Polo-like kinase 1 is overexpressed in colorectal cancer and participates in the migration and invasion of colorectal cancer cells.
ABSTRACT Polo-like kinase 1 (PLK1) is an important molecule in proliferation of many human cancers. The aim of study is to clarify the expression patterns and potential function of PLK1 in colorectal cancers.
Fifty-six colorectal cancers samples were collected and arranged onto a tissue array and the expression of PLK1 were detected by immunohistochemistry and correlated with clinico-pathological characteristics and expression of PCNA. Expression of PLK1 in 9 colorectal cancer cells lines was investigated by RT-PCR and Western blot, then SW1116 cells lines were treated with PLK1 siRNA and the efficiency was examined by Western blot. Transwell test was applied to detect the migration and invasion capability of cancer cells by counting the number of cells passing through the membranes. Cell proliferation and apoptosis were examined by Cell Counting Kit-8 (CCK-8) and Annexin-V Kit.
PLK1 was positively expressed in 73.2% (41/56) of colorectal cancers tissues, but in only 3.6% (2/56) of normal tissues, and was associated with Duke's stage (P<0.01), tumor size (P<0.01), invasion extent (P<0.05) and lymphatic metastasis (P<0.01). The expression of PLK1 was correlated with expression of PCNA (R=0.553, P<0.01). PLK1 was inhibited in SW1116 cells by treating with PLK1 siRNA oligos, which resulted in a decreased number of cells passing through the membrane as compared with control groups (P<0.01) at 24 hours after transfection. Cell proliferation was inhibited from 48 hours after transfection, while cells apoptosis was induced from 72 hours after transfection.
PLK1 could be a progression marker for colorectal cancer patients and PLK1 depletion can inhibit migration and invasion capability of colorectal cancer cells SW1116, suggesting that PLK1 might be involved in metastasis and invasion of colorectal cancer. Therapeutic strategies targeting PLK1 may be a new approach to colorectal cancer.
- [show abstract] [hide abstract]
ABSTRACT: Polo-like kinase (PLK) is a cell cycle-regulated, cyclin-independent serine/threonine protein kinase. Recent reports have shown a critical role for PLK during tumorigenesis. To explore whether PLK plays a general role as a tumor marker of endometrial carcinomas, we examined the expression of PLK mRNA and protein in endometrial carcinomas and normal endometrium, and analyzed the relationship between PLK protein expression and malignant potential. We found that PLK mRNA was expressed in all specimens from endometrial carcinoma patients using RT-PCR methods, although some specimens from normal endometria were negative. Immunohistochemically, most of the PLK was found in the cytoplasm (around the nucleus), and partly in the nucleus of endometrial carcinoma glands and also secreted tissues from endometrial carcinoma glands. PLK was expressed at the basement membrane of carcinoma glands and partly expressed in the head portion of papillary carcinoma tissues. There was a significant correlation between percentages of PLK-positive cells and histological grade of endometrial carcinoma (P<0.0001). However, the expression of proliferating cell nuclear antigen and Ki-67 was independent of PLK expression. Moreover, we noted that PLK is strongly expressed in invading carcinoma cells. PLK expression could reflect the degree of malignancy and proliferation in endometrial carcinoma. Thus, in addition to being of diagnostic value, modulation of PLK activity in the tumors by chemotherapeutic agents or gene therapy may prove to be of therapeutic value.Cancer Letters 08/2001; 169(1):41-9. · 4.26 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Polo-like kinase 1 (PLK1) is one of the serine threonine kinases that plays a role in cellular proliferation by activating the CDC25C-CDK1 (cdc2) loop. We recently demonstrated that cdc2 expression is associated with the biological aggressiveness of malignant lymphoma of the thyroid. In