Phenethyl caffeate benzoxanthene lignan is a derivative of caffeicacid phenethyl ester that induces bystander autophagy in WiDrcells

Vinod Vijayakurup • Carmela Spatafora •

Corrado Tringali • Padmakrishnan Chorakkode Jayakrishnan •

Priya Srinivas • Srinivas Gopala

Received: 15 December 2012 / Accepted: 26 October 2013 / Published online: 5 November 2013

� Springer Science+Business Media Dordrecht 2013

Abstract We recently reported that Phenethyl caffeate

benzoxanthene lignan (PCBL), a semisynthetic compound

derived from Caffeic Acid Phenethyl Ester (CAPE), indu-

ces DNA damage and apoptosis in tumor cells. In this

study, we further investigated whether PCBL induces

autophagy in WiDr cells. We also analyzed the pathways

regulating autophagy and the role of autophagy in PCBL-

induced cell death. Our acridine orange staining and LC3 II

expression results suggest that PCBL induces autophago-

somes in WiDr cells. The levels of LC3 II expression we

observed after co-treatment of PCBL with bafilomycin A1

and the reductions in p62 expression we observed after

PCBL treatment in WiDr cells demonstrate increased

autophagic flux, a reliable indicator of autophagic induc-

tion. The increased Beclin 1 expression in PCBL-treated

cells and the incapacity of PCBL to induce LC3 II in

3-methyladenine (3-MA)-treated cells we observed sug-

gests that PCBL-induced autophagy is class III PI3-kinase

dependent. PCBL did not alter phosphorylation of the

mTOR substrate p70 S6 kinase, indicating that PCBL-

induced autophagy was not mTOR regulated. Two

autophagy related proteins, Atg5 and Atg12, also remained

uninduced during PCBL treatment. The increased caspase

activity and expression levels of LC3 II and p62 we

observed in response to PCBL treatment in primary glioma

cells demonstrates that PCBL-induced apoptosis and

autophagy were not cell line specific. Pharmacological

inhibition of autophagy did not alter the antitumor efficacy

of PCBL in WiDr cells. This attests to the bystander nature

of PCBL-induced autophagy (in terms of cell death). In

toto, these data suggest that PCBL induces a class III

kinase dependent, but mTOR independent, bystander mode

of autophagy in WiDr cells.

Keywords Autophagy � Lignan � Caffeic acid �Cancer

Introduction

Autophagy is a catabolic process that degrades cellular

components through lysosomal machinery. During

autophagy, cytoplasmic constituents such as exhausted out

organelles or damaged proteins are sequestered into

double membrane vesicles called autophagosomes and are

subsequently delivered to lysosomes for degradation and

recycling [1]. Although it is generally considered a

mechanism to maintain cellular homeostasis, the role of

induced autophagy in tumor cells in response to chemo-

therapeutic drugs remains a topic of controversy [2]. It

can act as a pro-survival or death mechanism in tumor

cells depending on the cellular context and the mode and

intensity of chemotherapeutic insults [3–5]. Autophagy, as

a part of survival responses in tumor cells, is capable of

removing proteins and organelles that are critically

V. Vijayakurup � P. C. Jayakrishnan � S. Gopala (&)

Department of Biochemistry, Sree Chitra Tirunal Institute for

Medical Science and Technology, Thiruvanathapuram 695011,

India

e-mail: srinivasg@sctimst.ac.in

C. Spatafora � C. Tringali (&)

Dipartimento di Scienze Chimiche, Universita degli Studi di

Catania, Viale A. Doria 6, 95125 Catania, Italy

e-mail: ctringali@unict.it

P. Srinivas

Integrated Cancer Research Program V, Rajiv Gandhi Centre for

Biotechnology, Thiruvananthapuram 695014, India

123

Mol Biol Rep (2014) 41:85–94

DOI 10.1007/s11033-013-2840-8

damaged because of the stress induced by chemothera-

peutic agents [3]. On the other hand, under persistent

autophagic stimulation, uncontrolled autophagy can result

in cell death by digesting essential cytoplasmic compo-

nents in tumor cells [4]. Though there are numerous

reports to substantiate the role of autophagy in both cell

survival and death, autophagy induced in tumor cells in

response to drug-induced stress may not be an essential

cell death regulatory mechanism. The possibility that such

induced autophagy is a ‘bystander’ with no active role in

regulating cell death has been mentioned in recent

reviews on autophagy [5, 6]. Such unpredictable roles of

autophagy in tumor cell death make it an interesting

research topic, particularly when studying autophagy

induced during drug treatment.

