ORIGINAL PAPER

NAD+ Treatment Induces Delayed Autophagy in Neuro2a CellsPartially by Increasing Oxidative Stress

Jin Han • Shengtao Shi • Lan Min •

Teresa Wu • Weiliang Xia • Weihai Ying

Received: 7 April 2011 / Revised: 11 July 2011 / Accepted: 14 July 2011 / Published online: 11 August 2011

� Springer Science+Business Media, LLC 2011

Abstract NAD? plays important roles in various bio-

logical processes. In this study, we reported that treatment

of NAD? induces delayed autophagy in Neuro2a cells.

Moreover, the effects of NAD? on the autophagy in the

cells appear to be, at least partially, mediated by oxidative

stress. However, nicotinamide, a degradation product of

NAD?, does not affect the autophagy. Our experiments

have further indicated that the NAD?-induced autophagy

contributes to the NAD?-induced decrease in the survival

of these cells. In summary, our study has provided the first

evidence that NAD? treatment induces autophagy in can-

cer cells such as Neuro2a cells, which contributes to the

NAD?-induced decrease in cancer cell survival.

Keywords Autophagy � NAD? � Oxidative stress �Tumor cell survival � Nicotinamide

Introduction

Macroautophagy (hereafter called autophagy) is a catabolic

process for the degradation of cytoplasmic contents (e.g.

organelles, long-lived proteins, lipids) by using lysosomal

degradation machinery in an effort to recycle components

or energy [1]. While a basal level of autophagy supports

the homeostatic maintenance of metabolic conditions of

cells, excessive autophagy can lead to cell death. It has

been found that autophagy occurs in various types of

cancer cells after different anticancer treatments [2–4].

However, it remains unclear if the autophagy leads to an

increase or a decrease in the survival of cancer cells [5, 6].

As a classical energy molecule, NAD? plays important

roles in not only energy metabolism and mitochondrial

functions, but also in aging, gene expression, calcium

homeostasis, and cell death [7]. However, there has been

no sufficient information regarding the roles of NAD? in

the survival of cancer cells. Our latest study has shown that

NAD? treatment decreases the survival of multiple types of

cancer cells such as Neuro2a cells (neuroblastoma cells),

C6 glioma cells, and MCF-7 breast cancer cells [8]. It is of

interest that NAD? selectively decreases the survival of

cancer cells, while it does not impair the survival of mul-

tiple types of primary cell cultures such as astrocytes,

neurons and myocytes [9–11]. Thus, NAD? might become

a promising drug for cancer treatment. However, many

questions regarding the effects of NAD? on cancer cell

survival remain unclear: Can NAD? treatment induce

autophagy in cancer cells? What is the role of autophagy in

the NAD?-induced decrease in cell survival?

Recent findings have indicated that prolonged oxidative

stress induces autophagy in certain types of cancer cells

[12]. Our previous study has suggested that ROS con-

tributes to the NAD?-induced decrease in C6 glioma cell

survival [8]. In this study, we used Neuro2a cell, a mouse

neuroblastoma cell line, as a cell model to investigate the

effect of NAD? treatment on autophagy in cancer cells.

We also studied if oxidative stress is one of the possible

mechanisms by which NAD? may affect the autophagy.

Our studies have indicated that NAD? treatment induces

J. Han � S. Shi � L. Min � T. Wu � W. Xia (&) � W. Ying (&)

School of Biomedical Engineering and Med-X Research

Institute, Shanghai Jiao Tong University, 1954 Huashan Road,

Shanghai 200030, People’s Republic of China

W. Ying

e-mail: weihaiy@sjtu.edu.cn

W. Xia

e-mail: wlxia@sjtu.edu.cn

123

Neurochem Res (2011) 36:2270–2277

DOI 10.1007/s11064-011-0551-x

delayed autophagy in Neuro2a cells, at least partially by

generating oxidative stress, and this induced autophagy

contributes to the NAD?-induced decrease in cell

survival.

Materials and Methods

Materials

Reagents were purchased from Sigma Chemical Co. (St.

Louis, MO, USA) except where otherwise noted.

