PARP and RIP-1 are required for autophagy induced by 11 ...

Basic Research Paper 

Autophagy 7:6, 1-15; June 2011; © 2011 Landes Bioscience 

Basic Research Paper 

PARP and RIP-1 are required for autophagy 

induced by 11'-deoxyverticillin A, which precedes 

caspase-dependent apoptosis 

This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly. 

Nan Zhang, 1,2,† Yali Chen, 3,† Ruixuan Jiang, 4 Erwei Li, 3 Xiuling Chen, 2 Zhijun Xi, 1 Yinglu Guo, 1 Xingzhong Liu, 3 Yuguang Zhou, 2,5 

Yongsheng Che 3,5, * and Xuejun Jiang 2,3, * 

1 

National Research Center of Urological Cancer; Institute of Urology; Peking University First Hospital; 2 Biological Resource Center; 3 Key Laboratory of Systematic Mycology 

& Lichenology; 5 State Key Laboratory of Microbial Resources;Institute of Microbiology; Chinese Academy of Sciences; 4 Capital Medical University; 

6 

Beijing Institute of Pharmacology & Toxicology; Beijing, China 

The epipolythiodioxopiperazines (ETPs) are fungal secondary metabolites proven to trigger both apoptotic and necrotic 

cell death of tumor cells. However, the underlying mechanism of their regulatory role in macroautophagy and the 

interplay between autophagy and apoptosis initiated by the ETPs, remain unexplored. In the current work, we found that 

11'-deoxyverticillin A (C42), a member of the ETPs, induces autophagosome formation, accumulation of microtubuleassociated 

protein 1 light chain 3-ii (LC3-ii) and degradation of sequestosome 1 (SQSTM1/p62). In addition, the LC3-ii 

accrual and p62 degradation occur prior to caspase activation and coincide with PARP activation. Inhibition of autophagy 

©2011LandesBioscience. 

by either chemical inhibitors or by RNA interference single knockdown of essential autophagic genes partially reduces 

the cell death and the cleavage of both caspase 3 and PARP. Necrostatin-1, a specific inhibitor of necroptosis, inhibits both 

the augmentation of LC3-ii and the cleavage of caspase 3, which was confirmed by depletion of receptor-interacting 

protein 1 (RIP-1), a crucial necrostatin-1-targeted adaptor kinase mediating cell death and survival. Moreover, inhibition 

of PARP by either chemical inhibitors or RNA interference provides obvious protection for cell viability and suppresses 

Donotdistribute. 

the LC3-ii accretion caused by C42 treatment. Interestingly, double silencing of LC3 and p62 completely suppressed 

PARP cleavage and concurrently and maximally augmented the PAR formation induced by C42. Collectively, we have 

demonstrated that C42 enhances the cellular autophagic process, which requires both PARP and RIP-1 participation, 

preceding and possibly augmenting, the caspase-dependent apoptotic cell death. 

Introduction 

Macroautophagy (hereafter referred to as autophagy) was originally 

described as a cellular adaptation to starvation involving 

the vesicular sequestration of long-lived cytoplasmic proteins and 

organelles such as mitochondria. 1,2 The newly created doublemembrane 

autophagosome consequently fuses with lysosomes to 

form an autolysosome, in which the engulfed cytoplasmic material 

is hydrolyzed through the action of acid-dependent enzymes, 

ensuring that the amino acids and other macromolecular precursors 

can be recycled. 3 Although autophagy serves as a cell survival 

mechanism, if overactivated and allowed to go to excess, it will 

eventually lead to cell death by depletion of the cell’s organelles 

and critical proteins. 4-6 

Cell death plays essential roles in organ development, tissue 

homeostasis and degenerative diseases. Three major types of cell 

death have been described—apoptosis, 7 autophagy, 8,9 and necrosis, 

10,11 based on their distinct cell morphology. The apoptotic cell 

† 

These authors contributed equally to this work. 

Key words: 11'-deoxyverticillin A, autophagy, apoptosis, PARP, RIP 

death is characterized by the activation of caspases and formation 

of apoptotic bodies, 12,13 whereas the autophagic one is differentiated 

by large-scale sequestration of portions of the cytoplasm 

in autophagosomes, giving the cell a characteristic vacuolated 

appearance. 14 Accumulating evidence suggests the existence of 

several molecular connections among apoptosis, autophagy and 

necrosis. 15-17 In response to specific perturbations, the same input 

signal can cause cells to change from one cell death manifestation 

to another, 17,18 with a mixed type of cell death also observed 

in some cases. 19,20 In those cells undergoing persistent autophagy, 

hallmarks of apoptosis, such as caspase activation, necrotic cell 

death, organelle swelling and plasma membrane rupture, are 

often observed. 21 In nutrient-deprived or growth factor-withdrawn 

cells, autophagy protects the cells and allows their survival 

by inhibiting apoptosis. 22 In the presence of caspase inhibitors, 

autophagy also protects the cells from caspase-independent 

death. 16 Depending on the cellular setting, the same proteins can 

regulate both autophagic and apoptotic processes. For example, 

*Correspondence to: Yongsheng Che and Xuejun Jiang; Email: cheys@im.ac.cn and jiangxj@im.ac.cn 

Submitted: 09/22/10; Revised: 01/22/11; Accepted: 02/09/11 

DOI: 

www.landesbioscience.com Autophagy 1

p53, a potent inducer of apoptosis, also activates autophagy 

through its target gene, DRAM (damage-regulated modulator 

of autophagy). 23 Beclin 1, required for formation of the autophagic 

vesicle, also interacts with both Bcl-2 and Bcl-x L 

. 24 Calpainmediated 

cleavage of ATG5 is a critical pro-apoptotic event that 

enables the activation of caspase-dependent cell death. 25 Cho and 

Kim et al. have identified ATG6 as a link between apoptotic and 

autophagic cell death. 26 Induction of autophagy by ATG1 inhibits 

cell growth and induces apoptotic cell death. 27 It has been suggested 

that autophagy relates to apotosis by acting as a partner, 

an antagonist or an enabler of apoptosis depending on cell type, 

stimulus and environment. 28,29 

11'-deoxyverticillin A, a natural product isolated from the 

Cordyceps-colonizing fungus Gliocladium sp., 30 is a member of 

a class of fungal secondary metabolites known as epipolythiodioxopiperazines 

(ETPs). 31 The ETPs have been previously 

reported to have a broad range of biological activities including 

antimicrobial, antiparasitic, antiviral, immunosuppressive and/ 

or anti-inflammatory effects. 31 Gliotoxin is the first ETP reported 

and the best characterized one. Its toxicity to animal cell cultures 

has been studied extensively, and it is thought to be mediated in 

at least two ways: conjugation to, and consequent inactivation of, 

proteins with susceptible thiol residues and generation of reactive 

oxygen species (ROS) via redox cycling. 32 Results from previous 

studies have demonstrated that gliotoxin causes the apoptotic and 

colon carcinoma cell line HCT116 and adenocarcinoma colon 

cell lines SW480 and SW620. As shown in Figure 1A, C42 

caused cell viability loss of HCT116 in a time- and concentration-dependent 

manner and similar inhibitory effects were also 

observed in SW480 and SW620 cells (Sup. Fig. 1A and B). In 

addition, flow cytometry data indicated that the C42-induced 

cell death of HCT116 could be either apoptotic or necrotic (may 

be either necrosis or secondary necrosis) (Fig. 1B). 

Since the pan-caspase inhibitor Z-VAD-FMK (Z-V-FMK) 

can block caspase-dependent apoptosis, 36 we next examined 

the C42-dependent cell death in the presence of this inhibitor. 

