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Introduction

Gallbladder cancer (GBC) is the most common, aggressive malignancy of the bile duct worldwide, showing an increasing tendency in its incidence and mortality, and associated with extremely poor prognosis (1,2). The 5-year survival rate has improved due to recent advances in diagnostic and therapeutic approaches, but the prognosis is still poor for the patients with an advanced stage, high local recurrence and distant metastasis. Because of low surgical resection rate and serious side effects caused by ineffective chemotherapy and radiation therapy, the majority of patients now have unsatisfactory results (3,4). Therefore, new effective strategies for this lethal neoplasm is urgently needed.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor (TNF) family, has the ability to induce apoptosis in transformed, but not normal cells, via extrinsic signaling pathways (5,6). TRAIL initiates cell death upon binding to death receptor DR4 (TRAIL-R1) and/or DR5 (TRAIL-R2) on the surface of cancer cells, which subsequently causes the formation of DISC (death inducing signaling complex) and caspase cascades (7). Caspase-8 is activated in the death receptor signaling pathway due to its death domain (8). The active caspase-8 cleaves and directly activates downstream effector caspase-3 and PARP, and finally initiates caspase-dependent apoptosis (9). TRAIL therapy has emerged as a promising cancer therapeutic strategy. However, some tumor cells can downregulate the expression of TRAIL-receptors and acquire resistance to TRAIL-induced apoptosis (10,11). As such, TRAIL combination treatments that can upregulate TRAIL-receptor expression or inhibit downregulation were proved to be of therapeutic benefit (12–14).

The ubiquitin-proteasome system (UPS) plays a vital role in the regulation of protein levels in eukaryotic cells, and related to cell proliferation, survival, differentiation and programmed cell death (15,16). The activity of UPS is closely associated with the incidence and progression of many human malignant tumors. Now, targeting the proteasome is evidenced to be one of the most effective strategies for cancer treatment (17). MG132, a reversible peptide-aldehyde of specific proteasome inhibitor (Fig. 1A), can effectively inhibit the activity of 26S proteasome and cause accumulation of certain proteins harmful to the survival of tumor cells, inducing cell cycle arrest and cell apoptosis (18–20). Evidence has shown that MG132 could inhibit the proliferation of tumor cells in a dose-dependent manner in cell cycle transformation, and with the cell cycle protein Cyclin A, Cyclin B and P27 accumulation, and cell cycle arrests in G2/M phase (21). Likewise, it has been reported that MG132 induced apoptosis of tumor cells by caspase-related signal transduction pathway mediated by death receptor (18). To date, the effect of proteasome inhibitor MG132 on GBC still remains unclear, and also the effect on TRAIL-induced apoptosis in GBC remains unknown. Therefore, it may be more promising to uncover the mechanism that links proteasome dysfunction caused by MG132 and TRAIL-induced apoptosis to the treatment of GBC.

Figure 1 MG132 (A) effectively inhibits the proliferation of gallbladder cancer cells (B) and induces apoptosis (C). (A) The structure of MG132. (B) The GBC-SD cells were treated with various concentrations of MG132 for 24, 48 and 72 h. Effects of MG132 on cell proliferation were determined by CCK-8 assay. Data are expressed as the mean ± SD from three independent experiments. (C) The indicated cancer cells were treated with the given concentrations of MG132 for 48 h, and then the whole-cell proteins were extracted for western blotting (C). Data are expressed as the mean ± SD from three independent experiments.

In this study, we investigated the effect of the representative proteasome inhibitor MG132 on TRAIL-induced apoptosis in GBC-SD cells in vitro and in vivo, and further elucidated the mechanism of the cell apoptosis induction. Our data evidenced that MG132 can inhibit the proliferation of GBC-SD cells and induce apoptosis. The induction of apoptosis by MG132 was mainly through the upregulation of DR5 and subsequent caspase activation, which could promote TRAIL-induced apoptosis in GBC-SD cells. Therefore, the combinatorial treatment of MG132 with TRAIL could be a new and effective strategy for TRAIL-resistant GBC.

Materials and methods

Reagents

The proteasome inhibitor MG132 (Z-Leu-Leu-Leucinal, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO) as a 40-µM stock solution and stored at −20°C. DMSO was purchased from the Beyotime Institute of Biotechnology (Jiangsu, China). Antibodies against caspase-3, PARP, caspase-8, DR5 and DR4 were obtained from Sigma-Aldrich. DMEM cell culture medium and fetal bovine serum (FBS) were purchased from Gibco Co. Ltd. (USA).

