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Introduction
Prostate cancer is the most frequent non-cutaneous malignancy and
the second leading cause of cancer death among males in the western
world. It has long been recognized that cancer arises as a result
of somatic mutations; and a concept dramatically reinforced by the
demonstration that a cellular "proto-oncogene" is mutationally
involved in prostate cancer formation. To date a number of the genes
associated with prostate cancer formation and progression have been
identified, which are deregulated or abnormally over-expressed contributing
to tumor formation. Recent technological developments have paved
the way for the identification of the genes PRC17, Stat3, and Pim[1-5].
Identification of these genes provides a new target for prostate
cancer therapy.
The Pim serine/threonine kinase family was first identified as
a common proviral insertion site in T and B-cell lymphomas in mice[6].
To date, three family members have been identified: Pim-1, Pim-2,
and Pim-3. These related enzymes show substantial homology, but
differ in their tissue expression[7]. It is unknown to
what extent the various family members differ in their biochemical
effects. The Pim-2 gene encodes a cytoplasmic serine/thronine kinase
whose expression is regulated by hematopoietic cytokines[8,9].
There are multiple isoforms of Pim-2 protein resulting from the
use of the alternative translation start codon, CTG[10].
Among these isoforms, the short Pim-2 (34 kDa) form is the most
active at enhancing survival of FDCP1 cells after cytokine withdrawal.
The Pim-2 transgene induces lymphoid tumors, and exhibits potent
synergy with c-myc[9]. Recent studies have indicated
that Pim-2 kinase could phosphorylate the pro-apoptotic protein
Bard on serine 112, and thus inhibit cell apoptosis[11].
Although the contribution of Pim-2 to cancer formation and progress
is relatively unknown, Pim-2 has been recognized as a procongene,
and procongenes are associated with many types of cancer formation.
At present, more and more patients are being diagnosed with early
stage prostate cancer because of improvements in diagnostic techniques.
Although surgery and chemotherapy are effective on patients with
localized tumors, the prognosis of patients with advanced or metastatic
tumors is not ideal. It is clear that novel treatment approaches
to prostate cancer are urgently needed. It is now accepted that
antisense oligodeoxynucleotides have sequences that are complementary
to specific strands of RNA. Once delivered into a target cell, the
oligodeoxynucleotides hybridise with its RNA complement and inhibit
expression of the corresponding disease-relevant protein. More and
more data demonstrate that antisense therapy for cancer is a very
promising strategy. Consequently, the aim of our paper is to investigate
whether inhibition of Pim-2 expression using antisense oligodeoxy-nucleotides
can reduce proliferation of DU-145 cells.
Materials and methods
Antisense oligodeoxynucleotides The antisense sequences
used in these experiments were designed using a computational neural
network model[12]. BLAST confirmed that they were specific
for the Pim-2 gene. Eicosomer ASODN were synthesized in our laboratory
using an Applied Biosystems 391 DNA synthesizer and purified by
OPC(Oligonucleotide Purification Cartrigde) (Perkin-Elmer, Foster
City, CA, USA). Table 1 shows the sequence of ASODN.
Cell line and culture Human prostate cancer cell lines,
DU-145, were obtained from the Chinese Academy of Medical Sciences
(Beijing, China). Cells were cultured in RPMI-1640 medium (Invitrogen,
San Diego, CA, USA) supplemented with 10% fetal bovine serum (GIBCO
BRL, Grand Island, NY, USA), 100 kU/L benzylpenicillin and
100 mg/L streptomycin. All cultures were incubated at 37 °C
in a 5% CO2 atmosphere.
Cell viability The effects of ASODN on cellular viability
were determined using an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2
H-tetrazolium, inner salt] assay. In brief, 3×103
cells were seeded in 96-well microtiter plate and allowed to attach
overnight. Cells were then transfected with different concentrations
(0.1 μmol/L, 0.2 μmol/L, 0.4 μmol/L, 0.8 μmol/L)
of ASODN. After 48 h of incubation, 20 mL of MTS (Sigma Chemical
Company, St Louis, MO, USA) was added to each well. The 96-well
microtiter plate was incubated for 2 h at 37 °C, and a
490 nm absorbance value was determined using an MR 600 Microplate
reader (Wallac, Turku, Finland). Each assay was carried out in quadruplicate.
Cellular proliferation inhibition rate is calculated as: (Acontrol-Asample
)/(Acontrol-Ablank )×100%.
