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Introduction
Despite advances in cancer treatment in the past 2
decades, the prognosis of lung cancer patients has improved
minimally. Lung cancer is the leading cancer killer in both
men and women. There was an estimated 171 900 new cases
of lung cancer and an estimated 157 200 deaths from lung
cancer in the Unite States in 2003[1]. There are 2 types of
lung cancer cells, small cell lung cancer and non-small cell
lung cancer. Non-small cell lung cancer (NSCLC)
heterogeneously aggregates at least 3 distinct histological lung
cancers, including epidermoid or squamous carcinoma,
adenocarcinoma, and large cell carcinoma. NSCLC is the
most common histological cell type of all lung cancers, and
there is no curative treatment available for the advanced
stages of these diseases. Although chemotherapy is an
esta-blished treatment for advance NSCLC, it offers only a limited
survival benefit at the expense of substantial toxicity, drug
resistance, and poor target specificity. Thus, there is a need
for innovative strategies that effectively treat patients and
exhibit more favorable safety profiles in advanced NSCLC.
The ideal bioreductive drug should be administered as
an inactive prodrug that is only activated under low-oxygen
conditions by 1 or 2 electron
reductases[2_4]. Aziridine-substituted benzoquinone, such as mitomycin C, RH1, and
tirapazamine (TPZ), are 3 principal aziridinyl quinone classes
of hypoxia-specific cytotoxins that are being developed for
clinical use[5_7]. In the case of di-aziridinyl-substituted
quinone, this highly cytotoxic, bifunctional, alkylating agent
can cross-link DNA in cells which results in inducing
complicated cellular mechanisms leading to cell death by
apoptosis such as TPZ and CI-1010 or
necrosis[8,9].
Tumor tissue had lower oxidative reduction (redox)
potential relative to most normal tissues which could increase
the reductive activation of these quinone derivatives in
tumors[2]. Therefore, the selectivity of bioreductive drugs is
governed not only by the difference in oxygen tension
between tumors and normal tissues, but also by levels of
enzymes catalyzing bioreductive activation such as
DT-diaphorase[3,10_11]. This fact led to the 1990 publication of the
concept of "enzyme-directed bioreductive development" by
Workman and Walton[12].
In this study, one of these bis-aziridinylnaphthoquinone
analogues, designated as AZ4 (Figure 1) was found to
inhibit NSCLC cell growth in vitro. The mechanism of AZ4 to
the NSCLC cell H460 was investigated, including cell cycle
arrest and apoptosis.
Materials and methods
Synthesis of AZ1 to AZ4 AZ1 to AZ4 were prepared by
following our previously published
methods[13]. Stock solutions (10 mmol/L) were freshly prepared in DMSO (Sigma, St
Louis, MO, USA) and the series was diluted and directly
added into the cultured cells from 20 µmol/L to 0.2 µmol/L.
Cell culture The cell line H460 (human NSCLC) was
cultured in RPMI-1640 medium with 10% fetal bovine serum
(FBS), 2 mmol/L L-glutamine, 25 mmol/L
hydroxyethyl-1-piperazine-ethanesulfonic acid (HEPES). The normal cell
MRC-5 (human fetal lung fibroblast cell line) was cultured in
Dulbecco's modified eagle's medium (DMEM) with 10% FBS,
2 mmol/L L-glutamine, and miminal essential medium (MEM)
non-essential amino acid. The cell culture medium for those
two cell lines all contained penicillin-streptomycin and
fungizone. All the medium and supplements were purchased
from Gibco Laboratories (Grand Island, NY, USA). All of the
cells were incubated in a humidified atmosphere of 5%
CO2 at 37 °C. The amount of cells was counted after
trypsinization by a Neubauer hemocytometer (VWR, Scientific Corp,
Philadelphia, PA, USA).
Cytotoxicity assay (MTT assay) The MTT assay was
according to the method of Skehan et
al[14]. Two cell lines were seeded in 96-well flat-bottomed microtiter plates
(3000_5000 cells/well), respectively. The cells were incubated for
24 h with drugs, applied as serial 1:2 dilutions (100 µL/well)
ranging from 20 µmol/L down to 0.2 µmol/L or 0.1% DMSO
as control. Twenty microliters of MTT (5 mg/mL) was added
to each well and incubated for 4 h at 37 °C. The formazan
product was dissolved by adding 100 µL DMSO to each
well, and the plates were read at 550 nm. All measurements
were performed in triplicate and each experiment was repeated
at least 3 times. The IC50 was calculated from the 50% formazan
formation compared with the control without the addition of
drugs.
Cell cycle analysis The H460 cells were serum starved
without FBS overnight to synchronize cell phase, then the
cells were treated with various concentrations of AZ4 for 24 h.
