2-DG-Regulated RIP and c-FLIP Effect on Liver Cancer Cell Apoptosis Induced by TRAIL

Background

Liver cancer is one of the most common malignant tumors, with high morbidity and mortality [1,2]. It was confirmed that cancer cells survival depends on glucose metabolism, and inhibiting glycometabolism process might be an anti-cancer strategy [3,4]. 2-deoxy-D-glucose (2-DG) is a type of glycogen analogue phosphorylated by hexokinase. Its accumulation in the cells disrupts normal phosphorylation process of glucose metabolism. 2-DG can inhibit cell growth in a variety of cancers and increase chemotherapy drugs’ curative effect [5]. 2-DG can also restrain protein glycosylation, leading to increase endoplasmic reticulum tension and activate protein synthesis reaction [6]. In breast cancer, gastric cancer, and ovarian cancer, 2-DG is an important material to regulate receptor interacting protein (RIP) and cellular caspase 8 (FLICE)-like inhibitory protein (cFLIP) [7,8].

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family. It became a new resource for anti-cancer drugs because of its function in selective inducing cancer cells apoptosis [9,10]. TRAIL is highly expressed in several organs, such as spleen, lymph nodes, small intestine, prostate, and placenta. It also can be found in immune cells, such as natural killer cells, monocytes, B cells, and dendritic cells [11–13]. TRAIL can selectively kill heterogeneous tumors without damaging normal tissues [14]. TRAIL induces apoptosis mainly through binding with TRAIL receptors -1 and -2. After activating Caspase-8, it could activate the Caspases system directly or indirectly through the mitochondrial apoptosis pathway [15,16]. It was reported that TRAIL receptor expression level and a variety of regulatory mechanism in cells are closely related to cellular sensitivity to apoptosis induced by TRAIL. Materials that can induce apoptosis mainly include c-FLIP and pro-apoptotic and anti-apoptotic Bcl-2 family members and IAP family members (including XIAP, cIAP-1, and cIAP-2) [17]. XIAP can bind with Caspase-3/9 to inhibit their activity, while cIAP-1 and cIAP-2 could protect cells induced by TRAIL through RIP ubiquitin. Death receptor complex recruited to Caspase-8 when cIAP-1 and cIAP-2 were inhibited by Smac/DIABLO, leading to Caspase-8 activation and apoptosis [18]. Therefore, RIP is not only the effector of tumor necrosis factor signaling pathway, but also an essential factor in TNF-induced NF-κB activation. RIP can inhibit apoptosis and promote cell proliferation through activating NF-κB, eventually promoting cancer formation [19, 20]. This study tried to investigate the effect of 2-DG on RIP and c-FLIP expression in liver cancer cells to illustrate the mechanism of 2-DG regulating RIP and c-FLIP effect on TRAIL-induced liver cancer cell apoptosis. The study can not only help further understanding the mechanism of reversing TRAIL resistance, but also provide a basis for future clinical application of TRAIL.

Material and Methods

MAIN REAGENTS:

HepG2 cell line was bought from Shanghai Chuanxiang Biological Technology Co., LTD. Hep3B cell line was from Shanghai Bioleaf Biological Co., LTD. The 2-DG was from Sigma. Soluble recombinant TRAIL was from PeporTehc. The PI apoptosis detection kit was from the Keygen. The Caspase-8 detection kit was from Wuhan Boster Company. Caspase-3, RIP, and c-FLIP antibodies were bought from Abcam. β-actin and Caspase-8 antibodies were from Santa Cruz.

CELL CULTURE:

HepG2 and Hep3B cells were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum, 100 kU/L penicillin, and 100 mg/L streptomycin in a humid atmosphere containing 5% CO₂ at 37°C.

C-FLIP AND RIP SIRNA:

siRNA sequence was designed based on c-FLIP and RIP genetic sequence in the GeneBank and synthesized by QIAGEN. The sequences used were as follows. c-FLIP siRNA: forward, 5′-UCGGGGACUUGGCUGAACUUU-3′; reverse, 5′-AGUU CAGCCAAGUCCCCGACA-3′. RIP siRNA: forward, 5′-CCACUAG UCUGACGGAUAAUU-3′; reverse, 5′-UUAUCCGUCAGACUA UGGUAA-3′. We constructed recombinant plasmid pRNAT-U6/Neo/siRNA-c-FLIP and pRNAT-U6/Neo/siRNA-RIP to get better transfection effect. 7×10⁵ HepG2 or Hep3B cells were seeded in a 6-well plate. Empty vector, pRNAT-U6/Neo, pRNAT-U6/Neo/siRNA-c-FLIP, and pRNAT-U6/Neo/siRNA-RIP were transfected to the HepG2 and Hep3B cells through Lipofectamine 2000. After 24 h, Western blot was used to detect c-FLIP and RIP protein expression to determine the transfection efficacy.

