Abstract
Background: Statins can reduce the malignancies through stimulating apoptosis. We aimed to elucidate the role of lovastatin in HepG-2 cells. Methods: HepG-2 and non-tumor L-O2 cells were used as the cell models. CCK-8, flow cytometric analysis and carboxy fluorescein diacetate succinimidyl ester (CFDA-SE) labeling were performed to monitor the viability, apoptosis and proliferation. Results: We found that lovastatin exerted the most tumor suppressing effects on liver cancer cells among the three tested statins. Lovastatin treatment significantly reduced cell viability and proliferation, and induced apoptosis in HepG-2. However, drug resistance effects were observed in the non-tumor L-O2 cells. The apoptosis triggered by lovastatin was accompanied by high intracellular levels of ROS. Pretreatment with the ROS blocker N-acetyl-cysteine (NAC) could mitigate the lovastatin-induced cytotoxicity in HepG-2 cells. Mechanistically, lovastatin increased HepG-2 cell apoptosis by triggering mitochondrial and endoplasmic reticulum (ER) stress pathways through ROS accumulation. Conclusions: Lovastatin significantly induced cell apoptosis by activating ROS-dependent mitochondrial and ER stress pathways in HepG-2 cells.
Keywords: Lovastatin, apoptosis, reactive oxygen species, N-acetyl-cysteine, mitochondrial, ER stress pathways
Introduction
The 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) is a rate-limiting enzyme in the mevalonate pathway. Statins are inhibitors of HMGCR and are widely used for the treatment of hypercholesterolemia toreduce the risk of cardiovascular diseases [1,2]. Surprisingly, numerous reports have found the anti-tumor effects of the statins through inhibition of cell proliferation, promotion of apoptosis, or suppression of angiogenesis beyond cholesterol lowering effects [1,3]. In fact, large population-based clinical studies have shown that statins treatment have a protective effect against cancer and could significantly reduce both the incidence and mortality of overall cancers [4,5].Although the experimental and clinical data seemed controversial and poorly understood, statins still attract intensive attention for its therapeutic value in cancer treatment [6].
Interestingly, a significantly lower risk of liver cancer has been reported in patients with pre-existing liver disease or diabetes who have been taking statins, suggesting a benefit in terms of chemoprevention in persons at elevated risk of liver cancer [7,8]. Lovastatin, a member of the statin family, was previously reported to regulate the proliferation, apoptosis and chemo-resistance of tumor cells [9-11]. To our knowledge, there are still few studies investigating the effects of lovastatin on hepatocellular carcinoma (HCC) cells, except one early study showed that lovastatin alone slowed hepatoma tissue culture-4 (HTC-4) cell growth [12]. Another study demonstrated that lovastatin inhibited carcinogenesis in a rat model for HCC through inhibition of cell proliferation, which could at least in part be explained by the inhibition of ubiquinone synthesis [13].
In the present study, we mainly focused on the effects of lovastatin in HepG-2 cells, and compared its cytotoxic effects on HepG-2 cells and non-tumor L-O2 cells. We also intended to explore the potential underlying mechanism.
Materials and methods
Reagents
Atorvastatin, simvastatin and lovastatin were all ordered from Selleck (Houston, TX, USA) and diluted in dimethyl-sulfoxide (DMSO) to obtain concentrations of 0, 5, 10, 50 and 100 µM for cell culture work. N-acetyl-L-cysteine (NAC) was ordered from Sigma (St. Louis, MO, USA). Dulbecco’s Modified Eagle medium (DMEM), penicillin/streptomycin, 0.25% Trypsin-EDTA were acquired from Gibco/Invitrogen (Karlsruhe, Germany). Fetal bovine serum (FBS) was obtained from Biochrom (Berlin, Germany).
Cell culture
Human HCC cells HepG-2 and non-tumor hepatocytes L-O2 cells were both purchased from the Cell Resource of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Cells were suspended and maintained in a medium containing DMEM (high glucose), 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin in a humidified incubator at 37°C in 5% CO2.
