598849

research-article2015
IJI0010.1177/0394632015598849International Journal of Immunopathology and PharmacologyWang et al.

Original article

International Journal of

Piperlongumine induces apoptosis and Immunopathology and Pharmacology

2015, Vol. 28(3) 362­–373
© The Author(s) 2015
autophagy in human lung cancer cells Reprints and permissions:
sagepub.co.uk/journalsPermissions.nav

through inhibition of PI3K/Akt/mTOR DOI: 10.1177/0394632015598849

iji.sagepub.com

pathway

Feng Wang,1* Yong Mao,2* Qingjun You,3 Dong Hua2 and

Dongyan Cai2

Abstract
Piperlongumine (PL), a natural alkaloid present in the fruit of the Long pepper, is known to exhibit notable anti-cancer
effects. Nonetheless, the anti-tumor effect of PL in lung cancer cells still remains unclear. In the present study, we
reported the chemotherapeutic effects of PL using in vitro and in vivo models. We showed that PL displayed potent
anti-neoplastic activity against lung cancer A549 cells as well as corresponding docetaxel-resistant A549/DTX cells. In
addition, we found that PL induced apoptosis in both A549 and A549/DTX cells. PL also induced autophagy in A549/
DTX cells. Moreover, autophagy-specific inhibitors (3-methyladenine) or Beclin1 and Atg 5 small interfering RNAs
(siRNAs) enhanced PL-induced apoptosis, indicating that PL-mediated autophagy may protect A549/DTX cells from
undergoing apoptotic cell death. Furthermore, we observed the inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt/
mammalian target of rapamycin (mTOR) pathway by PL. Finally, PL inhibited the growth of A549/DTX xenograft tumors,
which was associated with inhibition of cell proliferation, induction of apoptosis of tumor cells and decreased expression
of p-Akt and p-mTOR in tumor xenograft tissues. In summary, our study demonstrated that PL induced apoptosis and
autophagy through modulation of the PI3K/Akt/mTOR pathway in human lung cancer cells. This study may provide a
rationale for future clinical application using PL as a chemotherapeutic agent for lung cancer.

Keywords
apoptosis, autophagy, lung cancer, PI3K/Akt/mTOR pathway, piperlongumine

Date received: 5 June 2015; accepted: 9 July 2015

Introduction
Lung cancer is the leading cause of cancer-related
mortality among malignancies worldwide.1In spite
of significant achievement in the treatment of lung
cancer over the last decade, the prognosis for
patients with advanced disease remains poor.2
Docetaxel, a semi-synthetic analog of paclitaxel,
was granted approval as a first-line chemotherapy
It is now well established that chemotherapy-
induced reduction in tumor load is a function of
1Department of Cardiothoracic Surgery, Ninth People’s Hospital,
Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
2Oncology Center, Affiliated Hospital of Jiangnan University, Wuxi, PR
China
3Department of Cardiothoracic Surgery, Affiliated Hospital of Jiangnan

