Δ133p53表达状态对rmhTNF效应的影响及机制研究

姜琪琪; 张红梅; 郭爱

doi:10.3969/j.issn.1007-3969.2015.04.008

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Δ133p53表达状态对rmhTNF效应的影响及机制研究

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- Δ133p53表达状态对rmhTNF效应的影响及机制研究
- Effect and mechanism of rmhTNF on gastric cancer cell lines with different Δ133p53 status
- 中国癌症杂志 2015年25卷第4期页码：287-293
- 作者机构：
  
  1. 潍坊医学院,山东,潍坊,261053
  2. 潍坊医学院附属医院消化内科,山东,潍坊,261031
- 作者简介：
- 基金信息：
  
  山东省优秀中青年科学家科研奖励基金(BS2010SW034)。
- DOI：10.3969/j.issn.1007-3969.2015.04.008
  中图分类号：
- 网络出版：2015-05-25，
  
  纸质出版：2015-05-25
- 稿件说明：
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姜琪琪,张红梅,郭爱,等. Δ133p53表达状态对rmhTNF效应的影响及机制研究[J]. 中国癌症杂志, 2015, 25(4): 287-293.

姜琪琪, 张红梅, 郭爱. Effect and mechanism of rmhTNF on gastric cancer cell lines with different Δ133p53 status[J]. China Oncology, 2015, 25(4): 287-293.
姜琪琪,张红梅,郭爱,等. Δ133p53表达状态对rmhTNF效应的影响及机制研究[J]. 中国癌症杂志, 2015, 25(4): 287-293. DOI： 10.3969/j.issn.1007-3969.2015.04.008.

姜琪琪, 张红梅, 郭爱. Effect and mechanism of rmhTNF on gastric cancer cell lines with different Δ133p53 status[J]. China Oncology, 2015, 25(4): 287-293. DOI： 10.3969/j.issn.1007-3969.2015.04.008.

摘要

背景与目的：p53异构体在胃癌发生中的作用报道较少。该研究旨在探讨p53异构体Δ133p53在重组改构人肿瘤坏死因子(recombinant mutant human tumor necrosis factor，rmhTNF)干预胃癌细胞系生物学效应中的作用，为胃癌诊断和治疗提供新的依据。方法：采用细胞增殖/毒性检测试剂盒(CCK-8)和流式细胞术，检测不同浓度的rmhTNF单独或联合氟尿嘧啶(5-FU)应用于MKN-45(表达Δ133p53)和SGC-7901(不表达Δ133p53)细胞，观察细胞抑制率和细胞凋亡情况。通过巢式逆转录多聚酶链反应(nested reverse transcriptase-polymerase chain reaction，nRT-PCR)和实时荧光定量多聚酶链反应(real-time polymerase chain reaction，RT-PCR)检测Δ133p53、Gadd45α和CyclinB1 mRNA的表达变化。结果：rmhTNF单独作用于Δ133p53表达阳性的MKN-45细胞有抑制作用，浓度为50、500 IU/mL的rmhTNF作用24 h后，细胞抑制率分别为24.82%、72.33%(t=-9.558，P0.01)，并可提高5-FU的抑制率，且具有显著的量效和时效关系，5-FU(25 μg/mL)、rmhTNF(50 IU/mL)+5-FU(25 μg/mL)、rmhTNF(500 IU/mL)+5-FU(25 μg/mL)作用于MKN-45细胞24 h后，抑制率分别为18.20%、48.66%、59.83%(F=82.742，P0.01)；rmhTNF(50 IU/mL)+5-FU(25 μg/mL)作用于MKN-45细胞24、48、72 h后，抑制率分别为48.66%、68.20%、85.23%(F=128.583，P0.01)。而对于Δ133p53表达阴性的SGC-7901细胞，浓度为50、500 IU/mL的rmhTNF单独抑制率为2.74%、3.25%，抑制作用不明显(t=-0.121，P0.05)。流式细胞术显示，rmhTNF不仅单独可引起MKN-45细胞凋亡，而且可显著增强5-FU促细胞凋亡作用，rmhTNF(50 IU/mL)、rmhTNF(50 IU/mL)+5-FU(25 μg/mL)、rmhTNF(500 IU/mL)+5-FU(25 μg/mL)作用于MKN-45细胞24 h后，凋亡率分别为7.21%、10.13%、15.28%(F=123.931，P0.05)。在MKN-45中，rmhTNF单独或联合5-FU可下调Δ133p53和CyclinB1基因，上调Gadd45α基因表达水平。nRT-PCR检测对照组及实验组Δ133p53基因相对表达量分别为0.886、0.499、0.330、0.161(F=240.927，P0.01)；Real-time PCR检测实验组Gadd45α基因相对表达量分别为1.227、1.694、3.394，Cyclin B1基因相对表达量分别为1.221、0.722、0.316。Δ133p53表达水平与CyclinB1呈正相关(r=0.977，P0.01)，与Gadd45α呈负相关(r=-0.950，P0.01)。结论：rmhTNF对表达Δ133p53胃癌细胞展现出显著的抑制效应，并能增加传统化疗药物5-FU的疗效，其中部分效应可能是通过调节p53下游分子CyclinB1和Gadd45α表达实现的，提示Δ133p53可能是rmhTNF治疗胃癌生物学效应的关键靶点。

