Molecular mechanisms of cell death
induced in glioblastoma by experimental
and antineoplastic drugs: New and old
drugs induce apoptosis in glioblastoma

Abstract
Glioblastoma multiforme (GBM) is one of the most aggressive astrocytic tumors; it is resistant to most
chemotherapeutic agents currently available and is associated with a poor patient survival. Thus, the devel￾opment of new anticancer compounds is urgently required. Herein, we studied the molecular mechanisms of
cell death induced by the experimental drugs resveratrol and MG132 or the antineoplastic drugs cisplatin and
etoposide on a human GBM cell line (D54) and on primary cultured mouse astrocytes (PCMAs). Caspases, Bcl-
2, inhibitors of apoptosis proteins (IAP) family members, and p53 were identified as potential molecular targets
for these drugs. All drugs had a cytotoxic effect on D54 cells and PCMAs, with a similar inhibitory concen￾tration (IC50) after 24 h. However, MG132 and cisplatin were more effective to induce apoptosis and autop￾hagy than resveratrol and etoposide. Cell death by apoptosis involved the activation of caspases-3/7, -8, and -9,
increased lysosomal permeability, LC3 lipidation, poly-(ADP-ribose) polymerase (PARP)-1 fragmentation, and
a differential expression of genes related with apoptosis and autophagy like Mcl-1, Survivin, Noxa, LC3,
and Beclin. In addition, apoptosis activation was partially dependent on p53 activation. Since experimental and
antineoplastic drugs yielded similar results, further work is required to justify their use in clinical protocols.
Keywords
Glioblastoma, antineoplastic drugs, resveratrol, MG132, apoptosis, autophagy, p53
1
Centro de Investigacion sobre Enfermedades Infecciosas, Instituto Nacional de Salud P ´ ublica (INSP), Cuernavaca, Morelos, M ´ exico ´ 2
Centro Nacional de Investigacion Disciplinaria en Salud Animal e Inocuidad, Instituto Nacional de Investigaciones Forestales, Agr ´ ´ıcolas y
Pecuarias, Jiutepec, Morelos, Mexico ´ 3
Unidad de Investigacion Biom ´ edica en C ´ ancer, Instituto de Investigaciones Biom ´ edicas, UNAM/Instituto Nacional de Cancerolog ´ ´ıa,
Ciudad de Mexico, M ´ exico ´ 4
Departamento de Investigacion en Salud Ambiental, Centro de Investigaci ´ on en Salud Poblacional, INSP, Cuernavaca, Morelos, M ´ exico ´ 5
Departamento de Ensen˜anza e Investigacion, Hospital Central Sur de Alta Especialidad Petr ´ oleos Mexicanos, Ciudad de M ´ exico, ´
Mexico ´ 6
Departamento de Genetica y Biolog ´ ´ıa Molecular, Centro de Investigacion y de Estudios Avanzados del Instituto Polit ´ ecnico Nacional, ´
Ciudad de Mexico, M ´ exico ´
Corresponding author:
A Lagunas-Mart´ınez, Centro de Investigacion sobre Enfermedades Infecciosas, Instituto Nacional de Salud P ´ ublica, Avenida Universidad ´
no. 655, Colonia Santa Mar´ıa Ahuacatitlan, cerrada los Pinos y Caminera, Cuernavaca, Morelos CP 62100, M ´ exico. ´

Introduction
Glioblastoma multiforme (GBM) is the most com￾mon, yet incurable, type of brain tumor. GBM, usu￾ally a heterogeneous population of cells, is highly
infiltrative, angiogenic, and resistat to standard
chemotherapy.1 The current treatment consists of
surgical resection, radiotherapy, and chemotherapy
with the alkylating agent temozolomide; however,
the overall survival rate is low.2 Thus, the develop￾ment of novel chemotherapeutic drugs for GBM
treatment is much needed to improve the prognosis
of these patients.
New drugs with antitumor properties are being
evaluated for GBM treatment, including resveratrol
and MG132. Resveratrol (3,5,40
-trihydroxystilbene)
is a plant-derived phytoalexin,3 while proteasome
inhibitors such as MG132 (Z-Leu-Leu-Leu-CHO) are
emerging as a new class of anticancer agents.4 Several
reports suggest that both compounds have a cytotoxic
effect on GBM, inducing apoptosis through caspase
activation and autophagy.4–11 However, further stud￾ies are required to determine its mechanism of action
and identify the key molecules involved in cell death,
such as p53. p53 is a tumor-suppressor protein that
regulates deoxyribonucleic acid (DNA) repair, meta￾bolism, and cell death; its activity is induced by DNA
damage or cellular stress.12 Some reports suggest that
p53 could play an important role in apoptosis induc￾tion by resveratrol in GBM cell lines.9,13,14
For years, toxic drugs like cisplatin and etoposide
have been used in cancer treatment. Cisplatin (cis￾diamminedichloroplatinum II) is a cross-linking
agent that binds purine bases in DNA. Cisplatin is
widely used against solid tumors.15 On the other
hand, etoposide is a topoisomerase II inhibitor, often
given as a first-line treatment against solid tumors in
combination with other drugs.16 In several GBM cell
lines, both antineoplastic drugs have been shown to
inhibit proliferation and induce apoptosis and
autophagy.17–23
This work is aimed to investigate the molecular
mechanisms underlying the activation of apoptosis
and autophagy by the experimental drugs resveratrol
and MG132 or the antineoplastic drugs currently used
for solid tumor treatment, cisplatin and etoposide. The
cytotoxic and proapoptotic effects of these drugs were
assessed in a human GBM cell line and in nontumoral
mouse astrocytes. In addition, we identified potential
targets of these agents, such as caspases, Bcl-2 or IAP
family members, and p53.
