3-Nitro-naphthalimide and nitrogen mustard conjugate NNM-25 induces hepatocellular carcinoma apoptosis via PARP-1/p53 pathway

Article abstract of DOI:10.1007/s10495-012-0712-7

Hepatocellular carcinoma (HCC) is one of the main causes of death in cancer. Some naphthalimide derivatives exert high anti-proliferative effects on HCC. In this study, it is confirmed that 3-nitro-naphthalimide and nitrogen mustard conjugate (NNM-25), a novel compound conjugated by NNM-25, displayed more potent therapeutic action on HCC, both in vivo and in vitro, than amonafide, a naphthalimide drug in clinical trials. More importantly, preliminary toxicological evaluation also supported that NNM-25 exhibited less systemic toxicity than amonafide at the therapeutic dose. The antitumor mechanism of conjugates of naphthalimides with nitrogen mustard remains poorly understood up to now. Here, we first reported that apoptosis might be the terminal fate of cancer cells treated with NNM-25. Inhibition of p53 by siRNA resulted in a significant decrease of NNM-25-induced apoptosis, which corroborated that p53 played a vital role in the cell apoptosis triggered by NNM-25. NNM-25 inhibited the PARP-1 activity, AKT phosphorylation, up-regulated the protein expression of p53, Bad, and mTOR as well as down-regulating the protein expression of Bcl-2 and decreasing mitochondrial membrane potential. It also facilitated cytochrome c release from mitochondria to cytoplasm, activated caspase 8, caspase 9, and caspase 3 in HepG2 cells in vitro, as also authenticated in H22 tumor-bearing mice in vivo. Collectively, the conjugation of naphthalimides with nitrogen mustard provides favorable biological activity and thus is a valuable strategy for future drug design in HCC therapy.

Full text of DOI:10.1007/s10495-012-0712-7

Apoptosis (2012) 17:725–734
DOI 10.1007/s10495-012-0712-7
ORIGINAL PAPER
3-Nitro-naphthalimide and nitrogen mustard conjugate NNM-25
induces hepatocellular carcinoma apoptosis via PARP-1/p53
pathway
•
Song-qiang Xie Ya-hong Zhang Qian Li
•
•
•
•
•
Feng-hua Xu Jiu-wang Miao Jin Zhao
Chao-jie Wang
Published online: 6 March 2012
Ó Springer Science+Business Media, LLC 2012
Abstract Hepatocellular carcinoma (HCC) is one of the
main causes of death in cancer. Some naphthalimide
derivatives exert high anti-proliferative effects on HCC. In
this study, it is confirmed that 3-nitro-naphthalimide and
nitrogen mustard conjugate (NNM-25), a novel compound
conjugated by NNM-25, displayed more potent therapeutic
action on HCC, both in vivo and in vitro, than amonafide, a
naphthalimide drug in clinical trials. More importantly,
preliminary toxicological evaluation also supported that
NNM-25 exhibited less systemic toxicity than amonafide at
the therapeutic dose. The antitumor mechanism of conju-
gates of naphthalimides with nitrogen mustard remains
poorly understood up to now. Here, we first reported that
apoptosis might be the terminal fate of cancer cells treated
with NNM-25. Inhibition of p53 by siRNA resulted in a
significant decrease of NNM-25-induced apoptosis, which
corroborated that p53 played a vital role in the cell apop-
tosis triggered by NNM-25. NNM-25 inhibited the PARP-1
activity, AKT phosphorylation, up-regulated the protein
expression of p53, Bad, and mTOR as well as down-reg-
ulating the protein expression of Bcl-2 and decreasing
mitochondrial membrane potential. It also facilitated
cytochrome c release from mitochondria to cytoplasm,
activated caspase 8, caspase 9, and caspase 3 in HepG2
cells in vitro, as also authenticated in H22 tumor-bearing
mice in vivo. Collectively, the conjugation of naphthali-
mides with nitrogen mustard provides favorable biological
activity and thus is a valuable strategy for future drug
design in HCC therapy.