Abstract

Background and purpose: Little about the function of p53 isoforms in gastric cancer was reported. This study was designed to explore the role of Δ133p53 in the effect of recombinant mutant human tumor necrosis factor (rmhTNF) on gastric cancer cells

and provide a new basis for the diagnostics and therapeutics of gastric carcinoma. Methods: MKN45 (with Δ133p53 expression) or SGC7901 (without Δ133p53 expression) cells were treated with rmhTNF of different concentrations only or combined with 5-FU (a traditional gastric cancer cellular killer)

and the growth inhibition rate and apoptosis was detected by CCK-8 and flow cytometry. mRNA expressions of Δ133p53

Gadd45α and CyclinB1 were measured by nested reverse transcription-polymerase chain reaction (nRT-PCR) or real-time polymerase chain reaction(RT-PCR). Results: On MKN-45 cells with positive Δ133p53 expression

the inhibitory effect of rmhTNF was significant

the inhibition rates of 50 and 500 IU/mL rmhTNF were 24.82%

72.33% after culturing for 24 h (t=-9.558

P0.01); also

the inhibitory effect of 5-FU was improved by rmhTNF remarkably in time- and dose-dependence

the inhibition rates of 5-FU (25 μg/mL)

rmhTNF (50 IU/mL) combined with 5-FU (25 μg/mL)

rmhTNF (500 IU/mL) combined with 5-FU (25 μg/mL) were 18.20%

48.66%

59.83%

separately

after culturing for 24 h (F=82.742

P0.01); the inhibition rates of rmhTNF (50 IU/mL) combined with 5-FU (25 μg/mL) were 48.66%

68.20%

85.23%

separately

after culturing for 24 h

48 h and 72 h (F=128.583

P0.01). However

on SGC-7901 cells with negative Δ133p53 expression

no growth inhibition was showed by rmhTNF only

the inhibition rates of 50 and 500 IU/mL were 2.74%

3.25% after culturing for 24 h (t=-0.121

P0.05). In apoptosis test

the apoptosis- enhancing effect of rmhTNF was significant on MKN45 cells

and the apoptosis-enhancing effect of 5-FU was further promoted significantly by rmhTNF

the apoptosis of rmhTNF (50 IU/mL)

rmhTNF (50 IU/mL) combined with 5-FU (25 μg/mL)

rmhTNF (500 IU/mL) combined with 5-FU (25 μg/mL) were 18.20%

48.66%

59.83%

separately

after culturing for 24 h (F=123.931

P0.05). In mRNA measurement

down-regulation of Δ133p53 and CyclinB1

up-regulation of Gadd45α were significant in MKN45 cells treated by rmhTNF alone or combined with 5-FU. In nRT-PCR analysis

the mRNA levels of Δ133p53 were relatively 0.886

0.499

0.330

0.161 (F=240.927

P0.01); In real-time PCR analysis

the mRNA levels of Gadd45α were 1.227

1.694

3.394

and the mRNA levels of CyclinB1 were 1.221

0.722

0.316

relatively. The expression of Δ133p53 was positively related to CyclinB1 (r=0.977

P0.01)

but negatively related to Gadd45α (r=-0.950

P0.01). Conclusion: In Δ133p53 positively expressed MKN45 cells

rmhTNF showed as an effective tumor inhibitor and an enhancer of 5-FU as well

and this effect might be helped by two p53 down-stream molecules CyclinB1 and Gadd45α. The results suggest that Δ133p53 might be a key target for the biological effect of rmhTNF against gastric cancer.

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Δ133p53异构体在5-FU抑制胃癌MKN45细胞系生长实验中的作用