Hepatocellular carcinoma (HCC) is the fifth most common cancer in men and the eighth most common cancer in women worldwide. It is also one of the most common cancers in China. HCC is one of the most crucial public health burdens worldwide and is associated with several risk factors such as genetic factors, lifestyle choices, and environmental conditions (Zhang et al., 2011b). Chemotherapy is one of the major methods of treating HCC, but chemotherapeutic drug resistance is a major problem in cancer chemotherapy. Multidrug resistance (MDR) has been implicated in the failure of chemotherapy in HCC (Xu et al., 2011). MDR is an intrinsic or acquired cross resistance to a variety of structurally and functionally unrelated drugs such as anthracyclines, epipodophyllotoxins, and vinca alkaloids and represents one of the major problems in the cancer therapy. To enhance the chemosensitivity of tumor cells, attention has been focused on MDR modulators.
Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) was originally identified as a gene associated with terminal differentiation and irreversible growth suppression of metastatic human melanoma cells (Yang et al., 2011). It binds to cell surface receptors to induce transcriptional activation of signal transducers and activators of transcription 3-phosphorylation (Dumoutier et al., 2001; Zhang et al., 2011a). Previous studies have shown that mda-7/IL-24 inhibits cell growth and induces apoptosis in melanoma, lung cancer, and other tumor types (Dent et al., 2010; Otkjaer et al., 2010; Wang et al., 2010; Wei et al., 2010; Xiao et al., 2010; Bhutia et al., 2011; Valero et al., 2011). It has also been shown that mda-7/IL-24 could reduce angiogenesis in tumors (Wang et al., 2007; Chang et al., 2009), suggesting that it is a tumor suppressor gene. A recent study has shown that mda-7/IL-24 could reverse MDR in human colorectal cancer (Emdad et al., 2007).
However, the potential role and molecular mechanism of mda-7/IL-24 in reversing the MDR of human HCC remain largely unknown. To clarify the role of mda-7/IL-24, we determined the expression of mda-7/IL-24 in drug-resistant HCC cell line BEL 7402/5-fluorouracil (5-FU). Further, we investigated the effects of overexpression of the mda-7/IL-24 gene in BEL 7402/5-FU cell line in vitro.
MATERIALS AND METHODS
The human HCC cell line BEL-7402/5-FU was obtained from American Type Culture Collection (Rockville, MD) and cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen), penicillin (100 U/mL), and streptomycin (100 mg/mL) at 37°C in 5% CO2.
The gene for mda-7/IL-24 (GenBank accession number NM_006850) coding region was cloned from human PBMCs from a healthy donor by reverse transcription-polymerase chain reaction (RT-PCR) using nested PCR. The primers used were: sense 1: 5′-GGAACACGAGACTGAGAGATG-3′, sense 2: 5′-TTGGATCCGAGAGATGAATTTTCAACAGAGG-3′, antisense 1: 5′-ACAGGGAACAAACCAGTGCCA-3′ and antisense 2: 5′-GCCTCGAGTCCTGGTCTAGACATTCAGAGC-3′. The restriction site sequence of BamH I and Xho I (TaKaRa) was added to the sense 2 and antisense 2 primers, respectively. The PCR product was digested with BamHI and XhoI and cloned into the same sites of the pcDNA3.1 vector (Invitrogen), which contains a selective marker (the neomycin phosphotransferase gene). The positive clones were determined by sequence analysis.
Transfection of mda-7/IL-24
An empty pcDNA3.1 vector and pcDNA3.1-mda-7/IL-24 expression plasmid was transfected into BEL-7402/5-FU cells by Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instruction. Cells were then screened with 800 μg/mL of G418 (Invitrogen) to generate mda-7/IL-24 overexpressing sublines (Ad-mda-7/IL-24-1 and Ad-mda-7/IL-24-2, respectively) and vector control sublines (Ad-mda-7/IL-24-V). These sublines were grown in RPMI-1640 with 10% fetal bovine serum and G418 at 37°C in 5% CO2. Mda-7/IL-24 protein expression was measured using Western blotting.
Reversal Effect Assays
The ability of mda-7/IL-24 to reverse drug resistance in BEL-7402/5-FU cells was evaluated by CellTiter-Glo assay (Promega). Briefly, cells were plated onto 96-well plates at a density of 1 × 104 cells/mL. The culture medium was replaced by new medium containing 0.05–6.4 mg/mL 5-fluorouracil (5-FU) or 0.002–0.032 mg/mL doxorubicin (DOX) after 48 hr and cells were lysed with the reagent of the assay kit for 10 min. Then, the mixtures were transferred onto opaque-walled 96-well plates, and luminescence produced from ATP-mediated chemical reaction was read by TriStar LB 941 (Berthold Technologies). The following formula was used to calculate the inhibition of cell growth: rate of inhibition (%) = [1−(mean A value of mda-7/IL-24 overexpressing group/mean A value of parent group)] × 100. The formulas of reversal fold (RF) and relative reversal rate (RRR) were RF = IC50 MDR cell/IC50 mda-7/IL-24 overexpressing MDR cell; RRR (%) = [(IC50 multidrug resistance cell-IC50 mda-7/IL-24 overexpressing multidrug resistance cell)/(IC50 multidrug resistance cell-IC50 parental cells)] × 100.