Keywords Hepatocellular carcinoma Á PARP-1 Á
Naphthalimide Á Alkylating agent Á Apoptosis
Introduction
Hepatocellular carcinoma (HCC) is the sixth most common
cancer in terms of incidence and the third most common
cause of death in cancer in the world [1]. The therapeutic
success of chemotherapeutic drugs is often limited by
severe adverse effects. Therefore, it is essential to develop
novel chemotherapeutic agents, which exert greater effi-
cacy to HCC with none or restricted toxicity to normal cells
[2]. Indeed, naphthalimides have shown potent anticancer
activity against a variety of murine and human cancer cell
lines, some of which, such as amonafide and mitonafide,
have been evaluated in clinical trials. However, most of
tested naphthalimides have been abandoned because of
poor therapeutic index and hematotoxicity [3]. In recent
years, we found that some naphthalimide derivatives dis-
played potent inhibitive effects on HCC. For example, the
naphthalimides conjugated with polyamines have been
designed to improve their efficacy against HCC with
reduced adverse effects [4, 5]. Previous report demon-
strated that the conjugates of naphthalimide and alkylating
agent were more effective than their respective naphthali-
mide and alkylating agent in S-180 and Ehrlich ascites
carcinoma models [6]. However, the detailed molecular
mechanism of their antitumor activity remains dimness up
to now.
S. Xie Á J. Miao
Institute of Chemical Biology, Henan University,
Kaifeng 475004, China
S. Xie Á Y. Zhang Á Q. Li Á F. Xu Á J. Miao Á J. Zhao Á
C. Wang (&)
The Key Laboratory of Natural Medicine and
Immuno-Engineering, Henan University, Kaifeng 475004, China
e-mail: wcjsxq@henu.edu.cn
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4-Amino-1,8-naphthalimide (4-AN) exerts potent anti-
from Beyotime (Shanghai, China). Primary antibodies
against: Bcl-2, Bad, caspase 9, caspase 3, caspase 8, PARP-
1, cytochrome c, p53, p-AKT, p-mTOR, as well as horse-
radish peroxidase-conjugated anti-mouse and anti-rabbit
secondary antibodies were all obtained from Santa Cruz
Biotechnologies (Santa Cruz, CA, USA). All other chem-
icals used in the experiments were commercial products of
reagent grade.
proliferative effects via poly(ADP-ribose) polymerase 1
(PARP-1) inhibition [7]. PARP-1 plays a pivotal role in
DNA damage and repair, post-translational modification,
etc. [8]. Several drugs, which were designed to inhibit
PARP-1 activity, are currently in clinical development [9].
Furthermore, the drug combination of PARP-1 inhibitors
and alkylating agents is also currently being investigated in
clinical trials for cancer therapy [10].
On account of these merits of naphthalimides and
nitrogen mustards, a series of conjugates were synthesized
as potential HCC therapeutic agents in our laboratory.
The synthetic route of 3-nitro-naphthalimide
and nitrogen mustard conjugate (NNM-25)
Tumor studies in vivo
After screening, we found that 3-nitro-naphthalimide and
nitrogen mustard (NNM-25), a novel conjugate of 3-nitro-
naphthalimide and nitrogen mustard, exerted favorable
anti-proliferative activity in hepatoma cells. The purpose of
the present study includes the evaluation of HCC inhibition
in vivo and in vitro, the preliminary systemic toxicity,
and the potential molecular mechanism of NNM-25. As
reported herein, our results indicated that NNM-25 could
potently inhibit HCC growth in vivo and in vitro via
apoptosis. Encouragingly, preliminary toxicological eval-
uation suggested that NNM-25 had no obvious systemic
toxicity at the therapeutic dose.
Kunming male mice (6–8 weeks old) were purchased from
the laboratorial animal center of Henan (Zhengzhou, China).
All animal procedures were performed following the pro-
tocol approved by the Institutional Animal Care and Use
Committee of Henan University. For solid tumor model, the
mice were injected sub-cutaneously in the right flank with
0.2 mL of cell suspension containing 5 9 107 of H22 cells.
Tumors were allowed to grow for approximate 7 days
before drug treatment. Tumor-bearing mice were randomly
assigned to one of the following three treatment groups
(n = 10 mice per group): control (physiologic saline),
NNM-25 (1.5 mg/kg) and amonafide (5 mg/kg). NNM-25
(purity 99.7%) was synthesized in our laboratory and the
structure was shown in Fig. 1a. The doses were chosen
according to their acute toxicity (1/2 LD10). For ascites
tumor model, the mice were injected in the abdominal cavity
with 0.2 mL of cell suspension containing 5 9 107 of H22
cells. Next day, the tumor-bearing mice were randomly
assigned to one of the following three treatment groups
(n = 20 mice per group): control (physiologic saline),
NNM-25 (1.5 mg/kg) and amonafide (5 mg/kg). Drug
treatments were administered through tail vein injection for
7 consecutive days. For the solid tumor study, the mice
were killed by aether anesthesia after 24 h of drug with-
drawal. Solid tumors were removed and weighed. The
inhibitory rate was calculated as follows: inhibitory rate
(%) = [(weightcontrol - weightdrug)/weightcontrol] 9 100.
