Citation: Cell Death and Disease (2014) 5, e1504; doi:10.1038/cddis.2014.

463
OPEN & 2014 Macmillan Publishers Limited All rights reserved 2041-4889/14
www.nature.com/cddis

Role of AMP-activated protein kinase in cross-talk

between apoptosis and autophagy in human
colon cancer
X Song1, S-Y Kim1, L Zhang2, D Tang1, DL Bartlett1, YT Kwon3 and YJ Lee*,1,2

Unresectable colorectal liver metastases remain a major unresolved issue and more effective novel regimens are
urgently needed. While screening synergistic drug combinations for colon cancer therapy, we identified a novel multidrug
treatment for colon cancer: chemotherapeutic agent melphalan in combination with proteasome inhibitor bortezomib and
mTOR (mammalian target of rapamycin) inhibitor rapamycin. We investigated the mechanisms of synergistic antitumor efficacy
during the multidrug treatment. All experiments were performed with highly metastatic human colon cancer CX-1 and HCT116
cells, and selected critical experiments were repeated with human colon cancer stem Tu-22 cells and mouse embryo fibroblast
(MEF) cells. We used immunochemical techniques to investigate a cross-talk between apoptosis and autophagy during the
multidrug treatment. We observed that melphalan triggered apoptosis, bortezomib induced apoptosis and autophagy,
rapamycin caused autophagy and the combinatorial treatment-induced synergistic apoptosis, which was mediated through an
increase in caspase activation. We also observed that mitochondrial dysfunction induced by the combination was linked
with altered cellular metabolism, which induced adenosine monophosphate-activated protein kinase (AMPK) activation,
resulting in Beclin-1 phosphorylated at Ser 93/96. Interestingly, Beclin-1 phosphorylated at Ser 93/96 is sufficient to induce
Beclin-1 cleavage by caspase-8, which switches off autophagy to achieve the synergistic induction of apoptosis. Similar
results were observed with the essential autophagy gene, autophagy-related protein 7, -deficient MEF cells. The multidrug
treatment-induced Beclin-1 cleavage was abolished in Beclin-1 double-mutant (D133A/D146A) knock-in HCT116 cells,
restoring the autophagy-promoting function of Beclin-1 and suppressing the apoptosis induced by the combination therapy.
These observations identify a novel mechanism for AMPK-induced apoptosis through interplay between autophagy and
apoptosis.
Cell Death and Disease (2014) 5, e1504; doi:10.1038/cddis.2014.463; published online 30 October 2014

Colorectal cancer ranks third in major causes of cancer- therapy. We previously investigated the mechanism of the
related mortality worldwide, appearing in ~ 150 000 new cases synergy between hyperthermia, biological agents (TNF-
in the United States annually, and ~ 20–50% of colorectal related apoptosis-inducing ligand/mapatumumab) and
cancer patients display hepatic metastases.1,2 Current stan- chemotherapeutic agent (oxaliplatin).8–10 However, the clinical
dard therapies for treating metastatic colon cancer include grade of those biological agents is no longer available after
chemotherapy and biological therapy followed by tumor newly merged companies decided not to produce them. We
resection, up front tumor resection followed by systemic then investigated potential replacement drugs, which are
therapy, radiofrequency ablation, thermal ablation, selective already Food and Drug Administration (FDA) approved. We
internal radiation therapy and hyperthermic isolated hepatic screened several FDA-approved drugs including melphalan,
perfusion (IHP) therapy.3–7 Although these therapies are chlorquine, bortezomib, carbamazepine, celecoxib, cetuximab
somewhat effective, more effective novel regimens are still and rapamycin using cytoxicity assay. We found that MBR
needed to improve the survival of patients with liver (melphalan+bortezomib+rapamycin) treatment has the best
metastases from colorectal cancer. cytotoxic effect on colon cancer cells and also on colon cancer
As unresectable liver metastases from colorectal cancer are stem cells. Currently, 2807 clinical trials are listed for colon
difficult to treat by single modality, we have spent several years cancer; of these, 225 studies and 195 studies are related to
developing a multimodality approach for hyperthermic IHP FOLFOX (folinic acid+fluorouracil+oxaliplatin) therapy and

