Clinical significance of expression of apoptotic signal proteins in gastric carcinoma tissue

Abstract

AIM: To evaluate the expressions of apoptotic signal proteins FADD, TRADD, FasL, Fas, and NFκB in gastric carcinoma tissues and their clinical significance.

METHODS: Western blot immune trace method was adopted to detect the expressions of apoptotic signal proteins FADD, TRADD, FasL, Fas, and NFκB in 55 tissue specimens of gastric carcinoma.

RESULTS: Five apoptotic signal proteins had different expressions in the gastric carcinoma samples and their expressions were not correlated to age (P = 0.085). Expressions of the FADD, FasL, Fas, and NFκB proteins reduced with increase of the volume of tumor with the exception of increased expression the TRADD protein (64.7-71.1%, P = 0.031). With gradual increase of the malignancy of gastric carcinoma tissues, expressions of the FADD, FasL, and Fas proteins decreased (78.6-28.0%, P = 0.008; 78.6-65.9%, P = 0.071; 100.0-46.3%, P = 0.014), while expressions of the TRADD and NFκB proteins increased (42.9-78.1%, P = 0.063; 78.6-79.1%, P = 0.134). With gradual increase of serum CEA, expression of the FADD protein decreased (62.5-34.0%, P = 0.073), but expressions of the TRADD, FasL, Fas, and NFκB proteins increased (0.0-80.8%, P = 0.005; 62.5-70.2%, P = 0.093; 0.0-70.2%, P = 0.003; 62.5-80.9%, P = 0.075). When compared to the tissues of gastric carcinoma without metastasis, the positive rate of expressions of the FADD and FasL proteins increased, whereas expressions of the TRADD, FADD, and NFκB proteins decreased. There was no significant difference between them (P = 0.095).

CONCLUSION: Gastric carcinoma is endurable to Fas-related apoptosis and apoptotic signal proteins are differently expressed in gastric carcinoma.

Key Words: Gastric cancer, Apoptosis, Signal protein, Western blot

INTRODUCTION

Gastric carcinoma is one of the most common causes of malignancy-related death in China[1-4]. Its therapy in clinics is a big challenge. Better remedial method possibly depends on advances in basic research[5-7]. Recent evidence suggests that apoptosis of cells is closely related to occurrence, progress and metastasis of the tumor[8-10]. At present, studies on the apoptosis of tumor cells are an important field of tumor therapy and tumor molecular biology[11-14]. The abnormalities in expressions of apoptotic signal proteins always influence the gastrocellular apoptosis, thus leading to the incidence and development of gastric carcinoma[15-17]. We adopted the Western blot immune trace method to detect the expressions of apoptotic signal proteins FADD, TRADD, FasL, Fas, and NFκB in gastric carcinoma tissues and their significance in gastric carcinoma was assessed.

MATERIALS AND METHODS

Samples

A total of 55 cases were selected from patients with primary gastric carcinoma excision in our hospital from January 2000 to April 2001, who were pathologically diagnosed as adenocarcinoma. There were 35 male and 20 female cases aged 34-78 years with an average age of 54 years. According to Edmondson grading III, the samples were graded as I-II grade in 14 cases and III-IV grade in 41 cases. All samples were submerged in liquid nitrogen within half an hour after the excision and placed in a -80°C refrigerator.

Protein extraction

Fifty-five tumor tissue specimens were homogenized, sonicated for 15 s twice in 500 µL of lysis buffer containing 1× phosphate-buffered saline (PBS), 1% Nonidet-P40, 0.5% sodium deoxycholate, 0.1% SDS and 0.1 mg/mL phenylmethylsulfonyl fluoride, and placed on ice for 30 min. The lysate was centrifuged at 13 000 g for 15 min at 4°C, the supernatant was collected (450 µL) and the protein concentration for each sample was determined by spectroph-otometry (Pharmacia) and using a DC protein assay kit (Bio-Rad).

