Salidroside induces apoptosis in human ovarian cancer SKOV3 and A2780 cells through the p53 signaling pathway

Introduction

Ovarian cancer (OC) is the most lethal gynecological malignancy (1). For epithelial OC (EOC), the prognosis for premenopausal women with early-stage EOC is favorable (2). In the past few decades, patients with EOC were treated with the latest chemotherapeutic drugs and surgical techniques, but the 5-year survival rate was still ≤40% (3). A previous study reported that EOC demonstrates genomic instability (4). During treatment, numerous patients with EOC have recurrence and become resistant to chemotherapy, indicating that new treatment strategies are required (5,6). Therefore, numerous studies have been performed to identify effective therapeutic agents and their associated mechanisms of action (6,7).

Salidroside, a p-hydroxyphenethyl-β-D-glucoside (or phenylpropanoid glycoside), is one of the major active ingredients extracted from Rhodiola rosea and has a long history of use in Chinese medicine (8–11). Salidroside has primarily been used as a brain tonic, a roborant or headache relief agent (8,12). Recently, salidroside has been studied in experimental animals for its protective effects against hypoxia, cold, radiation and heavy physical exercise (11). It has been demonstrated that salidroside has various pharmacological properties (13), including antiaging (14), anticancer (15), anti-inflammation, hepatoprotective and antioxidative effects (16).

The aim of the current study was to investigate the effects of salidroside on OC, and to determine whether it may be a new therapeutic candidate in the treatment of OC.

Materials and methods

Cell culture

Human ovarian cancer cell lines, SKOV3 and A2780 from the American Type Culture Collection (Manassas, VA, USA), were cultured in RPMI 1640 medium (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) containing 10% (v/v) heat-inactivated fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in an incubator containing humidified air with 5% (v/v) CO2.

Cell viability assay

SKOV3 and A2780 cells were seeded in 96-well plates at 5×103 cells/well and treated with salidroside at different concentrations (0, 50, 100, 500, 1,000 or 2,000 µmol/l) for 48 h, at 37°C in an incubator containing humidified air with 5% (v/v) CO2. A total of 10 µl MTT (5 mg/ml) was added to each well and incubated in the dark at 37°C for 4 h. The supernatant was removed and replaced with 150 µl dimethyl sulfoxide. The plates were oscillated for 10 min and the absorbance was measured at 490 nm.

Acridine orange/ethidium bromide (AO/EB) staining

SKOV3 and A2780 cells in the logarithmic growth phase were deposited in 2×103 cells each well of a 96-well plate. After 24 h, cells were subsequently treated with salidroside (0 or 1,000 µmol/l) for 48 h at 37°C in an incubator containing humidified air with 5% (v/v) CO2, stained with AO (100 µg/ml in PBS) and EB (100 µg/ml in PBS) at room temperature and analyzed by fluorescence microscopy at ×200 magnification (17). Five fields were randomly selected for study. Cell apoptosis (%) was defined as the number of apoptotic cells divided by the total number of cells (18,19).

Antibodies and western blotting

SKOV3 and A2780 cells [treated with salidroside (1,000 µmol/l; salidroside group) or PBS (control group) for 48 h at 37°C in an incubator containing humidified air with 5% (v/v) CO2] were collected and lysed in ice-cold radioimmunoprecipitation assay buffer (Roche Diagnostics, Basel, Switzerland). The protein concentration of the lysates was measured using a bicinchoninic acid Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.) according to the protocol of the manufacturer. Cell lysates were separated by 10% SDS-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes (Pall Corporation, Port Washington, NY, USA). The membranes were blocked using 5% skim milk then incubated with primary antibodies against β-actin (cat. no. 3700; 1:2,000 dilution; Cell Signaling Technology, Inc., Danvers, MA, USA), p53 (cat. no. 2524; 1:500 dilution; Cell Signaling Technology, Inc.), p21 Cip1/Waf1 (cat. no. 610233; 1:500 dilution; BD Biosciences, Franklin Lakes, NJ, USA), p16INK4a (cat. no. sc-53392; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bcl-2-associated X protein (Bax) (cat. no. 5020; 1:1,000 dilution; Cell Signaling Technology, Inc.), Bcl-2-associated death promoter (Bad) (cat. no. 9268; 1:500 dilution; Cell Signaling Technology, Inc.), and p-Bad (cat. no. 9291; 1:500 dilution; Cell Signaling Technology, Inc.), Bcl-2 (cat. no. 15071; 1:1,000 dilution; Cell Signaling Technology, Inc.), apoptosis-inducing factor (AIF) (cat. no. 5318; 1:1,000 dilution; Cell Signaling Technology, Inc.) and X-linked inhibitor of apoptosis (XIAP) (cat. no. 14334; 1:1,000 dilution; Cell Signaling Technology, Inc.) at 4°C overnight. The membranes were incubated with rabbit (cat. no. 7054; 1:5,000 dilution; Cell Signaling Technology, Inc.) or mouse (cat. no. 7056; 1:5,000 dilution; Cell Signaling Technology, Inc.) secondary antibodies at room temperature for 1.5 h. Immunoreactive bands were visualized using an enhanced chemiluminescence reagent (GE Healthcare, Chicago, IL, USA). Intensities of immunoreactive bands were determined by densitometric analysis using ImageJ software (version 1.61; National Institutes of Health, Bethesda, MD, USA) and normalized against β-actin.

