脳腫瘍(Brain Cancer)
ブラックシードのチモキノンの成分は神経膠芽腫細胞において細胞死を誘導することができます。グリア芽腫は脳腫瘍に一番見られる現象です。(細かい情報へ)
Thymoquinone Inhibits Autophagy and Induces Cathepsin-Mediated, Caspase-Independent Cell Death in Glioblastoma Cells
Ira O. Racoma,1 Walter Hans Meisen,2 Qi-En Wang,1 Balveen Kaur,2 and Altaf A. Wani1,*
Shawn B. Bratton, Editor
This article has beencited by other articles in PMC.
Abstract
Glioblastoma is the most aggressive and common type of malignant brain tumor in humans, with a median survival of 15 months. There is a great need for more therapies for the treatment of glioblastoma. Naturally occurring phytochemicals have received much scientific attention because many exhibit potent tumor killing action. Thymoquinone (TQ) is the bioactive compound of the Nigella sativa seed oil. TQ has anti-oxidant, anti-inflammatory and anti-neoplastic actions with selective cytotoxicity for human cancer cells compared to normal cells. Here, we show that TQ selectively inhibits the clonogenicity of glioblastoma cells as compared to normal human astrocytes. Also, glioblastoma cell proliferation could be impaired by chloroquine, an autophagy inhibitor, suggesting that glioblastoma cells may be dependent on the autophagic pathway for survival. Exposure to TQ caused an increase in the recruitment and accumulation of the microtubule-associated protein light chain 3-II (LC3-II). TQ also caused an accumulation of the LC3-associated protein p62, confirming the inhibition of autophagy. Furthermore, the levels of Beclin-1 protein expression were unchanged, indicating that TQ interferes with a later stage of autophagy. Finally, treatment with TQ induces lysosome membrane permeabilization, as determined by a specific loss of red acridine orange staining. Lysosome membrane permeabilization resulted in a leakage of cathepsin B into the cytosol, which mediates caspase-independent cell death that can be prevented by pre-treatment with a cathepsin B inhibitor. TQ induced apoptosis, as determined by an increase in PI and Annexin V positive cells. However, apoptosis appears to be caspase-independent due to failure of the caspase inhibitor z-VAD-FMK to prevent cell death and absence of the typical apoptosis related signature DNA fragmentation. Inhibition of autophagy is an exciting and emerging strategy in cancer therapy. In this vein, our results describe a novel mechanism of action for TQ as an autophagy inhibitor selectively targeting glioblastoma cells.
Introduction Glioblastoma is a grade IV glioma and remains the most aggressive and devastating cancer of the central nervous system[1]. It is the most common brain tumor diagnosed in adults, with about 9,000 new diagnoses annually in the United States alone. Adding to this statistic is the number of recurring tumors, which occurs in a vast majority of cases. The standard of care for newly diagnosed glioblastoma is surgical resection of the tumor, followed by radiation therapy with concomitant and adjuvant chemotherapy with the alkylating agent temozolomide (TMZ). Despite this and other medical advances in the treatment of glioblastoma, the median survival time for patients is approximately 15 months from the first diagnosis. The molecular alterations that promote tumorigenesis and sustained growth of glioblastoma also serve to promote resistance to apoptosis[2],[3]. In recurrent glioblastomas, anti-apoptotic Bcl-2 and Bcl-XL proteins of the Bcl-2 family are up-regulated, but the pro-apoptotic Bax and Bak proteins are down-regulated. This suggests that glioblastomas might naturally be under a selection pressure to develop resistance to apoptosis[2]. Another anti-apoptotic protein Bcl-2L12 is found to be up-regulated in almost all glioblastomas and contributes to apoptosis resistance by inhibiting caspase activation[2].