1
Department of Surgery, University of Pittsburgh, Hillman Cancer Center, Pittsburgh, PA 15213, USA; 2Department of Pharmacology and Chemical Biology, School of
Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA and 3Protein Metabolism Medical Research Center and Department of Biomedical Science, College of
Medicine, Seoul National University, Seoul 110-799, South Korea
*Corresponding author: YJ Lee, Department of Surgery, University of Pittsburgh, Hillman Cancer Center, Pittsburgh, PA 15213, USA. Tel: +1 412 623 3268; Fax: +1 412 623
7709; E-mail: leeyj@upmc.edu
Abbreviations: AICAR, aminoimidazole carboxamide ribonucleotide; AMPK, 5' adenosine monophosphate-activated protein kinase; ATP, adenosine-5'-triphosphate;
ATG, autophagy-related protein; BAX, Bcl-2-associated X protein; FDA, Food and Drug Administration; FITC, fluorescein isothiocyanate; IHP, isolated hepatic perfusion;
LC3, microtubule-associated protein 1A/1B-light chain 3; mTOR, mammalian target of rapamycin; PARP, poly (ADP-ribose) polymerase; PAGE, polyacrylamide gel
electrophoresis; SDS, sodium dodecyl sulfate; TRAIL, TNF-related apoptosis-inducing ligand
Received 30.4.14; revised 20.8.14; accepted 25.8.14; Edited by R Aqeilan
 Role of AMPK in a multimodality treatment
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FOLFIRI (folinic acid+fluorouracil+irinotecan) therapy, respec- cells detected by Annexin V/PI assay were observed in the
tively. There are only seven studies for IHP and, specifically, upper right quadrant of each plot. Our data clearly show that
there is no clinical trial with MBR for hyperthermic IHP therapy. treatment with MBR enhanced synergistic induction of
Apoptosis is a major cytotoxic mechanism of chemotherapy; apoptotic death. These synergistic effects were due to an
stress-induced apoptosis often proceeds through the intrinsic increased activation of caspases, and thus, the hallmark of
pathway where permeabilization of the mitochondrial outer apoptosis, poly (ADP-ribose) polymerase (PARP) cleavage
membrane releases cytochrome c and activates the caspase (Figures 1d and e). Similar results were observed in human
cascade.11,12 Melphalan hydrochloride (trade name Alkeran), colon cancer stem Tu-22 cells (Figure 1f).
which is commonly used in IHP, leads to double-stranded DNA
breaks and subsequent cell death through a caspase- MBR-induced mitochondrial dysfunction and AMPK
mediated, apoptotic pathway.13,14 activation. Stress-induced apoptosis often proceeds
Bortezomib, the first clinically available proteasome inhibitor, through the intrinsic pathway mediated by THE mitochondria.
possesses antitumor activity in a variety of human cancers In Figures 2a and b, the upper left quadrant of each plot
and is often used in the treatment of hematological displays cells with intact mitochondrial membrane potential,
malignancies. It can induce both proapoptotic effects, includ- whereas the lower right quadrant displays cells with impaired
ing the induction of Bik, Bim and Noxa proteins, and mitochondrial membrane potential. A significant shift
antiapoptotic effects, including the accumulation of Mcl-1 occurred to the lower right part of the quadrants in the
and HSP70, as well as autophagic formation.15–17 treatment with MBR. Figure 2c indicates that more cyto-
mTOR (mammalian target of rapamycin) is known to be well chrome c was released during multidrug treatment. We then
conserved and ubiquitously expressed in endothelial cells and assessed the ATP production as the mitochondria are known
is involved in cell energy metabolism, cell growth, apoptosis to have an important role in providing overall cellular energy
and autophagy. Several human cancers display mTOR supply. ATP production was markedly decreased during the
hyperactivation, thus making mTOR an attractive target in treatment with MBR in HCT116 cells (Figure 2d) and CX-1
cancer therapy.18 Sirolimus, known as rapamycin, an mTOR cells (data not shown). Figure 2e shows that MBR induced a
inhibitor, has relatively low cytotoxic activity. Better therapeutic large amount of phosphorylation (activation) of AMPK. We
outcomes should be obtained by using rapamycin in combina- then investigated whether Bcl-2-associated X protein (Bax)
tion with other anticancer agents.19,20 In this study, we was involved in the combinatorial treatment-induced apopto-
observed that melphalan triggered apoptosis, bortezomib sis. Data from Figure 2f clearly demonstrates that MBR-
induced both apoptosis and autophagy, rapamycin caused induced apoptosis and caspase activation were effectively
autophagy and the combinatorial treatment promoted mito- suppressed in Bax-deficient cells, indicating that the synergy
chondrial dysfunction and synergistic apoptosis. of MBR-associated apoptosis is partially mediated through
Hallmarks of cancer cells include uncontrolled growth, Bax. Interestingly, AMPK activation was also decreased in
evasion of apoptosis, immortality, ability to invade other HCT116 Bax− / − cells, implying that Bax contributes to
tissues and altered cellular metabolism.21 In our study, we AMPK activation. The hallmark of autophagy, microtubule-
investigated the effect of multidrug treatment on cellular associated protein 1A/1B-light chain 3 (LC3)-II, increased
metabolism. This study revealed that the combinatorial markedly in bortezomib-treated cells and mildly in rapamycin-
treatment altered cellular metabolism and induced energy treated cells but no significant increase with melphalan
sensor AMP-activated protein kinase (AMPK) activation at treatment. Notably, there was a significant decrease of LC3-
two stages in the process, resulting in Beclin-1 phosphoryla- II in the treatment of MBR compared with that of bortezomib
tion and autophagy starting with the early stage and Beclin-1 alone. Interestingly, LC3-II was increased in the treatment of
cleavage by caspase-8 and apoptosis concurrent with the MBR in HCT116 Bax− / − cells compared with that of HCT116
late stage. Our observations provided evidence that AMPK Bax+/+ cells, indicating that autophagy was increased in the
has an important role in cross-talk between autophagy and apoptosis-suppressed cells (Figure 2f). These results
apoptosis. indicate a cross-talk between autophagy and apoptosis
during treatment with MBR. Figure 2g shows that cytotoxicity
was significantly decreased in HCT116 Bax− / − compared
Results with HCT116 Bax+/+ (Po0.01).