Reagents and methods

Sheep antihuman FADD, TRADD, FasL, and Fas polyclonal antibodies, rabbit antihuman NFκB polyclonal antibodies, alkaline phophatase marking anti-sheep, anti-rabbit and protein molecular mass marker were also purchased from Santa Cruz Company. Western blot immune trace method was employed to determine the expression of apoptotic signal proteins. Twenty microliters of extracted protein for 120 g/L polyacrylamide gel electrophoresis. When it was semidry, electrical metastasis to the nitrocellulose film was made and enclosed with 100 mL/L bovine serum for 1 h. The first antibody (1:1 000) was put into PBST (1 g/L Tween20 in PBS) and stored overnight at 4°C. After being washed, the sample was enclosed again with 100 mL/L bovine serum for 30 min, and then the second antibody was put in at room temperature for 1 h. After being washed with PBS for 1 h, NBTBCIP development method was performed for coloration.

Assessment standards

The protein molecular mass marker was taken as the standard. In the respective different molecular mass scope, the sample with black stripes was considered positive and vice versa negative. The molecular mass of FADD, TRADD, FasL, Fas, and NFκB proteins was 23, 34, 31, 48, and 65 ku respectively.

Statistical analysis

Chi squared test, accurate 4-chess table method and correlation analysis were used to analyze the correlation of protein expressions with sex, age, tumor size, pathological grading and tumor metastasis. P < 0.05 was considered statistically significant.

RESULTS

Expression of apoptotic signal proteins in gastric carcinoma

The expression of FADD, TRADD, FasL, Fas, and NFκB proteins in gastric carcinoma tissue specimens was 36.4% (20/55), 69.1% (38/55), 69.1% (38/55), 60.0% (33/55) and 78.2% (43/55) respectively (Table 1). We carried out Western blot analysis to determine the accuracy of protein expression (Figure 1).

Table 1 Expressions of FADD, TRADD, FasL, Fas and NFκB proteins in gastric carcinoma tissue specimens, n (%).

Sex	n	FADD	TRADD	FasL	Fas	NFκB
Male	35	12 (34.3)	26 (74.3)	25 (71.4)	21 (60.0)	28 (80.0)
Female	20	8 (40.0)	12 (60.0)	13 (65.0)	12 (60.0)	15 (75.0)
	55	20 (36.4)	38 (69.1)	38 (69.1)	33 (60.0)	43 (78.2)

Figure 1 Western blot analysis of the accurate expression of apoptotic signal proteins. Lane 1: marker; lane 2: FADD; lane 3: TRADD; lane 4: FasL; lane 5: Fas; lane 6: NFκB.

Relation between apoptotic signal proteins and clinical characteristics

Different expressions of the five apoptotic signal proteins were not correlated to age (P = 0.085, Figure 2A). Expressions of the FADD, TRADD, FasL, Fas, and NFκB proteins reduced with increase of the tumor volume, with the exception of increased expression of the TRADD protein (64.7-71.1%, P = 0.031). Among them, the positive rate of the expression of the Fas protein in the tissue of gastric carcinoma with a lump ≤ 5 cm was higher than that of gastric carcinoma >5 cm and the difference was significant (P = 0.037, Table 2, Figure 2B).

Table 2 Relationship between expressions of the FADD, TRADD, FasL, Fas, NFκB proteins and clinical characteristics, n (%).

Clinical characteristics	n	FADD	TRADD	FasL	Fas	NFκB
Male	35	12 (34.3)	26 (74.3)	25 (71.4)	21 (60.0)	28 (80.0)
Female	20	8 (40.0)	12 (60.0)	13 (65.0)	12 (60.0)	15 (75.0)
Age/yr ≤ 45	22	8 (36.4)	14 (63.6)	16 (72.7)	13 (59.1)	16 (72.7)
> 45	33	13 (39.4)	24 (72.7)	22 (66.7)	20 (60.6)	27 (81.8)
Tumor
Size ≤ 5 cm	17	8 (47.1)	11 (64.7)	14 (82.4)	15 (88.2)a	14 (82.4)
> 5 cm	38	13 (34.2)	27 (71.1)	24 (63.2)	17 (44.7)	29 (76.3)
Grading I–II	14	11 (78.6)b	6 (42.9)	11 (78.6)	14 (100.0)c	11 (78.6)
III–IV	41	9 (22.0)	32 (78.1)	27 (65.9)	19 (46.3)	32 (78.1)
CEA ≤ 30 (µg/L)	8	5 (62.5)	0 (0.0)	5 (62.5)	0 (0.0)	5 (62.5)
> 30 (µg/L)	47	16 (34.0)	38 (80.9)d	33 (70.2)	33 (70.2)d	38 (80.9)
Tumor
transformation Yes	6	3 (50.0)	3 (50.0)	5 (83.3)	3 (50.0)	3 (50.0)
No	49	17 (34.2)	35 (71.4)	33 (67.4)	30 (61.2)	39 (79.6)