Recently, the mechanisms underlying the cell death

inducing effects of the novel semi-synthetic compound

bis(2-phenylethyl)-6,9,10-trihydroxybenzo[kl]xanthene-

1,2-dicarboxylate (or, more shortly, Phenethyl Caffeate

Benzo[kl]xanthene Lignan, PCBL) has reported by us [7].

The structure of PCBL is shown in Fig. 1. PCBL is a

molecule derived by the dimerization of the natural product

Caffeic Acid Phenethyl Ester (CAPE, a honeybee propolis

component). It belongs to the group of benzoxanthene

lignans, which are rarely encountered in nature. They have

consequently been studied only recently, once their bio-

mimetic synthesis at a sufficient quantity became practi-

cally successful using Mn-mediated oxidative coupling of

caffeic acid esters [8]. Among different benzoxanthene

lignans obtained through such chemical processes, PCBL is

the most promising antitumor agent because of its DNA

interacting capacity, antiproliferative activities [9], anti-

angiogenic properties [10], and its cell death inducing

effects in tumor cells [7]. In the current study, we have

extended these studies by analyzing the role of autophagy

in PCBL-induced cell death.

Materials and methods

Reagents

3-(4-5-Dimethylthiazol-2-yl)-2-5-diphenyl-tetrazolium bro-

mide (MTT), phenylmethanesulfonyl fluoride (PMSF),

Dulbecco’s Modified Eagle’s Medium (DMEM), fetal

bovine serum (FBS), B7 supplement, L-glutamine, basic

fibroblast growth factor (bFGF), epidermal growth factor

(EGF), trypan blue, 3-methyladenine (3-MA), bafilomycin

A1, and antibodies for b-actin and LC3 II were purchased

from Sigma-Aldrich (St. Louis, MO, USA). Cell lysis buffer

for protein extraction, antibodies against Atg5, Atg12,

phospho-p70 S6 kinase, and secondary anti rabbit antibodies

were purchased from Cell Signaling Technology Inc.

(Danvers, MA, USA). Antibodies against p62 and Beclin 1

were purchased from Abcam (Cambridge, MA, USA).

A West Pico Chemiluminiscence Detection kit used for the

development of western blots and a DyLight� 488 conju-

gated secondary antibody for immunoflourescence were

purchased from Pierce Biotechnology Inc. (Rockford, IL,

USA). A Caspase-Glo 3/7 assay kit was purchased from

Promega (Madison, WI, USA). PCBL was prepared through

oxidative coupling of Caffeic Acid Phenethyl Ester (CAPE)

using Mn(OAc)3 as the oxidative agent, following previ-

ously reported procedures [8].

Cell culture

WiDr colon adenocarcinoma cells were the kind gift of

Professor William R. Wilson, Auckland Cancer Society

Research Centre, University of Auckland, New Zealand.

The cells were grown as monolayer cultures in DMEM,

containing 10 % FBS and antibiotics (100 U/ml penicillin

and 100 lg/ml streptomycin) in a humidified atmosphere

of 5 % CO2 at 37 �C. Human glioma tissues were obtained

from patients undergoing surgical treatment at Sree Chitra

Tirunal Institute for Medical Sciences and Technology,

Thiruvanthapuram, India. Informed consent was obtained

from patients as approved by the institutional ethical

committee. The biopsy specimens were transported in

Hanks Balanced Salt Solution (HBSS). Tumor tissues were

washed with HBSS and minced to small fragments, which

were then dissociated into single cells with papain treat-

ment. The dissociated tumor cells were washed, resus-

pended and cultured in DMEM: F-12 supplemented with

10 % FBS, B 27 supplement, 2 mM of L-glutamine, 20 ng/ml

of bFGF, 20 ng/ml of EGF, and 100 U/ml of antibiotics

penicillin and streptomycin. The cells were incubated at

37 �C in 5 % CO2 and 95 % air. For all experiments,

DMEM containing 2.5 % FBS was used or the medium is

specified. Glioma cells after first passage were used for

experiments.

