Background
Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide and ranks as the third leading cause of cancer-related death, accounting for 748,300 new cases and 695,900 deaths worldwide per year. Half of these cases and deaths are estimated to occur in China [
1]. However, only approximately 30%–40% of patients are diagnosed in an early stage (0 or A) according to the Barcelona Clinic Liver Cancer staging system [
2], which defines patients who are suitable for potentially curative approaches, such as surgical therapies (resection and liver transplantation) and locoregional procedures (radiofrequency ablation). For patients who meet the criteria for the intermediate stage (multinodular HCC, relatively preserved liver function, absence of cancer-related symptoms, and no evidence of vascular invasion or extrahepatic spread), transcatheter arterial chemoembolization (TACE) has been established as the standard of care, and this treatment may achieve a partial response or complete necrosis [
3]. For patients with advanced HCC, sorafenib is the first agent discovered to result in favorable overall survival [
4]. Regional hepatic arterial infusion chemotherapy (HAIC) has also been used in patients with advanced HCC in cases in which TACE is not indicated or is ineffective [
5,
6].
The technique of TACE, including which drug is administrated, the scheduled followed after the first TACE or the follow-up imaging modalities, varies worldwide with no clear consensus. Among the agents commonly used in TACE and HAIC to inhibit cancer cell growth, 5-Fluorouracil (5-FU) is a widely used chemotherapeutic drug. It initiates apoptosis by targeting thymidylate synthase (TS) and direct incorporation of 5-FU metabolites into DNA and RNA. However, its efficacy in HCC is poor [
7], and the compound is associated with acquired and intrinsic resistance.
Sorafenib (BAY 43-9006, Nexavar) is an oral multikinase inhibitor that inhibits the serine-threonine kinases C-Raf and B-Raf, the receptor tyrosine kinase activity of vascular endothelial growth factor receptors -1, -2, and -3, platelet-derived growth factor receptor β, the receptor for the macrophage-colony stimulating factor (FLT3), Ret, and c-Kit. These kinases are involved in cell proliferation and tumor angiogenesis [
8,
9]. In addition, increasingly more studies have pointed out that signal transducer and activator of transcription 3 (STAT3) is a major kinase-independent target of sorafenib in HCC [
10,
11].
Recently, a phase II clinical trial has suggested that the combination of sorafenib and 5-fluorouracil is feasible, and the side effects are manageable for patients carefully selected for liver function and performance status [
12]. However, preclinical experimental data explaining interaction mechanisms are widely missing. One previous study in our institute found that resistance to 5-FU was significantly associated with basal p-ERK expression levels in HCC cell lines while sorafenib inhibited ERK phosphorylation in a dose-dependent manner [
13]. Chances are combination of sorafenib and 5-FU would exert a synergetic effect with the hypothesis that sorafenib could reverse the resistance to 5-FU of HCC cells by inhibiting p-ERK expressions. However, it is known that 5-FU is an S-phase-specific agent, whereas sorafenib causes G1-phase arrest in tumor cells [
14]. The latter implies that sorafenib treatment would decrease the proportion of cells in S phase. And in such situation, tumor cells might become less susceptible to the 5-FU action. Therefore, the effects of combined sorafenib and 5-FU co-administration are uncertain.
In the present study, we initiated an in vitro study in HCC cell lines MHCC 97H and SMMC-7721 to investigate the anticancer efficacy and molecular mechanisms of combined administration of sorafenib and 5-FU.
Methods
Drug preparations
Sorafenib (Nexavar), N-(3-trifluoromethyl-4-chlorophenyl)-N-(4-(2-methylcarbamoylyridin-4-yl)oxy-phenyl) urea, was purchased from BioVision, Inc. (Milpitas, CA, USA). The compound was dissolved in 100% dimethyl sulfoxide (DMSO; Sigma-Aldrich, St Louis, MO, USA) and diluted with Dulbecco's modified Eagle's medium (DMEM) or RPMI 1640 to the desired concentration; a final DMSO concentration of 0.1% (v/v) was present in cell studies. As solvent control, 0.1% DMSO alone was added to cultures. 5-Fluorouracil injection was purchased from Shanghai Xudong Haipu Pharmaceutical Co, Ltd. (Shanghai, China) and was diluted directly with cell culture medium to the desired concentration.
Cell lines
Human HCC tumor cell lines MHCC97H and SMMC-7721 were obtained from the Liver Cancer Institute of Fudan University (Shanghai, China) and cultured in DMEM or RPMI 1640 containing 10% v/v fetal bovine serum at 37°C in a humidified incubator containing 5% CO2. Unless otherwise indicated, cell culture reagents were purchased from GIBCO BRL (Grand Island, NE, USA).