regimen for NSCLC.3 However, chemoresistance University, Wuxi, PR China

remains a major obstacle constraining the clinical
application of this agent. Therefore, it is urgent to
develop novel anti-tumor agents with least side
effects to meet the unmet therapeutic demand of
lung cancer patients.
*These authors contributed equally to this work.
Corresponding author:
Qingjun You, Department of Cardiothoracic Surgery, Affiliated Hospital
of Jiangnan University, No. 200 Huihe Road, Wuxi 214062, PR China.
Email: wxsytsg@126.com
Wang et al. 363
apoptotic cell death (type I programmed cell death)
involving several well-characterized morphological
changes, including cell volume loss, chromatic con-
densation, and nuclear fragmentation. However,
other research has revealed how apoptosis is attenu-
ated in certain tumors that succeed in progressing to
states of high-grade malignancy and resistance to
therapy.4 Therefore, there has been a surge of activ-
ity around identification of novel pathways of cell
death, which could function in tandem in the pres-
ence or absence of efficient apoptotic machinery. In
this regard, recent evidence has highlighted the
existence of autophagy, or type II programmed cell
death, which is activated in response to growth fac-
tor deprivation or upon exposure to anti-cancer
agents.5 Autophagy is a dynamic process involving
the sequestration of cytoplasmatic portions and
intracellular organelles into vacuoles called
autophagosomes. These vesicles are fused with lys-
osomes to generate autophagolysosomes and mature
lysosomes, where the sequestered material is
degraded, leading to cell death. More importantly,
persistent autophagy in response to cellular stress
states serves as a potent death signal, as in the case
of chemotherapy-induced autophagy, a specific
non-apoptotic death pathway has been triggered off.
However, the role of autophagy in cancer is still
controversial. Recent studies suggest that autophagy
is required for cancer survival6 and tumorigenesis.7
Autophagy contributes to cytoprotective events that
help cancer cells survive under conditions of low
nutrient supply and resistance to anti-cancer treat-
ments and tumor suppression, not only by recycling
of metabolites but also possibly by removing dam-
aged organelles and growth factors and reducing
chromosome instability.8,9 Autophagy may be
upregulated under conditions of distress, eventually
leading to cellular demise. Contribution of autophagy
to programmed cell death seems to be context-
dependent in various experimental systems.10–12 It is
likely that apoptosis and autophagy, the two differ-
ent modes of cell death, are coordinated and regu-
lated. The cross-talk between the two pathways
could be supported by the fact that various proteins
encoded by the autophagy related genes (Atg) can
be cleaved by apoptosis activated proteases, namely
caspases and calpains.13 The intricate relationship
between apoptosis and autophagy poses a big chal-
lenge for cancer treatment.
Naturally-occurring substances are the most
dependable resource for therapeutic development.
A variety of FDA approved anti-neoplastic agents,
including docetaxel and topotecan, have demon-
strated clinical utility based on ongoing investiga-
tions of natural products. Piperlongumine (PL), a
natural alkaloid of the Long pepper (Piper longum),
exhibits numerous key biological activities. In
addition to its insecticidal and bactericidal capa-
bilities,14 recent literature points to the fact that PL
is capable of hindering the growth of sarcoma,
bladder, breast, melanoma, and lung tumours in
vitro and in vivo.15,16 The ability of PL to target the
cellular stress response through direct inhibition of
Glutathione S-transferase pi 1 (GSTP1) subse-
quently inducing of intracellular reactive oxygen
species17 accumulation promoting selective killing
of cancer cells was initially demonstrated by Raj et
al.16 In addition, PL has minimal high-dose acute
toxicity, and does not appear to significantly affect
any biochemical, hematologic, and histopathologic
parameters in animal models. Moreover, PL down-
regulates NF-kB activation17 and induces rapid
depletion of the androgen receptor in prostate can-
cer cells.18 Furthermore, it has been reported that
PL promotes autophagy in several cancer cell lines,
including human PC-3 prostate cancer cells, breast
cancer MCF-7 cells, renal carcinoma 786-O cells,
and human osteosarcoma U2OS cells.19,20 However,
the role of PL in lung cancer in this regard remains
to be defined. Therefore, in the current study, we
reported the anti-tumor effects and the underlying
biological mechanisms of PL in lung cancer cells.
Materials and methods
Antibodies and reagents
PL was obtained from Indofine Chemical Company
(Hillsborough, NJ, USA). Annexin V–fluorescein
isothiocyanate (FITC)/propidium iodide (PI) apop-
tosis detection kit was supplied by Biouniquer
Tech (Nanjing, PR China). Cyto-ID® Autophagy
detection kit was purchased from Enzo Life
Sciences (Farmingdale, NY, USA). Antibodies
specific for Bcl-2, Bax, PARP, PI3K, Akt, pho-Akt
(Thr308 and Ser 473), mTOR, pho-mTOR (Ser 2448),
and β-actin were purchased from Cell Signaling
Technology (Danvers, MS, USA). Ki-67 and the
secondary antibodies were purchased from Santa
Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
The protein assay kit was from Bio-Rad (Hercules,
CA, USA). Akt small interfering RNA (siRNA) I
kit was supplied by Cell Signaling (Beverly, MA,
364 International Journal of Immunopathology and Pharmacology 28(3)
USA). Atg5, beclin 1 and mTOR siRNA kits were
purchased from Genepharma (Shanghai, PR
China). Protein inhibitor cocktail tablets were pur-
chased from Roche Applied Science (Mannheim,
Germany). Other chemicals were obtained from
Sigma (St Louis, MO, USA).
Cell culture
The human lung cancer cell lines A549 was pur-
chased from American Type Culture Collection
(Rockville, MD, USA), and cultured as recom-
mended as monolayers in DMEM medium
(GibcoBRL Life Technologies, Grand Island, NY,
USA) supplemented with 10% fetal bovine serum
(FBS; GibcoBRL Life Technologies) and 1% pen-
icillin-streptomycin-neomycin (GibcoBRL Life
Technologies), in a humidified incubator at 37°C
in a 5% CO2 atmosphere. Docetaxel-resistant A549
cell line (A549/DTX) were established and pre-
served in 50 μg/L final concentration of docetaxel
in our laboratory. Stock solution of PL (50 mM)
was prepared in DMSO and all test concentrations
were prepared by diluting stock solution in tissue
culture medium. Cells treated with DMSO only
served as a vehicle control.
MTT assay
Cell viability was analyzed using 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. Cells were seeded in 96-well plates at a den-
sity of 1000–1500 cells/well and incubated for 24,
48, or 72 h. Approximately 20 μL of MTT (5 mg/
mL; Sigma, St Louis, MO, USA) was added to each