The cells were harvested and incubated with hypotonic
staining buffer [0.1% sodium citrate, 0.3% triton X-100, 0.01%
propidium iodide (PI), and 0.01% ribonuclease A] for 15 min
on ice in the dark. The DNA content was measured by a
Becton Dickinson FACScan using Cell Quest software and
analyzed using EXPO 32 and MultiCycle software (Beckman
Coulter, High Wycombe, UK).
Microscopy image of caspase 3, 8, and 9 activity in H460
cells The method was according the report of Komoriya
et al[15] with minor modifications. Briefly, the H460 cells were
incubated with fluorogenic caspase substrates
(PhiPhhiLux-G1D2, CaspaLux 8-L1D2, and CaspaLux 9-M1D2,
OncoIm-munin Inc, Gaithersburg, MD, USA), GDEVDGI, IETDGI, and
LEHDGI at 10 µmol/L respectively, while suspended in RPMI-
1640 plus 10% FBS, 10 mmol/L HEPES, and various
concentrations of AZ4 for 24 h, 30 h(for caspases 8 and 9 only), and
48 h incubation. Substrates were present at 10 µmol/L
throughout the course of the induction and imaging. The
cells were viewed on a Nikon fluorescence microscope
system (Tokyo, Japan). Samples were excited using a 488/518
nm krypton/argon laser, and fluorescent images were
acquired. The brightness/contrast settings were adjusted
so that the fluorescent signal of cells without AZ4 was near
the background. As the fluorogenic caspase substrates are
cleaved in apoptotic cells, cellular fluorescence shifts from
below to above the fluorescence of the bulk solution in the
same plane.
Western blot analysis of cyclin B, Cdc-2, p53, p21, and
the Bcl-2 protein The method used was that of Bacus
et al and slightly modified[16]. Briefly, the cells were collected
from a 100 mm cultured dish after being challenged by
various concentrations of the AZ4 compound at 24 and 48 h,
respectively. The cell pellets were spun down by
centrifugation at 1000×g for 20 min. The pellets were resuspended in
cold buffer (10 mmol/L HEPES, pH 7.9), 1.5 mmol/L
MgCl2, 10 mmol/L KCl, 0.5 mmol/L diethiotreitol (DTT), 0.5 mmol/L
phenylmethylsulfonyl fluoride, 1 mmol/L benzamidine, 30 mg/mL
leupeptin, 5 mg/mL aprotinin, and 5 mg/mL pepstatin A, and
incubated on ice for 5 min. The cells were broken down by
lysis buffer and a sonicator. Cell lysates (25 µg) was
separated by 12% SDS-PAGE and transferred onto polyvinylidene
difluoride membranes (Amersham, Buckingshamshire, UK).
The blots were incubated with blocking buffer (11 mmol/L
Tris-vase pH 7.4, 154 mmol/L NaCl, and 5% skim milk), washed
with washing buffer (11 mmol/L Tris-vase pH 7.4, 154
mmol/L NaCl, and 0.1% Tween-20), and incubated with specific
antibodies against specific proteins cyclin B, Cdc-2, p53, p21,
and Bcl-2. Each membrane was blocked in blocking buffer
prior to incubation with antibodies. The primary antibody
against cyclin B, Cdc-2, p53, p21, and Bcl-2, all belong to the
mouse antihuman monoclonal antibody (Imgenex Co, San
Diego, CA, USA). The secondary antibody (Jackson Immuno
Research Lab Inc, West Grove, PA, USA) conjugated with
horseradish peroxidase was added at an appropriate dilution
by blocking buffer. The dilution factors for the primary
antibodies were 1:500_1:1000 depending on the amount of
proteins, and the dilution factors for the secondary
antibodies were 1:2000. The primary β-actin antibody used was a
mouse monoclonal antibody with the dilution factor of
1:10 000 (Biogenesis, England, UK). Immunodetec-tion was
carried out using the enhanced chemiluminescence (NEN,
Boston, MA, USA) detection system. To quantify the
amount of protein expression achieved, we measured the
intensity of chemiluminescence of the second antibody
using a densitometer (BioRad Gel Doc 2000 software) and
analyzed using Gel Doc ( Hercules, CA, USA). The values in
the relative protein interested expression-quantifying table
represent the relative amount of protein expression in
respect to β-actin expression divided by its control.
