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Introduction

Hepatocellular carcinoma (HCC) accounts for 80–90% of primary liver cancers and is one of the most prevalent malignant tumors in the world (1). The incidence of HCC has steadily increased in Western countries over the past decade and the global incidence of HCC is predicted to continue to rise over the next few years (2,3). HCC has the third-highest rate of cancer-associated mortalities (4) and therefore studies into the effective treatment of HCC are essential.

Statins, cholesterol-lowering drugs, are one of the most commonly prescribed types of medications. Previous studies have focused on the use of statins as therapeutic agents for the treatment of solid and hematological cancers (5,6,7). Statins have been shown to elicit pleiotropic effects on various cell types and differentially modulate cellular functions, including cell migration, proliferation, survival and apoptosis, in normal as well as malignant cells (8). Simvastatin, a member of the statin family, was previously reported to regulate the migration, proliferation, apoptosis and growth of tumor cells (9,10); in addition, Wang et al (11) demonstrated that simvastatin induced caspase-dependent apoptosis and activated p53 in OCM-1 cells. Kochuparambil et al (10) found that the inhibitory effect of simvastatin on prostate cancer cell growth occurred via the inhibition of the Akt pathway. Previous studies have suggested that simvastatin regulated arteriogenesis following stroke and enhanced the differentiation of bone marrow stromal cells into endothelial cells via the Notch signaling pathway (12,13); this therefore indicated that the pharmacological manipulation of Notch signaling may be a promising novel strategy for the treatment of human disease.

Notch genes encode proteins which are activated via interaction with certain families of ligands (14,15); in mammals, there are four Notch receptors (Notch1-4), which have five corresponding ligands, including δ-like 1, 3 and 4 as well as Jagged 1 and 2. Interaction between Notch receptors and their ligands induces a two-step proteolysis, resulting in activation of the Notch receptors. Activated Notch receptors have a critical role in maintaining the balance between cell proliferation, differentiation and apoptosis (16); therefore perturbed Notch signaling may contribute to tumorigenesis. Notch1 is the primary member of the Notch family, which has been previously reported to regulate the growth, apoptosis, migration and invasion of tumor cells, including breast, pancreatic and cervical cancer cells (17–19). Qi et al 20) demonstrated that Notch1 regulated HCC cell proliferation and apoptosis via different mechanisms. Therefore, the aim of the present study was to investigate the effect of simvastatin on Akt signaling and, in turn, HCC cell proliferation and apoptosis, as well as to determine whether the mechanism of action of simvastatin proceeded via regulation of Notch1 expression.

Materials and methods

Cell culture

Human HCC HepG2 and Huh7 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in RPMI-1640 (Gibco-BRL, Carlsbad, CA, USA), as previously described (21), supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT, USA).

Simvastatin treatment

Simvastatin (Merck, Darmstadt, Germany) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) to obtain simvastatin concentrations of 0, 2, 4, 8 and 16 μM. HCC cells were plated and incubated with various concentrations of simvastatin (50 μl) for 24, 48, and 72 h at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Following treatment, cells were collected and washed with phosphate-buffered saline (PBS; Sigma-Aldrich) prior to further use.

Trypan blue assay

A trypan blue exclusion assay was used to determine the viability of HepG2 and Huh7 cells. In brief, HepG2 and Huh7 cells were stained using 0.04% (w/v) trypan blue solution (Invitrogen Life Technologies, Carlsbad, CA, USA) at room temperature for 7 min, during which irreversibly damaged cell membranes take up the anionic dye trypan blue; cells which excluded trypan were considered to have survived and trypan blue-positive cells were identified as dead. HepG2 and Huh7 cells were then visualized using microscopy (magnification, ×40). For each experiment, 200 HepG2 and Huh7 cells were analyzed from ten different fields of vision/dish.

MTT assay

HepG2 and Huh7 cells were seeded into 96-well plates at 1,000 cells per well and then treated with various concentrations of simvastatin. On the day cells were harvested, 100 μl spent culture medium was replaced with an equal volume of fresh medium. MTT (Sigma-Aldrich) was added to the cells at a final concentration of 0.5 mg/ml, and the plates were incubated for 4 h at 37°C. DMSO (100 μl) was then added to each well and plates incubated with agitation at room temperature for 10 min. The absorbance was measured at 570 nm using a spectrophotometer (Multiskan MK3; Thermo Fisher Scientific, Waltham, MA, USA).