Interestingly, pre-treatment of HCT116 (Fig. 1C) with the 

inhibitor provided only partial protection against C42-induced 

cell death. This finding was confirmed by colony growth in a 

survival assay, in which the inhibitor was nearly unable to rescue 

the C42-induced cell death (Fig. 1D). Results from additional 

experiments confirmed that porcine aortic endothelial 

(PAE) cells overexpressing the anti-apoptotic protein Bcl-x L 

(Sup. Fig. 1C, 2A and 2B) and p53 -/- HCT116 cells lacking the 

pro-apoptotic protein p53 (Sup. Fig. 1D) were both sensitive 

to C42, indicating that caspase-independent or non-apoptotic 

mechanisms also contribute to C42-induced cell death. In addition, 

we also found that 3-methyladenine (3-MA), a widely used 

inhibitor of autophagy, 37 inhibited the C42-induced cell death 


(Fig. 1E). To investigate the relationship between autophagy 

necrotic death of animal cells. 31,33 In one study, cell death caused 

by gliotoxin switched from apoptosis to necrosis at concentrations 

over 10 μM. 33 Leptosins, the ETPs isolated from a marine 

fungus, were cytotoxic to various tumor cells by inactivation of 

and C42-dependent cell death, two known activators of autophagy, 

rapamycin and trehalose, were used to promote the cellular 

autophagic processes. 38,39 As expected, the combination of 


either rapamycin or trehalose with C42 showed a measurable and 

the Akt/protein kinase B pathway or by inhibition of DNA topoisomerases 

I and II. 34 Chen and Ding reported that in HCT-116 

human colon cancer cells, either the p53 pathway or p38 MAPK 

signaling plays a role in mediation of 11,11'-dideoxyverticillin A 

induced G 2 

/M arrest. 35 

Although the above-mentioned natural products are cytotoxic 

against various tumor cells by inducing apoptosis or necrosis, their 

effects on the autophagic process have not been investigated. The 

purpose of this study is to understand the molecular mechanism 

for C42’s cytotoxicity to human tumor cells by exploring the two 

major categories of cell death. We found that it induces formation 

of intracellular vacuoles in human colon carcinoma cells and 

enhances the molecular hallmarks of autophagy, such as LC3 

processing and p62 degradation, while concomitantly activating 

caspases and leading to cleavage of PARP. Inhibition of PARP 

or RIP by either the chemical inhibitors or RNA interference 

knockdown reduces the LC3-II accrual, PARP activation and 

the cleavage of apoptotic hallmarks as well as cell death. Thus, 

activation of both PARP and RIP is required for C42-induced 

autophagy, which is closely associated with caspase-dependent 

cell death. 

Results 

11'-deoxyverticillin A (C42) induces cell death of human colon 

cell lines which is partially inhibited by 3-MA. In this study, 

the C42-induced cell death was first examined using the human 

more potent inhibitory effect on cell viability (Sup. Fig. 1E) and 

both agents were able to attenuate cell viability by themselves, 

suggesting that enhanced autophagy may directly result in cell 

death (Sup. Fig. 1E). 

C42 enhances autophagy in colon cells. Electron microscopy, 

which is believed to be one of the most convincing approaches, 40 

was used to determine whether C42 induces autophagy or not. 

Compared to the control, an obvious accrual of membrane vacuoles 

was found in the C42-treated HCT116 cells and cytosolic 

components or organelles were sequestered in some of those 

vacuoles. Autophagosome-like vacuoles with double-membrane 

structures (high magnification) and a segment of the doublemembrane 

being formed between a vacuole and mitochondrion 

(black arrowhead) were also observed (Fig. 2A). A swollen (white 

arrow) and degenerating mitochondrion (white arrow) and mitochondria 

inside of vacuole (black arrow) were also noted (Fig. 

2A and Sup. Fig. 2C). In addition, characteristics of apoptosis, 

such as the condensation and margination of chromation on 

the nuclear membrane, were found in these cells (Fig. 2A and 

white arrowhead), indicating that C42 induced autophagy and 

apoptosis in the same cell and directly induced the degradation 

of mitochondria. To further measure autophagosome formation 

in the C42-treated cells, HCT116 cells were transfected with a 

green fluorescent protein (GFP) and LC3 fusion protein (a specific 

marker of autophagosomes), 41 and visualized by confocal 

microscopy. In contrast to the C42-exposed cells, which showed 

increased punctate staining of GFP-LC3, GFP-LC3 staining 

2 Autophagy Volume 7 Issue 6



Figure 1. 11'-deoxyverticillin A (C42) induces programmed cell death and 3-MA application results in resistance to the C42-induced cell death. (A) 

hcT116 cells were treated with C42 (0.05–0.5 μM) for up to 48 h; cell viability was analyzed by MTS assay as described in Materials and Methods. Data 

are presented as mean ± SD and are representatives of three independent experiments. (B) Following treatment of HCT116 cells with C42 (0.25, 0.5 

μM) for 12 h, the apoptosis and necrosis induced were determined by flow cytometry. Apoptotic: AV-positive and PI-negative; necrotic: PI-positive; 

AV: annexin-V. The data are presented as mean ± SD from three independent experiments (C) HCT116 cells were treated with C42 (0.1–0.5 μM) for 24 

h in the presence or absence of Z-V-FMK (20 μM) before detection by MTS assay. (D) Colony survival assays in HCT116 cells were performed following 

the treatment of HCT116 cells with C42 (0.1 μM) for 6 h in the presence or absence of Z-V-FMK. Data represent the mean ± SD of three experiments, 

each performed in triplicate. (E) HCT116 cells were treated with C42 (0.1–0.5 μM) for 24 h in the presence or absence of 3-MA (2 mM), cell viability was 

analyzed by MTS assay. Ctrl: cells with equal amount of DMSO. 

remained diffuse in the control cells (Fig. 2B) (p < 0.01). Since 

the ratio of LC3-II to actin is an accurate indicator of autophagy, 

42 the expression of LC3-II in HCT116 (Fig. 2C), SW480 

(Sup. Fig. 2D) and SW620 (Sup. Fig. 2E) cells was analyzed. 

Treatment with C42 increased the ratio of LC3-II to actin relative 

to the controls in all the cells tested. 




Figure 2. C42 induces accumulation of LC3-ii and enhances autophagic flux. (A) Electron microscopy was performed on vehicle- (Ctrl) and C42-treated 

(0.25 μM, 6 h) HCT116 cells as described in Materials and Methods. White arrowheads: the condensation and margination of the chromatin on nuclear 

membrane; left of lower part: swollen and degenerating mitochondria (white arrows), a vacuole with mitochondria inside (black arrow), a segment of 

double-membrane structure between a vacuole and mitochondrion (black arrowhead); right of lower part: high-contrast image of the cell region indicated 

by white rectangle. (B) HCT116 cells were transfected with a plasmid expressing GFP-LC3. After 24 h, the cells were incubated for 6 h at 37°C in 

DMEM medium with DMSO (Ctrl) or C42 (0.25 μM). Following fixation, cells were stained with DAPI and visualized by confocal microscopy. The number 

of punctate GFP-LC3 in each cell was counted, and at least 50 cells were included for each group. The data were normally distributed and statistically 

analyzed using one-way ANOVA. The double asterisk denotes the group is statistically different from the control groups (p < 0.01). HCT116 cells were 

treated with C42 (0.05–0.5 μM) for 12 h (C and D) or in the presence of Baf A1 (10 nM) (E) and cell lysates were prepared and analyzed by immunoblotting 

using the indicated antibodies. Densitometry was performed for quantification and the ratios of LC3-ii, p62, phosphorylated mTOR and phosphorylated 

p70S6K (T389) to actin are presented below the blots. The ratios are representative of at least three independent experiments. 

Mammalian target of rapamycin (mTOR) inhibits autophagy 

and its kinase activity can be inferred by measuring the levels 

of phosphorylation of its substrates, such as p70S6 kinase. 43 To 

study whether or not C42-induced autophagy was associated 

with this known pathway, the phosphorylation state of mTOR 

and p70S6 kinase was examined. As expected, the phosphorylation 

of mTOR and p70S6K was attenuated in a concentrationdependent 

manner in response to the agent (Fig. 2D). To detect 


autophagic flux, the level of LC3-II was measured in 

the presence of bafilomycin A1 (Baf A1), which acts 

as a specific inhibitor of the vacuolar type H + -ATPase 

in cells, thus blocking acidification of organelles and 

subsequent fusion of the autophagosome with the 

lysosome. 44 Baf A1 addition resulted in further accumulation 

of LC3-II in the cells tested (Fig. 2E). Since 

p62/SQSTM1 is considered to be a selective substrate 

of autophagy and will be accumulated when autophagy 

is inhibited, 43 we also examined the levels of p62 

upon challenge with C42. As shown in Figure 2C 

and Supplemental Figure 2D and E, the degradation 

of p62 induced by C42 was concentration-dependent, 

indicating that C42 actually enhances the autophagic 

process. 

The mRNA level of LC3 was also measured by 

semiquantified RT-PCR and it was found that C42 

increased the level only at 0.5 μM compared to the 

control. Nevertheless, the augmentation of LC3 

mRNA is not significant and could be due to the 

reduced actin mRNA (Sup. Fig. 2F). Therefore, C42 

may affect the LC3 expression at high concentrations 

or at some time point via an unknown mechanism. 

Autophagy induced by C42 precedes caspase 


activation. Caspase activation was examined to 

explore the interplay between autophagy and apoptosis 

upon C42 challenge of HCT116 cells. After 1 h of 

treatment, we found that C42 induced the accumula- 


tion of LC3-II and degradation of p62 in a time- and 

concentration-dependent manner (Fig. 3A). Using 

antibodies against the early apoptosis hallmarks, 26,29 

caspase 9 and PARP-1, we noticed that LC3-II 

accrual and/or p62 degradation preceded the cleavage 

of caspases and PARP (Fig. 3A and Sup. Fig. 3A 

and B). In contrast to caspase activation and PARP-1 

cleavage detected at 4 h, the C42-induced formation 

of poly(ADP-ribose) (PAR) polymers appeared to 

occur concomitantly with the autophagic process from an early 

time-point of treatment (Fig. 3A). PAR, a direct result of PARP-1 

activation, is readily activated in response to DNA damage and 

well-associated with necrotic cell death. 10 Thus, we assume that 

C42, in addition to inducing caspase-dependent apoptosis, is also 

a powerful activator of PARP-1 and may induce caspase-independent 

or necrosis-like cell death in those cells if the stimulus 

is continually presented. On the other hand, activation of PARP 

is a cellular response to C42 stress and may be required for C42- 

induced autophagy. Results from additional experiment indicated 

that the mRNA level of PARP-1 is increased only at 0.5 μM of 

C42 as measured by semiquantified RT-PCR (Sup. Fig. 3C). 