Recent studies concerning a number of different tumors, including glioblastoma [4]?[6] have alluded to the fact that cancer cells are significantly more dependent on autophagy for survival than non-cancer cells [7]?[11]. Autophagy is a lysosomal-dependent degradation system that functions to maintain cellular homeostasis by recycling unneeded proteins, eliminating defective organelles, and sustaining cell growth during brief periods of starvation and other stressors [12], [13]. It has been suggested that many oncoproteins such as the previously mentioned anti-apoptotic members of the Bcl2 family, phosphatidylinositol 3-kinase, and Akt suppress any autophagy beyond basal levels. However once a tumor has formed, autophagy is activated as a means to generate ATP and overcome the metabolic stress of the tumor environment [7], [14]. Additionally, many anti-cancer drugs up-regulate autophagy, which can lead to recalcitrant tumors [9], [15], [16]. Recent studies have demonstrated that pharmacological or genetic inhibition of autophagy enhances the effects of conventional radio- and chemotherapy [7], [11], [17], suggesting that inhibition of autophagy might be a viable and auspicious strategy for cancer treatment. At the moment, chloroquine (CQ) and its derivative hydroxychloroquine (HCQ), which have both been used for years as anti-malarial and anti-rheumatoid arthritis drugs, are the only autophagy inhibitors in clinical trials for cancer therapy [4], [18]. CQ and HCQ are lysosomotropic agents, thereby preventing lysosome acidification and subsequent fusion of the autophagosome with the lysosome. However, long-term administration of chloroquine can result in retinopathies [19] which may limit its use as a chemotherapeutic.
There has been a growing interest in natural compounds with anti-cancer properties precisely because they are relatively non-toxic to healthy cells and are available in a readily-ingested form. The dietary phytochemical thymoquinone (TQ) is the primary bioactive component of Nigella sativa Linn seed (also known as black seed) oil. Nigella sativa has been used for centuries in Middle Eastern, Indian and European countries for culinary purposes and to promote good health [20], with the beneficial properties being attributed to TQ [21]. Its purported health benefits include anti-inflammatory, anti-oxidant and anti-hypertensive actions. We have shown that TQ induces apoptosis in HL-60 leukemia cells and MCF-7/DOX doxorubicin-resistant breast cancer cells [22], [23]. Additionally, a number of other studies have reported that the anti-tumor functions of TQ are specific for cancer cells [24], [25]. Pharmacokinetic studies have shown that mice and rats can consume large amounts of TQ without adverse effects [26]. Importantly, TQ readily crosses the blood-brain barrier due to its small size and lipophilicity [26], [27].
Here we report a novel mechanism of action for TQ which involves the inhibition of autophagy in glioblastoma cells via perturbation of the lysosomal membrane and cathepsin translocation from the lysosomal lumen to the cytosol, leading to caspase-independent apoptosis.
Materials and Methods
Cell culture and reagents
Human glioblastoma cells T98G and U87MG were kindly provided by Dr. Arnab Chakravarti, purchased from ATCC (ATCC numbers CRL-1690 and HTB-14, respectively). T98G and U87MG cells were grown in DMEM (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) and 0.05% penicillin/streptomycin (Life Technologies). Human glioblastoma cells Gli36ΔEGFR containing a mutant EGFR (vIII) [28], [29] were kindly provided by Dr. Balveen Kaur, and were grown in DMEM supplemented with 10% fetal bovine serum, 0.05% penicillin/streptomycin, 2 μg/ml puromycin (Sigma, St. Louis, MO) and 4 μg/ml blasticidin (Sigma). Normal human astrocytes (NHA) were grown in Complete Astrocyte Medium (kit), both purchased from ScienCell Research Laboratories (Carlsbad, CA) on poly-l-lysine coated plates (ScienCell Research Laboratories). All cells were grown at 37°C in a humidified 5% CO2 atmosphere. Thymoquinone, chloroquine, acridine orange and MTT were purchased from Sigma. Cathepsin inhibitor III (peptide sequence: Z-Phe-Gly-NHO-Bz-pOMe) was purchased from EMD Chemicals (Darmstadt, Germany). z-VAD-FMK was purchased from R&D Systems (Minneapolis, MN).