Notch1 small interfering (si)RNA plasmid transfection into HepG2 cells

Transfection of the Notch1 siRNA plasmid into HepG2 and Huh7 cells was performed as previously described (19). In brief, cells were plated in a 35-mm dish for 24 h prior to transfection in complete medium. Transfection was performed using Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer’s instructions. The Notch1-specific siRNA plasmid for HepG2 (sense, 5′-AAGGUGUCUUCCAGAUCCUGA-3′) (22) and the scrambled sequence (5′-AAAUGUGUGUACGUCUCCUCC-3′) were inserted into the pGPU6/green fluorescent protein/Neo plasmid (Shanghai GenePharma, Ltd, Shanghai, China). The efficiency of transfection was confirmed using western blot analysis. Clones, in which the expression of Notch1 was effectively suppressed, were selected and used for the in vitro study.

Detection of apoptosis

Cell apoptosis was analyzed using flow cytometric (FCM) analysis. In brief, following 48 h of simvastatin treatment at various concentrations (0, 2, 4, 8 and 16 μM), HepG2 and Huh7 cells were trypsinized using trypsin (Mingzhu Chemical Co., Shanghai, China), washed with PBS and then suspended with binding buffer. Cells apoptosis was detected by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (BD PharMingen, San Diego, CA, USA). Cells were incubated with 5 μl PI (Sigma-Aldrich) for 15 min at room temperature in the dark, prior to suspension in 500 μl binding buffer. The cells were then analyzed using FCM (Beckman-Coulter, Brea, CA, USA) for relative quantitative apoptosis.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Total RNA was isolated from transfected and non-transfected HepG2 cells using TRIzol® reagent (Life Technologies, Inc., Rockville, MD, USA) according to the manufacturer’s instructions. Reverse transcription was performed on 1 μg of total RNA from each sample using oligo (dT) primers and 200 units of SuperScript II (Life Technologies, Inc.) for extension. PCR amplification was performed using 1.25 U Ex Taq polymerase (Takara Bio, Inc., Dalian, China). All PCR products were resolved on 1.8% agarose gels containing ethidium bromide (Sigma-Aldrich). Primer pairs for human Notch1, p53, B cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), phosphorylated Akt (p-Akt), Akt and β-actin specific amplification of complementary DNA were as follows: Notch1 forward, 5′-CCGCAGTTGTGCTCCTGAA-3′ and reverse, 5′-ACCTTGGCGGTCTCGTAGCT-3′; p53 forward, 5′-GACCCAGGTCCAGATGAAGCT-3′ and reverse, 5′-ACCGTAGCTGCCCTGGTAGGT-3′; Bcl-2 forward, 5′-AGTTCGCCGAGATGTCCAGGCA-3′ and reverse, 5′-ACTTGTGGCCCAGATAGGCACC-3′; Bax forward, 5′-ACAGGGTTTCACCAGGATC-3′ and reverse, 5′-GCTGCCACCCGCAAGAAGAC-3′; p-Akt forward, 5′-GGAGAUCAUGCAGCAUCGC-3′ and reverse, 5′-GCGAUGCUGCAUGAUCUCC-3′; Akt forward, 5′-CTTTCCAGACCCACGACC-3′ and reverse, 5′-CTCCGAGTGCAGGTAGTCC-3′; β-actin forward, 5′-GAGGCACTCTTCCAGCCTTC-3′ and reverse, 5′-GGATGTCCACGTCACACTTC-3′.