Cell line and mice

The human gallbladder cancer cell line (GBC-SD) was purchased from the Committee on Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Female BALB/c mice, 7 weeks of age and weighing ~25 g, were obtained from the Science Department of Experimental Animals of Fudan University in China. All mice were housed in a specific pathogen-Free (SPF) level B animal facility. The study was approved by the Review Board of Fudan University Shanghai Cancer Center and Fudan Medical College.

Cell culture and experimental conditions

GBC-SD cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin in a humidified incubator containing 5% CO2 at 37°C. Then, the cells were seeded into 96-well plates at a density of 2,000 cells per well, incubated for 12 h, and further treated with different concentrations of MG132 (2.5, 5, 10, 20, 40, 80 and 160 µM) for 24, 48 and 72 h. Then, 10 µl of Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) reagent was added to each well of the plates and the plates were incubated for 1 h at 37°C. After removal of the supernatant, 150 µl of DMSO was added to each well. The optical density (OD) was measured at 450 nm using a spectrophotometric plate reader (Bio-Rad Co., Hercules, CA, USA) and the cell survival rate was determined according to the absorbance relative to that of untreated controls. Data are reported as the mean ± standard deviation of OD values obtained in each group. The concentrations of MG132 in this study were previously confirmed as being the most favorable for the following experiment.

Flow cytometric analysis of apoptosis

Apoptosis was performed with the Annexin V/PI apoptosis kit according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). GBC-SD cells were treated with MG132 (10 µM), TRAIL (100 ng/ml), and the combination of MG132 and TRAIL (10 µM + 100 ng/ml) for 48 h. After washing twice with cold PBS, the cells were resuspended in 100 µl binding buffer at a density of ×106 cells/ml. Then, 2.5 µl of Annexin V-FITC and 1 µl of PI working solution (100 µg/ml) were added to these cells and incubated for 30 min in the dark. The samples were analyzed by a flow cytometer (BD Biosciences, San Diego, CA, USA).

Western blot assay

GBC-SD cells were harvested, washed twice with cold PBS, and lysed in RIPA buffer (Beyotime, Shanghai, China) supplemented with protease inhibitor (Roche Applied Science, Indianapolis, IN, USA) at 4°C for 5 min. Total protein of the supernatant was determined by the bicinchoninic acid (BCA) assay kit (Beyotime) with BSA as a standard. Equal amounts of protein (40 µg/lane) from each group were separated by SDS-PAGE and transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% skim milk, and incubated with the primary antibodies against caspase-3, PARP, caspase-8, DR5, DR4, and GAPDH at 4°C overnight. Then, the membranes were incubated with a horseradish peroxidase-conjugated goat anti-rabbit/anti-mouse secondary antibody (1:5,000; Abcam, Cambridge, UK) for 1 h at room temperature, and visualized by a Gel Doc 2000 (Bio-Rad).

Real-time PCR

Primers were synthesized and purchased from Beijing Sunbiotech Co., Ltd. (Table I). Total RNA of GBC-SD cells was extracted after 48 h of incubation using the Purelink™ Micro-to-Midi purification system (Invitrogen Co.). Then, the cDNA was synthesized using 1 µl of total RNA with 0.5 µl AMV reverse transcriptase. PCR was carried out using 2.5 µl cDNA, 0.1 µl Ex Taq HS, 0.1 µl forward primer and 0.1 µl reverse primer. The PCR program consisted of an initial 2-min step at 94°C, and 35 cycles of 15 sec at 94°C, 40 sec at 60°C, and 1 min cycles at 72°C, followed by 72°C for 5 min. The results were determined using a UV gel imaging system and analyzed using Quantity One software (Bio-Rad Inc.), and presented as the ratio of target genes to internal control GAPDH.

Table I Primer sequences for detection of mRNA expression.

Table I

Primer sequences for detection of mRNA expression.