Clonogenic cell-survival assays
Cells were plated at a low density
and allowed to attach for 4?6 hours before treating with increasing
concentrations of TQ or chloroquine. Cells were allowed to grow until colonies
contained >50 cells per colony (7?10 days). After the growth period, cells
were fixed with 3.7% paraformaldehyde for 15 minutes at room temperature,
followed by staining with 1% methylene blue, also for 15 minutes at room
temperature. Colonies were defined as having >50 cells and were manually
counted. The surviving fraction (SF) for each treatment was calculated as a
ratio of the number of colonies counted to the number of cells plated
multiplied by plating efficiency. Lines were generated through best fit
non-linear regression automatic analysis with TableCurve software (Systat
Software Inc., San Jose, CA). Alternatively, cells were also plated at a low
density, allowed to attach for 4?6 hours prior to treating with TQ. Cells were
grown until untreated controls were confluent, then they were fixed with 3.7%
paraformaldehyde and stained with 1% methylene blue as described above, air
dried, photographed and evaluated for colony estimation.
Cell viability assay
Quantitation of cell viability was
determined by the MTT assay. Cells were seeded in 96-well plates, pre-treated
with Z-VAD-FMK (10 or 20 μM) or cathepsin inhibitor III cocktail (5 or 10 μM)
for 1 hour followed by TQ (20 μM or 40 μM) for 24 hours. MTT solution was then
added to the cultures to a final concentration of 0.5 mg/ml and incubated at
37°C for an additional 4 hours. The medium was aspirated and the formazan
crystals were solubilized with DMSO after which the absorbance was determined
by a uQuant microplate reader (Biotek Instruments, Winooski, VT) at 570 nm.
Apoptosis assay
Apoptosis was determined by flow cytometric analysis of PI and Annexin V stained cells. Cells were treated with increasing concentrations of TQ for 6 hours, washed and pelleted. Each sample was resuspended in 100 μl of staining solution composed of Annexin V (Molecular Probes, Carlsbad, CA) and propidium iodide (Sigma) in assay binding buffer, according to manufacturer's instructions. Cells were incubated in the dark at room temperature for 15 minutes and immediately analyzed by an Aria III flow cytometer (BD Biosciences, San Jose, CA).
DNA fragmentation analysis
Cells were treated with increasing concentrations of TQ for 24 hours. Detailed procedure for DNA fragmentation analysis has been described previously [23]. Briefly, adherent and floating cells were pooled together and lysed. RNA and protein were degraded by incubation with RNase and Proteinase K, respectively. DNA was precipitated with 100% ethanol and resuspended in TE buffer, pH 8. DNA was evaluated for fragmentation by resolving on a 1% agarose gel, stained with ethidium bromide and visualized under UV light.
Western blot analysis
Cells were treated with TQ, chloroquine or a combination of the two for 24 hours. Cells were harvested and lysed as previously described [23]. 50 μg of total protein for each sample was separated by SDS-PAGE as follows: LC3 was resolved with a 12% gel; p62/SQSTM1 and Beclin-1 were resolved with an 8% gel. Proteins were transferred to PVDF membranes and blocked with 5% non-fat dry milk in TBST buffer (blocking buffer) at 37°C for one hour. Membranes were incubated in primary antibody at 4°C overnight with rocking, followed by washing three times with TBST buffer then incubated with the appropriate HRP-conjugated secondary antibody at 37°C for one hour. The membranes were washed three times with TBST buffer and examined by chemiluminescence detection.
Antibodies against the following proteins were diluted in blocking buffer: β-actin, diluted 1/5000 (Santa Cruz Biotechnology, Santa Cruz, CA); microtubule associated light chain 3 (LC3), diluted 1/1000; p62/SQSTM1, diluted 1/1000; and Beclin-1, diluted 1/1000 (all from Cell Signaling Technology, Danvers, MA).
Acridine orange staining
Acidic lysosomes were labeled with acridine orange. Cells were grown on coverslips and treated with TQ or chloroquine for 1 or 6 hours. After treatment, cells were washed once with PBS containing Ca2+ and Mg2+ and stained with 1 μM acridine orange at 37°C for 15 minutes [30], [31]. Excess acridine orange was washed away with PBS and cells were immediately examined on a Nikon Eclipse 80i (Nikon Instruments Inc., Melville NY) wide field fluorescent microscope.