Fig. 1 Chemical structure of PCBL

86 Mol Biol Rep (2014) 41:85–94

123

Cell viability assay using MTT

Cell viability was measured by MTT assay as described

elsewhere [11]. WiDr cells were plated at a density of

1 9 104 cells per well in 96 well plates and incubated for

24 h. After incubation, WiDr cells were treated with differ-

ent concentrations of PCBL alone or in combination with

bafilomycin A1 or 3-MA. During combination treatments,

10 mM 3-MA or 10 nM bafilomycin A1 were pretreated for

1 h followed by PCBL treatment for 18 h. Following treat-

ment, MTT dissolved in the culture media was added to each

well (final concentration 1 mg/ml) and incubated at 37� C for

2� h. Acidified isopropanol was added to dissolve the MTT

crystals and the optical density was measured at 570 nm

(with 630 nm as reference wavelength) using a microplate

reader (BioTek Instruments, Winooski, VT, USA).

Western blot analysis

For western blotting, cells were lysed in cell lysis buffer

supplemented with a protease inhibitor cocktail. Lysates

were separated by 10 or 15 % SDS-PAGE and transferred to

a nitrocellulose membrane. The membrane was blocked with

Tris-buffered saline (pH 7.8) containing 0.5 % Tween 20 and

5 % skim milk to reduce non-specific binding. The blocked

membrane was probed with primary antibody against the

target protein followed by HRP-conjugated secondary anti-

body and the bands were visualized using West Pico

Chemiluminescence Detection Kit as per the instructions of

the manufacturer. The bands obtained were exposed to X-ray

films and documented using an image analysis system (Bio-

Rad Laboratories, Hercules, CA, USA).

Immunoflourescence microscopy

For immunocytochemistry, WiDr cells were plated on a 24

well plate (5 9 104 cells per well) and incubated for 24 h.

Following incubation, cells were treated with different

concentrations of PCBL for 8 h and fixed with 4 % para-

formaldehyde for 10 min. The fixed cells were blocked

with PBS containing 3 % Bovine Serum Albumin (BSA)

and 0.3 % Triton X-100 (PBST) for 1 h. The cells were

incubated with LC3 II antibody (1:600 dilution) overnight

at 4 �C. After washing, the cells were incubated with Dy-

Light 488-conjugated secondary antibody (1:800 dilution)

at room temperature and visualized using a fluorescent

microscope equipped with appropriate excitation and

emission filters (excitation/emission at 490/520 nm).

Acridine orange staining

Autophagic induction is characterized by increased acidic

autophagosomes, which can be detected by acridine orange

staining [12]. WiDr cells were seeded in 60 mm culture

dishes at a 70 % confluence and treated with PCBL

(25 lM) for 8 h. The cells were then rinsed twice with PBS

and incubated with acridine orange at a final concentration

of 1 mg/ml for 5 min. These cells were washed again with

PBS to remove the excess stain and observed under a

fluorescent microscope.

Trypan blue exclusion assay

For assessment of cell death, WiDr Cells were plated on a

24 well plate (5 9 104 cells per well), and incubated for

24 h. The cells were pre-treated with bafilomycin A1

(10 nM) for 1 h followed by co-treatment with or without

25 lM of PCBL for 12 or 24 h. After treatment, cells were

trypsinized and resuspended in PBS. Equal amount of cell

suspension was mixed with 0.4 % trypan blue solution and

incubated for 3 min. Stained (dead) and unstained (viable)

cells were counted in a hemocytometer and a quantitative

measure of viability was expressed as the percentage of

viable cells out of the total cell count.

Caspase assay

Caspase assay was performed using Caspase-Glo 3/7 Assay

kit per the manufacturer’s instructions. Briefly, 3 9 104

glioma cells were seeded into a 96 well plate and incubated

for 24 h and treated with or without 25 lM of PCBL for

12 h. After treatment, 100 ll of caspase reagent from the

kit was added to the cells without removing the medium.