After being weighted, the tumors were fixed in formalin and
stained with hematoxylin and eosin. Immunohistochemistry
(IHC) analysis was used to detect PARP-1 protein. Rabbit
anti-mouse PARP-1 polyclonal antibody (1:200 dilution)
Materials and methods
Cell lines and reagents
Three well-characterized human liver cancer cell lines
(HepG2, SMMC7721, and BEL7402), one mice liver
cancer cell line (H22), and one human liver non-tumoral
cell line (QSG7701) were maintained as an adherent
monolayer in RPMI-1640 medium (Gibco) supplemented
with 10% fetal bovine serum (20% for QSG7701
cells), 100 U/mL penicillin, and 100 lg/mL streptomy-
cin (Sigma) and incubated at 37°C in 5% CO2. RNase
A, rhodamine-123 (Rh-123), 3-(4,5-dimethylthiazol)-2,
5-diphenyltetrazolium bromide (MTT), Hoechst33342, and
propidium iodide (PI) were purchased from Sigma
(St. Louis, MO, USA). RPMI 1640 and fetal calf serum (FCS)
were purchased from Gibco (Grand Island, NY, USA).
Annexin V-FITC apoptosis detection kit was purchased
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Fig. 1 Antitumor activity and systemic toxicity of NNM-25 were
evaluated in vivo. a The chemical structure of NNM-25. b Mean
tumor weight after treated with NNM-25. c Specimen of sub-
cutaneously H22 xenograft tumor after treated with NNM-25 at the
end of the experiment. d Survival time of bearing H22 ascitic mice
treated with NNM-25, n = 10. e The weight change after treatment
with NNM-25. f Platelet, RBC, and WBC numbers were detected
after treatment NNM-25. g, h The organ index was evaluated after
NNM-25 treatment. Compared with control *p \ 0.05, **p \ 0.01,
n = 10
was used in standard indirect immunoperoxidase procedures
[11]. For each treatment group in ascites model, ten mice
were used for mean survival time (MST) analysis. The
extended lifespan rate was calculated as follows: extend rate
(%) = (MSTdrug/MSTcontrol) 9 100 [12]. The remaining
ten mice in each treatment group were used for apoptotic
mechanism study. The ascites of H22-bearing mice were
collected for apoptosis, MMP, intracellular fluorescence
intensity and western blotting assay.
precipitate as previously described [13]. Briefly, cells were
seeded into 96-well plates at 5 9 103 cells/well. After 12 h,
various concentrations (0.1, 1, 10 30, and 50 lM) of NNM-25
were subsequently added and incubated for 48 h and then
clearage by dimethyl sulfoxide. The inhibitory rate was cal-
culated from plotted results using untreated cells as 100%.
DNA binding assay
Solutions of ct DNA in 20 mM Tris–HCl buffer (pH 7.4)
gave a ratio of UV absorbance at 260 and 280 nm of 1.81:1,
indicating that the DNA was sufficiently free of protein. The
concentration of ct DNA was determined spectrophotomet-
rically assuming that the molar absorption is 6,600 M/cm
(260 nm). NNM-25 was then added into the mixture of ct
DNA and ethidium bromide (EB), and the final concentra-
tions of NNM-25 were 1, 2, 3, and 4 lM, respectively. The
final concentrations of DNA and EB were 11.3 and 47.8 lM,
respectively. All the above solutions were shaken for 30 min
at room temperature. Fluorescence wavelengths and inten-
sity areas of samples were measured at following conditions:
excitation 356 nm, emission 595 nm [4].
Systemic toxicity evaluation
To study the toxic effects of NNM-25, as indicated by loss of
body weight, food consumption, hematology, organ weight
and macroscopic evaluation, 30 mice without tumors were
randomly assigned to one of three treatment groups (n = 10
mice per group): control (physiologic saline), NNM-25
(1.5 mg/kg) and amonafide (5 mg/kg). The weight of mice
was recorded daily before tail vein injection. The experiment
was ended on day 8. Blood and bone marrow were collected,
and hematological profiling was undertaken by determining
platelet, red blood cell (RBC), white blood cell (WBC) and
bone marrow cells counts using a Coulter Counter T-890
(Coulter Electronics, USA). The organ index was investi-
gated for systemic toxicity evaluation. Organ index
(%) = (Organ weight/body weight) 9 100.