Western blot analysis

Cells were homogenized and lysed using radioimmunoprecipitation assay lysis buffer (100 mm NaCl; 50 mm Tris-HCl, pH 7.5; 1% TritonX-100; 1 mm EDTA; 10 mm β-glycerophosphate; 2 mm sodium vanadate; and protease inhibitor). Protein concentrations were assayed using the micro-bicinchoninic acid assay protein assay (Pierce Biotechnology, Inc., Rockford, IL, USA). Proteins (20–30 μg per lane) were separated using 10% SDS-PAGE and then electroblotted onto nitrocellulose membranes (GE Healthcare, Little Chalfont, UK). Non-specific binding was blocked by incubating with 5% non-fat milk in PBS with Tween 20 buffer (Sigma-Aldrich) at room temperature for 1 h. Cells were then incubated overnight at 4°C with a 1:1,000 dilution of primary antibodies for Notch1 (monoclonal mouse anti-human), Akt (polyclonal rabbit anti-human), p-Akt (polyclonal rabbit anti-human), p53 (polyclonal rabbit anti-human), β-actin (monoclonal mouse anti-human), Bcl-2 (monoclonal mouse anti-human) and Bax (monoclonal mouse anti-human), all purchase from Sigma-Aldrich, followed by incubation with the corresponding horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (Ig)G and rabbit anti-mouse IgG (1:2,000; Sigma-Aldrich) at room temperature for 1 h. Antigens were then detected using the standard enhanced chemiluminescence kit (Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Statistical analysis

All experiments were performed in triplicate, unless otherwise stated. Statistical analyses were performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). Values are presented as the mean ± standard deviation. Statistical comparisons between groups were performed using a one-way analysis of variance followed by a Student’s t test. P<0.05 was considered to indicate a statistically significant difference between values.

Results

Simvastatin inhibits HCC cell viability and proliferation

The cytotoxic effects of various concentrations (0, 2, 4, 8, and 16 μM) and incubation periods (24, 48, and 72 h) of simvastatin was examined in HepG2 and Huh7 cells using a trypan blue assay. As shown in Fig. 1, simvastatin demonstrated high levels of cytotoxicity in HepG2 and Huh7 cells in a time- and dose-dependent manner. Following 72 h of simavastin treatment, even at the lowest dose (2 μM), the viability and proliferation of HepG2 and Huh7 cells was significantly decreased by >34 and 36%, respectively, compared to the untreated group. These results therefore indicated that simvastatin may be a promising anti-cancer drug for the treatment of HCC.

Figure 1 Simvastatin decreases HepG2 and Huh7 cell viability and proliferation. HepG2 and Huh7 cells were treated with various concentration of simvastatin (0, 2, 4, 8 and 16 μM) for 24, 48 and 72 h. (A) Bar graph showing trypan blue-positive simvastatin-treated HepG2 cells. (B) Broken line showing MTT results of proliferation of simvastatin-treated HepG2 cells. (C) Bar graph showing trypan blue-positive simvastatin-treated Huh7 cells. (D) Broken line showing MTT results of proliferation of simvastatin-treated Huh7 cells. *P<0.05 and **P<0.01 compared with the control group.

Simvastatin induces apoptosis in HepG2 and Huh7 cells

Bcl-2 family genes have an important role in the regulation of apoptosis; Bcl-2 is an anti-apoptosis gene, while Bax is a pro-apoptosis gene (23). In normal cells the expression of Bcl-2 and Bax is maintained at an equilibrium; however, during apoptosis, the expression of Bcl-2 is downregulated, while Bax is overexpressed (24). In the present study FCM analysis was performed in order to determine whether apoptosis contributed to the simvastatin-induced reduction in HepG2 and Huh7 cell viability. As shown in Fig. 2A and B, HepG2 and Huh7 cells in the experimental groups demonstrated a marked increase in apoptosis in a time- and dose-dependent manner. In addition, Fig. 2C and D showed that the apoptosis-associated genes Bcl-2 and Bax had significantly altered expression in cells treated with simvastatin; mRNA expression levels of Bcl-2 were significantly decreased and expression levels of Bax were significantly increased in a time- and dose-dependent manner.

Figure 2 Simvastatin induces apoptosis and affects the expression of apoptosis-related genes in HepG2 and Huh7 cells. HepG2 and Huh7 cells were treated with various concentrations of simvastatin (0, 2, 4, 8 and 16 μM) for 48 h. Flow cytometric analysis of the apoptotic rate of simvastatin-treated (A) HepG2 and (B) Huh7 cells. (C) Reverse transcription quantitative polymerase chain reaction showing the mRNA expression level of Bcl-2 and Bax in simvastatin-treated (C) HepG2 and (D) Huh7 cells. *P<0.05 and **P<0.01 compared with the control group. FITC-A, fluorescein isothiocyanate; PE, R-phycoerythrin; Bcl-2, B cell lymphoma 2; Bax, Bcl-2-associated X protein.