MBR synergistically induced cytotoxicity and apoptosis. Interplay between autophagy and apoptosis during
To investigate the effect on cell viability of the application of treatment with MBR. To further investigate a relationship
MBR, human colorectal carcinoma HCT116 and CX-1 cells between apoptosis and autophagy during treatment with
were treated with a combination of 10 μg/ml melphalan, along MBR, we examined autophagy using as an autophagy-
with 50 nM bortezomib and/or 2.5 μg/ml rapamycin for 24 h. specific marker both processing of LC3-I into LC3-II using
Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)- immunoblot assay and also green fluorescent protein (GFP)-
5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazo- LC3 puncta formation using confocal microscopy. We
lium (MTS) assay. As shown in Figures 1a and b, synergistic observed that LC3-II (Figures 3a and b) and LC3 puncta
effect was observed in MBR compared with any single formation (Figure 3c) increased significantly in bortezomib
treatment or bitreatment in both cell lines (Po0.01). Our alone or in combination with melphalan or rapamycin.
observation was confirmed by combination index (CI) However, MBR treatment suppressed LC3-II or LC3 puncta
analysis; CI values were o1 (Table 1). In Figure 1c, apoptotic formation compared with bortezomib alone. It is known that

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Figure 1 MBR induced cytotoxicity and apoptosis. (a and b) HCT116 (a) and CX-1 (b) cells were treated with MBR for 24 h. Cell viability was analyzed by MTS assay. Error
bars represent S.D. from triplicate experiments. **Po0.01 represents a statistically significant difference. (c) HCT116 cells were treated with MBR for 24 h and cells were stained
with fluorescein isothiocyanate (FITC)-Annexin V and PI. Apoptosis was detected by the flow cytometric assay. (d–f) After treatment, the cleavage of caspase-8, caspase-9,
caspase-3 and PARP was detected by immunoblotting in HCT116 (d), CX-1 (e) and Tu-22 (f) cells. Actin was used to confirm the equal amount of proteins loaded in each lane

the classical autophagy pathway is dependent on Beclin-1, The role of AMPK in MBR-induced apoptosis. To further
autophagy-related protein 7 (ATG7), and so on.22 Figure 3d investigate a cross-talk between autophagy and apoptosis,
shows that MBR treatment-induced apoptosis was enhanced we hypothesized that enhancement of MBR-induced
in mouse embryo fibroblast (MEF) ATG7− / − cells. Similar apoptosis is mediated through the activation of AMPK. To
results were observed by MTS assay (Figure 3e). These test this hypothesis, we added an AMPK inhibitor, compound
results suggest that inhibition of autophagy enhances C, in the absence or presence of MBR. Figures 4a and b
multidrug-induced apoptosis. show that MBR-induced apoptosis was partially suppressed