Cells (5 × 105) were seeded in each well of a six-well plate and adhered overnight and were incubated with 5-FU for 24 hr. These cells were harvested, labeled with Annexin V, and PI using an apoptosis detection kit (BD PharMingen) according to the manufacturer's instructions and analyzed using FACScan (Becton Dickinson Company) subsequently.
Drug Accumulation and Efflux Assay
Drug accumulation and efflux were analyzed by flow cytometry (Navarro et al., 2012). Cells were incubated with 2 μmol/L DOX and 4 μmol/L Rhodamine-123 at 37°C for 60 min, respectively. After incubation, cells were washed twice with ice-cold phosphate buffered saline solution (PBS), resuspended in 400 μL PBS, and then analyzed by flow cytometry.
RNA Extraction and RT-PCR
Total cellular RNA was extracted with TRIZOL reagent according to the manufacturer's instruction. The RNA quality was assessed using agarose gel electrophoresis and was quantified spectrophotometrically. Five microgram of total RNA was used to perform RT for first-strand cDNA synthesis (TaKaRa). Real-time PCR analysis was performed using the Platinum SYBR green qPCR SuperMix-UDG System (Invitrogen) according to the manufacturer's instructions. The housekeeping gene β-actin was quantified as control. Primers of β-actin and target genes were as following: MDR1 (GenBank accession number NM_000927), Sense: 5′-CCCATCATTGCAATAGCAGG-3′, antisense: 5′-GTTCAAACTTCTGCTCCTAG-3′; multidrug resistance-related protein 1 (MRP1) (GenBank accession number NM_004996), Sense: 5′-TCTCTCCCGACATGACCGAGG-3′, antisense: 5′-CCAGGAATATGCCCCGACTTC-3′; lung resistance-related protein (LRP) (GenBank accession number NM_002332) Sense: 5′-GTCTTCGGGCCTGAGCTGGTGTCG-3′, antisense: 5′-CTTGGCCGTCTCTTGGGGGTCCTT-3′; β-actin (GenBank accession number NM_001101): Sense: 5′-AGCATCCTAGAACTCTGTGC-3′, 5′-ATTTCGGACCCCTGAACATA-3′. Quantification of expression of the target gene in samples was accomplished by measuring the fractional cycle number at which the amount of expression reaches a fixed threshold (CT). The relative quantification was given by the CT values, determined by triplicate reactions for test, and reference samples for target and for β-actin. Triplicate CT values were averaged and the β-actin CT subtracted to obtain ΔCT. The relative expression level of target gene was determined as 2–ΔCT.
Western Blot Analysis
Cells were lysed with protein lysis buffer (20 mM Tris-HCl pH 7.4, 5 mM EDTA, 1% Triton-X 100, 150 mM NaCl, 1% DTT, and 1% protease inhibitor cocktail (Sigma, St. Louis, MO). The proteins were separated by 12% sodium dodecyl sulfate polyacrylamide gel electropheresis in western blot assay (SDS-PAGE) and transferred to polyvinylidene difluoride membranes in western blot assay (PVDF) membranes (Pierce, Rockford, IL). Membranes were blocked in 5% skimmed milk in TBST (10 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 8.3) for 1 hr at room temperature, then incubated overnight with primary antibodies against mda-7 (R&D Systems, Minneapolis, MN) or LRP (Ser780, Ser795, and Ser807/811) or MDR1 (Cell Signaling Technology) or MRP1 and β-actin (Sigma-Aldrich, St Louis, MO), followed by incubation with horseradish peroxidase–conjugated appropriate secondary antibodies (Cell Signaling Technology). Immunoreactive bands were visualized using the enhanced chemiluminescence reagents (GE Healthcare).
Luciferase Reporter Assay
Cell transfection was carried out by lipofectamine 2000 (Invitrogen) following the manufacturer's instruction. Briefly, cells were plated in the 24-well plate at 1 × 105 cell/well. Cells were cotransfected with 400 ng of pAP-1-luc or pNFκB-luc, and 4 ng of pRL-SV40 as an internal control. Luciferase assays were performed using the Dual-luciferase Reporter Assay System (Promega) following the manufacturer's instruction after 24 hr.
