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Expression of Interleukin 1β in Gastric Cancer Tissue
and Its Effects on Gastric Cancer
Shenglu Yin*, Chao Lan, Hui Pei, Zhiqiang Zhu
The Department of the Emergency Center, the First Affiliated Hospital of Zhengzhou University,
Zhengzhou, Henan, 450052, China
*Correspondence: Shenglu Yin
The Department of the Emergency Center, the First Affiliated Hospital of Zhengzhou University,
Zhengzhou, Henan, 450052, China
Email : [email protected]
Abstract
Objective: This study detected expression level of interleukin 1β (IL-1β) in gastric cancer tissue
and para-carcinoma tissue and analyzed its relationship with clinical and pathological features, so
as to explore expression of IL-1β in gastric cancer tissue and its effect on gastric cancer.
Methods: This experiment was carried out in the lab of the affiliated provincial hospital of
Shandong University in April 2015. A total of 70 fresh primary gastric cancer tumors and
corresponding para-carcinoma tissues away from tumor incisal edge (≥ 5 cm) were selected.
Their total protein (TP) concentration was extracted with radio immunoprecipitation assay (RIPA)
lysis buffer and IL-1β content in tissues was detected with enzyme-linked immunosorbent assay
(ELISA), ratio of cell cytokine content and protein concentration was the relative cell cytokine
content, and the relationship between content of relative cytokines and clinical data was analyzed.
Conclusion: difference of content of IL-1β in gastric cancer tissues and para-carcinoma tissues
had no statistical significance, but content of IL-1β would rise with the increase of tumor through
analysis (4.2 > 2.9 ng/g) and there was a statistical difference (P < 0.05). Furthermore, content of
IL-1β reduced along with differentiation degree and infiltration depth (4.15 > 3.21ng/g, 3.45 > 3.06
ng/g), and the difference was not statistically significant (P > 0.05). Thus, it can be concluded that
IL-1β plays an important role in occurrence and development of gastric cancer tissue and
promotes treatment of patients with gastric cancer to some extent.
Keywords: gastric cancer; IL-1β; helicobacter pylori
Introduction
Gastric cancer, one of the most common malignant tumors in the world, has the second
death rate among malignant tumors [1], and in China, its morbidity ranks the second of national
malignant tumor registration area (322,300 /100,000) and death rate ranks the third (329,000
/100,000) [2]. Recently, death rate of gastric cancer is still the top in some countries and areas
although it has reduced morbidity, and with 5-year survival rate of only 5% ~ 10% [3-8], so
occurrence and development mechanism of gastric cancer is of great significance for the
prevention and treatment of gastric cancer. It have been confirmed that occurrence of gastric
cancer caused by various factors is a developing process that its own conditions and external
reasons participate in jointly, involving multiple gene mutation. In 1994, world health organization
(WHO) officially announced that helicobacter pylorus (HP) was a risk factor leading to gastric
cancer [9]. HP infection has individual differences in influencing the occurrence of gastric cancer,
which is related to genetic factors of infected person. Some studies verify that interleukin 1β (IL1β) infected with HP would promote gastric cancer [10], and a large number of epidemiology,
genetic and molecular biology studies confirm that persistent chronic inflammation plays a vital
role in activating, maintaining and promoting the growth of gastric cancer. IL-1β, a kind of
proinflammatory cytokine, regulates a variety of mechanisms, such as inflammatory immune
injury, gene epigenetics changes, etc.; and meanwhile, has strong effects in inhibiting gastric acid
secretion, raising bone marrow cells and promoting angiogenesis, which is closely related to
development of gastric mucosal lesion and atrophic gastritis infected by HP and occurrence of
gastric cancer [11].
IL-1β has been found to have a key role in occurrence and development of tumor, on the
one hand, it takes part in transformation, growth, invasion and metastasis of malignant tumors, on
the other hand, it is able to activate body’s immune system to limit tumor growth [12]. To date,
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effect of IL-1β on occurrence and development of tumor is still in dispute. This thesis aims to
discuss influence of IL-1β expression level on occurrence, development and treatment of gastric
cancer by analyzing the correlation of IL-1β level and clinical features in gastric cancer.
Materials and methods
Patient’s Clinical Data
This is original research that a total of 70 patients with primary gastric cancer who went
through operative treatment in the first affiliated hospital of Zhengzhou University from January to
December 2014, in which, 50 patients were male and the rest of them were female (average age:
61.5 ± 10.5 years), and none of them received any radio and chemo therapy before operation.