Gene name	Sequence	Amplicons (bp)
Caspase-8	F: 5′-CGACCTTTGGTAGGCCAATC-3′		356
	R: 5′-GCCAATTTGTATTGCCCAACTAT-3′
Caspase-3	F: 5′-GGTTCATCCAGTCCCTTTGC-3′		278
	R: 5′-GCGAGTGAGAATGTGCATAAATTC-3′
PARP	F: 5′-ACGCACAATGCCTATGAC-3′		442
	R: 5′-CCAGCGGAACCTCTACAC-3′
DR4	F: 5′-CTGAGCAACGCAGACTCGCTGTCCAC-3′		506
	R: 5′-TCCAAGGACACGGCAGAGCCTGTGCCAT-3′
DR5	F: 5′-GCCTCATGGACAATGAGATAAAGGTGGCT-3′		502
	R: 5′-CCAAATCTCAAAGTACGCACAAACGG-3′
GAPDH	F: 5′-TGTGTCCGTCGTGGATCTGA-3′		346
	R: 5′-CCTGCTTCACCACCTTCTTGA-3′

Xenograft tumor experiments

Subcutaneous injection of MG132 (10 µM), TRAIL (100 ng/ml), and their combination were performed to evaluate the growth of GBC-SD xenografts in athymic nude mice. All mice were housed in the SPF level B animal facility. GBC-SD cells in log-phase growth were subcutaneously injected into the right flank of the mice. On day 10, the mice were randomly divided into four groups (6 mice/group). The control group received an injection of vehicle (10% DMSO and 90% PBS) intraperitoneally (i.p.) each day. The other three groups were administered an i.p. injection of MG132 at 10 mg/kg, TRAIL at 80 µg/kg and the combination of MG132 and TRAIL, respectively, at 10 mg/kg and 80 µg/kg every day. Visible subcutaneous tumor volumes were measured every 3 days with calipers, and then calculated by the formula: volume = (length × width2) / 2, where length and width represent the length and width of the tumor, respectively. After 3 weeks of the treatment, all nude mice were sacrificed, and the tumor tissue was removed and weighed.

Statistical analysis

All data are presented as mean ± SD. Statistical analysis was conducted using SPSS 11.0 (SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) was used to evaluate within group data and one-way ANOVA was used to evaluate between group data. Least squares difference was used for pairwise comparisons between groups. The statistical significance was defined as P<0.05.

Results

MG132 inhibits the proliferation of GBC-SD cells and induces apoptosis

To investigate the effect of MG132 on the proliferation of cells, GBC-SD cells were treated with increasing concentrations of MG132 (2.5, 5, 10, 20, 40, 80 and 160 µM) for 24, 48 and 72 h. We found that MG132 played a potent cytotoxic role on GBC-SD cells in a time- and dose-dependent manner (P<0.01) (Fig. 1B). Moreover, this inhibitory rate in GBC-SD cells increased rapidly when the increasing concentrations of MG132 were between 0 and 10 µM, when the increasing inhibitory rate slowed down the concentrations of MG132 were between 10 and 160 µM. Hence, MG132 has a more sensitive killing effect on GBC-SD cells at the concentrations of 0–10 µM than at 10–160 µM, and we chose the low concentrations of MG132 (2.5, 5 and 10 µM) at 48 h for the following treatment. We further detected the hallmarks of apoptosis in GBC-SD cells treated with MG132, and found that the protein levels of cleavage of caspase-8, caspase-3 and PARP obviously increased in a dose-dependent manner, which indicated that MG132 activated these caspases in the above cells (Fig. 1C). Therefore, MG132 clearly induces apoptosis in GBC-SD cells, and has an important mechanism for its tumor-killing effect.

MG132 induces apoptosis by upregulating the levels of DR5 and DR4 in extrinsic apoptotic pathway in GBC-SD cells. The data in a previous study (?) clearly exhibited that MG132 induced caspase-dependent apoptosis, especially activiated caspase-8 which is an initiator caspase in the extrinsic apoptotic pathway. To further determine the mechanism on the activation of extrinsic apoptosis invovled in this experiment, we detected the expression of DR5 and DR4 in the experiment, which are well-known as TRAIL death receptors and can initiate extrinsic apoptotic pathway. During the given concentration range of 2.5–10 µM, MG132 obviously upregulated the protein levels of DR5 and DR4 in a dose-dependent manner in GBC-SD cells, and the expression of DR5 was more obvious than that of DR4 (Fig. 2A and B). Consistent with the protein expression of DR5 and DR4 in MG132-treated cells, the mRNA levels of DR5 and DR4 were also substantially increased when treated with MG132 in GBC-SD cells (Fig. 2C and D). These results indicate that MG132 induces DR5 and DR4-dependent apoptosis through extrinsic apoptotic pathway in GBC-SD cells.