Cell Cultures

Neuro2a cells were purchased from the Cell Resource

Center of Shanghai Institute of Biological Sciences, Chi-

nese Academy of Sciences. The cells were plated in 6- or

24-well cell culture plates at the initial density of 1 9 105

cells/ml in Dulbecco’s Modified Eagle Medium (contain-

ing 4,500 mg/l D-glucose, 584 mg/l L-glutamine, 110 mg/l

sodium pyruvate) (Thermo Scientific, Waltham, MA,

USA) that contained 1% penicillin and streptomycin

(GIBCO BRL, Grand Island, NY, USA) and 10% fetal

bovine serum (PAA, Linz, Austria). The cells were used 24

or 48 h after cell passage.

Western Blot

Neuro2a cells were washed with PBS once, harvested and

lysed in RIPA buffer (Millipore, Temecula, CA, USA)

containing Complete Protease Inhibitor Cocktail (Roche

Diagnostics, Mannheim, Germany), 2 mM PMSF, and

0.1% SDS. Lysates were centrifuged at 12,000g for 20 min

at 4�C. After quantifications of the total protein lysates

using BCA Protein Assay Kit (Pierce Biotechonology,

Rockford, IL, USA), *40 lg of total protein was elec-

trophoresed through a 12 or 14% SDS–polyacrylamide

gel and then transferred to 0.45 lm. Nitrocellulose Mem-

brane (Whatman, Hahnestrase, Dassel, Germany) or

0.2 lm PVDF membranes (Millipore, Billerica, MA,

USA), respectively on a semi-dry electro transferring unit

(Bio-Rad Laboratories, Hercules, CA, USA). Blots were

incubated overnight at 4�C with a rabbit polyclonal LC3 or

p62 antibody at dilution of 1:1,000 (Sigma, St. Louis, MO,

USA), then incubated with appropriate HRP-conjugated

secondary antibody (Epitomics, Hangzhou, Zhejiang

Province, China). Protein signals were detected using ECL

detection system (Pierce Biotechonology, Rockford, IL,

USA). A specific anti-b-actin antibody (Cat. # E1909,

Santa Cruz Biotechnology, Santa Cruz, CA, USA) was

used to normalize sample loading and transfer. The

intensity of the bands was quantified by densitometry using

a Gel-Pro Analyzer.

Beclin 1 Real-Time qPCR Assay

Total RNA was extracted from cells using Trizol Reagent

(Invitrogen, CA, USA). Total RNA (2 lg) was reverse

transcribed using a PrimeScript RT reagent kit (TaKaRa,

Dalian, Shandong Province, China). Reverse transcription-

PCR was performed before quantitative real-time qPCR.

Beclin 1 mRNA levels were then determined by real-time

qPCR using SYBR Premix Ex Taq (TaKaRa) and the fol-

lowing primers: Beclin 1 sense primer: GTGGCGGCTC

CTATTCCAT, antisense primer: CAGGCAAGACCCCA

CTTGAG; GAPDH sense primer: AGGTCGGTGTGAA

CGGATTTG, antisense primer: TGTAGACCATGTAG

TTGAGGTCA. The levels of targeted gene expression were

detected as follows: denaturing at 95�C for 10 s, followed by

40 cycles of 95�C for 5 s and 60�C for 30 s. Data were col-

lected after each annealing step. GAPDH was used as an

endogenous control to normalize the differences between

samples, and relative expression of the targeted genes was

calculated and expressed as 2-DDCt, as previously [13].

Monodansylcadaverine (MDC) Staining

As described previously [14], the cells were briefly incu-

bated with 50 lM MDC in DMEM for 15 min at 37�C,

then washed with PBS three times and fixed in 4% Para-

formaldehyde (PFA) for 30 min at room temperature. PFA

was then removed and cells were washed with PBS. The

fluorescence of incorporated intracellular MDC was

detected at excitation wavelength of 390 nm and emission

wavelength of 527 nm under a Leica fluorescence micro-

scope. Using a reference grid, the number of MDC-positive

vesicles per area was calculated for each treatment

group [15].