The contents of the wells were gently shaken for 2 h and

the luminescence emitted was measured using a lumino-

meter (Berthold Technologies GmbH & Co. KG, Bad

wildbad, Germany).

Statistical analysis

All statistical calculations were carried out using GraphPad

Prism software. All values are expressed as the

mean ± standard deviation (SD). The differences among

the mean values were analyzed with one-way ANOVA

followed by Tukey’s post hoc t test analysis.

Results

PCBL induces autophagy in WiDr cells

PCBL-treated WiDr cells exhibited vacuolated structures

(Fig. 2a), a morphological feature of autophagy. Autoph-

agy is a process characterized by increased acidic vesicular

organelles (AVOs), which can be detected by acridine

orange, a stain that accumulates in acid vesicles and

Mol Biol Rep (2014) 41:85–94 87

123

Fig. 2 PCBL increases the

level of autophagosomes in

WiDr cells. a Vacuolated

structures, probably

autophagosomes, observed in

PCBL-treated WiDr cells were

documented using a phase

contrast microscope. b WiDr

cells treated with or without

PCBL were stained with

acridine orange and observed

under a fluorescence

microscope. Cells treated with

PCBL show acridine orange-

stained acidic vesicles, an

indication of increased levels of

autophagosomes. c WiDr cells

were treated with different

concentrations of PCBL for 8 h,

fixed, and immunostained with

LC3 II antibody. A punctuated

distribution of LC3 II was

observed in PCBL-treated WiDr

cells. This suggests that PCBL

can induce autophagy. (Color

figure online)

88 Mol Biol Rep (2014) 41:85–94

123

Fig. 3 PCBL increases autophagic flux. a and b WiDr cells were

treated with PCBL for the indicated time periods and at the indicated

concentrations and were analyzed for LC3 II expression by western

blotting. WiDr cells were treated with PCBL (25 lM) for different

time periods or for 12 h with different concentrations of PCBL.

Results show time- and dose-dependent increase of LC3 II in PCBL-

treated cells c WiDr cells treated with bafilomycin A1 for 4 h were

analyzed for LC3 II expression by western blotting. Bafilomycin A1

blocks fusion of autophagosomes with lysosomes and induces

accumulation of LC3 II. Results show that the saturated concentration

needed for blocking autophagosome-lysosome fusion was 10 nM and

higher concentrations did not yield further accumulation of LC3 II.

d The Protein levels were expressed as fold changes over control after

normalising to b-actin. e WiDr cells were treated with bafilomycin

A1, PCBL, or PCBL?bafilomycin A1. f The PCBL treatment was

applied over 8 h and bafilomycin A1 co-treatment was applied only

during the last 4 h of PCBL treatment. Increased expression of LC3 II

was observed in cells co-treated with PCBL and bafilomycin A1

compared to cells treated with PCBL or bafilomycin A1 alone. g The

protein levels were expressed as fold changes over control after

normalising to b-actin. All of the above experiment were repeated

thrice. *** P value \0.001, **P value \0.01, ### P value \0.001.

Symbol asterisk (*) represents statistical significance between control

and treatment groups where as hash (#) represents statistical

significance between different treatment groups). h WiDr cells

treated with bafilomycin A1 for 4 h were analyzed for accumulation

of p62 by western blotting. Bafilomycin A1 treatment increased

accumulation of p62 in WiDr cells. i WiDr cells treated with PCBL

for 8 h and 12 h were analyzed for p62 expression by western

blotting. PCBL treatment decreased the expression of p62 in WiDr

cells

Mol Biol Rep (2014) 41:85–94 89

123

fluoresces in bright red. Vital staining of WiDr cells with

acridine orange showed an increase in AVO in the cells

exposed to 25 lM PCBL (Fig. 2b). The capacity of PCBL

to induce autophagy was further examined by analyzing the

intracellular distribution of LC3 II (autophagy marker)

upon PCBL treatment by immunofluorescence. A punctu-

ate distribution pattern of LC3 II, an indication of increased

autophagy, was observed in WiDr cells treated with dif-

ferent concentrations of PCBL (25, 37.5 and 50 lM)

(Fig. 2c).