well and incubated for 4 h. At the end of incubation,
supernatants were removed, and 150 μL of dimethyl-
sulfoxide (Sigma) was added to each well. The
absorbance value (optical density) of each well was
measured at 490 nm. All experiments were per-
formed thrice.
Colony formation assay
Cells were plated in 6-well culture plates at 600
cells/well. Each cell group had two wells. Cells
were treated with PL at the indicated dose for 48 h.
After incubation for another 10 days at 37°C, cells
were washed twice with phosphate buffered saline
and stained with hematoxylin solution. The number
of colonies containing >50 cells was counted under
a microscope. The colony formation efficiency was
calculated as (number of colonies/number of cells
inoculated) × 100%. All assays were independently
performed in triplicate.
Detection of apoptosis
PL-induced apoptotic cell death in A549 and A549/
DTX cells was quantitatively determined by flow
cytometry. Briefly, after treatment of cells with dif-
ferent concentrations of PL (6 and 10 µM) for 48 h,
cells were harvested, washed with PBS, and stained
with FITC Annexin V Apoptosis Detection Kit
(BD Biosciences), and analyzed on an Accuri C6
Flow Cytometer.
Cyto-ID staining assay
Cyto-ID is a proprietary reagent, which specifi-
cally labels autophagic vacuoles and co-localizes
with light chain 3 (LC3). Cyto-ID® Autophagy
detection kit (Enzo Life Sciences) was used accord-
ing to the manufacturer’s protocol. The fluores-
cence was measured by Tecan GeNios microplate
reader (Tecan, Finland).
Monodansylcadaverine staining assay
Monodansylcadaverine (MDC), a lysosomotropic
fluorescent compound, which selectively labels
autophagic vacuoles, was used to assess autophagy
induction as previously reported.21
Western blot analysis
A549 and A549/DTX cells were treated with PL
(0, 6, and 10 μM) for 48 h, cells were harvested,
washed with cold PBS, and lysed with ice-cold
lysis buffer supplemented with protease inhibitors.
Equivalent amounts of protein (30 μg) from each
cell lysate were resolved on SDS-PAGE. Gels were
electroblotted onto nitrocellulose membranes (0.45
μM; Bio-Rad), which were then incubated in
blocking solution (1×PBS, 0.1% Tween-20, 5%
non-fat dry milk powder) for 1 h at room tempera-
ture. Membranes were incubated with the primary
antibody at 4°C overnight. After additional TBST
washes, membranes were incubated with corre-
sponding horseradish peroxidase-conjugated sec-
ondary antibodies (Bio-Rad) for 1 h at room
temperature and detected by the enhanced chemi-
luminescence method (SuperSignal West Pico sub-
strate; Pierce; Rockford, IL, USA).
Wang et al. 365
Small interfering RNA transfection
A549 and A549/DTX cells were seeded in six-well
plates and transfected at 60% confluency with
siRNA duplexes against human Akt (100 nM),
mTOR (100 nM), Beclin1 (60 nM), Atg5 (60 nM),
or control siRNA by lipofectamine 2000
(Invitrogen) according to manufacturer’s protocol.
After 6 h incubation, the transfected cells were
exposed to arenobufagin for various time points,
followed by analysis of cell viability by MTT
assay, of apoptosis by flow cytometry, or of
autophagy by MDC staining assay. Transfection
efficiency was determined to be more than 75% by
fluorescein-labeled non-targeted siRNA control
(Ambion).
Tumor xenograft model
All procedures and experiments involving animals
in this study were performed in accordance with
the National Institutes of Health Guide for Care
and Use of Laboratory Animals. The study proto-
col was approved by the Animal Ethics Committee
at Shanghai Jiao Tong University School of
Medicine, PR China. Female athymic nude mice
aged 4–5 weeks were purchased from Shanghai
SLAC Laboratory Animal Co., Ltd. (Shanghai, PR
China) and were housed in the Animal Resource
Facility. Exponentially growing A549/DTX cells
(5×106 in 100 µL PBS) were injected subcutane-
ously in the right flank of each mouse. Tumor xen-
ografts were allowed to grow to an average size of
50–150 mm3 and were randomly assigned to three
different treatment groups (six mice per group): (1)
vehicle control (1% DMSO in physiological
saline); (2) PL 20 mg/kg; (3) PL 60 mg/kg. The
mice were administered PL via intraperitoneal
(i.p.) injections for 4 weeks (5 days/week). Tumor
size was measured on two axes with the aid of
Vernier calipers and tumor volume (mm3) was cal-
culated using the formula: 1/2(L × W2) where L is
the longest and W is the shortest axis. Mice were
euthanized at the end of the study and/or when
tumor size exceeded 2000 mm3.
TUNEL assay for apoptotic cells
The type of cell death (necrosis/apoptosis) was
evaluated by the terminal deoxynucleotidyl trans-
ferase-mediated deoxyuridine biotin nick-end
labeling (TUNEL) assay with an Apo-Direct kit
(Pharmingen, San Diego, CA, USA) as previously
reported.22 Briefly, after antigen retrieval, the
tumor sections (4 µm-thick) were fixed by incuba-
tion with 4% paraformaldehyde at 4°C. The per-
meabilized sections were incubated with terminal
deoxynucleotidyl transferase recombinant (rTdT)
enzyme-catalysed reaction and nucleotide mixture
for 60 min at 37°C in the dark. After immersion in
stop/wash buffer for 15 min at room temperature,
the sections were washed with PBS to remove
unincorporated fluorescein-12-dUTP and the
nuclei counterstained with hematoxylin.
Immunohistochemical detection of
Ki-67-positive, p-Akt-positive, and
p-mTOR-positive cells
Immunohistochemistry assays were performed
using antibodies against Ki67, p-Akt and p-mTOR.
After fixation, tumor sections (4 µm thick) were
deparaffinized and rehydrated. Following rehydra-
tion, antigen retrieval was carried out by placing
the slides in 10 mmol/L sodium citrate buffer (pH
6.0) at 95°C for 20 min followed by 20-min cool-
ing. The sections were then washed in PBS and
non-specific binding sites were blocked with 1%
bovine serum albumin with 2% goat serum in PBS
before incubation with antibody. After washing,
the sections were incubated with biotinylated sec-
ondary antibody followed by horseradish peroxi-
dase-conjugated streptavidin. The sections were
further incubated with 2,4-diaminobenzidine sub-
strate and counterstained with hematoxylin. Each
sample was examined separately and scored by
two pathologists.
Statistical analysis
Data are presented as mean ± SD unless otherwise
indicated. The statistical significance of the differ-
ence between the values of control and treatment
groups was determined by either Student t test or
simple one-way ANOVA followed by Tukey’s post
hoc test for multiple comparisons using Prism ver-
sion 5 (GraphPad Software, Inc.). Values of
P <0.05 were considered statistically significant.
Results
The effect of PL on cell growth was investigated
using MTT assay on human lung cancer A549 cells
and corresponding docetaxel-resistant A549/DTX
366 International Journal of Immunopathology and Pharmacology 28(3)