PARP-1 activity assay in cell-free system
Poly(ADP-ribose) polymerase 1 activity was assessed using a
commercially available kit (Trevigen, Gaithersburg, MD),
based on the measurement of radiolabeled NAD? incorpo-
ration. The assay was performed using purified PARP-1
protein, untreated or in the presence of graded concentrations
of NNM-25. The enzymatic reaction was carried out by
Cell proliferative assay in vitro
The anti-proliferative ability of NNM-25 was evaluated in
HepG2 cells, BEL7402 cells, SMMC7721 cells, H22 cells, and
QSG7701cells bythe conversion of MTT to a purple formazan
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incubating purified PARP-1 with 2 lCi 32P-NAD? (Amer-
sham, Milan, Italy), 10 lM NAD?, 1 9 PARP-1 buffer,
10 lg of nuclease-treated salmon testes DNA, 10 lg of his-
tones, according to the manufacturer’s instructions [14].
counterstained with Hoechst 33342 (1 lM) for 15 min at
dark. The change of MMP was analyzed by HCS [13].
Western blotting
After treatment, HepG2 cells and H22 cells were harvested
and washed with PBS. Cytosolic and mitochondrial fractions
were prepared as previously described [13]. Total cellular
protein was isolated using the protein extraction buffer (con-
taining 150 mM NaCl, 10 mM Tris (pH 7.2), 5 mM EDTA,
0.1% Triton X-100, 5% glycerol, and 2% SDS). Protein
concentration was determined using the protein assay kit
(Biyuntian, China). Equal amounts of proteins (50 lg/lane)
were fractionated using 8 or 12% SDS-PAGE and transferred
to PVDF membranes. The membranes were incubated with
primary antibodies. After washing with PBS, the membranes
were incubated with corresponding peroxidase-conjugated
goat anti-mouse or anti-rabbit secondary antibody, followed
by enhanced chemiluminescence staining through the
enhanced chemiluminescence system. b-Actin (45 kDa,
cytosolic protein) was used to normalize for protein loading.
Intracellular fluorescence intensity assay
The intracellular fluorescence intensity of NNM-25 was
detected by high content screening (HCS) analysis
(Thermo Scientific Cellomics ArrayScan Vti, Cellomics,
Pittsburgh, PA). Briefly, after cells were seeded into
96-well plates at 5,000 cells/well for 24 h, the cells were
stained with acridine orange (AO) (50 lg/mL) for 15 min,
then washed three times with PBS. NNM-25 (1.0, 10 or
30 lM) was added into 96-well plate and then the intra-
cellular fluorescence intensity of NNM-25 for 30 min was
detected using HCS. For H22 cells, which were collected
from the ascites of mice bearing H22 cells after treatment
with NNM-25, after AO staining, the intracellular fluo-
rescence intensity of NNM-25 was detected using HCS.
Cell numbers were determined by AO staining and intra-
cellular fluorescence intensity of NNM-25 was detected at
excitation 350 nm and emission 460 nm, AO was detected
at excitation 492 nm and emission 526 nm [13].
siRNA studies
HepG2 cells were seeded into 6-well plates at a density of
5 9 105 cells per well in RPMI-1640 medium supplemented
with 10% fetal bovine serum and grown to 90% confluent
overnight before transfection control siRNA (100 nM, CST
6568, Cell Signalling Technology) or p53 siRNA (100 nM,
CST 6562, Cell Signalling Technology) was transfected
using lipofectamine 2000 (Invitrogen) in 2 ml serum-free
medium per well according to the manufacturer’s instruc-
tions. Knock-down of p53 expression was confirmed by
western blot after 72 h transfection. For apoptotic evalua-
tion, the p53 siRNA-transfected HepG2 cells were co-incu-
bated with NNM-25 or amonafide for another 48 h, and then
cell apoptosis was evaluated by Annexin V-FITC apoptosis
detection kit by FACScan (Becton–Dickinson, USA).