All data in the study were analyzed by Statistical Package for Social Sciences software version 11.5 (SPSS). Values are expressed as mean ± SD of experiments performed in triplicate. Statistical significance was tested by Student's t-test for either paired or unpaired data as appropriate. Statistical significance was defined as P < 0.05.
Expression of Mda-7/IL-24 in Human Drug-Resistant HCC BEL-7402/5-FU Cell Lines
We detected the expression of mda-7/IL-24 in BEL-7402/5-FU parent cells, control cells, and mda-7/IL-24 overexpressing cells by Western blotting. The expression of mda-7/IL-24 was weakly detectable in the parent and control cells, whereas mda-7/IL-24 overexpressing cell lines showed an increased expression of mda-7/IL-24 compared to the parent and control cells (Fig. 1).
Mda-7/IL-24 Induced the Drug-Resistant Reversal Effect of BEL-7402/5-FU Cells
The reversal effect of the mda-7/IL-24 was evaluated by RF, as follows: RF > 1 indicates enhanced drug sensitivity, RF = 1 indicates no effect, and RF < 1 indicates decreased drug sensitivity; the greater the RF magnitude, the more significant the effect. Mda-7/IL-24 enhanced the sensitivity of BEL-7402/5-FU cells to 5-FU by 3.63-fold and 5.38-fold in Ad-mda-7/IL-24-1 and Ad-mda-7/IL-24-2, respectively. Chemosensitivity to DOX was enhanced by 3.5-fold and 4.25-fold in Ad-mda-7/IL-24-1 and Ad-mda-7/IL-24-2, respectively. The results showed that mda-7/IL-24 significantly increased the cytotoxicity of anticancer drugs in BEL-7402/5-FU cells. To assess whether mda-7/IL-24 produced complete or partial reversals of drug resistance, RRRs were calculated. RRR ≥ 100% indicated a complete reversal effect and RRR < 100% indicated a partial reversal effect. The RRRs for 5-FU were 78.44%–81.74%, and the RRRs for DOX were 90.12%–96.51%, respectively (Table 1). The results showed that mda-7/IL-24 exerted a partial reversal effect against chemotherapeutic drugs in BEL-7402/5-FU cells.
Table 1. Potency of mda-7/IL-24 in enhancing chemosensitivity of anticancer drugs in BEL-7402/5-FU cells
Reversal resistance of 5-FU
Reversal resistance of 5-FU
5-FU IC50 (mg/ml)
DOX IC50 (mg/ml)
IC50 values of 5-FU and DOX were determined by MTT assay in BEL-7402/5-FU cells. IC50 values are presented as the means ± SD from at least three independent experiments, compared to the control group, *P < 0.05. The RF and RRR are calculated. RF represents reversal effect of mda-7/IL-24, the greater the RF magnitude, the more significant the effect. RRR < 100% indicated that the modulator had a partial reversal effect.
0.0324 ± 0.0012
0.0051 ± 0.0002
5.1647 ± 1.2647
0.0264 ± 0.0019
4.8147 ± 1.1196
0.0271 ± 0.0029
0.9614 ± 0.0412*
0.0065 ± 0.0003*
1.6187 ± 0.1069*
0.0078 + 0.0004*
Mda-7/IL-24 Promoted Apoptosis of BEL-7402/5-FU Cells
To determine whether mda-7/IL-24 overexpression promotes apoptosis of the BEL-7402/5-FU cells, we detected the apoptotic rate using FACScan. The apoptotic rates were 43.4% ± 4.7% and 38.1% ± 6.5% in Ad-mda-7/IL-24-1 and Ad-mda-7/IL-24-2 groups, respectively, whereas the apoptotic rates were 10.3% ± 1.6% and 13.5% ± 1.1% in the parent cell group and the vector control group, respectively. The apoptosis of mda-7/IL-24 overexpressing group was significantly more than that of the parent cell group and the vector control group. There was no significant difference between the parent cell group and the vector control group (Fig. 2).
Mda-7/IL-24 Improved the Intracellular Accumulation of Rhodamine-123 and DOX of BEL-7402/5-FU Cells
The potential inhibitory effects mda-7/IL-24 on P-glycoprotein (P-gp) efflux was assessed by Rhodamine-123 and DOX exclusion assays (Zhu et al., 2012). The intracellular accumulation of Rhodamine-123 and DOX in mda-7/IL-24 overexpressing cells was significantly increased compared to the parent and control cells. The results implied that mda-7/IL-24 induced MDR reversal in drug-resistant cells by inhibiting P-gp function, increasing intracellular DOX accumulation, and decreasing efflux of DOX. This leads to an accumulation of antitumor drug to exert the cytotoxic effect (Fig. 3).