this study, we investigated PLK1 expression in thyroid lymphoma in order to elucidate its physiological significance in this disease. We immunohistochemically investigated PLK1 expression in 46 cases of thyroid lymphoma and 10 cases of chronic thyroiditis. Normal follicular cells did not express PLK1, whereas follicular cells in those in chronic thyroiditis and malignant lymphoma demonstrated a high level of PLK1 expression. In lymphocytes in chronic thyroiditis with strong lymphocyte infiltration, PLK1 was weakly expressed. In malignant lymphoma, PLK1 expression was observed in all cases, but PLK1 was more strongly expressed in cases with high cell proliferative activity and with cdc2 expression. These results suggest that PLK1 regulates the cell cycle progression of thyroid lymphoma cells in the G2-M-phase.Anticancer research 24(1):259-63. · 1.71 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The links between the cell cycle machinery and the cytoskeletal proteins controlling cytokinesis are poorly understood. The small guanine nucleotide triphosphate (GTP)-binding protein RhoA stimulates type II myosin contractility and formin-dependent assembly of the cytokinetic actin contractile ring. We found that budding yeast Polo-like kinase Cdc5 controls the targeting and activation of Rho1 (RhoA) at the division site via Rho1 guanine nucleotide exchange factors. This role of Cdc5 (Polo-like kinase) in regulating Rho1 is likely to be relevant to cytokinesis and asymmetric cell division in other organisms.Science 08/2006; 313(5783):108-11. · 31.20 Impact Factor
Polo-like kinase 1 is overexpressed in colorectal
cancer and participates in the migration and invasion
of colorectal cancer cells
Ding-pei HanABCDEF, Qian-lin ZhuCDE, Jiang-tao CuiBF, Pu-xiongzhi WangBCD,
Shun QuBD, Qi-feng CaoF, Ya-ping ZongD, Bo FengC, Min-hua ZhengG, Ai-guo LuAE
Department of General Surgery, Shanghai Ruijin Hospital, Shanghai Minimally Invasive Surgery Center, Shanghai,
Source of support: Natural Science Foundation of Shanghai, China, grant number: 08ZR1414000
Background: Polo-like kinase 1 (PLK1) is an important molecule in proliferation of many human cancers. The
aim of study is to clarify the expression patterns and potential function of PLK1 in colorectal cancers.
Material/Methods: Fifty-six colorectal cancers samples were collected and arranged onto a tissue array and the ex-
pression of PLK1 were detected by immunohistochemistry and correlated with clinico-pathologi-
cal characteristics and expression of PCNA. Expression of PLK1 in 9 colorectal cancer cells lines
was investigated by RT-PCR and Western blot, then SW1116 cells lines were treated with PLK1 siR-
NA and the efficiency was examined by Western blot. Transwell test was applied to detect the mi-
gration and invasion capability of cancer cells by counting the number of cells passing through
the membranes. Cell proliferation and apoptosis were examined by Cell Counting Kit-8 (CCK-8)
and Annexin-V Kit.
Results: PLK1 was positively expressed in 73.2% (41/56) of colorectal cancers tissues, but in only 3.6%
(2/56) of normal tissues, and was associated with Duke’s stage (P<0.01), tumor size (P<0.01), in-
vasion extent (P<0.05) and lymphatic metastasis (P<0.01). The expression of PLK1 was correlated
with expression of PCNA (R=0.553, P<0.01). PLK1 was inhibited in SW1116 cells by treating with
PLK1 siRNA oligos, which resulted in a decreased number of cells passing through the membrane
as compared with control groups (P<0.01) at 24 hours after transfection. Cell proliferation was
inhibited from 48 hours after transfection, while cells apoptosis was induced from 72 hours after
Conclusions: PLK1 could be a progression marker for colorectal cancer patients and PLK1 depletion can inhib-
it migration and invasion capability of colorectal cancer cells SW1116, suggesting that PLK1 might
be involved in metastasis and invasion of colorectal cancer. Therapeutic strategies targeting PLK1
may be a new approach to colorectal cancer.