In PAE cells (in which caspase 3 activation is easily detected 

compared to EGFR expressing cells), Bcl-x L 

overexpression attenuated 

the C42-induced caspase 3 cleavage (Fig. 3B), but failed 

to block its activation (Sup. Fig. 3D). Additionally, the forced 

overexpression of this anti-apoptotic protein showed no sign of 

affecting the C42-dependent LC3-II accumulation (Fig. 3B). 

Thus, Bcl-x L 

alone is insufficient to prevent the caspase activation 

Figure 3. C42 induces accumulation of LC3-ii or degradation of p62 that occurs prior 

to the activation of caspases and cleavage of PARP. HCT116 (A), PAE-EGFR (B) or PAE- 

Bcl-x L 

(B) cells were treated for up to 4 h; cell lysates were prepared and analyzed by 

immunoblotting using the indicated antibodies. The asterisk indicates a nonspecific 

band. 

and cell death and the overexpression of Bcl-x L 

rescues autophagosome 

formation. Notably, we were unable to observe a clear 

accumulation of LC3-II in C42-treated PAE-GFP cells (Sup. 

Fig. 3E), which are more sensitive to the agent because their caspase 

3 activation can be readily detected at an earlier stage (data 

not shown). We assume that C42 mainly activates the apoptosis 

pathway in those cells and hyperactivated apoptosis impairs 

autophagosome formation, whereas Bcl-x L 

and EGFR overexpression 

restore autophagy in C42-treated PAE cells. These findings 

provide supplementary evidence that C42-induced autophagy 

could be associated with the apoptotic process. 

3-MA partially inhibits the C42-induced activation of both 

caspases and PARP. To explore the possible association of C42- 

induced autophagy with apoptosis, both inhibitors and enhancers 

of autophagy were evaluated in the following experiments. 3-MA 

(Fig. 4A) inhibited (or delayed) cellular autophagy, whereas 

rapamycin (Sup. Fig. 4A) and Z-V-FMK (Fig. 4C) promoted 

the process in the cells as evidenced by both LC3-II accrual and 

p62 degradation. Notably, 3-MA inhibited both the cleavage and 


Figure 4. 3-MA inhibits C42-induced cleavage of PARP. HCT116 cells 

were treated with C42 (0.1 and 0.25 μM) in the presence or absence of 

3-MA (A and B, 2 mM) or Z-V-FMK (C, 20 μM) for 24 h before analysis by 

immunoblotting with the indicated antibodies. The ratios represent the 

results of three similar experiments. 

activation of PARP (Fig. 4A and Sup. Fig. 4B), whereas rapamycin 

showed opposite effects (Sup. Fig. 4A and C). Moreover, the 

inhibitory effect of 3-MA on activation of caspase 3 was concentration-dependent 

(Fig. 4B and Sup. Fig. 4E). Interestingly, 

application of Z-V-FMK remarkably increased the LC3-II level 

in a dose- and time-dependent manner in both HCT116 and the 

PAE-Bcl-x L 

overexpressing cells (Fig. 4C and Sup. Fig. 4E and 

F). Although Z-V-FMK induced accumulation of both p62 and 

LC3-II in a short time, it failed to block the drop of the C42- 

induced p62 level over an extended period (Fig. 4C and Sup. 

Fig. 4F). In addition, we found that the presence of Z-V-FMK 

enhanced C42-induced PAR formation (Sup. Fig. 4D). These 

findings suggest that this pan-caspase inhibitor could retard and 

transiently inhibit C42-induced autophagy in a short period but 

enhance the process in the long run, probably via a feedback 

mechanism. Under these circumstances, enhanced autophagy 

may directly lead to cell death or shift to caspase-independent 

apoptosis or necrosis-like cell death by hyperactivating PARP. 


Both PARP-1 RIP play a role in the C42-induced autophagy. 

It is well known that hyperactivated PARP-1 causes depletion 

of NAD + and ATP, eventually leading to irreversible cellular 

energy failure and necrotic cell death. 10 In addition, recent stud- 


ies have shown that PARP also participates in mediation of autophagy. 

45,46 We also observed that the PARP activation concurred 

with the C42-dependent autophagy, implying that the activation 

of PARP may be required for the C42-induced autophagy. To 

explore PARP’s regulatory role in the C42-triggered autophagy, 

both chemical inhibitor and RNA interference approaches were 

employed. The presence of 1,5-dihydroxyisoquinoline (ISO), 

a potent inhibitor of poly(ADP-ribose) synthetase, 47 provided 

apparent protection for the C42-treated cells in a concentration-dependent 

manner (Fig. 5A). Upon treatment with 3-aminobenzamide 

(3-AB), an inhibitor of PARP-1, 48 a similar cell 

viability rescue was observed (Sup. Fig. 5A). Pretreatment with 

ISO remarkably inhibited PAR formation and decreased LC3-II 

levels and caspase 3 activation induced by C42 (Fig. 5B and 

Figure 5 (See opposite page). PARP and RIP inhibition attenuate 

C42-induced accumulation of LC3-ii and result in resistance to C42- 

dependent cell death. (A) HCT116 cells were treated with C42 (0.1, 0.25 

and 0.5 μM) in the presence or absence of necrostatin-1 (Nec-1, 30 μM) 

or 1,5-isoquinolinediol (isO, 30 μM) for 12 h before detection by MTS 

assay. (B) Immunoblotting analysis was performed with the indicated 

antibodies following exposure to C42 (0.25 μM) in the presence or 

absence of Nec-1 (30 μM) or ISO (30 μM) for 12 h. HCT116 cells were 

transfected with the control (Mock), PARP (C) or RIP siRNA (D); after 36 

h of transfection, cells were treated with C42 (0.25 μM) for 12 h before 

immunoblotting analysis with the indicated antibodies. (E) Viability of 

cells (C and D) was analyzed by MTS assay. The data were statistically 

analyzed using Student’s t-test, which demonstrated that the difference 

between control siRNA and PARP siRNA (p = 0.02) or RIP-1 siRNA (p 

= 0.03) was significant. *indicates a significant difference between the 

groups at level p < 0.05. 




Figure 5. For figure legend, see page 6. 


Figure 6. NAC application and RIP-3 depletion suppress C42-induced 

LC3-ii accumulation and PARP cleavage. (A) HCT116 cells were transfected 

with the control (Mock) and RIP-3 siRNA, respectively; after 36 

h of transfection, cells were treated with C42 (0.25 μM) for 12 h before 

immunoblotting analysis with the indicated antibodies. (B) Immunoblotting 

analysis was performed with the indicated antibodies following 

exposure to C42 (0.25 μM) in the presence or absence of NAC (5 mM) 

for 12 h. Densitometry was performed for quantification and the ratios 

of LC3-ii to actin are presented in the graphs below the parts. The 

ratios are representative of two experiments. The asterisk indicates a 

nonspecific band. 

suggested that both RIP-1 and PARP-1 are implicated in the 

C42-induced autophagy, through which they may influence the 

caspase activation and cell death. 

RIP-3, a protein kinase with an N-terminal kinase domain 

similar to RIP-1, has been reported to participate in the mediation 

of apoptosis and necrosis. 51,52 To test whether this kinase 

is also involved in the C42-dependent autophagy and apoptosis 

or not, RNA interference was performed in HCT116 cells. 

Abrogation of RIP-3 with siRNA revealed an inhibitory effect 

on the C42-induced LC3-II accumulation and PARP cleavage 

(Fig. 6A). Interestingly, depletion of RIP-3 alone also increased 

the basal level of LC3-II, whereas abrogation of both RIP-1 and 

RIP-3 decreased both the basal and C42-induced LC3-II content 


(Fig. 6A Sup. Fig. 5C). Thus, RIP-1 RIP-3 may coordinate 

in mediating the C42-dependent autophagic process. 

Since ROS has been proven to mediate autophagy in multiple 

contexts and cell types, 53-56 and the EPTs are capabe of generat- 


ing ROS in the cell culture system, 32 C42 may affect autophagy 

Sup. Fig. 5B). Using a RNA interference approach, we found 

that knockdown of PARP-1 remarkably reduced the formation of 

PAR, the level of LC3-II and cleavage of PARP (Fig. 5C). The 

remaining PAR, we assume, could be due to incomplete RNA 

interference or the activation of another PARP family member. 

