Bel-7402 人肝癌细胞系

Abstract

AIM:To study the effect of a varying concentrations of ars enic trioxide on human hepatoma cell line BEL-7402 cultured in vitro and its mechanism of action.

METHODS:The BEL-7402 cells were treated with arsenic trioxide (at the concentrations of 0.5，1，2μmol/L, respectivel y) for 4 successive days. The cell growth and proliferation were observed by cell counting and cell-growth curve. Morphologic changes were studied with electronmicroscopy. Flow cytometry was used to assay cell-DNA distribution and the protein expression of Bcl-2 and Bax detected by immuno cytochemical method.

RESULTS:The cell growth was significantly inhibited by varying concentrations of arsenic trioxide as revealed by cell counting and cell-growt h curve, which was dose- and time-dependent. Arsenic trioxide treatment at 0.5, 1 and 2μmol/L resulted in a sub G1 cell peak, the apoptosis rate of the control group was 9.31% and that of 0.5μmol/L arsenic trioxide 15.53%, no significant difference was seen between the two. The apoptosis rates of 1, 2μmol/L arsenic trioxide were 19.10% and 21.87% respectively, which were much higher (both P＜0.05). Decrease of G0/G1 phase cells and increase of S phase cells were observed by flow cytometry, suggesting the inhibition effect of 0.5, 1, 2μmol/L arsenic trioxide on BEL-7402 cell lay in the G0/G1 phase. Morphologic changes such as intact cell membrane, nucleic condensation, apoptotic body formation were seen under transmission electronmicrescopy, whereas the 0.5mol/L arsenic trioxide-treated BEL-7402 cells showed decrease of nucleocytoplasmi c ratio, round nucleus, well-differentiated organelles in the cytoplasm. The processes and microvilli on the cell surface of the experimental groups under scanning electron microscopy were significantly decreased. High expressions of Bcl-2 and Bax were detected in 1 and 2μmol/L arseni c trioxide-treated cells, these were 46%, 87.33% and 83.08%, 95.83% respecti vely, among which that of Bax was more significant. Arsenic trioxide treatment at 0.5μmol/L resulted in a higher expression level of Bcl-2 and lower expression level of Bax, which were 8.81% and 3.83% respectiv ely, as compared with that of the control group (15.33%) (P1＜0.01, P2＜0.01).

CONCLUSION: Arsenic trioxide not only inhibited proliferation but also induced apoptosis of human hepatoma cell line BEL-7402. The induced-apoptosis effect of 1, 2μmol/L arsenic trioxide was related to the expression level of Bcl-2 and Bax.

INTRODUCTION

Arsenic trioxide is the main ingredient of traditional Chinese medicinal, pi shi. Zhang P, et al first reported the effect of Arsenic trioxide on promyelocytic leukemia (APL) with satisfactory results. The rate of complete remission in patients who had not received any treatment before reached 73.33%, and was 52.38% in patients with recurrence, the longest remission period was more than ten years, and intravenous route of administration was the choice, no toxic or adverse effects were seen[2-4]. There was no cross resistanc e between arsenic trioxide and other chemical drugs during the course of treatment of APL[5]. Shen[6]concluded that arsenic trioxide treatment was effective and relatively safe in APL patients refractory to ATRA and conventional chemotherapy. Inorganic arsenic trioxide was recently shown to induce apoptosis in NB4 promyelocytic leukemic cells[7]. The present study was so designed as to broaden the anti-tumor spectrum and to study the inhibitory effect of Arsenic trioxide on human hepatoma cell line and its mechanism of action, in order to provide some theoretical basis for its clinical use.

MATERIALS AND METHODS

Materials

Human hepatoma cell line BEL-7402 was purchased from the Cell Institute of Chinese Academy of Science. Arsenic trioxide was produced in Pharmaceutical Department of First Hospital of Harbin Medical University. RPMI 1640 was purchased from GIBCO. Propidium iodide and Rnase were from Sigma Chemical Co. A murine monoclonal antibody against human Bcl-2 and Bax oncoprotein and antimi ce rabbit polyclonal antibody were purchased from Maixin Co., Fuzhou.