Cell counting kit (CCK)-8 assay
To determine cell viability, the colorimetric CCK-8 assay was employed. Briefly, cells were counted and seeded into 96-well plates at a density of 3000-6000 per well for 24 hrs. Following the treatment with various concentrations of lovastatin for another 12 hrs, 10 µl CCK-8 solution (Dojindo, Kumamoto, Japan) was added into each well of the plates followed by 2 h-incubation. Then the absorbance at 450 nm was measured with a microplate reader. In each assay, five parallel wells were run and experiments were repeated in triplicate. The percentage of cell viability was calculated as follows: cell viability (%) = (mean absorbance in test wells)/(mean absorbance in control wells) × 100.
Annexin V/PI staining
To investigate the apoptosis, cells pretreated with lovastatin (5 µM) for 12 hrs were trypsinized, washed with PBS and then suspended with binding buffer. Apoptosis was detected by Annexin V/propidium iodide (PI) staining according to the manufacturer’s instructions (BD PharMingen, San Diego, CA, USA). Cells were with FITC-conjugated Annexin V and PI for 15 min at room temperature in the dark, prior to suspension in 500 µl binding buffer. The cells were finally analyzed by a flow cytometer.
Detection of cell proliferation by CFDA SE labeling
Cells were seeded in the 12-well plate and pretreated with lovastatin (5 µM) for 12 hrs. The ratio of proliferating cells was determined by labeling with carboxy fluorescein diacetate succinimidyl ester (CFDA SE) according to the manufacturer’s (Beyotime, Shanghai, China) instructions. After a 24 hr-incubation, cells were harvested, washed twice with ice-cold PBS, and subjected to flow cytometry. The fluorescence of CFDA SE-labeled non-proliferating cells was stable. The fluorescence intensity was decreased along with higher cell proliferation, and the peak moved to the left.
Determination of intracellular ROS
The changes of the intracellular reactive oxygen species (ROS) generation were measured by staining the cells with 2’,7’-dichlorfluorescein-diacetate (DCFH-DA). After treated by lovastatin and/or NAC, cells were incubated with 10 μmol/L DCFH-DA at 37°C for 30 min as per the manufacturer’s instruction (Beyotime, Shanghai, China). Then cells were washed with PBS and visualized under a microscopy. Alternatively, ROS levels were also detected by using flow cytometry analysis of 2’,7’-dichlorfluorescein (DCF) fluorescence.
Western blot
Cells were harvested and lysed using RIPA lysis buffer (Beyotime, Shanghai, China) containing protease inhibitors. Equal amount (20-30 μg) of each sample was subjected to 10% SDS-PAGE and then transferred onto nitrocellulose membranes (GE Healthcare, UK). After blocking for 1 h, membranes were incubated in the specific primary antibody solution at 4°C overnight and in the secondary antibody solution at room temperature for another 1 h. The target proteins were visualized and quantified using ECL detection kit (GE Healthcare Life Sciences) and analyzed using Image J software (1.43b; National Institutes of Health, Bethesda, MD, USA). Rabbit anti-human Caspase-3, Bax, Bcl-2, ATF-4, CHOP and mouse anti-human GAPDH, all purchased from CST were used as primary antibodies.
Statistical analysis
Results were shown as means ± SD. Student’s t-test was used for comparison between two groups. All experiments were performed in triplicate, and P<0.05 was considered to be statistically significant between values.
Results
To realize the inhibitory effects of statins on liver cancer cells, we employed three different statins, atorvastatin, simvastatin and lovastatin, and determined their cytotoxic effects on HepG-2 cells by CCK-8 assay. As shown in Figure 1, three statins all exhibited stronger cytotoxic effects along with their increasing concentrations (0, 1, 5, 10 and 50 µM). We observed that the half maximal inhibitory concentration (IC50) was 25.3 μM for atorvastatin, 11.7 μM for simvastatin, and 9.7 μM for lovastatin, respectively. This finding indicated that among these statins, lovastatin exerted the most tumor suppressing effect in HepG-2 cells. We then focused on the inhibitory roles of lovastatin in liver cancer cells and tried to explore the possible mechanisms.
