5-氟尿嘧啶联合柳氮磺胺吡啶对人胰腺癌细胞系BxPC-3体外增殖和凋亡的影响

Effects of 5-fluouracil combined with sulfasalazine on human pancreatic carcinoma cell line BxPC-3 proliferation and apoptosis in vitro.

作者信息

Huang Zhe, Guo Ke-Jian, Guo Ren-Xuan, He San-Guang

机构信息

Department of General Surgery, First Affiliated Hospital, China Medical University, Shenyang 110001, China.

出版信息

Hepatobiliary Pancreat Dis Int. 2007 Jun;6(3):312-20.

PMID:17548258

Abstract

BACKGROUND

Most pancreatic carcinomas are clinically insensitive to chemotherapeutics. The exact mechanisms of their apoptosis and multiple drug resistance are obscure at present. This study was undertaken to explore the influence of chemotherapy on anti-proliferation, apoptosis and the cell cycle, and lay a fundamental basis for further research into the apoptotic mechanisms and prevention of multiple drug resistance in pancreatic carcinoma.

METHODS

The human pancreatic carcinoma cell line BxPC-3 was cultured in vitro. The growth inhibition rate, cell cycle and apoptotic rate of cells treated with 5-fluorouracil (5-FU), sulfasalazine alone or a combination at different concentrations were evaluated with the MTT method and flow cytometry. Phase-contrast microscopy was used to observe morphological changes in the cells treated with 5-FU, sulfasalazine or both for 24 hours.

RESULTS

The growth inhibition rate of the BxPC-3 cells treated with 5-FU and sulfasalazine significantly increased in a time- and dose-dependent manner. The growth inhibition rate of the cells treated with 5-FU gradually increased, but decreased at different concentrations of sulfasalazine for a prolonged period. The apoptotic rate of the BxPC-3 cells induced by sulfasalazine (200 mg/L), 5-FU (100 mg/L) or both for 12 hours were (2.68+/-0.36)%, (6.59+/-0.90)%, and (10.52+/-0.55)%, respectively, compared with the corresponding control values were (3.17+/-0.08)%, (1.50+/-0.06)%, and (4.08+/-0.31)% [(t=2.33 (P>0.05), 9.78 and 17.56 (P<0.01)]. It increased to (7.63+/-0.68)%, (40.43+/-1.79)%, and (64.69+/-0.82)% for 48 hours, in comparison with the control that was (29.20+/-2.18)%, (5.61+/-0.13)%, and (12.02+/-0.52)% [t=17.06, 33.66 and 94.51 (P<0.01)]. The apoptotic rate, proportion of cells in S-phase and proliferative index rose after use of 5-FU (12.5, 25, 50, 75, and 100 mg/L) alone for 24 hours. However, the apoptotic rate at augmented concentrations of sulfasalazine for 24 hours slowly increased from (1.47+/-0.08)% to (3.45+/-0.28)%, the proportion of cells in G0/G1-phase increased from (35.13+/-0.32)% to (54.32+/-1.45)%, the proportion of cells in S-phase decreased from (45.37+/-1.48)% to (16.67+/-2.73)%, and the proliferative index gradually lowered. The proportion of G0/G1-phase cells treated by 5-FU (100 mg/L) and sulfasalazine (200 mg/L) increased from (43.31+/-1.52)% (12 hours) to (85.05+/-0.24)% (48 hours) compared with the corresponding controls [t=7.93 (12 hours), 21.30 (48 hours), P<0.01], and the proportion of cells in S-phase decreased from (11.63+/-1.11)% (12 hours) to (4.47+/-0.68)% (48 hours) in contrast to the controls [t=37.68 (12 hours), 8.60 (48 hours), P<0.01]. Most cells after the combined use of the two agents for 24 hours displayed pyknosis and oval shape by phase-contrast microscopy. The cells treated with 5-FU (100 mg/L) for 24 hours were pyknotic and oval shaped. A few of cells in the group treated with sulfasalazine (200 mg/L) were pyknotic at 24 hours.

CONCLUSIONS

Sulfasalazine may enhance the inhibitory proliferation and apoptosis effect on BxPC-3 cells induced by 5-FU, which is closely related to synergistically the cell cycle arrested in G0/G1-phase.

摘要

背景

大多数胰腺癌对化疗药物临床不敏感。目前其凋亡及多药耐药的确切机制尚不清楚。本研究旨在探讨化疗对胰腺癌细胞抗增殖、凋亡及细胞周期的影响，为进一步研究胰腺癌凋亡机制及预防多药耐药奠定基础。

方法

体外培养人胰腺癌细胞系BxPC-3。采用MTT法和流式细胞术评估不同浓度5-氟尿嘧啶（5-FU）、柳氮磺胺吡啶单独或联合处理后细胞的生长抑制率、细胞周期及凋亡率。用相差显微镜观察5-FU、柳氮磺胺吡啶或二者联合处理24小时后细胞的形态变化。

结果

5-FU与柳氮磺胺吡啶联合处理BxPC-3细胞的生长抑制率呈时间和剂量依赖性显著增加。5-FU处理组细胞生长抑制率逐渐升高，但在不同浓度柳氮磺胺吡啶长时间作用下降低。柳氮磺胺吡啶（200mg/L）、5-FU（100mg/L）单独或二者联合处理12小时后BxPC-3细胞的凋亡率分别为（2.68±0.36）%、（6.59±0.90）%和（10.52±0.55）%，相应对照组分别为（3.17±0.08）%、（1.50±0.06）%和（4.08±0.31）%[t=2.33（P>0.05），9.78和17.56（P<0.01）]。处理48小时后凋亡率分别增至（7.63±0.68）%、（40.43±1.79）%和（64.69±0.82）%，对照组分别为（29.20±2.18）%、（5.61±0.13）%和（12.02±0.52）%[t=17.06、33.66和94.51（P<0.01）]。单独使用5-FU（12.5、25、50、75和100mg/L）处理24小时后，细胞凋亡率、S期细胞比例及增殖指数升高。然而，不同浓度柳氮磺胺吡啶处理24小时后凋亡率从（1.47±0.08）%缓慢增至（3.45±0.28）%，G0/G1期细胞比例从（35.13±0.32）%增至（54.32±1.45）%，S期细胞比例从（45.37±1.48）%降至（16.67±2.73）%，增殖指数逐渐降低。5-FU（100mg/L）与柳氮磺胺吡啶（200mg/L）联合处理组G0/G1期细胞比例从（43.31±1.52）%（12小时）增至（85.05±0.24）%（48小时），与相应对照组相比[t=7.93（12小时），21.30（48小时），P<0.01]，S期细胞比例从（11.63±1.11）%（12小时）降至（4.47±0.68）%（48小时），与对照组相比[t=（37.68（12小时），8.60（48小时），P<0.01]。相差显微镜观察显示，二者联合处理24小时后多数细胞呈现核固缩及椭圆形。5-FU（100mg/L）处理24小时后细胞呈核固缩及椭圆形。柳氮磺胺吡啶（200mg/L）处理组24小时时有少数细胞呈核固缩。