Figure 1.  Effects of piperlongumine (PL) on lung cancer cell growth and apoptosis. (a) A549 and A549/DTX cells were treated with
0, 1, 3, 6, or 10 μM piperlongumine for 24, 48, or 72 h, cell viability was measured by MTT assay. Absorbance was read at 490 nm
with averages from triplicate wells. (b) Effects of PL on colony formation. Two days after treatment at the indicated concentrations
of PL, cells were cultured for another 10 days, and colonies counted. (c) A549 and A549/DTX cells were treated with different
concentrations of PL (0, 6, and 10 μM) for 48 h, followed by Annexin V-FITC/PI staining. (d) A549 and A549/DTX cells were cultured
in DMEM medium with 10% fetal bovine serum and treated with PL (0, 6, and 10 μM) for 48 h, whole-cell lysates were collected and
subjected to western blot analysis with the indicated antibodies. Data are presented as mean ± SD (n = 3) from three independent
experiments. * P <0.05, ** P <0.01 compared with the control group.

cells. As shown in Figure 1a, PL potently inhibited A549 and A549/DTX cells were sensitive to PL
the growth of both A549 and A549/DTX cells in a reflected by the results of cell viability assay.
dose- and time-dependent manner. Of note, both Colony formation assay further confirmed that PL
Wang et al. 367

Figure 2.  PL elicits autophagy in A549/DTX cells. (a) A549/DTX cells were incubated with PL (0, 3, 6, and 10 μM) for 48 h and
stained with Cyto-ID for 30 min at 37°C. Intracellular Cyto-ID fluorescence was analyzed by Tecan GeNios microplate reader.
(b) A549/DTX cells were incubated with PL (0, 3, 6, and 10 μM) for 48 h and stained with MDC. Intracellular MDC fluorescence
was analyzed by confocal fluorescence microscopy. (c) Immunoblot analysis of LC3 in A549/DTX cells treated by PL and
Pepstatin A (8 µM, 16 h). Data are presented as mean ± SD (n = 3) from three independent experiments. ** P <0.01 compared
with the control group. Scale bars are 100 μm.

inhibited the lung cancer cells growth in a dose-
dependent manner (Figure 1b).
Annexin V-FITC and PI staining were used to
examine whether the reduction in cell growth in
A549 and A549/DTX cells by PL treatment was
related to the induction of apoptosis, the lung can-
cer cells were treated with varying concentrations
of PL and the percentage of apoptotic cells were
assessed. As shown in Figure 1c, treatment of A549
and A549/DTX with PL for 48 h resulted in a dose-
dependent increase in the number of apoptotic cells
in both cell lines. Interestingly, A549/DTX cells
showed lower sensitivity to PL-mediated apoptosis
than A549 cells.
To further investigate the underlying mecha-
nisms involved in PL-induced effects on apoptosis
in A549 and A549/DTX cells, we determined the
expression of several key cellular proteins involved
in apoptosis. As shown in Figure 1d, treatment of
A549 and A549/DTX cells with PL (6 and 10 µM)
for 48 h resulted in a dose-dependent induction of
cleavage PARP. In addition, Bcl-2 level was
decreased, whereas Bax level was increased.
Because A549/DTX cells showed lower sensi-
tivity to PL-mediated apoptosis than A549 cells,
we examined whether PL induced autophagy,
which would have influenced the sensitivity of
cancer cells to chemotherapy-induced apoptosis.
After treatment with PL, Cyto-ID Green reagent
staining showed the relative fluorescence intensity
of cells was significantly increased, indicating the
occurrence of autophagy (Figure 2a). Similar data
were achieved through MDC staining (Figure 2b).
Lipidation of microtubule-associated protein 1
LC3, as an autophagy marker, coats autophago-
somes during autophagy and is converted to LC3-II
resulting in the appearance of the delayed electro-
phoretic mobility in gel. Treatment with PL (0, 3,
368 International Journal of Immunopathology and Pharmacology 28(3)

Figure 3.  Inhibition of autophagy enhances apoptosis in PL-treated A549/DTX cells. (a, b) A549/DTX cells were pretreated
with 3-MA (3 mM) for 30 min prior to a 48 h treatment with PL (6 µM). Cells were stained with MDC (a), or LC3 was analyzed
by western blot (b). (c) A549/DTX cells treated with the combination of PL (6 µM) and 3-MA (3 mM) or Beclin+/Atg5+ siRNA
duplexes. Cell apoptosis was analyzed by Annexin V-FITC/PI staining (c). Data are presented as mean ± SD (n = 3) from three
independent experiments. ** P <0.01 compared with the control group. Scale bars are 100 μm.