Lactate Dehydrogenase (LDH) Assay

As described previously [16], cell survival was quantified

by measuring LDH activity in cell lysates. In brief, cells

were lysed for 20 min in a lysing buffer containing 0.04%

Triton-X, 2 mM HEPES, 0.2 mM dithiothreitol, 0.01%

bovine serum albumin, and 0.1% phenol red, pH 7.5. 50 ll

cell lysates were mixed with 150 ll 500 mM potassium

phosphate buffer (pH 7.5) containing 1.5 mM NADH and

7.5 mM sodium pyruvate, and the A340nm change was

monitored over 90 s. Percentage cell survival was calcu-

lated by normalizing the LDH activities of samples to the

LDH activity of control (wash only) wells.

Neurochem Res (2011) 36:2270–2277 2271

123

Dihydroethidium (DHE) Assay

DHE assay was conducted as described previously [8].

After treatment the cell cultures were incubated in 5 lM

DHE for 30 min at 37�C. The cells were then washed once

with PBS, and the fluorescence signals were observed

under a Leica fluorescence microscope at excitation

wavelength of 545 nm and emission wavelength of

605 nm. Semiquantitative scoring was performed as fol-

lows: score: 0, no; 1, weak; 2, moderate; 3, high; 4, very

high; 5, extremely high intensity [17].

Statistical Analyses

All data are presented as mean ± SE. Data were assessed by

one-way ANOVA, followed by Tukey post hoc test. P val-

ues less than 0.05 were considered statistically significant.

Results

To investigate the relationship between NAD? treatment

and autophagy, mouse albino neuroblastoma (Neuro2a)

cells were used. Immunoblotting of LC3 I/II conversion

was used to assess the level of autophagy in Neuro2a cells

after NAD? treatment. The microtubule-associated protein

1 light chain 3 (LC3) has two isoforms: LC3 I and LC3 II.

During the elongation of autophagosome, the cytosolic

isoform LC3 I is conjugated to phosphatidylethanolamine

to form a membrane bound protein LC 3 II, which is

continually expressed on the completed autophagosome

until final fusion with lysosomes [18]. Therefore, the

increase in the ratio of LC 3 II/I is an indicator of

autophagy in cells. We found that treatment of the cells

with 1 or 10 mM NAD? for 48 h increased the ratios of LC

3 II/I in Neuro2a cells by nearly 10-fold (Fig. 1a, b). We

also assessed the effects of 1 mM NAD? treatment on the

ratio of LC 3 II/I at the time point of 6, 24 and 48 h. As

indicated in Fig. 1c and d, the ratio of LC 3 II/I in Neuro2a

cells after treatment of 1 mM NAD? for 24 h doubled in

comparison to the control cells, and this ratio was further

increased to 8 for the 48 h-treatment group (Fig. 1c, d).

We further determined if NAD? treatment may induce

autophagy of Neuro2a cells by assessing Beclin 1 gene

expression. Beclin 1, a mammalian homolog of the yeast

Atg6, is a component of the PI3K complex implicated in

Fig. 1 NAD? treatment dose-dependently and time-dependently

increased the ratio of LC 3 II/I in Neuro2a cells. a NAD? treatment

dose-dependently increased the ratio of LC 3 II/I in Neuro2a cells.

Neuro2a cells were treated with 0.01–10 mM NAD? for 48 h. The

ratio of LC 3 II/I in the cells was then assessed by Western blot. This

figure is representative of five independent experiments. b Quantifi-

cations of the results of the Western blot on the ratio of LC 3 II/I of

Neuro2a cells treated with NAD? for 48 h showed that treatment of

the cells with 1–10 mM NAD? significantly increased autophagy in

the cells. Data were collected from five independent experiments.

*P \ 0.05. c NAD? treatment time-dependently increased the ratio of

LC 3 II/I in Neuro2a cells. Neuro2a cells were treated with 1 mM

NAD? for 6, 24 or 48 h. The ratio of LC 3 II/I in the cells was then

assessed by Western blot. This figure is representative of five

independent experiments. d Quantifications of the results of the

Western blot on the ratio of LC 3 II/I in Neuro2a cells treated with

NAD? for 6, 24 and 48 h showed that treatment of the cells with

1 mM NAD? significantly increased autophagy in the cells. Data

were collected from five independent experiments. **P \ 0.01

2272 Neurochem Res (2011) 36:2270–2277

123

the initiation step of autophagosome formation [19]. We

used real-time qPCR to assess the mRNA level of Beclin 1

in NAD?-treated Neuro2a cells, and found that both 1 and

10 mM NAD? significantly increased Beclin 1 mRNA

levels to more than 2 folds compared to the control group

in 48 h after the treatment (Fig. 2).

