Zheng HC, Li YL, Sun JM, Yang XF, Li XH, Jiang WG, Zhang YC, Xin Y. Growth, invasion, metastasis, differentiation, angiogenesis and apoptosis of gastric cancer regulated by expression of PTEN encoding products. World J Gastroenterol 2003; 9(8): 1662-1666 [PMID: 12918097 DOI: 10.3748/wjg.v9.i8.1662]
Corresponding Author of This Article
Hua-Chuan Zheng, Lab. 4, Cancer Institute, The First Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning Province, China. zheng_huachuan@hotmail.com
Article-Type of This Article
Gastric Cancer
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Hua-Chuan Zheng, Jin-Min Sun, Xue-Fei Yang, Yin-Chang Zhang, Yan Xin, Lab. 4, Cancer Institute, The First Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning Province, China
Yi-Ling Li, Department of Digestive Diseases, The First Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning Province, China
Xiao-Han Li, Wei-Guo Jiang, Department of Pathology, The Second Affiliated Hospital of China Medical University, Shenyang 110004, Liaoning Province, China
ORCID number: $[AuthorORCIDs]
Author contributions: All authors contributed equally to the work.
Correspondence to: Hua-Chuan Zheng, Lab. 4, Cancer Institute, The First Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning Province, China. zheng_huachuan@hotmail.com
Received: March 5, 2003 Revised: March 12, 2003 Accepted: March 29, 2003 Published online: August 15, 2003
Abstract
AIM: To investigate expression of PTEN in gastric cancer and to explore its roles in tumorigenesis and progression of gastric cancer.
METHODS: Formalin-fixed and paraffin-embedded tissues of adjacent non-tumor mucosa and primary foci from 113 cases of gastric cancers were studied for the expression of PTEN and Caspase-3 and microvessel density (MVD) by streptavidin-peroxidase (S-P) immunohistochemistry with antibodies against PTEN, Caspase-3, and CD34. The relationship between PTEN and Caspase 3 expression and clinicopathological parameters of tumors was compared.
RESULTS: Primary gastric cancer cells expressed PTEN less frequently than adjacent epithelial cells of primary foci (54.9% vs 89.4%; P = 0.000, χ2 = 33.474). PTEN expression was significantly associated with invasive depth (P = 0.003, rs = 0.274), metastasis (P = 0.036, rs = 0.197), growth pattern (P=0.008, rs = 0.282), Lauren’s classification (P = 0.000, rs = 0.345), and histological classification (P = 0.005, rs = 0.262) of tumors, but not with tumor size (P = 0.639, rs = 0.045), Borrmann’s classification (P = 0.544, rs = 0.070) or TNM staging (P = 0.172, rs = 0.129). PTEN expression was negatively correlated with MDV in primary gastric cancer (P = 0.020, F = 5.558). Primary gastric cancer cells showed less frequent immunoreactivity to Caspase-3 than adjacent epithelial cells of primary foci (32.7% vs 50.4%; P = 0.007, χ2 = 7.286). Caspase-3 expression was dependent of PTEN expression in primary gastric cancer cells (P = 0.000, χ2 = 15.266).
CONCLUSION: Down-regulated expression of PTEN plays an important role in tumorigenesis, progression, growth, differentiation and angiogenesis of gastric cancer. Low expression of PTEN can decrease expression of Caspase-3 to disorder apoptosis of tumor cells, which might explain the molecular mechanisms of PTEN contributions to tumorigenesis and progression of gastric cancer.
Key Words: $[Keywords]
Citation: Zheng HC, Li YL, Sun JM, Yang XF, Li XH, Jiang WG, Zhang YC, Xin Y. Growth, invasion, metastasis, differentiation, angiogenesis and apoptosis of gastric cancer regulated by expression of PTEN encoding products. World J Gastroenterol 2003; 9(8): 1662-1666
Human suppressor gene, PTEN/MMAC1/TEP1 (phosphatase and tensin homology deleted from chromosome ten/mutated in multiple advanced cancer 1/TGF-β-regulated and epithelial cell enriched phosphatase 1), located on chromosome 10q23. 3 encodes a dual specific protein- phospholipid phosphatase that is involved in regulation of a variety of signal transduction pathways[1]. PTEN inhibits shc’s (src-homology collagen) phosphorylation following epidermal growth factor (EGF) stimulation and therefore blocks the activation of the Ras/MAP-kinase (MAPK) pathway[2]. Another mechanism that involves the protein phosphatase activity of PTEN is dephosphorylation and inactivation of focal adhesion kinase (FAK), thus playing a crucial role of PTEN in the interaction between extracellular matrix and cytoskeleton[3,4]. Besides its function as the protein phosphase, PTEN acts as a phospholipid phosphatase with phosphatidylinosiol 3,4,5-triphosphate (PIP3) as a substrate[5-7]. Recently, many studies have shown that there are several putative mechanisms relating to tumor suppression as follows: inhibiting cell invasion and metastasis by dephosphorylating FAK, inhibiting cell apoptosis and increasing cell growth by dephosphorylating PIP3, restraining cell differentiation by inhibiting MAPK signal pathway[5,8,9]. Mutation or abnormal expression of PTEN protein occurs commonly in multiple tumors and significantly correlates with tumorigenesis and progression of different malignancies[10-20]. It was reportedly suggested that deletion or mutation of PTEN could enhance the expression of vascular epithelial growth factor (VEGF) and matrix metalloproteinases (MMPs), which in turn closely correlated with tumor angiogenesis and metastasis[10-14]. Jones et al[15] found that activation of PTEN signal pathway could reduce expression of Caspase-3, resulting in inhibition of cellular apoptosis.