6, and 10 μM) for 48 h, led to a significant increase
in LC3-II (Figure 2c). This increase was further
enhanced by the pretreatment of the cells with pep-
statin A, a protease inhibitor, which inhibits LC3-II
turnover (Figure 2c). All these results supported
the idea that PL induced autophagy in A549/DTX
cells.
It has been reported that autophagy may facili-
tate cell survival in adverse microenvironment, and
inhibition of autophagy may trigger increased
induction of apoptosis in cells.23 Recent studies
suggested that autophagy may cooperate with
apoptosis to promote cell death.24,25 We, therefore,
explored whether inhibition of autophagy increased
apoptosis in A549/DTX cells under PL treatment.
This transition to apoptosis could be interrogated
using both pharmacological and genetic inhibitors
of different points of the autophagic pathway and
assessing the induction of apoptosis. Here, we
employed 3-methyladenine (3-MA), a specific
autophagy inhibitor to target the earliest stages of
autophagosome formation. Alternatively, inhibi-
tion of autophagy could be achieved by selective
inhibition of Beclin1 and Atg5 using RNA interfer-
ence. Both Beclin1 and Atg5 are common targets
for autophagy inhibition. Pretreatment with 3-MA
remarkably attenuated the formation of acidic
autophagic vacuoles in the presence of PL (Figure
3a) and blocked the appearance of LC3-II in gel
(Figure 3b). Addition of 3-MA also significantly
increased the proportion of apoptotic cells when
compared with those with PL alone (Figure 3c).
Wang et al. 369

Similar phenomena were observed in cells after

genetic knockdown of the essential autophagy
genes Beclin1/Atg5 (Figure 3c). In summary, these
results suggested that autophagy may protect cells
from PL-induced apoptotic cell death, and block-
ing autophagy by pharmacologic or molecular
inhibition improved the killing effect of PL in
A549/DTX cells through increased apoptosis.
Given the critical role of PI3K/Akt/mTOR
pathway in controlling cell survival/death in
response to external stimuli,26 we investigated
whether this pathway plays a central role in
PL-mediated cell death. As shown in Figure 4a,
when A549 and A549/DTX cells were treated
with various concentrations of PL (0, 3, 6, and 10
μM) for 48 h, the levels of PI3K, phosphorylation
of Akt at Thr308 and Ser473, and its downstream
factor phosphorylation of mTOR (Ser2448) were
effectively suppressed in a concentration-depend-
ent manner. To further identify the role of Akt and
mTOR in PL-mediated cell growth inhibition, we
employed the Akt inhibitor LY294002 and mTOR
blockers or siRNA to silence Akt and mTOR,
respectively. We then examined the impact of PL
on cell viability. The results showed that inactiva-
tion of Akt and mTOR tremendously sensitized
A549 and A549/DTX cells toward cytotoxicity of
PL (Figure 4b). These results together revealed Figure 4.  PL inhibits the aberrant activation of PI3K/Akt/
that PL inhibits PI3K/Akt/mTOR signaling path- mTOR pathway. (a) A549 and A549/DTX cells were treated
way in lung cancer cells. with PL (0, 3, 6, and 10 μM) for 48 h, and expressions of PI3K,
Akt, p-AktThr308, p-AktSer473, phosphorylation of mTOR
As these in vitro studies indicated that treatment (Ser2448) in total cell lysates were evaluated by western blot
of A549 and A549/DTX cells with PL reduced the analysis. (b) A549 and A549/DTX cells were treated with
cell growth and induces apoptosis of these cells, PL (3 μM) in the absence or presence of LY294002 (4 μM)
we sought to determine whether administration of and Akt siRNA (100 nM), or treated with mTOR blocker
rapamycin (50 μM) for 30 min or transfected with mTOR
PL inhibits in vivo tumor growth using a A549/ siRNA (100 nM) for 6 h prior to 48 h treatment with PL. Cell
DTX xenograft mouse model. As seen in Figure viability was measured by MTT assay. Data are presented as
5a, PL significantly suppressed tumor growth at mean ± SD (n = 3) from three independent experiments.
doses of 20 and 60 mg/kg for 28 days compared ** P <0.01 compared with the PL alone group.
with the control group. PL administration resulted
in significant reduction of tumor volume in the Uncontrolled tumor cell proliferation is a char-
nude mice. Animal weight loss is commonly used acteristic feature of most cancers. We therefore
as a surrogate marker of toxicity. The average body analyzed the A549/DTX tumor xenografts for the
weights of the PL-treated and non-PL-treated mice potential anti-proliferative effects of PL using
were comparable throughout the experimental immunohistochemical detection of Ki-67-positive
period. As seen in Figure 5b, there was no differ- cells. As shown in Figure 5c, PL decreased the
ence in body weight and any other abnormality in expression of Ki-67, a cell proliferation marker. In
food intake or behavior in PL treatment groups addition, to determine whether inhibition of tumor
compared with the control group. These data sug- growth by administration PL is caused by the apop-
gested that administration of PL at the concentra- tosis of tumor cells in xenograft tissues, the apop-
tion used in these studies was not associated with totic effect of PL on A549/DTX tumor tissues was
apparent gross toxicity. identified by expression of the DNA fragment by
370 International Journal of Immunopathology and Pharmacology 28(3)

