Download Zoledronic acid inhibits hepatocellular carcinoma cells growth in

Zoledronic acid inhibits growth of hepatocellular carcinoma cells
in vitro and in vivo
1

1
Quan LIU, 2Yonghui TAO, 1Ruizhen BAI, 1Shujian CHANG, 1＊Dong HUA
Oncology institute, The Fourth Affiliated Hospital of Soochow University, Wuxi
214062, China
2
Key Laboratory on Technology for Parasitic Disease Prevention and Control,
Ministry of Health, Jiangsu Institute of Parasitic Diseases, Wuxi, Jiangsu, People's
Republic of China.
＊
Correspondence to: Dr. Dong Hua (email: [email protected] ), Oncology
institute, The Fourth Affiliated Hospital of Suzhou University, Wuxi 214062, China
Tel:86-510-88682109
The first two authors contributed equally to this work.
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Abstract
Background
Growing preclinical evidence shows that zoledronic acid (ZOL) exhibits direct antitumor
activity in various cancer cell lines. However, the cytotoxic effects of ZOL on human
hepatocellular carcinoma (HCC) cells have not been established. In the present study, we
investigated the effect of ZOL on HCC both in vitro and in vivo.
Methods
Cytotoxicity and cell cycles were assessed with Sulforhodamine B colorimetric assay and
flow cytometry. Expression levels of cell cycle phase-linked proteins were examined.
The effect of ZOL on HCC in vivo was explored based on H22- s.c. (subcutaneous
injection) and H22- i.p. (intraperitoneal injection) mice model.
Results
ZOL inhibited the growth of SK-HEP-1 and H22 cells and induced S-phase arrest
through downregulating cdc2 protein and upregulating cyclin A. It inhibited the growth
of s.c tumors, and increased the survival of both H22- s.c. and H22- i.p. mice in vivo.
Conclusion
In conclusion, ZOL inhibits growth of HCC cells in vitro and in vivo. Additional studies
are warranted for management of HCC based on ZOL.
Keywords
Zoledronic acid; Hepatocellular carcinoma; S phase arrest
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BACKGROUND
Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality
worldwide, with an estimated one million new cases each year [1]. Most HCC cases are
diagnosed in the late stage, and the median survival following diagnosis is only
approximately 6 to 20 months [2]. Only 40% of HCC patients are eligible for potential
curative treatments (resection, transplantation, or local ablation). 20% of the patients are
eligible for chemoembolization, and for the remaining 40% patients with unresectble
lesions and unsuitable for locoregional therapy, the systemic therapy is appropriate [3].
The effect of chemoembolization and systemic therapy relies heavily on the use of
chemical drugs. Unfortunately, no specifically effective drug has been identified for the
HCC patients to date. This situation necessitates discovery and development of new
drugs to improve the therapeutic effect among HCC patients.
Zoledronic
acid
(ZOL)
is
heterocyclic imidazole bisphosphonate
a
used
third-generation
as
standard
nitrogen-containing
care
for
preventing
skeletal-related events (SREs) in patients with bone metastases [4]. Recently, growing
preclinical evidence shows that ZOL exhibits
remarkable antitumor activity in several
cancer cell lines [5-8] and animal models [9-12]. The antitumor ability has also been
corroborated in clinical trials. In postmenopausal breast cancer patients, ZOL combined
with standard adjuvant therapy reduces the risk of mortality by 29% (hazard ratio, 0.71;
p=0.017) compared with the adjuvant therapy alone [13]. Consequently, ZOL may exert
as a promising therapeutic effect in multiple cancers. However, the cytotoxic effects of
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ZOL on HCC have not been established so far. In the present study, we investigated
impact of ZOL on HCC both in vitro and in vivo.
METHODS
Cell culture
The SK-HEP-1 cell line was obtained from the American Type Culture Collection
(Manassas, Virginia, US). H22 cell lines were purchased from the Cell Bank of the
Chinese Academy of Sciences (Shanghai, China). All cancer cell lines were maintained
with DMEM medium. The medium was supplemented with 10% inactivated fetal bovine
serum (Life Technologies, Carlsbad, California, US) and penicillin/streptomycin (200
μM/l). Cultures were kept in an incubator at 37.0 ℃ in a water-saturated atmosphere with
5% CO2.