Gastric cancer is one of the commonest malignancies in China, and even in the world. In patients with gastric cancer, the natural disease process consists of carcinogenesis, metastasis and eventual death. However, the molecular aspects of carcinogenesis and progression of gastric cancer remain elusive[16-18]. In the current study, we evaluated the expression of PTEN in adjacent epithelial cells, primary gastric cancer cells and intented to find if there was any correlation between its expression and clinicopathological features and microvessel density (MVD) of gastric cancer, as well as between PTEN and Caspase-3 expression in primary foci in order to clarify its role in tumorigenesis and progression of gastric cancer.
MATERIALS AND METHODS
Patients
One hundred and thirteen cases of surgically resected specimens of gastric cancer were collected from the Second Affiliated Hospital of China Medical University from Sept, 1997 to Feb, 2001, including 83 men and 30 women. Their age ranged from 26 to 83 years, with the mean age of 57.1 years. Among them, 38 tumors were accompanied by lymph node or organ metastasis. None of the patients had received radiotherapy or chemotherapy before operation.
Preparation of tissue samples
Adjacent mucosa and primary lesions of each case were fixed in 4% formaldehyde solution, embedded in paraffin, incised into 4 μm sections and mounted on poly-lysine-coated slides. These sections were stained by hematoxylin-and-eosin method to confirm their histological diagnosis and other microscopic characteristics.
Evaluation of clinicopathological parameters
Clinical staging for each gastric carcinoma was evaluated according to the TNM system. Gross appearance of the tumors was described according to the Borrmann’s classification. Histomorphological architecture of the tumor samples was expressed on the basis of Lauren’s and Nakamura’s classifications. Growth patterns of gastric cancer were classified in the light of Zhang’s classification. Tumor diameter, invasive depth and metastasis were determined as well.
Immunohistochemistry
Representative and consecutive sections were studied with streptavidin-peroxidase immunohistochemistry (S-P kit from Zhongshan Biotech., China). Anti-PTEN, anti-CD34 and anti-Caspase-3 antibodies were purchased from Antibody Dignostica (USA), Zhongshan (China), and DAKO (Japan), respectively. All procedures were implemented according to the product recommendation. For negative controls, sections were processed as above but treated with PBS (0.01 mol/L, pH7.4) instead of primary antibodies.
Evaluation of PTEN and Caspase-3 immunostaining
The immunoreactivity to PTEN and Caspase-3 was localized in the cytoplasm. From 5 randomly selected representative fields of each section, two independent observers counted one hundred cells. According to the proportion of positive ones in counted cells, the degree of immunostaining was graded as follows: negative(-), ≤ 5%; weakly positive (+), 5%-25%; moderately positive (++), 25%-50%; and strongly positive (+++), ≥ 50%.
Microvessel density counting
Modified Weidner’s method was used to calculate MVD by anti-CD34 immunohistochemistry, which was described as follows. Microvessels in sclerotic areas within tumor, where microvessels were sparse and immediately adjacent areas of unaffected gastric tissue were considered as hot points in vessel counts. Observers selected five such areas and counted individual microvessels in a 400×field (i.e. 40×objective lens and 10×ocular lens, 0.1885 mm2 per field). Any brown staining endothelial cell or endothelial cell cluster that was clearly separated from adjacent microvessel, tumor cells, and other connective tissue elements was considered as a single, countable microvessel. They must agree on what constituted a single microvessel.
Statistical analysis
Statistical evaluation was performed using chi-square test to compare the rates between different groups, using Spearman test to analyze the rank data, and using the one-way ANOVA to differentiate the means of different groups. P < 0.05 was considered as statistically significant. SPSS 10.0 software was employed to analyze all data.
