Reactive oxygen species generation is essential for cisplatin-induced accelerated senescence in hepatocellular carcinoma

doi:10.1007/s11684-014-0327-1

Front. Med.

2014, Vol. 8

Issue (2) : 227-235 https://doi.org/10.1007/s11684-014-0327-1

RESEARCH ARTICLE

Reactive oxygen species generation is essential for cisplatin-induced accelerated senescence in hepatocellular carcinoma

Kai Qu¹,Ting Lin^1,²,Zhixin Wang¹,Sinan Liu¹,Hulin Chang¹,Xinsen Xu¹,Fandi Meng¹,Lei Zhou¹,Jichao Wei¹,Minghui Tai¹,Yafeng Dong^3,^*(

),Chang Liu^1,^2,^*(

)

1. Department of Hepatobiliary Surgery
2. Surgical Intensive Care Unit, the First Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an 710061, China
3. Department of Obstetrics and Gynecology, Kansas University, Kansas City, KS 66160, USA

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Abstract

Accelerated senescence is important because this process is involved in tumor suppression and has been induced by many chemotherapeutic agents. The platinum-based chemotherapeutic agent cisplatin displays a wide range of antitumor activities. However, the molecular mechanism of cisplatin-induced accelerated senescence in hepatocellular carcinoma (HCC) remains unclear. In the present study, the growth inhibitory effect of cisplatin on HepG2 and SMMC-7721 cells was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cellular senescence was then assessed by β-galactosidase assay. Senescence-related factors, including p53, p21, and p16, were evaluated by quantitative reverse transcription-polymerase chain reaction. Reactive oxygen species (ROS) was analyzed by flow cytometry. Our results revealed that cisplatin reduced the proliferation of HepG2 and SMMC-7721 cells in a dose- and time-dependent manner. Senescent phenotype observed in cisplatin-treated hepatoma cells was dependent on p53 and p21 activation but not on p16 activation. Furthermore, cisplatin-induced accelerated senescence depended on intracellular ROS generation. The ROS scavenger N-acetyl-L-cysteine also significantly suppressed the cisplatin-induced senescence of HepG2 and SMMC-7721 cells. In conclusion, our results revealed a functional link between intracellular ROS generation and cisplatin-induced accelerated senescence, and this link may be used as a potential target of HCC.

Keywords reactive oxygen species senescence cisplatin hepatocellular carcinoma

Corresponding Author(s): Yafeng Dong

Online First Date: 04 May 2014 Issue Date: 21 May 2014

Cite this article:

Ting Lin,Zhixin Wang,Sinan Liu, et al. Reactive oxygen species generation is essential for cisplatin-induced accelerated senescence in hepatocellular carcinoma[J]. Front. Med., 2014, 8(2): 227-235.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-014-0327-1
https://academic.hep.com.cn/fmd/EN/Y2014/V8/I2/227

Tab.1 Primer sequences for qRT-PCR

Fig.1 Effect of cisplatin on the growth of HepG2 and SMMC-7721 cells. (A) Cell viability of HepG2 cells treated with different concentrations of cisplatin (0.5, 1.0, 2.0, 5.0, 10, 20, and 40 µg/ml) for 48 h. (B) Cell viability of SMMC-7721 cells in response to cisplatin. The results are given as mean±SD from three experiments.

Fig.2 SA-β-gal staining detection of cisplatin-treated hepatoma cells. (A) SA-β-gal-positive cells exhibited an extended shape and enlarged nuclei compared with the negative cells. (B) SA-β-Gal staining was performed in hepatoma cells treated with different concentrations of cisplatin (0.5, 1.0, and 2.0 μg/ml) for 96 h.

Fig.3 Positive percentage of SA-β-gal staining in cisplatin-treated hepatoma cells. (A and B) Positive percentage of SA-β-gal staining in HepG2 and SMMC-7721 cells treated with 0.5, 1.0, and 2.0 μg/ml cisplatin for 96 h. (C and D) Positive percentage of SA-β-gal staining in 2.0 μg/ml cisplatin-treated HepG2 and SMMC-7721 cells from 0 h to 120 h. The results are presented as mean±SD from three experiments. *P<0.05 vs. 0 μg/ml cisplatin group; **P<0.01 vs. 0 μg/ml cisplatin group.

Fig.4 Expressions of p53, p21, and p16 in hepatoma cells treated with cisplatin. (A) mRNA expression of p53 in hepatoma cells treated with 2.0 μg/ml cisplatin was detected by real-time PCR. (B) Real-time PCR analysis of the mRNA expression of p21, which increased in hepatoma cells after the cells were treated with cisplatin. (C) mRNA expression of p16 was detected in cisplatin-treated hepatoma cells. The results are presented as mean±SD from three experiments. *P<0.05 vs. 0 h group; **P<0.01 vs. 0 h group; ***P<0.001 vs. 0 h group.

Fig.5 Cisplatin significantly induced ROS generation in hepatoma cells. (A) DCFH staining was performed to detect ROS levels in hepatoma cells treated with 2.0 μg/ml cisplatin for 96 h. (B) Analysis of the changes in ROS content of hepatoma cells treated with different concentrations of cisplatin. The results are presented as mean±SD from three experiments. *P<0.05 vs. control group; **P<0.01 vs. control group.

Fig.6 Removal of ROS by the antioxidant NAC attenuated cisplatin-induced senescence. SA-β-gal staining was performed to detect the changes in the cellular senescence of HepG2 (A) and SMMC-7721 (B) cells incubated with 1 mM exogenous NAC and/or 2.0 μg/ml cisplatin. The results are presented as mean±SD from three experiments. *P<0.05; **P<0.01.

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