Statistical Analysis
Statistical significance of differences observed between TQ-treated and TQ in combination with either Z-VAD-FMK or cathepsin inhibitor were determined using the Student's t-test. Differences were considered to be significant at p<0.05.
Results
TQ inhibits glioblastoma cell proliferation
TQ has shown to be effective in
killing a number of different tumor types. To test the effect of TQ on
glioblastoma cell proliferation, we subjected a heterogeneous set of
glioblastoma cells to a clonogenicity based cell survival assay, which
accurately assesses the inhibition of cell proliferation in response to a
cytotoxic or cytostatic agent. T98G (p53mut), U87MG (p53wt)
and Gli36ΔEGFR (p53mut) cells were plated at a low density and
incubated with TQ for 7?10 days. After the growth period, the colonies formed
were fixed and counted, and represented as a surviving fraction of the initial
number of cells seeded. TQ prevented glioblastoma cell proliferative capacity
in the three cell lines we tested, albeit to varying degrees (Figure
1A).
For example, Gli36ΔEGFR cells were found to be the most sensitive to TQ (IC50?=?2.4
μM), while T98G cells were the most resistant (IC50?=?10.3 μM) and
U87MG cells exhibited intermediate sensitivity (IC50?=?8.3 μM). The
sensitivity of each cell line does not appear to correlate with p53 status,
which is in agreement with earlier studies showing that TQ is cytotoxic to
cancer cells independent of p53 status [22], [32]. We also found that
normal human astrocytes were not sensitive to TQ by comparing their growth
against Gli36ΔEGFR cells. The survivability of diffusely growing NHA could not
be determined from colony formation as they fail to produce distinct countable
colonies. Their growth inhibition was determined from visual inspection of cell
populations against varying TQ concentrations. As can be seen in Figure
1B,
NHA proliferation was not affected at least up to 8 μM TQ with some easily
visible cell growth even at TQ concentrations up to 16 μM. On the other hand, 2
μM of TQ was enough to drastically inhibit growth of Gli36ΔEGFR cells, with no
visible cells at 4 μM. These results are in full agreement with the Gli36ΔEGFR
colony formation results in Figure
1A.
TQ inhibits glioblastoma
cell proliferation.
Autophagy inhibition
prevents glioblastoma cell proliferation
Once a tumor has formed, they have
been shown to be more dependent on autophagy as a means to survive the
metabolic stress of the tumor environment [33], [34]. Pharmacological or
genetic inhibition of autophagy is reported to induce cell death in cancer
cells. Therefore, we wanted to determine the sensitivity of glioblastoma cell
proliferation to pharmacologic inhibition of autophagy with chloroquine. We
performed a clonogenic assay as described above for TQ with increasing
concentrations of chloroquine and found that chloroquine reduced the proliferative
capacity of U87MG and Gli36ΔEGFR cells to a similar extent (IC50?=?3.6
μM and 4.4 μM, respectively) in each cell line (Figure
2).
Chloroquine inhibits
glioblastoma cell proliferation.
Thymoquinone inhibits
autophagic flux in glioblastoma cells
Autophagic flux is defined as the
complete process of autophagy beginning with formation of the autophagosome
around the cargo, followed by fusion of the autophagosome with the lysosome,
then ending with degradation and recycling of the cargo [12], [35]. To investigate the
effect of TQ on autophagic flux, we treated U87MG and Gli36ΔEGFR cells with TQ,
chloroquine or a combination of the two and subjected whole cell lysates to
Western blotting to determine changes in expression of LC3-II, p62 and
Beclin-1. Microtubule-associated protein 1 light chain 3, or LC3, a
ubiquitin-like protein, is synthesized in an unprocessed form called pro-LC3.