Sulforhodamine B colorimetric assay
The viability of SK-HEP-1 and H22 cells was evaluated by Sulforhodamine B
colorimetric assay (SRB) ( St. Louis, Missouri, US). 5000 cells/well were seeded into
96-well plates and left to adhere overnight. The cells were incubated for 48 h in the
absence or presence of 0.5-350 μM ZOL(Novartis Pharma AG, Basel, Switzerland). Cell
monolayers were fixed with 10% (wt/vol) trichloroacetic acid and stained for 30 min, and
after that the excess dye was removed by washing repeatedly with 1% (vol/vol) acetic
acid. The protein-bound dye was dissolved in 10 mM Tris base solution for optical
density determination at 495 nm using a microplate reader. The half-maximal maximal
inhibitory concentration (IC50) was determined using the nonlinear regression program
CalcuSyn (Biosoft, Cambridge, UK).
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Flow cytometry
Cells were incubated with or without ZOL as indicated and harvested. The cells were
fixed with 70% ethanol at 4 °C overnight. The fixed cells were washed with D-PBS and
resuspended in phosphate-buffered saline containing 500 μg/ml RNase A and 50 μg/ml
propidium iodide (Sigma-Aldrich, St. Louis, Missouri, US). DNA content was
determined by flow cytometry using a Coulter EPICS XL-MCL cytometer (Coulter Co.,
Miami, Florida, USA) and the cell cycle phase distribution was analyzed by ModFit 3.0
software (Verity Software House, Topsham, Maine, USA).
Western blotting
Total proteins were extracted by lysing cells with a buffer containing 50 mM Tris–HCl,
pH 7.4, 150 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA, 0.1% SDS and protease inhibitor
cocktail followed by ultracentrifugation at 13,000 rpm for 10 min. All fractions were
assayed for protein contents according to the protocol [14]. Equal amounts of proteins
were mixed with Laemmli sample buffer, resolved on SDS/PAGE (12%) and blotted onto
PVDF membranes (Millipore, Billerica, Massachusetts). Immunodetection was carried
out in 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20, and 5% non-fat dry
milk, followed by HRP-conjugated secondary antibodies at 1:20,000 dilution. Detection
was performed using an ECL kit.
H22 Solid tumor-bearing mouse model
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In vivo experiments were carried out in compliance with Guidelines on the Humane
Treatment of Lab Animals (Agreement No. [2006]398 ). H22 was maintained in DMEM
medium. H22 cells (1x107 ) were inoculated subcutaneously (sc.) into the right flanks of
4-6-week- Female Kunming mice (SLAC Laboratory animal, Shanghai). 30 of the
inoculated mice developed palpable tumors (60–90 mm3) by Day 10 after tumor cell
injection. These mice were randomly assigned to various treatment groups (control,
treated by ZOL 100μg/kg group, ZOL 400μg/kg group). ZOL was injected into the
mouse peritoneal cavity 100μg/kg or 400μg/kg every other day for a total of
four injections. PBS was used as a control. Tumors were measured using calipers. Tumor
size was measured with calipers every 3 days and tumor volumes were calculated (tumor
volume = 0.52 × length × width2). Mice were sacrificed when tumors reached 2,000 mm3.
Ascites tumor-bearing mouse model
Four- to six-week female Kunming mice were inoculated intraperitoneally (i.p.) For
1x107 H22 cell suspension on Day 0. After 3 days of tumor transplantation, ZOL was
administrated to the peritoneal cavity of H22-bearing mice at a dose of 100 μg/kg body
weight every other day for a total of four injections, and the survival days of mice were
recorded.
Statistical analysis
Results were presented as means ± SD. Statistical significance was determined by
one-way analysis of variance followed by the Student’s t test. Kaplan-Meier survival
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curves were determined by the Log-rank test. A value of P<0.05 was considered
statistically significant.
RESULTS
Antiproliferative activity assay
SK-HEP-1 and H22 cell lines were treated with increasing concentrations of ZOL for 48
h. The growth inhibitory effects of cells were examined by SRB assay. As shown in
Figure 1, after 48 h of ZOL treatment SK-HEP-1 and H22 showed significantly reduced
growth in a dose-dependent manner.
S phase arrest in HCC cells.
To elucidate the effect of ZOL on the cell cycle progression, flow cytometry analysis of
DNA content was performed on SK-HEP-1 and H22 cells (Figure 2). SK-HEP-1 and H22
cells were treated with different concentrations of ZOL. A clear cell cycle perturbation
effect of ZOL was observed. At 48 h after the treatment, with increased concentrations of
ZOL, the cells accumulated in the S phase, and gradually decreased in G1 and G2/M
phases (Figure 2). We speculated that ZOL arrested SK-HEP-1 and H22 cells in S phase.
Expression of phase markers and cell cycle regulators
In order to understand whether ZOL induced an S-phase cell cycle arrest in SK-HEP-1
and H22 cell lines, we investigated the expression of p53, cyclin B1, cyclin A and cdc2 in
SK-HEP-1 and H22 cell lines treated by ZOL. Cells were treated with 0.3% DMSO or 20
μM ZOL, and analyzed for protein expression levels after 48 h of treatment (Figure 3).
