Hepatocellular cancer is one of the most commonly diagnosed and lethal malignancy in China, and the high mortality rate is attributed in part to the development of chemoresistance. Although DNA synthesis-blocking anticancer drug such as 5-fluorouracil (5-FU) is initially effective, the most hepatocellular tumors are initially sensitive to chemotherapy and to the development of recurrent tumors that are resistant to 5-FU. The multidrug resistance (MDR) remains one of the major obstacles to successful therapy and is responsible for poor long-term overall patient survival. Hepatocellular cancer resistance to 5-FU is a complex phenomenon involving multiple defense gene expression such as multidrug resistance gene (MDR1), lung-resistance protein (LRP), multidrug resistance-associated protein (MRP1), glutathione-S-transferase-π (GST-π), and excision repair cross-complementing gene 1 (ERCC1) for augmentation of DNA repair and inhibition of apoptosis (Rabik and Dolan, 2007). The decreased cell p38 phosphorylation is a common feature of tumor cells, influencing the cellular signal transduction after drug treatment (Li et al., 2010).
Tissue factor pathway inhibitor-2 (TFPI-2) gene is located on chromosome 7q22 and consists of three Kunitz-type serine proteinase inhibitory domains (Wang et al., 2010). Recent reports suggest that TFPI-2 expression plays an important role in inhibiting cell metastasis and tumor invasion (Wong et al., 2007; Ran et al., 2009). Although the anticancer effects of TFPI-2 have been studied in various cancer cells, it remains unknown whether it produces reversal effects on drug-resistant cells such as 5-FU-resistant hepatocellular cancer cells. Therefore, in this study, we investigated the multidrug resistance reversal effect and the potential molecular mechanism of TFPI-2 in BEL-7402/5-FU through exploring the resistance index (RI) of 5-FU, function of the P-glycoprotein (P-gp) efflux pump, the expression of drug resistance gene, including MDR1, LRP, MRP1, GST-π, and ERCC1, and potential signal transduction pathways such as p38 activity.
MATERIALS AND METHODS
Cell Lines and Cell Culture
Human hepatocellular carcinoma BEL-7402 parental cells were obtained from American Type Culture Collection. The human hepatocellular carcinoma BEL-7402/5-FU cells which resistant 5-FU were induced form BEL-7402 by exposing increasing concentrations of 5-FU. Briefly, cells were placed in a six-well plate at 5 × 105 cells per well in medium with 2 μg/mL of 5-FU for 5 days, and the supernatant and dead cells were discarded. This procedure was repeated until cells could be cultured in medium with 200 μg/mL of 5-FU. Cells were cultured in RPMI-1640 containing 10% fetal bovine serum (FBS) in 37°C with 5% CO2 incubator. BEL-7402/5-FU cells were cultured in the afore-mentioned medium with 10 μg/mL of 5-FU until at least 2 weeks before experimentation.
Cell Transfection and Selection
Recombinant pEGFP-C1-TFPI-2 and empty vector were transfected into BEL-7402/5-FU cells using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer's instructions. Briefly, cells were placed in a six-well plate at 2 × 105 cells per well. Cells were transfected with 800 ng pEGFP-C1-TFPI-2 or empty vector. Cells were rested for 24 hr after transfection and were then cultured in RPMI-1640 containing 10% FBS in 37°C with 5% CO2 incubator for 24 hr. Positive cells were selected by 300 μg/mL of G418 to establish TFPI-2 overexpression and negative control sublines (empty vector-transfected cells). Expression of the TFPI-2 was determined by real-time PCR and Western blotting. Different selected clones were named as TFPI-2 overexpression BEL-7402/5-FU cell 1 and TFPI-2 overexpression BEL-7402/5-FU cell 2.