Materials and methods
Reagents
Dulbecco’s modified Eagle’s medium (DMEM; high￾glucose and L-glutamine), modified Eagle’s medium
(MEM) vitamin solution (100), antibiotic–antimy￾cotic, Trizol reagent, and moloney murine leukemia
virus (M-MLV) reverse transcriptase were purchased
from Invitrogen (Carlsbad, California, USA). MG132
was acquired from Calbiochem Millipore (San Diego,
California, USA), dissolved in dimethyl sulfoxide
(DMSO), and stored at 20C until used. Resveratrol,
cisplatin, etoposide, acridine orange (AO), poly-D￾lysine, pifithrin a (PFT-a), mouse monoclonal anti￾b-actin-peroxidase antibody, and goat-antimouse
horseradish peroxidase (HRP)-conjugated IgG were
purchased from Sigma-Aldrich (St Louis, Missouri,
USA). Resveratrol, cisplatin, and etoposide were dis￾solved in 70% ethanol, water, and DMSO, respec￾tively. Dissolution was performed according to the
manufacturer’s instructions. Cell Titer 96® AQueous
One Solution and caspase-Glo 3/7, 8, or 9 kits were
purchased from Promega (Madison, Wisconsin,
USA). Maxima SYBR green/ROX quantitative poly￾merase chain reaction (qPCR) Master Mix and Super￾Signal West Femto Maximum Sensitivity were
acquired from Thermo Scientific (Foster City, Cali￾fornia, USA). X-ray films were obtained from Kodak
(Rochester, New York, USA). Anti-p53 HRP￾conjugated human antibody was obtained from Santa
Cruz Biotechnology (Dallas, Texas, USA); antihuman
LC3A/B and PARP-1 were obtained from Cell Sig￾naling (Danvers, Massachusetts, USA).
Cell culture
The human glioblastoma cell line D54 was cultured in
DMEM supplemented with 10% fetal bovine serum
and 1 antibiotic–antimycotic (10,000 units of peni￾cillin, 10 mg of streptomycin, and 25 mg of amphoter￾icin B) at 37C under a 5% CO2 atmosphere. A
primary culture of mouse astrocytes (PCMAs) was
obtained from Balb/c neonatal mice.24 All experimen￾tal procedures on animals were performed in accor￾dance with applicable institutional guidelines. The
culture was previously characterized by the expres￾sion of the glial fibrillary acidic protein (GFAP) pro￾tein.24 Briefly, mouse brains were removed under
sterile conditions and placed in Hank’s solution, fol￾lowed by mechanical and enzymatic disaggregation
with 0.1% trypsin and 0.1% DNAse type I, under
2 Human and Experimental Toxicology XX(X)
agitation, for 10 min at 37C. Astrocytes were washed
by centrifugation and resuspended in DMEM supple￾mented with 10% fetal bovine serum, MEM vitamin
solution 10, and antibiotic–antimycotic. Astrocytes
were grown in Petri dishes pretreated overnight with
0.5 mg/mL of poly-D-lysine.
Cytotoxic effect of experimental and anti￾neoplastic drugs
To determine the IC50 of the drugs under study, either
D54 cells or PCMAs were seeded at a density of
10,000 cells/well in a 96-well plate and cultured in
the presence of different concentrations of resveratrol
(100, 250, or 500 mM), MG132 (20, 40, or 80 mM),
cisplatin (20, 40, or 80 mM), or etoposide (50, 100, or
250 mM) for 24 or 48 h. Additionally, 70% of ethanol
and DMSO were added to cell cultures in the same
volume as the experimental or antineoplastic drugs as
controls. Then, 20 mL/well of tetrazolium compound
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy￾phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)
reagent (Cell Titer 96® AQueous One Solution) was
added to the plates and incubated for 2 h at 37C
under a 5% CO2 atmosphere. To determine the cyto￾toxic effect of each drug, two absorbance measure￾ments were performed at 490 and 690 nm using a
microplate reader (LabSystems Multiskan, Thermo
Scientific). MTS values were normalized and
expressed as a percentage, with 100% viability corre￾sponding to control (untreated) cells.
Detection of caspase-3/7, -8, and -9 activity
The cells were seeded at a density of 10,000 cells/well
in a 96-well plate and treated with each of the drugs
under study at the IC50: resveratrol (100 mM), MG132
(40 mM), cisplatin (80 mM), or etoposide (250 mM for
D54 cells or 100 mM for PCMAs) and incubated for
24 h; these concentrations were used in later experi￾ments. To measure the activity of caspase-3/7, -8, and
-9, 100 mL of Caspase-Glo reagent was added to each
well. The plates were incubated at room temperature
for 30 min, and the luminescence of each sample was
measured in a GloMax plate-reading luminometer
(GloMax, Promega).
Lysosome integrity assessment
D54 cells and PCMAs (600,000 cells/well) were incu￾bated for 24 h in the presence of resveratrol, MG132,
cisplatin, or etoposide at the IC50. After detaching the
cells with a trypsin solution (0.1% trypsin, 0.025% tet￾rasodium EDTA, 0.1% glucose), they were washed and
collected by centrifugation at 2500 r/min for 5 min.
Next, the cells were resuspended in Hank’s solution
(140 mM NaCl, 27 mM KCl, 0.34 mM Na2HPO4,
0.44 mM KH2PO4, and 0.1% glucose) and incubated
with 5 mg/mL of AO for 15 min at 37C in the dark. The
cells were washed with phosphate-buffered saline (PBS
1), and 10,000 events were analyzed by flow cytome￾try in a FACS Aria II cytometer (BD Biosciences, San
Jose, California, USA). The FACSdiva software (BD
Biosciences) was used for data acquisition.
Real-time polymerase chain reaction
The cells were seeded at a density of 600,000 cells/well
in a six-well plate and incubated with the drugs under
study for 24 h. Total ribonucleic acid (RNA) was iso￾lated with Trizol reagent following the manufacturer’s
instructions. To obtain DNA-free RNA, total RNA was
treated with RNAse-free DNAse-I (Thermo Scientific).