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Table 1 Combination index for melphalan in combination with bortezomib and cleavage and switching the mode of cell death from
rapamycina autophagy to apoptosis.
Combination therapy Combination index
The kinetics of AMPK-α activation and Beclin-1
Melphalan Bortezomib Rapamycin HCT116 CX-1 phosphorylation and cleavage in the treatment of MBR.
(μg/ml) (nM) (μg/ml) Next, we examined the kinetics of MBR-induced apoptosis. As
10 50 2.5 0.33 0.52 shown in Figures 6a and b, MBR-induced apoptosis was
20 100 5 0.35 0.80 increased as time progressed. In CX-1 cells, AMPK was
40 200 10 0.49 0.95 phosphorylated (activated) very early at 1 h and then
a
decreased. Notably, activation of AMPK was increased again
Calculated by Compusyn software for HCT116 and CX-1 colon cancer cells
at around 16 h in a sustained manner. Beclin-1 phosphoryla-
tion at Ser 93/96 was markedly increased after 3 h. Beclin-1
cleavage increased after 12 h when apoptosis occurred and
by the addition of compound C, inhibiting the activation of then apoptosis was significantly increased. We also monitored
AMPK-α. This observation was confirmed by knocking down autophagy in CX-1 cells after MBR treatment, using as an
the expression of AMPK-α. Figure 4c shows that a combina- autophagy-specific marker processing of LC3-I into LC3-II.
tion of AMPK-α1 and AMPK-α2 small interfering RNA (siRNA) Interestingly, during the first 16 h, MBR treatment co-occurred
downregulated AMPK-α expression level and partially sup- with increased autophagy indicated by increased LC3-II levels.
pressed MBR-induced apoptosis, which was consistent with But then, reduced levels of LC3-II were observed at 24 h after
the effects of compound C. Our results suggest that MBR- treatment, implying that once caspases were fully activated,
induced AMPK activation has an important role in the cross- autophagy levels were diminished. Similar results were
talk between autophagy and apoptosis. To further investigate obtained in HCT116 cells, which were more sensitive towards
the treatment of MBR compared with CX-1 cells (Figure 6b). To
the role of AMPK activators metformin and AICAR (aminoi-
further test the differential role of AMPK activation at different
midazole carboxamide ribonucleotide) in MBR-induced cyto-
stage, we used compound C to inhibit transiently AMPK at
toxicity, cells were treated with metformin/AICAR in the
early stage (from 0 to 4 h and washed at 4 h with phosphate-
absence or presence of MBR. As shown in Figure 4d,
buffered saline) and late stage (from 10 to 24 h) separately. As
metformin alone did not affect CX-1 cell viability. However,
shown in Figures 6c and d, we observed that inhibiting AMPK
MBR-induced cytotoxicity was significantly enhanced in the
at early time point enhanced MBR-induced apoptosis, whereas
presence of 20 mM metformin. Similar results were observed
inhibiting AMPK at late time point abolished MBR-induced
in HCT116 cells (Figure 4e). Figures 4f and g show that
apoptosis in CX-1 and HCT116 cells.
AICAR alone did not affect cell viability in CX-1 and HCT116
We further examined the kinetics of autophagy during
cells but MBR-induced cytotoxicity was enhanced in the
treatment with MBR; GFP-LC3 puncta formation was inves-
presence of AICAR.
tigated. Figures 6e and f show that LC3 puncta formation was
detected at 3 h, and increased until 16 h and then decreased
AMPK activation-induced Beclin-1 phosphorylation at ~ 2-fold at 24 h compared with that of 16 h of MBR treatment.
Ser 93/96 and Beclin-1 cleavage. To examine the role of Moreover, Figure 6g shows that PARP cleavage, AMPK
AMPK in the cross-talk between autophagy and apoptosis, activation and Beclin-1 phosphorylation at Ser 93/96, as well
we further investigated AMPK downstream molecules. as Beclin-1 cleavage, consistently increased even at 36 h. We
Recently, AMPK was reported to phosphorylate Beclin-1 at also observed that LC3-II was markedly decreased at 28 and
Ser 91/94 for mouse or Ser 93/96 for human upon glucose 36 h. These data suggest that early AMPK activation-induced
starvation.23 C-terminal fragment of Beclin-1 localizes at the Beclin-1 phosphorylation and resulted in autophagy, whereas
mitochondria, inducing cytochrome c release and thus sustained late AMPK activation co-occurring with the activa-
enhancing the apoptosis.24 We observed Beclin-1 phosphor- tion of caspases induced Beclin-1 cleavage, enhancing
ylation at Ser 93/96 in HCT116 and CX-1 cells (Figures 5a apoptotic cell death.
and b), as well as Beclin-1 cleavage in HCT116 cells
(Figure 5c), during the treatment with MBR. Similar results Beclin-1 phosphorylation at Ser 91/94 is sufficient to
were observed in Tu-22 cells (Figure 5d). We also observed induce Beclin-1 cleavage and thus contributes to the
that AMPK inhibitor, compound C, inhibited Beclin-1 phos- synergistic induction of apoptosis. To evaluate the effect
phorylation at Ser 93/96 and Beclin-1 cleavage in both cell of Beclin-1 phosphorylation at Ser 91/94 on its cleavage, we
lines (Figures 5e and f). Figure 5g shows that 1 mM AICAR transiently transfected HCT116 cells with empty vector
alone, which activated AMPK, did not induce apoptosis in (pcDNA), wild-type murine Beclin-1, murine Beclin-1 S91-
CX-1 and HCT116 cells. However, AICAR enhanced MBR- /94A (residues 91 and 94 serine were replaced by alanine:
induced apoptosis. We also observed that MBR-induced dominant-negative mutant) and murine Beclin-1 S91/94D
PARP cleavage, Beclin-1 phosphorylation at Ser 93/96 and (residues 91 and 94 serine were replaced by aspartic acid:
Beclin-1 cleavage were enhanced in the presence of AICAR dominant-positive mutant) and then treated with MBR for
in both cell lines. AICAR alone increased the level of LC3-II 24 h. As shown in Figure 7a, phosphorylation of wild-type
(autophagy). However, a combinatorial treatment of MBR and murine Beclin-1, but not Beclin-1 S91/94A or Beclin-1
AICAR reduced MBR-induced LC3-II. These results suggest S91/94D, was elevated by treatment with MBR. Interestingly,
a strong correlation between AMPK activation, Beclin-1 Beclin-1 S91/94A reduced MBR-induced PARP cleavage and

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cell death (Figures 7a and b), indicating that Beclin-1 Cleavage of Beclin-1 occurred with caspase activation. As
phosphorylation at Ser 91/94 is a prerequisite for Beclin-1 caspase activation occurs during apoptotic signaling, we
cleavage. investigated if Beclin-1 and caspases bind with each other and

Figure 2 MBR-induced mitochondrial dysfunction and AMPK activation. (a and b) HCT116 (a) and CX-1 (b) cells were treated with MBR for 24 h, stained with JC-1 mitochondrial
membrane potential detection kit and analyzed by flow cytometry. (c) After treatment, cytochrome c release into the cytosol was determined by immunoblotting for cytochrome c in the
cytosolic fraction of HCT116 cells. Mitochondrial fraction was used as the control. Cox IV was used as a mitochondrial marker and actin as a cytosolic marker. (d) After treatment, ATP
levels were assessed by using an ATP Assay Kit (Perkin-Elmer, Akron, OH, USA) in HCT116 cells. Data represent relative ATP levels, with control cells set as 100% (mean ± S.D.,
n = 3, **Po0.01). (e) After treatment, the activation of AMPK-α was detected by immunoblotting in HCT116 and CX-1 cells. (f) Parental HCT116 (HCT116 Bax+/+) and Bax-knockout
HCT116 Bax− / − cells were treated with MBR for 24 h and immunoblotted with anti-PARP, anti-caspase-8, anti-caspase-9, anti-caspase-3, anti-phospho-AMPK-α, anti-AMPK-α or
anti-LC3 antibody. Actin was used to confirm the equal amount of proteins loaded in each lane. (g) HCT116 Bax+/+ and Bax− / − cells were treated with MBR for 24 h. Cell viability was
analyzed by MTS assay. Error bars represent S.D. from triplicate experiments. **Po0.01 represents a statistically significant difference