Proteolytic processing yields the 16 kDa form called LC3-I, which can then be
conjugated to phosphatidyl ethanolamine (PE) during autophagy to yield the 14
kDa form called LC3-II. LC3-II is widely used as a marker for complete
autophagosomes [35]?[37]. As shown in Figure
3A,
treatment with increasing concentrations of TQ for 24 hours resulted in a
dose-dependent increase of LC3-II. Treatment with chloroquine alone greatly
increased LC3-II levels. A combination of chloroquine and increasing
concentrations of TQ resulted in more LC3-II compared to TQ alone, which may be
due to either autophagy induction or autophagy inhibition. However, combination
treatment compared to chloroquine alone did not result in an increase of
LC3-II, suggesting that TQ is blocking autophagy.
TQ blocks autophagy in
glioblastoma cells.
To further explore the effect of TQ
on autophagic flux, we also measured the changes in p62 levels. p62, which is
also known as SQSTM1, is a ubiquitin-binding protein that is involved in
lysosome- or proteasome-dependent degradation of proteins. It incorporates into
the autophagosome via direct interaction with LC3-II and is degraded in the
process of autophagy. Inhibition of autophagy leads to increased levels of p62 [35], [36]. As shown in Figure
3B,
treatment with increasing concentrations of TQ for 24 hours resulted in a
dose-dependent increase of p62 levels. Furthermore, combination treatment with
chloroquine and increasing concentrations of TQ resulted in a dose-dependent
increase of p62 levels compared to either TQ or chloroquine alone, confirming
that TQ inhibits autophagic flux in these glioblastoma cells. In further
support of TQ as an autophagy inhibitor, we measured the levels of Beclin-1.
Beclin-1 levels are associated with autophagy induction, as it is needed to
initiate autophagosome formation [12]. After 1 hour treatment
with TQ, Beclin-1 levels do not change, providing additional evidence of TQ as
an autophagy inhibitor (Figure
3C).
Lysosomal membrane
permeabilization (LMP) and cathepsin activity is involved in
thymoquinone-induced cell death
Increased expression of LC3-II and
p62, taken together with stable levels of Beclin-1, strongly indicate that TQ
inhibits a later stage of autophagy, particularly at the point of
autophagosome-lysosome fusion. Upon inhibition of autophagy at this stage,
cells begin to display cytoplasmic vacuolization [38], which has been reported
by a number of studies [38]?[41]. Consistent with these
findings, chloroquine and TQ promote cytoplasmic vacuolization in both U87MG
and Gli36ΔEGFR cells at 6 hours, but can be seen as early as 1 hour in U87MG
cells (Figure
4A).
40 μM CQ induced less vacuolization in both cells lines compared to TQ. In
U87MG cells, both concentrations of TQ induced the same extent of
vacuolization. In Gli36ΔEGFR cells, 20 μM TQ caused more, but smaller vacuoles
than 10 μM TQ. An apparently anomalous dose-dependent change in vacuole size
has been observed previously in macrophages in which 100 μM chloroquine causes
smaller vacuolization than 30 μM [42].
Lysosomal membrane
permeabilization (LMP) and cathepsin activity is involved in thymoquinone-induced
cell death.
Defective autophagosome-lysosome
fusion can be attributed to a perturbation of lysosomal activity [43]. Therefore, we sought to
evaluate the effect of TQ on the lysosome by first examining lysosome membrane
permeability (LMP). Disruption of the lysosomal membrane can play a major role
in programmed cell death by mediating both caspase dependent and independent
cell death. To assess the effect of TQ on lysosomal membrane permeability
(LMP), lysosomes were labeled with acridine orange (AO). AO is a weakly basic
lysosomotropic dye that has differential staining capabilities. At a low pH, AO
will fluoresce orange-red, but at neutral pH it will fluoresce green. Untreated
cells prominently display punctate orange-red staining of lysosomes throughout
the cytoplasm, while in CQ-treated cells there is a marked decrease in
orange-red staining. After TQ treatment, while there is a modest decrease in
red acridine orange staining in U87MG cells,Gli36ΔEGFR cells exhibited a
considerable decrease of red staining(Figure
4B).
