Early apoptosis in intestinal and diffuse gastric carcinomas

Abstract

Key Words: stomach neoplasms/pathology, apoptosis, flow cytometry, Bcl-2 protein, phosphatidylserines

INTRODUCTION

Apoptosis, described by Kerr[1] in 1972, plays a key role in all types of regulated cellular processes in multicellular organisms. It is defined as a morphologic change, including fragmentation of the DNA, cell shrinkage, dilation of the endoplasmatic reticulum, cell fragmentation and formation of apoptotic bodies[2,3]. One of the abilities of apoptotic cell is to trigger its own engulfment by phagocytic cells prior to cell lysis, which is crucial to the avoidance of the tissue damage and inflammation associated with necrosis[4]. Apoptotic cells lose membrane phospholipid asymmetry and expose phosphatidylserine (PS) on the outer leaflet of the plasma membrane, macrophages then phagocytose apoptotic cells after specific recognition of the exposed PS[5]. Annexin V (AV), having a high affinity for PS in the presence of Ca²⁺ ions[6] and inhibition to PS-dependent procoagulant reactions[7], is proved to be a very useful general probe for early apoptosis-associated membrane change on live cells before the nuclear condensation events and fragmentation of the DNA[8].

Gastric carcinoma is estimated to be one of the most frequent cancers in the world. According to Lanren’s classification[9], gastric carcinoma can be divided into adenocarcinomas of diffuse and intestinal type, which differ in growth pattern, morphology, and some phenotypic markers. Recently, apoptotic index and apoptotic genes have been found different in both types[10,11]. In this study, we investigated the quantitations of early apoptosis in both intestinal and diffuse gastric carcinoma and their adjacent non-neoplastic tissues with regard to classification and differentiation, and whether the exposure of PS could be inhibited by Bcl-2 protein through various pathways.

MATERIALS AND METHODS

Patients

The 27 surgically resected specimens used for this study were obtained from consecutive patients with gastric cancer in the Department of General Surgery, Affiliated Hospital of Xuzhou Medical College during the period from April to September 1999. The patients received ineither chemotherapy nor radio therapy before undergoing gastrectomy. After tissue sections of 0.5 cm³ were excised from the carcinomatous and adjacent non-neoplastic tissues, they were embedded in ice box, and brought back to FCM laboratory. In addition, formalin fixed and paraffin embedded tissue specimens were used for pathological diagnosis accordding to the Lanren’s classification. All cases had adequate clinical information.

Preparation of single-cell suspension

The tissues (carcinomatous and adjacent non-neoplastic tissues) were thoroughly washed with an excess of ice-cold phosphate buffered saline (PBS), immersed in PBS and minced quickly to approximately 0.5 mm³ pieces using a pair of sharp scissors. After two more washings with PBS to remove red blood cells, these pieces were dispersed into single cells through a nylon screen (300 holes/cm³) by the press of a glass rod.

Detection of early apoptosis

The cell samples were washed with ice-cold PBS after centrifugation at 500 × g for 5 min at 4 °C. Supernatant was discarded, and finally the cell pellets were resuspended in ice-cold, diluted bind buffer to 2 × 10⁶ cells/mL. Five μL AV FITC (Immunotech, Cat. No.2375) solution and 5 μL propidium iodide (PI, Immunotech, Cat. No.2375) were added to 490 μL of the prepared cell suspension. And then the mixture was mixed gently and kept on ice and incubated for 10 min in the dark. Aliquots were directly aspirated into a FACSCalibur flow cytometer (USA BD Company) for analysis with simultaneous monitoring of green fluorescence for AV-FITC and red fluorescence for PI. The FCM software (Cell Quest) was used to calculate and analyze the AV+/PI- cells[12].

Statistical analysis

To analyse the correlation among early apoptosis, necrosis, viable cells in gastric carcinomatous and adjacent non-neoplastic tissues, the Student’s t test and the Pearson’s correlation coefficient were performed with State statistical software.