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Figure 3 Interplay between autophagy and apoptosis during treatment with MBR. HCT116 (a) and CX-1 (b) cells were treated with MBR for 24 h, and LC3-I and LC3-II were
detected by immunoblotting. (c) LC3 puncta formation was examined using confocal microscopy in HCT116 cells. (d) MEF ATG7+/+ and MEF ATG7 − / − cells were treated with
MBR for 24 h, and PARP, ATG7 and LC3 were detected by immunoblotting. Actin was used to confirm the equal amount of proteins loaded in each lane. (e) MEF ATG7+/+ and MEF
ATG7− / − were treated with MBR for 24 h. Cell viability was analyzed by MTS assay. Error bars represent S.D. from triplicate experiments. **Po0.01 represents a statistically
significant difference

thus execute their function during the treatment. Interestingly, decreasing autophagy; specifically, blocking Beclin-1 clea-
as shown in Figure 7c, Beclin-1 and its mutant types all bound vage promotes autophagy and suppresses the apoptosis
with caspase-8, whereas binding with caspase-3 was hardly induced by MBR.
detected. Of note, the binding affinity of caspase-8 with Beclin-
1 S91/94A was weaker than that of Beclin-1 WT and Beclin-1
S91/94D, which needs further study. We also observed that Discussion
the binding affinity of AMPK with Beclin-1 was increased after Multidrug treatment can potentially overcome the resistance
treatment of MBR in both Beclin-1 WT and its mutant types. developed by single agents as well as reduce their side
These observations were confirmed by immunoprecipitating effects.26,27 Here we have presented a novel combination
assay with endogenous Beclin-1 proteins (Figure 7d). Data treatment of melphalan in combination with the proteasome
from Figure 7d reveal that endogenous Beclin-1 indeed inhibitor bortezomib and the mTOR inhibitor rapamycin for
associated with AMPK and caspase-8 but not caspase-3. colon cancer cells, as well as colon cancer stem cells, and
Notably, the binding affinity of Beclin-1 and AMPK was demonstrated that this multidrug treatment-induced synergis-
increased after MBR treatment. tic apoptosis mediated by AMPK activation through facilitation
D133 and D146 of Beclin-1 are known to be cleaved by of Beclin-1 cleavage.
caspase-8 during apoptosis. HCT116 Beclin-1 knock-in In recent research, the relationship between cancer cell
(Beclin-1 KI) cell line with double-mutant (DM; D133A/ growth and cell metabolism has been emphasized, high-
D146A) was generated by homologous recombination.25 As lighting the role of AMPK as one of the main players.28 AMPK
shown in Figures 7e and f, MBR-induced Beclin-1 cleavage is a eukaryotic heterotrimeric (α, β and γ) protein kinase
was abolished in Beclin-1 KI cells, restoring Beclin-1's activated in response to environmental stresses such as
autophagy-promoting function as well as suppressing MBR- glucose deprivation and hypoxia, which produce changes in
induced apoptosis. cellular ATP levels, resulting in phosphorylation of AMPK at
Taken together, we summarized our observations in Thr 172. Chemotherapy with certain drugs can activate AMPK
Figure 7g. MBR treatment causes mitochondrial dysfunction to increase cell death and apoptosis.29,30 Rapamycin and
and AMPK activation. AMPK-mediated Beclin-1 phosphoryla- UPS (ubiquitin–proteasome system) inhibitors may upregu-
tion is sufficient to induce Beclin-1 cleavage and thus late AMPK activity.31,32 It has been found that AMPK is
contributes to the synergistic induction of apoptosis by involved in both cell survival and cell death pathways,

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Figure 4 The role of AMPK in the MBR-induced apoptosis. (a and b) CX-1 (a) and HCT116 (b) cells were pretreated with 10 μM AMPK inhibitor compound C followed by
treatment with MBR for 24 h. After treatment, PARP, caspase-8, caspase-9, caspase-3, p-AMPK and AMPK were detected by immunoblotting. (c) HCT116 cells were transfected
with nonsense sequence (control) or AMPK-α siRNA targeting AMPK-α mRNA. After 48 h, cells were treated with MBR. The levels of AMPK-α and PARP were detected by
immunoblotting. Actin was used as a loading control. (d and e) CX-1 (d) and HCT116 (e) cells were pretreated for 30 min with metformin (1–20 mM) followed by treatment with
MBR for 24 h. Cell viability was analyzed by MTS assay. Error bars represent S.D. from triplicate experiments. **Po0.01 represents a statistically significant difference. (f and g)
CX-1 (f) and HCT116 (g) cells were pretreated for 30 min with AICAR (0.5–5 mM) followed by treatment with MBR for 24 h. Cell viability was analyzed by MTS assay. Error bars
represent S.D. from triplicate experiments. *Po0.05 and **Po0.01 represents a statistically significant difference