To investigate whether NAD? influences autophagic

flux, the expression level of p62, a long lived scaffolding

protein involved in the transport of ubiquitinylated proteins

destined for proteosomal digestion was evaluated [20].

Consistent with increased autophagic flux following treat-

ment with NAD?, there was a dose-dependent decrease in

the level of p62 by immunoblotting (Fig. 3a, b). Also, to

determine if the NAD?-induced increase in the ratio of LC3

II/LC3 I may result from inhibition of the fusion of auto-

phagosomes with lysosome, we applied 0.1 lM Bafilomy-

cin A1 (BafA1) to block the fusion event. BafA1 is a

vacuolar ATPase inhibitor, which prevents autophagosome-

lysosome fusion by inhibiting the acidification of ATPase in

lysosomes [21]. As shown in Fig. 3c and d, co-treatment of

BafA1 with NAD? on Neuro2a cells for 24 h further

increased the ratio of LC3 II/LC3 I, compared to that in the

cells treated with NAD? alone. This result suggests that

inhibition of the fusion event can still increase the ratio of

LC3 II/LC3 I in cells treated with NAD?, thus arguing

against the possibility that the NAD?-induced increase in

the ratio of LC3 II/LC3 I results from inhibition of the

fusion of autophagosomes with lysosome.

Fig. 2 NAD? treatment dose-dependently increased Beclin 1 expres-

sion as assessed by real-time qPCR assay. Neuro2a cells were treated

with 0.01–10 mM NAD? for 48 h. Beclin 1 expression was then

assessed by real-time qPCR assay. Quantifications of the results

showed that treatment of the cells with 1–10 mM NAD? significantly

increased Beclin 1 expression of the cells. Data were collected from

three independent experiments. **P \ 0.01; *P \ 0.05

Fig. 3 NAD? treatment did not inhibit autophagic flux. a p62 protein

level was analyzed after 48 h of NAD? treatment (0.01–10 mM).

This figure is representative of three independent experiments.

b Quantifications of p62 protein level relevant to b-actin (loading

control) showed that treatment of the cells with 0.1–10 mM NAD?

significantly broke down p62 expression in the cells. Data were

collected from three independent experiments. **P \ 0.01.

c Neuro2a cells were treated with 1 mM NAD? for 24 h, and the

ratio of LC3 II/LC3 I increased. Co-treatment with 0.1 lM Bafilo-

mycin A1 further increased the ratio of LC3 II/LC3 I. This figure is

representative of three independent experiments. d Quantifications of

the results showed that co-treatment of the cells with 1 mM NAD?

and 0.1 lM Bafilomycin A1 significantly increased the ratio of LC3

II/LC3 I compared with 1 mM NAD? alone. Data were collected

from three independent experiments. *P \ 0.05; **P \ 0.01

Neurochem Res (2011) 36:2270–2277 2273

123

In order to identify whether NAD? induces autophagy

by generating its catabolic product, nicotinamide, Neuro2a

cells were treated with 1 or 10 mM nicotinamide for 48 h.

By conducting LC 3 II/I Western blot, we found that nic-

otinamide treatment failed to increase the ratio of LC 3 II/I

in comparison to NAD? treated groups (Fig. 4a, b). The

auto fluorescent compound MDC is commonly used to

stain autophagolysosomes (the fused autophagosome and

lysosome), and MDC localization serves as a positive

marker for autophagolysosomes, and hence is indicative of

autophagy [22]. MDC signals showed no significant dif-

ferences between the nicotinamide treated groups and the

control group, while both 1 and 10 mM NAD? treatment

upregualted the signals to about 3 folds (Fig. 4c, d). Taken

together, these results suggest that NAD? does not induce

autophagy in Neuro2a cells by generating nicotinamide.