key words:? Polo-like?kinase?•?PCNA?•?immunohistochemistry?•?colorectal?cancer?•?migration?•?invasion
Author’s address: Lu Ai-Guo, Department of General Surgery, Shanghai Ruijin Hospital, Shanghai Minimally Invasive Surgery Center,
197 Ruijin Er Rd, Shanghai 200025, China, e-mail: firstname.lastname@example.org
A Study Design
B Data Collection
C Statistical Analysis
D Data Interpretation
E Manuscript Preparation
F Literature Search
G Funds Collection
© Med Sci Monit, 2012; 18(6): BR237-246
Current Contents/Clinical Medicine • IF(2010)=1.699 • Index Medicus/MEDLINE • EMBASE/Excerpta Medica • Chemical Abstracts • Index Copernicus
Colorectal cancer is one of the most common gastrointesti-
nal cancers in Western countries and it ranks third in mor-
bidity and mortality of malignant cancer for both sexes .
In recent decades its morbidity and mortality have quickly
increased in some Asian countries . Although curative
surgery is improving and adjuvant/neoadjuvant treatment
strategies are now widely used , the 5-year survival is still
not satisfactory . Our aim in this study was to search for
biological markers of colorectal cancer with high sensitivity
and specificity, not only for the prediction of prognosis of
patients, but also for the direction of individual treatment.
Cancers are widely considered to be genetic diseases.
Abnormalities of cell proliferation and apoptosis resulting
from chromosomal instability (CIN) are involved in tumor-
igenesis . Moreover, the centrosome plays a key role in
stabilizing the chromosomal . In cancer cells, abnormal
centrosomes, such as supernumerary centrosomes, are of-
ten observed . We also know that dysregulated cell cycle
control contributes to tumorigenesis. If some proteins are
involved in both the formation of centrosomes and the cell
cycle, the proteins must be very important factors in tumor
development and may be potential oncogenes. The Polo-
like kinase 1 (PLK1) is one of these proteins.
PLK1, mammalian homologue of Polo (Drosophila) and
CDC5 (Saccharomyces cerevisiae), is a highly conservative ser-
ine/threonine kinase . Evidence shows that PLK1 plays
an important role in G2/M transition , bipolar spindle
formation and centrosome maturation . Knock-down
of the expression of PLK1 in human cancer cells can lead
to the failure of spindle assembly . It has been report-
ed that PLK1 is overexpressed in many human cancers, in-
cluding non-small-cell lung cancer , head and neck can-
cer , esophageal carcinoma , breast cancer ,
endometrial carcinoma , and non-Hodgkin’s lympho-
mas . It has been suggested that PLK1 might be used
as a diagnostic biomarker [12–14]. Moreover, overexpres-
sion of Plk1 in NIH3T3 cells could lead to oncogenic focus
formation and tumors in nude mice  and some studies
have found that depletion of PLK1 can induce apoptosis
and anti-proliferation effects in some cancers [19,20], sug-
gesting that PLK1 could be a good target for cancer therapy.
Some papers also showed overexpression of PLK1 in colorec-
tal cancer [21,22], but little is known about the other func-
tions of PLK1 in progression of cancer, especially in colorec-
tal cancer. In this study we analyzed the expression of PLK1
in colorectal cancer tissues, comparing it with the adjacent
normal tissues; and we postulate that PLK1 could be a cell
proliferation marker, just like proliferating cell nuclear an-
tigen (PCNA) . Finally, we knocked down the expres-
sion of PLK1 in colorectal cell line SW1116 by small RNA
interference method to observe its effect on cell motility,
proliferation and apoptosis.
