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Atorvastatin potentiates the chemosensitivity of human liver cancer cells to cisplatin via downregulating YAP1

Atorvastatin is a competitive inhibitor of β-hydroxy β-methylglutaryl-CoA reductase, which is involved in anticancer effects in numerous types of cancer, including in human liver cancer. However, its functions and underlying mechanisms of chemosensitivity in liver cancer remain to be elucidated. The present study investigated the effect of atorvastatin on cisplatin chemosensitivity and its molecular mechanisms, with a focus on the Yes1-associated transcriptional regulator (YAP1) protein. The present study demonstrated that atorvastatin significantly potentiated chemosensitivity to cisplatin in the liver cancer HepG2 and Huh-7 cell lines. Furthermore, cell survival and apoptosis in liver cancer cell lines were analyzed using MTT assay and flow cytometry, respectively. Atorvastatin suppressed HepG2 and Huh-7 cell viability in a dose-dependent manner, similar to cisplatin and paclitaxel. Subtoxic levels of atorvastatin significantly increased cisplatin-induced apoptosis in Huh-7 cells. Atorvastatin-promoted chemosensitivity was predominantly mediated by caspase 3, caspase 9 and poly-(ADP ribose)-polymerase activation, and YAP1 downregulation. Finally, YAP1 overexpression significantly reversed the susceptibility of Huh-7 cells to cisplatin. Overall, the results of the present study suggested the underlying mechanisms of atorvastatin chemosensitivity in inducing liver cancer cell apoptosis via downregulating YAP1 and implicated the potential application of atorvastatin-potentiated chemosensitivity in liver cancer therapy.


Introduction
Liver cancer is the third main cause of cancer-associated mortalities worldwide, with 782,500 new diagnosed cases and 745,500 deaths estimated each year (1). Hepatitis B virus (HBV) and hepatitis C virus (HCV) are the most critical known risk factors of liver cancer (1). Although surgery and chemotherapy have improved the survival time of patients with liver cancer, a considerable number of patients still undergo recurrence due to the resistance of cancer cells to chemotherapeutic drugs (2). However, the chemoresistance mechanisms of liver cancer remain unknown. Therefore, the identification of drugs that increase sensitivity to liver cancer chemotherapy is essential for the development of effective therapies, which will be beneficial for patients.
Atorvastatin, a competitive inhibitor of β-hydroxy β-methylglutaryl-CoA reductase, exerts beneficial effects on circulating lipid levels and is used for the treatment and prevention of coronary heart disease and stroke (3)(4)(5). Additionally, atorvastatin has been proposed as an anticancer drug candidate, since previous studies have demonstrated that atorvastatin exerts antiproliferative, pro-apoptotic and immunoregulatory effects (6)(7)(8)(9). However, the underlying mechanisms of atorvastatin-induced sensitization to chemotherapy in liver cancer has not been elucidated.
The present study investigated the synergistic effect of atorvastatin on cisplatin chemosensitivity and its associated molecular mechanisms. Additionally, the role of the Yes1-associated transcriptional regulator (YAP1) in liver cancer cells was evaluated. Furthermore, cell survival and apoptosis in liver cancer cell lines were analyzed using MTT assay and flow cytometry, respectively.

Drug treatment. Firstly
Cell viability assay. Cell viability of HepG2 and Huh-7 was tested in vitro using MTT assays. A total of 1x10 4 cells were seeded in 96-well plates. Following treatment, cells were incubated with MTT solution (Sigma-Aldrich; Merck KGaA) in PBS for 3 h at 37˚C according to the manufacturer's protocol. The purple formazan was solubilized using DMSO. The absorbance was read on a microplate reader at a wavelength of 490 nm (Molecular Devices, LLC). The combination index (CI) values between 100 µM atorvastatin and cisplatin in treating HepG2 and Huh-7 cells were calculated using the following formula: Cell viability of cisplatin + atorvastatin group / (cell viability of cisplatin group x cell viability of atorvastatin group). The cut-off of CI value to determine whether a synergistic effect was observed was 1.