Our previous study has suggested NAD? treatment

induced ROS generation in cancer cell lines [8]. To

understand the underlying mechanisms of NAD?-induced

autophagy, we tested whether or not ROS plays a role in

NAD?-induced autophagy in Neuro2a cells. Using DHE

staining assay, we found that treatment of Neuro2a cells

with 1 mM NAD? for 3 h markedly increased the DHE

signal within the cells, indicating increased ROS levels in

the cells (Fig. 5a, b). Moreover, pre-treatment of the cells

with antioxidant N-acetyl cysteine (NAC) at 100 lM

significantly attenuated the NAD?-induced increase in the

ratio of LC 3 II/I (Fig. 5c, d). Another two antioxidants,

Tempo and Trolox, produced similar effects on the LC 3 II/

I ratio (Data not shown). These results have collectively

suggested that oxidative stress contributes to the NAD?

treatment-induced autophagy.

To determine the role of the NAD?-induced autophagy

in Neuro2a cell survival, we determined the influence of

3-methyladenine (3-MA), an autophagy inhibitor [23], on

NAD?-induced decrease in the survival of Neuro2a cells.

We found that treatment with 10 mM NAD? resulted in

decreased survival of the cells, which could be partially

prevented by pre-treatment of the cells with 10 mM 3-MA,

as assessed by LDH assay (Fig. 6). These results suggests

that the NAD?-induced autophagy might contribute to the

NAD?-induced decrease in the survival of Neuro2a cells.

Discussion

The major observations of this study include: First, NAD?

treatment can induce delayed autophagy in Neuro2a cells;

second, oxidative stress contributes to the NAD? treat-

ment-induced autophagy; third, NAD? did not appear

to affect the autophagy in Neuro2a cells by generating

its degradation product, nicotinamide; and fourth, the

Fig. 4 Nicotinamide treatment did not affect the ratio of LC 3 II/I or

MDC staining. a Neuro2a cells were treated with 1–10 mM

nicotinamide or NAD? for 48 h, and the ratio of LC 3 II/I was

accessed by Western blot. This figure is representative of three

independent experiments. b Quantifications of the Western blot

results showed that treatment of the cells with 1–10 mM nicotinamide

did not affect the LC 3 II/I ratio in these cells, while 1–10 mM NAD?

significantly increased the ratio as formerly described. Data were

collected from three independent experiments. *P \ 0.05. c Neuro2a

cells were treated with 1–10 mM NAD? or nicotinamide for 48 h,

and MDC staining was subsequently conducted. This figure is

representative of three independent experiments. Scale bar 40 lm.

d Quantitative values for each treatment group showed that MDC

signals were significantly increased in 1–10 mM NAD? treated

groups, but there were no significant difference between the

nicotinamide treated and control groups. **P \ 0.01

2274 Neurochem Res (2011) 36:2270–2277

123

NAD?-induced autophagy contributes to the NAD?-

induced decrease in cell survival.

NAD? plays important roles in a variety of biological

functions and cell survival [24]. Our previous studies have

indicated that NAD? treatment decreases oxidative stress-

induced death in primary cultures of astrocytes and

neurons, and NAD? administration reduces ischemic brain

damage [9, 25]. Whether NAD? treatment poses any effect

on the survival or the biological functions of cancer cells,

however, has remained largely unexplored. Recently, we

have shown that in a number of cancer cell lines, NAD?

treatment significantly suppresses cell survival, as assessed

by classic measurements of cell injury including LDH

assay, PI staining and Trypan blue assay [8]. NAD? may

become a promising drug for treating cancer, because it

selectively decreases the survival of cancer cells without

damaging the survival of normal cells.