Therefore, the activation of PARP contributes to the C42- 

induced autophagy. 

Receptor-interacting protein (RIP), a protein with diverse 

functions, participates in the regulation of apoptosis, necrosis 

and autophagic cell death. 49 Application of Nec-1, a necroptosis 

inhibitor that blocks RIP kinase, 50 partially attenuated the C42- 

induced cell death, LC3-II accrual, caspase 3 cleavage and PARP 

activation (Fig. 5A and B). Abrogation of RIP-1 with siRNA 

showed a similar inhibitory effect on the aforementioned cleavage 

and activation (Fig. 5D). Moreover, silencing either PARP-1 

or RIP-1 provided partial, yet significant, protection for the 

C42-treated cells (p < 0.05) (Fig. 5E). Notably, RIP-1 depletion 

also affected the basal level of LC3, but failed to reduce the total 

LC3 content compared to the control (Fig. 5D). These findings 

via ROS production. To confirm this hypothesis, the widely used 

antioxidant, N-acetylcysteine (NAC), was used. As expected, 

NAC suppressed the C42-induced LC3-II accumulation and 

PARP cleavage (Fig. 6B), suggesting that ROS mediates the 

C42-induced autophagy and apoptosis. 

Knockdown of autophagy-related genes partially attenuates 

the C42-induced cleavage of caspase and PARP as well as 

cell death. To verify the association between the C42-dependent 

cell death and autophagy, siRNA was performed for the key 

autophagic genes LC3 and beclin 1. Knockdown of either LC3 

or beclin 1 inhibited the accumulation of LC3-II and degradation 

of p62 (Fig. 7A), confirming the interfering efficiency. 

Furthermore, the lack of expression of either LC3 or beclin 1 

resulted in a partial decrease in cell death (Fig. 7B), suggesting 

that autophagy is closely associated with or may augment, 

the C42-induced cell death. Notably, deletion of either LC3 or 

beclin 1 showed an inhibitory effect on PARP-1 cleavage, indicating 

that the C42-dependent autophagy is associated with 

caspase-dependent apoptosis (Fig. 7C and D and Sup. Fig. 6A). 

Although we noticed that beclin 1 depletion usually affected the 

level of LC3, the LC3 knockdown cells often maintained a relatively 

high content of Beclin 1 in a cell-type-dependent manner 

(Fig. 7A and Sup. Fig. 6B). The efficiency of siRNA could be 

the reason why the lack of beclin 1 exhibited a greater protective 

effect against the C42-induced cell death (Fig. 7B). In addition, 

double-perturbation of LC3 and beclin 1 offered greater protection 

against the C42-induced cell death (Fig. 7B). As expected, 




Figure 7. Autophagic genes silencing partially attenuates C42-dependent PARP cleavage and offers partial protection for the C42-treated cells. (A) 

hcT116 cells transfected with the control (Mock) or LC3 (siLC3) or Beclin 1 (siBec1) siRNA were exposed to C42 (0.25 μM) for 12 h and cells were lysed 

and analyzed by immunoblotting with the antibodies indicated to confirm siRNA efficiency. (B) MTS assays were used to determine cell viability (A). 

Immunoblotting was performed to detect the cleavage of PARP following siRNA knockdown of LC3 (C) and beclin 1 (D). Densitometry was performed 

for quantification and the ratios of cPARP (cleaved PARP) to actin are presented in the graphs besides the parts, while the ratios of LC3-ii to actin are 

shown below the parts. The ratios are representatives of three experiments. 

silencing of both LC3 and beclin 1 resulted in a greater inhibitory 

effect on PARP cleavage (Sup. Fig. 6A) compared to the singular 

beclin 1 perturbation. These findings were further confirmed in 

the PAE-Bcl-x L 

cells (Sup. Fig. 6B and C). In addition to inhibiting 

the activation of caspase 3, knockdown of either LC3 or beclin 

1 also affected the amount of RIP, suggesting that RIP is closely 

associated with the autophagic process (Sup. Fig. 6B). 

ATG5 is an essential protein required for autophagy at the stage 

of autophagosome precursor synthesis and deletion of the ATG5 

gene in yeast or mammalian cells usually blocks autophagy efficiently. 

57 In addition, ATG5 has been observed to be implicated 

in the apoptotic process. 25 Therefore, the possible involvement 

of ATG5 in the C42-dependent cell death was also investigated. 

Indeed, silencing of the ATG5 gene with siRNA remarkably 

reduced both the basal and C42-stimulated accumulation of 

LC3-II (Fig. 8A). The ATG5 lacking cells markedly decreased 

PARP-1 cleavage and were partially resistant to the C42-induced 

cell viability loss (Fig. 8A and B). In addition, these cells were 

defective in PAR formation when exposed to C42. Therefore, 

we assumed that the C42-induced autophagy could be associated 

with either the caspase-dependent or -independent apoptosis, 

likely with the involvement of ATG5 in both processes. These 

findings are consistent with previous reports that, in addition 

to its canonical function in autophagy, ATG5 is connected to a 

pathway that activates the apoptotic cascade. 

Although p62/SQSTM1 is considered as a selective substrate 

of autophagy, as a multidomain protein it has been found to interact 

with LC3. 58,59 Hence, the signaling adaptor p62 may actually 

participate in autophagy. Furthermore, p62 was reported to associate 

with the polyubiquitinated caspase 8 and trigger the extrinsic 

apoptosis pathway. 60 To examine whether p62 is involved in 

the C42-induced LC3 conversion and cell death, the cells were 

transfected with p62 siRNA. The singular perturbation of the 

p62 gene expression resulted in a decrease in LC3-II and cleavage 

of PARP (Fig. 8C) and resistance to the C42-triggered cell 

death (Fig. 8D). To test whether the cell death in either the LC3- 

or beclin 1-depleted setting is due to the accrual of p62, LC3 

and beclin 1 were each knocked down in combination with p62. 

Although singular knockdown of each gene offered significant 

protection for the cells, double-perturbation of LC3 and p62 or 

beclin 1 and p62, was unable to provide greater protection for the 

cells than p62 solitary silencing (Fig. 9A and B). Noticeably, the 




The ratios of PAR and cPARP to actin are presented in the graphs (left: cPARP to actin; right: PAR to actin). (B) MTS assays were used to analyze cell 

Figure 8. p62 is involved in both accumulation of LC3 and cell death induced by C42. (A) HCT116 cells were transfected with the control (Mock) or 

ATG5 siRNA; after 36 h of transfection, cells were treated with C42 (0.25 μM) for 12 h before immunoblotting analysis with the indicated antibodies. 

viability after depletion of ATG5. (C) HCT116 cells were transfected with the control (Mock) or p62 siRNA; after 36 h of transfection, cells were treated 

with C42 (0.25 μM) for 12 h before immunoblotting analysis with the indicated antibodies. The ratios LC3-ii or cPARP to actin are presented below the 

parts. (D) MTS assays were performed to analyze cell viability after silencing of p62. 

combined knockdown of LC3 and p62 offered a greater inhibitory 

effect on PARP-1 cleavage but resulted in the most increase 

in PAR formation (Fig. 9C); in contrast, double-depletion of 

beclin 1 and p62 did not show additional inhibition on PARP 

cleavage, but resulted in a higher suppression of PARP activation 

(at least in a short period) (Fig. 9D). Although knockdown 

of either LC3 or beclin 1 inhibited the cleavage of PARP, they 

affected the activation of PARP in different manners (Fig. 9C 

and D). Unlike LC3, knockdown of beclin 1 alone or in combination 

with p62 may temporally inhibit the caspase-independent 

apoptotic or necrotic pathway. Notably, it seems that there is an 

inverse correlation between the C42-dependent PARP cleavage 

and activation under autophagy-defective circumstances. Taken 

together, the above findings suggest that p62 is involved in 

mediating the C42-dependent LC3 processing and participates 

in cell death under this scenario, which is consistent with a previous 

report on p62 in activating the caspase pathways. 60 Since 

knockdown of the autophagic genes cannot completely prevent 

the C42-induced cell death, we assume that blocking autophagy 

only temporarily slows down or delays caspase-dependent apoptosis 

and may switch the cell to caspase-independent or necrosislike 

death. 