Resistance Index of 5-FU Assay
The ability of drug resistance reversal was evaluated in BEL-7402/5-FU cells as described by Zhu et al. (2012). Briefly, cells were seeded in 96-well plates at 5 × 103 cells per well. A range of concentrations of 5-FU (0.10, 0.22, 0.50, 1.00, 2.20, 5.00, 10.00, 22.00, 50.00, and 100.00 mg/mL) was applied to designated wells, and then buffer was applied to control wells. Each condition was repeated in 5-duplication. After 72 hr, the cells were incubated for 2 hr following the addition of 20 μL 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (2 mg/mL) to measure at 490 nm. The cell inhibitory rate was calculated according to the following formula: Inhibitory rate = 100 × (Acon − At)/Acon, where Acon is the A value of the control group and At is the A value of the treatment group. The 50% inhibitory concentration (IC50) was calculated by dose-response data from three independent experiments. The RI of 5-FU was calculated according to the following formula: RI = IC50res/IC50sen, where IC50res is the IC50 value in the 5-FU-resistant cell BEL-7402/5-FU and IC50sen is the IC50 in the 5-FU-sensitive cell BEL-7402. All experiments were repeated three times independently.
Cells were incubated with 5-FU (2 mg/mL) for 24 hr. The percentage of apoptosis was determined by Annexin V-FITC Apoptosis Detection kit (BD Biosciences) following the manufacture's instruction. Briefly, cells were washed with ice-cold PBS and resuspended in binding buffer. Annexin V-FITC and PI (BD Biosciences) were added to cells and incubated at room temperature in the dark for 15 min, and then the cells were analyzed by BD FACSCalibur flow cytometer (Becton Dickenson) with 488 nm excitation wavelength and 525 nm emission wavelength.
Intracellular Accumulation of Doxorubicin Assay
The intracellular doxorubicin accumulation was measured as described by Hong et al. (2005). Briefly, cells were harvested and resuspended at 1 × 106 cells per milliliter in PBS. Doxorubicin (5 μg/mL) was added to cells and incubated at 37°C in 5% CO2 for 2 hr. The cells were washed with cold PBS, and then the mean fluorescence intensities were detected by flow cytometry with 488 nm excitation wavelength and 575 nm emission wavelength.
Intracellular Accumulation of Rhodamine-123 Assay
The rhodamine-123 intracellular accumulation was measured as described by Xu et al. (2006). Briefly, cells were collected and incubated with 5 μg/mL rhodamine-123 at 37°C in 5% CO2 for 30 min. After incubation, cells were washed with cold PBS, and then the mean fluorescence intensity was detected by flow cytometry with 488 nm excitation wavelength and 525 nm emission wavelength.
Western Blot Analysis Assay
The cells were washed twice with cold PBS and pelleted by centrifugation (5 min, 300g) before homogenization in cold fractionation buffer (50 mM Tris-HCl, pH 7.4, 1 mM ethylene diamine tetraacetic acid (EDTA), 150 mM NaCl, 1% Triton X-100, 1 mM phenylmethyl sulfonylfluoride (PMSF), 10 μg/mL leupeptin, 10 μg/mL pepstatin A, 10 μg/mL aprotinin, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, and 50 mM sodium fluoride). The cell lysate was incubated on ice for 15 min and centrifuged at 20,000g for 30 min at 4°C. Protein concentrations were determined by bicinchoninic acid (BCA) protein assay kit. The cell lysate was collected and subjected to 12% SDS-PAGE and then transferred to a polyvinylidene fluoride membrane blocked by incubation with 5% bovine serum albumin in tween-tris-buffered saline (TTBS) at room temperature for 1 hr. Different primary antibodies [TFPI-2, 1:800; MDR1, 1:500; LRP, 1:800; MRP1, 1:200; GST-π, 1:500; ERCC1, 1:800; p-p38, 1:800; p38, 1:800 (all obtained from Santa Cruz); β-actin, 1:2,000 (from Sigma)] were applied at 4°C overnight followed by secondary antibody at room temperature for 1 hr. After washing, the blots were visualized by horseradish peroxidase (HRP) Western Blot Detection System.