Complementary DNA (cDNA) was synthetized using 1
mg of total RNA, 200 U of M-MLV reverse transcrip￾tase, and 0.5 mg of oligo dT primer. The cDNA
obtained was diluted 1:10, and 2 mL of each diluted
cDNA product was used to perform real-time (RT)
qPCR in a Maxima SYBR Green/ROX qPCR Master
Mix, following the manufacturer’s instructions. To per￾form the qPCR, 5 pg of each primer was used. The 18S
ribosomal gene was used to normalize the expression
of antiapoptotic (Mcl-1 and Survivin), proapoptotic
(Noxa), and autophagic genes (Beclin and LC3) in each
sample (Table 1).25 A Beclin mouse QuantiTect® pri￾mer assay (Qiagen, Germantown, Maryland, USA) was
also performed. Reaction conditions for all genes were
10 min at 95C, 40 cycles of 15 s at 95C, and 60 s at
60C. A melting curve analysis was conducted to
determine the specificity of the amplification products
and detect possible primer dimers. For each gene, a
standard curve was plotted using fivefold dilutions.
The efficiency of PCR amplification for the 18S ribo￾somal gene as well as for Mcl-1, Survivin, Beclin, LC3,
and Noxa was calculated by the standard curve method,
E10(1/slope) 1. Data were analyzed using the equa￾tion: 2DDCT.
26 All reactions were performed in a Ste￾pOne Plus Real-Time system (Thermo Fisher
Scientific, Waltham, Massachusetts, USA).
Western blot
After treatment, the cells were detached and collected
by centrifugation, as described above. The cells were
Contreras-Ochoa et al. 3
lysed in radioimmunoprecipitation assay buffer
(RIPA) buffer, as previously described.22 Fifty micro￾grams of total protein were separated through 10%
and 15% SDS-PAGE and transferred to nitrocellulose
membranes. The membranes were blocked for 1 h
with 5% skimmed milk and incubated overnight at
4C with anti-p53 HRP-conjugated human antibody
(diluted 1:500 in PBS), antihuman LC3B (diluted
1:2000), or antihuman PARP-1 (diluted 1:2000).
Immune complexes were visualized with the Super￾Signal West Femto Maximum Sensitivity Substrate
and recorded on X-ray film. Antimouse actin HRP￾conjugated monoclonal antibody was used as a loading
control. Antirabbit HRP-conjugated IgG (diluted 1:10
000) was used as a secondary antibody to detect LC3B
or PARP-1 signals on Western blot film, which were
densitometrically quantified with the ImageJ software
(Java2HTML version 1.5). Densitometric values of the
PARP-1, LC3B, or p53 proteins were normalized with
respect to actin concentration in each sample. The tar￾get protein/actin ratio was normalized with respect to
the vehicle (culture medium for cisplatin, DMSO for
etoposide and MG132, and ethanol for resveratrol).
The relative expression of control (untreated) cells or
the respective solvent was set as 100%. The total
relative expression of LC3B was determined as the
sum of LC3B-I (16 kDa) plus LC3B-II (14 kDa); the
sum of the relative density values for both bands was
considered as 100%. This value was normalized with
respect to actin concentration in the same sample
(LC3/actin), as described above.
Statistical analysis
Data were analyzed with the software STATA version
14 (StataCorp, College Station, Texas, USA). One￾way analysis of variance was performed to analyze
differences between treatments and between cell
types; p value <0.05 was considered as statistically
significant. Bonferroni correction was used to correct
the family-wise error by multiple comparisons.
Results
Experimental and antineoplastic drugs have
similar cytotoxic effects on D54 cells and PCMAs
To compare the cytotoxic effect induced by resvera￾trol, MG132, and the antineoplastic compounds (cis￾platin and etoposide), D54 cells and PCMAs were
incubated in the presence of several drugs at various
Table 1. Primer sequences and RT-PCR conditions.
h: human; m: mouse; F: forward; R: reverse; RT-PCR: real-time polymerase chain reaction.
4 Human and Experimental Toxicology
concentrations for 24 and 48 h. Experimental and
antineoplastic compounds induced a statistically sig￾nificant, dose-dependent cytotoxic effect in both cell
types after 24 and 48 h of treatment (Figure 1). After
24 h, IC50 was similar for both cell types treated with
resveratrol (100 mM), MG132 (40 mM), or cisplatin
(80 mM); in contrast, D54 cells were more resistant to
etoposide than PCMAs (250 and 100 mM, respec￾tively). The IC50 after 24 h of treatment was used in
later experiment.
The four compounds are effective to induce a cyto￾toxic effect in GBM cells and PCMAs; however, in
GBM, the experimental drugs MG132 and resveratrol
were statistically different compared with antineo￾plastic drugs (MG132 versus etoposide and cisplatin,
p < 0.01 or resveratrol versus cisplatin and etoposide,
p < 0.001). In addition, MG132 had a cytotoxic effect
at a lower dose than the other drugs.
Resveratrol, MG132 and antineoplastic drugs
induce apoptotic cell death through intrinsic and
extrinsic pathways
The possibility that experimental and antineoplastic
drugs induced apoptosis through executor caspases-3
and-7, and either extrinsic or intrinsic pathway
(caspase-8 and -9, respectively) was tested by treating
cells with the drugs under study at the IC50 for 24 h.
The activity of each caspase was compared with
appropriate controls. Even though resveratrol
Figure 1. Cytotoxic effect of experimental and antineoplastic drugs. D54 cells or PCMAs were treated with various drugs
at different concentrations for 24 or 48 h. Cell viability was determined with the MTS reagent. Viability percentage was
normalized with respect to untreated control cells; all doses evaluated had a statistically significant cytotoxic effect. The
charts show the mean result of three independent experiments, each one performed in duplicate + SE, p < 0.05. PCMA:
primary cultured mouse astrocytes; SE: standard error.
Contreras-Ochoa et al. 5
activates caspase-3/7, -8, and -9, no statistically sig￾nificant differences in the activity of any caspase were
observed in D54 cells (Figure 2(a)). Cisplatin, etopo￾side, and MG132 were the main activators of caspase-
3/7 in these cells (3.7-, 3.5-, and 2.9-fold increase,
respectively). The four drugs induced the activity of
this caspase in PCMAs; however, MG132 was the
primary activator of this protein (14.4-fold increase,
Figure 2(a)), followed by cisplatin.