Caspase-3 activity assay

A caspase-3 activity assay kit (cat no. C1115; Beyotime Institute of Biotechnology, Haimen, China) was used to test the activity of caspase-3 following treatment with salidroside (0 or 1,000 µmol/l) for 48 h in ovarian cancer (SKOV3 and A2780) cells. Caspase activity was expressed as a percentage of the control.

Statistical analysis

Data are presented as the mean ± standard error of the mean of three independent experiments. Statistical analysis was conducted using SPSS 19.0 software (IBM Corp., Armonk, NY, USA) and illustrated using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Statistical significance was determined using Student's t-test to compare two groups or analysis of variance with Tukey's post hoc test to compare multiple groups. P<0.05 was considered to indicate a statistically significant difference.

Results

Assessment of OC cell viability after treatment with salidroside

Across different concentrations (500, 1,000 or 2,000 µmol/l) of salidroside treatment, cell viability was significantly inhibited compared with the control (Fig. 1). The data revealed that salidroside treatment at 1,000 µmol/l for 48 h inhibited viability of SKOV3 and A2780 cells by ~50%. For this reason, in subsequent experiments 1,000 µmol/l was used as salidroside treatment.

Figure 1. Salidroside inhibits the viability of SKOV3 and A2780 cells. (A) SKOV3 and (B) A2780 cells were treated with salidroside. Relative cell viability was determined by MTT assay. *P<0.05 vs. control; n=6 independent experiments for each condition.

Salidroside induces apoptosis in SKOV3 and A2780 cells

Cell viability is a balance between cell proliferation and apoptosis (20). AO/EB staining revealed that salidroside significantly increased the rate of apoptosis in SKOV3 (Fig. 2A) and A2780 cells (Fig. 2B).

Figure 2. Salidroside induces apoptosis in SKOV3 and A2780 cells. Acridine orange/ethidium bromide staining was performed to detect changes in the nuclei of (A) SKOV3 and (B) A2780 cells. Red arrows indicate apoptotic cells. Magnification, ×200. Data are expressed as the mean ± standard error of the mean; n=3 for each group. *P<0.05 vs. control.

Salidroside activates pro-apoptotic signaling pathways

To explore the functional mechanisms of salidroside, the expression of Bax, Bcl-2, Bad, p-Bad, AIF and XIAP proteins was investigated by western blotting in SKOV3 (Fig. 3A-E) and A2780 (Fig. 3F-J) cells. The results indicated that treatment with salidroside significantly upregulated the ratio of Bax/Bcl-2 and the expression of Bad and AIF, but significantly downregulated p-Bad and XIAP expression in SKOV3 and A2789 cells. Meanwhile, caspase-3 activity was significantly increased by salidroside in SKOV3 (2.1-fold increase) and A2780 (2.5-fold increase) cells.

Figure 3. Salidroside alters p-Bad, Bad, Bax, Bcl-2, AIF and XIAP expression, and promotes caspase-3 activation. Western blotting was used to detect protein expression. Relative protein expression was normalized to β-actin. Caspase-3 activity was measured using a commercial assay kit. (A) Bax and Bcl-2 expression, (B) Bad and p-Bad expression, (C) caspase-3 activity, (D) AIF expression and (E) XIAP expression in SKOV3 cells treated with salidroside. (F) Bax and Bcl-2 expression, (G) Bad and p-Bad expression, (H) caspase-3 activity, (I) AIF expression and (J) XIAP expression in A2780 cells treated with salidroside. Data are expressed as the mean ± standard error of the mean; n=3 independent experiments for each group. *P<0.05 vs. control. Bax, Bcl-2-associated X protein; Bad, Bcl-2-associated death promoter; AIF, apoptosis-inducing factor; XIAP, X-linked inhibitor of apoptosis.