Material and Methods
Patients and tissue samples
Fifty-six colorectal cancer samples and matched adjacent
normal tissues from diagnosed patients were obtained by
surgery between March 2005 and May 2008 at Ruijin Hospital,
Shanghai Jiaotong University School of Medicine, China. The
adjacent normal tissues were taken more than 5 centimeters
away from the cancer tissues. Clinicopathological information
regarding these 56 colorectal cancer cases were as follows: 34
males, 22 females; age from 32 to 89 years old, median age 67
years old; carcinoma locations were 13 in right-hemicolon, 3
in left-hemicolon, 14 in sigmoid colon, 26 in rectum; carci-
noma differentiation was 5 well differentiated, 45 moderately
differentiated, 6 poorly differentiated; and pathological TNM
staging (American Joint Committee on Cancer ) was 13
in stage I, 10 in stage II, 29 in stage III, 4 in stage IV. All the
samples were formalin-fixed and paraffin-embedded before
performing tissue array and immunohistochemical analysis.
After defining the area of tumors, all the formalin-fixed and
paraffin-embedded samples were cored and arranged on a
tissue array block by Shanghai Outdo Biotech Co., Ltd; each
core was 1.5 millimeter in diameter, and each section was 4
micrometer thick. The slides were deparaffinized in xylene
and rehydrated in concentration gradient ethanols. For an-
tigen retrieval, the slides were boiled in 0.01 mol/L sodi-
um citrate buffer, pH 6.0 for 15 minutes, and then the en-
dogenous peroxidase activity was blocked in 3% hydrogen
peroxide for 30 minutes, followed by blocking non-specif-
ic binding in 1% BSA for 15 minutes. The slides were incu-
bated with primary antibody diluted 1:50 in Tris-buffered sa-
line pH 7.6 (TBS) with 1% BSA of mouse anti-human PLK1
(Santa Cruz, USA) for 2 hours at room temperature. Next,
after washing in TBS, the slides were incubated with second-
ary biotinylated antibody and peroxidase-labeled streptavi-
din for 10 minutes. Finally, 3, 3’-diaminobenzidine was used
as chromogenic agent for 20 minutes. Before being exam-
ined with light microscopy, the slides were counterstained
with Mayer’s hematoxylin. Negative controls were incubat-
ed without primary antibody. Slides were prepared follow-
ing the same protocol with mouse anti-human PCNA mono-
clonal antibody (Santa Cruz, USA).
Evaluation of immunohistochemistry
Staining intensity of the slides was detected independent-
ly by 2 pathologists who were blinded to our study. The in-
tensity of staining was scored as follows: 0, negative; 1, weak;
2, moderate; 3, strong. The percentage of positive cells was
scored as: 0, <5%; 1, 5–30%; 2; 30–75%; 3, >75%. The final
score was calculated by multiplication of the 2 parameters
(mean of the scores from the pathologists): -, 0–2; +, 3–5;
++, 6–7; +++, 8–9. The cases were grouped as PLK1 & PCNA
negative (≤5) and PLK1 & PCNA positive (>5).
Human colorectal cancer cell lines SW1116, SW480, SW620,
Colo205, DLD-1, and Lovo were cultured in RPMI 1640 (10%
fetal bovine serum) and HCT116, HT29, and Caco-2 were
cultured in DMEM (10% fetal bovine serum).
RNA extraction and PCR
The total cellular RNA of the 9 colorectal cancer cell lines
were isolated from cells by TRIzol reagent (Invitrogen,
Basic Research Med Sci Monit, 2012; 18(6): BR237-246
USA) one-step method. AMV Reverse Transcriptase (AMV
RT) system was obtained from Promega, USA. Primers
were: PLK1 sense 5’-TGTTCGCGGGCAAGATTGT-3’ and
antisense 5’-GGCTGCGGTGAATGGATATTTC-3’; GAPDH
sense 5’-GGACCTGACCTGCCGTCTAG-3’ and antisense
5’-GTAGCCCAGGATGCCCTTGA-3’. The PCR cycling con-
dition was 31 cycles, 95°C for 15s (denaturation), 55°C for
30s (annealing), 72°C for 30s (extension). The PCR products
were detected in 2% agarose gel under ultraviolet radiation.