Discussion 

Despite having long been recognized as the inducers of apoptosis 

and necrosis, the ETPs’ roles in autophagy remain unclear 

and the interplay between different cell death pathways induced 

Figure 9 (See opposite page). Combined silencing of LC3 and p62 switch the mode of cell death. (A) HCT116 cells were transfected with the control 

(Mock) or a variety of siRNAs (singular or combined), after transfection for 36 h, cells were treated with C42 (0.25 μM) for 24 h before immunoblotting 

analysis with anti-LC3 antibody. (B) MTS assays were performed to detect viability of cells (A). The data were statistically analyzed by using Student’s 

t-test. It revealed that the difference between the control siRNA and siRNAs of a variety of genes was significant. *indicates significant difference 

between the mock and various groups at level p < 0.05; **means significant difference at level p < 0.01. HCT116 cells were transfected with the control 

(Mock), LC3 (C), Beclin 1 (B) or p62 (C and D) siRNAs solely or combined, after 36 h of transfection, cells were treated with C42 (0.25 μM) for upon to 24 

h before immunoblotting analysis with the indicated antibodies. The ratios of PAR and PARP to actin were shown in the graph right of and below the 

parts in (C and D), respectively. The ratios are representatives of two similar experiments. 




www.landesbioscience.com Autophagy 11

have found that the activation of PARP plays a regulatory role 

in apoptosis induction, revealing that the PARP inhibitor 3-AB 

provides transient protection by switching necrosis to apoptosis 

in 1-methyl-3-nitro-1-nitrosoguanidinium (MNNG)-treated 

cells. In contrast to MNNG, preincubation with 3-AB results in 

decreased membrane blebbing and reduced formation of apoptotic 

bodies in melphalan-treated HL60 cells. 68 A recent study 

has shown that the inhibition of PARP-1 leads to prevention of 

the NAD + depletion and resistance against paclitaxel-induced 

caspase 3 activation and apoptotic cell death by activating the 

PtdIns3K/Akt pathway. 69 We have noted that the C42-induced 

formation of PAR was cell-type-dependent and the inhibition of 

PARP also yielded diverse results in a context-dependent manner. 

Nevertheless, perturbation of some autophagic genes obviously 

reduced the formation of PAR in all C42-treated cells, suggesting 

that autophagy may have a feedback effect on the activation 

of PARP. In other words, autophagy may also be involved in the 

C42-triggered caspase-independent or necrosis-like cell death. 

The pan-caspase inhibitor Z-V-FMK can block apoptotic 

cell death, but enhance death receptor-mediated necrotic cell 

death. 62 Wu and Shen reported that autophagy plays a protective 

role in Z-V-FMK-induced necrotic cell death. 16 We found that 

Z-V-FMK alone increased the level of p62 but decreased that of 

LC3-II. However, it failed to block the C42-dependent degra- 


dation of p62. Therefore, we propose that Z-V-FMK enhances 

by the ETPs has not been explored. In this study, the formation 

of autophagosomes was identified by fluorescence and 

electron microscopy upon treatment of the targeted cells with 

11'-deoxyverticillin A (C42). The EM photographs revealed the 

presence of the autophagic vacuoles and the characteristics of 

apoptosis. The C42-induced autophagy was further confirmed 

by immunoblotting for lipidation of LC3 and by monitoring the 

autophagic flux and was found to be closely associated with the 

activation of caspases and PARP, consistent with the reports in 

the literature that the accumulation of autophagic vacuoles can 

occur before cell death. 61,62 Our data demonstrated that the C42- 

induced autophagy preceded the caspase-dependent apoptosis in 

the cells tested. Inhibition of apoptosis enhanced the induction of 

autophagy markers but did not completely prevent the cell death 

from happening, whereas suppression of autophagy by chemical 

inhibitors or silencing the essential autophagic genes partially 

prevented or delayed the cleavage of caspases and PARP-1. Thus, 

we assumed that the C42-induced autophagy, once overactivated, 

could independently lead to cell death in addition to association 

with apoptotic cell death. We found that both PARP activation 

and RIP-1 expression were implicated in the C42-induced autophagic 

process, in agreement with previous reports in reference 

45, 46 and 63. It is interesting to note that RIP-1 also plays a 

role in mediating the cleavage and activation of PARP caused by 

C42. We also found that RIP-3, another RIP kinase participating 

in mediation of apoptosis and necrosis, 51,52 also played a role 

in the C42-dependent autophagy and apoptosis. Considering 

these results, we assume that both apoptosis and autophagy may 

cooperate to result in cell death. 

the C42-induced autophagic process, which may eventually lead 

to cell death. In addition, we assumed that, once the apoptotic 

pathway was inhibited, the cells would shift back to autophagic 


cell death. Furthermore, the overactivation of PARP caused by 

As the founding member of the PARP family, 64 PARP-1 is 

implicated in the apoptotic pathway and its cleavage can be 

evaluated to confirm the activation of caspase 3. In addition, 

PARP-1 can convert β-nicotinamide adenine dinucleotide 

(NAD + ) to polymers of PAR, which participate in regulating 

nuclear homeostasis upon DNA damage stress. 65 Such damage 

can cause overactivation of PARP, leading to the depletion of 

intracellular NAD + , suppression of ATP production and finally, 

cell death, especially the necrotic cell death. 10 However, several 

studies have shown that PARP activation also occurs during 

apoptosis and inhibition of PAR formation impairs the activation 

of apoptotic machinery, resulting in release of apoptosis-inducing 

factor (AIF). 11,63 Here, we clearly showed that both the PAR formation 

and C42-induced autophagy preceded the appearance of 

hallmarks of apoptosis and inhibitors of autophagy or silencing 

of essential autophagic genes suppressed both the cleavage and 

activation of PARP. Thus, autophagy apparently can act as a 

partner in the C42-dependent apoptotic cell death. Interestingly, 

chemical inhibition of PARP activation not only decreased the 

content of PAR, but also significantly attenuated caspase 3 activation, 

suggesting that the caspase-dependent and -independent 

apoptosis could be triggered by PARP overactivation upon stimulation 

by C42. Additionally, overactivated PARP induced by 

C42 could also cause the necrotic cell death. In HaCaT cells, 

inhibition of PARP influences the mode of sulfur mustardinduced 

cell death. 66 Induction of apoptosis by PARP inhibitors 

is also observed in HeLa cells. 67 Pogrebniak and Hasmann 

Z-V-FMK could switch the cells to caspase-independent or type 

III cell death upon treatment with C42. 

Nec-1, a specific inhibitor of RIP-1 kinase, inhibits programmed 

necrosis (necroptosis). 50 Results from a recent study 

showed that the presence of Nec-1 shifts shikonin-induced 

necroptosis to apoptosis. 70 However, we found that Nec-1 only 

partially decreased the LC3-II content and inhibited caspase 

cleavage and PARP activation induced by C42. Reduced RIP-1 

expression by RNAi also decreased the C42-induced LC3-II 

amount, PARP cleavage and cell death. Thus, C42 may act to 

directly affect autophagy, apoptosis and necrosis via the RIP-1 

protein or the RIP-1 complex formed with other proteins, 

although the precise mechanism(s) remains to be elucidated. In 

other words, RIP-1 or its complex is required for C42-induced 

apoptosis and Nec-1 may indirectly inhibit caspase-dependent 

apoptosis via the RIP-1 kinase. These findings are consistent 

with the notion that RIP-1 is a diversified functional protein, 

participating in regulation of apoptosis, necrosis and autophagic 

cell death. 71,72 

One intriguing finding of this study is that suppression of 

autophagy by chemical inhibitors or RNAi knockdown of the 

critical autophagic genes only partially inhibits the cleavage and 

activation of PARP. Conversely, inhibition of PARP activation 

also affects the cellular autophagic process. Thus, these findings 

suggest that a close link among different types of cell death may 

exist. We further assume that there may be a transfer between 

different modes of cell death induced by C42, as knockdown 


of both LC3 and p62 completely suppressed PARP cleavage and 

maximally augmented PAR formation. Accordingly, cell death 

may transfer from the caspase-dependent mode to caspase-independent 

apoptosis or necrosis under this scenario. Singular gene 

silencing merely provided the cell with partial protection, indicting 

that other related genes could have taken over the role to 

execute and complete the death process when one protein was 

missing and the stimulus continued to be presented. The above 

hypothesis was confirmed by combined gene silencing. Depletion 

of LC3 or beclin 1 usually concurred with defective degradation 

of p62, thus, the cells might utilize p62 to relay the death signaling 

in the continual presence of C42. 

ROS are highly reactive oxygen free radicals or nonradical 

molecules generated by multiple mechanisms in cells and the nicotinamide 

adenine dinucleotide phosphate (NADPH) oxidases 

and mitochondria are their major cellular sources. 73 To date, ROS 

are widely recognized as being implicated in various biological 

functions and diseases. 74 Depending on the cellular context, ROS 

can either promote cell survival or cell death. 54 Growing evidence 

indicates that ROS control autophagy in multiple contexts and 

cell types and changes in ROS and autophagy regulation contribute 

to cancer commencement and development. 53-56 Since 

the ETPs were reported to generate ROS via redox cycling, the 

regulatory effect of C42 on autophagy and apoptosis is likely to 

associate with those important signaling molecules. Therefore, 

(2762). Anti-PAR (551813) and anti-RIP-1 (610458) antibody 

were purchased from BD Pharmingen; antibodies to β-actin (sc- 

1616), p62 (sc-28359), GFP (sc-81045) and Beclin 1 (sc-11427) 

were purchased from Santa Cruz Biotechnology. Anti-RIP-3 

(ab56164) antibody was from Abcam. 