RESULTS

Clinical and pathological information

The 27 patients with gastric carcinomas (16 male and 11 female), including 9 intestinal and 18 diffuse tumors, with a mean age at operation of 57.4 ± 13.1 years (ranging from 31 to 88 years). There were 7 cases of early (restricted to mucosa and submucosa) and 20 cases of advanced gastric carcinomas, respectively. The mean tumor size was 4.4 cm ± 2.0 cm (ranging from 1.0 cm-8.0 cm). Lymph node metastasis was found in 16 cases.

Detection of early apoptosis with AV and PI

Figure 1 shows typical flow cytometer histograms for gastric carcinoma and adjacent non-neoplastic tissues. AV was an able to bind to viable cells of normal or abnormal tissues as it can not penetrate the phospholipid bilayer and PS did not expose to the outer leaflet. In apoptotic cells, however, the bilayer lose its symmetry when PS was flip flopped from the inner to outer leaflet. Once the cells were dead, the integrity of the plasma memebrane was lost and the penetration was enhanced promptly. The bivariate AV/PI analysis by cytogram showed that viable cells were negative for both AV and PI (LL), apopptotic cells were AV positive (LR), while dead cells were positive for both AV and PI (UR).

Figure 1 Bivariate AV/PI analysis of the gastric carcinoma (A) and adjacent non-neoplastic (B) tissues. The different labeling patterns in this assay identify the different cell subpopulations. i.e. region LL, viable cells (AV-/PI-), region LR, apoptotic cells (AV+/PI-), region UR, dead cells (AV+/PI+).

Early apoptosis in various histological types

By bivariate AV/PI analysis using flow cytometer, the percentages of early apoptosis of carcinomatous or non-plastic tissues were 16.7% ± 5.2% (¯x ± s) and 9.3% ± 3.9%, respectively. The apoptotic index of carcinoma was significantly higher than that of the adjacent non-neoplastic tissue, the difference being statistically significant (Table 1). However, the same result only appeared in carcinomatous tissues of diffuse and intestinal carcinomas, whereas no difference was found in adjacent non-neoplastic tissues (Figure 2).

Table 1 Percentages of three subpopulations in gastric carcinomas.

	AV+/PI-	AV+/PI+	AV-/PI-
Normal	9.3 ± 3.9	4.1 ± 2.5	85.7 ± 5.6
Vicious	16.7 ± 5.2a	6.1 ± 2.7b	76.7 ± 8.1b

^aP < 0.001,

^bP < 0.01 vs normal (adjacent non-plastic tissues).

Figure 2 Early apoptosis in diffuse and intestinal gastric carcinomas. 1 and 2 were EAIs in carcinomatous tissues of diffuse and intestinal tumor, 19.0% ± 3.9% and 12.0% ± 4.3% (P = 0.0002); 3 and 4 were in adjacent non-neoplastic tissues, 10.8% ± 3.3% and 7.3% ± 4.2% (P = 0.0516).

Correlation between early apoptosis and necrosis

The early apoptosis did not demonstrate a dose correlation with the necrosis, i.e. the correlation coefficients between AV+/PI- and AV+/PI+ in tumors or normal tissues were 0.3650 and 0.3877 respectively. And the early apoptosis was not correlated with sex, age, tumor size, lymph nodes metastasis, whereas closely correlated with depth of invasion.

DISCUSSION

In this report, we investigated the early apoptosis in diffuse and intestinal gastric carcinomatous and adjacent non-neoplastic tissues by AV/PI bivariate flow cytometry. Apoptosis, found in the normal tissues and in virtually all malignant tumors[13], is defined morphologically as the double-strand cleavage leading to the formation of fragments that are detected usually by agarose gel electrophresis (Ladder) or terminal deoxynucleotydil transferanse mediatied dUTP-digoxigenin nick end labelling technique (TUNEL). The results, however, derived from the Ladder or TUNEL methods only presents the latter process of apoptosis and can not be analyzed quantificationally[14-16].