Real-time quantitative PCR
After transcription, 1 µl cDNA was mixed with SYBR Green
PCR Master Mix (Applied Biosystems, USA) as the techni-
cal manual. PCR cycling condition was 40 cycles, 95°C for
15s (denaturation), 55°C for 30s (annealing), and 72°C
for 30s (extension). The reactions were carried out in 96-
well plates on ABI Prism 7000 (Applied Biosystems, USA).
The results of PCR products were quantitatively confirmed
by using melting curves and 2(–∆Ct) method; the expres-
sion amount of PLK1 relative to GAPDH was expressed as
100*2(–∆Ct); H2O was defined as negative control.
Silencing of PLK1 by small RNA interference
The siRNA oligos targeted PLK1 (NCBI Reference Sequence:
NM_005030.3) and negative controls (NC, scrambled se-
quence) were designed by Shanghai GenePharma Co.,
Ltd; the sequences of the siRNA oligos are listed in Table 1.
Before transfection, cells were cultured in 6-well plates over-
night; each well contained 2×105 cells. When the abundance
ratio of cells was up to about 60–70%, cells were trans-
fected with siRNA oligos by using Lipofectamine 2000™
(Invitrogen, USA) according the manufacturer’s instruc-
tions. The time of changing medium (6 hours after add-
ing siRNA oligos) is considered as 0 hour after transfection.
Cytoplasm total protein was extracted at 48 and 72 hours af-
ter transfection, and the efficiency of the siRNA oligos was
examined by Western blot and real-time PCR.
Total protein lysates (50 µg) were separated on 10% SDS-
PAGE gel and transferred onto PVDF membranes at 15 volt
for 1 hour. After being blocked in 5% skim milk for 2 hours
at room temperature, the membranes were incubated with
primary antibody of PLK1 (diluted 1:200) and GAPDH (di-
luted 1:2000) for 2 hours at room temperature, followed by
incubated with HRP-linked secondary antibody (Santa Cruz,
USA) for 1 hour at room temperature. Finally, the bands
were detected by using DAB reagent (Dako Corporation,
Migration and invasion assay
For migration assay, 6.5mm Transwell® with 8 µm pore
Polycarbonate Membrane Insert (Corning, USA) was applied.
At 0 hours after transfection, 200 µl of serum-free cell suspen-
sion containing 3×104 PLK1-siRNA-transfected cells were add-
ed into the upper chamber and 600 µl of medium containing
20% fetal bovine serum was added into the lower chamber.
After 24 hours of incubation, the cells were stained by methyl
violet and scraped off the cells on the bottom of upper cham-
ber. For invasion assay, the same steps were performed except
that the bottom of the upper chamber was covered with 100 µl
of diluted growth factor-reduced Matrigel (BD Corporation,
USA). The result was determined by counting the stained
cells passing through the pores in the membrane; data are ex-
pressed as means ±SD of counting 5 random fields of vision.
Cell proliferation assay
Cell Counting Kit-8 (CCK-8, Dojindo, Japan) uses a type of
highly water-soluble tetrazolium salt of WST-8. When WST-8
is reduced by dehydrogenases in cells it produces a yellow-
colored product (formazan), so the amount of the forma-
zan dye generated by the activity of dehydrogenases in cells
is directly proportional to the number of living cells. The
detection sensitivity of CCK-8 is higher than other tetrazo-
lium salts such as MTT, XTT, MTS or WST-1, according to
the CCK-8technical manual.
In this study, cells were cultured in 96-well plates with 100 µl medi-
um/well, and transfected with PLK1 & NC siRNA oligos accord-
ing the manufacturer’s instructions of Lipofectamine 2000™.
At 24, 48, 60, 72, 96, and 120 hours after transfection, 10 µl
CCK-8 was added. After incubation for 2 hours, absorbance
was measured at 450nm by a Microplate reader (µQuant, Bio-
Tek, USA). The PLK1-siRNA-treated group was compared with
NC group and Mock group in the proliferation curves chart.