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There was no significant change in the expression of Cyclin B1 and p53. Cdc2 was
markedly decreased after treatment with ZOL, while cyclin A was upregulated.
Zol inhibited tumor growth in vivo.
To evaluate the antitumor activity of the ZOL in vivo, xenograft H22 liver cancer model
was established in Kunming mice. Tumor volumes were recorded to assess the antitumor
effect in these treatment groups. As shown in Figure 4, the tumors in the PBS group grew
progressively during the course of the experiment. On the contrary, a lag was noticed in
the growth of tumor in the ZOL group until Day 19. At the sixth week after
administration, the average tumor volume of ZOL-treated mice was statistically smaller
than that of untreated mice (P < 0.05).
Survival of H22-s.c. and H22-i.p. mice
To determine the effect of ZOL on survival, H22- s.c. and H22- i.p. mice were treated
with 100 μg/kg body weight in the peritoneal cavity every other day for a total of
four injections starting from 10 days and 3 days after inoculation of tumor cells (Figure
5). Among the H22- s.c. mice, the survival time in the ZOL-treated group was
significantly longer than in the control group (P < 0.05), with five mice having a long
survival and the survival of the dead mice over 45 days after tumor inoculation. Among
the H22- i.p. mice, all animals (n = 10) in control group died within 10-25 days, whereas
those in the ZOL-treated group died within 15–35 days, with two mice having a long
survival.
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DISCUSION
In the present study, ZOL showed a significant inhibitory effect on the growth of
SK-HEP-1 and H22 cells in a dose-dependent manner. To elucidate the effect of ZOL on
the cell cycle progression, flow cytometry analysis of DNA content was performed on
SK-HEP-1 and H22 cells. (Figure 2). A clear dose-dependence of the S phase arrest
effect was noticed. This effect is in accordance with the previous studies on different
types of tumor cells, all suggesting arrest of ZOL-treated cells in the S-phase [15-17].
ZOL may fail to induce apoptosis. As shown in Figure 2, in both hepatocellular
carcinoma cell lines, ZOL treatment did not induce obvious apoptotic sub-G1 peak. We
also found no evident characteristics of apoptosis (such as cell shrinkage or cytoplasmic
blebs) in SK-HEP-1 cellular morphology after ZOL treatment (Appendix 1). Clearly,
further experiments are warranted to verify this assumption. To further elucidate
molecular mechanisms of the S phase arrest, we examined the cdc2, p53, cyclin A and
cyclin B1 expression in SK-HEP-1 and H22 cell lines treated with or without ZOL.
According to Figure 3, ZOL induced S-phase arrest probably through downregulating
cdc2 and upregulating cyclin A.
The direct antitumor effect of ZOL on solid tumors in animal models has been
demonstrated in several studies [11, 18, 19], and was further confirmed in our study
(Figure 4). The average tumor volume of ZOL-treated mice was statistically smaller than
that of untreated mice. However, it showed no significant difference between the 100 μg
/kg and 400 μg/kg groups. This may result from the plateau effect of 100 μg/kg and thus
no additional effect can be achieved with increased doses. Moreover, a life
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span-prolonging effect were found in ZOL-treated mice group compared to the control
group in H22- s.c. and H22- i.p. mice models (Figure 5). This effect of
prolonging survival was also observed in mice with osteosarcoma [12]. We hypothesize
that ZOL significantly inhibits HCC growth, thus extending the survival time. Our results
suggest that ZOL may be a promising drug for the treatment of HCC.
For most cancer types, pharmacokinetics of ZOL is the biggest obstacle that prevents us
from translating the preclinical antitumor activity into use in clinical settings. Research
shows that a routine clinical dose of 4 mg ZOL achieves a peak plasma concentration of
0.75–2 μM before it is rapidly absorbed by bones [20]. A single 30–100 μg/kg injection
in mice is equivalent to a single 4 mg human dose [21, 22]. Updated evidence has showed
the cytotoxic effects of ZOL in different cancer cell lines at concentrations of 10 μM of
ZOL or higher in vitro [23]. It means that the dose of ZOL should be elevated, otherwise
the direct antitumor effect of ZOL is not noticable. Obviously, it is unsuitable for human
body and may produce serious toxicity according to drug instructions.