Cellular apoptotic evaluation
Cell apoptosis was evaluated by Annexin V-FITC apop-
tosis detection kit using FACScan (Becton–Dickinson,
USA). Briefly, HepG2 cells were seeded in 6-well plates,
and exposed to NNM-25 for 48 h, then harvested and
stained according to manufacturer’s protocol. For H22
cells, on day 8 after treatment with NNM-25, amonafide or
physiologic saline, the H22 cells were extracted from
abdominal cavity of H22-bearing mice and then washed
with PBS for apoptosis, mitochondrial membrane potential
(MMP) and western blotting assay. Data acquisition and
analysis were controlled by CellQuest software [13]. In
addition, cell apoptosis was also detected by AO/EB dou-
ble staining. Briefly, after collected, cells were seeded
into 6-well plates and stained with AO (0.15 lM)/EB
(0.15 lM) for 30 min. After washing three times with PBS,
the fluorescent micrographs were taken using HCS.
Data analysis
All data are presented as mean SD and analyzed using
Students t test or analysis of variance (ANOVA) followed
by q test.
Measurement of mitochondrial membrane potential
Results
Mitochondrial membrane potential was assessed by the
retention of Rh-123, a membrane-permeable fluorescent
cationic dye. The uptake of Rh-123 by mitochondria is
proportional to the MMP. Briefly, cells were incubated
with Rh-123 (0.1 lg/mL) in the dark at room temperature
for 20 min. After being washed with PBS, cells were
Antitumor effect and systemic toxicity of NNM-25
in vivo
3-Nitro-naphthalimide and nitrogen mustard conjugate
significantly suppressed tumor growth in comparison to
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Apoptosis (2012) 17:725–734
729
control mice in solid tumor model (Fig. 1b, c). The tumor
inhibitory ratio was 40.3% (1.41 0.43) g for amonafide
and 68.6% (0.74 0.38) g for NNM-25, as compared to
control mice (2.36 0.52) g, respectively. The MST of the
control mice was (12.9 3.2) days in ascites model. After
treatment with NNM-25 or amonafide, the lifespan was
increased by 2.2-fold (28.5 6.5) days and 1.6-fold
(20.6 4.3) days, respectively (Fig. 1d).
brain, lung, and spleen was observed for both tested drugs
and control (Fig. 1h).
Anti-proliferative effects and tumor cell selectivity
of NNM-25
Previous data demonstrated that some conjugates of
nitrogen mustard and naphthalimide, such as napromustine,
only exerted potent antitumor effect in vivo but not in vitro
[6]. So we further evaluated whether NNM-25 exerted anti-
proliferative activity in vitro. Four HCC cell lines (HepG2,
Bel-7402, SMMC7721, and H22) and one normal hepato-
cyte line (QSG7701) were selected to assess the anti-pro-
liferative activity and tumor selectivity of NNM-25. The
IC50 value after 48 h treatment was (130.2 8.4),
(1.9 0.4), (2.8 0.9), (2.5 0.7) and (3.4 0.9) lM
for QSG7701 cells, HepG2 cells, SMMC7721 cells, H22
cells and Bel-7402 cells, respectively. This result implied
that NNM-25 exerted HCC selectivity (Fig. 2a). Accord-
ingly, the intracellular fluorescence intensity of NNM-25
was higher in liver cancer cells than in normal hepatocyte
(Fig. 2b). Similarly, NNM-25 was also efficiently uptake
by H22 cells in vivo (Fig. 2c). These results suggested that
the selectivity was nicely correlated with the intracellular
contents of NNM-25.
We did not observe weight loss in the NNM-25-treated
mice, whereas treatment with 5 mg/kg amonafide was
associated with weight loss, indicating the dose toxicity to
some extent (Fig. 1e). Moreover, we found that the food
consumption of tested mice was decreased after treatment
with amonafide, while NNM-25 treatment had no obvious
effect on the food consumption of mice in contrast with
control (data not shown). In addition, amonafide resulted in
a significant decrease in platelet, RBC, and WBC numbers
compared to control, while NNM-25 did not. At the same
time, NNM-25 had no obvious influence on bone marrow
cell numbers (Fig. 1f), suggesting that NNM-25 possessed
no evident hematotoxicity in this test. Additively, amona-
fide down-regulated spleen and thymus indexes more
potently than NNM-25, indicating that NNM-25 had no
influence on immune function but amonafide inhibited
immune function apparently (Fig. 1g). Finally, no signifi-
cant difference in the organic factors of heart, liver, kidney,
Fig. 2 The growth inhibitory effects, intracellular fluorescence
intensity, DNA binding and PARP-1 inhibitory activity were evalu-
ated after treatment with NNM-25. a Growth inhibitory effect of
NNM-25 after 48 h treatment. Each point represents the means SD
from four independent experiments. b The intracellular fluorescence
intensity of NNM-25 was detected by HCS after treatment with
NNM-25 for 30 min. Each point represents the means SD from
*p \ 0.05, **p \ 0.01, n = 10. d Fluorescence spectra of EB with
herring sperm DNA. Numbers 0–4 indicated the NNM-25 concentra-
tion: 0, 1, 2, 3, and 4 lM. e Activity of isolated, recombinant PARP-1
was inhibited by NNM-25. Each point represents the means SD
from four independent experiments. f Immunohistochemistry analysis
was used to detect PARP-1 protein expression in xenografts tumor
(Up, 4009), histological examination and morphological analysis of
tumoral xenografts (Down, H&E, 400 9). g The protein expression of
PARP-1 was detected by western blotting in HepG2 cell in vitro and
H22 cell in vivo
four independent experiments.