Furthermore, TQ appears to be more effective at destabilizing the lysosomal
membrane than the well-characterized lysosomotropic agent chloroquine. TQ
treatment also appears to affect lysosome localization. In untreated cells,
lysosomes can be seen dispersed throughout the cytosol and at the edges of the
cells, whereas treatment with TQ appears to cause the remaining intact
lysosomes to cluster around the nucleus. Cancer cells often display peripheral
localization of lysosomes, particularly those at the invasive edges of tumors [38] however, non-cancer
cells typically have a perinuclear arrangement of lysosomes.
A direct consequence of LMP is the
translocation of cathepsin proteases and other hydrolytic enzymes from the
lysosomal lumen to the cytosol [38], [44]. While all have optimal
activity at the low pH inside lysosomes, a few, including the cysteine
cathepsins, are still functional at neutral pH [45] and capable of signaling
to downstream mediators of cell death. To evaluate the contribution of
cathepsin activity to TQ-mediated cell death, U87 and Gli36ΔEGFR cells were
incubated with TQ, either with or without a cathepsin inhibitor cocktail (Cathepsin
inhibitor III, EMD Millipore), which primarily targets the cysteine cathepsin
B, for 24 hours. As shown in Figure
4C,
the presence of a cathepsin inhibitor significantly increased the percentage of
viable TQ-treated cells compared to TQ treatment alone.
Thymoquinone induces
caspase-independent apoptotic cell death in glioblastoma cells
One of the hallmarks of glioblastoma
cells is their resistance to apoptotic cell death following radiation and
chemotherapy [46]?[50]. This is thought to be a
direct result of upregulated survival signaling and increased expression of
anti-apoptosis proteins. As TQ has been shown to induce apoptosis in a variety
of cancer cells, we sought to determine whether TQ can also induce apoptosis in
glioblastoma cells by labeling cells with propidium iodide and Annexin V to
detect phosphatidyl serine (PS) flipping. PS is localized to the cytoplasmic
face of the cell membrane in healthy cells, but translocates to the
extracellular face of the cell membrane in apoptotic cells and thus can be
detected by conjugation to Annexin V. This translocation process occurs
relatively early in the cell death program. After treating cells with
increasing concentrations of TQ, a dose dependent increase in PI and Annexin V
double positive cells was observed. In U87MG cells, approximately 15% of the
cells are double positive for PI and Annexin V after treatment with TQ for 6
hours (Figure
5A).
The effect is even more dramatic in Gli36ΔEGFR cells, as approximately 50% of
the cells are undergoing apoptosis (Figure
5A).
Interestingly, the apoptotic program in glioblastoma cells appears to be
caspase independent. We pre-treated cells for 1 hour with the general caspase
inhibitor z-VAD-FMK prior to incubation with TQ for an additional 24 hours.
Cell viability was then measured with the MTT assay. As expected, TQ alone
reduced the cell viability of U87MG cells by approximately 43%, and of
Gli36ΔEGFR cells by approximately 53%. Once again, however, pre-treatment with
either 10 μM or 20 μM z-VAD-FMK failed to revert the viability of either cell
line in the presence of TQ (Figure
5B),
although this is within the effective concentration range of this general
caspase inhibitor [51]. We evaluated the degree
of DNA fragmentation in glioblastoma cells after treatment with increasing
concentrations of TQ. The degradation of DNA into fragments of multiples of 180
base pairs is carried out by certain caspase-activated DNases, and is an
essential feature of classical apoptotic cell death. After treating with
increasing concentrations of TQ, mostly high molecular weight virtually intact
DNA was recovered from U87MG and Gli36ΔEGFR cells, similar to what is seen in
the untreated controls (Figure
5C).
The results for both cell lines are in contrast to the extensive fragmentation
observed in TQ-treated doxorubicin-resistant MCF-7 cells, which we have
previously reported to die specifically by apoptosis in a caspase-dependent
manner upon TQ treatment [23].
TQ induces
caspase-independent apoptosis.