MTT ASSAY:

2-DG and TRAIL cytotoxic effects on liver cancer cell line HepG2 and Hep3B were determined 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT). 1×10⁴ HepG2 and Hep3B cells were seeded in 96-well plate for 24, 48, or 96 h. 20 μl 5 mg/mL MTT solution was added to each well and incubated for 4 h. We added 150 μl DMSO to each cell for 30 min at room temperature, and the plate was detected at 570 nm to get the absorbance value (optical density, OD).

MITOCHONDRIAL MEMBRANE POTENTIAL DETECTION:

Mitochondrial membrane potential was detected by JC-1. 2×10⁵ HepG2 and Hep3B cells were seeded in a 6-cell plate. After treated with 10 mM 2-DG or 200 ng/ml TRAIL for 24 h, the cells were added with 1 ml JC-1 dyeing liquid at 37°C for 20 min. Then the cells were washed twice by buffer and incubated in medium to be observed under a laser confocal microscope.

CELL APOPTOSIS DETECTION:

Cell apoptosis was measured by propidium iodine (PI). 4×10⁴ HepG2 and Hep3B cells were seeded in a 96-cell plate. After being treated with 10 mM 2-DG or 200 ng/ml TRAIL for 24 h, the cells were collected and added with 5 μl V-FITC and 5 μl PI for 15 min in the dark. Then the cells were detected by flow cytometry.

WESTERN BLOT:

After transfection, the cells were digested with lysis buffer. Total protein was separated by denaturing 10% SDS-polyacrylamide gel electrophoresis. After being incubated with c-FLIP, RIP, DR4, DR5, FADD, Caspase-3/8/9, ERK1/2, and DFF45 primary antibodies, the membrane was detected with chemiluminescence and calculated with Image J software. Protein levels were normalized to β-actin and changes were determined.

RT-PCR:

The cDNA was synthesized with 1 μg RNA from the samples by TRIzol. The primers were synthesized by Shanghai Boshang Biotechnology Co., LTD. XIAP forward primer: 5′-CGCCC TAGGCACCAGGGTGTG-3′, reverse primer: 5′-TCGGTGAGCAGCACA GGGTG-3′; c-IAP forward primer: 5′-GCCCAACCGCGCTGAGTACA -3′, reverse primer: 5′-TGCCTTCTATGGCCTCCACGA -3′; β-actin forward primer: 5′-GACATGCCGCCTGGAGAAAC-3′, reverse primer: 5′-AGCCCAGGATGCCCTTTAGT-3′. The cycling conditions consisted of an initial, single cycle of 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, 15 s at 60°C, and 30 s at 72°C.

STATISTICAL ANALYSIS:

All statistical analyses were performed using SPSS17.0 software (SPSS Inc., USA). Differences between multiple groups were analyzed by one-way ANOVA and Duncan’s multiple range test. Numerical data were presented as means and standard deviation (±SD). P<0.05 was considered as a significant difference.

Results

2-DG ENHANCED HEPG2 AND HEP3B CELLS APOPTOSIS INDUCED BY TRAIL:

HepG2 and Hep3B cell apoptosis rates were tested after being treated with 2-DG or TRAIL. It was found that single 2-DG or TRAIL reduced cell survival rate, and 2-DG group exhibited dose- and time-dependent effects (Figure 1A). Compared with the single TRAIL treatment group, cell survival rate decreased after treatment by 2-DG and TRAIL together (Figure 1B). Flow cytometry results showed that single 2-DG or TRAIL obviously promoted apoptosis (Table 1). Compared with the single treatment group, two liver cancer cells presented higher apoptosis rate after treatment by 2-DG and TRAIL together (Figure 1C). In addition, we used fluorescent probe JC-1 to detect the change of mitochondrial membrane potential. JC-1 showed green when the mitochondrial membrane was damaged, while it showed red in normal cells. Results revealed that liver cancer cells had obvious combined mitochondrial membrane damage after treatment by 2-DG and TRAIL (Figure 1D).

2-DG ENHANCED TRAIL INDUCED HEPG2 AND HEP3B CELL APOPTOSIS DEPENDENT ON CASPASES:

To investigate Caspase’s role in 2-DG enhanced cell apoptosis induced by TRAIL, we used Caspases inhibitor z-VAD-FMK. Table 2 shows that the HepG2 and Hep3B cell apoptosis rate decreased after treatment with z-VAD-FMK compared with 2-DG or TRAIL treatment, indicating that 2-DG enhanced TRAIL-induced HepG2 and Hep3B cell apoptosis dependent on Caspases.