Cell apoptosis was evaluated by using the Annexin V kit (BD
Corporation, USA). At 24, 48, and 72 hours after transfec-
tion, cells were incubated with Annexin V antibody and PI
according to the manufacturer’s instructions. Apoptosis ra-
tio of the 3 groups (PLK1-siRNA, NC, and Mock) was de-
tected by flow cytometry (FCM).
c2 test or Fisher’s exact test was used to examine the difference
in positive expressed rate of PLK1, PCNA between colorec-
tal cancer tissues and normal tissues; correlation of PLK1
Table 1. Sequences of small synthetic oligonucleotides unique to PLK1.
Med Sci Monit, 2012; 18(6): BR237-246Han D-p et al – Polo-like kinase 1 is overexpressed in colorectal cancer…
expression level and clinicopathological characteristics were
examined by c2 test or Fisher’s exact test and c2 test for trends;
Pearson correlation test was performed to examine the cor-
relation between PLK1 expression and PCNA expression.
One-way analysis of variance (ANOVA) was used to test the
difference in apoptosis rate between the 3 groups and the dif-
ferent effect on cell proliferation between the 3 groups at ev-
ery moment. P<0.05 was considered as statistically significant.
Expression of PLK1 and PCNA in colorectal cancer
tissues and adjacent normal tissues
In colorectal cancer tissues, PLK1 was cytoplasmically stained
and scored positively in 41 out of 56 cases (Table. 2, Figure 1B
and C); PCNA was stained and scored positively in 49 out of
56 cases (Table 2, Figure 1E and F). In adjacent normal tis-
sues, PLK1 and PCNA of almost all cases were stained neg-
atively (Figure 1A and D), but 2 and 6 cases of PLK1 and
PCNA group were stained positively. Positive expressions of
PLK1 and PCNA in colorectal cancer tissues were significant-
ly greater than in normal tissues – 73.2% (41/56) in cancer
tissues vs. 3.6% (2/56) in normal tissues of PLK1 (P<0.01),
87.5% (49/56) vs. 10.7% (6/56) of PCNA (P<0.01, Table 2).
Association between expression of PLK1 and
clinicopathological characteristics of colorectal cancers
Statistically significant associations were not observed be-
tween PLK1 expression and sex, age, histological differen-
tiation, tumor location and distant metastasis (Table 3).
However, there was a statistically significant association with
Duke’s stage (P<0.01), tumor size (P<0.01), invasion extent
(P<0.05) and lymphatic metastasis (P<0.01).
Correlation between expression of PLK1 and PCNA in
According to the immunohistochemistry results, 15/56
colorectal cancer cases showed +, 32/56 showed ++, and
Total PLK1 positive PLK1 negative
P-value PCNA positive PCNA negative
Normal tissues56 2 (3.6) 54 (96.4)
6 (10.7) 50 (89.3)
Cancer tissues56 41 (73.2) 15 (26.8) 49 (87.5) 7 (12.5)
Table 2. PLK1 and PCNA expression in colorectal tissues, n (%).
Figure 1. Expression of PLK1 and PCNA in colorectal tissues. (A) PLK1 negatively expressed in normal colorectal tissues; (B) PLK1 moderately
expressed in colorectal cancer tissues; (C) PLK1 strongly expressed in colorectal cancer tissues; (D) PCNA negatively expressed in normal
colorectal tissues; (E) PCNA moderately expressed in colorectal cancer tissues; (F) PCNA strongly expressed in colorectal cancer tissues.
Original magnifications ×200.
Basic ResearchMed Sci Monit, 2012; 18(6): BR237-246
≤67 years old
>67 years old
Tumor size (cm2)** 0.000
Tumor invasion** 0.021
0 (7.1) (0.0)
Table 3. Association between PLK1 expression and clinical characteristics of colorectal cancers, n (%).
* Fisher’s exact test; ** χ2 tests for trends.
Med Sci Monit, 2012; 18(6): BR237-246 Han D-p et al – Polo-like kinase 1 is overexpressed in colorectal cancer…