Autophagic induction in PCBL-treated WiDr cells were

further demonstrated by analyzing LC3 II expression using

western blotting. WiDr cells were treated with a concen-

tration of 25 lM of PCBL for different time periods (4, 8

and 12 h) or for 12 h with different concentrations of

PCBL (25, 37.5 and 50 lM). Our results show time- and

dose-dependent increase of LC3 II in PCBL-treated cells

(Fig. 3a, b). The autophagic flux during PCBL treatment,

which is an accurate indication of autophagic activity, was

also analyzed in this study using bafilomycin A1. Bafilo-

mycin A1 blocks the degradation of autophagosome cargo

in lysosomes, leading to the accumulation of autophago-

some components including LC3 II. Western blot analysis

of bafilomycin-A1-treated cells shows that the effects of

bafilomycin A1 (in terms of LC3 II accumulation and

autophagic inhibition in WiDr cells) saturated at a 10 nM

concentration (Fig. 3c, d), and this concentration was used

for further experiments analyzing autophagic flux. Treat-

ment of PCBL (25 lM) for 4 h followed by its co-treat-

ment with bafilomycin A1 for another 4 h in WiDr cells

shows increased accumulation of LC3-II compared to cells

treated with PCBL or bafilomycin A1 alone (Fig. 3e–g).

Such surplus accumulation of LC3 II induced by PCBL in

the presence of bafilomycin A1 demonstrates enhanced

autophagic flux in PCBL-treated WiDr cells. Furthermore,

we analyzed the expression dynamics of p62 (autophagic

substrate protein) in WiDr cells. Inhibition of autophagy by

bafilomycin A1 resulted in p62 accumulation confirms that

a considerable amount of p62 is degraded via autophagy in

WiDr cells (Fig. 3h). Since p62 degradation is enhanced by

increased autophagic flux, its expression should remain low

in autophagy-induced cells. As expected, PCBL treatment

reduced the expression of p62 in WiDr cells (Fig. 3i).

Taken together, these observations confirm that PCBL

induces autophagy.

PCBL-induced autophagy is class III PI3-kinase

dependent

To obtain information regarding the role of class III PI3-

kinase in PCBL-induced autophagy, we used 3-methylad-

enine (3-MA), which is a known inhibitor of class III PI3-

kinase. When administered in combination with 3-MA,

PCBL failed to induce LC3 II in WiDr cells (Fig. 4a).

Moreover, PCBL-induced autophagy in WiDr cells was

associated with increased expression of Beclin 1, an

autophagy related protein associated with the class III PI3-

kinase complex (Fig. 4b). Both of these results indicate a

positive involvement of class III PI3-kinase in PCBL-

induced autophagy.

PCBL treatment did not alter p70 S6k phosphorylation

To study the role of mTOR signaling in PCBL-induced

autophagy, we analyzed phosphorylation of p70 S6 kinase.

Active mTOR can phosphorylate its substrate p70 S6

kinase at ser 279 and thr 376 positions. Phosphorylation at

these sites, an indication of mTOR activity, was analyzed

by Western blotting. We found unaltered phosphorylation

of p70 S6 kinase at ser 279 or thr 376 in PCBL-treated

WiDr cells (Fig. 5a, b), indicating that PCBL does not alter

mTOR activity. These results further indicate that mTOR

signaling may not play a critical role in regulating PCBL-

induced autophagy.

PCBL treatment did not induce Atg5 and Atg12

expression

Atg5 and Atg12 proteins in their conjugated form (Atg5–

Atg12), having a molecular weight of about 55 kDa, are

required for the recruitment of LC3 II to autophagosomes.

Western blot analysis indicated that PCBL treatment did

not increase the expression of Atg5 or Atg12 (conjugated

form, 55 kDa) in WiDr cells (Fig. 6a, b). This result also

shows that Atg5–Atg12 conjugation did not increase fol-

lowing PCBL treatment.