Figure 5.  PL inhibits in vivo tumor xenograft growth in athymic nude mice. (a) Anti-tumor activity of PL in nude mice bearing A549/
DTX tumors. Vehicle control and PL (20 and 60 mg/kg, i.p.) were administered for 4 weeks (5 days/week). (b) Effect of PL on body
weight. (c) Tumors were excised and processed for immunostaining for Ki-67, TUNEL, p-Akt, and p-mTOR. Scale bars are 100 μm.
Data in the graphs represent the mean ± SD (6 mice per group). **P <0.01, versus vehicle control.

TUNEL assay. The results showed that greater
numbers of TUNEL-positive cells in the samples
from PL-treated as compared with the control
group. Furthermore, we determined the effect of
PL on PI3K/Akt/mTOR pathway in tumor xeno-
graft samples. Immunohistochemical analysis sug-
gested that administration PL resulted in decreased
the phosphorylation of Akt and mTOR in the tumor
xenografts compared with the non-PL-treated
group.
Discussion
Cancer cells evade programmed cell death to sup-
port malignant growth.27,28 Therefore, understand-
ing the mechanisms of programmed cell death and
designing therapeutic approaches to trigger cell
death in cancer cells are critical for effectively treat-
ing the disease. The treatment of lung cancer remains
a major challenge because of poor efficacy and
severe toxicities of standard and new chemotherapy.
There is an increased interest in seeking new thera-
pies for lung cancers from natural compounds.
Natural products have played an important role as
an effective source of anti-tumor agents. It is esti-
mated that up to 30–40% of the anti-cancer drugs
used globally are derived from plant sources.29 PL, a
natural alkaloid present in the fruit of the Long pep-
per, is a promising bioactive molecule that has dem-
onstrated anti-carcinogenic effects in some tumor
models.19,20,30,31 Moreover, it has been reported that
in a melanoma allografted mouse model, the anti-
tumor effect of PL was greater than that of the cyto-
toxic chemotherapeutic drug, cisplatin. Similarly,
PL outperformed paclitaxel in controlling a polyoma
middle T antigen-driven mouse model of spontane-
ous mammary tumorigenesis.32 However, the role of
PL in lung cancer remains unclear. We therefore
undertook a comprehensive analysis of the effects of
PL on lung cancer cells using both in vitro and in
vivo models. In this study, we demonstrated that PL
was effective against drug-sensitive A549 cells as
Wang et al. 371
well as docetaxel-resistant A549/DTX cells. Its anti-
tumor activity in vivo without weight loss or other
life-threatening toxicities in animals supports its
potential for further clinical investigation for lung
cancer treatment. Our data also showed that PL
induces apoptosis in A549 and A549/DTX cells,
accompanied by an increase of Bax/Bcl-2 expres-
sion ratio and cleavage of PARP. In accord with our
findings, some researchers reported that PL induces
apoptosis in human triple-negative breast cancer
cells and ovarian cancer cells.33,34
It is intriguing that the apoptotic rate in A549/
DTX cells was much lower than that of A549 cells
suggesting the presence of a cytoprotective mecha-
nism. We thus examined whether PL induced
autophagy. Autophagy is a catabolic process in
which cells respond to various stress stimuli, such
as hypoxia, nutrient starvation, and DNA damage.35
In this process, proteins or organelles, sequestered
by double-membrane structures, fuse with lys-
osomes and are subsequently degraded by lysoso-
mal hydrolases to be recycled to sustain
metabolism.36 However, the exact role of autophagy
in cancer treatment, and whether it protects cells
from cytotoxic effects of anti-cancer drugs by
blocking apoptosis or kills cells as an alternate
pathway of cell death is still controversial.12,37 Our
data revealed that PL could induce autophagy
accompanied by apoptosis in A549/DTX cells. We
found that inhibition of autophagy by specific
inhibitors (3-MA) or Beclin1+/Atg5+ siRNA mark-
edly increased apoptosis. Collectively, these results
indicate that autophagy might provide a protective
mechanism against PL-induced apoptosis.
Increasing evidence indicates that the cross-talk
between autophagy and apoptosis is made espe-
cially complicated by the fact that they share many
common regulatory molecules, such as p53, Bcl-2,
and the PI3K/Akt/mTOR signaling pathway.38,39 It
is well known that the PI3K/Akt/mTOR pathway
plays an important role in cell growth, survival, dif-
ferentiation, and metabolism.40 Inhibition of PI3K/
Akt/mTOR signaling pathway causes cell death
associated with apoptosis and/or autophagy.41,42