On the other hand, cisplatin was the most effective
inductor of caspase-8 activity (2.3-fold increase) in
D54 cells, while MG132 and cisplatin were the pri￾mary activators of this caspase in PCMAs (8.1- and
5.8-fold increase, respectively). Similarly, cisplatin
was the most active inducer of caspase-9 in D54 cells
(2.5-fold increase). PCMAs showed a fivefold
increase in the activation of this caspase after treat￾ment with cisplatin and MG132. In D54 cells, resver￾atrol failed to significantly activate any caspase. The
effectiveness of MG132, etoposide, and cisplatin to
activate caspase-3/7 was similar, while cisplatin
mainly activated caspase-8 and caspase-9. In PCMAs,
MG132 was the chief inductor of the extrinsic and
intrinsic apoptosis pathways, followed by cisplatin
and etoposide. In PCMAs and D54 cells, resveratrol
had the lowest effect among all tested drugs.
PARP-1 (a 116-kDa nuclear poly-ADP-ribose
polymerase) participates in DNA repair and maintains
Figure 2. Effect of experimental and antineoplastic drugs on apoptosis induction through caspase-8, caspase-9, and
caspase-3/7 activity in D54 cells and PCMAs. (a) Cells were incubated with the drugs at the IC50 for each drug for 24 h.
Control treatment with DMSO or ethanol was included. Caspase activity was measured using Caspase-Glo kits. The
charts show the mean result of three independent experiments + SE, p < 0.05. Statistically significant differences between
controls and treatments for each cell type are marked with an asterisk (*), whereas significant differences between cell
types are marked with (**). (b) Western blot of a representative experiment of PARP-1 fragmentation (89 kDa) induced
by experimental or antineoplastic drugs. (c) PARP-1 expression was densitometrically quantified, as described in
“Materials and methods” section. PCMA: primary cultured mouse astrocytes; SE: standard error; PARP: poly-(ADP￾ribose) polymerase; DMSO: dimethyl sulfoxide.
6 Human and Experimental Toxicology XX(X)
cell viability. PARP is a target of caspase-3. The
activation of caspase-3 cleavages the PARP amino￾terminal domain (24 kDa) from the carboxy-terminal
domain (89 kDa).27
In D54 cells, no significant increase in the concen￾tration of the 89-kDa fragment of PARP-1 was
observed after treatment with etoposide, cisplatin,
resveratrol, nor MG132 (Figure 2(b) and (c)). The
complete 116-kDa PARP-1 protein was not detected
in this work.
Experimental and antineoplastic drugs induced
autophagy in D54 cells and PCMAs
To determine whether other types of cell death were
activated in D54 cells or PCMAs by the drugs under
study, the activation of autophagy was assessed
through the permeability of lysosomal membranes
by flow cytometry. In D54 cells, all drugs induced
lower levels of autophagy cell death with respect to
apoptosis. MG132 was the most active autophagy
inducer (4.8-fold increase with respect to untreated
control cells), followed by resveratrol and cisplatin
(3.4- and 3.1-fold increase, respectively, Figure 3(a)
and (b)). Likewise, the chief inducer of changes in the
permeability of lysosomal membranes in PCMAs was
also MG132 (13-fold increase), followed by cisplatin
and resveratrol (5.2- and 4.6-fold increase, respec￾tively). No change in the permeability of lysosomal
membranes was observed in the control, DMSO, or
ethanol groups in either cell type. In addition, resver￾atrol, MG132, and etoposide induced lipidation of the
cellular autophagy marker LC3BI to LC3BII in D54
cells (Figure 3(b) and (c)).
These results indicate that MG132 was the most
effective lysosomal permeability inductor in PCMAs
Figure 3. Induction of autophagy in D54 cells or PCMAs by experimental or antineoplastic drugs. (a) After 24 h of
treatment, the cells were stained with acridine orange and analyzed by flow cytometry. A representative histogram is
shown for each cell type. (b) The chart shows the mean result of three independent experiments + SE, p < 0.05. Sta￾tistically significant differences between control and drugs for each cell type are indicated with an asterisk (*), whereas
significant differences between cell types are marked with (**). (c) Representative Western blot for the lipidated LC3B
protein in D54 cells. The image shows a representative film. (d) LC3B expression was densitometrically quantified, as
indicated above. The chart shows the mean result of three independent experiments + SE, p < 0.05. Significant differences
between treatments are marked with (*). PCMA: primary cultured mouse astrocytes; SE: standard error.
Contreras-Ochoa et al. 7
and D54 cells, whereas resveratrol, MG132, and eto￾poside were similarly effective to induce LC3B
lipidation.
Differential expression of apoptosis- and
autophagy-related genes induced by
experimental or antineoplastic drugs in
D54 cells and PCMAs
To identify whether apoptosis- or autophagy-related
genes were induced by treatments, qPCR assays were
performed. In D54 cells, resveratrol, MG132, and eto￾poside tripled the levels of Mcl-1 messenger RNA
(mRNA) (an antiapoptotic gene, Figure 4(a)). Addi￾tionally, an increase in the expression of the Survivin
gene (an antiapoptotic gene) was observed in cells
treated with cisplatin, while the mRNA levels of Noxa
(a proapoptotic gene) were slightly increased after
treatment with resveratrol and etoposide. On the other
hand, the expression levels of the autophagy-related
genes LC3 and Beclin were increased in cells treated
with MG132 (10- and 1.6-fold increase).
In PCMAs, cisplatin was the primary inducer of
Mcl-1 mRNA expression (2.5-fold increase). Cispla￾tin also increased the expression of the autophagy￾involved genes LC3 and Beclin (3.4- and 1.6-fold
increase, respectively) but failed to change the levels
of Survivin mRNA. In addition, etoposide and MG132
induced a remarkable increase in the expression of
Noxa mRNA (247- and 49-fold increase, respec￾tively). Both experimental and antineoplastic drugs
induced a differential expression of apoptosis- and
autophagy-related genes in D54 and PCMAs.