c The intracellular fluorescence
intensity of NNM-25 was detected by HCS after treatment with 7
consecutive days in H22 tumor-bearing mice. Compared with control
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DNA intercalating and PARP-1 inhibitive activity
of NNM-25
expression of PARP-1 was also significantly down-regu-
lated in H22 cells in vivo and in HepG2 cells in vitro
(Fig. 2g).
Previous studies demonstrated that naphthalimides exert
potent DNA intercalating properties and are DNA-dam-
aging agents [3]. To verify the hypothesis that NNM-25 is
also a DNA intercalating agent, we tested its DNA inter-
calating ability using DNA-EB assay. In a cell-free system,
the fluorescence intensity of DNA-EB was gradually
attenuated accompanied by the increasing concentration of
NNM-25 (Fig. 2d), indicating that NNM-25 was a more
powerful DNA intercalating agent than EB.
Cell apoptosis and related protein expression in vivo
and in vitro
Poly(ADP-ribose) polymerase 1 plays a critical role in
DNA damage and repair processes [8]. As NNM-25 exer-
ted potent DNA intercalating and PARP-1 inhibiting
activity, we further detected whether NNM-25 could
induce cell apoptosis. Compared with control cells, the
percentage of annexin V-positive cells was increased after
treatment with NNM-25 in HepG2 cells in vitro and in H22
cells in vivo (Fig. 3a). This apoptotic effect was also
authenticated by AO/EB staining (Fig. 3b). These data
suggested that NNM-25 could induce liver cancer cells
apoptosis in vitro and in vivo.
Some naphthalimides, such as 4-AN, exert potent anti-
tumor effect via inhibiting PARP-1 activity [8]. To directly
assess the inhibitory effect of NNM-25 on PARP-1 activ-
ity, we detected the activity of PARP-1 using recombinant
PARP-1 in a cell-free assay. As expected, PARP-1 activity
was markedly reduced by NNM-25 in a dose-dependent
manner (Fig. 2e). To provide evidences that the inhibition
of tumor growth by NNM-25 was related to PARP-1 in
vivo, a special study was accomplished to analyze PARP-1
in tumor tissues by IHC analysis. PARP-1 protein expres-
sion was evidently reduced after NNM-25 treatment
(Fig. 2f, up). Histological examination demonstrated that
the tumor cells were obviously decreased after treatment
with NNM-25 (Fig. 2f, down). Furthermore, the protein
Cell apoptosis is a complicated dead phenomenon and
many signal pathways are involved in this process. To
elucidate the molecular mechanism of NNM-25-mediated
cell apoptosis, we first detected the mitochondrial apoptotic
pathway. The MMP of HepG2 cells and H22 cells was
decreased significantly after treatment with NNM-25 in
vitro and in vivo (Fig. 3c, d), and accordingly facilitated
cytochrome c release from mitochondria to cytoplasm,
Fig. 3 The apoptosis and MMP were investigated in HepG2 cells in
vitro and H22 cells in vivo. a The apoptotic ratio was detected using
FACScan in HepG2 cells and H22 cells. Compared with control
*p \ 0.05, **p \ 0.01. b The cellular morphous was detected with
AO/EB staining using HCS in HepG2 cells and H22 cells (4009). c,
d The MMP was detected with Rh-123/Hoechst33342 staining using
HCS in HepG2 cells in vitro and H22 cells in vivo
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731
Fig. 4 The protein expression
was detected by western
blotting in HepG2 cells after
48 h treatment in vitro and H22
cells after 7 consecutive day
treatment in vivo. a The protein
expression was detected by
western blotting in HepG2 cells
in vitro. b The protein
expression was detected by
western blotting in H22 cells
after 7 consecutive days
treatment in vivo
respectively (Fig. 4a, b). Furthermore, the protein expres-
sion of Bad, p53, p-mTOR was up-regulated after
treatment with NNM-25. NNM-25 also induced the down-
regulation of Bcl-2 and p-AKT protein expression (Fig. 4a,
b). These results indicated that mitochondria and Bcl-2
were involved in NNM-25-mediated cell apoptosis in
HepG2 cells and H22 cells in vitro and in vivo. NNM-25
also activated caspase 8, caspase 9, and caspase 3, sug-
gesting that the proteins of caspase family were involved in
NNM-25-mediated cell apoptosis.