depending on the severity of energy stresses, drug action and emerged as an important stress response and cell death
cell types. Many researchers have reported that activation of regulatory mechanism. The process of conventional macro-
AMPK induces apoptosis in cancer cells by inhibition of fatty autophagy is dependent on Atg7 (ubiquitin-activating enzyme
acid synthase, phosphorylation of p53 or stimulation of the (E1)-like) and Beclin-1.39 We observed that MBR treatment-
mitochondrial apoptotic pathway.33–38 induced apoptosis was enhanced in MEF ATG7− / − cells,
In our current study, we observed significant AMPK indicating that blockage of autophagy promoted apoptosis in
activation in MBR treatment. AMPK inhibitor compound C MBR treatment. We also observed that autophagy decreased
and AMPK-α siRNA suppressed MBR-induced apoptosis, in the combination of MBR compared with bortezomib alone.
indicating the proapoptotic effect of AMPK in the combinatorial Thus, these results indicate the role of AMPK in cross-talk
treatment of MBR. This observation was also supported by between apoptosis and autophagy in the multidrug treatment.
experiments using AMPK activator metformin and AICAR. Beclin-1, a mammalian homolog of yeast Atg6, functions in
Interestingly, we observed that AMPK was activated at two autophagy by initiating autophagosome formation in combina-
stages. Early AMPK activation-induced autophagy, whereas tion with Vps34 (a class III PI-3 kinase that generates
late AMPK activation resulted in significant apoptosis. PtdIns3P) and with Vps15 and Atg14;40 Beclin-1 then
Autophagy, a catabolic degradation process, has recently regulates autophagosome maturation by binding to UVRAG

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Figure 5 AMPK-α activation-induced Beclin-1 phosphorylation at Ser 93/96 and Beclin-1 cleavage. (a and b) HCT116 (a) and CX-1 (b) cells were treated with MBR for
24 h and p-Beclin-1 (Ser 93/96) and Beclin-1 were detected by immunoblotting in HCT116 and CX-1 cells. (c) After treatment, Beclin-1 cleavage was detected by
immunoblotting in HCT116 cells. (d) Tu-22 cells were treated with MBR for 24 h and p-AMPK, AMPK, p-Beclin-1 (Ser 93/96) and Beclin-1 were detected by immunoblotting.
(e and f) HCT116 (e) and CX-1 (f) cells were pretreated with 10 μM compound C followed by MBR for 24 h, and Beclin-1 phosphorylation at Ser 93/96 and Beclin-1
cleavage were detected by immunoblotting. (g) CX-1 and HCT116 cells were pretreated for 30 min with 1 mM AICAR followed by treatment with MBR for 24 h. After treatment,
PARP, p-AMPK, AMPK, p-Beclin-1 (Ser 93/96), Beclin-1 and LC3 were detected by immunoblotting. Actin was used to confirm the equal amount of proteins loaded in
each lane

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Figure 6 The kinetics of AMPK-α activation and Beclin-1 phosphorylation and cleavage during treatment with MBR. CX-1 (a) and HCT116 (b) cells were treated with MBR for
various times (1–24 h). After treatment, cell lysates were immunoblotted with anti-PARP, anti-caspase-8, anti-caspase-9, anti-caspase-3, anti-phospho-AMPK-α, anti-AMPK-α,
anti-phospho-Beclin-1, anti-Beclin-1 or anti-LC3 antibody. Actin was used to confirm the equal amount of proteins loaded in each lane. (c and d) CX-1 (c) and HCT116 (d) cells
were treated with MBR in the presence of 10 μM compound C from 0 to 4 h (at early time point and washed with phosphate-buffered saline at 4 h) or from 10 to 24 h (at late time
point). After treatment, PARP, p-AMPK, AMPK and actin were detected by immunoblotting. (e) After treatment, LC3 punta formation was analyzed with confocal microscope in
HCT116 cells. (f) Quantification of GFP-LC3 puncta formations. **Po0.01 represents a statistically significant difference. (g) HCT116 cells were treated with MBR for various
times (12–36 h). After treatment, cell lysates were immunoblotted with anti-PARP, anti-phospho-AMPK-α, anti-AMPK-α, anti-phospho-Beclin-1, anti-Beclin-1 or anti-LC3 antibody.
Actin was used to confirm the equal amount of proteins loaded in each lane

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and Rubicon.41 Cross-talk between apoptosis and autophagy can then amplify mitrochondrion-mediated apoptosis through
was associated with caspase-mediated cleavage of Beclin-1, the cleaved C-terminal fragment.24 However, how Beclin-1
which both destroys its proautophagic activity22,25,42,43 and cleavage is regulated is largely unknown.

