG cells with FTY720–ATO combination resulted in a considerable decrease in detectable p62/SQSTM1 protein levels (see Figure 18, middle panel). Thus, an increase in AVOs displaying bright red fluorescence, GFP-LC3 puncta formation, LC3-II conversion and p62/SQSTM1 degradation demonstrate that induction of autophagy is the prime cause of cell death following treatment of the cells with FTY720–ATO combination.

FIGURE 14

Cells were stained with 1 µg/ml AO at 37°C for 20 minutes and fluorescent images were taken using an Olympus DP71 fluorescent microscope.

HMJ-607-fig14.jpg
FIGURE 15

Acridine orange-stained cells were quantitatively analysed by flow cytometry.

HMJ-607-fig15.jpg
FIGURE 16

U87MG cells stably expressing GFP-LC3 were treated with or without FTY720 (5 µM) for 24 hours in the presence or absence of ATO (2 µM). Following the treatment, images for GFP-LC3 puncta were taken using an Olympus DP71 fluorescent microscope.

HMJ-607-fig16.jpg
FIGURE 17

Puncta were counted, as described in Materials and methods. Data shown are means ± SD (n = 3).

HMJ-607-fig17.jpg
FIGURE 18

Cell were treated with or without FTY720 (5 µM) for 24 hours in the presence or absence of ATO (2 µM). Following the treatment, whole-cell lysates were prepared and a Western blot analysis of LC3-I, LC3-II and p62/SQSTM1 was carried out. Actin was used as a loading control.

HMJ-607-fig18.jpg

Akt (also known as protein kinase B) is a key cell survival and proliferative protein which belongs to the family of phosphatidylinositol 3-OH-kinase-regulated Ser/Thr kinases (Akt 1, 2, 3).23 Phosphorylation of Akt at its two major regulatory phosphorylation sites, Ser-473 and Thr-308, activates the protein.24 Recent investigations have shown that the inhibition of mTOR, one of the downstream targets of the Akt signalling pathway, is linked to the activation of autophagy.25 Thus, we sought to test whether or not FTY720–ATO combination treatment could induce autophagy through inhibition of the Akt/mTOR signalling pathway. As shown in Figure 19, FTY720–ATO combination treatment caused a marked reduction in Akt phosphorylation at Ser-473 along with the inhibition of mTOR phosphorylation at Ser-2448.

FIGURE 19

FTY720–ATO combination treatment markedly inhibits the activation of Akt and mTOR. Cells were treated with or without FTY720 (5 µM) for 24 hours in the presence or absence of ATO (2 µM). Following the treatment, whole-cell lysates were prepared and a Western blot analysis of p-Akt Ser473, Akt, p-mTOR Ser2448 and mTOR was carried out. Actin was used as loading control.

HMJ-607-fig19.jpg

Discussion

Although the therapeutic potential of FTY720 in the treatment of various types of cancers has been studied, the underlying mechanisms remain poorly understood.1318 The findings described in this study demonstrate that FTY720 is a potent antitumour agent against human malignant glioma cells. FTY720 at different concentrations is cytotoxic to U87MG and U118MG cells, yet did not cause PARP cleavage, suggesting that the classical apoptosis pathway is not the main mechanism of FTY720-induced cell death. In contrast, the features of autophagy, including increase in AVO formation, GFP-LC3-positive puncta formation and LC3-II conversion, were seen following FTY720 treatment.

Gliomas are very resistant to therapies that induce apoptosis. However, the growing body of evidence indicates that glioblastoma cells are less resistant to therapies that induce autophagy.1 The role of autophagy in cancer therapeutics is still controversial because autophagy also affords a means of survival for cancer cells by which cells clear damaged cytoplasmic proteins and organelles through lysosomal degradation and survive metabolic stress.26 On the other hand, autophagy has been considered as a mechanism for cell death in response to oxidative stress and various chemotherapeutic agents.27 Our data demonstrate that FTY720 is a potent inducer of autophagic cell death in human malignant glioma cells. FTY720-induced cell death was significantly inhibited by the autophagy inhibitor (Baf A1), whereas the caspase inhibitor (z-VAD-fmk) was unable to do so, suggesting that autophagy, rather than apoptosis, is the key signalling mechanism for the tumoricidal effect of FTY720.

The prime factor in designing a new anticancer drug is the understanding of the potential for combination treatment regimens. A combination of different antitumour agents is often advantageous in eliminating the non-specific side effects usually observed by an exceedingly high dose of a single regimen. Though ATO has been shown to induce autophagic cell death in glioma,9,10 several phase II clinical trials in cancer patients have shown that ATO alone has a very low efficacy in the treatment of solid tumours.28 These observations prompted us to examine the combined effect of ATO and FTY720 on the cell death mechanisms of malignant glioma. We believe that understanding the mechanism of FTY720 in combination with ATO may help in designing more effective therapy against malignant glioma. Our study demonstrated a synergistic augmentation of cytotoxic effects of ATO when combined with FTY720 in U87MG cells. An emerging body of evidence indicates that the Akt/mTOR pathway plays a crucial role in the regulation of apoptosis and autophagy.25 Data from this study indicate that FTY720–ATO combination treatment markedly inhibits Akt phosphorylation at Ser-473 and mTOR phosphorylation at Ser-2448.

Conclusion

Taken together, these findings provide a strong basis for using therapies that combine FTY720 and ATO for the treatment of malignant gliomas, which are resistant to pro-apoptotic therapies. Further experiments in animal models, however, are needed to fully understand the potential of FTY720 as a therapeutic agent to improve the clinical efficacy of ATO against malignant gliomas.

Acknowledgements

This work was financially supported by grants from the Sheikh Hamdan Award for Medical Sciences (MRG-60/2011–2012).

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