Lip-mediated transfection of antisense oligo-deoxynucleotides
Cells were plated in six-well plates at a density of 1×105
cells per well. Transfections were carried out after plating for
24 h, when cells reached a confluence of (50-80)%. Lipofectamine
2000 (Invitrogen) was used for transfection in this experiment and
transfection was carried out according to the manufacturer's instructions.
ASODN concentrations were selected as 0.1 μmol/L, 0.2 μmol/L,
0.4 μmol/L, 0.8 μmol/L, respectively. After transfection
(incu-bation for 6 h at 37 °C), the cells were washed
with phosphate-buffered saline (PBS) and incubated in fresh culture
medium.
Reverse transcription-polymerase chain reaction (RT-PCR) After
transfection for 48 h, total RNA was extracted using TRIzol
(Invitrogen) by a single-step phenol extraction. Subsequent RT-PCR
was carried out using a reverse transcription system (RT-PCR kit,
Promega, Madison, WI, USA). in brief, first strand cDNA was synthesized
using an Oligo(dT)15 primer at 42 °C for 30 min.
The PCR reaction for Pim-2 and β-actin was carried out in a single
reaction of 20 mL volume. The latter served as a control following
32 cycles of denaturing at 95 °C for 45 s, annealing at 55
°C for 40s, and extending at 72 °C for 40 s. Under this
reaction condition, the amplification showed linearity (data not
shown). PCR products were run on a 3.0% agarose gel and visualized
using ethidium bromide staining, and the intensities were measured
by scanning the gel with Gel Doc 1000 (Bio-Rad, Hercules, CA, USA).
Inhibition of Pim-2 mRNA was calculated according to the following
formula:
where Asample is the intensity of Pim-2 PCR product
in cells transfected with ASODN and lipofectamine, A0sample
is the intensity of Pim-2 PCR product in cells transfected with
lipofectamine alone, Acontrol is the intensity
of β-actin product in cells transfected with ASODN and lipofectamine,
and A0control is the intensity of β-actin product
in cells transfected with lipofectamine alone.
Western blot analysis After 72 h of transfection with
ASODN, cells were lysed in RIPA buffer [10 mmol Tris-HCL (pH 7.4),
1% deoxycholate, 1% NP40, 150 mmol NaCl, 0.1% SDS, 0.2 mmol phenylmethyl
sulfonyl fluoride, 1 mg/L aprotinin and 1 mg/L leupeptin] for 30
min on ice. The lysates were centrifuged at 12000×g
for 15 min to remove debris. Protein samples (30 μg) were separated
using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) gel and transferred onto hybond-polyvinylidene difluoride
(PVDF) membranes (Schleicher & Schuell Bio-sciences, Inc, Keene
N H, USA). Pim-2 protein was identified using anti-Pim-2 primary
and peroxidase-conjunct secondary antibody (Santa Cruz Biotechnology,
Santa Cruz, CA, USA). Finally, the reactive band was visualized
using an ECL-plus Detection Kit (Amersham Biosciences) and scanned
by Gel Doc 1000 (Bio-Rad).
Cell cycle and apoptosis analysis Cell cycle assays were
carried out as described previously[13]. in brief, cells
were harvested after transfection with 0.4 μmol/L ASODN for
48 h, and fixed with 70% ethanol at -20 °C overnight. Fixed
cells were washed twice again with PBS, and stained with 50
mg/L propidium iodide (PI) in the presence of RNase A. The stained
cells were analyzed for DNA content by fluorescence-activated cell
sorting (FACS) in a FACScan (SOBR model, Becton-Dickinson, San Jose,
CA, USA). Cell cycle fractions were quantified with CellQuest (Becton
Dickinson), and apoptosis was estimated using the fraction of sub-G1
phase cells.
Statistics Data were expressed as mean±SD. Statistical
analysis were carried out using Student's t-tests (two-tailed).
P<0.05 indicates statistical significance.
Results
Effects of ASODN on Pim-2 expression After transfection
of ASODN targeting Pim-2, semi-quantitative RT-PCR was used to determine
the inhibitory effects on Pim-2 mRNA transcription. ASODN 1-3 examined
in the present study exerted different inhibitory effects on mRNA
transcription (Figure 1A). The Pim-2 transcription
level was decreased by 64.5%, 56.8%, and 55.2% (P<0.05)
in DU-145 cells by ASODN1-3, respectively (Figure 1B). In addition,
the Pim-2 protein amount was diminished by treatment with ASODN
compared with the control (Figure 2).