Figure 2 MG132 induces apoptosis by upregulating DR5 and DR4 expression in GBC-SD cells. The GBC-SD cells were treated with the given concentrations of MG132 for 48 h. The expression levels of DR5 and DR4 were detected by western blotting (A) and RT-PCR analysis (C), and GAPDH was used as a loading control. The data of DR5 and DR4 proteins (B) and genes (D) expression are expressed as the mean ± SD (n=3). **P<0.01 compared with the 0 µM group; ##P<0.01 compared with the 2.5 µM group; ΔΔP<0.01 compared with the 5 µM group.

MG132 potentiates TRAIL-induced apoptosis through caspase-dependent pathway in GBC-SD cells. In the above experiments, we confirmed that MG132 can enhance the expression of DR5 and DR4 in GBC-SD cells, which have been proved to be major death receptors for the death ligand TRAIL. Therefore, we hypothesized that MG132 could enhance the apoptosis induced by TRAIL. To confirm this hypothesis, we compared the cytotoxic effect of MG132 alone, TRAIL alone, and their combination on GBC-SD cells. We found that the given concentration of MG132 or TRAIL could effectively inhibit the survival of GBC-SD cells, while the combination could play a more effective role in the killing of GBC-SD cells (Fig. 3A). Consistently, the apoptosis induced by the combination of MG132 and TRAIL was more potent than each single agent (Fig. 3B and C). Moreover, we determined whether the combination of MG132 and TRAIL induces apoptosis by activation of the caspase-dependent pathway. We compared the effects of MG132 alone, TRAIL alone, and their combination in inducing caspase-8, caspase-3 and PARP on GBC-SD cells, and found that the tested protein and mRNA levels induced by the combination were higher than those treated with either MG132 or TRAIL alone (Fig. 4). Hence, these data further support that MG132 enhances TRAIL-induced apoptosis through caspase-dependent pathway in GBC-SD cells.

Figure 3 MG132 enhances the killing effect of TRAIL on GBC-SD cells. (A) The GBC-SD cells were treated with different concentrations of MG132, TRAIL, and the combination of MG132 and TRAIL for 48 h, and then determined by CCK-8 assay. Data are expressed as the mean ± SD from three independent experiments. (B) The GBC-SD cells were treated with MG132 alone, TRAIL alone and their combination for 48 h, and then stained with Annexin V/PI double staining. The lower left quadrant (Annexin V−/PI−), lower right quadrant (Annexin V+/PI−) and upper right quadrant (Annexin V+/PI+) represent the percentage of non-apoptotic cells, early apoptotic cells and late apoptotic cells, respectively. (C) The ration of apoptotic cells is presented as the mean ± SD (n=3). **P<0.01 compared with the control group; ##P<0.01 compared with the MG132 group or TRAIL group.

Figure 4 MG132 potentiates TRAIL-induced apoptosis in GBC-SD cells by activating caspase-dependent pathway. The GBC-SD cells were treated with the given concentrations of MG132, TRAIL, and their combination for 48 h, and then the whole-cell proteins were extracted for western blotting (A) or RT-PCR analysis (C). The data of apoptosis-related proteins (B) and genes (D) activities are expressed as the mean ± SD (n=3). **P<0.01 compared with the control group; ##P<0.01 compared with the MG132 group or TRAIL group.

The combination of MG132 and TRAIL augments the killing effect of gallbladder cancer cells in vivo

To observe whether MG132 could enhance the killing effect of TRAIL on GBC-SD cells in vivo, we measured the weight and volume of GBC-SD xenografts in this experiment (Fig. 5A and C). Tumors excised from the mice are shown in Fig. 5B, and the tumor weights after 3 weeks of treatment showed an decreasing tendency from control to MG132 to TRAIL to combination groups (Fig. 5A). The differences between the groups were statistically significant (P<0.01). In addition, a significant reduction was also detected in tumor volumes treated with the combination compared to those treated with either MG132 or TRAIL alone (Fig. 5C). To examine whether the impact of MG132 and TRAIL on tumor growth inhibition was related to extrinsic apoptotic pathway, we detected the protein levels of DR5, DR4, caspase-8, caspase-3 and PARP in the gallbladder tumor tissues by western blot analysis, and found that the upregulation of DR5, DR4, cleaved caspase-3, caspase-8, and cleaved PARP induced by the combination were more obvious than those treated with either MG132 or TRAIL alone, which was in agreement with the results of the in vitro experiment (Fig. 5D).