According to gastric cancer tumor node metastasis (TNM) staging (2010) issued by American
Joint Committee on Cancer (AJCC), patients were divided as follows: I~II stage (25 cases), III~IV
stage (45 cases); radical operation treatment (65 cases), palliative operation treatment (5 cases).
Fresh tumor tissues and corresponding para-carcinoma tissues away from tumor incisal edge (≥
5 cm) were frozen with liquid nitrogen quickly after excision, and preserved in the refrigerator at 80 °C for standby application. This experiment was examined and approved by the ethics
committee of the First Affiliated Hospital of Zhengzhou University, and all patients signed
informed consent.
Sample Collection
Firstly, 1 ml powerful radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime
Company, Shanghai, China) was added into each tissue stored in the refrigerator at - 80 °C, then,
tissues were smashed with biological sample homogenizer (Bertin Precellys24, France) and
centrifuged at 4 °C. Afterwards, liquor was sub packaged and preserved at - 80 °C.
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Experimental Methods
First of all, protein concentration was tested based on protein assay (BCA) kit (Pierce,
America), then ratio of cell cytokine content and sample protein concentration (relative cell
cytokine content) was detected with enzyme-linked immunosorbent assay (ELISA). IL-1β
detection kit came from Invitrogen Company (America) and microplate reader was from Tecan
Safire 2 (Switzerland). Tissue protein was selected and unfrozen, standard substance performed
double dilution, then 50 μl was taken and added into commercialized ELISA plate, 100 μl biotin
antibodies was added in each hole and it was incubated at room temperature for 2 h. After liquid
was taken out of hole, it was washed continuously for 4 times with 400 μl scrubbing solution,
beaten repeatedly and sucked dry with filter paper, then 100 μl streptavidin- horseradish
peroxidase working liquid was added in every hole, and it was incubated at room temperature for
0.5 h. Afterwards, it was washed repeatedly for 4 times after liquid was taken out of hole, and
beaten to dry, then, hole was incubated at room temperature for 0.5 h after 100 μl color-substrate
solution was added, and finally, 100 μl stop buffer was added to terminate enzyme reaction.
Statistical Method
SPSS 13.0 software was used for statistical analysis, experimental data non-conforming
to normal distribution were performed Wilcoxon rank sum test and H test and expressed with
median; t test and one-way variance analysis were carried out on data conforming to normal
distribution and expressed with mean ± SD. The difference was statistically significant if P < 0.05.
Results
IL-1β Content in Tumor Tissue and Para-Carcinoma Tissue
Of 70 tumor tissues of patients with gastric cancer, IL-1β content (2.66 ng/g) had no
statistical difference in tumor tissue and para-carcinoma tissue (table 1).
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Relationship between IL-1β Content and Clinical and
Pathological Features in Tumor Tissues
Of 70 gastric cancer samples, IL-1β content rose as tumor increased, and the difference
was statistically significant (P < 0.05), but difference of difference ages, blood types, genders,
number of lymph node metastasis, tumor sites and TNM staging had no statistical significance (P
> 0.05, table 2).
Discussion
IL-1 gene was found to be located in human chromosome 2q13-14 in 1972, with total
length of about 430 kb, and it was known as lymphocyte-activating factor at the very start
because of being initially found as a kind of lymphocytic mitotic factor. Thereafter, studies
discovered that IL-1 consisted of IL-1α, IL-1β and IL-1 receptor antagonist, in which, IL-1β gene
was one of the most important member of the family, including 153 amino acid residues and
excluding disulfide bond, but two cysteine residues were essential for IL-1β activity [13]. Some
other researches found that [14-16] IL-1β was closely related to occurrence of diversified
malignant tumors, for instance, esophageal cancer, lung cancer, colon cancer, cervical cancer,
breast cancer and prostate cancer, and equally, expression of IL-1β in gastric cancer increased
obviously. Through testing three kinds of cytokines (IL-1β, TNF-α and IL-8) in serum of patients
with gastric cancer, Macrì et al. [17] figured out that expression levels of above three cytokines in
serum of patients with gastric cancer were higher than normal control group. Therefore, those
three indexes were believed to be gastric cancer markers used for detecting occurrence of gastric
cancer. IL-1β not only expresses highly in gastric cancer tissues, but also is notably associated
with clinical and pathological features of gastric cancer. Occurrence and development of gastric
cancer have an intimate connection with inflammation, HP infection plays a key role in it, and
elimination of HP infection has been widely accepted [18].