RNA Extraction and Quantitative Real-Time PCR Analysis Assay
First-strand cDNA synthesis was performed using total RNA (2 μg) with M-MLV reverse transcriptase. The mRNA expression of MDR1, ERCC1, GST-π, LRP, and β-actin was quantified with ABI Prism 7500 Sequence Detection System by SYBR Green Real-Time Master Mix. The forward and reverse PCR primers were as follows: MDR1 forward: 5′-GGAGCGGTTCTACGA-3′, reverse: 5′-ACGATGCCCAGGTGT-3′; MRP1 forward: 5′-GTCGGAACAAGTCGTGCCTG-3′, reverse: 5′-CAAAGCCTCCACCTCCTCA-3′; ERCC1 forward: 5′-ACCGTGAAGTCAGTCAACAAA-3′, reverse: 5′-TCGTGCAGGACATCAAACA-3′; GST-π forward: 5′-CTGGAAGGAGGAGGTGGTG-3′, reverse: 5′-GACGCAGGATGGTATTGGAC-3′; and LRP forward: 5′-GGCTCCTTCCGCTACGT-3′, reverse: 5′-GCCGAGACCGCTCAATAC-3′. The amount of PCR product formed in each cycle was evaluated on the basis of SYBR Green I fluorescence. All amplification reactions were performed using the 7500 PCR system with the following cycle conditions: one cycle at 95°C for 5 min, followed by 40 cycles at 95°C for 30 sec, 58°C for 30 sec, and 72°C for 30 sec. The mRNA level for each gene was normalized to β-actin, and the forward and reverse PCR primers of β-actin were as follows: forward: 5′-AAGGCTGTGGGCAAGG-3′ and reverse: 5′-TGGAGGAGTGGGTGTCG-3′. Initial copies (C0) of target genes were calculated by the following formula: C0 =10(Ct − intercept)/slope, where Ct is the threshold cycle indicating the fractional cycle number at which the amount of amplified target genes reaches the fixed threshold. The relative amount of target gene mRNA was calculated by the following formula: C0 target gene/C0 housekeeping gene (Dussault and Pouliot, 2006). The ratio of the density of each group was compared with the β-actin group.
Quantitative data are presented as the mean ± SD determined from the indicated number of experiments. Statistical analysis was based on one-way ANOVA for comparison of multiple groups.
Expression of TFPI-2 mRNA and Protein in Recombinant BEL-7402/5-FU Cells
To investigate the overexpression effect of TFPI-2 in BEL-7402/5-FU cells, we examined the expression of TFPI-2 in TFPI-2 overexpression BEL-7402/5-FU cells, BEL-7402/5-FU parent cells, and negative control BEL-7402/5-FU cells by real-time-PCR. The results showed significant upregulation of TFPI-2 mRNA in the TFPI-2 overexpression BEL-7402/5-FU cells when compared with the parent and negative control cells, as shown in Fig. 1A. The Western blot results showed the similar tendency with real-time PCR results, as shown in Fig. 1B.
Effect of TFPI-2 in Enhancing Cytotoxicity of 5-FU in BEL-7402/5-FU Cells
The cell growth rates of 5-FU on BEL-7402, BEL-7402/5-FU, and TFPI-2 overexpression BEL-7402/5-FU cells were evaluated by the MTS method. As determined by the dose-effect curve, as shown in Fig. 2A, the IC50 values of BEL-7402, BEL-7402/5-FU, and TFPI-2 overexpression BEL-7402/5-FU cells after 72 hr of treatment were 0.2028, 4.909, 5.081, 2.306, and 2.143 mg/mL, respectively. The RI was the important metric to evaluate the resistance of anticancer drug resistance cells and was determined by dividing the IC50 for the MDR tumor cells by that for the parental cells. In this report, we observed a 24.21-fold and 24.05-fold increase in resistance to 5-FU for BEL-7402/5-FU and TFPI-2 overexpression BEL-7402/5-FU negative control cells, respectively, when compared with parental BEL-7402 cells. We also observed an 11.37-fold and a 10.57-fold increase in resistance to 5-FU for TFPI-2 overexpression BEL-7402/5-FU cells (Table 1). The results confirm that TFPI-2 significantly increased the chemosensitivity of 5-FU in MDR tumor cells (Fig. 2B).