Flow cytometric analysis of apoptosis. Apoptosis was assessed using FITC-labeled Annexin-V (BD Biosciences) and propidium iodide (PI; Sigma-Aldrich; Merck KGaA) via flow cytometry. Briefly, following treatment for 24 h, Huh-7 cells were collected and stained with 500 µl solution containing Annexin V-FITC in the dark at room temperature for 30 min. This was followed by addition of PI for 5 min in the dark at room temperature. Flow cytometry (FACSCanto; Becton, Dickinson and Company) was used to detect fluorescent signals in the cells. Both early and late apoptotic cells were calculated using FlowJo 7.6 (FlowJo LLC).
Quantification of western blotting data, which was performed using ImageJ 2.0 (National Institutes of Health) was calculated as follows: i) Quantification of each protein density in triplicate; ii) quantification of β-actin density in triplicate; iii) dividing each protein density by the β-actin density to obtain the relative band density in triplicate; and iv) setting each replicate of relative density in the CK group as the control (as one), and the relative density in other groups was calculated based on the control.
Plasmid construction and transfection. The human YAP1 coding sequence was synthesized and subcloned into pcDNA3.1 (Addgene, Inc.). The integrity of the respective plasmid constructs was confirmed by DNA sequencing (data not shown). After 2x10 5 Huh-7 cells were seeded in 6-well plates overnight at 37˚C, cells were transfected with 0.8 µg plasmid using Lipofectamine ® 2000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Additionally, control pcDNA3.1 was synthesized and served as a negative control. Following incubation for 24 and 48 h at 37˚C, the overexpression efficiency of the plasmid was determined using western blotting, as aforementioned. At 24 h after transfection, Huh-7 cells transfected with the empty vector pcDNA3.1 were treated with 5 µg/ml cisplatin alone or 5 µg/ml cisplatin plus 100 µM atorvastatin and Huh-7 cells transfected with the pcDNA3.1-YAP1 were treated with 5 µg/ml cisplatin alone or 5 µg/ml cisplatin plus 100 µM atorvastatin for another 24 h at 37˚C. Subsequently, the flow cytometric analysis of apoptosis was performed.
Statistical analysis. Data are presented as the mean ± SD from ≥3 separate experiments. All statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, Inc.) and SPSS 13.0 (SPSS, Inc.) software packages. Statistical significance was determined using a two-sided unpaired Student's t-test or one-way ANOVA followed by Dunnett's or Tukey's multiple comparison test as appropriate. P<0.05 was considered to indicate a statistically significant difference.

Cytotoxicity of atorvastatin alone or in combination with cisplatin or paclitaxel on liver cancer cells.
To determine whether atorvastatin could inhibit liver cancer cell viability, two liver cancer cell lines, as well as cisplatin and paclitaxel, were used for experiments. Cytotoxicity was evaluated using an MTT assay. The results revealed that atorvastatin significantly suppressed HepG2 cell viability at 10, 30, 60, 70, 80, 90 and 100 µM, while Huh-7 cell viability was only inhibited at 80, 90 and 100 µM (Fig. 1A). Since 10 µM was the lowest concentration to inhibit cell viability and 100 µM was the highest (Fig. 1A), these concentrations were chosen for further experimentation. Additionally, the present study examined whether combined treatment of atorvastatin with cisplatin or paclitaxel exerted enhanced lethality on liver cancer cell lines. As shown in Fig. 1B-D, following co-treatment with the indicated concentrations of atorvastatin and cisplatin or paclitaxel for 24 h, cells were subjected to an MTT assay. The combination of atorvastatin and cisplatin significantly inhibited cell viability in HepG2 and Huh-7 cells, but only slightly with paclitaxel. Using 100 µM atorvastatin combined with 0-10 µg/ml or 0-20 µg/ml cisplatin significantly inhibited HepG2 or Huh-7 cell viability compared with 0 µM atorvastatin, respectively; additionally, 10 µM atorvastatin alone and 10 µM atorvastatin combined with 10 µg/ml cisplatin significantly inhibited HepG2 cell viability compared with 0 µM atorvastatin (Fig. 1B). However, only 100 µM paclitaxel combined with 100 µM atorvastatin or 100 µM atorvastatin alone significantly inhibited cell viability in both HepG2 and Huh-7 cells compared with 0 µM atorvastatin; additionally, 10 µM atorvastatin with 100 or 500 µM paclitaxel significantly inhibited HepG2 cell viability compared with 0 µM atorvastatin (Fig. 1C). Further experiments indicated that 4 µg cisplatin combined with 40 µM atorvastatin significantly inhibited HepG2 cell viability and 5 µg cisplatin combined with 100 µM atorvastatin significantly inhibited Huh-2 cell viability compared with the control Fig. 1D). These results indicated that atorvastatin may potentiate the chemosensitivity of liver cancer cells to cisplatin. Furthermore, CI values were calculated based on relative cell viability data, revealing that atorvastatin synergized with 5-20 µg/ml cisplatin in killing Huh-7 cells (CI values <1), but not HepG2 cells (CI values near or >1) (Table I).