P53 activation by experimental and
antineoplastic drugs in D54 cells
The p53 tumor-suppressor protein is a master regula￾tor of several cellular functions, including apoptosis.
Figure 4. Effect of experimental and antineoplastic drugs on the expression of apoptosis and autophagy-related genes.
mRNA levels of cell death-related genes were determined by RT-qPCR after 24 h of treatment. Relative expression was
calculated by the 2DDCT method. (a) D54 cell line and (b) PCMAs. The mean result of three independent experiments +
SE, p < 0.05, is shown. Statistically significant differences between treatments and the control group for each cell type are
marked with an asterisk (*). mRNA: messenger ribonucleic acid; PCMA: primary cultured mouse astrocytes; SE: standard
error; RT-qPCR: real-time quantitative polymerase chain reaction.
8 Human and Experimental Toxicology XX(X)
To determine whether cellular stress caused by the
antineoplastic or experimental drugs induced the
expression of p53, a Western blot analysis was per￾formed. A statistically significant increase in p53 lev￾els was observed in D54 cells treated with resveratrol,
etoposide, and MG132 (2.8-, 3-, and 2.5-fold, respec￾tively) with respect to control cells (p < 0.01;
Figure 5(a) and (b)). As expected, no increase in the
levels of the p53 protein was observed in cells treated
with DMSO or ethanol.
To determine whether the increased levels of the
p53 protein play a relevant role in apoptosis induction
in cells treated with the drugs under study, cells were
incubated with PFT-a, a reversible inhibitor of p53-
mediated apoptosis, and the activity of caspase-3/7
was evaluated as an indicator of apoptosis. A decrease
in caspase-3/7 activity was observed in cells pre￾treated with PFT-a for 2 h with respect to
resveratrol-, MG132-, cisplatin-, and etoposide￾treated cells (1.4, 3.4-, 2.5- and 2.2-fold decrease,
respectively) with no PFT-a pretreatment
(Figure 5(c)) indicating that p53 activation is partially
involved in apoptosis induction (p > 0.001).
Discussion
In this work, we investigated the molecular mechan￾isms underlying the activation of cell death by apop￾tosis and autophagy by experimental drugs
(resveratrol and MG132) or antineoplastic drugs cur￾rently used to treat solid tumors, such as cisplatin and
etoposide. These drugs showed a cytotoxic effect on
human glioblastoma D54 cells and on nontumoral
mouse astrocytes. Experimental and antineoplastic
drugs induced apoptosis and autophagy as measured
by the activation of caspase-3/7, -8, and -9, the
increase in lysosomal permeability, LC3 lipidation,
PARP-1 fragmentation, and differential expression
of apoptosis and autophagy-related genes. In addition,
apoptosis was found to be partially dependent on p53
activity.
The four drugs used in this work were chosen
because they have different molecular targets and
some are more toxic than others. The doses of these
drugs were established taking as reference different
concentrations reported in the literature in glioblas￾toma cell lines, which were used to perform dose–
response curve assays on D54 glioblastoma cell line.
Figure 5. Role of p53 in apoptosis induction by experimental or antineoplastic drugs. D54 cells were treated with the
drugs under study for 24 h. (a) Representative Western blot of p53 expression. (b) p53 expression was densitometrically
quantified, as indicated above. (c) The cells were cultivated either with or without pifithrin-a (10 mM) 2 h before drug
treatment and then incubated for 24 h. Caspase-3/7 activity was measured, as described in “Materials and methods”
section. The chart shows the mean result of three independent experiments + SE, p < 0.01 (p53 expression) or p > 0.001
(p53 activation). Statistically significant differences are marked with an asterisk (*). SE: standard error.
Contreras-Ochoa et al. 9
In addition, to determine whether the cytotoxic effect
of these drugs was specific to D54 cells, we carried
out the same experiments on PCMAs.
We found that experimental and antineoplastic
drugs induced a dose-dependent cytotoxic effect in
both cell types. Resveratrol, MG132, and cisplatin
were found to have the same IC50 in D54 and PCMAs.
Furthermore, MG132 produced a strong cytotoxic
effect using a lower concentration than the other
drugs. In contrast, D54 cells were more resistant to
etoposide.
Some authors found different IC50 values than
those reported in this study. These differences may
be due to the cell lines used (U87, A172, T98G,
U138MG, and U373), the incubation period, and the
methods used to assess cytotoxicity. In most reports,
dose values for experimental or antineoplastic drugs
used in in vitro cytotoxic assays on GBM or glioma
cell lines were in the range from 1 mM to 100 mM, and
treatments lasted from 24 h to 72 h.4,7,9,10,18,20,23
However, these drugs have only been evaluated on
rat or mouse primary culture astrocytes, with results
dissimilar to those reported in this study.10,28
The effect of resveratrol and MG132 has been eval￾uated in tumoral and nontumoral cells. Resveratrol
inhibited cell proliferation in normal and transformed
rat hepatocytes, with no differences between both cell
types.29 On the other hand, it has been proposed that
MG132 could have a synergistic action, enhancing the
cytotoxic effect of death ligands in hepatocellular car￾cinoma cells but not in nontumoral primary human
hepatocytes.30
These drugs have also been administered by the
intraperitoneal, intravenous, or intratumor route in in
vivo assays: cisplatin, 0.05, 1.5–3, and 7 mg/kg31–33;
etoposide, 3, 4, 80, 160, and 320 mg/kg34–36; and
resveratrol, 15, 25, and 50 mg/kg.37–39 These drugs
reduced tumor volume but failed to improve mouse
survival. Nevertheless, there is no evidence that
MG132 has been evaluated in vivo.