With the development of the study on the role and basic mechanisms underlying apoptosis, the externalization of phosphatidy lserine (PS), a kind of plasma membrane lipid, which results in the loss of pospholipid asymmetry, is a downstream event of early caspase activation[8,17] and recognized as an early and ubiquitous phenomenon event in apoptosis[5]. Based on the abilities of annexin V (AV) to bind to PS with high affinity and of propidium iodide (PI) to permeate the membrane losing integrity to stain DNA, AV and PI can be added simultaneously to the cell suspension to discriminate among viable, apoptotic and dead cells[18]. Viable cells with its membrane’s integrity and asymmetry were kept, and AV negative/PI negative were manifested (Figure 1, LL). After PS was exposed to the surface of membrane, apoptotic cells were presented with AV positive/PI negative (Figure 1, LR), whereas the necrotic cells with AV positive/PI positive (Figure 1, UR) were indicated when the membrane losts its integrity. In our study on fresh specimens, we found that the suspension from gastric carcinomatous and adjacent non-neoplastic tissues was identified easily to three subpopulations, among which the EAIs were different statistically. The percentages of apoptosis (AV positive/PI negative) were 16.7% ± 5.2% and 9.3% ± 3.9%, respectively, being higher than those reported in literature[19]. The reason might be that the experiments method are different and the sensitivity and specificity of AV/PI bivariate FCM are high, we therefore thick that the results from our study can reflect objectively the spontaneous occurrence of apoptosis.

The externalization of phosphatidylserine (PS) plays a potential role in cellular kinetic significance. It has been shown that tumorigenic cells expressed relatively larger amounts than that of normal kertinocytes by semiquantitative analysis[20] of PS in the outer leaflet of the cells. The same results were seen in our study. At the execution phase of apoptosis, PS was displayed at the outer membrane by flippases, scramblase and other proteinases. Possibly, with the participation of autologue cytophilic antibodies, components of the clotting cascade, and special phospholipases, macrophages discriminate between “self” and altered “self” by recognizing, phagocytosing, and disposing of effete or tumorigenic cells. Furthermore, it was found in cells that the binding of PS and AV could suppress phagocytosis[21]. That is, the expression of PS on the outer membrane leaflet of cells serves as a recognition moiety for macrophages.

There are various reports regarding the relationship between apoptotic indices and histological type in gastric carcinomas[22,23], the significance of apoptosis in human gastric carcinomas suggested that the higher occurrence of apoptosis in well differentiated carcinomas reflects their slow-growing nature, and poorly differentiated carcinomas escape from this process. However, our results differed from the reports above. According to Lauren’s classification, gastric carcinomas can be divided into adenocarcinomas of diffuse and intestinal type. One of the main difference between them is the degree of infiltration by macrocytes histologically, i.e. intestinal carcinomas grow with no or little infiltration whereas diffuse carcinomas do inversely. It might be that the heterogeneity of diffuse carcinomas is higher than that of intestinal carcinomas, the cells are prone to be initiated by multiple triggers of apoptosis, such as DNA damage, Fas ligand binding or withdrawal of growth factors[24-27].

In recent studies, at the trigger and execution phase of apoptosis, the expression of bcl-2, bax[28-30] was demonstrated to play important roles in cell life. Bcl-2 and Bax are members of the group proteins that regulate the apoptotic pathway, that is, Bcl-2 acts as an inhibitor of apoptosis, opposing Bax effects on cell life. Bcl-2 could protect cells from apoptosis in several ways, such as inhibition from transmembrane streaming of Ca²⁺ ion, blockage in releasing of cytochrome C, formation of heterodimers with Bax. In addition, Bcl-2 may regulate special PS-sensitive signal transduction pathway by protecting lipids from peroxidation[31,32]. Some clinicopathological studies demonstrated that Bcl-2 appears to be preferentially associated with the intestial type carcinomas and more prevalent in poorly differentiated ones[33-35].

In conclusion, the early apoptosis has been investigated in gastric carcinomatous and adjacent non-neoplastic tissurs by AV/PI bivariate flow cytometry. The early apoptosis is clearly related to tumor and its differentiation and depth of invasion.