Mda-7/IL-24 Suppresses the Expression of LRP, MDR1, and MRP1 in BEL-7402/5-FU Cells
MDR1, LRP, and MRP1 are associated with the MDR. Therefore, we determined the expression of LRP, MDR1, and MRP1 in the four cell lines using RT-PCR and Western blot to explore the effect of mda-7/IL-24 on the three genes. The results showed that the expression of LRP, MDR1, and MRP1 in mda-7/IL-24 overexpressing cells were decreased significantly compared to the parent and control cells on mRNA and protein levels (Fig. 4).
Mda-7/IL-24 Suppresses AP-1 and NF- κB Transcriptional Activation of BEL-7402/5-FU Cells
We examined the effect of mda-7/IL-24 on AP-1 and NF-κB transcriptional activation. The AP-1 and NF-κB transcriptional activation were detected by luciferase reporter assay. As shown in Fig. 5, the overexpression of mda-7/IL-24 suppressed AP-1 and NF-κB transcriptional activation in BEL-7402/5-FU cells.
Although mda-7/IL-24 has been widely identified as a candidate antitumor gene, but little is known regarding its role in MDR. The purpose of this study was to explore the potential role of mda-7/IL-24 on MDR reversal in human drug-resistant HCC cell line BEL-7402/5-FU. We detected the expression of mda-7/IL-24 in BEL-7402/5-FU parent cell line, control cell line, and mda-7/IL-24 overexpressing cell lines and found reduced expression of mda-7/IL-24 in parent and control cell lines. Meanwhile, there was an increased expression of mda-7/IL-24 in mda-7/IL-24 overexpressing cell lines. We further explored the effect and molecular mechanism of mda-7/IL-24 on the reversal of MDR.
Accumulative reports have shown that mda-7/IL-24 displays antitumor properties and inhibits cancer cell growth by inducing apoptosis and arresting cell cycle (Wang et al., 2007; Dent et al., 2010; Otkjaer et al., 2010; Wang et al., 2010; Valero et al., 2011). Previous studies have shown that LRP, MDR1, and MRP1 play important roles in tumor MDR. MDR1 is a member of ATP-binding cassette transporter family, encoding P-gp which is involved in pumping drugs from the inside to the outside of cancer cells, and preventing the intracellular accumulation of anticancer drugs (Cheng et al., 2011; Kanagasabai et al., 2011; Loo et al., 2012; Wang et al., 2012). As the member of ABC transporter superfamily, MRP1 also acts as the drug efflux pump rendering tumor cells resistant to cytostatic drugs (Haimeur et al., 2004). LRP encodes a major vault protein associated with drug resistance and has been found to predict a poor response to chemotherapy in many tumors (Catasús et al., 2011; Lu et al., 2011). Many researchers are trying to inhibit the expression of LRP, MDR1, and MRP1 to overcome the MDR phenotype by enhancing intracellular accumulation of anticancer drugs. In this study, we investigated the effect of mda-7/IL-24 on the expression of LRP, MDR1, and MRP1 and found a significant decreased expression of LRP, MDR1, and MRP1 in mda-7/IL-24 overexpressing cell lines compared to the parent and control cell lines, demonstrating that mda-7/IL-24 reversed the MDR of human HCC via decreasing the expression of MRPs MDR1, MRP1, and LRP.
Furthermore, we detected the effect of mda-7/IL-24 on efflux of chemotherapeutic drug. The results showed that the intracellular accumulation of DOX and Rhodamine-123 was significantly increased in mda-7/IL-24 overexpressing cells compared to the vector control or parent cells. This indicates that the overexpression of mda-7/IL-24 could increase the intracellular accumulation and concentration of anticancer drug in tumor cells. The results of MTT assay demonstrated that mda-7/IL-24 could significantly improve the chemotherapeutic sensitivity of tumor cells.
To further explore the molecular mechanism of antiresistance effect of mda-7/IL-24, we studied AP-1 and NF-κB transcriptional activation in four cell lines. AP-1 and NF-κB are major antiapoptotic/prosurvival transcriptional factors and their frequent alterations have been observed in human cancer cells. Accumulating evidence shows that AP-1 and NF-κB transcriptional activation play an important role in chemoresistance of cancer cells. Our results demonstrated that mda-7/IL-24 could overcome drug resistance in BEL-7402/5-FU cell lines by inhibiting AP-1 and NF-κB transcriptional activation.
In summary, we found that mda-7/IL-24 could reverse MDR of BEL-7402/5-FU cells by decreasing the expression of MRPs MDR1, MRP1, and LRP, increasing the intracellular accumulation of anticancer drug and apoptotic rate of cancer cells, and suppressing the AP-1 and NF-κB transcriptional activation. We considered that mda-7/IL-24 may act as a potential treatment for multidrug resistant HCC.