To determine whether drug cytotoxicity was due to
apoptosis, the activity of caspases-3/7, -8, and -9 was
measured. In D54 cells, resveratrol did not signifi￾cantly activate any caspase, so its cytotoxic mechan￾ism may not involve apoptosis, at least under the
conditions of this study. The other drugs induced
apoptosis, mainly cisplatin was the most efficient
activating both extrinsic and intrinsic apoptosis path￾ways. In PCMAs, both experimental and antineoplas￾tic drugs also induced apoptosis. MG132 caused a
strong caspase-mediated apoptosis, followed by
cisplatin and etoposide. Contrary to D54 cells, resver￾atrol induced a low but significant caspase-3/7 activa￾tion in PCMAs, suggesting no downregulation in the
activity of this pathway compared with reported in
some tumoral cell lines.
Using different drug concentrations and other
GBM cell lines, previous studies have demonstrated
that resveratrol, MG132, cisplatin, and etoposide
induce caspase-3/7 activity.8–10,18,19,21,23 Addition￾ally, an increase in caspase-8 activity induced by
MG132 in the U87MG cell line has been reported.6
The cleavage of caspase-9 has been reported in rat
primary culture astrocytes.40 However, no previous
evidence had been reported of caspase-8 or -9 activa￾tion induced by experimental or antineoplastic drugs
in D54 cells. To our knowledge, these results suggest
for the first time that resveratrol, MG132, cisplatin,
and etoposide induce apoptosis through both intrinsic
and extrinsic pathways in nontumoral PCMAs.
Caspase-3/7 induces proteolytic breakdown of
PARP-1. PARP-1 is involved in key cellular pro￾cesses, such as DNA replication, cell proliferation,
and apoptosis.40 We evaluated PARP-1 fragmentation
in D54 cells treated with experimental or antineoplas￾tic drugs and found increased levels of the 89-kDa
PARP-1 fragment, chiefly induced by resveratrol and
MG132, suggesting that these compounds are capable
of activating apoptosis. There are no previous reports
of PARP-1 fragmentation during apoptosis in D54
cells caused by any of the experimental or antineo￾plastic drugs evaluated in this study.
In D54 cells, we found that resveratrol induced
PARP-1 fragmentation but did not activate caspase-
3/7. This could be explained by the activation of other
caspases besides the caspase-3/7 executor; alterna￾tively, other proteases such as caspase-1 may be
involved in PARP-1 cleavage during apoptosis.27,41,42
To determine if transcription is a mechanism
required in apoptosis induction, we analyzed the
changes in the expression of genes related to apopto￾sis and autophagy. In D54 cells, resveratrol and etopo￾side induced the expression of Noxa, but only
cisplatin induced the expression of Survivin mRNA.
Resveratrol, MG132, and etoposide increased the lev￾els of Mcl-1 mRNA, while MG132 was the most
active inducer of LC3 and Beclin expression. In
PCMAs, the highest increase in the levels of Noxa
mRNA was observed after treatment with etoposide.
Cisplatin increased the expression of Mcl-1, LC3, and
Beclin mRNA. This differential expression of genes
could be due because the drugs have different cell
10 Human and Experimental Toxicology XX(X)
targets. These results suggest that induction of proa￾poptotic and proautophagic genes is required for the
cytotoxic effect of the drugs studied in D54 cells and
PCMAs. Previously, only one study has reported the
failure of etoposide to change the expression of Sur￾vivin mRNA in the GBM U251 cell line.22
In order to assess if the cytotoxic effect of these
compounds was due to the induction of other types of
death in addition to apoptosis, we evaluated autop￾hagy cell death. All drugs analyzed induced it, most
notably, MG132; this compound increased lysosomal
permeability in D54 cells, an event that coincided
with the increased expression of the LC3 and Beclin
genes and lipidation of the LC3BI protein to generate
LC3BII. Nevertheless, PCMAs were more sensitive to
lysosomal permeability caused by MG132 than D54
cells. Also, in PCMAs, resveratrol and cisplatin were
equally effective to induce this type of cell death,
while etoposide had the least effect. In this work,
LC3B lipidation was not analyzed in PCMAs.
In agreement with our results, other studies have
found autophagy induction in response to resveratrol
and etoposide in the U251MG, U87, and GBM9 cell
lines,7,18,43 suggesting that autophagy is induced in
glioblastoma cell lines by these compounds. Addi￾tionally, lipidation of the LC3BI protein to LC3BII
or an increase in the expression of the Beclin protein
was reported in U87 and U373 cell lines treated with
the experimental or antineoplastic drugs herein
studied.6,8,18,44,45
The p53 protein regulates several cell physiologi￾cal events, including cell death. We evaluated the
expression of this protein as a possible player in the
response to the cytotoxic effects of antineoplastic or
experimental drugs. The D54 cell line has a basal
level of p53 expression. The experimental and anti￾neoplastic drugs herein evaluated were equally effec￾tive to induce the expression of p53. An increase in
the levels of the p53 protein in response to etoposide
in D54 cells was previously reported.46 Other authors
found that resveratrol, cisplatin, and etoposide also
induced p53 expression in the U87, A172, U343, and
T98G GBM cell lines, as well as in rat astrocyte pri￾mary cultures.9,14,17,18,23,37,47 However, our study is
the first one to demonstrate the induction of p53 by
MG132 in D54 cells.
Furthermore, we found that p53 plays an important
role in the induction of apoptosis by experimental or
antineoplastic drugs, as shown by the decrease in the
activity of caspase-3/7 by 20–50% in cells treated
with these drugs after exposure to PFT-a, a reversible
p53 inhibitor. Unfortunately, no experimental evi￾dence is available about other master genes (like
p53) that regulates apoptosis or autophagy pathways.
Our results suggest that p53 is partially involved in
the apoptotic cell death induced by the experimental
and antineoplastic drugs.
Conclusions
The four compounds were equally effective to induce
a cytotoxic effect on a GBM cell line and on PCMAs.