PCBL induces autophagy and apoptosis in primary

glioma cells

The autophagic and apoptotic effects of PCBL are not cell

line specific. Our finding of decreased pro-caspase 7

Fig. 4 PCBL-induced autophagy is class III PI3-kinase dependent.

a WiDr cells were pre-treated with 3-MA for 1 h and subsequently

exposed to PCBL (25 lM) for 8 h and subjected to western blotting.

PCBL in combination with 3-MA did not induce LC3 II expression in

WiDr cells. b WiDr cells were treated with PCBL for the indicated

time periods and were analyzed for Beclin 1 expression by western

blotting. PCBL-treated WiDr cells showed increased expression of

Beclin 1

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expression and increased caspase 3/7 activity in glioma

cells treated with PCBL for 12 h demonstrates that PCBL

can induce apoptosis (Fig. 7a, b). PCBL-treated (25 lM)

glioma cells exhibited increased bright red fluorescent

structures when stained with acridine orange (Fig. 7c). This

suggests the presence of increased AVO in PCBL-treated

glioma cells. PCBL-treated glioma cells also showed

increased LC3 II accumulation and decreased p62 expres-

sion (Fig. 7d), a pattern expected in autophagy induced

cells. Taken together, these observations confirm the

capacity of PCBL to induce autophagy along with apop-

tosis in glioma cells.

Cell death induced by PCBL was unaltered

by inhibition of autophagy

The role of autophagy in PCBL-induced cell death was

analyzed by inhibiting autophagy in WiDr cells. Inhibition

of autophagy with bafilomycin A1 and 3-MA did not alter

the viability reduction or cell death induction caused by

PCBL in WiDr cells (Fig. 8a, b). The capacity of PCBL to

induce apoptosis was also not altered by inhibiting

autophagy. The pattern of cleaved PARP expression in

WiDr cells co-treated with bafilomycin A1 and PCBL was

similar to that of WiDr cells treated with PCBL alone

(Fig. 8c). Taken together, these results demonstrate that

PCBL-induced cell death is independent of its capacity to

induce autophagy.

Discussion

We recently reported the effects of PCBL on DNA damage

and apoptosis in tumor cells, specifically WiDr cells [7].

Autophagy has generally been considered a pathway that

has the potential to regulate the course of drug-induced cell

death. Hence, it is appropriate to investigate the capacity of

a novel synthetic compound to induce autophagy while

studying its antitumor properties. Since this investigation

pertains to the role of autophagy in PCBL-induced cell

Fig. 5 PCBL had no effect on p70 S6 kinase phosphorylation. WiDr

cells were treated with PCBL for different time periods and the

phosphorylation of the mTOR substrate p70 S6 kinase was analyzed

by western blotting. There was no decrease in phosphorylation at thr

389 or ser 371 residues of p70 S6 kinase (a and b)

Fig. 6 PCBL treatment did not increase Atg5 and Atg12 expression.

WiDr cells treated with 25 lM PCBL for different time periods were

analyzed for the expression of Atg5 (a) or Atg12 (b). Expression of

Atg5 and Atg12 remain uninduced in PCBL-treated cells

Fig. 7 PCBL induced apoptosis and autophagy in primary glioma

cells. a Glioma cells treated with PCBL for 12 h was analyzed for

pro-caspase 7 by western blotting. PCBL treatment decreased pro-

caspase 7 expression in glioma cells. b Glioma cells treated with

PCBL (25 lM) for 12 h were analyzed for caspase activity using a

caspase-Glo 3/7 assay kit. PCBL-treated cells exhibited increased

luminescence, which was graphically represented as caspase 3/7

activity. c Glioma cells were treated with PCBL for 12 h and stained

with acridine orange. Increased acridine orange staining of intracel-

lular vesicles was observed in PCBL-treated glioma cells. d PCBL-

treated glioma cells were analyzed for the expression of p62 and LC3

II by western blotting. PCBL-treated (25 lM) glioma cells showed

increased LC3 II and decreased p62 expression

Mol Biol Rep (2014) 41:85–94 91

123

death, we used PCBL at concentrations and treatment

regimens known to induce cell death in WiDr cells.