RESULTS
PTEN expression in adjacent epithelial cells and cancer cells of primary gastric cancer
Figure 1, Figure 2 and Figure 3 show that PTEN was positively expressed in the nuclei of adjacent epithelial cells, lymphocytes and cancer cells of primary foci of gastric cancer. In this study, epithelial cells and cancer cells were immunostained for PTEN protein in 89.4% and 54.9% of the tumors, respectively. There was a significant difference between them (P < 0.05) (Table 1).
Table 1 PTEN expression in adjacent epithelial cells and cancer cells of primary gastric cancer.
Figure 3 PTEN was negatively expressed in poorly-dif-ferentiated adenocarcinoma cells (-) and tumor infiltrated lymphocytes showed strong expression of PTEN (+++) (S-P, ×400).
Correlation between PTEN expression in primary cancer cells and clinicopathological features of gastric cancer
PTEN expression in primary cancer cells was significantly associated with invasive depth, metastasis, growth pattern, Lauren’s Classification, and histological classification of gastric cancer (P < 0.05). There was no close correlation between PTEN expression and tumor size, Borrmann’s Classification or TNM staging (P > 0.05). One ANOVA analysis showed PTEN expression was negatively correlated with MDV in primary gastric cancer (P < 0.05) (Table 2, Table 3, Figure 4).
Table 2 Relationship between expression of PTEN in primary foci and clinicopathological features of gastric cancer.
Clinicopathological features
n
PTEN expression
rs
P value
-
+
++
+++
PR(%)
Tumor size
0.045
0.639
< 4 cm
47
20
6
4
17
57.4
≥ 4 cm
66
31
7
7
21
53.0
Borrmann’s Classification
0.070
0.544
I/II
28
12
3
3
10
57.1
III/IV
59
30
5
6
18
49.2
Invasive depth
0.274
0.003
Above submucosa
26
7
4
2
13
73.1
Muscularis propria
34
14
2
4
14
58.8
Below subserosa
53
30
7
5
11
56.6
Metastasis
0.197
0.036
Negative
76
28
10
8
29
62.7
Positive
38
23
3
3
9
39.5
TNM staging
0.129
0.172
O
18
7
4
1
6
61.1
I
28
11
2
2
13
60.7
II
40
19
3
5
13
52.5
III
17
7
3
3
4
58.8
IV
10
7
1
0
2
30.0
Growth pattern
0.282
0.008
Mass
23
8
2
2
11
65.2
Nest
30
12
3
4
11
60.0
Diffuse
34
22
3
3
6
35.3
Lauren’s Classification
0.345
0.000
Intestinal type
36
8
2
6
20
77.8
Diffuse type
57
32
10
3
12
43.9
Mixed type
20
11
1
2
6
45.0
Histological classification
0.262
0.005
Differentiated
53
18
3
8
24
66.0
Undifferentiated
60
33
10
3
14
45.0
Table 3 Relationship between expression of PTEN expression and MVD in primary gastric cancer.
Figure 4 CD34 was located in cytoplasm and cellular mem-brane of vascular endothelial cells (S-P, ×400).
Relationship between PTEN and Caspase-3 expression in primary gastric cancer cells
Figure 5 and Figure 6 showed positive immunostaining of Caspase-3 in the cytoplasm of adjacent epithelial cells, infiltrating lymphocytes, and cancer cells of primary gastric cancer. Cancer cells showed less frequent immunoreactivity to Caspase-3 than adjacent epithelial cells (32.7% vs 50.4%; P < 0.05). Moreover, Caspase-3 expression was dependent of PTEN expression in primary gastric cancer cells (P < 0.05) (Table 4).
Table 4 Relationship between expression of PTEN and Caspase-3 in primary gastric cancer cells.
Figure 6 Caspase-3 was positively expressed in well-differ-entiated adenocarcinoma of stomach (++), and strongly in tu-mor infiltrated lymphocytes (+++) (S-P, ×400).
DISCUSSION
Deletion or down-regulation of tumor suppressing genes plays an important role in the multiple steps of tumorigenesis and progression of gastric cancer. PTEN, a tumor-suppressing gene, makes a great contribution to cellular differentiation, reproduction and apoptosis, as well as cellular adhesion and mobility[8,9]. Some studies showed down-regulation of PTEN protein expression was due to genetic changes such as mutation, loss of heterozygosity, hypermethylation in gastric cancer, prostate cancer and breast cancer[19-21]. Our results showed that expression of PTEN decreased in primary gastric cancer cells, compared with that in adjacent epithelial cells. This revealed that genetic changes of PTEN gene could play an important role in malignant transition of gastric epithelial cells.