Figure 1.
Comparison of tumor suppressing effects of three statins in HepG-2 cells. HepG-2 cells were treated with different concentrations (0, 1, 5, 10 and 50 µM) of atorvastatin, simvastatin or lovastatin for 12 hrs. Cell viability was assessed and bar graph showed the CCK-8 results measured by OD450 absorbance.
To determine the cytotoxic effects of lovastatin in HCC cells, we monitored the cell viabilities of HepG-2 and L-O2 cells upon increasing concentrations of lovastatin (0, 5, 10, 50 and 100 µM) treatment by CCK-8 assay. The results showed that a 12-hr-incubation of lovastatin induced a dose-dependent cytotoxicity in HepG-2 cells (Figure 2A). Even at the lowest dose (5 µM), the viability of HepG-2 cells was significantly decreased by nearly 40%. However, we observed a bit drug resistance in the non-tumor L-O2 cells, since its viability exhibited a modest decrease at a higher lovastatin dose (10 µM) by only 20% (Figure 2B). These findings indicated a higher cytotoxicity towards HCC cells than normal cells. Thereafter, we examined cell proliferation by using CFDA SE labeling followed by flow cytometry in these two lovastatin-treated cell lines. As shown in Figure 2C, treatment of HepG-2 cells with lovastatin significantly repressed cell proliferation (proliferating cells constituting of 80.24%), while L-O2 cells showed minor defects in proliferation (proliferating cells constituting of 89.84%). These data suggested that lovastatin inhibited cell viability and proliferation extremely in HCC cells while exerted somewhat minor effects on non-tumor L-O2 cells.
Figure 2.
Lovastatin reduced cell viability and proliferation in both L-O2 and HepG-2 cells. Cells were treated with different concentration of lovastatin (0, 5, 10, 50 and 100 µM) for 12 hrs. A. Bar graph showing CCK-8 results of L-O2 cell viability measured by OD450 absorbance. B. Bar graph showing CCK-8 results of HepG-2 cell viability measured by OD450 absorbance. The mean percentage of cell viability, relative to the untreated control (0 µM) ± SD was given. *P<0.05 and **P<0.01 compared with the 0 µM group. C. Flow cytometric analysis of the proliferating cell ratio determined by CFDA SE labeling. The ratios of proliferating cells were given.
To test whether apoptosis was also involved in lovastatin-induced cytotoxicity in HCC cells, annexin V/PI staining followed by flow cytometry was applied. As shown in Figure 3A, lovastatin significantly increased the percentage of both early-stage apoptotic cells (annexin V+PI-) and advanced-stage apoptotic cells (annexin V+PI+ cells) afer 12 h treatment, at a dose of 10 µM, in both L-O2 and HepG-2 cell lines. Moreover, we observed the apoptosis occurring more severely in HepG-2 cells, by more than 2-fold than that in L-O2 cells (P<0.05, Figure 3B). Together with the upper results, all data indicated that lovastatin exerted anti-tumor effects in HCC cells by suppressing cell proliferation and inducing cell apoptosis.
Figure 3.
Lovastatin induced HCC cell apoptosis. Cells were treated with 10 µM lovastatin for 12 h. A. Flow-cytometric analysis was used to determine the extent of cell apoptosis (annexin V/PI staining). B. The compiled result for (A) was shown. Typical pattern shown was representative of three independent experiments. *P<0.05, compared with the lovastatin treated L-O2 group.