^aP < 0.05 vs size >5 cm;

^bP < 0.01,

^cP < 0.05 vs grading III–IV;

^dP < 0.01 vs CEA ≤ 30 (μg/L).

Figure 2 Different expressions of five proteins before and after 45 years (A) and different tumor size (B).

With the increased of malignancy grade in adenocarcinoma tissues, expressions of the FADD, FasL, and Fas proteins decreased, while expressions of the TRADD and NFκB proteins increased. Expressions of the FADD and Fas proteins were significantly different between the pathological grades (P = 0.008 and P = 0.014, Figure 3A). With the increase of serum CEA, expression of the FADD protein in gastric carcinoma tissues decreased, while expressions of the TRADD, FasL, Fas and NFκB proteins increased. Expression of the TRADD and Fas proteins were positively correlated with the serum CEA level (r = 0.700, P = 0.005) and the difference was very significant (P = 0.003, Figure 3B). When compared to the tissues of gastric carcinoma with metastasis, the positive expression rate of the FADD and FasL proteins increased, whereas expressions of the TRADD, FAS and NFκB proteins decreased. There was no significant difference between them (P = 0.095).

Figure 3 Different expressions of the five proteins with different pathological grading (A) and different serum CEA (B).

DISCUSSION

Apoptotic abnormality is considered as an important mechanism underlying the development of gastric carcinoma[18,19] and even more crucial than the reproduction of cells of out control[20,21]. The expression of apoptotic signal proteins play different roles in the death of gastric cancer cells. The current studies[22,23] have denoted the main mechanism of the interaction of the apoptotic signal proteins to conduct down the apoptotic signal. FasL is polymerized with its corresponding Fas receptors[24,25]. By activating the FADD and caspase family, the apoptosis of cancer cells ensues; and by activating TRADD, RIP is motivated, causing NFκB to be activated, thereby participating in gene transcription of the survival of cells. The balance between counter signals and paths eventually determines the survival or apoptosis of cells.

In this study, the expression of Fas protein was downregulated with increase of the gastric carcinoma volume and the malignancy degree and positively correlated with the expression of CEA. It was reported[26] that gastric cancer cells inhibit expression of the downregulated FasL, thus escaping Fas-related apoptosis and immune monitoring of the host to enable cancer cells to reproduce continuously and increase their malignancy degree. Expression of the FADD protein is downregulated with increase of the malignancy degree of gastric carcinoma[27]. Gastric cancer cells might through the expression of the downregulated FADD enable caspases 3 and 8 necessary for apoptosis of Fas-related cells to be devitalized, resulting in inhibition of the apoptosis of gastric cancer cells. If FADD is injected, such cells can become sensitive to Fas and induce Fas-related apoptosis, suggesting that the function of FADD is extremely crucial.

The TRADD protein is able to vitalize the NFκB transcription factor and prolong the survival of cells. In the present study, the TRADD protein expression was positively correlated with CEA only when CEA possessed the value of 30 μg/L, suggesting that both of them can be used as indexes to detect gastric carcinoma. However, this can be determined only after investigating a large size of samples[28-30]. As the change of expressions of apoptotic signal proteins is not much related to metastasis of gastric carcinoma, the abnormalities of the function of these proteins only contribute to the reproduction and rise of malignancy degree of gastric cancer cells, whereas metastasis of gastric carcinoma might correlate to other factors.

In summary, gastric carcinoma is a tumor endurable to Fas-related apoptosis. Apoptotic signal proteins are differently expressed in gastric carcinoma.