treatment (Fig. 5b). Those data demonstrated that the
NNM-25 and amonafide-induced apoptotic mechanism is
not identical.
Discussion
The combinative administration of PARP-1 inhibitors and
alkylating agents has been proved to have synergistic
antitumor activities in many preclinical and clinical studies
[10]. Indeed, previous work [6] and present data of NNM-
25 confirmed that the conjugate of naphthalimides with
alkylating agent can also display better antitumor activity
than amonafide or mitonafide, naphthalimide derivatives
without nitrogen mustard, in the trials of the S-180 and
HCC tumoral xenograft growth inhibition and the life span
in vivo. Interestingly, the antitumor effect of NNM-25 is
obviously different from that of napromustine in vitro,
another conjugate of nitrogen mustard and naphthalimide.
NNM-25 is potent in tumor growth inhibition both in vivo
and in vitro, whereas napromustine is only effective in vivo
but not in vitro in the screen of several human cell lines [6].
Although amonafide is effective in clinical trials, it also
suffers from the dose-limited bone marrow toxicity [16].
Encouragingly, preliminary toxicological evaluation dem-
onstrated that NNM-25 had improved indices in terms of
systemic toxicity, especially hematotoxicity at the thera-
peutic dose. In addition, the observed minor body weight
gains of the mice treated with NNM-25, contrary to weight
losses by amonafide treatment compared with the control,
p53 is necessary for NNM-25-induced apoptosis
p53 has a major function in transducing stress to the
apoptotic machinery of the cell, consistent with the
important p53 status as a determinant of cellular response
to DNA-damaging drug [15]. To examine the role of p53 in
the control of NNM-25-induced apoptosis, we selected a
specific p53 small interference RNA (siRNA) to block p53
translation. Following the administration of this p53 siRNA
for 72 h, the protein expression of p53 was almost com-
pletely inhibited (Fig. 5a). Next, we examined whether the
p53 siRNA affects apoptosis. HepG2 cells were incubated
with p53 siRNA (100 nM for 72 h) prior to NNM-25 or
amonafide treatment, cell apoptosis was then assessed by
flow cytometry. Inhibition of p53 with the specific siRNA
led to a significant drop in apoptotic ratio after NNM-25
treatment, suggesting that p53 is necessary for NNM-
25-induced HepG2 cell apoptosis. However, the apoptotic
ratio was only moderately decreased after amonafide
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Fig. 5 p53 siRNA inhibited the protein expression of p53 and NNM-25-mediated apoptosis in HepG2 cells. a The protein expression was
detected by western blotting in HepG2 cells in vitro. b The apoptotic ratio was detected using FACScan in HepG2 cells
indicated that NNM-25 displayed activity without obvious
dose-limited toxicity. Although amonafide resulted in the
less food intake of tested mice, the difference in weight
variations might be also illustrated by the better cancerous
and normal cell selectivity of NNM-25 than that of
amonafide.
We previously found that AKT/mTOR signaling path-
way is involved in the naphthalimides, such as NPC-16,
induced apoptosis [13]. As expected, NNM-25 also inhib-
ited AKT phosphorylation and promoted mTOR phos-
phorylation, and caspases 3, 8, and 9 were involved in this
pathway. However, it seems that the differences between
NNM-25 and amonafide on p-AKT are not proportional to
that of apoptotic content, especially for H22 cells. Since
naphthalimides and alkylating agents can target DNA, it is
rational to speculate that DNA might be at least one of the
molecular targets of NNM-25. Unsurprisingly, NNM-25
could displace EB, one known DNA intercalator, from
DNA-EB complex in the fluorescence experiments as
previously reported [4]. This result is also in consistent
with that of naphthalmustine [17].