Since oxidative stress is closely associated with viability of cancer cells [14], we then investigated whether oxidative stress was involved in the cytotoxicity of lovastatin in HCC cells by measuring the important oxidative stress marker, intracellular ROS levels. L-O2 and HepG-2 cells were equally treated with 5 µM lovastatin for 1 hr and then detected by DCFH-DA probes as described in the method section. Our results showed that lovastatin significantly increased intracellular ROS level in HepG-2 cells, whereas induced less ROS generation in non-tumor L-O2 cells (Figure 4A and 4B). We also noticed that in the basic condition (DMSO treatment), intracellular ROS was more abundant in HepG-2 cells. The results suggested the potential involvement of oxidative stress in the cytotoxic action of lovastatin, especially in HCC cells.
Figure 4.
Lovastatin promoted intracellular ROS generation in HCC cells. A. Representative microscopy images of L-O2 and HepG-2 cells treated with DMSO or 5 µM lovastatin. Intracellular ROS levels were indicated with a fluorescent ROS probe, DCFH-DA. B. Flow cytometric analysis of the ROS levels as in (A). Mean FITC value reflected the ROS amount in indicated cells.
To further explore if the oxidative stress or ROS generation was the dominant factor contributing for lovastatin-induced cytotoxicity towards HCC cells, we incubated HepG-2 cells with lovastatin (5 µM) in the presence or absence of the thiol antioxidant N-acetylcysteine (NAC). As shown in Figure 5A and 5B, NAC exposure (300 µM, 1 hr) led to suppression of ROS level in basal HepG-2 cells. Moreover, NAC pretreatment could significantly block increase of ROS amount induced by lovastatin (Figure 5A and 5B). Besides, we found that cell viability inhibition induced by lovastatin was obviously suppressed by NAC (Figure 5C). Taken together, these results supported that oxidative stress was the main pathway in lovastatin-induced cytotoxicity in HCC cells, since its blocker, NAC significantly eased the cytotoxic effect of lovastatin in HepG-2 cells.
Figure 5.
NAC pretreatment mitigated lovastatin-induced cytotoxicity in HepG-2 cells. A. Representative microscopy images of HepG-2 cells treated with DMSO or 5 µM lovastatin, in absence or presence of NAC. Intracellular ROS levels were indicated with a fluorescent ROS probe, DCFH-DA, as in Figure 4. B. Flow cytometric analysis of the ROS levels as in (A). C. CCK-8 assay of cell viabilities after HepG-2 cells were treated with increasing concentration of lovastatin (0, 5, 10, and 50 µM) in absence or presence of NAC. The mean percentage of cell viability, relative to the untreated control (0 µM of lovastatin without NAC) ± SD was given. *P<0.05 compared within the 5 µM lovastatin treating groups.
To further investigate the molecular mechanism contributing to lovastatin-induced HepG-2 cell apoptosis, the expression of apoptosis related proteins was examined by western blot analysis. As shown in Figure 6A, the expressions of pro-apoptotic cleaved Caspase-3 and Bax protein were remarkably elevated, while expression of the anti-apoptotic Bcl-2 protein declined in HepG-2 cells following treatment with lovastatin, in a time-dependent manner. Since Caspase-3, Bax and Bcl-2 are well-known characteristics of mitochondrial apoptosis, these results indicated that lovastatin activated mitochondrial apoptotic pathway. On the other hand, we detected the expression of ATF-4 and CHOP, two markers of ER stress related apoptosis. We found that lovastatin also increased the expression of ATF-4 and CHOP (Figure 6A), indicating that ER stress was another key pathway involved in the lovastatin-induced apoptosis. However, the increase of Caspase-3, Bax, ATF-4 and CHOP expressions, and the decrease of Bcl-2 by lovastatin could all be reversed by addition of NAC, the ROS blocker (Figure 6B). These findings demonstrated that lovastatin induced HepG-2 cell apoptosis via ROS-dependent mitochondrial and ER stress pathways.
Figure 6.