Low expression of PTEN gene product was involved in clinicopathological stage and metastasis of various malignancies. We found that the positive rate of PTEN was lower in gastric cancer with metastasis than that without metastasis. Additionally, the positive rate of PTEN expression tended to decrease with increase of invasive depth. These results were similar to other kinds of tumors[22-29]. Mass-type gastric cancer showed more expression of PTEN than nest-type one, the latter more than diffuse-type one, demonstrating that PTEN expression was implicated in the growth pattern of gastric cancer. These results suggest that deletion or reduced expression of PTEN protein probably facilitates the invasive and metastatic ability of gastric cancer cells. Several studies revealed that PTEN could enhance mobility and metastasis of tumor cells by regulating MMPs and VEGF[10-14]. There was another report that PTEN dephosphorylated FAK so as to be involved in cellular adhesion[30]. Deletion or reduced expression of PTEN could result in decreased cellular adhesion, increased synthesis of MMPs and VEGF, which subsequently contributed to invasion and angiogenesis of tumor tissues. These biological effects possibly underlay the prelude of invasion and metastasis of tumor. Our results revealed that reduced expression of PTEN was implicated in progression of gastric cancer probably by decreased cellular adhesion, increased cellular mobility and angiogenesis, and could act as an objective and effective marker to reflect the pathobiological behaviors of gastric cancer.
In addition, undifferentiated carcinomas showed the lower expression of PTEN than differentiated ones, suggesting that down-regulated expression of PTEN was closely associated with differentiation of gastric cancer. Diffuse-type gastric cancer showed fewer expression of PTEN than intestinal-type one. It supported that there were different tumorigenetic pathways between diffuse- and intestinal-type gastric cancer. Diffuse-type gastric cancer, the main part of which was undifferentiated, showed diffusely invasive growth pattern. It was possible that down-regulation of PTEN expression could influence the function of cellular skeleton, mobility and adhesion of cancer cells.
Angiogenesis was necessary for tumor growth, invasion and metastasis, and tumor-host interaction induced several sorts of tumor angiogenetic factors such as VEGF and MMPs to stimulate the intratumoral and peritumoral neovascularization[31,32]. In this study, we found that mean MVD was negatively related to PTEN expression in primary gastric cancer. Huang et al[12] found that inhibition of endogenous endothelial PTEN by adenovirus-mediated over-expression of a dominant negative PTEN mutant in cultured endothelial cells potently enhanced a variety of VEGF-mediated cellular responses by PIP3-kinase signaling, including cell survival, mitogenesis, and migration. In contrast, these effects of VEGF were significantly inhibited by over-expression of wild-type PTEN. Moreover, overexpression of wild-type PTEN modulated endothelial tube formation in vitro and vascular sprouting in an ex vivo model of angiogenesis. On the other hand, decreased and loss of expression of wild-type PTEN would facilitate MMPs expression which played an important role in angiogenic processes, including proliferation of endothelial cells, breakdown of the extracellular matrix and endothelial cell migration[33]. These in vivo and in vitro observations suggested that down-regulated expression of PTEN in gastric cancer increased angiogenesis by up-regulating VEGF and MMPs.
Some investigators found that PTEN could promote cell apoptosis, which in turn inhibited tumor growth[34]. Additionally Caspase-3 played an important role in apoptosis as an effector. Krajewska et al[35] delineated for the first time some of the in vivo patterns of Caspase-3 expression in human tissues. Their observation that Caspase-3 was detectable in almost all types of cell implied its role in the regulation of cell life and death in a variety of types of cells as a proteinase. Our finding showed that Caspase-3 was less frequently expressed in gastric cancer cells than in adjacent epithelial cells. In addition, our study showed that expression of PTEN was positively correlated with expression of Caspase-3 in primary gastric cancer cells. Schwartzbauer et al[36] found increased PTEN expression by recombinant adenovirus in cultured neonatal rat primary cardiomyocytes caused cardiomyocyte apoptosis as evidenced by increased caspase-3 activity and cleaved poly (A) DP-ribose polymerase. Furthermore, several studies suggested PTEN could enhance Fas/FasL-mediated or cytochrome-c-mediated apoptosis, whose apoptotic pathway evoked Caspase-3[34,37]. Consequently, we could infer that low expression of PTEN could decrease expression of Caspase-3 to make tumor cells apoptosis dysfunction, which underlay the theoretic basis of contribution of PTEN to tumorigenesis and progression of gastric cancer.
In conclusion, expression of PTEN and Caspase-3 is down-regulated in tumorigenesis of gastric cancer. Decreased expression of PTEN is implicated in progression of gastric cancer by decreased cell adhesion and increased angiogenesis and cell mobility. Moreover, low expression of PTEN can reduce expression of Caspase-3 to make tumor cells apoptosis disorder, which forms molecular mechanisms of PTEN contribution to tumorigenesis and progression of gastric cancer. However, a further study is necessary to directly clarify regulatory roles of PTEN and MMP-7 expression in angiogenesis of gastric cancer.
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