However, this challenge posed by pharmacokinetics of ZOL may be overcome by
combined use with transarterial chemoembolization (TACE). TACE has become a
standard care for HCC patients who are not suitable for surgical or ablative treatment if
extrahepatic metastases and advanced liver disease are absent [24]. The rationale for
treatment of HCC by TACE is based on the dual blood supply of the liver. The nutrition
of liver parenchyma is predominantly supplied by portal veins, and the majority of the
blood supply to HCC is derived from the hepatic artery. TACE is designed to eliminate
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the tumor blood supply by particle embolization and directly infuse cytotoxic chemical
into the branch of the hepatic artery that feeds the tumor. Transarterial delivery of
cytotoxic drugs combined with embolization of arterial feeders are shown to reduce the
maximum plasma concentration and increase the concentration of cytostatic agent in the
tumor compared to
systemic treatments
[25]. Consequently, the hurdle of
pharmacokinetics will be resolved if ZOL can be applied in TACE, at least this will be
possible according to our findings.
Abbreviations
ZOL, Zoledronic acid; HCC, hepatocellular carcinoma; s.c., Subcutaneous injection; i.p.,
Intraperitoneal injection; TACE, Transarterial chemoembolization.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
Quan Liu, Yonghui Tao and Dong Hua designed and performed the experiments, Quan
Liu and Ruizhen Bai contributed to manuscript writing. Shujian chang analyzed the data.
All authors read and approved the final manuscript.
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FIGURE LEGENDS
Figure 1. Antiproliferative activity of ZOL
SK-HEP-1 and H22 cells were incubated for 48 h in the absence (control, the
inhibition rate = 0%) or presence of 0.5-350 μM ZOL. Viability of SK-HEP-1 (◆)
and H22 ( ■ ) cells were evaluated by SRB. ZOL exhibited a significant
antiproliferative activity against SK-HEP-1 and H22 with IC50 of 21.1 μM and 29.6
μM.
Figure 2. Effect of ZOL on the cell cycle distribution
Flow cytometry of DNA was performed on SK-HEP-1 (top) and H22 (bottom) cells.
SK-HEP-1 and H22 cells were treated with 0 (control), 25, 50 and 100μM of ZOL for
48 h and were stained with PI for flow cytometric analysis. A clear cell cycle
perturbation effect of ZOL was observed in a
dose-dependent manner. With
increased concentration of ZOL, the cells accumulated in the S phase, and G1 and
G2/M phase cells were gradually decreased. A representative figure is shown from
three separate experiments.
Figure 3. Effect of ZOL on the expression of cell cycle regulatory proteins
SK-HEP-1 and H22 cells were incubated with or without 20 μM ZOL. After 48 h of
treatment, cell lysates were evaluated for levels of Cyclin B1, Cyclin A, cdc2 and p53
expression by western blotting. Cyclin B1 and p53 showed no significant change in
expression, cdc2 was deeply decreased, and Cyclin A was upregulated in SK-HEP-1
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and H22 cell lines after treatment with ZOL for 48 h. A representative figure is shown
from three separate experiments.
Figure 4. Effect of ZOL on H22- s.c. mice
Kunming mice were subcutaneously implanted with 1x107 H22 cancer cells. When
tumors reached a size of 60-90 mm3 (~10 days), mice were intratumorally treated with
ZOL 100 μg/kg(■) or 400 μg/kg(▲) every other day for a total of four injections.
PBS injection was used as control (◆). Tumor volumes were monitored every 3
days and mice were killed when tumors reached 2,000 mm3. The average tumor
volume of ZOL-treated mice was statistically smaller than that of untreated mice. The
average tumor volume was not significant in the 100 μg /kg and 400 μg/kg groups.
Statistically significant difference is indicated (*) (P< 0.05).
Figure 5. Kaplan–Meier survival curve
ZOL prolonged the survival of both H22- s.c. (A) and H22- i.p. mice (B). Both ZOL
groups(◆) were treated with 100μg/kg intratumorally every other day for a total of
four injections. PBS injection (●) was used as control. Among the H22- s.c. mice,
the survival time in the ZOL-treated group was significantly longer than in the control
group (P < 0.05), with five mice having a long survival and the survival of the dead
mice over 45 days after tumor inoculation. Among the H22- i.p. mice, all animals (n =
10) in control group died within 10-25 days, whereas those in the ZOL-treated group
15
died within 15 – 35 days, with two mice having a long survival. Statistically
significant difference is indicated (*) (P< 0.05).
FIGURE
FIGURE 1
FIGURE 2
FIGURE 3
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FIGURE 4
FIGURE 5
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Appendix
Appendix 1. Effect of ZOL on SK-HEP-1 cellular morphology.
SK-HEP-1 cells were incubated for 48 hour in the absence (A) or presence of 21.1
μM ZOL (B). The cells were photographed through a phase-contrast microscope x100.
No evident characteristics of apoptosis (such as cell shrinkage or cytoplasmic blebs)
were observed.
Appendix 2. The gating of each FACS data.
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