Discussion
Glioblastoma is the most common and
the most aggressive malignant astrocytic brain tumor in adults. The median
survival time from the first diagnosis is approximately 15 months. Tumor
resection followed by ionizing radiation (IR) therapy and chemotherapy with the
alkylating agent temozolomide (TMZ) is the standard of care for glioblastoma,
however a majority of patients will experience a recurrence of the tumor around
the same location as the original tumor. Tumors recur for several reasons:
glioblastomas are highly infiltrative and diffuse, therefore de-bulking cannot
remove all of the initial cancer cells; they have a high degree of molecular
heterogeneity, therefore radiation and chemotherapy will not kill the entire
population of diverse cells of a single tumor. In fact, when combined with IR,
TMZ is only successful in the subset of patients who carry a methylated O6-methylguanine-DNA
methyltransferase (MGMT) promoter[52].
Thymoquinone (TQ) is the most abundant bioactive component of the Nigella sativa (black cumin) seed oil. We and others [22], [23], [32], [53] have previously shown that TQ induces apoptosis in several experimental cancer models. In the present study, we describe a novel mechanism of action for TQ in apoptosis-resistant glioblastoma cells and show that TQ inhibits the autophagic flux by inducing lysosome membrane permeabilization and subsequent translocation of lysosomal hydrolases to the cytosol (Figure 6).
Inhibition of late stage autophagy by TQ.
A clonogenic assay revealed that TQ inhibits growth in three different glioblastoma cells to different extents; however the p53 status of the cells does not appear to affect the mechanism by which TQ inhibits growth and subsequent colony formation. This is not surprising as TQ has been reported to be cytotoxic to p53-wild type [32] and p53-mutant [22] tumor cells. We also found that growth of normal human astrocytes is not affected by concentrations of TQ that are cytotoxic to glioblastoma cells. This is in agreement with previous reports showing that TQ is selective for cancer cells [24], [25], [54]. It is important to note that TQ readily crosses the blood brain barrier: in mouse models of petit mal epilepsy, TQ administered by intraperitoneal injection was shown to delay the onset and decrease the duration of seizures [26], and in rat models of ischemia-induced brain injury, TQ administered in drinking water prevented the depletion of several crucial endogenous neuronal antioxidants [27]. Given these precedents in in vivo models of two different neurological pathologies, and considering the chemical properties of TQ, it is reasonable to expect that TQ may provide a therapeutic benefit in both animal models of glioblastoma and in patients.
Here, we show that glioblastoma cells are sensitive to growth inhibition by chloroquine. Several studies have reported on the enhancing effect of chloroquine on traditional chemotherapies and on ionizing radiation therapy [7], [11], [17], including a small, randomized, double blind and placebo controlled trial in which the median survival for glioblastoma patients receiving chloroquine in addition to conventional radiation and chemotherapy was 24 months, compared to the placebo group for whom median survival was 11 months [4]. Chloroquine and its analog hydroxychloroquine accumulate in the lysosome as they become protonated within the acidic environment and cannot diffuse back out. The ability for chloroquine alone to cause a dose-dependent inhibition in glioblastoma cells strongly suggests that these cells are highly dependent on autophagy. Single-treatment with chloroquine was found to induce both apoptosis and necrosis, depending on the concentration, in A549 lung cancer cells [55].
TQ causes a time- and dose-dependent formation of cytoplasmic vacuoles. Many drugs, including chloroquine, neutral red, propranolol, atropine, and lidocaine, induce cytoplasmic vacuolization [42], [43]. Upon exposure to cytotoxic compounds, cells will attempt to sequester the drug into vacuoles to protect themselves. Vacuoles disappear after removal of the drug from the cell culture media, but prolonged vacuolization of the lysosome might lead to irreversible changes that result in cell death, particularly the release of a cationic form of the drug back into the cytosol. This implies that the lysosome membrane is now permeable [43].