Atorvastatin potentiates the chemosensitivity of liver cancer cells by inducing apoptosis. Subsequently, whether the sensitization effect of atorvastatin to cisplatin and paclitaxel involved the induction of apoptosis was examined. Huh-7 cells were subjected to flow cytometry analysis following treatment with 100 µM atorvastatin alone or in combination with 5 µg/ml cisplatin or 300 µM paclitaxel. The drug concentrations used for these experiments were determined due to the following: i) 100 µM atorvastatin, but not 10 µM atorvastatin, significantly enhanced the effect of cisplatin in suppressing relative Huh-7 cell viability (Fig. 1B) and 100 µM atorvastatin was therefore chosen for subsequent experiments; ii) 5 µg/ml cisplatin plus 100 µM atorvastatin achieved ~50% of Huh-7 cell inhibition rate (Fig. 1B), therefore 5 µg/ml cisplatin was chosen for subsequent experiments; and iii) paclitaxel at various concentrations plus 100 µM atorvastatin did not achieve 50% of Huh-7 cell inhibition rate (Fig. 1C), but 100 µM atorvastatin enhanced the effect of paclitaxel on Huh-7 cell inhibition at 100 µM, but not 500 µM paclitaxel, therefore 300 µM paclitaxel (the median between 100 and 500) was chosen for subsequent experiments.
As shown in Fig. 2A and B, atorvastatin significantly enhanced cisplatin-induced apoptosis in Huh-7 cells. The percentage of Annexin-V + cells increased from 16.37% (atorvastatin alone) and 23.12% (cisplatin alone) to 54.62% (atorvastatin combined with cisplatin). However, atorvastatin slightly enhanced paclitaxel-induced apoptosis in Huh-7 cells. The percentage of Annexin-V + cells increased from 16.37% (atorvastatin alone) and 14.35% (paclitaxel alone) to 32.35% (atorvastatin combined with paclitaxel) ( Fig. 2A and C). The present results suggested that atorvastatin significantly potentiated cisplatin sensitivity in Huh-7 cells via inducing apoptosis, while atorvastatin only slightly potentiated paclitaxel sensitivity in Huh-7 cells.

Apoptosis is involved in the synergistic effect of atorvastatin on cisplatin sensitivity in liver cancer cells.
There are two fundamental pathways of apoptosis, which are the extrinsic and intrinsic apoptosis pathways (10,11). Cleavage of caspases and PARP are hallmarks of intrinsic and extrinsic apoptosis pathways activation (12). As shown in Fig. 3A and B, co-treatment with atorvastatin and cisplatin in Huh-7 cells significantly increased the cleavage of caspases 3 and 9, and PARP compared with CK. Additionally, increasing evidence has demonstrated that increased YAP1 expression is involved in liver cancer progression and chemoresistance (13,14). To evaluate the effect of atorvastatin treatment on the expression of YAP1 in liver cancer, the Huh-7 cells treated with atorvastatin. As shown in Fig. 3C and D, atorvastatin treatment significantly inhibited YAP1 protein levels. The current observations indicated that the intrinsic and extrinsic apoptotic pathways and YAP1 may be involved in the synergistic effect of atorvastatin on cisplatin sensitivity in liver cancer cells.  Fig. 4A and B).