To further determine the effects of NAD? on cancer

cells, the effects of NAD? treatment on the autophagy of

cancer cells were investigated. Autophagy is a highly con-

served catabolic process where cytoplasmic components are

sequestered within double-membrane vesicles known as

autophagosomes, and delivered to lysosomes for degrada-

tion by hydrolases [6]. Reduced activity of autophagy is

observed along the progress of tumors, suggesting a tumor-

suppressing role of autophagy [26, 27]. Our current study,

by applying LC 3 II/I Western blot, real-time qPCR assay

for Beclin-1, and MDC staining, has provided the first

evidence that NAD? can dose-dependently induce autoph-

agy in Neuro2a cells. This result suggests a novel biological

effect of NAD? on cancer cells, which provides an impor-

tant piece of information for understanding the mechanisms

Fig. 5 NAD? treatment induced autophagy in Neuro2a cells by

increasing oxidative stress. a Neuro2a cells were treated with

1–10 mM NAD? for 3 or 6 h, and DHE staining was subsequently

conducted. This figure is representative of three independent exper-

iments. Scale bar 40 lm. b The semiquantitative analysis showed that

10 mM NAD? treated Neuro2a cells for 3 and 6 h markedly

increased DHE levels. **P \ 0.01; *P \ 0.05. c Pre-treatment of

the cells with 50–100 lM NAC prevented the NAD?-induced

increase in the ratio of LC 3 II/I. Neuro2a cells were pre-treated

with 50–100 lM NAC for 1 h, followed by co-treatment with 1 mM

NAD? for 24 h. The ratio of LC 3 II/I in the cells was then assessed

by Western blot. This figure is representative of four independent

experiments. d Quantifications of the Western blot results showed that

NAC treatment significantly attenuated the NAD? treatment-induced

increase in the ratio of LC 3 II/I in the cells. Data were collected from

four independent experiments. **P \ 0.01; *P \ 0.05

Fig. 6 The NAD?-induced autophagy in Neuro2a cells contributed

to the NAD?-induced decrease in cell survival. Neuro2a cells were

pre-treated with 10 mM 3-MA, followed by co-treatment with

1–10 mM NAD? for 24 h. Subsequently cell survival was assessed

by LDH assay. Data were collected from five independent experi-

ments. N = 15. **P \ 0.01

Neurochem Res (2011) 36:2270–2277 2275

123

underlying the effects of NAD? treatment on cancer cell

survival.

Our study has also indicated that oxidative stress medi-

ates the effect of NAD? on the autophagy of the cells: As

shown by the DHE assay, NAD? induces a marked increase

in ROS in the cells; and antioxidants such as NAC signifi-

cantly attenuates the effects of NAD? on the autophagy.

Previous studies have suggested that ROS can induce

autophagy [28]. Thus, it is not surprising that our study has

indicated an important role of oxidative stress in the NAD?-

induced autophagy. Nicotinamide is a major degradation

product of NAD?, which has been shown to decrease cell

death induced by such insults as oxidative stress [29]. Our

study has provided evidence arguing against the possibility

that NAD? induces the autophagy of Neuro2a cells by

producing nicotinamide: In contrast to the significant cell

loss induced by 0.1 mM NAD?, nicotinamide at even

10 mM did not affect autophagy in the cells.

Our study has also shown that inhibition of autophagy

attenuates the effects of NAD? on cell survival, thus sug-

gesting that NAD?-induced autophagy contributes to the

effect of NAD? on cell survival. This result also suggests

that in our experimental conditions autophagy contributes

to the declined survival of cancer cells. These observations

may be valuable for potential applications of NAD? for

treating cancer.

While NAD? increases autophagy in Neuro2a cells par-

tially by generating oxidative stress, it remains unclear how

NAD? treatment may lead to increased oxidative stress. It is

possible that NAD? might produce this effect by interacting

with certain receptors on the plasma membranes of the cells.

Future studies are warranted to further investigate the

mechanisms underlying the effects of NAD? on the

autophagy in cancer cells, and to determine if NAD? might

affect autophage in cancer cells in vivo.

Acknowledgments This study was supported by a Pujiang Scholar

Program Award 09PJ1405900 (to WY), a National Key Basic Research

‘973 Program’ Grant #2010CB834306 (to WY and WX), a Research

Grant of Shanghai Jiao Tong University for Interdisciplinary Research on

Engineering and Medicine (to WY), a Key Research Grant of Shanghai

Jiao Tong University for Interdisciplinary Research on Engineering

and Physical Sciences (to WY), grants to W X from NNSF China

(30900756), ‘‘Rising Star’’ Grant from Science and Technology

Commission of Shanghai (09QA1403400), a start-up grant from Min-

istry of Education China for returnees (K10MD06), and SJTU funding

(YG2009MS55) and SJTU SMC Morning Star program.

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