Simvastatin increases mRNA expression levels of Notch1 and p53

RT-qPCR was used in order to investigate the effects of simvastatin on the expression of Notch1 and p53 in HepG2 cells. As shown in Fig. 3, following incubation of HepG2 cells with various concentrations (0, 2, 4, 8 and 16 μM) of simvastatin for 48 h, mRNA expression levels of Notch1 and p53 were significantly increased in a dose-dependent manner.

Figure 3 Simvastatin increases mRNA levels of Notch1 and p53 in HepG2 cells. Reverse transcription quantitative polymerase chain reaction showing the mRNA expression levels of (A) Notch1 and (B) p53 in HepG2 cells treated with various concentrations of simvastatin (0, 2, 4, 8 and 16 μM) for 24, 48 and 72 h. *P<0.05 and **P<0.01 compared with control group.

Simvastatin-induced apoptosis is decreased following Notch1 gene knock-out

The results of the present study, as shown above, demonstrated that simvastatin increased mRNA expression of Notch1; therefore, Notch1 siRNA transfection experiments were performed which knocked out the Notch1 gene in HepG2 cells, in order to further investigate its role in simvastatin-induced apoptosis. As shown in Fig. 4A and B, Notch1 was successfully knocked out in HepG2 cells. Following Notch1 siRNA transfection, the inhibitory effect of simvastatin on proliferation in HepG2 cells was markedly decreased (Fig. 4C); in addition, mRNA expression levels of p53 and Bax were downregulated compared to those of the control and mock siRNA-transfected groups (P<0.05) (Fig. 4D).

Figure 4 Cell apoptosis induced by simvastatin is weakened and the expression of p53 and Bax decreased when the Notch1 gene was knocked out. (A) Western blot analysis demonstrates the successful knockout of Notch1 in HepG2 cells. (B) Reverse transcription quantitative polymerase chain reaction of Notch1 mRNA expression levels in HepG2 cells following Notch1 knockout. (C) Broken line showing MTT results of proliferation of HepG2 cells following Notch1 knockout. (D) Western blot analysis of protein expression levels of p53, Bcl-2 and Bax in HepG2 cells. Bcl-2, B cell lymphoma 2; Bax, Bcl-2-associated X protein; siNotch1, cells transfected with Notch1 small interfering RNA plasmid; siMock, cells transfected with an empty vector; control, untransfected cells.

Simvastatin inhibits the phosphorylation of Akt via upregulation of Notch1 expression in HepG2 cells

Western blot analysis and RT-qPCR were performed in order to investigate the mechanism of simvastatin-induced HepG2 cell apoptosis by detecting the differential expression of the cell survival-associated gene Akt. As shown in Fig. 5, simvastatin inhibited the phosphorylation of Akt in the control and mock siRNA-transfected groups; however, following Notch1 siRNA transfection, the inhibitory effect of simvastatin on p-Akt levels was significantly decreased (Fig. 5). This therefore indicated that simvastatin regulated the phosphorylation of Akt via increasing the expression of Notch1, which, in turn, resulted in HCC cell apoptosis.

Figure 5 Simvastatin decreases phosphorylation levels of Akt via upregulation of Notch1 expression in HepG2 cells. (A) Western blot analysis was used to determine protein expression levels of Akt and p-Akt in HepG2 cells following Notch1 knockout. (B) Reverse transcription quantitative polymerase chain reaction demonstrated mRNA expression levels of Akt and p-Akt in HepG2 cells following Notch1 knockout. **P<0.01 compared with the control and siMock groups. p-Akt, phosphorylated Akt; siNotch1, cells transfected with Notch1 small interfering RNA plasmid; siMock, cells transfected with an empty vector; control, untransfected cells.