Effects of ASODN on cell viability As shown in Figure 3,
all of the ASODN targeting Pim-2 gene treatment significantly decreased
cell viability, and the inhibition rate was dose-dependent.
Effects of ASODN on DU-145 cell growth arrest and apoptosis
Flow cytometry was used to quantify changes in cell cycle and
apoptosis 48 h after treatment with Pim-2 ASODN. All of the
ASODN targeting Pim-2 increased the fraction of cells undergoing
growth arrest at G0/G1 in DU-145 cells, and
the inhibitory effect of ASODN1 was greater than the others (Table 2).
It should be noted that no apparent apoptosis occurred in cells
after treatment with any of the ASODN (Figure 4).
Discussion
The balance between cell death and cell proliferation determines
cells survive or die. Although the detailed mechanism and function
of Pim-2 in cells are still unknown, research suggests that Pim-2
acts as an important factor to mediate cell survival. Consequently,
ASODN against Pim-2 are anticipated to restrain cell proliferation.
In this experiment, three ASODN targeting Pim-2 were designed and
synthesized by our laboratory. Results indicated that all three
ASODN could specifically reduce the mRNA transcription and protein
expression level of Pim-2 in DU-145 cells (Figures 1, 2).
Prostate cancer is the most frequently diagnosed cancer in men.
The incidence of prostate cancer rose in the late 1980s and early
1990s as a result of increased life expectancy, earlier and more
accurate diagnosis, and increased public awareness of the disease.
Nowadays, prostate cancer is believed to be the second leading cause
of cancer related death in men. Thus, there is an urgent need to
develop efficient therapy approaches to prostate cancer. Data presented
here indicate that our ASODN could repress DU-145 cells growth to
varying extents, and inhibitory effects were dose-dependent (Figure 3).
Despite extensive investigation, the physiological substrates of
the Pim-2 kinase remain unknown. However, ASODN against Pim-2 induced
marked G1 phase cell cycle arrest in DU-145 prostate
cancer cells (Table 2). This result implies that a number of proteins
that are associated with cell cycle control are substrates of Pim-2
kinase. Similarly, Pim-1, its homological gene, was found to phosphorylate
the cell cycle inhibitor p21Cip1/WAF1 and abrogate G1
phase cell cycle arrest[14]. Taken together, these results
offer evidence that the Pim serine/threonine kinase family show
substantial homology, which can mediate the activity of a number
of cell cycle related proteins. This experiment, implies that, in
DU-145 cells, Pim-2 kinase is not the essential factor determining
cell survival. In addition, abrogation of G1 phase cell
cycle arrest by Pim-2 serine/threonine kinase may be an important
mechanism for prostate cell proliferation.
Antisense therapeutics for cancer are, after decades of
difficulties, finally close to fulfilling their promise in the clinic.
Antisense compounds targeting certain genes, such as HER-2, VEGF,
IGF-IR, and protein kinase C, were reported to efficiently inhibit
target gene expression[15-18]. Advances in defining molecular
abnormalities in prostate cancer offer the hope of improving both
the diagnosis and therapy of the disease. It is now well established
that the progression of epithelial cells in the prostate from normal
to dysplastic or adenomatous epithelium to carcinoma in situ
and finally to invasive carcinoma is the result of the sequential
accumulation of genetic abnormalities involving oncogenes and tumor
suppressor genes. A number of genetic mutations are found in prostate
cancer, including activating mutations of the Pim-2 oncogene and
loss of the tumor suppressor gene P53[1-5].
In the present study, our antisense compounds could downregulate
the expression of target mRNA level in vitro, as well as
display certain antitumor activity. It is possible that antisense
or other small molecule approaches to inhibit Pim-2 signaling may
play a role in the treatment of patients with prostate cancer. In
addition, a significant fraction of non-prostate tumors, including
many colon, gastric, and testis carcinomas express elevated Pim-2
levels[19,20]. Whether or not antisense ODN targeting
Pim-2 can also show antitumor activity against such neoplasm requires
further study.
In summary, ASODN designed and synthesized by our laboratory against
Pim-2 can efficiently suppress target gene expression and inhibit
the growth of DU-145 cells, providing a new promising therapy target
for prostate cancer.
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