MG132 was more effective to induce cell death by
apoptosis and autophagy. The main mechanism of
action for cisplatin was apoptosis induction. In con￾trast, resveratrol failed to activate any type of cell
death. Etoposide was as effective as cisplatin to acti￾vate caspase-3/7, and it induced LC3B lipidation in a
similar extent to MG132. The molecular mechanisms
of apoptosis and autophagy induction involved the
participation of players, such as caspase-3/7, -8, and
-9, LC3B, Beclin, Noxa, and the p53 protein. Finally,
our results suggest Leupeptin that the experimental drugs,
resveratrol and MG132, are similarly effective as the
antineoplastic drugs, cisplatin and etoposide.
Acknowledgments
The D54 cell line was kindly donated by Dr Alfonso Due￾n˜as of the Instituto de Investigaciones Biom´edicas, UNAM.
The authors thank the Red Tem´atica Farmoqu´ımicos
editing of this manuscript.
Author contributions
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest
with respect to the research, authorship, and/or publication
of this article.
Funding
The author(s) disclosed receipt of the following financial
support for the research, authorship, and/or publication of
this article: This work was supported in part by grants from
CONACyT (168896) and FOSISS (261875) to JD-C.
ORCID iD
Contreras-Ochoa et al. 11
References
1. Stavrovskaya AA, Shushanov SS and Rybalkina EY.
Problems of glioblastoma multiforme drug resistance.
Biochemistry (Mosc) 2016; 81: 91–100.
2. Polivka Jr, Polivka J, Holubec L, et al. Advances in
experimental targeted therapy and immunotherapy for
patients with glioblastoma multiforme. Anticancer Res
2017; 37: 21–33.
3. Kulkarni SS and Cant´o C. The molecular targets of
resveratrol. Biochim Biophys Acta 2015; 1852:
1114–1123.
4. Zanotto-Filho A, Braganhol E, Battastini AM, et al.
Proteasome inhibitor MG132 induces selective apop￾tosis in glioblastoma cells through inhibition of PI3K/
Akt and NFkappa B pathways, mitochondrial dysfunc￾tion, and activation of p38-JNK1/2 signaling. Invest
New Drugs 2012; 30: 2252–2262.
5. Shu XH, Wang LL, Li H, et al. Diffusion efficiency
and bioavailability of resveratrol ddministered to rat
Brain by different routes: therapeutic implications.
Neurotherapeutics 2015; 12: 491–501.
6. Zeng RX, Zhang YB, Fan Y, et al. p62/SQSTM1 is
involved in caspase-8 associated cell death induced by
proteasome inhibitor MG132 in U87MG cells. Cell
Biol Int 2014; 38: 1221–1226.
7. Filippi-Chiela EC, Thom´e MP, Bueno e Silva MM,
et al. Resveratrol abrogates the temozolomide-induced
G2 arrest leading to mitotic catastrophe and reinforces
the temozolomide-induced senescence in glioma cells.
BMC Cancer 2013; 13: 147.
8. Filippi-Chiela EC, Villodre ES, Zamin LL, et al.
Autophagy interplay with apoptosis and cell cycle reg￾ulation in the growth inhibiting effect of resveratrol in
glioma cells. PLoS One 2011; 6: e20849.
9. Lin H, Xiong W, Zhang X, et al. Notch-1
activation-dependent p53 restoration contributes to
resveratrol-induced apoptosis in glioblastoma cells.
Oncol Rep 2011; 26: 925–930.
10. Zanotto-Filho A, Braganhol E, Battastini AM, et al.
Proteasome inhibitor MG132 induces selective apop￾tosis in glioblastoma cells through inhibition of PI3K/
Akt and NFkappa B pathways, mitochondrial dysfunc￾tion, and activation of p38-JNK1/2 signaling. Invest
New Drugs 2012; 30: 2252–2262.
11. Zanotto-Filho A, Braganhol E, Schro¨der R, et al.
NFkB inhibitors induce cell death in glioblastomas.
Biochem Pharmacol 2011; 81: 412–424.
12. Fu T, Min H, Xu Y, et al. Molecular dynamic simula￾tion insights into the normal state and restoration of
p53 function. Int J Mol Sci 2012; 13: 9709–9740.
13. Li H, Liu Y, Jiao Y, et al. Resveratrol sensitizes
glioblastoma-initiating cells to temozolomide by indu￾cing cell apoptosis and promoting differentiation.
Oncol Rep 2016; 35: 343–351.
14. Sato A, Okada M, Shibuya K, et al. Resveratrol pro￾motes proteasome-dependent degradation of Nanog
via p53 activation and induces differentiation of
glioma stem cells. Stem Cell Res 2013; 11: 601–610.
15. Dasari S and Tchounwou PB. Cisplatin in cancer ther￾apy: molecular mechanisms of action. Eur J Pharma￾col 2014; 740: 364–378.
16. Najar IA and Johri RK. Pharmaceutical and pharma￾cological approaches for bioavailability enhancement
of etoposide. J Biosci 2014; 39: 139–144.
17. Carminati PO, Donaires FS, Marques MM, et al. Cis￾platin associated with LY294002 increases cytotoxi￾city and induces changes in transcript profiles of
glioblastoma cells. Mol Biol Rep 2014; 41: 165–177.
18. Biasoli D, Kahn SA, Corn´elio TA, et al. Retinoblas￾toma protein regulates the crosstalk between autop￾hagy and apoptosis, and favors glioblastoma
resistance to etoposide. Cell Death Dis 2013; 4: e767.
19. Chen J, Sun X, Yang W, et al. Cisplatin enhanced
sensitivity of glioblastoma multiforme U251 cells to
adenovirus-delivered TRAIL in vitro. Tumour Biol
2010; 31: 613–622.
20. Taghavi MS, Akbarzadeh A, Mahdian R, et al. Cispla￾tin downregulates BCL2L12, a novel apoptosis-related
gene, in glioblastoma cells. In Vitro Cell Dev Biol
Anim 2013; 49: 465–472.
21. Qi Q, Liu X, Li S, et al. Synergistic suppression of
noscapine and conventional chemotherapeutics on
human glioblastoma cell growth. Acta Pharmacol Sin
2013; 34: 930–938.