Results
Cytotoxic activity evaluation The chemical structures of
AZ1 to AZ4 are shown in Figure 1. The chemical structure of
AZ1 to AZ4 was composed of 2 aziridinylnaphthoquinones
bridging by ethylene glycol units. AZ1 had the shortest
ethylene glycol units; in retrospect, AZ4 contained the 4
ethylene glycol units. The cytotoxic activities of AZ1 to
AZ4 against NSCLC H460 cells were evaluated; the human
fetal lung fibroblast cell line (MRC5) was used as a normal
cell control, and their IC50 values are listed in Table 1. The
bis-aziridinylnaphthoquinone AZ1 to AZ4 were effective
cytotoxic agents against H460 cells with
IC50 values ranging from 1.23 to 7.21 µmol/L. The normal lung fibroblast cells
MRC-5 were less sensitive to AZ1 to AZ4 with
IC50 values of 2.8 to 12.7 µmol/L, respectively. The correlation between the
cytotoxic activities and the linker distance between 2
aziridinylnaphthoquinone moieties against H460 cells was
directly in proportion, but not with the MRC-5 cells. Initial
characterization indicated that AZ4 was the most effective
compound at inhibiting the growth of H460 cells and was
less effective with MRC5 cells.
Induction of the G2-M cell cycle arrest and the effects on
the expression of cell cycle-related proteins in H460 cells
treated with AZ4 An analysis of DNA content was used to
determine whether cell arrest was induced by AZ4. A DNA
content analysis by a flow cytometer indicated that H460
cells had a significant population of cell arrest in
G2-M treated with AZ4 in 24 h compared with 0.1% DMSO alone (Figure
2). The peak area under the G2-M phase started to ascend as
low as 0.5 µmol/L with AZ4. The cell cycle-related proteins
associated with mitotic, cyclin B and Cdc-2, in the AZ4-treated
H460 cells for 24 h were also investigated (Figure 3, Table 2).
The expression pattern of cyclin B in the cells remained
basically unaltered with increased concentrations of AZ4
treatment; however, the expression pattern of Cdc-2 was
decreased relative to the control in a dose-dependent manner.
The expression of Cdc-2 decreased by 47% at 2.0 µmol/L
with AZ4. We proposed that the growth arrest on H460 cells
induced by AZ4 might be altered with reduced protein
expression of Cdc-2, but not cyclin B.
AZ4 triggers H460 cell apoptosis via caspase 3, 8, and
9-dependent routes and is associated with altered
expression of p53 and Bcl-2 proteins We investigated whether
apoptosis of H460 cells induced by AZ4 was mediated by
caspase 3, 8, and 9 activation for 48 and 30 h after treatment.
The activation of caspases 3, 8, and 9 was assessed by cell
permeable fluorogenic caspase substrate, GDEVDGI, IETDGI, and LEHDGI, respectively. The induction of
caspases 3, 8, and 9 would cleave the cell permeable to
fluorogenic substrate individually, and then release the green
fluorogenic substance. From those results, there was a large
increase of the green peripheral clump as indicated by the
arrows in Figures 4 and 5. The upstream enzyme of caspases
3 and 8 were activated in H460 cells induced by AZ4, but not
incaspase 9 (data not shown). According to the visual
observa-tion, the green peripheral clump appeared as low as
the concentration at 0.625 µmol/L AZ4 at 30 h when
observing caspase 8 (Figure 4) and 48 h for caspase 3 (Figure 5).
However, enzyme activation of caspase 3 was not found to
be as high as treated with the 2 µmol/L AZ4 (data not shown).
The apoptosis related proteins, tumor suppressor
protein p53, and the anti-apoptotic protein Bcl-2 are two
important components of the apoptotic pathway which act as
regulators of apoptosis. From the results of the Western blot,
AZ4 treatment altered both p53 and Bcl-2 expression in H460
cells in a dose-dependent manner after 48 h incubation (Figure
6, Table 3). The AZ4 induced overexpression of the p53
protein 25% more than the control when the H460 cells were
exposed to 2.0 µmol/L AZ4. It is of interest that Bcl-2 protein
expression was strongly reduced by 39% compared with the
control when the H460 cells were treated with 2.0 µmol/L
AZ4. In the time-dependent effect, the expression of p53
and p21 proteins were increased to a maximum at 24 h, and
then decreased at 48 h (Figure 7, Table 4).
Discussion
Bioreductive drugs such as AZQ, mitomycin C, RH1 and
TPZ, have been developed to exploit the oxygen deficiency
in the hypoxic fraction of solid tumors on the premise that
hypoxic cells should show a greater propensity for
reductive metabolism than well-oxygenated
cells[3,5_7]. Our pervious study on different series of
bis-aziridinylnaphtho-quinone compounds showed that these compounds exhibit
more potent responses toward the solid tumors than the
circulation tumors[14]. These results are supported by other
reports indicating that there are differences in the reductive
metabolism between the solid tumors and the circulation
tumors[3]. Considering the importance of all the cellular
reductases (eg NADPH cytochrome P450 reductase,
cytochrome b5 reductase, NADP(H) oxidoreductase, NQO1) in
response to the whole cellular reductive metabolism; these
reductases are also responsible for the bioactivation of AZ4.