Salidroside activates p53 signaling pathways

To define whether p53 signaling was involved in salidroside-induced apoptosis, the protein levels of p53, p21Cip1/Waf1 and p16INK4a were evaluated. They were identified to be significantly upregulated in SKOV3 (Fig. 4A-C) and A2780 (Fig. 4D-F) cells after treatment with salidroside compared with the control. These results indicated that the p53/p21Cip1/Waf1/p16INK4a pathway serves a critical function in salidroside-induced apoptosis in SKOV3 and A2780 cells.

Figure 4. Salidroside induces the expression of p53, p21Cip1/Waf1 and p16INK4a in SKOV3 and A2780 cells. Protein levels of (A) p53, (B) p21Cip1/Waf1 and (C) p16INK4a in SKOV3 cells. Protein levels of (D) p53, (E) p21Cip1/Waf1 and (F) p16INK4a in A2780 cells. Data are expressed as the mean ± standard error of the mean; n=3 for each group. *P<0.05 vs. control.

Discussion

In the present study, in vitro experiments demonstrated that salidroside exerts potent anti-proliferative effects on SKOV3 and A2780 cells by inducing apoptosis. Furthermore, the mechanisms underlying the anticancer effects of salidroside on OC were investigated. The results indicated that activation of the caspase-3-dependent pathway and the p53 signaling pathway were involved in mediating these salidroside-induced effects. Therefore, the present results may provide an experimental basis for the potential role of salidroside in treating OC.

The current results are consistent with previous reports demonstrating that salidroside exerts anticancer effects in breast carcinoma (15), human fibrosarcoma (21,22) and neuroblastoma (23). The mechanism of action of salidroside has been reported to involve autophagy (24). However, the effect of salidroside in OC is not yet fully understood.

The present data suggested that salidroside has antiproliferative and pro-apoptotic effects on OC cells. It was revealed that salidroside inhibited the viability of SKOV3 and A2780 cells. Furthermore, AO/EB staining indicated that salidroside induced apoptosis in OC cells.

The effect of salidroside on the regulation of gene expression has been studied previously. Numerous studies have revealed that Bax, Bcl-2 and caspase-3 are involved in apoptosis in SKOV3 and A2780 cells (25). Liu et al (26) identified that caspase-mediated cleavage of Beclin1 inhibits autophagy and promotes apoptosis in SKOV3 cells. Furthermore, andrographolide radiosensitizes human SKOV3 cells (27), and miRNA-149 modulates the chemosensitivity of A2780 cells by modulating Bax protein expression (28). In the present study, it was demonstrated that salidroside could induce Bcl-2 and Bax expression, and upregulate caspase-3 in SKOV3 and A2780 cells. In addition, the ratio of Bcl-2/Bax was /decreased, indicating that salidroside promotes apoptosis in OC. Bad is known to regulate apoptosis by forming heterodimers with Bax and Bcl-2 (29). XIAP inhibits activation of caspases by binding to them, preventing apoptosis of tumor cells (30). In the present study, Bad protein was significantly increased while p-Bad and XIAP levels were significantly decreased following treatment with salidroside. These results demonstrate that salidroside could induce apoptosis via caspase-3-dependent apoptosis signaling in OC (31,32). Furthermore, p53 can influence apoptosis by regulating Bcl-2 (33). Previous studies have revealed that >50% of tumors are associated with p53 gene mutation; wild-type p53 gene therapy has been suggested to strengthen sensitivity to cisplatin in SKOV3 cells (34–36). In the present study, it was demonstrated that salidroside has the ability to induce OC apoptosis. Salidroside also promoted the expression of p53, p21Cip1/Waf1 and p16 INK4a expression in SKOV3 and A2780 cells. It was identified that salidroside promotes the expression of caspase-3 and activation of the p53/p21Cip1/Waf1/p16INK4a pathway.

In summary, salidroside was demonstrated to reduce cell viability and promote apoptosis in OC. Furthermore, it was identified that salidroside activates caspase-3 and the p53/p21Cip1/Waf1/p16INK4a pathway. Therefore, salidroside is a promising new approach for the treatment of OC, but its underlying mechanism needs to be explored further.

Competing interests

The authors declare that they have no competing interests.

References