The present results indicated that PL regulates
PI3K, Akt, and mTOR. Either Akt-specific inhibi-
tor LY294002 or Akt gene silencing by siRNA pre-
treatment caused the decrease in cell viability.
Furthermore, mTOR inhibitor rapamycin or mTOR
siRNA pretreatment enhances the cytotoxicity of
PL, which further proved the critical role of PI3K/
Akt/mTOR pathway.
Although in vitro cell culture models are a good
system for preliminary screening of the effects of
chemotherapeutic agents; the observations must be
verified in vivo using animal models prior to their
potential consideration of their use in humans. We
therefore used an in vivo model of xenografts of
A549/DTX tumor cells in athymic nude mice to
verify the chemotherapeutic potential of PL against
lung cancer cell growth. Our study provided evi-
dence that administration of PL inhibited the
growth of A549/DTX lung tumor xenografts with-
out any apparent sign of toxicity in the athymic
nude mice. These data are in accordance with the
decreased proliferation documented by Ki67
immunostaining. Additionally, PL induced apopto-
sis as indicated by TUNEL staining in tumor tis-
sues, at least in part, by targeting PI3K/Akt/mTOR
cell survival pathway. These in vivo results are
consistent to our in vitro study.
In conclusion, the present study demonstrates
that PL inhibits the PI3K/Akt/mTOR pathway and
invokes strong anti-cancer activity against lung
cancer by inducing apoptosis as well as autophagy.
Thus PL appears to be an attractive bioactive phy-
tochemical for lung cancer chemoprevention and/
or treatment. Our studies provide a rationale for the
development of PL as chemotherapeutic agent
against lung cancer in the clinical setting. K-ras
mutant lung tumors might benefit from PL. Further
studies evaluating the anti-tumor effects of PL for
normal and other K-ras mutant lung tumors are
needed to advance this treatment approach to a
clinical setting.
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
This work was supported by Key Talent Project of Jiangsu
Province (No. RC2011032), Major Projects of Hospital
Management Center in Wuxi City (No. YGZX201212),
and Project of National Science Foundation (No.
81401186).
References
1. Jemal A, Bray F, Center MM, et al. (2011) Global
cancer statistics. CA: A Cancer Journal for Clinicians
61: 69–90.
2. Gettinger S and Lynch T (2011) A decade of advances
in treatment for advanced non-small cell lung cancer.
Clinics in Chest Medicine 32: 839–851.
372 International Journal of Immunopathology and Pharmacology 28(3)
3. Uygun K, Aksu G, Cicin I, et al. (2008) The efficiency
of single agent docetaxel in patients with platinum-
refractory non-small cell lung carcinoma. Medical
Oncology 25: 408–414.
4. Adams JM and Cory S (2007) The Bcl-2 apoptotic
switch in cancer development and therapy. Oncogene
26: 1324–1337.
5. Tsujimoto Y and Shimizu S (2005) Another way to
die: Autophagic programmed cell death. Cell Death
and Differentiation 12 Suppl 2: 1528–1534.
6. Yang S, Wang X, Contino G, et al. (2011) Pancreatic
cancers require autophagy for tumor growth. Genes &
Development 25: 717–729.
7. Guo JY, Chen HY, Mathew R, et al. (2011) Activated
Ras requires autophagy to maintain oxidative metabo-
lism and tumorigenesis. Genes & Development 25:
460–470.
8. Edinger AL and Thompson CB (2003) Defective
autophagy leads to cancer. Cancer Cell 4: 422–424.
9. Zhou S, Zhao L, Kuang M, et al. (2012) Autophagy
in tumorigenesis and cancer therapy: Dr. Jekyll or Mr.
Hyde? Cancer Letters 323: 115–127.
10. Gozuacik D and Kimchi A (2007) Autophagy and cell
death. Current Topics in Developmental Biology 78:
217–245.
11. Kourtis N and Tavernarakis N (2009) Autophagy
and cell death in model organisms. Cell Death and
Differentiation 16: 21–30.
12. Maiuri MC, Zalckvar E, Kimchi A, et al. (2007) Self-
eating and self-killing: Crosstalk between autophagy
and apoptosis. Nature Reviews Molecular Cell
Biology 8: 741–752.
13. Norman JM, Cohen GM and Bampton ET (2010) The
in vitro cleavage of the hAtg proteins by cell death
proteases. Autophagy 6: 1042–1056.
14. Yang YC, Lee SG, Lee HK, et al. (2002) A piperi-
dine amide extracted from Piper longum L. fruit
shows activity against Aedes aegypti mosquito lar-
vae. Journal of Agricultural and Food Chemistry 50:
3765–3767.
15. Bezerra DP, Pessoa C, Moraes MO, et al. (2008) In
vivo growth inhibition of sarcoma 180 by piperlon-
guminine, an alkaloid amide from the Piper species.
Journal of Applied Toxicology 28: 599–607.
16. Raj L, Ide T, Gurkar AU, et al. (2011) Selective kill-
ing of cancer cells by a small molecule targeting the