Fungal material and extracts preparation. The strain of 

the Cordyceps-colonizing fungus was isolated by one of the 

authors (X.L.) from the samples of Cordyceps sinensis (Berk.) 

Sacc. collected in Linzhi, Tibet, in March, 2004. The fungus 

was identified as Gliocladium sp. and assigned the accession No. 

XZC04-CC-302 in X.L.’s culture collection at the Institute of 

Microbiology, Chinese Academy of Sciences, Beijing. The crude 

extract was prepared as described previously in reference 75. 

Plasmids and siRNAs. The GFP-LC3 plasmid is a kind gift of 

Dr. Tamotsu Yoshimori (Osaka University, Japan). 

The siRNA specific for human MAP1LC3 (sc-43390), 

PARP-1 (sc-29437), RIP-1 (sc-36426), RIP-3 (sc-61482), ATG5 

(sc-41445) and p62 (sc-29679) were purchased from Santa Cruz 

Biotechnology along with the control siRNA. siGENOME 

SMART pool BECN1 (Dharmacon, 8678) targeting beclin 1 was 

bought from Dharmacon. 

Cell culture and western blot analysis. Standard single-cell 

cloning and G418 selection procedures were used to establish 

stable cell lines of porcine aortic endothelial cells, that express 


wild-type (WT) EGFR as described previously in reference 76. 

understanding of the mechanisms of C42-induced autophagy via 

ROS generation will provide significant implications for development 

of the ETPs into therapeutic anticancer agents. 

In summary, the ETPs are an interesting class of fungal 

The WT GFP-EGFR- and GFP-Bcl-x L 

-expressing PAE cell 

lines were grown in F12 medium containing 10% fetal bovine 

serum, antibiotics and glutamine and supplemented with 


G418. The human adenocarcinoma of the colon cells SW480 

toxins that inhibit farnesyl transferase, an enzyme required for 

normal functioning of the Ras oncogene. Compounds sharing 

such a property have been explored as putative anticancer agents. 

Here, we demonstrate that 11'-deoxyverticillin A (C42) can activate 

and enhance the cellular autophagic process in addition to 

triggering apoptosis. These results provide new insights into the 

function and the mode of action of C42, allowing us to further 

understand the molecular mechanisms by which C42 exerts its 

anticancer activity. Future work in this direction could yield 

valuable information in the development of these compounds 

into effective cancer therapeutic strategies through modulation 

of autophagy. 

Materials and Methods 

Chemicals and antibodies. 11'-Deoxyverticillin A was isolated 

from the solid-substrate fermentation culture of the Cordycepscolonizing 

fungus Gliocladium sp. The following reagents were 

purchased from Sigma-Aldrich: rapamycin (R0395), trehalose 

(T9531), 3-methyladenine (M9281), necrostatin-1 (N9037), 

3-aminobenzamide (A0788), N-acetyl-L-cysteine (NAC, 

A7250), isoquinolinediol (I138) and polyclonal antibodies against 

LC3 (L7543) and ATG5 (A0731). Z-VAF-FMK (FMK001) 

was purchased from R & D systems. The following antibodies 

were from Cell Signaling Technology: caspase 3 (9662), cleaved 

caspase 3 (9664), caspase 9 (9508), PARP (9542), p70S6K 

(Thr389; 9205), phospho-mTOR (Ser2448; 2971) and Bcl-x L 

were maintained in DMEM (HyClone, SH30022. 01B) supplemented 

with 10% fetal bovine serum (GIBCO, 16000), plus 

antibiotics. Human adenocarcinoma of the colon cells SW620 

were maintained in RPMI-1640-medium (HyClone, SH30809. 

01B), whereas human colon carcinoma cells HCT116 were 

grown in McCoy’s 5A (KeyGEN, KGM5ASY) supplemented 

with FBS and antibiotics. Cells were split overnight and grown 

to 50% confluency before adding 11'-dideoxyverticillin A 

(C42). 

For siRNA interference, cells were grown to 40% confluence 

in their respective medium without antibiotics and transfected 

using DharmaFECT (Dharmacon, T2001) according to the 

manufacturer’s instructions. After transfection for 36 h, cells 

were directly treated with C42 without being split. Whole cell 

lysates were prepared with lysis using Triton X-100/glycerol buffer, 

which contained 50 mM Tris-HCl, pH 7.4, 4 mM EDTA, 

2 mM EGTA and 1 mM dithiothreitol, supplemented with 1% 

Triton X-100 and protease inhibitors and then separated on a 

SDS-PAGE gel (15, 10 or 8% according to the molecular weights 

of the proteins of interest) and transferred to PVDF membrane. 

Western blotting was performed using appropriate primary antibodies 

and horseradish peroxidase-conjugated suitable secondary 

antibodies, followed by detection with enhanced chemiluminescence 

(Pierce Chemical, 34080). 

Several x-ray films were analyzed to verify the linear range of 

the chemiluminescence signals and the quantifications were carried 

out using densitometry. 


Cell proliferation assay (MTS). Cells were plated in 96-well 

plates (10,000 cells per well) in 100 μl complete culture medium. 

After overnight culture, the medium was replaced with complete 

medium that was either drug-free or contained C42 or other 

chemicals. The cells were cultured for various periods and cellular 

viability was determined with CellTiter 96 ® Aqueous Non- 

Radioactive Cell Proliferation Assay (G1111, Promega). 

For siRNA, each well was plated with 5,000 cells. After 36 h 

of transfection, C42 was directly added to the cells and cellular 

viability was determined. 

Flow cytometry assay. HCT116 cells were treated with C42 

(0.25 and 0.5 μM, respectively), trypsinized and harvested 

(keeping all floating cells), washed with PBS buffer, followed by 

incubating with a fluorescein isothiocyanate-labeled annexin V 

(FITC) and propidium iodide (PI) according to the instructions 

of an Annexin-V-FITC Apoptosis Detection Kit (Biovision Inc., 

K101-100) and analyzed by flow cytometry (FACSAria, Becton 

Dickinson). Percentages of the cells with annnexin V-positive 

and PI-negative stainings were considered as apoptotic, whereas 

PI-positive staining was considered to be necrotic. 

Colony growth assay. Cells were treated with C42 for 6 h, 

split, seeded at 300 cells/ml and cultured for two weeks to allow 

colony growth of surviving cells. 

Fluorescence microscopy. HCT116 cells were transfected with 

the GFP-LC3 expressing plasmid and after 24 h, cells were treated 

according to the manufacturer’s protocol and its integrity was 

confirmed by electrophoresis on ethidium bromide-stained 1% 

agarose gel. Total cellular RNA (1 μg) was reverse transcribed 

at 37°C for 15 min in 20 μl of PrimeScript TM RT reagent Kit 

(TaKaRa, DRR037A). Reactions were stopped by heat inactivation 

for 5 s at 85°C. Primer sequences used for amplification were 

as fellows: LC3 upstream primer, 5'-ATG CCG TCG GAG AAG 

ACC-3', downstream primer, 5'-TTA CAC TGA CAA TTT 

CAT CC-3'; PARP-1 upstream primer, 5'-CTA AAG GCT CAG 

AAC GAC C-3', downstream primer, 5'-GAA GGA GGG CAC 

CGA ACA-3'; β-actin upstream primer, 5'-GCC TGA CGG 

CCA GGT CAT CAC-3', downstream primer, 5'-CGG ATG 

TCC ACG TCA CAC TTC-3'. PCR reactions with LC3 primers 

were initiated with a 5 min denaturation at 95°C in a final 

volume of 20 μl. The cycle profile was 95°C, 60°C and 72°C for 

30 s for up to 25 cycles. The PCR amplification products were 

determined in DNA agarose gels, which were photographed and 

quantitatively scanned using Photoshop software. The expression 

of β-actin was used as the internal control. 

Statistical analysis. Normally distributed data are shown as 

mean ± SD and were analyzed using one-way analysis of variance 

and the Student-Newman-Keuls post-hoc test. 


Acknowledgements 

We thank Alexander Sorkin from UCHSC for providing EGFR 

plasmid, Dr. Tamotsu Yoshimori from Osaka University for the 

GFP-LC3 plasmid, Dr. Quan Chen from the Institute of Zoology 

for the Bcl-x L 

plasmid and Dr. Xin Ye from the Institute of 


Microbiology, CAS for p53 -/- HCT116 cells. We are also grateful 

with C42, the fluorescence of GFP-LC3 was viewed and the 

images were acquired via confocal microscopy (Leica, TCS SP5). 

Electron microscopy. Electron microscopy was performed as 

described previously in reference 77. Briefly, HCT116 cell samples 

were washed three times with PBS, trypsinized and collected 

by centrifuging. The cell pellets were fixed with 4% paraformaldehyde 

overnight at 4°C, post-fixed with 1% OsO 4 

in cacodylate 

buffer at room temperature for 1 h and dehydrated stepwise 

with ethanol. The dehydrated pellets were rinsed with propylene 

oxide for 30 min and then embedded in Spurr resin for sectioning. 