Effect of TFPI-2 in Enhancing Apoptosis of 5-FU in BEL-7402/5-FU Cells
The induction of tumor cell apoptosis is a major mechanism of chemotherapy. On the basis of the potency of 5-FU on cell growth inhibition of four cell lines, we chose to use 5-FU at concentrations of 2 mg/mL. The cells were treated with 5-FU for 24 hr, and Annexin V/PI costaining assay was used to quantify the apoptosis rate. The results showed that 5-FU induced 46.3% and 50.8% of TFPI-2 overexpression BEL-7402/5-FU cells apoptosis and 18.6% and 19.3% of parent and negative control cells apoptosis, respectively, suggesting that TFPI-2 significantly increased the chemosensitivity of 5-FU in MDR tumor cells (Fig. 3).
Reversal Effect of TFPI-2 on Intracellular Accumulation of Doxorubicin and Rhodamine-123 in BEL-7402/5-FU Cells
The efflux of anticancer drugs of tumor cells result in a decrease of intracellular drug concentration, and it is believed to be one of the common causes of MDR. The fluorescence of doxorubicin was used to measure intracellular drug concentration by flow cytometry. The results showed that the intracellular accumulation of doxorubicin was increased to 2.84-fold and 3.06-fold in TFPI-2 overexpression BEL-7402/5-FU cells when compared with parent and negative control cells. The results suggest that TFPI-2 could promote the accumulation of the anticancer drugs through inhibiting drug efflux from BEL-7402/5-FU cells. The efflux of anticancer drugs from tumor cells depends on P-gp (Oga et al., 2012). Rhodamine-123 is a well-established P-gp substrate to investigate the activity of the P-gp by the detection of intracellular fluorescence by flow cytometry (Clark et al., 1996; Li et al., 2012). The results indicated that TFPI-2 overexpression BEL-7402/5-FU cells enhanced the accumulation of rhodamine-123 to 2.50-fold and 2.85-fold levels when compared with parent and negative control cells (Fig. 4). The results suggest that TFPI-2 could increase the accumulation of the anticancer drugs through inhibiting the function of P-gp in BEL-7402/5-FU cells.
Inhibitory Effect of TFPI-2 on Expression of Drug-Resistant Genes in BEL-7402/5-FU Cells
MDR results from the abnormal expression of drug-resistant genes such as MDR1, LRP, MRP1, GST-π, and ERCC1. To investigate the mechanism of TFPI-2 reversal effect of MDR in BEL-7402/5-FU cells, we used real-time PCR to examine the mRNA expression of MDR1, LRP, MRP1, GST-π, and ERCC1. The results showed significant downregulation of above-mentioned genes in the TFPI-2 overexpression BEL-7402/5-FU cells when compared with parent and negative control cells, as shown in Fig. 5A. The Western blot results showed a similar decreased tendency of above-mentioned drug-resistant genes in protein level with real-time PCR results, as shown in Fig. 5B.
Enhancement Effect of TFPI-2 on p38 mitogen-activated protein kinases (MAPK) Activation in BEL-7402/5-FU Cells
Activation of p38 MAPK could influence the cellular response and drug treatment effect, and higher p38 phosphorylation level could enhance anticancer drug treatment effect through MDR inhibition (Jiao and Wen, 2011).We investigated the effect of TFPI-2 on p38 MAPK activity in BEL-7402/5-FU cells by Western blot assay. As shown in Fig. 6, phosphorylation of p38 MAPK in TFPI-2 overexpression BEL-7402/5-FU cells was significantly increased when compared with parent and negative control cells.
Accumulative research showed that resistance to anticancer drug treatment represents one of the major limitations to the efficacy of chemotherapy in malignancies. This study demonstrated that TFPI-2 could reverse MDR through downregulation of drug-resistance genes in 5-FU-resistant hepatocellular cancer BEL-7402/5-FU cells.