22. Jin F, Zhao L, Guo YJ, et al. Influence of etoposide on
anti-apoptotic and multidrug resistance-associated pro￾tein genes in CD133 positive U251 glioblastoma
stem-like cells. Brain Res 2010; 1336: 103–111.
23. Park CM, Park MJ, Kwak HJ, et al. Induction of
p53-mediated apoptosis and recovery of chemosensi￾tivity through p53 transduction in human glioblastoma
cells by cisplatin. Int J Oncol 2006; 28: 119–125.
24. Contreras-Ochoa CO, Lagunas-Mart´ınez A, Belkind–
Gerson J, et al. Toxoplasma gondii invasion and repli￾cation within neonate mouse astrocytes and changes in
apoptosis related molecules. Exp Parasitol 2013; 134:
256–265.
25. Scherz-Shouval R, Weidberg H, Gonen C, et al.
p53-dependent regulation of autophagy protein LC3
supports cancer cell survival under prolonged
12 Human and Experimental Toxicology XX(X)
starvation. Proc Natl Acad Sci USA 2010; 107:
18511–18516.
26. Schmittgen TD and Livak K. Analyzing real-time PCR
data by the comparative C (T) method. Nat Protocol
2008; 3: 1101–1108.
27. Chaitanya GV, Steven AJ and Babu PP. PARP-1 clea￾vage fragments: signatures of cell-death proteases in
neurodegeneration. Cell Commun Signal 2010; 8: 31.
28. Lu X, Ma L, Ruan L, et al. Resveratrol differentially
modulates inflammatory responses of microglia and
astrocytes. J Neuroinflammation 2010; 7: 1–14.
29. Rubiolo JA, L´opez-Alonso H, Mart´ın-V´azquez V,
et al. Resveratrol inhibits proliferation of primary rat
hepatocytes in G0/G1 by inhibiting DNA synthesis.
Folia Biol (Praha) 2012; 58: 166–172.
30. Ganten TM, Koschny R, Haas TL, et al. Proteasome
inhibition sensitizes hepatocellular carcinoma cells,
but not human hepatocytes, to TRAIL. Hepatology
2005; 42: 588–597.
31. Coluccia D, Figueiredo CA, Wu MY, et al. Enhancing
glioblastoma treatment using cisplatin-gold-nano￾particle conjugates and targeted delivery with
magnetic resonance-guided focused ultrasound.
Nanomedicine 2018; 14: 1137–1148.
32. Zhang WF, Yang Y, Li X, et al. Angelica polysacchar￾ides inhibit the growth and promote the apoptosis of
U251 glioma cells in vitro and in vivo. Phytomedicine
2017; 33: 21–27.
33. Redjal N, Zhu Y and Shah K. Combination of systemic
chemotherapy with local stem cell delivered S-TRAIL
in resected brain tumors. Stem Cells 2015; 33:
101–110.
34. Bello L, Carrabba G, Giussani C, et al. Low-dose che￾motherapy combined with an antiangiogenic drug
reduces human glioma growth in vivo. Cancer Res
2001; 61: 7501–7506.
35. Cheema TA, Kanai R, Kim GW, et al. Enhanced anti￾tumor efficacy of low dose etoposide with oncolytic
herpes simplex virus in human glioblastoma stem cell
xenografts. Clin Cancer Res 2011; 17: 7383–7393.
36. Smith SJ, Rahman CV, Clarke PA, et al. Surgical
delivery of drug releasing poly (lactic-co-glycolic
acid)/poly(ethylene glycol) paste with in vivo effects
against glioblastoma. Ann R Coll Surg Eng 2014; 96:
495–501.
37. Clark PA, Bhattacharya S, Elmayan A, et al. Resvera￾trol targeting of AKT and p53 in glioblastoma and
glioblastoma stem-like cells to suppress growth and
infiltration. J Neurosurg 2017; 126: 1448–1460.
38. Shu XH, Wang LL, Li H, et al. Diffusion efficiency
and bioavailability of resveratrol administered to rat
brain by different routes: therapeutic implications.
Neurotherapeutics 2015; 12: 491–501.
39. Guo W, Li A, Jia Z, et al. Transferrin modified
PEG-PLA-resveratrol conjugates: in vitro and in vivo
studies for glioma. Eur J Pharmacol 2013; 718:
41–47.
40. Goldbaum O, Vollmer G and Richter-Landsberg C.
Proteasome inhibition by MG-132 induces apoptotic
cell death and mitochondrial dysfunction in cultured
rat brain oligodendrocytes but not in astrocytes. Glia
2006; 53: 891–901.
41. Soldani C and Scovassi AI. Poly (ADP-ribose)
polymerase-1 cleavage during apoptosis: an update.
Apoptosis 2002; 7: 321–328.
42. Germain M, Affar EB, D’Amours D, et al. Cleavage of
auto modified poly (ADP-ribose) polymerase during
apoptosis. Evidence for involvement of caspase-7. J
Biol Chem 1999; 274: 28379–28384.
43. Zanotto-Filho A, Braganhol E, Klafke K, et al. Autop￾hagy inhibition improves the efficacy of curcumin/
temozolomide combination therapy in glioblastomas.
Cancer Lett 2015; 358: 220–231.
44. Liu X, Sun K, Wang H, et al. Knockdown of retino￾blastoma protein may sensitize glioma cells to cisplatin
through inhibition of autophagy. Neurosci Lett 2016;
620: 137–142.
45. Xue S, Xiao-Hong S, Lin S, et al. Lumbar
puncture-administered resveratrol inhibits STAT3
activation, enhancing autophagy and apoptosis in
orthotopic rat glioblastomas. Oncotarget 2016; 7:
75790–75799.
46. Kim MS, Blake M, Baek JH, et al. Inhibition of
histone deacetylase increases cytotoxicity to antic￾ancer drugs targeting DNA. Cancer Res 2003; 63:
7291–7300.
47. Meley D, Spiller DG, White MR, et al. p53-mediated
delayed NF-kB activity enhances etoposide-induced
cell death in medulloblastoma. Cell Death Dis 2010