Figure 7 Beclin-1 phosphorylation at Ser 91/94 was a prerequisite for Beclin-1 cleavage and the cleavage of Beclin-1 contributed to the synergistic induction of apoptosis.
HCT116 cells were transiently transfected with empty vector (pcDNA), Myc-Beclin-1 WT, Myc-Beclin-1 S91/94A and Myc-Beclin-1 S91/94D; 48 h later cells were treated with
MBR for 24 h. (a) PARP, Myc, phosphorylated Beclin-1 and Beclin-1 were detected by immunoblotting. Actin was shown as an internal standard. (b) Cell viability was determined
by MTS assay. *Po0.05 represents a statistically significant difference. (c) Cell lysates were immunoprecipitated with anti-Myc antibody or IgG and then immunoblotted with anti-
caspase-8/3, anti-AMPK-α or anti-Myc antibody (upper panels). The presence of caspase-8/3, AMPK-α and Myc in the lysates was examined (lower panels). (d) HCT116 cells
were treated with MBR for 24 h and cell lysates were immunoprecipitated with anti-Beclin-1 antibody or IgG and then immunoblotted with anti-caspase-8/3, anti-AMPK-α or anti-
Beclin-1 antibody (upper panels). The presence of caspase-8/3, AMPK-α and Beclin-1 in the lysates was examined (lower panels). (e) Parental HCT116 (HCT116 Beclin-1 WT)
and HCT116 Beclin-1 KI (DM; D133A/D146A) cells were treated with MBR and immunoblotted with anti-PARP, anti-Beclin-1 or anti-LC3 antibody. Actin was used to confirm the
equal amount of proteins. (f) After treatment, cell viability was determined by MTS assay. *Po0.05 and **Po0.01 represent a statistically significant difference. (g) A schematic
diagram of the role of AMPK in cross-talk between apoptosis and autophagy

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 Role of AMPK in a multimodality treatment
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Beclin-1 phosphorylation sites reported include: Thr 119,44 (Danvers, MA, USA). Anti-LC3 and anti-actin antibody were from Sigma-Aldrich.
Ser 14,45 Ser 93/9623 and Ser 234/295.46 Recently, AMPK Anti-Beclin-1 and anti-cytochrome c antibody were from BD PharMingen (San Jose,
was reported to phosphorylate directly Beclin-1 at Ser 93/96 CA, USA).
for activating proautophagy Vps34 complex and subsequently
MTS assays. MTS studies were carried out using the Promega CellTiter 96
inducing autophagy.23 The question remains as to how AMPK- AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA).
mediated phosphorylation of Beclin-1 at Ser 93/96 regulates CX-1 cells (1 × 105) were grown in RPMI-1640 medium containing 10% fetal bovine
caspase-8-associated cleavage of Beclin-1. Here, we serum in tissue culture-coated 96-well plates and treated with drugs for 24 h. Cells
reported that Beclin-1 phosphorylation at Ser 93/96 is a were then treated with 20 μl MTS/phenazine methosulfate solution for 1–4 h at
prerequisite for Beclin-1 cleavage and thus contributes to the 37 °C. Absorbance at 490 nm was determined using an enzyme-linked
immunosorbent assay plate reader.
synergistic induction of apoptosis. AMPK inhibitor compound
C inhibited both Beclin-1 phosphorylation at Ser 93/96 and CI analysis. CIs were calculated using CompuSyn software program
Beclin-1 cleavage. In addition, Myc-tagged murine Beclin-1 (ComboSyn Inc., Paramus, NJ, USA). Base on CI values, extent of synergism/
S91/94A mutant was resistant to cleavage and suppressed antagonism is determined. In general, CI value below 1 suggests synergy, whereas
MBR-induced apoptosis. Moreover, the binding affinity of CI value above 1 indicates antagonism between the drugs. CI values in the range of
Beclin-1 S91/94A with caspase-8 was decreased, providing 0.9–1.10 would mainly indicate additive effects, those between 0.9 and 0.85 would
one possible mechanism of AMPK-mediated Beclin-1 clea- suggest slight synergy, those in the range of 0.7–0.3 are indicative of moderate
synergy and those o0.3 would suggest strong synergy.
vage through its phosphorylation and formation of an AMPK–
Beclin-1–caspase-8 complex. Beclin-1 DM (D133A/D146A) Annexin V binding. Cells were treated with drugs, harvested by trypsinization,
knock-in HCT116 cells partially abolished the apoptosis, washed with serum-free medium and suspended in binding buffer (Annexin
confirming the role of Beclin-1 cleavage in the multidrug V-fluorescein isothiocyanate (FITC) Staining Kit; BD PharMingen). This cell
treatment. suspension was stained with mouse anti-human Annexin V antibody and propidium
Taken together, we present here that a novel multidrug iodide (PI) and immediately analyzed by flow cytometry.
treatment of chemotherapeutic agent in combination with
Immunoprecipitation. Briefly, cells were lysed in CHAPS lysis buffer with
proteasome inhibitor and mTOR inhibitor induced robust protease inhibitor cocktail (Calbiochem). The lysate (0.5–1 mg) was incubated with
apoptosis in colon cancer cells as well as colon cancer stem 1.5 μg of anti-Myc/Beclin-1 antibody or rabbit/mouse IgG (Santa Cruz, Dallas, TX,
cells, an apoptotic process that is linked with altered cellular USA) at 4 °C overnight, followed by the addition of protein G PLUS-agarose beads
metabolism and AMPK activation. We believe that these (Santa Cruz) and rotation at room temperature for 2 h followed by immunoblot
results demonstrate for the first time that the induction of analysis.
apoptosis by AMPK is associated with Beclin-1 cleavage
Immunoblot analysis. Cells were lysed with Laemmli lysis buffer and boiled
through Beclin-1 phosphorylation at Ser 93/96. Melphalan, for 10 min. The protein content was measured with BCA Protein Assay Reagent
bortezomib and rapamycin are all commonly used FDA- (Pierce, Rockford, IL, USA), separated by sodium dodecyl sulfate-polyacrylamide
approved drugs and could be considered for colorectal hepatic gel electrophoresis (SDS-PAGE) and electrophoretically transferred to nitrocellulose
metastases treatment in clinics. membrane. The nitrocellulose membrane was blocked with 5% nonfat dry milk in
PBS-Tween-20 (0.1%, v/v) for 1 h and incubated with primary antibody at room
temperature for 2 h. Horseradish peroxidase-conjugated anti-rabbit or anti-mouse
Materials and Methods IgG was used as the secondary antibody. Immunoreactive protein was visualized by
Cell cultures and transfection. Human colorectal carcinoma CX-1 cells, the chemiluminescence protocol.
which were obtained from Dr. JM Jessup (National Institutes of Health, Bethesda,
MD, USA),47 were cultured in RPMI-1640 medium (Gibco-BRL, Grand Island, NY, JC-1 mitochondrial membrane potential assay. After drug treatment,
USA) containing 10% fetal bovine serum (HyClone, Logan, UT, USA). The human cells were stained with JC-1 Mitochondrial Membrane Potential Detection Kit
colorectal carcinoma HCT116 Bax-containing (Bax+/+) and Bax-deficient (Bax− / −) (Invitrogen, Carlsbad, CA, USA) for 10 min and analyzed by flow cytometry.
cell lines were kindly provided by Dr. B Vogelstein (Johns Hopkins University, Fluorescence intensity was measured with the FACScan flow cytometer
Baltimore, MD, USA); these cell lines were cultured in McCoy’s 5A medium (Gibco- (Beckman Coulter, Hialeah, FL, USA). Results were analyzed with
BRL) containing 10% fetal bovine serum.48 Human colon cancer stem Tu-22 cells CellQuest software (Becton Dickinson Immunocytometry Systems, San Jose,
were established by Dr. E Lagasse (University of Pittsburgh, Pittsburgh, PA, USA) CA, USA).
and cultured in DMEM/F12 medium (Gibco-BRL) containing 0.5% fetal bovine
serum (HyClone) and 1% insulin, transferrin and selenium (Fisher Scientific, ATP assay. The ATP content in whole-cell extracts was determined with a
Pittsburgh, PA, USA).49 MEF ATG7+/+ and MEF ATG7− / − cell lines, a kind gift from luminescent ATP Detection Kit (ATPlite, Perkin-Elmer, Akron, OH, USA) according to
Dr. Masaaki Komatsu (The Tokyo Metropolitan Institute of Medical Science, Tokyo, the manufacturer’s instructions. The luminescence intensity was measured by using
Japan), were cultured in DMEM medium (Gibco-BRL) containing 10% fetal bovine a microplate reader (Synergy 2; BioTek Instruments, Winooski, VT, USA). In parallel,
serum. All the cells were kept in a 37 °C humidified incubator with 5% CO2. For the cell numbers in whole-cell samples were counted by Trypan blue exclusion
transient transfection, cells were transfected with Lipofectamine 2000 (Life assay. The results were expressed as relative ATP level compared with controls
Technologies, Carlsbad, CA, USA) and were treated 48 h after transfection. after normalizing for cell numbers.