Huh-7 cells were transfected with empty vector pcDNA3.1 or pcDNA3.1-YAP1 plasmid and incubated for 24 h. Subsequently, cells were treated with 5 µg/ml cisplatin and 100 µM atorvastatin for another 24 h. Following treatment, flow cytometry was performed to determine the apoptotic cell percentage by co-staining with Annexin V-FITC and PI (Fig. 4C). YAP1 overexpression significantly attenuated the apoptosis mediated by the combination of atorvastatin and cisplatin (Fig. 4D). Overall, the present results indicated that atorvastatin may sensitize liver cancer cells to cisplatin, at least partially via inhibiting YAP1.

Discussion
To date, increasing evidence has associated the YAP1 oncogene to tumorigenesis of several types of cancer, including pancreatic ductal adenocarcinoma, lung cancer, colon cancer, prostate cancer and liver cancer (15)(16)(17)(18)(19). YAP1 is the downstream effector of the Hippo signaling pathway, and in cooperation with the TEA domain transcription factor 1, increased YAP1 expression stimulates a number of target genes responsible for cell viability and apoptosis (20,21). Several studies have demonstrated that increased YAP1 expression is associated with elevated drug resistance in numerous cancer cells, such as neuroblastoma, esophageal cancer and colorectal cancer cells (22)(23)(24)(25). The present study investigated the mechanism of the synergistic effects of YAP1 with cisplatin. Firstly, the present study revealed that atorvastatin inhibited liver cancer cell viability in a dose-dependent manner. Secondly, the present study demonstrated that sub-cytotoxic levels of atorvastatin sensitized HepG2 and Huh-7 cells to different concentrations of cisplatin and paclitaxel using an MTT assay. Subsequently, the synergistic effect of atorvastatin on cisplatin or paclitaxel sensitivity was analyzed, revealing that this mechanism involved apoptosis induction in Huh-7 cells subjected to flow cytometry analysis following treatment with atorvastatin alone or in combination with cisplatin or paclitaxel. The present results suggested that atorvastatin may regulate the intrinsic and extrinsic apoptotic pathways to increase cell sensitivity to cisplatin and paclitaxel. In addition, western blotting was performed to evaluate the protein expression levels of cleaved caspase 3 and 9, and PARP, which were all upregulated in Huh-7 cells co-treated with atorvastatin and cisplatin compared with CK.
Finally, the YAP1 protein was further investigated, since increased YAP1 expression is best known as a regulator of cell viability, survival and chemoresistance (26)(27)(28)(29). The present study demonstrated that YAP1 levels were decreased by atorvastatin treatment in Huh-7 cells. Furthermore, transfecting Huh-7 cells with pcDNA3.1-YAP1 expression plasmid significantly reversed the apoptosis mediated by the combination of atorvastatin with cisplatin. Therefore, the current data revealed that atorvastatin may potentiate the chemosensitivity of liver cancer cells to cisplatin by regulating YAP1 expression.
Despite the findings of the present study, there are still some limitations. First, the combined effect of atorvastatin plus cisplatin or paclitaxel on cell apoptosis, and protein expression levels, such as YAP1, were detected in a single cell line; therefore, further validation in multiple cell lines is required in future studies. Second, the deeper molecular mechanism of atorvastatin plus cisplatin treatment via YAP1 requires further exploration. Third, due to lack of funding, in vivo validation was not performed in the present study and should therefore be performed in future studies. In conclusion, the current results demonstrated that elevated levels of YAP1 in liver cancer may serve a role in cancer cell chemoresistance. Although other downstream target genes may also be involved in regulating apoptosis following atorvastatin treatment, the present data illustrated that atorvastatin may potentiate chemosensitivity to cisplatin in liver cancer cells by regulating YAP1, which may serve a role as an apoptosis suppressor. Therefore, the results of the present study indicated that atorvastatin plus cisplatin therapy may be a potential strategy for the treatment of chemoresistant liver cancer.