Figure 5 MG132 in vivo enhances the killing effect of TRAIL (A–C) on GBC-SD cells by activating the extrinsic apoptotic pathway (D). GBC-SD cells were subcutaneously injected into the right flank of the nude mice, and then the mice were treated with MG132 alone, TRAIL alone, their combination or vehicle (10% DMSO and 90% PBS) intraperitoneally every day for 3 weeks. (A and B) Tumors were excised from the animals and weighed. (C) Tumor dimensions were periodically measured with calipers. The data are illustrated as the mean ± SD (n=6). (D) The apoptosis-related proteins of tumor tissues were extracted for western blotting. Data are expressed as the mean ± SD from three independent experiments. **P<0.01 compared with the control group; ##P<0.01 compared with the MG132 group or TRAIL group.

Discussion

In this study, we demonstrated that the proteasome inhibitor MG132 can effectively inhibit the proliferation of GBC-SD cells and induce cell apoptosis in vitro and in vivo. Apoptosis of GBC-SD cells induced by MG132 were mainly through the activation of extrinsic apoptosis pathway. In addition, we evidenced that MG132 can enhance TRAIL-induced apoptosis in GBC-SD cells in vivo and in vitro. As far as we know, this is the first study to indicate that MG132 induces apoptosis in GBC-SD cells by activating the extrinsic apoptotic pathway.

Death receptor DR5 and DR4 are pro-apoptotic receptors of TRAIL ligand, which can trigger the extrinsic apoptotic signaling when interacting with TRAIL (7). To date, TRAIL-receptors, especially DR5 and DR4, have emerged as key mediators of cell apoptosis (12,22). In this study, we found that MG132 can upregulate the expression of DR5 and DR4 protein and mRNA in GBC-SD cells in a dose-dependent manner, and the expression level of DR5 was significantly stronger than that of DR4. Then it was investigated whether upregulation of DR5 caused by MG132 could potentiate TRAIL-induced apoptosis in GBC-SD cells.

The ubiquitin-proteasome system (UPS) is a key regulator of protein degradation in cellular biological processes including apoptosis (23). It has been confirmed that death receptor-mediated apoptotic pathway is regulated by UPS (24). In this study, we found that proteasome inhibitor MG132 can increase the expression level of DR5 protein in a dose-dependent manner. Thus, we speculate that the upregulation of DR5 protein in tumor cells may be attributed to the dysfunction of proteasome caused by MG132. Furthermore, the expression of DR5 also increased at the transcriptional level. In this study, we observed that MG132 can enhance the expression level of DR5 mRNA in a dose-dependent manner. Hence, the transcriptional regulation of DR5 expression is another mechanism for the upregulation of DR5 expression. In conclusion, the upregulation of DR5 expression caused by MG132 may be due to enhanced protein stability and gene transcription, which is consistent with previous studies (25–27).

At present, TRAIL has been proven to have significant potential in cancer therapy for its good tumor specificity and favorable safety (28). TRAIL-based therapies are being increasingly explored also in clinical trials (29,30). Unfortunately, many tumor cells are resistant to TRAIL-induced apoptosis. Combination therapy has been evidenced to be an effective way to overcome tumor resistance. Some studies have demonstrated that treatment with proteasome inhibitors can upregulate TRAIL-receptors expression on tumor cell surface, which promoted sensitization to TRAIL-induced apoptosis (31,32). In this study, we found that MG132 in vitro and in vivo can enhance TRAIL-mediated apoptosis in GBC-SD cells, and this process involves upregulation of DR5 and caspase-dependent pathways (Fig. 6). Thus, we speculate that MG132 can enhance TRAIL-induced apoptosis via upregulating DR5 expression in GBC-SD cells, which may overcome TRAIL resistance in cancer therapy.

Figure 6 Schematic diagram of the signaling pathway induced by the combined treatment with MG132 and TRAIL.

In conclusion, we have established that proteasome inhibitor MG132 can potentiate TRAIL-induced apoptosis both in vitro and in vivo, and uncovered that MG132 induces apoptosis by its effect on the upregulation of DR5. In this study, we have provided experimental evidence demonstrating a possible tumor suppressor role of MG132 in GBC-SD cells, and its increasing effect on TRAIL-mediated activities.

Acknowledgments

The authors would like to thank Xin Wang for technical support. This study was supported by the Minhang District Natural Science Foundation of Shanghai (2012MHZ025), the Public Health Bureau Youth Foundation of Shanghai (20134Y089), the Natural Science Foundation of Shanghai (12nm0502202 and 114119a4700), the Pudong New Area Science and Technology Development Fund (PKJ2012-Y24), and the Pudong New Area Health System discipline lead development program (PWRd2013-10).

Abbreviations:

References