Reagents and antibodies. Bortezomib and melphalan were from Millennium Measurement of cytochrome c release. To determine the release of
Pharmaceuticals (Cambridge, MA, USA) and GlaxoSmithKline (Coraopolis, PA, cytochrome c from the mitochondria, cells growing in 100 mm dishes were used.
USA), respectively. Rapamycin, metformin, aminoimidazole carboxamide ribonu- After drug treatment, mitochondrial and cytosolic fractions were prepared using
cleotide and protease inhibitor cocktail were obtained from Sigma-Aldrich (St. Louis, Mitochondrial Fractionation Kit (Active Motif, Carlsbad, CA, USA) following company
MO, USA). AMPK inhibitor (compound C) was from Calbiochem (Darmstadt, instructions with reagents included in the kit. Cytosolic fractions were subjected to
Germany). Myc-Beclin-1 WT, Myc-Beclin-1 S91/94A and Myc-Beclin-1 S91/94D SDS-PAGE gel electrophoresis and analyzed by immunoblotting using anti-
were kindly provided by Dr. Kun-Liang Guan (University of California at San Diego, cytochrome c antibody.
La Jolla, CA, USA). Anti-Myc, anti-Bax, anti-caspase-8, anti-caspase-9, anti-
caspase-3, anti-phosphorylated AMPK-α/AMPK-α, anti-phosphorylated Beclin-1 Knockdown of AMPK-α with siRNA oligomers. To generate AMPK-α
(Ser 93/96), anti-COX-IV and anti-PARP antibody were from Cell Signaling knockdown cells, cells were transfected with 10 nM of siRNA AMPK-α1 and

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