In this study, IC50 values of 5-FU was significantly decreased in TFPI-2 overexpression BEL-7402/5-FU cells when compared with parent and negative control cells; however, they were higher than IC50 values in BEL-7402 cells. Therefore, we concluded that TFPI-2 could partially reverse the drug resistance of BEL-7402/5-FU cells. Furthermore, TFPI-2 significantly reduced the RI for 5-FU of BEL-7402/5-FU cells, showing an increased cytotoxicity of 5-FU in TFPI-2 overexpression BEL-7402/5-FU cells.
The reduction of the intracellular concentration of anticancer drug is one of the major causes of MDR. P-gp, which is encoded by the MDR1 gene, has been found to induce the efflux of anticancer drug from MDR tumor cells (Filomeni et al., 2008; Achari et al., 2011). In our study, we investigated the inhibition of TFPI-2 overexpression on P-gp function in BEL-7402/5-FU cells through doxorubicin and rhodamine-123 accumulation assays. Meanwhile, the real-time PCR and Western blot assay result also showed a decrease in the mRNA and protein expression of MDR1 in TFPI-2 overexpression BEL-7402/5-FU cells. Accordingly, we considered that TFPI-2 reverses drug resistance by inhibiting the P-gp function and that MDR1 expression results in increasing intracellular drug concentration.
Recent studies show that there are numerous molecular mechanisms involved in MDR. LRP has been identified to be the major human vault protein and has been reported to be the marker for tumor drug resistance (Losert et al., 2012) contributing to the mass of vault particles. Some studies considered that vaults act as the shuttles between the 5-FU and nucleus through the nuclear pore complex (Turner, 2012). Therefore, the LRP could change the anticancer drug distribution results in MDR in tumor cells. MRP1 is a member of the ABC superfamily, which is the membrane transport protein. MRP1 localized both plasma membrane and intracellular organelles membranes, leading to cause the intracellular and cytoplasmic sequestration of drugs to prevent to reaching the drug targets (Carew, 2011). GST is an isoenzyme of multigene family, which is classified as Phase II detoxification enzymes. GST-π is an enzyme related to cell protection against toxic electrophiles and oxidative stress products, leading to drug resistance (Laborde, 2010). ERCC1 is an important rate-limiting DNA repair protein in the nucleotide excision repair, and it is important for interstrand crosslinks in the DNA repair and recombination processes (Ozkan et al., 2012). In some reports, ERCC1 expression was shown to be increased in the drug-resistant tumor cells (Chai et al., 2012; Krawczyk et al., 2012). To explore the potential mechanism of TFPI-2 reversal effect of drug resistance, we detected the mRNA and protein expression of LRP, MRP1, GST-π, and ERCC1 and found a decreased expression of LRP, MRP1, GST-π, and ERCC1 in TFPI-2 overexpression BEL-7402/5-FU cells. Thus, we believe that the ability of TFPI-2 to reverse 5-FU resistance might result from reduced MDR1 expression, increasing the 5-FU intracellular concentration or toxicity.
The mechanism of p38 MAPK downstream signal pathways has long been studied, including the involvement of apoptosis (Wu et al., 2011) and MDR1 expression regulation (Bulavin and Fornace, 2004). In this study, our results showed an increased p38 MAPK phosphorylation level in TFPI-2 overexpression BEL-7402/5-FU cells. Thus, we concluded that the MDR reversal induced by downregulation of MDR1 may be caused by the enhancement of p38 MAPK activity in TFPI-2 overexpression BEL-7402/5-FU cells.
In summary, our results demonstrate that TFPI-2 has significant reversal effects on 5-FU resistance in human hepatocellular cancer BEL-7402/5-FU cells. The mechanism of action of the highly potent MDR reversal modulator is mainly related to the downregulation of the expression of LRP, MRP1, GST-π, ERCC1, and the functions of P-gp via upregulation of p38 MAPK activity. These results indicate that TFPI-2 may serve as a potential therapeutic target of 5-FU-resistant human hepatocellular cancer.
Table 1. Potency of TFPI-2 in enhancing cytotoxicity of 5-FU in BEL-7402/5-FU cells