Cell viability assay
Cells were plated in 96-well microtiter plates (4,000 per well) in 100 μL of serum-containing medium and incubated overnight at 37°C in the culture incubator. On the following day, the medium was replaced with fresh medium containing sorafenib, 5-FU, or a combination of the two agents at various concentrations. Treatment with sorafenib was done for 24 h at concentrations of 0, 0.25, 0.5, 1, 4, 8, 16, 32, 64, or 128 μM; that with 5-FU was for 48 h at concentrations of 0, 0.1, 1, 2, 4, 8, 16, 32, 64, 128, or 256 mg/L. Cell viability was measured using the Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's instructions. The half maximal inhibitory concentration (IC50) values were calculated by nonlinear regression analysis using GraphPad Prism version 5.0 software (GraphPad Software, Inc., San Diego, CA, USA).
Combination index (CI) values were calculated using the median effect analysis method. A synergistic effect is defined as CI < 1, an additive effect as CI = 1, and an antagonistic effect as CI > 1.
Each condition was tested six times, and the results were confirmed in at least three independent experiments.
To further investigate combined effects of sorafenib and 5-FU on cell proliferation, growth inhibition, cell cycle distribution and pathways activities, six treatment groups were designed as follows: group control (0.1% DMSO); group S (treatment with 8 μM sorafenib for 24 h); group F (treatment with 4 mg/L 5-FU for 48 h); group (S + F) (concurrent treatment with 8 μM sorafenib and 4 mg/L 5-FU for 48 h); group S + F (8 μM sorafenib pretreatment for 24 h followed by 4 mg/L 5-FU treatment for another 48 h); group F + S (4 mg/L 5-FU treatment followed by 8 μM sorafenib for another 24 h).
Cell cycle assays
Exponentially growing cells were starved in serum-free medium for 24 h, after which they were grown in medium containing 10% serum with the compounds 8 μM sorafenib for 24 h or 4 mg/L 5-FU for 48 h, either alone or in combination patterns. Cell cycle analyses and quantification of genomic DNA fragmentation were performed using the Cell Cycle Detection Kit (KeyGEN, Nanjing, China) according to the manufacturer’s protocol. Cell cycle distributions were analyzed by flow cytometry with a Becton Dickinson FACS Calibur.
Western blot analysis
To prepare whole-cell protein extracts, cells were washed twice with phosphate-buffered saline and then lysed with a modified radio-immunoprecipitation assay buffer (50 mM Tris–HCl pH 7.4, 1% v/v NP-40, 0.25% v/v sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mg/mL of protease inhibitors (leupeptin and pepstatin), 1 mM Na3VO4, and 1 mM NaF) on ice for 30 min. Insoluble material was removed by centrifugation at 12,000 p/min for 15 min at 4°C. The protein concentration of cell lysates was measured using the Bradford Protein Assay Kit (Beyotime, Shanghai, China), and 30 μg of protein samples were loaded on 10% polyacrylamide gels containing sodium dodecyl sulfate and separated by electrophoresis at a constant voltage of 70 V for 2 h and transferred onto 0.45-μm polyvinylidene fluoride membranes (Millipore Corporation, Billerica, MA, USA) at a constant voltage of 100 V for 3 h at 0°C. The membranes were probed with the specific primary antibodies followed by a horseradish peroxidase-conjugate secondary antibody (1:5,000) and detected by enhanced chemiluminescence (ECL kit from Pierce, Rockford, IL, USA). The following primary antibodies were used: anti-C-RAF (1:1,000), anti-phospho-C-RAF (1:1,000), anti-ERK1/2 (1:1,000), and anti-phospho-ERK1/2 (Thr202/Tyr204) (1:1,000) from Cell Signaling Technology, Inc. (Danvers, MA, USA); anti-STAT-3 (1:1,000) and anti-phospho-STAT-3 (Tyr705) (1:1,000) from Abcam (Cambridge, MA, USA); and anti-cyclin D1 (1:1000) and anti-β-actin from Beyotime. Unless otherwise indicated, immunoblot reagents were purchased from Beyotime.
Statistical analysis
Statistical analysis was performed with SPSS 17.0 software (SPSS, Chicago, IL, USA). Measured values are expressed as mean ± standard deviation. Analysis of variance and least significant difference were used to evaluate statistical significance of differences between groups, and a P value of <0.05 was considered statistically significant.
Discussion
Though few basic scientific studies have provided substantial evidence about the activity of 5-FU in combination with sorafenib in HCC, combined effects of the two agents on other solid tumors are controversial. Thomas and colleagues [
16] have shown that single-agent therapy with sorafenib or 5-FU is equally effective in human colorectal cancer, and combination therapy shows no additional effect. On the other hand, a recent study demonstrates that combination therapy of 5-FU and sorafenib exerts a synergistic antitumor effect in renal cell carcinoma [
17]. As sorafenib and 5-FU are both commonly used in HCC patients, it is meaningful and instructive to investigate the combined effects in HCC cells.
We find that both sorafenib and 5-FU display antitumor effects in the HCC cell lines MHCC97H and SMMC-7721. Combined effects of the two agents are schedule-dependent: concurrent treatment shows similar efficacy, while pretreatment with sorafenib exacerbates inhibitory effects, but 5-FU pretreatment followed by sorafenib ameliorates inhibitory effects compared with 5-FU monotherapy. According to variations in IC50 values, we find that HCC cells become less sensitive to 5-FU after pretreatment with sorafenib, yet more sensitive when 5-FU pretreatment is followed by sorafenib. That is to say, sequential treatment of 5-FU followed by sorafenib seems to be the optimal schedule for combined administration of the two agents.
