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

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

Introduction

Cellular senescence, a process that leads to irreversible cell division arrest, was first described by Hayflick et al. [1] in human fibroblasts cultured in vitro. Since its initial discovery, cellular senescence has been observed in various mammalian tissue in culture and in vivo [2]. Accelerated cellular senescence can be triggered by many different cellular mechanisms, including recognition by cellular sensors of DNA double-strand breaks, leading to the activation of cell cycle checkpoint responses and recruitment of DNA repair foci. In contrast to normal somatic cells, most tumor cells exhibit extended or infinite life spans. Evidence has suggested that senescence escape is involved in the transformation and immortalization of tumor cells.

Chemotherapy-induced senescence is among the key determinants of tumor response to therapy [3]. Studies have demonstrated that chemotherapy drugs affect cell cycle progression, particularly in mitosis and DNA replication; these drugs can also induce senescence-like morphological changes in tumor cells [4]. Platinum compounds, a group of widely used chemotherapeutic drugs, induce DNA damage that can trigger cellular senescence. Cisplatin, the first platinum compound used as a drug, induces the accelerated senescence of lung cancer cells [5] and nasopharyngeal carcinoma cells [6]. However, cisplatin-induced senescence and its molecular mechanism in hepatocellular carcinoma (HCC) have not been clearly demonstrated.

The relationship between intracellular reactive oxygen species (ROS) and accelerated senescence has been reported in several studies [7]. Oxystress-inducing treatments can trigger tumor cells to undergo senescence rapidly [8]. Considering that cisplatin can potently induce oxidative damage, we assume that intracellular ROS generation may also be responsible for cisplatin-induced senescence in HCC. To explore this hypothesis in the present study, we used two HCC cell lines expressing wild-type p53 (HepG2 and SMMC-7721 cells). These cell lines were treated with cisplatin to construct models of accelerated senescence. After detecting intracellular ROS levels in these senescent cells, we evaluated the anti-senescent activity of the ROS scavenger N-acetyl-L-cysteine (NAC). Our results demonstrated that intracellular ROS generation mainly functioned in cisplatin-induced accelerated senescence in HCC cells.

Materials and methods

Materials and agents

Fetal calf serum and RPMI-1640 medium were purchased from GIBCO (Canada). Cisplatin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and propidium iodide were purchased from Sigma (USA). Annexin V-FITC apoptosis detection kit was purchased from BD Biosciences (USA). Senescence-associated β-galactosidase (SA-β-Gal) staining kit was purchased from GENMED (USA). Total RNA kit and polymerase chain reaction (PCR) primer were purchased from TaKaRa (Japan).

Cell culture

The human HCC cell line HepG2 was obtained from the American Type Culture Collection (ATCC no. HB-8065). SMMC7721 cells were provided by the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). HepG2 cells (5.0×10⁴ cells/ml) were cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37β°C with 5% CO₂. SMMC7721 was maintained in Dulbecco’s modified Eagle’s medium and cultured under the same conditions.

Intracellular ROS assay

Dichloro-dihydro-fluorescein diacetate (DCFH-DA) was cleaved intracellularly by non-specific esterases and oxidized by ROS, forming highly fluorescent 2,7-dichlorofluorescein (DCF). HepG2 cells (5.0×10⁴ cells/well) were seeded onto six-well plates and treated with cisplatin for 48 h. DCFH-DA working solution was added directly to the medium until a final concentration of 10 μM was reached. The cells were then incubated at 37°C for 15 min, washed once with 1×phosphate buffered saline (PBS), and stored in an ice bath for immediate DCF detection by flow cytometry (Becton Dickinson FACS Calibur, USA). Afterward, the cells were analyzed using Cell Quest software (BD Biosciences).

MTT assay for cell viability

HepG2 and SMMC-7721 cells (5.0×10⁴ cells/well) were seeded onto 96-well plates and incubated with cisplatin at various concentrations (0, 0.5, 1.0, 2.0, 5.0, 10, 20, and 40 µg/ml) for 48 h at 37°C in an atmosphere containing 50 ml/L CO₂. MTT solution (20 µl; 5 g/L) was then added to each well and incubated for another 4 h. Supernatants were removed and formazan crystals were dissolved in 200 µl of dimethylsulfoxide. Optical density was determined at 490 nm by using a POLARstar OPTIMA microplate reader (BMG Labtechnologies, Ortenberg, Germany).

SA-β-Gal assay

The cells were seeded onto six-well plates (5.0×10⁴ cells/well) and incubated with test substances at 37°C at various time points with 50 ml/L CO₂. After the medium was removed, the cells were washed in 1×PBS and fixed in 2% formaldehyde/0.2% glutaraldehyde for 3 min to 5 min at room temperature. These cells were washed twice with 1×PBS and incubated at 37°C with fresh SA-β-Gal staining solution containing 0.4 g/L X-gal, 40 mM citric acid/sodium phosphate (pH 6.0), 5 mM potassium ferrocyanide, 150 mM NaCl, and 2 mM MgCl₂. Staining was achieved in 2 h to 4 h.