The results from Table 1 suggest that AZ4 is a novel class of
bis-aziridinylnaphthoquinone and is cytotoxic against H460
cells.
As shown in Figure 2, the cell cycle arrest at the
G2-M phase was observed at 0.5 µmol/L AZ4. The
G2-M phase-arresting proteins of H460 cells induced by AZ4 was related
with the reduced expression of the Cdc-2 protein, but not
cyclin B (Figure 3).
Apoptosis plays an important role in the removal of
aberrant cells that might otherwise cause the development of
tumors[17,18]. Normally, the process of cell reproduction is an
ordered process known as cell cycle. The tumor suppressor
gene p53 is a multifunctional protein mainly responsible for
maintaining genomic integrity, and is the most frequently
mutated gene in human tumors[19]. In response to DNA
damage, aberrant growth signals or the chemotherapeutic
drug p53 is stabilized and induces apoptosis and/or cell cycle
arrest[20_22]. Therefore, p53 is a tumor suppressor gene with
key regulator effects on both cell cycle and
apoptosis. p53 also exerts its control on apoptosis by interacting with other
important apoptotic molecules, such as members of the
Bcl-2 family[23].
The protein from the Bcl-2 gene family, which is an
anti-apoptotic associated protein, plays an important role in the
regulation of apoptosis[24]. In the apoptotic pathway, some
drugs will activate the cysteine protease family, such as
caspases which specifically cleave their substrates after
aspartic acid, then activate or inactivate their cellular protein
targets by a process of limited proteolysis. The caspases
play central roles in the execution of
apoptosis[25_27]. Pro-apoptotic therapeutic strategies require, in turn, the exact
knowledge of the very specific downstream pathway of
apoptosis and of each single step in the targeted cell. In
some cells, the signaling cascade is mediated by the caspases.
Several proteins are known to regulate or interfere with this
step of apoptosis, that is, proteins of the Bcl protein family,
acting either anti-apoptotically (Bcl-2, Bcl-x etc) or
pro-apoptotically (bax, bid etc)[28], and the
pro-apoptotically-acting transcription factor p53.
The antitumor mechanism of AZ4 to lung cancer cell H460
was mediated with cell cycle arrest and the apoptosis
pathway. According to the results shown in Figures 2 and 3,
the cytotoxic activity of H460 cells induced by AZ4 was
associated with G2-M phase arrest at 24 h. Then, the H460
cells steered into the apoptotic pathway after prolonged
treatment with AZ4 for 48 h. We observed significant apoptosis
phenomena in H460 cells associated with the activation of
caspase 3 which mediated the caspase 8 enzymes, but not
caspase 9, the upregula-tion of p53 protein, and Bcl-2 down
regulation by various concentrations of AZ4 (Figures 4_6).
Bcl-2 was inactivated and made the G2-M cells of H460
induced by AZ4 more susceptible to apoptosis which is
supported by Yamamoto et al who proposed that stress response
kinases phosphorylate Bcl-2 during cell cycle progression
as a normal physiological process to inactivate Bcl-2 at
the phase of G2-M[29].
To observe the timing relationship of protein
expression from cell cycle arrest to apoptosis, the time-
dependent effect (Figure 7), the expression of p53 and p21
proteins were increased to the maximum at 24 h, then
decreased at 48 h. p21 is a member of the Cip/Kip family of
proteins, which promote cell cycle arrest by binding to and
inhibiting cyclin-dependent kinases. The latter, when
coupled with specific cyclins, facilitates the orderly
procession of the cell cycle. p21 is also tightly regulated at the
transcriptional level by p53 and probably serves as the
effector of the p53 cell cycle
control[30]. This is in agreement with the above results, which support cell cycle arrest at
24 h, then apoptosis at 48 h.
In conclusion, AZ4 displayed selective cytotoxic
activity against H460 cells and low cytotoxic activity when
cultured with non-neoplastic human adherent lung fibroblasts.
The cytotoxic effect induced by AZ4 on H460 cells may
correlate with the induction of the G2-M cell cycle at 24 h which
increased p53, p21, and Bcl-2 protein expression and
decreased the expression of the Cdc-2 protein. The cells were
then steered the apoptotic pathway for 48 h treatment, where
the anti-apoptotic protein Bcl-2 and cell checkpoint proteins
p53 and p21decreased, while the apoptosis enzymes caspase
3 and 8 were activated. It is believed that this cytotoxic
mechanism study holds promise for the development of a
new generation of potent antitumor agents.
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