Manov and colleagues [
18] found that sorafenib, when combined with doxorubicin, increased survival and reduced doxorubicin-induced autophagy by inhibiting MEK/ERK and inducing degradation of cyclin D1 in the HCC cell line Hep3B. Based on these results, they believe that the use of MEK/ERK inhibitors in combination with chemotherapeutics might have possible antagonistic effects. Our results tend to lead to a similar conclusion. Thus, we have tried to understand the mechanism by examining some of the sorafenib-related pathways, like the STAT3 and RAF/MEK/EKR cascade. In addition, we have analyzed cell cycle distribution and expression of proteins associated with cell cycle progression, as it is known that 5-FU is an S-phase-specific chemotherapeutic drug.
Our data reveal that sorafenib efficiently blocks STAT3 and RAF/MEK/EKR pathways, showing down regulation of p-C-RAF, p-ERK, and p-STAT3, while 5-FU shows almost no effect. No changes were observed for total C-RAF, ERK and STAT3 proteins by any of the treatments. Furthermore, sorafenib slows cell cycle progression by inducing a G1-phase arrest, which results in a reduction of the S-phase subpopulation. Sorafenib significantly down regulates cyclin D1 expression in HCC cells, while 5-FU has an opposite effect. Since expression levels of cyclin D1 in combination groups were as well down-regulated, we believe that sorafenib plays a dominant role in regulating cell cycle distributions and cyclin D1 expressions in combined treatments of sorafenib and 5-FU.
Signaling through RAF/MEK/ERK plays a crucial role in cell proliferation, differentiation, malignant transformation, and apoptosis [
19,
20]. It has been thoroughly demonstrated that sorafenib exhibits remarkable antitumor activity in HCC in vitro and in vivo, through targeting the RAF/MEK/EKR cascade [
21,
22]. Our results agree well with these reports.
The STAT3 proteins have dual roles as cytoplasmic signaling proteins and nuclear transcription factors that activate a diverse set of genes, including some that are importantly implicated in tumor cell proliferation, survival, invasion, cell-cycle progression, tumor angiogenesis, and tumor cell evasion of the immune system [
23‐
25]. Recently, sorafenib has been shown to suppress tumor growth by decreasing STAT3 phosphorylation in a group of human malignancies [
26‐
29], including HCC [
11,
30]. As the results we obtained from tests of STAT3 activation after sorafenib treatment are in line with previous studies, we have gained further insight into the mechanism of anti-cancer effects of sorafenib.
It is well known that key genes in cell-cycle control, such as cyclin D1, an important regulator of G1-to-S phase progression [
31], are regulated by STAT3 [
25,
26]. In addition, some studies have demonstrated that cyclin D1 is regulated by both the RAF/ MEK/ ERK and phosphoinositide-3 kinase (PI3K)/Akt pathways [
32,
33]. Interestingly, some recent studies point out that sorafenib inhibits growth and metastasis of HCC in part by blocking the MEK/ERK/STAT3 and PI3K/Akt/STAT3 signaling pathways [
11]; and that sorafenib-induced Tyr705 STAT3 dephosphorylation is mediated by Raf inhibition, as the Raf-inhibitor ZM336372 also results in Tyr705 STAT3 dephosphorylation [
34]. Therefore, we have reasons to believe that STAT3 somehow functions downstream of RAF/MEK/ERK signaling.
A recent study has indicated that 5-FU resistance in oral squamous cell carcinoma (OSCC) cell lines HSC-3 and CA9-22, both of which are hypoxia-sensitive (HS), is due to suppressed growth rate and G1-phase accumulation [
35]. Similarly, we find that sorafenib causes a G1-phase arrest of HCC cells and, as well, decreases sensitivity to 5-FU, leading to an antagonistic effect of the two agents in the sorafenib-pretreatment strategy.
To summarize, combination effects of sorafenib and 5-FU vary between the different treatment orders. On the whole, antitumor effects are highest in 5-FU pretreatment strategies, and they are lowest following sorafenib pretreatment patterns. Since 5-FU is an S-phase-specific chemotherapeutic drug, it works less efficiently after exposure to sorafenib because of reduction in the proportion of S-phase cells. In contrast, sorafenib exerts further antitumor effects after 5-FU treatments, as the mechanism of sorafenib is cell cycle-independent.
Our in vitro study is limited to the cellular level, and in vivo studies are needed that cover sequential therapy of cell cycle-dependent chemotherapeutic drugs and molecular-targeted drugs. Still, our results do provide some important clues that may help guide drug selection and therapeutic strategy used in clinical treatments.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
LD, ZR, QJ, WW, HS, and YW contributed to the study design, analysis, and interpretation of data. YW and ZR conceived the study. LD and QJ performed the experiments. LD and HS participated in statistical analysis. LD drafted the manuscript. ZR and WW carried out the revision and provided important suggestions. All authors read and approved the final manuscript.