RNA isolation and qRT-PCR analysis

Total RNA was isolated from cultured cells by using an RNAfast200 kit (Fastagen Biotech, Shanghai, China). cDNA was synthesized from the total RNA (2 μg) by using random hexamer primers and a RevertAid^TM first-strand cDNA synthesis kit (Fermentas, USA) according to the manufacturer’s instructions. PCR was performed using an Applied Biosystems Prism 7300 with the SYBR green dye (TaKaRa). qRT-PCR was performed according to methods described previously [9]. mRNA expression was assayed in triplicate and normalized to β-actin mRNA expression. Relative levels were calculated using the comparative-Ct method (∆∆Ct method). Primer pairs for different Prx isoforms and β-actin (Sangon Biotech, Co., Ltd., Shanghai, China) are listed in Table 1.

Statistical analysis

Data were expressed as mean±standard deviation (SD) and analyzed by SPSS 11.0 software (SPSS Inc., Chicago, IL, USA). Data were analyzed using Student’s t-test. P<0.05 was considered statistically significant. All of the experiments were repeated thrice.

Results

Cisplatin inhibits cellular proliferation in human hepatoma cells

The growth of HepG2 and SMMC-7721 cells was evaluated using the MTT assay after these cells were treated with cisplatin at various concentrations (0.5, 1.0, 2.0, 5.0, 10, 20, and 40 µg/ml). In the MTT assay, hepatoma cells were treated with 5.0 µg/ml cisplatin for 48 h, and the result showed that cell viability decreased by 30% to 60% (Fig. 1). The IC₅₀ values of cisplatin in HepG2 and SMMC-7721 cells were 3.8 and 9.7 µg/ml, respectively. By contrast, no difference in cell viability was observed in the cells treated with cisplatin at concentrations ranging from 0.5 µg/ml to 2.0 µg/ml.

Cisplatin induces an accelerated senescent phenotype of human hepatoma cells

Cell viability was not significantly affected by low-dose cisplatin (0.5 μg/ml to 2.0 μg/ml). However, cisplatin may have caused senescence in hepatoma cells because the sublethal concentrations of chemotherapeutic agents have induced senescence in some tumors [5,6]. In this study, SA-β-gal, one of the most commonly used biomarkers for senescent and aging cells, was used to detect senescent phenotype induced by 0.5, 1.0, or 2.0 μg/ml cisplatin. SA-β-gal-positive cells usually show an extended cell shape with enlarged or multiple nuclei. In our study, SA-β-gal staining was positive in the cytoplasm of these cells (Fig. 2A). The highest positive rate of SA-β-Gal staining was observed in the group treated with 2.0 µg/ml; by contrast, the control group and the 1.0 µg/ml cisplatin-treated group showed low staining rates (P<0.05; Figs. 2B, 3A, and 3B, respectively). HepG2 and SMMC-7721 cells were treated with 2.0 µg/ml cisplatin for 120 h and then collected at an interval of 24 h for analysis by SA-β-Gal staining. The result showed that cisplatin accelerated senescence in a time-dependent manner; the highest percentage in HepG2 and SMMC-7721 cells was reached at 96 h (47.7%±9.0% and 45.3%±7.6%; Fig. 3C and 3D, respectively).

Cisplatin affects the expressions of p53, p21, and p16 genes in hepatoma cells

Senescence is usually mediated by p53/p21 or p16 pathway [10]. As such, we examined the effect of cisplatin on the expressions of p53, p21, and p16 in HepG2 and SMMC-7721 cells. HepG2 and SMMC-7721 cells were treated with 2.0 µg/ml cisplatin for 120βh and collected at an interval of 24 h for qRT-PCR detection. As the senescence level of hepatoma cells increased, cisplatin treatment significantly increased the mRNA expression levels of p53. The maximum level was reached at 120 h (Fig. 4A). p21, as the downstream gene activated by p53, also increased after HepG2 and SMMC-7721 cells were treated with 2.0 µg/ml cisplatin (Fig. 4B). By contrast, the changes in the mRNA expression levels of p16 in cisplatin-treated HepG2 and SMMC-7721 cells were negligible (Fig. 4C).