During autophagy, autophagosomes fuse with lysosomes

forming autophagolysosomes, and its components, includ-

ing LC3 II, of the autophagosome membrane become

subsequently degraded in the lysosomes [13]. Though

increased acridine orange fluorescence and LC3 II

expression provide evidence of increased autophagosomes

in PCBL-treated cells, these findings cannot be accepted as

definitive evidence of increased autophagy because an

increased levels of autophagosomes or LC3 II in the

cytosol at a particular time can either be because of

increased autophagy or reduced clearance of

autophagosomes (blockage of autophagy) [14]. Hence,

instead of attempting to take a snap shot of autophago-

somes, assaying its increased turnover (autophagic flux)

may be a more reliable indicator of autophagy in response

to a particular treatment [15]. The guidelines provided by

Rubinsztein et al., to analyze autophagic flux in response to

drug treatment were used in our study [16]. An increased

level of LC3 II or autophagosomes when autophagosome

degradation is blocked by bafilomycin A1 reflects the basal

rate of autophagosome formation (autophagy flux) in cells.

The additional accumulation of autophagosomes and LC3

II following treatment with the test compound in the pre-

sence of bafilomycin A1 indicates a true autophagosomal

Fig. 8 Autophagic inhibition fails to alter PCBL-induced cell death.

a WiDr cells were treated with different concentrations of PCBL (0,

12.5, 25, 37.5, or 50 lM) in combination with 3-methyladenine

(5 mM) or bafilomycin A1 (10 nM) for 18 h and analyzed for cell

viability by MTT assay. 3-MA and bafilomycin A1 were applied for

1 h before the addition of PCBL. The antitumor efficacy of PCBL was

not altered in cells treated with bafilomycin A1 or 3-methyladenine.

ns indicates a lack of statistical significance. b WiDr cells were

treated with PCBL (25 lM), bafilomycin A1 (10 nM), or the

combination of both for 12 or 24 h and were analyzed for cell

viability by trypan blue assay. WiDr cells were pre-treated with

bafilomycin A1 for 1 h before the addition of PCBL, when treated in

combination. c WiDr cells treated with 25 lM PCBL and/or 10 nM

bafilomycin A1 for 12 h were analyzed for cleaved PARP levels by

western blotting. Bafilomycin A1 was pre-treated for 1 h before the

addition of PCBL for the inhibition of autophagy. The expression of

cleaved PARP was increased in PCBL-treated cells but was not

further altered because of bafilomycin A1 treatment. ***P value

\0.005, ns non-significant

92 Mol Biol Rep (2014) 41:85–94

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increase because of the influence of the test compound. If a

compound is inducing autophagy, in the presence of ba-

filomycin A1, it should induce a surplus accumulation of

autophagosomes and LC3 II when compared to treatment

with either bafilomycin A1 or the compound alone. We

observed such additional accumulation of PCBL-induced

LC3 II in the presence of bafilomycin A1, suggesting that

PCBL induces autophagy. We also analyzed expression of

p62, another protein marker of autophagy, to provide fur-

ther evidence regarding PCBL-induced autophagy. The

protein p62 serves as a link between LC3 and the cargo

substrates that typically degrade in autophagolysosomes.

During the process of cargo protein targeting, p62 itself

gets degraded in autophagolysosomes, reducing its

expression during increased autophagy [17]. Accumulation

of p62 in bafilomycin-A1-treated cells suggests that a

sufficient amount of p62 was being targeted and degraded

in autophagolysosmes during autophagy in WiDr cells, as

reported in other cell types [18]. Reduced expression of

p62 thus suggests increased protein degradation through

autophagy in PCBL-treated WiDr cells.

Signaling mediated by mTOR and class III PI3-kinase

has been reported to be involved in regulating autophagy.

Signaling by mTOR can provide a gauge of cellular

nutrient stress and can regulate autophagy accordingly.