Images of thin sections were observed under a transmission 

electron microscope (JEM1230, Tokyo, Japan). 

RNA extraction and RT-PCR analysis. Total cellular RNA 

was extracted using TRIzol reagent (Invitrogen, 15596-018) 

to Dr. Daniel J. Klionsky from the University of Michigan for his 

advice in preparing the manuscript. This study was supported by 

grants from the National Natural Science Foundation of China 

(NSFC, grants 90408024, 30900731, 30672097, 30870057 and 

30925039) and the Ministry of Science and Technology of China 

(2009CB522302 and 2009ZX09302-004). 

Note 

Supplemental materials can be found at: 

www.landesbioscience.com/journals/autophagy/article/15103 

References 

1. Klionsky DJ, Emr SD. Autophagy as a regulated pathway 

of cellular degradation. Science 2000; 290:1717-21. 

2. Mizushima N. Autophagy: process and function. Genes 

Dev 2007; 21:2861-73. 

3. Levine B, Klionsky DJ. Development by self-digestion: 

molecular mechanisms and biological functions of 

autophagy. Dev Cell 2004; 6:463-77. 

4. Codogno P, Meijer AJ. Autophagy and signaling: their 

role in cell survival and cell death. Cell Death Differ 

2005; 12:1509-18. 

5. Edinger AL, Thompson CB. Death by design: apoptosis, 

necrosis and autophagy. Curr Opin Cell Biol 2004; 

16:663-9. 

6. Levine B, Kroemer G. Autophagy in the pathogenesis 

of disease. Cell 2008; 132:27-42. 

7. Jacobson MD, Weil M, Raff MC. Programmed cell 

death in animal development. Cell 1997; 88:347-54. 

8. Levine B, Yuan J. Autophagy in cell death: an innocent 

convict? J Clin Invest 2005; 115:2679-88. 

9. Mizushima N, Levine B, Cuervo AM, Klionsky DJ. 

Autophagy fights disease through cellular self-digestion. 

Nature 2008; 451:1069-75. 

10. Ha HC, Snyder SH. Poly(ADP-ribose) polymerase is a 

mediator of necrotic cell death by ATP depletion. Proc 

Natl Acad Sci USA 1999; 96:13978-82. 

11. Yu SW, Wang H, Poitras MF, Coombs C, Bowers WJ, 

Federoff HJ, et al. Mediation of poly(ADP-ribose) 

polymerase-1-dependent cell death by apoptosis-inducing 

factor. Science 2002; 297:259-63. 

12. Baehrecke EH. How death shapes life during development. 

Nat Rev Mol Cell Biol 2002; 3:779-87. 

13. Budihardjo I, Oliver H, Lutter M, Luo X, Wang X. 

Biochemical pathways of caspase activation during 

apoptosis. Annu Rev Cell Dev Biol 1999; 15:269-90. 

14. Baehrecke EH. Autophagy: dual roles in life and death? 


15. Luo S, Rubinsztein DC. Apoptosis blocks Beclin 

1-dependent autophagosome synthesis: an effect rescued 

by Bcl-x L 

. Cell Death Differ 2010; 17:268-77. 

16. Wu YT, Tan HL, Huang Q, Kim YS, Pan N, Ong 

WY, et al. Autophagy plays a protective role during 

zVAD-induced necrotic cell death. Autophagy 2008; 

4:457-66. 

17. Yu L, Alva A, Su H, Dutt P, Freundt E, Welsh S, 

et al. Regulation of an ATG7-beclin 1 program of 

autophagic cell death by caspase-8. Science 2004; 

304:1500-2. 

18. Zong WX, Ditsworth D, Bauer DE, Wang ZQ, 

Thompson CB. Alkylating DNA damage stimulates a 

regulated form of necrotic cell death. Genes Dev 2004; 

18:1272-82. 

19. Guillon-Munos A, van Bemmelen MX, Clarke PG. 

Role of phosphoinositide-3-kinase in the autophagic 

death of serum-deprived PC12 cells. Apoptosis 2005; 

10:1031-41. 

20. Maiuri MC, Zalckvar E, Kimchi A, Kroemer G. Selfeating 

and self-killing: crosstalk between autophagy and 

apoptosis. Nat Rev Mol Cell Biol 2007; 8:741-52. 

21. Zucchini-Pascal N, de Sousa G, Rahmani R. Lindane 

and cell death: at the crossroads between apoptosis, 

necrosis and autophagy. Toxicology 2009; 256:32-41. 


22. Colell A, Ricci JE, Tait S, Milasta S, Maurer U, 

Bouchier-Hayes L, et al. GAPDH and autophagy preserve 

survival after apoptotic cytochrome c release in the 

absence of caspase activation. Cell 2007; 129:983-97. 

23. Crighton D, Wilkinson S, O’Prey J, Syed N, Smith P, 

Harrison PR, et al. DRAM, a p53-induced modulator 

of autophagy, is critical for apoptosis. Cell 2006; 

126:121-34. 

24. Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, 

Mizushima N, et al. Bcl-2 antiapoptotic proteins 

inhibit Beclin 1-dependent autophagy. Cell 2005; 

122:927-39. 

25. Yousefi S, Perozzo R, Schmid I, Ziemiecki A, Schaffner 

T, Scapozza L, et al. Calpain-mediated cleavage of Atg5 

switches autophagy to apoptosis. Nat Cell Biol 2006; 

8:1124-32. 

26. Cho DH, Jo YK, Hwang JJ, Lee YM, Roh SA, Kim 

JC. Caspase-mediated cleavage of ATG6/Beclin-1 links 

apoptosis to autophagy in HeLa cells. Cancer Lett 

2009; 274:95-100. 

27. Scott RC, Juhasz G, Neufeld TP. Direct induction of 

autophagy by Atg1 inhibits cell growth and induces 

apoptotic cell death. Curr Biol 2007; 17:1-11. 

28. Eisenberg-Lerner A, Bialik S, Simon HU, Kimchi A. 

Life and death partners: apoptosis, autophagy and 

the cross-talk between them. Cell Death Differ 2009; 

16:966-75. 

29. Zalckvar E, Yosef N, Reef S, Ber Y, Rubinstein AD, 

Mor I, et al. A systems level strategy for analyzing the 

cell death network: implication in exploring the apoptosis/autophagy 

connection. Cell Death Differ 2010; 

17:1244-53. 

30. Dong JY, He HP, Shen YM, Zhang KQ. Nematicidal 

epipolysulfanyldioxopiperazines from Gliocladium 

roseum. J Nat Prod 2005; 68:1510-3. 

40. Mizushima N. Methods for monitoring autophagy. Int 

J Biochem Cell Biol 2004; 36:2491-502. 

41. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, 

Kirisako T, Noda T, et al. LC3, a mammalian homologue 

of yeast Apg8p, is localized in autophagosome 

membranes after processing. EMBO J 2000; 19:5720-8. 

42. Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, 

Aliev G, Askew DS, et al. Guidelines for the use and 

interpretation of assays for monitoring autophagy in 

higher eukaryotes. Autophagy 2008; 4:151-75. 

43. Hay N, Sonenberg N. Upstream and downstream of 

mTOR. Genes Dev 2004; 18:1926-45. 

44. Yamamoto A, Tagawa Y, Yoshimori T, Moriyama 

Y, Masaki R, Tashiro Y. Bafilomycin A1 prevents 

maturation of autophagic vacuoles by inhibiting fusion 

between autophagosomes and lysosomes in rat hepatoma 

cell line, H-4-II-E cells. Cell Struct Funct 1998; 

23:33-42. 

45. Huang Q, Wu YT, Tan HL, Ong CN, Shen HM. A 

novel function of poly(ADP-ribose) polymerase-1 in 

modulation of autophagy and necrosis under oxidative 

stress. Cell Death Differ 2009; 16:264-77. 

46. Munoz-Gamez JA, Rodriguez-Vargas JM, Quiles-Perez 

R, Aguilar-Quesada R, Martin-Oliva D, de Murcia 

G, et al. PARP-1 is involved in autophagy induced by 

DNA damage. Autophagy 2009; 5:61-74. 

47. Wieler S, Gagne JP, Vaziri H, Poirier GG, Benchimol 

S. Poly(ADP-ribose) polymerase-1 is a positive regulator 

of the p53-mediated G 1 

arrest response following 

ionizing radiation. J Biol Chem 2003; 278:18914-21. 

48. Ding W, Liu W, Cooper KL, Qin XJ, de Souza Bergo 

PL, Hudson LG, et al. Inhibition of poly(ADP-ribose) 

polymerase-1 by arsenite interferes with repair of oxidative 

DNA damage. J Biol Chem 2009; 284:6809-17. 