stress response to ROS. Nature 475: 231–234.
17. Han SS, Son DJ, Yun H, et al. (2013) Piperlongumine
inhibits proliferation and survival of Burkitt lym-
phoma in vitro. Leukemia Research 37: 146–154.
18. Golovine KV, Makhov PB, Teper E, et al. (2013)
Piperlongumine induces rapid depletion of the andro-
gen receptor in human prostate cancer cells. Prostate
73: 23–30.
19. Makhov P, Golovine K, Teper E, et al. (2014)
Piperlongumine promotes autophagy via inhibition of
Akt/mTOR signalling and mediates cancer cell death.
British Journal of Cancer 110: 899–907.
20. Wang Y, Wang JW, Xiao X, et al. (2013)
Piperlongumine induces autophagy by targeting p38
signaling. Cell Death & Disease 4: e824.
21. Gao M, Yeh PY, Lu YS, et al. (2008) OSU-03012, a
novel celecoxib derivative, induces reactive oxygen
species-related autophagy in hepatocellular carci-
noma. Cancer Research 68: 9348–9357.
22. Prasad R, Vaid M and Katiyar SK (2012) Grape proan-
thocyanidin inhibit pancreatic cancer cell growth in
vitro and in vivo through induction of apoptosis and by
targeting the PI3K/Akt pathway. PLoS One 7: e43064.
23. Xu ZX, Liang J, Haridas V, et al. (2007) A plant trit-
erpenoid, avicin D, induces autophagy by activation
of AMP-activated protein kinase. Cell Death and
Differentiation 14: 1948–1957.
24. Bareford MD, Hamed HA, Tang Y, et al. (2011)
Sorafenib enhances pemetrexed cytotoxicity through
an autophagy-dependent mechanism in cancer cells.
Autophagy 7: 1261–1262.
25. Dalby KN, Tekedereli I, Lopez-Berestein G, et al.
(2010) Targeting the prodeath and prosurvival func-
tions of autophagy as novel therapeutic strategies in
cancer. Autophagy 6: 322–329.
26. Yap TA, Garrett MD, Walton MI, et al. (2008)
Targeting the PI3K-AKT-mTOR pathway: pro-
gress, pitfalls, and promises. Current Opinion in
Pharmacology 8: 393–412.
27. Choi KS (2012) Autophagy and cancer. Experimental
& Molecular Medicine 44: 109–120.
28. Hu YL, Jahangiri A, Delay M, et al. (2012) Tumor
cell autophagy as an adaptive response mediating
resistance to treatments such as antiangiogenic ther-
apy. Cancer Research 72: 4294–4299.
29. Newman DJ, Cragg GM and Snader KM (2003) Natural
products as sources of new drugs over the period 1981–
2002. Journal of Natural Products 66: 1022–1037.
30. Bharadwaj U, Eckols TK, Kolosov M, et al. (2015)
Drug-repositioning screening identified piperlongu-
mine as a direct STAT3 inhibitor with potent activity
against breast cancer. Oncogene 34: 1341–1353.
31. Liu Y, Chang Y, Yang C, et al. (2014) Biodegradable
nanoassemblies of piperlongumine display enhanced
anti-angiogenesis and anti-tumor activities. Nanoscale
6: 4325–4337.
32. Burgess DJ (2011) Anticancer drugs: Selective oxy-
cution? Nature Reviews Drug Discovery 10: 658.
33. Gong LH, Chen XX, Wang H, et al. (2014)
Piperlongumine induces apoptosis and synergizes with
cisplatin or paclitaxel in human ovarian cancer cells.
Oxidative Medicine and Cellular Longevity 2014:
906804.
34. Shrivastava S, Kulkarni P, Thummuri D, et al. (2014)
Piperlongumine, an alkaloid causes inhibition of PI3 K/
Akt/mTOR signaling axis to induce caspase-dependent
Wang et al. 373
apoptosis in human triple-negative breast cancer cells.
Apoptosis 19: 1148–1164.
35. Rabinowitz JD and White E (2010) Autophagy and
metabolism. Science 330: 1344–1348.
36. Klionsky DJ and Emr SD (2000) Autophagy as a regu-
lated pathway of cellular degradation. Science 290:
1717–1721.
37. Eisenberg-Lerner A, Bialik S, Simon HU, et al.
(2009) Life and death partners: Apoptosis, autophagy
and the cross-talk between them. Cell Death and
Differentiation 16: 966–975.
38. Turcotte S and Giaccia AJ (2010) Targeting can-
cer cells through autophagy for anticancer therapy.
Current Opinion in Cell Biology 22: 246–251.
39. Zhang DM, Liu JS, Deng LJ, et al. (2013)
Arenobufagin, a natural bufadienolide from toad
venom, induces apoptosis and autophagy in human
hepatocellular carcinoma cells through inhibition of
PI3K/Akt/mTOR pathway. Carcinogenesis 34: 1331–
1342.
40. Hennessy BT, Smith DL, Ram PT, et al. (2005)
Exploiting the PI3K/AKT pathway for cancer drug
discovery. Nature Reviews Drug Discovery 4: 988–
1004.
41. Shrivastava A, Kuzontkoski PM, Groopman JE, et al.
(2011) Cannabidiol induces programmed cell death
in breast cancer cells by coordinating the cross-talk
between apoptosis and autophagy. Molecular Cancer
Therapeutics 10: 1161–1172.
42. Wang K, Liu R, Li J, et al. (2011) Quercetin
induces protective autophagy in gastric cancer cells:
Involvement of Akt-mTOR- and hypoxia-induced
factor 1alpha-mediated signaling. Autophagy 7:
966–978.