Discussion

Previous studies have indicated the potential use of statins as cancer therapeutics; one study demonstrated simvastatin was shown to decrease the cellular activity of thioredoxin reductases in HepG2 cells, inhibiting cancer cell proliferation (25). Cancer cells express elevated levels of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and low-density lipid receptor in order to facilitate the increased demand for isoprenoids and lipids (26); this therefore results in the increased sensitivity of cancer cells to statins compared with that of normal cells. However, the mechanisms by which the anti-cancer effects of statins proceed remains to be elucidated. Numerous studies have demonstrated that statins induced apoptosis in cancer cells via various pathways in vitro (27–31); however, there were discrepancy between the results of in vivo and in vitro studies. The biological mechanisms by which statins induce apoptosis and exert anti-proliferative effects in HCC cells are complex. The results of the present study revealed a novel mechanism by which simvastatin exerted cytotoxic effects in the HepG2 and Huh7 cancer cell lines.

Notch1 protein is an essential molecule in the Notch signaling pathway, which has an important role in maintaining the balance between proliferation and differentiation. Previous studies have shown that activated Notch1 inhibited growth in human papillomavirus-positive cervical carcinoma cells and prostate cancer cells, as well as induced cell cycle arrest in small cell lung cancer cells (32–34). In addition, a study in Notch1-deficient mice demonstrated that Notch1 functioned as a tumor suppressor in mouse skin via inhibition of β-catenin signaling (35). Another previous study provided novel evidence for the potential role of Notch1 signaling in modulating the state of human liver carcinoma (36,37). The results of the present study, showed that simvastatin inhibited HepG2 and Huh7 cell viability and proliferation, as well as promoted apoptosis; in addition, the anti-tumor effect of simvastatin was found to be associated with the Notch1 gene. This therefore indicated that simvastatin may inhibit HCC cell growth via regulation of Notch1 expression. These results were consistent with a previous study which showed that Notch1 arrested the cell cycle of HCC via cyclin A, cyclin D1, cyclin E and cdk4 (38).

The aim of the present study was to investigate the role of Notch1 signaling in simvastatin-induced apoptosis in HepG2 and Huh7 cell by assessing the effect of Notch1 signaling on the expression of the apoptosis-associated proteins p53, Bax, and Bcl-2. The results showed that expression levels of p53 and Bax expression were significantly increased, whereas Bcl-2 expression was significantly decreased in a time- and dose-dependent manner. Previous studies have demonstrated that the overexpression of mutant or wild-type p53 may downregulate Bcl-2 expression, resulting in apoptotic cell death (39). The results of the present study therefore indicated that following cell cycle arrest, Notch1 signaling induced apoptosis via a p53-dependent reduction in Bcl-2 pathway signaling.

Numerous studies have indicated that Akt kinase may be a noval target for cancer therapy (40–43). Experimental models have revealed an association between the regulation of survivin expression and increased Akt activity (44). In prostate cancer cells, survivin expression was shown to be regulated by insulin-like growth factor 1 (IGF-1)-stimulated Akt-mTOR signaling (45), which was impaired following simvastatin treatment (10). Therefore, the present study investigated whether simvastatin-induced apoptosis in HepG2 and Huh7 cells was associated with the Akt pathway. The results demonstrated that simvastatin reduced levels of phosphorylated Akt in HCC cells. However, following Notch1 gene knock out, simvastatin had no effect on p-Akt expression. These results suggested that the mechanism of simvastatin-induced HCC cell apoptosis may proceed via regulation of the expression of the Notch1 gene, which has an important role in the Akt-dependent signaling pathway.

In conclusion, the results of the present study demonstrated that simvastatin treatment induced apoptosis in human HCC cell lines HepG2 and Huh7; in addition, simvastatin decreased cell viability and proliferation in a time- and dose-dependent manner. The anti-tumor effect of simvastatin was found to be associated with p53, Bcl-2 and Bax expression levels, as well as the Akt-dependent signaling pathway. The present study reinforced the anti-tumor effect of statins and provided evidence for the potential beneficial effects of simvastatin in HCC therapy in humans.

Acknowledgements

The present study was supported by the Science and Technology Plan Projects of Social Development Plans for Public Relations of Shaanxi Province (nos. S2009SF471 and 2012SF2-13).

References