As far as we know, the apoptosis produced by the
conjugate of naphthalimides with alkylating agents, and
the related apoptotic mechanism are not reported, which
are important to elicit how the conjugation between
naphthalimides and alkylating agents results in the syner-
gistic cell killing effect on tumors. Indeed, obvious apop-
tosis is observed for both NNM-25 and amonafide.
Interestingly, NNM-25 with better bioactivity and less
toxicity triggered more potent apoptosis than amonafide. It
is therefore necessary to establish the relevant apoptotic
mechanism.
Poly(ADP-ribose) polymerase 1 is a nuclear enzyme
that plays a critical role in DNA damage response
123

Apoptosis (2012) 17:725–734
733
processes. PARP-1 inhibition has been successfully
employed as a novel therapeutic strategy to enhance the
cytotoxic effects of DNA-damaging agents [18]. Because
PARP-1 inhibitor can enhance the antitumor effect of
alkylating agents and 4-AN is a PARP-1 inhibitor [19], so
we supposed that the encouraging biological results of
NNM-25, with the similar naphthalimide segment to 4-AN,
are also related to its PARP-1 inhibitive capacity. Thus, the
PARP-1 protein expression and activity were detected
using a cell-free assay, and IHC analysis, western blotting
in vivo and in vitro. Our results demonstrated that NNM-25
significantly inhibited PARP-1 protein expression in H22
xenograft tumor and ascites H22 cell in vivo, and also
inhibited PARP-1 activity in a cell-free system in vitro. In
addition, NNM-25 inhibited the activity and protein
expression of PARP-1 more effectively than the control
drug amonafide, which is in accordance with the more
potent inhibition of NNM-25 against HCC in vitro and in
vivo than amonafide. Huang et al. [20] discovered that
PJ34, an inhibitor of PARP-1, exerted suppressive HepG2
cell growth in vivo and in vitro, and also induced cell
apoptosis via caspase pathways. The observed H22 cell
apoptosis in vivo and HepG2 cell in vitro significantly
induced by NNM-25 are in accordance with above reports.
DNA damage-triggered signaling and execution of
apoptosis depend on the p53 status, death-receptor
responsiveness, and most importantly, DNA repair capacity
[21]. p53 could repair the impaired DNA to protect cells
from DNA damage, and also facilitate cell apoptosis once
the DNA damage could not be soundly repaired [22]. In
addition, p53 participates directly in the intrinsic apoptosis
pathway by interacting with the multidomain members of
the Bcl-2 family to induce mitochondrial outer membrane
permeabilization [23]. Bcl-2 family proteins are also
often involved in mitochondria-mediated apoptosis [13].
Accordingly, we turned our attention to Bad and Bcl-2,
which are two classical pro-apoptotic and anti-apoptotic
Bcl-2 family members, respectively. Our results demon-
strated that NNM-25-mediated apoptosis was associated
with these facilitative and inhibitive apoptotic factors.
Indeed, NNM-25 increased the expression of p53 and Bad,
decreased the expression of Bcl-2, degraded MMP and
promoted cytochrome c release from mitochondria to
cytoplasm, and activated caspase-3, -8, and -9. Further-
more, inhibition of p53 by specific siRNA led to a signif-
icant decrease in apoptotic ratio after NNM-25 treatment,
suggesting that p53 is a vital factor for NNM-25-induced
HepG2 cell apoptosis. However, the comparatively mild
effect of p53 siRNA on amonafide triggered apoptosis
indicated that the introduction of alkylating agents to
naphthalimides did change the apoptotic mechanism.
Collectively, this study showed that NNM-25 exerted
potent effects on HCC in vivo and in vitro compared to
amonafide. At the same time, the improved systemic tox-
icity at the therapeutic dose added more evidences that the
conjugation of naphthalimides with nitrogen mustards
provides favorable biological activity and thus is a valuable
strategy for future drug discovery in HCC therapy. To our
knowledge, the NNM-25 induced HCC cell apoptosis,
which is different from that of amonafide, is first reported.
And the present results help to establish a complex sig-
naling pathway of apoptosis involving AKT/mTOR and
PPAR-1/p53, which gives a clue for further exploration of
the detailed molecular mechanism of conjugates formed by
naphthalimides with nitrogen mustards.
Acknowledgments This study was funded by the National Natural
Science Foundation of China (No. 90913001) for Chao-jie Wang,
Projects of Science and Technology of Henan (No. 0821022700;
102300410095) and China Postdoctoral Science Foundation Funded
Project (No. 20090450092; 201003395) for Song-qiang Xie.
Conflict of interest None.
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