Risk factors of gastric cancer contain environment, host genetic factor and interaction of
them. As to environment, HP infection rate reaches to about 70% in common people in China and
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increases at the rate of 2%, which is recognized as a significant pathogenic factor of chronic
gastritis and peptic ulcer. IL-1β that works by activating stomach prostaglandin synthesis directly
affects physiological responses of the stomach, and potential physiopathologic mechanism inside
stomach will be activated when it is invaded by HP. Most researches focus on the effect of
bacterial virulence on gastric cancer, but it cannot completely explain various clinical results of
HP infection, and in which, host genetic factor is also vital. Relationship between genetic element
IL-1β gene polymorphism and gastric cancer has been a popular study point, IL-1β gene
encoding is highly polymorphic, and there are two in -31 and -51 in promoter region, on behalf of
transformation of C-T and T-C, respectively.
With the continuous development of modern medical research techniques, people’s
understanding to IL-1β and HP is deepening. IL-1β influences the occurrence and development of
gastric cancer mainly through inhibiting gastric acid secretion, inducing changes of gene
epigenetics, collecting bone marrow cells, promoting angiogenesis, luring adhesive factor and
other inflammatory factors release, etc. IL-1β is of strong gastric acid secretion inhibitory effect, 1
g molecule of IL-1β gastric acid secretion inhibition capacity is equal to 100 times of the same
dose of omeprazole (proton pump inhibitor) and 6,000 times of cimetidine. It is assumed that IL1β is capable of increasing the risk of gastric cancer due to lipogastry caused by strong acid
suppression effect of it. A large number of findings confirm that the incidence of gastric cancer in
HP infected person before lesions is significantly higher than that without infection, because cell
apoptosis and proliferation increase in HP infected gastric antral mucosa and increasing extent is
correlated with degree of inflammation. During the occurrence and development of gastric cancer,
HP is likely to lead to imbalance of the proliferation and apoptosis of gastric mucosa epithelial cell
and trigger abnormal gastric mucosa by promoting the abnormal expression of certain genes, and
finally result in gastric cancer.
Relationship between inflammation and tumor has become the research hot spot since
Balkwill et al [19] originally found a relevance existing in chronic inflammation and canceration. A
study [20] have verified that chronic inflammation plays a role in three stages of start, proliferation
and progress of the tumor formation, furthermore, current research focus has transferred from the
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nature of immune cells infiltration to cytokines and main role of its soluble medium in tumor
activation. IL-1β as a core element in inflammatory medium is a key factor affecting occurrence
and development of gastric cancer, but its mechanism in gastric cancer is still not clear
completely at present. Hence, further study on IL-1β can provide a novel thought for revealing the
pathogenesis of gastric cancer.
To sum up, obviously high IL-1 expression level in tumor tissues is intimately associated
with occurrence and development of tumor. Results obtained from this study demonstrated that
IL-1β as a core element in inflammatory medium is a key factor affecting occurrence and
development of gastric cancer, but its mechanism in gastric cancer is still not clear completely at
present. Hence, looking for cancer promotion mechanism inducing IL-1β expression is expected
to become a new target for the treatment of gastric cancer to further control occurrence,
development and metastasis of gastric cancer.
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Table 1 IL-1β content in tumor tissue and para-carcinoma tissue
Cell
Sample
Cell cytokine
Cell cytokine content
Pair comparison
cytokines
size
content in cancer
in para-carcinoma
of Z value of
tissue
tissue
rank sum test
3.45
2.66
-3.20
IL-1β
70
10
P value
<0.01
Table 2 Relationship between IL-1β content and clinical and pathological features in tumor
tissues
Clinical and pathological
Sample size
IL-1β
features
Age
IL-1β level
Test statistics
P value
-0.09
0.92
-0.69
0.48
1.56
0.66
-0.87
0.38
-1.15
0.24
0.80
0.84
-2.16
0.03
0.64
0.72
>60
37
3.81
≤60
33
3.50
Male
52
3.59
Female
18
4.04
A
25
3.59
B
13
3.06
O
22
4.91
AB
10
2.79
I~II stage
24
4.15
III stage
46
3.21
Invasion
T1~T2
12
3.45
depth
T3~T4
58
3.06
Number of
N0(0)
15
3.31
lymph node
N1(1-2)
9
3.13
metastasis
N2(3-6)
28
3.59
N3(≥7)
18
5.30
Diameter of
≥5cm
45
3.21
tumor
<5cm
25
3.80
Tumor site
Antrum of
50
3.45
Gender
Blood type
Differentiated
degree
stomach
11
Gastric
10
6.00
10
3.16
25
3.15
body
Cardia of
stomach
TNM staging
I~II
12
-0.30
0.76