Effect of HCV NS3 protein on p53 protein expression in hepatocarcinogenesis

Abstract

AIM To investigate hepatocarcinogenesis by detecting the effect of HCV NS₃ protein on p53 protein expression in hepatocellular carcinoma (HCC) and pericarcinomatous liver tissue (PCLT).

METHODS The expression of HCV NS₃ and p53 protein was det ected with immunohistochemical technique (SP method) in specimens of HCC and PCLT from 47 patients with negative HBV.

RESULTS The positive rate of HCV NS₃ protein was lower in HCC (62%) than in PCLT (83%) (P < 0.025). The better differentiaton of cancer cells, the stronger expression of HCV NS₃ protein (P < 0.025). The positive rate of p53 protein in HCC (81%) was higher than in PCLT (47%) (P < 0.025). The worse differentiaton of cancer cells, the stronger expression of p53 protein (P < 0.05). The p53 protein expression was not correlated with the HCV NS₃ protein expression in HCC (P > 0.5), whereas their expression was closely related to PCLT (P < 0.01), and the expression rate of p53 protein in the cases of positive HCV NS₃ protein was higher than that in the cases of negative HCV NS₃ protein.

CONCLUSION HCV NS₃ protein may exert its hepatocarcinogenic effect in early stage on host cells by endogenous pathway which may bring about mutation of p53 gene and transformation of hepatocytes.

Key Words: liver neoplasms; oncogenes; hepatitis virus; p53 protein

INTRODUCTION

Hepatocellular carcinoma (HCC) is one of the most common human cancers in the world. Recently, the HCV infection was found to be an etiological factor to HCC. HCV is a RNA virus bearing no reverse transcriptase activity. Therefore, in stead of “pro-moter insertion”, or “insertion mutagenesis”, the HCV NS₃ expression of HCV gene may play an es-sential role in transfomation of hepatocytes[1]. p53 gene, an oncogene, has been intensively investiga-ted recently. Mutation of this gene was found to be related to HCC in a variety of studies. Inorder to understand the relationship among p53 protein, HCV NS₃ protein and HCC, we studied the effect of HCV NS₃ protein on expression of p53 protein in HCC and pericarcinomatous liver tissue (PCLT).

MATERIALS AND METHODS

Tissue samples

HCC and PCLT were obtained by surgical resection from 47 patients in Xiangya Hospital and the Se-cond Affiliated Hospital of Hunan Medical University, China. Forty patients were males and 7 fe-males. Their age ranged 33 to 67 years (mean, 52 years), and all patients were negative for HBsAg serological marks. The tissues were fixed in 10% formalin and embedded in paraffin.

Reagents

Anti-HCV NS₃ protein MAb was purchased from GIB Comp. (Beijing, China), anti-p53 protein MAb and SP detection kit from Maixing Comp. (Fuzhou, China).

Immunohistochemistry

Five μm tissue sections were deparaffinized and washed in 0. 05 mol/L PBS, handled with 20 g/L H₂O₂ and treated with microwave. According to SP method, the tissues were detected with immunohis-tochemical technique. HCV RNA(+) biopsy liver tissues and breast cancer tissues were used as positive control of HCV NS₃ protein and p53 protein respec-tively. PBS was used as substitutes of Mabs for negative control groups.

Histological assessment

Semi-quantity analysis was performed as Formonitz[2] described.

Statistical analysis

The difference between each group was analyzed by Chi-square test.

RESULTS

Expression and distribution of HCV NS3 protein in HCC and PCLT

Among 47 cases, the positive rate of HCV NS₃ protein in HCC was 62% (29/47), and the positive cells were clustered or diffused in HCC. The positive signal was localized in cytoplasm. The expression strength of HCV NS₃ protein in HCC was rela-ted to the degree of carcinoma cell differentiation (P < 0.05). The better differentiaton of cancer cells, the stronger expression of HCV NS₃ protein (P < 0.05). The positive rate (83%) and the expression strength of HCV NS₃ protein in HCC were higher than those in PCLT. The distribution of positive cells in PCLT appeared large patchy or dif-fused.

Expression and distribution of p53 protein in HCC and PCLT

Of the 47 cases, the positive rate of p53 protein in HCC was 81% (38/47), and the positive cells ap-peared focal or patchy in HCC. The positive signal was in the nuclei. The expression strength of p53 protein in HCC was correlated with the degree of carcinomatous cell differentiation (P < 0.05). The worse differe ntiaton of cancer cells, the stronger expression of p53 protein (P < 0.05). The positive rate (47%) and the expression strength of p53 protein in HCC were lower than those in PCLT. The positive cells in PCLT was scattering in distribution.(Table 1)

Table 1 The expression strength of HCV NS protein and p53 protei n in HCC and PCLT.

	n	HCV NS3 protein					p53 protein
		-	+	++	+++	Positive rate(%)	-	+	++	+++	Positive rate(%)
HCC
I	8	2	0	3	3	6/8	4	2	2	0	4/8
II	10	2	1	2	5	8/10	2	3	4	1	8/10
III	18	8	3	6	1	56	2	0	6	10	89
IV	11	6	3	2	0	46	1	1	3	6	91
PCLT
Normal	3	2	1	0	0	1/3	3	0	0	0	0
Hepatitis	23	4	2	5	12	83	13	8	2	0	44
Cirrhosis	21	2	2	6	11	91	9	7	5	0	57

Relationship between p53 protein expression and HCV NS₃ protein

In HCC, the expression rate of p53 protein in 29 cases of positive HCV NS₃ protein was 83% (24/29), and in 18 cases of negative HCV NS₃ prote in was 78% (14/18), the difference between the for-mer and the latter being not significant (P > 0.1). In PCLT, the expression rate of p53 protein in 39 cases of positive HCV NS₃ protein was 54% (21/39), and in 8 cases of negative HCV NS₃ protein was only 13% (1/8), the difference between the former and the latter being significant (P < 0.05).

DISCUSSION

Chronic infection with HCV is strongly associated with the development of HCC. HCV causes HCC by expressing protein, especially HCV NS₃ protein[1,3]. Nevertheless, the exact molecular mechanism re-mains quite unknown. Our results showed that the positive rate and expression strength of HCV NS₃ protein in PCLT were higher than those in HCC, and the expression strength of HCV NS₃ protein in HCC was related to the degree of carcinoma cell dif-ferentiation. The better differentiaton of cancer cells, the stronger expression of HCV NS₃ protein. It is indicated that the cellular internal environment for HCV replication is disturbed with cancer growth. HCV may be eliminated at the final stage of hepato cytes transformation because the virus without reverse transcriptase is unable to integrate into the host hepatocytes genome. On the other hand, the positive rate and expression strength of p53 protein in PCLT were lower than those in HCC. The expression strength of p53 protein in HCC was related to the degree of carcinoma cell dif-ferentiation. The worse differentiaton of cancer cells, the stronger expression of p53 protein. It is suggested that p53 protein may play a role in the morphological change and the differentiated degree of cancer cells in HCC, thus detecting p53 protein expression is of benefit to HCC prognosis.

Effect of HCV NS₃ protein on mutation of p53 gene was not confirmed, although HCV infection may result in mutation of p53 gene[4]. This study revealed that there was no relationship between p53 protein and HCV NS₃ prote in in HCC. In PCLT, p53 protein expression was positive in 21 of 39 cases of pos itive HCV NS₃ protein while only one case of p53 protein expression in 8 cases of negative HCV NS₃ protein was positive. HCV NS₃ protein may exert its hepatocarcinogenic effect in early stage on host cells by endogenous pathway which may bring about mutation of p53 gene and transformation of hepatocytes.