Lovastatin induced HepG-2 cell apoptosis by triggering intrinsic mitochondrial and ER stress pathways through ROS accumulation. A. HepG-2 cells were incubated with 5 µM lovastatin for indicated times (0-24 h), and the expressions of cleaved Caspase-3, Bax, Bcl-2, ATF-4, CHOP were then examined by Western blot. GAPDH was as the loading control (upper). The quantified bar graph was given (below). B. Expressions of cleaved Caspase-3, Bax, Bcl-2, ATF-4 and CHOP in indicated cells were detected by Western blot (upper) and normalized against GAPDH (bottom). Data in (A) and (B) are presented as the mean ± SD.
Discussion
As is known, HCC accounts for a majority of primary liver cancers and is one of the most frequent as well as aggressive malignancies in the world [15]. The primary therapy option for HCC patients at their early stage is surgical resection. Afterwards, patients will accept chemotherapy. The side effects of chemotherapeutic drugs and the chemo-resistance of tumor cells are among the main reasons leading to the bad outcome. Therefore it’s of great importance to develop new drugs for HCC.
Statins have attracted intense attention for their therapeutic value in anti-tumor treatments extending beyond their primary lipid-lowering effects as single agents or in combined use with other chemotherapeutic agents. It was previously shown that lovastatin induced HCC cell apoptosis by activating mitochondrial apoptotic signals [16]. However, systemically studies characterizing the effects of lovastatin in HCC cells are still absent, and the underlying molecular mechanisms remain largely unclear.
In the present study, we found that lovastatin exerted the most tumor suppressing effects on HepG-2 cells by decreasing cell viability among the three tested satins. Lovastatin treatment significantly led to inhibition of cell viability, cell proliferation and induction of apoptosis. We surprisingly found that a relative lower concentration of lovastatin (5 µM) resulted more obvious cytotoxicity in HepG-2 cells, while did not cause inhibition of cell viability in non-tumor L-O2 cells. This finding suggested that lovastatin might be safely used for HCC patients because of the drug resistance in normal cells. We further found that lovastatin induced cell death in HCC cells was accompanied by high intracellular levels of ROS, and NAC pretreatment could ease the lovastatin-induced cytotoxicity in Hep-G2 cells.
The increasing knowledge about ROS-related apoptosis have identified several signaling pathways, in which the intrinsic mitochondrial apoptosis pathway is the most mentioned [17]. Excess cellular levels of ROS may lead to decrease of mitochondrial membrane potential and release of cytochrome C into the cytoplasm, thereby activating Caspase-9 and processing Caspase-3 and else, leading to apoptosis [18]. Although we did not examined the release of cytochrome C, our results revealed a time-dependent activation of Caspase-3 in HepG-2 cells following lovastatin treatment. On the other hand, the apoptosis defects are mainly determined by a defective balance among pro- and anti-apoptotic members of the Bcl-2 family. Herein, we observed that Bax was increased but Bcl-2 was decreased in lovastatin-treated HepG-2 cells, indicating that the mitochondrial pathway was involved in lovastatin-induced cell apoptosis. Besides, we learned from the literature that ROS also activates ER stress apoptosis pathway [17,19]. In the present study, treatment with lovastatin markedly increased ATF-4 and CHOP expressions, indicating the involvement of ER stress in lovastatin-induced apoptosis in HepG-2 cells. Importantly, combination treatment with NAC, the ROS blocker, significantly reversed the expression of abovementioned four molecules, indicating that lovastatin induced cell apoptosis via mitochondrial and ER stress pathways in an ROS-dependent manner.
In summary, our results demonstrated that lovastatin induced apoptosis by promoting ROS generation in Hep-G2 cells, which could be reversed by NAC treatment. Mechanistically, we verified that ROS-activated mitochondrial and ER stress apoptosis pathways served as the major mechanism for lovastatin-induced apoptosis. The present study reinforced the anti-tumor effect of lovastatin, and provided additional evidence for the potential beneficial effects of lovastatin in HCC therapy in humans.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (81100826).
Disclosure of conflict of interest
None.
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