Autophagy, once activated, can compensate for nutrient

deficits by degrading and recycling cellular components for

energy needs. Under normal nutrient conditions, mTOR

suppresses autophagy by phosphorylating Atg13 and

thereby blocking its interaction with ULK, a process

required for the initiation of autophagy [19, 20]. Signaling

by mTOR is generally considered to be a potent regulator

of autophagy, but drugs that can induce autophagy inde-

pendent of the mTOR pathway has also been reported

frequently [21]. PCBL failed to reduce mTOR activity,

suggesting that autophagy induced by PCBL was not reg-

ulated through the mTOR pathway. It is interesting to note

that drugs that induce autophagy independent of the mTOR

pathway possess therapeutic benefits against neurodegen-

erative disorders such as Parkinson’s and Huntington’s

disease. Most of these drugs can induce autophagy by

reducing intracellular inositol or inositol 1,4,5-trisphos-

phate levels [22]. Another well established positive regu-

lator of autophagy is class III PI3-kinase, a molecular

complex present in mammals, whose function is to produce

phosphatidylinositol-3-phosphate from phosphatidyl group

[19]. Phosphoinositol-3-phosphate formed by the activation

of class III PI3-kinase is supposed to help the assembly of

autophagosomes. Autophagy dependent on class III PI3-

kinase is termed canonical. The need to specify autophagy

as ‘canonical’ arises from reports of the existence of

autophagy that does not require the activity of class III PI3-

kinase for its action [23]. Such forms of autophagy, termed

non canonical, do not depend on Beclin 1 and are insen-

sitive to 3-MA, a class III PI3-kinase inhibitor, as reported

with resveratrol-induced autophagy [24]. Autophagy

induced by PCBL is class III PI3-kinase dependent, as

PCBL-induced autophagy is 3-MA sensitive and associated

with increased expression of Beclin 1. This provides evi-

dence for the canonical nature of PCBL-induced autoph-

agy. Atg5–Atg12 conjugative protein is responsible for the

conjugation between LC3 protein and phosphatidyl etha-

nolamine in autophagosome membranes and thus plays an

important role in the assembly of autophagosomes [19].

The expression of Atg5 and Atg12 proteins in their con-

jugative form in PCBL-treated cells prompted us to agree

with the view of Klionsky et al. [13] that increased Atg5–

Atg12 conjugation is not necessary to induce autophagy.

Our observations of Atg5 and Atg12 expression also sup-

ports the argument cautioning against the use of autophagy

related proteins other than LC3 II and p62 in assaying

autophagy.

Because of the overt tendency to use the term ‘auto-

phagic cell death’ to address the increased autophagy

associated with cell death, recent reviews stressed the need

to demarcate clearly between ‘cell death with autophagy’

and ‘cell death by autophagy’. The term ‘cell death with

autophagy’ is proposed to address a passive form of

autophagy induced in a dying cell with no role in regulating

cell death. Similarly ‘cell death by autophagy’ can be used

as a term to describe cell death where autophagy acts as a

prominent cell death mechanism [5, 6]. Comparisons of

cell death induced by PCBL in normal and autophagy

inhibited WiDr cells suggests that autophagy induced by

PCBL is ‘cell death with autophagy’. Our previous report

demonstrated that apoptosis was the main mode of cell

death induced by PCBL [7]. Given that PCBL induces

autophagy along with apoptosis, ‘apoptosis with autophagy

as a bystander’ is probably the apt terminology for

describing such cell death. Indeed, autophagy is not a

specialized mechanism for cell death. It can even be a pro-

survival pathway. Such pro-survival effects of autophagy

are capable of providing resistance against the cell death

induced by classic chemotherapeutic agents, thereby

reducing their effectiveness. The neutral modes of

autophagy induced in WiDr cells by PCBL also imply that

the activated autophagy mounts no resistance to PCBL-

induced cell death. Considering the perplexing, yet over-

whelming reports about the role of autophagy in cell death

induced by antitumor agents, it is worthwhile to gather

information regarding the role of autophagy in cell death

induced by a novel compound with promising antitumor

potential.

In summary, we demonstrated that PCBL-induced cell

death was associated with canonical, mTOR-independent

autophagy. However, autophagy associated with PCBL has

Mol Biol Rep (2014) 41:85–94 93

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only a bystander role in terms of WiDr cell death. From our

observations, autophagy may not possess a critical death

regulatory role in PCBL-induced cell death. However, this

report, in general perspective, raises concerns against the

canonical dogma that couples autophagy associated with

cell death as a prominent cell death regulator. Moreover,

this is the first work demonstrating autophagy induced by

PCBL.

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