We then demonstrate that TQ causes lysosome membrane permeability (LMP) via a decrease in red acridine orange staining. An obvious corollary to LMP is the leakage of lysosomal proteases, such as cathepsins, from the lysosome to the cytosol. While most cathepsins are non-functional at the relatively neutral pH of the cytosol, several, namely cathepsins B and D, are still active and capable of signaling to downstream mediators of cell death such as the mitochondria [44]. In some cell types, released cathepsins can induce apoptotic cell death [56], but in apoptosis-resistant cells they mediate caspase-independent cell death [56], [57]. Furthermore, cathepsin B is overexpressed in gliomas and serves as a strong prognostic marker for primary tumors of the CNS [58]. While this change allows for a potentiation of angiogenic and metastatic capacity of tumors, it also confers a susceptibility to lysosome membrane permeabilization [56] by chemotherapeutic agents. It is known that microtubule poisons, such as paclitaxel and vincristine, are capable of inducing LMP [38], [44], [57] and interfering with lysosomal trafficking [59]. TQ was recently demonstrated to target microtubules, causing a time and dose-dependent degradation of α and β-tubulin in U87MG glioblastoma and Jurkat T lymphoblastic leukemia cells [54]. Taken together, these reports support our findings that TQ induces LMP and cathepsin translocation in glioblastoma cells.
In agreement with other reports that
TQ induces apoptosis in cancer cells, we also observed apoptosis induction by
detection of PS flipping. Interestingly, the general caspase inhibitor
z-VAD-FMK was unable to preserve cell viability, strongly pointing to a
caspase-independent process. This was confirmed by the lack of DNA
fragmentation after TQ treatment, as DNases are activated by caspases.
Mechanistic studies of novel glioblastoma therapies involving the natural
compound asiatic acid [47] and a retroviral
approach using mutant survivin [48] have also reported that
z-VAD-FMK could not protect glioblastoma cell death from these various insults.
In addition to caspase-independent apoptosis, increasing evidence is pointing
to the existence of other mechanisms of cell death, for example apoptosis-like
cell death, in which caspase activation and other markers of classical
apoptosis are completely absent [44], [57], and can help explain
why inhibition of caspases does not always protect cells from cytotoxic
stimuli. A number of non-caspase proteases such as lysosomal cathepsins are
capable of executing programmed cell death. Additionally, release of
mitochondrial proteins other than cytochrome c such as endonuclease G and
apoptosis inducing factor (AIF) also play a role in caspase-independent
programmed cell death [60].
Other labs have reported that the
cytotoxicity of TQ is due to the in vitro generation of reactive oxygen
species by virtue of its quinone chemical structure [61]. However, in these
studies, N-acetyl-cysteine (NAC) was used to inhibit the pro-oxidant effects of
TQ. This might have confounded their results because TQ is also an arylating
quinone [62], and NAC might have
formed an adduct with TQ before TQ had the opportunity to redox cycle. Further
complicating the issue is that arylating quinones are capable of inducing cell
death independent of ROS by initiating the unfolded protein response, and this
phenomenon can be inhibited by pre-treating cells with NAC [63]. Nevertheless, ROS are
known to induce LMP on their own [38], [44], and
arylating/alkylating agents can disrupt microtubules [56] which results in LMP.
Presently, we do not know which of these mechanisms is prevalent in TQ-induced
LMP, and is deserving of further exploration.
Ionizing radiation and temozolomide have both been shown to increase a cytoprotective autophagy response in glioblastoma cells, leading to resistant tumors. In addition, many other chemotherapeutics, such as rapamycin, tamoxifen, and etoposide, induce a protective autophagic response in cancer cells. Therefore, inhibitors of autophagy, both alone and in combination with standard therapies, may provide a viable and promising new strategy in cancer treatment. The immunosuppressant FTY720 and the anti-schistome lucanthone (Miracil D), have both been identified as autophagy inhibitors in mantle cell lymphoma [64] and breast cancer [11], respectively. To the best of our knowledge, this report represents the first finding of TQ as an autophagy inhibitor, and provides a platform for which to extend studies in the treatment of glioblastoma with TQ.
Funding Statement
This work was supported by National Institutes of Health (NIH) grants CA93413 and ES2388. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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