2-DG IMPACT ON RIP AND C-FLIP EXPRESSION IN HEPG2 AND HEP3B CELLS:

RT-PCR and Western blot showed that XIAP and c-IAP mRNA and protein expression in HepG2 and Hep3B cells significantly decreased after treatment with 2-DG for 24 h (Figure 2A), and RIP and c-FLIP protein also declined (Figure 2B), suggesting that 2-DG inhibits XIAP, c-IAP, RIP, and c-FLIP expression in HepG2 and Hep3B cells.

DOWN-REGULATING RIP AND C-FLIP CAN ENHANCE HEPG2 AND HEP3B CELL APOPTOSIS INDUCED BY TRAIL:

We further investigate the role of RIP and c-FLIP in cell apoptosis induced by TRAIL. As shown in Figure 3A, down-regulating RIP and c-FLIP expression significantly enhanced HepG2 and Hep3B cell apoptosis induced by TRAIL or 2-DG, while combined treatment showed higher apoptosis rate than in the TRAIL treatment group (Figure 3B). The TRAIL-induced HepG2 and Hep3B cells apoptosis rate increased obviously after 2-DG reduced RIP and c-FLIP expression (Figure 3C).

Discussion

Tumor cell survival depends on glycolysis metabolism. They need to absorb glucose and glycolysis at high speed to survive. Thus, glycolysis inhibitors become a potential anti-cancer drug [2]. 2-DG is a synthesized glucose analogue, which is not only an endoplasmic reticulum stress inhibitor, but also is a hexokinase inhibitor. It can suppress cancer cell uptake glycogen to inhibit cancer cell growth and metabolism and has been considered as a potential anticancer agent [3]. Studies have confirmed that there is a certain correlation between 2-DG and TRAIL. 2-DG can up-regulate GRP78 and TRAIL-R2 expression in melanoma cells. It can promote cell apoptosis induced by TRAIL, but its mechanism is still unclear [4]. We used liver cancer cells to investigate the effect of 2-DG on RIP and c-FLIP expression, and its role in cell apoptosis induced by TRAIL.

Apoptosis is also called programmed cell death, and it can maintain normal cell development and homeostasis. It can eliminate the cells unable to repair, mutation, aging, or infected by pathogens. Cytokines, virus, and antitumor drugs can induce cell apoptosis [21,22]. TRAIL is an important ligand of the tumor necrosis factor (TNF) family that can induce cancer cells apoptosis with no effect on normal cells [23,24]. TRAIL inducing cell apoptosis through the death receptor pathway is an important antitumor mechanism. After binding with TRAIL receptor, TRAIL may cause FADD domain combined with pro-Caspase-8 to form DISC [25]. Pro-Caspase-8 became the activated form in DISC that can activate Caspase-9. It further activated Caspase-3 and DNA fragment factor 45 (DFF45) to promote apoptosis occurrence. In this process, some molecules, such as c-FLIP, RIP, and TRADD, were recruited to DISC to participate in apoptosis regulation [26,27]. This study confirmed that 2-DG and TRAIL reduced cell mitochondrial membrane potential and induced cell apoptosis, and that 2-DG depended on Caspases to enhance cell apoptosis induced by TRAIL.

c-FLIP is a kind of apoptosis-inhibiting protein similar to Caspase-8 [28]. Research confirmed that c-FLIP can bind with FADD to suppress Caspase-8 activation and restrain cell apoptosis [29,30]. Therefore, overexpressed c-FLIP can inhibit tumor cell apoptosis. Stanger et al. found that overexpressed RIP can activate NF-κB signaling pathway by yeast two-hybrid analysis [31]. Recent studies suggested that cancer cell c-FLIP and RIP expression levels are closely related to Caspase-8 activation, and overexpressed c-FLIP or RIP may suppress Caspase-8 activation [32]. Several studies have confirmed that RIP and c-FLIP activity were closely related to caspase-8 [33]. ER stress can regulate multiple caspases, including caspase-8 [34]. As an inhibitor of ER stress, 2-DG may affect RIP and c-FLIP expression through caspase-8. Further research is needed on the impact of 2-DG on caspase-8 activity and to explore its effect on RIP and c-FLIP. Our results showed that the c-FLIP and RIP are up-regulated in liver cancer cell HepG2 and Hep3B, and that 2-DG treatment can significantly decrease c-FLIP and RIP protein expression. In the HepG2 and Hep3B cells with RIP, c-FLIP gene silencing or down-regulation by 2-DG, the TRAIL-induced cell apoptosis rate increased significantly.

Conclusions

2-DG can down-regulate RIP and c-FLIP expression, thus increasing the liver cancer cell apoptosis induced by TRAIL. Therefore, 2-DG might be an important molecular target for inducing liver cancer cell apoptosis and liver cancer gene therapy.