49. Yu L, Wan F, Dutta S, Welsh S, Liu Z, Freundt E, 

59. Pankiv S, Clausen TH, Lamark T, Brech A, Bruun 

JA, Outzen H, et al. p62/SQSTM1 binds directly to 

Atg8/LC3 to facilitate degradation of ubiquitinated 

protein aggregates by autophagy. J Biol Chem 2007; 

282:24131-45. 

60. Jin Z, Li Y, Pitti R, Lawrence D, Pham VC, Lill JR, et 

al. Cullin3-based polyubiquitination and p62-dependent 

aggregation of caspase-8 mediate extrinsic apoptosis 

signaling. Cell 2009; 137:721-35. 

61. Espert L, Denizot M, Grimaldi M, Robert-Hebmann 

V, Gay B, Varbanov M, et al. Autophagy is involved in 

T cell death after binding of HIV-1 envelope proteins 

to CXCR4. J Clin Invest 2006; 116:2161-72. 

62. Gonzalez-Polo RA, Boya P, Pauleau AL, Jalil A, 

Larochette N, Souquere S, et al. The apoptosis/autophagy 

paradox: autophagic vacuolization before apoptotic 

death. J Cell Sci 2005; 118:3091-102. 

63. Yu SW, Andrabi SA, Wang H, Kim NS, Poirier GG, 

Dawson TM, et al. Apoptosis-inducing factor mediates 

poly(ADP-ribose) (PAR) polymer-induced cell death. 

Proc Natl Acad Sci USA 2006; 103:18314-9. 

64. Ame JC, Spenlehauer C, de Murcia G. The PARP 

superfamily. Bioessays 2004; 26:882-93. 

65. Schreiber V, Dantzer F, Ame JC, de Murcia G. 

Poly(ADP-ribose): novel functions for an old molecule. 


66. Kehe K, Raithel K, Kreppel H, Jochum M, Worek F, 

Thiermann H. Inhibition of poly(ADP-ribose) polymerase 

(PARP) influences the mode of sulfur mustard 

(SM)-induced cell death in HaCaT cells. Arch Toxicol 

2008; 82:461-70. 

67. Ghosh U, Bhattacharyya NP. Induction of apoptosis by 

the inhibitors of poly(ADP-ribose)polymerase in HeLa 

cells. Mol Cell Biochem 2009; 320:15-23. 


68. Pogrebniak A, Schemainda I, Pelka-Fleischer R, Nussler 

31. Gardiner DM, Waring P, Howlett BJ. The epipolythiodioxopiperazine 

(ETP) class of fungal toxins: distribution, 

mode of action, functions and biosynthesis. 

Microbiology 2005; 151:1021-32. 

32. Bernardo PH, Brasch N, Chai CL, Waring P. A novel 

redox mechanism for the glutathione-dependent reversible 

uptake of a fungal toxin in cells. J Biol Chem 2003; 

278:46549-55. 

33. Beaver JP, Waring P. Lack of correlation between early 

intracellular calcium ion rises and the onset of apoptosis 

in thymocytes. Immunol Cell Biol 1994; 72:489-99. 

34. Yanagihara M, Sasaki-Takahashi N, Sugahara T, 

Yamamoto S, Shinomi M, Yamashita I, et al. Leptosins 

isolated from marine fungus Leptoshaeria species inhibit 

DNA topoisomerases I and/or II and induce apoptosis 

by inactivation of Akt/protein kinase B. Cancer Sci 

2005; 96:816-24. 

35. Chen Y, Miao ZH, Zhao WM, Ding J. The p53 pathway 

is synergized by p38 MAPK signaling to mediate 

11,11'-dideoxyverticillin-induced G 2 

/M arrest. FEBS 

Lett 2005; 579:3683-90. 

36. Ekert PG, Silke J, Vaux DL. Caspase inhibitors. Cell 

Death Differ 1999; 6:1081-6. 

37. Seglen PO, Gordon PB. 3-Methyladenine: specific 

inhibitor of autophagic/lysosomal protein degradation 

in isolated rat hepatocytes. Proc Natl Acad Sci USA 

1982; 79:1889-92. 

38. Heitman J, Movva NR, Hall MN. Targets for cell cycle 

arrest by the immunosuppressant rapamycin in yeast. 

Science 1991; 253:905-9. 

39. Sarkar S, Davies JE, Huang Z, Tunnacliffe A, 

Rubinsztein DC. Trehalose, a novel mTOR-independent 

autophagy enhancer, accelerates the clearance of 

mutant huntingtin and alpha-synuclein. J Biol Chem 

2007; 282:5641-52. 

et al. Autophagic programmed cell death by selective 

catalase degradation. Proc Natl Acad Sci USA 2006; 

103:4952-7. 

50. Degterev A, Hitomi J, Germscheid M, Ch’en IL, 

Korkina O, Teng X, et al. Identification of RIP-1 kinase 

as a specific cellular target of necrostatins. Nat Chem 

V, Hasmann M. Poly ADP-ribose polymerase (PARP) 

inhibitors transiently protect leukemia cells from alkylating 

agent induced cell death by three different effects. 

Eur J Med Res 2003; 8:438-50. 

69. Szanto A, Hellebrand EE, Bognar Z, Tucsek Z, Szabo 

Biol 2008; 4:313-21. 

51. Feng S, Yang Y, Mei Y, Ma L, Zhu DE, Hoti N, et al. 

Cleavage of RIP3 inactivates its caspase-independent 

apoptosis pathway by removal of kinase domain. Cell 

Signal 2007; 19:2056-67. 

52. Zhang DW, Shao J, Lin J, Zhang N, Lu BJ, Lin SC, et 

al. RIP3, an energy metabolism regulator that switches 

TNF-induced cell death from apoptosis to necrosis. 

Science 2009; 325:332-6. 

53. Huang J, Brumell JH. NADPH oxidases contribute to 

autophagy regulation. Autophagy 2009; 5:887-9. 

54. Mazure NM, Pouyssegur J. Hypoxia-induced autophagy: 

cell death or cell survival? Curr Opin Cell Biol 

2010; 22:177-80. 

55. Scherz-Shouval R, Shvets E, Fass E, Shorer H, Gil 

L, Elazar Z. Reactive oxygen species are essential for 

autophagy and specifically regulate the activity of Atg4. 

EMBO J 2007; 26:1749-60. 

56. Zhang H, Kong X, Kang J, Su J, Li Y, Zhong J, et al. 

Oxidative stress induces parallel autophagy and mitochondria 

dysfunction in human glioma U251 cells. 

Toxicol Sci 2009; 110:376-88. 

57. Cecconi F, Levine B. The role of autophagy in mammalian 

development: cell makeover rather than cell death. 

Dev Cell 2008; 15:344-57. 

58. Gao Z, Gammoh N, Wong PM, Erdjument-Bromage 

H, Tempst P, Jiang X. Processing of autophagic protein 

LC3 by the 20S proteasome. Autophagy 2010; 6:126-37. 


A, Gallyas F Jr, et al. PARP-1 inhibition-induced activation 

of PI-3-kinase-Akt pathway promotes resistance 

to taxol. Biochem Pharmacol 2009; 77:1348-57. 

70. Han W, Xie J, Li L, Liu Z, Hu X. Necrostatin-1 reverts 

shikonin-induced necroptosis to apoptosis. Apoptosis 

2009; 14:674-86. 

71. Declercq W, Vanden Berghe T, Vandenabeele P. RIP 

kinases at the crossroads of cell death and survival. Cell 

2009; 138:229-32. 

72. Festjens N, Vanden Berghe T, Cornelis S, Vandenabeele 

P. RIP-1, a kinase on the crossroads of a cell’s decision 

to live or die. Cell Death Differ 2007; 14:400-10. 

73. Trachootham D, Lu W, Ogasawara MA, Nilsa RD, 

Huang P. Redox regulation of cell survival. Antioxid 

Redox Signal 2008; 10:1343-74. 

74. Giannoni E, Buricchi F, Grimaldi G, Parri M, Cialdai 

F, Taddei ML, et al. Redox regulation of anoikis: 

reactive oxygen species as essential mediators of cell 

survival. Cell Death Differ 2008; 15:867-78. 

75. Liu L, Liu S, Jiang L, Chen X, Guo L, Che Y. 

Chloropupukeananin, the first chlorinated pupukeanane 

derivative and its precursors from Pestalotiopsis 

fici. Org Lett 2008; 10:1397-400. 

76. Jiang X, Huang F, Marusyk A, Sorkin A. Grb2 regulates 

internalization of EGF receptors through clathrin-coated 

pits. Molecular biology of the cell 2003; 14:858-70. 

77. Li M, Jiang X, Liu D, Na Y, Gao GF, Xi Z. Autophagy 

protects LNCaP cells under androgen deprivation conditions. 

Autophagy 2008; 4:54-60.

PARP and RIP-1 are required for autophagy induced by 11 ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?