Cisplatin induces intracellular ROS generation in senescent hepatoma cells

ROS functions with intracellular Ca²⁺ signaling pathways and may regulate the balance between cell proliferation and cell cycle arrest. Studies have shown that ROS can regulate the entry of many primary and tumor cells into senescence [11]. To investigate the mechanism underlying the significant induction of accelerated senescence by cisplatin, we determined intracellular ROS generation by FACS analysis (Fig. 5A). A significant, dose-dependent increase in intracellular ROS levels was observed in hepatoma cells treated with cisplatin at 48 h. The highest ROS levels were obtained in hepatoma cells treated with 2.0 μg/ml cisplatin (Fig. 5B); with this cisplatin treatment, ROS content was four times higher than that in the control group. A consistent expression pattern of intracellular ROS generation and accelerated senescence induction in hepatoma cells were observed, suggesting the important function of ROS production in cisplatin-induced accelerated senescence.

ROS scavenger NAC suppresses cisplatin-induced senescence in hepatoma cells

The free-radical scavenger NAC can neutralize ROS as soon as these toxic substances are generated, indicating the function of ROS in biological processes. To demonstrate the relationship between cisplatin-induced ROS production and accelerated senescence, we treated the hepatoma cells with 2.0 μg/ml cisplatin in the presence or absence of 1 mM exogenous NAC. After the cells were co-incubated with 1 mM NAC, senescence induced by cisplatin treatment was abrogated (Fig. 6). In contrast to 2.0 µg/ml cisplatin alone, the combined treatment of cisplatin and 1 mM NAC reduced the positive rate of SA-β-gal staining by approximately 20%. In the absence of cisplatin, NAC treatment exhibited a negligible effect on accelerated senescence. Hence, ROS exhibited a significant function in the cisplatin-induced accelerated senescence of hepatoma cells.

Discussion

Accelerated senescence is described as the physiological process of terminal growth arrest [12]. Clinical and preclinical studies have indicated that many anti-tumor manipulations, including chemotherapeutic drugs and radiation, can induce tumor senescence [13,14]. Studies have further shown that senescence is involved in the transformation of liver cancer; treatment-induced senescence is also among the key determinants of tumor response to therapy in vitro and in vivo [15]. Although several cellular senescent phenomena in several cancers [6,8,16,17] have been reported, the chemotherapeutic induction of cellular senescence in HCC is rarely investigated [18]. In the present study, accelerated senescence was detected in low-dose cisplatin-treated hepatoma cell lines HepG2 and SMMC-7721. Approximately 50% of senescent cells were harvested after these cells were treated with 2.0 μg/ml cisplatin compared with other treatment groups. Our results are consistent with those presented in previous studies; in previous studies, the relative importance of different pathways of cell death and senescence in tumor cell response to anticancer drugs may vary depending on cell type, nature of drugs, and conditions of drug exposure. Cell apoptosis induction is generally observed at high drug doses, whereas low-dose chemotherapeutic drugs elicit a more pronounced cytostatic effect (i.e., senescence) [3]. In the present study, senescence was relatively more prominent at low cytotoxic drug doses (1.0 μg/ml to 5.0 μg/ml cisplatin). In another study, senescence can be readily induced and maintained for a long period of time in the patients’ plasma by continuous infusion [19]. Therefore, senescence may be a significant determinant of tumor response to continuous infusion protocols.

p53/p21 and p16 function as the main regulators of senescence in various tumor types; these regulators can sufficiently promote tumor regression by triggering senescence [20,21]. DNA damage induced by clinically relevant concentrations of cisplatin can induce irreversible growth arrest by activating p53/p21 or p16 [22,23]. In our previous studies, oxaliplatin (a third-generation platinum-derived chemotherapy agent) significantly induces p53 expression but fails to affect p16 expression [9]. In the present study, our data also confirmed that p53 and p21 exhibited an important function in the onset of senescence, whereas p16 may not be involved in cisplatin-induced senescence in hepatoma cells. This result is consistent with that in a previous study regarding human fibroblast senescence, in which differential functions of p21 and p16 in cellular senescence and differentiation are observed [24].

ROS is a mediator of intracellular signaling cascades, which can induce the collapse of mitochondrial membrane potential and trigger a series of mitochondrion-associated events, such as apoptosis and senescence [25]. Platinum compounds are related to ROS production, and cisplatin can potentially induce ROS generation in hepatoma cells [26]. To determine whether or not cisplatin-induced accelerated senescence depends on ROS generation, we detected intracellular ROS levels in hepatoma cells treated with cisplatin. Our results demonstrated that the cisplatin-induced accelerated senescence was associated with the generation of ROS; by contrast, antioxidant NAC functioned as a ROS scavenger and suppressed the cisplatin-induced senescent phenotype.

In conclusion, hepatocellular senescence induced by sublethal concentrations of cisplatin could be involved in intracellular ROS generation. The molecular mechanism of this process should be evaluated further to elucidate the principles of chemotherapy and prevent chemotherapy-induced toxicity in patients with HCC.

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